Muscles, Ligaments and Tendons Journal 2018;8 (4):465-472 465

Debora Burini1

Sara Salucci1

Francesco Fardetti2,3

Alessandro Beccarini2

Vittorio Calvisi3

Pietro Gobbi1

Elisabetta Falcieri1

Davide Curzi1

1 Department of Biomolecular Sciences, Universityof Urbino Carlo Bo, Urbino, Italy

2 Porta Sole Hospital, Perugia, Italy3 Department of Life, Health and Environmental

Sciences, University of L’Aquila, L’Aquila, Italy

Corresponding author:Davide CurziDepartment of Biomolecular SciencesUniversity of Urbino Carlo BoCà Le Suore St., “E. Mattei” Campus61029 Urbino, ItalyTel: 0722 304311 E-mail: davide.curzi@uniurb.it

Summary

Introduction: Chondrocalcinosis is a pathologicalcondition characterized by the presence of calci-um pyrophosphate dihydrate (CPPD) crystal de-position in the soft tissues. Even if knee articularcartilage is the most involved anatomical area,different kind of tissue and joint can be affectedby this disorder. Methods: The aim of this manuscript is to analyzeat histological and ultrastructural level the crystaldeposition in shoulder soft tissue subjected tomechanical stress of patients affected by CPPDdisease. Moreover, the cellular behavior in thesame specimens has been investigated by meansof transmission electron microscopy at variabledistances from crystal deposits. Results: An interesting relationship between CP-PD and cellular impairment appears in humeralarticular cartilage, joint capsule and long head ofbiceps brachii tendon sheath, where respectivelychondrocytes and fibroblasts, close to crystal de-posits, reveal numerous cell damages, such aschromatin condensation, dilation of organelles or

cell membrane rupture. Conclusion: Considering that cells far to the crys-tals are healthy, their behavior appears to be dif-ferent from that of neighboring cells, then ourpreliminary results suggest a possible cause-ef-fect relationship between events. Level of evidence: basic science study.

KEY WORDS: shoulder, chondrocalcinosis, articularcartilage, long head of biceps brachii, joint capsule,calcium crystals.

Introduction

Calcium pyrophosphate dihydrate crystal depositiondisease (CPPD) is an articular disorder, character-ized by the presence of radiographic articular fibro- orhyaline cartilage calcifications. This condition is alsocalled chondrocalcinosis. CPPD is the third mostcommon form of arthritis in elderly and epidemiologi-cal studies highlight that 4 to 7% of European andUSA populations are affected by chondrocalcinosis1,2.The prevalence shows no gender difference and CP-PD onset is strictly correlated to aging, to date identi-fied as the most relevant risk factor together with pre-vious joint trauma3. In fact, Neame et al. have de -monstrated an increased prevalence of knee CPPD inpatients aged 80-84 (17.5%), respect to those aged55-59 (3.7%), without difference between man andwoman4. Moreover, CPPD is frequently associated tomechanical stress induced by repetitive trauma, asconfirmed by its relationship with sport practice or in-juries5. CPPD and osteoarthritis (OA) often coexist in thesame joint, probably inducing a symptom worsening.The presence of calcium crystals has been observedin cartilage specimens in 25-30% of patients affectedby OA, following knee arthroplasty6,7. The most frequent anatomical site of CPPD is knee,followed by wrist, hip, pubis symphysis and shoul-der8. From a radiological point of view, crystal deposi-tion has been observed in the connective tissues(mainly fibrocartilage and hyaline cartilage) of differ-ent joint anatomical structures, such as menisci, artic-ular capsule, tendon, ligament, synovial bursae, syn-ovial fluid, synovial membrane, articular cartilage9.Meniscus fibrocartilage is the most affected tissue(from 86.3 to 95% of patients affected by CPPD intwo different studies), followed by articular cartilage(from 45 to 56.8%) and articular capsule fibrous con-

