Colin Przybylowski^1,2, Mohamed Ammar³, Courtney LeBlon⁴, Sabrina S. Jedlicka^2,3,5*

Show more
¹ Integrated Degree in Engineering, Arts & Sciences (IDEAS) Program, Lehigh University, Bethlehem, PA, USA.
² Bioengineering Program, Lehigh University, Bethlehem, PA, USA.
³ Department of Materials Science & Engineering, Lehigh University, Bethlehem, PA, USA.
⁴ Department of Mechanical Engineering & Mechanics, Lehigh University, Bethlehem, PA, USA.
⁵ Center for Advanced Materials & Nanotechnology, Lehigh University, Bethlehem, PA, USA.
Show less

Abstract: Biointerface design that targets osteogenesis is a growing area of research with significant implications in biomedicine. Materials known to either support or stimulate osteogenesis are composed of a biomimetic ceramic material, such as bioactive glass. Bioactive glass is osteoproductive, and the potential for osteoproductivity can be enhanced by the addition of proteins or other additives designed to alter functionality. In addition, soluble growth factors are often added to osteogenic culture on bioactive glasses, further intensifying the effects of the material. In this paper, synthetic peptide combinations, covalently bound to a three-dimensional bioactive glass network, are used to mimic the effects of the whole fibronectin and bone morphogenetic proteins (BMP) 2 and 9. Peptide-silanes possessing critical binding sequences from each of these proteins are synthesized and used to decorate the surface of three-dimensional (3D) nano-macroporous bioactive glass. MC3T3 preosteoblast cells are then assessed for differentiation on the materials in the absence of soluble differentiation cues. MC3T3 preosteoblasts undergo enhanced differentiation on the peptide-silane samples over the standard nano-macroporous bioactive glass, and the differentiation capacity of the cells exposes only to peptide-silane surfaces approaches that of cells grown in chemical differentiation induction media.

Keywords: Bioactive Glass, Bioactive Peptide, MC3T3, Osteogenesis

Cite this paper: Przybylowski, C. , Ammar, M. , LeBlon, C. , Jedlicka, S. (2015) Osteogenesis of MC3T3 Preosteoblasts on 3D Bioactive Peptide Modified Nano-Macroporous Bioactive Glass Scaffolds. Journal of Biomaterials and Nanobiotechnology, 6, 146-159. doi: 10.4236/jbnb.2015.63015.

References

[1]   Hattar, S., Asselin, A., Greenspan, D., Oboeuf, M., Berdal, A. and Sautier, J.-M. (2005) Potential of Biomimetic Surfaces to Promote in Vitro Osteoblast-Like Cell Differentiation. Biomaterials, 26, 839-848.
http://dx.doi.org/10.1016/j.biomaterials.2004.03.026

[2]   Hench, L.L., Xynos, I.D., Edgar, A.J., Buttery, L.D.K., Polak, J.M., Zhong, J.-P., et al. (2002) Gene Activating Glasses. Journal of Inorganic Materials, 17, 897-909.
http://www.jim.org.cn/EN/Y2002/V17/I5/897

[3]   Xynos, I.D., Edgar, A.J., Buttery, L.D.K., Hench, L.L. and Polak, J.M. (2000) Ionic Products of Bioactive Glass Dissolution Increase Proliferation of Human Osteoblasts and Induce Insulin-Like Growth Factor II mRNA Expression and Protein Synthesis. Biochemical and Biophysical Research Communications, 276, 461-465.
http://dx.doi.org/10.1006/bbrc.2000.3503

[4]   Barboza, E.P., Caúla, A.L., de Oliveira Caúla, F., de Souza, R.O., Neto, L.G., Sorensen, R.G., et al. (2004) Effect of Recombinant Human Bone Morphogenetic Protein-2 in an Absorbable Collagen Sponge with Space-Providing Biomaterials on the Augmentation of Chronic Alveolar Ridge Defects. Journal of Periodontology, 75, 702-708.
http://dx.doi.org/10.1902/jop.2004.75.5.702

