Characterizing Cellular Expansion In Response To Long Bone Distraction Osteogenesis
Ankit Salhotra, B.S., Harsh N. Shah, B.S., M.P.H, Courtney A. Stockman, B.S., Derrick C. Wan, M.D., Michael T. Longaker, M.D., M.B.A..
Stanford University, Stanford, CA, USA.
PURPOSE: Distraction Osteogenesis (DO) is a commonly used procedure to correct for skeletal abnormalities. However, the cellular contribution responsible for inducing bone formation during DO is not well understood. We hypothesize that the clonal expansion observed during distraction is due to skeletal stem cells.
METHODS: Actin;R26VT2/GK3 (rainbow) is a multicolor, ubiquitous, genetic driver that marks cells under the actin promoter. Upon tamoxifen, induction cells are randomly and genetically marked with one of ten color combinations. Subsequent daughter cells are marked with the same color as their parent population enabling for lineage tracing. 10-week old male rainbow mice underwent distraction surgery that consisted of a five-day latency period followed by a twice-daily distraction rate of 0.15 mm for 10 days, ending with a consolidation period of 28 days. At post-operative day (POD 43) the distracted tibias of the rainbow mice (n=3) were harvested and imaged using confocal microscopy. Furthermore, the functional assessment of fracture and distracted skeletal stem cells (SSCs) was determined using proliferation (n=5) and osteogenic assays (n=5). Wild-type 10-week-old male mice were divided into two groups: fracture and distraction. At POD 10, fracture and distracted tibias were harvested for fluorescent activated cell sorting (FACS) of SSCs.
RESULTS: Rainbow labeling of an uninjured tibia shows successful genetic marking of the diaphysis. During distraction, a clonal expansion of cells is observed preceded by successful bone formation. SSCs sorted from distraction calluses at POD 10 form robust colonies and larger bone nodules in comparison to fracture calluses in vitro. In addition, quantification analysis indicates distraction SSCs form more colonies (***p <0.001) and have increased incidence of in vitro bone formation (****p<0.0001).
CONCLUSIONS: We demonstrate successful rainbow labeling of the mouse tibia and show clonal expansion of skeletal stem cells in response to distraction. Future studies will delineate the role of SSCs in response to distraction.
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