Oscillatory perfusion seeding and culturing of osteoblast-like cells on porous beta-tricalcium phosphate scaffolds.

Perfusion culture systems have proven to be effective bioreactors for constructing tissue engineered bone in vitro, but existing circuit-based perfusion systems are complicated and costly for conditioned culture due to the large medium volume required. A compact perfusion system for artificial bone fabrication using oscillatory flow is described here. Mouse osteoblast-like MC 3T3-E1 cells were seeded at 1.5 x 10(6) cells/100 microL and cultured for 6 days in porous ceramic beta-tricalcium phosphate scaffolds (10 mm in diameter, 8 mm in height) by only 1.5 mL culture media per scaffold. The seeding efficiency, cell proliferation, distribution and viability, and promotion of early osteogenesis by both a static and an oscillatory perfusion method were evaluated. The oscillatory perfusion method generated higher seeding efficiency, alkaline phosphatase activity, and scaffold cellularity (by DNA content) after 6 days of culture. Stereomicroscopic observation of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining and Calcein-AM/propidium iodide double staining also demonstrated homogeneous seeding, proliferation, and viability of cells throughout the scaffolds in the oscillatory perfusion system. By contrast, the static culture yielded polarized seeding and proliferation favoring the outer and upper scaffold surfaces, with only dead cells in the center of the scaffolds. Thus, these results suggest that the oscillatory flow condition not only allow a better seeding efficiency and homogeneity, but also facilitates uniform culture and early osteogenic differentiation. The oscillatory perfusion system could be a simple and effective bioreactor for bone tissue engineering.

[1]  Jason W. Triplett,et al.  Osteoblasts and osteocytes respond differently to oscillatory and unidirectional fluid flow profiles , 2007, Journal of cellular biochemistry.

[2]  S. Usami,et al.  Roles of MAP kinases in the regulation of bone matrix gene expressions in human osteoblasts by oscillatory fluid flow , 2006, Journal of cellular biochemistry.

[3]  S. Donahue,et al.  Mechanical stimulation of MC3T3 osteoblastic cells in a bone tissue-engineering bioreactor enhances prostaglandin E2 release. , 2005, Tissue engineering.

[4]  Christopher R Jacobs,et al.  Effects of short-term recovery periods on fluid-induced signaling in osteoblastic cells. , 2005, Journal of biomechanics.

[5]  Antonios G. Mikos,et al.  Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics , 2005, Annals of Biomedical Engineering.

[6]  A. Goldstein,et al.  Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner. , 2005, Bone.

[7]  Roger Zauel,et al.  3-D computational modeling of media flow through scaffolds in a perfusion bioreactor. , 2005, Journal of biomechanics.

[8]  Antonios G Mikos,et al.  Flow perfusion culture induces the osteoblastic differentiation of marrow stroma cell-scaffold constructs in the absence of dexamethasone. , 2005, Journal of biomedical materials research. Part A.

[9]  Ian A. Coe,et al.  Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation , 2004, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[10]  A. Goldstein,et al.  Hydrodynamic shear stimulates osteocalcin expression but not proliferation of bone marrow stromal cells. , 2004, Tissue engineering.

[11]  Antonios G Mikos,et al.  Influence of the in vitro culture period on the in vivo performance of cell/titanium bone tissue-engineered constructs using a rat cranial critical size defect model. , 2003, Journal of biomedical materials research. Part A.

[12]  Junzo Tanaka,et al.  Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials. , 2003, Tissue engineering.

[13]  Robert E Guldberg,et al.  Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro. , 2003, Tissue engineering.

[14]  Antonios G. Mikos,et al.  Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Wendt,et al.  Oscillating perfusion of cell suspensions through three‐dimensional scaffolds enhances cell seeding efficiency and uniformity , 2003, Biotechnology and bioengineering.

[16]  C. Rubin,et al.  Fluid pressure gradients, arising from oscillations in intramedullary pressure, is correlated with the formation of bone and inhibition of intracortical porosity. , 2003, Journal of biomechanics.

[17]  C. Jacobs,et al.  Mechanosensitivity of bone cells to oscillating fluid flow induced shear stress may be modulated by chemotransport. , 2003, Journal of biomechanics.

[18]  Yng-Jiin Wang,et al.  Osteogenic enrichment of bone-marrow stromal cells with the use of flow chamber and type I collagen-coated surface. , 2003, Journal of biomedical materials research. Part A.

[19]  K. Lau,et al.  Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. , 2003, Bone.

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

[21]  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.

[22]  L. Lanyon,et al.  Mechanical strain and fluid movement both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different signaling pathways. , 2002, Bone.

[23]  A. Mikos,et al.  Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. , 2001, Biomaterials.

[24]  H J Donahue,et al.  Osteopontin Gene Regulation by Oscillatory Fluid Flow via Intracellular Calcium Mobilization and Activation of Mitogen-activated Protein Kinase in MC3T3–E1 Osteoblasts* , 2001, The Journal of Biological Chemistry.

[25]  H J Donahue,et al.  Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow. , 2000, Journal of biomechanical engineering.

[26]  D J Mooney,et al.  Dynamic seeding and in vitro culture of hepatocytes in a flow perfusion system. , 2000, Tissue engineering.

[27]  J A Frangos,et al.  Steady and Transient Fluid Shear Stress Stimulate NO Release in Osteoblasts Through Distinct Biochemical Pathways , 1999, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[28]  D. Burr,et al.  Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. , 1998, American journal of physiology. Cell physiology.

[29]  Gordana Vunjak-Novakovic,et al.  Bone Tissue Engineering Using Human Mesenchymal Stem Cells: Effects of Scaffold Material and Medium Flow , 2004, Annals of Biomedical Engineering.

[30]  Cato T Laurencin,et al.  Human osteoblast-like cells in three-dimensional culture with fluid flow. , 2003, Biorheology.