Matrix Metalloproteinase-dependent Activation of Latent Transforming Growth Factor-β Controls the Conversion of Osteoblasts into Osteocytes by Blocking Osteoblast Apoptosis
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m207205200
ISSN1083-351X
AutoresM.A. Karsdal, Lykke Larsen, Michael T. Engsig, Henriette Lou, Mercedes Ferreras, André Lochter, Jean‐Marie Delaissé, Niels T. Foged,
Tópico(s)Bone health and treatments
ResumoUpon termination of bone matrix synthesis, osteoblasts either undergo apoptosis or differentiate into osteocytes or bone lining cells. In this study, we investigated the role of matrix metalloproteinases (MMPs) and growth factors in the differentiation of osteoblasts into osteocytes and in osteoblast apoptosis. The mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts were either grown on two-dimensional (2-D) collagen-coated surfaces, where they morphologically resemble flattened, cuboidal bone lining cells, or embedded in three-dimensional (3-D) collagen gels, where they resemble dendritic osteocytes constituting a network of cells. When MC3T3-E1 osteoblasts were grown in a 3-D matrix in the presence of an MMP inhibitor (GM6001), the cell number was dose-dependently reduced by approximately 50%, whereas no effect was observed on a 2-D substratum. In contrast, the murine mature osteocyte cell line, MLO-Y4, was unaffected by GM6001 under all culture conditions. According to TUNEL assay, the osteoblast apoptosis was increased 2.5-fold by 10 μm GM6001. To investigate the mechanism by which MMPs mediate the survival of osteoblasts, we examined the effect of GM6001 on MC3T3-E1 osteoblasts in the presence of extracellular matrix components and growth factors, including tenascin, fibronectin, laminin, collagenase-cleaved collagen, gelatin, parathyroid hormone, basic fibroblast growth factor, vascular epidermal growth factor, insulin-like growth factor, interleukin-1, and latent and active transforming growth factor-β (TGF-β). Only active TGF-β, but not latent TGF-β or other agents tested, restored cell number and apoptosis to control levels. Furthermore, we found that the membrane type MMP, MT1-MMP, which is produced by osteoblasts, could activate latent TGF-β and that antibodies neutralizing endogenous TGF-β led to a similar decrease in cell number as GM6001. Whereas inhibitors of other protease families did not induce osteoblast apoptosis, an inhibitor of the p44/42 mitogen-activated protein kinase showed the same but non-synergetic effect as GM6001. These findings suggest that MMP-activated TGF-β maintains osteoblast survival during trans-differentiation into osteocytes by a p44/42-dependent pathway. Upon termination of bone matrix synthesis, osteoblasts either undergo apoptosis or differentiate into osteocytes or bone lining cells. In this study, we investigated the role of matrix metalloproteinases (MMPs) and growth factors in the differentiation of osteoblasts into osteocytes and in osteoblast apoptosis. The mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts were either grown on two-dimensional (2-D) collagen-coated surfaces, where they morphologically resemble flattened, cuboidal bone lining cells, or embedded in three-dimensional (3-D) collagen gels, where they resemble dendritic osteocytes constituting a network of cells. When MC3T3-E1 osteoblasts were grown in a 3-D matrix in the presence of an MMP inhibitor (GM6001), the cell number was dose-dependently reduced by approximately 50%, whereas no effect was observed on a 2-D substratum. In contrast, the murine mature osteocyte cell line, MLO-Y4, was unaffected by GM6001 under all culture conditions. According to TUNEL assay, the osteoblast apoptosis was increased 2.5-fold by 10 μm GM6001. To investigate the mechanism by which MMPs mediate the survival of osteoblasts, we examined the effect of GM6001 on MC3T3-E1 osteoblasts in the presence of extracellular matrix components and growth factors, including tenascin, fibronectin, laminin, collagenase-cleaved collagen, gelatin, parathyroid hormone, basic fibroblast growth