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Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces (TiO2) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected TiO2-seeded cells osteogenesis by interfering with selective calcium-related osteogenic pathways, and greatly enhanced hBM-MSCs osteogenic features such as the expression of early/late osteogenic genes and protein production (e.g., ALP, COL-I, osteocalcin and osteopontin) and ALP activity. Finally, PEMF-treated cells resulted to secrete into conditioned media higher amounts of BMP-2, DCN and COL-I than untreated cell cultures. These findings confirm once more the osteoinductive potential of PEMF, suggesting that its combination with TiO2 nanostructured surface might be a great option in bone tissue engineering applications. [ABSTRACT FROM AUTHOR]

The culture of progenitor mesenchymal stem cells (MSC) onto osteoconductive materials to induce a proper osteogenic differentiation and mineralized matrix regeneration represents a promising and widely diffused experimental approach for tissue-engineering (TE) applications in orthopaedics. Among modern biomaterials, calcium phosphates represent the best bone substitutes, due to their chemical features emulating the mineral phase of bone tissue. Although many studies on stem cells differentiation mechanisms have been performed involving calcium-based scaffolds, results often focus on highlighting production of in vitro bone matrix markers and in vivo tissue ingrowth, while information related to the biomolecular mechanisms involved in the early cellular calcium-mediated differentiation is not well elucidated yet. Genetic programs for osteogenesis have been just partially deciphered, and the description of the different molecules and pathways operative in these differentiations is far from complete, as well as the activity of calcium in this process. The present work aims to shed light on the involvement of extracellular calcium in MSC differentiation: a better understanding of the early stage osteogenic differentiation program of MSC seeded on calcium-based biomaterials is required in order to develop optimal strategies to promote osteogenesis through the use of new generation osteoconductive scaffolds. A wide spectrum of analysis has been performed on time-dependent series: gene expression profiles are obtained from samples (MSC seeded on calcium-based scaffolds), together with related microRNAs expression and in vivo functional validation. On this basis, and relying on literature knowledge, hypotheses are made on the biomolecular players activated by the biomaterial calcium-phosphate component. Interestingly, a key role of miR-138 was highlighted, whose inhibition markedly increases osteogenic differentiation in vitro and enhance ectopic bone formation in vivo. Moreover, there is evidence that Ca-P substrate triggers osteogenic differentiation through genes (SMAD and RAS family) that are typically regulated during dexamethasone (DEX) induced differentiation. [ABSTRACT FROM AUTHOR]

Objective. Bradykinin (BK) induces differentiation of lung fibroblasts into myofibroblasts, which play an important role in extracellular matrix remodeling in the airways of asthmatic patients. It is unclear whether this process is affected by antiasthma therapies. Here, we evaluated whether a glucocorticoid, budesonide (BUD), and a long-acting β2-agonist, formoterol (FM), either alone or in combination, modified BK-induced lung fibroblast differentiation, and affected the BK-activated intracellular signaling pathways. Methods. Human fetal lung fibroblasts were incubated with BUD (0.001-0.1 μM) and/or FM (0.0001-0.1 μM) before exposure to BK (0.1 or 1 μM). Fibroblast differentiation into α-smooth-muscle-actin-positive (α-SMA+) myofibroblasts, BK2 receptor (B2R) expression, extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation (p-ERK1/2), intracellular Ca2+ concentration ([Ca2+]i), and p65 nuclear factor kappa B translocation were evaluated. Results. BUD (0.1 μM) and FM (0.1 μM), either alone or in combination, completely inhibited BK-induced α-SMA protein expression and decreased the numbers of α-SMA+ fibroblasts, with a clear reduction in α-SMA stress fibers organization. BUD also completely inhibited the increase of B2R, whereas FM with or without BUD had no effect. BK-induced increases of [Ca2+]i and p-ERK1/2 were significantly reduced to similar levels by BUD and FM, either alone or in combination, whereas p65 translocation was completely inhibited by all treatments. Conclusion. Both BUD and FM, either alone or in combination, effectively inhibited the BK-induced differentiation of fibroblasts into α-SMA+ myofibroblasts and the intracellular signaling pathways involved in fibroblast activation. These results suggest that BUD and FM combination therapy has potential to inhibit fibroblast-dependent matrix remodeling in the airways of asthmatic patients. [ABSTRACT FROM AUTHOR]

