In vitro evaluation of a basic fibroblast growth factor-containing hydrogel toward vocal fold lamina propria scar treatment

Abstract

Scarring of the vocal fold lamina propria can lead to debilitating voice disorders that can significantly impair quality of life. The reduced pliability of the scar tissue-which diminishes proper vocal fold vibratory efficiency-results in part from abnormal extracellular matrix (ECM) deposition by vocal fold fibroblasts (VFF) that have taken on a fibrotic phenotype. To address this issue, bioactive materials containing cytokines and/or growth factors may provide a platform to transition fibrotic VFF within the scarred tissue toward an anti-fibrotic phenotype, thereby improving the quality of ECM within the scar tissue. However, for such an approach to be most effective, the acute host response resulting from biomaterial insertion/injection likely also needs to be considered. The goal of the present work was to evaluate the anti-fibrotic and anti-inflammatory capacity of an injectable hydrogel containing tethered basic fibroblast growth factor (bFGF) in the dual context of scar and biomaterial-induced acute inflammation. An in vitro co-culture system was utilized containing both activated, fibrotic VFF and activated, pro-inflammatory macrophages (MΦ) within a 3D poly(ethylene glycol) diacrylate (PEGDA) hydrogel containing tethered bFGF. Following 72 h of culture, alterations in VFF and macrophage phenotype were evaluated relative to mono-culture and co-culture controls. In our co-culture system, bFGF reduced the production of fibrotic markers collagen type I, α smooth muscle actin, and biglycan by activated VFF and promoted wound-healing/anti-inflammatory marker expression in activated MΦ. Cumulatively, these data indicate that bFGF-containing hydrogels warrant further investigation for the treatment of vocal fold lamina propria scar. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1258-1267, 2018.

Keywords: 3D cell culture; basic fibroblast growth factor; immunomodulation; vocal fold lamina propria; vocal fold scar.

Figure 1

Overall experimental design for the study. (A) First, scar and biomaterial-induced vocal fold phenotypes (VFF⁺ and MΦ⁺) were experimentally created through treatment of VFF⁻ and MΦ ⁻ with activation media (AM) containing TGF-β1 and LPS for 5 days. (B) Following confirmation of this activation protocol in 2D, more complex 3D studies were performed. The experimental groups analyzed to determine if bFGF tethered to PEGDA hydrogels can transition VFF⁺ towards a more normal phenotype in the presence of MΦ⁺ are depicted schematically.

Figure 2

Gene expression of a panel of markers representing pro-inflammatory, wound healing, or anti-inflammatory phenotypes in macrophages after 5 day activation with LPS/TGF-β1 (MΦ⁺) relative to MΦ ⁻. * denotes a significant difference, p < 0.05.

Figure 3

Relative protein levels of fibrotic tissue markers (Col 1, αSMA and Biglycan) and the anti-fibrotic marker MMP-1 in VFF following 72 h culture in various 3D PEGDA hydrogel experimental groups. *,#,$ denote a significant difference from the VFF⁺, bFGF/VFF⁺, and VFF⁺/MΦ⁺ groups, respectively.

Figure 4

Relative gene expression of a panel of markers representing pro-inflammatory, wound healing, or anti-inflammatory phenotypes in MΦ following 72 h culture in various 3D PEGDA hydrogel experimental groups. *,#,$ denote a significant difference from the MΦ⁺, bFGF/MΦ⁺, and MΦ⁺/VFF⁺ groups, respectively.