Fabrication Of ACL Scaffold Based On Col-I/PVA Opolymerization Filature Technology

Anterior cruciate ligament of the knee controls the normal motion and acts as a knee jointstabilizer to prevent tibia from moving forward. However, the ACL injury can hardly repairitself, which will greatly influence stability of the joint. ACL tears will lead to instability ofthe knee joint and further degeneration of the knee articular cavity, which will affect the jointmovement, and even cause disability. Especially for osteoarthritis patients, the ligamentdamage has been proved to be one of the most important early incentive. Recently, ACLinjury is treated mainly through autograft, allograft or artificial ligament reconstruction, whichall exist deficiency. The source for Autograft reconstruction is limited, and complication ofthe donor site is serious. Allograft reconstruction solved the problem of autolograft, but it isrestricted in use due to the risk of infectious disease transfer and the immunoreaction.Artificial ligament reconstruction is a new treatment method in recent years, which overcomesthe source limitation and infectious disease risk, but the artificial ligament used in clinicalapplication is permanent ligament, which will probably lead to synovial inflammation andfatigue fracture. The ligament advanced reinforcement system (LARS) produced bypolyethylene terephthalate (PET) fibers has a great short-term effects in the clinicalapplication, but LARS lacks long-term study. In recent years, the development of tissueengineering provides ACL injury treatment with new strategies and methods.In recent times, many scholars have engaged in studying on artificial ligament or tissueengineering ligament at home and abroad. Many high polymer materials have been used in theartificial ligament or tissue engineering ligaments study. In laboratory study, these materialshave showed good biocompatibility and physical properties. But there are some problems, sothat not a real tissue engineering ligament and excellent artificial ligament have been adoptedin the clinical application.There are some popular materials for tissue engineering ligament asfollows:1. Natural polymer materials: collagen and silk (SF);2. Synthetic polymer materials:Polyglycolicacido(PGA), polylactic(PLA), polyvinylalcohol(PVA) and so on.Some encouraging results has got from these polymer materials in the lab studies, but they still havedefects for clinical application:1. The material’s bioactivity is not good enough;2. Thematerial’s biocompatibility(BC) is not good enough;3. The joint between regeneratedorganization and the native organization is not good enough;4. The speed of the materials’degradation doesn’t match that of the tissue regeneration;5. The mechanical properties ofmaterials need to be improved.In conclusion, in accordance with the principle of bionics, the study makes use of themain ingredients of native ACL extracellular matrix(ECM): Type I Collagen (Col-I) andpolyvinylalcohol (PVA), which are made into the Col-I/PVA copolymerization fiber to braidscaffold type artificial ligament.In this study, the Col-I/PVA copolymerization fiber has goodmechanical properties, and it has no significant difference in maximum load withpolyethylene glycol terephthalate (PET) fiber, it is better than PET in modulus of elasticity;vitro degradation test shows that the Col-I/PVA copolymerization fiber’s degradation speedcan meet the speed of new tissue replacement. Col-I/PVA copolymerization fiber has showeda good biocompatibility in leach liquor MTT test of Col-I/PVA copolymerization fiber, Micefibroblasts(L-929) coculture with Col-I/PVA copolymerization fibers and mice subcutaneousimplantation short-term (9W) test.The large animal experiments testified that our scaffoldtype artificial ligament can afford the effect of stabilizing knee joint. Part of the ligamenttissues can be regenerated after24weeks. The regenerated ligaments are similar to normalACL in the extracellular matrix (ECM) based on histological and immunohistochemicalanalysis. The immunohistochemistry staining shows that type I collagen is strongly positive,type III collagen and tenascin-C are weakly positive, and the regenerated tissues are ligaments.Biomechanical test shows that the maximum load of the regenerated ligament is472.43±15.20N, which has a slight difference compared with normal ACL630.07±76.11N, but canabsolutely afford the knee’s daily activities such as walking, descending stairs