The Research Of Biofilm-Induced Capsular Contracture Following Breast Implants

[Background and Objective]Capsular contracture (CC) is one of the most common complications associated with breast implants. Numerous studies implicated subclinical (biofilm) infection around breast implants as an important cause of CC.Staphylococcus epidermidis (SE) is both a human commensal and an opportunistic pathogen. They would gain access to the breast implant at the time of placement and, once in contact with the prosthetic surface, form a biofilm causing inflammation. Inflammation disorders may lead to the proliferation and phenotypic transition of fibroblasts, which would lead to the imbalance of the collagen synthesis and degradation in the extracellular matrix (EMC), the production of large numbers of cytokines,and in the end,the establishment of CC.The purpose of this study is to understand the situation of biofilm formation by SE clinically isolated in the breast surgery on the surface of silicon materials, and investigate the influences of SE’s icaA, icaD, and aap genes on bacterial biofilm (BF) formation. By determining the activation of fibroblasts (Fb) by different concentrations of SE, understand the influences of SE on the proliferation and phenotypic transition of Fb, so as to investigate the mechanism of SE infection-related capsular contracture. To construct a Staphylococcus epidermidis (SE) infection-related capsular contracture (CC) animal model using Kunming small-ear pigs, and investigate the correlation between SE biofilm formation and capsular contracture. [Methods]1、Biofilm on breast implants surface and the role of icaA, icaD and aap in Staphylococcus epidermidis isolates in Breast Surgery44strains of SE were isolated from clinical samples of symptomless female patients admitted by the breast surgery department from December2011to January2013. Of the44strains of SE,26were cultured in flushing fluid of breast ducts,11were cultured in the capsule of prosthesis and dilator, and7were cultured in post-surgery drainage fluid of breast cancer. After the species was identified as the SE strains, genome DNA of the bacteria were extracted. Expressions of BF formation-related genes, icaA, icaD, and aap were determined by PCR, and the SE strains were divided into an icaA+icaD+/aap+group (group A), an icaA+icaD+/aap-group (group B), an icaA-icaD-/aap+group (group C), and an icaA-icaD-/aap-group (group D) according to PCR results. The4groups of SE were used for in vitro formation of BF on the surface of silicon materials. The silicon material was incubated in SE culture for6,12,24,30, and36hours before determining amount of surface adhesion by semi-quantitive crystal violet assay, so as to understand the change of adhesion amount as time passed and to draw a BF amount-time curve. Thickness of the biofilm after12and24h of incubation were measured by CLSM, and the ultrastructure of the BF after24h of incubation were observed by SEM, so as to understand the capability of SE to form BF on silicon surface.2、Influences of Stphylococcus epidermidis on the proliferation and Phenotypic Transition of Fibroblasts:(1). Normal breast tissue was obtained from Diannan small-ear pig for Fb primary culture and identified by immunocytochemistry using vimentin monoclonal antibody.(2). SE lysate was prepared by ultrasound pyrolysis apparatus and repeated freezing and thawing method, protein concentration of the SE lysate was determined by BCA method, and the conditioned medium containing the lysate was prepared to three concentrations of100μug/ml,50μg/ml, and25μg/ml.(3) MTT assay was used to determin the influences of normal culture medium, conditioned medium containing different concentrations of SE biofilm-positive standard SE RP62A and clinical isolates SE101lysate, and conditioned medium containing different concentrations of SE biofilm-negative standard SEAT12228and clinical isolates SE101lysate on the growth situation of Fb; TGF-β1levels in the cell supernatant cultured with different medium were determined by Elisa assa; and a-SMA expressions of Fb under different culture conditions were determined by RT-PCR, western blot, and immunocytochemistry.3、The experimental research of Biofilm-Induced Capsular Contracture in a Porcine Model(1). Three healthy adult female Diannan small-ear pigs were used, weighted35kg,39kg, and40kg respectively and with5-6pairs of nipple, which were randomly divided into group A (control), group B (ATCC12228group), and group C (RP62A group).(2). Biomaterial model construction of small-ear pigs (silicone breast dilator): cavities were stripped from the bottom of mammary glands, and lml PBS were injected in cavities of group A, and group B and C were injected with lml freshly prepared bacterial suspensions (1.2x105CFU/ml) of two different experimental strains:SE ATCC12228and SE RP62A. Each of the cavities was filled with a10-ml sterile cylindrical sterile silicone dilator (12for group A,10for group B and12for group C with34in total). Blood samples were obtained from precaval vein on the third day, and at3,6,9,and12weeks after implantation for blood cell analysis.(3). The capsule around the tissue expansion was taken for analysis after the experimental animals were fed in a clean environment for3months. Capsule tension was measured using an applanation tonometer, and capsule weight was measured using an electronic scale. Histological examination of the capsule:1) the microscopic structure characteristics of the capsule were observed after H-E staining under a light microscope, and capsule thickness was measured using a microscopic scale;2) capsule collagen characteristics were observed after Van-Gieson staining;3) MFb stained by a-SMA immunocytochemistry was observed under a light microscope;4) type I and III collagen was observed after Sirius red staining by polarizing microscope; 5) Biofilm formation on silicon dilator surface was observed by scanning electronic microscopy and the capsule bacteria was identified using the API system for bacterial identification.