Calcium deposition in osteoarthritic meniscus and meniscal cell culture

Abstract

Introduction: Calcium crystals exist in the knee joint fluid of up to 65% of osteoarthritis (OA) patients and the presence of these calcium crystals correlates with the radiographic evidence of hyaline cartilaginous degeneration. This study sought to examine calcium deposition in OA meniscus and to investigate OA meniscal cell-mediated calcium deposition. The hypothesis was that OA meniscal cells may play a role in pathological meniscal calcification.

Methods: Studies were approved by our human subjects Institutional Review Board. Menisci were collected during joint replacement surgeries for OA patients and during limb amputation surgeries for osteosarcoma patients. Calcium deposits in menisci were examined by alizarin red staining. Expression of genes involved in biomineralization in OA meniscal cells was examined by microarray and real-time RT-PCR. Cell-mediated calcium deposition in monolayer culture of meniscal cells was examined using an ATP-induced (45)calcium deposition assay.

Results: Calcium depositions were detected in OA menisci but not in normal menisci. The expression of several genes involved in biomineralization including ENPP1 and ANKH was upregulated in OA meniscal cells. Consistently, ATP-induced calcium deposition in the monolayer culture of OA meniscal cells was much higher than that in the monolayer culture of control meniscal cells.

Conclusions: Calcium deposition is common in OA menisci. OA meniscal cells calcify more readily than normal meniscal cells. Pathological meniscal calcification, which may alter the biomechanical properties of the knee meniscus, is potentially an important contributory factor to OA.

Figure 1

Normal and osteoarthritis menisci, stained with alizarin red. Menisci were derived from (a) a 39-year-old female osteosarcoma patient, (b) a 43-year-old male osteosarcoma patient, (c) a 42-year-old male osteoarthritis (OA) patient, and (d) a 49-year-old female OA patient. The normal menisci exhibited a smooth, white and glistening surface, with no signs of degeneration (a and b). OA menisci showed discoloration and a rough surface. Degeneration was apparent (c and d). (e), (f) There were no calcium depositions in the normal menisci; (g) to (j) calcium deposition was present in all OA menisci. Representative images of grade 0 (e and f), grade 1 (g), grade 2 (h), grade 3 (i) and grade 4 (j) of alizarin red staining are shown.

Figure 2

ATP-induced calcium deposition. ATP-induced calcium deposition in monolayer cultures of osteoarthritis (OA) meniscal cells derived from five OA patients (right-hand group) was significantly higher than that in the monolayer cultures of normal control meniscal cells derived from three osteosarcoma patients (left-hand group). The difference between the two groups was statistically significant (*P < 0.005). Count per minute (CPM) data were normalized against total protein levels.

Figure 3

Comparison of osteoarthritis meniscal cell-mediated and osteoarthritis chondrocyte-mediated calcium deposition. (a) ATP-induced calcium deposition in the monolayer cultures of osteoarthritis (OA) meniscal cells derived from four OA patients (OA-M) (see Table 3) was 60% greater than present in the monolayer cultures of OA hyaline articular chondrocytes (OA-C). The difference was statistically significant (*P < 0.05). (b) Calcium phosphocitrate (CaPC) inhibited ATP-induced calcium deposition in the monolayer cultures of OA meniscal cells in a dose-dependent manner (*P < 0.001). Count per minute (CPM) data were normalized against total protein levels.