Study On Age-Related Changes Of Skin Structure And Wound Healing Ability And Its Mechanisms In Rats

ObjectiveStudy on rats of different ages:(1)Age-related changes of skin structure in rats;(2)Age-related changes of wound healing ability and their mechanism in rats,to elaborate the mechanism of delayed wound healing in elderly patients,and provide a theoretical basis and scheme for targeted treatment.(3)Developing hydrogel material coated with EGF,and the age-related mechanism of EGF on wound healing was explored through sustained release of EGF;(4)A simple and stable scald model with different depths in rats of different ages was established to study the age-related mechanism of scald healing.Methods1.Study on age-related changes of skin structure in rats.Juvenile(1 month old),young(2 months old),middle-aged(12 months old)and aged(22 months old)rats were selected.Skin biopsy tissues with a diameter of 8 mm were taken from the thoracic and dorsal regions of the rats.Biopsy skin tissues were fixed with 4%paraformaldehyde or cryopreserved at-80 ~oC.(1)HE staining of rats of different ages;(2)Immunofluorescence(IF):CD271 and EGFR;(3)Western blotting(WB)staining:CD271 EGFR/JAK2/STAT3/SOCs3.2.Study on age-related decrease of wound healing ability and its mechanism in ratsJuvenile(1 month old),young(2 months old),middle-aged(12 months old)and aged(22 months old)rats were selected to prepare two 1 cm longitudinal full-thickness skin tangential wound models on the thorax and back of each rat.The wound healing was observed on days 0,3,5,7 and 9.The biopsy tissues were taken on days 0,3 and 9 after injury and fixed with 4%paraformaldehyde or cryopreserved at-80 ~oC.Fixed samples were used to analyze the expression of CD271 and EGFR in pathological sections by IF staining.The frozen tissue proteins were extracted for WB detection of JAK/STAT/SOCs signaling pathway expression changes.(1)1 cm longitudinal full-thickness skin tangential injury model preparation;(2)HE staining of the wound on day 3;(3)Immunofluorescence(IF):CD271 and EGFR;(4)WB detection:EGFR,JAK2,STAT3,SOCs3.3.Study on development of slow-release EGF hydrogel and its effect and mechanism on wound healing.Sodium carboxymethyl cellulose(CMC)and gelatin(GL)were used to develop a polysaccharide hydrogel with water locking and slow release of EGF.The polymerization of hydrogel was coated with RhEGF.Four full-thickness skin defects of with a 8 mm diameter were prepared on the middle and back of juvenile(1 month old),young(2 months old),middle-aged(12 months old)and old(22 months old)rats.The wounds were treated with NS,EGF,CMC-GL and CMC-GL-GEF respectively.The wound healing was observed on days days 1,3,7,10,13 and 16.The biopsy tissues were fixed with 4%paraformaldehyde or cryopreserved at-80 ~oC.Fixed samples were used for histopathological analysis.The frozen tissue protein was extracted for WB.(1)Developing sustained-release EGF hydrogel(CMC-GL-EGF);(2)Hydrogel Electron Microscopy(SEM),Rheological and EGF Release Tests;(3)Biosafety testing of hydrogels;(4)The preparation and treatment of trauma model;(5)Histopathological analysis of HE and Masson;(6)IHC:collagen II and III,anti-factor VIII staining;(7)WB detection:EGF,bFGE,TGF-beta 1,vEGF.4.Developing a simple burn model of different depths in rats of different agesJuvenile(1 month old),young(2 months old),middle-aged(12 months old)and old(22 months old)rats were taken and scalded on their back.A self-made stainless steel metal column with good thermal conductivity was used as a scald module,which was heated in boiling water and applied on rat skin for different time to create scalds with different depths.(1)Under the condition of constant temperature and pressure,the condition of the scald models in rats of different ages with 1 cm in diameter:4weeks,depth II:3 s,III:5 s;8 weeks,superficial II:3 s,deep II:5 s,III:7 s,9 s;12 and 22 months,superficial II:3 s,5 s,deep II:7 s,9 s,III:11 s,13 s.(2)Under constant temperature and pressure,a metal column with a diameter of 1 cm,1.5 cm and height of 2 cm was used.After heated in boiling water bath,the rats were scalded for 5 seconds.The scald depth of the model was the same,and all of them were deep second degree scalds.Results1.Age-related changes of skin structure in rats(1)The histological structure of the dorsal skin of normal rats was arranged orderly.The thickness of the whole skin layer gradually increased with age before the middle age(12 months old),and then decreased after the middle age.The thickness of the epidermis showed a trend of age-related thinning.(2)The number of CD271+cells in skin epidermis,hair follicles and sebaceous glands of rats did not change(One-Way ANOVA,p>0.05),the expression of EGFR membrane protein decreased with age(One-Way ANOVA,p<0.05).