gms | German Medical Science

Objectives: Compromised vascular supply and insufficient neovascularization are primary clinical challenges for bone repair and regeneration. Osteoblasts require close proximity to oxygenated capillaries and co-mobilize with new blood vessels to induce bone healing. Thus, injuries to bones with low peripheral vascular supply (e.g., tibia) and large bone defects with an obliterated vascular bed are prone to non-union. Cysteine-rich angiogenic inducer of 61kD (Cyr61) is a matricellular growth factor whose expression is regulated by mechanical cues during fracture repair and its delivery can enhance bone healing; however, its cellular mechanism(s) of action in fracture repair are unknown. Here, we tested the effects of recombinant Cyr61 delivery on vascularized bone repair and angiogenesis.

Methods: We evaluated the effects of Cyr61 delivery on bone regeneration in vivo, with or without ambulatory mechanical loading by varying fixation plates stiffness (stiff vs. compliant external fixator, MouseExFix, RISystem; n= 6–8) in C57BL/6J female mice aged 12–16 weeks. Cyr61 (1 µg/defect) was delivered in a photocrosslinked gelatin-fibrin hydrogel. All procedures were conducted in accordance with UPenn IACUC regulations (protocol no: 806482).MicroCT and histological analysis were performed 14 days post-fracture (dpf). In parallel, we evaluated the effects of Cyr61 treatment on vascular network formation in a 3D in vitro angiogenesis assay combing human umbilical vein endothelial cells (HUVECs) and human mesenchymal stromal cells (hMSCs). Images were taken at 3 and 5 days and the tube number and mean length were analyzed.

Results and conclusion: Cyr61 treatment had no effect on fracture callus bone volume and cartilage formation at 14 dpf, regardless of fixation stiffness. However, Cyr61 treatment increased formation of interfragmentary vascularized tissue and significantly increased endomucin-positive vessel area under stiff fixation, compared to the vehicle control, but did not alter tissue formation or vascularization under compliant fixation. In vitro, Cyr61 treatment enhanced tubular network formation, with significantly elevated tube length at both 3 and 5 days. Together, we found that Cyr61 delivery promoted angiogenesis during fracture repair, but only under stiff fixation conditions. Previously, we showed that large interfragmentary strains, if applied early in the bone healing process, mechanically disrupt vascular ingrowth. Thus, while potently angiogenic, both in vitro and in vivo, Cyr61 did not enhance vessel formation under mechanical conditions that were unfavorable for functional neovessel growth. Together with our in vitro findings, we postulate that Cyr61 delivery represents a potential pro-angiogenic therapeutic for vascularized bone healing under conditions of limited blood vessel supply, such as tibial fractures and large bone defects, but does not promote cartilage callus formation or mineralization per se.