Study On The Effects Of Micro Negative Pressure On The Biological Behavior And Signaling Pathway Of Wound Repair Cells

BackgroundWith the rapid development of molecular biology,material science and other disciplines,a large number of new ideas and methods on wound management have emerged,among which the emergence of Negative Pressure wound Therapy（NPWT）—a technique that is widely used clinically—has greatly improved the speed and quality of wound healing,bringing about revolutionary changes to the field of wound care.In clinical practice,we have found that the effects of different value ranges of negative pressure on wound vary according to phases of healing.Traditionally,pressure of-80mm Hg to-120mm Hg has a stronger promotional effect on granulation tissue growth than epithelialization,while minor pressure such as-1 mm Hg to-5mm Hg,tends to act inversely,which can accelerate the epithelialization process under the premise that granulation hyperplasia is not obvious.In recent years,domestic scholars have found that the rate of wound re-epithelialization can be significantly accelerated by using Micro Negative Pressure（MNP）therapy,which has been confirmed both in clinical work and in animal experiments.But the specific mechanism has not yet been explained.Previous studies have demonstrated that there is mechanical force on negative pressure-applied wound surface,and the local mechanical microenvironment changes can regulate cellular behavior through activation of different signal transduction pathways.In the field of wound repair,clinicians and researchers have gradually focused more on the attempt to improve wound healing by regulating physical signals such as electricity and force.Therefore,we intend to explore the effects of MNP environment on the biological activities of repair cells in the process of wound healing,so as to reveal the cytological mechanism of MNP expediting wound healing and the possible signal transduction pathway involved.ObjectiveThis study aims to explore the cytological mechanism of wound epithelialization promoted by MNP via observing the impacts of MNP on the proliferation and migration activities of the repair cells during the process of wound healing.In addition,by examining intracellular molecular changes caused by MNP,we intend to further study the signal transduction pathway involved,hoping to establish experimental basis for revealing the mechanism of wound healing in depth,and if possible,to provide new insights into clinical optimization of wound management.PartⅠEstablishment of MNP environment—Design of the cell culture system under MNPMethodsBy reference to the operating principle of the pressure regulating chamber of water-sealed bottle used in closed thoracic drainage system,we made some modifications to the existing laboratory and clinical equipment and materials,transforming them into a new“cell culture system under specific MNP”,with CO₂ cell incubator,cell culture apparatus,pressure trimmer and negative pressure pump included.Specifically,a liquid column with adjustable liquid level—pressure regulating chamber—was arranged between the negative pressure pump and the cell culture apparatus,which could buffer the negative pressure initially generated by the pump,so that the pressure under which cells were incubated did not exceed that maintained by the height of the liquid column.ResultsThe MNP environment for cell culture was successfully simulated by the modified device,which kept pressure of-5cm H₂O to-10cm H₂O.It can be used for cultivating various cells under this condition,which provides the most basic support for cell models incubated in MNP environment needed in the subsequent experiments.PartⅡDetection of the effects of MNP on cellular biological behavior related to wound repairMethodsThe cell proliferation and migration of HFBs and HaCaTs incubated under MNP and normal pressure were detected by using Alamar Blue staining and scratch test at different time nodes,respectively.Results1.There was no apparent discrepancy in the proliferation rates of cells between the MNP group and the control group at the 1st,3rd,5th and 7th day of incubation,neither in HFBs nor in HaCaTs.2.In HFBs,there was no significant difference in cell migration rates at the 12th hour between the MNP group and the control group,but a clear division emerged at the24th hour.In HaCaTs,obvious differences in cell migration rates were noticed both at the12th and 24th hour between the two groups.PartⅢStudy on the signaling pathway of MNP affecting cell migration related to wound repairMethodsThe PEX100 microarray was used to screen the differential proteins in the cells incubated in MNP and normal pressure environment,and the proteins related to cell migration pathway were identified by bioinformatic analysis and Western Blot.Then relevant literature was reviewed to predict a migration signaling pathway as well as its upstream and downstream molecules,and the target pathway was further verified site-by-site with immunofluorescence or Western Blot.Results1.Compared with control group,Akt and Rho were increased in HFBs and HaCaTs incubated under MNP,and results from bioinformatic analysis indicated that the migration signaling pathway was Akt-related.2.The Ca²⁺in HFBs and HaCaTs increased with time in the MNP group,while microfilament showed a trend of first rising and then declining,but these two components in cells of the control group did not change significantly.3.After addition of mechanical calcium channel inhibitor,the Ca²⁺in HFBs and HaCaTs of the MNP group decreased with time,and the previous dynamic change of the microfilament weakened.In the control group,the Ca²⁺increased significantly at first and then slightly decreased,presenting an overall increasing trend,while the microfilament had no obvious change.ConclusionIn this study,we utilized a modified device to incubate cells under the mimetic micro negative pressure environment（-5～-10cm H₂O）,and explored the specific mechanism of MNP promoting wound healing for the first time.It was confirmed that MNP could promote the migration of HFBs and HaCaTs in the process of wound repair,but had no significant effect on the proliferation of these cells.Further,we preliminarily defined that MNP could work through the"MNP—Ca²⁺—Akt-Rho—cytoskeleton remodeling"signaling pathway to promote cell migration and accelerate epithelialization,thereby speeding up wound healing.We believe that our study fills a gap in the current understanding of the cellular regulatory mechanism of MNP in the process of wound healing and helps to provide new targets for seeking better wound treatment in the future.