| Skin is an important body protection organ which blocking the invasion of pathogens and preventing body fluids loss,regulating body temperature and other functions.Once damaged,the ideal situation is to be repaired and reduce the scar as soon as possible.Extracellular matrix(ECM)plays a very important role in the healing process of skin wounds,and it has a large number of nanometer-scale fibrous structural components,which can induce cell behavior,including reproduction,migration and so on.While dermal fibroblasts and epidermal keratinocytes are the main functional cells in wound healing.Once the skin is injured,fibroblasts migrate to the wound and contract to promote healing,at the same time keratinocytes migrate to the wound and proliferate to quickly cover the injured tissue.The abnormal behavior of these two cells will cause delayed wound healing and form an abnormal scar.Therefore,it is crucial to search the ways of accelerating wound healing while avoiding scar formation,as well as a method to monitor this process.This will provide a reference for clinical studies of skin wounds healing.In recent years,researchers have found that nano-scaled morphology could simulate the body ECM,affecting a series of cell behavior--"contact guidance",enabling cells align and elongate along the nano-grooves direction.In addition,nano morphology can control and induce cell migration and proliferation of the direction and speed,playing an important role in wound healing process.More and more researchers have focused on the study of cellular behavior on various three-dimensional structures.However,they are usually performed using fluorescence microscopy,scanning electron microscopy(SEM)and atomic force microscopy(AFM).These methods involve complex steps that are destructive to cells.More importantly,these methods rely on endpoint detection,without providing dynamic information.In order to overcome these limitations,ECIS(Electric Cell-substrate impedance sensing)has been developed and is being applied to the monitoring of cell behaviors.It is a real-time,non-destructive and quantitative detection method.Giaever and Keese cultured cells in a small area of the gold electrode,with the cell adhesion and spreading,the current is physically blocked,resulting in increased impedance.Subsequently,ECIS is applied more widely,including cell adhesion,cell proliferation,cell migration,cytotoxicity,drug screening and so on.In this paper,two kinds of human skin cells were cultured on the nano-grooves substrate.The proliferation and migration of the cells were monitored by ECIS.The impedance signals were analyzed with the assistance of microscope observation,and the correlation between them was established.Chapter One:IntroductionThis chapter mainly introduces the related contents of human skin fibroblasts and human skin keratinocytes,and then introduces the domestic and foreign researches on the process of skin wound repairing,including cell proliferation and cell migration on two-dimensional and three-dimensional substrates.This paper mainly introduces the ECIS method,including its principle,application on cell behavior,drug screening and so on.This chapter illustrates the advantage of using ECIS method to monitor the healing process of skin wound,and finally puts forward the research purpose and significance of this paper and the innovation.Chapter Two:Design of nano-grooves structure capable of inducing directional behavior of skin cellsIn the healing of skin wounds,the directional behavior of cells plays a very important role in accelerating wound healing.In this chapter,nano-grooves with a line width of 200 nm;a period of 400 nm and a depth of 75 nm were prepared by nanoimprinting on the gold substrate to simulate the extracellular matrix.Human skin fibroblasts(HFF)and human skin keratinocytes(HaCaT)were cultured on nano-grooves.Cell photographs were collected by ordinary optical microscopy.The cell elongation and spreading direction were analyzed statistically.The results show that the HFF cells have good elongation on the nano-grooves and can spread along the direction of the nano-grooves,and the morphology of the HaCaT cells does not change significantly,and the spreading direction is also irregular,indicating that the prepared nano-grooves can induce the orientation of fibroblasts.Chapter three:ECIS method to monitor the migration of skin cells on nano-groovesIn this chapter,an ECIS device based on nano-grooves was constructed to monitor the changes of AC impedance signals during the migration of two cells on the nano-grooves.The impedance of HFF cells and HaCaT cells showed an increasing trend,while the impedance of the HFF cells on the nano-grooves is greater than that of the flat electrodes due to their different sizes of wounds on the nano-grooves and flat electrodes,whereas the HaCaT cells are larger on the flat electrodes.The changes in wound size were observed by microscopy,and it was found that the wound healing of the two cells on the nano-grooves was faster than that of the flat electrode.After the AC impedance response is associated with the wound healing rate,it is found that there is a good linear relationship between them,and when heal the same pencentage of wound,the impedance of the HFF cells obtained on nano-grooves is smaller than the flat,and the HaCaT cells are similar in the two electrodes.Chapter four:ECIS method to monitor the proliferation of skin cells on nano-groovesIn this chapter,human skin fibroblasts and human skin keratinocytes were cultured on nano-grooves and the proliferation process of these two cells was monitored by ECIS.The results showed that the impedance of the two cells on nano-grooves in the proliferation increased with time,and was decreased compared with that of the flat electrode.The number of cells in the nano-grooves was smaller compared with flat electrode.HFF cells show good orientation behavior on the nano-grooves.On the other hand,the reduction of their proliferation has a positive effect on reducing the formation of scars.After the changes in impedance response of the proliferation process are related to the number of cells,and there is a certain linear relationship between them.The same number of cells in the nano-groove caused smaller impedance than flat electrode.The study of this paper will provide data and technical support for the clinical study of human skin wound healing,and it will promote the development of regenerative medicine,plastic surgery and other fields. |
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