Factors affecting bone cell growth and differentiation under differing culture conditions
Helmke, Christopher D.
Gustin, Michael C.
Master of Arts thesis
Marrow stromal cell (MSC) differentiation into osteoblasts is an important part of the bone growth and remodeling process. This process can be exploited to help solve the problem of bone wound healing. Because of problems with bone grafts, implantation of biodegradable 3D scaffolds seeded with MSCs has been suggested. However, differences in osteoblast differentiation in 2D versus 3D cultures remain unclear. In this study, rat marrow stromal cells (MSCs) were grown both on plastic and in 3D polymer scaffolds and their differentiation into osteoblasts studied. MSCs cultured in a synthetic 3D matrix differentiate faster into osteoblasts than those grown on plastic; the osteoblast differentiation markers alkaline phosphatase and osteocalcin peak in mRNA expression first in 3D in vitro cultures. The culture conditions of MSCs grown in 3D scaffolds were studied to determine the optimal conditions for osteoblastic differentiation. Factors such as cell density, scaffold seeding method, scaffold thickness and secreted soluble factors were investigated. Soluble factors secreted by the differentiated cells into the culture medium were found to be critical for timely differentiation. Lack of such factors promoted cellular proliferation over differentiation. Constant perfusion of cell culture medium through the scaffolds enhanced osteoblastic differentiation. Mature osteoblasts have been shown to undergo chemotaxis, and it is possible that their progenitor cells (MSCs) could as well. Very little is known about MSC chemotaxis. It is possible that they, like osteoblasts, can be recruited to a site where bone formation is needed. Under agarose chemotaxis assays were performed to investigate MSC chemotaxis toward osteoblast matrix factors or other cell types. MSCs did not appear to move under any of the conditions studied.
Molecular biology; Cell biology; Biochemistry