Koichi Imai

In vitro embryotoxicity testing of metals for dental use by differentiation of embryonic stem cell test

ABSTRACT    We examined embryotoxicity using the embryonic stem cell test (EST) protocol. Tests were conducted using standard reagents for the atomic absorption measurement of 11 metal ions, silver, cobalt, chromium, copper, mercury, nickel, palladium, antimony, tin, vanadium, and zinc from among metals comprising dental alloys. In addition, for four metals like silver, cobalt, chromium, and nickel, the tests were also conducted using a test solution extracted from powder in the cell culture medium. The embryotoxic potential was obtained from a biostatistics‐based prediction model, which was calculated from three endpoints, the ID50, IC50ES and IC503T3. Data with the standard reagents showed that chromium and mercury ions corresponded to class 3, that is, having a strong embryotoxicity, while antimony, tin, and vanadium ions exhibited a weak embryotoxicity. The other metal ions demonstrated no embryotoxicity. On the other hand, when extracts of metal powder in cell culture solutions were used, silver exhibited a weak embryotoxicity while all other metals exhibited no embryotoxicity. In the future, it will be important to clarify the embryotoxicity of the many dental materials that are in use today. In addition, it is necessary to develop substances to ensure they have no toxicity before use in dental applications.

Local Controlled Release of Polyphenol Conjugated with Gelatin Facilitates Bone Formation

Catechins are extensively used in health care treatments. Nevertheless, there is scarce information about the feasibility of local administration with polyphenols for bone regeneration therapy, possibly due to lack of effective delivery systems. Here we demonstrated that the epigallocatechin-3-gallate-conjugated gelatin (EGCG/Gel) prepared by an aqueous chemical synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholinium chloride (DMT-MM) gradually disintegrated with time and facilitated bone formation in a critical size defect of a mouse calvaria. Conjugation of EGCG with the Gel generated cross-linking between the two molecules, thereby leading to a retardation of the degradation of the EGCG/Gel and to a delayed release of EGCG. The prepared EGCG/Gels represented significant osteogenic capability compared with that of the uncross-linked Gel and the cross-linked Gel with uncombined-EGCG. In vitro experiments disclosed that the EGCG/Gel induced osteoblastogenesis of a mouse mesenchymal stem cell line (D1 cells) within 14 days. Using fluorescently-labeled EGCG/Gel, we found that the fraction of EGCG/Gel adsorbed onto the cell membrane of the D1 cells possibly via a Gel-cell interaction. The interaction might confer the long-term effects of EGCG on the cells, resulting in a potent osteogenic capability of the EGCG/Gel in vivo. These results should provide insight into local controlled release of polyphenols for bone therapy.

Development of technique for in vitro embryotoxicity of dental biomaterials

Summary The Embryonic Stem Cell Test (EST) developed in Germany in 1997 is known as a screening test method capable of predicting the presence of unknown chemicals influencing normal human development. Firstly, we investigated the embryotoxicity of 24 types of monomer including dental monomers and dental alloy-component metal elements using this test. Monomers including Bis-GMA contained in base resin of composite resin exhibited weak embryotoxicity, and the toxicity level varied among dental alloy-component metal elements. It was clarified that metal ions eluted from currently sold dental alloys show no embryotoxicity. Then, we investigated a method that also considers human metabolic activity, which is not possible with the EST, in the results of embryotoxicity. In addition, an evaluation method using a hybrid culture system for hepatocytes and mouse ES cells and a method using oviduct or uterus cells for feeder cells were also investigated.