(3)WB test showed that there was no significant difference in CD271expression;EGFR expression decreased with age;SOCs3 expression increased with age,and JAK2 expression decreased with age.STAT3expression in young rats was significantly higher than that in other groups.2.Decrease of wound healing ability and its age-related mechanism in rats(1)The wound healing ability of the aged rats was significantly reduced.The young rats healed by day 5,the young group and the middle-aged group healed by day 7,but the middle-aged group healed poorly,and the wound still showed clear marks.The aged group healed by day 9.(2)Histopathological analysis showed that the neoepithelial thickness of wound on days 3 decreased with age,and there was a significant difference(One-Way ANOVA,p<0.05).The healing ability of skin thickness on wound on day 7 decreased gradually to normal,and the thickness of skin in juvenile rats was still greater than that in other groups.(3)IF test showed that there was no significant difference in the distribution of CD271~+cells in the skin of aged rats after trauma detection(One-Way ANOVA,p>0.05).The expression of EGFR was significantly activated three days after trauma as evident with the proliferation of cells expressing EGFR,the thickening of new epithelium,the decrease of thickness of new epithelium in age-related manner,and the decrease of age-related degree in normal tissue.The expression of EGFR in intDen/Area after trauma was similar to that in normal skin of young rats(One-Way ANOVA,P<0.05).(4)WB test showed that the expression of SOCs3 increased in wounds on day 3 after trauma,which showed age-related trends.There was no significant difference in STAT3 expression.Delayed wound healing occurred in aged rats;JAK2/STAT3 signaling pathway was activated during wound healing,and endogenous cytokine signaling inhibitors(SOCs)were down-regulated,but the expression increased with age.3.Development of controlled-release EGF hydrogel and its effect on wound healing and its mechanism(1)With the increase of GL concentration,hydrogels are more and more difficult to spread on the surface.When CMC concentration was 50 mg/ml and GL concentration was 25 mg/mL,G'was greater than G'in the range of angular velocity 1-100 rap/min,and the curve showed a steady upward trend.CMC-GL crosslinking was stable in gel state in the system,and had a soft smearing performance on the wound surface.(2)Because of its three-dimensional network structure and excellent biocompatibility,EGF hydrogels can encapsulate proteins or cells to ensure their physiological activity.SEM observation showed that CMC-GL-EGF hydrogel has a large number of irregular holes and three-dimensional network structure.The rheological test showed that in the range of angular velocity,G'was greater than G',and the average rate was larger than that of gel CMC-GL-2.It was proved that the addition of EGF will not destroy the cross-linking structure inside the gel,and it may be because the negatively charged EGF dispersed in the interconnected channel of CMC-GL-2 and the negatively charged gelatin had weak electrostatic interaction and hydrogen bonding effect to increase the crosslinking density of the whole system,so that the CMC-GL-EGF system had better rheological properties than CMC-GL-2gel.EGF encapsulated inside the gel could effectively ensure its protein activity and slow and orderly release.In vitro simulated body fluids(PBS),EGF in hydrogels could be released slowly,and the absorbance value(OD 280=?)could be measured according to the maximum amount of EGF coating.The amount of EGF released from the gel at 8 h can be calculated.Controlled-release EGF hydrogel could promote delayed wound healing in aged rats as soon as possible.(3)CMC-GL and CMC-GL-EGF hydrogels had good biological safety.The co-incubation of CMC-GL and CMC-GL-EGF hydrogels with RBCs at different concentrations showed a lower hemolysis rate(<5%).Color of supernatant:The blood cells in NaCl group,CMC-GL-2 group and CMC-GL-EGF hydrogel group were not ruptured.The color of supernatant was colorless and transparent.The blood cells treated with Tris-X100 had ruptured and released hemoglobin.The color of supernatant was bright red.L929 cells showed lower cytotoxicity under different concentrations of CMC-GL and CMC-GL-EGF hydrogels,and the cell survival rate was more than 90%.