The Animal Experimental Study And Clinical Application Study Of Knee Creuciated Ligament Reconstruction Using Allogenous Graft

PART ONEThe animal experiments of knee cruciate ligament reconstruction with allogenous tendon graftsBackgroundReconstruction of the knee joint cruciate ligament using tendon allografts is the hot topic in recent decades.A certain amount of clinical papers have been published at home and abroad.This method attracts more attention mainly due to no harm of the knee joint in harvesting grafts.Reconstructing cruciate ligament with allografts must be supported by animal experiments as the allograft are the materials preserved in certain condition for a period of time and this kind of allograft is not the'same as autogenous tendon.Animal experiments are less and no papers have been published in china.Reconstruction of the knee joint cruciate ligament by tendon allograft receives much concern as follows:(1)Whether the biomechanical strength of tendon allografts is changed after frozen and cold storage? Which factor could lead to disintegration of collagen fibers and then reduce the biomechanical strength of tendon allograft in the freezing and thawing process of the tendon? How to protect the tendon effectively?(2) Whether immunological rejection is initiated by tendon allograft?(3) Whether there are differences in reparation and healing of the cruciate ligaments between tendon allograft and autograft?The animal study of tendon allograft transplantation is a fundamental work for establishment of human tendon allograft bank.Moreover,the results of animal experiments will provide crucial data and theoretical basis for adjustment and improvement of tendon allograft preparation technology.In addition,the healing process in the animals will supply important references for operation and rehabilitation schemes in clinical application.Objective(1) To study the process of tendon allograft preparation,and to investigate the effects of tendon allograft preparation on the biomechanical strength and histomorphology of the tendon.(2) To observe the immunological changes of cruciate ligament after tendon allograft transplantation.(3) To investigate the tendon healing process of the cruciate ligament after tendon allograft transplantation and to provide theoretical basis of tendon allograft by comparison with autograft.Methods(1) Operation model Discission and Reconstruction of the anterior crucial ligament by transplantation of New Zealand white rabbits 1/2BPTB.(2) Tendon preparation method 1/2BPTB were detached and washed three times with 0.9%normal sodium and soaked in protective solution(10%calf serum + MEM) for 10-15 min,then,were placed in frozen stock tube.①Deep- frozen(-80℃)Method The tube were put in 4℃for 30 min,then in -20℃for 4 h,and then were transferred to a deep-frozen refrigerator gradually down to -80℃.②Programmable freezing and liquid nitrogen store method The tube were placed in a programmable freezer (CBS2100,USA) lower to 4℃at a speed of 1℃/min,then lower to -80℃at a speed of 1℃/min,finally lower to -180℃at the speed of 1℃/min according to the programmed procedure.The tube was stored in -180℃for further investigation.③Tendon thawing method The tendon allografts were put in 0.9%normal sodium (40℃) for 15 min,then were placed in gentamicin solution(400U/ml) and washed for 5-10 min when its elasticity recovering.The tendon could be transplanted after rinsing with 0.9%normal sodium. (3) Effects of different cryopreservative methods on patellar tendon allograft 48 1/2 BPTBs were detached from 12 white rabbits.The BPTBs were randomly divided into -80℃deep-frozen group,programmable freezing and liquid nitrogen store group and control group.In every group,8 BPTBs were detected with biomechanical test,6 BPTBs with cell viability analysis,and 2 BPTBs with morphological observation.(4) Preparation of 1/2BPTB for transplantation 64 1/2 BPTBs were detached from 16 white rabbits,then,were preserved with -80℃deep-frozen method or liquid nitrogen, respectively,for 2 weeks for further transplantation.(5) The experiments of reconstruction of the knee joint cruciate ligament by BPTB transplantation 48 white rabbits were randomly divided into -80℃deep-frozen group, programmable freezing and liquid nitrogen store group and control group.Cytotoxic effect,cell viability analysis,biomechanical strength of grafts and morphological changes were detected after 3 and 8 weeks of transplantation.Results(1) The histological changes of tendon with different cryopreservative methods were much similar.At early stage,a lot of fibroblasts and proliferating capillaries invaded into the transplanted tendon,collagen tissue were damaged and replaced by neonatal collagen gradually.Finally,the neonatal collagen line up in order and form undee collagen fibers bundles.