BackgroundIt is believed that phosphocitrate (PC) exerts its disease-modifying effects on osteoarthritis (OA) by inhibiting the formation of crystals. However, recent findings suggest that PC exerts its disease-modifying effect, at least in part, through a crystal-independent action. This study sought to examine the disease-modifying effects of PC and its analogue PC-β-ethyl ester (PC-E) on partial meniscectomy-induced OA and the structure-activity relationship.MethodsCalcification- and proliferation-inhibitory activities were examined in OA fibroblast-like synoviocytes (FLSs) culture. Disease-modifying effects were examined using Hartley guinea pigs undergoing partial meniscectomy. Cartilage degeneration was examined with Indian ink, safranin-O, and picrosirius red. Levels of matrix metalloproteinase-13 (MMP-13), ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5), chemokine (C-C motif) ligand 5 (CCL5), and cyclooxygenase-2 (Cox-2) were examined with immunostaining. The effects of PC-E and PC on gene expressions in OA FLSs were examined with microarray. Results are expressed as mean ± standard deviation and analyzed using Student’s t test or Wilcoxon rank sum test.ResultsPC-E was slightly less powerful than PC as a calcification inhibitor but as powerful as PC in the inhibition of OA FLSs proliferation. PC significantly inhibited cartilage degeneration in the partial meniscectomied right knee. PC-E was less powerful than PC as a disease-modifying drug, especially in the inhibition of cartilage degeneration in the non-operated left knee. PC significantly reduced the levels of ADAMTS5, MMP-13 and CCL5, whereas PC-E reduced the levels of ADAMTS5 and CCL5. Microarray analyses revealed that PC-E failed to downregulate the expression of many PC-downregulated genes classified in angiogenesis and inflammatory response.ConclusionsPC is a disease-modifying drug for posttraumatic OA therapy. PC exerts its disease-modifying effect through two independent actions: inhibiting pathological calcification and modulating the expression of many genes implicated in OA. The β-carboxyl group of PC plays an important role in the inhibition of cartilage degeneration, little role in the inhibition of FLSs proliferation, and a moderate role in the inhibition of FLSs-mediated calcification.

Background:Chondrocytes have been traditionally thought to be responsible for calcium crystal deposits within osteoarthritic knees. Increasing recent experimental evidence suggests that menisci may also play a role. However, the calcifying potential of chondrocytes and meniscal cells derived from same OA patients, and the genes associated with meniscal calcification have never been fully examined.Objective:Examine and compare the calcifying potential of articular chondrocytes and meniscal cells derived from same OA patients and identify the calcium crystal type(s) and selected gene expression in OA menisci.Methods:Chondrocytes and meniscal cells were isolated from articular cartilage and menisci of OA patients undergoing total knee arthroplasty. Chondrocyte- and meniscal cell-mediated calcification was examined using both monolayer and micromass culture-based assays. Crustal types were examined with histological staining. Levels of Type X Collagen, MMP-13, and ANKH in OA menisci were examined using immunohistochemistry.Results:Primary human OA meniscal cells produced calcified deposits at a similar rate compared to OA chondrocytes in-vitro. Histological examinations indicate that both BCP crystals and CPPD crystals are present in the meniscal tissue. Type X collagen, MMP-13, and ANKH were found in human OA menisci and their levels increased with OA severity. In addition, type X collagen was co-localized with calcium crystals.Conclusion:These findings suggest that OA meniscal cells have a similar calcifying potential as OA chondrocytes, supporting a pathogenic role of OA menisci in OA.

Phosphocitrate (PC) inhibited meniscal calcification and the development of calcium crystal-associated osteoarthritis (OA) in Hartley guinea pigs. However, the mechanisms remain elusive. This study sought to examine the biological activities of PC in the absence of calcium crystals and test the hypothesis that PC is potentially a meniscal protective agent. We found that PC downregulated the expression of many genes classified in cell proliferation, ossification, prostaglandin metabolic process, and wound healing, including bloom syndrome RecQ helicase-like, cell division cycle 7 homolog, cell division cycle 25 homolog C, ankylosis progressive homolog, prostaglandin-endoperoxide synthases-1/cyclooxygenase-1, and plasminogen activator urokinase receptor. In contrast, PC stimulated the expression of many genes classified in fibroblast growth factor receptor signaling pathway, collagen fibril organization, and extracellular structure organization, including fibroblast growth factor 7, collagen type I, alpha 1, and collagen type XI, alpha 1. Consistent with its effect on the expression of genes classified in cell proliferation, collagen fibril organization, and ossification, PC inhibited the proliferation of OA meniscal cells and meniscal cell-mediated calcification while stimulating the production of collagens. These findings indicate that PC is potentially a meniscal-protective agent and a disease-modifying drug for arthritis associated with severe meniscal degeneration.

Phosphocitrate inhibits cartilage degeneration, however, the prospect of phosphocitrate as an oral disease modifying drug might be limited. The purpose of this study was to investigate the biological effects and disease-modifying activity of a phosphocitrate "analog," CM-01 (Carolinas Molecule-01), and test the hypothesis that CM-01 is a disease modifying drug for osteoarthritis therapy. The effects of CM-01 on calcium crystal-induced expression of matrix metalloproteinase-1 and interleukin-1 beta, cell-mediated calcification and production of proteoglycan by chondrocytes were examined in cell cultures. Disease-modifying activity was examined using Hartley guinea pig model of posttraumatic osteoarthritis. Cartilage degeneration in untreated and CM-01 treated guinea pigs was examined with Indian ink and Safranin-O-fast green. Levels of matrix metalloproteinase-13, ADAM metallopeptidase with thrombospondin type 1 motif 5, chemokine (C-C motif) ligand 5, and cyclooxygenase 2 were examined with immunostaining. CM-01 inhibited crystal-induced expression of matrix metalloproteinase-1 and interleukin-1β, reduced cell-mediated calcification, and stimulated the production of proteoglycan by chondrocytes. In Hartley guinea pigs, CM-01 not only reduced damages in articular surface but also reduced resorption of calcified zone cartilage. The reduction in cartilage degeneration was accompanied by decreased levels of matrix metalloproteinase-13, ADAM metallopeptidase with thrombospondin type 1 motif 5, chemokine (C-C motif) ligand 5 and cyclooxygenase 2. These findings confirmed that CM-01 is a promising candidate to be tested as an oral drug for human OA therapy. CM-01 exerted its disease-modifying activity on osteoarthritis, in part, by inhibiting the production of matrix-degrading enzymes and a molecular program resembling the endochondral pathway of ossification. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:309-317, 2018.

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