| Background:Prosthetic breast reconstruction is a vital method of oncoplastic surgery among people with breast cancer.However,capsular contracture(CC)after the operation has been a tricky issue,for which staphylococcus epidermidis(SE)implantation has been the main reason for CC.SE would form a biofilm(BF)on the surface of the implant if it enters the deep tissue along with the implant during the implantation of the medical device,and thus leading to a capsular contracture(CC).The BF formation of SE is regulated by various genes,of which the expression would changed by microenvironment or disorder of regulating factors.It will induced to express in virto in the presence of ethanol,changes of hormone levels inside human body would also lead to changes of characteristics in vivo.Increasing levels of sex hormones would lead to growth of bacterial numbers and virulence at specific sites,resulting in host-associated infections.As a hormone-dependent tumor,breast cancer patients have been confirmed that estrogen levels were higher than normal population by a large number of epidemiological studies.While it has not been reported that how well the prosthetic surface bacterial BF formation capacity is,and it is unclear that the relationship between estrogen and bacterial growth,biofilm formation,and capsular contracture.Therefore,we envision whether their estrogen levels will affect the implant in the breast prosthesis surface characteristics of SE in a long-term high estrogen level breast cancer populations,and eventually made a contribution to in the process of prosthesis CC.Aim of this study is to investigate effects of estradiol(E2)on growth and BF formation of SE on breast implants in vitro,to determine effects of E2 on the gene expression of aap,icaA,SarA,Lux,RNAⅢ and icaR.Moreover,an animal model of SE infection in mammary glandular material was established in order to explore effects of E2 level on SE infection in vivo.Section Ⅰ:Effect of estradiol on the biofilm formation of Staphylococcus epidermidis plantinged on the surface of breast implantsObjectiveTo observe effects of estradiol in different level on growth,biofilm formation ability and biofilm-structure by SE plantinged on the surface of breast implants.MethodTo simulate the estradiol levels in people with breast cancer,participants in this study were divided into control group and experimental group according to different estradiol concentrations(0,50,125,250,500 pmol/L).Breast silica gel was selected as carrier,and the SE standard strains(ATCC35984 positive,ATCC12228 negative)as well as clinical strains(SE101 biofilm phenotype positive,SE40 biofilm phenotype negative)were incubated for 4h,6h,12h,24h,48h and 72h.Growth curve was drawn according to OD value;and initial adhesion and forming ability of biofilm were evaluated by crystal violet staining semi-quantitative detection.SE(ATCC35984 and ATCC 12228)were used as experimental materials according to previous experimental data.The growth of SE was detected by XTT colorimetry,and we compared different initial concentration of bacteria on the growth of SE biofilm formation process.The biofilm thickness of ATCC35984 was measured by confocal laser scanning microscopy(CLSM)at different concentrations of estradiol.The biofilm thickness was measured at 6h,12h and 24h after incubation with silica gel.Scanning electron microscopy(SEM)was used to observe the bacterial biofilm ultrastructure of SE standard strains and clinical strains at different concentrations of estradiol at different time points.Results1.Bacterial growth curves:In the blank group without estradiol,four strains of ATCC35984,ATCC 12228,SE101 and SE40 entered the logarithmic growth phase at the forth hour all the same.In 125pmol/L estradiol group,four strains of SE enter the logarithmic growth phase in the third hour,and the growth ability of the SE strain in estradiol group is better than that in the blank group.2.SE initial adhesion ability:The ability of ATCC35984 and SE101 to form initial adhesion on silica gel was enhanced with the increase of estradiol concentration,and the adhesion ability was the strongest at 125pmol/L(P<0.05).The adhesive ability of estradiol increased with no increase.While estradiol had no effect on the ability of ATCC12228 and SE40 adhere to the biomaterial surface.3.SE biofilm formation ability:At different estradiol concentrations,the biofilm formed by SE ATCC35984 and SE101 on the surface of silica gel material gradually increased with the culture time.The biofilm reached the maximum thickness at 24h,and the biofilm formed at 48h and 72h decreased compared with that at 24h(P<0.05).At the early stage of biofilm formation(at 4-6h),the biofilm thickness of ATCC35984 decreased with the increase of estradiol concentration,and the biofilm was higher in low estradiol group(≤50pmol/L)than that of high estradiol group,but the thickness of biofilm formed from 12h increased significantly with the increase of estradiol concentration,and the biofilm formed under the action of 125mmolol/L