Joel D. Boerckel

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal μ-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. μ-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

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.

Human stem cell delivery for treatment of large segmental bone defects

Local or systemic stem cell delivery has the potential to promote repair of a variety of damaged or degenerated tissues. Although various stem cell sources have been investigated for bone repair, few comparative reports exist, and cellular distribution and viability postimplantation remain key issues. In this study, we quantified the ability of tissue-engineered constructs containing either human fetal or adult stem cells to enhance functional repair of nude rat critically sized femoral defects. After 12 weeks, defects treated with cell-seeded polymer scaffolds had significantly higher bone ingrowth and torsional strength compared to those receiving acellular scaffolds, although there were no significant differences between the cell sources. Next, stem cells were labeled with fluorescent quantum dots (QDs) in an attempt to noninvasively track their distribution after delivery on scaffolds. Clear fluorescence was observed at implantation sites throughout the study; however, beginning 7–10 days after surgery, signals were also observed at contralateral sites treated with acellular QD-free scaffolds. Although immunostaining for human nuclei revealed retention of some cells at the implantation site, no human cells were detected in the control limb defects. Additional histological analysis of implantation and control defect tissues revealed macrophages containing endocytosed QDs. Furthermore, QD-labeling appeared to diminish transplanted cell function resulting in reduced healing responses. In summary, augmentation of polymeric scaffolds with stem cells derived from fetal and adult tissues significantly enhanced healing of large segmental bone defects; however, QD labeling of stem cells eliminated the observed therapeutic effect and failed to conclusively track stem cell location long-term in vivo.

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.

Spatiotemporal delivery of bone morphogenetic protein enhances functional repair of segmental bone defects

Osteogenic growth factors that promote endogenous repair mechanisms hold considerable potential for repairing challenging bone defects. The local delivery of one such growth factor, bone morphogenetic protein (BMP), has been successfully translated to clinical practice for spinal fusion and bone fractures. However, improvements are needed in the spatial and temporal control of BMP delivery to avoid the currently used supraphysiologic doses and the concomitant adverse effects. We have recently introduced a hybrid protein delivery system comprised of two parts: a perforated nanofibrous mesh that spatially confines the defect region and a functionalized alginate hydrogel that provides temporal growth factor release kinetics. Using this unique spatiotemporal delivery system, we previously demonstrated BMP-mediated functional restoration of challenging 8mm femoral defects in a rat model. In this study, we compared the efficacy of the hybrid system in repairing segmental bone defects to that of the current clinical standard, collagen sponge, at the same dose of recombinant human BMP-2. In addition, we investigated the specific role of the nanofibrous mesh tube on bone regeneration. Our results indicate that the hybrid delivery system significantly increased bone regeneration and improved biomechanical function compared to collagen sponge delivery. Furthermore, we observed that presence of the nanofiber mesh tube was essential to promote maximal mineralized matrix synthesis, prevent extra-anatomical mineralization, and guide an integrated pattern of bone formation. Together, these results suggest that spatiotemporal strategies for osteogenic protein delivery may enhance clinical outcomes by improving localized protein retention.

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.

Effects of In Vivo Mechanical Loading on Large Bone Defect Regeneration

Fracture healing is highly sensitive to mechanical conditions; however, the effects of mechanical loading on large bone defect regeneration have not been evaluated. In this study, we investigated the effects of functional loading on repair of critically sized segmental bone defects. About 6‐mm defects were created in rat femora, and each defect received 5 µg recombinant human bone morphogenetic protein‐2 (rhBMP‐2), delivered in alginate hydrogel. Limbs were stabilized by either stiff fixation plates for the duration of the study or compliant plates that allowed transfer of compressive ambulatory loads beginning at week 4. Healing was assessed by digital radiography, microcomputed tomography, mechanical testing, histology, and finite element modeling. Loading significantly increased regenerate bone volume and average polar moment of inertia. The response to loading was location‐dependent with the polar moment of inertia increased at the proximal end of the defect but not the distal end. As a result, torsional stiffness was 58% higher in the compliant plate group, but failure torque was not altered. In single samples assessed for histology from each group, a qualitatively greater amount of cartilage and a lesser degree of remodeling to lamellar bone occurred in the loaded group compared to the stiff plate group. Finally, principal strain histograms, calculated by FE modeling, revealed that the compliant plate samples had adapted to more efficiently distribute loads in the defects. Together, these data demonstrate that functional transfer of axial loads alters BMP‐induced large bone defect repair by increasing the amount and distribution of bone formed within the defect.

