Promotion of articular cartilage matrix vesicle mineralization by type I collagen

Abstract

Objective: Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals occur in up to 60% of osteoarthritic joints and predict an increased severity of arthritis. Articular cartilage vesicles (ACVs) generate CPPD crystals in the presence of ATP and BCP crystals with added beta-glycerophosphate. While ACVs are present in normal articular cartilage, they mineralize primarily in cartilage from osteoarthritic joints. The aim of this study was to explore the hypothesis that ACV mineralization is regulated by components of the surrounding extracellular matrix.

Methods: Porcine ACVs were embedded in agarose gels containing type II and/or type I collagen and/or proteoglycans. Mineralization was measured as (45)Ca accumulation stimulated by ATP or beta-glycerophosphate and reflects both nucleation and growth. Synthetic CPPD and BCP crystals were embedded in similar gels to isolate the effect of matrix components on crystal growth.

Results: After establishing baseline responsiveness of ACVs to ATP and beta-glycerophosphate in agarose gels, we examined the ability of ATP and beta-glycerophosphate to stimulate mineral formation in gels containing various matrix components. Type II collagen suppressed the ability of ATP to stimulate mineralization, while a combination of type II plus type I collagen increased the effect of ATP and beta-glycerophosphate on mineralization. Type I collagen affected ACV mineralization in a dose-responsive manner. Neither type of collagen significantly affected crystal growth or levels of mineralization-regulating enzymes. Proteoglycans suppressed mineral formation by ACVs in gels containing both type I and type II collagen.

Conclusion: Cartilage matrix changes that occur with osteoarthritis, such as increased quantities of type I collagen and reduced proteoglycan levels, may promote ACV mineralization.

Figure 1.

Generation of both calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals by articular cartilage vesicles (ACVs). ACVs were incubated for 5 days in agarose gels with either 1 mM ATP or 1 mM β-glycerophosphate (B-GP). The crystals were isolated and analyzed by synchrotron Fourier transform infrared spectroscopy (FTIR) microscopy. Representative results are shown. In the upper portion of the figure, the thick line represents the FTIR spectrum of the crystal shown in the inset at the upper left (ACVs incubated with β-glycerophosphate), as compared with a BCP crystal standard (thin line). In the lower portion of the figure, the thick line represents the FTIR spectrum of the crystal shown in the inset at the middle left (ACVs incubated with ATP), as compared with a monoclinic CPPD (M-CPPD) crystal standard (thin line). The area containing the characteristic CPPD peaks is enlarged and is shown at the middle right as ×2.

Figure 2.

Effect of heat treatment on mineralization of articular cartilage vesicles (ACVs). ACVs were heat-treated (121°C with 15 pounds of pressure for 15 minutes) or were left untreated. Untreated and heat-treated ACVs were embedded in agarose gels and incubated for 5 days in calcifying salt solution that had been trace-labeled with⁴⁵Ca alone (solid bars) or with 1 mM ATP (shaded bars) or 1 mM β-glycerophosphate (hatched bars). Levels of⁴⁵Ca in ACVs were measured by liquid scintigraphy. Values are the mean and SD (n = 6 samples). Heat treatment decreased the total mineral formation and abolished the ability of ACVs to mineralize in the presence of ATP or β-glycerophosphate (P < 0.0001).

Figure 3.

Effect of type II collagen, type II plus type I collagen, and proteoglycans (PGs) on the growth of calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals. Synthetic CPPD (solid bars) or BCP (hatched bars) crystals were embedded in agarose gels (control) or gels containing type II collagen (CII), type II plus type I collagen (CII + CI), with or without proteoglycans. CPPD crystal–containing gels were incubated with calcifying salt solution (CSS) containing⁴⁵Ca and pyrophosphate, while BCP crystal–containing gels were incubated with CSS containing⁴⁵Ca and phosphate. After 5 days of incubation,⁴⁵Ca in the crystal fraction was measured. Values are the mean and SD (n = 10 samples).

Figure 4.

Effect of various concentrations of type I and type II collagen on mineralization of articular cartilage vesicles (ACVs). ACVs were treated with various doses of type II collagen (A) or type I collagen (B) in⁴⁵Ca-containing calcifying salt solution, either alone (solid bars), with ATP (shaded bars), or with β-glycerophosphate (hatched bars). Controls included agarose without added collagen.⁴⁵Ca accumulation was measured by liquid scintigraphy. Values are the mean and SD (n = 6 samples). Type II collagen inhibited calcium pyrophosphate dihydrate (CPPD) crystal formation (P < 0.003), whereas type I collagen stimulated both CPPD and basic calcium phosphate crystal formation (P < 0.001). Levels of type II and type I collagen in ACVs were measured by Western blotting (insets). In the Western blots, lane 1 shows positive controls for type II collagen in A and for type I collagen in B; lane 2 shows ACVs.

Figure 5.

Collagen and proteoglycan binding proteins on articular cartilage vesicles (ACVs). Proteins from ACVs were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes. Western blots were performed with antibodies to annexin V (A), CD44 (B), and link protein (C). Expected molecular weights for these proteins are shown at the left of each blot.