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ABSTRACT: Although severe extremity trauma is often inclusive of skeletal and vascular damage in combination, segmental bone defect repair with concomitant vascular injury has yet to be experimentally investigated. To this end, we developed a novel rat composite limb injury model by combining a critically-sized segmental bone defect with surgically-induced hind limb ischemia (HLI). Unilateral 8 mm femoral defects were created alone (BD) or in combination with HLI (BD+HLI), and all defects were treated with rhBMP-2 via a hybrid biomaterial delivery system. Based on reported clinical and experimental observations on the importance of vascular networks in bone repair, we hypothesized that HLI would impair bone regeneration. Interestingly, the BD+HLI group displayed improved radiographic bridging, and quantitative micro-CT analysis revealed enhanced bone regeneration as early as week 4 (p<0.01) that was sustained through week 12 (p<0.001) and confirmed histologically. This effect was observed in two independent studies and at two different doses of rhBMP-2. Micro-CT angiography was used to quantitatively evaluate vascular networks at week 12 in both the thigh and the regenerated bone defect. No differences were found between groups in total blood vessel volume in the thigh, but clear differences in morphology were present as the BD+HLI group possessed a more interconnected network of smaller diameter vessels (p<0.001). Accordingly, while the overall thigh vessel volume was comparable between groups, the contributions to vessel volume based on vessel diameter differed significantly. Despite this evidence of a robust neovascular response in the thigh of the BD+HLI group, differences were not observed between groups for bone defect blood vessel volume or morphology. In total, our results demonstrate that a transient ischemic insult and the subsequent recovery response to HLI significantly enhanced BMP-2-mediated segmental bone defect repair, providing additional complexity to the relationship between vascular tissue networks and bone healing. Ultimately, a better understanding of the coupling mechanisms may reveal important new strategies for promoting bone healing in challenging clinical scenarios.
Bone 05/2013; · 4.02 Impact Factor
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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 pre-clinical 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 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 non-healing defect in the femur), or composite injury combining the bone and muscle defects. Bone defects were treated with 2μg of rhBMP-2 delivered in pre-gelled alginate injected into a cylindrical perforated nanofiber mesh. Bone regeneration was quantitatively assessed using μCT, and limb function was assessed using gait analysis and muscle strength measurements. At 12 weeks post-surgery, 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 two weeks, 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.
Tissue Engineering Part C Methods 09/2012; · 4.64 Impact Factor
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ABSTRACT: The use of tissue grafting for the repair of large bone defects has numerous limitations including donor site morbidity and the risk of disease transmission. These limitations have prompted research efforts to investigate the effects of combining biomaterial scaffolds with biochemical cues to augment bone repair. The goal of this study was to use a critically-sized rat femoral segmental defect model to investigate the efficacy of a delivery system consisting of an electrospun polycaprolactone (PCL) nanofiber mesh tube with a silk fibroin hydrogel for local recombinant bone morphogenetic protein 2 (BMP-2) delivery. Bilateral 8 mm segmental femoral defects were formed in 13-week-old Sprague Dawley rats. Perforated electrospun PCL nanofiber mesh tubes were fitted into the adjacent native bone such that the lumen of the tubes contained the defect (Kolambkar et al., 2011b). Silk hydrogels with or without BMP-2 were injected into the defect. Bone regeneration was longitudinally assessed using 2D X-ray radiography and 3D microcomputed topography (μCT). Following sacrifice at 12 weeks after surgery, the extracted femurs were either subjected to biomechanical testing or assigned for histology. The results demonstrated that silk was an effective carrier for BMP-2. Compared to the delivery system without BMP-2, the delivery system that contained BMP-2 resulted in more bone formation (p<0.05) at 4, 8, 12 weeks after surgery. Biomechanical properties were also significantly improved in the presence of BMP-2 (p<0.05) and were comparable to age-matched intact femurs. Histological evaluation of the defect region indicated that the silk hydrogel has been completely degraded by the end of the study. Based on these results, we conclude that a BMP-2 delivery system consisting of an electrospun PCL nanofiber mesh tube with a silk hydrogel presents an effective strategy for functional repair of large bone defects.
Journal of the mechanical behavior of biomedical materials. 07/2012; 11:123-31.
