Assistant Professor South Dakota School of Mines and Technology, United States
Introduction:: Implants have revolutionized the medical field. They play pivotal roles in the body depending on their function. However, several case reports indicate implant failures due to various causes including fibrotic capsule formation (FCF). Fibroblasts are thought to be the regulators of deposition of excess of the ECM (in particular dense collagen fibrils) during FCF that form around foreign bodies (Noskovicova et al., 2021).
Hyaluronic acid (HA) is a glycosaminoglycan in the ECM that is known to regulate collagen organization. This is a function of HA that could be utilized to address fibrotic capsule formation. HA binding peptides are a class of bioactive sequences that are able to form strong bonds with HA thus retain HA in the sites they are present (Singh et al., 2014). In this study, we developed a nanofibrillar interface presenting HABP to attract and retain HA at the implant tissue interface. We showed fibroblasts growing on HABP containing nanofibers exhibit lower alpha smooth muscle actin and organize collagen differently.
Materials and Methods:: Material analysis: Electrospinning of PCL membranes were produced following our earlier methods and the fiber diameter was tuned accordingly. HABP and control peptide sequences were synthesized using solid phase peptide synthesis protocols published before. PCL fiber membranes were further functionalized with HABP with a terminal cysteine amino acid using aminolysis protocol and further conjugation with Sulfo-SMCC.[2] Reaction of the peptide with PCL fibers will be confirmed with Ninhydrin assay and SEM will be performed to confirm protection of fiber topography after peptide conjugation, FTIR, XP and Contact angle analysis will be performed to confirm surface chemistry.
Primary Cell Culture and Cell derived ECM deposition: Human primary dermal fibroblasts (HDFs) were cultured in DMEM media with (FBS), and penicillin-streptomycin. Fibroblast derived matrices were generated by.
Cell Viability and Cytotoxicity of synthesized peptide molecules: To assess viability of cells on scaffolds, Calcein-AM (1 M) and Propidium Iodide (0.2 M) were added onto cells at 37 ˚C and incubated for 15 minutes followed by immediate imaging with a fluorescence microscope. Physical properties of collagen fibrils (alignment, density) will be analyzed from SEM images using NIH ImageJ. The cells will be analyzed for ECM deposition using Alcian Blue for HA and Type I Collagen immunostaining. Furthermore a PCR array targeting woung healing associated genes was performed to assess the therapeutic efficacy of HABP on fibroblast behavior.
Results, Conclusions, and Discussions:: PCL scaffolds are produced, and the fiber morphology is intact, the average fiber diameter was measured as 0.96±0.36 µm. Viability measurements between Ctrl and HABP group did not indicate statistically significant difference. Moreover, more than 90% viability was observed, with the total number of dead cells and live cells being similar for both groups. a-SMA expression was diminished on both uncoated and HABP coated nanofibers whilst the collagen analysis showed collagen fibers deposited on HABP nanofibers were longer, thinner and more randomly organized as opposed to collagen fibers deposited onto uncoated PCL scaffolds. We report these results would be beneficial in designing better strategies to address fibrotic capsule formation around soft implants particularly breast implants.
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References (Optional): : Corrales-Ureña, Y. R., Souza-Schiaber, Z., Lisboa-Filho, P. N., Marquenet, F., Noeske, P.-L. M., Gätjen, L., & Rischka, K. (2020). Functionalization of hydrophobic surfaces with antimicrobial peptides immobilized on a bio-interfactant layer. RSC Advances, 10(1), 376-386.
Noskovicova, N., Hinz, B., & Pakshir, P. (2021). Implant Fibrosis and the Underappreciated Role of Myofibroblasts in the Foreign Body Reaction. Cells, 10(7), 1794.
Singh, A., Corvelli, M., Unterman, S. A., Wepasnick, K. A., McDonnell, P., & Elisseeff, J. H. (2014). Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid. Nature materials, 13(10), 988-995.