Chondrocalcinosis: a morphofunctional study ofcrystal deposition in mechanically stressedshoulder soft tissues

Original article

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nective tissue (about 30%)4,8. From a histologicalpoint of view, crystal depositions in human connectivetissue were found close to degenerating collagenfibers in the matrix surrounded hypertrophic chondro-cytes. This kind of matrix was characterized by elec-tron-dense amorphous material, including proteogly-cans and debris of cellular components10. Moreover,crystal deposition induced fibrosis, angiogenesis andneutrophils accumulation in the ligament flavum11. Unlike knee, epidemiological data on glenohumeraljoint crystals distribution are unknown and a limitedliterature describes this pathological condition onshoulder tissue. Frequently it is difficult to discernCPPD and OA in this anatomical area, due to theirsimilar radiographic features, such as subchondralbone sclerosis and osteophytes12. The presence ofcalcifications has been observed, by means of mag-netic resonance imaging and X-rays technique, inhumeral articular cartilage, tendons, capsule, syn-ovial fluid, synovial membrane and bursae13,14. Although the mechanisms of connective tissue calcifi-cation are not completely understood even for themost common and studied crystal deposition dis-eases, an impaired chondrocyte behavior seems tobe involved in articular cartilage calcification of pa-tients affected by OA15. In particular, similarly to otherkinds of crystals, calcium pyrophosphate dihydrate

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precipitates may induce cell death, involving thenecroptosis pathway16. The aim of this work was to investigate at microscop-ic and histological level the crystal deposition onshoulder soft tissues of patients affected by CPPD.Electron microscopy analysis has been performed inthe same specimens to investigate the cellular behav-ior close to crystal and far from them. This manu-script allows correlate the crystal microscopic locationand cell behavior, suggesting an interesting relation-ship.

Materials and methods

Patients and specimen handlingThe study meets the ethical standards of the journal.In particular, all experimental procedures were car-ried out according to the journal guidelines17. Specimens from humeral articular cartilage, joint cap-sule, long head of biceps brachii and loose bodieswere withdrawn from 6 patients with CPPD, aged67±5, during shoulder arthroplasty. For each patient,pre-surgery X-ray images have been performed toconfirm the typical degenerative features of CPPD. Inparticular, shoulders revealed a diffuse calcium crys-tal deposit in the soft tissue (Figs. 1A, B). Fragments

Figure 1. Radiographs of right and left shoulders of patients affected by CPPD: anteroposterior (A) and outlet-view (C). Athigh magnification (B, D), the presence of widespread calcium crystal accumulations in soft tissues is observable (arrow).

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from each tissue were fixed with 2.5% glutaraldehydesolution in a 0.1 M phosphate buffer for 3 h and thenminced into smaller specimens (3 mm3), which werefixed in the same solution for a supplementary hour.After washing, samples were post-fixed with 1% os-mium tetroxide solution in the same buffer for 1 h,rinsed and dehydrated in a graded series of etha -nol18.

Environmental scanning electron microscopy(ESEM) and microanalysisFixed samples from each tissue were dried andmounted on conventional SEM stubs, and then ob-served with a FEI QUANTA 200 environmental scan-ning electron microscope (mode: low vacuum; detec-tor: secondary electrons; high voltage: 30 kV). Toconfirm the diagnosis of chondrocalcinosis, an energydispersive X-ray spectroscopic microanalysis (EDS)has been performed in the same conditions of obser-vation19.

Histological analysisOther fixed fragments were embedded in araldite andsectioned by an LKB 2088 ultramicrotome. Semithinsections were stained with 1% toluidine blue in dis-tilled water for 2 minutes. After washing, they werestained with 2% alizarin red in distilled water for 2minutes. To better fix alizarin red staining, specimensections were immersed for 20 seconds in acetone,acetone-xylene (1:1) solution and xylene, respectively.Then, samples were washed and observed by meansof Olympus optical microscope at 10x, 20x, 40x or100x (oil)20.

