Dispositivo Medico ChondroGrid · ricostruzione del tessuto cartilagineo articolare. Per queste...

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Dispositivo Medico ChondroGrid®

Report di valutazione clinica: profilo di efficacia e sicurezza

Revisione 01 del 24.07.2016

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Sommario

Introduzione ......................................................................................................................................... 4

Standard seguiti per la realizzazione del documento. ...................................................................... 5

Autori................................................................................................................................................ 5

Caratteristiche del dispositivo .............................................................................................................. 6

Il dispositivo ChondroGrid ............................................................................................................... 6

Il meccanismo d’azione: il collagene e le sue potenzialità terapeutiche .......................................... 6

Le indicazioni d’uso e l’applicazione ............................................................................................... 7

Il ventaglio di indicazioni ................................................................................................................. 7

Un’alternativa sicura, almeno parziale, all’impiego degli antidolorifici .......................................... 8

La qualità farmaceutica della materia prima .................................................................................... 9

La sterilizzazione terminale e l’integrità dei peptidi ...................................................................... 10

Il dispositivo ChondroGrid: classificazione CE ............................................................................. 10

Bioteck: il Fabbricante ................................................................................................................... 11

Materiali e metodi di reperimento delle informazioni ....................................................................... 12

Reperimento degli articoli significativi .......................................................................................... 13

Creazione di un database bibliografico di riferimento ................................................................... 15

Analisi del database di riferimento ................................................................................................. 15

ChondroGrid: Profilo di sicurezza ..................................................................................................... 16

Considerazioni generali sulla sicurezza dell’impiego del collagene in ambito biomedico ............ 16

Valutazione dei rischi connessi all’uso di ChondroGrid ................................................................ 17

Possibile trasmissione di patogeni animali ..................................................................................... 18

Possibile trasmissione di encefalopatie spongiformi ...................................................................... 18

Possibile immunogenicità o mancanza di biocompatibilità ........................................................... 18

Test di citotossicità..................................................................................................................... 20

Test di sensibilizzazione allergica .............................................................................................. 20

Test di reattività intracutanea ..................................................................................................... 20

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Test di tossicità sistemica acuta ................................................................................................. 21

Pirogenicità ................................................................................................................................ 21

Test di tossicità subacuta............................................................................................................ 21

Test di genotossicità ................................................................................................................... 22

Test d’impianto .......................................................................................................................... 22

Valutazione del rischio residuo complessivo ................................................................................. 23

Valutazione da parte della autorità competenti degli altri Paesi Europei: ..................................... 23

Gli standard tecnici impiegati nella progettazione e fabbricazione del dispositivo ....................... 24

ChondroGrid: profilo di efficacia ...................................................................................................... 26

Effetti biologici del collagene e contestualizzazione dell’impiego di ChondroGrid ..................... 26

Studi in vitro ................................................................................................................................... 30

Studi in vivo ................................................................................................................................... 32

Studi clinici..................................................................................................................................... 33

Conclusioni ........................................................................................................................................ 36

Riepilogo dei risultati ......................................................................................................................... 37

Razionale biologico dell'utilizzo del dispositivo ............................................................................ 37

Razionale clinico dell'utilizzo del dispositivo ................................................................................ 37

Livello di evidenza relativo alla sicurezza dell'impiego clinico del dispositivo ............................ 37

Tabelle Riassuntive ............................................................................................................................ 39

Sicurezza del dispositivo ChondroGrid .......................................................................................... 39

Efficacia del dispositivo ChondroGrid. .......................................................................................... 40

Domande Frequenti – Frequently Asked Questions (FAQ) ............................................................... 42

Bibliografia ........................................................................................................................................ 45

Appendici ......................................................................................................................................... 130

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Introduzione

ChondroGrid è un dispositivo medico innovativo, destinato all’uso professionale, per il trattamento

sintomatologico e funzionale di numerose affezioni a carico delle articolazioni, dei tendini e dei

legamenti. ChondroGrid è innovativo in quanto è progettato e realizzato per permettere al paziente

di beneficiare al meglio delle proprietà terapeutiche del collagene, una proteina ampiamente diffusa

nel regno animale, nonché quella maggiormente rappresentata nel corpo umano ed il principale

costituente della matrice cartilaginea, dei tendini e dei legamenti.

Le due caratteristiche principali di ChondroGrid che permettono l’ottenimento del migliore effetto

terapeutico consistono a) nell’impiego di collagene in forma idrolizzata, a basso peso molecolare e

b) nella modalità di somministrazione, che avviene tramite iniezione intra- o peri-articolare.

Avere idrolizzato il collagene realizza due importanti vantaggi: a) il collagene in forma idrolizzata

si può facilmente sospendere in fase acquosa e b) esso può diffondere rapidamente, essendo i

peptidi molecole di dimensione limitata, negli spazi intra- e peri-articolari, distribuendosi su tutta la

superficie raggiunta dal liquido stesso.

La modalità di somministrazione, attraverso iniezione, permette di portare direttamente il collagene

idrolizzato al sito di interesse, ove è necessario che esso esplichi la sua azione. ChondroGrid,

quindi, rende possibile un’azione molto semplice: arricchire di collagene tutto l'ambiente intra-

articolare o peri-articolare soggetto a trauma o degenerazione o altra patologia.

Poiché le componenti articolari (matrice cartilaginea) e peri-articolari (tendini e legamenti) hanno,

come loro costituente principale, il collagene stesso e poiché, come sarà più oltre spiegato in

dettaglio, il collagene e i peptidi di origine collagenica sono in grado di unirsi fra loro

spontaneamente, il collagene idrolizzato sarà in grado immediatamente di fornire rinforzo

strutturale alla componente lesionata.

Inoltre, è stato provato che la presenza del collagene in forma peptidica è in grado, ad esempio nelle

cartilagini affette da osteoartrosi, di ridurre l’infiammazione e quindi il dolore a livello

dell’articolazione, contribuendo allo stesso tempo a migliorare il trofismo cellulare.

Scopo del presente report è fornire al lettore gli elementi necessari alla comprensione del

meccanismo d’azione del dispositivo ChondroGrid, e di illustrarne – nel contesto di una puntuale e

dettagliata valutazione clinica – i profili di sicurezza e di efficacia.

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Standard seguiti per la realizzazione del documento.

La valutazione clinica del dispositivo medico ChondroGrid descritta nel presente report è stata

eseguita secondo le indicazioni contenute nelle linee guida MEDDEV 2.7.1 Rev.3 del Dicembre

2009, pubblicate dal Direttorato Generale della Commissione Europea, settore beni di consumo,

cosmetici e dispositivi medici: “Guidelines on medical devices. Clinical evaluation: a guide for

manufacturers and notified bodies” (Linee guida sui dispositivi medici. Valutazione clinica: una

guida per i fabbricanti e gli organismi notificati).

La valutazione è basata su di un’analisi esaustiva dei dati clinici disponibili sul dispositivo in

relazione al suo uso inteso, al fine di evidenziare come il dispositivo soddisfi i requisiti di sicurezza

ed efficacia espressi dal suo fabbricante, sulla base di dati scientifici e clinici oggettivi,

evidenziando come l’impiego del dispositivo ChondroGrid per il suo inteso realizzi una situazione

per il paziente nella quale il beneficio supera abbondantemente il rischio residuo.

Autori

Il presente report è stato redatto da Clariscience S.r.l. – www.clariscience.com – azienda di

consulenza scientifica specificamente rivolta ai fabbricanti e distributori di dispositivi medici.

All'interno di Clariscience opera personale di estrazione biomedica ed esperti in affari regolatori

con esperienza più che decennale nel settore.

http://www.clariscience.com/

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Caratteristiche del dispositivo

Il dispositivo ChondroGrid

ChondroGrid è un dispositivo medico composto costituito da collagene idrolizzato a basso peso

molecolare, di origine bovina, liofilizzato, per iniezione intra- e peri-articolare.

CHondroGrid è indicato per il trattamento della sintomatologia dolorosa e della perdita di

funzionalità delle maggiori articolazioni (ginocchio, spalla, anca, polso e caviglia) e delle strutture

muscolo-tendinee e legamentose ad esse annesse, causate da patologie degenerative o dovute a esito

traumatico o a sovraccarico.

In Appendice A è riportato il foglietto illustrativo del dispositivo.

Il meccanismo d’azione: il collagene e le sue potenzialità terapeutiche

Il meccanismo d’azione di ChondroGrid si basa sulle molteplici caratteristiche possedute dal

collagene, struttura proteica come si è detto ubiquitaria la cui funzione biologica è sia strutturale che

funzionale. Da un punto di vista strutturale, in quanto proteina fibrillare in grado di autoassemblarsi

in modo gerarchico in fibre mano a mano più complesse (Kadler et al., 2008) esso garantisce la

creazione di strutture estremamente elastiche e, allo stesso tempo resistenti: sia nella costituzione

della matrice amorfa che compone la cartilagine, sia nelle strutture organizzate di connettivi

specializzati quali tendini e legamenti, sia infine in tessuti di sostegno quale quello osseo. A livello

funzionale, la sua azione si esplica in molteplici modi. Alcuni di questi, struttura-mediati,

consistono nella capacità di interazione con molecole diffusibili quali piccole proteine o ioni: in

questo senso il collagene può agire da filtro ionico o molecolare, facilitando la diffusione di alcuni

ioni o molecole e impedendo quella di altre (in funzione sia della carica che della dimensione

molecolare); esempio limite di questa proprietà del collagene di interagire con altri composti è la

capacità che esso dimostra nel tessuto osseo di autocatalizzare la propria mineralizzazione,

favorendo la formazione e deposizione sulla sua stessa fibra di cristalli di apatite ossea. Altri effetti,

invece, sono basati su precisi meccanismi biomolecolari e prevedono l'interazione tra porzioni della

molecola collagenica (anche di pochi amminoacidi) con specifici recettori localizzati a livello della

membrana cellulare di differenti tipi cellulari, siano essi precursori di cellule differenziate o cellule

già al termine del processo differenziativo. Si può quindi a ragione parlare del collagene, sia in

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senso lato che nello specifico ambito della medicina rigenerativa, come di una molecola ad azione

pleiotropica.

Il meccanismo d’azione del dispositivo Chondrogrid, quando iniettato per via intra-articolare

consiste nel distribuirsi del collagene a basso peso molecolare sulla superficie dell’articolazione

rinforzando la matrice cartilaginea, deteriorata dai processi patologici in atto, la cui impalcatura

sono formate da un fitto intreccio di fibre collagene. Il dispositivo svolge quindi un’azione

meccanica di rinforzo diretto di strutture collageniche indebolite e/o deteriorate, migliorando la

mobilità e contribuendo a ridurre la sintomatologia dolorosa a carico dell’articolazione. Il

meccanismo per via peri-articolare è simile: il collagene idrolizzato a basso peso molecolare agisce

quale rinforzo diretto all’impalcatura collagenica danneggiata delle strutture peri-articolari, quali

tendini e/o legamenti, la cui componente più abbondante è il collagene stesso - contribuendo ad una

riduzione del dolore e ad una più veloce ripresa funzionale.

Le indicazioni d’uso e l’applicazione

Le più comuni indicazioni di utilizzo di ChondroGrid sono il trattamento sintomatologico e

funzionale di: osteoartrosi, artrosinoviti acute o croniche secondarie ad artrosi o artrite reumatoide,

esiti di traumi o lesioni, affaticamento da sovraccarico a carico delle articolazioni sopra indicate.

ChondroGrid è indicato inoltre in caso di meniscopatie e in preparazione e come terapia di

mantenimento a seguito di interventi di meniscectomia, di ricostruzione legamentosa o di pulizia e/o

ricostruzione del tessuto cartilagineo articolare. Per queste indicazioni l’applicazione è eseguita per

via intra-articolare.

Per esiti di traumi e/o lesione peri-articolari o a carico di strutture muscolo-tendinee-ligamentose si

procede invece tramite iniezione peri-articolare e infiltrazione delle strutture coinvolte.

Il ventaglio di indicazioni

ChondroGrid, date le indicazioni per l’uso ed il meccanismo d’azione presentati nei paragrafi

precedenti, è indicato per il trattamento della sintomatologia dolorosa a carico di differenti

articolazioni in differenti distretti corporei, quali:

- l’anca

- le articolazioni vertebrali lombo-sacrali

- il ginocchio

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- la spalla

- le articolazioni minori, quali il gomito, la caviglia, il polso

sia che il dolore abbia origine dall’invecchiamento, da difetti posturali, patologie croniche o

cronico-degenerative, o traumi. Il dispositivo ha pertanto un ampio ventaglio di indicazioni, e può

essere d’ausilio nel trattamento di patologie particolarmente comuni, la cui incidenza nella

popolazione è destinata ad aumentare a causa dell’incremento della vita media cui si assiste, e si

assisterà, nel corso degli anni. Ulteriormente, per lo meno nei Paesi occidentali e industrializzati si

osserva un aumento di attenzione della popolazione alla qualità della vita, aumento che spinge i

pazienti a chiedere trattamenti più efficaci per tutte quelle patologie che sono potenzialmente

debilitanti e che possono mettere a rischio il benessere complessivo della persona.

Un’alternativa sicura, almeno parziale, all’impiego degli antidolorifici

In questo contesto, il dispositivo ChondroGrid fornisce all’utilizzatore medico la possibilità di

ridurre in modo sicuro l’impiego dei farmaci antidolorifici da banco o da prescrizione. Infatti,

ChondroGrid:

- agisce in modo locale nelle zone dolenti (e non per via sistemica)

- esibisce un tropismo naturale verso le strutture danneggiate (il collagene lega

spontaneamente altre strutture collageniche per formare strutture composte quali fibrille e

fibre)

- inoltre, e soprattutto, il dispositivo non presenta effetti collaterali o allergenicità (salvo il

caso in cui il paziente presenti un’ipersensibilità al collagene, per quanto rara) in quanto gli

xeno-collageni, un volta idrolizzati e sottoposti ad eliminazione dei telopeptidi N- e C-

terminali, perdono le loro proprietà immunogeniche (Cooperman L et al., 1984; De Lustro F

et al., 1987; DeLustro F et al., 1990).

L’impiego del dispositivo può quindi contribuire a ridurre la somministrazione di farmaci,

analgesici e FANS, che pur attenuando la sintomatologia dolorosa non esercitano alcuna azione sui

fattori scatenanti, ed espongono inoltre il paziente a potenziali effetti avversi quali la tossicità

gastrointestinale, il possibile aumento di effetti a carico del sistema cardiovascolare, renale ed

epatico. ChondroGrid, invece, fornisce in modo diretto piccole porzioni ed aminoacidi specifici

della struttura collagenica stessa, il che contribuisce al recupero strutturale e funzionale delle

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strutture intra- (cartilagine) e peri- (tendini e legamenti) articolari, migliorando così la

sintomatologia dolorosa e la funzionalità in modo sicuro ed efficace.

La qualità farmaceutica della materia prima

Da un punto di vista prettamente molecolare, il collagene (o, meglio, le famiglie dei collageni) non

differisce in modo sostanziale tra le diverse specie di Mammiferi, mostrando la sequenza

amminoacidica del protocollagene solo minime variazioni da una specie all’altra (Wada H et al.,

2006; Stover DA et al., 2011). Il dispositivo, dunque, potrebbe essere teoricamente fabbricato a

partire da collagene proveniente da qualunque specie di Mammifero. ChondroGrid è fabbricato a

partire da gelatina di collagene bovino, di grado farmaceutica e di altissima purezza. La scelta

dell’impiego di gelatina di collagene di origine bovina risiede nel fatto che la tecnologia alla base

della sua estrazione è oggi ampiamente collaudata in termini di qualità di prodotto finito, le cui

caratteristiche sono tali da superare tutti i requisiti estremamente stringenti che si applicano in

ambito farmaceutico.

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La sterilizzazione terminale e l’integrità dei peptidi

Il dispositivo ChondroGrid è sterilizzato, alla fine del processo per la sua fabbricazione, attraverso

irraggiamento beta (elettroni accelerati) a 25 kGy. Questo processo assicura che il prodotto finale

sia sterile, con un fattore di sicurezza conforme agli standard internazionali di riferimento uguale a

10-6. Gli elettroni accelerati sono radiazioni ionizzanti, ovvero radiazioni in grado di degradare gli

acidi nucleici batterici e/o virali, uccidendo così i microorganismi. Potrebbe quindi sorgere il

dubbio che tali radiazioni ionizzanti possano anche degradare la miscela peptidica di cui è composto

ChondroGrid. Questo non accade: il fabbricante ha infatti verificato, attraverso opportuni test

cromatografici, che il trattamento di sterilizzazione attraverso irraggiamento beta non è in grado di

degradare i peptidi presenti in ChondroGrid, come illustrato dall’immagine che segue.

Figura1. Il grafico mostra il profilo di eluizione in HPLC (High Performance Liquid Chromatography) di ChondroGrid

prima (rosso) e dopo (blu) la sterilizzazione attraverso irraggiamento beta. Ogni picco nel grafico corrisponde ad un

particolare peptide. Si può osservare come non vi sia differenza nella distribuzione dei picchi tra le due curve, a

dimostrazione del fatto che nessun peptide subisce degradazione a causa del processo di sterilizzazione.

Il dispositivo ChondroGrid: classificazione CE

Il dispositivo è un dispositivo impiantabile, di origine animale e destinato ad essere assorbito

completamente a seguito di impianto. Per questo motivo è classificato in classe III secondo la

direttiva di riferimento europea 93/42/EEC e s.m.i., relativa ai dispositivi medici. Il dispositivo è

registrato presso il Repertorio dei Dispositivi Medici del Ministero della Salute con numero di

iscrizione 1412112/R.

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Bioteck: il Fabbricante

ChondroGrid è prodotto da Bioteck S.p.A., azienda italiana leader mondiale nella fabbricazione di

dispositivi medici impiantabili di origine animale per rigenerazione tessutale. Bioteck opera in

questo settore dal 1995. Azienda a compagine sociale interamente italiana, applica processi

biotecnologici avanzati per la creazione di sostituti tessutali e dispositivi coadiuvanti per la

rigenerazione tessutale all’avanguardia. Attualmente, Bioteck è presente in più di 50 Paesi. I

dispositivi medici fabbricati da Bioteck sono impiegati con successo in diversi ambiti,

dall’Ortopedia, alla Neurochirurgia, alla Chirurgia Oro-maxillo-facciale.

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Materiali e metodi di reperimento delle informazioni

Le informazioni utilizzate per la redazione del presente report sono state raccolte attraverso due

canali.

Per tutto quanto riguarda il processo di fabbricazione di ChondroGrid, la tecnologia impiegata per

la sua realizzazione, le caratteristiche degli ambienti di fabbricazione, i test di biocompatibilità

eseguiti prima della sua immissione in commercio, si è analizzata e valutata la documentazione

tecnica, messa gentilmente a disposizione dal fabbricante, che lo stesso ha presentato in sede di

procedura di marcatura CE all’Organismo Notificato di riferimento (CE 0373, Istituto Superiore di

Sanità). Tali informazioni, analizzate, sono confluite principalmente nella definizione del profilo di

sicurezza del dispositivo.

Per quanto riguarda invece l’approfondimento relativo al profilo di efficacia del dispositivo si è

proceduto, oltre ad analizzare le informazioni fornite dal fabbricante, ad una specifica ricerca

bibliografica in merito, secondo le modalità di seguito descritte.

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Reperimento degli articoli significativi

Al fine di identificare i dati ritenuti pertinenti in letteratura è stata eseguita una ricerca bibliografica

interrogando il maggiore database biomedicale mondiale, PubMed, gestito dal National Institute of

Health (NIH) americano (www.pubmed.com ).

La ricerca, visto l'elevato numero di lavori sull'impiego del collagene in ambito biomedico e la

conseguente mole di articoli da analizzare, ha seguito un approccio "top-down" ove si è operato

inserendo nel motore di ricerca parole chiave sempre più stringenti in una procedura di "narrowing

down" al fine di identificare gli articoli realmente pertinenti secondo un processo di natura logico-

deduttiva.

Per ogni parola/frase chiave, constatata la dimensione del corrispondente insieme di articoli

corrispondenti, si è deciso di a) eseguire un "narrowing down" con parole chiave più stringenti

oppure b) analizzare gli abstract degli articoli reperiti nel database per identificare gli articoli

inerenti. Si è quindi proceduto al recupero, ove possibile, degli articoli stessi e all’analisi della

bibliografia in essi citata per il reperimento di ulteriori articoli inerenti. La soglia di cut-off relativa

all'operatività a) o b) è stata decisa arbitrariamente in 150 pubblicazioni. E' da notare che, nella

strategia di ricerca "narrowing down" anche i risultati delle ricerche ove non si sono analizzati gli

abstract relativi portano con sé un'informazione significativa, ovvero il numero di lavori inerenti.

Gli abstract degli articoli ritenuti pertinenti sono presentati in Bibliografia. Gli articoli scientifici

contenuti nel database di riferimento sono stati letti e valutati criticamente in funzione alla loro

relazione con ChondroGrid, identificando in primo luogo le analogie o le differenze di

composizione chimica tra i prodotti e/o i composti testati nelle pubblicazioni con quelle del

dispositivo; in secondo luogo – per gli studi su animale e clinici – si sono anche valutate le analogie

o le differenze con il dispositivo del Fabbricante per quanto riguarda la modalità di impiego del

dispositivo e, chiaramente, le indicazioni cliniche proposte dal Fabbricante in funzione dei risultati

attesi.

http://www.pubmed.com/

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La tabella seguente riporta le parole chiave utilizzate e i risultati della ricerca dopo il narrowing

down.

Parola/frase chiave

Numero di articoli reperiti

Numero di articoli inerenti

Ulteriori articoli reperiti dall’analisi della bibliografia degli articoli inerenti

Totale articoli inerenti

Low molecular weight hydrolyzed collagen

19 13 6 18

Intra-articular collagen injection

296 5 0 5

Tabella 1. Articoli reperiti sul sito http://www.ncbi.nlm.nih.gov/pubmed. La ricerca è stata eseguita in data 20 giugno 2016.

Ulteriormente, sono stati consultati database già in possesso degli autori raccoglienti le

pubblicazioni più significative, apparse su riviste indicizzate ed impattate, riguardanti la struttura

molecolare e le azioni biologiche principali del collagene in quanto componente delle matrici

extracellulari dei tessuti di origine connettivale.

http://www.ncbi.nlm.nih.gov/pubmed

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Creazione di un database bibliografico di riferimento

Gli articoli scientifici considerati inerenti al dispositivo del Fabbricante, sono stati quindi

raggruppati in un database generale, consultabile in bibliografia, e possono essere distinti per

tipologia di studio reperito come descritto nella tabella che segue.

Tipologia di studio Sottotipologia Numero di articoli inerenti

Studio in vitro 21

Studio su animale 25

Studio clinico (*) Case reports 1

Cross sectional surveys 1

Case-control studies 1

Cohort studies 2

Randomised controlled trials with non-definitive results (a point estimate that suggests a clinically significant effect but with confidence intervals overlapping the threshold for this effect)

0

Randomised controlled trials (RCT) with definitive results (confidence intervals that do not overlap the threshold clinically significant effect)

7

Systematic reviews and meta-analyses

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Articoli reperiti e analizzati 84

Tabella 2. Composizione del database di riferimento.

(*) La classificazione degli studi clinici utilizzata è quella descritta in: Guyatt GH, Sackett DL, Sinclair JC, Hayward R, Cook DJ,

Cook RJ. Users' guides to the medical literature. IX. A method for grading health care recommendations. JAMA 1995; 274:1800-4.

Analisi del database di riferimento

Gli articoli scientifici e clinici contenuti nel database di riferimento sono stati letti e valutati

criticamente in funzione alla loro relazione con il dispositivo, identificando in primo luogo le

analogie o le differenze di composizione chimica tra i prodotti e/o i composti testati nelle

pubblicazioni con quelle del dispositivo stesso; in secondo luogo – per gli studi su animale e clinici

– si sono anche valutate le analogie o le differenze con il dispositivo ChondroGrid per quanto

riguarda la modalità di impiego e, chiaramente, le indicazioni cliniche proposte in funzione dei

risultati attesi.

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ChondroGrid: Profilo di sicurezza

Considerazioni generali sulla sicurezza dell’impiego del collagene in ambito biomedico

Un’analisi ad ampio respiro dell’impiego del collagene come biomateriale mostra che esso investe

vari ambiti quali l’impiego come materiale con attività osteogenica, per la sua attività pro-

coagulante, come rivestimento per la guarigione di ferite e ustioni. Recentemente è stato usato

anche come veicolo per numerosi farmaci; in questi casi il collagene viene usato sotto forma di

microsfere e reti la cui struttura è costituita appunto da tale sostanza; da ricordare inoltre l’esistenza

di membrane costituite da collagene sintetico in oftalmologia e i composti costituiti da collagene

liposomiale destinato al rilascio di farmaci (Wakitani et al., 1989). Il collagene è anche utilizzato

dall’ingegneria biomedica come substrato fisico per la creazione in vitro di pelle, vasi sanguigni e

delle valvole cardiache. I film di collagene sono usati anche come matrice per la valutazione della

calcificazione tissutale negli studi sulle neoplasie (Lee et al., 2001). Sono descritti in letteratura usi

del collagene iniettabile anche in ambito urologico (reflusso vescicouretrale, trattamento

dell’incontinenza urinaria) e in ambito gastroenterico (trattamento del reflusso gastroesofageo

trattamento dell’insufficienza della glottide) (Aragona et al., 1998). Il collagene è usato in ambito

chirurgico in vari campi quali la chirurgia cardiovascolare, la neurochirurgia, la chirurgia plastica,

nonché nella fase ricostruttiva delle ferite e come emostatico. Il collagene di tipo I è usato

comunemente nella costituzione dei dispositivi medici impiantabili (Yunus Basha et al., 2015), ad

esempio in interventi di rigenerazione ossea, sotto forma di materiale spugnoso, anche per la

costituzione di matrici atte a prolungare la permanenza delle proteine morfogenetiche dell’osso

(bone morphogenetic proteins, BMPs), o come scaffold per dare sostegno all’invasione dei vasi e

delle cellule osteoprogenitrici (Geiger et al., 2003) anche in combinazione con l’idrossiapatite per la

costituzione innesti ossei (Wahl et al., 2006). Non sono riportati in letteratura problemi di

interazione del collagene con farmaci o eccipienti utilizzati per la loro fabbricazione (Lucey P et al,

2014).