[5]   Bergeron, E., Marquis, M.E., Chrétien, I. and Faucheux, N. (2007) Differentiation of Preosteoblasts Using a Delivery System with BMPs and Bioactive Glass Microspheres. Journal of Materials Science: Materials in Medicine, 18, 255- 263.
http://dx.doi.org/10.1007/s10856-006-0687-4

[6]   Best, S.M., Porter, A.E., Thian, E.S. and Huang, J. (2008) Bioceramics: Past, Present and for the Future. Journal of the European Ceramic Society, 28, 1319-1327.
http://dx.doi.org/10.1016/j.jeurceramsoc.2007.12.001

[7]   Blaker, J.J., Gough, J.E., Maquet, V., Notingher, I. and Boccaccini, A.R. (2003) In Vitro Evaluation of Novel Bioactive Composites Based on Bioglass (R)-Filled Polylactide Foams for Bone Tissue Engineering Scaffolds. Journal of Biomedical Materials Research Part A, 67A, 1401-1411.
http://dx.doi.org/10.1002/jbm.a.20055

[8]   Blaker, J.J., Nazhat, S.N. and Boccaccini, A.R. (2004) Development and Characterisation of Silver-Doped Bioactive Glasscoated Sutures for Tissue Engineering and Wound Healing Applications. Biomaterials, 25, 1319-1329.
http://dx.doi.org/10.1016/j.biomaterials.2003.08.007

[9]   Chen, Q.Z.Z., Thompson, I.D. and Boccaccini, A.R. (2006) 45S5 Bioglass—Derived Glass—Ceramic Scaffolds for Bone Tissue Engineering. Biomaterials, 27, 2414-2425.
http://dx.doi.org/10.1016/j.biomaterials.2005.11.025

[10]   Hench, L.L. (2009) Genetic Design of Bioactive Glass. Journal of the European Ceramic Society, 29, 1257-1265.
http://dx.doi.org/10.1016/j.jeurceramsoc.2008.08.002

[11]   Hench, L.L. (1998) Bioceramics. Journal of the American Ceramic Society, 81, 1705-1728.

http://dx.doi.org/10.1111/j.1151-2916.1998.tb02540.x

[12]   Hench, L.L., Xynos, I.D. and Polak, J.M. (2004) Bioactive Glasses for in Situ Tissue Regeneration. Journal of Biomaterials Science-Polymer Edition, 15, 543-562.
http://dx.doi.org/10.1163/156856204323005352

[13]   Knabe, C., Stiller, M., Berger, G., Reif, D., Gildenhaar, R., Howlett, C.R. and Zreiqat, H. (2005) The Effect of Bioactive Glass Ceramics on the Expression of Bone-Related Genes and Proteins in Vitro. Clinical Oral Implants Research, 16, 119-127.
http://dx.doi.org/10.1111/j.1600-0501.2004.01066.x

[14]   Xynos, I.D., Hukkanen, M.V.J., Batten, J.J., Buttery, L.D., Hench, L.L. and Polak, J.M. (2000) Bioglass 45S5 Stimulates Osteoblast Turnover and Enhances Bone Formation in Vitro: Implications and Applications for Bone Tissue Engineering. Calcified Tissue International, 67, 321-329.
http://dx.doi.org/10.1007/s002230001134

[15]   Neo, M., Nakamura, T., Ohtsuki, C., Kokubo, T. and Yamamuro, T. (1993) Apatite Formation on 3 Kinds of Bioactive Material at an Early Stage in Vivo—A Comparative Study by Transmission Electron Microscopy. Journal of Biomedical Materials Research, 27, 999-1006.
http://dx.doi.org/10.1002/jbm.820270805