(2) In comparision with control group,the peak loads of patellar tendon and cell viability preserved by different cryopreservative methods were not obviously impaired. However,programmable freezing and liquid nitrogen store group could achieve better performance.(3) There was no obvious immunologic rejection of the tendon transplantation in the whole process,and the immunological response was reduced with time passing.(4) 3 weeks after transplantation,there was no differences in peak load between -80℃deep-frozen group and programmable freezing and liquid nitrogen store group.8 weeks after transplantation,the peak load and cell activity in programmable freezing and liquid nitrogen store group is superior to -80℃deep-frozen group.Conclusions (1) -80℃deep-frozen method and programmable freezing and liquid nitrogen store method can effectively preserve tendon allografts,and the latter has better effects.(2) The cytotoxic effect caused by the transplantation of the tendon stored with -80℃deep-frozen method and programmable freezing and liquid nitrogen store method were unconspicuous.However,the reject reaction and stimulus response of the joint need to be verification in human.(3) The healing process of cruciate ligaments of knee joint after tendon allograft transplantation was similar with but slower than that of autograft transplantation. PART TwoThe clinical observation of the Cruciate ligaments reconstruction by allogenic tendon transplantation under arthroscopeBackgroundKnee is lower extremity weight-bearing and athletics joint.It is the most important and complex joint.The muscles round the knee absorb most stress and shear force which motion caused.While the articular ligament and fibrous capsule absorb little.During motion process,knee subject prodigious power.If it is bigger than its stabilization power,the ligament will be teared.The overbalance of static and dynamic structure caused by teared ligament lead to the instability of the knee during motion process.Since traffic accident and overexercise more and more,Cruciate ligament lesion increase together.Cruciate ligament lesion is frequent in knee injury which result to knee ache and unsteadiness,then arthrochalasis,retrogression,and meniscus and cartilage injury,some even irreversible joint damage.So it is important for orthopedist and athletic expert to treat the injury.It is effective using surrogate reconstructing the cruciate construction and function to treat the cruciate lesion.Arthroscope technology proved to be good way in reconstruction of cruciate ligament because less operation wound and better therapeutic efficacy.Auto tendon transplantation is original in cruciate ligament reconstruction,the most are Bone-Patellar tendon-Bone(BPTB) and Semitendon & Graces (ST+Gr).When BPTB was removed,anterior area pain of knee,clicking and difficult to kneel occurred(20%-40%).Even reforming 2 transsection was used,the symptom is 10%.It lost 20%muscle force aider ST+Gr was incised,and it take 1-1.5 years for the injury to recovery.We can conclude that the auto tendons transplantation is limit because it cause to new injury of the knee,especially in multiligament injury. The recovery process of allogenic tendon after transplantation is same to auto tendon.It avoid the new injury of auto tendon transplantation.The material is unlimited and satisfied mutiligament reconstruction after injury.However,the safety and clinical therapeutic result of allogenic tendon transplantation need to be proved since the tendon through exosomatic freezing,conservation,and defrosting process and so on.The operation of Cruciate ligaments reconstruction by allogenic tendon transplantation needs high quality tendon resource coincidence to operation standard. Some other countries and nations have aUogenic tendon warehouse,but we can not use those,and we do not know the standard process of allogenic tendon making.This title invest the technology and approach of establishing human allogenic tendon warehouse and formulate flow-sheet and supervising system after concluded correlated document and experience combinating the animal test data.The allogenic tendon is different to auto on material,such as 1/2BPTB,1/3 quadriceps femoris tendon.It is can not be obtained of tibialis anterior and posterior tendon,long and short peroneal tendons and achilles tendon.The operation is different duo to different transplantation materials.This title also investigate the operation ways.Objective1.investigating and improving the making manufacture technology and manipulate process of human allogenic tendon,establish the institution of administering tendon warehouse.Providing the coincident allogenic tendons for clinical reconstruction operations of cruciate ligaments.2.Approaching specific operation way under arthroscope according to different allogenic tendons.3.Observing the safety after allogenic tendon transplantation in human body through the analyses of clinical follow-up,concluding the clinical therapeutic efficacy of reconstructing anterior cruciate ligament by allogenic tendon.Materials and methods 1.to establish the allogenic tendon warehouse.