The Study On The Biocompatibility Of Thermosensitive Hydroxybutyl Chitosan

Part?The Cellular Study on the Biocompatibility of Thermosensitive Hydroxybutyl ChitosanBackgrounds and Objectives: To aim at a new temperature sensitive chitosan derivative(hydroxybutyl chitosan hydrogel),which is firstly prepared by our team in domestic;To make preliminary study on the cytology behavior of the material by observing its impact on the growth of fibroblasts and vascular endothelial cells in vitro.So we can provide cytology experiment basis for clinical application of the new material.Methods:1.The effect of HBC extraction medium on L929 cells We immersed the HBC hydrogel in 37 degrees DMEM culture solution within 24 hours in order to get extract medium.We put 25%,50%,75%,100% concentration of extraction medium into four test groups,and set up negative control group,positive control group of phenol.The cells were cultured on 3 pieces of 96-hole plate,in 10 parallel control groups.We put the cells in the 37 ?,5%CO2 incubator and observed L929 cells growth under inverted microscope in 1 days,2 days,4 days,so that we could determinate the OD value of L929 by MTS method.The survival rate of L929 cells in different concentration groups was calculated.Then we put 50%,100% concentration of extraction medium into two test groups,set up negative control group,and tested L929 cells apoptosis in Streaming cytological examination in three days.2.The effect of HBC extraction medium on HUVEC cells and HLF cells We put 25%,50%,75%,100% concentration of extraction medium into four test groups and set up negative control group.We cultured HUVEC cells and HLF cells on 96-hole culture plates,in 10 parallel control groups.100 ul test fluid was put into each hole.The cells were cultured in the 37 ?,5%CO2 incubator and observed under inverted microscope in 1 days,2 days,4 days,so that we could determinate the OD value of HUVEC cells by MTS method.The survival rate of HUVEC cells in different concentration groups was calculated.Results: In HBC extract L929 cell cytotoxicity test,compared with positive phenol group,the difference of dead cells in the 25%,50%,75%,100% concentration of extraction group and negative control was remarkable(P<0.001).We observed that HBC extract could inhibit the growth of L929 cells and promoted vascular endothelial cells apoptosis.We found that L929 cellular differentiation could be inhibited during G1 phase to S phase by HBC extract in Streaming cytological examination(P<0.05).The existence of cells apoptosis was verified.The HBC extract had no cytotoxicity,and 100% concentration of extraction had mild effect on promoting HUVEC growth(P<0.001),compared with negative control group.Similar cell apoptosis was also observed in HLF cells test in 4 days.Conclusion: The thermosensitive chitosan derivative material HBC has no significant cytotoxicity,but it can promote general cells growth and inhibt the fibroblasts growth.Part?The Animal Study on the Biocompatibility of Thermosensitive Hydroxybutyl ChitosanBackgrounds and Objectives: In this study,we synthesised the a new type of thermosensitive injectable gel system by hydroxybutyl chitosan.The biological safety was preliminarily evaluated by the acute toxicity test,pyrogen test,intracutaneous stimulation and the immune cytotoxic test.So we can provide zoology experiment basis for clinical application of the new material.Methods:1.Acute toxicity test We put the HBC hydrogel into normal saline(NS)after 24 hours and filtered out the precipitation to get HBC saline extracts.The clean healthy SD rats were randomly divided into experimental group and control group,with 5 rats in each group.HBC saline extract was injured into the rats through the tail vein at the dose injection speed of 0.1mL/s,50mL/kg.2.Pyrogen reaction test HBC saline extract was injured into seven clean New Zealand rabbits through ear vein at the speed of 0.1mL/s,the dose of 2.5mL/kg.The changes of their body temperature were recorded.3.Intracutaneous stimulation test Four kinds of liquid,which was 100% HBC extract,50% HBC extract,saline,phenol solution,was injured into the back skin of five clean New Zealand rabbits.The changes of their skin were recorded.4.Immune cytotoxic test 80 rats were randomly divided into low,medium and high dose group and the control group.Drug was given by peritoneal injection twice a week,lasting for 2 weeks.Then we detected the serum of rats and compared immunological indexes.The rat femur bone marrow tissue was taken and observed after being dyed with Giemsa stain.Results: The HBC extract did not cause any mortality or side effects after injected into rats' bodies in one week.The acute toxicity test result was negative.The HBC extract injected into rabbits' blood vessel did not cause high fever,the pyrogen test result was negative.The HBC extract was injected into the dorsal skin of rabbits,there was no significant change of skin color in test group compared with negative group.But there was significant skin color change in the positive control group.In serum immunology experiments,the difference of immune globulin IgA,IgG,complement C3,killer(CD3 + CD8 +)T cells was remarkable(P<0.05)and the difference of globulin IgM,complement C4,CD3 + T cells,auxiliary type(CD3 +,CD4 +)T cells was not remarkable.Immunoserology experiment shows that the immunity of rats was improved after HBC injection of SD rats.Bone marrow smear shows that there was no significant difference between three test groups(low dose group,middle dose group,high dose group)and the control group.The phenomena of bone marrow hyperplasia or bone marrow inhibition was not observed in recovery and no recovery bone marrow smear.Conclusion: In animal test,HBC hydrogel showes good biocompatibility and can improve the immunity of rats.It is expected to become an ideal medical biological material.