[16]   Maquet, V., Boccaccini, A.R., Pravata, L., Notingher, I. and Jérme, R. (2003) Preparation, Characterization, and in Vitro Degradation of Bio-Resorbable and Bioactive Composites Based on Bioglass-Filled Polylactide Foams. Journal of Biomedical Materials Research Part A, 66A, 335-346.
http://dx.doi.org/10.1002/jbm.a.10587

[17]   Marques, A.C., Almeidaa, R.M., Thiema, A., Wang, S.J., Falk, M. and Jain, H. (2009) Sol-Gel-Derived Glass Scaffold with High Pore Interconnectivity and Enhanced Bioactivity. Journal of Materials Research, 24, 3495-3502.
http://dx.doi.org/10.1557/jmr.2009.0440

[18]   Garcia, A.J., Ducheyne, P. and Boettiger, D. (1998) Effect of Surface Reaction Stage on Fibronectin-Mediated Adhesion of Osteoblast-Like Cells to Bioactive Glass. Journal of Biomedical Materials Research, 40, 48-56.
http://dx.doi.org/10.1002/(SICI)1097-4636(199804)40:1<48::AID-JBM6>3.0.CO;2-R

[19]   Zhang, K., Wang, Y.B., Hillmyer, M.A. and Francis, L.F. (2004) Processing and Properties of Porous Poly(L-lactide)/ Bioactive Glass Composites. Biomaterials, 25, 2489-2500.
http://dx.doi.org/10.1016/j.biomaterials.2003.09.033

[20]   Arcos, D., Ragel, C.V. and Vallet-Regi, M. (2001) Bioactivity in Glass/PMMA Composites Used as Drug Delivery System. Biomaterials, 22, 701-708.
http://dx.doi.org/10.1016/S0142-9612(00)00233-7

[21]   Boccaccini, A.R. and Maquet, V. (2003) Bioresorbable and Bioactive Polymer/Bioglass Composites with Tailored Pore Structure for Tissue Engineering Applications. Composites Science and Technology, 63, 2417-2429.
http://dx.doi.org/10.1016/S0266-3538(03)00275-6

[22]   Yao, J., Radina, S., Leboy, P.S. and Ducheyne, P. (2005) The Effect of Bioactive Glass Content on Synthesis and Bioactivity of Composite Poly (Lactic-Co-Glycolic Acid)/Bioactive Glass Substrate for Tissue Engineering. Biomaterials, 26, 1935-1943.
http://dx.doi.org/10.1016/j.biomaterials.2004.06.027

[23]   Kontonasaki, E., Sivropoulou, A., Papadopoulou, L., Garefis, P., Paraskevopoulos, K.M. and Koidis, P. (2006) Attachment and Proliferation of Human Periodontal Ligament Fibroblasts on Fibronectin-Coated Bioactive Glass Modified Ceramics. Bioceramics, 18, 727-730.

[24]   Kaufmann, E., Ducheyne, P. and Shapiro, I.M. (2000) Evaluation of Osteoblast Response to Porous Bioactive Glass (45S5) Substrates by RT-PCR Analysis. Tissue Engineering, 6, 19-28.
http://dx.doi.org/10.1089/107632700320856

[25]   Lu, H.H., El-Amin, S.F., Scott, K.D. and Laurencin, C.T. (2003) Three-Dimensional, Bioactive, Biodegradable, Polymer- Bioactive Glass Composite Scaffolds with Improved Mechanical Properties Support Collagen Synthesis and Mineralization of Human Osteoblast-Like Cells in Vitro. Journal of Biomedical Materials Research Part A, 64A, 465-474.
http://dx.doi.org/10.1002/jbm.a.10399

[26]   Moura, J., Teixeira, L.N., Ravagnani, C., Peitl, O., Zanotto, E.D., Beloti, M.M., et al. (2007) In Vitro Osteogenesis on a Highly Bioactive Glass-Ceramic (Biosilicate). Journal of Biomedical Materials Research Part A, 82A, 545-557.
http://dx.doi.org/10.1002/jbm.a.31165

