| ObjectiveThe skin is the largest organ in the body,covering the entire body surface and making up about 16%of the body's mass.The skin is composed of epidermis,dermis,subcutaneous tissue and accessories,which mainly protect the body against the invasion of external harmful substances,thermoregulation and so on.However,as an organ in close contact with the surrounding environment,the skin is prone to burn injury in daily life.Skin has lost its original tissue structure and function after large area deep burn,the stem cells and tissues with repair potential in the skin have been severely damaged,and the potential self-repair ability of the skin has also been weakened or lost.At present,there are still many problems in the treatment of large area deep burn wounds,such as insufficient sources of available auto-skin grafts,the risk of immune rejection and infectious diseases caused by foreign or allogeneic skin,the inability to be permanently replaced,the high price and the impact on appearance and function of scar contracture after wound healing.Clinical data have confirmed that the use of scab ablative surgery and other methods to remove the necrotic layer of burns and retain the denaturing dermis after burn is conducive to promoting burn wound healing,and the quality of wound healing is closer to normal skin,but the healing mechanism is complex,which is still not fully explained at present.In deep ?° or mixed ?° burns,part of the dermis has not fully necrosis,only part cell dysfunction,and morphological structure changes,but when after the local microenvironment is improved,the above phenomenon will improve or disappear,damaged dermis can reverse back to normal morphology and function,the research results show that the modified after burns under certain conditions has returned to normal dermis leather structure morphology and function of potential.In clinical practice,scab cutting will often remove the denatured dermis and completely necrotic tissue together for infection control,which will not only cause waste of denatured dermis that may be reversed,but also often lead to obvious scar hyperplasia after wound healing,resulting in the appearance and functional dysfunction of burn patients.Previous studies have confirmed that the biological properties of the modified burned dermal matrix treated with acellular technology are satisfactory,and it may be a potential substitute material for burned dermis.There are still a lot of unknowns that need to be clarified with further research.The prepare timing influence of dermal sampling,how to better treat the burned denatured dermal matrix to optimize its physical properties,improve the effect of transplantation and make clear the cytokines in the denatured dermal matrix prepared at different periods after treatment are the questions concerned.Series based on the early burn denatured dermis matrix research,this study intends to cut early callus abandoned burns degeneration leather recycling,application of combined to take off the 0.25%trypsin,0.5%Triton-100 cell processing,Laser micropore technology to deal with,preparation of cell-free burn into shock microporous degeneration dermal matrix(Laser micropore denatured acellular dermal matrix,LPDADM),and to biological performance testing and in vivo LPDADM preparetions,combined with autologous blade thickness skin transplantation,The LPDADM protein factor was detected by using protein chip technology,so as to evaluate and discuss the biological properties and transplantation effect,sampling opportunity and repair mechanism after transplantation.This study is the improvement and deepening of a series of studies on modified dermal matrix of burns,and the research results will provide the theoretical basis for the further basic research,clinical application and tissue engineering of modified dermal matrix.Methods1.Preparation of LPDADM and performance testSPF kunming male mice for deep ?° burns animal model.Thermostatic water bath scald method was used for modeling.Set time points after injury put to death the experimental mice,take the burned area under the skin,remove dead skin completely trimmed and necrotic tissue,cut out part of the organizational line histopathological examination,keep in line with the deep ?° burn samples.After treatment with 0.25%trypsin and 0.5%Triton x-100 for 15min,the liquid was titrated with PBS until it was completely clear.The processing of dermal matrix can be divided into two groups at random,either group[marked as experimental group,another group as control group(DADM,Denatured acellular dermal matrix)],the experimental group templates with specific laser punching technology,punch on decellularized modified dermal matrix,to 1.0mm pore clearance design,aperture size is 135 ?m,the preparation into LPDADM.The color,texture,elastic toughness and ductility of DADM and LPDADM were observed and compared.HE staining and optical microscope were used to observe the cell morphology and fiber arrangement,and then the corresponding physical properties were tested.2.Study the protein factors expressions of LPDADM were in prepared different time point using protein chip techniqueSamples were collected at 24,48,and 72 hours after burn without infection in experimental animals and at the time of confirmed burn wound infection.After the prepared LPDADM,cytokine protein chip detection was carried out to study the differences in the expression of cytokines in LPDADM prepared by sampling at different periods.3.Experimental study on subcutaneous implantation of LPDADMThe histocompatibility and immune rejection of two groups of modified dermal matrix in vivo were observed by subcutaneous implantation.Two subcutaneous sacs were taken on both sides of the back of mice.One LPDADM was implanted in the one side capsule of the experimental group,and one DADM was implanted in the other side capsule of the control group.The two implanted modified dermal matrix did not touch each other.Observation indexes:(1)the appearance,texture,elasticity,fiber coating and surface capillary growth of dermal matrix were observed in the first,second,third and fourth weeks after implantation.