Brent A. Uhrig

Effects of protein dose and delivery system on BMP-mediated bone regeneration.

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.

Mechanical regulation of vascular growth and tissue regeneration in vivo

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.

A silk hydrogel-based delivery system of bone morphogenetic protein for the treatment of large bone defects.

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.

Oxidized alginate hydrogels for bone morphogenetic protein-2 delivery in long bone defects

Autograft treatment of large bone defects and fracture non-unions is complicated by limited tissue availability and donor site morbidity. Polymeric biomaterials such as alginate hydrogels provide an attractive tissue engineering alternative due to their biocompatibility, injectability, and tunable degradation rates. Irradiated RGD-alginate hydrogels have been used to deliver proteins such as bone morphogenetic protein-2 (BMP-2), to promote bone regeneration and restoration of function in a critically sized rat femoral defect model. However, slow degradation of irradiated alginate hydrogels may impede integration and remodeling of the regenerated bone to its native architecture. Oxidation of alginate has been used to promote degradation of alginate matrices. The objective of this study was to evaluate the effects of alginate oxidation on BMP-2 release and bone regeneration. We hypothesized that oxidized-irradiated alginate hydrogels would elicit an accelerated release of BMP-2, but degrade faster in vivo, facilitating the formation of higher quality, more mature bone compared to irradiated alginate. Indeed, oxidation of irradiated alginate did accelerate in vitro BMP-2 release. Notably, the BMP-2 retained within both constructs was bioactive at 26days, as observed by induction of alkaline phosphatase activity and positive Alizarin Red S staining of MC3T3-E1 cells. From the in vivo study, robust bone regeneration was observed in both groups through 12weeks by radiography, micro-computed tomography analyses, and biomechanical testing. Bone mineral density was significantly greater for the oxidized-irradiated alginate group at 8weeks. Histological analyses of bone defects revealed enhanced degradation of oxidized-irradiated alginate and suggested the presence of more mature bone after 12weeks of healing.

Functional analysis of limb recovery following autograft treatment of volumetric muscle loss in the quadriceps femoris

Severe injuries to the extremities often result in muscle trauma and, in some cases, significant volumetric muscle loss (VML). These injuries continue to be challenging to treat, with few available clinical options, a high rate of complications, and often persistent loss of limb function. To facilitate the testing of regenerative strategies for skeletal muscle, we developed a novel quadriceps VML model in the rat, specifically addressing functional recovery of the limb. Our outcome measures included muscle contractility measurements to assess muscle function and gait analysis for evaluation of overall limb function. We also investigated treatment with muscle autografts, whole or minced, to promote regeneration of the defect area. Our defect model resulted in a loss of muscle function, with injured legs generating less than 55% of muscle strength from the contralateral uninjured control legs, even at 4 weeks post-injury. The autograft treatments did not result in significant recovery of muscle function. Measures of static and dynamic gait were significantly decreased in the untreated, empty defect group, indicating a decrease in limb function. Histological sections of the affected muscles showed extensive fibrosis, suggesting that this scarring of the muscle may be in part the cause of the loss of muscle function in this VML model. Taken together, these data are consistent with clinical findings of reduced muscle function in large VML injuries. This new model with quantitative functional outcome measures offers a platform on which to evaluate treatment strategies designed to regenerate muscle tissue volume and restore limb function.

Recovery from hind limb ischemia enhances rhBMP-2-mediated segmental bone defect repair in a rat composite injury model

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 8mm 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.

Characterization of a composite injury model of severe lower limb bone and nerve trauma

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

Muscle Injury Attenuates BMP-2 Mediated Tissue Regeneration in a Novel Rat Model of Composite Bone and Muscle Injury

Musculoskeletal diseases and injuries are a major burden on society, representing the most common cause of pain and impaired function worldwide. Composite injuries involving bone and the surrounding soft tissue comprise one of the most challenging musculoskeletal conditions to return to normal function. During repair of these injuries there is a loss of the synergistic interactions between adjacent tissues resulting in impaired bone regeneration. Additionally, local soft tissue ischemia may also be a contributing factor to increased infection rates observed in severe composite tissue injuries. Muscle has been implicated as a source for re-vascularization, osteoprogenitor cells and osteogenic factors, as well as a contributor to the biomechanical stimuli; however, associated studies have mostly been qualitative in nature, offering little insight into the mechanistic nature of the relationship of soft tissue to bone regeneration. Small animal models of critically sized bone defects are an efficient means to test engraftment strategies of novel constructs and therapeutics particularly in terms of functional restoration of a limb. Our lab previously developed a critically-sized rat segmental defect model with which we have quantitatively assessed bone regeneration using numerous constructs and therapeutic treatments [1]. Our objective was to develop a composite injury model by combining this segmental defect model with a muscle injury adjacent to the bone defect. We hypothesized that animals with a composite injury would have attenuated BMP-2 mediated tissue regeneration as compared to animals with a single tissue injury.Copyright