Evaluation of the biological effects of amelogenin on human oral keratinocytes

Amelogenins are clinically used in periodontal regeneration as main components of root surface modifying agents, even without specifically preventing the premature colonization of the healing tissue defect by means of a physical barrier membrane. The objective of this study was to investigate the effects of human amelogenin on the proliferation, migration, and morphology of Immortalized Human Oral Keratinocytes (iHOKs).

Immortalized Human Oral Keratinocytes were expanded in Keratinocyte Growth Medium-2 (KGM-2). Full-length recombinant amelogenin protein was diluted in KGM-2 in five concentrations (10 ng/ml, 100 ng/ml, 1.000 ng/ml, 5.000 ng/ml and 10.000 ng/ml). iHOKs were cultured in medium supplemented with the amelogenin dilutions. Samples without amelogenin served as control. Cell metabolism and cell proliferation together with cell migration were evaluated at day 7, 14, 21.

At day 7, iHOKs treated with 10,000 ng/ml showed a significant decrease in keratinocytes´ proliferation. The metabolic activity at this timepoint was significantly lower for concentrations ≥ 1000 ng/ml. At days 14 and 21, both the addition of 5000 ng/ml and even more 10,000 ng/ml amelogenin reduced significantly the proliferation of keratinocytes. The effects on the metabolic activity for these timepoints were visible already with 100 ng/ml. Treatment of iHOKs with amelogenin of ≥ 1000 ng/ml led to inhibitory effects on cell migration already after 24 h.

The full-length recombinant amelogenin has a significant biological impact on iHOKs. The increasing dose dependent inhibitory effects of amelogenin shown on iHOKs might explain the disruption of the apical migration of the junctional epithelium during regenerative healing.

Amelogenin, presents time- and dose-dependent inhibitory effects on the growth of keratinocytes, which might explain the biological rationale behind its application in periodontal regeneration.