gms | German Medical Science

Objectives: Fracture healing is known to be highly mechano-sensitive and its success is determined by the amount of shear acting across fracture fragments. Micro-vascular re-establishment across a fracture gap is essential for healing but also dependent on mechano-sensation of inner-vascular forces such as hemodynamic forces and fluid shear stress within the vascular lumen. Endothelial YAP/TAZ are important regulators in the mechano-sensation and build-up of vascular networks. How the endothelial YAP/TAZ depends upon the macro-mechanics in regeneration and thereby determines success of vascular and collagen network regeneration remains largely unknown. This study aimed to unravel the role of endothelial YAP/TAZ and their ability to regulate the re-build of micro-vascular networks as prerequisite of successful bone healing.

Methods: A comparative approach of a successful vs. a delayed healing model was used by employing a rigid fixator that results in successful healing after 21 days and a semi-rigid fixator that results in substantially delayed healing at 21 days. For loss of function experiments the Yapfl/fl Tazfl/fl Cdh5-iCreERT2 mouse strain was used, and tamoxifen was injected 7 days prior to setting the osteotomy. Soft callus formation, cell organization and ECM deposition within the fracture gap in control and EC YAP/TAZ dKO mice at an early phase of healing at 7 days post-osteotomy were compared to a later stage at 14 days post-osteotomy. The development of vascular structures was analyzed by immune-histological staining of EC markers Emcn and CD31. Fibrillar collagen was visualized by SHG imaging.

Results and conclusion: At 7 days post-osteotomy a complete micro-vascular network with CD31 and Emcn positive vessels spanned the osteotomy gap in control and EC YAP/TAZ dKO mice under rigid fixation. Vessel growth in the osteotomy gap was significantly reduced under semi-rigid fixation compared to rigid in both, control and EC YAP/TAZ dKO mice. At 14 days, a distinct process of endochondral ossification has been established as a pre-requirement for bone bridging and healing. Strikingly, we found that mean vessel lumen thickness in the osteotomy gap is significantly upregulated in the EC YAP/TAZ dKO condition compared to control under rigid fixation. Our results suggest that the macroscopically and externally imposed mechanics (e.g. by choosing an appropriate fracture fixation) dominate the local cell-based mechano-transduction mediated via endothelial YAP/TAZ during vascular development. Further, these findings suggest that endothelial specific YAP/TAZ deletion leads to increased bone vasculature in the hypoxic microenvironment of an osteotomy gap, which may open up novel therapeutic approaches to promote angiogenesis.