Patrick Hwang^1,2†, Chung Min Shin^3†, Jennifer A. Sherwood¹, DongHo Kim⁴, Vineeth M. Vijayan⁵, Krishna C. Josyula², Reid C. Millican¹, Donald Ho⁶, Brigitta C. Brott^1,7, Vinoy Thomas⁸, Chul Hee Choi⁴, Sang‑Ha Oh³, Dong Woon Kim^9* and Ho‑Wook Jun^1,2*

¹Endomimetics, LLC, Birmingham, AL 35242, USA. 
²Department of Biomedical Engineering, University of Alabama at Birmingham, 806 Shelby, 1825 University Boulevard, Birmingham, AL 35294, USA. 
³Department of Plastic and Reconstructive Surgery, Chungnam National University College of Medicine, Daejeon 35015, Republic of Korea. 
⁴Department of Microbiology, Chungnam National University College of Medicine, Daejeon 35015, Republic of Korea. 
⁵Department of Biomedical Engineering, Alabama State University, Montgomery, AL 36104, USA. 
⁶Department of Pediatric Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA. 
⁷Department of Medicine and Division of Cardiovascular Disease, University of Alabama at Birmingham, Birmingham, AL 35233, USA. 
⁸Department of Material Science and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA. 
⁹Department of Anatomy and Cell Biology, Brain Research Institute, College of Medicine, Chungnam National University College of Medicine, Daejeon 35015, Republic of Korea. 

^†Patrick Hwang and Chung Min Shin contributed equally to this work.
^*Correspondence: Dong Woon Kim, Ho‑Wook Jun

Background: Capsular contracture is a critical complication of silicone implantation caused by fibrotic tissue formation from excessive foreign body responses. Various approaches have been applied, but targeting the mechanisms of capsule formation has not been completely solved. Myofibroblast differentiation through the transforming growth factor beta (TGF-β)/p-SMADs signaling is one of the key factors for capsular contracture development. In addition, biofilm formation on implants may result chronic inflammation promoting capsular fibrosis formation with subsequent contraction. To date, there have been no approaches targeting multi-facted mechanisms of capsular contracture development.

Methods: In this study, we developed a multi-targeting nitric oxide (NO) releasing bionanomatrix coating to reduce capsular contracture formation by targeting myofibroblast differentiation, inflammatory responses, and infections. First, we characterized the bionanomatrix coating on silicon implants by conducting rheology test, scanning electron microcsopy analysis, nanoindentation analysis, and NO release kinetics evaluation. In addition, differentiated monocyte adhesion and S. epidermidis biofilm formation on bionanomatrix coated silicone implants were evaluated in vitro. Bionanomatrix coated silicone and uncoated silicone groups were subcutaneously implanted into a mouse model for evaluation of capsular contracture development for a month. Fibrosis formation, capsule thickness, TGF-β/SMAD 2/3 signaling cascade, NO production, and inflammatory cytokine production were evaluated using histology, immunofluorescent imaging analysis, and gene and protein expression assays.

Results: The bionanomatrix coating maintained a uniform and smooth surface on the silicone even after mechanical stress conditions. In addition, the bionanomatrix coating showed sustained NO release for at least one month and reduction of differentiated monocyte adhesion and S. epidermidis biofilm formation on the silicone implants in vitro. In in vivo implantation studies, the bionanomatrix coated groups demonstrated significant reduction of capsule thickness surrounding the implants. This result was due to a decrease of myofibroblast differentiation and fibrous extracellular matrix production through inhibition of the TGF-β/p-SMADs signaling. Also, the bionanomatrix coated groups reduced gene expression of M1 macrophage markers and promoted M2 macrophage markers which indicated the bionanomatrix could reduce inflammation but promote healing process.

Conclusions: In conclusion, the bionanomatrix coating significantly reduced capsular contracture formation and promoted healing process on silicone implants by reducing myfibroblast differentiation, fibrotic tissue formation, and inflammation. A multi-targeting nitric oxide releasing bionanomatrix coating for silicone implant can reduce capsular contracture and improve healing process. The bionanomatrix coating reduces capsule thickness, α-smooth muscle actin and collagen synthesis, and myofibroblast differentiation through inhibition of TGF-β/SMADs signaling cascades in the subcutaneous mouse models for a month.