3-D Fibrin Scaffold Improves Stemness of Human Peripheral Blood Endothelial Progenitor Cells

Aims Fibrin is a natural biopolymer appealing for cell-based regenerative therapies, because it can support growth, migration and differentiation of different cell types. Endothelial progenitor cells (EPC) represent a very interesting alternative cell source for mature endothelial cells; the fact that can easily isolated from the peripheral blood, thereby eliminating donor morbidity, makes them ideal in applications in the field of regenerative medicine. We have demonstrated that fibrin can support EPC viability and growth. Aim of this study was to evaluate if fibrin can affect EPC differentiation and stem cell markers expression. Methods Fibrin was prepared mixing commercially available (Kedrion S.p.A. Lucca, Italy) fibrinogen (9 mg/ml) and thrombin (25 U/ml). Clot ultrastructure was investigated by scanning electron microscopy (SEM) and cryogenic SEM (CRYO-SEM) to measure fibre diameter and density. Clot elasticity was evaluated by atomic force microscopy (AFM), measuring the tip-sample force by cantilever displacement. EPC were obtained from peripheral blood and cultured on fibrin at the concentration of 1x106cell/cm2. Fibronectin coating was used as a control. Metabolic activity was assessed after 7 and 14 days by WST1 assay and viability by confocal microscopy (calcein incorporation). The expression of both endothelial (CD31, KDR, vWF, Ve-Cadherin) and stem cell markers (nanog, oct-4) was assessed by flow cytometry, confocal microscopy and Real Time RT-PCR. Results SEM analysis revealed a nanometric fibrous structure, with mean fiber diameter of 165?4 nm and mean density of 95.9?0.2 %. CRYO-SEM suggested a reticulate structure with mesh-size up to 10 ?m. Fibrin clot elasticity was 1.78 MPa, as in literature. WST1 assay showed that fibrin increased EPC metabolic activity as compared to fibronectin (fibrin: 0.606?0.056 a.u. vs. fibronectin: 0.311?0.067). Calcein staining demonstrated that EPC were still viable at 14 days. Flow cytometry showed the expression of endothelial markers (CD31=41.8?8.4%; vWF=32.3?3.0%; KDR=89.3?3.7%; VE-Cadherin=41.2?3.8%), confirmed also by confocal microscopy and Real Time RT-PCR. Interestingly, nanog and oct-4 (embryonic stem cell markers) expression was significantly greater on fibrin (p<0.001) as compared to fibronectin. Conclusions These findings suggest that fibrin it is not only a suitable scaffold for EPC growth and viability but also induces EPC differentiation. The observation that Nanog, known as the most important marker of stemness, is maintained longer than on fibronectin, may offer a surplus value to stem cell-based therapies

Development of a new technology for 3-D nanostructured scaffolds with potential cardiovascular applications

Aims The in situ release and maintaining of cells to promote revascularization is a new goal of cardiovascular therapy. Endothelial progenitor cells (EPC) may contribute to the process of vascular repair. Medical devices realized according to tissue engineering are composed by a cellular component and by an artificial component, usually made of a biocompatible polymer. Scaffolds may be coated with bio-polymers like fibrin to enhance cell adhesion and growth. Aim of this study was to realize nanocomposite 3D scaffolds composed by a synthetic polymer coated with fibrin to support EPC growth and to promote in vivo angiogenesis. Methods 3D PEtU-PDMS scaffolds were studied in vitro for their biocompatibility (viability and proliferation tests; citokine release). In vivo biocompatibility was studied by intramuscular implant in a rabbit model. The scaffolds were fabricated by spray-phase inversion technique. 25U/mL thrombin was sprayed during the fabrication process. The composite scaffold was then incubated o.n. at 37?C with 18mg/mL fibrinogen. The scaffold morphology was analysed by stereo-microscopy and by scanning electron microscopy (SEM). EPC obtained from peripheral blood were cultured for 1 week on the scaffolds at the concentration of 1x106 cell/ml. Fibronectin coating was used as a control. Cell viability was assessed by confocal laser (Calcein-AM incorporation). To test in vivo angiogenesis, EPC-seeded scaffolds were subcutaneously implanted into the back of rats for 14 days. After harvesting, the scaffolds were examined histologically and immunohistochemically to evaluate inflammatory response and neovascularization. Results In vitro and in vivo biocompatibility data demonstrated absence of any citotoxic effect, immunocompatibility and a slight inflammatory reaction without any sign of encapsulation and implant rejection. Morphological analyses showed an homogeneus fibrin coating of the scaffolds, tightly bound and interconnected to the PEtU-PDMS surface. SEM showed the presence of a well organized layer of fibres in a nm scale (mean diameter ~140nm). Cell viability and phenotype were not affected when EPC were seeded on PEtU-PDMS/fibrin scaffolds. The histological observation of explanted scaffolds revealed a slightly inflammatory response and a significant increased numbers of neovessels in tissues surrounding the EPC-seeded scaffold as compared to the scaffold without cells. Conclusions Our data suggest that PEtU-PDMS/fibrin scaffold obtained with a new spray manufacturing technology can support in vitro EPC growth and promote in vivo neovascularisation. Further studies are currently under way in an ischemic hindlimb rat model

A dynamic clamping approach using in silico IK1 current for discrimination of chamber-specific hiPSC-derived cardiomyocytes

: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach&nbsp;improves the translational relevance of the hiPSC-CM model for studying mechanisms underlying inherited or acquired atrial arrhythmias in human CM, and for screening anti-arrhythmic agents

Spontaneous synchronous bilateral hemothorax as the only finding in primary pleural angiosarcoma: a case report and a literature review.

