Construction Of The Plantar Skin And Soft Tissue Scaffolds And The Cultivation Of The Induction Function

The foot is an important organ of the body, and the plantar skin soft tissue defects in foot trauma are common. Bums, mechanical trauma, and chronic diseases caused by ulcers (such as diabetes) are the main causes of the plantar skin soft tissue defects and loss of function.From the perspective of histological structure, there are some differences between the plantar skin and other parts of the body. The plantar skin is dense with a thick cuticle and strong subcutaneous tissue and the fat pad so as to relieve the oppression of the buffer plantar pressure on the nerves and blood vessels. Plantar skin tissue in the vertical go-shaped fibers and the aponeurosis of the plantar muscles are connected to be effective in limiting excessive skin move, forming a "skin connection". At the same time, the fat pad in plantar skin tissue is full of scattered small and tough connective tissue fibers to fix the skin and limit fat to shift. Their orientation responds to the main pressure direction. Therefore, the plantar skin has the function of hard wearing, pressure wearing and load bearing, and has the characteristics of non-slipping and different thickness in different parts, which favors walking and weight-bearing stability.The current domestic and foreign treatments of plantar skin soft tissue defects are mainly through the transplant of flaps. A number of reports in the literature concern the choice of the flap donor site, the restore of blood supply and the establishment of sense. These studies are about the transplant from the healthy parts of patients to repair defects of the plantar skin soft tissue. Although the sensory recovery, foot thickness and pressure wearing are improved to a certain extent, but the flaps (except the exterior and interior flaps) lack "skin connection"’structure unique to the plantar skin, causing recurring ulcers after flap transplant and the "plantar slipping" phenomena when the flaps bear weight in walking. There have been no good solutions till now. In addition, it does secondary damage to the donor site artificially. This treatment model of’wound healing wound" is bound to be replaced by the new approach of "no wound healing wound" of tissue engineering.Abroad from1975onwards, Rheinwald and Green first achieved a large amplification of the epidermal cells in vitro, and in1979received a complete epidermal cell patch, so that the epidermis transplants become possible. In1981, O’Connor for the first time successfully cultivated in vitro with autologous epidermal cell patch in the transplantation of skin wounds. In the early1980s Burke and Yannas developed successful products of the cell tissue engineered dermal-Integra (Integra life sciences) and they were commercialized in1996and obtained the clinical use license by U.S. FDA. Cells in complex tissue engineering leather produced by the Advanced tissue sciences Dermagraft treats chronic diabetic foot ulcer significantly faster than traditional coveringsIn China, skin tissue engineering research has attracted people’s attention. Universities, research institutes and medical institutions have also made bold attempts to explore the tissue engineered skin and achieved positive results. Cao Yilin, etc. adopted Poly lactide as the main material, combined with patients’ autologous cells, developed a tissue engineering skin. Ma Jian biao, etc. used chitosan as the main material to cultivate a sponge-like artificial leather which can promote human fibroblast growth. Jin yan of the Fourth Military Medical University utilized human fibroblasts and epidermal cells to plant in collagen gels, successfully constructing a similar Apligraf-like tissue-engineered bilayered skin substitute, which has been approved by the State Food and Drug Administration (SFDA) for clinical use. In addition, the acellular dermal scaffold of Xia Zhaofan and Suzhou University’s study of building skin regeneration products from silk protein have certain characteristics. But so far, skin regeneration products with tissue induction features are still in the laboratory research stage.Although scholars have used tissue engineering principles to build human skin equivalents to solve the problem of skin defects, and made some breakthroughs, and accumulated a lot of basic data on skin tissue engineering. However, reports on the tissue engineering restoration of plantar skin soft tissue defects are still very few.This project rebuilds the plantar skin and soft tissue through the principles of tissue engineering. First, we use micrologicaltechnique and scanning electron microscope to analyze the structural characteristics of plantar fibers and understand the biomechanical properties of the foot. On this basis, we use natural non-toxic polymers to build the plantar skin soft tissue scaffold, and carry out performance tests on the functions of the scaffold to meet the biological requirements on the foot’s function. Meanwhile we culture cells and stent in vitro and implant the stent into animals, and explore the induction mechanism of the bracket on the cells, tissues in vitro and in vivo, and provide experimental basis for the tissue engineering of plantar skin defects. This project has important scientific and practical significance on the study of reshaping the foot function.The research includes the following aspects.1. The microstructure of the plantar and the construction of scaffoldsObjective:To understand the plantar soft tissue structures as well as fiber and fat cells’distribution at different levels, the growing direction of the fiber and the composition of the fiber; to measure heel thickness and biomechanical indicators, analyze the plantar soft tissue