Regulated Production of Mineralization-competent Matrix Vesicles in Hypertrophic Chondrocytes

Cell Culture

Chondrocytes were isolated from the hypertrophic region of day 19 embryonic chick tibia growth plate cartilage by trypsin followed by collagenase digestion as described previously (49). Cells were plated at an initial density of 3 × 10⁶ cells into 100-mm tissue culture dishes in Dulbecco's modified high glucose Eagle's medium (HG-DMEM; GIBCO BRL, Gaithersburg, MD) containing 10% defined fetal bovine serum (Hyclone, Logan, UT), 2 mM l-glutamine, and 50 U/ml penicillin and streptomycin (complete medium). Chondrocytes reached confluency in ∼7 d. Once confluent, cells were treated with 50 μg/ml of freshly prepared vitamin C and 2.5 mM phosphate. Medium was changed daily.

RNA Isolation and Analysis

Total RNAs were isolated from different zones of tibia growth plate cartilage from day 19-old chick embryos and from cultured cells by the method of Chomczynski and Sacchi (17). For Northern blot analysis, 10 μg of total RNA was denatured by glyoxalation, fractionated on 1% agarose gels, and transferred to Hybond-N membranes by capillary blotting as described previously (46, 50). Blots were stained with 0.04% methylene blue to verify that each sample has been transferred efficiently. Blots were hybridized in 6× SSC, 5× Denhardt's solution, 100 μg/ml sheared salmon sperm DNA, 2% SDS, and 50% formamide at 45°C overnight with ³²P-labeled cDNA probes. The cDNA clones used to prepare probes for type X collagen, annexin V, and type I collagen mRNAs are pDLr10 (40), pACII.1 (52), and pCOL3 (72), respectively. Blots were washed at high stringency (0.1× SSC, 0.1% SDS at 60°C) and exposed to Kodak X-ray films at −70°C.

Immunofluorescence Staining and Alizarin Red S Staining

This procedure was carried out as previously described (33, 35). Briefly, longitudinal 8-μm-thick frozen sections from day 19 chick embryonic tibia growth plate cartilage were fixed for 10 min in ice-cold acetone. Before immunostaining, the fixed sections were decalcified with 0.1 M EDTA, pH 7.4, for 1 h and pretreated with sheep testicular hyaluronidase (2 mg/ml; Sigma Chemical Co., St. Louis, MO) for 30 min at 37°C. Cells were washed with PBS, pH 7.4, and fixed with 70% ethanol. The sections or fixed cells were incubated with a mixture of monoclonal mouse anti-chick type I collagen IgG and polyclonal rabbit anti–chick type X collagen IgG or a mixture of monoclonal mouse anti–chick type I collagen IgG and polyclonal rabbit anti–chick annexin V IgG for 3 h at room temperature, followed by incubation with a mixture of rhodamine-conjugated goat anti– mouse IgG and FITC-conjugated goat anti–rabbit IgG (Cappel, Durham, NC) for 1 h at room temperature.

To localize calcium deposits, sections were stained with 0.5% alizarin red S solution, pH 4.0, for 5 min at room temperature. To verify that alizarin staining was specific, sections were decalcified and then stained with alizarin red S. Stained sections were washed three times with water and ethanol. Sections and cells were examined under a Zeiss microscope and viewed in both epifluorescence and phase modes.

Isolation of Matrix Vesicles and Ca²⁺ Uptake Studies

Matrix vesicles were isolated from cultured chondrocytes by enzymatic digestion as described previously (20, 34). Briefly, adherent chondrocytes were washed with PBS and then incubated in PBS containing 0.1% trypsin (type III; Sigma Chemical Co.) at 37°C for 30 min. The trypsin was removed by washing with PBS, and the cells were then treated with crude collagenase (500 U/ml; type IA; Sigma Chemical Co.) at 37°C for 3 h. Matrix vesicles were harvested by differential ultracentrifugation as described previously (20). Three dishes (100 mm) of confluent chondrocyte cultures were used to isolate matrix vesicle fractions containing between 300 and 500 μg of total proteins. For Ca²⁺ uptake studies, matrix vesicles (50 μg of total proteins) were sedimented by centrifugation and resuspended in 500 μl of synthetic cartilage lymph; synthetic cartilage lymph, pH 7.4, contained 2 mM Ca²⁺ and 1.42 mM P_i, in addition to 104.5 mM Na⁺, 133.5 mM Cl⁻, 63.5 mM sucrose, 16.5 mM Tris, 12.70 mM K⁺, 5.55 mM d-glucose, 1.83 mM HCO₃₋, 0.57 mM Mg²⁺, and 0.57 mM SO₄ ²⁻. The matrix vesicle suspension was incubated at 37°C, centrifuged, and resuspended in 50 μl of 0.1 M HCl. Aliquots of the suspensions were analyzed for calcium using the microcolorimetric method of Baginski et al. (11).

Fourier Transform Infrared Spectroscopy

Matrix vesicle samples were freeze dried and stored desiccated. Each sample was analyzed by Fourier transform infrared spectroscopy (FT-IR; Magna IR 550 spectrometer; Nicolet Instrument Technologies, Madison, WI), operated in the diffuse reflectance mode. Vesicle preparations were milled in an agate mortar and layered on KBr (ratio of KBr to sample, 300:1, wt/ wt); routinely, 300 interferograms were collected at 4 cm⁻¹ resolution; background due to organic matrix was subtracted using a chondrocyte membrane preparation. Spectra were coadded and the resultant interferograms Fourier transformed, and second derivative spectra (1,200–500 cm⁻¹) were obtained using a software package (Omnic; Nicolet Instrument Technologies).

Electron Microscopy

Matrix vesicles were sedimented by centrifugation, and the resulting pellets were fixed in Karnovsky's fixative, postfixed in 2% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in epoxy resin. Ultrathin sections were cut on an Ultratome (LKB Instruments, Inc., Stockholm, Sweden), picked up on formvar-coated grids, and contrasted with uranyl acetate and lead citrate. Specimens were evaluated in a transmission electron microscope (100CX II; JEOL USA, Peabody, MA) operated at 80 kV.

To analyze matrix vesicle preparations for calcium and sulfur, freezedried matrix vesicle pellets were mounted on SEM specimen stubs and coated with carbon. The samples were analyzed using a scanning electron microscope (T330A; JEOL USA) and EDS microanalysis system (Delta I; Kevex Corp., Foster City, CA).

Dot Blot Analysis

To analyze for cartilage proteoglycan (aggrecan) in matrix vesicle preparations, aliquots of matrix vesicle fractions (2 μg of total protein) were dotted onto nitrocellulose filters. After blocking with low fat milk powder the filters were immunostained with polyclonal antibodies against chicken aggrecan followed by secondary horseradish-conjugated anti–rabbit IgG (Rockland Inc., Gilbertsville, PA) and α-chloronaphthol as color substrate. The optical density of the color reaction was determined using a densitometer. To determine the amount of aggrecan in the matrix vesicle fractions, purified aggrecan in different concentrations (1–20 μg) was applied onto nitrocellulose filters and immunostained with the aggrecan antibodies.

Antibodies

The preparation and specificity of a polyclonal antibody against chicken annexin V were described elsewhere (42). The preparations of antibodies against chick aggrecan and types X, II, and I collagen were described elsewhere (1, 23, 38, 47). These antibodies have been shown to be specific and do not cross-react with other collagens or matrix proteins (1, 23, 38, 47).

SDS–polyacrylamide Gel Electrophoresis and Immunoblotting

Samples were dissolved in 3% SDS sample buffer with dithiothreitol, denatured at 100°C for 3 min, and analyzed by electrophoresis in 8 or 10% (wt/vol) polyacrylamide gels. Gels were stained with Coomassie blue, or proteins were electroblotted onto nitrocellulose filters (Schleicher & Schuell, Inc., Keene, NH). After blocking with low fat milk protein, blotted proteins were immunostained with the appropriate antibodies using peroxidase-conjugated secondary antibodies and α-chloronaphthol as a color substrate.

Other Methods

To measure the protein content and alkaline phosphatase activity in the cell layer, cells were washed in PBS and then suspended in 10 mM Tris/ HCl, pH 7.5, 0.1% Triton X-100, 0.5 mM MgCl₂. Protein content was analyzed by the BCA assay from Pierce (Rockford, IL). Alkaline phosphatase activity was determined using p-nitrophenyl phosphate as substrate (63). To measure calcium and phosphate levels, the suspension was centrifuged, and the pellet was resuspended in 0.1 M HCl. Inorganic phosphate was determined by a modified method of Ames (4).

Calcification and Production of Matrix Vesicles in Chondrocyte Cultures

In a first series of experiments we investigated whether mineralization in chondrocyte cultures and the formation of matrix vesicles were affected by vitamin C and phosphate treatment. Chondrocytes isolated from the hypertrophic zone of day 19 embryonic chick tibia growth plate cartilage were cultured in control medium or medium containing 50 μg/ml vitamin C and 2.5 mM phosphate. After 14 d, mineralization in vitamin C/phosphate-treated chondrocyte cultures was observed (Fig. 1 B); bright field microscopy failed to show mineral in the matrix of untreated chondrocytes (Fig. 1 A). Vitamin C increased the activity of alkaline phosphatase; in contrast, phosphate did not stimulate alkaline phosphatase activity (Fig. 2 A). Cultures supplemented with vitamin C or phosphate led to a 4-fold elevation in the calcium and phosphate content of the cell layer; together both reagents led to a more than 12-fold increase in ion accumulation (Fig. 2, B and C). After 7 d, little calcification was evident in vitamin C and phosphatesupplemented cultures (data not shown).

Since the vitamin C and phosphate treatment significantly increased the rate and extent of calcification of chondrocyte cultures, we determined next whether this treatment affected the composition of matrix vesicles. Matrix vesicles were isolated from the cell layers of day 14 control nonmineralizing and vitamin C/phosphate-treated mineralizing cultures by mild trypsin and collagenase digestion followed by ultracentrifugation. Table I summarizes the general composition of the vesicles. The total protein content of vesicles from control cultures was about half that of vesicles from mineralizing cultures. Most important was the finding that the alkaline phosphatase activity and the Ca²⁺ and P_i content of the vesicles from mineralizing cultures were over 300- and 10-fold higher than those in vesicles from nonmineralizing control cells, respectively (Table I).

Given the quite different characteristics of vesicles from control and mineralizing cultures, we asked whether the vesicles would also differ in their ability to take up Ca²⁺. Vesicles isolated from control cultures showed no significant Ca²⁺ uptake after 24 h incubation in synthetic cartilage lymph (2% of the total amount of Ca²⁺ present in the reaction mixture), while vesicles isolated from mineralizing cultures accumulated ∼30% of the total amount of Ca²⁺ in the reaction mixture (Table I). Similar rates of ∼30% Ca²⁺ uptake over 24 h test period were also found in matrix vesicles isolated from calcified cartilage (30, 34).

Ca²⁺ uptake by matrix vesicles is probably mediated by annexin V (30, 34, 59); in addition, annexin V mediates binding of types II and X collagen to matrix vesicles (32, 34, 68). Binding of these collagens to matrix vesicles stimulates Ca²⁺ uptake by these particles (30, 34). Thus, we asked whether the differences in Ca²⁺ uptake seen above reflected differences in annexin V and types II and X collagen content in the vesicles. Vesicle samples were subjected to SDS-PAGE and immunoblotting analyses. Fig. 3 (lanes b and d, control matrix vesicles; lanes a and c, mineralizing matrix vesicles) shows that there were several major differences in the protein profile of vesicles from control (Fig. 3, lane b) versus mineralizing cultures (Fig. 3, lane a). For example, a major band with an apparent molecular mass of 120 kD was present in control vesicles (Fig. 3, lane b) but absent in mineralizing vesicles (lane a). Immunoblot analysis using antibodies against annexin V revealed that indeed only matrix vesicles from mineralizing chondrocytes contained annexin V (Fig. 3, lane c), while vesicles from control cultures showed no appreciable amounts of this protein (Fig. 3, lane d). Immunoblot analyses using antibodies against types I, II, and X collagen revealed that only mineralizing matrix vesicles contained types II (Fig. 4, lane a) and X collagen (Fig. 4, lane c), while no immunostaining was obtained in control matrix vesicles (Fig. 4, lane b and d). No appreciable amounts of type I collagen were detected in both mineralizing (Fig. 4, lane e) and control matrix vesicles (Fig. 4, lane f).

Ultrastructural analysis revealed that matrix vesicles freshly isolated from vitamin C/phosphate-treated cultures varied in size, shape, and content. In general, they were round to oval with diameters ranging from 30 to 100 nm. A few preformed crystallites were observed in these freshly isolated matrix vesicles (Fig. 5 A). After 24 h incubation in synthetic cartilage lymph at 37°C, numerous needle-like crystals were formed in association with matrix vesicles (Fig. 5 B); by 48 h, these vesicle preparations were heavily calcified (Fig. 5 C). Strikingly, no needle-like crystals were formed in association with matrix vesicles isolated from control cultures (Fig. 5 D).

To determine the nature of the crystals, vesicle samples incubated in synthetic lymph for 24 h were analyzed by Fourier transform infrared spectroscopy. Fig. 6 A shows the second derivative spectra of the ν₁ν₃ phosphate region (1,200–950 cm⁻¹) of matrix vesicle preparations obtained from mineralizing cultures. Peaks can be seen at 960, 1,025, 1,102, 1,065, and 1,157 cm⁻¹, consistent with apatite structure. In contrast, the spectra of the ν₁ν₃ region of control vesicles exhibited no such peaks but rather a nondescript pattern consistent with lack of a mineral phase (Fig. 6 C). Fig. 6, B and D shows the second derivative structure in the ν₄ domains (640–500 cm⁻¹). Peaks at 626 and 539 cm⁻¹, also characteristic of HPO₄ apatites, were clearly evident in mineralizing vesicle samples (Fig. 6 B) but were markedly reduced in control samples (Fig. 6 D). Both samples contained prominent bands at 561, 576, and 602 cm⁻¹ which are representative of phosphate ions (Fig. 6, B and D).

Previous studies have suggested that proteoglycans are involved in the nucleation of cartilage mineralization (25, 26, 53). Thus, it is possible that the different calcification properties of matrix vesicles isolated from control and mineralizing chondrocyte cultures may be due to differences in vesicle-associated proteoglycan content. To analyze this possibility we carried out two quantitative analyses of proteoglycan content. In the first set of experiments, aliquots of the matrix vesicle preparations (2 μg of total protein) were dotted onto nitrocellulose filters and immunostained with antibodies against the cartilage proteoglycan aggrecan (Fig. 7 B); as a control, purified aggrecan at concentrations from 1 to 20 μg/dot was also applied to the filters (Fig. 7 A). Vesicle preparations from both control and vitamin C/phosphate-treated cultures exhibited immunostaining for aggrecan (Fig. 7 B). However, densitometric analysis revealed that similar amounts of aggrecan were present in both preparations (192 μg/mg total protein in vesicles from control cultures; 141 μg/mg total protein in vesicles from mineralizing cultures). In a second set of experiments we analyzed the sulfur and calcium content in the vesicle preparations using scanning electron microscopy and EDS microanalysis. A similar sulfur content was observed in vesicles from control cultures (Fig. 7 C, peak labeled with S) and mineralizing cultures (Fig. 7 D, peak labeled with S). Interestingly, strong calcium peaks were only detected in vesicles from mineralizing cultures (Fig. 7 D, peaks labeled with Ca) but were absent in vesicles from control cultures (Fig. 7 C). In addition, mineralizing matrix vesicles contained more phosphate than control vesicles (Fig. 7, C and D, peaks labeled with P). These findings indicate that differences in proteoglycan content cannot account for the differences in mineralizing properties of vesicles produced by control and mineralizing chondrocytes.

Annexin V and Type I Collagen Expression

We asked next whether the presence of annexin V-rich, mineralization-competent matrix vesicles in mineralizing cultures reflected increased gene expression of this protein. We also asked whether possible changes in annexin V expression may be accompanied by changes in type I collagen expression, given the possible prominent role of this matrix protein in mineral propagation (9, 10, 13, 36).