In Vivo Model for Evaluating the Effects of Mechanical Stimulation on Tissue-Engineered Bone Repair

It has long been known that the bone adapts according to the local mechanical environment. To date, however, a model for studying the effects of functional mechanical loading on tissue-engineered bone repair in vivo has not yet been established. We have developed a rat femoral defect model, in which ambulatory loads are transduced through the implanted tissue-engineered construct to elucidate the role of the mechanical environment in functional restoration of a large bone defect. This model uses compliant fixation plates with integrated elastomeric segments, which allow transduction of ambulatory loads. Multiaxially and uniaxially compliant plates were characterized by mechanical testing and evaluated using in vivo pilot studies. In the first study, experimental limbs were implanted with multiaxial plates, which have a low stiffness in multiple loading modes. In the second study, experimental limbs were stabilized by a uniaxial plate, which allowed only axial deformation of the defect. X-ray scans and mechanical testing revealed that the multiaxial plates were insufficient to stabilize the defect and prevent fracture under ambulatory loads as a result of low flexural and torsional stiffness. The uniaxial plates, however, maintained integrity of the defect when implanted over a 12 week period. Postmortem microCT scans revealed a 19% increase in bone volume in the axially loaded limb compared with the contralateral standard control, and postmortem mechanical testing indicated that torsional strength and stiffness were increased 25.6- and 3.9-fold, respectively, compared with the control. Finite element modeling revealed high strain gradients in the soft tissue adjacent to the newly formed bone within the implanted construct. This study introduces an in vivo model for studying the effects of physiological mechanical loading on tissue-engineered bone repair. Preliminary results using this new in vivo model with the uniaxially compliant plate showed positive effects of load-bearing on functional defect repair.

Evolution of Bone Grafting: Bone Grafts and Tissue Engineering Strategies for Vascularized Bone Regeneration

The regeneration of bone in segmental defects has historically been a challenge in the orthopedic field. In particular, a lack of vascular supply often leads to nonunion and avascular necrosis. While the gold standard of clinical care remains the autograft, this approach is limited for large bone defects. Therefore, allograft bone is often required for defects of critical size though a high complication rate is directly attributable to their limited ability to revitalize, revascularize, and remodel resulting in necrosis and re-fracture. However, emerging insights into the mechanisms of bone healing continue to expand treatment options for bony defects to include synthetic materials, growth factors, and cells. The success of such strategies hinges on fabricating an environment that can mimic the body’s natural healing process, allowing for vascularization, bridging, and remodeling of bone. Biological, chemical, and engineering techniques have been explored to determine the appropriate materials and factors for potential use. This review will serve to highlight some of the historical and present uses of allografts and autografts and current strategies in bone tissue engineering for the treatment for bony defects, with particular emphasis on vascularization.

Functional restoration of critically sized segmental defects with bone morphogenetic protein-2 and heparin treatment.

BackgroundBone defects and fracture nonunions remain a substantial challenge for clinicians. Grafting procedures are limited by insufficient volume and donor site morbidity. As an alternative, biomaterial scaffolds functionalized through incorporation of growth factors such as bone morphogenetic proteins (BMPs) have been developed and appear to regenerate the structure and function of damaged or degenerated skeletal tissue.Objectives/purposesOur objectives were therefore to determine whether: (1) the addition of heparin alone to collagen scaffolds sufficed to promote bone formation in vivo; (2) collagen-heparin scaffold improved BMP-mediated bone regeneration; and (3) precomplexed heparin and BMP-2 delivered on collagen scaffold could restore long bone biomechanical strength.MethodsWe created bilateral surgical defects in the femora of 20 rats and filled the defects with PCL scaffolds with one of five treatments: collagen matrix (n = 5), collagen/heparin matrix (n = 7), collagen matrix + BMP-2 (n = 9), collagen/heparin matrix + BMP-2 (n = 9), or collagen matrix + BMP-2/heparin complex (n = 9). Bone formation was observed with radiographs and micro-CT analysis and biomechanical testing was used to assess strength.ResultsThe addition of heparin alone to collagen did not promote bone ingrowth and the addition of heparin to collagen did not improve BMP-mediated bone regeneration. Delivery of precomplexed BMP-2 and heparin in a collagen matrix resulted in new bone formation with mechanical properties similar to those of intact bone.Clinical RelevanceOur findings suggest delivery of precomplexed BMP-2 and heparin may be an advantageous strategy for treatment of clinically challenging bone defects.