Hypoxia-induced epithelial-mesenchymal transition and fibrosis for the development of breast capsular contracture.

Fibrosis has been considered as a major cause of capsular contracture. Hypoxia has widely emerged as one of the driving factors for fibrotic diseases. The aim of this study was to examine the association between hypoxia-induced fibrosis and breast capsular contracture formation. Fibrosis, epithelial-mesenchymal transition (EMT), expression levels of hypoxia-inducible factor-1α (HIF-1α), vimentin, fibronectin, and matrix metalloproteinase-9 (MMP-9) in tissues from patients with capsular contracture were determined according to the Baker classification system. Normal breast skin cells in patients with capsular contracture after implant-based breast surgery and NIH3T3 mouse fibroblasts were cultured with cobalt chloride (CoCl2) to mimic hypoxic conditions. Treatment responses were determined by detecting the expression of HIF-1α, vimentin, fibronectin, N-cadherin, snail, twist, occludin, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and -2, as well as phosphorylated ERK. The expression levels of HIF-1α, vimentin, fibronectin, and fibrosis as well as EMT were positively correlated with the severity of capsular contracture. MMP-9 expression was negatively correlated the Baker score. Hypoxia up-regulated the expression of HIF-1α, vimentin, fibronectin, N-cadherin, snail, twist, TIMP-1 and -2, as well as phosphorylated ERK in normal breast skin cells and NIH3T3. Nonetheless, the expression levels of MMP-9 and occludin were down-regulated in response to CoCl2 treatment. This study is the first to demonstrate the association of hypoxia-induced fibrosis and capsular contracture.

Long-term studies on the integration of acellular porcine dermis as an implant shell and the effect on capsular fibrosis around silicone implants in a rat model.

Acellular dermal matrices have recently increasingly been used in alloplastic breast reconstruction with silicone breast implants. Among these matrices, acellular porcine dermis (APD) is frequently applied, but long-term data on tissue integration and capsular fibrosis formation are still missing. Silicone prostheses with (group A) and without (group B) APD as an implant-covering shell were implanted in male Lewis rats. At 3, 12, and 52 weeks after implantation, the constructs were explanted. Molecular biological and immunohistochemical analyses were performed afterwards. On comparing the collagenous layer and the newly formed myofibroblast-rich layer around the implants of both groups, it became apparent that in group A, these layers were thinner, followed by a lower expression of TGFß1 after 12 and 52 weeks. Further, in this group, at the endpoint of 52 weeks, a lower amount of CD68-positive cells in the collagenous and myofibroblast-rich layers were observed and the expression of TNFα was reduced, while the number of Ki67-positive cells was significantly higher with time. Furthermore, MMP1 expression in group A was lower than that in group B, and the calculated ratio of MMP1:TIMP1 expression was higher. The long-term results clearly show a reduction in inflammatory and fibrotic tissue reaction when APD is used to cover silicone prostheses. These experimental data will be of considerable importance for implant-based breast surgery, as they indicate a potential benefit in the reduction of capsular fibrosis formation of an interposition of APD between the recipient and the silicone implant.

Non-linear optical microscopy and histological analysis of collagen, elastin and lysyl oxidase expression in breast capsular contracture.

BACKGROUND: Capsular contracture is one of the most common complications in surgical interventions for aesthetic breast augmentation or post-mastectomy breast reconstruction involving the use of silicone prostheses. Although the precise cause of capsular contracture is yet unknown, the leading hypothesis is that it is caused by long-term unresolved foreign body reaction towards the silicone breast implant. To authors' best knowledge, this is the first study that elucidates the presence of lysyl oxidase (LOX)-an enzyme that is involved in collagen and elastin crosslinking within fibrous capsules harvested from patients with severe capsular contracture. It was hypothesized that over-expression of LOX plays a role in the irreversible crosslinking of collagen and elastin which, in turn, stabilizes the fibrous proteins and contributes to the progression of capsular contracture. METHODS: Eight fibrous capsules were collected from patients undergoing capsulectomy procedure, biomechanical testing was performed for compressive Young's moduli and evaluated for Type I and II collagen, elastin and LOX by means of non-linear optical microscopy and immunohistology techniques. RESULTS: Observations revealed the heterogeneity of tissue structure within and among the collected fibrous capsules. Regardless of the tissue structure, it has been shown that LOX expression was intensified at the implant-to-tissue interface. CONCLUSION: Our results indicate the involvement of LOX in the initiation of fibrous capsule formation which ultimately contributes towards the progression of capsular contracture.

