Production and evaluation of electrospun polyaniline/biopolymer composite nanofibres for medical applications

The aim of this study is the production of a nanofibrous electroactive mat and the investigation of its potential use in tissue engineering, and more specifically for wound dressing purposes. The limitations regarding electrospinnability of the conducting polymer will be identified and addressed and the factors related to its biological properties will be evaluated. To this end, conducting polymer, polyaniline (PANI) was chosen as the electroactive component and blend electrospinning was identified as the most suitable method to produce continuous nanofibres containing PANI. Various biocompatible polymers and solvent systems were investigated for their suitability to assist in electrospinning and PEO (polyethylene oxide) and CH (chitosan) were chosen as carrier polymers for blend electrospinning of PANI. Consequently, CSA (Camphor-10-sulfonic acid (β)) doped PANI/PEO and CSA doped PANI/CH conducting nanofibrous mats were produced by electrospinning. The electrospinning windows for both blends were determined by using full factorial experimental designs. The combined effects of the humidity, voltage and flow rate on the fibre morphology and diameter were examined for both blends, demonstrating that the ambient humidity is the critical factor affecting the electrospinning process and determining the electrospinning window for a conducting polymer. Low humidity favors the formation of defect free fibres while high humidity either hinders fibre formation or causes the formation of defects on the fibres. In the case of PANI/PEO blends, different levels of PANI doping were investigated, and high level of doping with CSA was found to lead to the formation of crystalline structures. Data fitting was used to explore the behavior of conducting polymers using the case of PANI/PEO electrospinning and very good agreement between experimental and theoretical predictions was obtained for only a limited range of experimental conditions, whereas deviation was observed for all other sets of conditions. In the case of PANI/CH, the effect of different ratios of conducting polymer in the blend (0:1, 1:3, 3:5 and 1:1) was examined, as for the electrospinnability, resulting 3 nanofibrous morphology, mat contact angle, electrical conductivity, antibacterial activity and cellular biocompatibility. The incorporation of PANI in the electrospinning blend, affected the electrospinnability of the solution, making it more susceptible to RH deviations, and contributed to the decrease of nanofibre diameter. Higher PANI content was found to result in more hydrophobic and more conducting mats. The method that was used to stabilize the PANI/CH mats was also found to affect antibacterial activity and conductivity. The produced blend mats, exhibited antibacterial activity which was higher against Gram positive B. subtilis and lower against gram negative E. coli. The cellular biocompatibility was assessed with human osteoblasts and fibroblasts, in terms of cell proliferation rate as well as cell attachment and morphology. Cells of both cell lines adhered well and showed good growth rates on nanofibrous substrates of all blend ratios when compared to standard tissue culture plastic. Finally, amongst the PANI containing mats, the one of 1:3 PANI:CH ratio, was identified as the best to support osteoblast and fibroblast cell proliferation when compared to the pure chitosan

Conductive PANI fibres and determining factors for the electrospinning window

Polyaniline doped with CSA / PEO conductive nanofibres were produced by electrospinning. The electrospinning window was determined by using a three level, full factorial experimental design. The combined effects of the humidity, voltage and flow rate on the fibre morphology and diameter were examined demonstrating that the ambient humidity is the critical factor affecting the electrospinning process and determining the electrospinning window for a conductive polymer. Low humidity favors the formation of defect free fibres while high humidity either hinders fibre formation or causes the formation of defects on the fibres either due to jet discharge or due to water absorption and phase separation. High level of doping with CSA led to the formation of crystalline structures. Data fitting was used to explore the behavior of conductive polymers in electrospinning and very good agreement between experimental and theoretical predictions was obtained for only a limited range of experimental conditions, whereas deviation was observed for all other sets of conditions

Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 days

Introduction: Successful long-term expansion of skeletal muscle satellite cells (MuSCs) on a large scale is fundamental for cultivating animal cells for protein production. Prerequisites for efficient cell expansion include maintaining essential native cell activities such as cell adhesion, migration, proliferation, and differentiation while ensuring consistent reproducibility. Method: This study investigated the growth of bovine MuSC culture using low-volume spinner flasks and a benchtop stirred-tank bioreactor (STR). Results and discussion: Our results showed for the first time the expansion of primary MuSCs for 38 days in a bench-top STR run with low initial seeding density and FBS reduction, supported by increased expression of the satellite cell marker PAX7 and reduced expression of differentiation-inducing genes like MYOG, even without adding p38-MAPK inhibitors. Moreover, the cells retained their ability to proliferate, migrate, and differentiate after enzymatic dissociation from the microcarriers. We also showed reproducible results in a separate biological benchtop STR run.Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 dayspublishedVersio

Bioprocess development for scalable production of cultivated meat

Traditional farm-based products based on livestock are one of the main contributors to greenhouse gas emissions. Cultivated meat is an alternative that mimics animal meat, being produced in a bioreactor under controlled conditions rather than through the slaughtering of animals. The first step in the production of cultivated meat is the generation of sufficient reserves of starting cells. In this study, bovine adipose-derived stem cells (bASCs) were used as starting cells due to their ability to differentiate towards both fat and muscle, two cell types found in meat. A bioprocess for the expansion of these cells on microcarriers in spinner flasks was developed. Different cell seeding densities (1,500, 3,000, and 6,000 cells/cm 2) and feeding strategies (80%, 65%, 50%, and combined 80%/50% medium exchanges) were investigated. Cell characterization was assessed pre- and postbioprocessing to ensure that bioprocessing did not negatively affect bASC quality. The best growth was obtained with the lowest cell seeding density (1,500 cells/cm 2) with an 80% medium exchange performed (p '.0001) which yielded a 28-fold expansion. The ability to differentiate towards adipogenic, osteogenic, and chondrogenic lineages was retained postbioprocessing and no significant difference (p '.5) was found in clonogenicity pre- or postbioprocessing in any of the feeding regimes tested

Biocompatibility Assessment of Conducting PANI/Chitosan Nanofibers for Wound Healing Applications

As electroactive polymers have recently presented potential in applications in the tissue engineering and biomedical field, this study is aiming at the fabrication of composite nanofibrous membranes containing conducting polyaniline and at the evaluation of their biocompatibility. For that purpose, conducting polyaniline–chitosan (PANI/CS) defect free nanofibres of different ratios (1:3; 3:5 and 1:1) were produced with the electrospinning method. They were characterized as for their morphology, hydrophilicity and electrical conductivity. The membranes were then evaluated for their cellular biocompatibility in terms of cell attachment, morphology and cell proliferation. The effect of the PANI content on the membrane properties is discussed. Increase in PANI content resulted in membranes with higher hydrophobicity and higher electrical conductivity. It was found that none of the membranes showed any toxic effects on osteoblasts and fibroblasts, and that they all supported cell attachment and growth, even to a greater extent than tissue culture plastic. The membrane with the PANI/CS ratio 1:3 supports better cell attachment and proliferation for both cell lines due to a synergistic effect of hydrophilicity retention due to the high chitosan content and the conductivity that PANI introduced to the membrane

Biocompatibility assessment of conducting PANI/Chitosan nanofibers for wound healing applications

As electroactive polymers have recently presented potential in applications in the tissue engineering and biomedical field, this study is aiming at the fabrication of composite nanofibrous membranes containing conducting polyaniline and at the evaluation of their biocompatibility. For that purpose, conducting polyaniline–chitosan (PANI/CS) defect free nanofibres of different ratios (1:3; 3:5 and 1:1) were produced with the electrospinning method. They were characterized as for their morphology, hydrophilicity and electrical conductivity. The membranes were then evaluated for their cellular biocompatibility in terms of cell attachment, morphology and cell proliferation. The effect of the PANI content on the membrane properties is discussed. Increase in PANI content resulted in membranes with higher hydrophobicity and higher electrical conductivity. It was found that none of the membranes showed any toxic effects on osteoblasts and fibroblasts, and that they all supported cell attachment and growth, even to a greater extent than tissue culture plastic. The membrane with the PANI/CS ratio 1:3 supports better cell attachment and proliferation for both cell lines due to a synergistic effect of hydrophilicity retention due to the high chitosan content and the conductivity that PANI introduced to the membrane