Angiosarcoma is a rare malignancy of vascular origin, mostly originating from skin, soft tissues, and breast, but rarely also from the pleura. We present the case of a 55-year-old man who referred to our hospital for a spontaneous bilateral hemothorax. The CT angiography did not show any source of active bleeding; plus, no pleural or lung masses were observable. Cytological and microbiological analyses made on a sample of pleural fluid resulted negative. Despite numerous blood transfusions and thoracenteses, the patient deceased from hemorrhagic shock ten days later and the diagnosis of primary pleural epithelioid angiosarcoma was obtained only by autopsy. Additionally, we present a review of the literature about primary pleural angiosarcomas

Development of a new technology for 3-d nanostructured scaffolds with potential cardiovascular applications

Aims The in situ release and maintaining of cells able to promote revascularization is a new goal of cardiovascular therapy. Medical devices realized according to tissue engineering are composed by a cellular component and by an artificial component (scaffold) supporting the cells, usually made of a biocompatible polymer. Scaffolds may be coated with bio-polymers like fibrin in order to enhance cell adhesion and growth. Increasing in vitro and in vivo evidence indicates that endothelial progenitor cells (EPC) may contribute to the process of vascular repair. The goal of this study was to realize nanocomposite 3D scaffolds composed by a synthetic polymer coated with fibrin able to support EPC growth and to promote in vivo angiogenesis. Methods 3D poly(ether)urethane–polydimethylsiloxane (PEtU-PDMS) scaffolds were studied in vitro for their biocompatibility by viability and proliferation tests on L929 cells and citokine release determination on monocytes. In vivo biocompatibility studies were performed by intramuscular implant in a rabbit model. The scaffolds were fabricated using PEtU-PDMS and fibrin, by spray-phase inversion technique. Briefly, to reach a deep permeation of fibrin into the wall thickness, a thrombin solution (25 U/mL) was sprayed, during the fabrication process. At the end, the composite thrombin-PEtU-PDMS scaffold was incubated overnight at 37°C with a fibrinogen solution (18 mg/mL). The surface morphology of the 3D nanostructured scaffold was analysed by stereo-microscopy observation, after the protein-specific Ponceau Red staining and by scanning electron microscopy (SEM) observation. EPC were obtained from peripheral blood of healthy donors and cultured for 1 week on the scaffolds at the concentration of 1x106 cell/ml in endothelial growth medium containing 5 FBS and specific growth factors. Cell viability was assessed by confocal laser (Calcein-AM incorporation). As a control a 20 µg/ml fibronectin coating was used. To test in vivo angiogenesis, EPC-seeded scaffolds were subcutaneously implanted into the back of rats for 14 days. After harvesting, the implanted scaffolds were examined histologically (H&E staining) and immunohistochemically to evaluate inflammatory response and neovascularization. Results In vitro and in vivo biocompatibility data demonstrated absence of any citotoxic effect, good immunocompatibility and a slight inflammatory reaction without any sign of encapsulation and implant rejection. Morphological analyses showed that the scaffolds presented an homogeneus fibrin coating, with suitable thickness, tightly bound and interconnected to the PEtU-PDMS surface below. Besides, SEM observation showed the presence of well organized layer of fibrin fibres in a nanometric scale (mean diameter ~ 140 nm). Cell viability and phenotype were not affected when EPC were seeded on PEtU-PDMS/fibrin scaffolds instead of fibronectin. The histological observation of explanted scaffolds revealed a slightly inflammatory response and a significant increased numbers of neovessels in tissue surrounding the implanted EPC-seeded scaffold as compared to the control (scaffold without cells) Conclusions Our data suggest that PEtU-PDMS/fibrin nanostructured scaffold obtained with a new spray manufacturing technology can support in vitro EPC growth and promote in vivo neovascularisation. Further studies are currently under way in an ischemic hindlimb rat model

Comparison of digital PCR systems for the analysis of liquid biopsy samples of patients affected by lung and colorectal cancer

Background and aims: Highly sensitive technologies are available for the molecular characterization of solid tumors, including digital PCR (dPCR). Liquid biopsy, based on the analysis of cell-free DNA (cfDNA), is often used to assess EGFR or RAS alterations in lung and colorectal cancers. Our study aimed to compare the results of two different dPCR platforms for the detection of mutations in cfDNA. Methods: Plasma samples from lung and colorectal cancer patients collected as per routine procedures have been tested. cfDNA Was extracted from plasma, and samples were screened on the droplet digital PCR (ddPCR, BioRad) and solid dPCR QIAcuity (Qiagen). Results: A total of 42 samples were analyzed, obtained from 20 Non-Small Cell Lung Cancer (NSCLC) patients carrying an EGFR or a KRAS mutation on tissue at diagnosis, and from 22 samples of colorectal cancer (CRC) patients, 10 of which presenting a KRAS mutation. EGFR mutation detection was 58.8% for ddPCR and 100% for dPCR (κ&nbsp;=&nbsp;0.54; 95% CI, 0.37-0.71), compared to tissue results. The detection rate for RAS mutations was 72.7% for ddPCR and 86.4% for dPCR (κ&nbsp;=&nbsp;0.34; 95% CI, 0.01-0.68), compared to tissue results. Conclusions: This study showed moderate agreement between dPCR and ddPCR. Sampling effect or threshold settings may potentially explain the differences in the cfDNA data between the two different platforms