The role of angiogenesis, inflammation and estrogen receptors in breast implant capsules development and remodeling.

BACKGROUND: Capsular contracture is the most common complication following breast implant placement. The multiple factors unbalancing the physiological response to the foreign body have not been fully elucidated. The aim of this study was to investigate the role of neo-angiogenesis, inflammation and estrogen receptors in peri-prosthetic tissue development and remodeling. METHODS: The study enrolled 31 women who underwent expander substitution with definitive implant. Specimens were stained with hematoxylin/eosin, Masson trichrome, immunohistochemistry and immunofluorescence for alpha-smooth muscle actin, estrogen receptor-α (ER-α), estrogen receptor-ß (ER-ß), Collagen type I and III, CD31 (as a marker of neo-angiogenesis) and vascular endothelial growth factor (VEGF). Inflammatory infiltration was quantified and analyzed. Transmission electron microscopy was performed for ultrastructural evaluation. RESULTS: Myofibroblasts, mainly localized in the middle layer of capsular tissue, expressed VEGF, ER-α and ER-ß. ER-ß expression positively correlated with Collagen type I deposition (p= 0.025). Neo-angiogenesis was predominant in the middle layer. CD31 expression positively correlated with Collagen type I expression (p=0.009) and inflammatory infiltration grade (p= 0.004). The degree of inflammatory infiltration negatively correlated with the time from implantation (p = 0.022). DISCUSSION: The middle layer is key in the development and remodeling of capsular tissue. Myofibroblasts produce VEGF, that induces neo-angiogenesis. New vessels formation is also correlated to the inflammatory response. Collagen deposition is associated with ER-ß expression and neo-angiogenesis. These findings may prelude to targeted pharmacologic therapies able to control such interactions, thus hampering the self-sustaining loop promoting the progression of physiologic fibrosis toward pathologic contracture.

Thy1 (CD90) Expression Is Elevated in Radiation-Induced Periprosthetic Capsular Contracture: Implication for Novel Therapeutics.

BACKGROUND: Capsular contracture is a devastating complication of postmastectomy implant-based breast reconstruction. Unfortunately, capsular contracture rates are drastically increased by targeted radiotherapy, a standard postmastectomy treatment. Thy1 (also called CD90) is important in myofibroblast differentiation and scar tissue formation. However, the impact of radiotherapy on Thy1 expression and the role of Thy1 in capsular contracture are unknown. METHODS: The authors analyzed Thy1 expression in primary human capsular tissue and primary fibroblast explants by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. Thy1 was depleted using RNA interference to determine whether Thy1 expression was essential for the myofibroblast phenotype in capsular fibroblasts. Furthermore, human capsular fibroblasts were treated with a new antiscarring compound, salinomycin, to determine whether Thy1 expression and myofibroblast formation were blocked by salinomycin. RESULTS: In this article, the authors show that radiation therapy significantly increased Thy1 mRNA and protein expression in periimplant scar tissue. Capsular fibroblasts explanted from scar tissue retained the ability to make the myofibroblast-produced scar-forming components collagen I and α-smooth muscle actin. Depletion of Thy1 decreased the fibrotic morphology of capsular fibroblasts and significantly decreased α-smooth muscle actin and collagen levels. Furthermore, the authors show for the first time that salinomycin decreased Thy1 expression and prevented myofibroblast formation in capsular fibroblasts. CONCLUSIONS: These data reveal that ionizing radiation-induced Thy1 overexpression may contribute to increased capsular contracture severity, and fibroblast scar production can be ameliorated through targeting Thy1 expression. Importantly, the authors' new results show promise for the antiscarring ability of salinomycin in radiation-induced capsular contracture. CLINCAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Toll-Like Receptor 4 Expression in Human Breast Implant Capsules: Localization and Correlation with Estrogen Receptors.

