| ObjectiveRupture of anterior cruciate ligament is the common sports injury. The only way to restorethe function and stability of the knee is ACL reconstruction. The common ACL substitutionsare autograft ligament(tendon), allograft ligament(tendon) and man-made ligament(tendon).All of them have a lot of drawbacks, theno secondary hurt of the donor site, the improvement of preservation and sampling, theallograft ligament(tendon) is increasingly used in ACL reconstruction. When the allograftligament is implanted inIt is composed of avascular necrosis, revascularization, cells proliferation and remodeling.muneresponse. When the allograft ligament is implanted, superficial glycoprotein of the tendon cellrepresent the Major Histocompatibility antigen(MHC-1), it can induce the immune responsebetween the implant and the host. The immune response influences the bone-tendon healingprocess, delays and weakens the healing, even leads to the failure. The extracellularmatrix(ECM) is composed of type collagen protein. The difference of the same tissueECM among the races is very small and can hardly induce immune response. Cell-free oracellular technique can remove the immune-response induced element and retain the originalbiochemical and mechanical character. Recombined human bone morphogeneticprotein-2(rh-BMP-2) had been certified to have the ability to promote the tendon-bonehealing in other study. We use tributyl phosphate(TBP) to remove the cell element of allograftligament, and test the histology and mechanical of the acellular ligament. Therefore we use theacellular ligament which was recombined with human bone morphogeneticprotein-2(rh-BMP-s ACL. We check the reconstructed ACLregularly by histology observation, CT scan and mechanical test.Method1. acellular ligament administration We use tributyl phosphate(TBP) to remove the cell element of allograft rabbit ligament.First step is to immerse the allograft ligament into the10mm Tris solution(which includeserpin, metalloproteinase inhibitor, and penicillin/streptomycin) for36h; Second step is toimmerse the allograft ligament into the50mm Tris solution(which include Triton X-100,solution; the fourth step is to immerse the ligament into TBP and50mm Tizmmar for48h.2. Histology and mechanical test of the acellular ligamentWe treat the acellular ligament with Hematoxylin-Eosin stain, DAPI fluorescent stain toidentify the acellular effect of TBP. We test the GAG content of the acellular ligament byDMMB to identify the effect of TBP on GAG content. We test the DNA content of theacellular ligament by animal tissue DNA kit to identify the effect of TBP on DNA content. Wetest the collagen content of the acellular ligament by hydroxyproline kit to identify the effectof TBP on collagen content. We test the max tension load and modulus of elasticity to identifythe effect of TBP on mechanical character.3. Iconography observation after ACL reconstruction with acellular ligament and rh-BMP-2We reconstruct rabbit ACL with acellular ligament and rh-BMP-2. We scan thereconstructed ACL and bone tunnel with mulspiral-CT postoperative1m,3m and6m. We andmeasure the width of different part of bone tunnel, such as extrance, middle and exit part, insagittal section. We estimated the width of the tunnel with mean width. Comparing the resultwith the control group, we try to identify the effect of alleluar ligament and rh-BMP-2whichwere combined to reconstruct the ACL on tunnel widening.4. Histology observation and mechanical test after ACL reconstruction with acellular ligamentand rh-BMP-2We reconstruct rabbit ACL with acellular ligament and rh-BMP-2.1,3and6monthsafter surgery, We cut the bone tunnel into the slides by hard tissue section and stain with picricacid to identify the cartilage growth in tendon-bone interface. We cut the ACL into the slides and stain with Hematoxylin-Eosin at1m,3m,6m operatively. It is to identify the cellsproliferation. Comparing with the control group, we try to identify the effect of alleluarligament and rh-BMP-2which were combined to reconstruct the ACL on tendon-bone healing.3and6months after surgery we cut off the knee joint and put it on the biological materialtesting machine for mechanical test. We record the max tension load and modulus of elasticitywhen the reconstructed ACL was pulled out from the tunnel.Result1. Histology and mechanical test of the acellular ligamentThere is no cell element seen by HE stain in acellular ligament administered by TBP. Theresult of DAPI fluorescent staining is negative. It indicates that the acellular is effective. Withthe DMMB method, there is no GAG content change between the ligament and the acellularligament. With the hydroxyproline kit, there is no collagen content change between theligament and the acellular ligament. We find that there is no difference on the max tensionload and modulus of elasticity between the ligament and the acellular ligament.2. Iconography observation after ACL reconstruction with acellular ligament and rh-BMP-2We can rebuilt the image of rabbit ACL reconstruction model undergoing CT plain scan.There is no width difference of the bone tunnel between the two groups1month after surgery.3month after surgery, there is osteogenic response seen in the bone tunnel in acellularbone tunnel is wider than before, but is no statistical significance.6month after surgery, thereis more osteogenic response seen in the bone tunnel in acellular ligament group, and still nois little osteogenic response seen in control group, the width of the bone tunnel is more wider3. Histology observation and mechanical test after ACL reconstruction with acellular ligament and rh-BMP-21month after surgery, the width of the tendon-bone interface in acellular ligament groupget narrow. There is a few sharpey fiber and chondrocytes seen in the interface. But the widthof the control group is wide, there is no sharpey fiber and chondrocytes.3month after surgery,the width of the tendon-bone interface in acellular ligament group get narrower than before.Sharpey fiber became more obviously and arranged in order. Chondrocytes became more andgrew mature. And the width of the control group is narrower, but still wider than the trialgroup s, there is a little sharpey fiber and chondrocytes seen.6months after surgery, the widthof the tendon-bone interface in acellular ligament group is hardly seen, a large number ofsharpery fiber generate and arrange in order, chondrocytes grew even mature. And the widthof the control group is narrower, but it still exists, there is more sharpey fiber andCompared with the controlgroup, there are few lymphocytes seen in acellular ligament1month afer surgery. There aremore collegan fibers and chondrocytes seen in acellular ligament group3months afer surgery,but few collegan fibers and chondrocytes in control group.6months after surgery, we finda large number of the collegan fibers and chondrocytes in acellular group. The fiberarrangered orderly and tightly, and chondrocytes get mature. But the collegan fibers andchondrocytes in control group were still few, and the collegan fiber arranged loose.Postoperative36months, the acellular group score higher than the control group in theelastic modulus and the maximum load index during the anti-pullout test, and the difference isstatistically significant.Conclusion1. TBP can effectively remove the cell element of the tendon and retain the original biologicaland mechanical character.2. CT plain scan can rebuild the image of knee joint well. There is no widening sign of thebone tunnel in acellular ligament group at3and6month after surgery. We can find obviousosteogenic response in the tendon-bone interface. 3. After ACL reconstruction, the interface healing is good in acellular ligament group,osteogenic response and sharpey fiber were seen at136month postoperatively. The gapwidth of tendon-bone interface gets gradually narrowing. The speed of the cell proliferation isquicker than the control group.4. After ACL reconstruction, the acellular group is better in the elastic modulus and themaximum load index test during the biomechanical test, anti-pullout test, postoperative3,6months.
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