Experimental Study Of Cryopreserved Meniscal Allograft In Rabbits

Background and ObjectiveMeniscus is a main part of normal knee joint, it lies between the tibial plateau and femoral condyle. It plays a key role in absorbing shock,conducting loading,joint stabilization,lubrication and nutrition etc. After the severe injury of meniscus or total meniscectomy, the articular cartilage loses the protection from meniscus and the cartilage degeneration will be accelerated, then the knee joint's unstability and osteoarthritis will emerge.The blood supply of the outer layer of meniscus comes from the end branches of genicular arterial plexus, so the self healing is possible after moderate injury of that part; while the inner part of meniscus hardly has any vascular blood supply, and it can not heal by itself after injury, under this circumstance, surgical procedure should be taken. The traditional managements of meniscus injury include meniscus suturation or meniscectomy, but the long-term follow-up results indicate that early signs of articular degeneration would still appear even after those operations. So, facing with the absence of meniscus or severe meniscal injury which can not be repaired, we should reconstruct the meniscus to prevent the occurrence of osteoarthritis.Meniscal allograft transplantation is becoming more and more popular, and its therapeutic effect is also very affirmative. Following with the numerous experimental studies, more and more surgeons are using this technique in clinical practice. Researches indicate that after meniscal allograft transplantation, the chondrocytes and fibroblasts in the donor meniscus will gradually substituted by the chondrocytes and fibroblasts from receptor. During the remodeling phase of the meniscal microstructure, the amount of cells of the receptor is increasing and the meniscal biomechanical frame structure is more fragile than normal, and under unsutable stress, the meniscus inclined to degeneration or rupture injury then cause the failure of transplantation, meanwhile, long term stabilization will greatly increase the possibility of osteoarthritis after operation. How to promote the microstructure reconstruction of allograft meniscus, and consequently improve the prognosis of allograft transplantation, then effectively slow down or prevent the articular degeneration is still the research focus of scholars.The local vascular reconstruction and cellular differentiation and proliferation play a key role in the prognosis of meniscal allograft transplantation. Especially for the later one, weather the mesenchymal cells can differentiate into the function active fibrocartilage cells and fibroblasts then proliferation directly determine the outcome of the allograft meniscal reconstruction. Insulin can induct many biological functions through the insulin growth factor receptor, it can regulate the cellular metabolic process and promote the proliferation of many kinds of cells including the chondrocytes, meanwhile insulin is relatively economic and easy to obtain, so it has a great clinical practical value.This research intend to develop experimental model of rabbit meniscal allograft transplantation; by comparing the prognosis of allograft meniscus of simple transplantation and transplantation with the induction of insulin, we try to find out whether insulin can improve the prognosis of meniscal allograft transplantation, and we can make a good preparation for further clinical practice. Research on this aspect is still not reported by now. Materials and Methods1. Experimental animal: 30 female Chinese rabbits were included, weight between 2.5-3.0 kg, the average age was 18 months.2. Preparation and conservation of meniscus:Take the all together 20 medial menisci of the double knees of 10 rabbits under aseptic operation, number them, put them into the aseptic bottle separately, store them in the -80℃refrigerator for use.3. Meniscal allograft transplantation:Randomly divide these 20 menisci into two groups, group A and B, 10 menisci in each group, before transplantation, the 10 menisci of group A were rapidly thawed in 36℃isotonic sodium chloride, and insulin was added into the thawing solution of group B, the thawing time is about 1 hour.Chose rabbits which proximate the donor rabbits in weight, and divide them into two group A and B, cut the medial meniscus, then begin the simple meniscal allograft transplantation and allograft transplantation with the induction of insulin separately.After operation, leave two rabbits each cage, and the rabbits were allowed to move freely in the cage. For group B, 0.3ml of 1% insulin was injected into the knee joint cavity of transplantation, one time each week, and consequently inject 5 times.4. After 8 weeks of operation, kill all the rabbits by air embolism. Mutilate the knee joint, open the joint cavity, observe the transplanted menisci and joint inner structure, 10% formalin fixation,decalcification,cut the splice,paraffin wax fixation, stained with hematoxylin & eosin,immunohistochemistry staining, observe the results.Results1. Macroscopic findings: after 8 weeks of operation, the transplanted menisci showed macroscopic attachment to the capsule and synovial tissue in all rabbits studied, in some menisci, the posterior horns were not attached to the articular surface of the tibia but were attached to the surrounding synovial membrane, and in 3 menisci of group A and 2 in group B, the graft showed slight shrinkage,; synovial membrane showed moderate congestion; no obvious fluid in the joint cavity, among them,; there was statistical variation between these two groups on macroscopic scoring.2. Histological findings: after 8 weeks of transplantation, light microscopy showed coverage of the free inner edge of the graft with the highly vascular synovial tissue and cell proliferation in both groups. Fibrocartilage like cells grew into the inner layer of allograft meniscus, there were still some pyknotci nuclear cells in the middle of the section of the allograft meniscus; the collagen fibers were relatively regular in outer layer of the allograft menisci. There was statistical variation between these two groups on microscopic scoring.3. Immunohistochemistry stainings: high expression of TIMP-1 in both groups, after analysis of gray values in both groups by image processing system, comparison of the data showed that there was statistical variance between these two groups.Conclusion:1. We successfully develop the experimental rabbit model of meniscal allograft transplantation, the allograft menisci are conserved by deep frozen technique, the transplantation method is simply suturing the allograft meniscus with articular capsule and result indicate good healing.2. Histological findings show that new bom cells grow into the inner layer of transplanted meniscus from outside layer, and there is likely metaplasia of fibrocartilage cells from synovial cells; TIMP-1 locates in the cytoplasm, it is an important regulatory factor which maintain the normal metabolism and invertion of cartilage substance, immunohistochemistry staining shows positive TIMP-1 expression of the new born chondrocytes, and indicates that these cells have the ability to secrete collagen substances.3. Insulin can promote the proliferation of fibrocartilage cells in allograft menisci, and it can help the early reconstruction of structure and function of transplanted menisci and it can effectively prevent the early degeneration of knee after meniscal transplantation. However, its concrete mechanism of action still needs further investigation.