The employment of composite scaffolds with a well-organized architecture and multi-scale porosity... more The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(3-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 mm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell–material interaction are simultaneously requested.
Synthetic nerve conduits represent a promising strategy to enhance functional recovery in periphe... more Synthetic nerve conduits represent a promising strategy to enhance functional recovery in peripheral nerve injury repair. However, the efficiency of synthetic nerve conduits is often compromised by the lack of molecular factors to create an enriched microenvironment for nerve regeneration. Here, we investigate the in vivo response of mono (MC) and bi-component (BC) fibrous conduits obtained by processing via electrospinning poly(ε-caprolactone) (PCL) and gelatin solutions. In vitro studies demonstrate that the inclusion of gelatin leads to uniform electrospun fiber size and positively influences the response of Dorsal Root Ganglia (DRGs) neurons as confirmed by the preferential extensions of neurites from DRG bodies. This behavior can be attributed to gelatin as a bioactive cue for the cultured DRG and to the reduced fibers size. However, in vivo studies in rat sciatic nerve defect model show an opposite response: MC conduits stimulate superior nerve regeneration than gelatin containing PCL conduits as confirmed by electrophysiology, muscle weight and histology. The G-ratio, 0.71 ± 0.07 for MC and 0.66 ± 0.05 for autograft, is close to 0.6, the value measured in healthy nerves. In contrast, BC implants elicited a strong host response and infiltrating tissue occluded the conduits preventing the formation of myelinated axons. Therefore, although gelatin promotes in vitro nerve regeneration, we conclude that bi-component electrospun conduits are not satisfactory in vivo due to intrinsic limits to their mechanical performance and degradation kinetics, which are essential to peripheral nerve regeneration in vivo.
In this study, the design and fabrication of porous scaffolds, made of blends of polylactic-co-ca... more In this study, the design and fabrication of porous scaffolds, made of blends of polylactic-co-caprolactone (PLC) and polylactic acid (PLA) polymers, for tissue engineering applications is reported. The scaffolds are prepared by means of a bio-safe thermally induced phase separation (TIPS) approach with or without the addition of NaCl particles used as particulate porogen. The scaffolds are characterized to assess their crystalline structure, morphology and mechanical properties, and the texture of the pores and the pore size distribution. Moreover, in vitro human mesenchymal stem cells (hMSCs) culture tests have been carried out to demonstrate the biocompatibility of the scaffolds. The results of this study demonstrate that all of the scaffold materials processed by means of TIPS process are semi-crystalline. Furthermore, the blend composition affected polymer crystallization and, in turn, the nano and macro-structural properties of the scaffolds. Indeed, neat PLC and neat PLA crystallize into globular and randomly arranged sub micro-size scale fibrous conformations, respectively. Concomitantly, the addition of NaCl particles during the fabrication route allows for the creation of an interconnected network of large pores inside the primary structure while resulted in a significant decrease of scaffolds mechanical response. Finally, the results of cell culture tests demonstrate that both the micro and macro-structure of the scaffold affect the in vitro hMSCs adhesion and proliferation.
In the last decade, bicomponent fibers have been proposed to fabricate bio-inspired systems for t... more In the last decade, bicomponent fibers have been proposed to fabricate bio-inspired systems for tissue repair, regenerative medicine, medical healthcare and clinical applications. In comparison with monocomponent fibers, key advantage concerns their ability of self-adapting to the physiological conditions through an extended pattern of signals - morphological, chemical and physical ones - confined at the single fiber level. Hydrophobic/hydrophilic phases may be variously organized by tuneable processing modes (i.e., blending, core/shell, interweaving) thus offering different benefits in terms of biological activity, fluid sorption and molecular transport properties (first generation). The possibility to efficiently graft cell-adhesive proteins and peptide sequences onto the fiber surface mediated by spacers or impregnating hydrogels allows to trigger cell late activities by a controlled and sustained release in vitro of specific biomolecules (i.e., morphogens, growth factors). Here, we introduce an overview of current approaches based on bicomponent fiber use as extra cellular matrix analogs with cell-instructive functions and hierarchal organization of living tissues.