Chitosan & conductive PANI/Chitosan composite nanofibers - Evaluation of antibacterial properties

Within the healthcare industry, including the treatment of chronic wounds, the challenge of antimicrobial resistance continues to grow. As such, there is a need to develop new treatments that can reduce the bioburden in wounds. The present study is focused on the development of conductive polyaniline (PANI) / chitosan (CH) nanofibrous electrospun membranes and evaluates their antibacterial properties. The blended fibre membranes combine the materials’ respective properties such as electrical conductivity, biocompatibility and antibacterial activity. To this end, experimental design was used to determine the electrospinning windows of both pure chitosan and PANI/CH blends of different ratios (1:3, 3:5, 1:1). The effect of key environmental and process parameters (relative humidity and applied voltage) was determined, as well as the effect of the PANI/CH ratio in the blend, and the molecular interactions between PANI and Chitosan that led to jet stability. The nanofibrous mats were evaluated regarding their morphology and antibacterial effect against gram positive and gram negative bacterial strains, namely B. subtilis and E. coli. High PANI content mats show increased bactericidal activity against both bacterial strains. This study suggests that electrospun PANI/CH membranes are promising candidates for healthcare applications such as wound dressings

Development of a Chemically Defined Medium for in vitro Expansion of Primary Bovine Satellite Cells

The use of fetal bovine serum (FBS) in animal cell culture media is widely spread since it provides a broad spectrum of molecules that are known to support cell attachment and growth. However, the harvest and collection procedures of FBS raise ethical concerns and serum is an ill-defined and expensive component. This is especially problematic when it comes to regulatory approval for food applications like cultured meat. The aim of this study is to develop a chemically defined, cost efficient serum-free and animal-free medium that supports the attachment and expansion of bovine myoblasts while maintaining their differentiation capacity. Bovine satellite cells were harvested and isolated from a fresh sample of skeletal muscle tissue and cultured in planar systems. The efficacy of the tested formulations was assessed with metabolic assays and cell counting techniques. Optical microscopy was used to observe cellular morphology and statistical analysis was applied. Based on a comprehensive literature analysis, a defined serum-free medium (SFM) composition was developed consisting of DMEM/F12 as basal medium, supplemented with L-ascorbic acid 2-phosphate, fibronectin, hydrocortisone, GlutaMAX(™), albumin, ITS-X, hIL-6, α-linolenic acid, and growth factors such as FGF-2, VEGF, IGF-1, HGF, and PDGF-BB. To our knowledge, this is the first defined serum-free and animal free medium formulation specific for bovine myoblasts to date. We conclude that the SFM formulation supported exponential cell growth up to 97% of the serum—containing golden standard growth medium. All reagents used in this study are chemically defined

Biocompatibility Assessment of Conducting PANI/Chitosan Nanofibers for Wound Healing Applications

As electroactive polymers have recently presented potential in applications in the tissue engineering and biomedical field, this study is aiming at the fabrication of composite nanofibrous membranes containing conducting polyaniline and at the evaluation of their biocompatibility. For that purpose, conducting polyaniline–chitosan (PANI/CS) defect free nanofibres of different ratios (1:3; 3:5 and 1:1) were produced with the electrospinning method. They were characterized as for their morphology, hydrophilicity and electrical conductivity. The membranes were then evaluated for their cellular biocompatibility in terms of cell attachment, morphology and cell proliferation. The effect of the PANI content on the membrane properties is discussed. Increase in PANI content resulted in membranes with higher hydrophobicity and higher electrical conductivity. It was found that none of the membranes showed any toxic effects on osteoblasts and fibroblasts, and that they all supported cell attachment and growth, even to a greater extent than tissue culture plastic. The membrane with the PANI/CS ratio 1:3 supports better cell attachment and proliferation for both cell lines due to a synergistic effect of hydrophilicity retention due to the high chitosan content and the conductivity that PANI introduced to the membrane