Il collagene idrolizzato è considerato in primis un alimento, per ragioni storiche connesse al suo

impiego, e per questo tossicità ed effetti collaterali sono già stati ampiamente valutati. La FDA lo

definisce infatti come “Generally Recognized As Safe” (GRAS), ponendolo nella categoria più

elevata di sicurezza, la stessa categoria degli aminoacidi essenziali, ma anche del sale, dello

zucchero, delle vitamine. Nessun ente nazionale o internazionale impone restrizioni all’uso del

collagene, riconoscendo l’impiego del collagene idrolizzato come additivo alimentare sicuro e come

integratore autorizzato in ambito medico. La tossicità del collagene si osserva a dosi così elevate

17

(Takeda et al., 1982) da potere affermare con sicurezza che non sussiste la possibilità di una

tossicità sistemica nell’impiego del dispositivo ChondroGrid. Ulteriori studi sul collagene

idrolizzato condotti su Salmonella, E. coli e midollo osseo di Hamster attraverso il Test di Ames

non hanno mostrato né effetti di mutagenicità né carcinogenicità (come si vedrà poi tali risultati

sono confermati da tutta la batteria di test di biocompatibilità condotti sul dispositivo

ChondroGrid).

Il collagene impiantato, idrolizzato o meno, è degradato dall’azione di una serie di enzimi

collagenolitici rilasciati da diverse popolazioni cellulari, compresi i granulociti neutrofili e i

macrofagi (Patino et al., 2002). I casi di infiammazione indotta dal collagene, tuttavia, sono

estremamente rari. In ambito di impianto dermico, ad esempio, è stata riscontrata un frequenza di

infiammazione indotta da collagene in soli 72 casi su oltre 1 milione presi in considerazione (Cukier

et al, 1993). L’impiego ormai più che decennale di numerosi dispositivi medici costituiti da

collagene per la rigenerazione e la riparazione dei tessuti sia molli che duri confermano come

l’impianto non crei fenomeni né di irritazione locale né di tossicità sistemica: evidenziando che

un’eventuale risposta immunitaria nei confronti dei loro costituenti collagenici non predispone

comunque a fenomeni di ipersensibilità successivi (DeLustro F et al., 1990). Come per tutte le

proteine, esiste anche per il collagene ed i suoi derivati la possibilità, per quanto rara (Charriere G et

al. 1989), di causare sensibilizzazione e reazioni allergiche in singoli pazienti predisposti. Tale

eventualità, opportunamente segnalata nella documentazione che accompagna il dispositivo, deve

essere accertata attraverso un’accurata anamnesi nei confronti del paziente.

Valutazione dei rischi connessi all’uso di ChondroGrid

I possibili rischi connessi all’impiego del dispositivo sono stati valutati attraverso l’esecuzione di

opportuni test e l’attenta analisi del processo produttivo, dalla materia prima di origine animale fino

alla sterilizzazione terminale.

Per quanto riguarda i due rischi maggiori connessi all’origine animale, e specificamente bovina,

della materia prima si devono considerare due fattori: 1) il processo di produzione dell’idrolizzato

di collagene è un processo virus- e prione-inattivante e 2) la materia prima da cui è estratto

l’idrolizzato proviene da Paesi non considerati a rischio di BSE. Inoltre, gli animali le cui parti sono

impiegate per la produzione dell’idrolizzato di collagene sono soggetti a tutti i controlli europei

previsti per la tracciabilità degli animali destinati al consumo umano. In sintesi, dato un lotto di

prodotto è sempre possibile risalire all’animale da cui la materia prima derivava, e viceversa. Più in

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particolare, per i diversi rischi connessi alla sicurezza del dispositivo valgono le considerazioni che

seguono:

Possibile trasmissione di patogeni animali

Il processo produttivo prevede il trattamento con basi ed acidi forti a concentrazioni tali

da inattivare qualunque virus/patogeno eventualmente presente nella materia prima.

Ulteriormente, il processo terminale di sterilizzazione a raggi beta a 25 kGy introduce un

ulteriore fattore di riduzione della carica microbiologica pari a 10-6 (che rappresenta già

un fattore di sicurezza adeguato in funzione degli standard tecnici di riferimento, quali

lo standard ISO11137:2007).

Possibile trasmissione di encefalopatie spongiformi

Il processo produttivo, sempre per la presenza di basi e acidi forti ad alta concentrazione,

è in grado di inattivare eventuali prioni presenti nella materia prima (cfr. ad esempio

Grobben AH et al, 2004).

In più, gli animali utilizzati per l’estrazione del collagene provengono da Paesi

considerati intrinsecamente sicuri per la presenza di BSE. Tutto questo è certificato

dall’EDQM (European Directorate for the Quality of Medicine) con apposita

certificazione, disponibile su richiesta

Possibile immunogenicità o mancanza di biocompatibilità

è noto che il collagene è utilizzato da tempo in medicina come materiale impiantabile, e

si è dimostrato intrinsecamente sicuro nella sua lunga storia d’impiego (a meno di

ipersensibilità individuale al collagene, peraltro rare e facilmente diagnosticabili –

Charriere G et al. 1989).

Il dispositivo è stato inoltre oggetto di una serie di prove di biocompatibilità nel quadro

concettuale di quanto prescritto dalla ISO10993, Biological evaluation of medical

devices – Part 1: Evaluating and testing e più precisamente:

19

Prova Standard tecnico Esito

Test di Citotossicità ISO 10993-5

Superati positivamente

Test di Pirogenicità/Lal Test Eu.Ph. § 2.6.14

Test di Mutagenesi (Ames) ISO 10993-3 ISO 10993-12

Test di Reattività cutanea (intradermica) ISO 10993-10

Test di Ipersensibilità ritardata ISO 10993-10

Test di Tossicità subacuta ISO 10993-2 ISO 10993-11 ISO 10993-12

Test di Tossicità sistemica acuta ISO 10993-11

Test di impianto ISO 10993-6

Ciascun test è descritto con maggior dettaglio nei paragrafi che seguono:

20

Test di citotossicità

Obiettivo del test: tramite l’uso della tecnica di coltura cellulare, il test di citotossicità

determina l’eventuale lisi cellulare, la morte cellulare, l’inibizione alla crescita cellulare

e altri effetti indotti dal rilascio di eventuali sostanze dal dispositivo medico.

Metodo utilizzato: Cytotoxicity test MEM elution - UNI EN ISO 10993-5: Tests for in

vitro cytotoxicity; Protocollo interno PI 07 “Test di citotossicità in vitro”.

Test di sensibilizzazione allergica

Obiettivo del test: il test di sensibilizzazione allergica valuta il potenziale sensibilizzante

dell’estratto del dispositivo medico sottoposto ad analisi tramite contatto diretto con la

cute. Il test di sensibilizzazione allergica è attendibile poiché valuta un’eventuale

sensibilizzazione indotta dal rilascio di sostanze sensibilizzanti dopo un’esposizione

prolungata.

Metodo utilizzato: Guinea Pig Magnusson Kligman Maximisation Test - ISO 10993-10:

Tests for irritation and delayed-type hypersensitivity

Test di reattività intracutanea

Obiettivo del test: il test di reattività intracutanea valuta la reazione locale cutanea

all’estratto di un dispositivo medico; questo test è utilizzabile allorché la determinazione

di reattività dermica e mucosale risulti essere inapplicabile.

Metodo utilizzato: Intracutaneous reactivity test in the rabbit - ISO 10993-10: Tests for

irritation and delayed-type hypersensitivity.

21

Test di tossicità sistemica acuta

Obiettivo del test: il test di tossicità sistemica acuta valuta i potenziali effetti dannosi di

singole o multiple esposizioni, durante l’arco delle 72 h, agli estratti del dispositivo

medico in analisi, su di un modello animale. Il test è indicato nel caso in cui il contatto

col dispositivo permetta il rilascio di sostanze o di prodotti di degradazione.

Metodo utilizzato: Acute systemic toxicity test in the mouse - ISO 10993-11: Tests for

systemic toxicity.

Pirogenicità

Obiettivo del test: il test di pirogenicità rileva reazioni pirogeniche indotte dagli estratti

dei dispositivi medici.

Metodo utilizzato: Pyrogen test in the rabbit – Ph. Eur., § 2.6.14 Bacterial endotoxins.

Method D (Kinetic-chromogenic test).

Test di tossicità subacuta

Obiettivo del test: Il Test di tossicità subacuta valuta gli effetti di esposizione multipla o

di contatto prolungato con l’estratto del dispositivo medico su di un modello animale

per un periodo protratto di giorni. Questo studio dovrebbe garantire una base razionale

per stabilire un’ analisi del rischio in ambito umano.

Metodo utilizzato: Rabbit sub-acute systemic toxicity test- ISO 10993-11: Tests for

systemic toxicity; Rabbit sub-acute systemic toxicity

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Test di genotossicità

Obiettivo del test: Tale batteria di test usa colture cellulari di mammiferi e non, oppure

anche altre tecniche per determinare mutazioni genetiche, alterazioni nel numero dei

cromosomi o delle strutture cromosomiche, e del DNA; viene indagata anche tossicità

genetica causata dagli estratti dei dispositivi medici.

Metodo utilizzato: Ames test - UNI EN ISO 10993-3: Tests for genotoxicity,

carcinogenicity and reproductive toxicity; OECD 471.

Metodo utilizzato: MLA Test - UNI EN ISO 10993-3: Tests for genotoxicity,

carcinogenicity and reproductive toxicity; OECD 476.

Test d’impianto

Obiettivo del test: Questo test determina l’effetto locale patologico dell’impianto del

dispositivo medico su tessuti vivi, similari a quelli ai quali è destinato il dispositivo, sia

a livello macroscopico/necroscopico che a livello istologico.

Metodo utilizzato: Implantation, 28 days, in the rabbit - UNI EN ISO 10993-6: Tests for

local effects after implantation.

23

Valutazione del rischio residuo complessivo

Il rischio residuo complessivo relativo all’utilizzo del dispositivo per il suo uso inteso è

stato oggetto di analisi del rischio secondo gli standard tecnici:

• ISO 22442:2015 Animal tissues and their derivatives utilized in the

manufacture of medical devices

• ISO 14971:2012 Medical devices - Application of risk management to

medical devices

concludendo che il rischio residuo complessivo è risultato trascurabile in

rapporto al beneficio che il dispositivo apporta al paziente.

Valutazione da parte della autorità competenti degli altri Paesi Europei:

Il dispositivo soddisfa inoltre i requisiti espressi nel Regolamento Europeo 722/2012

(COMMISSION REGULATION (EU) No 722/2012 - of 8 August 2012 - concerning

particular requirements as regards the requirements laid down in Council Directives

90/385/EEC and 93/42/EEC with respect to active implantable medical devices and

medical devices manufactured utilising tissues of animal origin).

Si noti che, in virtù del suddetto Regolamento, il profilo di sicurezza ed il rapporto

beneficio/rischio del dispositivo ChondroGrid sono stati valutati con esito positivo non

solo dall’Organismo Notificato che ha rilasciato le certificazione CE di conformità ai

requisiti della Direttiva sui dispositivi medici (Istituto Superiore di Sanità, Organismo

Notificato 0373) ma anche da ciascuna Autorità Competente (ovvero, Ministeri della

Salute) dei Paesi che seguono: Austria, Belgio, Bulgaria, Croazia, Repubblica Ceca,

Cipro, Danimarca, Estonia, Finlandia, Francia, Germania, Grecia, Ungheria, Islanda,

Lituania, Lussemburgo, Malta, Olanda, Norvegia, Polonia, Portogallo, Romania,

Slovacchia, Spagna, Svezia, Slovenia, Spagna, Svizzera, Turchia, Gran Bretagna.

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Gli standard tecnici impiegati nella progettazione e fabbricazione del dispositivo

Ad ulteriore supporto della sicurezza intrinseca al dispositivo ChondroGrid, si citano di seguito gli

standard tecnici seguiti nella progettazione e fabbricazione del dispositivo. Gli standard tecnici

definiscono dei metodi condivisi a livello mondiale per verificare, da un punto di vista tecnico, i

requisiti di sicurezza ed efficacia relativi ai dispositivi medici, in ogni loro parte. L’adozione di

precisi standard tecnici è ulteriore garanzia che gli specifici requisiti siano effettivamente verificati,

e che la conformità del dispositivo alla Direttiva europea di riferimento – in termini di sicurezza ed

efficacia - sia effettivamente provata

Standard tecnico Titolo

ICH guidelines, 06-2003 Q1A (R2) Stability Testing of New Drug Substances and Products

ASTM F1929-98:2004 Standard Test Method for Detecting Seal Leaks in Porous Medical Packaging by Dye Penetration

ASTM F1608-00:2009 Standard Test Method for Microbial Ranking of Porous Packaging Materials ( Exposure Chamber Method).

ASTM F1886-09:2009 Standard Test Method for Determining Integrity of Seals for Flexible Packaging by Visual Inspection

ASTM F1980-07:2011 Standard Guide for Accelerated Aging of Sterile Barrier Systems for Medical Devices

EU-GMP:2008, Annex 1 Good manufacturing practice (GMP) Guidelines

ISO 11737-1:2006 Sterilization of medical devices -- Microbiological methods -- Part 1: Determination of a population of microorganisms on products

ISO 11737-2:2009 Sterilization of medical devices -- Microbiological methods -- Part 2: Tests of sterility performed in the definition, validation and maintenance of a sterilization process

ISO 14698-1:2003 Cleanrooms and associated controlled environments -- Biocontamination control -- Part 1: General principles and methods

ISO 14698-2:2003 Cleanrooms and associated controlled environments -- Biocontamination control -- Part 2: Evaluation and interpretation of biocontamination data

ISO 187:1990 Paper, board and pulps -- Standard atmosphere for conditioning and testing and procedure for monitoring the atmosphere and conditioning of samples

ISO/ASTM 51261:2002 Practice for calibration of routine dosimetry systems for radiation processing

ISO/ASTM 51707:2005 Guide for estimation of measurement uncertainty in dosimetry for radiation processing

Italian Ph. Ed. XII, § 2.6.1 Italian Pharmacopea

OECD 471 Ed. 1997 Bacterial Reverse Mutation Test

OECD 476 Ed. 1997 In vitro Mammalian Cell Gene Mutation Test

Ph. Eur. Ed. VII, § 2.6.1 European Pharmacopea

Ph. Eur. Ed. VII, § 2.6.14 European Pharmacopea

UNI CEI EN 1041:2013 Information supplied by the manufacturer of the medical devices

UNI CEI EN ISO 14971:2012 Medical devices - Application of risk management to medical devices

UNI CEI EN ISO 15223-1:2012 Medical devices - Symbols to be used with medical device labels, labelling and information to be supplied - Part 1: General requirements

UNI EN 556-1:2002 Sterilization of medical devices - Requirements for medical devices to be designated "STERILE" - Part 1: Requirements for terminally sterilized medical devices

UNI EN 556-2:2005 Sterilization of medical devices - Requirements for medical devices to be designated "STERILE" - Part 2: Requirements for aseptically processed medical devices

UNI EN 868-5:2009 Packaging for terminally sterilized medical devices - Part 5: Sealable pouches and reels of porous materials and plastic film construction - Requirements and test methods

UNI EN ISO 10993-1:2009 Biological evaluation of medical devices -- Part 1: Evaluation and testing within a risk management process

25

UNI EN ISO 10993-10:2002 Biological evaluation of medical devices -- Part 10: Tests for irritation and skin sensitization

UNI EN ISO 10993-11:2006 Biological evaluation of medical devices -- Part 11: Tests for systemic toxicity

UNI EN ISO 10993-12:2007 Biological evaluation of medical devices -- Part 12: Sample preparation and reference materials

UNI EN ISO 10993-2: 2006 Biological evaluation of medical devices -- Part 2: Animal welfare requirements

UNI EN ISO 10993-5:2009 Biological evaluation of medical devices -- Part 5: Tests for in vitro cytotoxicity

UNI EN ISO 10993-6:2009 Biological evaluation of medical devices -- Part 6: Tests for local effects after implantation

UNI EN ISO 11137-1:2006 Sterilization of health care products -- Radiation -- Part 1: Requirements for development, validation and routine control of a sterilization process for medical devices

UNI EN ISO 11137-2:2012 Sterilization of health care products -- Radiation -- Part 2: Establishing the sterilization dose

UNI EN ISO 11137-3:2006 Sterilization of health care products -- Radiation -- Part 3: Guidance on dosimetric aspects

UNI EN ISO 11138-1:2006 Sterilization of health care products -- Biological indicators -- Part 1: General requirements

UNI EN ISO 11607-1:2009 Packaging for terminally sterilized medical devices -- Part 1: Requirements for materials, sterile barrier systems and packaging systems

UNI EN ISO 11607-2:2006 Packaging for terminally sterilized medical devices -- Part 2: Validation requirements for forming, sealing and assembly processes

UNI EN ISO 13485: 2012 Medical devices -- Quality management systems -- Requirements for regulatory purposes

UNI EN ISO 14644-1:2001 Cleanrooms and associated controlled environments -- Part 1: Classification of air cleanliness by particle concentration

UNI EN ISO 14644-2:2001 Cleanrooms and associated controlled environments -- Part 2: Monitoring to provide evidence of cleanroom performance related to air cleanliness by particle concentration

UNI EN ISO 14644-3:2006 Cleanrooms and associated controlled environments -- Part 3: Test methods

UNI EN ISO 22442-1:2008 Medical devices utilizing animal tissues and their derivatives -- Part 1: Application of risk management

UNI EN ISO 9001:2008 Quality management systems -- Requirements

26

ChondroGrid: profilo di efficacia

L'efficacia di un dispositivo medico è comprovata da tutto l'insieme di dati che dimostrano come,

per l'uso inteso del dispositivo, la sua applicazione conduca efficacemente al risultato atteso. I dati

possono essere diretti, derivanti cioè dall'applicazione pre-clinica e clinica dello specifico

dispositivo oggetto d'esame, o indiretti – a loro volta suddivisibili concettualmente in a) evidenza

dell'equivalenza del dispositivo oggetto di esame con uno o più dispositivi equivalenti e b) analisi

della letteratura inerente l'applicazione dei costituenti di cui il dispositivo è composto. Nel presente

report si è voluto dare un quadro generale dei dati disponibili, comprendendo anche quelli indiretti,

in quanto si ritiene – nel caso particolare del dispositivo in esame – che essi permettano anche di

meglio comprendere quello che è il meccanismo d'azione del dispositivo e quindi di fornire al

lettore un quadro maggiormente esaustivo ed utile a comprendere l'effettiva utilità del preparato.

Effetti biologici del collagene e contestualizzazione dell’impiego di ChondroGrid

Gli effetti biologici del collagene sulle strutture coinvolte nella patologia degenerative, traumatica o

da sovraccarico e sulle cellule che le compongono sono molteplici. Importante è la funzione che il

collagene esercita di filtro molecolare, come componente della matrice extracellulare, modulando

la diffusione di molecole diffusibili (Toroian D et al. 2007). A livello di orientamento di

popolazioni cellulari è noto che il collagene possiede sequenze segnale in grado di favorire

l'adesione ad esso di diversi tipi di popolazioni cellulari (Ruoslahti E et al. 1987). Vi è un indubbio

effetto sull'adesione di cellule mesenchimali, anche di derivazione midollare (Liu G et al, 2004,

Chan CK et al. 2009; Beitzel K et al. 2014). Tale adesione, che gioca un ruolo chiave nei primi

eventi di ripopolamento del sito lesionato, avviene attraverso il riconoscimento, come si è detto, di

specifiche sequenze amminoacidiche presenti nella catena collagenica. Tali sequenze sono

riconosciute da una specifica famiglia di recettori a livello della membrana cellulare delle cellule

mesenchimali, le integrine. Il legame integrina-collagene, oltre a permettere l'adesione della cellula

alla matrice funge inoltre da segnale per ulteriori eventi intracellulari, prime fra tutti le

modificazioni delle strutture citoscheletriche (Shen B et al. 2012; Das M et al.2014) responsabili

della migrazione cellulare. A livello sopramolecolare, il collagene costituisce il componente

principale delle matrici extracellulari, tanto che esso è largamente impiegato in ingegneria tessutale

nella produzione di sostituti tessutali che, mimino la matrice extracellulare naturale al fine di

favorire e guidare lo sviluppo di nuovo tessuto funzionale (Cunniffe GM et al. 2011, Aigner T et al,

27

2003) ed è stato oggetto per questo di numerosi studi relativi alla sua applicazione in ingegneria

tessutale nella rigenerazione di diversi tessuti, (Lee YB et al. 2010; Chen Z et al. 2011; Sumita Y et

al. 2006, Zhang Y et al. 2006; Gungormus, et al 2004;(Ruszczak Z, 2003), (Davidenko N et al.

2010),data anche la sua abbondanza nel regno animale ed il fatto che le porzioni antigeniche

specie-specifiche, che consistono nelle sole porzioni terminali della molecola (Davison PF et al.

1967; Michaeli D et al. 1969; Furthmayr et al. 1971), possono facilmente essere eliminate per

digestione enzimatica tramite pepsina. E’ in questo contesto più ampio che si colloca l’impiego

medico del collagene idrolizzato iniettabile: il collagene, anche nella sua forma idrolizzata, gioca un

ruolo fondamentale nel trattamento sintomatologico e funzionale delle cartilaginopatie e nelle

affezioni muscolo-tendinee in quanto le strutture anatomiche coinvolte ne sono costituite in maggior

parte: così che gli eventi di danno degenerativo o traumatico coinvolgono sempre, in qualche

misura, questa molecola. La cartilagine articolare è un tessuto altamente specializzato, non

vascolarizzato e privo di terminazioni nervose, la cui matrice è sintetizzata da cellule specializzate, i

condrociti. A livello sopramolecolare, la matrice cartilagine consiste di due componenti principali,

una fibrillare ed una non-fibrillare. La componente fibrillare è una fitta rete costituita

principalmente di collagene di tipo II, e in minore quantità di collagene di tipo IX e XI. La

componente non fibrillare consiste di monomeri di aggrecano, acido ialuronico, proteine in forma di

aggregati polianionici. E’ la componente fibrillare collagenica che conferisce alla cartilagine le sue

proprietà tensili di resistenza alla compressione, mentre le molecole di aggrecano ne modulano,

assorbendo e rilasciando la componente liquida, le proprietà viscoelastiche (Aigner et al, 2002). Nel

corso dell’instaurarsi della patologia artrosica, si osserva sia la degradazione delle componenti

molecolari sia la destabilizzazione delle strutture sopramolecolari, compresa la distruzione della

matrice collagenica visibile prima solo microscopicamente e poi anche a livello macroscopico. La

degradazione avviene sia per usura meccanica che per degradazione enzimatica, in presenza di

processi anabolici della matrice che divengono alterati, così come il profilo di espressione genica

dei condrociti. L’importanza di un aumentato apporto di collagene nel momento dell’instaurarsi

della patologia, anche nelle prime fasi di destabilizzazione ancora osservabili solo per via

microscopica, è evidenziata dal fatto che gli stessi condrociti, in questa condizione, aumentano la

loro attività di sintesi di collagene di tipo II (Aigner et al., 2002). E’ chiaro quindi che già nelle

prime nelle prime fasi della patologia, ove si assiste al cosiddetto “network loosening” della

struttura fibrillare collagenica, l’apporto di collagene esogeno è da considerarsi favorevole in

quanto rende possibile il rinforzo della struttura di matrice fibrillare in corso di deterioramento,

mimando un evento riparativo (la biosintesi di collagene da parte dei condrociti) naturale. Nel

tessuto tendineo/legamentoso la sequenza degli eventi al sito di danno è tradizionalmente divisa in

28

tre fasi sovrapposte (Yang et al. 2013, Hope e Saxby, 2007) - (1) infiammazione, (2) la

proliferazione / riparazione, e (3) rimodellamento Nella fase infiammatoria, il coagulo di sangue

che si forma immediatamente dopo la rottura dei vasi del tendine attiva il rilascio di fattori

chemiotattici e serve come impalcatura preliminare per l'invasione. Cellule infiammatorie, tra cui

neutrofili, monociti e linfociti migrano dai tessuti circostanti nel sito della ferita, dove digeriscono

tramite fagocitosi i detriti necrotici (Voleti et al., 2012). Inoltre, in questa fase inizia il reclutamento

e l'attivazione di tenociti inizia, che raggiungeranno un picco nella fase successiva. La seconda fase,

detta fase proliferativa o riparativa, inizia circa due giorni dopo il verificarsi del danno. I fibroblasti

del paratenon o della circostante guaina sinoviale sono reclutati verso la zona lesionata e

proliferano, in particolare nel epitenon (Garner et al., 1989). Allo stesso modo, tenociti intrinseci

localizzati nell'endotenon ed epitenon migrano verso il sito lesionato ed iniziano a proliferare.

