Modulation of marrow stromal cell differentiation in bone tissue engineering constructs
Holtorf, Heidi Lynn
Mikos, Antonios G.
Doctor of Philosophy
Better understanding of the factors that affect marrow stromal cell differentiation will allow researchers to optimize the design of bone tissue engineering constructs toward healing large bone defects in human patients. This research characterizes the effects of scaffold properties and culture supplements on the osteoblastic differentiation of marrow stromal cells (MSCs) seeded on solid, porous scaffolds and cultured in a flow perfusion bioreactor. This bioreactor creates a culture environment similar to that experienced by osteoblasts in vivo by minimizing diffusional constraints and providing mechanical stimulation to the cells through fluid shear. For these studies, MSCs were seeded on scaffolds, cultured under static or flow perfusion conditions, and assayed for DNA, alkaline phosphatase activity, osteopontin, and calcium to assess osteoblastic differentiation. Light and electron microscopy were used to visualize cell morphology and matrix deposition. The results show that brief exposure of MSCs to dexamethasone, a chemical stimulus typically required for osteoblast differentiation, was required prior to seeding on a titanium fiber mesh scaffold for ectopic bone formation to occur in a subcutaneous implantation site. However, in the absence of dexamethasone, either flow perfusion culture or decellularized bone-like extracellular matrix deposited on titanium fiber mesh induced osteoblastic differentiation in MSCs. Altering the diameter of titanium fibers composing the mesh affected the osteoblastic differentiation of seeded MSCs in flow perfusion culture; wider fibers were conducive to early osteoblast differentiation while thinner fibers were conducive to later differentiation and matrix deposition. Alternatively, coating the titanium scaffold surface with the adhesion peptide RGD resulted in increased cell adhesion strength leading to delayed osteoblastic differentiation in vitro, but had no effect on bone formation in vivo. Flow perfusion culture of MSCs seeded on porous calcium phosphate ceramic scaffolds resulted in better cell distribution within the scaffold and enhanced osteoblastic differentiation compared to static culture. These results show that scaffold geometry influences cell behavior in a flow perfusion bioreactor, emphasizing the importance of scaffold design in bone tissue engineering. In addition, undifferentiated marrow stromal cells can be induced toward the osteoblastic phenotype by signals other than dexamethasone, including bone-like extracellular matrix and fluid flow mediated shear stress.
Cell biology; Biomedical engineering