Transmission electron microscopy (TEM)The stained semithin sections were used to identifyareas where to perform TEM analysis. Thin sectionswere then obtained from these chosen regions,stained with uranyl acetate and lead citrate solutionsand then observed with Philips CM10 transmissionelectron microscope (voltage: 80 kV)21.

Results

Humeral articular cartilageChondrocalcinotic humeral articular cartilage dis-played a general degradation. In transverse sections(Fig. 2A), numerous clefts occurred from the articularsurface to the deeper tissue layers, and some areasof cartilage surface layer appeared completely de-stroyed. Calcium crystal depositions (red stained)were visible on the superficial zones, as well as in themiddle layers of tissue (Fig. 2B). These crystals ap-pear to be generally collected in deposits and rarelycan be found alone. When this happens, it affects thedeeper levels of the tissue and not the superficialones.The cellular distribution was deeply modified, as con-firmed by the almost complete absence of cells onthe superficial layers. In the middle zone, TEM im-

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ages revealed the presence of rare healthy chondro-cytes, which showed a regular organization of nucle-us, nuclear pores and chromatin, as well a diffuse en-doplasmic reticulum and numerous glycogen gran-ules (Fig. 2C). However, several chondrocytes dis-played necrotic features, such as vacuolized cyto-plasm, degraded organelles, plasma and nuclearmembrane discontinuities, with lack of chromatin con-densation (Fig. 2D).

Long head of biceps brachii tendon sheathOptical images of the long head of biceps brachii ten-don revealed a high tissue mineralization. In particu-lar, the outer fibrous sheath, that, together with theinner synovial ones, forms the tendon sheath, ap-peared characterized by crystal accumulations, aswell as numerous single crystals scattered in the tis-sue. Analyzing at optical microscopy the tissue areasclose to these sediments, fibroblasts showed an evi-dent vacuolization, suggesting a compromised cell vi-tality (Fig. 3A). TEM micrographs confirmed the im-paired cell morphology. Fibroblasts showed an evi-dent cell shrinkage with visible cytoplasmic alter-ations characterized by numerous vacuoles, many ofwhich containing cellular material inside. In addition,chromatin condensation appeared (Fig. 3C). On theother hand, tissue areas far from crystal depositshowed preserved healthy cells (Fig. 3B). In most ofthese cells, the cytoplasmic vacuoles were complete-ly absent and, when present, they had a clear re-duced dimension if compared to those close to crystalsediments.

Joint capsuleOptical transverse sections of articular capsule dis-played wide crystal accumulations on the synovialmembrane, and the presence of single crystals scat-tered within the tissue (Fig. 4A, B). The capsule in-tegrity appeared compromised by tissue clefts, ex-tending from the joint cavity to the tissue deeper lay-ers. From optical images, a relationship between sin-gle crystal and clefts locations was observable. Inparticular, the sediments in the depth layers of tissueappeared neighboring to the cleft wall. At ultrastructural level, the fibroblast-like synoviocyte,if compared to few preserved cells (Fig. 4C), revealedthe presence of autophagic vacuoles and swollen or-ganelles (Fig. 4D).

Loose bodyOptical microscope images revealed wide calcifiedtissue area in the intra-articular loose body. Given thenature of this anatomical structure, which is usuallycomposed by cartilage or cartilage and bone, the cal-cification presence is apparently not noteworthy22.However, given the pathology of the patient, under-standing the nature of these crystals was necessaryand the crystal analysis confirmed results comparableto bone tissue (data not shown). Morphological ob-servations were not able to identify single or accumu-lated calcium pyrophosphate dihydrate crystals in this

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tissue (Fig. 5A), however in the high calcified zonesthe identification of specific kinds of crystals ap-peared really hard. Even though the loose body is afree fragment in the articular space, its cells showeda high vitality if compared to those of other tissues.TEM micrographs confirmed the chondrocyte mor-phofunctional integrity. In particular, cells showed aregular arrangement of nuclear and cytoplasmic com-ponents (Fig. 5B).

