Chitosan And Its Derivatives As Drug Delivery For Bone Regeneration

Bone regeneration is a complex and delicate physiological process, not only can be seen in the process of fracture healing, but also the physiological bone remodeling with the person’s life. In clinic, trauma, infection, tumor resection and bone dysplasia often lead to large bone defect, and the reconstruction of large bone defect is still one of the problems in clinical treatment. At present, there are various treatment strategies, including autologous bone transplantation, allograft bone transplantation, bone tissue engineering(growth factor, bone conduction scaffold and osteoblast) and distraction osteogenesis. Autologous bone graft sources limited and will cause damage to donor site, the biomechanics of bone graft substitutes and difficult to comparable with normal bone tissue. In order to overcome the limitations of these methods, many studies concentrated on tissue engineering "local repair strategies and gene therapy, even a" systemic "enhanced bone repair to achieve the purpose of normal bone tissue regeneration.Gene therapy is the delivery of normal gene or therapeutic genes into the human body through a certain way to target cells to correct a defect in the gene or play a role in treatment, which plays an important role in biomedical technology for the purpose of disease treatment. In the second chapter, we evaluated if chitosan-polyethylenimine(CS-PEI) nanoparticle can effectively deliver h BMP-2 locally with lower or no toxicity and promote osteoblast differentiation and new bone formation in vitro and in vivo. Data demonstrated that the synthesized CS-PEI/h BMP-2 nanoparticle at W/W ratio of 20 to 1, which was the smallest size(162 nm) and highest Zeta potential(24 m V), effectively transfected MC3T3-E1 cells without cytotoxicity in vitro, and had the ability to promote cell proliferation. Interestingly, the CS-PEI/h BMP-2 nanoparticle eliminated disadvantages of lower transfection efficiency from chitosan and cytotoxicity from PEI. RT-QPCR data showed that MC3T3-E1 cells treated with CS-PEI/h BMP-2 nanoparticle dramatically expressed higher levels of BMP-2 and significantly increased gene expressions of COL I on days 3 and 14, SP7 on days 3, 7 and 14, and ALP on day 14. Alizarin red staining demonstrated that CS-PEI/h BMP-2 nanoparticle-treated MC3T3-E1 cells significantly increased cell mineralization. These in vitro data suggest that the CS-PEI/h BMP-2 nanoparticle can effectively induce osteogenic differentiation of MC3T3-E1 cells in vitro. Western blot further demonstrated that transgene BMP-2 indeed phosphorylated Smad 1/5/8, which indicates that CS-PEI/h BMP-2 nanoparticle affects cell differentiation through BMP-2 signal pathway. Importantly, in vivo data showed that CS-PEI/h BMP-2 nanoparticle clearly promoted new bone formation at the bone defect area 12 weeks post-implantation. This indicates that synthesized CS-PEI/h BMP-2 nanoparticle has the potential to become a useful therapeutic vector for bone defect treatment with further modification.Rapamycin(Rapamycin, RAPA) could block T lymphocytes and other cells from G1 phase to S phase of the process, while blocking T and B lymphocytes of calcium dependent and calcium dependent signaling pathway, by blocking the signal transduction of different cell factor receptor. RAPA is an effective inducer of autophagy. it can significantly stimulate BMP/SMAD signal andincrease expression of early markers of RUNX2. In the third chapter, we introduce the RAPA into CS-PEI/ h BMP-2 treatment system, the Real time-PCR results showed that the CS-PEI/ h BMP-2+RA could enhance the expression of bone related genes compare with other groups(CS-PEI/BMP-2, RA and control) at each time point. Alizarin red staining and quantitative results showed that CS-PEI/BMP-2+RA group can increase the quantity of calcium nodule formation of MC3T3-E1 cells. Thus, we can prove that CS-PEI/BMP-2+RA group had the better effect on osteogenic differentiation in vitro compared with CS-PEI/ h BMP-2 group and RA group. In the critical bone defect model of rat skull, gelatin sponge composited CS-PEI/ h BMP-2+RA was implanted into the bone defect. After 12 weeks, the results observed by CBCT showed that the large pieces of bone formation could be found clearly in the group of CS-PEI/BMP-2+RA, covering almost the whole defect. CS-PEI/BMP-2 group and RA group also has different degree of new bone formation, thus, compared to genes and drugs we know that CS-PEI/BMP-2+RA could induce the promotion of bone tissue regeneration in vivo, and not show obvious toxicity in vivo.EPO(hemopoietin, EPO) can promote bone regeneration and angiogenesis. To facilitate repair of irregular bone defect in space, in the fourth chapter, we used chitosan, gelatin(GA) and sodium glycerophosphate(GP) as raw material, chitosan thermosensitive hydrogel(CS/GP/GA) was prepared by ionic crosslinking, which is at room temperature(20℃) for liquid and turn into solid state properties in body temperature(37℃). The results of ELISA method showed that the release rate of EPO-CS/GP/GA had burst release rate the reached 40% within 2h, the release rate of 72 h reached 78 % and reached 94% within 15d; The effect of EPO-CS/GP/GA on the proliferation of BMSCs cells were detected by MTT at each time point. The results showed that, Co cultured after 24 hours,the proliferation rate of group BMSCs cells was significantly increased treated by EPO-CS/GP/GA, and, similar to EPO without any treatment effect; Differentiation of BMSCs cells was detected by Real time-PCR. The results show that the EPO released from EPO-CS/GP/GA could promote the osteogenic related factor expression in these three time points 3D and 7d and 14d; in addition, we established New Zealand rabbit maxillary sinus augmentation model, and the EPO-CS/GP/GA gel contain 200 units EPO were implanted into rabbit maxillary sinus.After 12 weeks, observed by CBCT, EPO-CS/GP/GA group could elevate the rabbit maxillary sinus and the amount of new bone formation and osteoblast quantity than the pure gel group and PBS group, which proves that EPO-CS/GP/GA can effectively promote the rabbit maxillary sinus lifting.