factor, vascular epidermal growth factor, insulin-like growth factor, interleukin-1, and latent and active transforming growth factor-β (TGF-β). Only active TGF-β, but not latent TGF-β or other agents tested, restored cell number and apoptosis to control levels. Furthermore, we found that the membrane type MMP, MT1-MMP, which is produced by osteoblasts, could activate latent TGF-β and that antibodies neutralizing endogenous TGF-β led to a similar decrease in cell number as GM6001. Whereas inhibitors of other protease families did not induce osteoblast apoptosis, an inhibitor of the p44/42 mitogen-activated protein kinase showed the same but non-synergetic effect as GM6001. These findings suggest that MMP-activated TGF-β maintains osteoblast survival during trans-differentiation into osteocytes by a p44/42-dependent pathway. The skeleton is a dynamic tissue that is continuously remodeling to sustain calcium homeostasis, repair microfractures, and react to strain and stress of the skeleton. The remodeling process is a complex process and relies on the coupling between bone resorption and formation that involves osteoclasts, osteoblasts, and osteocytes. The constant regeneration of bone emphasizes the delicate balance between bone resorption and bone formation, which otherwise may lead to pathological conditions such as osteoporosis or osteopetrosis. The investigation of the cellular actions of the major players of bone remodeling may therefore contribute significantly to the discovery of new and better drugs for the treatment of osteoporosis (1Weinstein R.S. Manolagas S.C. Am. J. Med. 2000; 108: 153-164Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The major pharmaceutical interventions for treatment and prevention of osteoporosis have focused on reduction of osteoclast numbers and their resorptive activity. However, osteoporotic patients with a very low bone mass who already have passed below the threshold for fractures are unlikely to rise above this critical limit by intervention with an anti-resorptive therapy. As a consequence, an increasing number of studies have investigated the regulation of bone matrix synthesis, a complex interplay of osteoblasts, osteocytes, and osteoclasts in the so-called basic multicellular unit (2Parfitt A.M. J. Cell. Biochem. 1994; 55: 273-286Crossref PubMed Scopus (752) Google Scholar). The rate of supply of new osteoblasts and osteoclasts, and the timing of the death of osteoblasts, osteocytes, and osteoclasts by apoptosis are critical determinants of the initiation of new basic multicellular units and the extension or reduction of the lifetime of existing ones (1Weinstein R.S. Manolagas S.C. Am. J. Med. 2000; 108: 153-164Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Of particular interest for the regulation of anabolic processes in bone are the effect of growth factors and cytokines produced in the bone microenvironment on osteoblast apoptosis (3Manolagas S.C. Endocr. Rev. 2000; 21: 115-137Crossref PubMed Scopus (1978) Google Scholar, 4Plotkin L.I. Weinstein R.S. Parfitt A.M. Roberson P.K. Manolagas S.C. Bellido T. J. Clin. Invest. 1999; 104: 1363-1374Crossref PubMed Scopus (776) Google Scholar, 5Jilka R.L. Weinstein R.S. Bellido T. Roberson P. Parfitt A.M. 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The matrix metalloproteinases (MMPs) 1The abbreviations used are: MMP, matrix metalloproteinase; ECM, extracellular matrix; 2-D, two-dimensional; 3-D, three-dimensional; αMEM, minimal essential medium; TGF-β, transforming growth factor-β; PTH, parathyroid hormone; DAPI, 4′,6-diamidine-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; VEGF, vascular epidermal growth factor; MAPK, mitogen-activated protein kinase; MT1-MMP, membrane type 1 MMP 1The abbreviations used are: MMP, matrix metalloproteinase; ECM, extracellular matrix; 2-D, two-dimensional; 3-D, three-dimensional; αMEM, minimal essential medium; TGF-β, transforming growth factor-β; PTH, parathyroid hormone; DAPI, 4′,6-diamidine-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; VEGF, vascular epidermal growth factor; MAPK, mitogen-activated protein kinase; MT1-MMP, membrane type 1 MMPconstitute a family of more than 25 secreted and cell surface enzymes that process or degrade numerous peri- and extracellular proteins (7Birkedal-Hansen H. 