Bone Abstracts

Bone fractures can be a serious and frequent problem for patients suffering from osteoporosis, metastatic bone cancer and congenital bone disorders. The promotion of new bone formation and mineralization can facilitate healing and stronger union of fractured bones. Our laboratory has identified important developmental roles of sphingomyelin phosphodiesterase 3 (SMPD3), which include the promotion of apoptosis of hypertrophic chondrocytes and mineralization of cartilage and bone extracellular matrix. However, the transcriptional regulation of this gene and its role during fracture healing is still unknown.

Objectives:
1. To elucidate the transcriptional regulation of Smpd3 in skeletal cells

2. To investigate the role of SMPD3 in fracture healing

Methods:
1 - ATDC5 and MC3T3 cell lines were used to investigate the transcriptional regulation of Smpd3 in chondrocytes and osteoblasts, respectively. These cells were transfected with pSox9 or pOsx expression vectors. For Smpd3 promoter studies, a 1.9-kb mouse Smpd3 proximal promoter was cloned into the pGL4.10 (luc2) vector. This construct was then co-transfected with or without pSox9 or pOsx. 2 - To investigate the role of SMPD3 in fracture healing, we generated a conditional knockout mouse, Smpd3^flox/flox;Osx-Cre, which lacks Smpd3 in both chondrocytes and osteoblasts. Rodded immobilized fracture surgeries were performed in the tibia of these mice. The bones were then analyzed at 1 and 4 weeks post-surgery by X-ray, micro-CT, histology and histomorphometry.

Results:
1 - A significant upregulation of Smpd3 was seen in the presence of pSox9 and pOsx in ATDC5 and MC3T3 cells, respectively. Furthermore, Smpd3 promoter activity was significantly upregulated in the presence of pSox9 and pOsx. 2 - Histological analyses showed a prominent callus at the site of fracture in Smpd3^flox/flox;Osx-Cre mice, whereas the fractures induced in the WT mice healed well. Histomorphometric analysis showed a significant increase in osteoid volume in Smpd3^flox/flox;Osx-Cre bones compared to WT bones.

Conclusion: Our data provides compelling evidence that SMPD3 activity, regulated by Sox9 and Osx, is critical for bone fracture healing. To test SMPD3’s potential as a therapeutic agent to improve fracture healing, studies have been initiated to encapsulate Smpd3 and inject it into the fracture sites in WT and SMPD3-deficient mice.

Disclosure: The authors declared no competing interests.