Three-dimensional growth of differentiating MC3T3-E1 pre-osteoblasts on porous titanium scaffolds.

The present work assesses the potential of three-dimensional porous titanium scaffolds produced by a novel powder metallurgy process for applications in bone engineering through in vitro experimentation. Mouse MC3T3-E1 pre-osteoblasts were used to investigate the proliferation (DNA content), differentiation (alkaline phosphatase activity and osteocalcin release) and mineralisation (calcium content) processes of cells on titanium scaffolds with average pore sizes ranging from 336 to 557 microm, using mirror-polished titanium as reference material. Scanning electron microscopy was employed to qualitatively corroborate the results. Cells proliferate on all materials before reaching a plateau at day 9, with proliferation rates being significantly higher on foams (ranging from 123 to 163 percent per day) than on the reference material (80% per day). Alkaline phosphatase activity is also significantly elevated on porous scaffolds following the proliferation stage. However, cells on polished titanium exhibit greater osteocalcin release toward the end of the differentiation process, resulting in earlier mineralisation of the extracellular matrix. Nevertheless, the calcium content is similar on all materials at the end of the experimental period. Average pore size of the porous structures does not have a major effect on cells as determined by the various analyses, affecting only the proliferation stage. Thus, the microstructured titanium scaffolds direct the behaviour of pre-osteoblasts toward a mature state capable of mineralising the extracellular matrix.

[1]  S. Graves,et al.  Formation of mineralized nodules by bone derived cells in vitro: a model of bone formation? , 1993, American journal of medical genetics.

[2]  G. Gronowicz,et al.  An in vitro model for mineralization of human osteoblast-like cells on implant materials. , 1999, Biomaterials.

[3]  D. Dean,et al.  Prostaglandins mediate the effects of titanium surface roughness on MG63 osteoblast-like cells and alter cell responsiveness to 1 alpha,25-(OH)2D3. , 1998, Journal of biomedical materials research.

[4]  M. Zimmerman,et al.  Three-dimensional printing and porous metallic surfaces: a new orthopedic application. , 2001, Journal of biomedical materials research.

[5]  L. Claes,et al.  Proliferation and differentiation parameters of human osteoblasts on titanium and steel surfaces. , 2001, Journal of biomedical materials research.

[6]  Miqin Zhang,et al.  Three-dimensional macroporous calcium phosphate bioceramics with nested chitosan sponges for load-bearing bone implants. , 2002, Journal of biomedical materials research.

[7]  T. Albrektsson,et al.  Osteoinduction, osteoconduction and osseointegration , 2001, European Spine Journal.

[8]  P. Krebsbach,et al.  Isolation and Characterization of MC3T3‐E1 Preosteoblast Subclones with Distinct In Vitro and In Vivo Differentiation/Mineralization Potential , 1999, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[9]  L D Zardiackas,et al.  Structure, metallurgy, and mechanical properties of a porous tantalum foam. , 2001, Journal of biomedical materials research.

[10]  Jef A. Helsen,et al.  Metals as Biomaterials , 1998 .

[11]  C. Lohmann,et al.  Migration, Matrix Production and Lamellar Bone Formation of Human Osteoblast-Like Cells in Porous Titanium Implants , 2002, Cells Tissues Organs.

[12]  Antonios G. Mikos,et al.  Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  H. Cheung,et al.  Growth of osteoblasts on porous calcium phosphate ceramic: an in vitro model for biocompatibility study. , 1989, Biomaterials.

[14]  K. Mustafa,et al.  Production of transforming growth factor beta1 and prostaglandin E2 by osteoblast-like cells cultured on titanium surfaces blasted with TiO2 particles. , 2003, Clinical oral implants research.

[15]  L. Bonewald,et al.  Effects of combining transforming growth factor beta and 1,25-dihydroxyvitamin D3 on differentiation of a human osteosarcoma (MG-63). , 1992, The Journal of biological chemistry.

[16]  T. Albrektsson,et al.  Characteristics of the surface oxides on turned and electrochemically oxidized pure titanium implants up to dielectric breakdown: the oxide thickness, micropore configurations, surface roughness, crystal structure and chemical composition. , 2002, Biomaterials.

[17]  R M Pilliar,et al.  The optimum pore size for the fixation of porous-surfaced metal implants by the ingrowth of bone. , 1980, Clinical orthopaedics and related research.

[18]  H. Aro,et al.  Pore diameter of more than 100 μm is not requisite for bone ingrowth in rabbits , 2001 .

[19]  J. Ong,et al.  Morphological behavior of osteoblast-like cells on surface-modified titanium in vitro. , 2002, Biomaterials.

[20]  D. Pioletti,et al.  Effect of different Ti-6Al-4V surface treatments on osteoblasts behaviour. , 2002, Biomaterials.

[21]  M. Mabuchi,et al.  Processing and mechanical properties of autogenous titanium implant materials , 2002, Journal of materials science. Materials in medicine.

[22]  Michel Assad,et al.  Porous titanium-nickel for intervertebral fusion in a sheep model: part 1. Histomorphometric and radiological analysis. , 2003, Journal of biomedical materials research. Part B, Applied biomaterials.

[23]  Y. Amagai,et al.  In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria , 1983, The Journal of cell biology.

[24]  C. Lohmann,et al.  Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition. , 1998, Biomaterials.

[25]  K. Chihara,et al.  Smad3 Promotes Alkaline Phosphatase Activity and Mineralization of Osteoblastic MC3T3‐E1 Cells , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[26]  L L Hench,et al.  Biomaterials: a forecast for the future. , 1998, Biomaterials.

[27]  Shigeyuki Ebisu,et al.  Effects of α-TCP and TetCP on MC3T3-E1 proliferation, differentiation and mineralization , 2003 .

[28]  N. Z. Zur Nieden,et al.  In vitro differentiation of embryonic stem cells into mineralized osteoblasts. , 2003, Differentiation; research in biological diversity.

[29]  S F Hulbert,et al.  Potential of ceramic materials as permanently implantable skeletal prostheses. , 1970, Journal of biomedical materials research.

[30]  D. Hutmacher,et al.  Scaffolds in tissue engineering bone and cartilage. , 2000, Biomaterials.

[31]  J B Lian,et al.  Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. , 1989, Physiological reviews.

[32]  T. Bateman,et al.  Effect of nitinol implant porosity on cranial bone ingrowth and apposition after 6 weeks. , 1999, Journal of biomedical materials research.

[33]  M. B. Bickell,et al.  Selection of Materials , 1967 .

[34]  Michael Tanzer,et al.  Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial. , 1999, The Journal of bone and joint surgery. British volume.

[35]  Antonios G Mikos,et al.  Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh. , 2003, Journal of biomedical materials research. Part A.

[36]  David L. Cochran,et al.  Osteoblasts generate an osteogenic microenvironment when grown on surfaces with rough microtopographies. , 2003, European cells & materials.