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Metab. 1999; 17: 61-65Crossref PubMed Scopus (121) Google Scholar, 22Cheng B. Kato Y. Zhao S. Luo J. Sprague E. Bonewald L.F. Jiang J.X. Endocrinology. 2001; 142: 3464-3473Crossref PubMed Scopus (125) Google Scholar) (kindly provided by L. Bonewald, University of Texas Health Science Center, San Antonio, TX) was routinely maintained on collagen-coated dishes in αMEM supplemented with 2.5% fetal bovine serum, 2.5% calf serum (Invitrogen, #26170-043), 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma). For experiments, MC cells were plated for cultivation in two dimensions (2-D) at a density of 30,000 cells per cm2 into 96-well tissue culture plates (Costar). Alternatively, the cells were seeded for cultivation in 3-D collagen I gel (Nitta Collagen) or Matrigel (R&D Systems) at 1 × 105 cells/ml, and cultured in 5% heat-inactivated fetal calf serum in α-MEM for 5 days unless otherwise mentioned. Unless otherwise indicated, growth factors and inhibitors were added at the time of plating into the culture medium at the following concentrations: TGF-β1 (R&D Systems) at 2.5 ng/ml, basic fibroblast growth factor (R&D Systems) at 2 ng/ml, PTH (R&D Systems) at 10 nm, GM6001 (AM Scientific) at 10 μm, aprotinin (Calbiochem) at 10 μm, pepstatin (Calbiochem) at 10 μm, and E-64 (Calbiochem) at 10 μm. Primary mouse osteoblasts were isolated from 1-day-old mouse calvarias, cultured for 7 days, and used for experiments (23Sprague S.M. Krieger N.S. Bushinsky D.A. Kidney Int. 1994; 46: 1199-1206Abstract Full Text PDF PubMed Scopus (42) Google Scholar). To quantify the cell number, the Alamar Blue assay (Trek Diagnostics) was used according to the manufacturer's instructions. For Annexin, 4′,6-diamidine-2-phenylindole (DAPI), and propidium iodide assays, MC cells were seeded as described under cell culture and stained according to the manufacturer's description by the Annexin-V-FLOUS staining kit (Roche Molecular Biochemicals, catalog no. 1858777). For detection of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), the MC cells were cultured on chamber slides, fixed in 4% formaldehyde, and dehydrated in a sucrose gradient. Samples were frozen at −80 °C until sectioning in a cryostat at −20 °C. Sections were mounted on coverslips and stained according to the manufacturer's descriptions by the in situ Cell Death Detection Kit (Roche Molecular Biochemicals, catalog no. 1684809). Human pro-MMP-2 was a kind gift from Drs. F. Frankenne and J. M. Foidart (University of Liege, Belgium). Mouse pro-MMP-9 was purified from cell culture media of transformed baby hamster kidney cells, by means of two consecutive chromatographic steps on gelatin-Sepharose and concanavalin A-Sepharose columns (Amersham Biosciences) (24Morodomi T. Ogata Y. Sasaguri Y. Morimatsu M. Nagase H. Biochem. J. 1992; 285: 603-611Crossref PubMed Scopus (178) Google Scholar). The catalytic domain of rabbit MMP-14 was purified from conditioned medium of transfectedEscherichia coli. 2M. A. Karsdal, L. Larsen, M. T. Engsig, H. Lou, M. Ferreras, A. Lochter, J.-M. Delaissé, and N. T. Foged, unpublished. Pro-MMPs were activated with 4-aminophenyl mercuric acetate. The molar concentration of active MMPs (25Knight C.G. Methods Enzymol. 