Date of Award

5-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Dr. Jiro Nagatomi

Committee Member

Dr. Karen Burg

Committee Member

Dr. Delphine Dean

Committee Member

Dr. Kyle Jeray

Committee Member

Dr. Ken Webb

Abstract

Changes in the mechanical loading on the skeleton have been shown to influence bone density. In bone disorders, such as disuse osteoporosis, the reduction of loading on bone leads to reduced bone mineral density and thus increased risk of fracture. Previous studies have suggested this may be caused by a change in the mechanical environment, specifically hydrostatic pressures, of the bone cells, resulting in a change in cellular activity. However, the pressure parameters and mechanisms involved in the promotion/suppression of osteogenesis in 3D in vitro culture of bone cells are not well understood. This doctoral thesis sought to investigate the role cyclic pressure frequency has on the osteogenic differentiation of mesenchymal stem cells (MSC) in a 3D alginate microbead in vitro culture. Specifically, the objective of this research was to test the hypothesis that the osteogenic differentiation of MSCs in 3D was enhanced by cyclic pressure applied at a critical threshold frequency. To test this hypothesis an alginate scaffold and 3D culture conditions were first developed and then used to assess the effects of cyclic pressure at different frequencies on MSC differentiation. To develop a 3D culture system the effects of RGD modification on mouse MSC survival and differentiation in alginate microbeads were first examined. MSCs were encapsulated in unmodified or RGD-modified alginate and cell proliferation and the expression of several osteogenic markers were monitored over a 28 day culture. While RGD modification did facilitate cell adhesion, it did not affect MSC viability or proliferation. However, RGD did promote the development of mature osteoblasts and was therefore used in the remaining experiments. Both an increased matrix stiffness and low seeding density were found to enhance the osteogenic differentiation of MSCs in 3D, most likely through a change in integrin binding and nutrient supply, respectively. The addition of cyclic pressure (5-35 kPa for 1 h/day) also resulted in increased osteogenesis of MSCs in 3D, but only when applied at 0.5 Hz and not 0.1 Hz. In summary, we have demonstrated that RGD, a high matrix stiffness, and low seeding density can promote the early upregulation of osteogenic markers and maturation of osteoblasts. Additionally, the results of the present study provide evidence that the promotion of the osteogenesis of MSCs in 3D by cyclic pressure is affected by frequency and that a threshold likely exists around 0.5 Hz. The current findings will help progress the field of bone cell mechanobiology and can be used in the treatment of bone degenerative diseases.

Share

COinS