In Vitro And In Vivo Study Of Biodegradable Porous Polyester Scaffolds With Hydroxyapatite Coating

Restoration of large tissue defects has been a classic challenge topic in regenerative medicine area, and tissue engineering offers a new route to solve the problem. In tissue engineering, degradable polyesters have been widely applied to prepare porous scaffolds due to its good biocompatibility, excellent processing property and adjustable degradability. So it is quite meaningful to study polyester-based biomaterials and translate them from lab to bench.In spite of many reports of polyesters, some fundamental issues have not yet been extensively studied. For instance, many researchers worried about the side effects of degradation products of polyesters, but which lacks focused and unambiguous experimental evidence about the extent and the key reason of the side effects. Hydroxyapatite is the main component of natural bone. Inspired by it, introducing hydroxyapatite into polyesters has been used to enhance the restoration of large bone defects. It is usually carried out to prepare hydroxyapatite/polyester composites via a mixing technique, while coating hydroxyapatite on pore surfaces of porous polyester scaffolds could be an alternative way, which can not only maintain the good properties of polyester scaffolds, but also trigger osteoconductive and osteoinductive abilities of hydroxyapatite on the interior surfaces. Then, it is quite important to develop some techniques to coat hydroxyapatite on pore surfaces effectively and efficiently. Another fundamental question could be addressed such as interactions between cells and bioceramics. Surface patterning has recently been developed as a powerful tool to study cell-material interactions. While patterning techniques of polymers and metals have been well improved, the preparation method of bioceramic patterns remains a technical bottleneck.This Ph.D. thesis aimed for investigating some fundamental science of biomaterials, including in vitro evaluation of cytotoxicity of degradation products of aliphatic polyester, development of coating techniques of inorganics on interior pore surfaces of porous polyester scaffolds, and a methodology study of cell-material interactions.The main innovative achievements in this Ph.D. thesis are listed as follows:(1) A focused evaluation of in vitro effects of degradation products of porous polyester scaffolds on viability and differentiation of mesenchymal stem cells.The porous polyester scaffolds were degraded in phosphate buffer solution in vitro for 24 weeks, and the degradation media were collected to study their effects on mesenchymal stem cells. The results indicated that degradation media didn’t induce severe cytotoxicity. Through adding lactic acid directly into cell culture media, the effects of lactic acid on mesenchymal stem cells were investigated. It was confirmed unambiguously that pH was the the main factor for cytotoxicity induced by lactic acid, and D,L-lactic acid was found a bit more cytotoxic than L-lactic acid at high concentrations, yet the critical tolerance of rat mesenchymal stem cells to both lactic acids were about 20 mmol/L. It implies that one does not need to worry the side effects of acidic degradation products on cells and tissues in local milieu, unless the porous polyester scaffolds be of very high mass and implanted at the sites with little body fluids exchange.(2) A rapid method for coating nano-hydroxyapatite on pore surfaces of polyester scaffolds and the in vivo restoration of bone defects by the porous scaffolds.A method combining oxygen plasma treatment and modified simulated body fluid incubation was suggested, which rapidly precipitated a nano-hydroxyapatite coating on pore surfaces of porous polyester scaffolds in just 9 hours. In contrast, the conventional method takes about 1 week. Our method is time-saving and easy for quality control. Porous polyester scaffolds with or without hydroxyapatite coating were of good biocompatibility and the scaffolds with hydroxyapatite coating enhanced growth of osteoblastic cells and mesenchymal stem cells significantly. In cooperation research, critical radius defects of rabbits were successfully restored by our porous scaffolds loaded with autogenous mesenchymal stem cells. Radiology and histology evaluation indicated that whole bone cortex was regenerated, and the medullary cavity was, after 12 weeks, reformed in the rabbits implanted of porous scaffolds with hydroxyapatite coating. So it can be confirmed that the hydroxyapatite coating on pore surfaces did enhance the bone defects restoration. No notable body rejection and aseptic inflammation were found in vivo, which is consistent with the in vitro cytotoxicity studies of the degradation products. So in some cases, one does not need to worry the side effects of acidic degradation products.(3) A method to prepare regular hydroxyapatite micropatterns.A regular hydroxyapatite micropattern was prepared by combining photolithography, surface grafting of negatively charged chemicals, and modified simulated body fluid incubation. Cell localization was further achieved after the background was passivated by polyethylene glycol). Such a method can be applied for further studies of the interactions between cells and bioceramics.In summary, this Ph.D. thesis studied the preparation of biodegradable porous polyester scaffolds, especially developed an innovative way to coat a hydroxyapatite coating on interior pore surfaces effectively and efficiently, and restored the segmental bone defects successfully using the porous scaffold loaded with mesenchymal stem cells. The effects of polyester degradation products on mesenchymal stem cells were also investigated, and both qualitative and quantitative results on cytotoxicity of degradation products demonstrated satisfactory biocompatibility of polyesters under normal conditions. Besides that, some technical breakthroughs were made to prepare bioceramics micropatterns for further study of interactions between cells and bioceramics. The new insight and techniques in this Ph.D. thesis might be helpful for guiding research and development of biomaterials for regenerative medicine.