Calcium pyrophosphate dihydrate crystal depositionSingle crystal ultrastructure and accumulation mor-phology were respectively evaluated by ESEM andTEM in each tissue (Fig. 6A, B). The crystal chemicalnature was investigated by EDS spectrum (Fig. 6C),showing the relationship between calcium and phos-phorous peaks, in line with the stoichiometric ratio ofCPPD crystals.

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Discussion

Although the diagnoses of CPPD of shoulder soft tis-sues are increasing, many questions regarding thispathological condition, such as its origin or its devel-opment, still have no answers. Numerous scientificworks have analyzed this condition at macroscopiclevel through radiological investigation, but, to thebest of our knowledge, this is the first study which ex-amined, by means of various microscopy techniques(previous described), CPPD crystal deposition in dif-ferent human shoulder anatomical structures, evalu-ating a possible relationship between cell behaviorand crystal deposits location. If cell death is the origin or the consequence of crys-tal deposition is still under discussion. The molecularmechanisms of CPPD crystal formation have not yetbeen fully understood, but the most quoted hypothe-sis in human cartilage argues that chondrocyte dys-

Figure 2. Light microscopy (A, B) and TEM (C, D) images of chondrocalcinotic cartilage cross-sections. In A and B the accu-mulations of CPPD crystals appear red stained. In A, red stained superficial (sca) and deep crystal accumulations (dca) areobservable. TEM micrographs of cartilage middle layers reveal preserved chondrocytes far to crystal deposits, which showan undamaged nucleus (nu), an abundant rough endoplasmic reticulum (rer) and several glycogen granules (g). Close tocrystal deposits, necrotic chondrocytes with discontinuous nuclear (arrow heads) and plasma membrane (arrows), appear.Magnification: A: 20x; B: 100x. Bars: C, D: 1 μm.

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function in maintaining extracellular matrix turnoverwould be the starting point. In particular, these cellsseem to produce excessive extracellular inorganic py-rophosphate in the tissue. These molecules arestored in synovial fluid through the direct contact be-tween tissues and the weeping lubrication mecha-nism of cartilage. In the synovia, a significant in-crease of inorganic pyrophosphate induces CPPDcrystal deposits2,3,22, which in turn are allocated, asshown by our results, in the different soft tissues indirect or indirect contact with the synovial fluid, suchas humeral articular cartilage, inner synovial mem-brane of joint capsule and long head of biceps brachiitendon sheath. Our preliminary results suggest a keyrole of tissue wear, related to mechanical stress andto crystal deposits themselves. In fact, even if the cal-cification process may develop within the same tis-sue, as well as for other pathological conditions,these results reveal a clear relationship betweencrystal location and tissue clefts, suggesting the abili-ty of these crystals to insert themselves into the cleftsthrough the synovial fluid, and then their capacity to

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pass from that space to the close tissue areas. Com-pared to other shoulder soft tissue, morphologicaland chemical techniques were not able to reveal CP-PD crystals in the loose body. Nevertheless, the os-teochondral nature of this structure could have affect-ed our analysis. In particular, the identification of CP-PD crystals in the large calcified area appeared par-ticularly complex. According to the literature23, de-spite the loose body is a free fragment in the jointcavity, its cartilaginous area was characterized by nu-merous healthy chondrocytes, confirming the synovialfluid ability to regularly make contact with it. Al-though our observations have not confirmed it, beingthe synovial fluid the carrier of crystals, this contactsuggests a probable crystals deposit in this tissue. The crystal deposition effects on each tissue are notyet clear. Some studies suggest a relationship be-tween them and acute inflammation, which probablydepends on the crystals interaction with neutrophils inthe synovial fluid24,25. CPPD crystal accumulation inarticular cartilage could also alter its mechanicalproperties, contributing to joint damage2. There are

