Investigating How the Properties of Electrospun Poly(lactic acid) Fibres Loaded with the Essential Oil Limonene Evolve over Time under Different Storage Conditions.

Essential oils have been identified as effective natural compounds to prevent bacterial infections and thus are widely proposed as bioactive agents for biomedical applications. Across the literature, various essential oils have been incorporated into electrospun fibres to produce materials with, among others, antibacterial, anti-inflammatory and antioxidant activity. However, limited research has been conducted so far on the effect of these chemical products on the physical characteristics of the resulting composite fibres for extended periods of time. Within this work, electrospun fibres of poly(lactic acid) (PLA) were loaded with the essential oil limonene, and the impact of storage conditions and duration (up to 12 weeks) on the thermal degradation, glass transition temperature and mechanical response of the fibrous mats were investigated. It was found that the concentration of the encapsulated limonene changed over time and thus the properties of the PLA-limonene fibres evolved, particularly in the first two weeks of storage (independently from storage conditions). The amount of limonene retained within the fibres, even 4 weeks after fibre generation, was effective to successfully inhibit the growth of model microorganisms Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The results of this work demonstrate the importance of evaluating physical properties during the ageing of electrospun fibres encapsulating essential oils, in order to predict performance modification when the composite fibres are used as constituents of medical devices.

Electrospinning of honey and propolis for wound care.

Phytochemicals and naturally derived compounds, such as plant extracts and bee products, are regarded as complementary and alternative medicines for the treatment of skin wounds, due to their antibacterial, anti-inflammatory, and antioxidant properties. In recent years, it has been shown that dressings impregnated with honey (particularly Manuka honey) are effective for the topical treatment of wounds and burns, and some of them are currently used in clinics. This has stimulated the development of more advanced dressings based on polymeric nanofibres that can release honey and other bee products (like propolis) to promote wound healing. In this review, the current literature on the electrospinning of honey and propolis is analyzed and the effectiveness of the resulting dressings to inhibit bacterial growth and stimulate cellular proliferation and tissue repair is discussed.

Exploring the Mechanical Properties and Performance of Type-I Collagen at Various Length Scales: A Progress Report.

Collagen is the basic protein of animal tissues and has a complex hierarchical structure. It plays a crucial role in maintaining the mechanical and structural stability of biological tissues. Over the years, it has become a material of interest in the biomedical industries thanks to its excellent biocompatibility and biodegradability and low antigenicity. Despite its significance, the mechanical properties and performance of pure collagen have been never reviewed. In this work, the emphasis is on the mechanics of collagen at different hierarchical levels and its long-term mechanical performance. In addition, the effect of hydration, important for various applications, was considered throughout the study because of its dramatic influence on the mechanics of collagen. Furthermore, the discrepancies in reports of the mechanical properties of collagenous tissues (basically composed of 20-30% collagen fibres) and those of pure collagen are discussed.

Fracture behaviour and toughening mechanisms of dry and wet collagen.

The growing interest to the use of collagen films for biomedical applications motivates the analysis of their fracture behaviour in different environments. Studies revealed the decreased mechanical strength and stiffness as well as increased plasticity in water compared to collagen specimens tested in air. However, the fracture behaviour of pure collagen films in both air and water has not been reported so far. In this paper, the entire process of mode-I loading of single-edge notched tension (SENT) specimens was recorded and analysed. In case of in-air (dry) specimens, cracks propagated rapidly in a brittle fashion while large plastic deformations were observed in aqua prior to failure due to crack opening and a blunting mechanism in wet specimens. The fracture-toughness parameters for pure collagen in air and in aqua were estimated using linear-elastic (KI and GI) and elasto-plastic (JI) fracture-mechanics approaches, respectively, following the force-displacement response and deformational behaviour. GIC and JI were 1365 ± 112 J/m2 and 2500 ± 440 J/m2, respectively. Scanning electron microscopy was used to observe the structural changes linked to collagen fibrils in the crack-tip area and the fracture surface. For in-air specimens, the former mostly exhibited extrinsic toughening (usually at micro scale) acting behind the crack-tip, while in-aqua intrinsic toughening acting ahead of a crack tip was found. Fractography of in-air specimens showed no occurrence of voids while multiple micro-voids were found for in-aqua specimens. STATEMENT OF SIGNIFICANCE: The fracture toughness and crack propagation of both mineralised (bone, dentine) and non-mineralised (skin) tissues has been extensively investigated over the past decades. Though these tissues are rich in collagen, the fracture properties of pure collagen have not been quantified yet at macroscale. Considering the applications of collagen films in tissue regeneration, it is essential to perform investigations of their fracture behaviour in both dry and wet conditions. Determining the effect of environment on the fracture behaviour of collagen and understanding its toughening mechanism are essential for prevention of failures during application. Moreover, this would give an insight for fabrication of tougher collagen-based biomaterials for biomedical uses.

Electrospinning of stimuli-responsive polymers for controlled drug delivery: pH- and temperature-driven release.

Stimuli-responsive polymers have attracted academic interest over the last 60 years due to their potential use in developing systems with a range of functionalities that can be activated by external artificial triggers. The diversity of responses and stimuli, which can be achieved through careful polymer selection and processing, opens up applications in nearly every sector. In particular, the use of responsive polymers for the controlled delivery of drugs and bioactive compounds has been heavily researched. This review provides an overview of the recent advancements in electrospinning of temperature- and pH-responsive polymers to create networks of nanofibres with controlled drug delivery profiles for biomedical applications.

Electrospinning and Additive Manufacturing: Adding Three-Dimensionality to Electrospun Scaffolds for Tissue Engineering.

The final biochemical and mechanical performance of an implant or scaffold are defined by its structure, as well as the raw materials and processing conditions used during its fabrication. Electrospinning and Additive Manufacturing (AM) are two contrasting processing technologies that have gained popularity amongst the fields of medical research i.e., tissue engineering, implant design, drug delivery. Electrospinning technology is favored for its ability to produce micro- to nanometer fibers from polymer solutions and melts, of which, the dimensions, alignment, porosity, and chemical composition are easily manipulatable to the desired application. AM, on the other hand, offers unrivalled levels of geometrical freedom, allowing highly complex components (i.e., patient-specific) to be built inexpensively within 24 hours. Hence, adopting both technologies together appears to be a progressive step in pursuit of scaffolds that better match the natural architecture of human tissues. Here, we present recent insights into the advances on hybrid scaffolds produced by combining electrospinning (melt electrospinning excluded) and AM, specifically multi-layered architectures consisting of alternating fibers and AM elements, and bioinks reinforced with fibers prior to AM. We discuss how cellular behavior (attachment, migration, and differentiation) is influenced by the co-existence of these micro- and nano-features.

Resonance vibration interventions in the femur: Experimental-numerical modelling approaches.

MOTIVE: External vibration excitation might be key to many novel non-surgical interventions for pathologies in the musculoskeletal system and in other parts of the human organism. Lack of understanding about vibration patterns, their controllability, and reproducibility are three limitations of ongoing research. This study establishes a bovine vibration model and animal model replacements for future research. METHODS: We used biological samples (n=5) and one polyurethane sample of the bovine femur. Mechanical resonance was measured experimentally and analysed numerically by finite element method. MAIN RESULTS: The experiments obtained 5 distinct mode shapes for the biological sample set, with standard deviation < 7.5%. