Callus resolution and fracture healing is not hindered in cathepsin-k null mice

Cathepsin K (CatK) is a cysteine protease expressed predominantly in osteoclasts which degrades demineralized bone matrix. Adult CatK deficient mice have high bone mass in both cortical and cancellous regions as the result of significant reduction in osteoclastic bone resorption as well as enhanced bone formation. Here, a mouse femur fracture model was employed to assess fracture healing in CatK knock-out (KO) versus wild type (WT) mice. Pre-pin stabilized, mid-diaphysial femoral fractures (Fx) were made with an Einhorn device in 8-11 week old mice. Callus formation and resolution were assessed in vivo via a dental x-ray at the time of Fx and at 4, 7, 14, 21, and 28 days. Based on blind scoring of Fx radiographs at 14 days, KO and WT mice were randomized into 3 groups (n= 6-8/group) which were sacrificed at for 14, 18 and 28 days post-Fx, respectively. Similar procedures were applied in a separate experiment to assess callus strength at 28 and 42 days. The fractured and intact contra-lateral femora were harvested and the calluses were assessed ex vivo by caliper, DEXA, μCT and histomorphometry. Original cortical bone within the forming callus was manually excluded from μCT slices allowing assessment of bone formed post fracture. The callus size as assessed by caliper, DEXA-derived area, histomorphometry and μCT-derived volume was similar in KO vs. WT at 14 days with a strong trend towards smaller callus size in KO at 18 and 28 days post-Fx. The calluses from KO mice had statistically significantly (p<0.05, one way ANOVA, Fisher's PLSD) greater overall BMD at each timepoint relative to WT controls as assessed by both DEXA and μCT. Callus BMC as measured by μCT demonstrated a higher trend in KO mice at 14 days and gradually declined from 14 to 28 days during callus resolution in both KO vs. WT bones. Histomorphometric analysis of calluses revealed higher bone area/total callus area, % in KO vs. WT at all time points. Cartilage area/total callus area, % was similar in KO vs. WT at all time points. Furthermore, CatK KO calluses had overall higher bone mass at all time points. Biomechanical testing and histomorphometric analyses are onging to determine callus turnover and strength during the healing process. We conclude that callus resolution and healing of fracture is not hindered in Cat K deficient mice.