Surface functionalization of titanium surfaces grafted with hyaluronic acid through alendronate self-assembling monolayers

摘要

Introduction: H6Al4V alloy is widely used to manufacture dental and orthopaedic implants due to its biocompatibility, corrosion resistance, low modulus and good fatigue strength, among other desirable properties. The surface characteristics of implants govern rapid and stable integration with bone. In a bid to improve osseointegration different approaches have been investigated such as air blasting, acid etching, sol-gel and chemical treatments and more recently, grafting of extracellular matrix (ECM) compounds are being used to enhance biological properties This study presents the modification of Ti6Al4V alloy via chemisorption of alendronate self-assembling monolayers (SAMs) from aqueous solutions. These have the ability to bind biomimetic ECM such as partially oxidized hyaluronic acid (HA) derivatives to induce differentiation of human mesenchymal stem cells (hMSC) into bone cell phenotypes and promote bone formation. Experimental Methods: Hyaluronic acid was converted to an aldehyde derivative (HA-Ald) by controlled oxidation of glucuronic units with sodium periodate (10 mol%) and neutralized with ethylene glycol. The product was exhaustively dialysed against water, freeze-dried and characterised by 1H NMR. Alkali etched Ti6Al4V discs were used to form SAM's by solvent casting on the surface from aqueous alendronate solution followed by evaporation. Ald-HA was grafted afterwards on SAMs by Schiff base reaction through the free amines from alendronate and aldehyde groups of Ald-HA followed by gently washing with DI water and isopropanol. SEM microscopy accompanied by energy dispersive X-ray analysis (EDX) was used to obtain high resolution imaging and composition. Surface chemistry was further determined by RAMAN and X-ray photoelectron spectroscopy (XPS). Cell viability and osseoinductive capacity of the substrates was studied in vitro by assessing the expression of differentiation markers of hMSCs cultured onto the surface treated discs. Results and Discussion: Biomimetic ECM grafted on Ti6Al4V were effectively obtained and the modified surfaces were found to exhibit higher wettability than the native Ti6Al4V, with regular surface topography and tailored surface chemistry. XPS analysis revealed formation of a homogenous distribution of alendronate along the surface through Ti-O-P bonds. The alendronate self-assembly monolayers formed on the Ti alloy surfaces led to the availability of the pendant amino groups permitting further surface modification. The Ald-HA was formed by a Schiff-base reaction between the amino group of the alendronate and aldehyde groups of the partially oxidized hyaluronic acid. Cell morphology (SEM and actin staining using Phalloidin) on the treated Ti surfaces (Figure 1), showed a more polygonal morphology indicative of a differentiating stem cell compared to the normal elongated stem cell morphology observed on the untreated Ti surface, however hMSC's cultured on treated Ti6Al4V surfaces showed lower cell attachment and proliferation than observed on non-treated surfaces. Total RNA yield obtained from the treated Ti discs were also higher than the untreated group and results from ongoing qRT-PCR analysis will give an indication of the differentiation potential of the cells on the biomimetic Ti surfaces.