[27]   Oonishi, H., Kushitani, S., Yasukawa, E., Iwaki, H., Hench, L.L., Wilson, J., et al. (1997) Particulate Bioglass Compared with Hydroxyapatite as a Bone Graft Substitute. Clinical Orthopaedics and Related Research, 334, 316-325.
http://www.ncbi.nlm.nih.gov/pubmed/9005929
http://dx.doi.org/10.1097/00003086-199701000-00041

[28]   Sepulveda, P., Jones, J.R. and Hench, L.L. (2001) Characterization of Melt-Derived 45S5 and Sol-Gel-Derived 58S Bioactive Glasses. Journal of Biomedical Materials Research, 58, 734-740.
http://dx.doi.org/10.1002/jbm.10026

[29]   Silver, I.A., Deas, J. and Erecinska, M. (2001) Interactions of Bioactive Glasses with Osteoblasts in Vitro: Effects of 45S5 Bioglass, and 58S and 77S Bioactive Glasses on Metabolism, Intracellular Ion Concentrations and Cell Viability. Biomaterials, 22, 175-185.
http://dx.doi.org/10.1016/S0142-9612(00)00173-3

[30]   Varanasi, V.G., Saizb, E., Loomer, P.M., Ancheta, B., Uritani, N., Ho, S.P., et al. (2009) Enhanced Osteocalcin Expression by Osteoblast-Like Cells (MC3T3-E1) Exposed to Bioactive Coating Glass (SiO2-CaO-P2O5-MgO-K2O-Na2O System) Ions. Acta Biomaterialia, 5, 3536-3547.
http://dx.doi.org/10.1016/j.actbio.2009.05.035

[31]   Verrier, S., Blakera, J.J., Maquet, V., Hench, L.L. and Boccaccini, A.R. (2004) PDLLA/Bioglass Composites for Soft-Tissue and Hard-Tissue Engineering: An in Vitro Cell Biology Assessment. Biomaterials, 25, 3013-3021.
http://dx.doi.org/10.1016/j.biomaterials.2003.09.081

[32]   Vogel, M., Voigta, C., Gross, U.M. and Müller-Mai, C.M. (2001) In Vivo Comparison of Bioactive Glass Particles in Rabbits. Biomaterials, 22, 357-362.
http://dx.doi.org/10.1016/S0142-9612(00)00191-5

[33]   Yuan, H.P., de Bruijn, J.D., Zhang, X.D., van Blitterswijk, C.A. and de Groot, K. (2001) Bone Induction by Porous Glass Ceramic Made from Bioglass (45S5). Journal of Biomedical Materials Research, 58, 270- 276.
http://www.ncbi.nlm.nih.gov/pubmed/11319740
http://dx.doi.org/10.1002/1097-4636(2001)58:3<270::AID-JBM1016>3.0.CO;2-2

[34]   Gao, T.J., Arob, H.T., Ylnen, H. and Vuorio, E. (2001) Silica-Based Bioactive Glasses Modulate Expression of Bone Morphogenetic Protein-2 mRNA in Saos-2 Osteoblasts in Vitro. Biomaterials, 22, 1475-1483.
http://dx.doi.org/10.1016/S0142-9612(00)00288-X

[35]   Kim, H.W., Lee, H.H. and Knowles, J.C. (2008) Nanofibrous Glass Tailored with Apatite-Fibronectin Interface for Bone Cell Stimulation. Journal of Nanoscience and Nanotechnology, 8, 3013-3019.
http://www.ncbi.nlm.nih.gov/pubmed/18681040
http://dx.doi.org/10.1166/jnn.2008.106

