Introduction and Specific Aim¶ Currently, titanium (Ti) implants are used successfully for tooth replacements in mandibular or maxillary bone (86% and 76% success rate, respectively [1]) and for bone replacement in the cranium (nearly 93% success rate [2]). However, faster bone integration and improvements in implant success rate and longevity are still needed. A new family of bioactive glasses (50-59 wt. % SiO ₂) may enhance the osteointegration potential of Ti [3-15]. The bioactive glasses acts as a functionally graded interface that remains well adherent to titanium [15] while also forming a hydroxyapatite surface layer and releasing calcium and silicon ions to osteoblasts for direct bone bonding [14, 16].¶ Interestingly, it has been discovered that these ions released to osteoblasts may play an active role in osteoblast function. Foppiano and colleagues [17] and Hench and colleagues [18-27] found these ions enhance osteoblast gene expression, yet, no significant effort on downstream matrix protein production has been established. Thus, this study tested the hypothesis that bioactive glass ions enhance osteogenesis and that a direct connection exists between enhanced gene expression and enhanced matrix protein synthesis. The key osteogenic markers to be investigated here are collagen type 1 alpha 1 (Col1α1) and alpha 2 (Col1α2), core binding factor a (Cbfa1/Runx2), alkaline phosphatase (ALP), and osteocalcin.¶ Materials and Methods¶ Melt-derived experimental bioactive glass (6P53-b) and commercial Bioglass (45S5) were used in this study by soaking glass specimens in cell culture medium to make glass conditioned medium (Table 1). The 6P53 b and 45S5 glass conditioned mediaum were measured for their ion concentrations using inductively coupled plasma mass spectrometry (ICP MS) prior to addition to mouse osteoblast progenitors (MC3T3 E1.4, at confluence in multi-well plates). The cells were induced to differentiate by adding ascorbic acid (50 μg mL^-1) to GCM and control media. Cells were lysed for both mRNA and protein for sample analysis. Gene expression (Col1α1, Col1α2, Runx2, osteocalcin) was measured using quantitative reverse transcription-polymer chain reaction (qRT PCR) and amplification curve results were analyzed using a sigmoidal curve fitting method [28]. Protein expression (ALP, osteocalcin) was measured using enzyme-linked immunosorbent assay (ELISA). Statistical comparisons (p < 0.05 for statistical significance) were made between GCM- and control-treated cells using two-way ANOVA (independent variables: time, treatment; dependent variables: gene, protein expression) and Tukey's for individual group comparisons. Results It was observed that increased silicon and calcium concentrations were contained in 45S5 and 6P53-b GCM as compared to control media (α-MEM) (Table 2). GCM treatment enhanced osteocalcin expression (Figure 1). The expression of osteocalcin peaked (Figure 1A) for cells treated with ascorbic acid and 6P53 b GCM (40x control after 10 days) and 45S5 GCM (70x control after 6 days). These significantly increased osteocalcin protein levels correlated with the increased osteocalcin mRNA levels within 7 days of treatment. While MC3T3-E1.4 cells differentiated, osteocalcin gene expression reached a maximum after 5 days in 6P53 b GCM (14x control) and after 3 days in 45S5 GCM (19x control) (Figure 1B). Other markers measured included: Runx2 levels were approximately 2x control after 7 days exposure to GCM, which confirmed the results of Foppiano and colleagues [17]. Alkaline phosphatase activity was also 2x control for both GCM treated cells after 10 days of exposure. Finally, Col1α1 expression was enhanced after 1 day of exposure (45S5 GCM: 3x control; 6P53 b GCM: 4x control) and throughout the course of differentiation (day 5, Col1α2, 45S5 GCM: 3.15x control; 6P53 b GCM: 2.35x control).¶ Conclusions¶ This study tested the hypothesis that bioactive glass ions enhance osteogenesis and that there is a direct connection between enhanced gene expression and protein matrix production. 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