Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL) composite nanofibers were fabricated with two different HAp concentrations—1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10% HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type I collagen expression decreased on HAp scaffolds. These results indicate that cells are sensitive to minor changes in mineral content within nanofibers, even at just 1% w/w, and elucidating the sensing mechanism may lead to optimized osteogenic scaffold designs.
References
[1]
Drosse, I.; Volkmer, E.; Capanna, R.; de Biase, P.; Mutschler, W.; Schieker, M. Tissue engineering for bone defect healing: an update on a multi-component approach. Injury 2008, 39, 9–20.
[2]
Heino, T.J.; Hentunen, T.A. Differentiation of osteoblasts and osteocytes from mesenchymal stem cells. Curr. Stem. Cell Res. Ther. 2008, 3, 131–145, doi:10.2174/157488808784223032.
[3]
Haynesworth, S.E.; Goshima, J.; Goldberg, V.M.; Caplan, A.L. Characterization of cells with osteogenic potential from human marrow. Bone 1992, 13, 81–88.
[4]
Prockop, D. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997, 276, 71–74, doi:10.1126/science.276.5309.71.
[5]
Jorgensen, N.R.; Henriksen, Z.; Sorensen, O.H.; Civitelli, R. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: Validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids 2004, 69, 219–226, doi:10.1016/j.steroids.2003.12.005.
[6]
Johnson, M.R.; Lee, H.J.; Bellamkonda, R.V.; Guldberg, R.E. Sustained release of BMP-2 in a lipid-based microtube vehicle. Acta Biomater. 2009, 5, 23–28, doi:10.1016/j.actbio.2008.09.001.
[7]
Laflamme, C.; Rouabhia, M. Effect of BMP-2 and BMP-7 homodimers and a mixture of BMP-2/BMP-7 homodimers on osteoblast adhesion and growth following culture on a collagen scaffold. Biomed. Mater. 2008, 3, doi:10.1088/1748-6041/3/1/015008.
[8]
Laurencin, C.T.; Attawia, M.A.; Lu, L.Q.; Borden, M.D.; Lu, H.H.; Gorum, W.J.; Lieberman, J.R. Poly(lactide-co-glycolide)/hydroxyapatite delivery of BMP-2-producing cells: A regional gene therapy approach to bone regeneration. Biomaterials 2001, 22, 1271–1277, doi:10.1016/S0142-9612(00)00279-9.
[9]
Lee, J.Y.; Choo, J.E.; Choi, Y.S.; Suh, J.S.; Lee, S.J.; Chung, C.P.; Park, Y.J. Osteoblastic differentiation of human bone marrow stromal cells in self-assembled BMP-2 receptor-binding peptide-amphiphiles. Biomaterials 2009, 30, 3532–3541, doi:10.1016/j.biomaterials.2009.03.018.
[10]
Olivares-Navarrete, R.; Raz, P.; Zhao, G.; Chen, J.; Wieland, M.; Cochran, D.L.; Chaudhri, R.A.; Ornoy, A.; Boyan, B.D.; Schwartz, Z. Integrin alpha2beta1 plays a critical role in osteoblast response to micron-scale surface structure and surface energy of titanium substrates. Proc. Natl. Acad. Sci. USA 2008, 105, 15767–15772.
[11]
Marletta, G.; Ciapetti, G.; Satriano, C.; Pagani, S.; Baldini, N. The effect of irradiation modification and RGD sequence adsorption on the response of human osteoblasts to polycaprolactone. Biomaterials 2005, 26, 4793–4804, doi:10.1016/j.biomaterials.2004.11.047.
[12]
Dalby, M.J.; Gadegaard, N.; Tare, R.; Andar, A.; Riehle, M.O.; Herzyk, P.; Wilkinson, C.D.; Oreffo, R.O. The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. Nat. Mater. 2007, 6, 997–1003, doi:10.1038/nmat2013.
[13]
Biggs, M.J.; Richards, R.G.; Gadegaard, N.; Wilkinson, C.D.; Oreffo, R.O.; Dalby, M.J. The use of nanoscale topography to modulate the dynamics of adhesion formation in primary osteoblasts and ERK/MAPK signalling in STRO-1+ enriched skeletal stem cells. Biomaterials 2009, 30, 5094–5103, doi:10.1016/j.biomaterials.2009.05.049.