morphology, provide fundamental biological data to construct plantar soft tissue scaffolds. Methods:using surgical microscope to conduct microsurgical anatomy; HE staining and VG staining, slice, optical microscopy; biomechanical test instrument measurements and scanning electron microscopy method.Results:Through microsurgical anatomy observation, plantar subcutaneous tissue fibers are divided into superficial and deep layers. The superficial fibrous septa are small and dense while the deep fibrous septa are big and sparse. These fibrous septa and the skin and the fascia of plantar are closely linked, with a number of fibers impenetrating the fibrous septa to link skin and calcaneal fascia. HE staining showed that the plantar soft tissue from the surface to the depth consists of four layers:the epidermis, dermis, basement membrane and connective tissue. A lot of fibers in the connective tissue layer are in longitudinal, transverse and oblique rows, weaving a three-dimensional network structure, with a number of fat cells located in them; the VG staining showed that most of these fibers are collagen fibers. Scanning electron microscopy found that the fibers can be seen criss-crossing within the plantar after the degreasing and the honeycomb rooms shed of the fat cells. These small rooms are arranged in a regular pipe-like structure with interconnected pores, which is consistent with HE staining. For Six body plantars soft tissue thickness was measured. The average thickness is18.6±1.7mm. We use biomechanical analyzer to test stress on five parts of different thickness conduct quadratic curve fitting. R2are respectively0.982,0.977,0.993,0.992and0.989, indicating fitting well.B, D and F for the skin and connective tissue samples of different thickness of the plantar, where B, D, fitting curve is basically the same features, indicating that the thicker the plantar soft tissue, the stronger ability to absorb external forces. On the contrary, such as H, J, respectively, are from the dermis and epidermis of the plantar. By external forces, pressure-displacement fitting curve is relatively flat, indicating that the thinner, the poorer ability to absorb external forces.Conclusion:Plantar soft tissue consist of the superficial and deep fibers, with the superficial layer being dense and of high tension while the deep layer loose and of gradually increasing tension responding to the increase of external force. The fibrous septa are mainly composed of collagen fibers, some of which are directly connected with the skin and calcaneal fascia to prevent heel skin from moving excessively and maintain the stability of the arch to keep the balance of the body. Meanwhile, according to the structural characteristics of plantar soft tissue, we design scaffolds for tissue engineering, and use polymer protein to make a biodegradable stent, which can induce internal growth of cells of the plantar. The collagen fibers grow along the micron (100-150μm) channel within the scaffold. The channel surface is rough, connected with micrometer-sized holes (20-40μm), which can promote cell adhesion and the transmission of message between each other.2.Construction of stents and the cultivation in vitro of cell induction functionObjective:To simulate the structural feature of two layers of plantar soft tissue, via electrospinning method and self-made mold, we prepare grafted silk fibroin polylactide three-dimensional porous scaffolds and gelatin-silk fibroin polymer scaffold. We measured the stent pro/hydrophobicity. mechanical properties, degradability, morphology and cell compatibility performance. Constructed in line with the foot biomechanics, the organization has good cell compatibility of biodegradable polymer stents, understanding of cell proliferation in the stent within the growth of cultured in vitro data for the repair of the plantar soft tissue trauma.Methods:First developed by the spinning system and self-made multi-channel (diameter100μm) mold preparation of glutaraldehyde-grafted PLA-silk fibroin (PLA-SP) nanoscale electrospinning stent and glutaraldehyde cross-linkingchannel gelatin-silk fibroin protein (Gel-SP) polymer scaffold. Determination of the stents water absorption and degradation rate of drying and mass loss; biomechanics was determined by measuring the tensile strength of electrospun scaffold and the pressure of the polymer scaffold; scanning electron microscopy analysis of the morphology of the stent; cells and scaffold co-cultured cells adhesion rate of proliferation rates were measured by MTT activity, immunofluorescence methods, evaluation of the compatibility of the cells and scaffold.Results:the use of the electrospinning system0.11g/ml PLA/DMF+CH2Cl2and0.13g/ml the PLA+SP (solid ratio of1/2)/TFA in the electrospinning voltage of20kV, with diameters were410±193.lnm and250.7±101.5nm evenly distributed, no bead fiber. Mechanical tests, the use of the quadratic curve fitting, and R2were0.991and0.972, indicating that well fitted by analysis, difference of PLA-SP with PLA between the two groups were statistically significant (F=181353.1,P<0.001) The PLA-SP electrospun scaffold has better tensile performance and scalability. Degradation of the performance test, the degradation rate of the two samples was statistically significant (F=.20.506,P=0.011).PLA-SP group is higher significantly than PLA group.The degradation curves of the two samples,0.11g/ml PLA/DMF+CH2Cl2, porous fibers scaffolds over time to extend the mass loss increased, the degradation rate leveled off in30days to60days.0.13g/ml (PLA-SP)(1:2)/TFA porous fibrous scaffold mass loss in7days with PLA is quite at a later time, the mass loss is greater than the PLA fiber bracket, shows that, after grafting hydrophilic ability of the