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, weCorrespondence to: Robert E. Guldberg, robert.guldberg@me.gatech.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBiomaterials. Author manuscript; available in PMC 2012 January 1. 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.

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.

An 8-mm rat segmental defect model was used to evaluate quantitatively the ability of longitudinally oriented poly(L-lactide-co-D,L-lactide) scaffolds with or without growth factors to promote bone healing. BMP-2 and TGF-b3, combined with RGD-alginate hydrogel, were co-delivered to femoral defects within the polymer scaffolds at a dose previously shown to synergistically induce ectopic mineralization. A novel modular composite implant design was used to achieve reproducible stable fixation, provide a window for longitudinal in vivo micro-CT monitoring of 3D bone ingrowth, and allow torsional biomechanical testing of functional integration. Sequential micro-CT analysis showed that bone ingrowth increased significantly between 4 and 16 weeks for the scaffold-treated defects with or without growth factors, but no increase with time was observed in empty defect controls. Treatment with scaffold alone improved defect stability at 16 weeks compared to nontreatment, but did not achieve bone union or restoration of mechanical function. Augmentation of scaffolds with BMP-2 and TGF-b3 significantly increased bone formation at both 4 and 16 weeks compared to nontreatment, but only produced bone bridging of the defect region in two of six cases. Histological evaluation indicated that bone formed first at the periphery of the scaffolds, followed by more limited mineral deposition within the scaffold interior, suggesting that the cells participating in the initial healing response were primarily derived from periosteum. This study introduces a challenging segmental defect model that facilitates quantitative evaluation of strategies to repair critically sized bone defects. Healing of the defect region was improved by implanting structural polymeric scaffolds infused with growth factors incorporated within RGD-alginate. However, functional integration of the constructs appeared limited by continued presence of slow-degrading scaffolds and suboptimal dose or delivery of osteoinductive signals. ß

Porous scaffold biomaterials may offer a clinical alternative to bone grafts; however, scaffolds alone are typically insufficient to heal large bone defects. Numerous studies have demonstrated that osteoinductive growth factor or gene delivery significantly improves bone repair. However, given the important role of vascularization during bone regeneration, it may also be beneficial to incorporate factors that promote vascular ingrowth into constructs. In this study, a strategy combining structural polycaprolactone-20% tricalcium phosphate (PCL-TCP) composite scaffolds with platelet-rich plasma (PRP) was tested. Following bilateral implantation of constructs into 8 mm rat nonunion femoral defects, 3D vascular and bone ingrowth were quantified at 3 and 12 weeks using contrast-enhanced microcomputed tomography (micro-CT) imaging. At week 3, PRP-treated femurs displayed 70.3% higher vascular volume fraction than control femurs. Interestingly, bone volume fraction (BVF) was significantly higher for the empty scaffold group at the early time point. At 12 weeks, BVF measurements between the two groups were statistically equivalent. However, a greater proportion of PRP-treated femurs (83%) achieved bone union as compared to empty scaffold controls (33%). Consistent with this observation, biomechanical evaluation of functional integration also revealed a significantly higher torsional stiffness observed for PRP-treated defects compared to empty scaffolds. Ultimate torque at failure was not improved, however, perhaps due to the slow resorption profile of the scaffold material. Histological evaluation illustrated infiltration of vascularized connective tissue and bone in both groups. Given that bone ingrowth into untreated defects in this model is minimal, PCL-TCP scaffolds were clearly able to promote bone ingrowth but failed to consistently bridge the defect. The addition of PRP to PCL-TCP scaffolds accelerated early vascular ingrowth and improved longer-term functional integration. Taken together, the results of this study suggest that the use of PRP, alone or in combination with other bioactive components, may be an effective approach to augment the ability of porous biomaterial scaffolds to repair orthotopic defects.

Healing large bone defects and non-unions remains a significant clinical problem. Current treatments, consisting of auto-and allografts, are limited by donor supply and morbidity, insufficient bioactivity and risk of infection. Biotherapeutics, including cells, genes and proteins, represent promising alternative therapies, but these strategies are limited by technical roadblocks to biotherapeutic delivery, cell sourcing, high cost, and regulatory hurdles. In the present study, the collagen-mimetic peptide, GFOGER, was used to coat synthetic PCL scaffolds to promote bone formation in criticallysized segmental defects in rats. GFOGER is a synthetic triple helical peptide that binds to the α2β1 integrin receptor involved in osteogenesis. GFOGER coatings passively-adsorbed onto polymeric scaffolds, in the absence of exogenous cells or growth factors, significantly accelerated and increased bone formation in non-healing femoral defects compared to uncoated scaffolds and empty defects. Despite differences in bone volume, no differences in torsional strength were detected after 12 weeks, indicating that bone mass but not bone quality was improved in this model. This work demonstrates a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. This biomaterial coating strategy represents a cost effective and facile approach translatable into a robust clinical therapy for musculoskeletal applications.