Study On The Degradation Rate Of Sodium Hyaluronate In Vitro And Its Clinical Significance

Objectives:Hyaluronic acid(ha)is a type of mucopolysaccharide naturally occurring in the human body.It is formed from Glc A and Glc NAc monosaccharides by alternating glycosidic linkage of ?-(1?4)and ?-(1?3).Hyaluronic acid molecules in the skin form polar bonds and hydrogen bonds with water molecules,which combine with a large amount of water and have a good moisturizing effect,thus being dubbed as "natural moisturizing factor(NMF)".Natural hyaluronic acid is easily diffused in tissues,and is easily degraded by oxidative substances or enzymes in the human body,with a rapid clearance rate,which limits its clinical application.In order to overcome this shortcoming and improve viscoelasticity,natural hyaluronic acids are often chemically modified by crosslinking agents to obtain cross-linked sodium hyaluronate gels,of which 1,4-butanediol diglycidyl ether(BDDE)is the crosslinking agent used in most of the leading sodium hyaluronate fillers in the market.Due to its special physical and chemical properties,sodium hyaluronate has been widely used in medical cosmetology,especially in soft tissue filling.However,with the increase of the amount of sodium hyaluronate injection,the number of complications also increases at the same time.Once it occurs,it affects the appearance or is life-threatening.Hyaluronidase can catalyze the degradation of hyaluronic acid to form two monosaccharides,glucuronic acid and n-acetylglucosamine.When complications occur after sodium hyaluronate injection,it is often used by clinicians for treatment.The purpose of this experiment is to observe the dissolution rate at several time points during the degradation process of sodium hyaluronate in a certain period of time when it is dissolved by high concentration hyaluronidase in vitro,to understand the kinetics characteristics of sodium hyaluronate degradation,and to assist clinicians in the treatment of injection complications.Method and process:The experiment was divided into two parts: degradation of sodium hyaluronate and detection of glucuronic acid.The experimental groups were divided into 2,4,8,12,16,and 24-hour enzymatic hydrolysis groups,and 24-hour non-enzymatic groups,with 3 samples in each group.In the degradation experiment,Sodium hyaluronate gel was diluted to a concentration of 4 mg/g in phosphate buffered saline(PBS),and a hyaluronidase reagent at a concentration of 2U/m L was prepared.On the day of experiment,33.333 g of sodium hyaluronate solution was taken,and 66.666 g of hyaluronidase reagent was added for reaction.The reaction time of the enzymatic hydrolysis groups were 2,4,8,12,16,and 24 hours,respectively.After the reaction,the mixture was filtered through a 0.45 ?m filter,and the weight of the filtered solution was weighed.Then,the capacity of the filtered solution was adjusted according to the weight of the filtered solution/the adjusted solution weight = 0.666,and then stored in cold storage for later use.The glucuronic acid test included preparation of test solution and standard solution,carbazole reaction and uv analysis.We took 0.666 g of each adjusted solution of hydrolysis reaction,diluted with 0.400 g of HCI solution,and added water to obtain a test solution with dilution factor F=8.2.000 g of 24-hour non-enzymatic solution was taken and added 0.400 g HCl solution and water to obtain the test solution with dilution factor F=3.The original glucuronic acid solution with a concentration of 1000 g/g was prepared,and the intermediate solution with a concentration of 200 g/g was obtained after dilution.Then a standard glucuronic acid solution T5/T4/T3/T2/T1 with a concentration of 80/60/40/20/12 g/g was available by diluting the intermediate solution with water.The carbazole reaction reagent included reagent A and reagent B,3.133 g of pure sodium pentahydrate tetraborate was dissolved in 458.000 g of pure sulfuric acid at a concentration of 0.033 mol/L to obtain reagent A,and 0.1254 g of pure carbazole was dissolved in 78.900 g of pure ethanol at a concentration of 0.0075 mol/L to obtain reagent B.Each of the diluted test solution and the standard solution was subjected to a carbazole reaction three times,and then ultraviolet analysis was carried out to measure the absorbance of each solution at 530 nm.Making a standard curve from the absorbance of a standard solution,the corresponding glucuronic acid concentration was obtained according to the absorbance of the tested solution.The average concentration of sodium hyaluronate in each group was used to calculate the weight of sodium hyaluronate in the test solution.The ratio of this weight to the initial weight was the degradation rate of sodium hyaluronate.Results:In the temperature of 37 ? in vitro experiments,when the concentration of hyaluronidase was 2U/m L,33.333 g diluted sodium hyaluronate at a concentration of 4mg/g was completely degraded about 5.87mg?25.03mg?68.81mg?100.88mg?116.28mg? 130.67 mg respectively,with a reaction time of 2,4,8,12,16,and 24 hours in each group.The average degradation rate was 4%,19%,52%,76%,87% and 98%respectively.In the absence of hyaluronidase,approximately 6.35 mg of 33.333 g sodium hyaluronate solution at a concentration of 4 mg/g was completely degraded at a degradation rate of 5% when exposure to air for 24 hours.Conclusion:The degradation of sodium hyaluronate by hyaluronidase is highly efficient and highly specific,and the degradation rate increases with the extension of time and is almost completely degraded within 24 hours.It is suitable for the treatment of adverse consequences after injection and filling of sodium hyaluronate in clinical practice.