Heat or radiofrequency plasma glow discharge treatment of a titanium alloy stimulates osteoblast gene expression in the MC3T3 osteoprogenitor cell line

Abstract

PurposeThe purpose of this study was to determine whether increasing the Ti6Al4V surface oxide negative charge through heat (600℃) or radiofrequency plasma glow discharge (RFGD) pretreatment, with or without a subsequent coating with fibronectin, stimulated osteoblast gene marker expression in the MC3T3 osteoprogenitor cell line.MethodsQuantitative real-time polymerase chain reaction was used to measure changes over time in the mRNA levels for osteoblast gene markers, including alkaline phosphatase, bone sialoprotein, collagen type I (α1), osteocalcin, osteopontin and parathyroid hormone-related peptide (PTH-rP), and the osteoblast precursor genes Runx2 and osterix.ResultsOsteoprogenitors began to differentiate earlier on disks that were pretreated with heat or RFGD. The pretreatments increased gene marker expression in the absence of a fibronectin coating. However, pretreatments increased osteoblast gene expression for fibronectin-coated disks more than uncoated disks, suggesting a surface oxide-mediated specific enhancement of fibronectin's bioactivity. Heat pretreatment had greater effects on the mRNA expression of genes for PTH-rP, alkaline phosphatase and osteocalcin while RFGD pretreatment had greater effects on osteopontin and bone sialoprotein gene expression.ConclusionsThe results suggest that heat and RFGD pretreatments of the Ti6Al4V surface oxide stimulated osteoblast differentiation through an enhancement of (a) coated fibronectin's bioactivity and (b) the bioactivities of other serum or matrix proteins. The quantitative differences in the effects of the two pretreatments on osteoblast gene marker expression may have arisen from the unique physico-chemical characteristics of each resultant oxide surface. Therefore, engineering the Ti6Al4V surface oxide to become more negatively charged can be used to accelerate osteoblast differentiation through fibronectin-dependent and independent mechanisms.

Heat and radiofrequency plasma glow discharge pretreatment of a titanium alloy promote bone formation and osseointegration

“…The effects of fibronectin on trabeculae‐implant contacts were generally greater for RFGD‐pretreated implants and, in some cases, greater for heat‐pretreated implants compared to untreated specimens. When viewed within the context of our in vitro studies [Rapuano et al, ), our current results suggest that RFGD pretreatment and, to a lesser extent, heat pretreatment increased trabecular bonding partly by augmenting adsorbed fibronectin's intrinsic capacity to stimulate the differentiation of osteoblasts on the implant surface. In an osteogenesis‐favorable environment, osteblastic cells on the implant surface are thought to be capable of producing a repertoire of paracrine growth factors that would induce differentiation of osteoprogenitor cells in the surrounding tissue to form a mineralized matrix [Kieswetter et al, ].…”

“…This supposition is supported by FTIR findings showing areas of new trabecular bone formation near fibronectin‐coated implants signified by higher acid phosphate levels and lower mineral:matrix ratios [Spevak et al, ] compared to the surrounding trabecular or cortical bone. Moreover, we have shown that the pretreatments enhance matrix gene/osteoblast marker gene expression [Rapuano et al, ], cell‐directed mineralization [Rapuano et al, ], and trabecular bonding/implant shear strength (Figs. ) even in the absence of an exogenous fibronectin coating.…”

“…The differences between the two pretreatments of in their effects on osteoblast marker gene expression [Rapuano et al, ], cell‐directed mineralization [Rapuano et al, ], and osseointegration may have arisen from the unique physicochemical characteristics of each resultant oxide surface. We found that heat and RFGD pretreatments increased the Ti6Al4V oxide's net surface charge with the latter pretreatment exerting a greater effect [MacDonald et al, ].…”

“…Pretreating the surface oxide with heat or radiofrequency plasma glow discharge (RFGD) increased the oxide's negative net charge [MacDonald et al, ], markedly increased the number of osteoblasts that attached to surface adsorbed fibronectin [Rapuano and MacDonald, ], and increased the exposure of fibronectin's integrin binding domain to enhance its binding to α 5 β 1 integrins [Rapuano et al, ]. Later studies showed that fibronectin coating, heat and RFGD pretreatments each increased the expression of osteoblast gene markers, suggesting a stimulation of osteoblast differentiation [Rapuano et al, ]. Pretreatments also increased osteoblast gene expression for fibronectin‐coated disks more than uncoated disks, suggesting a pretreatment‐induced specific enhancement of fibronectin's bioactivity [Rapuano et al, ] putatively via conformational changes induced by the modified oxide [Rapuano et al, ].…”

“…Later studies showed that fibronectin coating, heat and RFGD pretreatments each increased the expression of osteoblast gene markers, suggesting a stimulation of osteoblast differentiation [Rapuano et al, ]. Pretreatments also increased osteoblast gene expression for fibronectin‐coated disks more than uncoated disks, suggesting a pretreatment‐induced specific enhancement of fibronectin's bioactivity [Rapuano et al, ] putatively via conformational changes induced by the modified oxide [Rapuano et al, ].…”