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Unraveling the Mechanisms of Hypertrophy-Induced Matrix Mineralization and Modifications in Articular Chondrocytes

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Abstract

Chondrocyte hypertrophic differentiation is a main event leading to articular cartilage degradation in osteoarthritis. It is associated with matrix remodeling and mineralization, the dynamics of which is not well characterized during chondrocyte hypertrophic differentiation in articular cartilage. Based on an in vitro model of progressive differentiation of immature murine articular chondrocytes (iMACs) into prehypertrophic (Prehyp) and hypertrophic (Hyp) chondrocytes, we performed kinetics of chondrocyte differentiation from Prehyp to Hyp to follow matrix mineralization and remodeling by immunofluorescence, biochemical, molecular, and physicochemical approaches, including atomic force microscopy, scanning electron microscopy associated with energy-dispersive X-ray spectroscopy (SEM–EDS), attenuated total reflection infrared analyses, and X-ray diffraction. Chondrocyte apoptosis was determined by TUNEL assay. The results show the formation of a mineral phase 7 days after Hyp induction, which spreads within the matrices to form poorly crystalline carbonate-substituted hydroxyapatite after 14 days, then the proportions of crystalline relative to amorphous content increases over time. Hyp differentiation also induced a matrix turnover that occurs over the first 7 days, characterized by a decrease in type II collagen and aggrecan and the concomitant appearance of type X collagen. This is accompanied by an increase in the enzymatic activity of MMP-13, the main collagenase in cartilage. The number of apoptotic chondrocytes slightly increased with Hyp differentiation and SEM–EDS analyses detected phosphorus-rich structures that could correspond to apoptotic bodies. Our findings highlight the mechanisms of matrix remodeling events leading to the mineralization of articular cartilage that may occur in osteoarthritis.

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References

  1. Yan J, Qin W, Xiao B et al (2020) Pathological calcification in osteoarthritis: an outcome or a disease initiator? Biol Rev 95:960–985. https://doi.org/10.1111/brv.12595

    Article  PubMed  Google Scholar 

  2. Wang X, Qin Wu, Zhang Ru et al (2023) Stage-specific and location-specific cartilage calcification in osteoarthritis development. Ann Rheum Dis 82:393. https://doi.org/10.1136/ard-2022-222944

    Article  CAS  PubMed  Google Scholar 

  3. Zuscik MJ, Hilton MJ, Zhang X et al (2008) Regulation of chondrogenesis and chondrocyte differentiation by stress. J Clin Invest 118:429–438. https://doi.org/10.1172/JCI34174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Mackie EJ, Ahmed YA, Tatarczuch L et al (2008) Endochondral ossification: how cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol 40:46–62. https://doi.org/10.1016/j.biocel.2007.06.009

    Article  CAS  PubMed  Google Scholar 

  5. Kronenberg HM (2003) Developmental regulation of the growth plate. Nature 423:332–336. https://doi.org/10.1038/nature01657

    Article  CAS  PubMed  Google Scholar 

  6. Anderson HC, Garimella R, Tague SE (2005) The role of matrix vesicles in growth plate development and biomineralization. Front Biosci J Virtual Libr 10:822–837. https://doi.org/10.2741/1576

    Article  CAS  Google Scholar 

  7. Kirsch T, Swoboda B, Nah H-D (2000) Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic cartilage. Osteoarthritis Cartilage 8:294–302. https://doi.org/10.1053/joca.1999.0304

    Article  CAS  PubMed  Google Scholar 

  8. Pullig O, Weseloh G, Ronneberger D-L et al (2000) Chondrocyte differentiation in human osteoarthritis: expression of osteocalcin in normal and osteoarthritic cartilage and bone. Calcif Tissue Int 67:230–240. https://doi.org/10.1007/s002230001108

    Article  CAS  PubMed  Google Scholar 

  9. Rim YA, Nam Y, Ju JH (2020) The role of chondrocyte hypertrophy and senescence in osteoarthritis initiation and progression. Int J Mol Sci 21:2358. https://doi.org/10.3390/ijms21072358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Singh P, Marcu KB, Goldring MB, Otero M (2019) Phenotypic instability of chondrocytes in osteoarthritis: on a path to hypertrophy. Ann N Y Acad Sci 1442:17–34. https://doi.org/10.1111/nyas.13930

