An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects
Introduction
Autologous and allogeneic bone grafting are the most widely used treatment modalities for fracture non-unions and large bone defects [1], [2]. However, these techniques are associated with a number of drawbacks, including the limited graft material available for autografts and the high failure rate of allografts [3], [4], [5]. These limitations have stimulated the search for improved techniques for bone repair, and tissue engineering/regenerative medicine (TE/RM) strategies have demonstrated significant potential in developing bone graft substitutes [6], [7]. These approaches promote tissue repair by providing a combination of physical and biochemical cues through structural scaffolds and biologics [8], [9], [10].
Much of bone TE/RM research is focused on the use of three-dimensional scaffolds having adequate strength to support in vivo loading [11], [12], [13]. However, these structural scaffolds are difficult to design and fabricate at high porosity. They usually do not provide an optimal environment for cellular function and many suffer from slow resorption kinetics, thereby impeding functional restoration of the damaged tissue. We previously demonstrated, for example, that poly(l/dl-lactide) scaffolds infused with recombinant human bone morphogenetic protein-2 (rhBMP-2) promoted bone ingrowth but failed to fully restore the mechanical properties of long bone defects [11]. Thin, two-dimensional membranes have been used to promote bone repair by placing them along the periosteal surface to demarcate the osseous from the non-osseous region [14], [15], [16], [17]. This technique, termed guided bone regeneration, has been applied successfully in the oral and maxillofacial fields to regenerate lost alveolar and skull bone [18], [19], [20]. However, few studies have investigated the use of polymer membranes in the treatment of large defects in load-bearing bones, and none have quantitatively evaluated the restoration of limb function [21], [22], [23].
Electrospun nanofiber meshes have recently emerged as a new generation of scaffold membranes, possessing a number of features suitable for tissue regeneration [24], [25]. They have fibers of the same size-scale of extracellular matrix (ECM) components (fiber diameters ranging from nanometer to sub-micrometer) and a large surface area, which may improve cellular attachment, morphology, migration and function. Nanofiber meshes have been shown to support osteogenic differentiation of progenitor and stem cells in vitro [26], [27], [28], [29], and have been tested in calvarial defect models in vivo [30], [31]. However, their efficacy in guiding long bone regeneration in vivo remains to be investigated.
Though a scaffold provides a template for guiding bone regeneration, biologic factors such as cells, growth factors or genes are typically required to effectively regenerate challenging bone defects [11], [32]. Osteoinductive growth factors like rhBMP-2 have demonstrated some clinical success for bone healing, but large doses are needed [33], [34]. Delivery systems that provide sustained release and improved local retention may provide efficacy at lower protein dose, thereby minimizing complications and making the therapy more cost effective [35], [36], [37], [38]. Alginate hydrogels, made from brown algae derived polysaccharides, have been established as a scaffolding material [39] and a spatiotemporal delivery vehicle for a wide range of proteins [40], [41], [42]. Though mammalian cells lack receptors for alginate polymers, the alginates can be covalently coupled with adhesion peptides to promote cellular attachment [43]. In addition, the degradation rate of these hydrogels can be increased by Gamma-irradiation, resulting in lower molecular weight polymers. These modified alginates have been demonstrated to be better suited for TE/RM applications by allowing faster ingrowth of cells and tissue [39], [44].
The primary objective of this study was to develop and test a hybrid growth factor delivery system for bone repair that utilizes an injectable alginate hydrogel for protein delivery and an electrospun nanofiber mesh for guiding bone regeneration. To test this system, we evaluated its ability to deliver rhBMP-2 for the repair of critically-sized segmental bone defects in vivo. For control group comparisons, we also examined the ability of the nanofiber mesh alone, and in combination with alginate hydrogel, to heal the bone defects without rhBMP-2. Furthermore, the effect of a perforated nanofiber mesh design on bone repair was investigated. We hypothesized that rhBMP-2 delivery in the nanofiber mesh/alginate system would promote bone ingrowth and fully restore the mechanical properties of 8 mm segmental bone defects in the rat model. We further hypothesized that the perforated nanofiber mesh design would accelerate bone ingrowth due to enhanced early defect vascularization. We tested our hypothesis in an in vivo test bed model that utilizes quantitative techniques to assess differences in bone and vascular regrowth and restoration of mechanical function.
Section snippets
Fabrication of nanofiber mesh tubes
Poly(ε-caprolactone) (PCL) pellets (Sigma–Aldrich, St. Louis, MO) were dissolved in a 90:10 volume ratio of hexafluoro-2-propanol (HFP):dimethylformamide (DMF) (Sigma–Aldrich) to obtain a 12% (w/v) polymer solution. DMF was first slowly added to HFP to prevent excessive heat generation, and mixed well on a stir plate for 5 min. The PCL pellets were then added to the solvent solution, and gently stirred for 16–24 h. The solution was visually inspected to ensure a homogeneous and clear solution.
Nanofiber mesh tube characterization and placement
The nanofibers obtained by electrospinning were observed to be smooth and bead-free (Fig. 1A). The fibers ranged in diameter from 51 nm to 974 nm with 82% of the fibers between 50 nm and 150 nm. The mean and the median fiber diameter were calculated to be 154 nm and 107 nm respectively. Despite the high porosity of these meshes (80–90%), the effective pore size was observed to be less than 5 μm. After 5 h of electrospinning, the mesh was found to be approximately 300–400 μm thick. This
Discussion
The treatment of large osseous defects remains a challenge for orthopaedic surgeons. To address this problem, we have developed a growth factor delivery technique for the functional repair of large bone defects using an electrospun nanofiber mesh tube and alginate hydrogel. Tubular scaffolds constructed from nanofiber meshes were placed around segmental defects. Alginate hydrogel containing 5 μg rhBMP-2 was injected into the tubes and constrained within the defect site by the mesh tube. Our
Conclusions
A hybrid growth factor delivery system utilizing an electrospun nanofiber mesh and alginate hydrogel was presented in this study. This system resulted in complete bony bridging of challenging segmental bone defects in a rat model. Perforations accelerated the deposition of mineralized tissue and resulted in functional repair, possibly due to interactions of the surrounding soft tissues with the regenerating bone. The mesh tube alone, or in combination with alginate hydrogel, did not generate a
Acknowledgements
This study was supported with funding from NIH R37 DE013033, NIH R01 AR051336, the Armed Forces Institute for Regenerative Medicine (AFIRM), and the Center for Advanced Engineering and Soldier Survivability (CABSS). The authors would like to thank Dr. Megan Oest for assistance with the segmental defect model and Dr. Craig L. Duvall for advice on μCT-based angiography. We thank Angela Lin and Dr. Laura O’Farrell for assistance with μCT analysis and animal welfare, respectively. We gratefully
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