Attenuated Human Bone Morphogenetic Protein-2–Mediated Bone Regeneration in a Rat Model of Composite Bone and Muscle Injury

Abstract

Extremity injuries involving large bone defects with concomitant severe muscle damage are a significant clinical challenge often requiring multiple treatment procedures and possible amputation. Even if limb salvage is achieved, patients are typically left with severe short-and long-term disabilities. Current preclinical animal models do not adequately mimic the severity, complexity, and loss of limb function characteristic of these composite injuries. The objectives of this study were to establish a composite injury model that combines a critically sized segmental bone defect with an adjacent volumetric muscle loss injury, and then use this model to quantitatively assess human bone morphogenetic protein-2 (rhBMP-2)-mediated tissue regeneration and restoration of limb function. Surgeries were performed on rats in three experimental groups: muscle injury (8-mm-diameter full-thickness defect in the quadriceps), bone injury (8-mm nonhealing defect in the femur), or composite injury combining the bone and muscle defects. Bone defects were treated with 2 mg of rhBMP-2 delivered in the pregelled alginate injected into a cylindrical perforated nanofiber mesh. Bone regeneration was quantitatively assessed using microcomputed tomography, and limb function was assessed using gait analysis and muscle strength measurements. At 12 weeks postsurgery, treated bone defects without volumetric muscle loss were consistently bridged. In contrast, the volume and mechanical strength of regenerated bone were attenuated by 45% and 58%, respectively, in the identically treated composite injury group. At the same time point, normalized muscle strength was reduced by 51% in the composite injury group compared to either single injury group. At 2 weeks, the gait function was impaired in all injury groups compared to baseline with the composite injury group displaying the greatest functional deficit. We conclude that sustained delivery of rhBMP-2 at a dose sufficient to induce bridging of large segmental bone defects failed to promote regeneration when challenged with concomitant muscle injury. This model provides a platform with which to assess bone and muscle interactions during repair and to rigorously test the efficacy of tissue engineering approaches to promote healing in multiple tissues. Such interventions may minimize complications and the number of surgical procedures in limb salvage operations, ultimately improving the clinical outcome.

Effect of Cell Origin and Timing of Delivery for Stem Cell-Based Bone Tissue Engineering Using Biologically Functionalized Hydrogels

“…37,51,52 The time point chosen in our delayed delivery study was selected based on the assumption that the presence of endogenous tissue in the defect site may facilitate nutrient and waste exchange of the delivered stem cells and facilitate recruitment of host progenitors before the degradation of BMP-2. However, given our results showing significantly less mineralized tissue in delayed samples compared with their immediate counterparts, our delivery time may have been too late.…”

“…Samples were placed in a custom holder for micro-CT scanning. Mineralized matrix composition of the constructs was determined using a VivaCT scanner (Scanco Medical 17,[36][37][38][39] This corresponds to a mineral density of approximately 380 mgHA/ cm 3 . The scans were segmented such that only bone inside the nanofiber mesh was included in the analysis.…”

Pathophysiology of Volumetric Muscle Loss Injury

“…Preclinical models of extremity VML have been developed in various species, including mice [Machingal et al, 2011;Page et al, 2011;Rossi et al, 2011;Corona et al, 2012], rats [Merritt et al, 2010;Wu et al, 2012;Corona et al, 2013a, b;Willett et al, 2013;Aurora et al, 2014;Garg et al, 2014a, c;Li et al, 2014;Pilia et al, 2014;VanDusen et al, 2014;Garg et al, 2015], dogs [Turner et al, 2012], and pigs [unpubl. data] ( fig.…”

“…Li et al [2014] demonstrated that 2 weeks after injury reductions in muscle mass and volume measured by MRI were similar ( ≈ 25%) in rat quadriceps VML, indicating that at least at earlier time points after injury muscle mass and volume deficits are surrogates. At prolonged time points, histologically there are apparent increases in ECM content in the remaining muscle mass [Willett et al, 2013;Garg et al, 2014a], and, therefore, measurements of muscle mass likely underestimate the amount of muscle contractile protein loss as the VML injury progresses. The inclusion among future studies of in vivo imaging modalities that allow discrimination of cellular migration and quantification of the tissue phenotype within VMLinjured muscle [Hatori et al, 2015;Webster et al, 2015] would greatly improve our understanding of secondary pathophysiological manifestations of VML injury.…”

“…1 b). For example, quadriceps and tibialis anterior (TA) muscle VML models that predict an initial 10 and 20% loss of muscle mass have presented strength deficits of ∼ 65% [Willett et al, 2013] and 33% [Corona et al, 2013a], respectively. Greater strength deficits than percent VML have been observed in various animal models (muscle types and species), at prolonged time points ( fig.…”