Disruption of thrombospondin‐2 accelerates ischemic fracture healing

Abstract

Thrombospondin-2 (TSP2) is a matricellular protein that is highly up-regulated during fracture healing. TSP2 negatively regulates vascularity, vascular reperfusion following ischemia, and cutaneous wound healing. As well, TSP2-null mice show increased endocortical bone formation due to an enhanced number of mesenchymal progenitor cells and show increased cortical thickness. Mice deficient in TSP2 (TSP2-null) show an alteration in fracture healing, that is unrelated to their cortical bone phenotype, which is characterized by enhanced vascularization with a shift towards an intramembranous healing phenotype; thus, we hypothesized that there would be enhanced ischemic fracture healing in the absence of TSP2. We investigated whether an absence of TSP2 would enhance ischemic fracture healing utilizing Laser doppler, mCT and histological analysis. Ischemic tibial fractures were created in wildtype (WT) and TSP2-null mice and harvested 10, 20, or 40 days post-fracture. TSP2-null mice show enhanced vascular perfusion following ischemic fracture. At day 10 post-fracture, TSP2-null mice have 115% greater bone volume than WT mice. This is associated with a 122% increase in vessel density, 20% increase in cell proliferation, and 15% decrease in apoptosis compared to WT. At day 20, TSP2-null mice have 34% more bone volume, 51% greater bone volume fraction, and 37% more bone tissue mineral density than WT. By 40 days after fracture the TSP2-null mice have a 24% increase in bone volume fraction, but other parameters show no significant differences. These findings indicate TSP2 is a negative regulator of ischemic fracture healing and that in the absence of TSP2 bone regeneration is enhanced. Keywords: thrombospondin-2; ischemia; bone fracture healing Blood supply during fracture healing is crucial for proper bone repair. Vasodilation following fracture promotes edema and inflammation at the site of injury, and contributes to the development of the fracture hematoma. Inflammatory cells that occupy the hematoma produce cytokines and growth factors that then drive neovascularization which is essential for the next phases of repair. Intact vessels are required for new vessel formation to occur during the normal healing process.1,2 However, with a fracture injury there is almost always some disruption of established blood vessels. Thus, fractures with severely disrupted vascularization (ischemia) present a complex medical challenge. Up to 46% of patients with impaired vascular flow have complications during fracture repair, leading to delayed healing or nonunion that necessitates multiple surgeries that typically include staged bone grafting or possibly even amputation. [3][4][5] An important, yet largely untapped, therapeutic strategy to improve ischemic bone regeneration is to enhance vascularization at the fracture site. This can be achieved by either activating angiogenic pathways or by blocking angiogenesis inhibitors. Blocking angiogenic inhibitors has received relatively little attention as a therapeutic target in f...

A computational model to explore the role of angiogenic impairment on endochondral ossification during fracture healing

“…The predicted pattern of healing in the model of mildly impaired angiogenesis was similar to that observed experimentally during ischemic fracture healing by Miedel et al (Miedel et al, 2013), while the pattern of healing predicted in the models of moderately and severely impaired angiogenesis was similar to the ex- perimental observations of Lu et al (Lu et al, 2007). In their study, Miedel et al (Miedel et al, 2013) found that, although ischemia during fracture healing resulted in delayed healing, by day 20, bony union between the cortical surfaces had been achieved. This behaviour was observed in the model of mild vascular impairment whereby bridging of the fracture gap had occurred by day 21.…”

“…The developed models of ischemia also provide a potential explanation for the conflicting results reported by studies examining the pattern of healing within fractured ischemic limbs (Miedel et al, 2013;Lu et al, 2007). The predicted pattern of healing in the model of mildly impaired angiogenesis was similar to that observed experimentally during ischemic fracture healing by Miedel et al (Miedel et al, 2013), while the pattern of healing predicted in the models of moderately and severely impaired angiogenesis was similar to the ex- perimental observations of Lu et al (Lu et al, 2007).…”

“…In order to test the model hypothesis that oxygen levels regulate chondrocyte hypertrophy and endochondral ossification during fracture healing, model predictions were compared to experimental measurements of the ratio of cartilage to hypertrophic cartilage in normal and ischemic fractures (Taylor et al, 2009;Miedel et al, 2013). In the case of normal fracture healing, only the day 10 experimental results were available as very little cartilage was observed experimentally by day 20 (Taylor et al, 2009).…”

“…Computational mechanobiological models of fracture healing under normal and impaired angiogenesis were developed and compared to experimental murine models of normal and ischemic fracture healing described in detail elsewhere (Taylor et al, 2009;Miedel et al, 2013). Briefly, for normal fracture healing, closed transverse fractures were created in both the right and left tibiae of 9 to 10 week-old mice.…”

“…Ischemic fractures are typically characterised by an altered temporal and spatial distribution of tissue phenotypes within the callus when compared to nonischemic fractures (Miedel et al, 2013;Lu et al, 2007). Observations range from increased cell apoptosis, reduced cell proliferation and decreased bone formation (Lu et al, 2007), to the persistence of cartilage within the ischemic fracture callus (Miedel et al, 2013).…”