| BackgroundJoint ligament injury is the most common disease In daily life, in the United States,about6per thousand people pain for anterior cruciate ligament injury each year accordingto statistics, and half of the patients require surgical reconstruction. With the rapiddevelopment of articular surgery, concerning on research of artificial ligament materialhave been growing. In which the relative studies of material safety and biologicalalternative have become the hot issues being, and material mediated cell migration is thebasis of other cells biological functions which the material affect.Biological alternative procedure of artificial material depends on its migration inducing properties of the biological cells in the implantation area, which is the premisebase of the biological material having impact on cell biological function. In vivo cellmigration process undergoes four steps: firstly, cell creates a new adhesion withextracellular matrix (ECM), makes the cell-ECM been fixed, secondly, the front of cellextends lamellipodia along the direction of migration, and adheres with anterior ECM tomaintain the stretching state, subsequently, the intracellular actin and myosin play roles inregulating the contraction of cell body, promoting the cell moves forward, finally theposterior part of cell separates with ECM and lifts forward by contracting the posterior,finishes the whole migration process. Numerous studies show that different nature ofbiological material and design on biological scaffold engineering has a certain impact onvariety of biological functions such as cell growth and migration. This prompted us that,researches on the relationship between biological effects of material and spatial structureof biological scaffold with cytology will help to further improve the bio-safety andbio-alternative, and to provide a better method for the treatment of clinical disease.Artificial ligament triturated by polyethylene terephthalate (PET) repairs ligamentinjury is an important joint surgical reconstruction technique. Our previous study foundthat after modified by chitosan-graft, the biocompatibility and wettability of artificialligament made by PET material had been greatly improved, prompt that modificatednew PET material has a good potential for artificial ligament, but the effect of the wovenknit on the biological behavior of the cells needs to be further researched. After artificialligament implantation, bone marrow mesenchymal stem cells (BMSC) is one of the maincells in the implanted area, which can accomplish cell differentiation under stress. Whilethe migration process of BMSC toward artificial ligament is affected by the micro-porestructure, tension and biocompatibility and many other factors of knitted material.Therefore, the study of cell migration and related mechanisms in biological materials is ofgreat significance.Rho family of small molecules guanosine triphosphatase is one of the most importantmolecular signaling pathways mediating a variety of cell biological functions, and is alsothe common signaling molecules integrating lots of intracellular signaling pathways, acts like a molecular switch. Studies have shown that the role of the Rho family plays anindispensable role in the cytoskeleton, gene transcription, regulation of cell adhesion andmultiple signaling pathways regulating. Being one of the Ras superfamily members, Rhofamily is an important upstream class of signal transduction molecules of MAPK system,in which studies on the main role and mechanism on RhoA, Cdc42and RAC3memberscould be relatively clear. In the cell migration process, RhoA plays a very important role,the RhoA/ROCK pathway mediated is known as the dynamic motor in cell movement.RhoA belongs to monomeric G protein, the structure sequence of whic h is relativelyconservative. RhoA is regulated by the intrinsic activation mechanism, Rho GTPasesexists in the form of combined GDP (non-active) or binded GTP (active), to play aregulatory role through the mutual conversion between. Activated RhoA switc hes on thepathway through activating the downstream effective molecular ROCK. ROCK is aserine/threonine kinase, the active form phosphorylates downstream substrate. During theprocess of cell migration, activated ROCK would phosphorylate the downstream s ubstratemyosin light chain phosphotase (MLCP) or phosphorylates myosin light chain directly.These two pathways increase the phosphorylated level of the intracellular myosin lightchain, stimulate the cross-linking of myosin and muscle actin, enhance the contraction ofactin. ROCK can also activates LIM kinase, LIM kinase activation allows actindepolymerization and cutting factor inactivation. In addition, ROCK can activate cofilinand therefore increase and stabilize actin. From the signal pathways descr ibed above, cellseventually caused to migrate.Purpose1. To establish a cell migration model applied to knitted biological materia in vitro;2. To investigate the effect of RhoA/ROCK pathway on different nature and weavingmethod of the material-guided mouse BMSC migration;Methods1.316L medical stainless steel was designed to produce the cell migration model. Themodel was placed into6-well cell culture plates, mouse BMSC was seeded through the sample hole of model. After cultured for24h, the model was withdrawaled andcontinued to culture for24h,48h and72h;2. When the model was confirmed can be effectively used to quantify the cell migrationprocess, the model then was utilized to observe the migration ability of BMSC ongroup3000D and5000D modified knitted PET. The PET was stained by giemsa andthe migration area was calculated;3. To set up the migration model of mouse BMSC cell cultured in the PET material knit,divided according to the different materials in to3000D A weaving group,3000D Bweaving group,5000D A and5000D B weaving group. After cultured96h, Giemsastaining and Western blot were used to observe cell migration process and theexpression of RhoA protein.4. concentrations were0,5,10,20mol/L ROCK kinase inhibitor Y27632treatme ntmice BMSC5000D A knitting method of PET material culture96h, Giemsa stainingand Western blot was used to observe the speed of cell migration on RhoA ROCK andp-MLC protein expression.Results1. The model was removed after the cells cultured for24h, the central area of six-wellplate was blank which is equal to the bottom of the model. BMSC gradually migratefrom the peripheral to the central area with time;2. After the cell migration model guided by modified PET knitted material wassuccessfully established, the result of Giemsa staining showed that The migrationareas were gradually inceased with time between groups, and the migration area of3000D group was significantly greater than5000D group(P <0.05);3. Giemsa staining showed that the migration area of5000D A group was significantlylarger than other goups(P <0.05), then followed by3000D A group, the5000DBgroup was better than3000D B group. Western blot results suggested that thechanging trends of RhoA protein expression was similar with Giemsa staining;4. After the cell migration model guided by5000D A material was successfully established, Giemsa staining and Western blot analysis found that with theconcentration of Y27632inhibitor increased, the cell migration area graduallydecreased and the expression of RhoA protein, ROCK protein and p-MLC proteinexpression was decreased (P <0.05).Conclusion1.316L Medical stainless steel model can be applied to basic research for the artificialknitted material guided cell migration;2. The linear density of modified PET knitted material has an effect on migration abilityof murine BMSC;3. Characteristic differences and knitting method variety of biological material result inmaterial with different microporous structure, affecting the process of cell migration,RhoA/ROCK signaling pathway may be one of the key regulatory moleculesmediated those changes above. |
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