Study On The Effect Of Thermosensitive Chitosan Hydrogel Loaded With TNF-α On Apoptosis Of Vertebral Growth Plate Chondrocytes Of Piglet

Background:Early-onset scoliosis(EOS)seriously affects children’s growth as well as physical and mental development.Congenital hemivertebra deformity is a common pathogenic factor and progressing rapidly.The treatment of EOS includes non-operative treatment and surgical treatment.For children with rapid progress and severe deformities,the curative effect of non-operative treatment is poor and it is difficult to ensure the patients’ compliance,while the choice of surgical plan and timing is difficult to control,faced with high risk of complications and adverse effects on children’s physical and mental health caused by repeated operations.Chitosan/β-glycerophosphate hydrogel has the characteristics of temperature-sensitive sol-gel transition and drug sustained release,while TNF-α can promote the apoptosis of vertebral growth plate chondrocytes at medium and high concentrations,and promote bone formation and bone fusion at low concentrations.The construction of in situ implantable drug delivery system is expected to slow down or even control the deformity progress of EOS patients by local injection of drugs,ensure the coordinated development of chest and lung function,reduce the risk of complications and psychological problems caused by multiple operations,and reduce the economic burden.Objective:Prepare thermosensitive chitosan/β-glycerophosphate hydrogel loaded with different concentrations of TNF-α.Culture vertebral growth plate chondrocytes of piglet in vitro to explore the effect of drug-loaded hydrogel on chondrocyte apoptosis and extracellular matrix,and to explore the appropriate drug loading concentration,in order to provide theoretical reference for following-up animal experiments and clinical applications.Method:1.Prepare chitosan/β-glycerophosphate hydrogel system,and measure the gelling time at 37℃ by test tube inversion method.Observe the growth of chondrocytes transfected with green fluorescent protein(GFP)on the hydrogel,then draw the drug release curve of TNF-a-loaded chitosan hydrogel.2.Prepare the thermosensitive chitosan/β-glycerophosphate hydrogels loaded with different concentrations(25,50,75 and 100ng/ml)of TNF-α and detect the expression of caspase,aggrecan and collagenⅡa mRNAs of chondrocytes cultured in the chamber for 24 hours.Detect the apoptosis rate of chondrocytes by flow cytometry,then select an appropriate drug concentration.Results:1.The prepared chitosan/β-glycerophosphate sol was milky white and translucent,and the sol-gel transition completed in 5 minutes at 37℃.The chondrocytes transfected with GFP grew well and the hydrogel system had good cytocompatibility under microscope.The drug-loaded hydrogel prepared by embedding TNF-α into chitosan solution and then mixed with sodiumβ-glycerophosphate had a good sustained release effect,and the release rate of TNF-α(%)was 63.068±2.790 after 24 hours.2.The results of casepase-3 mRNA expression detected by PCR showed that the level of caspase-3 mRNA in the chondrocytes treated with different concentrations was significantly higher than that in the control group(P<0.05).When chitosan hydrogel was loaded with low and middle concentration(25-75ng/ml)of TNF-α,the caspase-3 mRNA level increased with the increasing of TNF-α concentration,and when the TNF-α concentration reached 100ng/ml,the caspase-3 mRNA level decreased slightly.3.The results of aggrecan and collagen Ⅱα mRNA expression detected by PCR showed that compared with the control group,the levels of aggrecan and collagen Ⅱ αmRNA in the chondrocytes treated with different concentrations were significantly lower than those in the control group(P<0.05).The expression level of TNF-α in chitosan hydrogel was slightly higher when the concentration of TNF-α was 25ng/ml,and the decreasing degree of aggrecan and collagen Ⅱ α mRNA levels decreased with the increasing of TNF-α concentration in medium and high concentration(50-100ng/ml).4.The results of flow cytometry showed that the apoptosis rate of chondrocytes increased with the increasing of the concentration of TNF-α in chitosan hydrogel.Conclusions:1.The thermosensitive chitosan/β-glycerophosphate hydrogel has good cytocompatibility and can complete the sol-gel transition in 5 minutes at 37℃.The drug release curve is relatively smooth and the sustained release effect is good,so it is a good drug carrier.2.Chitosan hydrogel loaded with concentration of 75ng/ml TNF-α can effectively induce apoptosis of growth plate chondrocytes and reduce the expression of matrix,affecting its homeostasis and interfering with the growth of growth plate cartilage,which can be used as a new means of growth arrest for further research.