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. 2001 Apr;60(4):399–406. doi: 10.1136/ard.60.4.399

Basic calcium phosphate crystals activate human osteoarthritic synovial fibroblasts and induce matrix metalloproteinase-13 (collagenase-3) in adult porcine articular chondrocytes

G McCarthy 1, P Westfall 1, I Masuda 1, P Christopherson 1, H Cheung 1, P Mitchell 1
PMCID: PMC1753595  PMID: 11247873

Abstract

OBJECTIVE—To determine the ability of basic calcium phosphate (BCP) crystals to induce (a) mitogenesis, matrix metalloproteinase (MMP)-1, and MMP-13 in human osteoarthritic synovial fibroblasts (HOAS) and (b) MMP-13 in cultured porcine articular chondrocytes.
METHODS—Mitogenesis of HOAS was measured by [3H]thymidine incorporation assay and counts of cells in monolayer culture. MMP messenger RNA (mRNA) accumulation was determined either by northern blot analysis or reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA from chondrocytes or HOAS treated with BCP crystals. MMP-13 secretion was identified by immunoprecipitation and MMP-1 secretion by western blot of conditioned media.
RESULTS—BCP crystals caused a 4.5-fold increase in [3H]thymidine incorporation by HOAS within 20 hours compared with untreated control cultures (p⩽0.05). BCP crystals induced MMP-13 mRNA accumulation and MMP-13 protein secretion by articular chondrocytes. In contrast, in HOAS, MMP-13 mRNA induced by BCP crystals was detectable only by RT-PCR, and MMP-13 protein was undetectable. BCP crystals induced MMP-1 mRNA accumulation and MMP-1 protein secretion by HOAS. MMP-1 expression was further augmented when HOAS were co-incubated with either BCP and tumour necrosis factor α (TNFα; threefold) or BCP and interleukin 1α (IL1α; twofold).
CONCLUSION—These data confirm the ability of BCP crystals to activate HOAS, leading to the induction of mitogenesis and MMP-1 production. MMP-13 production in response to BCP crystals is substantially more detectable in porcine articular chondrocytes than in HOAS. These data support the active role of BCP crystals in osteoarthritis and suggest that BCP crystals act synergistically with IL1α and TNFα to promote MMP production and subsequent joint degeneration.



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Figure 1  .

Figure 1  

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Basic calcium phosphate (BCP) crystal induced accumulation of matrix metalloproteinase-13 (MMP-13) messenger RNA (mRNA) in adult articular porcine chondrocytes. Time course. Confluent cultures of chondrocytes, in 60 mm plates, were incubated in serum-free media for 24 hours before being stimulated with BCP crystals (24 µg/cm2). At various times after treatment, cultures were harvested and total RNA was isolated. Northern blot analysis was performed using a specific probe for MMP-13, followed by autoradiography. The probe was then removed by washing and the blot was re-probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control. The positions of the 18S and 28S ribosomes are shown. B = BCP crystals; C = unstimulated control cultures.

Figure 2  .

Figure 2  

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Basic calcium phosphate (BCP) crystal induced stimulation of matrix metalloproteinase-13 (MMP-13) protein synthesis and secretion in adult articular porcine chondrocytes. One millilitre samples of media, conditioned for 24 or 48 hours in the presence of 50 µCi/ml of [35S]methionine, from confluent cultures of chondrocytes untreated or treated with various quantities of BCP crystals, were incubated with protein A-Sepharose and pre-immune rabbit serum at 4°C for one hour. Samples were processed as in "Materials and methods" and incubated overnight with 10 µl of MMP-13 antiserum. Samples were washed with immunoprecipitation buffer and examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by autoradiography. B = BCP crystals; C = unstimulated control cultures. Numbers to the left refer to the molecular weight in kDa.

Figure 3  .

Figure 3  

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Mitogenic effects of basic calcium phosphate (BCP) crystals in human osteoarthritic synovial fibroblasts. Confluent, quiescent cultures of synovial fibroblasts grown in 24 well plates were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 0.5% fetal bovine serum (FBS). Twenty four hours later, fresh DMEM containing either 0.5% control (C) or 10% FBS (FBS) or BCP crystals (BCP) (25 µg/cm2) was added. At the times indicated, cultures were pulse labelled with [3H]thymidine (1 µCi/ml) for one hour. The plates were then processed, and thymidine incorporation was determined as described in "Materials and methods". All values (SEM), n=4. Significant mitogenic response to BCP at 20, 22.5, and 25 hours compared with C (p⩽0.05).

Figure 4  .

Figure 4  

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Basic calcium phosphate (BCP) crystal induced accumulation of matrix metalloproteinase-1 (MMP-1) messenger RNA (mRNA) in human osteoarthritic synovial fibroblasts. Time course. Confluent cultures of synovial fibroblasts, in 100 mm plates, were incubated in 0.5% fetal bovine serum-Dulbecco's modified Eagle's medium for 24 hours before being stimulated with BCP crystals (18 µg/cm2). Control cultures were either untreated or treated with tumour necrosis factor α (10 ng/ml) or interleukin 1α (100 U/ml). At various times after treatment, cultures were harvested and total RNA was isolated. Northern blot analysis was performed using a specific probe for MMP-1, followed by autoradiography. The probe was then removed by washing and the blot was re-probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control. The positions of the 18S and 28S ribosomes are shown. C = unstimulated control cultures; B = BCP crystals; IL1 = interleukin 1α; TNF = tumour necrosis factor α.

Figure 5  .

Figure 5  

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Basic calcium phosphate (BCP) crystal induced stimulation of matrix metalloproteinase-1 (MMP-1) protein synthesis and secretion in human osteoarthritic synovial fibroblasts. Confluent, quiescent human osteoarthritic synovial fibroblasts incubated in Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum (FBS) were stimulated with either BCP crystals (18 µg/cm2), interleukin 1α (100 U/ml) or tumour necrosis factor α (10 ng/ml). Untreated cultures were in 0.5% FBS. The culture medium was analysed by western blotting using a specific monoclonal antibody to MMP-1. Numbers refer to hours after treatment. Molecular weight markers are shown to the left. C = unstimulated control cultures; B = BCP crystals; IL1 = interleukin 1α; TNF = tumour necrosis factor α.

Figure 6  .

Figure 6  

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Basic calcium phosphate (BCP) crystal induced stimulation of matrix metalloproteinase-1 (MMP-1) protein synthesis and secretion in human osteoarthritic synovial fibroblasts (HOAS). Affect of co-incubation with tumour necrosis factor α (TNFα) or interleukin 1α (IL1α). Confluent, quiescent HOAS incubated in Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum (FBS) were stimulated with either BCP crystals (18 µg/cm2) and IL1α (100 U/ml) or TNFα (10 ng/ml). For comparison, some cultures were treated with both IL1α and TNFα. Untreated cultures were in 0.5% FBS. The culture medium was analysed by western blotting using a specific monoclonal antibody to MMP-1. Numbers refer to hours after treatment. Molecular weight markers are shown to the left. C = unstimulated control cultures; B = BCP crystals.

Figure 7  .

Figure 7  

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Reverse transcriptase-polymerase chain reaction (RT-PCR) determination of matrix metalloproteinase-13 (MMP-13) mRNA in human osteoarthritic synovial fibroblasts. Total RNA was isolated from synovial fibroblasts and subjected to RT-PCR analysis for MMP-13 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR amplification was performed for 40 cycles as described in "Materials and methods". Numbers refer to time in hours after treatment with BCP crystals (18 µg/cm2), tumour necrosis factor α (TNFα; 10 ng/ml), or interleukin 1α (IL1α; 100 U/ml).

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