Entrambe le fonti di tenociti sono importanti nella sintesi della matrice extracellulare e nella

creazione di una rete interna neovascolare (James et al., 2008). Allo stesso tempo, i livelli di

neutrofili diminuiscono mentre i macrofagi continuano a rilasciare fattori di crescita che dirigono il

reclutamento e l'attività dei diversi tipi cellulari (Voleti et al., 2012). In questa fase iniziale di

guarigione, la matrice sintetizzata dai tenociti è composta di una maggiore quantità di collagene di

tipo III (Juneja et al., 2013). Le concentrazioni di contenuto d'acqua e glicosamminoglicani in

questa fase sono particolarmente elevate (Sharma e Maffulli, 2006). Infine, 1-2 mesi dopo il

verificarsi della lesione ha inizio la fase di rimodellamento. Tenociti e fibre collagene si allineano

alla direzione del carico e viene sintetizzata una maggiore percentuale di collagene di tipo I è

(Abrahamsson, 1991), con una corrispondente riduzione della cellularità, del collagene di tipo III e

del contenuto di glicosamminoglicani (Sharma e Maffulli, 2006). Dopo 10 settimane, il tessuto

fibroso diviene gradualmente un tessuto tendineo simil-cicatriziale, un processo che continua per

anni. Il tessuto riparato non riacquista mai completamente le proprietà biomeccaniche che aveva

prima del danno e le caratteristiche biochimiche e ultrastrutturali rimangono anormali anche a 12

mesi dalla lesione (Miyashita et al., 1997). Appare dunque chiaro che, tra gli eventi principali che

conducono alla guarigione dei tendini e dei legamenti, vi è la sintesi di matrice extracellulare che,

come si è visto ha come componente principale il collagene di tipo I. Anche in questo caso, quindi,

l’apporto di collagene esogeno, in modo mirato e localizzato al sito di danno, realizza una

condizione favorevole al rinforzo strutturale della parte affetta. Ne è conferma la presenza di studi

in cui altri dispositivi impiantabili a base di collagene, anche se non in forma diffusibile, sono stati

utilizzati con successo come adiuvanti nel trattamento di patologie a carico della cartilagine (Riesle

J et al. 1998; Aigner T et al. 2003) e dei tendini (Chen JL et al, 2010).

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I risultati di questi studi concordano tutti nell'evidenziare che l'applicazione locale al sito di innesto

di collagene esogeno induce la riduzione della sintomatologia algico-infiammatoria causata dalla

lesione. come permesso da ChondroGrid, il quale dà all’operatore, essendo iniettabile, un modo

semplice e rapido per arricchire il sito di impianto di un componente fondamentale della matrice

extracellulare, fornendo così

a) supporto meccanico diretto alle strutture sofferenti, come già detto e

b) un substrato già ottimale all'azione degli elementi cellulari coinvolti nella

rigenerazione tessutale al sito lesionato, favorendo la modificazione degli eventi

riparativi in eventi rigenerativi.

Esempi che illustrano questa tipologia di modificazione si possono ritrovare in diversi studi

preclinici e clinici inerenti la rigenerazione di tessuti anche differenti fra loro: esempi di effetti

positivi nell'utilizzo di collageni sono reperibili nell'ambito della rigenerazione del tessuto nervoso

(Itoh S et al. 2001, Fukushima K et al. 2008), dei dischi intervertebrali (Sato M et al. 2003; Sakai D

et al. 2006; Halloran DO et al. 2008; Lee KI et al. 2012) di lesioni dermiche refrattarie a trattamento

(Nakanishi A et al. 2005); di lesioni muscolari sperimentalmente indotte (Kin S et al. 2007); di

difetti cartilaginei ed ossei (Jeong IH et al. 2013, Hamanishi M et al. 2013; Kagawa R et al. 2012;

Yamaoka H et al. 2010; Sato M et al. 2007; Masuoka K et al. 2006; Stone KR et al. 1997).

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Studi in vitro

Per quanto riguarda gli studi in vitro, considerati inerenti, riguardanti il collagene, e in particolare il

collagene idrolizzato di tipo I, si è constatato che l’applicazione di collagene idrolizzato su colture

cellulari di condrociti (le cellule deputate alla produzione e al mantenimento della cartilagine)

induce una stimolazione della produzione del collagene stesso, probabilmente come possibile

meccanismo di regolazione a feedback positivo della stessa produzione di collagene o come segnale

di presenza di collagene degradato e, quindi, come segnale di danno e di necessità di procedere alla

conseguente sintesi riparativa (Oesser et al, 2003); inoltre, particolari sequenze peptidiche

certamente presenti negli idrolizzati di collagene a basso peso molecolare, quali il dipeptide Prolina-

Idrossiprolina (Pro-Hyp), hanno mostrato un effetto pro-differenziante su colture condrocitarie e

l’induzione di un aumento della sintesi di glicosaminoglicani, un componente fondamentale della

matrice extracellulare cartilaginea (Nakatani et al, 2009). Questo effetto, misurabile in un

incremento del 300% quando era impiegato il dipeptide come sostanza test, era osservato dagli

stessi autori, anche se in misura minore, quando la sostanza test era l’intero idrolizzato collagenico

(+200%). Nell’ambito dello stesso studio, Nakatani e colleghi hanno quindi proceduto all’impiego

del dipeptide e dell’idrolizzato collagenico in un modello animale di osteoartrosi inducibile

(attraverso eccesso di fosfato in dieta), verificando che entrambi i composti erano in grado di inibire

sia la perdita condrocitaria che l’assottigliamento della cartilagine articolare. Risultati del tutto

simili sono stati ottenuti nel 2010 dal gruppo di Ohara (Ohara et al, 2010) su cellule sinoviali in

coltura dove il dipeptide Prolina-Idrossiprolina si è mostrato in grado di stimolare la produzione di

acido ialuronico (un altro costituente fondamentale della matrice extracellulare) da parte di questo

tipo cellulare. Studi simili sono stati condotti anche con una particolare forma di collagene di tipo I,

ottenuto da pepsinizzazione di tessuti connettivali porcini (Furuzawa-Carballeda J et al, 2009(1)), su

colture cartilaginee e sinoviali ottenute da biopsie di pazienti affetti da osteoartrosi. Gli autori, oltre

ad evidenziare una stimolazione sia della proliferazione condrocitaria che della produzione di

proteine della matrice extracellulare, hanno anche verificato la diminuzione del livello di numerosi

marcatori biochimici dell’infiammazione. Nel caso specifico dell’osteoartrosi, infatti, che come

noto è una patologia infiammatoria caratterizzata da un progressivo deterioramento della cartilagine

articolare e delle articolazioni sinoviali, le principali citochine maggiormente coinvolte nella

fisiopatologia della malattia risultano essere l’interleuchina (IL)-1β, il tumor necrosis factor (TNF)-

α, e l’IL-6. Altri fattori biomolecolari coinvolti includono le IL-15, IL-18, IL-21, ed il leukemia

inhibitory factor (LIF) Recentemente, si sono compiuti molti progressi nella comprensione dello

sviluppo della patologia e dei meccanismi responsabili del suo decorso. Una nuova strategia

terapeutica consiste nello sviluppare agenti in grado di modificare la progressione strutturale della

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patologia e, quindi, di agire a livello del lenimento della sintomatologia. Recenti studi in vitro

hanno visto coinvolti anche gli idrolizzati di collagene: in uno studio su condrociti di pazienti affetti

da osteoartrosi, l’esposizione anche a collagene idrolizzato non ha mostrato effetti sulla vitalità

cellulare, indicando un’assenza di citotossicità del composto, mentre a seguito di stimolazione con

IL1β, una condizione simulante uno stato infiammatorio, il collagene idrolizzato contribuiva a

ridurre i marcatori tipici dell’infiammazione quali l’NO, l’IL-6, la metalloproteinasi 3 (Comblain F

et al, 2015).Uno studio ulteriore degli stessi autori ha mostrato come ulteriori potenziali bersagli

molecolari possono essere il l’IL-1β stimulated chemokine ligand 6, la metalloproteinase di matrice

MM13, la bone morphogenetic protein-2 (BMP2) e la stanniocalcina-1. In tutti i casi la presenza

dell’idrolizzato contribuiva a diminuire sia l’espressione genica che la sintesi proteica di questi

fattori. Per alcuni fattori, l’effetto era specificamente quello inverso a quello dell’IL-1β,

confermando quindi un potenziale effetto antinfiammatorio diretto dell’idrolizzato (Comblain et al,

2016). Allo stesso tempo, studi meno recenti, hanno dimostrato che un idrolizzato di collagene,

ulteriormente idrolizzato (ad esempio se assunto per via orale) origina una concentrazione ematica

di peptide contenenti prolina-idrossiprolina: questi sono in grado, in vitro, di stimolare cellule

sinoviali a produrre acido ialuronico e, in un modello animale di artrosi, di stimolare

significativamente la sintesi di proteoglicani a livello delle articolazioni epifisarie. E’ ragionevole

supporre che la somministrazione intra-articolare di un idrolizzato di collagene renda disponibili,

immediatamente e poi nel corso del tempo mano a mano che i polipeptidi sono idrolizzati dagli

enzimi tessutali una certa quantità di dipeptidi Pro-Hyp che potrebbe portare ai benefici effetti

osservati nei modelli animali in cui il dipeptide risulta presente a livello ematico, e quindi

disponibile agli elementi cellulari coinvolti (Ohara et al, 2010).

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Studi in vivo

Gli studi su animale reperiti riguardano sia il profilo di sicurezza che l’attività anti-infiammatoria

del collagene idrolizzato specialmente in modelli animali di osteoartrosi. Per quanto riguarda il

profilo di sicurezza, si è osservato che studi sull’immunogenicità del collagene di tipo I iniettabile

di origine animale sono presenti in letteratura a partire dal 1987 (De Lustro F et al, 1987), con

risultati che mostrano la scarsa o nulla antigenicità di questa tipologia di preparati. Un elegante

studio su animale di Oesser et al, 1999 mostra chiaramente come il collagene idrolizzato mostri un

particolare tropismo per le cartilagini articolari: quando gelatina di collagene marcato

radioattivamente è stata somministrata alle cavie per via orale si è osservato un accumulo

significativamente maggiore di collagene nelle articolazioni di quanto osservato nel gruppo

controllo. In relazione agli studi sui modelli di osteoartrosi, oltre ai già citati studi di Nakatani e

Ohara ricordiamo anche il lavoro di Naraoka et al. (2013), che hanno mostrato attraverso indagine

istologica ed immunoistochimica come l’iniezione intra-articolare ripetuta di un tripeptide

collagenico di tipo Gly-X-Y in un modello animale affetto da osteartrosi artificialmente indotta

permetta una significativa riduzione della degradazione della cartilagine articolare e l’incremento

del numero di condrociti positivi per i test immunoistochimici di nuova sintesi di collagene di tipo

II, a dimostrare che esiste almeno una possibile frazione peptidica proveniente dal collagene

idrolizzato che potrebbe indurre fenomeni riparativi da parte del sistema condrocitario.

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Studi clinici

Gli studi clinici analizzati forniscono innanzitutto evidenza che il collagene iniettabile è poco o per

nulla immunogenico (Cooperman L et al, 1984) in linea con quanto osservato su animale (De

Lustro F et al, 1987). Il primo uso clinico di collagene iniettabile per alleviare la sintomatologia

dolorosa delle articolazioni risale al 1982 (Batyrov TU et al., 1982). Più recentemente sono stati

apparsi studi clinici che dimostrano come l’iniezione intra-articolare di collagene sia efficace per il

trattamento sintomatico delle patologie degenerative articolari. Uno studio di Furuzawa-Carballeda

e collaboratori nel 2009 (Furuzawa-Carballeda J et al, 2009(2)) su 53 pazienti affetti da osteoartrosi

del ginocchio, divisi in gruppo controllo (27 pazienti) a cui veniva iniettato un placebo e gruppo test

(26 pazienti) a cui veniva iniettato collagene di tipo I, mostra che l’iniezione di collagene di tipo I

porta alla riduzione significativa di tutti i parametri di misura della sintomatologia dolorosa e

funzionale, quali il Western Ontario and McMaster University Osteoarthritis Index, il Lequesne

index, e l’analogo visivo del dolore (scala VAS). Anche parametri secondari, quali il punteggio

globale del paziente e del ricercatore, e la quantità di farmaci, tra cui gli antidolorifici, assunti

migliorano significativamente quando il paziente è trattato tramite iniezione intra-articolare di

collagene. Lo stesso studio è stato recentemente ripetuto dagli stessi autori (Furuzawa-Carballeda et

al, 2012) in un trial clinico randomizzato in doppio cieco, confermando i risultati precedentemente

descritti. Risultati simili erano stati ottenuti dallo stesso gruppo nel 2006 (Furuzawa-Carballeda et

al., 2006) in uno studio clinico randomizzato su pazienti affetti da artrite reumatoide. Ulteriormente,

vi è notevole evidenza che integratori alimentari a base di collagene realizzano una condizione per

cui a seguito dell’attività gastrica e dell’assorbimento intestinale si osserva un picco ematico di

peptidi del collagene, tra cui il dipeptide Pro-Hyp, con effetto tropico e biologico sulla popolazione

condrocitaria delle cartilagini come già precedentemente descritto. Si noti che dei tre enzimi

proteolitici operanti nel succo gastrico, pepsina, tripsina e chemotripsina, la pepsina è l’enzima

principalmente impiegato per la produzione dei collageni idrolizzati, e tra questi per la produzione

del dispositivo ChondroGrid. L’enzima pepsina è più specificamente una proteasi idrolitica acida in

grado di recidere il legame amidico tra gli aminoacidi che formano le catene polipeptidiche

preferenzialmente a valle, in direzione N terminale, degli aminoacidi aromatici quali fenilalanina,

triptofano e tirosina (Cox et al., 2008). La sua azione, così come quella degli altri enzimi, a livello

delle catene polipeptidiche del collagene realizza una miscela di peptidi a diverso peso molecolare

che vengono successivamente ulteriormente degradati e assorbiti a livello intestinale per entrare

infine nel circolo ematico. L’introduzione per via orale di integratori a base di collagene realizza

quindi una condizione simile, sebbene con diverse cinetiche di esposizione delle strutture e delle

componenti cellulari coinvolte ai frammenti collagenici, simile a quella che si realizza attraverso

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l’iniezione diretta intra-articolare di collagene idrolizzato che – tramite quest’ultima modalità –

viene portato in situ a maggiori concentrazioni e con un più ampio spettro di pesi molecolari (cioè:

dimensioni) di frammenti peptidici. In questo senso i risultati degli studi condotti su paziente

pubblicati sull’effetto antiinfiammatorio dell’assunzione orale di integratori a base di collagene

sono una dimostrazione indiretta dell’efficacia di ChondroGrid. Si consideri a questo riguardo la

revisione di letteratura di Bello ed Oesser del 2006, ove già si riepilogava come il collagene

idrolizzato assunto per via orale, in seguito ad assorbimento intestinale, è in grado di accumularsi

nella cartilagine, stimolando un aumento significativo della sintesi della matrice extracellulare da

parte dei condrociti, suggerendo una possibile spiegazione per i dati osservati di riduzione del

dolore e miglioramento della funzionalità in pazienti affetti sia da artrosi che da altre patologie

articolari. Ulteriori studi sono stati condotti sull’uso del collagene in pazienti sia geriatrici che

sportivi, affetti da osteoartrite e osteoartrosi a carico delle articolazioni; per citare i più noti si

ricordino Moskowitz et al. (2000) e Clark et al. (2008): in questi studi il collagene idrolizzato risulta

essere somministrato per via orale alla dose di 10 g al dì, per periodi variabili dai 3 fino ai 6 mesi,

con miglioramento della sintomatologia dolorosa rispetto al gruppo di controllo negativo. Più

recentemente, uno studio clinico randomizzato di Schauss AG et al, 2012 ha mostrato come pazienti

affetti da osteoartrosi che assumevano collagene per via orale evidenziavano una significativa

riduzione della patologia dolorosa dopo 70 giorni di trattamento e un miglioramento funzionale

(misurato attraverso la scala WOMAC), nonché della propria attività fisica generale, già dopo 35

giorni di trattamento. Analogo risultato era stato ottenuto da Benito Ruiz e colleghi (2009) in un

trial clinico randomizzato su 250 pazienti affetti da osteoartrite del ginocchio cui sono stati

somministrati 10 g di collagene idrolizzato per os una volta al giorno per 6 mesi: ove i pazienti che

assumevano collagene mostravano un miglioramento significativo del dolore articolare, misurato

attraverso la scala VAS e la sottoscala WOMAC relativa al dolore. I soggetti col maggiore danno

articolare risultavano essere quelli con il maggiore beneficio. Un trial clinico randomizzato in

doppio cieco con gruppo di controllo su pazienti affetti da osteoartrosi del ginocchio ha dato

recentemente ulteriore conferma di questi risultati (Kumar S et al., 2015): i pazienti del gruppo di

studio, trattati per via orale con collagene idrolizzato estratto o da derma suino o da osso bovino

sperimentavano, dopo 13 settimane, un significativo miglioramento dei punteggi ottenuti secondo le

tre scale utilizzate: WOMAC, VAS e QOL (Quality of Life), sia rispetto alla baseline sia rispetto il

gruppo controllo. Questi studi, in congiunzione a quelli già precedentemente citati relativi

all’iniezione intra-articolare di collagene, suggeriscono che sia plausibile supporre che l’iniezione

locale di collagene idrolizzato possa rappresentare un trattamento avente quanto meno degli

outcome simili e potenzialmente più favorevoli all’ingestione, data la maggiore concentrazione

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locale ottenibile tramite l’iniezione in situ, ferme restando le differenze sulla via di

somministrazione che portano le due condizioni a non essere direttamente confrontabili.

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Conclusioni L’analisi della letteratura reperita porta a concludere che a) gli studi in vitro dimostrano che cellule

cimentate con idrolizzato collagenico non subiscono alcun tipo di danno ma, anzi, tutti i parametri

di vitalità e trofismo cellulare misurati negli studi considerati risultano aumentati (e, in studi

specificamente mirati, marcatori dell’infiammazione cellulare risultano significativamente

diminuiti); il razionale biologico secondo il quale ChondroGrid è in grado esercitare le prestazioni

previste è quindi coerente e robusto. A loro volta, b) gli studi su animale confermano a livello

istologico che l’applicazione del collagene idrolizzato migliora il metabolismo cellulare in modelli

sperimentali delle patologie indicate come indicazioni d’uso del dispositivo ChondroGrid, e che si

ottiene una riduzione dei marcatori considerati coerente con la prestazione attesa in ambito clinico.

Risulta di particolare interesse l’osservazione che i peptidi che compongono gli idrolizzati di

collagene mostrano un particolare tropismo per le strutture articolari quali la cartilagine, ove –

oltretutto – si accumulano. Inoltre, c) applicazioni cliniche di preparati simili, se non identici, a

ChondroGrid per le stesse applicazioni ed indicazioni permettono di ottenere i risultati clinici dati

come risultati attesi per ChondroGrid stesso (trattamento con esito positivo della sintomatologia

dolorosa e funzionale). Gli scriventi concludono, quindi, che la valutazione clinica di sicurezza ed

efficacia del dispositivo ChondroGrid dia esito completamente positivo. Il presente report sarà

aggiornato quando saranno disponibili i risultati degli studi clinici post-market che attualmente il

fabbricante ha comunicato essere in procinto di attivazione.

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Riepilogo dei risultati

Razionale biologico dell'utilizzo del dispositivo

Il dispositivo ChondroGrid permette all’operatore di apportare direttamente alla struttura coinvolta

nell’affezione dolorosa sia essa di origine degenerativa, traumatica o da sovraccarico, un’elevata

concentrazione di collagene idrolizzato a basso peso molecolare, ovvero una miscela di diversi

peptidi collagenici, sotto forma di sospensione. Poiché al diminuire della dimensione molecolare

aumentano i coefficienti di diffusione delle molecole stesse in fase acquosa, la miscela peptidica

contenuta i ChondroGrid è in grado di diffondere efficacemente e in modo uniforme in tutto il

volume raggiunto dall’iniezione, distribuendosi su tutta la superficie della struttura anatomica

coinvolta. La particolare proprietà di autoassemblaggio del collagene fa sì che il collagene

idrolizzato, legandosi alle strutture esposte – anch’esse costituite per la maggior parte di collagene,

eserciti un’azione di supporto e sostegno meccanico. Ulteriori evidenze, presentate nel presente

report, suggeriscono effetti ancillari di tipo biologico quali la stimolazione di alcuni tipo cellulari

alla produzione di ulteriore collagene, o di acido ialuronico o di glicosaminoglicani, e l’inibizione

dell’attività o dell’espressione di alcune citochine mediatrici dei processi infiammatori.

Razionale clinico dell'utilizzo del dispositivo

Visti gli effetti biologici che il dispositivo è in grado di esercitare, risulta evidente che l'impiego

clinico realizza una condizione al sito di iniezione estremamente favorevole per la creazione di uno

stato di trofismo locale in grado di migliorare la percezione dolorosa – e quindi favorire il recupero

funzionale – del paziente. i

Livello di evidenza relativo alla sicurezza dell'impiego clinico del dispositivo

Il dispositivo ChondroGrid può ritenersi intrinsecamente sicuro. La sicurezza del dispositivo è

comprovata direttamente, in quanto è dimostrato attraverso test condotti da enti terzi che

ChondroGrid è completamente biocompatibile secondo lo standard di riferimento, accettato a livello

mondiale, ISO 10993. Ulteriormente, la possibilità che il dispositivo possa essere veicolo di

patologie trasmissibili da animale all'uomo, ad esempio a causa di agenti patogeni virali e prioni, è

nulla in quanto la materia prima animale subisce un’esposizione prolungata sia a basi che acidi forti

condizioni alle quali è stato dimostrato nessun virus possa mantenere la propria capacità replicativa,

né possano permanere molecole prioniche in forma mutata. Prioni patogenici la cui presenza è

comunque da ritenere estremamente improbabile nella materia d’origine come certificato dalle

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Autorità Competenti europee con apposito certificato EDQM. Infine, conferma della sicurezza

dell'efficacia del dispositivo è apportata dalla certificazione CE che esso ha ottenuto da Organismo

Notificato per la valutazione della Conformità dei Dispositivi Medici (Istituto Superiore di Sanità,

Organismo Notificato CE 0373) alle normative europee di riferimento.

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Tabelle Riassuntive

Sicurezza del dispositivo ChondroGrid

ChondroGrid è stato oggetto di saggi e test che hanno valutato diversi parametri relativi alla

sicurezza, secondo stringenti standard internazionali:

Parametro Test Standard

Biocompatibilità

Citotossicità ISO 10993-5

Reattività intracutanea ISO10993 -10

Tossicità sistemica ISO10993-11

Pirogenicità Eu.Ph. § 2.6.14

Tossicità subacuta ISO10993-11

Impianto ISO10993-6

Possibilità di trasmissione di

encefalopatie spongiformi

La materia prima (tessuti animali) utilizzata per la

fabbricazione di ChondroGrid proviene da Paesi non a

rischio di BSE: la sicurezza è certificata anche

dall’European Directorate for the Quality of Medicines and

Healthcare (EDQM).

Il processo produttivo di ChondroGrid prevede inoltre alcuni

passaggi (impiego di basi e acidi forti) che sono in grado di

eliminare un’eventuale contaminazione prionica del

materiale d’origine.

Sterilità Sterilità ISO11137

Inattivazione virale Gli stessi passaggi del processo produttivo prima menzionati

in merito all’inattivazione e rimozione di possibili prioni

sono in grado anche di eliminare un’eventuale

contaminazione virale presente nel materiale d’origine.

Ulteriormente, il prodotto subisce la sterilizzazione

terminare per irraggiamento beta.

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Efficacia del dispositivo ChondroGrid.

I dati a sostegno dell'efficacia di ChondroGrid possono essere riassunti come segue:

Parametro di efficacia Livello di evidenza

Azione a livello cellulare Gli studi in vitro mostrano che il collagene idrolizzato è in

grado di stimolare il trofismo cellulare, ad esempio

condrocitario, inducendo un aumento sia della sintesi del

collagene stesso, sia dei glucosaminoglicani.

Le cellule sinoviali sono in grado, quando in contatto con

peptidi derivati da collagene idrolizzato di produrre

maggiori quantità di acido ialuronico.

I condrociti, in particolare, sono stimolati ad una maggiore

proliferazione.

Non si osserva alcun tipo di citotossicità indotta dal

collagene idrolizzato.

Azione a livello di signaling Il collagene idrolizzato inibisce in modo significativo

l’espressione di alcune interleuchine e di altri marcatori

dell’infiammazione.

Azione clinica – studi

preclinici

Gli studi preclinici mostrano che l’iniezione di collagene

idrolizzato intra- e peri-articolare è, prima di tutto, sicura

non osservandosi nei modelli sperimentali sensibilizzazione,

irritazione, tossicità né acuta né ritardata.

Ulteriormente, gli studi preclinici mostrano che il collagene

in forma di peptidi, come è ChondroGrid, ha un forte

tropismo per le strutture articolari e peri-articolari, ove si

accumula preferenzialmente, concentrandosi naturalmente

nei siti oggetto del trattamento.

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Gli studi pre-clinici suggeriscono inoltre che il collagene

idrolizzato, grazie all’azione di specifici peptidi nella

miscela, possa avere anche ulteriori azioni quali la

stimolazione di attività riparativa e rigenerativa da parte di

alcuni specifici elementi cellulari.

Azione clinica – casistica su

paziente

Per quanto riguarda la sicurezza di ChondroGrid, anche gli

studi su paziente mostrano che l’iniezione di collagene intra-

e peri-articolare non dà reazioni immunitarie indesiderate,

né a breve né a lungo termine.

Per quanto concerne l’efficacia di ChondroGrid, gli studi

reperiti, alcuni dei quali anche randomizzati e in doppio

cieco, mostrano inequivocabilmente che quando il collagene

idrolizzato, come è ChondroGrid, raggiunge le articolazioni

di pazienti affetti da osteoartrosi, si assiste ad una

significativa diminuzione della sintomatologia dolorosa e a

un più rapido recupero funzionale, a prescindere dalla via di

somministrazione.

I pazienti in cura con questo tipo di approccio terapeutico,

inoltre, assumono una minore quantità di farmaci analgesici

ed antinfiammatori.