modified PLA fiber stent. Different solid content than gelatin-the silk fibroin polymer stents water absorption was statistically significant (F=2447.191, P<0.001). Do multiple comparisons test found that the B and C were water absorption, the difference was not statistically significant (p>0.05), the difference A and B. and C was statistically significant (p<0.05), showed that the graft modification of5:1water absorption of the stent relative to the10:1and1:0significantly improved to achieve3.65g/cm2. Stress tests, using quadratic curve fitting, and R2, respectively, for the0.956,0.978and0.993, indicating that the fitting better. The three groups were statistically significant (F=8972.991,P<0.001). Do multiple comparisons test, A, B, C groups differences were statistically significant differences between (P<0.001). Description of the different solid content than the porosity of the scaffolds is not the same,5:1porosity also the strongest ability to absorb external forces, compressive strength.10:1followed by1:0worst. Degradation of performance testing, three groups of sample degradation speeds were statistically significant (F=63.746,P<0.001), and further to do multiple comparisons test found that A, B, and C groups were statistically significant differences between (P<0.001).1:0degradation of the fastest, followed by10:1.5:1slow. Note for the water-soluble gelatin and silk fibroin, the preparation of the stent after graft modification, the surface hydrophilic properties reduce the degradation will slow down. Cells and scaffold training, five groups of samples,24h adhesion rate were statistically significant (F=108.651, P<0.001).Do multiple comparisons test found that five groups were statistically significant differences between (P<0.05). Five groups of samples,96h proliferation rate were statistically significant (F=40.076, P<0.001).Gel, Gel-SP and PLA, the PLA-SP analysis of variance between the two groups, the time factor to deal with factors that differences were statistically significant p<0.05. Description planting24h fibroblast Gel-SP stand adhesion rate the Gel bracket, culture96h after Gel-SP stand on the fibroblast proliferation rate significantly faster than the Gel scaffold; epithelial cells stand in the PLA-SP the adhesion rate and proliferation rate higher than that of the PLA scaffold. The above data demonstrate significantly improved after the grafted stent, cell compatibility. MTT activity in the PLA, PLA-SP, three groups of samples were statistically significant (F=2791.419, P<0.001). Do multiple comparisons test found that three groups were statistically significant differences between (P<0.05), indicating the compatibility of silk fibroin modified polylactic acid stents and epithelial cells significantly better than in the pure polylactide stent. The scanning electron microscope image shows the growth of epithelial cells in the PLA and PLA-SP spinning stand, the epithelial cells along the fiber direction of the growth of cells in the proliferation rate of PLA-SP stand faster than PLA. Confocal microscopy showed that cells in the unmodified PLA porous scaffolds is not easy spreading, globular, and aggregation and uneven distribution in the surface grafted silk fibroin porous scaffolds epithelial cells spreading someimproved the distribution of the stent more uniform cell spreading. Statistical analysis software SPSS13.0. scaffold-water absorption, bio-mechanical properties, degradability, MTT activity between the two groups were compared using repeated measures ANOVA (Repeated Measures).24h adhesion rate and96h proliferation rate between the two groups were compared using one-way varianceanalysis (One-Way ANOVA).If the overall difference was statistically significant, multiple comparisons (LSD).a=0.05for the test standards. Test were two-sided.Conclusion:The solid content ratio5:1Gel-SP bracket and PLA-SP spinning stent has better biomechanical properties and cell compatibility can be used as the plantar soft tissue build scaffolds.3.Modified bracket to cultivate cells, tissues and body of the induced functionObjective:Gel-SP by different solid content ratio stent implantation in SD rats subcutaneous tissue, and observe the stent cells in vivo compatibility and induced cells, the organization’s growth.Methods:6Weight200-250g SD rats, sterile stents (Gel-SP solid content ratio1:0,5:1,10:1) implanted in the subcutaneous tissue of the same rats. The rats were sacrificed at different times, subjects, were observed by HE staining and scanning electron microscopy to understand the different times of cells in the scaffolds to move and the degradation of stents in animals.Results:7days after stent implantation did not cause rats had significant inflammatory response, only a few fibroblasts immersed in the bracket secretion of extracellular matrix, the implantation of the stent did not change significantly;17days, a large number of inflammatory cells(monocytes, macrophages, etc.) to move into the bracket, and part of angiogenesis; A large number of fibroblasts regiment moved to within the21days after stent, the stent gradually began to degrade, inflammatory cells started to decrease;29days5:1than10:1bracket degradation speed faster, and fat cells to move into; in33days later, the stent internal vascularization.5:1stent obvious;41days after stent degradation significantly, vascular many long into the stent, the stent within the organization a lot of survival.Conclusion:Gel-SP solid content ratio5:1stent cells compatible with good, does not cause significant inflammatory response, and can be induced into fiber cells and macrophages to move into the release of macrophage cellsrole of growth factors, chemokines, endothelial cells in the scaffolds to generate new small blood vessels.