| Objective:Tendon defect in the clinical is the most frequent hand traumadisease. To improve symptoms and restore functions in the greatest, thetreatment often needs to use allograft tendon to repair the different degree ofdefect to restore the important function which has been lost. Various materialscan be used for transplantation to repair the different levels of tendon defects,which according to the different preparation methods can be divided into:autogenous tendon, allograft tendon and tissue-engineered tendon, etc.Autogenous tendon (inversion, prolonging and autologous fascia) and allografttendon transplantations are often used in these repair methods. Because of thedisadvantages such as limited materials sources, new traumas added andinfluences on the function of the site, the traditional method cannot satisfy themore serious tendon defect at the same time. This fact makes peoplecontinuously explore new methods of tendon preparation to provide new ideasfor the treatment and rehabilitation of tendon defect. Since the1950s, thedeep research of allograft tendon has been developing rapidly, and it hasachieved fruitful results. Allograft tendon transplantation has become the mainmethod of treatment of tendon defect. Yet, the allograft tendon thatsimultaneously has all the advantages of good biological activity, strongmechanics performance and low immunogenicity is an important and difficultproblem that waits for solving. The problems after transplantation such as,heavy tendon adhesion, weak mechanical properties and strong immunerejection all can fail it. Researches that relevant scholars have done show thathamstring antigenicity mainly exists in the cell membrane of the tendon cellswhile hamstring collagen fiber itself has low antigenicity. How to removetendon immunogenicity, prevent tendon adhesion and retain graft biological properties is the focus of future research. The experiment takes leghorn asexperimental materials object, using vitrification method and chemicalextraction for preparation of allogeneic tendon, analyzing and discussing thebiological performance of allogeneic tendon prepared by vitrification methodand chemical extraction method through form the three aspects index ofhistological observation, biomechanical testing, immunogenicity (the ratiobetween periphery blood CD4~+, CD8~+T lymphocyte count and CD4~+/CD8~+detection and so on, in order to prepare an ideal allogeneic tendon and tofurther provide beneficial theoretical support and clinical guidance forallogeneic tendon preparation and transplantation.Methods:This experiment selected48health male white6-monthleghorns with same batch and same feeding conditions (offered by Hebei YiDaAnimal Experiment Center). These leghorns were (2.0~2.2)Kg weight,average (2.10±0.053)Kg. After numbered and marked, the leghorns wererandomly divided into three groups as A (vitrification method group), B(chemical extraction group) and C (blank control group), each group of16.The experiment used the third toes of leghorn’s double foots for surgery. Theanimals were given a injection of1.0%of ketamine (15.0mg/kg) anddiazepam (1.0mg/kg) in the middle-upper-part thigh muscle, after anesthesia,double lower limbs were fixed at prone position,disinfected and draped.Hemostatic by rubber and then the surgery began. Procedure: in the palm sidetook a Bruner’s incision, from the first toe articulatory near end to tiptoe about5.0cm, cut longitudinally the skin and fully separate the shallow deep tendonand tendon button of equine toe from the skin. Broken the two tendon buttonat the joint of tendon then took about5-cm deep bend tendon from the nexttoe.Washed the deep bend tendon over and over again until the outermembrane down, then placed the tendon in D-Hank ’s fluid (4℃) in freezer,refrigerating as standby. Observed respectively the three group of tendonwhich had been dealed with as above(vitrification method chemical extractionand blank control). ⅠForm histological observation: Respectively observedand analysed their morphology in general and lens observation(HE and Masson staining); ⅡBiomechanical testing: took respectively a test for thetensile fracture strength (Pmax), tensile fracture power (Wmax) and tensileelongation rupture rate (δ Max) of the three group of tendon; ⅢImmunogenicity detection: used the detection instrument with flow, got theproportion of CD4~+, CD8~+T lymphocytes in peripheral blood and calculatedthe proportion of CD4~+/CD8~+ratio(buried Group A, B, C three groups oftendon by combination of two in allogeneic animal’s body, extractedperipheral blood for test in the first, second, third, sixth weekend).Results:(1) The form histological observation:①In general:The tendonin blank control group was milk white long cable structure, with soft texture,smooth surface, and good elastic toughness. all testing tendons were completewithout defect. There was no obvious differences among the three group.②The lens observation: Hamstring tendon cells in the blank control group, alongthe collagen fiber around present a beaded longitudinal distribution, andseveral collagen fibers closely linked in the beam pattern. Tendons in thevitrification method group were visible that a little collagen fibers havingsigns of fracture, that gaps among the collagen fibers were broader than thosein the blank control group, and that the cell number was less than that in theblank control group. The tendon collagen fiber gaps in Chemical extractiongroup was slightly wider than those in vitrification group. The collagen fibershad a little fracture, without the signs of residual cells;(2) The biomechanical testing: The differences among the groups at theaspects of tendon tensile fracture strength (Pmax), tendon tensile failure power(Wmax) and tendon tensile elongation rupture rate (δ Max) was notstatistically significant, indicating that there was no obvious difference (P>0.05), showing that the biological mechanics performance of tendons werewell preserved in both methods;(3) Immunogenicity testing: Among groups, the difference of CD4~+,CD8~+T lymphocytes content and CD4~+/CD8~+ratio was statistically significantat1st,2nd weekend, so the difference was significant(P <0.05); At the3rd,6th weekend, the difference of CD4~+, CD8~+T lymphocytes content and CD4~+/CD8~+ratio between Group A and B was not statistically significant. Thecontents and ratios of Group A, B group were lower than those of Group Csuggests that the methods in Group A and B had not seen obvious difference(P>0.05), that the difference between Group A and C, Group B and C wasnotable(P<0.05). All above indicate that using the vitrification method and thechemical extraction method significantly reduced or removed the tendonimmunogenicity and reduced or even avoided the immune rejection happeningafter transplantation.Conclusion:1.Vitrification method is better than chemical extraction forpreparation of tendon at retaining the original tendon cells;2.Vitrification method and chemical extraction for preparation of tendonobviously reduce or remove the hamstring immunogenicity, and both retaingood biological mechanics performance. |
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