42

Domande Frequenti – Frequently Asked Questions (FAQ)

Cosa contiene ChondroGrid?

ChondroGrid è composto esclusivamente di collagene idrolizzato e liofilizzato a basso peso

molecolare, altamente purificato, estratto da cute bovina.

Cosa è il collagene idrolizzato?

Si tratta di collagene che è stato sottoposto ad un processo di frammentazione attraverso

l’impiego di metodi enzimatici e chimico-fisici. Come risultato si producono peptidi di

diverso peso molecolare, ovvero con diversa lunghezza.

Perché è importante il collagene idrolizzato?

I peptidi, avendo dimensione minore rispetto quella dell’intera molecola, diffondono

facilmente nei liquidi, e quindi si prestano ad essere iniettati negli spazi intra- e peri-articolari

ove, per diffusione, raggiungono l’intera superficie dell’articolazione o dei tendini e dei

legamenti.

E' possibile una sensibilità individuale al collagene idrolizzato?

E' da considerarsi un evento estremamente raro.

Quali sono le indicazioni d'uso di ChondroGrid?

Chondrogrid è indicato per il trattamento sintomatologico e funzionale di: osteoartrosi,

artrosinoviti acute o croniche secondarie ad artrosi o artrite reumatoide, esiti di traumi o

lesioni, affaticamento da sovraccarico a carico delle diverse articolazioni nonché in caso di

meniscopatie e in preparazione e come terapia di mantenimento a seguito di interventi di

meniscectomia, di ricostruzione legamentosa o di pulizia e/o ricostruzione del tessuto

cartilagineo articolare. Inoltre è indicato nel trattamento degli esiti di traumi e/o lesione peri-

articolari o a carico di strutture muscolo-tendinee-legamentose.

43

Quale è il meccanismo d'azione di ChondroGrid®?

ChondroGrid agisce apportando un’alta quantità di collagene idrolizzato biodisponibile al

sito di iniezione. Per via intra-articolare, il collagene idrolizzato a basso peso molecolare si

distribuisce sulla superficie dell’articolazione rinforzando la matrice cartilaginea, deteriorata

dai processi patologici in atto, la cui impalcatura è formata da un fitto intreccio di fibre

collagene. Per via peri-articolare il collagene idrolizzato a basso peso molecolare agisce

quale rinforzo diretto all’impalcatura collagenica danneggiata delle strutture peri-articolari,

quali tendini e/o legamenti, contribuendo ad una riduzione del dolore e ad una più veloce

ripresa funzionale.

Come deve essere utilizzato ChondroGrid?

Secondo uno specifico protocollo terapeutico. Se iniettato per via intra-articolare il

trattamento prevede tre iniezioni: due eseguite a distanza di 15 giorni l’una dall’altra, la terza

dopo un mese dall’ultimo trattamento. Se utilizzato per iniezione peri-articolare, il

trattamento prevede due iniezioni a distanza di 30 giorni l’una dall’altra. Per infiltrazione in

strutture muscolo-tendinee-legamentose, due iniezioni ad un intervallo di circa 10 giorni

l’una dall’altra.

Quali sono i limiti di ChondroGrid?

Sebbene alcuni studi indichino che il collagene idrolizzato di basso peso molecolare possa

anche indurre la rigenerazione delle strutture danneggiate, l’impiego di ChondroGrid deve

essere comunque inteso, allo stato attuale della conoscenza, come coadiuvante nella terapia

sintomatologica e funzionale nelle diverse indicazioni fornite.

ChondroGrid deve essere utilizzato da solo?

Si, ChondroGrid esplica la sua azione da solo, grazie all’azione del collagene idrolizzato che

esso contiene.

ChondroGrid è sicuro?

Si. E' un dispositivo che ha superato tutti i test di sicurezza previsti dalla normativa europea

sui dispositivi medici (marcatura CE).

44

Dove viene fabbricato ChondroGrid ?

ChondroGrid è un dispositivo medico fabbricato in Italia, presso Bioteck, l’azienda leader

italiana nella produzione di dispositivi medici per rigenerazione tessutale.

45

Bibliografia1

Scand J Plast Reconstr Surg Hand Surg Suppl. 1991;23:1-51.

Matrix metabolism and healing in the flexor tendon. Experimental studies on rabbit tendon.

Abrahamsson SO.

I. The rabbit flexor tendon within the synovial sheath contains segments with fibrocartilage-like

areas. These segments have a higher proteoglycan and a lower collagen and non-collagen protein

synthesis compared to the segment with "true" tendon tissue. Cell proliferation is also lower within

the proximal segment than in the intermediate and distal segments. These regional variations should

be considered when interpreting experimental data. They may also be of importance for the variable

healing capacity of different flexor tendon regions. II. Recombinant human insulin-like growth

factor, insulin and fetal calf serum stimulate matrix synthesis and cell proliferation in a dose

dependent manner in flexor tendon explants cultured for three days. rhIGF-I was more potent than

insulin in stimulating cell proliferation and matrix synthesis. rhIGF-I also stimulated matrix

synthesis to a higher degree than FCS. III. In long-term culture of flexor tendon explants, the

addition of rhIGF-I to the culture medium stimulates matrix synthesis, but does not influence turn-

over rates. The total hexosamine and collagen contents in tendons cultured in medium with rhIGF-I

remain at the same level, while non-collagen protein content decreases. There are no major

differences in matrix metabolism between tendons cultured in medium supplemented with FCS or

with rhIGF-I only. rhIGF-I may therefore be used as a growth factor supplement in serum-free

culture of tendon tissue. IV. Dehydration inhibits in vitro matrix synthesis and cell proliferation in

tendon explants. These effects are counteracted by keeping the exposed tendon segments moist with

physiological saline solution during preparation. The sensitivity of tendon tissue to dehydration

should be considered during tendon surgery. V. Tendon explants, cultured in a diffusion chamber,

survive and exhibit an intrinsic capacity for healing. In healing tendon segments incubated for three

weeks, protein synthesis remains unchanged and collagen synthesis decreases, whereas the rate of

cell proliferation increases as compared with native tendons. VI. Endotenon cells of the rabbit

1 La bibliografia riporta gli abstract delle pubblicazioni citate nel documento ordinati in ordine

alfabetico per primo autore. Le pubblicazioni complete sono disponibili su richiesta.

46

flexor tendon can restore the injured tendon surface and bridge the tendon gap. The rabbit flexor

tendon is a morphologically and biochemically heterogeneous tissue with an intrinsic capability for

healing. Tendon tissue is susceptible to dehydration and during exposure quickly looses its viability.

The metabolic and proliferative capacity of the tendon is stimulated by growth factors and rhIGF-I

may be of importance in tendon healing.

47

Cell Mol Life Sci. 2002 Jan;59(1):5-18.

Molecular pathology and pathobiology of osteoarthritic cartilage.

Aigner T, McKenna L.

The biochemical properties of articular cartilage rely on the biochemical composition and integrity

of its extracellular matrix. This matrix consists mainly of a collagen network and the proteoglycan-

rich ground substance. In osteoarthritis, ongoing cartilage matrix destruction takes place, leading to

a progressive loss in joint function. Beside the degradation of molecular matrix components,

destabilization of supramolecular structures such as the collagen network and changes in the

expression profile of matrix molecules also take place. These processes, as well as the pattern of

cellular reaction, explain the pathology of osteoarthritic cartilage degeneration. The loss of

histochemical proteoglycan staining reflects the damage at the molecular level, whereas the

supramolecular matrix destruction leads to fissuring and finally to the loss of the cartilage.

Chondrocytes react by increasing matrix synthesis, proliferating, and changing their cellular

phenotype. Gene expression mapping in situ and gene expression profiling allows characterization

of the osteoarthritic cellular phenotype, a key determinant for understanding and manipulating the

osteoarthritic disease process. Adv Drug Deliv Rev. 2003 Nov 28;55(12):1569-93.

48

Collagens--major component of the physiological cartilage matrix, major target of cartilage

degeneration, major tool in cartilage repair.

Aigner T, Stöve J.

Collagens serve important mechanical functions throughout the body and in particular in the

connective tissues. Additionally, collagens exert important functions as cellular microenvironment

and partly via binding and release of cellular growth mediators. In articular cartilage, fibrillar

collagens are providing most of the biomechanical properties of the extracellular matrix essential

for its functioning. The collagenous matrix is one main target of destructive processes in general

degenerative joint disease and focal matrix lesions. The development of an adequate collagen

framework represents the major aim of therapeutic cartilage repair. In this respect, collagenous

matrices or collagen-imitating scaffolds are more and more emerging as highly suitable vehicles for

cell and (growth) factor transport into cartilage lesion. Thus, collagens are not only major

constituents of connective tissues in terms of integrity and function, they are also major targets of

tissue destruction and regeneration and might become major tools to achieve tissue repair.

49

Eur Urol. 1998;33(2):129-33.

Immunologic aspects of bovine injectable collagen in humans. A review.

Aragona F, D'Urso L, Marcolongo R.

No abstract available

50

Arthroscopy. 2014 Mar;30(3):289-98.

Properties of biologic scaffolds and their response to mesenchymal stem cells.

Beitzel K, McCarthy MB, Cote MP, Russell RP, Apostolakos J, Ramos DM, Kumbar SG, Imhoff

AB, Arciero RA, Mazzocca AD.

PURPOSE: The purpose of this study was to examine, in vitro, the cellular response of human

mesenchymal stem cells (MSCs) to sample types of commercially available scaffolds in comparison

with control, native tendon tissue (fresh-frozen rotator cuff tendon allograft). METHODS: MSCs

were defined by (1) colony-forming potential; (2) ability to differentiate into tendon, cartilage,

bone, and fat tissue; and (3) fluorescence-activated cell sorting analysis (CD73, CD90, CD45).

Samples were taken from fresh-frozen human rotator cuff tendon (allograft), human highly cross-

linked collagen membrane (Arthroflex; LifeNet Health, Virginia Beach, VA), porcine non-cross-

linked collagen membrane (Mucograft; Geistlich Pharma, Lucerne, Switzerland), a human platelet-

rich fibrin matrix (PRF-M), and a fibrin matrix based on platelet-rich plasma (ViscoGel; Arthrex,

Naples, FL). Cells were counted for adhesion (24 hours), thymidine assay for cell proliferation (96

hours), and live/dead stain for viability (168 hours). Histologic analysis was performed after 21

days, and the unloaded scaffolds were scanned with electron microscopy. RESULTS: MSCs were

successfully differentiated into all cell lines. A significantly greater number of cells adhered to both

the non-cross-linked porcine collagen scaffold and PRF-M. Cell activity (proliferation) was

significantly higher in the non-cross-linked porcine collagen scaffold compared with PRF-M and

fibrin matrix based on platelet-rich plasma. There were no significant differences found in the

results of the live/dead assay. CONCLUSIONS: Significant differences in the response of human

MSCs to biologic scaffolds existed. MSC adhesion, proliferation, and scaffold morphology

evaluated by histologic analysis and electron microscopy varied throughout the evaluated types of

scaffolds. Non-cross-linked porcine collagen scaffolds showed superior results for cell adhesion and

proliferation, as well as on histologic evaluation. CLINICAL RELEVANCE: This study enables the

clinician and scientist to choose scaffold materials according to their specific interaction with

MSCs.

51

Curr Med Res Opin. 2006 Nov;22(11):2221-32.

Collagen hydrolysate for the treatment of osteoarthritis and other joint disorders: a review of

the literature.

Bello AE, Oesser S.

BACKGROUND: There is a need for an effective treatment for the millions of people in the United

States with osteoarthritis (OA), a degenerative joint disease. The demand for treatments, both

traditional and non-traditional, will continue to grow as the population ages. SCOPE: This article

reviews the medical literature on the preclinical and clinical research on a unique compound,

collagen hydrolysate. Articles were obtained through searches of the PubMed database

(www.pubmed.gov) through May 2006 using several pairs of key words (collagen hydrolysate and

osteoarthritis; collagen hydrolysate and cartilage; collagen hydrolysate and chondrocytes; collagen

hydrolysate and clinical trial) without date limits. In addition, other sources of information, such as

abstracts presented at scientific congresses and articles in the German medical literature not

available on PubMed, were reviewed and included based on the authors' judgment of their relevance

to the topic of the review. FINDINGS: According to published research, orally administered

collagen hydrolysate has been shown to be absorbed intestinally and to accumulate in cartilage.

Collagen hydrolysate ingestion stimulates a statistically significant increase in synthesis of

extracellular matrix macromolecules by chondrocytes (p < 0.05 compared with untreated controls).

These findings suggest mechanisms that might help patients affected by joint disorders such as OA.

Four open-label and three double-blind studies were identified and reviewed; although many of

these studies did not provide key information--such as the statistical significance of the findings--

they showed collagen hydrolysate to be safe and to provide improvement in some measures of pain

and function in some men and women with OA or other arthritic conditions. CONCLUSION: A

growing body of evidence provides a rationale for the use of collagen hydrolysate for patients with

OA. It is hoped that ongoing and future research will clarify how collagen hydrolysate provides its

clinical effects and determine which populations are most appropriate for treatment with this

supplement.

52

Int J Food Sci Nutr. 2009;60 Suppl 2:99-113.

A randomized controlled trial on the efficacy and safety of a food ingredient, collagen

hydrolysate, for improving joint comfort.

Benito-Ruiz P, Camacho-Zambrano MM, Carrillo-Arcentales JN, Mestanza-Peralta MA, Vallejo-

Flores CA, Vargas-López SV, Villacís-Tamayo RA, Zurita-Gavilanes LA.

INTRODUCTION: Current options to promote joint comfort are limited to medicines that can

reduce pain but can also have adverse effects. Collagen, a major component of joint cartilage, is

found in the diet, particularly in meat. Its hydrolysed form, collagen hydrolysate (CH), is well

absorbed. CH may stimulate the joint matrix cells to synthesize collagen, so helping to maintain the

structure of the joint and potentially to aid joint comfort. METHODS: In a randomized, double-

blind, controlled multicentre trial, 250 subjects with primary osteoarthritis of the knee were given

10 g CH daily for 6 months. RESULTS: There was a significant improvement in knee joint comfort

as assessed by visual analogue scales to assess pain and the Womac pain subscale. Subjects with the

greatest joint deterioration, and with least intake of meat protein in their habitual diets, benefited

most. CONCLUSION: CH is safe and effective and warrants further consideration as a food

ingredient.

53

Biomed Mater. 2009 Jun;4(3):035006.

Early adhesive behavior of bone-marrow-derived mesenchymal stem cells on collagen

electrospun fibers.

Chan CK, Liao S, Li B, Lareu RR, Larrick JW, Ramakrishna S, Raghunath M.

A bioabsorbable nanofibrous scaffold was developed for early adhesion of mesenchymal stem cells

(MSCs). Collagen nanofibers with diameters of 430 +/- 170 nm were fabricated by electrospinning.

Over 45% of the MSC population adhered to this collagen nanofiber after 30 min at room

temperature. Remarkably, collagen-coated P(LLA-CL) electrospun nanofibers were almost as

efficient as collagen nanofibers whereas collagen cast film did not enhance early capture when it

was applied on cover slips. The adhesive efficiency could be further increased to over 20% at 20

min and over 55% at 30 min when collagen nanofibers were grafted with monoclonal antibodies

recognizing CD29 or CD49a. These data demonstrate that the early adhesive behavior is highly

dependent on both the surface texture and the surface chemistry of the substrate. These findings

have potential applications for early capture of MSCs in an ex vivo setting under time constraints

such as in a surgical setting.

54

J Am Acad Dermatol. 1989 Dec;21(6):1203-8.

Reactions to a bovine collagen implant. Clinical and immunologic study in 705 patients.

Charriere G, Bejot M, Schnitzler L, Ville G, Hartmann DJ.

A small percentage of patients treated with bovine collagen implants have adverse reactions

involving both the cellular and humoral types of immune response. We report a clinical follow-up

of 705 subjects treated with a new bovine collagen implant, Atelocollagen (Koken Co. Ltd., Tokyo,

Japan). Sensitization to the implant was evaluated in all subjects by skin testing, and humoral

response was monitored in 166 subjects by measuring the level of circulating antibodies directed

against bovine collagen. Twenty-seven patients (3.8%) exhibited a positive response to a skin test,

and of the remaining 656 patients, an adverse reaction to the implant developed in 2.3%. We found

a strong correlation between the presence of antibodies to collagen and a positive response to skin

testing (92%) or an adverse reaction (100%). In the case of a borderline clinical response to bovine

collagen implantation, anticollagen serologic tests appeared to be a useful tool for the identification

of clinically reactive patients.

55

Biomaterials. 2010 Dec;31(36):9438-51.

Efficacy of hESC-MSCs in knitted silk-collagen scaffold for tendon tissue engineering and

their roles.

Chen JL, Yin Z, Shen WL, Chen X, Heng BC, Zou XH, Ouyang HW.

Human embryonic stem cells (hESC) and their differentiated progenies are an attractive cell source

for transplantation therapy and tissue engineering. Nevertheless, the utility of these cells for tendon

tissue engineering has not yet been adequately explored. This study incorporated hESC-derived

mesenchymal stem cells (hESC-MSCs) within a knitted silk-collagen sponge scaffold, and assessed

the efficacy of this tissue-engineered construct in promoting tendon regeneration. When subjected

to mechanical stimulation in vitro, hESC-MSCs exhibited tenocyte-like morphology and positively

expressed tendon-related gene markers (e.g. Collagen type I & III, Epha4 and Scleraxis), as well as

other mechano-sensory structures and molecules (cilia, integrins and myosin). In ectopic

transplantation, the tissue-engineered tendon under in vivo mechanical stimulus displayed more

regularly aligned cells and larger collagen fibers. This in turn resulted in enhanced tendon

regeneration in situ, as evidenced by better histological scores and superior mechanical performance

characteristics. Furthermore, cell labeling and extracellular matrix expression assays demonstrated

that the transplanted hESC-MSCs not only contributed directly to tendon regeneration, but also

exerted an environment-modifying effect on the implantation site in situ. Hence, tissue-engineered

tendon can be successfully fabricated through seeding of hESC-MSCs within a knitted silk-collagen

sponge scaffold followed by mechanical stimulation.

56

Adv Drug Deliv Rev. 2003 Nov 28;55(12):1595-611.

Brain Res. 2011 Jan 12;1368:71-81.

Synergism of human amnion-derived multipotent progenitor (AMP) cells and a collagen

scaffold in promoting brain wound recovery: pre-clinical studies in an experimental model of

penetrating ballistic-like brain injury.

Chen Z, Lu XC, Shear DA, Dave JR, Davis AR, Evangelista CA, Duffy D, Tortella FC.

One of the histopathological consequences of a penetrating ballistic brain injury is the formation of

a permanent cavity. In a previous study using the penetrating ballistic-like brain injury (PBBI)

model, engrafted human amnion-derived multipotent progenitor (AMP) cells failed to survive when

injected directly in the injury tract, suggesting that the cell survival requires a supportive matrix. In

this study, we seated AMP cells in a collagen-based scaffold, injected into the injury core, and

investigated cell survival and neuroprotection following PBBI. AMP cells suspended in AMP cell

conditioned medium (ACCS) or in a liquefied collagen matrix were injected immediately after a

PBBI along the penetrating injury tract. Injured control rats received only liquefied collagen matrix.

All animals were allowed to survive two weeks. Consistent with our previous results, AMP cells

suspended in ACCS failed to survive; likewise, no collagen was identified at the injury site when

injected alone. In contrast, both AMP cells and the collagen were preserved in the injury cavity

when injected together. In addition, AMP cells/collagen treatment preserved some apparent brain

tissue in the injury cavity, and there was measurable infiltration of endogenous neural progenitor

cells and astrocytes into the preserved brain tissue. AMP cells were also found to have migrated

into the subventricular zone and the corpus callosum. Moreover, the AMP cell/collagen treatment

significantly attenuated the PBBI-induced axonal degeneration in the corpus callosum and

ipsilateral thalamus and improved motor impairment on rotarod performance. Overall, collagen-

based scaffold provided a supportive matrix for AMP cell survival, migration, and neuroprotection.

57

Curr Med Res Opin. 2008 May;24(5):1485-96.

24-Week study on the use of collagen hydrolysate as a dietary supplement in athletes with

activity-related joint pain.

Clark KL, Sebastianelli W, Flechsenhar KR, Aukermann DF, Meza F, Millard RL, Deitch JR,

Sherbondy PS, Albert A.

BACKGROUND: Collagen hydrolysate is a nutritional supplement that has been shown to exert an

anabolic effect on cartilage tissue. Its administration appears beneficial in patients with

osteoarthritis. OBJECTIVE: To investigate the effect of collagen hydrolysate on activity-related

joint pain in athletes who are physically active and have no evidence of joint disease. DESIGN

AND SETTING: A prospective, randomized, placebo-controlled, double-blind study was

conducted at Penn State University in University Park, Pennsylvania. Parameters including joint

pain, mobility, and inflammation were evaluated with the use of a visual analogue scale during a

24-week study phase. STUDY PARTICIPANTS: Between September 2005 and June 2006, 147

subjects who competed on a varsity team or a club sport were recruited. Data from 97 of 147

subjects could be statistically evaluated. INTERVENTION: One hundred and forty-seven subjects

(72 male, 75 female) were randomly assigned to two groups: a group (n = 73) receiving 25 mL of a

liquid formulation that contained 10 g of collagen hydrolysate (CH-Alpha) and a group (n = 74)

receiving a placebo, which consisted of 25 mL of liquid that contained xanthan. MAIN

OUTCOME MEASURES: The primary efficacy parameter was the change in the visual analogue

scales from baseline during the study phase in relation to the parameters referring to pain, mobility,

and inflammation. RESULTS: When data from all subjects (n = 97) were evaluated, six parameters

showed statistically significant changes with the dietary supplement collagen hydrolysate (CH)

compared with placebo: joint pain at rest, assessed by the physician (CH vs. placebo (-1.37 +/- 1.78

vs. -0.90 +/- 1.74 (p = 0.025)) and five parameters assessed by study participants: joint pain when

walking (-1.11 +/- 1.98 vs. -0.46 +/- 1.63, p = 0.007), joint pain when standing (-0.97 +/- 1.92 vs. -

0.43 +/- 1.74, p = 0.011), joint pain at rest (-0.81 +/- 1.77 vs. -0.39 +/- 1.56, p = 0.039), joint pain

when carrying objects (-1.45 +/- 2.11 vs. -0.83 +/- 1.71, p = 0.014) and joint pain when lifting (-

1.79 +/- 2.11 vs. -1.26 +/- 2.09, p = 0.018). When a subgroup analysis of subjects with knee

arthralgia (n = 63) was performed, the difference between the effect of collagen hydrolysate vs.

placebo was more pronounced. The parameter joint pain at rest, assessed by the physician, had a

statistical significance level of p = 0.001 (-1.67 +/- 1.89 vs. -0.86 +/- 1.77), while the other five

parameters based on the participants' assessments were also statistically significant: joint pain when

walking (p = 0.003 (-1.38 +/- 2.12 vs. -0.54 +/- 1.65)), joint pain when standing (p = 0.015 (-1.17

58

+/- 2.06 vs. -0.50 +/- 1.68)), joint pain at rest with (p = 0.021 (-1.01 +/-1.92 vs. -0.47 +/- 1.63)),

joint pain when running a straight line (p = 0.027 (-1.50 +/- 1.97 vs. -0.80 +/- 1.66)) and joint pain

when changing direction (p = 0.026 (-1.87 +/- 2.18 vs. -1.20 +/- 2.10)). CONCLUSION: This was

the first clinical trial of 24-weeks duration to show improvement of joint pain in athletes who were

treated with the dietary supplement collagen hydrolysate. The results of this study have implications

for the use of collagen hydrolysate to support joint health and possibly reduce the risk of joint

deterioration in a high-risk group. Despite the study's size and limitations, the results suggest that

athletes consuming collagen hydrolysate can reduce parameters (such as pain) that have a negative

impact on athletic performance. Future studies are needed to support these findings.

59

PLoS One. 2015 Mar 23;10(3):e0121654.

Curcuminoids extract, hydrolyzed collagen and green tea extract synergically inhibit

inflammatory and catabolic mediator's synthesis by normal bovine and osteoarthritic human

chondrocytes in monolayer.

Comblain F, Sanchez C, Lesponne I, Balligand M, Serisier S, Henrotin Y.

The main objective of this study was to assess the in vitro effects of curcuminoids extract,

hydrolyzed collagen and green tea extract in normal bovine chondrocytes and osteoarthritic human

chondrocytes cultured in monolayer. This study also investigated the synergic or additive effects of

these compounds. Enzymatically isolated primary bovine or human chondrocytes were cultured in

monolayer until confluence and then incubated for 24 hours or 48 hours in the absence or in the

presence of interleukin-1β and with or without curcuminoids extract, hydrolyzed collagen or green

tea extract, added alone or in combination, at different concentrations. Cell viability was neither

affected by these compounds, nor by interleukin 1β. In the absence of interleukin-1β, compounds

did not significantly affect bovine chondrocytes metabolism. In human chondrocytes and in the

absence of interleukin 1β, curcuminoids extract alone or in combination with hydrolyzed collagen

and green tea extract significantly inhibited matrix metalloproteinase-3 production. In interleukin-

1β-stimulated bovine chondrocytes, interleukin-6, inducible nitric oxide synthase, cyclooxygenase2,

matrix metalloproteinase 3, a disintegrin and metalloproteinase with thrombospondin type I motifs

4 and a disintegrin and metalloproteinase with thrombospondin type I motifs 5 expressions were

decreased by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea

extract. The combination of the three compounds was significantly more efficient to inhibit

interleukin-1β stimulated matrix metalloproteinase-3 expression than curcuminoids extract alone. In

interleukin-1β-stimulated human chondrocytes, nitric oxide, interleukin-6 and matrix

metalloproteinase 3 productions were significantly reduced by curcuminoids extract alone or in

combination with hydrolyzed collagen and green tea extract. These findings indicate that a mixture

of curcuminoids extract, hydrolyzed collagen and green tea extract has beneficial effects on

chondrocytes culture in inflammatory conditions and provide a preclinical basis for the in vivo

testing of this mixture.

