Journal of Biological Chemistry · 2004 · 233 citations · 50 references
DentistryBioengineeringOral BiologyBiomaterialsBone MetabolismDentin BiologyBone MineralizationBone TissueDentin MineralizationDentin StructureOsteogenesisTooth MineralizationApatite FormationCollagen BindingCollagen Fibril DiameterCollagen-binding ProteinsExtracellular Polymeric MatrixMineralized Extracellular MatrixMineralized Matrix FormationNoncollagenous Proteins
During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein expressed during the initial stages of mineralized matrix formation in bone and dentin. Here we show that DMP1 bound specifically to type I collagen, with the binding region located at the N-telopeptide region of type I collagen. Peptide mapping identified two acidic clusters in DMP1 responsible for interacting with type I collagen. The collagen binding property of these domains was further confirmed by site-directed mutagenesis. Transmission electron microscopy analyses have localized DMP1 in the gap region of the collagen fibrils. Fibrillogenesis assays further demonstrated that DMP1 accelerated the assembly of the collagen fibrils in vitro and also increased the diameter of the reconstituted collagen fibrils. In vitro mineralization studies in the presence of calcium and phosphate ions demonstrated apatite deposition only at the collagen-bound DMP1 sites. Thus specific binding of DMP1 and possibly other noncollagenous proteins on the collagen fibril might be a key step in collagen matrix organization and mineralization. During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein expressed during the initial stages of mineralized matrix formation in bone and dentin. Here we show that DMP1 bound specifically to type I collagen, with the binding region located at the N-telopeptide region of type I collagen. Peptide mapping identified two acidic clusters in DMP1 responsible for interacting with type I collagen. The collagen binding property of these domains was further confirmed by site-directed mutagenesis. Transmission electron microscopy analyses have localized DMP1 in the gap region of the collagen fibrils. Fibrillogenesis assays further demonstrated that DMP1 accelerated the assembly of the collagen fibrils in vitro and also increased the diameter of the reconstituted collagen fibrils. In vitro mineralization studies in the presence of calcium and phosphate ions demonstrated apatite deposition only at the collagen-bound DMP1 sites. Thus specific binding of DMP1 and possibly other noncollagenous proteins on the collagen fibril might be a key step in collagen matrix organization and mineralization. Mineralized tissues such as bone and dentin are hierarchically organized biocomposites possessing unique mechanical properties (1Weiner S. Wagner H.D. Annu. Rev. Mater. Sci. 1998; 28: 271-298Crossref Scopus (2225) Google Scholar). Understanding the biomineralization process is of great value for synthesis of bioengineered bone and dentin-like materials with optimum structural properties. Type I collagen accounts for 90% of the total protein in the organic matrix of bone and dentin (1Weiner S. Wagner H.D. Annu. Rev. Mater. Sci. 1998; 28: 271-298Crossref Scopus (2225) Google Scholar). It not only provides the structural framework with viscoelastic properties but also defines compartments for ordered mineral deposition. Studies have demonstrated that the apatite crystals are first nucleated in the gap region, and the growing mineral platelets are highly organized in a staggered manner within the collagen fibrils (2Traub W. Arad T. Weiner S. Matrix. 1992; 12: 251-255Crossref PubMed Scopus (93) Google Scholar, 3Jager I. Fratzl P. Biophys. J. 2000; 79: 1737-1746Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). It is well established that type I collagen matrix does not have the capacity to induce matrix-specific mineral formation from metastable calcium phosphate solutions that do not spontaneously precipitate. Several in vitro studies have demonstrated that ordered mineralization of apatite on collagen fibril is impossible without additives (4Saito T. Arsenault A.L. Yamauchi M. Kuboki Y. Crenshaw M.A. Bone. 1997; 21: 305-311Crossref PubMed Scopus (146) Google Scholar, 5Bradt J. Mertig M. Teresiak A. Pompe W. Chem. Mater. 