The Effect Of Several Methods To Reduce Immunogenicity On Biological Characteristics Of Allogenic Tendons

Objective Tendon transplantation is the most commonly used method for reconstruction of tendon defects.The autologous tendon has been recognized as the "gold standard" because of its good “bone-tendon integration” ability,no immune rejection and no risk of disease transmission.However,autologous tendons may lead to complications in the donor site.Thus,allogeneic tendon has gradually been used as an alternative considering its many advantages,such as no donor-site related complications and no restrictions on the amount of transplantation.Nonetheless,the use of allogeneic tendons also increases the risk of immune rejection.There are currently a variety of treatment options.This study aimed to compare the impact of different treatment methods [deep cryogenic freezing,95% alcohol,trinbutyl phosphate(TBP)and sodium dodecyl sulfate(SDS)] on the biological characteristics of allogeneic tendons.Methods Cadaver tendon freshly collected within 24 hours were randomly divide into five groups,one of which was designed as a blank control sample without any treatment.One each of the other four groups was stored at-80 ℃ for 90 days,decellularized with 95% alcohol(96h),decellularized with 1% TBP(48h)and decellularized with1% SDS(24h),respectively.10 samples were used for cyclic loading test and tensile fracture test,to draw displacement-load curve and stress-strain curve,and to calculate the following biomechanical indicators: cyclic creep,limit load,maximum displacement,maximum stress,maximum strain,elastic modulus,yield strength and energy absorption.Five samples were taken from each group to tested the DNA content.For the above biomechanical indicators and DNA content data,Shapiro-Wilk test and modified Bartlett’s test were used to test its normality and homogeneity of variance.For the indicators that meet normality and homogeneity of variance,one-way analysis of variance and post hoc analysis by Turkey method were used to conduct the statistical analysis.The rest indicators were tested by Kruskal-Wallis test and post hoc analysis with Dunnett’s method.Histomorphology was observed by HE staining,DAPI staining,toluidine blue staining and transmission electron microscopy.Results The cyclic loading test found that the overall difference in cyclic creep among the groups was significant(F=4.328,p=0.005),and the post hoc analysis showed that the cyclic creep of the tendon treated with 95% alcohol was lower than that of the fresh tendon(p=0.002).Statistical analysis showed that there was no significant difference in the indicators of the tensile fracture test(p> 0.05).Dunnett ’s multiple comparison only found that the elastic modulus of tendon treated by SDS was significantly lower than that of fresh tendon(p=0.044).Compared with fresh tendons,the residual DNA content in the cryogenic freezing group,95% alcohol group,TBP group and SDS group were significantly reduced(p<0.001).According to the HE and toluidine blue staining,there was only a small amount of scattered nuclear components,while tendons in the 95% alcohol group,SDS group,and TBP group had almost no residual cell nucleus.The gap between collagen fibers of the 95% alcohol group,SDS group and the TBP group was larger than that of the fresh group and the cryogenic freezing group.DAPI staining showed that there was only a small amount of blue fluorescent nuclei remaining in the cryopreservation group,but no obvious blue fluorescent staining nuclei existed in the 95% alcohol group,SDS group and TBP group.Transmission electron microscopy showed that 95% alcohol,SDS and TBP resulted in a slight increase in the collagen fiber gap and an increase in the ratio of fine / crude fibers compared with fresh tendons and cryogenic frozen tendons.Conclusions Deep cryogenic freezing,95% alcohol decellularization,SDS decellularization and TBP decellularization can effectively remove the immunogenic components in the allogeneic tendon,which significantly reduces the number of residual cells and DNA content in the tendon.Among them,cryogenic treatment has almost no damage to tendon histology and biomechanics;95% alcohol,SDS and TBP decellularization methods have different degrees of damage to tendon histology and biomechanics.