(1) selection donor:well and active donor and extremity after traumatic amputation.(2) bolting donor:1) age,20-25 years old males.2) exclude the donors with infection,blood transmissible disease and malignant tumor especially bone and muscle system.3) Blood analysis.Including HBV,HCV,RPR,HIV-Ab and blood bacterial culture and so on.(3) To draw the material:drawing the material in 2 hours aider the material was donated,some maybe prolong to 12 Hs.We draw the tendons on operation table under asepsis condition,then wash it 2-2 times by SPSS,dishing in safety fluid(calf serum and men solution) 10-15 minutes,and capsulating in asepsis plastic pouchs.(4) Freezing:the temperature cooling to -180℃step by step by freezing machine,then restored it in nitrogen canister.We can use the tendon 10 days later.(5) Administering the tendon warehouse:1)label and illustration.After preparing the transplantation materials,we titled them,labeled the number,size,volume,quant, preparation time,expiry data and announcements,stock condition,open and use ways according to its source,position,preparative method and sterilization way.2) To establish the institution of record and manipulation,including bolting donors,obtaining, preparing and distributing the materials.3) Monitoring the record by special messenger.2.The group of anterior cruciate ligament reconstruction under arthroscope.(1) 27 patients by 1/2BPTB,15 by double tendon of tibialis anterior and posterior tendon, long and short peroneal tendons and achilles tendon.The reconstruction was operated all under arthroscope according to isometric rules.The BPTB were fixed by Ti screw or absorption interface screw.Double tendon was fixed by introfix plate at femur and absorption interface screw at tibia.(2) unified rehabilitation procedure after operation. (3) The indexes of result:the range extension and flexion,Lysholm score,Lachman test,pivot shift test,anterior drawer test,and subjective evaluation the same to before operation.(4) observing the wound condition,arthrocele,fluidify and infection and so on.3.The group of posterior cruciate ligament reconstruction under arthroscope.(1) 21 patients by double achilles tendon,the operation rules and ways is the same to the 2^nd group.The rehabilitation and indexes is the same to the 2^nd group except for the posterior drawer test.Results1.The tendons which was defrosted 15 minutes in 40℃SPSS looks fresh with enough strength however lower than normal.The bacterial culture of poly-point is negative in operation.2.All the patients have no infection after operation,however,some with gently or medium arthrocele and fluidify,but the syndrome extinct 1-3 months later.3.37 of 42 anterior cruciate ligament reconstruction patients was follow-up mean 1 year and 2 months(6months-2years and 3 months).the follow up indexes is the same to anterior operations.Lysholm score increased from 58.2±6.3 to 92.1±4.8.the motion range is the same to the other knee.25 Patients were satisfied.10 were improved,2 had advcanticement.Objective check with KT2000 showed that -2～4mm,compared to normal.All the patients had not receive the revisional operation.4.18 of 21 posterior cruciate ligament reconstruction patients was follow-up mean 11 months(6months-1years and 9 months).the follow up indexes is the same to anterior operations.Larson score increased from 59(37～72) to 92(85～98).Lyscholm score increase from 56(39-76) to 91(81-99).The increase mean ranges are 33 by Larson score and 35 Lyscholm score.The PDT and Lzchman test are all positive while they are all negative except 2 weakly positive by PDT and 1 by Lachman test after operations.11 Patients were satisfied.6 were improved,1 had advcanticement.Conclusions1.Clinical using proved that the allogenic tendon our preparing are safety without any bacterial contamination and reflex of irritation and infection when they were transplanted in human body.2.The Cruciate ligaments reconstruction by allogenic tendon transplantation under arthroscope with less operation injuery have good clinical therapeutic efficacy to treat joint unstable due to cruciate ligament lesion.