1999; 11: 2694-2701Crossref Scopus (283) Google Scholar). As a result, much attention has been drawn to the noncollagenous proteins (NCPs) 1The abbreviations used are: NCP, noncollagenous protein; AFM, atomic force microscopy; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; TEM, transmission electron microscopy; DMP, dentin matrix protein; PBS, phosphate-buffered saline; GST, glutathione S-transferase; BSA, bovine serum albumin; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone. 1The abbreviations used are: NCP, noncollagenous protein; AFM, atomic force microscopy; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; TEM, transmission electron microscopy; DMP, dentin matrix protein; PBS, phosphate-buffered saline; GST, glutathione S-transferase; BSA, bovine serum albumin; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone. that are tightly bound to the collagen fibers in mineralized tissues. Bone and/or dentin-specific NCPs are mostly acidic in nature and are rich in glutamic acid, aspartic acid, and phosphoserines (6Boskey A.L. Connect. Tissue Res. 1996; 35: 357-363Crossref PubMed Scopus (174) Google Scholar, 7Butler W.T. Ritchie H. Int. J. Dev. Biol. 1995; 39: 169-179PubMed Google Scholar). They possess high calcium binding capacity and hydroxyapatite affinity (8Chen Y. Bal B.S. Gorski J.P. J. Biol. Chem. 1992; 267: 24871-24878Abstract Full Text PDF PubMed Google Scholar, 9Stetler-Stevenson W.G. Veis A. Calcif. Tissue Int. 1987; 40: 97-102Crossref PubMed Scopus (77) Google Scholar, 10Wallwork M.L. Kirkham J. Chen H. Chang S.X. Robinson C. Smith D.A. Clarkson B.H. Calcif. Tissue Int. 2002; 71: 249-255Crossref PubMed Scopus (34) Google Scholar). In vitro mineralization analyses suggest that these NCPs can greatly influence the apatite deposition rate and morphology of crystals, therefore they can be considered as nucleators or inhibitors of mineralization (11Hunter G.K. Hauschka P.V. Poole A.R. Rosenberg L.C. Goldberg H.A. Biochem. J. 1996; 317: 59-64Crossref PubMed Scopus (518) Google Scholar). Results from genetic disease analyses and transgenic animal models show that dysfunctional NCPs cause impaired mineralization in vivo (12Xiao S. Yu C. Chou X. Yuan W. Wang Y. Bu L. Fu G. Qian M. Yang J. Shi Y. Hu L. Han B. Wang Z. Huang W. Liu J. Chen Z. Zhao G. Kong X. Nat. Genet. 2001; 27: 201-204Crossref PubMed Scopus (280) Google Scholar, 13Sreenath T. Thyagarajan T. Hall B. Longenecker G. D'Souza R. Hong S. Wright J.T. MacDougall M. Sauk J. Kulkarni A.B. J. Biol. Chem. 2003; 278: 24874-24880Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). All of these results suggest an active regulatory role for NCPs during the biomineralization process. Several NCPs have been reported to bind to the collagen surface (8Chen Y. Bal B.S. Gorski J.P. J. Biol. Chem. 1992; 267: 24871-24878Abstract Full Text PDF PubMed Google Scholar, 14Dahl T. Sabsay B. Veis A. J. Struct. Biol. 1998; 123: 162-168Crossref PubMed Scopus (69) Google Scholar). Phosphophoryn in particular was shown to bind at the e-band in the middle of the gap region (15Traub W. Jodaikin A. Arad T. Veis A. Sabsay B. Matrix. 1992; 12: 197-201Crossref PubMed Scopus (80) Google Scholar). Other studies have demonstrated that NCPs bind to charge group constellations favoring binding at the gap zone boundaries in collagen fibrils (16Fujisawa R. Nodasaka Y. Kuboki Y. Calcif. Tissue Int. 1995; 56: 140-144Crossref PubMed Scopus (76) Google Scholar), where the mineral is initiated in vivo (2Traub W. Arad T. Weiner S. Matrix. 1992; 12: 251-255Crossref PubMed Scopus (93) Google Scholar). However, there is still no in-depth study pertaining to the specific nature of collagen-NCP interaction and how it modulates mineralization. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein (17George A. Sabsay B. Simonian P.A. Veis A. J. Biol. Chem. 1993; 268: 12624-12630Abstract Full Text PDF PubMed Google Scholar) and is now known to be present in the mineralized matrix of both dentin and bone (18Qin C. Brunn J.C. Jones J. George A. Ramachandran A. Gorski J.P. Butler W.T. Eur. J. Oral Sci. 2001; 109: 133-141Crossref PubMed Scopus (97) Google Scholar). In situ hybridization experiments have demonstrated that the initial expression of DMP1 coincides with bone and dentin mineralization (19Kamiya N. Takagi M. Histochem. J. 2001; 33: 545-552Crossref PubMed Scopus (31) Google Scholar, 20George A. Silberstein R. Veis A. Connect. Tissue Res. 1995; 33: 67-72Crossref PubMed Scopus (61) Google Scholar), indicating that DMP1 is actively involved in regulating the temporal and spatial aspects of mineral initiation. This assumption was further confirmed by preliminary results showing that recombinant DMP1 can initiate apatite nucleation in vitro (21He G. Dahl T. Veis A. George A. Connect. Tissue Res. 2003; 44 (suppl.): 240-245Crossref PubMed Google Scholar). To understand DMP1-mediated spatial regulation of matrix-mediated mineralization, we hypothesize that specific collagen fibril-DMP1 interaction is necessary for mineral induction. In this study we have demonstrated that DMP1 binds to the N-telopeptide of fibrillar type I collagen in vitro in a regular, periodic manner. Further, the functional collagen binding domains in DMP1 have also been identified and characterized. These results provide the molecular basis underlying the initial steps involved in mineralized tissue formation, namely the specific binding of NCPs on collagen template. Solid-phase Binding Assay of DMP1 with Monomeric Collagen— Lathyritic rat skin collagen was prepared as described previously (22Payne K.J. Veis A. Biopolymers. 1988; 27: 1749-1760Crossref PubMed Scopus (515) Google Scholar). The collagen was then dissolved in 0.01 m HCl at a 1 mg/ml concentration. Collagen binding property (23Dickeson S.K. Mathis N.L. Rahman M. Bergelson J.M. Santoro S.A. J. Biol. Chem. 1999; 274: 32182-32191Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) was determined by coating microtiter plates (Immulon 4, Dynex Technologies) with 5 μg of type I collagen in 100 μl of 0.1 m acetic acid/well overnight at 4 °C. Wells were washed three times with PBS and blocked with 0.5% (w/v) bovine serum albumin in PBS at 37 °C for 2 h before the addition of varying concentrations of the GST-fused recombinant DMP1 (24Srinivasan R. Chen B. Gorski J.P. George A. Connect. Tissue Res. 1999; 40: 251-258Crossref PubMed Scopus (51) Google Scholar). After incubation at room temperature for 2 h with gentle shaking, the wells were extensively washed with PBST (PBS containing 0.1% (v/v) Tween 20). They were then with for 2 The plates were washed three times with and then with was to the After incubation at room temperature for 1 the wells were washed with The was by 100 μl of m containing 1 mg/ml phosphate to the After of incubation the at was a were in and with prepared DMP1 from wells blocked with without collagen coating were as the and from of other protein in well was on the from wells with known of the The were determined from the are as the value from a Solid-phase Binding Assay of DMP1 with rat was and as described previously A. J.P. Veis A. Sci. 1999; Scholar). The was by on the by the μg of recombinant DMP1 in 100 μl of PBS was 4 wells overnight at 4 °C. After with μl of 0.1% Tween the wells were blocked with μl of in for 2 The wells were then with μl of times and with 100 μl of N-telopeptide for 2 h in a After with μl of the wells were with for 1 h with a at The wells were then washed with with phosphate and with a microtiter at Wells were in and the were DMP1 the functional in DMP1 that with type I collagen, the of DMP1 was two and by with a of the of the were then the and expressed in The and by was by at 4 °C The recombinant were and of Collagen Binding in DMP1 by and μg of in 100 was with 2 μg of and overnight at 37 °C. After the reaction was by the addition of and the was on a with 100 The was then washed extensively with to The and the were to of the was the and with 1 μl of