60

PLoS One. 2016 Jun 8;11(6):e0156902.

Identification of Targets of a New Nutritional Mixture for Osteoarthritis Management

Composed by Curcuminoids Extract, Hydrolyzed Collagen and Green Tea Extract.

Comblain F, Dubuc JE, Lambert C, Sanchez C, Lesponne I, Serisier S, Henrotin Y.

OBJECTIVE: We have previously demonstrated that a mixture of curcuminoids extract, hydrolyzed

collagen and green tea extract (COT) inhibited inflammatory and catabolic mediator's synthesis by

osteoarthritic human chondrocytes. The objective of this study was to identify new targets of COT

using genomic and proteomic approaches. DESIGN: Cartilage specimens were obtained from 12

patients with knee osteoarthritis. Primary human chondrocytes were cultured in monolayer until

confluence and then incubated for 24 or 48 hours in the absence or in the presence of human

interleukin(IL)-1β (10-11M) and with or without COT, each compound at the concentration of 4

μg/ml. Microarray gene expression profiling between control, COT, IL-1β and COT IL-1β

conditions was performed. Immunoassays were used to confirm the effect of COT at the protein

level. RESULTS: More than 4000 genes were differentially expressed between conditions. The key

regulated pathways were related to inflammation, cartilage metabolism and angiogenesis. The IL-1β

stimulated chemokine ligand 6, matrix metalloproteinase-13, bone morphogenetic protein-2 and

stanniocalcin1 gene expressions and protein productions were down-regulated by COT. COT

significantly decreased stanniocalcin1 production in basal condition. Serpin E1 gene expression and

protein production were down-regulated by IL-1β. COT reversed the inhibitory effect of IL-1β.

Serpin E1 gene expression was up-regulated by COT in control condition. CONCLUSION: The

COT mixture has beneficial effect on osteoarthritis physiopathology by regulating the synthesis of

key catabolic, inflammatory and angiogenesis factors. These findings give a scientific rationale for

the use of these natural ingredients in the management of osteoarthritis.

61

J Am Acad Dermatol. 1984 Apr;10(4):638-46.

The immunogenicity of injectable collagen. I. A 1-year prospective study.

Cooperman L, Michaeli D.

With the growing use of collagen-based biomaterials, questions have been raised regarding the

immunogenicity of this protein in humans. Currently a bovine collagen implant is in widespread use

for the correction of dermal contour deficiencies ( Zyderm Collagen Implant (hereafter referred to

as "the implant"); Collagen Corporation, Palo Alto, CA). To investigate potential immunologic

consequences of this material, sixty-one subjects were evaluated in a 1-year prospective study. Two

of the sixty-one subjects (3%) experienced localized, self-limiting inflammatory responses to the

implant material; only in these two subjects could elevated levels of anti-implant collagen

antibodies be measured by radioimmunoassay. These antibodies did not cross-react with human

dermal collagen nor did they result in elevated levels of circulating immune complexes. Routine

blood and urine testing failed to reveal any results of clinical significance. Thus, this protein

displayed only weak antigenic activity in this study population.

62

Cox M, Nelson DR, Lehninger AL (2008).

Lehninger principles of biochemistry.

San Francisco: W.H. Freeman.

No abstract available

63

Ann Intern Med. 1993 Jun 15;118(12):920-8.

Association between bovine collagen dermal implants and a dermatomyositis or a

polymyositis-like syndrome.

Cukier J, Beauchamp RA, Spindler JS, Spindler S, Lorenzo C, Trentham DE.

OBJECTIVE: To determine whether an excess incidence of dermatomyositis or polymyositis or

both exist in patients treated with injectable bovine collagen implants and to characterize the

clinical picture. DESIGN: Historical cohort study (July 1980 through June 1988). PATIENTS:

Patients were identified from personal experience or adverse reaction reports received by the

manufacturer. SETTING: An 8-year period in the United States during which approximately

345,000 patients received implants. RESULTS: Eight patients with dermatomyositis and an

additional patient with polymyositis were identified from approximately 345,000 patients receiving

injectable bovine collagen implants from July 1980 through June 1988. The nine patients with

dermatomyositis or polymyositis were diagnosed an average of 6.4 months (range, 0.7 to 24.9

months) after collagen implant or skin test exposure or both. Eight of the nine patients had a

delayed-type hypersensitivity response at the test or treatment sites or both, and five of six patients

tested were found to have increased serum antibodies to collagen. Compared with the general

population, the incidence of dermatomyositis or polymyositis among collagen-treated patients was

statistically increased (standardized incidence ratio, 5.05; 95% CI, 2.31 to 9.59; P < 0.0001). A

similar analysis of the eight dermatomyositis case patients produced a standardized incidence ratio

of 18.8 (CI, 8.1 to 37.0; P < 0.0001). Using a Monte Carlo simulation, an interval of 6.4 months or

less from exposure to onset of disease was found to be an extremely rare event, occurring less than

72 times per one million simulation trials (CI, 57 to 91). CONCLUSIONS: Because these data

suggest that an immunologic response to bovine type I or type III collagen or both caused this

dermatomyositis or polymyositis-like syndrome, the risks versus benefits for the cosmetic use of

collagen implants should be reassessed.

64

JOM. 2011;April:66-73.

Collagen Scaffolds for Orthopedic Regenerative Medicine.

Cunniffe GM, O'Brien FJ

Collagen and collagen-based scaffolds offer distinct advantages when selected as biomaterials for

use across a broad spectrum of regenerative medicine applications. However, relatively poor

mechanical properties are often perceived to limit their usefulness for orthopedic applications.

These problems can be overcome through enhanced crosslinking mechanisms or through the

addition of a second, stiffer phase such as hydroxyapatite, thus allowing tailored composite

scaffolds to meet specific tissue requirements. This overview will highlight the current state of the

art of these scaffolds, and consider the exciting prospects and future directions of collagen-based

technologies for orthopedic regenerative medicine.

65

Biochim Biophys Acta. 2014 Feb;1838(2):579-88. doi: 10.1016/j.bbamem.2013.07.017. Epub 2013

Jul 24.

Mechanisms of talin-dependent integrin signaling and crosstalk.

Das M, Subbayya Ithychanda S, Qin J, Plow EF.

Cells undergo dynamic remodeling of the cytoskeleton during adhesion and migration on various

extracellular matrix (ECM) substrates in response to physiological and pathological cues. The major

mediators of such cellular responses are the heterodimeric adhesion receptors, the integrins.

Extracellular or intracellular signals emanating from different signaling cascades cause inside-out

signaling of integrins via talin, a cystokeletal protein that links integrins to the actin cytoskeleton.

Various integrin subfamilies communicate with each other and growth factor receptors under

diverse cellular contexts to facilitate or inhibit various integrin-mediated functions. Since talin is an

essential mediator of integrin activation, much of the integrin crosstalk would therefore be

influenced by talin. However, despite the existence of an extensive body of knowledge on the role

of talin in integrin activation and as a stabilizer of ECM-actin linkage, information on its role in

regulating inter-integrin communication is limited. This review will focus on the structure of talin,

its regulation of integrin activation and discuss its potential role in integrin crosstalk. This article is

part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane

channels, receptors and transporters.

66

Acta Biomater. 2010 Oct;6(10):3957-68.

Collagen-hyaluronic acid scaffolds for adipose tissue engineering.

Davidenko N, Campbell JJ, Thian ES, Watson CJ, Cameron RE.

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that

supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous

collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying

technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride.

All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%).

Physical and chemical analysis showed no signs of collagen denaturation during the formation

process. The values of thermal characteristics indicated that crosslinking occurred and that its

efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of

the strut material in water, the collagen-HA matrix as a whole tended to swell more and show

higher dissolution resistance than pure collagen samples. The compressive modulus and elastic

collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support

the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a

significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites

displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for

8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust,

freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.

67

J Exp Med. 1967 Aug 1;126(2):331-46.

The serologic specificity of tropocollagen telopeptides.

Davison PF, Levine L, Drake MP, Rubin A, Bump S.

Tropocollagen preparations from carp, buffalo fish, rats, calves, sheep, and humans have been

studied by electron microscopy and serologic methods. Tropocollagens from each species appeared

identical by electron microscopy but they were readily distinguished (except between sheep and

calves) by C'-fixation tests with rabbit antisera against the various tropocollagens. Tests with calf

tropocollagen antiserum showed no distinction between tropocollagen isolated from different

tissues nor between individuals of the same or different strains. The major immunogenic sites in

native tropocollagen are the telopeptides, and these are present on both alpha1- and alpha2-chains.

The C'-fixing activity was lost with heat denaturation of the tropocollagen, but could be recovered

in a concentration-dependent process on cooling. The fact that pure and enzyme-treated collagen

can provoke serologic reaction implies that collagenous sutures and prostheses used in surgery may

lead to sensitization and rejection, a fact which may merit clinical concern.

68

Plast Reconstr Surg. 1987 Apr;79(4):581-94.

Reaction to injectable collagen: results in animal models and clinical use.

DeLustro F, Smith ST, Sundsmo J, Salem G, Kincaid S, Ellingsworth L.

Since its commercial release, Zyderm collagen implant has been used to treat more than 200,000

subjects in the United States for soft-tissue contour defects and more than 250,000 patients

internationally (including the United States). Approximately 3 percent of subjects' skin tested with

Zyderm collagen experience localized hypersensitivity reactions to collagen, whereas

approximately 1 percent of treated patients demonstrate symptoms of hypersensitivity at treatment

sites. Of the latter treatment responses reported since the conclusion of clinical trials with Zyderm,

56 percent occurred following the first treatment, 28 percent following the second, 10 percent

following the third, and 6 percent following subsequent exposures. The data indicate that most

patients receive a median of three treatments (mean = 4.4) with Zyderm collagen, but most patients

who are likely to develop sensitivity to Zyderm collagen appear to respond immunologically to the

test implant or first treatment exposure. Examining these treatment responses, 45 percent of the

patients reported an onset of symptoms within 10 days and 22 percent at more than 30 days

following the last treatment with Zyderm collagen. Erythema was the sole symptom in 24 percent of

cases, whereas erythema plus induration comprised an additional 42 percent. Antibodies against

Zyderm collagen were detected in the sera of 88 percent of these subjects using an ELISA, but no

reactivity was observed against human collagen. Sera from patients reporting only systemic

symptoms were not found to have anticollagen antibodies. These data suggest that the relative risk

of a hypersensitivity reaction to Zyderm collagen does not increase with multiple exposures, since

patients who are going to develop an immune response to bovine collagen react with greatest

frequency to initial injections of collagen. In animal models, Zyderm collagen was shown to be less

immunogenic than other medical devices which are composed of bovine collagen. Specifically,

comparative studies were conducted in which Zyderm collagen and hemostatic agents were

implanted in the guinea pig subcutaneum: sera from animals treated with collagen-derived

hemostatic devices possessed significant levels of anti-implant antibodies (titers greater than 640),

whereas animals treated with Zyderm collagen mounted minimal responses (titers less than 40).

Additional studies were conducted in which implant materials were compared in a guinea pig

parietal (bony defect) model and in a rabbit hemostasis model: in both, Zyderm collagen

demonstrated lower immunogenicity than commercial bovine collagen hemostatic agents.

Histologic results from these studies showed Zyderm

69

Clin Orthop Relat Res. 1990 Nov;(260):263-79.

Immune responses to allogeneic and xenogeneic implants of collagen and collagen derivatives.

DeLustro F, Dasch J, Keefe J, Ellingsworth L.

Whereas xenogeneic collagen has provided a safe and effective biomaterial for numerous medical

applications, there are few instances in which data permit the correlation of the immunologic profile

of well-defined devices with their clinical sequelae. A major exception is the use of injectable

bovine dermal collagen for soft-tissue contour correction. The low incidence of hypersensitivity has

been studied in the context of clinical efficacy and safety with several devices. The findings indicate

that such immunity usually results in the manifestation of local symptoms of dermal inflammation

at sites of treatment that resolve as the implant is resorbed by the host. In contrast, more

immunogenic hemostatic agents may elicit a more frequent or vigorous immune response that is not

clinically visible or relevant in that application. Recent experiences with collagen-based devices for

the repair and regeneration of bone have also demonstrated that the presence of immunity to their

collagenous or non-collagenous components does not necessarily predict adverse clinical sequelae.

Indeed, numerous specific data indicate that this immunity can exist as an epiphenomenon with no

effect on osteogenesis. To get a true composite picture of biocompatibility, significant steps must be

taken to characterize biomaterials properly and to ensure that immunologic, clinical, histologic, and

other pertinent laboratory data are viewed in relation to one another and not in isolation.

70

J Med Dent Sci. 2008 Mar;55(1):71-9.

The axonal regeneration across a honeycomb collagen sponge applied to the transected spinal

cord.

Fukushima K, Enomoto M, Tomizawa S, Takahashi M, Wakabayashi Y, Itoh S, Kuboki Y,

Shinomiya K.

We developed a honeycomb-shaped lyophilized Type I atelocollagen (Honeycomb Collagen: HC)

with different pore sizes, and the effectiveness of the honeycomb shape on nerve regeneration was

examined. We analyzed neurite outgrowth of dorsal root ganglion (DRG) explants on HC, both in

vitro and, with direct implantation of HC into the defects of adult rat spinal cords, in vivo. The

neurites of DRGs on HC extended linearly through the pores. HC with a 400 microm-pore size

enhanced neurite extension, and YIGSR laminin peptide coating to the HC extended more neurites

than fibronectin coating. The HC scaffolds coated with YIGSR were implanted into 2 mm-defects

of spinal cords at the level of T8-9. Four weeks after implantation, the implants had degraded and

been replaced with self-tissues, repairing the injured site. Neurofilament-positive fibers were

observed in the implantation area and passed the borders between the HC and spinal cord stumps.

Functionally, a motor-evoked potential was observed in the quadriceps femoris muscle 10 weeks

after implantation. The electrophysiological examination showed reconstruction of axon tracts over

the implant. This result indicates that our developed honeycomb shape is advantageous for host

spinal cord compared to the random pored sponge shape, and that it promotes axonal regeneration

after spinal cord injury.

71

FEBS Lett. 1971 Feb 9;12(6):341-344.

Different antigenic determinants in the polypeptide chains of human collagen.

Furthmayr H, Beil W, Timpl R.

No abstract available.

72

Clin Exp Rheumatol. 2006 Sep-Oct;24(5):514-20.

Polymerized-type I collagen for the treatment of patients with rheumatoid arthritis. Effect of

intramuscular administration in a double blind placebo-controlled clinical trial.

Furuzawa-Carballeda J, Fenutria-Ausmequet R, Gil-Espinosa V, Lozano-Soto F, Teliz-Meneses

MA, Romero-Trejo C, Alcocer-Varela J.

OBJECTIVE: To determine the efficacy, tolerance and safety of intramuscular injections of

porcine type I collagen-PVP in patients with RA in a long term-therapy. METHODS: The study

was a double blind placebo-controlled and included 30 patients with active RA (ACR). Patients

were treated with intramuscular injections of 2 ml of collagen-PVP (3.4 mg of collagen) or 2 ml of

placebo during 6 months. The follow up was done during the next 6 months. The primary endpoints

included the Ritchie index (RI), swollen joint count, disease activity score (DAS), erythrocyte

sedimentation rate (ESR), and C-reactive protein (CRP). The secondary endpoints included

morning stiffness, pain intensity on a visual analogue scale (VAS), and Spanish-health assessment

questionnaire (HAQ-DI). Improvement was determined using American College of Rheumatology

response criteria (ACR20, 50 and 70). RESULTS: Collagen-PVP was safe and well tolerated. There

were no adverse events. Patients had a statistically significant improvement (p < 0.05) in collagen-

PVP-treated vs. placebo at 6 months of treatment in: swollen joint count (7.1 +/- 0.8 vs. 16.0 +/-

1.6), RI (8.1 +/- 0.8 vs. 15.2 +/- 1.5), morning stiffness (9.2 +/- 3.1 vs. 29.1 +/- 5.9 min), HAQ-DI

(50.0 +/- 10.8 vs. 22.9 +/- 10.3), DAS (3.0 +/- 0.2 vs. 4.9 +/- 0.3), ACR20 (78.6 vs. 71.4%), ACR50

(57.1 vs. 0%) and ACR70 (7.1 vs. 0%) and CRP (1.1 +/- 0.4 vs. 2.5 +/- 0.7). Patients treated with

collagen-PVP required lower doses of methotrexate vs. placebo (12.6 +/- 0.6 vs. 14.2 +/- 0.7 at 6

months and 12.3 +/- 0.8 vs. 15.4 +/- 0.6 at 12 months; p < 0.05). Serological or haematological

parameters remained unchanged. CONCLUSION: Collagen-PVP has been shown to be a safe and

well-tolerated drug for the long-term treatment of RA. Combination of collagen-PVP plus

methotrexate was more efficacious than methotrexate alone. This biodrug can be useful in the

treatment of RA.

73

Eur J Clin Invest. 2009 Jul;39(7):591-7.

Effect of polymerized-type I collagen in knee osteoarthritis. I. In vitro study.

Furuzawa-Carballeda J, Muñoz-Chablé OA, Barrios-Payán J, Hernández-Pando R.

BACKGROUND: Polymerized-type I collagen (polymerized-collagen) is a down-regulator of

inflammation and a tissue regenerator biodrug. The aim of this study was to evaluate its effect in co-

cultures of cartilage and synovial tissue from patients with knee osteoarthritis (OA). MATERIALS

AND METHODS: Cartilage and synovial tissue from five patients with OA were co-cultured for 7

days in the presence or absence of 1% polymerized-collagen. To determine proteoglycans content,

tissues were stained with alcian blue technique. Pro- and anti-inflammatory cytokines [interleukin

(IL)-1beta, IL-8, IL-10, IL-12, tumour necrosis factor (TNF)-alpha, and interferon (IFN)-gamma]

and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants by ELISA and

results were normalized by total protein concentration. Cartilage oligomeric matrix protein

(COMP), type II collagen, TNF-alpha, IL-10 and Ki-67 expression were determined by

immunohistochemistry. RESULTS: Polymerized-type I collagen induced an increase of 3- to 6fold

cell proliferation (Ki-67), proteoglycans content, and COMP and type II collagen expression,

whereas it inhibited IL-1beta and TNF-alpha production. IL-10 levels were up-regulated in treated

vs. untreated cultures. No differences were found on IL-8 or TIMP-1 levels in supernatants from

polymerized-collagen-treated co-cultures when compared with untreated cultures. IL-12 and IFN-

gamma were undetectable. CONCLUSION: The addition of polymerized-type I collagen to

cartilage and synovial tissue co-cultures induced up-regulation of chondrocytes proliferation and

cartilage extracellular matrix proteins production (COMP, type II collagen and proteoglycans) as

well as an anti-inflammatory cytokine (IL-10) and the down-modulation of pro-inflammatory

cytokines (IL-1beta and TNF-alpha). It is possible that this mechanism might contribute to induce

tissue regeneration and down-regulation of inflammation in OA.

74

Eur J Clin Invest. 2009 Jul;39(7):598-606.

Effect of polymerized-type I collagen in knee osteoarthritis. II. In vivo study.

Furuzawa-Carballeda J, Muñoz-Chablé OA, Macías-Hernández SI, Agualimpia-Janning A.

BACKGROUND: Polymerized-Type I Collagen (Polymerized-Collagen) is an anti-inflammatory

and a tissue regenerator biodrug. The aim of the study was to evaluate the efficacy and safety of

intra-articular injections of Polymerized-Collagen in patients with knee osteoarthritis (OA).

METHODS AND DESIGN: Patients (n=53) were treated with 12 intra-articular injections of 2 mL

of Polymerized-Collagen (n=27) or 2 mL of placebo (n=26) during 6 months. Follow up period was

6 months. The primary endpoints included Western Ontario and McMaster University Osteoarthritis

Index, Lequesne index, and pain intensity on a visual analogue scale (VAS). Secondary outcomes

were patient global score, investigator global score and drug evaluation. Clinical improvement was

determined if the decrease in pain exceeds 20 mm on a VAS and patients achieved at least 20% of

improvement from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type

II (CTXII) and serum high-sensitivity C-reactive protein (hsCRP) were determined by enzyme

immunoassays. Statistical analysis was performed by intention to treat. RESULTS: Polymerized-

Collagen was safe and well tolerated. Patients had a statistically significant improvement (P<0.05)

from baseline vs. Polymerized-Collagen and vs. placebo at 6 months in: Lequesne Index (13.1+/-0.5

vs. 7.1+/-0.7 vs. 9.6+/-0.8; P=0.027), WOMAC (9.0+/-0.5 vs. 4.0+/-0.6 vs. 5.80+/-0.8; P=0.032),

patient VAS (60.0+/-2.6 vs. 20.6+/-2.4 vs. 36.1+/-4.5; P=0.003), physician VAS (49.8+/-1.9 vs.

16.8+/-2.9 vs. 29.8+/-2.9; P=0.002), patient global score (1.08+/-0.1 vs. 2.7+/-0.1 vs. 1.9+/-0.2;

P=0.028) and analgesic usage (30.1+/-9.4 vs. 11.0+/-3.4 vs. 17.9+/-4.9; P=0.001). This

improvement was persistent during the follow up. A threefold increase in CTXII was determined in

placebo group. No differences were found on hs CRP and incidence of adverse events between

groups. CONCLUSION: Polymerized-Collagen is safe and effective in the treatment of knee OA.

75

ScientificWorldJournal. 2012;2012:342854.

Polymerized-type I collagen downregulates inflammation and improves clinical outcomes in

patients with symptomatic knee osteoarthritis following arthroscopic lavage: a randomized,

double-blind, and placebo-controlled clinical trial.

Furuzawa-Carballeda J, Lima G, Llorente L, Nuñez-Álvarez C, Ruiz-Ordaz BH, Echevarría-Zuno

S, Hernández-Cuevas V.

OBJECTIVES: Polymerized-type I collagen (polymerized collagen) is a downmodulator of

inflammation and cartilage regenerator biodrug. AIM: To evaluate the effect of intraarticular

injections of polymerized collagen after arthroscopic lavage on inflammation and clinical

improvement in patients with knee osteoarthritis (OA). METHODS: Patients (n = 19) were treated

with 6 intraarticular injections of 2 mL of polymerized collagen (n = 10) or 2 mL of placebo (n = 9)

during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on

a visual analogue scale (VAS), WOMAC, analgesic usage, the number of Tregs and

proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were

Likert score and drug evaluation. Clinical and immunological improvement was determined if the

decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters

from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII) and

erythrocyte sedimentation rate (ESR) were determined. RESULTS: Polymerized collagen was safe

and well tolerated. Patients had a statistically significant improvement (P < 0.05) from baseline

versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs

IL-1β, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in

polymerized collagen versus placebo. No differences were found on incidence of adverse events

between groups. CONCLUSION: Polymerized collagen is safe and effective on downregulation of

inflammation in patients with knee OA.

76

Plast Reconstr Surg. 1989 May;83(5):875-9.

Identification of the collagen-producing cells in healing flexor tendons.

Garner WL, McDonald JA, Koo M, Kuhn C 3rd, Weeks PM.

A monoclonal antibody to procollagen type I (anti-pC) which specifically stains cells synthesizing

collagen was used to study the healing of chicken flexor tendons in vivo. Healing was assessed

using routine hematoxylin and eosin histology and immunoperoxidase staining with anti-pC.

Studies showed that epitenon cells proliferate 3 days after injury and are producing collagen by 7

days after injury. Tenocytes do not begin producing collagen until 14 to 21 days after injury. From

3 to 5 weeks, the entire substance of the tendon becomes filled with collagen-synthesizing cells.

These cells may have originated from the tendon sheath, the epitenon, or the endotenon; however,

the evidence presented in this study suggests that the epitenon is a major source of these cells.

77

Urol. 1998;33(2):129-33.


Collagen sponges for bone regeneration with rhBMP-2.

Geiger M, Li RH, Friess W.

In the US alone, approximately 500,000 patients annually undergo surgical procedures to treat bone

fractures, alleviate severe back pain through spinal fusion procedures, or promote healing of non-

unions. Many of these procedures involve the use of bone graft substitutes. An alternative to bone

grafts are the bone morphogenetic proteins (BMPs), which have been shown to induce bone

formation. For optimal effect, BMPs must be combined with an adequate matrix, which serves to

prolong the residence time of the protein and, in some instances, as support for the invading

osteoprogenitor cells. Several factors involved in the preparation of adequate matrices, specifically

collagen sponges, were investigated in order to test the performance in a new role as an implant

providing local delivery of an osteoinductive differentiation factor. Another focus of this review is

the current system consisting of a combination of recombinant human BMP-2 (rhBMP-2) and an

absorbable collagen sponge (ACS). The efficacy and safety of the combination has been clearly

proven in both animal and human trials.

78

Biotechnol Appl Biochem. 2004 Jun;39(Pt 3):329-38.

Inactivation of the bovine-spongiform-encephalopathy (BSE) agent by the acid and alkaline

processes used in the manufacture of bone gelatine.

Grobben AH, Steele PJ, Somerville RA, Taylor DM.