Abstract: Transforming growth factor (TGF)-β may play a significant role in nasal polyposis pathogenesis, possibly through fibroblast activation. We studied the effects of two TGF-β isoforms (TGF-β1 and TGF-β2) on nasal polyposis fibroblasts by evaluating cell proliferation and differentiation into myofibroblasts. In addition, the inhibitory activity of different concentrations of fluticasone propionate (F.P.) was tested in this in vitro system. Primary nasal polyp tissue-derived fibroblasts were stimulated with different concentrations (1, 10 and 20ng/ml) of TGF-β1 and TGF-β2 for different incubation periods (24, 48 and 72h) and cell proliferation [3H thymidine ([3H]TdR) incorporation] and α-smooth muscle actin (α-SMA) expression (immunocytochemistry) was evaluated. The lowest concentration of TGF-β1 (1ng/ml) induced a significant increase in [3H]TdR incorporation at 48 and 72h (p <0.05, each comparison), while in the presence of TGF-β (10ng/ml) and TGF-β2 (1ng/ml) the enhancement in cell proliferation was significant only after 48h (p <0.05, each comparison with the unstimulated cells). In contrast, a significant increase in α-SMA expression was observed in the presence of the two highest concentration of both TGF-β isoforms, at 48 and 72h for TGF-β1 (p <0.05, each comparison), but only at 72h for TGF-β2 (<0.05, each comparison). Finally, at all concentrations tested, F.P. significantly inhibited the TGF-β1 and TGF-β2-induced 3HTdR incorporation (p <0.01, each comparison) and the α-SMA expression (p <0.05, each comparison). Thus, in vitro different concentrations of TGF-β1 and TGF-β2 appear to sequentially stimulate primary nasal polyp tissue-derived fibroblast proliferation and myofibroblast differentiation. These activities are effectively inhibited by F.P. [Copyright &y& Elsevier]

Exposure to Pulsed Electromagnetic Field (PEMF) has been shown to affect proliferation and differentiation of human mesenchymal stem cells derived from bone marrow stroma (BM-hMSC). These cells offer considerable promise in the field of regenerative medicine, but their clinical application is hampered by major limitations such as poor availability and the time required to differentiate up to a stage suitable for implantation. For this reason, several research efforts are focusing on identifying strategies to speed up the differentiation process. In this work we investigated the in vitro effect of PEMF on Ca2+-related mechanisms promoting the osteogenic differentiation of BM-hMSC. Cells were daily exposed to PEMF while subjected to osteogenic differentiation and various Ca2+-related mechanisms were monitored using multiple approaches for identifying functional and structural modifications related to this process. The results indicate that PEMF exposure promotes chemically induced osteogenesis by mechanisms that mainly interfere with some of the calcium-related osteogenic pathways, such as permeation and regulation of cytosolic concentration, leaving others, such as extracellular deposition, unaffected. The PEMF effect is primarily associated to early enhancement of intracellular calcium concentration, which is proposed here as a reliable hallmark of the osteogenic developmental stage. [ABSTRACT FROM AUTHOR]

Abstract: Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B2 receptor expression by Western blot analysis; cell proliferation by [3H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively. Constitutive bradykinin B2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B2 receptor expression in HBAFb. Bradykinin, via bradykinin B2 receptor, significantly increased fibroblast proliferation at lower concentration (10−11 M) and α-SMA expression/polymerization at higher concentration (10−6 M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation. These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients. [Copyright &y& Elsevier]