Activity of two hyaluronan preparations on primary human oral fibroblasts

Abstract

Background and objective: The potential benefit of using hyaluronan (HA) in reconstructive periodontal surgery is still a matter of debate. The aim of the present study was to evaluate the effects of two HA formulations on human oral fibroblasts involved in soft tissue wound healing/regeneration.

Material and methods: Metabolic, proliferative and migratory abilities of primary human palatal and gingival fibroblasts were examined upon HA treatment. To uncover the mechanisms whereby HA influences cellular behavior, wound healing-related gene expression and activation of signaling kinases were analyzed by qRT-PCR and immunoblotting, respectively.

Results: The investigated HA formulations maintained the viability of oral fibroblasts and increased their proliferative and migratory abilities. They enhanced expression of genes encoding type III collagen and transforming growth factor-β3, characteristic of scarless wound healing. The HAs upregulated the expression of genes encoding pro-proliferative, pro-migratory, and pro-inflammatory factors, with only a moderate effect on the latter in gingival fibroblasts. In palatal but not gingival fibroblasts, an indirect effect of HA on the expression of matrix metalloproteinases 2 and 3 was detected, potentially exerted through induction of pro-inflammatory cytokines. Finally, our data pointed on Akt, Erk1/2 and p38 as the signaling molecules whereby the HAs exert their effects on oral fibroblasts.

Conclusion: Both investigated HA formulations are biocompatible and enhance the proliferative, migratory and wound healing properties of cell types involved in soft tissue wound healing following regenerative periodontal surgery. Our data further suggest that in gingival tissues, the HAs are not likely to impair the healing process by prolonging inflammation or causing excessive MMP expression at the repair site.

Keywords: gene expression; growth factors; hyaluronic acid; oral soft tissue wound healing; pro-inflammatory cytokines.

Figure 1

Effects of hyaluronan (HA) preparations on the metabolism and behavior of primary human palatal (HPF) and gingival (HGF) fibroblasts. A, The two HA preparations exert no negative effects on the viability of primary oral fibroblasts. Viability of HPF and HGF cells incubated with various concentrations (0‐4 mg/mL) of each of the two HA preparations was assessed by measuring the metabolic capacity using the CellTiter‐Blue^® Cell Viability Assay. Viable cells retain the ability to reduce the indicator dye resazurin into highly fluorescent resorufin. Experimental values were normalized to those of control cells (0 mg/mL HA; 100% viability). Data represent means ± SD from three independent experiments. B and C, The two HA preparations strongly increase the proliferative and migratory abilities of primary oral fibroblasts. B, Proliferation rates of HA‐treated HPF and HGF cells were assessed by BrdU incorporation into newly synthesized DNA immediately after plating (0 h) as well as at 24, 48, 72, and 96 h. Means ± SD from three independent experiments and significant differences to control (ctrl) cells at the time point 0, ***P < 0.001, **P < 0.01 are shown. C, Migration of HPF and HGF cells toward HA was evaluated by transwell migration assay using filters with 8 μm pore size. Cell migration was quantified by measuring the area on the lower side of the filter covered with cells. Means ± SD from three independent experiments and significant differences to control cells, ***P < 0.001, **P < 0.01 are shown. Representative images of the staining in each of the experimental groups are shown below the bar graph. Scale bar, 500 μm

Figure 2

The two hyaluronan (HA) preparations trigger expression of COL3A1 and TGFB3 genes characterizing scarless wound healing. Effect of HA1 and HA2 on COL1A1 (A), COL3A1 (B), TGFB1 (C), and TGFB3 (D) mRNA levels in primary human palatal (HPF) and gingival (HGF) fibroblasts. Oral fibroblasts were treated with each of the two HA preparations for 24 h before total RNA was extracted and analyzed by qRT‐PCR. Values normalized to GAPDH are expressed relative to the values of untreated control (ctrl) cells. Data represent means ± SD from three independent experiments. Significant differences to the respective control unless otherwise indicated, ***P < 0.001, **P < 0.01, *P < 0.05

Figure 3

Hyaluronan (HA) induces expression of genes encoding growth factors and cytokines essential for the wound healing process in primary human palatal (HPF) and gingival (HGF) fibroblasts. Effect of HA1 and HA2 on PDGFB (A), FGF2 (B), EGF (C), IL1A (D), IL1B (E), and TNF (F) mRNA levels in HPF and HGF cells. Oral fibroblasts were treated with each of the two HA preparations for 24 h before total RNA was extracted and analyzed by qRT‐PCR. Values normalized to GAPDH are expressed relative to the values of untreated control (ctrl) cells. Data represent means ± SD from three independent experiments. Significant differences to the respective control unless otherwise indicated, ***P < 0.001, **P < 0.01, *P < 0.05

Figure 4

Hyaluronan (HA) induces cell type‐specific differences in the expression of MMP2 and MMP3 genes. Effect of HA1 and HA2 on MMP1 (A), MMP8 (B), MMP2 (C), and MMP3 (D) mRNA levels in primary human palatal (HPF) and gingival (HGF) fibroblasts. Oral fibroblasts were treated with each of the two HA preparations for 24 h before total RNA was extracted and analyzed by qRT‐PCR. Values normalized to GAPDH are expressed relative to the values of untreated control (ctrl) cells. Data represent means ± SD from three independent experiments. Significant differences to the respective control unless otherwise indicated, ***P < 0.001, **P < 0.01, *P < 0.05

Figure 5

Pro‐inflammatory cytokines exhibit a stimulatory and dose‐dependent effect on MMP2 and MMP3 gene expression in primary human oral fibroblasts. Effect of recombinant IL‐1α, IL‐1β, andTNF‐α on the expression of MMP genes in primary human palatal (HPF) and gingival (HGF) fibroblasts. Cells were treated with increasing concentrations (0, 5, and 10 ng/mL) of each of the three pro‐inflammatory cytokines for 24 h before total RNA was extracted. Expression of MMP1 (A), MMP8 (B), MMP2 (C), and MMP3 (D) mRNAs was analyzed by qRT‐PCR. Values normalized to GAPDH are expressed relative to the values of untreated cells (0 ng/mL HA). Means ± SD from three independent experiments and significant differences to untreated cells, ***P < 0.001, **P < 0.01, *P < 0.05 are shown

Figure 6

The two hyaluronan (HA) preparations significantly enhance phosphorylation of Akt, Erk1/2, and p38 kinases in primary human oral fibroblasts. Immunoblot analyses of phospho‐Akt (pAkt) (A), phospho‐Erk1/2 (pErk1/2) (B), and phospho‐p38 (pp38) (C) proteins in whole‐cell extracts from HA‐treated primary human palatal (HPF) and gingival (HGF) fibroblasts. Blots for total Akt, Erk1/2, and p38 proteins as well as the vinculin loading control are also shown. The bar charts represent densitometric quantifications of the immunoblots. pAkt, pErk1/2, and pp38 levels are normalized to the respective total proteins used as internal controls. Data represent means ± SD from three independent experiments. Significant differences to the respective control (ctrl) cells of each of the two cell types, **P < 0.01, *P < 0.05