1995; 248: 85-101Crossref PubMed Scopus (74) Google Scholar) was determined by using the MMP inhibitor BB94 (kindly provided by Dr. H. Van Wart, Roche Bioscience, Palo Alto, CA). Collagen type I (Nitta Collagen) or latent TGF were incubated with 4-aminophenyl mercuric acetate-activated MMP for 5 or 24 h unless otherwise indicated in a buffer containing 50 mm Tris-Cl, 0.15 m NaCl, 10 mm CaCl2, 50 μmZnSO4, 0.05% Brijj, pH 7.5). Activation of latent TGF-β was followed by Western blotting. Western blotting was performed on total cell lysates in radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.4, 30 mm NaCl, 5 mm EDTA, 1% Nonidet P40, 1% deoxycholic acid, 0.1% SDS) containing 10 mm NaF and 50 mmNa3VO4. Samples were resolved on 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes (Bio-Rad). The membranes were blocked overnight at 4 °C with TBS-T (50 mm Tris-HCl, pH 7.5, 0.1% Tween 20, 100 mm NaCl) containing 5% milk powder. Membranes were then incubated for 1 h at ambient temperature with antibodies against TGF-β. After washing vigorously with TBS-T for 1 h, membranes were incubated for 1 h at ambient temperature with horseradish peroxidase-conjugated rabbit anti-mouse antibodies (Dako) and developed with an enhanced chemiluminescence kit (ECLTM, AmershamBiosciences), according to the manufacturer's instructions. To study cell shape, cells were fixed with 5% glutaraldehyde in phosphate-buffered saline, washed extensively with water, and stained with 0.5% toluidine blue (Sigma) in 2.5% Na2CO3. Cells were examined by light microscopy (Olympus IX70, ×20 magnification). All graphs show one representative experiment of at least three, each with four individual replications. All graphs show the mean of four replications and standard deviations. All statistical calculations have been performed by the Student's two-tailed un-pairedt test assuming normal distribution with equal variance. Statistical significance is given by number of asterisks (*,p < 0.05, **, p < 0.01). When the mouse calvaria osteoblast cell line MC3T3-E1 (MC) or primary osteoblasts isolated from calvariae of 1-day-old mice were cultured on 2-D type I collagen-coated surfaces they morphologically resembled flattened, cuboidal bone lining cells, whereas, when embedded in 3-D type I collagen gels, they resembled dendritic osteocytes constituting a network of cells (Fig. 1 A). When MC cells or osteoblasts were grown in the 3-D collagen matrix in the presence of the MMP inhibitor, GM6001, the cell morphology was severely affected. Cell spreading was inhibited after 24 h of culture, and apoptotic vesicles and pycnotic nuclei were apparent after 2 days. In contrast, cell morphology was not affected by GM6001 when cells were grown on the 2-D collagen-coated substratum (Fig.1 A). To evaluate the exact amount of cell death due to MMP inhibitor treatment AlamarBlue assessment of cell number in MC cultures was performed (Fig. 1 B). The cell number was significantly lower after 2 days of culture and reduced by approximately 50% compared with the untreated control at day 5 of culture (Fig. 1 B). The effect of GM6001 was dose-dependent, with an IC50 value of 0.1 μm (Fig. 1 B). Primary osteoblasts displayed a similar pattern of sensitivity to MMP inhibition as the MC cell line when assessed by AlamarBlue (Fig.1 C). The MMP inhibitor BB-94 showed the same effect as GM6001 on both MC cells and primary mouse osteoblasts (data not shown). To evaluate if the observed effect on cell death was restricted to a bone-mimicking microenvironment consisting of 3-D type I collagen, MC cells were also cultured in another 3-D gel material, Matrigel. Matrigel consists of solubilized basement membrane, of which the major constituents are laminin, type IV collagen, heparan sulfate proteoglycans, and entactin and among other growth factors TGF-β and basic fibroblast growth factor (26Kleinman H.K. McGarvey M.L. Liotta L.A. Robey P.G. Tryggvason K. Martin G.R. Biochemistry. 1982; 21: 6188-6193Crossref PubMed Scopus (963) Google Scholar, 27McGuire P.G. Seeds N.W. J. Cell. Biochem. 