Bone regeneration around N-acetyl cysteine-loaded nanotube titanium dental implant in rat mandible

Abstract

New strategies involving drugs loading onto implant surfaces are required to enhance osseointegration and shorten healing time after implantation. In this study, we examined the feasibility of N-acetyl cysteine (NAC)-loaded nanotube titanium (NLN-Ti) implants as a potential drug delivery system. To determine the effect of NLN-Ti in in vitro and in vivo, viability and ROS formation was assessed and enzyme-linked immunosorbant assay (ELISA), Western blot, micro-computed tomography (μ-CT), hematoxylin and eoxin (H&E) staining and immunohistochemical (IHC) analysis were done. In vitro, cell viability was increased and inflammatory responses and reduced oxidative stress-related defense were decreased with MC 3T3-E1 cells exposed to a sustained release of NAC from NLN-Ti implants. Following NLN-Ti implant installation, μ-CT revealed an increase of newly formed bone volume and bone mineral density in the mandibles of Sprague Dawley rats. Relatively well formed new bone was demonstrated in close contact to the NLN-Ti implant surface by H&E staining. IHC revealed significantly higher expression of bone morphogenetic protein-2, -7 and heme oxygenase-1, and reduced expression of receptor activator of nuclear factor-kappa B ligand. The data indicate that NLN-Ti implants enhance osseointegration and highlight the value of the small animal model in assessing diverse biological responses to dental implants.

Introduction

Enhanced osseointegration and shortened healing time are required to ensure a direct bone-to implant anchorage. However, some metabolic diseases including diabetes mellitus adversely affect the biological performance of titanium (Ti) implants, such as osteoconductive capacity [1], [2]. Various techniques have been used to modify surface roughness, topography, chemistry and electrical charge to improve the biocompatibility and osseointegration of Ti implants because these factors are important in the biological and clinical success of implants [3], [4]. Among the modifications to the Ti surface, the nanotube structure fabricated by anodic oxidation has demonstrated accelerated osteoblast adhesion or proliferation, and can be used as a carrier for drugs and anti-bacterial agents [5], [6]. Furthermore, the nanotube structure is an excellent biomaterial with a high surface area-to-volume ratio and controllable dimensions for improved biocompatibility. The use of different-sized nanotubes has demonstrated an enhanced osteoblast function [7], [8]. Although nanotubes have demonstrated benefits for bone cell–material interaction, the biological performance of nanotube structures concerning aspects like delivery of drugs, chemical compounds and bio-molecules from the implant to the bone needs to be investigated for their use in clinical applications and dental implants.

Post-implantation, the generation of inflammation and its outcomes are major obstacles for implant success. It is also important to prevent the occurrence of subsequent implant failure due to a chronic inflammatory response to implant-derived wear particles or osteogenic cell stress [9], [10]. Some pathological inflammatory conditions occur from insufficiency of the cellular anti-oxidant capacity and lead to excessive production of reactive oxygen species (ROS) as well as the inflammation of the bone. ROS have been reported to be involved in bone resorption by reducing the bone mineral density [11]. Although Ti has an excellent biocompatibility, it also increases intracellular ROS levels. The harmful effects of ROS cause potential biological damage such as chronic inflammation and reduced bone regeneration [12], [13]. Hence, surface treatment of implants with anti-oxidants that are used as pharmacological agents is considered to reduce the inflammation via ROS scavenging, thereby enhancing bone regeneration and healing around the implant.

N-acetyl cysteine (NAC) is a cell-permeable glutathione derivative and a cysteine analog drug with multiple therapeutic applications. NAC promotes the glutathione redox cycle as it is an amino acid derivative and an anti-oxidant [14]. NAC also acts as a direct ROS scavenger by increasing the cellular glutathione levels. The glutathione redox cycle is regarded as the most important regulatory mechanism for controlling oxidative stress [15]. A previous study reported that NAC-loaded pure Ti surfaces had the potential to eliminate ROS [12]. Hence, surface treatment of implants with NAC may reduce implant-induced inflammation and promote faster bone regeneration.

The conventional protocol of drug loading onto an implant surface is challenging in terms of achieving the desired cell and bone growth. Several techniques proposed for the modification of implant surfaces involve alteration of surface topography. The topography of a surface is related to the degree of biocompatibility. We previously reported that modification of the surface topography of TiO₂ nanotubes with anodization increased osteoblast cell responses such as proliferation and mineralization [16]. However, the effect of drug delivery and drug release from the TiO₂ nanotubes is unclear in vitro and after in vivo implantation.

In this study, we examined whether the NAC-loaded nanotube Ti (NLN-Ti) implants can be used as a potential drug delivery system and can ensure sustained release of NAC from the implant to the bone in Sprague Dawley rat mandibles after implantation.