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Non-porous monodispersed strontium containing bioactive glass (Si2O-CaO-SrO) nanoparticles (Sr- BGNPs), were synthesised using a modified Stöber process. Silica nanoparticles (Si-NPs) with diameters 90 ± 10 nm were produced through hydrolysis and polycondensation reactions of the silicon alkoxide precursor, tetraethyl orthosilicate (TEOS), prior to the incorporation of cations; calcium (Ca) and strontium (Sr), into the silica networks through heat treatment (calcination). Sr was substituted for Ca on a mole basis from non- (0SrBGNPs) to fullsubstitution (100SrBGNPs) in order to increase the amount of network modifiers in the Si-NPs. The different ratios of Si: Ca; 1:1.3 and 1:8.0, presented various elemental compositions (i.e. 77–92 mol% of SiO

We report that the release of manganese ions from bioactive glass provoked human mesenchymal stem cell (hMSC) differentiation down a bone pathway, whereas hMSCs exposed to the Mn-free glass did not differentiate. Bioactive glasses (BGs) are widely used for bone regeneration, and allow the incorporation of different ions with therapeutic properties into the glass network. Amongst the different ions with therapeutic benefits, manganese (Mn) has been shown to influence bone metabolism and activate human osteoblasts integrins, improving cell adhesion, proliferation and spreading. Mn has also been incorporated into bioceramics as a therapeutic ion for improved osteogenesis. Here, up to 4.4 mol% MnO was substituted for CaO in the 58S composition (60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5) and its effects on the glass properties and capability to influence the osteogenic differentiation were evaluated. Mncontaining BGs with amorphous structure, high specific surface area and nanoporosity were obtained. The presence of Mn 2+ species was confirmed by X-ray photoelectron spectroscopy (XPS). Mn-containing BGs presented no cytotoxic effect on human mesenchymal stem cells (hMSCs) and enabled sustained ion release in culture medium. hMSCs osteogenic differentiation stimulation and influence on the mineralisation process was also confirmed through the alkaline phosphatase (ALP) activity, and expression of osteogenic differentiation markers, such as collagen type I, osteopontin and osteocalcin, which presented higher expression in the presence of Mn-containing samples compared to control. Mn incorporation offers great promise for obtaining glasses with superior properties for bone tissue regeneration.

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A major challenge in bone tissue engineering is to develop patient-specific, defect-site specific grafts capable of triggering specific cell signaling pathways. We could programmably fabricate the 3D printed bone constructs via direct ink writing of silk-gelatin-bioactive glass (SF-G-BG) hybrids using two different compositions of melt-derived bioactive glasses (with and without strontium) and compared against commercial 45S5 Bioglass. Physico-chemical characterization revealed that released ions from bioactive glasses inhibited the conformational change of Bombyx mori silk fibroin protein (from random coil to β-sheet conformation), affecting printability of the SF-G-BG ink. In-depth molecular investigations showed that strontium containing SF-G-BG constructs demonstrated superior osteogenic differentiation of mesenchymal stem cells (TVA-BMSCs) over 21 days towards osteoblastic (marked by upregulated expression of runt related transcription factor, alkaline phosphatase, osteopontin, osteonectin, integrin bone sialoprotein, osteocalcin) and osteocytic (marked by podoplanin, dentin matrix acidic phosphoprotein, sclerostin) phenotype compared to other BG compositions and silk-gelatin alone. Moreover, ionic release from bioactive glasses in the silk-gelatin ink triggered the activation of signaling pathways (BMP-2, BMP-4 and IHH), which are critical in regulating bone formation in vivo. Overall, the presence of strontium containing bioactive glass in silk-gelatin matrices provided appropriate cues in regulating the development of custom-made 3D in vitro human bone constructs.