Antimicrobial Effects of Amelogenins on Oral Microorganisms

Abstract

Enamel matrix derivative (EMD) has been used during periodontal surgery for the regeneration of periodontal tissues. It is known that periodontal flap operation with Emdogain® Gel (Emd®-Gel), mainly composed of EMD, shows early healing and less inflammation compared to open flap debridement alone. One reason for this is thought to be that EMD has antimicrobial activity, however, the antimicrobial effect of EMD has not been clearly evaluated, because EMD is composed of multiple proteins such as amelogenin (AMEL), enamelin, seathlin and proteases. Especially, AMEL, which consists mainly of EMD (more than 90%), exists as an assembly of various molecular masses. The purpose of this study was to evaluate the antimicrobial effects of AMEL on oral microorganisms. EMD was extracted from the tooth germs of immature enamel in porcine fresh mandible. Twenty-five kDa porcine AMEL and its derivatives (20kDa, 13kDa and 6kDa) were purified by Sephadex G-100 gel filtration. Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans and Candida albicans were used for the antimicrobial test. Emd®-Gel, PGA, AMEL fractions and histatin 5 were used as test solutions. Each test solution was added to a calibrated suspension of microbes, and antimicrobial effects were examined based on the calculated number of colony forming units on an agar medium. Emd®-Gel showed antimicrobial effects on all microorganisms, and PGA also showed a strong antimicrobial effect on periodontal disease-related bacteria, but not on C. albicans. An antimicrobial action against P. gingivalis was found in 25kDa, 20kDa and 6kDa AMEL fractions of 250μg/ml, but the activities were low. Antimicrobial effect of AMEL was not found on P. intermedia and A. actinomycetemcomitans. Therefore, the antimicrobial effects of Emd®-Gel on periodontal disease-related bacteria seem to be due to the PGA. Meanwhile, all AMEL fractions showed a strong antimicrobial effect of concentration dependence on C. albicans. Antimicrobial effects of Emd®-Gel on C. albicans seem to be mainly caused by AMEL. However, it is possible that enamelin and seathlin may have antimicrobial effects, because EMD is composed of these proteins other than AMEL. Further studies are needed to elucidate the property of AMEL and other proteins in EMD to develop antimicrobial peptide and examine the availability of clinical application.