A validation study was carried out to determine the capacity of the traditional acid and alkaline

processes used in the manufacture of bovine bone gelatine to remove and/or inactivate the

transmissible agent that causes BSE (bovine spongiform encephalopathy). Using an accurately

scaled down laboratory process that precisely mimicked the minimum conditions of the industrial

processes, gelatine (gelatin) was manufactured from industrial starting material that had been spiked

with mouse brain infected with the 301V strain of mouse-passaged BSE agent. Clearance factors

were determined by titrating the infectivity levels of the infected mouse brain tissue, the gelatine

extracts, and the final sterilized gelatine solution. The infectivity level of the spiked starting

material was 10(8.4) mouse intracerebral ID(50)/kg (ID(50) is the dose at which half of the

challenged animals were infected). Clearance factors of 10(2.6) and 10(3.7) ID(50) were

demonstrated for the first stages of the acid and alkaline processes respectively during which the

bones are converted to crude gelatine. It was further demonstrated that the complete acid and

alkaline processes both reduced infectivity to undetectable levels, giving clearance factors of

>/=10(4.8) ID(50) for the acid process, and >/=10(4.9) ID(50) for the alkaline process.

79

Dent Traumatol. 2004 Dec;20(6):334-7.

The effect on osteogenesis of type I collagen applied to experimental bone defects.

Gungormus M.

The purpose of the present investigation was to evaluate the effects of type I collagen sponge on the

healing of bone defects. In this study, six adult male rabbits were used. After the induction of

general anesthesia with intraperitoneal kethamine, the anterior surfaces of tibias of the rabbits were

surgically exposed, and two holes with 4 mm in diameter were prepared on each tibia for the

investigation. Only one hole in each tibia was filled with type I collagen, the other unfilled hole was

used as control. During the study, radiopacity changes in the radiographs of the tibias of the rabbits

were evaluated. The animals were killed on the 28th day, and histologic sections of the tibias were

prepared. On the 28th day, it was histopathologically observed that collagen cavities were filled

with new bone. In addition, it was determined that there was an increase in radiopacity of the defect

areas from 14 to 28 days in both groups, and there were statistically a significant difference between

control and collagen groups (P = 0.0001). In this study, consequently, it was determined that type I

collagen sponge in the experimental cavities provides a more rapid regeneration of bone defects

compared with non-filled cavities.

80

JAMA. 1995 Dec 13;274(22):1800-4.

Users' guides to the medical literature. IX. A method for grading health care

recommendations. Evidence-Based Medicine Working Group.

Guyatt GH, Sackett DL, Sinclair JC, Hayward R, Cook DJ, Cook RJ.

No abstract available.

81

Biomaterials. 2008 Feb;29(4):438-47.

An injectable cross-linked scaffold for nucleus pulposus regeneration.

Halloran DO, Grad S, Stoddart M, Dockery P, Alini M, Pandit AS.

Incorporation of scaffolds has long been recognized as a critical element in most tissue engineering

strategies. However with regard to intervertebral disc tissue engineering, the use of a scaffold

containing the principal extracellular matrix components of native disc tissue (i.e. collagen type II,

aggrecan and hyaluronan) has not been investigated. In this study the behavior of bovine nucleus

pulposus cells that were seeded within non-cross-linked and enzymatically cross-linked,

atelocollagen type II based scaffolds containing varying concentrations of aggrecan and hyaluronan

was investigated. Cross-linking atelocollagen type II based scaffolds did not cause any negative

effects on cell viability or cell proliferation over the 7-day culture period. The cross-linked scaffolds

retained the highest proteoglycan synthesis rate and the lowest elution of sulfated

glycosaminoglycan into the surrounding medium. From confined compression testing and volume

reduction measurements, it was seen that the cross-linked scaffolds provided a more stable structure

for the cells compared to the non-cross-linked scaffolds. The results of this study indicate that the

enzymatically cross-linked, composite collagen-hyaluronan scaffold shows the most potential for

developing an injectable cell-seeded scaffold for nucleus pulposus treatment in degenerated

intervertebral discs.

82

J Orthop Sci. 2013 Jul;18(4):627-35.

Treatment of cartilage defects by subchondral drilling combined with covering with

atelocollagen membrane induces osteogenesis in a rat model.

Hamanishi M, Nakasa T, Kamei N, Kazusa H, Kamei G, Ochi M.

BACKGROUND: The coverage of the atelocollagen membrane at the chondral defect after

subchondral drilling might improve the beneficial effects for cartilage repair because of the

prevention of scattering and accumulation of cells and growth factors from bone marrow within the

chondral defect. On the other hand, it might block cells and factors derived from the synovium or

cause high pressure in the chondral defect, resulting in prevention of cells and growth factors

gushing out from the bone marrow, which leads to disadvantages for cartilage repair. METHOD:

We tested this hypothesis in a 2-mm-diameter chondral defect created in the articular cartilage of

the patellar groove in a rat models. Defects were left untreated, or were drilled or drilled and

covered with an atelocollagen membrane; healing was evaluated by histology and gene expression

analysis using real-time polymerase chain reaction and immunohistochemistry. RESULTS:

Membrane coverage induced bone tissue ingrowth into the punched chondral defect. At 1 week,

expression of TGFβ, Sox9, Runx2, osteocalcin, Col1a1, and Col2a1 in the drilling group was

significantly higher than in the covering group. At 4 weeks, expressions of TGFβ, Runx2, and

Col1a1 were all significantly higher in the drilling group, while Sox9, osteocalcin, and Col2a1 were

significantly higher in the covering group. Immunohistochemistry demonstrated Sox9, osteocalcin,

and type II collagen on the bony reparative tissue in the covering group. CONCLUSIONS: These

results suggest that the atelocollagen membrane coverage resulted in inhibition of cartilage repair.

83

Foot Ankle Clin. 2007 Dec;12(4):553-67, v.

Tendon healing.

Hope M, Saxby TS.

An understanding of the processes of tendon healing and tendon-to-bone healing is important for

the intraoperative and postoperative management of patients with tendon ruptures or of patients

requiring tendon transfers in foot and ankle surgery. Knowledge of the normal process allows

clinicians to develop strategies when normal healing fails. This article reviews the important work

behind the identification of the normal phases and control of tendon healing. It outlines the failed

response in tendinopathy and describes tendon-to-bone healing in view of its importance in foot and

ankle surgery.

84

J Reconstr Microsurg. 2001 Feb;17(2):115-23.

A study of induction of nerve regeneration using bioabsorbable tubes.

Itoh S, Takakuda K, Ichinose S, Kikuchi M, Schinomiya K.

A silicone tube (S-tube) was packed with CPLA (copolymer of poly-L-lactide) fibers (S-

tube+CPLA) or collagen fibers (S-tube+fiber). Two types of tube were prepared from a collagen

sheet (Col-tube) and a bioabsorbable atelocollagen membrane for guided tissue regeneration (GTR-

tube). They were packed with collagen fibers or films (Col-tube+fiber, GTR-tube+fiber and GTR-

tube+film). Bridge grafting (15 mm in length) was performed with these tubes in a rat sciatic nerve

model. Specimens were harvested after 8 weeks. Minifascicles were formed in the open space

between the CPLA fibers in the S-tube+CPLA group. Regenerated axons were also formed in the

degenerated collagen fibers in the S-tube+Col group. Immunocytochemistry evaluation revealed

that Schwann cells invaded the space in the absorbing collagen fibers. Histologic analysis of the

regenerated axons in the groups with Col-tubes or GTR-tubes revealed that both the Col-tube and

the GTR-tube packed with collagen fibers were effective in providing a scaffold for regenerating

nerve tissue

85

J Hand Surg Am. 2008 Jan;33(1):102-12.

Tendon: biology, biomechanics, repair, growth factors, and evolving treatment options.

James R, Kesturu G, Balian G, Chhabra AB.

Surgical treatment of tendon ruptures and lacerations is currently the most common therapeutic

modality. Tendon repair in the hand involves a slow repair process, which results in inferior repair

tissue and often a failure to obtain full active range of motion. The initial stages of repair include

the formation of functionally weak tissue that is not capable of supporting tensile forces that allow

early active range of motion. Immobilization of the digit or limb will promote faster healing but

inevitably results in the formation of adhesions between the tendon and tendon sheath, which leads

to friction and reduced gliding. Loading during the healing phase is critical to avoid these adhesions

but involves increased risk of rupture of the repaired tendon. Understanding the biology and

organization of the native tendon and the process of morphogenesis of tendon tissue is necessary to

improve current treatment modalities. Screening the genes expressed during tendon morphogenesis

and determining the growth factors most crucial for tendon development will likely lead to

treatment options that result in superior repair tissue and ultimately improved functional outcomes.

86

Cells Tissues Organs. 2013;198(4):278-88.

Autologous collagen-induced chondrogenesis using fibrin and atelocollagen mixture.

Jeong IH, Shetty AA, Kim SJ, Jang JD, Kim YJ, Chung YG, Choi NY, Liu CH.

For articular cartilage defect treatment, many treatment modalities have been developed. We

evaluate the cartilage repair potential of an atelocollagen and fibrin mixture transplanted to cartilage

defects. A circular, articular cartilage defect 4 mm in diameter was made in the trochlear region in

each of 20 New Zealand white rabbits. The 10 rabbits in the control group were kept without

treatment and the 10 rabbits in the experimental group underwent injection of atelocollagen mixed

with fibrin. At week 12 following surgery the cartilage was observed and histologically compared

in both groups. The surface of the newly generated cartilage was very smooth and even, and we also

noted that the entire area was completely regenerated in the experimental group. The control group

showed incomplete and irregular cartilage formation in the defect. Regarding the histological

scoring, comparison of the two groups differed significantly (p < 0.001). Injection of a mixture of

atelocollagen and fibrin used to treat articular cartilage defects of the knee appears to be an effective

method for cartilage regeneration.

87

Connect Tissue Res. 2013;54(3):218-26.

Cellular and molecular factors in flexor tendon repair and adhesions: a histological and gene

expression analysis.

Juneja SC, Schwarz EM, O'Keefe RJ, Awad HA.

Flexor tendon healing is mediated by cell proliferation, migration, and extracellular matrix synthesis

that contribute to the formation of scar tissue and adhesion. The biological mechanisms of flexor

tendon adhesion formation have been linked to transforming growth factor β (TGF-β). To elucidate

the cellular and molecular events in this pathology, we implanted live flexor digitorum longus grafts

from the reporter mouse Rosa26(LacZ/+) in wild-type recipients, and used histological β-

galactosidase (β-gal) staining to evaluate the intrinsic versus extrinsic cellular origins of scar, and

reverse transcription-polymerase chain reaction to measure gene expression of TGF-β and its

receptors, extracellular matrix proteins, and matrix metalloproteinases (MMPs) and their regulators.

Over the course of healing, graft cellularity and β-gal activity progressively increased, and β-gal-

positive cells migrated out of the Rosa26(LacZ/+) graft. In addition, there was an evidence of influx

of host cells (β-gal-negative) into the gliding space and the graft, suggesting that both graft and host

cells contribute to adhesions. Interestingly, we observed a biphasic pattern in which Tgfb1

expression was the highest in the early phases of healing and gradually decreased thereafter,

whereas Tgfb3 increased and remained upregulated later. The expression of TGF-β receptors was

also upregulated throughout the healing phases. In addition, type III collagen and fibronectin were

upregulated during the proliferative phase of healing, confirming that murine flexor tendon heals by

scar tissue. Furthermore, gene expression of MMPs showed a differential pattern in which

inflammatory MMPs were the highest early and matrix MMPs increased over time. These findings

offer important insights into the complex cellular and molecular factors during flexor tendon

healing.

88

Curr Opin Cell Biol. 2008 Oct;20(5):495-501.

Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and

nucleators.

Kadler KE, Hill A, Canty-Laird EG.

Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-

ECM interface. There are more than 30 collagens and collagen-related proteins but the most

abundant are collagens I and II that exist as D-periodic (where D = 67 nm) fibrils. The fibrils are of

broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair,

fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils

in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation

in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin,

fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-

containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular

mechanisms are in place to control what is otherwise a protein self-assembly process. This review

puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the

site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.

89

J Bone Miner Metab. 2012 Nov;30(6):638-50.

Chronological histological changes during bone regeneration on a non-crosslinked

atelocollagen matrix.

Kagawa R, Kishino M, Sato S, Ishida K, Ogawa Y, Ikebe K, Oya K, Ishimoto T, Nakano T, Maeda

Y, Komori T, Toyosawa S.

Cleavage of the antigenic telopeptide region from type I collagen yields atelocollagen, and this is

widely used as a scaffold for bone regeneration combined with cells, growth factors, etc. However,

neither the biological effect of atelocollagen alone or its contribution to bone regeneration has been

well studied. We evaluated the chronological histological changes during bone regeneration

following implantation of non-crosslinked atelocollagen (Koken Co., Ltd.) in rat calvarial defects.

One week after implantation, osteogenic cells positive for runt-related transcription factor 2

(Runx2) and osteoclasts positive for tartrate-resistant acid phosphatase (TRAP) were present in the

atelocollagen implant in the absence of bone formation. The number of Runx2-positive osteogenic

cells and Osterix-positive osteoblasts increased 2 weeks after implantation, and bone matrix

proteins (osteopontin, OPN; osteocalcin, OC; dentin matrix protein 1, DMP1) were distributed in

newly formed bone in a way comparable to normal bone. Some resorption cavities containing

osteoclasts were also present. By 3 weeks after implantation, most of the implanted atelocollagen

was replaced by new bone containing many resorption cavities, and OPN, OC, and DMP1 were

deposited in the residual collagenous matrix. After 4 weeks, nearly all of the atelocollagen implant

was replaced with new bone including hematopoietic marrow. Immunohistochemistry for the

telopeptide region of type I collagen (TeloCOL1) during these processes demonstrated that the

TeloCOL1-negative atelocollagen implant was replaced by TeloCOL1-positive collagenous matrix

and new bone, indicating that new bone was mostly composed of endogenous type I collagen.

These findings suggest that the atelocollagen itself can support bone regeneration by promoting

osteoblast differentiation and type I collagen production.

90

ASAIO J. 2007 Jul-Aug;53(4):506-13.

Regeneration of skeletal muscle using in situ tissue engineering on an acellular collagen

sponge scaffold in a rabbit model.

Kin S, Hagiwara A, Nakase Y, Kuriu Y, Nakashima S, Yoshikawa T, Sakakura C, Otsuji E,

Nakamura T, Yamagishi H.

Because of the limited ability of skeletal muscle to regenerate, resection of a large amount of

muscle mass often results in incomplete recovery due to nonfunctional scar tissue. The aim of this

study was to regenerate skeletal muscle using in situ tissue engineering in a rabbit model. In 18

male rabbits, a muscle defect (1.0 x ~1.0 x ~0.5 cm) was created in the vastus lateralis of both legs.

A piece of cross-linked atelocollagen sponge was then inserted into the defect in one leg, whereas

the defect in the other leg was left untreated. Both defects were finally covered with fascia. Twenty-

four weeks after surgery, the defect that had been filled with the cross-linked atelocollagen sponge

scaffold showed mild concavity and slight adhesion to the fascia, while the control side showed

severe scar formation and shrinkage. Histologically, the regenerating myofibers at the site

containing the collagen sponge were greater in number, diameter, and length than those at the

control site. These results indicate that cross-linked atelocollagen sponge has the potential to act as

a scaffold for muscle tissue regeneration.

91

J Sci Food Agric. 2015 Mar 15;95(4):702-7.

A double-blind, placebo-controlled, randomised, clinical study on the effectiveness of collagen

peptide on osteoarthritis.

Kumar S, Sugihara F, Suzuki K, Inoue N, Venkateswarathirukumara S.

Author information

Abstract

Background: Recent studies show that enzymatically hydrolysed collagen, the collagen peptide, is

absorbed and distributed to joint tissues and has analgesic and anti-inflammatory properties. A

double-blind, placebo-controlled, randomised trial with collagen peptides isolated from pork skin

(PCP) and bovine bone (BCP) sources was carried out to study the effectiveness of orally

supplemented collagen peptide to control the progression of osteoarthritis in patients diagnosed with

knee osteoarthritis. Improvement in treatment was assessed with reduction in Western Ontario

McMaster Universities (WOMAC), visual analogue scale (VAS) and quality of life (QOL) scores

from baseline to 13 weeks (Visit 7). Safety and tolerability were also evaluated. Results: There was

significant reduction from baseline to Visit 7 in the primary end points of WOMAC and VAS

scores and in the secondary end point of QOL score in subjects with PCP and BCP groups, while in

subjects with placebo group the end point indices remained unaltered. Furthermore, all the score

levels of WOMAC, VAS and QOL decreased significantly (P < 0.01) in the study group compared

to placebo group in Visit 7. Conclusion: The study demonstrated that collagen peptides are potential

therapeutic agents as nutritional supplements for the management of osteoarthritis and maintenance

of joint health.

92

Int J Pharm. 2001 Jun 19;221(1-2):1-22.

Biomedical applications of collagen.

Lee CH, Singla A, Lee Y.

Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and

safety due to its biological characteristics, such as biodegradability and weak antigenecity, made

collagen the primary resource in medical applications. The main applications of collagen as drug

delivery systems are collagen shields in ophthalmology, sponges for burns/wounds, mini-pellets and

tablets for protein delivery, gel formulation in combination with liposomes for sustained drug

delivery, as controlling material for transdermal delivery, and nanoparticles for gene delivery and

basic matrices for cell culture systems. It was also used for tissue engineering including skin

replacement, bone substitutes, and artificial blood vessels and valves. This article reviews

biomedical applications of collagen including the collagen film, which we have developed as a

matrix system for evaluation of tissue calcification and for the embedding of a single cell

suspension for tumorigenic study. The advantages and disadvantages of each system are also

discussed.

93

Spine (Phila Pa 1976). 2012 Mar 15;37(6):452-8.

Tissue engineering of the intervertebral disc with cultured nucleus pulposus cells using

atelocollagen scaffold and growth factors.

Lee KI, Moon SH, Kim H, Kwon UH, Kim HJ, Park SN, Suh H, Lee HM, Kim HS, Chun HJ,

Kwon IK, Jang JW.

STUDY DESIGN: In vitro experiment using rabbit nucleus pulposus (NP) cells seeded in

atelocollagen scaffolds under the stimulation of growth factors. OBJECTIVE: To demonstrate the

effect of anabolic growth factors in rabbit NP cells cultured in atelocollagen type I and type II.

SUMMARY OF BACKGROUND DATA: Atelocollagen provides intervertebral disc (IVD) cells

for a biocompatible environment to produce extracellular matrix. IVD cells with exogenous

transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) also

render an increase in matrix synthesis. However, the effect of anabolic growth factors in NP cells

cultured in atelocollagens was not elucidated before. METHODS: Rabbit NP cell was harvested,

enzymatically digested, and cultured. The NP cells were seeded to atelocollagen type I and type II

scaffolds, and then cultures were exposed to TGF-β1 (10 ng/mL) and/or BMP-2 (100 ng/mL). DNA

synthesis was measured using [4H]-thymidine incorporation. Newly synthesized proteoglycan was

measured using [35S]-sulfate incorporation. Reverse transcription-polymerase chain reactions (RT-

PCRs) for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin were

performed. RESULTS: Rabbit NP cells cultured in atelocollagen type I scaffold showed an increase

(1.7 to 2.4-fold) in DNA synthesis in response to TGF-β1 and/or BMP-2 (P < 0.05), whereas NP

cultures in atelocollagen type II demonstrated a 30% increase in DNA synthesis only with

combination of both growth factors compared with control (P < 0.05). Rabbit NP cells in

atelocollagen type II scaffold with TGF-β1 and combination of both growth factors exhibited robust

5.3- and 5.4-fold increases in proteoglycan synthesis (P < 0.05), whereas any cultures in

atelocollagen type I failed to show any significant increase compared with control. Rabbit NP cells

in atelocollagen type I and type II scaffolds with TGF-β1 and/or BMP-2 demonstrated the

upregulation of aggrecan, collagen type I, and collagen type II mRNA expression compared with

saline control (P < 0.05). The response in transcriptional level was more robust in atelocollagen

type II than in type I. In any event, there is no recognizable expression of osteocalcin (P < 0.05).

CONCLUSION: NP cells in atelocollagens under the stimulation of TGF-β1 and BMP-2 exhibited

anabolic responses in transcriptional and translational levels. Hence, such an approach can provide

a suitable engineered tissue for IVD regeneration with potential for robust refurbishment of matrix.

94

Exp Neurol. 2010 Jun;223(2):645-52.

Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture.

Lee YB, Polio S, Lee W, Dai G, Menon L, Carroll RS, Yoo SS.

Time-released delivery of soluble growth factors (GFs) in engineered hydrogel tissue constructs

promotes the migration and proliferation of embedded cells, which is an important factor for

designing scaffolds that ultimately aim for neural tissue regeneration. We report a tissue

engineering technique to print murine neural stem cells (C17.2), collagen hydrogel, and GF

(vascular endothelial growth factor: VEGF)-releasing fibrin gel to construct an artificial neural

tissue. We examined the morphological changes of the printed C17.2 cells embedded in the

collagen and its migration toward the fibrin gel. The cells showed high viability (92.89+/-2.32%)

after printing, which was equivalent to that of manually-plated cells. C17.2 cells printed within

1mm from the border of VEGF-releasing fibrin gel showed GF-induced changes in their

morphology. The cells printed in this range also migrated toward the fibrin gel, with the total

migration distance of 102.4+/-76.1microm over 3days. The cells in the control samples (fibrin

without the VEGF or VEGF printed directly in collagen) neither proliferated nor migrated. The

results demonstrated that bio-printing of VEGF-containing fibrin gel supported sustained release of

the GF in the collagen scaffold. The presented method can be gainfully used in the development of

three-dimensional (3D) artificial tissue assays and neural tissue regeneration applications.

95

Chin J Traumatol. 2004 Dec;7(6):358-62.

Effect of type I collagen on the adhesion, proliferation, and osteoblastic gene expression of

bone marrow-derived mesenchymal stem cells.

Liu G, Hu YY, Zhao JN, Wu SJ, Xiong Z, Lu R.

OBJECTIVE: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by

type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived

mesenchymal stem cells (MSCs). METHODS: The third generation MSCs isolated from mature

rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm

x 1.2 cm x 2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal

calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and

proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the

incorporation rate of [(3)H]-TdR. In order to examine MSCs differentiation, the expression of

osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN)

mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further

more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM).

RESULTS: Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher

than controls (6 h, 2144 cpm+/-141 cpm vs. 1797 cpm+/-118 cpm, P=0.017; 8 h, 2311 cpm+/-113

cpm vs. 1891 cpm+/-103 cpm, P=0.01). The cells which cultured on PLGA coated with type I

collagen showed significantly higher cell proliferation than controls on the 7 th day (1021 cpm+/-

159 cpm vs. 451 cpm+/-67 cpm, P=0.002), the 14th day (1472 cpm+/-82 cpm vs. 583 cpm+/-67

cpm, P<0.001) and 21 th day (1728 cpm+/-78 cpm vs. 632 cpm+/-55 cpm, P<0.001). Osteoblasts

markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21

th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well

distributed on the polymer coated by type I collagen while cylindric or round cells in controls.

CONCLUSIONS: Type I collagen is effective in promoting the adhesion, proliferation and

differentiation of MSCs on PLGA.

96

Facial Plast Surg. 2014 Dec;30(6):615-22.

Complications of collagen fillers.

Lucey P, Goldberg DJ.

As the skin ages, a deficiency in collagen occurs, thus injectable collagen products have become a

sensible and popular option for dermal filling and volume enhancement. Several types of collagen

have been developed over the years, including animal sources such as bovine and porcine collagen,

as well as human-based sources derived from pieces of the patient's own skin, cadaver skin, and

later cultured from human dermal fibroblasts. While collagen overall has a relatively safe, side

effect profile, there are several complications, both early and late onset, that practitioners and

patients should be aware of. Early complications, occurring within days of the procedure, can be

divided into non-hypersensitivity and hypersensitivity reactions. The non-hypersensitive reactions

include injection site reactions, discoloration, maldistribution, infection, skin necrosis, and the very

rare but dreaded risk of vision loss, whereas the hypersensitivity reactions present usually as

delayed type IV reactions, but can also rarely present as an immediate type I reaction. Late

complications, occurring within weeks to even years after injection, include granuloma formation,

foreign body reactions, and infection secondary to atypical mycobacteria or biofilms. This review

will give a detailed overview of the complications secondary to cutaneous collagen injections.

97

J Biomed Mater Res B Appl Biomater. 2006 Oct;79(1):25-34.

Tissue engineering of articular cartilage with autologous cultured adipose tissue-derived

stromal cells using atelocollagen honeycomb-shaped scaffold with a membrane sealing in

rabbits.

Masuoka K, Asazuma T, Hattori H, Yoshihara Y, Sato M, Matsumura K, Matsui T, Takase B,

Nemoto K, Ishihara M.

Adipose tissue derived stromal cells (ATSCs), which were isolated from adipose tissue of rabbit,

have shown to possess multipotential, that is, they differentiate into osteoblasts and adipocytes in

plate-culturing and into chondrocytes in an established aggregate culture using defined

differentiation-inductive medium. The aim of this study was to evaluate the utility of ATSCs in

tissue engineering procedures for repair of articular cartilage-defects using the atelocollagen

honeycomb-shaped scaffold with a membrane sealing (ACHMS-scaffold). We intended to repair

full-thickness articular cartilage defects in rabbit knees using autologously cultured ATSCs

embedded in the ACHMS-scaffold. ATSCs were incubated within the ACHMS-scaffold to allow a

high density and three-dimensional culture with control medium. An articular cartilage defect was

created on the patellar groove of the femur, and the defect was filled with the ATSCs-containing

ACHMS-scaffold, ACHMS-scaffold alone, or empty (control). Twelve weeks after the operation,

the histological analyses showed that only the defects treated with the ATSCs-containing ACHMS-

scaffold were filled with reparative hyaline cartilage, highly expressed Type II collagen. These

results indicate that transplantation of autologous ATSCs-containing ACHMS-scaffold is effective

in repairing articular cartilage defects.

98

Science. 1969 Dec 19;166(3912):1522-4.

