ESPE Abstracts

Background: Bone development and remodeling are controlled by the phosphoinositid-3-kinase (PI3K) signaling pathway. We investigated the effects of downregulation of phosphatase and tensin homolog (Pten), a negative regulator of PI3K signaling, in osteoprogenitor cells.

Methods: Femura, tibiae and bone marrow stromal cells (BMSCs) from mice with Cre-inducible Pten knockdown in cells expressing the transcription factor Osterix/Sp7 (Pten cKO) and Cre negative control mice were compared. Bone phenotyping was performed via µCT and 3-point bending test. Number of osteoclasts and osteoblasts was determined by tartrate resistant acid phosphatase (TRAP) immunohistochemistry. Proliferation of BMSCs was measured by counting Hoechst and Ki-67 stained cells. In vitro adipogenic and osteogenic differentiation was determined via Oil Red O and alkaline phosphatase staining, respectively. Bone turnover was assessed by ELISA detecting CTX and P1NP and intraperitoneal Calcein injections 2 and 5 days prior to sacrifice.

Results: We detected a higher trabecular bone volume/total volume (BV/TV) and higher bone mineral density (BMD) in Pten cKO of both sexes, while BV/TV and BMD was lower in cortical bone. Biomechanical analysis revealed a significantly higher maximum force (3.7fold, P=0.0003 for males) and elasticity of Pten cKO femura. Pten cKO bones from male mice displayed less osteoclasts (0.7fold) and more osteoblasts (2.0fold, P=0.0006) per bone surface compared to controls. Bone turnover markers P1NP and CTX were significantly increased both in Pten cKO male and female mice. The percentage of bone marrow fat was higher in male mice (1.4fold, P=0.007), but not in female Pten cKO mice compared to controls. An increase in the proliferation rate of male (1.8fold), but not female BMSCs from Pten cKO mice compared to controls was detected. While there was no significant difference in adipogenic differentiation in vitro, osteogenic differentiation capacity was significantly enhanced in BMSCs from both, male and female, Pten cKO mice. Evaluation of bone dynamics revealed significantly higher P1NP and CTX serum levels in Pten cKO mice compared to controls. A trend towards increased mineralized surface per bone surface and bone formation rate was observed in Pten cKO but not Cre negative bones of 38 week old mice.

Conclusion: Conditional Pten knockout in osteoprogenitor cells increases both, bone stability and elasticity, and leads to increased proliferation and osteogenic differentiation of bone marrow stromal cells. Bone regeneration is preserved in older Pten cKO mice.