Electro-magnetic field promotes osteogenic differentiation of BM-hMSCs through a selective action on Ca(2+)-related mechanisms

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

Development of label-free biophysical markers in osteogenic maturation

The spatial and temporal changes of morphological and mechanical properties of living cells reflect complex functionally-associated processes. Monitoring these modifications could provide a direct information on the cellular functional state. Here we present an integrated biophysical approach to the quantification of the morphological and mechanical phenotype of single cells along a maturation pathway. Specifically, quantitative phase microscopy and single cell biomechanical testing were applied to the characterization of the maturation of human foetal osteoblasts, demonstrating the ability to identify effective label-free biomarkers along this fundamental biological process

Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients

Background: Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms. Methods: The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13). Results: In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter. Conclusions: This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD. © 2012 Pace et al

Controlled single-cell cyclic compression and transcription analysis: a pilot study

An innovative platform for the study of the molecular mechanisms at the basis of mechanotransduction has been implemented, developing an experimental approach capable of providing controlled dynamic compression stimuli and retrieving the biomolecular response with single-cell sensitivity. The system provides the ability to perform compression-release cycles on single cells with controlled forces in the nN range and a user-defined repetition rate. Experimental procedures to perform qPCR from a small set of single cells were finely tuned. The experimental platform was tested in the context of bone (cell line hFOB 1.19), a physiological environment highly subjected to mechanical stimuli. Target genes were identified in the literature, based on their involvement in the osteogenesis process or in the bone response to mechanical stimuli. qPCR analysis shows an increase in expression of the chosen targets, and confirms the effectiveness of the presented approach for studying living single cells response to dynamic compression

Osteogenic Differentiation of MSC through Calcium Signaling Activation: Transcriptomics and Functional Analysis.

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

Cell adhesion.

<p>SEM image representing mesenchymal stem cells adhering to the HA surface. Bar is 10 micron.</p

Up/down-regulated genes grouped into main biological processes.

<p>Up/down-regulated genes grouped into main biological processes.</p

Cardinality of mRNA Datasets.

<p>Cardinality of mRNA Datasets.</p

miRNA Heatmaps.

<p>Heatmaps of differentially expressed miRNA, obtained using logFC = 1 and pValue<0.05 thresholds, measured for cases A, B and C.</p