Localization of antigenic determinants in the polypeptide chains of collagen.

Michaeli D, Martin GR, Kettman J, Benjamini E, Leung DY, Blatt BA.

After being injected with collagen from rat or guinea pig skin, rabbits form high titer, species-

specific antibodies to collagen. Antibody is directed primarily against the alpha2 chain of collagen

with little reaction with the alpha1 chain. The amino terminal peptide of cyanogen bromide

cleavage of the alpha2 chain of guinea pig skin collagen, alpha2-CBl, effectively inhibited the

antigen-antibody reaction, an indication that a major antigenic determinant of collagen is located at

the amino terminal of the alpha2 chain.

99

Arch Orthop Trauma Surg. 1997;116(8):454-62.

Histological and biomechanical observations of the rabbit patellar tendon after removal of its

central one-third.

Miyashita H , Ochi M, Ikuta Y.

Using 35 Japanese white rabbits, a study was made of tissue regeneration and the mechanical

properties of the patellar tendon after removal of its central one-third. After removal of the central

one-third of the patellar tendon on one side, in experiment 1 the strength of the entire patellar

tendon including the regenerated tissue was compared with that of the patellar tendon on the

opposite side with the central one-third removed at the time of killing, and in experiment 2 the

strength of only the regenerated tissue was compared with that of the patellar tendon on the opposite

side with two-thirds of the medial and lateral sides removed at the time of death. In experiment 1,

the maximum load showed no significant difference between the operated side and the control. In

one half of the cases, the strength of the operated side including the regenerated tissue was weak,

suggesting weakening of the patellar tendon on the residual bilateral sides. In experiment 2, the

maximum load of the regenerated tissue was significantly lower than that of the control, the former

being 25% of the latter even at 6 months. Histologically, the characteristics of the cells and collagen

fibers gradually approached those of normal tissue, but the crimp pattern of the collagen fibers and

fibrils was evidently smaller than that of the control. These results indicate that regenerated tissue

was still mechanically weak and immature at 6 months.

100

Semin Arthritis Rheum. 2000 Oct;30(2):87-99.

Role of collagen hydrolysate in bone and joint disease.

Moskowitz RW.

OBJECTIVES: To review the current status of collagen hydrolysate in the treatment of

osteoarthritis and osteoporosis. METHODS: Review of past and current literature relative to

collagen hydrolysate metabolism, and assessment of clinical investigations of therapeutic trials in

osteoarthritis and osteoporosis. RESULTS: Hydrolyzed gelatin products have long been used in

pharmaceuticals and foods; these products are generally recognized as safe food products by

regulatory agencies. Pharmaceutical-grade collagen hydrolysate (PCH) is obtained by hydrolysis of

pharmaceutical gelatin. Clinical studies suggest that the ingestion of 10 g PCH daily reduces pain in

patients with osteoarthritis of the knee or hip; blood concentration of hydroxyproline is increased.

Clinical use is associated with minimal adverse effects, mainly gastrointestinal, characterized by

fullness or unpleasant taste. In a multicenter, randomized, double-blind, placebo-controlled trial

performed in clinics in the United States, United Kingdom, and Germany, results showed no

statistically significant differences for the total study group (all sites) for differences of mean pain

score for pain. There was, however, a significant treatment advantage of PCH over placebo in

German sites. In addition, increased efficacy for PCH as compared to placebo was observed in the

overall study population amongst patients with more severe symptomatology at study onset.

Preferential accumulation of 14C-labeled gelatin hydrolysate in cartilage as compared with

administration of 14C-labeled proline has been reported. This preferential uptake by cartilage

suggests that PCH may have a salutary effect on cartilage metabolism. Given the important role for

collagen in bone structure, the effect of PCH on bone metabolism in osteoporotic persons has been

evaluated. Studies of the effects of calcitonin with and without a collagen hydrolysate-rich diet

suggested that calcitonin plus PCH had a greater effect in inhibiting bone collagen breakdown than

calcitonin alone, as characterized by a fall in levels of urinary pyridinoline cross-links. PCH

appeared to have an additive effect relative to use of calcitonin alone. CONCLUSIONS: Collagen

hydrolysate is of interest as a therapeutic agent of potential utility in the treatment of osteoarthritis

and osteoporosis. Its high level of safety makes it attractive as an agent for long-term use in these

chronic disorders.

101

J Dermatol. 2005 May;32(5):376-80.

Atelocollagen sponge and recombinant basic fibroblast growth factor combination therapy

for resistant wounds with deep cavities.

Nakanishi A, Hakamada A, Isoda K, Mizutani H.

Recent advances in bioengineering have introduced materials that enhance wound healing. Even

with such new tools, some deep ulcers surrounded by avascular tissues, including bone, tendon, and

fascia, are resistant to various therapies and easily form deep cavities with loss of subcutaneous

tissue. Atelocollagen sponges have been used as an artificial dermis to cover full-thickness skin

defects. Topical recombinant human basic fibroblast growth factor has been introduced as a growth

factor to induce fibroblast proliferation in skin ulcers. We applied these materials in combination in

two patients with deep resistant wounds: one with a cavity reaching the mediastinum through a

divided sternum and one with deep necrotic wounds caused by electric burns. These wounds did not

respond to the topical basic fibroblast growth factor alone. In contrast, the combination therapy

closed the wounds rapidly without further surgical treatment. This combination therapy is a potent

treatment for resistant wounds with deep cavities.

102

Osteoarthritis Cartilage. 2009 Dec;17(12):1620-7.

Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular

cartilage in vitro and in vivo.

Nakatani S, Mano H, Sampei C, Shimizu J, Wada M.

OBJECTIVE: To investigate the direct effect of prolyl-hydroxyproline (Pro-Hyp) on chondrocytes

under in vivo and in vitro conditions in an attempt to identify Pro-Hyp as the bioactive peptide in

collagen hydrolysate (CH). METHODS: The in vivo effects of CH and Pro-Hyp intake on articular

cartilage were studied by microscopic examination of sections of dissected articular cartilage from

treated C57BL/6J mice. In this study, mice that were fed diets containing excess phosphorus were

used as an in vivo model. This mouse line showed loss of chondrocytes and reduced thickness of

articular cartilage, with abnormality of the subchondral bone. The in vitro effects of CH, Pro-Hyp,

amino acids and other peptides on proliferation, differentiation, glycosaminoglycan content and

mineralization of chondrocytes were determined by MTT activity and staining with alkaline

phosphatase, alcian blue and alizarin red. Expression of chondrogenesis-specific genes in ATDC5

cells was determined by semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-

PCR). RESULTS: In vivo, CH and Pro-Hyp inhibited the loss of chondrocytes and thinning of the

articular cartilage layer caused by phosphorus-induced degradation. In the in vitro study, CH and

Pro-Hyp did not affect chondrocyte proliferation but inhibited their differentiation into mineralized

chondrocytes. A combination of amino acids such as proline, hydroxyproline and prolyl-

hydroxyprolyl-glycine did not affect chondrocyte proliferation or differentiation. Moreover, CH and

Pro-Hyp caused two and threefold increases, respectively, in the staining area of glycosaminoglycan

in the extracellular matrix of ATDC5 cells. RT-PCR indicated that Pro-Hyp increased the aggrecan

mRNA level approximately twofold and decreased the Runx1 and osteocalcin mRNA levels by

two-thirds and one-tenth, respectively. CONCLUSION: Pro-Hyp is the first bioactive edible

peptide derived from CH to be shown to affect chondrocyte differentiation under pathological

conditions.

103

Arthritis Res Ther. 2013 Feb 22;15(1):R32.

Periodic knee injections of collagen tripeptide delay cartilage degeneration in rabbit

experimental osteoarthritis.

Naraoka T, Ishibashi Y, Tsuda E, Yamamoto Y, Kusumi T, Toh S.

INTRODUCTION: Collagen peptides have been reported to possess various biological activities for

various cell types. The purposes of this study were, first, to examine the therapeutic effects of

collagen tripeptide (Ctp) in rabbit osteoarthritis and, second, to explore a synergetic effect with

hyaluronan (HA). METHODS: Osteoarthritis was induced by anterior cruciate ligament transection

of the right knee in 72 Japanese white rabbits and they were divided into four groups (control, Ctp,

HA and Ctp/HA). Each material was injected weekly into the knee, and knee joint samples were

collected 5, 10 and 15 weeks after surgery. Macroscopic and histomorphological analyses of

cartilage were conducted. Expression of type II collagen and matrix metalloproteinase-13 was also

analyzed immunohistochemically. A Tukey's honestly significant difference test was used to

evaluate the statistical significance of difference in the macroscopic, histological and

immnohistochemical results. RESULTS: All treatment groups exhibited slightly higher resistance to

the progression of osteoarthritis than the control group macroscopically 15 weeks after surgery.

Histologically, intra-articular injection of Ctp significantly reduced cartilage degradation 10 weeks

after surgery, and Ctp/HA significantly reduced it 5 weeks after surgery in comparison with the

control. Immunohistochemically, both Ctp-treated and Ctp/HA-treated groups had significantly

increased type II collagen-positive chondrocytes at the fifth week after the surgery, although the

numbers of matrix metalloproteinase-13-positive chondrocytes were not affected. CONCLUSION:

Periodical injections of Ctp and Ctp/HA delayed progression of cartilage degeneration of early

osteoarthritis induced by anterior cruciate ligament transection in rabbits. This effect appears to be

exerted by promotion of type II collagen synthesis predominantly.

104

J Nutr. 1999 Oct;129(10):1891-5.

Oral administration of (14)C labeled gelatin hydrolysate leads to an accumulation of

radioactivity in cartilage of mice (C57/BL).

Oesser S, Adam M, Babel W, Seifert J.

Several investigations showed a positive influence of orally administered gelatin on degenerative

diseases of the musculo-skeletal system. Both the therapeutic mechanism and the absorption

dynamics, however, remain unclear. Therefore, this study investigated the time course of gelatin

hydrolysate absorption and its subsequent distribution in various tissues in mice (C57/BL).

Absorption of (14)C labeled gelatin hydrolysate was compared to control mice administered (14)C

labeled proline following intragastric application. Plasma and tissue radioactivity was measured

over 192 h. Additional "gut sac" experiments were conducted to quantify the MW distribution of

the absorbed gelatin using SDS-electrophoresis and HPLC. Ninety-five percent of enterally applied

gelatin hydrolysate was absorbed within the first 12 h. The distribution of the labeled gelatin in the

various tissues was similar to that of labeled proline with the exception of cartilage, where a

pronounced and long-lasting accumulation of gelatin hydrolysate was observed. In cartilage,

measured radioactivity was more than twice as high following gelatin administration compared to

the control group. The absorption of gelatin hydrolysate in its high molecular form, with peptides of

2.5-15kD, was detected following intestinal passage. These results demonstrate intestinal absorption

and cartilage tissue accumulation of gelatin hydrolysate and suggest a potential mechanism for

previously observed clinical benefits of orally administered gelatin.

105

Cell Tissue Res. 2003 Mar;311(3):393-9. Epub 2003 Feb 25.

Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured

with degraded collagen.

Oesser S, Seifert J.

The functional integrity of articular cartilage is dependent on the maintenance of the extracellular

matrix (ECM), a process which is controlled by chondrocytes. The regulation of ECM biosynthesis

is complex and a variety of substances have been found to influence chondrocyte metabolism. In the

present study we have investigated the effect of degraded collagen on the formation of type II

collagen by mature bovine chondrocytes in a cell culture model. The culture medium was

supplemented with collagen hydrolysate (CH) and biosynthesis of type II collagen by chondrocytes

was compared to control cells treated with native type I and type II collagen and a collagen-free

protein hydrolysate. The quantification of type II collagen by means of an ELISA technique was

confirmed by immunocytochemical detection as well as by the incorporation of (14)C-proline in the

ECM after a 48 h incubation. Chondrocytes in the control group were maintained in the basal

medium for 11 days. The presence of extracellular CH led to a dose-dependent increase in type II

collagen secretion. However, native collagens as well as a collagen-free hydrolysate of wheat

proteins failed to stimulate the production of type II collagen in chondrocytes. These results clearly

indicate a stimulatory effect of degraded collagen on the type II collagen biosynthesis of

chondrocytes and suggest a possible feedback mechanism for the regulation of collagen turnover in

cartilage tissue.

106

Biosci Biotechnol Biochem. 2010;74(10):2096-9.

Effects of Pro-Hyp, a collagen hydrolysate-derived peptide, on hyaluronic acid synthesis using

in vitro cultured synovium cells and oral ingestion of collagen hydrolysates in a guinea pig

model of osteoarthritis.

Ohara H, Iida H, Ito K, Takeuchi Y, Nomura Y.

Proline-hydroxyproline (Pro-Hyp) stimulated hyaluronic acid production in cultured synovium

cells. It was detected in guinea pig blood after oral ingestion of collagen hydrolysates. Oral

administration of collagen hydrolysates increased the amount of proteoglycans in the epiphyses. It

also reduced the morphological changes associated with osteoarthritic cartilage destruction of the

knee joint. The results suggest that collagen hydrolysates have therapeutic potential for treatment of

osteoarthritis.

107

J Oral Implantol. 2002;28(5):220-5.

Collagen as an implantable material in medicine and dentistry.

Patino MG, Neiders ME, Andreana S, Noble B, Cohen RE.

Collagen is a highly versatile material, extensively used in the medical, dental, and pharmacological

fields. Collagen is capable of being prepared into cross-linked compacted solids or into lattice-like

gels. Resorbable forms of collagen have been used to dress oral wounds, for closure of graft and

extraction sites, and to promote healing. Collagen-based membranes also have been used in

periodontal and implant therapy as barriers to prevent epithelial migration and allow cells with

regenerative capacity to repopulate the defect area. It has been hypothesized that membrane

regenerative techniques facilitate the natural biological potential by creating a favorable

environment for periodontal and peri-implant regeneration. Due to the enormous potential of

collagen-based regenerative barriers, clinicians may benefit from a review of potential applications

of implantable collagen and knowledge of collagen preparation and membrane types as well as from

as awareness of the functional and degradation properties of those materials.

108

J Cell Biochem. 1998 Dec 1;71(3):313-27.

Collagen in tissue-engineered cartilage: types, structure, and crosslinks.

Riesle J, Hollander AP, Langer R, Freed LE, Vunjak-Novakovic G.

The function of articular cartilage as a weight-bearing tissue depends on the specific arrangement of

collagen types II and IX into a three-dimensional organized collagen network that can balance the

swelling pressure of the proteoglycan/water gel. To determine whether cartilage engineered in vitro

contains a functional collagen network, chondrocyte-polymer constructs were cultured for up to 6

weeks and analyzed with respect to the composition and ultrastructure of collagen by using

biochemical and immunochemical methods and scanning electron microscopy. Total collagen

content and the concentration of pyridinium crosslinks were significantly (57% and 70%,

respectively) lower in tissue-engineered cartilage that in bovine calf articular cartilage. However,

the fractions of collagen types II, IX, and X and the collagen network organization, density, and

fibril diameter in engineered cartilage were not significantly different from those in natural articular

cartilage. The implications of these findings for the field of tissue engineering are that differentiated

chondrocytes are capable of forming a complex structure of collagen matrix in vitro, producing a

tissue similar to natural articular cartilage on an ultrastructural scale.

109

Science. 1987 Oct 23;238(4826):491-7.

New perspectives in cell adhesion: RGD and integrins.

Ruoslahti E, Pierschbacher MD.

Rapid progress has been made in the understanding of the molecular interactions that result in cell

adhesion. Many adhesive proteins present in extracellular matrices and in the blood contain the

tripeptide arginine-glycine-aspartic acid (RGD) as their cell recognition site. These proteins include

fibronectin, vitronectin, osteopontin, collagens, thrombospondin, fibrinogen, and von Willebrand

factor. The RGD sequences of each of the adhesive proteins are recognized by at least one member

of a family of structurally related receptors, integrins, which are heterodimeric proteins with two

membrane-spanning subunits. Some of these receptors bind to the RGD sequence of a single

adhesion protein only, whereas others recognize groups of them. The conformation of the RGD

sequence in the individual proteins may be critical to this recognition specificity. On the

cytoplasmic side of the plasma membrane, the receptors connect the extracellular matrix to the

cytoskeleton. More than ten proved or suspected RGD-containing adhesion-promoting proteins

have already been identified, and the integrin family includes at least as many receptors recognizing

these proteins. Together, the adhesion proteins and their receptors constitute a versatile recognition

system providing cells with anchorage, traction for migration, and signals for polarity, position,

differentiation, and possibly growth.

110


Effect of collagen matrices on dermal wound healing.

Ruszczak Z.

Dermal substitution and wound healing are areas of medicine in which there have been many recent

advances, but neither the commercially available products nor the products currently described in

experimental studies are able to fully substitute for natural living skin. There is an overall consensus

that to heal wounds, the substitution of connective tissue matrix, the main component of each

wound, is necessary. Both artificial and natural polymers have been used to reconstitute dermis.

Nowadays, collagen has been discovered again. Collagen is a natural substrate for cellular

attachment, growth and differentiation, and promotes cellular proliferation and differentiation. Once

dermis reconstruction is done, the covering of the wound surface with both in vitro expanded

epidermis and autologous split-skin transplants is significantly easier and has an improved chance

of success. Nowadays, many commercial and experimental products have been introduced to

improve cutaneous wound healing. This review discusses some of both acellular and cell-containing

products used in the treatment of skin wounds.

111

Biomaterials. 2006 Jan;27(3):335-45.

Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to

the degenerated intervertebral disc.

Sakai D, Mochida J, Iwashina T, Hiyama A, Omi H, Imai M, Nakai T, Ando K, Hotta T.

Intervertebral disc (IVD) degeneration, a common cause of low back pain in humans, is a

relentlessly progressive phenomenon with no currently available effective treatment. In an attempt

to solve this dilemma, we transplanted autologous mesenchymal stem cells (MSCs) from bone

marrow into a rabbit model of disc degeneration to determine if stem cells could repair degenerated

IVDs. LacZ expressing MSCs were transplanted to rabbit L2-L3, L3-L4 and L4-L5 IVDs 2 weeks

after induction of degeneration. Changes in disc height by plain radiograph, T2-weighted signal

intensity in magnetic resonance imaging (MRI), histology, immunohistochemistry and matrix

associated gene expressions were evaluated between normal controls (NC) without operations,

sham operated with only disc degeneration being induced, and MSC-transplanted animals for a 24-

week period. Results showed that after 24 weeks post-MSC transplantation, degenerated discs of

MSC-transplanted group animals regained a disc height value of about 91%, MRI signal intensity of

about 81%, compared to NC group discs. On the other hand, sham-operated group discs

demonstrated the disc height value of about 67% and MRI signal intensity of about 60%.

Macroscopic and histological evaluations confirmed relatively preserved nucleus with circular

annulus structure in MSC-transplanted discs compared to indistinct structure seen in sham.

Restoration of proteoglycan accumulation in MSC-transplanted discs was suggested from

immunohistochemistry and gene expression analysis. These data indicate that transplantation of

MSCs effectively led to regeneration of IVDs in a rabbit model of disc degeneration as suggested in

our previous pilot study. MSCs may serve as a valuable resource in cell transplantation therapy for

degenerative disc disease.

112

Med Biol Eng Comput. 2003 May;41(3):365-71.

Tissue engineering of the intervertebral disc with cultured annulus fibrosus cells using

atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS scaffold).

Sato M, Kikuchi M, Ishihara M, Ishihara M, Asazuma T, Kikuchi T, Masuoka K, Hattori H,

Fujikawa K.

The objective of the study was to investigate the regeneration of intervertebral discs after laser

discectomy using tissue engineering procedures. Annulus fibrosus (AF) cells from the intervertebral

discs of Japanese white rabbits were cultured in an atelocollagen honeycomb-shaped scaffold with a

membrane seal (ACHMS scaffold), to produce a high-density, three-dimensional culture for up to 3

weeks. Although the DNA content in the scaffold increased at a lower rate than that in the

monolayer culture, expression of type II collagen and glycosaminoglycan accumulation in the

scaffold were at higher levels than in the monolayer. The AF cells that had been cultured in the

scaffold for 7 days were allografted into the lacunae of intervertebral discs of recipients (40 rabbits,

14-16 weeks old; average weight, 3.2 kg), whose nucleus pulposus (NP) had been vaporised with an

ICG dye-enhanced laser. The allografted cultured AF cells survived and produced hyaline-like

cartilage. Furthermore, the narrowing of the intervertebral disc space of the cell-containing scaffold

insertion groups was significantly inhibited after 12 post-operative weeks.

113

J Biomed Mater Res B Appl Biomater. 2007 Oct;83(1):181-8.

Effects of growth factors on heparin-carrying polystyrene-coated atelocollagen scaffold for

articular cartilage tissue engineering.

Sato M, Ishihara M, Ishihara M, Kaneshiro N, Mitani G, Nagai T, Kutsuna T, Asazuma T, Kikuchi

M, Mochida J.

The specific aim of our investigation is to study the potential use of a collagen/heparin-carrying

polystyrene (HCPS) composite extracellular matrix for articular cartilage tissue engineering. Here,

we created a high-performance extracellular matrix (HpECM) scaffold to build an optimal

extracellular environment using an HCPS we originally developed, and an atelocollagen

honeycomb-shaped-scaffold (ACHMS-scaffold) with a membrane seal. This scaffold was coated

with HCPS to enable aggregation of heparin-binding growth factors such as FGF-2 and TGF-beta1

within the scaffold. Three-dimensional culture of rabbit articular chondrocytes within the HpECM-

scaffold and subsequent preparation of a tissue-engineered cartilage were investigated. The results

showed remarkably higher cell proliferative activity within the HpECM-pretreated-FGF-2 scaffold

and the sustenance of phenotype within the HpECM-pretreated-TGF-beta1 scaffold. It was thought

that both FGF-2 and TGF-beta1 were stably immobilized in the HpEMC-scaffold since HCPS

generated an extracellular environment similar to that of heparan sulfate proteoglycan within the

scaffold. These results suggest that an ACHMS-scaffold immobilized with HCPS can be a HpECM

for cartilage regeneration to retain the heparin-binding growth factors within the scaffolds.

114

J Agric Food Chem. 2012 Apr 25;60(16):4096-101.

Effect of the novel low molecular weight hydrolyzed chicken sternal cartilage extract, BioCell

Collagen, on improving osteoarthritis-related symptoms: a randomized, double-blind,

placebo-controlled trial.

Schauss AG, Stenehjem J, Park J, Endres JR, Clewell A.

Osteoarthritis (OA) is a significant source of pain and disability. Current medical and surgical

treatments can be costly and have serious side effects. The aim of this randomized, double-blind,

placebo-controlled trial was to investigate the tolerability and efficacy of BioCell Collagen (BCC),

a low molecular weight dietary supplement consisting of hydrolyzed chicken sternal cartilage

extract, in the treatment of OA symptoms. Patients (n = 80) in the study had physician-verified

evidence of progressive OA in their hip and/or knee joint. Joint pain had been present for 3 months

or longer at enrollment, and pain levels were 4 or higher at baseline as assessed by Physician Global

Assessment scores. Subjects were divided into two groups and administered either 2 g of BCC or

placebo for 70 days. Other outcome measurements included visual analogue scale (VAS) for pain

and Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores taken on days 1,

35, and 70. The tolerability profile of the treatment group was comparable to that of the placebo.

Intent-to-treat analysis showed that the treatment group, as compared to placebo, had a significant

reduction of VAS pain on day 70 (p < 0.001) and of WOMAC scores on both days 35 (p = 0.017)

and 70 (p < 0.001). The BCC group experienced a significant improvement in physical activities

compared to the placebo group on days 35 (p = 0.007) and 70 (p < 0.001). BCC was well tolerated

and found to be effective in managing OA-associated symptoms over the study period, thereby

improving patient's activities of daily living. BCC can be considered a potential complement to

current OA therapies.

115

J Musculoskelet Neuronal Interact. 2006 Apr-Jun;6(2):181-90.

Biology of tendon injury: healing, modeling and remodeling.

Sharma P, Maffulli N.

Tendon disorders are frequent, and are responsible for much morbidity both in sport and the

workplace. Although the presence of degenerative changes does not always lead to symptoms, pre-

existing degeneration has been implicated as a risk factor for acute tendon rupture. The term

tendinopathy is a generic descriptor of the clinical conditions in and around tendons arising from

overuse. The terms "tendinosis" and "tendinitis/tendonitis" should only be used after

histopathological examination. Disordered healing is seen in tendinopathy, and inflammation is not

typically seen. In acute injuries, the process of tendon healing is an indivisible process that can be

categorized into three overlapping phases for descriptive purposes. Tendon healing can occur

intrinsically, via proliferation of epitenon and endotenon tenocytes, or extrinsically, by invasion of

cells from the surrounding sheath and synovium. Despite remodeling, the biochemical and

mechanical properties of healed tendon tissue never match those of intact tendon. Tendon injuries

account for considerable morbidity, and often prove disabling for several months, despite what is

considered appropriate management. Chronic problems caused by overuse of tendons probably

account for 30% of all running-related injuries, and the prevalence of elbow tendinopathy in tennis

players can be as high as 40%. The basic cell biology of tendons is still not fully understood, and

the management of tendon injury poses a considerable challenge for clinicians. This article

describes the structure of tendons, and reviews the pathophysiology of tendon injury and healing.

116

Curr Opin Cell Biol. 2012 Oct;24(5):600-6.

Inside-out, outside-in, and inside-outside-in: G protein signaling in integrin-mediated cell

adhesion, spreading, and retraction.

Shen B, Delaney MK, Du X.

