Tissue Engineering Micro-tissue Repair Of Achilles Tendon Injury In Rats

ObjectiveTo explore the preparation of extracellular matrix of rat tail tendon,and to construct micro-tissue with adipose mesenchymal stem cells to repair rat Achilles tendon injury and evaluate the effect of this biomaterials on repairing tendon injury,and provide experimental basis for clinical treatment of tendon injury.MethodsIn vivo:20 SD rats(150-200g)tails as experimental material,rat’s tail tendons were extracted respectively,repeated freezing and thawing 3 times after thorough cleaning with PBS,and then processed with 1% SDS at room temperature for 24 hours,next,removed the cell residue with a mass of pbs for 1 weeks,and finally disinfection standby.The pathological analysis of the material was carried out to observe the cell residue.At the same time,its α-gal content was detected.In addition,complexed rat tail tendon extracellular matrix with adipose mesenchymal stem cells to observe the effects of the material on cell proliferation.At the same time,the cytotoxicity of the biomaterials was detected by CCK8 Kit.In vitro: The extracellular matrix of rat tail tendon was cultured with compound adipose stem cells,and was obtained after 7 days,which was used to promote tendon repair in rat Achilles tendon transection.The expression of related factors such as ColⅠ,ColⅢ,FBN,VEGF and TNMD were observed by RT-PCR in 2 weeks.The effects of the biomaterials on the repair of Achilles tendon injury were evaluated by general observation,pathological staining,mechanical examination,gait examination and imaging observation at 4 weeks and 8 weeks.ResultsIn vivo:The gross observation showed that the volume of the treated tail tendon increased,and the shape was milky white homogenate.The results of HE and DAPI staining showed that the nucleus of the treated tail tendon was significantly reduced,the number of cells decreased markedly,and the cells residue in each high magnification lens was not more than 5.The residual α-gal content of the treated tail tendon was statistically different from that of the normal tail tendon.The number of living cells increased significantly after 7 days of co-culture.And the results of CCK-8 toxicity test showed that the biocompatibility of this treated tail tendon was good and no cytotoxicity was found.In vitro: At 2 weeks,the PCR results showed that the expression of Col I,Col III and TNMD in the micro-tissue group was higher than that in other control groups,but the expression of VEGF was lower than that in other control groups,while there was no significant difference in FBN expression;At 4 weeks,the surface of the regenerative tendon in the micro-tissue group was smooth and glossy,HE and Masson staining showed good tissue continuity,and each group showed different levels of inflammatory response,and the micro-tissue group was lighter than the other groups,at the same time collagen fibrosis and angiogenesis were observed in micro-tissue group.At 8 weeks,the inflammatory responses of each group were reduced,and the collagenous fibers in the micro-tissue group were more orderly than before,and the new capillaries in each group decreased.Compared with the control group,the collagen fibers in the micro-tissue group were more dense and the cells were spindle-shaped and arranged in a regular pattern.The mechanical results showed that the ultimate load were: normal group 63.95±4.42 N,blank group 25.14±6.17 N,cell group 37.34±4.01 N,micro-carrier group 46.29±3.25 N,microtissue group 56.77±7.69N;And the tensile strength were: normal group 5.09±0.35 MPa,blank group 0.65±0.16 MPa,cell group 1.90± 0.20 MPa,micro-carrier group 1.20 ± 0.08 MPa,micro-tissue group 4.52±0.61 MPa.The catwalk results showed that the mean intensity of the micro-tissue group was greater than that of other control groups at 2 weeks,but there was no statistically difference compared with the micro-carrier group.At 4 weeks and 8 weeks,the mean intensity of the micro-tissue group was statistically significant compared with that of the control groups.At 2 weeks,the average paw area of the microtissue group was larger than that of the blank group,and the difference was statistically significant,but there was no difference between the micro-carrier group and the cell group.At 4 weeks and 8 weeks,the average paw area of the micro-tissue group was larger than that of the control groups,and the difference was statistically significant.B-Ultrasound results showed: at 4 weeks,the control groups showed different levels of tissue disorder,edema and inflammatory response.The micro-tissue group showed a good continuity of the Achilles tendon,which was significantly improved compared with the blank group and the cell group.The volume of the Achilles tendon was slightly increased and the contour was blurred.At 8 weeks,the control groups had a decrease in edema and inflammatory reaction,but the arrangement of tissues was still disordered.The continuity of the Achilles tendon in the micro-tissue group was good,the tissue was arranged orderly,and the Bultrasonic showed a little thin-form and low echo between the parallel and the strong echoes.Conclusions(1)The extracellular matrix of rat tail tendon was successfully prepared by the decellular method mentioned this experiment,and it had good biocompatibility with adipose mesenchymal stem cells,and could be used as a biomaterial to repair tendon injury.(2)The micro-tissue formed by the compound adipose stem cells and this kind of biomaterials can promote the healing of the Achilles tendon in rats,and it has a certain reference value for the study of tendon injury repair.