or jogging.Part one Col-I/PVA copolymerization fiber manufacture andperformance and biocompatibility testIn this study, in order to reconstruct ACL, based on the principle of bionic, we makeCol-I/PVA copolymerization fiber by adopting Col-I and PVA wet spinning, use the Col-I/PVA copolymerization fiber to braid the scaffold type artificial ligament, and test thephysical and chemical properties of materials, biocompatibility of Col-I/PVAcopolymerization fiber was tested by means of leach liquor MTT test of Col-I/PVAcopolymerization fiber, Mice fibroblasts(L-929) coculture with Col-I/PVA copolymerizationfibers and mice subcutaneous implantation short-term (9W) test.1. Materials and methods1.1Type I collagen protein (pigskin extraction, homemade) and PVA (Model AH-26,Medicine group chemical Co., LTD, China) are made into Col-I/PVA hybrid according to theconcentration of50:50. After the process of filtering and de-foaming, wet spinning is adoptedto make Col-I/PVA copolymerization fiber under the influence of appropriate traction incoagulation bath which takes saturated sodium sulfate solution as coagulator.(Col-I/PVAcopolymerization fibers are provided by Fengmao Fine Chemicals Co., Ltd., China)1.2The related test for the vitro degradation test and Mechanical properties test ofCol-I/PVA copolymerization fiber1.2.1. Vitro degradation test for the Col-I/PVA copolymerization fiber’s degradationspeed1.2.2. Mechanical properties test on experiment group Col-I/PVA copolymerization fiberand control group PET fiber1.3Four groups were Divided:100%concentration Col-I/PVA copolymerization fiberleach liquor group,50%concentration Col-I/PVA copolymerization fiber leach liquor group,control group and the positive control group. Mice fibroblasts(L-929) was used to test theabsorbance in1,2,3,4,5,6,7d by MTT assay, and the relative growth rate(RGR)was calculatedto determine the level of cytotoxicity.1.4Mice fibroblasts (L-929) were cocultured with Col-I/PVA copolymerization fibers,and the growth situation of cells in the Col-I/PVA copolymerization fibers was observedthrough inverted microscope and scanning electron microscope(SEM).Col-I/PVAcopolymerization fibers and PET fiber were subcutaneously implanted in KM mice. The micewere sacrificed at1stweek,3rd week,9thweek postoperatively, and cut into sections of5μm, and stained by hematoxylin and eosin (H.E), Masson for histological observation.2. Result2.1The Col-I/PVA copolymerization fiber is50Denier (D), and it is flexible to braid. 2.2According to vitro degradation test, the degradation rate of Col-I/PVAcopolymerization fiber was8.28%in1stweek,15.14%in2ndweek,16.26%in3rdweek,16.88%in4thweek,17.17%in6thweek and17.44%in8thweek.2.3Based on mechanical test, as for of the Col-I/PVA copolymerization fiber,maximum load:88.6433±0.69189, tensile strength:112.8667±0.88288, modulus ofelasticity:0.4667±0.04631, Group elongation at break:17.2733±1.41608, breaking strength:56.51±13.54123; as for the PET fiber, maximum load:96.1075±0.9393, tensile strength:151.07±1.47561, modulus of elasticity:123.48±14.76304, Group elongation at break:21.32±7.68921, breaking strength:39.2125±5.18963. In conclusion, only the elasticity modulusof Col-1/PVA copolymerization fiber group and PET fiber group (p <0.05) has significantdifference, while the other parameters (p>0.05) has no significant differences.2.4MTT assay: the relative growth rate(RGR) of cells in the two experimental groups(50%,100%leach liquor group) in different time points is90%~123.61%. The cytotoxicityof Col-I/PVA copolymerization fiber is0～I level.2.5Cells and material coculture: Col-I/PVA Copolymer fiber and L929cells wereco-cultured for4days, the result of which shows that cells on the fibers grew well andappositionally with a normal spindle shape.2.6Envelops were formed around both Subcutaneously implanted Col-I/PVAco-polymerizing filature and PET fiber at1st,3rd and9th week, and the transparency ofenvelops was increasing with the lengthening of time. The inflammatory cells aroundenvelops were mainly neutrophilic granulocytes, macrophages and lymphocytes, which wereobviously decreasing with the extension of time.3ConclusionCol-I/PVA copolymerization fiber can be braided, and its degradation rate and excellentmechanical properties conform to the needs of the scaffold type artificial ligament. Col-I/PVAco-polymerizing fiber has good Biocompatibility. Its Cytotoxicity and histocompatibility arewithin the permission of clinical