The integrin family of cell adhesion receptors mediates bi-directional signaling: 'inside-out'

signaling activates the ligand binding function of integrins and 'outside-in' signaling mediates

cellular responses induced by ligand binding to integrins leading to cell spreading, retraction,

migration, and proliferation. Integrin signaling requires both heterotrimeric G proteins and

monomeric small G proteins. This review focuses on recent development in the roles of G proteins

in integrin outside-in signaling. The finding of direct interaction between the heterotrimeric G

protein subunit Gα13 and integrin β subunits reveals a new mechanism for integrin signaling, and

also uncovers a crosstalk between the signaling pathways initiated by G protein-coupled receptors

(GPCRs) and integrins. This crosstalk, which may be referred to as 'inside-outside-in' signaling,

dynamically regulates contractility and greatly promotes integrin outside-in signaling.

117

J Bone Joint Surg Am. 1997 Dec;79(12):1770-7.

Regeneration of meniscal cartilage with use of a collagen scaffold. Analysis of preliminary

data.

Stone KR, Steadman JR, Rodkey WG, Li ST.

A collagen scaffold was designed for use as a template for the regeneration of meniscal cartilage

and was tested in ten patients in an initial, Food and Drug Administration-approved, clinical

feasibility trial. The goal of the study was to evaluate the implantability and safety of the scaffold as

well as its ability to support tissue ingrowth. The study was based on the findings of in vitro and in

vivo investigations in dogs that had demonstrated cellular ingrowth and tissue regeneration through

the scaffold. Nine patients remained in the study for at least thirty-six months, and one patient

voluntarily withdrew after three months for personal reasons. The collagen scaffold was found to be

implantable and to be safe over the three-year period. Histologically, it supported regeneration of

tissue in meniscal defects of various sizes. No adverse immunological reactions were noted on

sequential serological testing. On second-look arthroscopy, performed either three or six months

after implantation, gross and histological evaluation revealed newly formed tissue replacing the

implant as it was resorbed. At thirty-six months, the nine patients reported a decrease in the

symptoms. According to a scale that assigned 1 point for strenuous activity and 5 points for an

inability to perform sports activity, the average score was 1.5 points before the injury, 3.0 points

after the injury and before the operation, and 2.4 points at six months postoperatively, 2.2 points at

twelve months, 2.0 points at twenty-four months, and 1.9 points at thirty-six months. According to a

scale that assigned 0 points for no pain and 3 points for severe pain, the average pain score was 2.2

points preoperatively and 0.6 point thirty-six months postoperatively. One patient, who had had a

repair of a bucket-handle tear of the medial meniscus and augmentation with the collagen scaffold,

had retearing of the cartilage nineteen months after implantation. Another patient had debridement

because of an irregular area of regeneration at the scaffold-meniscus interface twenty-one months

after implantation. Magnetic resonance imaging scans demonstrated progressive maturation of the

signal within the regenerated meniscus at three, six, twelve, and thirty-six months. These findings

suggest that regeneration of meniscal cartilage through a collagen scaffold is possible. Additional

studies are needed to determine long-term efficacy.

118

Mol Biol Evol. 2011 Jan;28(1):533-42.

Comparative vertebrate evolutionary analyses of type I collagen: potential of COL1a1 gene

structure and intron variation for common bone-related diseases.

Stover DA, Verrelli BC.

Collagen type I alpha 1 (COL1a1), which encodes the primary subunit of type I collagen, the main

structural and most abundant protein in vertebrates, harbors hundreds of mutations linked to human

diseases like osteoporosis and osteogenesis imperfecta. Previous studies have attempted to predict

the phenotypic severity associated with type I collagen mutations, yet an evolutionary analysis that

compares historical and recent selective pressures, including across noncoding regions, has never

been conducted. Here, we use a comparative genomic and species evolutionary analysis

representing ∼450 My of vertebrate history to investigate functional constraints associated with

both exons and introns of the >17-kb COL1a1 gene. We find that although the COL1a1 amino acid

sequence is highly conserved, there are both spatial and temporal signatures of varying selective

constraint across protein domains. Furthermore, sites of high evolutionary constraint significantly

correlate with the location of disease-associated mutations, the latter of which also cluster with

respect to specific severity classes typically categorized in clinical studies. Finally, we find that

COL1a1 introns are significantly short in length with high GC content, patterns that are shared

across highly diverged vertebrates, and which may be a signature of strong stabilizing selection for

high COL1a1 gene expression. In conclusion, although previous studies focused on COL1a1 coding

regions, the current results implicate introns as areas of high selective constraint and targets of

bone-related phenotypic variation. From a broader perspective, our comparative evolutionary

approach provides further resolution to models predicting mutations associated with bone-related

function and disease severity.

119

Biomaterials. 2006 Jun;27(17):3238-48.

Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering.

Sumita Y, Honda MJ, Ohara T, Tsuchiya S, Sagara H, Kagami H, Ueda M.

Tooth structure can be regenerated by seeding dissociated tooth cells onto polyglycolic acid fiber

mesh, although the success rate of tooth production is low. The present study was designed to

compare the performance of collagen sponge with polyglycolic acid fiber mesh as a 3-D scaffold

for tooth-tissue engineering. Porcine third molar teeth at the early stage of crown formation were

enzymatically dissociated into single cells, and the heterogeneous cells were seeded onto collagen

sponge or the polyglycolic acid fiber mesh scaffolds. Scaffolds were then cultured to evaluate cell

adhesion and ALP activity in vitro. An in vivo analysis was performed by implanting the constructs

into the omentum of immunocompromised rats and evaluating tooth production up to 25 weeks.

After 24h, there were a significantly higher number of cells attached to the collagen sponge scaffold

than the polyglycolic acid fiber mesh scaffold. Similarly, the ALP activity was significantly higher

for the collagen sponge scaffold was than the polyglycolic acid fiber mesh scaffold after 7 days of

culture. The area of calcified tissue formed in the collagen sponge scaffold was also larger than in

the polyglycolic acid fiber mesh scaffold. The results from in vivo experiments show conclusively

that a collagen sponge scaffold allows tooth production with a higher degree of success than

polyglycolic acid fiber mesh. Taken together, the results from this study show that collagen sponge

scaffold is superior to the polyglycolic acid fiber mesh scaffold for tooth-tissue engineering.

120

J Toxicol Sci. 1982 Dec;7 Suppl 2:63-91.

Acute and subacute toxicity studies on collagen wound dressing (CAS) in mice and rats.

Takeda U, Odaki M, Yokota M, Sasaki H, Niizato T, Kawaoto H, Watanabe H, Ito T, Ishiwatari N,

Hayasaka H, Seki M, Koeda T.

Single administration of collagen wound dressing (CAS) made from bovine derm in the form of

finely ground powders was given to mice and rats via i.p., s.c. and p.o. routes and via i.v. route in

the form of physiological saline extracts and it was continuously injected into mice for 28 days via

s.c. route to study its acute and subacute toxicity. Examinations were made of on general

conditions, body weight, food and water consumption, hematology, serum biochemistry, organ

weight, and gross and microscopic findings. Results showed no marked toxicity except for local

irritation which was seen only after parenteral administration. We concluded on the basis of these

animal experiments that there should be no problem in regard to safety after somewhat more

extensive therapeutic application of CAS as a wound dressing in clinical practice.

121

J Biol Chem. 2007 Aug 3;282(31):22437-47.

The size exclusion characteristics of type I collagen: implications for the role of

noncollagenous bone constituents in mineralization.

Toroian D, Lim JE, Price PA.

The mineral in bone is located primarily within the collagen fibril, and during mineralization the

fibril is formed first and then water within the fibril is replaced with mineral. The collagen fibril

therefore provides the aqueous compartment in which mineral grows. Although knowledge of the

size of molecules that can diffuse into the fibril to affect crystal growth is critical to understanding

the mechanism of bone mineralization, there have been as yet no studies on the size exclusion

properties of the collagen fibril. To determine the size exclusion characteristics of collagen, we

developed a gel filtration-like procedure that uses columns containing collagen from tendon and

bone. The elution volumes of test molecules show the volume within the packed column that is

accessible to the test molecules, and therefore reveal the size exclusion characteristics of the

collagen within the column. These experiments show that molecules smaller than a 6-kDa protein

diffuse into all of the water within the collagen fibril, whereas molecules larger than a 40-kDa

protein are excluded from this water. These studies provide an insight into the mechanism of bone

mineralization. Molecules and apatite crystals smaller than a 6-kDa protein can diffuse into all

water within the fibril and so can directly impact mineralization. Although molecules larger than a

40-kDa protein are excluded from the fibril, they can initiate mineralization by forming small

apatite crystal nuclei that diffuse into the fibril, or can favor fibril mineralization by inhibiting

apatite growth everywhere but within the fibril.

122

Annu Rev Biomed Eng. 2012;14:47-71.

Tendon healing: repair and regeneration.

Voleti PB, Buckley MR, Soslowsky LJ.

Injury and degeneration of tendon, the soft tissue that mechanically links muscle and bone, can

cause substantial pain and loss of function. This review discusses the composition and function of

healthy tendon and describes the structural, biological, and mechanical changes initiated during the

process of tendon healing. Biochemical pathways activated during repair, experimental injury

models, and parallels between tendon healing and tendon development are emphasized, and cutting-

edge strategies for the enhancement of tendon healing are discussed.

123

Evol Dev. 2006 Jul-Aug;8(4):370-7.

Molecular evolution of fibrillar collagen in chordates, with implications for the evolution of

vertebrate skeletons and chordate phylogeny.

Wada H, Okuyama M, Satoh N, Zhang S.

Vertebrates have seven types of fibrillar collagens that are encoded by 11 genes. Types I, V, and

XXIV collagens are components of mineralized bone, whereas types II, XI, and XXVII collagens

are components of cartilage. In this study, we traced the molecular evolutionary history of chordate

collagen genes and examined how gene duplications gave rise to the collagen genes used for

skeletons. Our analyses of deuterostome collagen genes, including one amphioxus gene that we

identified in this study, suggest that the common ancestors of deuterostomes possessed three

fibrillar collagen genes. Expression analyses of chordate fibrillar collagen genes suggest that in the

ancestors of chordates, fibrillar collagen was co-opted to the formation of the notochord sheath

independently in three clades. Our results also imply that co-option of collagen genes to cartilage

occurred in clade A (col2A1), clade B (col11A1, 11A2), and clade C (COL27A1). Similarly, some

fibrillar collagen genes have been co-opted for mineralized bone independently from clade A genes

(col1A1, 1A2, 5A2), clade B genes (col5A1), and clade C genes (COL24A1). These frequent co-

options for notochord, cartilage, and mineralized bone must have been accompanied by the rapid

evolution of cis-regulatory elements for transcription. In addition, we found that one of the ascidian

fibrillar collagen genes possesses an amino acid insertion at the identical site of the C-terminal

noncollagenous domain in vertebrate fibrillar collagen genes. This observation raises a suspicion

about the relatively well-accepted phylogeny of the close relationship between amphioxus and

vertebrates.

124

Eur Cell Mater. 2006 Mar 28;11:43-56.

Collagen-hydroxyapatite composites for hard tissue repair.

Wahl DA, Czernuszka JT.

Bone is the most implanted tissue after blood. The major solid components of human bone are

collagen (a natural polymer, also found in skin and tendons) and a substituted hydroxyapatite (a

natural ceramic, also found in teeth). Although these two components when used separately provide

a relatively successful mean of augmenting bone growth, the composite of the two natural materials

exceeds this success. This paper provides a review of the most common routes to the fabrication of

collagen (Col) and hydroxyapatite (HA) composites for bone analogues. The regeneration of

diseased or fractured bones is the challenge faced by current technologies in tissue engineering.

Hydroxyapatite and collagen composites (Col-HA) have the potential in mimicking and replacing

skeletal bones. Both in vivo and in vitro studies show the importance of collagen type,

mineralisation conditions, porosity, manufacturing conditions and crosslinking. The results outlined

on mechanical properties, cell culturing and de-novo bone growth of these devices relate to the

efficiency of these to be used as future bone implants. Solid free form fabrication where a mould

can be built up layer by layer, providing shape and internal vascularisation may provide an

improved method of creating composite structures.

125

J Bone Joint Surg Br. 1989 Jan;71(1):74-80.

Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel.

Wakitani S, Kimura T, Hirooka A, Ochi T, Yoneda M, Yasui N, Owaki H, Ono K.

In an attempt to repair articular cartilage, allograft articular chondrocytes embedded in collagen gel,

were transplanted into full-thickness defects in rabbit articular cartilage. Twenty-four weeks after

the transplantation, the defects were filled with hyaline cartilage, specifically synthesising Type II

collagen. These chondrocytes were autoradiographically proven to have originated from the

transplanted grafts. Assessed histologically the success rate was about 80%, a marked improvement

over the results reported in previous studies on chondrocyte transplantation without collagen gel. By

contrast, the defects without chondrocyte transplantation healed with fibrocartilage. Immunological

enhancement induced by transplanted allogenic chondrocytes or collagen was not significant at

eight weeks after treatment, so far as shown by both direct and indirect blastformation reactions.

Thus, allogenic transplantation of isolated chondrocytes embedded in collagen gel appears to be one

of the most promising methods for the restoration of articular cartilage.

126

J Biomed Mater Res A. 2010 Apr;93(1):123-32.

The application of atelocollagen gel in combination with porous scaffolds for cartilage tissue

engineering and its suitable conditions.

Yamaoka H, Tanaka Y, Nishizawa S, Asawa Y, Takato T, Hoshi K.

For improving the quality of tissue-engineered cartilage, we examined the in vivo usefulness of

porous bodies as scaffolds combined with an atelocollagen hydrogel, and investigated the suitable

conditions for atelocollagen and seeding cells within the engineered tissues. We made tissue-

engineered constructs using a collagen sponge (CS) or porous poly(L-lactide) (PLLA) with human

chondrocytes and 1% hydrogel, the concentration of which maximized the accumulation of

cartilage matrices. The CS was soft with a Young's modulus of less than 1 MPa, whereas the porous

PLLA was very rigid with a Young's modulus of 10 MPa. Although the constructs with the CS

shrank to 50% in size after a 2-month subcutaneous transplantation in nude mice, the PLLA

constructs maintained their original sizes. Both of the porous scaffolds contained some cartilage

regeneration in the presence of the chondrocytes and hydrogel, but the PLLA counterpart

significantly accumulated abundant matrices in vivo. Regarding the conditions of the chondrocytes,

the cartilage regeneration was improved in inverse proportion to the passage numbers among

passages 3-8, and was linear with the cell densities (10(6) to 10(8) cells/mL). Thus, the rigid porous

scaffold can maintain the size of the tissue-engineered cartilage and realize fair cartilage

regeneration in vivo when combined with 1% atelocollagen and some conditioned chondrocytes.

127

Birth Defects Res C Embryo Today. 2013 Sep;99(3):203-22. doi: 10.1002/bdrc.21041.

Tendon and ligament regeneration and repair: clinical relevance and developmental

paradigm.

Yang G, Rothrauff BB, Tuan RS.

As dense connective tissues connecting bone to muscle and bone to bone, respectively, tendon and

ligament (T/L) arise from the somitic mesoderm, originating in a recently discovered somitic

compartment, the syndetome. Inductive signals from the adjacent sclerotome and myotome

upregulate expression of Scleraxis, a key transcription factor for tenogenic and ligamentogenic

differentiation. Understanding T/L development is critical to establishing a knowledge base for

improving the healing and repair of T/L injuries, a high-burden disease due to the intrinsically poor

natural healing response. Current treatment of the three most common tendon injuries-tearing of the

rotator cuff of the shoulder, flexor tendon of the hand, and Achilles tendon-include mostly surgical

repair and/or conservative approaches, including biophysical modalities such as rehabilitation and

cryotherapy. Unfortunately, the fibrovascular scar formed during healing possesses inferior

mechanical and biochemical properties, resulting in compromised tissue functionality. Regenerative

approaches have sought to augment the injured tissue with cells, scaffolds, bioactive agents, and

mechanical stimulation to improve the natural healing response. The key challenges in restoring full

T/L function following injury include optimal combination of these biological agents as well as

their delivery to the injury site. A greater understanding of the molecular mechanisms involved in

T/L development and natural healing, coupled with the capability of producing complex

biomaterials to deliver multiple biofactors with high spatiotemporal resolution and specificity,

should lead to regenerative procedures that more closely recapitulate T/L morphogenesis, thereby

offering future patients the prospect of T/L regeneration, as opposed to simple tissue repair.

128

Mater Sci Eng C Mater Biol Appl. 2015 Dec 1;57:452-63.

Design of biocomposite materials for bone tissue regeneration.

Yunus Basha R, Sampath Kumar TS, Doble M.

Several synthetic scaffolds are being developed using polymers, ceramics and their composites to

overcome the limitations of auto- and allografts. Polymer-ceramic composites appear to be the most

promising bone graft substitute since the natural bone itself is a composite of collagen and

hydroxyapatite. Ceramics provide strength and osteoconductivity to the scaffold while polymers

impart flexibility and resorbability. Natural polymers have an edge over synthetic polymers because

of their biocompatibility and biological recognition property. But, very few natural polymer-

ceramic composites are available as commercial products, and those few are predominantly based

on type I collagen. Disadvantages of using collagen include allergic reactions and pathogen

transmission. The commercial products also lack sufficient mechanical properties. This review

summarizes the recent developments of biocomposite materials as bone scaffolds to overcome these

drawbacks. Their characteristics, in vitro and in vivo performance are discussed with emphasis on

their mechanical properties and ways to improve their performance.

129

Biochem Biophys Res Commun. 2006 May 26;344(1):362-9.

Novel chitosan/collagen scaffold containing transforming growth factor-beta1 DNA for

periodontal tissue engineering.

Zhang Y, Cheng X, Wang J, Wang Y, Shi B, Huang C, Yang X, Liu T.

The current rapid progression in tissue engineering and local gene delivery system has enhanced our

applications to periodontal tissue engineering. In this study, porous chitosan/collagen scaffolds were

prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding

human transforming growth factor-beta1 (TGF-beta1). These scaffolds were evaluated in vitro by

analysis of microscopic structure, porosity, and cytocompatibility. Human periodontal ligament

cells (HPLCs) were seeded in this scaffold, and gene transfection could be traced by green

fluorescent protein (GFP). The expression of type I and type III collagen was detected with RT-

PCR, and then these scaffolds were implanted subcutaneously into athymic mice. Results indicated

that the pore diameter of the gene-combined scaffolds was lower than that of pure chitosan/collagen

scaffold. The scaffold containing Ad-TGF-beta1 exhibited the highest proliferation rate, and the

expression of type I and type III collagen up-regulated in Ad-TGF-beta1 scaffold. After implanted

in vivo, EGFP-transfected HPLCs not only proliferated but also recruited surrounding tissue to

grow in the scaffold. This study demonstrated the potential of chitosan/collagen scaffold combined

Ad-TGF-beta1 as a good substrate candidate in periodontal tissue engineering.

130

Appendici

Appendice A: Foglietto Illustrativo del Dispositivo

CHondroGrid®

Dispositivo sterile monouso Descrizione Collagene idrolizzato a basso peso molecolare, liofilizzato, per iniezione intra- e peri-articolare. Composizione Collagene idrolizzato a basso peso molecolare di origine bovina. Indicazioni CHondroGrid® è indicato per il trattamento della sintomatologia dolorosa e della perdita di funzionalità delle maggiori articolazioni (ginocchio, spalla, anca, polso e caviglia) e delle strutture muscolo-tendinee e legamentose ad esse annesse, causate da patologie degenerative o dovute a esito traumatico o a sovraccarico. Le più comuni indicazioni di utilizzo di CHondroGrid ® sono il trattamento sintomatologico e funzionale di: osteoartrosi, artrosinoviti acute o croniche secondarie ad artrosi o artrite reumatoide, esiti di traumi o lesioni, affaticamento da sovraccarico a carico delle articolazioni sopra indicate. CHondroGrid ® è indicato inoltre in caso di meniscopatie e in preparazione e come terapia di mantenimento a seguito di interventi di meniscectomia, di ricostruzione legamentosa o di pulizia e/o ricostruzione del tessuto cartilagineo articolare. Per queste indicazioni l’applicazione è eseguita per via intra-articolare. Per esiti di traumi e/o lesione peri-articolari o a carico di strutture muscolo-tendinee-ligamentose si procede invece tramite iniezione peri-articolare e infiltrazione delle strutture coinvolte. Meccanismo d’azione Per via intra-articolare: il collagene idrolizzato a basso peso molecolare si distribuisce sulla superficie dell’articolazione rinforzando la matrice cartilaginea, deteriorata dai processi patologici in atto, la cui impalcatura è formata da un fitto intreccio di fibre collagene. CHondroGrid ® svolge quindi un’azione meccanica di rinforzo diretto di strutture collageniche indebolite e/o deteriorate, migliorando la mobilità e contribuendo a ridurre la sintomatologia dolorosa a carico dell’articolazione. Per via peri-articolare: il collagene idrolizzato a basso peso molecolare agisce quale rinforzo diretto all’impalcatura collagenica danneggiata delle strutture peri-articolari, quali tendini e/o legamenti, contribuendo ad una riduzione del dolore e ad una più veloce ripresa funzionale. Protocollo terapeutico Iniezione intra-articolare Il trattamento prevede tre iniezioni: due eseguite a distanza di 15 giorni l’una dall’altra, la terza dopo un mese dall’ultimo trattamento. Iniezione peri-articolare Il trattamento prevede due iniezioni a distanza di 30 giorni l’una dall’altra. Infiltrazione in strutture muscolo-tendinee-ligamentose Due iniezioni ad un intervallo di circa 10 giorni l’una dall’altra. Materiale necessario all’uso del dispositivo (non compreso nella confezione) Una fiala di acqua sterile per iniezioni (2 ml o più). Una siringa sterile (5 ml o più) Due aghi sterili

Istruzioni per l’uso Attenzione: procedere allo svuotamento del liquido articolare o del versamento prima di iniettare CHondroGrid ®.

1. Aprire la fiala di acqua sterile per iniezioni. 2. Aspirarne 2 ml usando la siringa da 5 ml e relativo ago. 3. Aprire il blister, estrarre flacone di liofilizzato e iniettare attraverso il tappo in gomma l’acqua sterile per iniezioni. 4. Agitare fino a completa solubilizzazione. 5. Aspirare nella siringa la soluzione ottenuta. 6. Sostituire l’ago usato con il secondo ago sterile. 7. Somministrare il prodotto avendo cura di avere eseguito una corretta asepsi.

Iniezione intra-articolare Iniettare all’interno dello spazio articolare, se necessario sotto guida strumentale, ad esempio ecografia, specialmente in caso di trattamento dell’anca e della spalla. Infiltrazione peri-articolare e in strutture muscolo-tendinee-legamentose Individuare il punto o i punti di massima dolorabilità ed eventualmente segnarli con una penna dermografica. L’iniezione può essere eseguita sotto guida strumentale in modo da essere certi di raggiungere il sito di lesione. Avvertenze e precauzioni CHondroGrid ® deve essere utilizzato esclusivamente da personale medico qualificato, seguendo procedure strettamente asettiche. CHondroGrid ® non deve essere utilizzato in associazione ad altri composti iniettabili. E’ tuttavia indicato in pazienti precedentemente trattati con iniezione di acido ialuronico o PRP autologo. Nel caso sia richiesta una ulteriore terapia di supporto alla matrice cartilaginea può essere associato a trattamento per via orale con integratori a base di collagene idrolizzato a basso peso molecolare, quali ad esempio CHONDROVITA® o simili. Si raccomanda di non sottoporre l’articolazione trattata a carichi eccessivi nelle ore immediatamente successive all’infiltrazione. Non usare CHondroGrid ® se la confezione risulta aperta o danneggiata. Non iniettare CHondroGrid ® per via endovascolare. Non utilizzare dopo la data di scadenza. La data di scadenza si riferisce al prodotto in confezione integra e conservato in modo corretto. Tenere fuori dalla portata dei bambini. Controindicazioni ed effetti indesiderati Pazienti con accertata ipersensibilità al collagene. Infezione articolare o peri-articolare, emartro, eritema nella zona da infiltrare, chiazze psoriasiche nella zona da infiltrare. L’iniezione intra- e peri-articolare può causare occasionalmente dolore transitorio, leggero gonfiore, reazione cutanea superficiale e arrossamento dovuto all’azione meccanica dell’ago. Queste reazioni sono generalmente di breve durata e si risolvono spontaneamente entro pochi giorni ponendo l’arto a riposo e con l’applicazione di ghiaccio. Contenuto della confezione ed etichette paziente Flacone di vetro in singolo blister di PETG. Foglietto illustrativo. L'etichetta apposta sul blister consiste di sei etichette, staccabili singolarmente, che fungono anche da etichetta paziente per l'apposizione in cartella clinica o altra documentazione. Sterilizzazione e conservazione Il prodotto è sterilizzato tramite irraggiamento a raggi beta a 25 kGy. Conservare al riparo dall’esposizione diretta ai raggi solari, in luogo fresco e asciutto, ad una temperatura compresa tra 4° e 40°C. In condizioni di conservazione corrette l’integrità della confezione e quindi la sterilità del prodotto sono garantite per 2 anni dalla data di produzione (vedi data di scadenza sull’etichetta esterna). Fabbricante Bioteck SpA, Via E. Fermi 49, 36057 Arcugnano (VI), Italia. Fabbricato nello stabilimento di Via G. Agnelli, 3 - 10020 Riva presso Chieri (Torino), Italia. Iscrizione al repertorio dei dispositivi medici e classificazione CND RDM (numero di iscrizione al repertorio): 1412112/R CND (classificazione): P900402 - PRODOTTI RIASSORBIBILI PER RIEMPIMENTO E RICOSTRUZIONE

Rev 20160629 del 29.06.2016

Appendice B: Certificazioni CE di ChondroGrid

Dispositivo Medico ChondroGrid · ricostruzione del tessuto cartilagineo articolare. Per queste...

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Transcript of Dispositivo Medico ChondroGrid · ricostruzione del tessuto cartilagineo articolare. Per queste...