1989; 40: 215-227Crossref PubMed Scopus (96) Google Scholar). Therefore, Matrigel does not resemble bone, but still retains the features of a 3-D matrix. MMPs were only important for survival of MC cells cultured in the type I collagen matrix and not in Matrigel (Fig. 2 A). Thus, MMPs are not essential for the survival in 3-D per se but specifically important when osteoblasts are surrounded by type I collagen. To further investigate the role of proteases in osteoblast survival in the bone microenvironment, we tested the effect of inhibitors of serine, cysteine, and aspartic proteases on MC cells. However, none of the protease inhibitors reduced the number of osteoblasts in 3-D type I collagen gels as measured by AlamarBlue (Fig. 2 B). Furthermore, the morphological characteristics of apoptosis such as blebbing processes, apoptotic vesicles, condensed cytoplasm, and pycnotic nuclei were only induced in osteoblasts treated by the MMP inhibitor and not by inhibitors of the other protease families To investigate whether MMP inhibitor-treated osteoblasts die by apoptosis rather than necrosis, MC cells were cultured in 3-D type I collagen gels and stained for Annexin V together with DAPI and propidium iodide or for TUNEL. According to DAPI staining (Fig.3 A), the nuclei of cells treated with GM6001 were smaller and less abundant than those of vehicle-treated cells. In contrast to vehicle-treated control cells, GM6001-treated cells allowed uptake of propidium iodide, which only traverses the plasma membrane of cells that have lost their membrane potential, i.e. that either are late stage apoptotic or have died by necrosis. Cells treated with GM6001 were clearly positive for Annexin V, which indicates that they undergo apoptosis (Fig.3 A). However, because some cells were positive for both Annexin V and propidium iodide, it is difficult to conclude whether they died from apoptosis or necrosis, even when the classic morphological phenotypes of apoptosis were taken into account. Therefore, to further determine osteoblast cell death characteristics, TUNEL staining was applied to 3-D type I collagen gels after sectioning. After 5 days of culture in the presence of GM6001, we detected a 2.5-fold increase of apoptotic cells compared with vehicle-treated control cells (Fig. 3 B). Furthermore, the increase in apoptotic cells from days 2 to 5 was 5-fold higher when MC cells were treated with GM6001 (Fig. 3 B). Thus, we conclude that MMP inhibitor-induced cell death of osteoblasts in 3-D type I collagen is due to apoptosis. To further investigate the mechanism by which MMPs control the survival of osteoblasts, we examined the effect of GM6001 in the presence of cleaved type I collagen fragments and extracellular matrix components. We hypothesized that the MMP-dependent survival may in part be explained by a proteolytic action on type I collagen that would expose an otherwise cryptic peptide sequence that is able to mediate survival, e.g. a peptide containing the integrin binding RGD motif (28Morimura N. Tezuka Y. Watanabe N. Yasuda M. Miyatani S. Hozumi N. Tezuka K. J. Biol. Chem. 2001; 276: 42172-42181Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 29Jones P.L. Crack J. Rabinovitch M. J. Cell Biol. 1997; 139: 279-293Crossref PubMed Scopus (305) Google Scholar) and that mice, carrying a collagenase-resistant form of type I collagen, have altered skeletal remodeling resulting in empty lacunae in osteocytes in calvariae as a result of osteocyte apoptosis (30Zhao W. Byrne M.H. Wang Y. Krane S.M. J. Clin. Invest. 2000; 106: 941-949Crossref PubMed Scopus (114) Google Scholar). We therefore examined if the incorporation of denatured collagen (gelatin) or proteolytically generated collagen fragments would have a positive effect on cell morphology and cell number in the presence of an MMP inhibitor. Collagen was denatured by heating or cleaved with either a collagenase (MMP-13) resulting in the classic ¾-¼ fragments, a gelatinase (MMP-9) or bacterial collagenase with unspecific activity against type I collagen. All incubations were checked by SDS-PAGE followed by silver staining to detect both ¾ and ¼ fragments and unspecific cleavage of type I collagen (data not shown). These fragments were then incorporated into the type I collagen gel at a 1% w/w basis. However, neither heat-denatured collagen nor collagen fragments promoted osteoblast survival in the presence of GM6001 according to assessment by AlamarBlue (Fig. 4 A) and/or cell morphology (data not shown). ECM components may, in combination with RGD sequences released through MMP-directed proteolysis of collagen, mediate