This study introduces a novel mandrel-less electrodeposition method and a novel electrode prototypes useful for tissue engineering applications. More specifically, in this study a novel fabrication protocol to electrospun fibers-based chords, with length spanning from 2 cm to virtually no limitations on longitudinal length, is presented. For the first part of this study, Poly(ester urethane) urea (PEUU) was used to fabricate, by electrodeposition technique, continuous microfiber wires with controlled micro-architecture and tunable mechanics. A novel apparatus to process degradable or non-degradable polymers was used. Soft tissue injuries are common in daily clinical and surgical practice. Outcomes of degradable and non-degradable suture materials are often affected by mechanical mismatch, excessive fibrosis and inflammation. Microfiber wire morphology and mechanical properties have been characterized by scanning electron microscopy and uniaxial tensile test respectively. Furthermore, the in vitro response of mouse bone marrow-derived macrophages (BMM) to PEUU degradation products, PEUU electrospun (ES) and casted (Cast) 2D scaffold configuration and PEUU ES wires was evaluated by immunoblotting and immunolabeling. Moreover, the host response to electrospun wires in vivo was tested: fifteen rats, randomized in 5 groups, received a 2 cm infra-scapular incision and the skin was closed using PEUU microfiber and cast wires and the most common suture materials used (polyglycolic acid, polydioxanone and polypropylene). After one month, mechanical and histological evaluation of explants and suture wires was performed. In vitro results have shown an anti-inflammatory macrophage response associated to PEUU ES 2D scaffolds and 3D wires. In vivo, PEUU ES wires group showed better mechanical performance compared to the other groups, a favorable collagen remodeling comparable to the healthy group and a mild host response reaction. These results suggest that microfiber wires reduce macrophage pro-inflammatory response and improve collagen deposition, which make it an ideal candidate for soft tissue suture applications. The second part of this study is focused on creating a biomimetic engineered chordae tendineae (BECT) using Poly(carbonate urethane) urea (PCUU). The mandrel-less electrodeposition methodology is used to mimic the macro and micro architecture of native chordae tendineae (CT). The voltage generated by the two facing electrodes will induce a deposition of highly aligned microscopic fibers that duplicate native tissue anatomy and function structure. Current methods for CT replacement do not recapitulate the microarchitecture nor the mechanics of the native CT and do not promote healthy tissue regeneration. Human and porcine CT were harvested and tested to evaluate mechanical and histological native tissue structure. PCUU was used to fabricate BECT via mandrel-less electrodeposition. Moreover, BECT were micro-integrated with NIH-3T3 rat fibroblasts. Micro-integrated BECT were divided in 3 groups: static culture in plate, tension culture in plate and dynamic culture using a stretch bioreactor. All groups were conditioned for 1 week. Finally, scanning electron microscopy (SEM), uniaxial tensile testing and histological evaluation were performed to compare native tissue and BECT micro-architecture and mechanical properties. BECT mimicked native CT shape, diameter, and length. SEM analysis showed highly aligned fiber microstructure recapitulating the arrangement observed in native CT. BECT mechanical characterization showed a lower elastic modulus than native tissue that increased with dynamic conditioning for cell micro-integrated BECT. The histology of cell-seeded BECT demonstrated cell adhesion and infiltration after one week. This mandrel-less electrodeposition method allows the bio-fabrication of BECT that demonstrate the ability to recapitulate native CT structure with use of a scaffold. Micro-integration preliminary data provided evidence of cell proliferation and viability, demonstrating an early proof-of concept for potential host cell recruitment.

(2021). Mandrel-less fabrication of biomimetic electrospun microfiber wires for tissue engineering applications.

Mandrel-less fabrication of biomimetic electrospun microfiber wires for tissue engineering applications

ADAMO, Arianna
2021-03-04

Abstract

This study introduces a novel mandrel-less electrodeposition method and a novel electrode prototypes useful for tissue engineering applications. More specifically, in this study a novel fabrication protocol to electrospun fibers-based chords, with length spanning from 2 cm to virtually no limitations on longitudinal length, is presented. For the first part of this study, Poly(ester urethane) urea (PEUU) was used to fabricate, by electrodeposition technique, continuous microfiber wires with controlled micro-architecture and tunable mechanics. A novel apparatus to process degradable or non-degradable polymers was used. Soft tissue injuries are common in daily clinical and surgical practice. Outcomes of degradable and non-degradable suture materials are often affected by mechanical mismatch, excessive fibrosis and inflammation. Microfiber wire morphology and mechanical properties have been characterized by scanning electron microscopy and uniaxial tensile test respectively. Furthermore, the in vitro response of mouse bone marrow-derived macrophages (BMM) to PEUU degradation products, PEUU electrospun (ES) and casted (Cast) 2D scaffold configuration and PEUU ES wires was evaluated by immunoblotting and immunolabeling. Moreover, the host response to electrospun wires in vivo was tested: fifteen rats, randomized in 5 groups, received a 2 cm infra-scapular incision and the skin was closed using PEUU microfiber and cast wires and the most common suture materials used (polyglycolic acid, polydioxanone and polypropylene). After one month, mechanical and histological evaluation of explants and suture wires was performed. In vitro results have shown an anti-inflammatory macrophage response associated to PEUU ES 2D scaffolds and 3D wires. In vivo, PEUU ES wires group showed better mechanical performance compared to the other groups, a favorable collagen remodeling comparable to the healthy group and a mild host response reaction. These results suggest that microfiber wires reduce macrophage pro-inflammatory response and improve collagen deposition, which make it an ideal candidate for soft tissue suture applications. The second part of this study is focused on creating a biomimetic engineered chordae tendineae (BECT) using Poly(carbonate urethane) urea (PCUU). The mandrel-less electrodeposition methodology is used to mimic the macro and micro architecture of native chordae tendineae (CT). The voltage generated by the two facing electrodes will induce a deposition of highly aligned microscopic fibers that duplicate native tissue anatomy and function structure. Current methods for CT replacement do not recapitulate the microarchitecture nor the mechanics of the native CT and do not promote healthy tissue regeneration. Human and porcine CT were harvested and tested to evaluate mechanical and histological native tissue structure. PCUU was used to fabricate BECT via mandrel-less electrodeposition. Moreover, BECT were micro-integrated with NIH-3T3 rat fibroblasts. Micro-integrated BECT were divided in 3 groups: static culture in plate, tension culture in plate and dynamic culture using a stretch bioreactor. All groups were conditioned for 1 week. Finally, scanning electron microscopy (SEM), uniaxial tensile testing and histological evaluation were performed to compare native tissue and BECT micro-architecture and mechanical properties. BECT mimicked native CT shape, diameter, and length. SEM analysis showed highly aligned fiber microstructure recapitulating the arrangement observed in native CT. BECT mechanical characterization showed a lower elastic modulus than native tissue that increased with dynamic conditioning for cell micro-integrated BECT. The histology of cell-seeded BECT demonstrated cell adhesion and infiltration after one week. This mandrel-less electrodeposition method allows the bio-fabrication of BECT that demonstrate the ability to recapitulate native CT structure with use of a scaffold. Micro-integration preliminary data provided evidence of cell proliferation and viability, demonstrating an early proof-of concept for potential host cell recruitment.
tissue engineering; mandrel-less deposition; electrospinning; cardiac tissue; sutures; host response; macrophages; collagen
(2021). Mandrel-less fabrication of biomimetic electrospun microfiber wires for tissue engineering applications.
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Open Access dal 05/02/2022

Descrizione: Arianna Adamo PhD thesis
Tipologia: Tesi di dottorato
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10447/479155
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