| The skin consists of epidermis,dermis,and subcutaneous tissue,forming a protective balrrier to the external environment.The sebaceous gland and sweat gland s are equally protective.When the skin is subjected to damage,such as chemical hazards,and bacterial fungal invasion,the skin loses barrier and function,meanwhile,it also triggers physical stress response to promote wound healing.For irregular,large-area and inconsistent skin wound areas,we are using allograft or xenogeneic acellular matrix dressings which are tissue engineering products for mesh grafts for transplantation,with low immunogenicity and good tissue compatibility.Mechanical is close to normal skin and is an ideal scaffold material.However,its source is limited,the fit of dressings can cause cavities,long treatment,and highly complications.At the same time,only the dermis matrix is not enough to repair the wound.In order to accelerate the wound healing,it is still necessary to repair the epidermis and the dermis at the same time.Therefore,in this article,we use an innovative cell-spray device for cell spray grafting to promote regeneration of the epidermis after injury.In combination with cross-discipline of tissue engineering,a self-developed spray device for autologous skin keratinocytes cells and decellularized porcine dermal matrix powder can be used for skin wound treatment.Explore the significance of cell spray system as a tissue engineering product for skin wound repair.Therefore,the subject can be decomposed into three parts.In the first part,the cells are combined with the appropriate concentration of extracellular matrix to spray,and the biological function analysis of droplet size,cell distribution,activity,proliferation and differentiation potentialis performed.Identification,by adjusting the cell density,extracellular matrix composition,concentration,so that the biological function of the sprayed cells is optimized.The second part focuses on the establishment of a gentle,efficient method for the isolation of human keratinocytes stem cells(hKSCs)to construct a three-dimensional culture program that is proliferation in vitro,and eventually can enter clinical treatment procedures.In the third part,the preparation of decellular porcine skin extracellular matrix powder(ECMP)with high purity and biological activity was prepared by in vitro biological enzymolysis and cryogenic mechanical grinding as a liquid carrier for cell transplantation.Epidermal cells and fibroblasts participate in the repair of injury,and at the same time,they explore the supportive role of ECMP in the growth of epidermal and fibroblasts and their promotion of skin wound repair.1.Biological test of cells after sprayWe designed different types of cell loading sprays to demonstrate that the spraying process has no effect on cell number,distribution,and activity;isolated and cultured human's keratinocytes cells had no effect on the related functions such as proliferation,migration after spraying.After long-term culture in vitro,the function of cells is not much different from normal inoculation.The formation of vascularization in vitro of HUVECs cells and spheronization function of HaCaT cell were maintained after spraying;After the three dimensions cultured of MSCs were sprayed,the morphology of the spraying cells still maintained in a short spindle shape with normal expression;the above experimental results proved that the spraying from another aspect.The process has no effect on the performance of the cell.The results of Live/Dead staining and immunofluorescence showed that the spray device can achieve atomization and even distribution of droplets;the cells in the droplets after spraying have high activity,and the same atomization effect is uniform distributed.After resuspension of different cell types with different concentrations of extracellular matrix and loading for the spray,the results showed that the cells did not clump together and they could quickly adhere and stretch.2.Isolation and culture of epidermal stem cells as a reserve of seed cellsWe have explored and innovated a rapid,mild enzyme combination of digestion methods to obtain highly active cells.Next,histological fluorescent staining of the isolated cells was confirmed as epidermal stem cells.A three-dimensional culture system with no fibroblasts and no serum was established for epidermal stem cells.The results showed that the doubling time within 6th generations of in vitro expansion was stably maintained at 52 hours,and a sufficient number of epidermises were obtained for transplantation into clinical cells at a later stage.In this manner,the epidermal stem cells expanded under culture conditions are in very good state.Through transmission electron microscopy,secretory vesicles can be clearly seen,and a large number of desmosomes unique of the epidermis are formed.Histological examination revealed that the cell spheres highly expressed the regeneration-associated protein K14,proliferating proteins P63,Ki67,and epithelial cells connected to E-cad and Claudinl.The long-term culture and identification of epidermal stem cells in vitro revealed that the cells could form new epidermal in vitro after spraying.After identification,the cell of the lowest layer showed high expression of the regeneration marker proteins ITGA6 and K14,indicating that the sprayed cells were implementation of proliferation and differentiation just like normal skin expression.A full-thickness skin wound model was established on the back of NOD/SCID fully immunodeficient mice.After injury,epidermal stem cells were loaded on the skin spray gun and sprayed on the wound surface.After transplantation,the wound was observed at the wound site.In vivo transplantation experiments of NOD/SCID mice demonstrated that the isolated and cultured human epidermal stem cells sprayed wounds on the 7th day and began to recover rapidly compared with the control group,demonstrating that the transplanted cells have high activity and have a long survival time in vitro.Secretion stimulates the wound to accelerate healing.3.Decellularized Dermal Matrix Facilitate Human Keratinocytes and Fibroblasts-Mediated Wound RepairThe obtained decellular skin was lyophilized and stored at-80 ?.After different frozen grinding process,the ECMP was obtained.And 60Co irradiation was used to get a sterile ECMP.Images of ECMP were taken for calculating their size distribution to finally determine the suitable frozen grinding program.To determine the appropriate range of ECMP concentration,the cell proliferation was measured after co-culture of cells with ECMP at a series of concentrations;The migration-promoting effect of ECMP to fibroblasts were detected by the migration speed of cells in Transwell chamber.The proliferation of primary human epidermal keratinocytes(hKSCs)was further studied.The expression of immature and differentiated markers of hKSCs co-cultured with ECMP was finally detected by immunofluorescence.The above data show that the spraying device used in this systerm has made a thorough and detailed study on the biological function of cells after spraying,and verified that not only the cell viability but also the proliferation and differentiation of cells in vitro during the spraying process have no effect.Ensuring the successful established of cell transplantation system.The identification of the biological characteristics of the epidermal stem cells(hKSCs)obtained showed that the cells have the same amplification potential in vitro and lay the foundation for the scale-up amplification.At the same time,medium with serum-free and feeder-free were achieved;clinical grade Knockout SR was used as a serum substitute to stably obtain functionally competent cells.In wound healing,extracellular matrix can maximize the retention of various nutrient factors secreted during cell culture as cells proliferate and migrate,and accelerate the epidermalization of wounds.After the establishment of this project,it was found that the obtained ECMP particle size was less than 15?m under the 7 cycles of the cryogenic grinding procedure,which can meet the requirements of the application.ECMP coating at the culture plate with 500 ?g/cm2,which can significantly promote fibroblasts.Migrate and promote epidermal cell proliferation and maintain its dryness. |
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