BACKGROUND: Capsular contracture is the most common complication following breast augmentation and reconstruction. Myofibroblasts, which are specialized fibroblasts with contractile activity, are involved in its pathogenesis. Toll-like receptor 4 stimulation in fibroblasts induces transcription of genes involved in extracellular matrix remodeling and tissue repair; furthermore, it enhances sensitivity to transforming growth factor-ß1 and promotes transition to myofibroblasts. 17ß-Estradiol, by binding to its main receptors, α and/or ß, increases the expression of toll-like receptor 4 and the production of proinflammatory mediators by macrophages; moreover, it promotes extracellular matrix production and myofibroblasts contraction and differentiation. The aim of the study was to investigate the expression of toll-like receptor 4 in breast implant capsules and its relationship with estrogen receptors. METHODS: The study enrolled 30 women who underwent expander removal following breast reconstruction. Specimens were stained with hematoxylin and eosin, Masson trichrome, immunohistochemistry, and immunofluorescence for toll-like receptor 4, α-smooth muscle actin (a marker of myofibroblasts), estrogen receptor-α, and estrogen receptor-ß. RESULTS: Toll-like receptor 4 was expressed by fibroblasts and myofibroblasts of capsular tissue. Its expression positively correlated with estrogen receptor-ß expression (p = 0.012). A positive correlation was found between estrogen receptor-ß and α-smooth muscle actin expression (p = 0.037). CONCLUSIONS: This study demonstrates the expression of toll-like receptor 4 in myofibroblasts of capsular tissue and its correlation with estrogen receptor-ß positivity. Activation of toll-like receptor 4 and estrogen receptor-ß, and their interplay, may be involved in myofibroblast differentiation and in the profibrotic pathogenic process underlying capsular contracture.

Histologic, Molecular, and Clinical Evaluation of Explanted Breast Prostheses, Capsules, and Acellular Dermal Matrices for Bacteria.

BACKGROUND: Subclinical infections, manifest as biofilms, are considered an important cause of capsular contracture. Acellular dermal matrices (ADMs) are frequently used in revision surgery to prevent recurrent capsular contractures. OBJECTIVE: We sought to identify an association between capsular contracture and biofilm formation on breast prostheses, capsules, and ADMs in a tissue expander/implant (TE/I) exchange clinical paradigm. METHODS: Biopsies of the prosthesis, capsule, and ADM from patients (N = 26) undergoing TE/I exchange for permanent breast implant were evaluated for subclinical infection. Capsular contracture was quantified with Baker Grade and intramammary pressure. Biofilm formation was evaluated with specialized cultures, rtPCR, bacterial taxonomy, live:dead staining, and scanning electron microscopy (SEM). Collagen distribution, capsular histology, and ADM remodeling were quantified following fluorescent and light microscopy. RESULTS: Prosthetic devices were implanted from 91 to 1115 days. Intramammary pressure increased with Baker Grade. Of 26 patients evaluated, one patient had a positive culture and one patient demonstrated convincing evidence of biofilm morphology on SEM. Following PCR amplification 5 samples randomly selected for 16S rRNA gene sequencing demonstrated an abundance of suborder Micrococcineae, consistent with contamination. CONCLUSIONS: Our data suggest that bacterial biofilms likely contribute to a proportion, but not all diagnosed capsular contractures. Biofilm formation does not appear to differ significantly between ADMs or capsules. While capsular contracture remains an incompletely understood but common problem in breast implant surgery, advances in imaging, diagnostic, and molecular techniques can now provide more sophisticated insights into the pathophysiology of capsular contracture. LEVEL OF EVIDENCE: 4 Therapeutic.

The impact of triamcinolone acetonide in early breast capsule formation in a rabbit model.

