Bioactive glass induced osteogenic differentiation of human adipose stem cells is dependent on cell attachment mechanism and mitogen-activated protein kinases

Abstract

Bioactive glasses (BaGs) are widely utilised in bone tissue engineering (TE) but the molecular response of cells to BaGs is poorly understood. To elucidate the mechanisms of cell attachment to BaGs and BaG-induced early osteogenic differentiation, we cultured human adipose stem cells (hASCs) on discs of two silica-based BaGs S53P4 (23.0 Na 2 O -20.0 CaO -4.0 P 2 O 5 -53.0 SiO 2 (wt-%)) and 1-06 (5.9 Na 2 O -12.0 K 2 O -5.3 MgO -22.6 CaO -4.0 P 2 O 5 -0.2 B 2 O 3 -50.0 SiO 2 ) in the absence of osteogenic supplements. Both BaGs induced early osteogenic differentiation by increasing alkaline phosphatase activity (ALP) and the expression of osteogenic marker genes RUNX2a and OSTERIX. Based on ALP activity, the slower reacting 1-06 glass was a stronger osteoinducer. Regarding the cell attachment, cells cultured on BaGs had enhanced integrinβ1 and vinculin production, and mature focal adhesions were smaller but more dispersed than on cell culture plastic (polystyrene). Focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK)-induced c-Jun phosphorylations were upregulated by glass contact. Moreover, the BaG-stimulated osteoinduction was significantly reduced by FAK and mitogenactivated protein kinase (MAPK) inhibitors, indicating an important role for FAK and MAPKs in the BaGinduced early osteogenic commitment of hASCs. Upon indirect insert culture, the ions released from the BaG discs could not reproduce the observed cellular changes, which highlighted the role of direct cell-BaG interactions in the osteopotential of BaGs. These findings gave valuable insight into the mechanism of BaG-induced osteogenic differentiation and therefore provided knowledge to aid the future design of new functional biomaterials to meet the increasing demand for clinical bone TE treatments.

Bioactive glass ions for in vitro osteogenesis and microvascularization in gellan gum‐collagen hydrogels

“…As previously reported, the decrease of cell number has been associated with borosilicate BaG ionic species induced restriction of cell proliferation and accompanying strong osteogenesis (Ojansivu et al, ). Moreover, boron ion induced effect on cell morphology has also been indicated by previous research (Brown et al, ; Rodriguez, Gonzalez, Rios, & Cambiazo, ), together with observed changes to cytoskeleton, cell adhesion, and signaling pathways (Ojansivu et al, , ). To the point, we observed a similar effect with borosilicate bioactive glass 2‐06 based BaG EM‐OM condition with strong osteocalcin production along with cubic cell morphology producing less oriented structures and truncated tubular growth.…”

Human mesenchymal stem cells differentiate into an osteogenic lineage in presence of strontium containing bioactive glass nanoparticles

“…The differentiation of pluripotent murine embryonic stem cells to osteogenic cells was also stimulated, indicated by increased mineralized nodule formation, ALP activity, and osteoblast-associated gene expression (runx2/cbfa-1) when the cells were grown in a culture medium containing dissolution products from sol-gel 58S BG (60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5) [58]. Osteogenic differentiation of human adipose stem cells (hASCs) cultured on two compositions of bioactive glass discs, S53P4 BG (23. SiO2, wt%), was induced, as the ALP activity and the expression of osteogenic marker genes runx2a and osterix were stimulated [59,60]. Dissolution products of a sol-gel glass with higher phosphate content (60 mol% SiO2, 28 mol% CaO, 12 mol% P2O5) also provoked mineralisation and increased mRNA expression of the osteogenic differentiation markers ALP, osteopontin and osteonectin from human dental pulp stem cells (hDPSCs) [61].…”

Diopside‐tricalcium phosphate bioactive ceramics for osteogenic differentiation of human adipose stem cells

“…Such defects can be identified as the microcracks in TCP that propagate and coalesce under the effect of external stresses. In the single Schwab, & Spengler, 2009;Goldstein, 1987 and vinculin related intracellular mechanisms in hASCs leading to enhanced proliferation, Col-I production and osteogenesis (Ojansivu et al, 2018). Observations of the cross sections of the specimens after cell culture demonstrate the preferential colonization of the initial TCP zones in the composites.…”

“…Besides ionic compositions in growth medium, an important factor for osteogenic induction and ECM production of MSCs is the surface properties of the growth platform. Our recent results indicate that rougher bioactive glass surface, compared to smoother cell culture well, induces integrin and vinculin related intracellular mechanisms in hASCs leading to enhanced proliferation, Col‐I production and osteogenesis (Ojansivu et al, ). Observations of the cross sections of the specimens after cell culture demonstrate the preferential colonization of the initial TCP zones in the composites.…”

“…Blocking was done with 1% bovine serum albumin (Sigma‐Aldrich, St. Louis, MO) for 1 hr at +4°C. For further evaluation of the late osteogenic differentiation, maturation of extracellular matrix (ECM) and cellular actin organization with collagen‐I immunocytochemical staining and phalloidin staining were conducted as described elsewhere (Ojansivu et al, , ). Shortly, the primary antibody, mouse monoclonal anti‐collagen‐I (dilution 1:2000; Abcam, Cambridge, UK) and the secondary antibody donkey anti‐mouse Alexa fluor 488 IgG (Invitrogen, Thermo Fisher Scientific; dilution 1:800) were used.…”