[36]   Leach, J.K., Kaiglerb, D., Wang, Z., Krebsbach, P.H. and Mooney, D.J. (2006) Coating of VEGF-Releasing Scaffolds with Bioactive Glass for Angiogenesis and Bone Regeneration. Biomaterials, 27, 3249-3255.
http://dx.doi.org/10.1016/j.biomaterials.2006.01.033

[37]   Bacakova, L., Filova, E., Kubies, D., Machova, L., Proks, V., Malinova, V., et al. (2007) Adhesion and Growth of Vascular Smooth Muscle Cells in Cultures on Bioactive RGD Peptide-Carrying Polylactides. Journal of Materials Science-Materials in Medicine, 18, 1317-1323.
http://dx.doi.org/10.1007/s10856-006-0074-1

[38]   De Giglio, E., Sabbatinib, L., Colucci, S. and Zambonin, G. (2000) Synthesis, Analytical Characterization, and Osteoblast Adhesion Properties on RGD-Grafted Polypyrrole Coatings on Titanium Substrates. Journal of Biomaterials Science, Polymer Edition, 11, 1073-1083.
http://dx.doi.org/10.1163/156856200743580

[39]   Morgan, A.W., Roskova, K.E., Lin-Gibson, S., Kaplan, D.L., Becker, M.L. and Simon Jr., C.G. (2008) Characterization and Optimization of RGD-Containing Silk Blends to Support Osteoblastic Differentiation. Biomaterials, 29, 2556- 2563.
http://dx.doi.org/10.1016/j.biomaterials.2008.02.007

[40]   Itoh, D., Yoneda, S., Kuroda, S., Kondo, H., Umezawa, A., Ohya, K., et al. (2002) Enhancement of Osteogenesis on Hydroxyapatite Surface Coated with Synthetic Peptide (EEEEEEEPRGDT) in Vitro. Journal of Biomedical Materials Research, 62, 292-298.
http://dx.doi.org/10.1002/jbm.10338

[41]   Moursi, A.M., Damsky, C.H., Lull, J., Zimmerman, D., Doty, S.B., Aota, S., et al. (1996) Fibronectin Regulates Calvarialosteoblast Differentiation. Journal of Cell Science, 109, 1369-1380.
http://www.ncbi.nlm.nih.gov/pubmed/8799825

[42]   Yang, X.B., Roacha, H.I., Clarke, N.M.P., Howdle, S.M., Quirk, R., Shakesheff, K.M., et al. (2001) Human Osteoprogenitor Growth and Differentiation on Synthetic Biodegradable Structures after Surface Modification. Bone, 29, 523- 531.
http://dx.doi.org/10.1016/S8756-3282(01)00617-2

[43]   Massia, S.P. and Hubbell, J.A. (1991) An RGD Spacing of 440 nm Is Sufficient for Integrin Alpha V Beta 3-Mediated Fibroblast Spreading and 140 nm for Focal Contact and Stress Fiber Formation. Journal of Cell Biology, 114, 1089- 1100.
http://www.ncbi.nlm.nih.gov/pubmed/1714913
http://dx.doi.org/10.1083/jcb.114.5.1089

[44]   Chen, D., Zhao, M. and Mundy, G.R. (2004) Bone Morphogenetic Proteins. Growth Factors, 22, 233-241.
http://www.ncbi.nlm.nih.gov/pubmed/15621726
http://dx.doi.org/10.1080/08977190412331279890

[45]   Cheng, H.W., Jiang, W., Phillips, F.M., Haydon, R.C., Peng, Y., Zhou, L., et al. (2003) Osteogenic Activity of the Fourteen Types of Human Bone Morphogenetic Proteins (BMPs). Journal of Bone and Joint Surgery, American Volume, 85-A, 1544-1552.
http://www.ncbi.nlm.nih.gov/pubmed/12925636