mg/ml in with was for mapping in Peptide were identified on the by the of DMP1 on two clusters in DMP1 were to collagen binding The used C. and DMP1 was two and The were in a and used as the template for further as to DMP1 with The was with and which was by and DMP1 was two The were in a and used as the template for further as The was with and which was by process is the as for that the template used was All of the were and The recombinant proteins were expressed and as Fibrillogenesis fibril assembly was by at as a of at °C A. Veis A. PubMed Scopus Google Scholar). The was used to initiate K.J. Biopolymers. PubMed Scopus Google Scholar). 4 μl of a containing mg/ml type I collagen in 0.01 m HCl was with μl and of in phosphate in a 0.1 mg/ml collagen with and 5 DMP1 and Transmission reconstituted collagen fibrils were prepared at °C for as μl of collagen was surface for After with the was and collagen fibrils were by with a with a were The diameter and of the fibrils were by was prepared to the of and Eur. J. Biol. Google Scholar) a 4 to a of were on a of fibrillar collagen for 2 h at room The were washed with and on a of surface overnight at 4 °C. The were then washed with and with for 1 bovine serum albumin prepared to the was used as a for collagen binding The were with an electron at In in vitro apatite nucleation was as described previously (21He G. Dahl T. Veis A. George A. Connect. Tissue Res. 2003; 44 (suppl.): 240-245Crossref PubMed Google Scholar). were a two of an with containing calcium and the other phosphate 1 The and the an was the ions the surface The were to a and were The nucleation was for 5 with collagen fibrils as Binding of DMP1 to Type I binding was used as the of for of between collagen and noncollagenous proteins (8Chen Y. Bal B.S. Gorski J.P. J. Biol. Chem. 1992; 267: 24871-24878Abstract Full Text PDF PubMed Google Scholar, S.K. Mathis N.L. Rahman M. Bergelson J.M. Santoro S.A. J. Biol. Chem. 1999; 274: 32182-32191Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The binding shown in 1 that the DMP1 bound to collagen in a and manner an and binding at μg type I To the specific responsible for this DMP1 was two and expressed binding demonstrated that only the of DMP1 affinity to the collagen with an 2 and binding at μg of type I collagen DMP1 to the of the Collagen an to study the specific on the collagen that was responsible for interaction with binding assays were with N-telopeptide Results from this study that the N-telopeptide demonstrated specific binding affinity with DMP1 with a of The binding was to be μg of which an This that in the collagen fibril assembly the N-telopeptide region be a for specific binding of the Collagen Binding on the on DMP1 that are responsible for collagen the DMP1 was and a demonstrated that three bound on the collagen two and at the of DMP1 demonstrated high binding affinity with collagen. were to be of affinity to the To further this were in the collagen binding clusters by with as shown in and three proteins were expressed as recombinant of these recombinant proteins and the other recombinant protein in both of the collagen binding These recombinant proteins were then for binding and was with and demonstrated collagen binding but binding was μg of collagen for and for These were that of DMP1 collagen binding affinity was with These results further confirmed that both and were responsible for binding collagen of the collagen binding in protein were with collagen binding by site-directed as described and binding of the with Type I collagen. The binding demonstrated that both clusters impaired the of collagen DMP1 Type I Collagen Fibrillogenesis and the the role of DMP1 during collagen fibril formation was initiated by the In a collagen the is by a by a as the of the of with the during which the of the do not to by a fibril growth and a region J. Biol. Chem. Full Text PDF PubMed Google Scholar). the present the of fibril assembly demonstrated that in the of the and was However, the to and 2 1 and 5 DMP1 was in experiments The of the growth and the increased with the presence of These were to be with accelerated fibril formation in the presence of of analyses have been in at and possess the of and at G. L. C. 2002; PubMed Scopus Google Scholar, Biophys. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). by AFM, fibrils reconstituted in the presence of DMP1 to be well with periodic However, the of the reconstituted fibrils a of of the presence of The in the that the diameter of the fibrils were in the presence of In the of DMP1 the diameter of the fibrils was at However, 1 DMP1 was to 0.1 mg/ml collagen the diameter of the fibrils was with a and fibrils Thus these results that the addition of DMP1 the of collagen fibril of the diameter of the collagen fibrils by collagen fibrils prepared without DMP1 and with 1 DMP1 were a surface and by were and the diameter and of the fibrils were by The diameter of reconstituted collagen fibrils in the of DMP1 was to be and in the presence of 1 DMP1 was DMP1 in the of the Collagen of DMP1 on collagen fibrils was by as this is the used to collagen-NCP (16Fujisawa R. Nodasaka Y. Kuboki Y. Calcif. Tissue Int. 1995; 56: 140-144Crossref PubMed Scopus (76) Google Scholar). of on with reconstituted collagen fibrils and confirmed that was at the of the gap region the was located in the gap These well with results showing specific binding of DMP1 to the as this region is located at the of the gap As a with were to be and not with type I collagen fibrils In vitro mineralization experiments were with established (21He G. Dahl T. Veis A. George A. Connect. Tissue Res. 2003; 44 (suppl.): 240-245Crossref PubMed Google Scholar). The of this in vitro is that it specific mineral during the In the bound with collagen with and without were the and to mineralization for 5 The were then to Results that no mineral deposition was on the collagen surface that the of collagen in mineralized tissues is to as a the mineral were on collagen surface with DMP1 This that DMP1 can and initiate mineralization on the surface of the collagen Type I collagen is the matrix protein in bone and dentin P. of Bone Scholar). It is expressed by and at stages during and the framework that and mineralization M. H. Dev. Biol. 1988; PubMed Scopus Google Scholar). on type I collagen matrix have been shown to with of expression such as bone and M. Kuboki Y. J. Biochem. 2001; PubMed Scopus Google Scholar). The acidic noncollagenous proteins identified in the mineralized matrix of bone and dentin S. H. J. 2001; PubMed Scopus Google Scholar) are known to as or inhibitors of mineral deposition in in vitro experiments (11Hunter G.K. Hauschka P.V. Poole A.R. Rosenberg L.C. Goldberg H.A. Biochem. J. 1996; 317: 59-64Crossref PubMed Scopus (518) Google Scholar). Several of these NCPs have been shown to possess a collagen binding These NCPs bone and acidic matrix protein (8Chen Y. Bal B.S. Gorski J.P. J. Biol. Chem. 1992; 267: 24871-24878Abstract Full Text PDF PubMed Google Scholar, 14Dahl T. Sabsay B. Veis A. J. Struct. Biol. 1998; 123: 162-168Crossref PubMed Scopus (69) Google Scholar, W. Jodaikin A. Arad T. Veis A. Sabsay B. Matrix. 1992; 12: 197-201Crossref PubMed Scopus (80) Google Scholar, R. Nodasaka Y. Kuboki Y. Calcif. Tissue Int. 1995; 56: 140-144Crossref PubMed Scopus (76) Google Scholar, J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, H. L. H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, P. B. H. J. H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Sci. 2000; PubMed Scopus Google Scholar, R. Kuboki Y. Calcif. Tissue Int. 1992; PubMed Scopus (76) Google Scholar). However, the role of NCPs in collagen mineralization is not well understood. The of DMP1 in the matrix of bone and dentin to specific with collagen be demonstrated as DMP1 and type I collagen are expressed during the stages of mineralization. we reported that DMP1 as an for apatite nucleation in vitro and calcium phosphate with a morphology that the crystals at the mineralization of bone and dentin G. Dahl T. Veis A. George A. Nat. Mater. 