| BackgroundThe mechanism by which negative pressure wound therapy(NPWT)promotes wound healing has not been clearly elucidated.Recently,researchers have found that cells are not only regulated by the spatial-temporal changes of biochemical signal,but also respond to the spatial-temporal changes of mechanical environment such as the structure,stiffness and 3D strain field of the extracellular matrix,activating different signal transduction pathways and changing cell behavior.In the field of tissue regeneration and wound repair,the importance of the regulation of mechanical environment has received increasing attention.Under NPWT,it remains elusive how the mechanical microenvironment(e.g.,stiffness,strain gradients)changes both in time and space during wound healing.Previous studies have shown that regulation of mechanical environment will promote YAP/TAZ nucleus localization,which plays a vital role in cell migration and proliferation.However,whether NPWT can promote the migration and proliferation of wound repair cells by regulating YAP/TAZ signal through changing the mechanical microenvironment to promote wound healing still needs further study.ObjectiveFor this,the mechanism of tissue stiffness and 3D strain promoting wound healing through YAP/TAZ signal was firstly investigated by observing the changes of wound mechanical microenvironment in the treatment of NPWT and further studied by constructing a 3D cellular mechanical microenvironment simulating NPWT in vitro.Therefore,a systematic method of wound biomechanic is developed to provide a new vision for relevant studies and a theoretical support for the optimization of NPWT dressings and individualized treatment.Methods Part I Temporal and spatial changes of the skin mechanical microenvironment of the rabbit dorsal full-thickness wound in the treatment of NPWT1.(1)Twenty-four New Zealand white rabbits were randomly divided into NPWT group(-50,-80,-125,-150 mmHg)and control group(N=6 per group).The wound healing was observed and compared everyday.(2)Two rabbits were randomly selected,and 3D MR reconstruction was performed on 1 and 5 days after the surgery,respectively,to build finite element numerical models,and shear wave elastography(SWE)was performed to measure the skin stiffness of the dorsal wounds of each group at the same time.(3)After the above experimental parameters were fitted by using ABQUES,boundary conditions were set and the above negative pressure loads were applied.The 3D stress-strain distribution of 0,5 and 10 mm ROI from the edge and the bottom of the wound was analyzed by the nonlinear hyperelastic finite element method on days 1 and 5 after treatmeat.SPSS 19.0 was used for statistical analysis of the data.(4)Assemble the 3D digital speckle strain detection system which was based on Digital Image Correlation(DIC).(5)Spray speckle.(6)Data collection and statistical analysis.Collect the data of the equivalent strain under the NPWT(-50,-80,-125 and-150 mmHg)dynamic loading on days 1 and 5 after treatmeat,respectively.The data with the experimental one was compared with the strain data of FEA,the value was represented as mean and standard deviation,P values were determined by one-way analysis of variance with Sidak's multiple comparisons test.Significance was defined as *P<0.05,**P<0.01,***P<0.001,****P<0.0001.Part ? The regulation of YAP/TAZ for promoting wound healing of rabbit dorsal full-thickness wound in the treatment of NPWT2.(1)The sponge dressing was cut into 1×2cm strips and sutured on the wound surface.Then on days 1 and 5 after treatmeat,the wound edge and the underlying tissue connected with the sponge dressing were cut,fixed with formalin and embedded with paraffin before HE staining,and histological observation was conducted under the microscope.(2)On days 1 and 5 after treatmeat,the wound edge and the underlying tissue of the sponge dressing were cut,fixed with formalin and embedded with paraffin,followed by Masson staining for histological observation under the microscope.(3)The localization and expression of YAP,TAZ,NMosyin IIB and PCNA were detected by immunofluorescence staining.(4)Nuclear localization and expression of YAP and TAZ in each group were detected by Western blotting.(5)The expressions of YAP and TAZ mRNA in each group were detected by quantitative PCR.Part ? In-vitro construction of 3D cell mechanical environment under different circumstances of NPWT3.(1)Parameter fitting of hyperelastic material of polytetrafluoroethylene(PTFE)elastic membrane.(2)Preparation of finite element geometric and physical model of PTFE elastic membrane.(3)Strain and displacement field were analyzed by FEA and a customized spherical gradient strain cell automatic stretching device was assembled according to the data.(4)Preparation of gelatin-?-CD composite 3D hydrogels.(5)SWE was applied to measure the stiffness of hydrogels.Results1.(1)Compared with the control group,wound healing time in the-50,-80 and-125 mmHg NPWT groups was shortened(P<0.05),while there was no significant difference in the-150 mmHg group(P>0.05).(2)SWE showed that the stiffness of the young's modulus(tissue stiffness)of the wound skin in the-50,-80,-125,-150 mmHg group and the control group were 9.5± 1.4,9.8± 2.1,10.5± 2.3,11.5± 2.3 and 21.8± 3.3 kPa on day 1 after treatment,and 21± 3.1,22± 2.3,22.5± 2.4,23.1± 2.2 and 31.7± 2.6 kPa on day 5,respectively.There was no statistical difference between the NPWT negative pressure setting groups(P>0.05),and the stiffness of wound skin in each group was higher on day 5 than that on day 1(****P<0.0001),while the control group was higher than the average value of the NPWT group on days 1 and 5,the difference was statistically significant.(****P<0.0001).(3)The 3D strain of the skin at the edge decreased during wound healing.(4)The 3D strain gradient of the skin at the wound edge formed and decreased during wound healing.(5)The 3D digital speckle strain analysis showed that NPWT with-50 to-150 mmHg setting generated an outwardly decreasing strain gradient in the horizontal direction of wound.There is no statistically significant difference between the horizontal strain of DIC and the strain obtained from FEA under the loading of-50,-80,-125 and 150 mmHg on days 1 and 5(P>0.05).2.(1)NPWT with-50,-80 and-125 mmHg setting promoted the formation of granulation tissue and the synthesis of collagen in the rabbit dorsal wound.(2)Immunofluorescence showed that PCNA and NM IIB were expressed at the contact sites of wound surface and sponge dressing,and the expression of PCNA was increased in the-50,-80 and-125 mmHg groups on day 5.(2)WB showed that the nuclear expression of YAP/TAZ of-50,-80 and-125 mmHg groups increased on day 5 compared with the control group and the-150 mmHg group,and the difference was statistically significant(P < 0.01).(3)PCR showed that the expression of YAP/TAZ mRNA in the-50,-80 and-125 mmHg groups was higher on day 1 than that in the-150 mmHg group and was higher on day 5 than that in the control group and the-150 mmHg group,with statistically significant difference(P<0.0001).3.(1)A non-uniform strain gradient tension device based on elastic culture plate was established.Strain gradient can be accurately adjusted.(2)Construct a 3D hydrogel culture environment simulating wound stiffness.Conclusion1.The spatial-temporal changes of mechanical environment in wounded skin,including tissue stiffness and 3D strain field with gradients under NPWT,was firstly expounded by combining MR 3D imaging with SWE,nonlinear FEA and digital image correlation.During wound healing,the stiffness of the wounded skin increased,which contributed to cell migration and accelerated wound healing.The strain and its spatial gradient decreased during wound healing under NPWT,which should be considered but is often overlooked in NPWT.2.NPWT within proper pressure range may activate YAP/TAZ nuclear localization by controlling the appropriate strain gradient field,whicn may be benefit to the proliferation,migration and other behavioral of repair cells,promoting granulation tissue formation and vascularization.3.The 3D cellular mechanical microenvironment in different circumstances of NPWT with adjustable accuracy was built for the first time. |
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