[46]   Lee, K.S., Kim, H.J., Li, Q.L., Chi, X.Z., Ueta, C., Komori, T., et al. (2000) Runx2 Is a Common Target of Transforming Growth Factor Beta1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotentmesenchymal Precursor Cell Line C2C12. Molecular and Cellular Biology, 20, 8783- 8792.
http://www.ncbi.nlm.nih.gov/pubmed/11073979
http://dx.doi.org/10.1128/MCB.20.23.8783-8792.2000

[47]   Wozney, J.M. (1992) The Bone Morphogenetic Protein Family and Osteogenesis. Molecular Reproduction and Deve- lopment, 32, 160-167.
http://dx.doi.org/10.1002/mrd.1080320212

[48]   Lee, J., Lee, J. and Murphy, W. (2010) Modular Peptides Promote Human Mesenchymal Stem Cell Differentiation on Biomaterial Surfaces. Acta Biomateriala, 6, 21-28.
http://dx.doi.org/10.1016/j.actbio.2009.08.003

[49]   Jedlicka, S.S., Rickus, J.L. and Zemyanov, D.Y. (2007) Surface Analysis by X-Ray Photoelectron Spectroscopy of Sol-Gel Silica Modified with Covalently Bound Peptides. Journal of Physical Chemistry B, 111, 11850-11857.

http://dx.doi.org/10.1021/jp0744230

[50]   Jedlicka, S.S., Little, K.M., Nivens, D.E., Zemlyanov, D. and Ri, J.L. (2007) Peptide Ormosils as Cellular Substrates. Journal of Materials Chemistry, 17, 5058-5067.
http://dx.doi.org/10.1039/b705393b

[51]   Vueva, Y., Gama, A., Teixeira, A.V., Almeida, R.M., Wang, S.J., Falk, M.M., et al. (2010) Monolithic Glass Scaffolds with Dual Porosity Prepared by Polymer-Induced Phase Separation and Sol-Gel. Journal of the American Ceramic Society, 93, 1945-1949.
http://dx.doi.org/10.1111/j.1551-2916.2010.03697.x

[52]   Wang, S., Falk, M.M., Rashad, A., Saad, M.M., Marques, A.C., Almeida, R.M., et al. (2011) Evaluation of 3D Nano- Macro Porous Bioactive Glass Scaffold for Hard Tissue Engineering. Journal of Materials Science-Materials in Medicine, 22, 1195-1203.
http://www.ncbi.nlm.nih.gov/pubmed/21445655
http://dx.doi.org/10.1007/s10856-011-4297-4

[53]   Wang, S.J. and Jain, H. (2010) High Surface Area Nano-Macroporous Bioactive Glass Scaffold for Hard Tissue Engineering. Journal of the American Ceramic Society, 93, 3002-3005.
http://dx.doi.org/10.1111/j.1551-2916.2010.03970.x

[54]   Hamid, R., Rotshteyn, Y., Rabadi, L., Parikh, R. and Bullock, P. (2004) Comparison of Alamar Blue and MTT Assays for High Through-Put Screening. Toxicology in Vitro, 18, 703-710.
http://dx.doi.org/10.1016/j.tiv.2004.03.012

[55]   Benesch, J., Mano, J.F. and Reis, R.L. (2008) Proteins and Their Peptide Motifs in Acellular Apatite Mineralization of Scaffolds for Tissue Engineering. Tissue Engineering Part B-Reviews, 14, 433-445.
http://dx.doi.org/10.1089/ten.teb.2008.0121

[56]   Takeuchi, A., Ohtsuki, C., Kamitakahara, M., Ogata, S.-I., Miyazaki, T. and Tanihara, M. (2008) Biomimetic Deposition of Hydroxyapatite on a Synthetic Polypeptide with Beta Sheet Structure in a Solution Mimicking Body Fluid. Journal of Materials Science: Materials in Medicine, 19, 387-393.
http://www.ncbi.nlm.nih.gov/pubmed/17607510
http://dx.doi.org/10.1007/s10856-007-3179-2

