Effect of varying physical properties of porous, surface modified bioactive glass 45S5 on osteoblast proliferation and maturation.

The objective of this study was to determine the effect of porous bioactive glass (45S5) substrate characteristics on the expression and maintenance of the osteoblastic phenotype. We cultured ROS 17/2. 8 cells on substrates with different pore size and porosity for periods up to 14 days and analyzed the characteristics of the cells and extracellular matrix. Results of the study show that the glass substrates supported the proliferation and growth of osteoblast-like cells. Although the morphologies of the cells differed on the various substrates, their shape and the extent of membrane ruffling suggested that they maintained high levels of metabolic activity. Cells on all substrates expressed high levels of alkaline phosphatase activity and produced extracellular matrices that mineralized to form nonstoichiometric, carbonated, calcium-deficient apatites. An important finding was that at a given porosity of 44%, the pore size neither directed nor modulated the in vitro expression of the osteoblastic phenotype. In contrast, porosity did affect cellular function. We noted that at an average pore size of 92 microm, as the porosity increased from 35 to 59%, osteoblast activity was reduced. As designed in this experiment, an increase in the porosity led to a corresponding increase in total surface area of the specimens. With increasing porosity and surface area, glass reactions in the media may persist for longer durations at higher intensities, thereby affecting local media composition. As such, we suggest that extensive conditioning treatments before cell seeding can reduce this effect. Our results also revealed that the expression of the osteoblastic phenotype is enhanced by the ongoing glass dissolution. The reaction pathway at the origin of this effect still needs to be elucidated. Taken together, the findings support the overall hypothesis that in vitro cell activity can be controlled by a careful selection of substrate properties.

Initial events at the bioactive glass surface in contact with protein-containing solutions.

Upon implantation, bioactive glass undergoes a series of reactions that leads to the formation of a calcium phosphate-rich layer. Most in vitro studies of the changes that occur on the surface of bioactive glass have employed the use of buffer solutions with compositions reflecting the ionic composition of interstitial fluid. Although these studies have documented the physical and chemical changes associated with bioactive glass immersed in aqueous media, they do not reveal the effect of serum proteins and cells that are present at the implantation site. In the present study, we document, using atomic force microscopy (AFM) and Rutherford backscattering spectrometry (RBS), significant differences in the reaction layer composition, thickness, morphology, and kinetics of formation arising from the presence of serum proteins. The data reveal that the uniform and rapid adsorption of serum proteins on the surface may serve to protect the surface from further direct interaction with the aqueous media, slowing down the transformation reactions. This finding is in agreement with previous studies that have shown that the presence of serum proteins significantly delays the formation of hydroxyapatite at the surface of bioactive glass. These data also support the hypothesis that initial reaction layers in vivo interact with cells in order to produce the tissue-bioactive glass interface typically observed on ex vivo specimens.

Evaluation of osteoblast response to porous bioactive glass (45S5) substrates by RT-PCR analysis.

Previous studies have shown that neonatal rat calvaria osteoblasts elaborate substantial amounts of extracellular material with bone-like characteristics when cultured on porous bioactive glass substrates in vitro. However, the osteoblastic response to this material has not been fully characterized. The objective of this study was to characterize osteoblast response to porous bioactive glass substrates following the expression of the classical markers for osteoblast differentiation. In this study we synthesized porous bioactive glass substrates, seeded them with osteoblast-like cells (ROS 17/2.8) and followed the temporal expression of alkaline phosphatase (AP) activity, as well as the expression of mRNA for collagen type I (Coll-1), osteonectin (OSN), osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP). The data confirm that porous bioactive glass substrates are capable of supporting the in vitro growth and maturation of osteoblast-like cells. At a porosity of 42% and an average pore size of 80 microm, the substrates promote the expression and maintenance of the osteoblastic phenotype. The results additionally suggest that there is both a solution-mediated and a surface-controlled effect on cell activity.