Impaired bone healing following treatment of established nonunion correlates with serum cytokine expression

Abstract

Delayed union and nonunion are a significant concern in long bone fractures and spinal fusions. Treatment of nonunion often entails multiple revision surgeries that further increase the financial, physical, and emotional burden on patients. The optimal treatment strategy for nonunions remains unclear in many cases, and the risk of complications after revision procedures remains high. This is in part due to our limited understanding of the biological mechanisms that inhibit proper bone healing and lead to nonunion. And yet, few preclinical models directly investigate how healing is impacted after establishment of nonunion, with most instead primarily focusing on treatment immediately after a fresh bone injury. Here, we utilized a critical size femoral defect model in rats where treatment was delayed 8 weeks post-injury, at which time nonunion was established. In this study, acute and delayed treatments with bone morphogenetic protein-2 (BMP-2) were assessed. We found that delayed treatment resulted in decreased bone formation and reduced mechanical strength compared to acute treatment, even when BMP-2 dose was increased by 2.5 times the acute treatment dose. Interestingly, serum cytokine analysis at 12 weeks post-treatment revealed signs of chronic immune dysregulation after delayed treatment. In particular, non-responders (rats that did not exhibit defect bridging) demonstrated higher overall expression of inflammatory cytokines, including TNFa and IL-1b, compared to responders. These findings suggest that re-establishing long-term immune homeostasis may be critical for successful bone healing, particularly after nonunion. ß

Evaluation of global gene expression in regenerate tissues during Masquelet treatment

“…If a chronic nonunion represents an adaptive immune environment, one possible future outcome of our data would be the identification of indicators of nonunion that could be monitored in at‐risk patients with any type of fracture. Indeed, systemic levels of inflammatory cytokines remain elevated up to 12 weeks after the establishment of a nonunion using the rat critical‐size defect model 35 …”

Early systemic immune biomarkers predict bone regeneration after trauma

“…Here we used a previously established femoral bone defect model of chronic nonunion in rats (25) to investigate systemic immune dysregulation and how it relates to functional bone regeneration. In this model, treatment with bone morphogenetic protein 2 (BMP-2) is delivered 8 wk after initial creation of the bone defect (delayed treatment), which is the time needed to establish nonunion, as defined by radiographic mineralized capping of bone ends.…”

Heparin-mediated delivery of bone morphogenetic protein-2 improves spatial localization of bone regeneration

“…Although robust bone regeneration has been achieved in animal models using a variety of BMP-2 delivery vehicles, these results cannot easily be translated to humans due to the dramatically higher BMP-2 doses required to induce bone repair in humans (0.1 to 1 mg BMP-2/kg body weight) ( 7 , 9 )] and do not recapitulate the heterotopic ossification and soft tissue inflammation that are potential consequences of clinical BMP-2 delivery. In addition, challenging conditions typically associated with chronic fracture nonunion in humans cannot easily be treated with low doses of BMP-2 ( 37 ). The 8-mm rat femoral defect described here exhibits consistent bony bridging following treatment with low BMP-2 doses (0.01 to 0.02 mg BMP-2/kg body weight) delivered within an alginate hydrogel ( 18 , 35 ).…”