T. V. Boyko1,2, O. Marecic1, E. Y. Seo1, C. K. Chan1, T. Leavitt1, 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: DEL1 is a secreted protein, which has been shown to be involved in bone fracture healing. In previous experiments we have demonstrated that Del1 gene knockout (KO) mice healed fractures with 15% less bone when compared to wildtype (WT) mice. Increased apoptosis was also seen in fracture calluses of KO mice. Separately, we identified the mouse skeletal stem cell (mSSC) and 7 other unique subpopulations of skeletal progenitor cells that are capable of self-renewal and giving rise to all three components of the skeleton: bone, cartilage and stroma. One subpopulation, the Bone Cartilage Stroma Progenitor Cells (BCSPs), are the primary skeletal stem cell population involved in fracture repair. Following fracture, BCSPs transition to another sub-type, f-BCSPs, that has greater osteogenic potential. We hypothesized that Del1 deletion leads to decreased fracture callus due to an effect on BCSP biology.
Methods: Femurs were fractured in KO and WT mice. Fracture calluses were harvested on post-operative day 7. BCSPs, mSSCs and f-BCSPs were isolated by Fluorescent Activated Cell Sorting (FACS) following staining for signature cell surface markers. Antibody staining for Annexin V was used to determine extent of apoptosis. KO and WT BCSPs were cultured in vitro and colony-forming units (CFUs) were counted 14 days after plating.
Results: FACS analysis revealed that the BCSP populations in KO and WT femurs were equivalent prior to fracture (p=0.498, n=3 each). There is an increase of skeletal progenitors in the callus following fracture, but fewer BCSPs were found in KO mice compared to WT (11,700 cells/1 million events vs 77,706 cells/1 million events, p<0.01, n=4 each). Additionally, mSSCs showed a similar pattern in the KO mice with an attenuated increase after fracture (12,452 cells/1 million events in KO vs 57,451 cells /1 million events in WT, p<0.02, n=4 each). The percentage of apoptotic cells was found to be higher in both BCSPs (23.98% KO vs. 6.56% WT, p<0.001, n=4 each) as well as for mSSCs (14.83% KO vs. 4.74% WT, p<0.002, n=4 each). Following culture in vitro, KO BCSPs showed no difference in proliferation compared to WT, but did form significantly fewer CFUs (16.3 vs. 24 CFUs, p<0.05, n=6 each). Examining f-BCSPs showed an equivalent percentage in KO compared to WT.
Conclusion: Following fracture, skeletal progenitors expand to create bone. In KO mice, this expansion is attenuated leading to decreased bone formation. BCSPs still transition to a more osteogenic phenotype, but there are just fewer of them. These data suggest DEL1 may have a therapeutic role in promoting fracture healing or regenerating bone.