Calcified Tissue International

, Volume 104, Issue 3, pp 331–343 | Cite as

Basic Calcium Phosphate Crystals Induce Osteoarthritis-Associated Changes in Phenotype Markers in Primary Human Chondrocytes by a Calcium/Calmodulin Kinase 2-Dependent Mechanism

  • Jing Rong
  • Bregina Pool
  • Mark Zhu
  • Jacob Munro
  • Jillian Cornish
  • Geraldine M. McCarthy
  • Nicola Dalbeth
  • Raewyn PoulsenEmail author
Original Research


Chondrocytes in osteoarthritis undergo a phenotype shift leading to increased production of cartilage-degrading enzymes. There are similarities between the phenotype of osteoarthritic chondrocytes and those of growth plate chondrocytes. Hydroxyapatite can promote chondrocyte differentiation in the growth plate. Basic calcium phosphate (BCP) crystals (which consist of hydroxyapatite, octacalcium apatite and tricalcium phosphate) are frequently found in osteoarthritic joints. The objective of this study was to determine whether BCP crystals induce disease-associated changes in phenotypic marker expression in chondrocytes. Primary human chondrocytes isolated from macroscopically normal cartilage were treated with BCP for up to 48 h. Expression of indian hedgehog (IHH), matrix metalloproteinase 13 (MMP13), interleukin-6 (IL-6) and type X collagen (COLX) were higher, and expression of sry-box 9 (SOX9) lower, in BCP-treated chondrocytes (50 µg/mL) compared to untreated controls. COLX protein was also present in BCP-treated chondrocytes. Intracellular calcium and levels of phosphorylated and total calcium/calmodulin kinase 2 (CaMK2) were elevated following BCP treatment due to BCP-induced release of calcium from intracellular stores. CaMK2 inhibition or knockdown ameliorated the BCP-induced changes in SOX9, IHH, COLX, IL-6 and MMP13 expression. BCP crystals induce osteoarthritis-associated changes in phenotypic marker expression in chondrocytes by calcium-mediated activation of CaMK2. The presence of BCP crystals in osteoarthritic joints may contribute to disease progression.


Crystal arthropathy Chondrocyte hypertrophy Hydroxyapatite 



This study was funded by an Arthritis New Zealand project grant (Grant No. R267) and a Sir Charles Hercus Health Research Fellowship, Health Research Council New Zealand (Grant No. 16/022) to RCP.


Support from funders was financial only. Funders were not involved in any aspect of the study, the preparation of the manuscript or the decision to publish.

Compliance with Ethical Standards

Conflict of interest

Jing Rong, Bregina Pool, Mark Zhu, Jacob Munro, Jillian Cornish, Geraldine M. McCarthy, Nicola Dalbeth, Raewyn Poulsen declare that they have no conflicts of interest.

Human and Animal Rights and Informed Consent

Human tissue used in this study was obtained from patient donors with informed consent and with the approval of the Health and Disability Ethics Committee (New Zealand).

Supplementary material

223_2018_494_MOESM1_ESM.tif (40 kb)
Supplementary Figure 1: Expression of a type II collagen and b aggrecan was detectable in chondrocytes from macroscopically normal (undamaged) and damaged cartilage isolated from all patient donors at passage 1. (TIF 40 KB)
223_2018_494_MOESM2_ESM.tif (46 kb)
Supplementary Figure 2: Expression of a SOX9, b RUNX2 and c MMP13 in chondrocytes isolated from damaged cartilage exposed to 50µg/mL BCP crystals for 24h. Data shown are the pooled results from three separate patient donors (n=3) and are expressed as mean ± standard deviation. (TIF 46 KB)


