The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation
Introduction
Segmental bone defects and non-unions resulting from trauma, resection or pathology represent significant clinical challenges world-wide, for the orthopaedic, reconstructive and maxillofacial surgeons [1], [2]. To date, bone grafting techniques that use materials such as autografts, allografts or metallic implants face considerable limitations for bone repair due to the lack of available bone tissue, disease transmission, cell-mediated immune responses, and significant costs. These difficulties have resulted in the search for alternative bone graft substitutes to be used in repairing non-union fractures, spinal fusions and bone tumour resections.
The development and regeneration of natural bone tissue is a complex, coordinated temporal process involving a myriad of molecular, cellular, biochemical and mechanical cues [3]. Advances in the field of regenerative medicine have led to the possibility of successfully repairing and restoring the function in damaged or diseased tissues [4], [5]. The release of growth factors and osteoprogenitor cell populations by biocompatible scaffolds have been shown to enhance the regenerative capacity of bone [6], [7], [8] as a consequence, in part, of replicating the natural bone environment by provision of an appropriate synthetic extracellular matrix scaffolds [9], presentation of growth factors [10] and osteoprogenitor cells. The presentation of these cells and cues, at appropriate time intervals offers, the opportunity to mimic the repair sequences in bone.
However, critical for regenerative tissue is the generation and existence of a functional microvascular network within the engineered constructs to provide oxygen and nutrients to facilitate growth, differentiation, and tissue functionality [3], [11]. Thus, the lack of an adequate microvascular network will result in the hypoxic cell death of the implanted engineered tissue. Factors that are involved in angiogenesis are also fundamental to the osteogenic response in fracture healing. VEGF165, a potent growth factor involved in angiogenesis has been extensively investigated and shown to be involved in osteogenesis and bone repair [12]. During endochondral bone formation VEGF165 modulates angiogenesis, chondrocyte apoptosis, cartilage remodelling, osteoblast migration and endochondral growth plate ossification [13], [14], [15], [16]. Moreover, BMP-2 has been shown to upregulate VEGF165 [17]. Conversely, inhibition of angiogenesis prevents fracture healing [18] and inhibition of VEGF165 decreases angiogenesis, callus mineralisation and bone healing [19].
A number of studies have shown that the presentation of individual growth factors encapsulated in scaffolds lead to successful engineering of bone like constructs. Indeed the release of active BMP-2 & VEGF165 respectively from polymer scaffolds such as PDLLA has been shown to induce HBMSC osteogenesis [6], [20]. However, mimicking the signalling cascade, release and presentation of these growth factors in a fracture repair by developing composite scaffolds that can release a number of growth factors at varying kinetic rates in situ will, ultimately, enhance the regenerative capacity of bone. Recently, scaffolds have been developed to release dual sets of growth factors for regeneration of tissue [10]. Simmons et al. [21] showed that TGF-β3 and BMP-2 released from a scaffold and transplanted bone marrow stromal cells enhanced bone formation in vivo. Additionally, Huang et al. [7] fabricated Poly(lactic-co-glycolic acid) scaffolds which were able to release VEGF and a plasmid DNA encoding BMP-4 resulting in the generation of ectopic bone by HBMSC within these scaffolds when implanted into the subcutaneous tissue of SCID mice.
The presentation of multiple growth factors released at different kinetic rates from 3D biodegradable scaffolds within close proximity of osteoprogenitor cells offers an attractive strategy for the augmentation and repair of bone defects. Using a supercritical CO2 and alginate entrapment technique, composite scaffolds consisting of alginate and poly(d,l-lactic acid) (PLA) were generated to allow different biodegradation rates and release of selected factors at different rates [22]. For this study VEGF165 was encapsulated in the alginate fibres and BMP-2 was encapsulated into PDLLA inducing an accelerated rate of release of VEGF165 and slower rate of release of BMP-2. In this current study we have used a critical sized femur defect model (load–bearing) to investigate the potential bone regeneration capacity of such a combination therapy of human bone marrow stromal cells (HBMSC) transplanted onto the composite alginate-VEGF165/PDLLA-BMP-2 scaffolds.
Section snippets
Materials
Recombinant human BMP-2 was kindly provided by Professor Walter Sebald, University of Wurzburg, Germany. Recombinant Human VEGF165 was purchased from Tebu-bio, UK. Vibrant® carboxy fluorescein diacetate, succinimidyl ester (CFDA SE) cell tracer kit and Ethidium Homodimer-1 were purchased from Molecular Probes, UK. BMP-2 and VEGF165 ELISA kits were purchased from R&D Systems, UK. Phosphate Buffered Saline (PBS) was purchased from Lonza Biologics, UK; endothelial cell culture growth supplement
The release of VEGF165 and BMP-2 from alginate-VEGF165/PDLLA-BMP-2 scaffolds
A VEGF165 and BMP-2 specific ELISA kit was used to quantitate the release of each growth factor respectively from the composite scaffolds in vitro culture. After 7 days in culture there was an increased release of VEGF165 (750.4 ± 596.8 ρg/ml) compared to BMP-2 (136.9 ± 123.4 ρg/ml) from the alginate-VEGF165/PDLLA-BMP-2 composite scaffolds. Over a 28 day culture period the release kinetics of the two growth factors from the alginate-VEGF165/PDLLA-BMP-2 composite scaffolds showed a cumulative release
Discussion
Natural bone regeneration is a complex, highly coordinated temporal process in which a number of environmental stimuli play critical roles in the proliferation, differentiation, and osteogenesis of progenitor cells. For successful regeneration of bone tissue the coordinated actions of progenitor cells, kinetic release of growth factors, re vascularisation of the de novo tissue and a suitable scaffold template to accommodate these coordinated actions is essential. In this study we have attempted
Conclusion
In summary, our current studies demonstrate the ability to deliver growth factors locally from biodegradable composite scaffolds at varying kinetic rates in close proximity to seeded HBMSC can enhance the reparative mechanism of critical sized bone defects; thus, mimicking the in vivo bone repair environment. Strategies to enable the coordinated, multiple releases of growth factors such as VEGF165 and BMP-2 from composite scaffolds offer an opportunity to mimic, in part, the conditions present
Acknowledgements
The authors would like to thank members of the Bone and Joint Research Group for useful discussions and, in particular, Ms Kate Murawski for her technical assistance. We would like to thank Dr Larry Fisher for provision of Type I collagen antibody and Professor Walter Sebald for the provision of BMP-2 and to the midwives at the Princess Anne hospital for the collection of umbilical cords. This work was supported by the Engineering and Physical Sciences Research Council (grant number
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