In vitro evaluation of anti-fibrotic effects of select cytokines for vocal fold scar treatment

Abstract

Scarring of the vocal fold lamina propria (LP) can cause considerable voice disorders due to reduced pliability in scar tissue, attributed in part to abnormal extracellular matrix (ECM) deposition produced by the fibrotic vocal fold fibroblast (fVFF). Cytokines with anti-fibrotic potential have been investigated to limit abnormal LP ECM, but are limited by the need for repeat injections. Moreover, the potentially significant role played by activated macrophages (AMOs) is usually not considered even though the interaction between AMO and fibrotic fibroblasts is known to regulate scar formation across different tissues. AMO are also regulated by cytokines that are used for LP scar removal, but little is known about AMO behaviors in response to these cytokines within the context of LP scar. In the present study, we evaluated anti-fibrotic effects of hepatocyte growth factor (HGF), interleukin-10 (IL-10) and interleukin-6 (IL-6) in a 3D, in vitro fVFF-AMO co-culture system using poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Data from all cytokines was synthesized into a heat-map that enabled assessment of specific associations between AMO and fVFF phenotypes. Cumulatively, our results indicated that both HGF and IL-10 are potentially anti-fibrotic (reduction in fibrotic markers and enhancement in normal, anti-fibrotic VFF markers), while IL-6 displays more complex, marker specific effects. Possible associations between AMO and fVFF phenotypes were found and may highlight a potential desirable macrophage phenotype. These data support the therapeutic potential of HGF and IL-10 for LP scar treatment, and shed light on future strategies aimed at targeting specific AMO phenotypes. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1056-1067, 2019.

Keywords: 3D cell culture; cytokine treatment; fibroblasts; macrophages; vocal fold scar.

FIGURE 1.

Overall experimental design for the study. (A) Scar and biomaterial-induced vocal fold phenotypes (fVFF and AMO) were experimentally induced with activation media (AM) containing TGF-β1 and LPS for 4 days. (B) Effects of various cytokines on fVFF and AMO were tested utilizing 3D PEGDA hydrogels. The transition of fVFF to a more normal phenotype was examined in mono- and co-cultures containing select, tethered cytokines (HGF, IL-10, and IL-6). Data from all treatments (HGF, IL-10, and IL-6) are examined to find patterns of associations between fVFF and AMO phenotypes.

FIGURE 2.

Relative protein expression of a panel of markers indicating fVFF and AMO phenotype following 72 h culture in various 3D PEGDA hydrogel experimental groups with or without addition of HGF. (A) Fibrotic and anti-fibrotic/healthy markers were utilized to assess fVFF phenotype. (B) Pro-inflammatory, wound healing, or anti-inflammatory/pro-resolving markers were utilized to assess AMO phenotype. * denotes a significant main effect resulting from HGF treatment relative to no treatment. # denotes a significant main effect of the co-culture relative to mono-culture.

FIGURE 3.

Relative protein expression of a panel of markers indicating fVFF and AMO phenotype following 72 h culture in various 3D PEGDA hydrogel experimental groups with or without addition of IL-10. (A) Fibrotic and anti-fibrotic/healthy markers were utilized to assess fVFF phenotype. (B) Pro-inflammatory, wound healing, or anti-inflammatory/pro-resolving markers were utilized to assess AMO phenotype. * denotes a significant main effect resulting from IL-10 treatment relative to no treatment. # denotes a significant main effect of the co-culture relative to mono-culture.

FIGURE 4.

Relative protein expression of a panel of markers indicating fVFF and AMO phenotype following 72 h culture in various 3D PEGDA hydrogel experimental groups with or without addition of IL-6. (A) Fibrotic and anti-fibrotic/healthy markers were utilized to assess fVFF phenotype. (B) Pro-inflammatory, wound healing, or anti-inflammatory/pro-resolving markers were utilized to assess AMO phenotype. * denotes a significant main effect resulting from IL-6 treatment relative to no treatment. # denotes a significant main effect of the co-culture relative to mono-culture.

FIGURE 5.

Heat-map summarizing cytokine effects on fVFF (top) and AMO (bottom) phenotypic markers. Color code was determined based off significance values as denoted in “Materials and Methods” section. Red and blue colors represent an increase or decrease of the protein in response to cytokine treatment, respectively.

FIGURE 6.

Reorganized heat-map to illustrate potential associations between fVFF and AMO phenotypes. Color code was determined based off significance values as denoted in “Materials and Methods” section. In this heat-map, red and blue colors indicate a change in the direction of the markers associated with a given phenotype (i.e. pro-fibrotic markers) in response to cytokine treatment.