estradiol was the thickest,the difference was statistically significant(P<0.05).SE101 also formed the thickest biofilm in 125pmol/L group.When the concentration of estradiol was more than or equal to 250pmol/L,the thickness of biofilm decreased,but it was still higher than that of low estradiol(≤50pmol/L).ATCC12228 and SE40 showed no significant biofilm formation in different concentrations of estradiol(OD<0.12).4.Power of bacterial growth:The OD values of ATCC12228 and ATCC35984 all increased with the time of culture.At the same time point,all OD values increased with the increase of estradiol concentration(P<0.05).At 4～6h,the growth of the two bacteria in the estradiol group was in the accelerated phase.The growth of ATCC 12228 was gentle at 12-48h and accelerated at 72h;the growth of ATCC35984 was gentle at 12-24h,in 48h when entered the accelerated growth period.When the concentration of estradiol ≥125pmol/L,the OD value of the two bacteria at 4h was significantly higher than 0 and 50pmol/L estradiol group(P<0.05),with the passage of time,estradiol>125pmol/L group was always higher than that of the group ≤50 pmol/L(P<0.05).5.The bacterial growth and biofilm formation of ATCC35984 with different initial concentration of bacterium:The growth of bacteria was detected by XTT.The OD value of 1 ×108CFU/mL bacterial solution group was always higher than 1 ×107CFU/mL broth with the time of incubation group.With the increase of estradiol concentration,the growth rate of 1×107CFU/mL and 1×108CFU/mL bacteria solution increased with the increase of estradiol concentration,but different from that of 1×107CFU/mL bacteria group,the difference of OD value with 1×108CFU/mL in all the estradiol groups at 4-6h was not statistically significant(P>0.05).The growth of 1×108CFU/mL bacteria solution in ≤50pmol/L estradiol groups were gentle at 12-24h,and accelerated at 48h,while the growth rate of bacteria in ≥125pmol/L estradiol groups was accelerated at the same time(P<0.05).The thickness of biofilm formation of SE ATCC35984 was increased with time,and the OD value of 1 ×108CFU/mL bacterium group was significantly higher than that of 1 ×107CFU/mL at 4h and 6h(P<0.05).At 12h,the OD value of 1×108CFU/mL bacterial solution group and 1 ×107CFU/mL group had no significant difference(P>0.05);at 24～72h,the OD value in the group of 1 ×107CFU/mL was significantly higher than that in the 1 ×108CFU/mL group(P<0.05).The OD of 1 ×107CFU/mL bacterial solution group was the highest at 24h,and the OD value of 1 ×108CFU/mL bacterium group was the highest at 48h,and the OD value of the two groups was lower than 72h.At the same time point,the OD values of 1 ×107CFU/mL and 1×108CFU/mL in 125pmol/L estradiol group were higher than those in other estradiol groups(P<0.05).6.The biofilm thickness and metabolic status of the bacteria were not significantly different from the estradiol group at 6h(P>0.05).The biofilm thickness of estradiol≥125pmol/L group was greater than that of ≤50pmol/L group at 12-24h,and the bacterial biofilm thickness of ≥125pmol/L groups was significantly higher than that of the other estradiol groups(P<0.05).The biofilm thickness of each group was increased with the passage of time(P<0.05),and reached the maximum at 24 hours.Bacterial biofilm metabolism was observed by dead or viable bacteria staining,and the surface of the biofilm was dominated by viable bacteria and dead bacteria at the bottom.When incubated for 12 hours,the biofilms formed were mainly live bacteria,and a large number of dead bacteria appeared at 24 hours.7.Bacterial biofilm ultrastructure:ATCC12228 and SE40 had no biofilm structure.When the concentration of estradiol was 125pmol/L,bacterial mass could be seen at 4 hours in ATCC35984 and SE101.At 6h,a large amount of bacterial mass was covered on the surface of silica gel.At 12h,fibrous tissue connected with bacterial groups was formed;24 hours to see the honeycomb mature bacterial biofilm structure;48h and 72h when the decomposition of biofilm structure.The biofilm formed by 125pmol/L of estradiol was the most abundant,the most abundant and the most abundant.Conclusions1.Estradiol can promote the growth of SE,and bacterial growth motility estradiol concentration-dependent,suggesting that estradiol may play a role in maintaining the survival of bacteria.2.In the process of staphylococcal biofilm formation,high concentration of estradiol can significantly enhance the initial adhesion of bacteria and promote the formation of biofilm,which may be one of the factors that patients with high estradiol levels are much easier to form bacterial biofilm.3.Biofilm-forming positive strains in the appropriate high concentration of estradiol,have denser biofilm,and the structure of the multicellular population is more highly organized,which may be one of the reasons that it is difficult to deal with the postoperative infection of breast cancer patientsSection Ⅱ:Study on effects of genes associated with the biofilm formation of SE related to estradiolObjectiveTo explore possible