Biocompatibility of a synthetic extracellular matrix on immortalized vocal fold fibroblasts in 3-D culture

Abstract

In order to promote wound repair and induce tissue regeneration, an engineered hyaluronan (HA) hydrogel - Carbylan GSX, which contains di(thiopropionyl) bishydrazide-modified hyaluronic acid, di(thiopropionyl) bishydrazide-modified gelatin and polyethylene glycol diacrylate - has been developed for extracellular matrix (ECM) defects of the superficial and middle layers of the lamina propria. The purpose of this study was to evaluate the biocompatibility of Carbylan GSX in a previously established immortalized human vocal fold fibroblast (hVFF) cell line prior to human clinical trials. Immortalized hVFF proliferation, viability, apoptosis and transcript analysis for both ECM constituents and inflammatory markers were measured for two-dimensional and three-dimensional (3-D) culture conditions. There were no significant differences in morphology, cell marker protein expression, proliferation, viability and apoptosis of hVFF cultured with Carbylan GSX compared to Matrigel, a commercial 3-D control, after 1 week. Gene expression levels for fibromodulin, transforming growth factor-beta1 and tumor necrosis factor-alpha were similar between Carbylan GSX and Matrigel. Fibronectin, hyaluronidase 1 and cyclooxygenase II expression levels were induced by Carbylan GSX, whereas interleukins 6 and 8, Col I and hyaluronic acid synthase 3 expression levels were decreased by Carbylan GSX. This investigation demonstrates that Carbylan GSX may serve as a natural biomaterial for tissue-engineering of human vocal folds.

Figure 1

Electrophoresis of PCR products for each pair of real-time PCR primers. PCR products for each gene match a single band of predicted size as in Table 1 confirming the primer specificity for each gene.

Figure 2

Morphologic appearance and fibroblast marker (hPH) distribution of immortalized hVFFs in various culture environments. After 72h of in vitro culture, fluorescent images of hVFF on polystyrene (A), on a thin layer of Carbylan GSX (2D condition) or in Carbylan GSX (3D condition) were captured by an inverted confocal microscope equipped with dual excitation lasers for green and blue fluorescence.

Figure 3

The distribution of hPH (A) and ICAM-1 (B) in hVFF cultured in 3D Matrigel and Carbylan GSX environments. After 72h of in vitro culture, the fluorescent images of hVFF in Matrigel and Carbylan GSX (3D condition) were captured by an inverted confocal microscope equipped with dual excitation lasers for red, green and blue fluorescence.

Figure 3

The distribution of hPH (A) and ICAM-1 (B) in hVFF cultured in 3D Matrigel and Carbylan GSX environments. After 72h of in vitro culture, the fluorescent images of hVFF in Matrigel and Carbylan GSX (3D condition) were captured by an inverted confocal microscope equipped with dual excitation lasers for red, green and blue fluorescence.

Figure 4

Proliferation rates of immortalized hVFF cells in various culture conditions. The cells were initially seeded at 2×10³/well of 96-well plate and cell number was quadruplicated determined by CellTiter-Glo Luminescence Cell Viability assay over the period of 7 days. The ATP RLU is proportional to the number of viability cells. (A) Cell proliferation rates on polystyrene, 2D Carbylan GSX and in 3D Carbylan GSX; (B) Cell proliferation rates in 3D Matrigel and Carbylan GSX: similar proliferation rates were shown for Carbylan GSX and Matrigel. * cell proliferation rate in 2D Carbylan shown significantly lower comparing to polystyrene (p<0.01); ** cell proliferation rate in 3D Carbylan GSX was significanty lower comparing to polystyrene (p<0.01).

Figure 4

Proliferation rates of immortalized hVFF cells in various culture conditions. The cells were initially seeded at 2×10³/well of 96-well plate and cell number was quadruplicated determined by CellTiter-Glo Luminescence Cell Viability assay over the period of 7 days. The ATP RLU is proportional to the number of viability cells. (A) Cell proliferation rates on polystyrene, 2D Carbylan GSX and in 3D Carbylan GSX; (B) Cell proliferation rates in 3D Matrigel and Carbylan GSX: similar proliferation rates were shown for Carbylan GSX and Matrigel. * cell proliferation rate in 2D Carbylan shown significantly lower comparing to polystyrene (p<0.01); ** cell proliferation rate in 3D Carbylan GSX was significanty lower comparing to polystyrene (p<0.01).

Figure 5

Live/Dead viability/apoptosis assay of immortalized hVFFs seeded in various culture conditions at Day 3 and Day 7. Y-axis indicates the live cell % in total cell population. (A) Total live cell percentage on polystyrene and 2D Carbylan GSX, and in 3D Carbylan GSX; (B) Total live cell percentage in 3D Matrigel and Carbylan GSX. * p<0.05, ** p<0.01

Figure 6

Gene expression levels (mRNA) of COX2, IL-6, IL-8 and TNF-α for immortalized hVFFs cultured in/on Carbylan GSX or Matrigel at Day 7. Y-axis demonstrates fold of control (polystyrene or Matrigel) of each target gene (mRNA concentration ng/μl), normalized by housekeeping gene, β-actin mRNA (ng/μl). (A) Polystyrene was used as control and mRNA level on polystyrene after seven days of culture was defined as 1.0. Y-axis showed the fold change of mRNA levels in 2D and 3D Carbylan GSX; (B) Matrigel was used as control and mRNA level in Matrigel after seven days of culture was defined as 1.0. The y-axis showed the fold change of mRNA levels in 3D Carbylan GSX. ** p<0.01

Figure 7

ECM related gene expression levels (mRNA) of collagen type I α-2 (Col I), fibronectin (FN), fibromodulin (FMOD), hyaluronic acid synthase 3 (HAS3), hyaluronidase 1 (HYAL1) and TGF-β1 in immortalized hVFFs responding to Carbylan GSX and Matrigel at Day 7. Y-axis shows the fold of control (polystyrene or Matrigel) of each target gene (mRNA concentration ng/μl), normalized by housekeeping gene, β-actin mRNA (ng/μl). (A) Tissue culture polystyrene was used as control and mRNA level on polystyrene after seven days of culture was defined as 1.0. Y-axis demonstrates fold change of mRNA levels in 2D and 3D Carbylan GSX: Carbylan GSX; (B) Matrigel was used as control and mRNA level in Matrigel after seven days of culture was defined as 1.0. Y-axis demonstrates fold change of mRNA levels in 3D Carbylan GSX. * p<0.05, ** p<0.01.