[14]
Olivares-Navarrete, R.; Hyzy, S.L.; Hutton, D.L.; Erdman, C.P.; Wieland, M.; Boyan, B.D.; Schwartz, Z. Direct and indirect effects of microstructured titanium substrates on the induction of mesenchymal stem cell differentiation towards the osteoblast lineage. Biomaterials 2010, 31, 2728–2735.
[15]
Ruckh, T.T.; Kumar, K.; Kipper, M.J.; Popat, K.C. Osteogenic differentiation of bone marrow stromal cells on poly(epsilon-caprolactone) nanofiber scaffolds. Acta Biomater. 2010.
[16]
Wutticharoenmongkol, P.; Pavasant, P.; Supaphol, P. Osteoblastic phenotype expression of MC3T3-E1 cultured on electrospun polycaprolactone fiber mats filled with hydroxyapatite nanoparticles. Biomacromolecules 2007, 8, 2602–2610, doi:10.1021/bm700451p.
[17]
Zhang, P.; Hong, Z.; Yu, T.; Chen, X.; Jing, X. In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-co-glycolide) and hydroxyapatite surface-grafted with poly(l-lactide). Biomaterials 2008.
[18]
Lee, J.H.; Rim, N.G.; Jung, H.S.; Shin, H. Control of osteogenic differentiation and mineralization of human mesenchymal stem cells on composite nanofibers containing poly[lactic-co-(glycolic acid)] and hydroxyapatite. Macromol. Biosci. 2010, 10, 173–182.
[19]
Gupta, D.; Venugopal, J.; Mitra, S.; Giri Dev, V.R.; Ramakrishna, S. Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts. Biomaterials 2009, 30, 2085–2094.
[20]
Ramakrishna, S.; Fujihara, K.; Teo, W.-E.; Lim, T.-C.; Ma, Z. An Introduction to Electrospinning and Nanofibers; World Scientific: Singapore, 2005; p. 382.
[21]
Rutledge, G.C.; Fridrikh, S.V. Formation of fibers by electrospinning. Adv. Drug Deliv. Rev. 2007, 59, 1384–1391, doi:10.1016/j.addr.2007.04.020.
[22]
Theron, S.A.; Zussman, E.; Yarin, A.L. Experimental investigation of the governing parameters in the electrospinning of polymer solutions. Polymer 2004, 45, 2017–2030.
[23]
Sinha, V.R.; Bansal, K.; Kaushik, R.; Kumria, R.; Trehan, A. Poly-e-caprolactone microspheres and nanospheres: An overview. Int. J. Pharm. 2004, 278, 1–23.
[24]
Zeng, J.; Xu, X.; Chen, X.; Liang, Q.; Bian, X.; Yang, L.; Jing, X. Biodegradable electrospun fibers for drug delivery. J. Control. Release 2003, 92, 227–231, doi:10.1016/S0168-3659(03)00372-9.
[25]
Pulkkinen, M.; Malin, M.; Tarvainen, T.; Saarim？ki, T.; Sepp？l？, J.; J？rvinen, K. Effects of block length on the enzymatic degradation and erosion of oxazoline linked poly-e-caprolactone. Eur. J. Pharm. Sci. 2007, 31, 119–128, doi:10.1016/j.ejps.2007.03.001.
[26]
Sangsanoh, P.; Waleetorncheepsawat, S.; Suwantong, O.; Wutticharoenmongkol, P.; Weeranantanapan, O.; Chuenjitbuntaworn, B.; Cheepsunthorn, P.; Pavasant, P.; Supaphol, P. In vitro biocompatibility of schwann cells on surfaces of biocompatible polymeric electrospun fibrous and solution-cast film scaffolds. Biomacromolecules 2007, 8, 1587–1594, doi:10.1021/bm061152a.
[27]
Palmer, L.C.; Newcomb, C.J.; Kaltz, S.R.; Spoerke, E.D.; Stupp, S.I. Biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel. Chem. Rev. 2008, 108, 4754–4783.
[28]
Anderson, H.C.; Sipe, J.B.; Hessle, L.; Dhanyamraju, R.; Atti, E.; Camacho, N.P.; Millan, J.L. Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. Am. J. Pathol. 2004, 164, 841–847.
[29]
Genge, B.R.; Wu, L.N.; Wuthier, R.E. In vitro modeling of matrix vesicle nucleation: Synergistic stimulation of mineral formation by annexin A5 and phosphatidylserine. J. Biol. Chem. 2007, 282, 26035–26045.
[30]
Nikolov, S.; Raabe, D. Hierarchical modeling of the elastic properties of bone at submicron scales: The role of extrafibrillar mineralization. Biophys. J. 2008, 94, 4220–4232, doi:10.1529/biophysj.107.125567.
[31]
Norman, J.; Shapter, J.G.; Short, K.; Smith, L.J.; Fazzalari, N.L. Micromechanical properties of human trabecular bone: A hierarchical investigation using nanoindentation. J. Biomed. Mater. Res. A 2008, 87, 196–202.
[32]
McCreadie, B.R.; Goulet, R.W.; Feldkamp, L.A.; Goldstein, S.A. Hierarchical structure of bone and micro-computed tomography. Adv. Exp. Med. Biol. 2001, 496, 67–83.
[33]
White, D.J.; Puranen, S.; Johnson, M.S.; Heino, J. The collagen receptor subfamily of the integrins. Int. J. Biochem. Cell Biol. 2004, 36, 1405–1410, doi:10.1016/j.biocel.2003.08.016.
[34]
Denhardt, D.T.; Noda, M. Osteopontin expression and function: Role in bone remodeling. J. Cell Biochem. Suppl. 1998, 30-31, 92–102, doi:10.1002/(SICI)1097-4644(1998)72:30/31+<92::AID-JCB13>3.0.CO;2-A.
[35]
Kazanecki, C.C.; Uzwiak, D.J.; Denhardt, D.T. Control of osteopontin signaling and function by post-translational phosphorylation and protein folding. J. Cell Biochem. 2007, 102, 912–924, doi:10.1002/jcb.21558.
[36]
McKee, M.D.; Nanci, A. Osteopontin: An interfacial extracellular matrix protein in mineralized tissues. Connect. Tissue Res. 1996, 35, 197–205, doi:10.3109/03008209609029192.
[37]
Cui, W.; al, e. In situ growth of hydroxyapatite within electrospun poly(DL-lactide) fibers. J. Biomed. Mater. Res. Part A 2007, 82, 831–841.
[38]
Fang, B.; Wan, Y.Z.; Tang, T.T.; Gao, C.; Dai, K.R. Proliferation and osteoblastic differentiation of human bone marrow stromal cells on hydroxyapatite/bacterial cellulose nanocomposite scaffolds. Tissue Eng. Part A 2009, 15, 1091–1098, doi:10.1089/ten.tea.2008.0110.
[39]
Guarino, V.; Taddei, P.; Di Foggia, M.; Fagnano, C.; Ciapetti, G.; Ambrosio, L. The influence of hydroxyapatite particles on in vitro degradation behaviour of pcl based composite scaffolds. Tissue Eng. Part A 2009, 15, 3655–3668, doi:10.1089/ten.tea.2008.0543.
Venugopal, J.R.; Low, S.; Choon, A.T.; Kumar, A.B.; Ramakrishna, S. Nanobioengineered electrospun composite nanofibers and osteoblasts for bone regeneration. Artif Organs 2008, 32, 388–397, doi:10.1111/j.1525-1594.2008.00557.x.
[42]
Kim, H.-W.; Lee, H.-H.; Knowles, J.C. Electrospinning biomedical nanocomposite fibers of hydroxyapaite/poly(lactic acid) for bone regeneration. J. Biomed. Mater. Res. Part A 2006, 79, 643–649.
[43]
Jackson, S.M.; Demer, L.L. Peroxisome proliferator-activated receptor activators modulate the osteoblastic maturation of MC3T3-E1 preosteoblasts. FEBS Lett. 2000, 471, 119–124, doi:10.1016/S0014-5793(00)01372-7.
