To Study The Cytotoxicity Of Chitosan Thermosensitive Hydrogel To Rat Osteoblasts

BackgroundBone defects and nonunion caused by the trauma, inflammation, cancer and other reasons have been the area of orthopedic problems, which material can be used in bone grafting has been the focus of research. As technology advances, using the methods of bone tissue engineering or gene therapy to repair bone defects and nonunion has attracted people's great attention.We plant the seed cells in a good biocompatibility biological materials which can be gradually degraded and absorbed by the body , when the seed cells amplify and the form the structure of cell / biomaterial, which can be planted back into the body, so that damaged tissue be repaired ,this is a common method of tissue engineering. Use the method of injection, injectable hydrogel formed scaffold in situ, which provide three-dimensional space for the cells to survival ,at the same time this method also reduce the damage caused to surrounding tissue during the implantation.In recent years, the research of hydrogel biomaterials used in tissue engineering has developed rapidly. Many studies show that chitosan hydrogel is a kind of biomaterials which has good biocompatibility, degradation in vivo, this kind of biological material used as tissue engineering scaffolds has a wide range of applications. Currently,there are more studies report that thermosensitive chitosan compound chondrocytes to repair cartilage defects using the form of injected, but there are relative lack of research show that thermosensitive chitosan compound osteoblasts to build bone tissue which used in the minimally invasive treatment of bone nonunion and bone defects.Objectiveto observe the growth of osteoblasts in the thermosensitive chitosan hydrogel, and to study the cytotoxicity of chitosan thermosensitive hydrogel to osteoblasts (OB) for its application in the minimally invasive treatment of nonunion and bone defects。MethodsIn this expriment, firstly,we successfully use of tissue culture method to isolate and cultivate a Sprague Dawley(SD) rat osteoblasts, and then we identify its histological source;secondly, we prepared chitosan hydrogel, with good properties of thermosensitivety, we implanted thermosensitive chitosan hydrogel into rats'thigh muscle to test the compatibility of organizations;and we used the thermosensitive chitosan hydrogel as a scaffold, the scaffold compound osteoblasts whice were cultured together to form bone tissue in vitro, then we observed the morphology,and amplification of osteoblasts in the thermosensitive chitosan hydrogel,;Finally, we prepared four different concentrations of chitosan thermosensitive hydrogel extracts, and extracts were used on the osteoblast toxicity expriment. The expriment were divided into three groups that complete medium (negative control group), different concentrations of chitosan thermosensitive hydrogel extract group (experimental group), pure lead material extract (positive control group). We cultured the osteoblasts in different concentrations of chitosan thermosensitive extracts in vitro for 24h, 48h, 72h, 96h, respectively, used the method of MTT assay to determine the relative growth rate(RGR)of ob and determined the level of cytotoxicity through it.ResultsAfter thermosentive chitosan hydrogel was being implanted into rats'thigh muscle,the inflammatory occurred gradually, thermosentive chitosan hydrogel'degradation time and the inflammatory reaction time are less than 8 weeks;Osteoblasts were cultured in chitosan thermosensitive hydrogel ,we viewed under a microscope and found that cell morphology was round in 24 hours,then the osteoblasts extruded its horns and amplified after 48 hours;The RGF of osteoblasts cultured in different concentrations of chitosan thermosensitive extracts in vitro in each group was 92% to112% at different time points, and the cytotoxic of all kind of concentrations of chitosan thermosensitive extracts were 0 or 1,which completely meet the standards of safety evaluation of biological materials. Compare the data of negative control and experimental group,p>0.05, they have significant difference;compare the data of positive control group and experimental group,p<0.05, they have significant difference;amongst the data of experimental group ,they have no significant difference , p> 0.05.ConclusionThermosensitive chitosan hydrogel is nontoxic to osteoblasts, with the good compatibility between the two, which shows that temperature-sensitive chitosan hydrogel is a good carrier of osteoblasts, and it will provide a theoretical basis for minimally invasive tissue engineering nonunion and bone defect.