Silica-based bioactive glasses modulate expression of bone morphogenetic protein-2 mRNA in Saos-2 osteoblasts in vitro
Introduction
Silica-based bioactive glasses have attracted a great deal of interest as promising bone substitutes. Studies on their effects range widely from basic material sciences and cell biology to clinical investigations. Initially, the working principle of the bioactive glasses or glass ceramics was considered to be a chemical reaction of the silica gel layer forming on the surface of the bioactive glass, resulting in bone bonding [1], [2]. A layer of hydroxycarbonate apatite (HCA) was observed to be rapidly deposited on the surface of the silica gel as a result of exchange between calcium and silica ions when bioactive glass came into contact with living tissues or was immersed in simulated body fluid. HCA crystallites were found to nucleate and bond to osteoblast cell products, both collagens and noncollageneous proteins, thus forming a direct chemical bond between bioactive glass and bone structure [3]. With this unique property, bioactive glasses and glass ceramics have been clinically used in orthopaedic, oral, maxillofacial, and otological surgery for bone and cartilage reconstruction.
Recently, the concept of biological surface modification has opened new insights into biomaterial engineering. It is well known that protein absorption is an early in vivo event in the interaction between implanted biomaterial and living tissue. Thereafter, cellular responses will determine the role of the biomaterial in the tissue reaction and/or functional replacement as well as the fate of the biomaterial itself, such as biocompatibility, degradation and bioactivity [4]. Moreover, it has been demonstrated that bioactive glass has no osteoinductivity. Among the different proteins absorbed on bioactive glasses or glass ceramics, fibronectin has been thought to be an important molecule for cell adhesion and to trigger further biological reactions in surrounding cells [5]. However, fibronectin is not specific for signaling cells towards osteogenic differentiation. It has been assumed that other cytokines or growth factors adsorbed on the surface of bioactive glass could also influence the cell – cell and cell – bioactive glass interactions, and result in biological bonding between bone and bioactive glass. Understanding the effects of bioactive glasses on cell behavior and expression of osteogenic molecules forms the basis for further biological surface modification of bioactive glasses.
Bone morphogenetic proteins (BMPs) have been recognized to act as morphogens and mitogens for osteogenesis during embryonic development and in postnatal bone formation [6], [7]. BMPs also play multiple roles in cell signaling and cell–cytokine interactions. Since bioactive glass is a biomaterial with specific bone affinity, we hypothesized that bioactive glass may affect cell behavior by regulating the expression of BMPs. The purpose of this study was to compare and determine whether two different kinds of bioactive glasses influence the expression of BMP-2 mRNA in an osteoblastic cell line with proven BMPs expression in vitro.
Section snippets
Bioactive Glasses
Two bioactive glasses of different compositions were prepared for the experiments: bioactive glass (BAG) containing 6% Na2O, 12% K2O, 20% CaO, 4% P2O5, 5% MgO and 53% SiO2, and biocompatible glass (BCG) containing 6% Na2O, 12% K2O, 15% CaO, 4% P2O5, 5% MgO and 58% SiO2 (wt%). For control, inert glass (G0) was made by melting commercial glassware. BAG and BCG glasses were made by mixing analytical-grade Na2CO3, K2CO3, MgO, CaCO3, H3BO3 and CaHPO4·2H2O and commercial Belgian quartz sand. The
Northern Analyses
The mRNA levels of characteristic osteoblastic markers in total RNA isolated from Saos-2 cells cultured on BAG, BCG and G0 glass-made plates and dishes were determined using cDNA probes for proα1(I) collagen and alkaline phosphatase (Fig. 1). The results demonstrated that mRNA levels of proα1(I) collagen and alkaline phosphatase were higher in cells cultured on BAG and BCG plates than on control plates at days 4, 7 and 14 (Fig. 2A and C). A similar finding was made with cells cultured in BAG
Discussion
In the present study, the two bioactive glasses with different silica contents both demonstrated a capacity to induce Saos-2 cells to express an osteogenic phenotype. The high mRNA level of collagen type I, a major organic component of mineralized bone matrix, indicated an active functional status of Saos-2 cells grown on BAG and BCG bioactive glasses. Although the mRNA levels of collagen type I were not identical in Saos-2 osteoblasts grown on BAG and BCG bioactive glasses, the change in the
Conclusion
The results of the present study clearly show that expression of the osteogenic phenotype, i.e. mRNA for alkaline phosphatase and collagen type I, was enhanced in Saos-2 osteoblasts cultured on plates and dishes made of bioactive glass. Two bioactive glasses with different Si contents stimulated BMP-2 mRNA level of Saos-2 osteoblasts in the culture. This finding suggests that the Si component of the bioactive glass selectively activates some osteogenic genes of osteoblasts. Enhanced BMP-2
Acknowledgments
The authors are grateful to Prof. K. Väänänen and Dr. H.B. Zhao for the confocal microscopy facilities at the Department of Anatomy, and Dr. Q. Qu for ALP staining. The technical assistance of Jukka Morko and Merja Lakkisto in the hybridization experiments, and of Liisa Peltonen and Marita Potila in cell cultures is also acknowledged. The research project was financially supported by the Academy of Finland (Project No 37311) and partially by Medical Research EVO-Grant of Turku University
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