Chapter 179 CHAPTER CONCLUSIONS AND RECOMMENDATIONS This thesis presents a successful application of electrospinning and supercritical CO2 foaming techniques in the fabrication of biocompatible and biodegradable controlled release formulations for the delivery of BMP-2 protein and BMP-2 plasmid. Fabrication methods and detailed characterization of the BMP-2 protein-loaded and BMP-2 plasmidloaded formulations have been reported in this thesis. Three-dimensional fibrous scaffolds fabricated by electrospinning contain microscaleinterconnected pores, resembling the extracellular matrix (ECM) and the threedimensional nature of the scaffolds allows for the infiltration and proliferation of cells. Due to the limit of fibrous scaffolds in mechnical strength, they are not capable of being a graft directly. As a result, the osteoconductive role of fibrous scaffolds in bone regeneration is not investigated. However, BMP-2 protein loaded in fibrous scaffolds performs well in the osteoinduction, and therefore enhances the bone healing in tibia bone defect model (see Chapters and 6, and Appendix A1). The incorporation of HAp nanoparticles in fibrous scaffolds significantly enhanced the mechanical strength of scaffolds. Moreover, the HAp nanoparticles incorporated in scaffolds were acting as a regulator of BMP-2 release rate. Specifically, higher content of HAp can speed up the Chapter 180 disintegration of the scaffolds, and subsequently enhance the release of BMP-2 protein from scaffolds, as shown in the in vitro release profiles (Figures 3.6 and 5.5). Animal experiments demonstrated that the bioactivity of BMP-2 protein released from PLGA/HAp scaffolds was maintained and in vivo and satisfactory performance of bone healing was observed. For the case of BMP-2 plasmid delivery, the optimization of dosage forms on cytotoxicity and transfection efficiency is crucial. Naked DNA has low transfection efficiency so DNA loaded chitosan particles are used in DNA delivery due to their high transfection efficiency, but the strong immunological reaction of cells posed by them is undesirable. In order to preserve the advantage of polymeric particles and reduce immunological effect at the same time, a new DNA release system is developed which makes possible sustained DNA release and efficient transfection with negligible immunological effects. BMP-2 plasmid was successfully encapsulated into polymeric fibrous matrices in three different ways, and the animal experimental results indicate that the bioactivity of BMP-2 plasmid released from all the three kinds of scaffolds was well maintained, which helped improve the formation of new bone and the healing of segmental defects in vivo. Groups A, B and C released DNA or DNA nanoparticles in different ways and time frames (Chapter 5), thus their performances in bone healing were diverse (Chapter 6). Nevertheless, all three kinds of scaffolds meet the requirements set for “good” DNA delivery devices, including low cytotoxicity and high transfection efficiency. The conceptual advantages of group C scaffolds (see Chapters and 6), which release plasmid for longer than eight weeks, were not well demonstrated in the tibia bone defect model (see Chapter 181 Appendix A1) as the bone defect will heal by itself in six-week period. To explore this, a larger critical-sized defect will be required in the future study. Preliminary results on critical-sized femur bone defect model suggest that all the three kinds of fibrous scaffolds developed in this research (groups A, B and C) are promising for healing of critical-sized bone defect (refer to Appendix A2). For the three-dimensional PLGA foams produced via supercritical carbon dioxide, their most attractive advantage is the formation of stable and porous structures, but the surfaces will be negatively charged. In the latter part of this thesis (Chapters and 8), two ways of modifications are employed to investigate the effects of -NH2 incorporation on cell adherence, cell viability and in vitro expression. The results suggest that PLGA/chitosan composites fabricated by the combination of spray drying and supercritical CO2 foaming techniques are promising for gene delivery. The chitosan incorporated was coated on the surface of PLGA matrix in an amorphous form, which could help to slow down the disintegration of PLGA matrix, consequently retarding the release of DNA from the polymer matrix. The resultant sustained release of DNA led to low cytotoxicity and sustained expression of gene. Moreover, cell attachment and cell viability are enhanced with the aid of chitosan coating on the PLGA matrix. PLGA porous foams can also be post-functionalized by Lysine-based peptides. The adsorption capacity and release behavior of DNA were found to be highly dependent on the charge properties of the foam surfaces. Because of the presence of ionic interaction between the carboxyl groups of DNA and the amine groups added to the foams F1 and F2, Chapter 182 the release rates of DNA from the K4- and K20-functionalized foams are more sustained in comparison to the blank foam. The positively charged surface of the functionalized foams appeared to be favorable for loading DNA and displayed sustained release of DNA, possibly due to a balance of electrostatic interaction and hydrophilic interaction between DNA and the surface of Lysine pepetide modified foams. The sustained release of DNA from PLGA/chitosan and PLGA/Lysine foams led to negligible cytotoxicity and sustained expression of DNA, which is favorable for DNA delivery and tissue engineering applications. In conclusion, this research has shown potential applications of electrospinning and supercritical CO2 foaming techniques in the fabrication of three dimensional scaffolds for controlled release and bone regeneration. These two techniques can be easily extended to the fabrication of other biomedical devices, such as drug delivery systems for treatment of osteoporosis, brain tumor, central nervous system (CNS) and so on. However, several specific disadvantages are still observed, and therefore call for further works for improvement. Particularly, fibrous scaffolds used for BMP-2 protein delivery (Chapters and 5) and BMP-2 plasmid delivery (Chapters and 6) are not strong enough in mechanical strength, limiting their use as bone graft. As a result, this function was served by the dead bone segment and intra-medullary wire in the current animal model (see Appendix A1) and scaffolds were just utilized to serve as a source of protein or DNA. In future studies, the improvement of PLGA fibrous scaffolds on mechanical properties is necessary. For the cases of PLGA/chitosan and PLGA/Lysine composites, their release profiles have not been optimized so far. The release of DNA from PLGA/chitosan Chapter 183 scaffolds is low, consequently it fails to produce high enough DNA concentration and subsequent significant expression in site. In contrast, the release of DNA from PLGA/Lysine scaffolds is fast but not sustainable, which is not favorable for tissue engineering. Moreover, the DNA from both PLGA/chitosan and PLGA/Lysine composites is in form of naked DNA and its transfection efficiency is extremely low, which would hinder their applications in biomedical engineering. In future investigations, DNA/polycationic complexes, such as DNA/chitosan, DNA/PEI and DNA/PLL, can be incorporated into or onto the foams in some way, on the conditions that the release of DNA/polycationic complex from the foams is significant and sustainable. Thus a comprehensive animal investigation on the PLGA/chitosan scaffolds and PLGA/Lysine scaffolds is deserved. As a versatile technique, electrospinning can be used for the fabrication of core/shell structured microfibers and microspheres (for the case of electrohydrodynamic atomization or EHDA) for co-release or even sequential release of multi-drugs (chemical drugs, proteins, DNA or combinations of them) in the future. Bone regeneration is a complicated process involving many growth factors and different growth factors have diverse roles in different stages of bone regeneration, therefore a sequential release of growth factors from a single release device is highly demanded. This will result in a more effective treatment for bone defects as compared to the treatment by a singale growth factor offered by existing delivery systems. . delivery and tissue engineering applications. In conclusion, this research has shown potential applications of electrospinning and supercritical CO 2 foaming techniques in the fabrication of. C ONCLUSIONS AND RECOMMENDATIONS This thesis presents a successful application of electrospinning and supercritical CO 2 foaming techniques in the fabrication of biocompatible and biodegradable controlled. well maintained, which helped improve the formation of new bone and the healing of segmental defects in vivo. Groups A, B and C released DNA or DNA nanoparticles in different ways and time