  1. 1.
    Goldring MB (2012) Articular cartilage degradation in osteoarthritis. HSS J 8:7–9. CrossRefGoogle Scholar
  2. 2.
    van der Kraan PM, van den Berg WB (2012) Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration? Osteoarthritis Cartilage 20(3):223–232. CrossRefGoogle Scholar
  3. 3.
    Taschner MJ, Rafigh M, Lampert F, Schnaiter S, Hartmann C (2008) Ca2+/calmodulin-dependent kinase II signaling causes skeletal overgrowth and premature chondrocyte maturation. Dev Biol 317(1):132–146. CrossRefGoogle Scholar
  4. 4.
    Li Y, Ahrens MJ, Wu A, Liu J, Dudley AT (2011) Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes. Development 138(2):359–370. CrossRefGoogle Scholar
  5. 5.
    Jung GY, Park YJ, Han JS (2010) Effects of HA released calcium ion on osteoblast differentiation. J Mater Sci: Mater Med 21(5):1649–1654. Google Scholar
  6. 6.
    Sugita S, Hosaka Y, Okada K, Mori D, Yano F, Kobayashi H, Taniguchi Y, Mori Y, Okuma T, Chang SH, Kawata M, Taketomi S, Chikuda H, Akiyama H, Kageyama R, Chung U-i, Tanaka S, Kawaguchi H, Ohba S, Saito T (2015) Transcription factor Hes1 modulates osteoarthritis development in cooperation with calcium/calmodulin-dependent protein kinase 2. Proc Natl Acad Sci 112(10):3080–3085. CrossRefGoogle Scholar
  7. 7.
    Fuerst M, Bertrand J, Lammers L, Dreier R, Echtermeyer F, Nitschke Y, Rutsch F, Schafer FK, Niggemeyer O, Steinhagen J, Lohmann CH, Pap T, Ruther W (2009) Calcification of articular cartilage in human osteoarthritis. Arthritis Rheum 60(9):2694–2703. CrossRefGoogle Scholar
  8. 8.
    Nguyen C, Bazin D, Daudon M, Chatron-Colliet A, Hannouche D, Bianchi A, Come D, So A, Busso N, Busso N, Liote F, Ea HK (2013) Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage. Arthritis Res Ther 15(5):R103. CrossRefGoogle Scholar
  9. 9.
    Ea H-K, Chobaz V, Nguyen C, Nasi S, van Lent P, Daudon M, Dessombz A, Bazin D, McCarthy G, Jolles-Haeberli B, Ives A, Van Linthoudt D, So A, Lioté F, Busso N (2013) Pathogenic role of basic calcium phosphate crystals in destructive arthropathies. PLoS ONE 8(2):e57352. CrossRefGoogle Scholar
  10. 10.
    Halverson PB (1992) Arthropathies associated with basic calcium phosphate crystals. Scan Microsc 6(3):791–796 (discussion 796–797)Google Scholar
  11. 11.
    Nalbant S, Martinez JA, Kitumnuaypong T, Clayburne G, Sieck M, Schumacher HR Jr (2003) Synovial fluid features and their relations to osteoarthritis severity: new findings from sequential studies. Osteoarthritis Cartilage 11(1):50–54CrossRefGoogle Scholar
  12. 12.
    Fuerst M, Niggemeyer O, Lammers L, Schafer F, Lohmann C, Ruther W (2009) Articular cartilage mineralization in osteoarthritis of the hip. BMC Musculoskelet Disord 10:166. CrossRefGoogle Scholar
  13. 13.
    McCarthy GM, Christopherson PA, Mitchell PG (1997) Basic calcium phosphate crystals and tumor necrosis factor a induce matrix metalloprotease 13 (collagenase-3) in adult porcine articular chondrocytes. Arthritis Rheum 40(9):580–580Google Scholar
  14. 14.
    Mitchell PG, Struve JA, McCarthy GM, Cheung HS (1992) Basic calcium phosphate crystals stimulate cell proliferation and collagenase message accumulation in cultured adult articular chondrocytes. Arthritis Rheum 35(3):343–350. CrossRefGoogle Scholar
  15. 15.
    McCarthy GM, Westfall PR, Masuda I, Christopherson PA, Cheung HS, Mitchell PG (2001) Basic calcium phosphate crystals activate human osteoarthritic synovial fibroblasts and induce matrix metalloproteinase-13 (collagenase-3) in adult porcine articular chondrocytes. Ann Rheumatic Dis 60(4):399–406. CrossRefGoogle Scholar
  16. 16.
    McCarthy GM, Mitchell PG, Cheung HS (1991) The mitogenic response to stimulation with basic calcium-phosphate crystals is accompanied by induction and secretion of collagenase in human fibroblasts. Arthritis Rheum 34(8):1021–1030. CrossRefGoogle Scholar
  17. 17.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675CrossRefGoogle Scholar
  18. 18.