[Results]1、Of the44clinically isolated SE strains:①as shown by gel electrophoresis results of PCR products of the DNA extracted,23(52.27%,23/44) were icaA+icaD+, and25(56.81%,25/44) were app+;13were icaA+icaD+/aap+(group A),12were icaA+icaD+/aap-(group B),16were icaA-icaD-/aap+(group C), and3were icaA-icaD-/aap-(group D).②Of the44SE strains,29formed BF, including13of group A,7of group B,9of group C, and0of group D.③All icaA, icaD, and aap genes played important roles in BF formation (P<0.01), as shown by the semi-quantitative adhesion test:after6h of incubation, no significant difference in adhesion ability was detected among the4groups (P>0.05); from12h to36h, adhesion abilities of group A, B and C were significantly higher in than that in group D, and group A was significantly higher than group B and C (P<0.05); no significant difference was detected between group B and C (P>0.05). Confocal microscopy showed that at12or24h, the BF thickness were greater in group A, B and C than in group D, and greater in group A than in group B and C; no significant difference was detected between group B and C (P>0.05). Scanning electron microscopy showed that after24h of incubation, mature BF structure could be seen on the silicone surface in group A, B and C but not D; and BF of group A was the most widely distributed, and had the most compact and well-organized structure; BF of group B and C had similar structures; no obvious BF was formed in group D.2、Influences of Stphylococcus epidermidis on the proliferation and Phenotypic Transition of Fibroblasts:①The immumohistochemical staining of vimentin monoclonal antibodies of cultured cells were positive wtih typical morphology of Fb.②The results of MTT assay showed that the light absorption value was significantly higher in the biofilm-positive SE groups (standard strain SE RP62A and isolated strain SE101) than normal control group and biofilm-negative SE groups of different concentrations (standard strain SE RP62A and isolated strain SE101), P<0.01; no difference was detected between biofilm-negative SE groups and normal control (P>0.05).③The TGF-β1levels in the supernatant of the biofilm-positive SE groups were higher than normal control and biofilm-negative SE groups, P<0.01, and it was positively correlated with SE lysate concentration; the supernatant TGF-β1levels in the groups of biofilm-positive SE groups had no significant difference with the normal control group, P>0.05; however, with the increase of lysate concentration, statistical differences were found, P<0.05.④Immunocytochemistry showed that Fb treated by lysate of biofilm-positive SE transformed into a-SMA-positive myofibroblasts (MFb); some of the Fb treated by lysate of biofilm-negative SE also transformed; no Fb cultured in normal medium transformed.⑤Western blot and RT-PCR showed that a-SMA expression was higher in biofilm-positive SE groups than in normal control and biofilm-negative SE groups, P<0.01. All5groups of cells had increased level of a-SMA expression in the5th day at varying extent. The4treated groups maintained stable level of a-SMA expression on the7th day as the5th day, P>0.05; but a-SMA expression of the untreated group significantly decreased compared to that of the5th day, P<0.01.3、The experimental research of Biofilm-Induced Capsular Contracture in a PorcineModelSpecimen analysis:34silicon dilators in total were implanted in the3pigs,2of the12papilla of group A were CC-positive (16.7%), and positive rate was2/10(20%) for group B and8/12(66.7%) for group C. Capsule structure of the three groups was similar with obvious dense layer and loose layer, and capsule tension showed no statistical difference among the three groups (P>0.05). Capsule weight and thickness were greater in group C than in group A and B (P<0.01). Areal density of type I collagen was lower in control group and SE AT12228group than in SE RP62A group (P<0.05), and no significant difference was detected among the three for type III collagen (P>0.05). Infection with SE RP62A strain was closely related to biofilm formation and occurrence of CC, positive rates of biofilm formation and CC (Grade Ⅲ/Ⅳ) were significantly higher in group C than in group A and B (P<0.05). Univariate analysis showed a22-fold and8-fold increase in risk of biofilm formation in group C (inoculated with biofilm-positive SE strain) compared with group A and B; meanwhile incidence of CC was7times and5.6times higher in group C than group A and B. Of the12cases of biofilm positive samples,10also had CC, and univariate analysis suggested a47.25-fold increase in CC due to biofilm formation.[Conclusion]1、icaA, icaD and aap genes are of great significance in BF formation on silicon surface by SE, and SE strains simultaneously expressing icaA, icaD and aap have the strongest BF-forming ability.2、Biofilm-positive SE strains can effectively promote Fb proliferation and TGF-β1secretion, and can stimulate a-SMA expression and transformation of Fb to MFb; while biofilm-negative SE strains have no significant effect on Fb proliferation, and weaker effect in promoting transformation of Fb to MFb than biofilm-positive SE strains.3、After the tissue expansion (prosthesis) is implanted, occurrence of CC is closely related to the formation of SE biofilm on the tissue expansion surface and (or) its capsule.