Characterization of human vocal fold fibroblasts derived from chronic scar

Abstract

Objectives/hypothesis: In vitro modeling of cell-matrix interactions that occur during human vocal fold scarring is uncommon, as primary human vocal fold scar fibroblast cell lines are difficult to acquire. The purpose of this study was to characterize morphologic features, growth kinetics, contractile properties, α-smooth muscle actin (α-SMA) protein expression and gene expression profile of human vocal fold fibroblasts derived from scar (sVFF) relative to normal vocal fold fibroblasts (nVFF).

Study design: In vitro.

Methods: We successfully cultured human vocal fold fibroblasts from tissue explants of scarred vocal folds from a 56-year-old female and compared these to normal fibroblasts from a 59-year-old female. Growth and proliferation were assessed by daily cell counts, and morphology was compared at 60% confluence for 5 days. Gel contraction assays were evaluated after seeding cells within a collagen matrix. α-SMA was measured using western blotting and immunocytochemistry (ICC). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to assess differential extracellular matrix gene expression between the two cell types.

Results: sVFF were morphologically indistinguishable from nVFF. sVFF maintained significantly lower proliferation rates relative to nVFF on days 3 to 6 (day 3: P = .0138; days 4, 5, and 6: P < .0001). There were no significant differences in contractile properties between the two cell types at any time point (0 hours: P = .70, 24 hours: P = .79, 48 hours: P = .58). ICC and western blot analyses revealed increased expression of α-SMA in sVFF as compared with nVFF at passages 4 and 5, but not at passage 6 (passage 4: P = .006, passage 5: P = .0015, passage 6: P = .8860). Analysis of 84 extracellular matrix genes using qRT-PCR revealed differential expression of 15 genes (P < .01).

Conclusions: nVFF and sVFF displayed differences in proliferation rates, α-SMA expression, and gene expression, whereas no differences were observed in contractile properties or morphology. Further investigation with a larger sample size is necessary to confirm these findings.

FIGURE 1

nVFF (A) and sVFF (B) at passage 5. Both cell types exhibit spindle shape typical of fibroblasts (40X magnification). No differences can be noted between nVFF and sVFF.

FIGURE 2

Growth curves of nVFF and sVFF at passage 5. Means and standard deviations are the results for four replicated. Statistical significance is observed between groups on days 3-6 (Day 3: p = 0.0138, Days 4,5 & 6: p < .0001) as indicated by the asterisk.

FIGURE 3

Representative Western blots showing α-SMA expression and GAPDH for nVFF and sVFF at passages 4, 5, and 6. Graphs contain corresponding densitometric analysis of α-SMA which was normalized to GAPDH for each passage. Significance in upregulation was observed at passages 4 and 5 for sVFF (p = 0.0006 and p = 0.0015 respectively).

FIGURE 4

IHC images showing α-SMA expression in passage 5 nVFF (A) and sVFF (B) at 200x magnification. Green is indicative of α-SMA expression in representative cells whereas blue (DAPI) highlights the nucleus of the cell. sVFF exhibited greater expression of α-SMA than nVFF.

FIGURE 5

Contraction of fibroblast-populated collagen lattice with no cells (control), sVFF and nVFF. Each lattice (three replicates for each condition) was measured upon initial release of the collagen matrix, at 24 and 48 hours. Each point on the graph represents the average and standard deviation for three geometrical areas measured at each time point. Difference in contraction between sVFF and nVFF was not significant (p = 0.70, p = 0.79, p = 0.58) for any of the time points measured.