The design of functionalized polymers that can elicit specific biological responses and the devel... more The design of functionalized polymers that can elicit specific biological responses and the development of methods to fabricate new devices that incorporate biological cues are of great interest to the biomedical community. The realization of nanostructured matrices that exhibit biological properties and that comprise fibers with diameters of similar scale to those of the natural extracellular matrix (ECM) would enable the provision of tailored materials for tissue engineering. Accordingly, the goal of this work is to create a biologically active functionalized electrospun matrix capable of guiding neurite growth for the regeneration of nerve tissue. In this study, nanoscale electrospun membranes made of poly ε-caprolactone enhanced with gelatin from calf skin were investigated to validate their biological response under in vitro culture of PC-12 nerve cells. Preliminary observations from SEM studies supported by image analysis highlighted the nanoscale texture of the scaffold with fiber diameters equal to 0.548 ± 0.140 μm. In addition, contact angle measurements confirmed the hydrophilic behavior of the membranes, ascribable to the gelatin content. We demonstrate that the balance of morphological and biochemical properties improves all the fundamental biological events of nerve regeneration, enhancing cell adhesion, proliferation, and differentiation in comparison with PCL nanofibrous scaffolds, as well as supporting the neurite outgrowth.
Biological studies indicate that numerous materials present in living tissues owe their success t... more Biological studies indicate that numerous materials present in living tissues owe their success to an optimal combination of properties and adaptive structures, rather than to extreme properties per se. Through studying natural tissues and by biomimesis, new polymer and composite materials may be designed to emulate the structural and functional responses of bone. These materials must ensure biochemical affinity with host tissue through judicious mixing of specific chemical cues. Also, they must mimic the response under load exhibited by natural bone through complex organisation of material phases, i.e. embedding of collagen fibres in the extracellular substance. Fibre and particulate reinforced polymers are increasingly significant in the development of new biomedical materials, since they can be engineered more accurately than monolithic structures. Meanwhile, design of nanocomposites with specific morphological and chemical signals is emerging as a powerful approach to the mimesi...
The employment of composite scaffolds with a well-organized architecture and multi-scale porosity... more The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(3-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 mm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell–material interaction are simultaneously requested.
Synthetic nerve conduits represent a promising strategy to enhance functional recovery in periphe... more Synthetic nerve conduits represent a promising strategy to enhance functional recovery in peripheral nerve injury repair. However, the efficiency of synthetic nerve conduits is often compromised by the lack of molecular factors to create an enriched microenvironment for nerve regeneration. Here, we investigate the in vivo response of mono (MC) and bi-component (BC) fibrous conduits obtained by processing via electrospinning poly(ε-caprolactone) (PCL) and gelatin solutions. In vitro studies demonstrate that the inclusion of gelatin leads to uniform electrospun fiber size and positively influences the response of Dorsal Root Ganglia (DRGs) neurons as confirmed by the preferential extensions of neurites from DRG bodies. This behavior can be attributed to gelatin as a bioactive cue for the cultured DRG and to the reduced fibers size. However, in vivo studies in rat sciatic nerve defect model show an opposite response: MC conduits stimulate superior nerve regeneration than gelatin containing PCL conduits as confirmed by electrophysiology, muscle weight and histology. The G-ratio, 0.71 ± 0.07 for MC and 0.66 ± 0.05 for autograft, is close to 0.6, the value measured in healthy nerves. In contrast, BC implants elicited a strong host response and infiltrating tissue occluded the conduits preventing the formation of myelinated axons. Therefore, although gelatin promotes in vitro nerve regeneration, we conclude that bi-component electrospun conduits are not satisfactory in vivo due to intrinsic limits to their mechanical performance and degradation kinetics, which are essential to peripheral nerve regeneration in vivo.
In this study, the design and fabrication of porous scaffolds, made of blends of polylactic-co-ca... more In this study, the design and fabrication of porous scaffolds, made of blends of polylactic-co-caprolactone (PLC) and polylactic acid (PLA) polymers, for tissue engineering applications is reported. The scaffolds are prepared by means of a bio-safe thermally induced phase separation (TIPS) approach with or without the addition of NaCl particles used as particulate porogen. The scaffolds are characterized to assess their crystalline structure, morphology and mechanical properties, and the texture of the pores and the pore size distribution. Moreover, in vitro human mesenchymal stem cells (hMSCs) culture tests have been carried out to demonstrate the biocompatibility of the scaffolds. The results of this study demonstrate that all of the scaffold materials processed by means of TIPS process are semi-crystalline. Furthermore, the blend composition affected polymer crystallization and, in turn, the nano and macro-structural properties of the scaffolds. Indeed, neat PLC and neat PLA crystallize into globular and randomly arranged sub micro-size scale fibrous conformations, respectively. Concomitantly, the addition of NaCl particles during the fabrication route allows for the creation of an interconnected network of large pores inside the primary structure while resulted in a significant decrease of scaffolds mechanical response. Finally, the results of cell culture tests demonstrate that both the micro and macro-structure of the scaffold affect the in vitro hMSCs adhesion and proliferation.