(4)Compared with the NS group,both hydrogel/epidermal growth factor(GEL/EGF)and epidermal growth factor(EGF)could promote wound healing,and only the use of GEL could prevent wound contraction.On day 3 after injury,the NS group showed signs of bleeding and inflammation.On day 7 after different treatments,the healing rates were(52.05%±4.32),(55.17%±2.45),(50.92%±2.77),(69.34%±1.53)in the aged group;(68.41%±0.79),(73.40%+2.81),(56.23%±4.49),(79.62%±0.69)in the middle-aged group;(84.91%±0.31),(85.79%±0.28),(79.39%±2.13),(86.09%±0.46)in the young group;(85.12%±0.35),(86.91%±0.36),(79.93%±2.37),(86.13%±0.32)in the juvenile group.The results suggested that the effect of exogenous EGF on wound healing was age-related.(5)On days 7 and 14,H&E staining showed that there was no significant difference in granulation tissue quality and epidermal/dermal maturity between EGF and GEL-EGF groups.The healing response of GEL-EGF(high)group was enhanced.On day 14,the wound maturation was accelerated,and the epidermis was well formed,accompanied by tight positive keratinization.However,there were many well-organized granulation tissues in GEL-EGF group.In addition,the wounds treated with NS and GEL showed more inflammatory granulation tissue and incomplete epithelialization of epidermis,covering the scab until day 14 after injury.4.Developing a simple burn model of different depths in rats of different ages(1)Under constant temperature and pressure,the preparation conditions of rat scald model were:1 month old,deep II degree:3 s,III degree:5 s;2month old,superficial II degree:3 s,deep II degree:5 s,III degree:7 s,9 s;12and 12 months old,superficial II degree:3 s,5 s,deep II degree:7 s,9 s,III degree:11 s,13 s.(2)Under constant temperature and pressure,a metal column with diameter of 1 cm/1.5 cm and height of 2 cm was used.After heated in boiling water bath,the rats were scalded for 5 seconds.The scald depth of the model was the same,and all of them were deep second degree scalds.Conclusions1.The number of keratinocyte progenitor cells from the basal part of the epidermis does not change significantly,but the expression of EGFR decreases with age,indicating that the proliferation and differentiation ability of dry cells in the epidermis decreases,leading to the thinning of the epidermis thickness with age.Signal transduction of JAK2/STAT3/SOCs3 shows age-related changes in normal skin tissues.The expression of SOCs3 has age-relacted increasion.Signal transduction of JAK2/STAT3/SOCs3 is inhibited by inhibiting JAK2 expression.2.The wound healing ability of SD rats decreases with age,and the wound healing ability of natural aging SD rats decreases significantly.Normally,there was no significant age-related difference in CD271+cells in the basal epidermis.During wound healing in rats,CD271+cells complete re-epithelializes through proliferation and migration,and differentiate into keratinocytes at the same time.In this process,there is no age-related change in the proportion of CD271+cells in neonatal epithelium.However,under normal conditions,the expression of EGFR in epidermis cells decreases with age,which is partially activated during wound healing in rats,and EGFR+cells proliferates and epidermis thickness increases.In this process,there is no significant change in EGFR expression in the epidermal cells of juvenile and young groups,but the expression of EGFR in middle-aged and aged groups increases under traumatic stimulation,approaching the level of juvenile and young groups,and the thickness of EGFR+epidermal cell layer increases with age-related decrease.The expression of SOCs3 increases after trauma,and the expression of SOCs3 also shows age-related changes.Age-related wound healing ability decrease was associated with increased SOCs3 expression.Increased expression of SOCs3 inhibits EGFR/JAK2/STAT3 signal transduction and decreases the age-related proliferation of epidermal EGFR+cells,thus affecting wound healing.3.The CMC-GL hydrogel coated with EGF has good sustained release effect.In the treatment of delayed wound healing in aged rats with hydrogel sustained-release EGF,wound healing can be effectively treated by sustained drug release at a certain dose.At the same time,we speculate that EGF stimulates the synthesis and secretion of EGF,bFGF and vEGF by sustained release of wound permanent cells,and enhances the synthesis,cell migration and proliferation of basement membrane and extracellular matrix components.4.The scald models of rats of different ages established in this experiment can be used to prepare a simple animal model with accurate scald depth for rats of different ages by adjusting the burn time and the diameter of the scald module.It is a good model for studying the correlation between scald repair mechanism and age and evaluating new scald treatment methods.To study the mechanism of epidermal cells in delayed wound healing in the elderly,and to provide experimental basis for treating delayed wound healing and promoting wound healing in the elderly.