survival of smooth muscle cells (29Jones P.L. Crack J. Rabinovitch M. J. Cell Biol. 1997; 139: 279-293Crossref PubMed Scopus (305) Google Scholar). The viability of these cells is hypothesized to be sustained by an MMP-dependent pathway where RGD sequences facilitate endogenous production of tenascin, which thereby mediates survival. We therefore investigated the effect of fibronectin, laminin, and tenascin on osteoblast survival in the presence of an MMP inhibitor in 3-D type I collagen gels. None of the ECM molecules tested affected cell survival in the presence or absence of GM6001 measured either by cell number (Fig. 4 B) or by cell morphology (data not shown). Therefore, we conclude that MMP-dependent survival of osteoblasts does not seem to be due to proteolytically generated type I collagen fragments or ECM molecules. Growth factors such as PTH, TNF-α, and TGF-β1 have previously been shown to modulate osteoblasts apoptosis (7Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral. Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2633) Google Scholar, 31Hock J.M. Krishnan V. Onyia J.E. Bidwell J.P. Milas J. Stanislaus D. J. Bone Miner. Res. 2001; 16: 975-984Crossref PubMed Scopus (187) Google Scholar). To further examine the effect of MMP action on osteoblast survival in 3-D collagen gels, we investigated the effect of a battery of known bone growth factors on MC cells in type I collagen gels,i.e. PTH, VEGF, TGF-β1, interleukin-1, and insulin-like growth factor. None of these growth factors had an effect on cell number in the absence of GM6001, and only TGF-β restored cell number and morphology to control levels in the presence of an MMP inhibitor (Fig. 5 A). MMPs can activate latent TGF-β (32Maeda S. Dean D.D. Gay I. Schwartz Z. Boyan B.D. 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Sohn K.Y., Jo, J.S. Ryoo H.M. J. Cell. Biochem. 1996; 61: 609-618Crossref PubMed Scopus (207) Google Scholar), we investigated whether both active and latent TGF-β could prevent MMP inhibitor-mediated osteoblast apoptosis. Active TGF-β, but not latent TGF-β, restored cell number to control levels in the presence of GM6001 (Fig. 5 B). To further corroborate that endogenously produced and activated TGF-β is responsible for blocking osteoblasts apoptosis, we monitored cell number in the presence of TGF-β function-blocking antibodies. Addition of function-blocking anti-TGF-β antibodies dose-dependently reduced the cell number to the same extent as GM6001 (Fig. 5 C). Control antibodies had no significant effect on the cell number. TGF-β did not increase the cell number compared with vehicle-treated control cultures (Fig. 5, Aand B). To investigate further whether TGF-β blocks MMP inhibitor-induced apoptosis, we assessed by TUNEL-staining apoptosis of MC cells cultured in 3-D type I collagen gels in the absence and presence of TGF-β or/and GM6001. The increase in apoptosis induced by GM6001 was prevented by TGF-β (Fig. 5 D). TGF-β alone had some anti-apoptotic effects compared with controls, as previously described in serum starvation experiments (6Jilka R.L. Weinstein R.S. Bellido T. Parfitt A.M. Manolagas S.C. J. Bone Miner. Res. 1998; 13: 793-802Crossref PubMed Scopus (467) Google Scholar). Taken together these results clearly demonstrate that TGF-β rescues the apoptosis induced by loss of MMP activity, suggesting that MMPs support cell survival through activation of latent TGF-β. The survival of trans-differentiating osteoblasts but not of mature osteocytes depends on MMP activity and is correlated to TGF-β-induced p44/42 MAPK activation. To compare the role of MMP activity in trans-differentiating osteoblasts to that in mature osteocytes, we cultured the osteocyte cell line MLO-Y4 (21Bonewald L.F. J. Bone Miner. Metab. 1999; 17: 61-65Crossref PubMed Scopus (121) Google Scholar, 22Cheng B. Kato Y. Zhao S. Luo J. Sprague E. Bonewald L.F. Jiang J.X. Endocrinology. 2001; 142: 3464-3473Crossref PubMed Scopus (125) Google Scholar) on 2-D type I collagen-co
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