The overall objective of this project is to develop a clinically and commercially viable tissue engineered bone construct using nanocrystalline ceramic scaffolds for proliferating and differentiating adipose-derived progenitor cells into cells of the osteogenic lineage. The use of a nanostructured hydroxyapatite (nano-HAP) scaffold offers several advantages for bone tissue engineering: 1) emerging evidence indicates that nanophase substrates enhance the adhesion and adhesion-dependent functions of anchorage-dependent cells; 2) the ceramic nature of nano-HAP affords chemical stability to the scaffold and therefore confers the advantage of long-term shape integrity; 3) the scaffolds are mechanically robust and can support and protect the bone tissue construct during the implantation procedure and within the body, thus allowing integration of culture and implant steps. Increasing evidence shows that human adipose tissue contains mesenchymal stem cells (MSCs) capable of differentiating into osteoblasts. Unlike bone marrow cells, adipose cells are easy to obtain and result in minimal patient discomfort. This Phase I STTR project leverages the unique material properties of nano-HAP against powerful cell property analysis and design tools drawn from metabolic engineering to identify optimal scaffold parameters and medium conditions for in vitro expansion and differentiation of adipose MSCs into osteoblasts. This work hypothesizes that the surface topography of nano-HAP promotes interactions between cell membrane and matrix proteins that result in greater stability of adherent cells, and thus improve adhesion dependent cell functions.
The specific aims are: 1) Expansion of adipose-derived adult stem cells on idealized 2D nano-surface; 2) Induction of differentiated osteogenic function markers; and 3) Directed modulation of osteoblast function by surface property modification.
These specific aims will be achieved using the following research design and methods. First, parallel cell culture experiments will compare adhesion and proliferation of human adipose progenitor cells on nano-HAP and other commonly used scaffolding material. These experiments will also evaluate the effects of varying nano-HAP grain and pore sizes with or without matrix protein pretreatment. Second, dose response experiments will be followed by a factorial design experiment to systematically explore the known differentiation factor space. A newly developed bioinformatics tool will be used to functionally relate differentiation factor doses and combinations with extent and speed of differentiation. Emphasis will be placed on selective differentiation into osteoblasts by controlling for differentiation into adipocytes. Finally, metabolic flux analysis will be performed to investigate the hypothesis that intermediary metabolism may be directed to support differentiated osteoblast function, including matrix protein deposition and bone formation (mineralization). Nano-HAP surface properties, metabolic flux distribution, and osteoblast activity will be functionally related using the aforementioned bioinformatics tool. This project is expected to identify specific scaffold properties, medium factors, or enzyme targets for enhancing osteoblast function through further metabolic engineering. ? ? ?
