Bone Abstracts

Periosteum contains mesenchymal progenitors and is essential for fracture healing. Signaling mechanisms governing periosteal reaction to injury remain largely unidentified. We previously investigated how PI3 kinase signaling affects the skeletal system using Cbl^YF/YF knock-in (YF) mice wherein PI3K signaling is perturbed by abolition of interaction between Cbl, an E3 ubiquitin-ligase/adaptor protein, and p85 subunit of PI3K. YF mice displayed increased bone volume under homeostatic conditions and formed larger bony callus during fracture repair. Given these data and because PI3K regulates osteoblast differentiation, we hypothesized that aberrant PI3K signaling regulates fracture healing by modulating osteogenic commitment of periosteal progenitors upon injury. To visualize periosteal cells, WT-Osterix^RFP or YF-Osterix^RFP mice were generated and used as per IACUC protocols. Creation of stabilized femoral fractures in these mice and comparative histological analyses at days 1 and 3 post fracture revealed no changes in intact femoral periosteum. Interestingly, Osx^RFP+ periosteal thickness in YF-Osx^RFP mice was significantly increased over WT-Osx^RFP in D1 fractured femurs (WT-28 μm, YF-57 μm, P<0.05, n=3) and D3 fractured femurs (WT-90 μm,YF-189 μm, P<0.05, n=6). Osx^RFP+ periosteal cells in D3 fractured femurs were 1.6-fold higher in YF-Osx^RFP over WT-Osx^RFP. Osteogenic potential of injured periosteum was detected by alkaline phosphatase staining. ALP⁺ periosteum was increased in D1 fractured femurs (WT-35 μm, YF-50 μm, n=3) but showed threefold enhancement in D3 fractured femurs in YF-Osx^RFP over WT-Osx^RFP (WT-58 μm, YF-170 μm, P<0.05, n=6). Since PI3K signaling regulates cell proliferation, we examined periosteal proliferation upon injury by EdU labeling. 3 days post fracture, significantly more EdU⁺ periosteum was observed in YF-Osx^RFP than WT-Osx^RFP (WT-4%, YF-22%, P<0.05; n=3). Flow cytometric analysis of isolated periosteal cells revealed that in YF mice, fractures led to enhanced expression of MSC markers, Sca1 (Intact-5.5%, Fx-13.5%, P<0.05), CD29 (Intact-9.6%, Fx-20%, P<0.05) and CD105 (Intact-6.6%, Fx-12.1%, P<0.05) over intact femurs with no significant changes seen in WT (n=4). In conclusion, perturbed PI3K signaling, upon injury, promotes expanded, highly proliferative, Osterix⁺ periosteum with more MSC-like cells and enhanced osteogenic potential.