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ABSTRACT: Severe extremity trauma often results in large zones of injury comprising multiple types of tissue and presents many clinical challenges for reconstruction. Considerable investigation is ongoing in tissue engineering and regenerative medicine therapeutics to improve reconstruction outcomes; however, the vast majority of musculoskeletal trauma models employed for testing the therapeutics consist of single-tissue defects, offering limited utility for investigating strategies for multi-tissue repair. Here we present the first model of composite lower limb bone and nerve injury, characterized by comparison to well-established, single-tissue injury models, using biomaterials-based technologies previously demonstrated to show promise in those models. Quantitative functional outcome measures were incorporated to facilitate assessment of new technologies to promote structural and functional limb salvage following severe extremity trauma. Nerve injury induced significant changes in the morphology and mechanical properties of intact bones. However, BMP-mediated segmental bone regeneration was not significantly impaired by concomitant nerve injury, as evaluated via radiographs, microcomputed tomography (μCT) and biomechanical testing. Neither was nerve regeneration significantly impaired by bone injury when evaluated via histology and electrophysiology. Despite the similar tissue regeneration observed, the composite injury group experienced a marked functional deficit in the operated limb compared to either of the single-tissue injury groups, as determined by quantitative, automated CatWalk gait analysis. As a whole, this study presents a challenging, clinically relevant model of severe extremity trauma to bone and nerve tissue, and emphasizes the need to incorporate quantitative functional outcome measures to benchmark tissue engineering therapies. Copyright © 2012 John Wiley & Sons, Ltd.
Journal of Tissue Engineering and Regenerative Medicine 06/2012; · 3.28 Impact Factor
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ABSTRACT: New vascular network formation is a critical step in the wound healing process and a primary limiting factor in functional tissue regeneration. Like many tissues, neovascular networks have been shown in vitro to be highly sensitive to mechanical conditions; however, the effects of matrix deformations on neovascular network formation and remodeling in engineered tissue regeneration in vivo have not been evaluated. We quantified the effects of early and delayed functional loading on neovascular growth in a rat model of large bone defect regeneration using compliant fixation plates that were unlocked to allow transfer of ambulatory loads to the defect either at the time of implantation (early), or after 4 wk of stiff fixation (delayed). Neovascular growth and bone regeneration were quantitatively evaluated 3 wk after the onset of loading by contrast-enhanced microcomputed tomography and histology. The initial vascular response to bone injury featured robust angiogenesis and collateral vessel formation, increasing parameters such as vascular volume and connectivity while decreasing degree of anisotropy. Application of early mechanical loading significantly inhibited vascular invasion into the defect by 66% and reduced bone formation by 75% in comparison to stiff plate controls. In contrast, delaying the onset of loading by 4 wk significantly enhanced bone formation by 20% and stimulated vascular remodeling by increasing the number of large vessels and decreasing the number of small vessels. Together, these data demonstrate the mechanosensitivity of neovascular networks and highlight the capacity of biomechanical stimulation to modulate postnatal vascular growth and remodeling.
Proceedings of the National Academy of Sciences 08/2011; 108(37):E674-80. · 9.68 Impact Factor
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ABSTRACT: Delivery of recombinant proteins is a proven therapeutic strategy to promote endogenous repair mechanisms and tissue regeneration. Bone morphogenetic protein-2 (rhBMP-2) has been used to promote spinal fusion and repair of challenging bone defects; however, the current clinically-used carrier, absorbable collagen sponge, requires high doses and has been associated with adverse complications. We evaluated the hypothesis that the relationship between protein dose and regenerative efficacy depends on delivery system. First, we determined the dose-response relationship for rhBMP-2 delivered to 8-mm rat bone defects in a hybrid nanofiber mesh/alginate delivery system at six doses ranging from 0 to 5 μg. Next, we directly compared the hybrid delivery system to the collagen sponge at 0.1 and 1.0 μg. Finally, we compared the in vivo protein release properties of the two delivery methods. In the hybrid delivery system, bone volume, connectivity and mechanical properties increased in a dose-dependent manner to rhBMP-2. Consistent bridging of the defect was observed for doses of 1.0 μg and greater. Compared to collagen sponge delivery at the same 1.0 μg dose, the hybrid system yielded greater connectivity by week 4 and 2.5-fold greater bone volume by week 12. These differences may be explained by the significantly greater protein retention in the hybrid system compared to collagen sponge. This study demonstrates a clear dose-dependent effect of rhBMP-2 delivered using a hybrid nanofiber mesh/alginate delivery system. Furthermore, the effective dose was found to vary with delivery system, demonstrating the importance of biomaterial carrier properties in the delivery of recombinant proteins.
Biomaterials 08/2011; 32(22):5241-51. · 7.40 Impact Factor