BACKGROUND: The etiology and clinical treatment of capsular contracture remain unresolved as the causes may be multifactorial. Triamcinolone acetonide applied in the pocket during surgery was reported to be ineffective in prevention of capsular contracture. However, if injected 4-6 weeks after surgery or as a treatment for capsular contracture, decreased applanation tonometry measurements and pain were observed. It was assumed that intraoperative application of triamcinolone was not effective because its effect does not last long enough. However, betadine, antibiotics, and fibrin were found to be effective in preventing capsular contracture with intraoperative applications and are more effective in the early phases of wound healing than in later stages. The role of triamcinolone acetonide in capsule formation is unknown. The purpose of this study was to determine if triamcinolone acetonide modulates breast capsule formation or capsular contracture in the early phases of wound healing in a rabbit model. METHODS: Rabbits (n=19) were implanted with one tissue expander and two breast implants and were killed at 4 weeks. Implant pocket groups were (1) Control (n=10) and (2) Triamcinolone (n=9). Pressure/volume curves and histological, immunological, and microbiological evaluations were performed. Operating room air samples and contact skin samples were collected for microbiological evaluation. RESULTS: In the triamcinolone group, a decreased capsular thickness, mild and mononuclear inflammation, and negative or mild angiogenesis were observed. There were no significant differences in intracapsular pressure, fusiform cell density, connective tissue, organization of collagen fibers, and microbiological results between the groups. There was no significant difference in the dialysate levels of IL-8 and TNF-α, but correlation between IL-8 and TNF-α was observed. CONCLUSION: Triamcinolone acetonide during breast implantation influences early capsule formation and may reduce capsular contracture. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors at www.springer.com/00266.

The impact of Smad3 loss of function on TGF-ß signaling and radiation-induced capsular contracture.

BACKGROUND: Capsular contracture remains a major problem following prosthetic breast implantation, especially in patients undergoing irradiation. Recent studies suggest that such radiation injuries are a cascading process of cytokine activation, with transforming growth factor (TGF)-ß acting as the "master switch." Because TGF-ß signals through phosphorylation of Smad3, a plausible approach to abate TGF-ß-induced capsular contracture would be to interrupt Smad3 signaling. To test this hypothesis, capsular contracture formation in wild-type and Smad3 knockout mice was compared using micro-computed tomographic and histologic examination. METHODS: On day 0, 48 mice were implanted with bilateral silicone gel implants. Postoperatively, animals were imaged using live-scan micro-computed tomographic scanning. Animals in the radiation arm then received a 10-Gy directed radiation dose. On postoperative days 21, 28, 35, and 42, animals were imaged again. Histologic evaluation was performed at necropsy. RESULTS: Irradiated implants in the wild-type mice demonstrated shape and contour deformation on micro-computed tomographic scanning beginning on postoperative day 21 and progressing through day 42. Conversely, micro-computed tomographic scanning of irradiated implants in knockout mice demonstrated few changes from day 0 through day 42. Corresponding histologic specimens from wild-type mice demonstrated irregular capsules composed of disorganized collagen that became thicker from day 21 to day 42. Irradiated knockout specimen maintained thin capsules from day 21 through day 42. CONCLUSIONS: In this work, inhibiting TGF-ß signaling led to a reduction in radiation-induced capsular contracture as measured by micro-computed tomographic and histologic evaluation. The results of this study suggest a promising target for the prevention of capsular contracture through the development of anti-Smad3/TGF-ß-based therapies.

Tumour necrosis factor-α expression is associated with increased severity of periprosthetic breast capsular contracture.

BACKGROUND: The pathogenesis of periprosthetic capsular contracture following breast implant surgery is unclear. The aim of this study was to identify the expression of tumour necrosis factor-α (TNF-α), collagen type III α(1) (COL3A1), transforming growth factor-ß(1) (TGF-ß(1)) and connective tissue growth factor (CTGF) in different Baker grades of breast capsular contracture. METHODS: Seven periprosthetic breast capsule specimens were collected from 6 patients. TNF-α, COL3A1, TGF-ß(1) and CTGF gene expression were analysed using real-time quantitative polymerase chain reaction. Immunohistolocalisation of TNF-α was performed on paraffin-embedded sections. Significant correlations were analysed using the Pearson correlation coefficient. RESULTS: TNF-α expression was associated with increased Baker grade of capsular contracture (Pearson correlation, r = 0.558; p = 0.02). COL3A1 gene expression was reduced with increasing severity of contracture (r = -0.490; p = 0.05). There were no significant correlations between TGF-ß(1) and CTGF expression with Baker grade. Positive TNF-α staining in breast capsules was localised to fibroblasts, macrophages, and extracellularly close to the prosthesis. CONCLUSION: The increased expression of TNF-α may play a key role in the inflammatory response associated with capsular contracture. The corresponding decrease in COL3A1 may contribute to the change in capsular physical properties seen in capsular contracture.