In vitro release of silver ions and expression of osteogenic genes by MC3T3-E1 cell line cultured on nano-hydroxyapatite and silver/strontium-coated titanium plates.

Abstract

Attempts are ongoing to improve the surface properties of dental implants by application of different coatings, aiming to enhance osseointegration, and decrease the adverse effects of titanium and its alloys used in dental implants. Coating of implant surface with hydroxyapatite (HA) is one suggested strategy for this purpose due to its high biocompatibility and similar structure to the adjacent bone. This study aimed to quantify the release of silver ions and expression of osteogenic genes by MC3T3-E1 cells cultured on nano-HA and silver/strontium (Ag/Sr)-coated titanium plates via the electrochemical deposition method. Plates measuring 10 × 10 × 0.9mm were fabricated from Ti-6Al-4V alloy, and polished with silicon carbide abrasive papers before electrochemical deposition to create a smooth, mirror-like surface. After applying homogenous nano-HA coatings with/without silver/strontium on the surface of the plates, the composition of coatings was confirmed by energy-dispersive X-ray spectroscopy (EDS), and their morphological properties were analyzed by scanning electron microscopy (SEM). The coated specimens were then immersed in simulated body fluid (SBF), and the concentration of released sliver ions was quantified by spectroscopy at 7-14days. The MC3T3-E1 osteoblastic cell line was cultured in osteogenic medium for 7-14days, and after RNA extraction and cDNA synthesis, the expression of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN); osteogenic genes was quantified by polymerase chain reaction (PCR) using SYBR Green Master Mix kit. The expression of genes and the released amount of silver ions were compared between the two groups using the Mann-Whitney U test. The two groups were not significantly different regarding silver ion release at 14days (P > 0.05). However, silver ion release was significantly higher from nano-HA coatings with silver/strontium at 7days (P = 0.03). The difference in expression of RUNX2 (P = 0.04), OPN (P = 0.04), and OCN (P = 0.03) genes was also significant between nano-HA coating groups with and without silver/strontium at 7days, and the expressions were higher in nano-HA with silver/strontium group, but this difference was not significant at 14days. Addition of silver and strontium to specimens coated with nano-HA increased the release of silver ions within the non-toxic range, and enhanced the expression of osteogenic genes particularly after 7days.