2003; PubMed Scopus Google Scholar). this protein can be considered as a protein for the and of mineralization As collagen matrix forms the for mineral it is of to study the between collagen and DMP1 and the role of this in mineralization. In this we by a of that DMP1 specifically with type I collagen with a of clusters at the of DMP1 were identified as by of the and domains with confirmed that the binding was acidic noncollagenous proteins have been identified with collagen binding there are no on the domains that are responsible for interaction with collagen. The two identified collagen binding domains in DMP1 clusters of acidic which might suggest that the binding between DMP1 and collagen are by we also the in DMP1 responsible for mineral G. Dahl T. Veis A. George A. Nat. Mater. 2003; PubMed Scopus Google Scholar). These are located between the two collagen binding sites. we suggest that binding of DMP1 on the collagen might DMP1 to an for mineral induction. Collagen fibril formation of the collagen It is known that two the and the fibril growth The is the for of a of in J. Biol. Chem. Full Text PDF PubMed Google Scholar). to the DMP1 was shown to collagen with a which that DMP1 the assembly of collagen fibrils. The addition of DMP1 was to in fibrils with the studies have shown that and can the collagen process J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, H. L. H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, P. B. H. J. H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Sci. 2000; PubMed Scopus Google Scholar). to NCPs in the mineralized matrix of bone and bone was to type I collagen R. Kuboki Y. Calcif. Tissue Int. 1992; PubMed Scopus (76) Google Scholar), and collagen Sauk Biophys. PubMed Scopus Google Scholar) but the diameter of the reconstituted collagen fibrils B.H. A.R. Oral Biol. 1993; PubMed Scopus Google Scholar). Thus noncollagenous proteins might as collagen fibrils and an ordered which might be necessary to controlled mineral deposition T. Veis A. Connect. Tissue Res. 2003; PubMed Scopus Google Scholar). The N-telopeptide region of the collagen fibril was identified as the DMP1 binding The N-telopeptide of the of which have been highly in a of The of collagen fibrils the to molecular and formation, in the J.P. A. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. George A. and Google Scholar). studies of collagen in have demonstrated an initial nucleation located the of the gap region, which to the of the mineral the gap region A. P.A. PubMed Scopus Google Scholar, C. A. J. Biol. PubMed Scopus Google Scholar). and studies have also shown that apatite and from the gap region in the collagen fibrils (2Traub W. Arad T. Weiner S. Matrix. 1992; 12: 251-255Crossref PubMed Scopus (93) Google Scholar, W. Arad T. Weiner S. Sci. S. A. PubMed Scopus Google Scholar, S. R. A.L. Fratzl P. Bone. 1999; PubMed Scopus Google Scholar). analyses confirmed the of DMP1 on reconstituted collagen fibrils. DMP1 bound to the of the gap region of the collagen where the N-telopeptide is it is that DMP1 binds to the N-telopeptide of the collagen fibril and collagen and mineral nucleation during tissue formation. Binding of DMP1 on the collagen fibril an functional role in the mineralization process. was in a and for mineralization property in it was that only initiated deposition on the collagen there was no calcium phosphate deposition on the collagen results have been the collagen surface was with bone protein G.K. Goldberg H.A. J. Mater. Res. 2001; PubMed Scopus Google Scholar) and (4Saito T. Arsenault A.L. Yamauchi M. Kuboki Y. Crenshaw M.A. Bone. 1997; 21: 305-311Crossref PubMed Scopus (146) Google Scholar). In of these mineralization was initiated specifically on the collagen surface with noncollagenous These results further the specific role of noncollagenous proteins during the mineralization process. In the present study that the interaction between DMP1 and collagen matrix be an step in the biomineralization process in matrix-mediated mineralization. Understanding the involved in be in the of materials with mechanical properties both and
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