Author(s): Rundle CH, Wang X, Sheng MH, Wergedal JE, Lau KH,
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Abstract This study sought to determine the role of the pro-apoptotic gene, Bax, in fracture healing by comparing femoral fracture healing in Bax knockout (KO) and wild-type C57BL/6J (background strain) mice. Bax KO fractures were larger, had more bone mineral content, had approximately 2-fold larger cartilage area per callus area in the first and second weeks of fracture healing, and showed an increased osteoclast surface area in the third and fourth weeks of fracture healing compared to C57BL/6J fractures. The increased cartilage area in the Bax KO fracture callus was due to increases in number of both pre-hypertropic and hypertropic chondrocytes. TUNEL analysis showed no significant differences in the number of either chondrocyte or non-chondrocyte apoptotic cells between Bax KO and C57BL/6J fractures at 7 or 14 days post-fracture, indicating that the increased number of chondrocytes in Bax KO fractures was not due to reduced apoptosis. Analysis of expression of apoptotic genes revealed that although the expression levels of Bcl-2 and Bcl-xL were not different between the Bax KO and C57BL/6J mice at 7 or 14 days post-fracture, the expression of BH3-domain only Bak and "Bik-like" pro-apoptotic gene increased approximately 1.5-fold and approximately 2-fold, respectively, in Bax KO fractures at 7 and 14 days post-fracture, compared to C57BL/6J fractures, suggesting that up-regulation of the Bak and Bik-like pro-apoptotic genes in Bax KO mice might compensate for the lack of Bax functions in the context of apoptosis. Analysis by in vivo incorporation of bromodeoxyuridine into chondrocytes within the fracture tissues indicated a highly significant increase in chondrocyte proliferation in Bax KO fractures compared to C57BL/6J fractures at day 7. The increased expression of collagen 2alpha1 and 9alpha1 gene in Bax KO fractures during early healing was consistent with an increased chondrocyte proliferation. In conclusion, this study demonstrates for the first time that Bax has an important role in the early stage of fracture healing, and that the increased callus size and cartilage area in Bax KO fractures was due to increased chondrocyte proliferation and not to reduced apoptosis or increased chondrocyte hypertrophy. The unexpected effect of Bax deficiency on chondrocyte proliferation implicates a novel regulatory function for Bax on chondrocyte proliferation during fracture repair.
This article was published in Bone
and referenced in Journal of Steroids & Hormonal Science