An in vitro assessment of the response of THP-1 macrophages to varying stiffness of a glycol-chitosan hydrogel for vocal fold tissue engineering applications

Abstract

The physical properties of a biomaterial play an essential role in regulating immune and reparative activities within the host tissue. This study aimed to evaluate the immunological impact of material stiffness of a glycol-chitosan hydrogel designed for vocal fold tissue engineering. Hydrogel stiffness was varied via the concentration of glyoxal cross-linker applied. Hydrogel mechanical properties were characterized through atomic force microscopy and shear plate rheometry. Using a transwell setup, macrophages were co-cultured with human vocal fold fibroblasts that were embedded within the hydrogel. Macrophage viability and cytokine secretion were evaluated at 3, 24, and 72 hr of culture. Flow cytometry was applied to evaluate macrophage cell surface markers after 72 hr of cell culture. Results indicated that increasing hydrogel stiffness was associated with increased anti-inflammatory activity compared to relevant controls. In addition, increased anti-inflammatory activity was observed in hydrogel co-cultures. This study highlighted the importance of hydrogel stiffness from an immunological viewpoint when designing novel vocal fold hydrogels.

Keywords: fibroblast; hydrogel; immunomodulation; macrophage; stiffness.

FIGURE 1

Transwell setup for the ([Mφ + VFF + hydrogel]) culture. Mφ were seeded on the basolateral transwell membrane and VFFs were embedded in the hydrogel. For (Mφ + hydrogel), VFFs were excluded. For (Mφ + VFF), the hydrogel was excluded. For (Mφ), VFFs, and the hydrogel were excluded. DMEM, Dulbecco’s modified Eagle’s medium; Mφ, macrophages; VFFs, vocal fold fibroblasts

FIGURE 2

The (a) storage modulus and (b) loss modulus of the three glyoxal cross-linked glycol-chitosan hydrogels assessed using shear plate rheometry. Glyoxal cross-linker concentrations used were 0.005, 0.01, and 0.02%

FIGURE 3

(a) Interaction between culture type and Time on TNF-α detected (co-culture: N = 36; monoculture N = 36); (b) Main effect of hydrogel presence on TNF-α concentrations (hydrogel absent control group: N = 18; hydrogel present groups: N = 54). Data are graphed as concentration normalized to total protein levels (pg/g protein). The bars and error bars represent the mean and standard error of the data respectively. *p < .05, ***p < .001, ****p < .0001

FIGURE 4

Three-way interaction between cross-linker concentration, Time and culture type on TNF-α detected in hydrogel cultures. Data is graphed as concentration normalized to total protein levels (pg/g protein). The bars and error bars represent the mean and standard error of the data respectively (N = 9 per co/monoculture group). *p < .05, **p < .01, ***p < .001, ****p < .0001

FIGURE 5

(a) Interaction between hydrogel presence and time on IL-10 detected (Absent: N = 18; Present: N = 54); (b) Interaction between culture type and time on IL-10 detected (co-culture: N = 36; monoculture = 36). Data are graphed as concentration normalized to total protein levels (pg/g protein). The bars and error bars represent the mean and standard error of the data respectively. ***p < .001, ****p < .0001

FIGURE 6

Three-way interaction between cross-linker concentration, time and culture type on IL-10 detected in hydrogel cultures. Data is graphed as concentration normalized to total protein levels (pg/g protein). The bars and error bars represent the mean and standard error of the data respectively (N = 9 per co/monoculture group). **p < .01, ***p < .001, ****p < .0001

FIGURE 7

Live/dead confocal images of Mφ on the underside of the basal membrane following culture with glyoxal cross-linked glycol-chitosan hydrogels. Panel A displays Mφ from (Mφ + VFF + hydrogel). Panel B displays Mφ from (Mφ + hydrogel). Live cells fluoresce green, dead cells fluoresce red. Cell density and distribution varied to an extent across the membrane surface and between samples. All images were taken at ×100 original magnification (scale bar = 100 μm). Mφ, macrophages; VFFs, vocal fold fibroblasts

FIGURE 8

Interaction between culture type and hydrogel presence for Mφ viability (%). The bars and error bars represent the mean Mφ viability and standard error of the data respectively (Absent: N = 18; Present: N = 54). **p < .01. Mφ, macrophages

FIGURE 9

Representative examples of the quadrant gates drawn to determine upregulations of (a) CD80 or (b) CD206 in comparison to relevant FMO controls. The example shown is for a hydrogel co-culture (0.005% glyoxal) sample. Quadrant names refer to whether that given population expressed none, one, or both markers. Numbers refer to the percentage of cells from the multistained samples only that were found in a given quadrant population. Contour plots (5% levels) in FlowJo were examined prior to gate drawing to assist with defining upregulated populations. Color key: Red = Multistain (A, B); Blue = FMO (80) (A) FMO (206) (B); Orange = FMO (33) (A, B). FMO, fluorescence minus one