[57]   Lian, J.B., Javed, A., Zaidi, S.K., Lengner, C., Montecino, M., van Wijnen, A.J., et al. (2004) Regulatory Controls for Osteoblast Growth and Differentiation: Role of Runx/Cbfa/AML Factors. Critical Reviews in Eukaryotic Gene Expression, 14, 1-41.
http://www.ncbi.nlm.nih.gov/pubmed/15104525

[58]   Ducy, P., Geoffroy, V. and Karsenty, G. (1996) Study of Osteoblast-Specific Expression of one Mouse Osteocalcin Gene: Characterization of the Factor Binding to OSE2. Connective Tissue Research, 35, 7-14.
http://www.ncbi.nlm.nih.gov/pubmed/9084638
http://dx.doi.org/10.3109/03008209609029169

[59]   Gaur, T., Lengner, C.J., Hovhannisyan, H., Bhat, R.A., Bodine, P.V.N., Komm, B.S., et al. (2005) Canonical WNT Signaling Promotes Osteogenesis by Directly Stimulating Runx2 Gene Expression. Journal of Biological Chemistry, 280, 33132-33140.
http://www.ncbi.nlm.nih.gov/pubmed/16043491
http://dx.doi.org/10.1074/jbc.m500608200

[60]   Hanada, K., Dennis, J.E. and Caplan, A.I. (1997) Stimulatory Effects of Basic Fibroblast Growth Factor and Bone Morphogenetic Protein-2 on Osteogenic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells. Jour- nal of Bone and Mineral Research, 12, 1606-1614.
http://www.ncbi.nlm.nih.gov/pubmed/9333121
http://dx.doi.org/10.1359/jbmr.1997.12.10.1606

[61]   Banerjee, C., Javed, A., Choi, J.Y., Green, J., Rosen, V., van Wijnen, A.J., et al. (2001) Differential Regulation of the Two Principal Runx2/Cbfa1 N-Terminal Isoforms in Response to Bone Morphogenetic Protein-2 during Development of the Osteoblast Phenotype. Endocrinology, 142, 4026-4039.
http://www.ncbi.nlm.nih.gov/pubmed/11517182
http://dx.doi.org/10.1210/endo.142.9.8367

[62]   Ito, Y. and Miyazono, K. (2003) RUNX Transcription Factors as Key Targets of TGF-Beta Superfamily Signaling. Current Opinion in Genetics & Development, 13, 43-47.
http://dx.doi.org/10.1016/S0959-437X(03)00007-8

[63]   Hoffmann, A., Preobrazhenska, O., Wodarczyk, C., Medler, Y., Winkel, A., Shahab, S., et al. (2005) Transforming Growth Factor-Beta-Activated Kinase-1 (TAK1), a MAP3K, Interacts with Smad Proteins and Interferes with Osteogenesis in Murinemesenchymal Progenitors. Journal of Biological Chemistry, 280, 27271-27283.
http://dx.doi.org/10.1074/jbc.M503368200

[64]   Li, J., Tsuji, K., Komori, T., Miyazono, K., Wrana, J.L., Ito, Y., et al. (1998) Smad2 Overexpression Enhances Smad4 Gene Expression and Suppresses CBFA1 Gene Expression in Osteoblasticosteosarcoma ROS17/2.8 Cells and Primary Rat Calvaria Cells. Journal of Biological Chemistry, 273, 31009-31015.
http://dx.doi.org/10.1074/jbc.273.47.31009

[65]   Stein, G.S. and Lian, J.B. (1993) Molecular Mechanisms Mediating Proliferation Differentiation Interrelationships during Progressive Development of the Osteoblast Phenotype. Endocrine Reviews, 14, 424-442.
http://www.ncbi.nlm.nih.gov/pubmed/8223340
http://dx.doi.org/10.1210/edrv-14-4-424

[66]   Stein, G.S. and Lian, J.B. (1993) Cellular and Molecular Biology of Bone. Academic Press, San Diego.