In the last decade, bicomponent fibers have been proposed to fabricate bio-inspired systems for t... more In the last decade, bicomponent fibers have been proposed to fabricate bio-inspired systems for tissue repair, regenerative medicine, medical healthcare and clinical applications. In comparison with monocomponent fibers, key advantage concerns their ability of self-adapting to the physiological conditions through an extended pattern of signals - morphological, chemical and physical ones - confined at the single fiber level. Hydrophobic/hydrophilic phases may be variously organized by tuneable processing modes (i.e., blending, core/shell, interweaving) thus offering different benefits in terms of biological activity, fluid sorption and molecular transport properties (first generation). The possibility to efficiently graft cell-adhesive proteins and peptide sequences onto the fiber surface mediated by spacers or impregnating hydrogels allows to trigger cell late activities by a controlled and sustained release in vitro of specific biomolecules (i.e., morphogens, growth factors). Here, we introduce an overview of current approaches based on bicomponent fiber use as extra cellular matrix analogs with cell-instructive functions and hierarchal organization of living tissues.
The design of functionalized polymers that can elicit specific biological responses and the devel... more The design of functionalized polymers that can elicit specific biological responses and the development of methods to fabricate new devices that incorporate biological cues are of great interest to the biomedical community. The realization of nanostructured matrices that exhibit biological properties and that comprise fibers with diameters of similar scale to those of the natural extracellular matrix (ECM) would enable the provision of tailored materials for tissue engineering. Accordingly, the goal of this work is to create a biologically active functionalized electrospun matrix capable of guiding neurite growth for the regeneration of nerve tissue. In this study, nanoscale electrospun membranes made of poly ε-caprolactone enhanced with gelatin from calf skin were investigated to validate their biological response under in vitro culture of PC-12 nerve cells. Preliminary observations from SEM studies supported by image analysis highlighted the nanoscale texture of the scaffold with fiber diameters equal to 0.548 ± 0.140 μm. In addition, contact angle measurements confirmed the hydrophilic behavior of the membranes, ascribable to the gelatin content. We demonstrate that the balance of morphological and biochemical properties improves all the fundamental biological events of nerve regeneration, enhancing cell adhesion, proliferation, and differentiation in comparison with PCL nanofibrous scaffolds, as well as supporting the neurite outgrowth.
Biological studies indicate that numerous materials present in living tissues owe their success t... more Biological studies indicate that numerous materials present in living tissues owe their success to an optimal combination of properties and adaptive structures, rather than to extreme properties per se. Through studying natural tissues and by biomimesis, new polymer and composite materials may be designed to emulate the structural and functional responses of bone. These materials must ensure biochemical affinity with host tissue through judicious mixing of specific chemical cues. Also, they must mimic the response under load exhibited by natural bone through complex organisation of material phases, i.e. embedding of collagen fibres in the extracellular substance. Fibre and particulate reinforced polymers are increasingly significant in the development of new biomedical materials, since they can be engineered more accurately than monolithic structures. Meanwhile, design of nanocomposites with specific morphological and chemical signals is emerging as a powerful approach to the mimesi...
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Papers by Vincenzo Guarino
certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the
middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper,
fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are
composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(3-caprolactone) matrix, and
were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding
technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing
peaks at ca 10 and 200 mm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In
vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35
days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation
of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached
a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal
cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented
migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on
composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA
degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed
promise whenever tuneable porosity, controlled degradability and guided cell–material interaction are
simultaneously requested.
certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the
middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper,
fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are
composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(3-caprolactone) matrix, and
were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding
technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing
peaks at ca 10 and 200 mm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In
vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35
days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation
of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached
a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal
cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented
migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on
composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA
degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed
promise whenever tuneable porosity, controlled degradability and guided cell–material interaction are
simultaneously requested.