Silica-based bioactive glasses modulate expression of bone morphogenetic protein-2 mRNA in Saos-2 osteoblasts in vitro

doi:10.1016/S0142-9612(00)00288-X

Biomaterials

Volume 22, Issue 12, 15 June 2001, Pages 1475-1483

https://doi.org/10.1016/S0142-9612(00)00288-X Get rights and content

Abstract

A chemical exchange of the silica gel layer forming on the surface of bioactive glasses is thought to be the principal reaction for bone–bioactive glass bonding. The contribution of biological molecules on cell–bioactive glass interaction is largely unknown. To further analyze the mechanisms involved in efficient bone bonding to bioactive glass, Saos-2 osteoblastic cells with proven osteogenic phenotype were cultured for 4, 7 and 14 days on two bioactive glasses with different Si contents. Culture plates and dishes made of bioactive (BAG, 53% SiO₂), biocompatible (BCG, 58% SiO₂) and control (G0) glasses were extensively conditioned with phosphate buffer and DMEM medium before seeding the cells. Northern hybridization was used for analysis of mRNA levels of collagen type I (Col-I), alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2). A significant increase was observed in Col-I mRNA levels in cells grown on the two bioactive glasses when compared with those grown on controls at 4 and 7 days (p<0.04). The mRNA level for ALP in the cultures of bioactive glasses-made plates and dishes was also increased over control at 7 days (p<0.02) and remained this way between BAG and G0 at 14 days. Striking differences in BMP-2 mRNA levels existed between BAG and G0 plates and dishes at 7 days (p<0.05). BMP-2 mRNA level in BAG group was higher than in BCG group at 4, 7 and 14 days, but without statistical significance. Saos-2 osteoblastic cells with strong ALP staining were mostly seen on BAG plates under a light microscope. In confocal microscopy, a bright FITC-stained F-actin ring was present in the cytoplasm of cells grown on BAG dish, demonstrating an active functional status. Stimulation of the expression of BMP-2 and other bone mRNAs by bioactive glasses in osteoblastic cells suggests biological involvement of bone related growth factors, peptides and cytokines in bone – bioactive glass bonding.

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

References (27)

F.B. Bagambisa et al.
Cellular and molecular biological events at the implant interface
J Cranio Maxillo Fac Surg
(1994)
M. Metsaranta et al.
Specific hybridization probes for mouse type I, II, III and IX collagen mRNA
Biochim Biophys Acta
(1991)
W.C.A. Vrouwenvelder et al.
Better histology and biochemistry for osteoblasts cultured on titanium-doped bioactive glassbioglass 45S5 compared with iron-, titanium-, fluorine- and boron-containing bioactive glasses
Biomaterials
(1994)
L. Nissinen et al.
Bone morphogenetic protein-2 is a regulator of cell adhesion
Exp Cell Res
(1997)
W.C.A. Vrouwenvelder et al.
Biomaterials
(1992)
L.L. Hench et al.
Direct chemical bond of bioactive glass–ceramic materials to bone and muscle
J Biomed Mater Res (Symp)
(1973)
L.L. Hench et al.
Surface-active biomaterials
Sciences
(1984)
T. Kokubo
Surface chemistry of bioactive glass–ceramics
J Noncrystal Solids
(1995)
K.D. Lobel et al.
In vitro adsorption and activity of enzymes on reaction layers of bioactive glass substitutes
J Biomed Mater Res
(1998)
J. Wozney et al.
Novel regulators of bone formationmolecular clones and activities
Science
(1988)

E. Riley et al.

Bone morphogenetic protein -2

Clin Orthop

(1996)

E. Murry et al.

Characterization of a human osteoblastic osteosarcoma cell line(Saos-2) with high bone alkaline phosphatase activity

J Bone Miner Res

(1987)

M.M. Wong et al.

Long-term effects of physiological concentrations of dexamethasone on human bone-derived cells

J Bone Miner Res

(1990)

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Silica-based bioactive glasses modulate expression of bone morphogenetic protein-2 mRNA in Saos-2 osteoblasts in vitro

Abstract

Introduction

Section snippets

Bioactive Glasses

Northern Analyses

Discussion

Conclusion

Acknowledgments

J Cranio Maxillo Fac Surg

Biochim Biophys Acta

Biomaterials

Exp Cell Res

Biomaterials

Direct chemical bond of bioactive glass–ceramic materials to bone and muscle

J Biomed Mater Res (Symp)

Surface-active biomaterials

Sciences

Surface chemistry of bioactive glass–ceramics

J Noncrystal Solids

In vitro adsorption and activity of enzymes on reaction layers of bioactive glass substitutes

J Biomed Mater Res

Novel regulators of bone formationmolecular clones and activities

Science

Bone morphogenetic protein -2

Clin Orthop

Characterization of a human osteoblastic osteosarcoma cell line(Saos-2) with high bone alkaline phosphatase activity

J Bone Miner Res

Long-term effects of physiological concentrations of dexamethasone on human bone-derived cells

J Bone Miner Res