(2)inflammatory cell infiltration,collagen arrangement,cell infiltration and vascular implantation of the two groups of denaturated dermal matrix were observed under light microscope at week 1,2,3 and 4 after implantation.4.Study on the effect of LPDADM combined with autologous blade thickness skin transpiantationThis part consists of animal experiment and clinical experiment.Animal parts:mice be divided into two groups,after anesthesia,in the back cut out a piece of skin 1×2 cm2 size and transplanted LPDADM,DADM and allograft respectively in two groups of animal skin defect area,and then cut out the skin to eliminate the skin cuticle covers stitching on the dermal substitute materials and packing pressure,respectively,in 10 days,14 days and 21 days after operation to observe wound in general situation,histology,cell morphology and contrast observed under electron microscope to observe the wound healing rate.Clinical experimental part:Adult patients admitted to our department who needed autologous skin repair for deep burns were randomly treated with allogeneic acellular dermal matrix LPDADM,HADM combined with autologous blade thickness skin transplantation and autologous blade thickness skin transplantation for deep burn wounds.The general situation of the wound was observed regularly after the operation and the wound healing rate was compared with the results of major laboratory tests.To judge the application effect of LPDADMResults1.Gross specimens and microscopic observationTo the naked eye,the newly prepared DADM is porcelain white,soft in texture,with certain elasticity and toughness.LPDADM presents neat and dense pores,without carbonization.No epithelial structure and cell survival was observed,dermal collagen hyaline degeneration was observed in DADM and LPDADM,but slight collagen fiber breakage was observed locally in LPDADM2.Biomechanical properties24h group,the elastic modulus(MPa),maximum load(N)and tensile length(cm)of LPDADM were 71.60±2.30;118.00±3.54;2.48±0.23,respectively.48h group,the results were 70.20±2.85;107.30±6.15;2.52±0.18;72h group the results were 71.80±2.40;123.55±4.75;2.33±0.31;The elastic modulus,maximum load and tensile length of LPDADM obtained from wound infection samples were 67.40±3.53;101.00±3.22;2.10±0.27,respectively.there was no significant difference in each group of indicators(P>0.05),but DADM[elastic modulus(MPa)3 5.80±2.17,maximum load(N)67.40±3.51,tensile length(cm)2.66±0.05]without laser microporous treatment showed significant change,the difference was statistically significant(P<0.05).3.The protein factors expressions result of LPDADM were prepared in different time periods3.1 The protein component analysis of LPDADM showed that LPDADM protein factors were expressed differently at different time periods(24h,48h,72h and when the wound was infected).With the time delay,the expression of most protein factors showed a gradually increasing trend,such as IL-15,CCL-8,GM-CSF,MMP-3,etc.3.2 LPDADM obtained from infected wounds showed increased expression of all protein factors except MMP-24/MT5-of MMP,VE-cadherin,TCK-1,IL6 and VEGF.4.Results of LPDADM in vivo studyThe implanting in the animal body showed that the two groups of modified dermal matrix did not shrink or curl after implanting,nor did obvious exudation,redness and other phenomena occur.At 2 weeks in LPDADM group,a layer of fiber film with fine vascular network distributed on the surface of implanting modified dermal matrix could be seen.HE results showed that there were differences in cell infiltration and capillary implantation in the LPDADM group,and the degree of vascular implantation and fiber fusion in the experimental group was more obvious,but no inflammatory cell infiltration was observed in either group.The results of LPDADM combined with autologous thick-edge skin animal study showed that the skin in the LPDADM group was fleshy red on the 10th day after the operation,and the skin was basically alive,while the local dry character was scattered in black and the local color was darker in the control group.There was a statistically significant difference in wound healing rate 14 days after surgery(P<0.05).Scar hyperplasia was more obvious in DADM group.HE staining showed more obvious vascularization of LPDADM and mild inflammatory response.The DADM group had more inflammatory cells and a lower degree of vascularization.The results of electron microscope showed that the new tissue in DADM group was difficult to grow,few fibroblasts were transplanted,and inflammatory cells were the main ones.In the LPDADM group,a small number of inflammatory cells were transplanted,and the transplanted fibroblasts showed irregular morphology of large nuclei under electron microscope,and the rough endoplasmic reticulum was full of cell fluid.The results of clinical test of LPDADM combined with autologous blade thickness skin showed that,compared with the HADM group,the healing wound surface of the LPDADM group was slightly larger.In the LPDADM group,the softness(1.18±0.34 points),thickness(1.24±0.47 points)and wound healing time(28.05±5.23 days)were significantly different from the statistical results of their corresponding indicators,and the test results showed statistical significance(P<0.05).There were no significant differences in blood routine,biochemistry,cellular immunity and cytokines after allodermal replacement material transplantation in 2 groups(P>0.05).Conclusion1.The elastic modulus,maximum load and tensile length of denaturated acellular dermal matrix after treatment with laser micropore technology were increased;Has good physical performance.2.The protein component analysis showed that the protein factor expression was different in LPDADM at different time points.The protein factors that are beneficial to wound healing in the denaturated acellular dennal matrix of laser microporous prepared at the early stage and without wound infection are higher than those prepared at the time of wound infection,which may be more suitable for transplantation.3.After implantation of burned denaturated acellular dermal matrix treated with laser micropore technology,it was found that it had good biocompatibility and low immune rejection reaction,which was more conducive to receptor cell infiltration,inflammation disappeared and capillary implantation than burned denaturated acellular dermal matrix.4.LPDADM combined with autologous blade thickness skin transplantation can safety promote wound healing,improve the quality of wound healing. |
PDF Full Text Request