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. 2022 Feb;26(2):1773-1783.
doi: 10.1007/s00784-021-04152-8. Epub 2021 Aug 30.

Combination of enamel matrix derivative and hyaluronic acid inhibits lipopolysaccharide-induced inflammatory response on human epithelial and bone cells

Liza L Ramenzoni  1   2 , Laura Annasohn  3   4 , Richard J Miron  5 , Thomas Attin  3 , Patrick R Schmidlin  3   4
Affiliations

Combination of enamel matrix derivative and hyaluronic acid inhibits lipopolysaccharide-induced inflammatory response on human epithelial and bone cells

Liza L Ramenzoni et al. Clin Oral Investig. 2022 Feb.

Abstract

Objectives: The aim of this study was to evaluate the in vitro effect of enamel matrix derivative (EMD) and hyaluronic acid (HA) and their synergistic combination on lipopolysaccharides (LPS)-induced inflammation in human keratinocytes and osteoblasts.

Material and methods: Cells were challenged with LPS (1 μg/ml) and cultured in the following treatment groups with EMD (30 mg/ml) and HA (30 mg/ml): LPS, EMD, HA, EMD + HA, EMD + LPS, HA + LPS, and EMD + HA + LPS. Cell viability, inflammatory cytokine expression, and cell migration were determined using colorimetric assay, quantitative real-time polymerase chain reaction (qPCR), and scratch wound healing assay, respectively.

Results: Cell viability was decreased when exposed to LPS compared to the controls. Overall, LPS treatment expressed upregulation on inflammatory cytokine tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6). EMD and HA reduced up to 3.0-fold the cytokine expression caused by LPS (p < 0.05). EMD and HA statistically induced higher migration in osteoblasts and keratinocytes, respectively. Migration was impaired by LPS, whereas it significantly increased after addition of EMD and HA.

Conclusions: EMD and HA are advantageous biomaterials that individually generate strong directional migratory keratinocyte and osteoblast response. Their combination also enhances cell viability, and anti-inflammatory and migratory abilities to promote healing specially under LPS inflammatory stimulus. Future in vivo and animal research is necessary to further characterize the effect of EMD and HA on periodontal regeneration.

Clinical relevance: The use of EMD in conjunction with HA resulted in a reduction of inflammation and improvement of tissue healing at wound sites. Both biomaterials combined may potentially improve the effectiveness of bone regeneration in periodontal bone defects, pointing to the potential clinical relevance of both materials in regenerative periodontal surgery.

Keywords: Bone regeneration; Cell viability; Enamel matrix derivative; Hyaluronic acid; Oral wound healing; Pro-inflammatory cytokines.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Evaluation of cellular viability of keratinocytes (HEGK) (A) and osteoblasts (HOAS) (B). Graphic bars represent the percentage, with respect to control cells (untreated, 100%), of viable cells after 24 h exposure treatment to: (1) LPS (1 μg/ml); (2) EMD (30 mg/ml); (3) HA (30 mg/ml); (4) EMD (30 mg/ml) + HA (30 mg/ml); (5) EMD (30 mg/ml) + LPS (1 μg/ml); (6) HA (30 mg/ml) + LPS (1 μg/ml); (7) EMD (30 mg/ml) + HA (30 mg/ml) + LPS (1 μg/ml). Data show the mean ± SE (n = 3). Statistically significant with respect to the control according to one-way ANOVA; * p < 0.05. Y-axis = optical density
Fig. 2
Fig. 2
Expression of mRNA for inflammatory cytokines in HEGK challenged with EMD, HA, and LPS and their combinations. qPCR, with normalization to GAPDH using the Ct method analysis, are shown as means ± SD. A IL-1β; B TNF-α; C IL-6. The symbol * indicates a statistically significant increase in cytokine mRNA expression in comparison to non-challenged cells (two-way repeated measure ANOVA). * p < 0.05
Fig. 3
Fig. 3
Expression of mRNA for inflammatory cytokines in HOAS challenged with EMD, HA, and LPS and their combinations. qPCR, with normalization to GAPDH using the Ct method analysis, are shown as means ± SD. (A) IL-1β; (B) TNF-α; (C) IL-6. The symbol ✽ indicates a statistically significant increase in cytokine mRNA expression in comparison to non-challenged cells (two-way repeated-measures ANOVA). * p < 0.05
Fig. 4
Fig. 4
HEGK cell migration (scratch wound healing assay). Migratory ability of HEGK cells after culture with (1) LPS (1 μg/ml); (2) EMD (30 mg/ml); (3) HA (30 mg/ml); (4) EMD (30 mg/ml) + HA (30 mg/ml); (5) EMD (30 mg/ml) + LPS (1 μg/ml); (6) HA (30 mg/ml) + LPS (1 μg/ml); (7) EMD (30 mg/ml) + HA (30 mg/ml) + LPS (1 μg/ml). Images were recorded 24 h after wounding. Controls showed no or incomplete healing patterns and regarded as 0% wound closure. Representative images are shown from 3 independent experiments with brighter gray defined as areas lacking cells or wound area. ImageJ values of percentage wound closure mean ± SD, * p < 0.05. Scale bar: 50 μm
Fig. 5
Fig. 5
HOAS cell migration (scratch wound healing assay). Migratory ability of HEGK cells after 24 h culture with (1) LPS (1 μg/ml); (2) EMD (30 mg/ml); (3) HA (30 mg/ml); (4) EMD (30 mg/ml) + HA (30 mg/ml); (5) EMD (30 mg/ml) + LPS (1 μg/ml); (6) HA (30 mg/ml) + LPS (1 μg/ml); (7) EMD (30 mg/ml) + HA (30 mg/ml) + LPS (1 μg/ml). ImageJ values of percentage wound closure mean ± SD, * p < 0.05. Scale bar: 50 μm
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