Selected genes for testing DNA microarray outcomes in quantitative real-time PCR.

<p>Selected genes for testing DNA microarray outcomes in quantitative real-time PCR.</p

Cellular viability and proliferation after QMR treatment.

<p>A) Histograms represent the % of cellular viability after two cycles of QMR treatment at the different settings compared to the sham-exposed controls determined by flow cytometry. Data were shown as mean ± SD of three independent experiments; B) Percentages of cellular proliferation on the controls were obtained by WST-1 assay after 72 hours. Data were represented as mean ± SD of n = 6 independent experiments. No statistical differences were found between conditions.</p

MSC morphology and flow cytometry analysis after QMR stimulation.

<p>A) The images were obtained after 10 minutes of QMR stimulation at Day 5 (first cycle of treatment) and at B) Day 12 (second cycle of treatment). Scale bar = 100 μm. Total magnification = 100X. One representative experiment was shown. C) Five colour combination of monoclonal antibodies was used to verify MSC identity according to the above listed surface markers of a representative sample. Grey line = unstained control (CTL-). Blue line = sham-exposed control (CTL). Green line = QMR setting 80. Red line = QMR setting 40.</p

<i>In-vitro</i> analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells

<div><p>Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4–64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling.</p></div

Adipogenic, osteogenic and chondrogenic differentiation after QMR cycles of stimulation.

<p>Panels display one representative experiment showing the final outcome in MSC multilineage differentiation after 21 days of induction. QMR-treated (at 40 and 80 nominal powers) and untreated samples (CTL+) were induced to differentiation. Osteogenic differentiation after one cycle (A) and two cycles (B) of QMR stimulation was assessed using Alizarin Red. Adipogenic (C, D) and chondrogenic (E, F) differentiation were detected using Oil Red O and Alcian Blue stainings, respectively. Scale bar = 100 μm. Total magnification = 100x.</p

Relative gene expressions using quantitative real-time PCR.

<p>Expression of 8 genes selected by cDNA microarray was illustrated after n = 6 independent experiments using TBP as representative reference gene; mean ± SD; * p<0.05; ** p<0.01.</p

Best enrichment gene lists.

<p>Analysis of functional gene enrichment using ToppFun tool (application of ToppGene Suite) for A) up-regulated and B) down-regulated DEG between 40 QMR setting and control with significant enrichment (dotted line) for FDR B&H q-value <0.01); C) Comparative analysis of up-regulated (green bar) and down-regulated (red bar) functional gene enrichments using IPA software with significant enrichment (dotted line) for -log2 (B-H p-value) >2.</p

Decellularized esophageal tubular scaffold microperforated by quantum molecular resonance technology and seeded with mesenchymal stromal cells for tissue engineering esophageal regeneration

Current surgical options for patients requiring esophageal replacement suffer from several limitations and do not assure a satisfactory quality of life. Tissue engineering techniques for the creation of customized “self-developing” esophageal substitutes, which are obtained by seeding autologous cells on artificial or natural scaffolds, allow simplifying surgical procedures and achieving good clinical outcomes. In this context, an appealing approach is based on the exploitation of decellularized tissues as biological matrices to be colonized by the appropriate cell types to regenerate the desired organs. With specific regard to the esophagus, the presence of a thick connective texture in the decellularized scaffold hampers an adequate penetration and spatial distribution of cells. In the present work, the Quantum Molecular Resonance® (QMR) technology was used to create a regular microchannel structure inside the connective tissue of full-thickness decellularized tubular porcine esophagi to facilitate a diffuse and uniform spreading of seeded mesenchymal stromal cells within the scaffold. Esophageal samples were thoroughly characterized before and after decellularization and microperforation in terms of residual DNA content, matrix composition, structure and biomechanical features. The scaffold was seeded with mesenchymal stromal cells under dynamic conditions, to assess the ability to be repopulated before its implantation in a large animal model. At the end of the procedure, they resemble the original esophagus, preserving the characteristic multilayer composition and maintaining biomechanical properties adequate for surgery. After the sacrifice we had histological and immunohistochemical evidence of the full-thickness regeneration of the esophageal wall, resembling the native organ. These results suggest the QMR microperforated decellularized esophageal scaffold as a promising device for esophagus regeneration in patients needing esophageal substitution

Image2_Decellularized esophageal tubular scaffold microperforated by quantum molecular resonance technology and seeded with mesenchymal stromal cells for tissue engineering esophageal regeneration.jpg

Current surgical options for patients requiring esophageal replacement suffer from several limitations and do not assure a satisfactory quality of life. Tissue engineering techniques for the creation of customized “self-developing” esophageal substitutes, which are obtained by seeding autologous cells on artificial or natural scaffolds, allow simplifying surgical procedures and achieving good clinical outcomes. In this context, an appealing approach is based on the exploitation of decellularized tissues as biological matrices to be colonized by the appropriate cell types to regenerate the desired organs. With specific regard to the esophagus, the presence of a thick connective texture in the decellularized scaffold hampers an adequate penetration and spatial distribution of cells. In the present work, the Quantum Molecular Resonance® (QMR) technology was used to create a regular microchannel structure inside the connective tissue of full-thickness decellularized tubular porcine esophagi to facilitate a diffuse and uniform spreading of seeded mesenchymal stromal cells within the scaffold. Esophageal samples were thoroughly characterized before and after decellularization and microperforation in terms of residual DNA content, matrix composition, structure and biomechanical features. The scaffold was seeded with mesenchymal stromal cells under dynamic conditions, to assess the ability to be repopulated before its implantation in a large animal model. At the end of the procedure, they resemble the original esophagus, preserving the characteristic multilayer composition and maintaining biomechanical properties adequate for surgery. After the sacrifice we had histological and immunohistochemical evidence of the full-thickness regeneration of the esophageal wall, resembling the native organ. These results suggest the QMR microperforated decellularized esophageal scaffold as a promising device for esophagus regeneration in patients needing esophageal substitution.</p