    Rachow JW, Ryan LM, McCarty DJ, Halverson PC (1988) Synovial fluid inorganic pyrophosphate concentration and nucleotide pyrophosphohydrolase activity in basic calcium phosphate deposition arthropathy and Milwaukee shoulder syndrome. Arthritis Rheum 31(3):408–413CrossRefGoogle Scholar
  19. 19.
    Shimazaki A, Wright MO, Elliot K, Salter DM, Millward-Sadler SJ (2006) Calcium/calmodulin-dependent protein kinase II in human articular chondrocytes. Biorheology 43(3–4):223–233Google Scholar
  20. 20.
    Caron MMJ, Emans PJ, Coolsen MME, Voss L, Surtel DAM, Cremers A, van Rhijn LW, Welting TJM (2012) Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthritis Cartilage 20(10):1170–1178. CrossRefGoogle Scholar
  21. 21.
    Dai Y, Wang H, Ogawa A, Yamanaka H, Obata K, Tokunaga A, Noguchi K (2005) Ca2+/calmodulin-dependent protein kinase II in the spinal cord contributes to neuropathic pain in a rat model of mononeuropathy. Eur J Neurosci 21(9):2467–2474. CrossRefGoogle Scholar
  22. 22.
    Liao M-H, Xiang Y-C, Huang J-Y, Tao R-R, Tian Y, Ye W-F, Zhang G-S, Lu Y-M, Ahmed MM, Liu Z-R, Fukunaga K, Han F (2013) The disturbance of hippocampal CaMKII/PKA/PKC phosphorylation in early experimental diabetes mellitus. CNS Neurosci Ther 19(5):329–336. CrossRefGoogle Scholar
  23. 23.
    Zhou X, Li J, Yang W (2014) Calcium/calmodulin-dependent protein kinase II regulates cyclooxygenase-2 expression and prostaglandin E2 production by activating cAMP-response element-binding protein in rat peritoneal macrophages. Immunology 143(2):287–299. CrossRefGoogle Scholar
  24. 24.
    Litherland GJ, Hui W, Elias MS, Wilkinson DJ, Watson S, Huesa C, Young DA, Rowan AD (2014) Glycogen synthase kinase 3 inhibition stimulates human cartilage destruction and exacerbates murine osteoarthritis. Arthritis Rheumatol 66(8):2175–2187. CrossRefGoogle Scholar
  25. 25.
    Miclea RL, Siebelt M, Finos L, Goeman JJ, Lowik CW, Oostdijk W, Weinans H, Wit JM, Robanus-Maandag EC, Karperien M (2011) Inhibition of Gsk3beta in cartilage induces osteoarthritic features through activation of the canonical Wnt signaling pathway. Osteoarthritis Cartilage 19(11):1363–1372. CrossRefGoogle Scholar
  26. 26.
    Kuhl M, Sheldahl LC, Malbon CC, Moon RT (2000) Ca2+/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J Biol Chem 275(17):12701–12711CrossRefGoogle Scholar
  27. 27.
    Nalesso G, Wagner K, Bertrand J, Sherwood JC, Thomas BL, Eldridge SE, Pitzalis C, Dell’Accio F (2016) Pharmacological blockade of CaMKII is detrimental for osteoarthritis progresssion. Osteoarthritis Cartilage 24:S149–S149CrossRefGoogle Scholar
  28. 28.
    Swulius MT, Waxham MN (2008) Ca2+/calmodulin-dependent protein kinases. Cell Mol Life Sci 65(17):2637–2657. CrossRefGoogle Scholar
  29. 29.
    Halverson PB, Greene A, Cheung HS (1998) Intracellular calcium responses to basic calcium phosphate crystals in fibroblasts. Osteoarthritis Cartilage 6(5):324–329. CrossRefGoogle Scholar
  30. 30.
    McCarthy GM, Cheung HS, Abel SM, Ryan LM (1998) Basic calcium phosphate crystal-induced collagenase production: role of intracellular crystal dissolution. Osteoarthritis Cartilage 6(3):205–213. CrossRefGoogle Scholar
  31. 31.
    Nguyen C, Lieberherr M, Bordat C, Velard F, Come D, Liote F, Ea HK (2012) Intracellular calcium oscillations in articular chondrocytes induced by basic calcium phosphate crystals lead to cartilage degradation. Osteoarthritis Cartilage 20(11):1399–1408. CrossRefGoogle Scholar
  32. 32.
    Malagodi MH, Chiou CY (1974) Pharmacological evaluation of a new Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8): studies in smooth muscles. Eur J Pharm 27(1):25–33. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Medicine, School of MedicineUniversity of AucklandAucklandNew Zealand
  2. 2.Department of Surgery, School of MedicineUniversity of AucklandAucklandNew Zealand
  3. 3.School of MedicineUniversity College DublinDublinIreland
  4. 4.Faculty of Medical & Health SciencesUniversity of AucklandAucklandNew Zealand

Personalised recommendations