    Article  CAS  PubMed  Google Scholar 

  11. Wuthier RE, Chin JE, Hale JE et al (1985) Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture. J Biol Chem 260:15972–15979. https://doi.org/10.1016/S0021-9258(17)36354-8

    Article  CAS  PubMed  Google Scholar 

  12. Shen G (2005) The role of type X collagen in facilitating and regulating endochondral ossification of articular cartilage. Orthod Craniofac Res 8:11–17. https://doi.org/10.1111/j.1601-6343.2004.00308.x

    Article  CAS  PubMed  Google Scholar 

  13. Iyama K, Ninomiya Y, Olsen BR et al (1991) Spatiotemporal pattern of type X collagen gene expression and collagen deposition in embryonic chick vertebrae undergoing endochondral ossification. Anat Rec 229:462–472. https://doi.org/10.1002/ar.1092290405

    Article  CAS  PubMed  Google Scholar 

  14. Gerstenfeld LC, Landis WJ (1991) Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system. J Cell Biol 112:501–513. https://doi.org/10.1083/jcb.112.3.501

    Article  CAS  PubMed  Google Scholar 

  15. Kergosien N, Sautier J-M, Forest N (1998) Gene and protein expression during differentiation and matrix mineralization in a chondrocyte cell culture system. Calcif Tissue Int 62:114–121. https://doi.org/10.1007/s002239900404

    Article  CAS  PubMed  Google Scholar 

  16. van Eegher S, Perez-Lozano M-L, Toillon I et al (2021) The differentiation of prehypertrophic into hypertrophic chondrocytes drives an OA-remodeling program and IL-34 expression. Osteoarthritis Cartilage 29:257–268. https://doi.org/10.1016/j.joca.2020.10.013

    Article  PubMed  Google Scholar 

  17. Gosset M, Berenbaum F, Thirion S, Jacques C (2008) Primary culture and phenotyping of murine chondrocytes. Nat Protoc 3:1253–1260. https://doi.org/10.1038/nprot.2008.95

    Article  CAS  PubMed  Google Scholar 

  18. Farndale RW, Sayers CA, Barrett AJ (1982) A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res 9:247–248. https://doi.org/10.3109/03008208209160269

    Article  CAS  PubMed  Google Scholar 

  19. Fleet ME, Liu X, King PL (2004) Accommodation of the carbonate ion in apatite: An FTIR and X-ray structure study of crystals synthesized at 2–4 GPa. Am Mineral 89:1422–1432. https://doi.org/10.2138/am-2004-1009

    Article  CAS  Google Scholar 

  20. Boskey A, Camacho NP (2007) FT-IR imaging of native and tissue-engineered bone and cartilage. Biomaterials 28:2465–2478. https://doi.org/10.1016/j.biomaterials.2006.11.043

    Article  CAS  PubMed  Google Scholar 

  21. Pleshko N, Boskey A, Mendelsohn R (1991) Novel infrared spectroscopic method for the determination of crystallinity of hydroxyapatite minerals. Biophys J 60:786–793

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Paschalis EP, DiCarlo E, Betts F et al (1996) FTIR microspectroscopic analysis of human osteonal bone. Calcif Tissue Int 59:480–487. https://doi.org/10.1007/BF00369214

    Article  CAS  PubMed  Google Scholar 

  23. Boraldi F, Lofaro FD, Quaglino D (2021) Apoptosis in the extraosseous calcification process. Cells 10:131. https://doi.org/10.3390/cells10010131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Dreier R (2010) Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Res Ther 12:216. https://doi.org/10.1186/ar3117

    Article  PubMed  PubMed Central  Google Scholar 

  25. van der Kraan PM, van den Berg WB (2012) Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration? Osteoarthritis Cartilage 20:223–232. https://doi.org/10.1016/j.joca.2011.12.003