application. Part two ACL scaffold manufacture,large animal experiment and theregenerated ligament detectionIn this study, to confirm the reconstruction performance of Col-I/PVA co-polymerizingfiber ligament, we used the Chinese guangxi Obama miniature pigs to establish ACL acuteinjury large animal model, and adopted scaffold type artificial ligament to make ACLreconstruction of the large animal model, and then tested the pigs’ ACL gross anatomy andthe relative content of main extracellular matrix.1. Materials and methods1.1Braiding of the Col-I/PVA co-polymerizing fiber ligament1.2Mechanical teat on Col-I/PVA co-polymerizing fiber ligament1.3Establishment of ACL acute injury large animal model, and ACL reconstructionbased on the large animal model by means of scaffold type artificial ligament1.4Postoperative evaluation2. Results2.1The maximum load and elasticity modulus of regenerated ligament have nosignificant differences with those of Col-I/PVA copolymerization fiber ligament. Theregenerated ligament is similar to the native ACL of Chinese guangxi Obama miniature pig.2.2Imaging observation:4weeks postoperatively, DR examination shows that thescaffold is fixed firm with screws. CT thin slice scanning shows that the bone tunnel ofexperiment group is not expanding and has obvious mineralized tissue. The MRI scanningshows that the regenerated ligament has grown in the scaffold. The regenerated ligament issimilar to the native posterior cruciate ligament (PCL) according to the imaging observation2.3Gross observation of the regenerated ligament: Based on gross observation ofexperiment group, there is no joint effusion and synovitis, the fixed screws are firm, theregenerated ligament has generated in the original ACL location, and the basic shape andcolor are similar to those of the native ACL; As for the control group, there is no jointeffusion and synovitis, the resected ACL isn’t repaired, and the ACL nub is wrapped up bysynovial tissue.2.4Histological assessment, immunohistological analysis and ultrastructure observation:According to experiment group after24weeks，H.E staining shows that the regenerated tissues and cells in the articular cavity scaffold are similar to those of the native ACL. H.Eand fast-green staining of ligament-bone junction show that the interface was filled withabundant connective tissue whose collagen fibers are projecting from regenerated ligamentinto bone tissue. The immunohistochemistry staining shows that type I collagen was stronglypositive and type III collagen was weakly positive and tenascin-C was positive. Scanning bySEM shows that a large amount of new fibrous tissues come into existence.3. ConclusionsThis study leads us to believe that Col-I/PVA co-polymerizing fiber has excellentbiocompatibility and mechanical properties. Miniature pig models were applied to reconstructACL by scaffold type artificial ligament. At24week postoperatively, fibroblast, ECM andmechanical property of the regenerated ligaments were similar to those of the native ACL. Itimplies that Col-I/PVA co-polymerizing fiber scaffold has great potential in clinicalapplications.Full-text conclusionIn this study, the scaffold type artificial ligament which base on Col-I/PVAco-polymerizing fiber is more bionic. Copolymerization of type I collagen and PVA not onlykeeps the type I collagen’s good biocompatibility, but also integrates the PVA’s mechanicalproperties.Col-I/PVA co-polymerizing fiber has excellent mechanical properties, which is inaccordance with the requirements of the scaffold type artificial ligament. It has no significantdifferences with PET fiber in mechanical properties except modulus of elasticity.Vitrodegradation test shows that the degradation rate of Col-I/PVA co-polymerizing fiber conformsto the needs of the scaffold type artificial ligament.The cytotoxicity and histocompatibilitytest prove that the Col-I/PVA co-polymerizing fiber have excellent biocompatibility.In largeanimal experiment, the artificial ligament can play the role of a replacement. Theimmunohistochemistry staining shows that type I collagen and tenascin-C were positive andtype III collagen weakly positive.According to the mechanical properties test, the mechanicalproperties of regenerated ligament approach those of the native ACL of ChineseGuangxi-bama miniature pig.In further study, we will test on the material ratio and braidmethod, making the scaffold type artificial ligament more bionic, and have better mechanical properties.