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.

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

DI STEFANO, ROSSELLA;CHIELLINI, FEDERICA;BALBARINI, ALBERTO
2008-01-01

Abstract

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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/126900
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