Fibrin gel: a new scaffold for cardiovascular applications

Aims: Peripheral blood endothelial progenitor cells (EPC) are promising therapies for irreversible myocardial damage, heart failure and peripheral ischemia disease. Natural biopolymers as fibrin are appealing in tissue engineering, because fibrin is biocompatible and bioresorbable. In vitro studies indicate that fibrin can support the growth migration and proliferation of several cells types. Up to date numerous studies have proved the potential of fibrin based injectable cell delivery systems. No studies are available with fibrin as scaffold for EPC. The goal of this study was to investigate if fibrin is a suitable matrix for EPC culture as compared with fibronectin and if different concentrations of fibrinogen (Fb) and thrombin (Th) can influence fibrin structure and EPC behaviour. Methods: Fibrin (Kedrion S.p.a. Lucca, Italy) was prepared mixing Fb (final 4.5-9-18-36 mg/ml) and Th (final 6-12.5-25-50 U/ml). The scaffolds were maintained for 1 hour at 37?C, 5% CO2 before cell seeding. The ultrastructure of fibrin was investigated by scanning electron microscopy (SEM), cryogenic SEM (CRYO-SEM) and atomic force microscopy (AFM) that allow the hydratating analysis of the sample, to evaluate fibre diameter and density. EPC were obtained from peripheral blood of healthy donors and cultured for 1 week on fibrin at the concentration of 1x106 cell/ml in endothelial growth medium. EPC seeded on fibronectin were used as control. Metabolic cell activity on the different scaffolds was assessed after 7 and 14 days by WST1 while cell viability by confocal microscopy (Calcein AM incorporation). Results: Fibrin polymerization rate ranged between 17 and 68 seconds and increased at higher Fb or Th concentrations. Both AFM and SEM analysis revealed a nanometric fibrous structure, with a decrease in fiber diameter with higher fibrinogen concentrations (4.5 mg/ml: 166?4 nm. vs. 36 mg/ml: 119?3 nm, p<0.005, n=5). Different concentrations of Th didn\u27t affect fibre diameter and density. CRYO-SEM suggested a reticulate structure with mesh-size up to 10?m. WST1 assay showed that EPC metabolic activity was better with lower fibrinogen concentrations (4.5 mg/ml: 0.890?0.134 a.u. vs. 36 mg/ml 0.234?0.046 a.u., p<0.05, n=5), while Th had no significant effect. Calcein staining demonstrated that EPC were viable at 14 days and even organised in cluster. Conclusions: Fibrin combines important properties of an ideal biological scaffold, like the nanometric structure, important for the growth and migration of cells. Fibrin is also an ideal scaffold for EPC but the ratio between fibrinogen and thrombin is important for cell viability

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

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

Scratch closure rate.

<p>Quantification of the effect of PL on scratch closure. A progressive closure was observed with PL treatment, starting from 18 hours. Scratch wounded cells incubated with 10% and 20% PL for 48 and 72 hours showed a significant higher scratch closure as compared to 5% PL, comparable to complete medium (positive control). *: p<0.05; **: p<0.005, all vs. 0% PL. α: p<0.05 vs. 5% PL. Bars represent the means ± SD of values obtained from three independent experiments each one with three replicates per group of treatment. Closure rates are expressed as percentage of scratch closure after 6, 18, 24, 48 and 72 hours compared to the initial area.</p

HUVEC viability and proliferation.

<p>HUVEC viability (<b>A</b>) and proliferation (<b>B</b>) were assessed by XTT assay and BrdU incorporation assay, respectively, following 48 hours of incubation with different PL concentrations (1, 5, 10 and 20%). The percentage of cell viability and proliferation were calculated versus the complete medium (assumed as 100%). Data are means ± SD of values obtained from three independent experiments with six replicates each. * p<0.01 vs. serum-free medium, 1 and 5% PL. .</p

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

Scratch closure assay.

<p>Representative phase contrast micrographs of 0 and 20% PL-treated keratinocytes at 0, 24 and 72 hours are shown. Scale bar: 200 μm.</p

Angiogenesis assay.

<p>The effect on HUVECs of 10% or 20% PL-pre-treatment in serum-free medium for 48 hours, followed by seeding on Matrigel, is shown. The following morphometric parameters were evaluated after 8 hours of angiogenesis assay by AngioJ analysis: area % (<b>A</b>), branching points (<b>B</b>), total tubule length (<b>C</b>). Data are expressed as mean±SD of three independent experiments carried out in duplicate. The 20% PL significantly increased morphometric parameters (p<0.05 vs. 0% PL) comparably to the complete medium. A non significant increase was observed with 10% PL.</p

Activation of intracellular pathways and inflammatory response.

<p>Time-course of ERK1/2 and NFκB phosphorylation after 10% or 20% PL treatment of HUVECs. Data are expressed as mean±SD of the ratio between the absorbance referred to the phosphorylated form and to the corresponding total protein. (<b>A</b>) Phosphorylation of ERK1/2. *: p<0.05; **: p<0.005, all vs. 0% PL. #: p<0.05 vs. both 15 and 30 min. α: p<0.001 vs. complete medium. (<b>B</b>) Phosphorylation of NFκB at Ser 468. *: p<0.05; **: p<0.01, all vs. 0% PL. #: p<0.005 vs. both 1 and 24 hours. α: p<0.05; αα: p<0.01, all vs. complete medium. (<b>C</b>) Phosphorylation of NFκB at Ser 536. *: p<0.05; **: p<0.0001, all vs. 0%PL. &: p<0.05 vs. 10% PL. #: p<0.05; ##: p<0.005, all vs. 1 hour. °: p<0.01 vs. 24 hours. α: p<0.05 vs. complete medium.</p