Preliminary Research Of Plantar Soft Tissue In Vitro Reconstruction

Plantar soft tissue trauma is relatively common in clinical.At present surgical method of repair and reconstruction of plantar soft tissue is relatively mature, but still dissatisfied with postoperative functional recovery. In the plantar soft tissue reconstruction, the greater the use of small leg flap repair. Due to the lack of skin and deep fascia flap between the longitudinal collagen fiber bundles, making local soft tissue after repair poor stability, resulting in recovered patients with unstable standing, walking slip. Resumption of plantar soft tissue-specific function, especially the stability, is difficult to solve the problem of clinical.Tissue engineering is a subject that the application of life sciences and engineering principles and methods, research and development for the repair, enhancement or improvement of many human tissues and organs function and morphology after injury. Tissue engineering change the traditional treatment model that with wound healing wounds, for the eventual realization of a traumatic injury and repair has opened up new avenues.In this research, we apply theory and technology of tissue engineering to repair the plantar soft tissue trauma, to resolve local soft tissue stability problems.Plantar soft tissue structure have fibroblasts, white blood cells, fat cells and a small number of undifferentiated mesenchymal cells and so on. Fibroblasts mainly produce collagen fibers, elastic fibers and reticular fibers. There are the large numbers of Collagen fibers in the plantar soft tissue which the most biochemical composition is typeⅠcollagen and typeⅢ. In these fibers, the type I collagen fiber is the majority. Therefore, in plantar soft tissue in vitro fibroblast activity and collagen I secretion is vital. A lot of research data shows that PTGF-BB, TGF-β, IL-13 and other cytokines promote fibroblast chemo taxis, proliferation and collagen secretion. Therefore, this experiment selected fibroblast cells as seed cells. In order to fibroblast can high secrete type I collagen, we selected PTGF-BB, TGF-β1, IL-13 three-cell factor study in this experiment.Another important study of tissue engineering is scaffold material. An ideal scaffold material should meet:①mimic the natural extra cellular matrix structure and biological function.②have a good biocompatibility may be adhesion and proliferation of the cells.③degradation rate and the rate of tissue regeneration in consistent.④The appropriate mechanical properties can support cells.⑤has good microstructure, such as the appropriate pore size, high porosity and connected to the hole shape.⑥Specific three-dimensional shape.⑦Large surface area and appropriate physical and chemical properties of surface. The collagen fiber of natural extra cellular matrix size between 50 and 500 nm, so nanofibers scaffold materials that can best mimic in vivo extra cellular matrix characteristics. At present, the electrostatic spinning is the only way that can direct, continuous make the polymer nanofibers.In this experiment, we apply PCL/gelatin composite nano-fibers as a scaffold material that make by electrostatic spinning. PCL/gelatin nanofibers not only has large surface area, small pore size, the high porosity, good uniformity, and the diameter is close to the extracellular matrix.It is the greatest degree imitate the natural structure of the extracellular matrix. Also it avoid leading to the PCL alone slower degradation, poor surface biological activity, and single-gelatin degradation too quickly. By adjusting the PCL, gelatin different proportions to achieve that we need the degradation rate and the surface of biological activity.Experimental select SD rats,2 days after birth, skin of the back to do primary culture in order to obtain seed cells. By using different cytokines act on the seed cell to select the best cytokines that promote secretion collagen I of fibroblasts. We seed cells on PCL/gelatin scaffold which Observation nano-material bio-activity and the secretion of collagen. For subsequent in vivo experiments and clinical application of rats provide a reference. We research includes the following two parts:Objective:Different concentrations of IL-13, TGF-β1, PDGF-BB induce dermal fibroblasts to secrete type I collagen in vitro. To select cytokines that stimulate fibroblasts high-secreted type I collagen.Methods:Using dorsal skin of 2d born SD rats for primary culture of dermal fibroblasts. Applications of immunofluorescence were used for purity determination. Experiments were divided into four different groups namely, PDGF-BB(30ng/ml), IL-13(100ng/ml), TGF-β1 (lOng/ml) and a control group. MTT, ELISA were used to determine the growth condition of dermal fibroblast cell as well as the concentration of type I collagen at T=24h, T=48h and T=72hResults:Primary cultured dermal fibroblast cells were determine to be≥90% in purity. PDGF-BB, IL-13, TGF-β1 can stimulate dermal fibroblast cell proliferation and secretion of type I collagen. At T=48h and T=72h, PDGF-BB Group dermal fibroblast cell proliferation and secretion of type I collagen were significantly higher than IL-13 group, TGF-β1 group and negative control group (P<0.05). Conclusion:30ng/ml of PDGF-BB shows more prominent effect in the stimulation of dermal fibroblasts cell proliferation and secretion of type I collagen protein.Objective:Observation of dermal fibroblasts in PCL/gelatin composite nano-scaffold and testing the concentration of collagen type I in the culture medium and thus evaluation of PCL/gelatin composite nanofibers biocompatibility.Methods:Electrostatic spinning make the PCL/gelatin composite nanofibers. The dermal fibroblast cells were seeded into the material surface. Using scanning electron microscopy observe dermal fibroblast cells in the growth of surface and using the ELISA measure concentration of collagen type I in medium. This experiment is divided into three groups:the scaffold material group, PDGF-BB group, blank control group.Results:PCL/gelatin nanofibers which PCL, gelatin mixed solution by electrostatic spinning technology, showing three-dimensional network connectivity with each other; fiber diameter and distribution more uniform and the diameter about 700nm. Dermal fibroblasts into the PCL/gelatin scaffold surface attach solid, showing a long spindle-shaped or polygonal, stretch fully and suddenly slender cell. Between cells and scaffold materials, cells and cells, there were more dendritic protrusions cross-linked with each other, and also the cells within the fibrous material. The concentration of collagen type I in medium test results statistics show that among all groups was no significant statistical difference after 24h (P> 0.05). Concentration of type I collagen of PDGF-BB group in medium is significantly higher than scaffolds and control group after 48h,72h (P<0.05). PDGF-BB group and the control group of concentration of collagen type I in medium are not differ significantly at different times (P> 0.05).Conclusion:PCL/gelatin composite nano-material has good biocompatibility and surface activity, can serve as a subcutaneous plantar soft tissue scaffold.