    Article  PubMed  Google Scholar 

  26. Dorozhkin SV (2010) Amorphous calcium (ortho)phosphates. Acta Biomater 6:4457–4475. https://doi.org/10.1016/j.actbio.2010.06.031

    Article  CAS  PubMed  Google Scholar 

  27. Gebauer D (2018) Nucleation of Minerals: Precursors, Intermediates and Their Use in Materials Chemistry. MDPI

  28. Foley B, Greiner M, McGlynn G, Schmahl WW (2020) Anatomical variation of human bone bioapatite crystallography. Crystals. https://doi.org/10.3390/cryst10100859

    Article  Google Scholar 

  29. Gibson G, Lin DL, Roque M (1997) Apoptosis of terminally differentiated chondrocytes in culture. Exp Cell Res 233:372–382. https://doi.org/10.1006/excr.1997.3576

    Article  CAS  PubMed  Google Scholar 

  30. Magne D, Bluteau G, Faucheux C et al (2003) Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosis-associated mineralization: possible implication of apoptosis in the regulation of endochondral ossification. J Bone Miner Res Off J Am Soc Bone Miner Res 18:1430–1442. https://doi.org/10.1359/jbmr.2003.18.8.1430

    Article  CAS  Google Scholar 

  31. Wang M, Sampson ER, Jin H et al (2013) MMP13 is a critical target gene during the progression of osteoarthritis. Arthritis Res Ther 15:R5. https://doi.org/10.1186/ar4133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Inada M, Wang Y, Byrne MH et al (2004) Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci 101:17192–17197. https://doi.org/10.1073/pnas.0407788101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wu CW, Tchetina EV, Mwale F et al (2002) Proteolysis Involving matrix metalloproteinase 13 (collagenase-3) is required for chondrocyte differentiation that is associated with matrix mineralization. J Bone Miner Res 17:639–651. https://doi.org/10.1359/jbmr.2002.17.4.639

    Article  CAS  PubMed  Google Scholar 

  34. Nerlich AG, Wiest I, von der Mark K (1993) Immunohistochemical analysis of interstitial collagens in cartilage of different stages of osteoarthrosis. Virchows Arch B 63:249–255. https://doi.org/10.1007/BF02899269

    Article  CAS  PubMed  Google Scholar 

  35. Martin I, Jakob M, Schäfer D et al (2001) Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints. Osteoarthr Cartil 9:112–118. https://doi.org/10.1053/joca.2000.0366

    Article  CAS  Google Scholar 

  36. Casagrande D, Stains JP, Murthi AM (2015) Identification of shoulder osteoarthritis biomarkers: comparison between shoulders with and without osteoarthritis. J Shoulder Elbow Surg 24:382–390. https://doi.org/10.1016/j.jse.2014.11.039

    Article  PubMed  PubMed Central  Google Scholar 

  37. Mwale F, Billinghurst C, Wu W et al (2000) Selective assembly and remodelling of collagens II and IX associated with expression of the chondrocyte hypertrophic phenotype. Dev Dyn 218:648–662. https://doi.org/10.1002/1097-0177(200008)218:4%3c648::AID-DVDY1022%3e3.0.CO;2-P

    Article  CAS  PubMed  Google Scholar 

  38. Kwan KM, Pang MK, Zhou S et al (1997) Abnormal compartmentalization of cartilage matrix components in mice lacking collagen X: implications for function. J Cell Biol 136:459–471. https://doi.org/10.1083/jcb.136.2.459

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kong RYC, Kwan KM, Lau ET et al (1993) Intron-exon structure, alternative use of promoter and expression of the mouse collagen X gene, Col10a-1. Eur J Biochem 213:99–111. https://doi.org/10.1111/j.1432-1033.1993.tb17739.x

    Article  CAS  PubMed  Google Scholar 

  40. Kirsch T, Swoboda B, von der Mark K (1992) Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and × collagen and matrix mineralization. Differentiation 52:89–100. https://doi.org/10.1111/j.1432-0436.1992.tb00503.x

    Article  CAS  PubMed  Google Scholar 

  41. Fuerst M, Bertrand J, Lammers L et al (2009) Calcification of articular cartilage in human osteoarthritis. Arthritis Rheum 60:2694–2703. https://doi.org/10.1002/art.24774