Figure 3. Light microscopy (A) and TEM (B, C) images of the outer fibrous layer of long head of biceps brachii tendonsheath. In A, red stained crystal accumulations (ca) and single crystal (sc) are observable near to high vacuolated cells (ar-row head). At high magnification, fibroblasts far to crystal deposits display a healthy morphology (B), characterized by a reg-ular disposition of chromatin in the nucleus (nu) and undamaged mitochondria (m). On the other hand, cells close to sedi-ments show a compromised ultrastructure (C), with a starting chromatin condensation and numerous vacuoles in cytoplasm(arrow head), some of which seems containing cellular material inside (arrow). Magnification: A: 40x. Bars: B, C: 0,5 μm.

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no evidences, instead, about a possible cell death as-sociated to CPPD. There are only rare studies re-ferred to other pathologies, for instance calcium ox-alate stones seem to induce death in renal epithelialcells, but the mechanism is unclear25.In this study, a relationship between crystal sedi-ments location and cell death has been observed. Inhumeral cartilage superficial zone, where calcium de-posits are more significant, an almost total absenceof chondrocyte is observable. Even if their death canbe easy related to tissue wear, near single CPPDcrystals, in cartilage middle layer numerous necroticcells have been revealed. Similarly, in the long headof biceps brachii tendon sheath, impaired and healthyfibroblasts are major concentrated, respectively,close and far away from the calcium crystals. The

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synovial membrane of joint capsule displayed a com-parable condition, where several impaired cells arespecifically located close to CPPD crystal accumula-tions. Taken together, these preliminary findings sug-gest a connection between cell death and CPPDcrystals. However, in order to understand their causeand effect relationship, future studies will be neces-sary and the possible existence of a positive feed-back system between the two events should be con-sidered as a hypothesis worth examining.

Compliance with ethical standards

FundingThe Authors received no specific funding for this

Figure 4. Light microscopy (A, B) and TEM (C, D) images of synovial membrane and joint capsule. In A, red stained superfi-cial crystal accumulation (sca) appears at the synovial membrane, while in B, the presence of single crystals (sc) in thedeeper layers of joint capsule is observable. Synoviocytes, supported by collagen fibers (cf), show a regular chromatin dis-position in the nucleus (nu), far from sediments, as well as undamaged mitochondria in the cytoplasm. Contrary, synovio-cytes close to crystal deposits display an electron dense nucleus and numerous autophagic vacuoles (av) in the cytoplasm,suggesting a compromised cell vitality. Magnification: A: 20x; B: 40x. Bars: C, D: 0,5 μm.

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work, but the work was only supported by “Fondi Val-orizzazione” and “Fondi Ricerca” DiSB, Carlo Bo

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Urbino University, which are a form of personal re-search funds.

Figure 5. Light microscopy (A) and TEM (B) images of loose body. Numerous and wide calcified area (ca) from bone origin(red stained) surrounded from cartilaginous tissue are observable (A). A preserved chondrocyte appears, displaying an un-damaged nucleus (nu) and nucleolus (n) and, in the cytoplasm, a high concentration of glycogen granules (g) and rough en-doplasmic reticulum (rer) is observable. Magnification: A: 20x. Bars: B: 1 μm.

Figure 6. ESEM (A) and TEM (B) images of a single calcium pyrophosphate dihydrate crystal and an accumulation of nu-merous crystals in cartilaginous tissue respectively. In C, EDS spectrum confirms the nature of crystals. Bars: A: 2 μm; B: 5μm.© C

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Conflict of interestThe Authors declare that they have no conflict of in-terest.

Acknowledgements

We wish to thank Dr. Rita De Matteis and Dr. SabrinaBurattini from Department of Biomolecular Sciencesof Urbino University, for their advice in histologicalanalysis and technical help respectively. Also thankto Dr. Patrizia Ammazzalorso, head of PesaroARPAM department, for ESEM analysis assistance.This work has been possible thanks to the DISB 2017Enhancement Project of Urbino University.

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