THE DEVELOPMENT OF A MESENCHYMAL STEM CELL BASED BONE GRAFT SYSTEM

Greater than 5.5 million fractures are sustained by Americans each year, accounting for more than 500,000 bone graft surgeries. Bone grafts are the second most transplanted material, surpassed only by blood. The current \u27gold standard\u27 for bone grafting involves harvesting bone material from the patient\u27s iliac crest, due to its osteoinductive properties. Unfortunately, the surgery required to harvest material from the iliac crest causes additional pain for the patient, relies on a limited amount of available bone tissue, and results in, on average, a 30% rate of donor site morbidity. Both natural and synthetic substitutes have been developed to avoid complications and alleviate the pain associated with harvesting grafts from a patient\u27s healthy bone; however, these substitutes are costly and lack the osteoinductive properties desired by surgeons for proper repair. Bone tissue engineering solutions combine scaffold materials with viable stem cells or bone cells in products that can be implanted into bony defects. Incorporating cells and growth factors within a bone graft prior to implantation improves upon its ability to induce bone formation and decreases the time needed for native tissue to proliferate and integrate with the graft. The long-term goal of this research is to develop a mesenchymal stem cell-based bone graft system with the potential of someday eliminating the use of autogenic tissue. The first part of this research project was centered on the order of events of physiological remodeling: bone resorption by osteoclasts followed by the deposition of mineralized matrix by osteoblasts. These events indicate that the cells responsible for resorbing bone mineral may also possess the ability to recruit and/or induce osteoblast development and activation. A series of studies were developed to gain further insight into the role that osteoclasts may play on the differentiation of mesenchymal stem cells (MSCs) when incorporated into a bone graft system. The results demonstrated that osteoclasts can positively influence expression of bone cell markers during the initial and final stages of osteoblast differentiation without having a significant effect on markers linked to the middle stage. The observed effects on osteoblasts could prove to be advantageous to bone tissue engineering applications. A second set of studies were performed to examine the aspirate material obtained from the femoral shaft using the Reamer/Irrigator/Aspirator (RIA) device (Synthes, USA; Paoli, PA) as an untapped source of MSCs that could be incorporated into a bone graft system. The fat layer of aspirate, which has traditionally not been a topic of research or clinical interest, was the focus of this work. Viable, proliferating cells were successfully and consistently isolated, using the same techniques, from the fatty layer of RIA aspirate of multiple patient samples. Further characterization performed on the fat layer from multiple patient samples showed that the fatty layer of aspirate is mainly composed of four main fatty acids: oleic, palmitic, linoleic, and stearic. Finally, synthesis of the osteoblast cell markers alkaline phosphatase and calcium by cells isolated from the RIA fat layer was observed in both two-dimensional tissue culture polystyrene and three-dimensional ceramic bone graft granule cultures. The results of this work suggest that the fatty layer of RIA aspirate may be a new, untapped source for autologous progenitor cells with bone forming capabilities. Previously considered waste, the lipid rich fat layer of aspirate collected during reaming of the medullary canal may be a source of adult mesenchymal stem cells that could be used to stimulate new bone growth at the site of fracture repair