T. V. Boyko1,2, Z. Wang1, M. T. Longaker1, G. P. Yang1,3 1Stanford University,Surgery,Palo Alto, CA, USA 2State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 3VA Palo Alto Healthcare Systems,Surgery,Palo Alto, CA, USA
Introduction: We have identified the mouse skeletal progenitor cells consisting of 8 distinct subpopulations capable of self-renewal and giving rise to all three components of the skeleton: bone, cartilage and stroma. The periosteum consists of a thin layer of cells that have been shown to expand and contribute to bone fracture healing. Separately, we recently reported the strong expression of Del1 in cartilage and its potential role in osteoarthritis. Del1 was also strongly expressed in skeletal progenitors, and histology had shown strong expression in the periosteum. We hypothesized that the expression of Del1 in the periosteum served as a marker for the presence of skeletal progenitors and that periosteal cells would be found to consist primarily of skeletal progenitor cells.
Methods: Del1-LacZ mice were anesthetized, the left femur exposed, and the periosteum was injured by scraping with the side of a pair of scissors. The right femur was untouched and used as a control. On post-operative day 7, mice were harvested and processed for histology. To obtain cells for Fluorescent Activated Cell Sorting (FACS) analysis uninjured femurs were stripped of periosteum, which was digested to obtain a cell suspension. Cells were then stained for surface markers of skeletal progenitor cells and FACS was performed to analyze for presence of skeletal progenitor cells.
Results: Strong expression of Del1 could be seen in uninjured periosteum. Using LacZ staining, we demonstrated that these cells underwent expansion with differentiation into hypertrophic cartilage and bone at the injury site. To identify skeletal progenitors within the periosteum, we harvested cells and subjected them to FACS analysis. FACS showed that the largest subpopulation of skeletal progenitors in the periosteum were Bone Cartilage Stroma Progenitor cell population (BCSPs), the skeletal progenitors found to contribute most to fracture healing.
Conclusion: We have previously shown that mouse skeletal progenitor cells play an integral role in bone fracture healing. It has long been known that the periosteum contributes cells to bone repair. We show here that the periosteum contains a large number of skeletal progenitor cells and that the largest population were BCSPs, the cells most involved in fracture repair. Furthermore, we have identified Del1 expression as a strong marker of skeletal progenitor cells. Separately, we have shown that Del1 deletion leads to fracture healing with a decreased bony callus. We conclude that Del1 represents a novel regulator of skeletal progenitor cell biology.