| Objective: To select the suitable cryoprotective agents by evaluating the effects of four cryoprotective agents(ethylene glycol,propylene glycol,glycerol,dimethylsulfoxide(DMSO))on cryopreservation by vitrification of flexor digitorum profundus tendon,which can provide the basis for the cryopreservation by vitrification of allogeneic tendon.Methods: In this study,30 flexor digitorum profundus tendon abandoned after amputation in The Fifth Affiliated Hospital of ZMU from October 2018 to October 2020 were collected,each of which was about 5 cm.According to the type of cryoprotective agents,they were randomly divided into five groups(four experimental groups and one control group),six in each group,namely,30% ethylene glycol group,30% propylene glycol group,30% glycerol group,30% DMSO group and the control group.The experimental group used different types of cryoprotective agents,and the control group did not add cryoprotective agents,and both groups were cryopreserved for 14 days.The tendon specimens of each group were observed under light microscope and transmission electron microscope.The content of hydroxyproline(HYP)in tendon was determined by alkaline hydrolysis;Tension tester was used to measure the tensile fracture strength(Pmax)and tensile elongation at break(δmax)of each group of tendons.Statistical methods were used to compare the hydroxyproline content and tensile force values,and whether the difference between groups was statistically significant was analyzed.Results:1.HE staining results: In the control group,collagen fibers were obviously broken,arranged in disorder,gap widened,the number of tendon cells decreased,tendon cell structure and tissue structure were seriously damaged;In the ethylene glycol group,collagen fibers were arranged neatly,distributed parallelly along the longitudinal axis,partially crossed,and the number of tendon cells was abundant.They were arranged uniformly like beads along the collagen fibers.In the propylene glycol group,collagen fibers showed signs of fracture,irregular arrangement,no obvious widening gap.In the glycerol group,collagen fibers were obviously fractured,sparsely arranged,and the gap widened.In the DMSO group,collagen fibers were closely connected,arranged neatly,and the gap was not wide,without fracture.The number and morphology of tendon cells were normal.2.The Results of transmission electron microscopy: In the control group,collagen fibers were different in thickness,disordered arrangement,widened gap,slightly distorted deformation,nuclear condensation was obvious,nuclear membrane structure was fuzzy,the number of sarcoplasmic reticulum in cytoplasm was significantly reduced,mitochondrial membrane rupture and lysosome increased.In the ethylene glycol group,the distribution of collagen fibers was regular and basically distributed in the same plane.A large number of sarcoplasmic reticulums were observed in the cytoplasm,the mitochondrial structure was clear,the bilayer membrane was obvious,and the chromatin in the nucleus was uniform.In the propylene glycol group,the collagen fibers were regular,and the surface occasionally swelled.A small amount of glycogen particles were distributed between collagen fibers,and some mitochondria were destroyed.In glycerol group,collagen fibers arranged in parallel,gap widened,chromatin condensed,nuclear membrane structure blurred,sarcoplasmic reticulum decreased,ribosome and lysosome increased;DMSO group showed unclear boundary between collagen fibers,increased mitochondria in cytoplasm,sparse distribution of sarcoplasmic reticulum and concentrated nucleus and cytoplasm.3.Detection results of hydroxyproline content: Compared with the control group,the hydroxyproline content in ethylene glycol group,propylene glycol group,DMSO group and glycerol group was significantly increased(P﹤0.05);Compared with the glycerol group,the hydroxyproline content in the ethylene glycol group and DMSO group was significantly increased(P ﹤ 0.05),and there was no significant difference in the hydroxyproline content in the propylene glycol group(P > 0.05).Compared with propylene glycol group,hydroxyproline content in ethylene glycol group and DMSO group increased significantly(P ﹤ 0.05).Compared with DMSO group,the content of hydroxyproline in ethylene glycol group was significantly increased(P﹤0.05).4.Test results of the biomechanical testing:Compared with the control group,the maximum load and elongation of ethylene glycol group,propylene glycol group,glycerol group and DMSO group were significantly increased(P﹤0.05);Compared with glycerol group,the maximum load of propylene glycol group,DMSO group and ethylene glycol group was significantly increased(P﹤0.05).Compared with propylene glycol group,the maximum load of DMSO group and ethylene glycol group was significantly increased(P﹤0.05).Compared with DMSO group,the maximum load of ethylene glycol group was significantly increased(P ﹤ 0.05).Compared with glycerol group,the elongation of ethylene glycol group and DMSO group increased significantly(P﹤0.05),and there was no significant difference in the elongation of propylene glycol group(P > 0.05).Compared with the propylene glycol group,the elongation of the ethylene glycol group was significantly increased(P ﹤ 0.05),and there was no significant difference in the elongation of the DMSO group(P > 0.05).Compared with DMSO group,the elongation of ethylene glycol group had no significant difference(P > 0.05).Conclusion:1.Ethylene glycol,propylene glycol and DMSO had protective effects on the cryopreservation by vitrification of flexor digitorum profundus tendon,while glycerol had no obvious cryopreservation effect.2.Ethylene glycol may be more suitable for cryopreservation by vitrification of flexor digitorum profundus tendon as a cryoprotective agents. |
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