    Article  CAS  PubMed  Google Scholar 

  42. Chen S, Birk DE (2013) The regulatory roles of small leucine-rich proteoglycans in extracellular assembly. FEBS J 280:2120. https://doi.org/10.1111/febs.12136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Chen CC, Boskey AL (1986) The effects of proteoglycans from different cartilage types on in vitro hydroxyapatite proliferation. Calcif Tissue Int 39:324–327. https://doi.org/10.1007/BF02555199

    Article  CAS  PubMed  Google Scholar 

  44. Nishida Y, Shinomura T, Iwata H et al (1994) Abnormal occurrence of a large chondroitin sulfate proteoglycan, PG-M/versican in osteoarthritic cartilage. Osteoarthr Cartil 2:43–49. https://doi.org/10.1016/S1063-4584(05)80005-6

    Article  CAS  Google Scholar 

  45. Li S, Zhan J-K, Wang Y-J et al (2019) Exosomes from hyperglycemia-stimulated vascular endothelial cells contain versican that regulate calcification/senescence in vascular smooth muscle cells. Cell Biosci 9:1. https://doi.org/10.1186/s13578-018-0263-x

    Article  PubMed  PubMed Central  Google Scholar 

  46. Wojtas M, Lausch AJ, Sone ED (2020) Glycosaminoglycans accelerate biomimetic collagen mineralization in a tissue-based in vitro model. Proc Natl Acad Sci 117:12636–12642. https://doi.org/10.1073/pnas.1914899117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

ILJ and BF acknowledge the financial support from “Interface pour le vivant” PhD program (Sorbonne Université) and from the Institute of Materials Science (iMAT) of the Alliance Sorbonne Université, respectively.

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Authors and Affiliations

  1. Laboratoire de Réactivité de Surface, LRS, CNRS, Sorbonne Université, 4, Place Jussieu, 75005, Paris, France

    Ilhem Lilia Jaabar, Brittany Foley, Alberto Mezzetti & Jessem Landoulsi

  2. INSERM, Centre de Recherche Saint-Antoine, CRSA, Sorbonne Université, 34 Rue Crozatier, 75012, Paris, France

    Ilhem Lilia Jaabar, Francis Berenbaum & Xavier Houard

  3. Laboratoire de Biomécanique & Bioingénierie, CNRS, Université de Technologie de Compiègne, BP 20529, 60205, Compiègne Cedex, France

    Brittany Foley

  4. Laboratoire Interfaces et Systèmes Electrochimiques, LISE, CNRS,, Sorbonne Université, 75012, Paris, France

    Françoise Pillier

  5. Rheumatology Department, AP-HP Saint-Antoine Hospital, 184, Rue du Faubourg Saint-Antoine, 75012, Paris, France

    Francis Berenbaum

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  1. Ilhem Lilia Jaabar
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Contributions

Research design: ILJ, JL, and XH. Data acquisition and analysis: ILJ, BF, AM, FP, FB, JL, and XH. Drafting the article: ILJ, JL, and XH. Critical revision of the article: ILJ, BF, AM, FP, FB, JL, and XH. Final approval of the article: ILJ, BF, AM, FP, FB, JL, and XH.

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Correspondence to Jessem Landoulsi or Xavier Houard.

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Conflict of interest

FB reports personal fees from 4P Pharma, 4Moving Biotech, Grunenthal, GSK, Heel, Nordic Bioscience, Novartis, Servier, TRB Chemedica, Viatris outside the submitted work. ILJ, BF, AM, FP, JL and XH declare that they have no competing interest.

Human and Animal Rights and Informed Consent

All experiments with murine chondrocytes were performed according to the protocols approved by French and European ethics committees (Comité d’Ethique en Expérimentation Animale n°5 Charles Darwin de la Région Ile de France).

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Jaabar, I.L., Foley, B., Mezzetti, A. et al. Unraveling the Mechanisms of Hypertrophy-Induced Matrix Mineralization and Modifications in Articular Chondrocytes. Calcif Tissue Int (2024). https://doi.org/10.1007/s00223-024-01229-w

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