The Study Of Bone Regeneration

ObjectiveThe clinical application for bone substitutes is as filler for defects caused by surgical removal of tumors and cysts, trauma, osteolytic defects, or surgical debridement of infections. The ideal bone substitute requires good biocompatibility and no toxicity or induction of inflammation, has good osteoconductivity and/or osteoinductivity, and would degrade gradually and be replaced by host bone.nano-hydroxyapatite / collagen/Poly-L-lactic acid(nHAC/PLA) is a kind of bone scaffold material which has similar composition and structure with natural bone. In this studay, we implanted MG-63 cells into this scaffold material and cocultured in vitro. The adherence, proliferation and biofunction of MG-63 cells were investigated in order to provide proof for the clinical application of nHAC/PLA.During the previous research work of bone tissue engineering, it is difficult to observe the situation of seed cells in the scaffold materials by normal methods, due to the three-dimensionalporous structure and light proof characters of the scaffold materials. This makes us in trouble with the study of seed cells' growth regularity. Until now there is no better method to deal with this problem. CFDA-SE is a kind of vital dye and it can stay inside the nucleus stably without causing apoptosis. In this study, we labeled MG-63 cells with CFDA-SE to test the effect of this dye material to the adherence of cells and observe the dynamic growth situation of MG-63 cells on the scaffold.Moreover, vasculogenesis of bone tissue is also an important part of bone regeneration and reconstruction. Osteoblast and vescular endothelial cell, which are most related to bone regeneration and vasculogenesis, as well as the interaction of these two kinds of cells are the hotspot of correlated field. It was reported that VEGF, which was produced by vescular endothelial cells, can induce vasculogenesis and take part in the progress of wound healing. ET-1 receptor is on the surface of osteoblast. It can bond to ET-1 which was produced by vescular endothelial cells and improve the function of osteoblast. In this study we cocultured human umbilical vein endothelial cell and MG-63 cells directly in vitro to investigate the effect of human umbilical vein endothelial cells to MG-63 cells in order to provide some basic proof to the research of vasculogenesis in bone tissue engineering.After bone substitutes were implanted into bodys, its metabolism was affected by the immune system of the host. The clinical failure of artificial bone application which was caused by immunological rejection or chronic inflammation is very common. Immune cells and phagocytes influence the bone destruction associated with inflammation or immunological rejection by production of inflammatory mediators. After phagocytosis of apoptotic cells, however, phagocytes exhibit anti-inflammatory responses. Phosphatidylserine (PS), which is translocated on the surface of apoptotic cells during apoptosis, is recognized by the receptors on phagocytes to initiate uptaking of apoptotic cells and changing phagocytes to the anti-inflammatory phenotypes. Furthermore, PS-liposome (PSL) can mimic the effects of apoptotic cells. Therefore, it is reasonable to speculate that PSL may suppress the bone destruction associated with inflammation and immunological rejection. In the present study, we have thus attempt to examine the effect of PSL on inflammatory bone destruction using adjuvant arthritis (AA) model in rat. We also examined the effects of PSL on the secretion of cytokines from macrophages culture system and the osteoclast formation in bone marrow cells culture system.Methods1. The effect of nano-hydroxyapatite / collagen/Poly-L-lactic acid scaffold material to the adhesion and biological function of human osteoblast.Human osteoblast were seeded and co-cultured on nHAC/PLA scaffold material in vitro. The culture process was observed by inverted phase contrast microscope, HE stains to study the effects of nHAC/PLA scaffold material to the early adhesion of human osteoblast. At the same time, the cell proliferation index, ALP activity, and mRNA expression of osteocalcin (OC) and collogen type I(Col-1) were detected to evaluate the effects of nHAC/PLA scaffold material to the biological functions of human osteoblast.2. Adhesion of CFDA SE labeled human osteoblast to nHAC/PLA scaffold material. Human osteoblast were labeled with CFDA SE and then seeded on nHAC/PLA scaffold material. The adhesion efficiency was caculated after 24h's culture, with the labeled human osteoblast as a control. The cell-nHAC/PLA composites were observed under Confocal laser scanning microscope on the 1^st 3^rd, , and 7^th days after seeding3. The study of human umbilical vein endothelial cells and human osteoblast co-culture in vitro.MG-63 Cells and HUVECs were co-cultured directly in vitro. function of human osteoblasts was assessed by the alkaline phosphatase(ALP) activity and OC assay.4. The effect of PSL to inflammatory/immunoreactive bone resorption.Using adjuvant arthritis (AA) model in rat examined the effects of PSL on inflammatory/immunoreactive bone destruction and the effects of PSL on the osteoclast formation of bone marrow cells culture system.Results1. Cells were observed to spread and proliferate throughout the inner-pores of the scaffold material. There are significant difference of the proliferation index of co-cultured cells and the ALP activity of co-cultured cells compare to the contral group. The mRNA expression of Col-1 and OC were also affacted compared to the contral group.2. The adhesion efficiencies of CFDA SE labeled and unlabeled human osteoblast were (97.99±1.43)% and (98.57±1.17)% respectively, there was no significant difference between the test and control group(P>0 05).3. Both MG-63 cells and HUVECs could grow well after co-cultured directly. The ALP activity and OC content of MG-63 cells increased significantly in co-culture system.4. PSL can significantly inhibit the bone destruction of AA rat. The mRNA espression of pro-inflammatory cytokine such as IL-1β, TNF-αet al in the local tissue of PSL treatment group were significantly lower than the control group. While the mRNA expression of anti-inflammatory cytokine such as IL-10 et al in the local tissue of PSL treatment group were significantly higher than the control group. And PSL can significantly inhibit the formation of osteoclast in bone marrow cells culture system. Conclutions1. nHAC/PLA scaffold material was a good kind of bone tissue engineering scaffold material. It can improve the bio-function of human osteoblast nHAC/PLA scaffold material has no effect to the spread and adherence of Mg-63 cells. It can be used as bone tissue engineering scaffold material.2. Application of vital fluorescence dye CFDA SE to human osteoblast shows no effect on the adhesive ability of MG-63 cells, CFDA SE label can be used as a good method to observe the adhesion of human osteoblast on nHAC/PLA scaffold material.3. HUVECs can increase the biological activity of Human Osteoblast when co-cultured directly with it.4. PSL can inhibit inflammatory/immunoreactive bone resorption by adjusting the function of macrophages.