[44]
Still, K.; Grabowski, P.; Mackie, I.; Perry, M.; Bishop, N. The peroxisome proliferator activator receptor alpha/delta agonists linoleic acid and bezafibrate upregulate osteoblast differentiation and induce periosteal bone formation in vivo. Calcif. Tissue Int. 2008, 83, 285–292, doi:10.1007/s00223-008-9175-9.
Berger, J.; Moller, D. The mechanisms of action of PPARs. Annu. Rev. Med. 2002, 53, 409–435, doi:10.1146/annurev.med.53.082901.104018.
[47]
Lecka-Czernik, B.; Suva, L.J. Resolving the two “bony” faces of PPAR-gamma. PPAR Res. 2006, doi:10.1155/PPAR/2006/27489.
[48]
Lin, T.-H.; Yang, R.-S.; Tang, C.-H.; Lin, C.-P.; Fu, W.-M. PPARg inhibits osteogenesis via the down-regulation of the expression of COX-2 and iNOS in rats. Bone 2007, 41, 562–574.
[49]
Syversen, U.; Bakke, I.; Aune, G.; Thommesen, L. PPAR-Alpha agonists increase bone mineral density in female rats. J. Bone Miner. Res. 2003, 18, S2–S56.
[50]
De Jong, D.S.; Vaes, B.L.; Dechering, K.J.; Feijen, A.; Hendriks, J.M.; Wehrens, R.; Mummery, C.L.; van Zoelen, E.J.; Olijve, W.; Steegenga, W.T. Identification of novel regulators associated with early-phase osteoblast differentiation. J. Bone Miner. Res. 2004, 19, 947–958.
[51]
Badami, A.S.; Kreke, M.R.; Thompson, M.S.; Riffle, J.S.; Goldstein, A.S. Effect of fiber diameter on spreading, proliferation, and differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates. Biomaterials 2006, 27, 596–606, doi:10.1016/j.biomaterials.2005.05.084.
[52]
Bashur, C.A.; Dahlgren, L.A.; Goldstein, A.S. Effect of fiber diameter and orientation on fibroblast morphology and proliferation on electrospun poly(D,L-lactic-co-glycolic acid) meshes. Biomaterials 2006, 27, 5681–5688, doi:10.1016/j.biomaterials.2006.07.005.
[53]
Tsiridis, E.; Upadhyay, N.; Giannoudis, P. Molecular aspects of fracture healing: Which are the important molecules? Injury 2007, 38, 11–25.
[54]
Chen, L.; Jiang, W.; Huang, J.; He, B.C.; Zuo, G.W.; Zhang, W.; Luo, Q.; Shi, Q.; Zhang, B.Q.; Wagner, E.R.; et al. Insulin-like growth factor 2 (IGF2) potentiates BMP9-induced osteogenic differentiation and bone formation. J. Bone Miner. Res. 2010, 25, 2447–2459, doi:10.1002/jbmr.133.
Ekholm, E.C.; Ravanti, L.; Kahari, V.; Paavolainen, P.; Penttinen, R.P. Expression of extracellular matrix genes: transforming growth factor (TGF)-beta1 and ras in tibial fracture healing of lathyritic rats. Bone 2000, 27, 551–557, doi:10.1016/S8756-3282(00)00359-8.
[57]
Hirakawa, K.; Hirota, S.; Ikeda, T.; Yamaguchi, A.; Takemura, T.; Nagoshi, J.; Yoshiki, S.; Suda, T.; Kitamura, Y.; Nomura, S. Localization of the mRNA for bone matrix proteins during fracture healing as determined by in situ hybridization. J. Bone Miner. Res. 1994, 9, 1551–1557.
[58]
Schuckert, K.H.; Jopp, S.; Teoh, S.H. Mandibular defect reconstruction using three-dimensional polycaprolactone scaffold in combination with platelet-rich plasma and recombinant human bone morphogenetic protein-2: De novo synthesis of bone in a single case. Tissue Eng. Part A 2009, 15, 493–499.
[59]
Thorwarth, M.; Rupprecht, S.; Falk, S.; Felszeghy, E.; Wiltfang, J.; Schlegel, K.A. Expression of bone matrix proteins during de novo bone formation using a bovine collagen and platelet-rich plasma (prp)—an immunohistochemical analysis. Biomaterials 2005, 26, 2575–2584.
Weiss, S.; Zimmermann, G.; Baumgart, R.; Kasten, P.; Bidlingmaier, M.; Henle, P. Systemic regulation of angiogenesis and matrix degradation in bone regeneration—distraction osteogenesis compared to rigid fracture healing. Bone 2005, 37, 781–790, doi:10.1016/j.bone.2005.06.014.
[62]
Henle, P.; Zimmermann, G.; Weiss, S. Matrix metalloproteinases and failed fracture healing. Bone 2005, 37, 791–798, doi:10.1016/j.bone.2005.06.015.
[63]
Coleman, J.E. Structure and mechanism of alkaline phosphatase. Annu. Rev. Biophys. Biomol. Struct. 1992, 21, 441–483, doi:10.1146/annurev.bb.21.060192.002301.
[64]
Orimo, H.; Shimada, T. The role of tissue-nonspecific alkaline phosphatase in the phosphate-induced activation of alkaline phosphatase and mineralization in SaOS-2 human osteoblast-like cells. Mol. Cell. Biochem. 2008, 315, 51–60, doi:10.1007/s11010-008-9788-3.
[65]
Kulterer, B.; Friedl, G.; Jandrositz, A.; Sanchez-Cabo, F.; Prokesch, A.; Paar, C.; Scheideler, M.; Windhager, R.; Preisegger, K.H.; Trajanoski, Z. Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation. BMC Genomics. 2007, 8, doi:10.1186/1471-2164-8-70.
[66]
Park, S.-H.; Kim, T.-L.; Ku, Y.; Chung, C.-P.; Han, S.-B.; Yu, J.-H.; Lee, S.-P.; Kim, H.-W.; Lee, H.-H. Effect of hydroxyapatite-coated nanofibrous membrane on the responses of human periodontal ligament fibroblast. J. Ceram. Soc. Jpn. 2008, 116, 31–35, doi:10.2109/jcersj2.116.31.
[67]
Venugopal, J.; Low, S.; Choon, A.; Kumar, A.; Ramakrishna, S. Electrospun-modified nanofibrous scaffolds for the mineralization of osteoblast cells. J. Biomed. Mater. Res. Part A 2007, 85, 408–417.
[68]
Salih, E.; Ashkar, S.; Gerstenfeld, L.C.; Glimcher, M.J. Protein kinases of cultured osteoblasts: Selectivity for the extracellular matrix proteins of bone and their catalytic competence for osteopontin. J. Bone Miner. Res. 1996, 11, 1461–1473.
[69]
Katayama, Y.; House, C.M.; Udagawa, N.; Kazama, J.J.; McFarland, R.J.; Martin, T.J.; Findlay, D.M. Casein kinase 2 phosphorylation of recombinant rat osteopontin enhances adhesion of osteoclasts but not osteoblasts. J. Cell Physiol. 1998, 176, 179–187, doi:10.1002/(SICI)1097-4652(199807)176:1<179::AID-JCP19>3.0.CO;2-2.
[70]
Wang, J.; Glimcher, M.J.; Mah, J.; Zhou, H.Y.; Salih, E. Expression of bone microsomal casein kinase II, bone sialoprotein, and osteopontin during the repair of calvarial defects. Bone 1998, 22, 621–628, doi:10.1016/S8756-3282(98)00057-X.
[71]
Liu, F.; Malaval, L.; Aubin, J.E. Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation. J. Cell Sci. 2003, 116, 1787–1796, doi:10.1242/jcs.00376.
[72]
Hamada, K.; Hirose, M.; Yamashita, T.; Ohgushi, H. Spatial distribution of mineralized bone matrix produced by marrow mesenchymal stem cells in self-assembling peptide hydrogel scaffold. J. Biomed. Mater. Res. A 2008, 84, 128–136.
[73]
Robey, P.G.; Fedarko, N.S.; Hefferan, T.E.; Bianco, P.; Vetter, U.K.; Grzesik, W.; Friedenstein, A.; van der Pluijm, G.; Mintz, K.P.; Young, M.F.; et al. Structure and molecular regulation of bone matrix proteins. J. Bone Miner. Res. 1993, 8, 483–487.
[74]
Young, M.F. Bone matrix proteins: Their function, regulation, and relationship to osteoporosis. Osteoporos. Int. 2003, 14, 35–42.
[75]
Qin, C.; Baba, O.; Butler, W.T. Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis. Crit. Rev. Oral Biol. Med. 2004, 15, 126–136, doi:10.1177/154411130401500302.
[76]
Keykhosravani, M.; Doherty-Kirby, A.; Zhang, C.; Brewer, D.; Goldberg, H.A.; Hunter, G.K.; Lajoie, G. Comprehensive identification of post-translational modifications of rat bone osteopontin by mass spectrometry. Biochemistry 2005, 44, 6990–7003.
[77]
Goldberg, H.A.; Hunter, G.K. The inhibitory activity of osteopontin on hydroxyapatite formation in vitro. Ann. NY. Acad. Sci. 1995, 760, 305–308, doi:10.1111/j.1749-6632.1995.tb44642.x.
[78]
Pampena, D.A.; Robertson, K.A.; Litvinova, O.; Lajoie, G.; Goldberg, H.A.; Hunter, G.K. Inhibition of hydroxyapatite formation by osteopontin phosphopeptides. Biochem. J. 2004, 378, 1083–1087.
[79]
Rajachar, R.M.; Truong, A.Q.; Giachelli, C.M. The influence of surface mineral and osteopontin on the formation and function of murine bone marrow-derived osteoclasts. J. Mater. Sci. Mater. Med. 2008, 19, 3279–3285, doi:10.1007/s10856-008-3455-9.
[80]
Sodek, J.; Zhu, B.; Huynh, M.H.; Brown, T.J.; Ringuette, M. Novel functions of the matricellular proteins osteopontin and osteonectin/SPARC. Connect. Tissue Res. 2002, 43, 308–319.
[81]
Taller, A.; Grohe, B.; Rogers, K.A.; Goldberg, H.A.; Hunter, G.K. Specific adsorption of osteopontin and synthetic polypeptides to calcium oxalate monohydrate crystals. Biophys. J. 2007, 93, 1768–1777, doi:10.1529/biophysj.106.101881.
Meyers, V.E.; Zayzafoon, M.; Douglas, J.T.; McDonald, J.M. RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity. J. Bone Miner. Res. 2005, 20, 1858–1866.
[84]
Khatiwala, C.B.; Kim, P.D.; Peyton, S.R.; Putnam, A.J. ECM compliance regulates osteogenesis by influencing MAPK signaling downstream of RhoA and ROCK. J. Bone Miner. Res. 2009, 24, 886–898.
[85]
Yoshida, T.; Clark, M.F.; Stern, P.H. The small GTPase RhoA is crucial for MC3T3-E1 osteoblastic cell survival. J. Cell Biochem. 2009, 106, 896–902, doi:10.1002/jcb.22059.
[86]
Chan, M.C.; Nguyen, P.H.; Davis, B.N.; Ohoka, N.; Hayashi, H.; Du, K.; Lagna, G.; Hata, A. A novel regulatory mechanism of the bone morphogenetic protein (BMP) signaling pathway involving the carboxyl-terminal tail domain of BMP type II receptor. Mol. Cell. Biol. 2007, 27, 5776–5789.
[87]
Zambuzzi, W.F.; Bruni-Cardoso, A.; Granjeiro, J.M.; Peppelenbosch, M.P.; de Carvalho, H.F.; Aoyama, H.; Ferreira, C.V. On the road to understanding of the osteoblast adhesion: Cytoskeleton organization is rearranged by distinct signaling pathways. J. Cell Biochem. 2009, 108, 134–144, doi:10.1002/jcb.22236.
[88]
Robling, A.G.; Castillo, A.B.; Turner, C.H. Biomechanical and molecular regulation of bone remodeling. Annu. Rev. Biomed. Eng. 2006, 8, 455–498, doi:10.1146/annurev.bioeng.8.061505.095721.