molecular mechanism of estradiol in SE biofilm.MethodReal-time PCR was used to detect the mRNA expression of aap,icaA,SarA,Lux,RNAⅢ and icaR genes in SE after cultured for 12h,24h,48h and 72h with estradiol concentrations of 0,50,125 and 250pmol/L.To observe the expression of all the genes in different estradiol concentrations.Results1.Aap gene was positively regulated in both the formation period(12h)and the mature stage(24h)of biofilm,and the effect was most significant under the concentration of 125pmol/L of E2.2.The expression of icaA was not up-regulated at 12h after biofilm formation.However,at 24h,the icaA gene started to show a slight up-regulation at 125pmol/L of estradiol.At 48h,the expression of icaA gene was significantly up-regulated in 250pmol/L estradiol.3.At 12h,sarA was negatively regulated in all estradiol concentrations,but increased with the increase of estradiol concentration,and was significantly up-regulated at 125pmol/L at 24h and significantly up at 250pmol/L at 48h.4.LuxS was inhibited in both SE biofilm formation(12h)and new biofilm establishment(72h),and inhibition of LuxS was more significant with the increase in estradiol concentration.5.The mRNA level of RNAⅢ was significantly up-regulated at 125pmol/L group at 48h of biofilm formation and significantly inhibited with the increase in estradiol concentration at 72h.6.The expression of icaR.was down-regulated with the increase in estradiol concentration at 12h after the biofilm formation,and was up-regulated at 24h and 48h,and was significantly up-regulated at 125pmol/L at 24h.Conclusions1.At the early stage of biofilm formation,procedures were dominated by positive regulatory genes,while at the late stage of biofilm maturation,positive regulatory genes became weaker and negative regulatory genes began to work.In the biofilm collapse period,negative regulatory genes dominated the step.2.Both positive regulatory genes and negative regulatory genes were strengthened in high concentration of estradiol,suggesting that estradiol can promote the formation of biofilm by not only positive but also negative regulations.Section Ⅲ:Effects of estradiol on bacterial biofilm in ovariectomized rats and its relationship with capsule contractureObjectiveTo investigate effects of estradiol on bacterial biofilm formation and encapsulated contracture in vivo,and to evaluate effects of estradiol on the ability to clear the infection in animals by establishing different models of estradiol serum level in rats.MethodThe animal models with different serum levels of estradiol were established by ovariotomy and estradiol injection.Silicone prosthesis was implanted and infected with SE(ATCC12228 or ATCC35984).After 60 days of infection,the rats were sacrificed.The infection rate of the main reproductive organs and the colonies of the infected bacteria were measured.The infiltration of the inflammatory cells was observed under microscope.The surface biofilm formation on the silica gel were observed by scanning electron microscopy.The structure of the contracted capsule was observed by pathology.Results1.The serum levels of estradiol was 46.886(±3.965)pmoL/L for ovariotomy rats,and was 152.381(±10.455)pmoL/L in high E2 group,and was 84.962(±24.735)pmoL/L in low E2 group(P<0.05).2.In high E2 group,ATCC35984 infected mice increased organ infection rate,the number of colonies was significantly higher than the normal group and low E2 group(P<0.05).3.In high E2 group,the rate of capsular contracture was up to 80%(Grade Ⅲ/Ⅳ),and a lot of dense fibrous tissue and synovial metaplasia were seen under the microscope.Under scanning electron microscope,ATCC35984 infection,and significantly mature bacterial biofilm formation.4.The levels of serum IFN-γ and TNF-α in the low E2 group were significantly higher than those in the high E2 group at the 1st,3rd and 7th day after infection with ATCC35984.The levels of IL-6 and Ig-G in E2 group were significantly higher than those in the low E2 group(P<0.05).5.The levels of CD3,CD4 and CD8/CD8 in the high E2 group decreased from the first day of infection to the lowest level on the third day after infection,then gradually increased.The level of CD8 increased from day 1 of infection to the highest level of infection 3 days,then gradually decreased(P<0.05).Conclusions1.Construction of rat models with different estradiol serum levels can be achieved by castration surgery.2.High concentration of estradiol can improve the incidence of prosthetic capsule contracture as well as promote the proliferation of collagen fibers,which will form thick and dense capsule.It may be one of the most important reasons for prosthetic capsule contracture.3.High concentrations of estradiol can also induce the infiltration of massive inflammatory cells,so that serious inflammatory reaction happened to surrounding tissue.This will further increase the immune imbalance of the infected host,increasing the risk of infection spread.The above mechanism is one of the possible causes of host immune system disorders. |