Chondrocytes were cultured for 7 and 14 d in the absence or presence of vitamin C and phosphate, and total cellular RNAs were then isolated and examined by Northern blot analysis. Indeed, annexin V gene expression increased several fold in the vitamin C/phosphate-treated cultures already by day 7 (Fig. 8, Day 7, AV) and remained high by day 14 (Fig. 8, Day 14, AV). Likewise, gene expression of type X collagen, a marker of hypertrophic chondrocytes, was also markedly stimulated (Fig. 8, α1(X)). Interestingly at day 14, vitamin C/phosphate-treated cultures exhibited high levels of type I collagen expression compared to control cultures (Fig. 8, Day 14, α1(I)), while only low levels of type I collagen expression were detected after 7 d of treatment (Fig. 8, Day 7, α1(I)). Thus, during induction of mineralization in chondrocyte cultures, expression of annexin V and type X collagen genes increases first and is followed by increased expression of type I collagen.

To ascertain whether the above changes in gene expression were accompanied by corresponding changes at the protein level, cultures were immunostained with antibodies against annexin V, type X collagen, or type I collagen. Control cultures were immunopositive for type X collagen but exhibited no staining for type I collagen on day 7 (not shown) and day 14 (Fig. 9, B and C), even though low levels of α1(I) mRNA were detectable on day 14 (Fig. 8, Day 14, α1(I)); the control cells were also immunopositive for annexin V (Fig. 9 A). Cultures treated with vitamin C and phosphate were strongly immunopositive for annexin V and type X collagen on day 7, while only a few cells were positive for type I collagen (Fig. 9, D–F). After 14 d of treatment, the cultures not only exhibited strong annexin V and type X collagen immunoreactivity (Fig. 9, G and H) but also strong type I collagen immunoreactivity, resulting in >90% of the cells being surrounded by a type I collagen-rich matrix (Fig. 9 I).

Annexin V and Type I Collagen Expression in Tibia Growth Plate Cartilage

Our in vitro studies above show that chondrocytes undergoing mineralization first upregulate annexin V gene expression followed by onset of type I collagen expression and matrix calcification. We determined next whether a similar sequence of events occurs in vivo. To address this question we examined annexin V and type I collagen gene expression in the developing chick tibia growth plate cartilage in vivo by Northern blots and immunohistochemistry.

The proliferative, upper and lower hypertrophic zones of tibia growth plate cartilage were microsurgically isolated from day 19 chick embryos; the resulting tissue fragments were processed for RNA isolation and Northern blot analyses. We found that the upper hypertrophic zone contained large amounts of annexin V transcripts while minimal levels were present in the proliferative and lower hypertrophic zones (Fig. 10, AV). In contrast, mRNA encoding type I collagen was undetectable in the proliferative and upper hypertrophic zones but was extremely prominent in the lower hypertrophic zone (Fig. 10, α1(I)). To ascertain that the different growth plate zones had been microdissected accurately, blots were hybridized with a type X collagen cDNA probe. The upper hypertrophic cartilage exhibited large amounts of type X collagen transcripts, while no detectable type X collagen transcripts were found in the proliferative zone, and minimal levels were present in the lower hypertrophic zone (Fig. 10, α1(X)).

To ascertain if type I collagen mRNA is expressed by chondrocytes, we used immunostaining to localize type X collagen, type I collagen, and annexin V in embryonic tibia growth plate cartilage. Double immunostaining using antibodies against types X and I collagen revealed that type I collagen was restricted to a narrow zone of chondrocytes that bordered the chondro–osseous junction, which also showed strong immunostaining for type X collagen. In contrast, hypertrophic chondrocytes away from the border were only type X collagen positive, while adjacent osteoid tissue was immunopositive for type I collagen but not type X collagen (Fig. 11, A and B). Immunostaining with antibodies against annexin V showed strong staining of the matrix only in a few cell layers close to the chondro–osseous junction, while in other areas of the hypertrophic zone staining for annexin V was restricted to the cell surface (Fig. 11 C). Immunostaining for annexin V was also observed in the osteoid matrix (Fig. 11 C). Staining of sections derived from similar areas with alizarin red S revealed a strong staining of the narrow type I collagen, type X collagen, and annexin V–positive zone, demonstrating the codistribution of type I collagen and annexin V in the matrix of calcified cartilage (Fig. 11 D).

In summary, annexin V is expressed more widely than, and before, type I collagen in hypertrophic cartilage. However, both proteins are abundant in the restricted area of hypertrophic cartilage engaged in calcification.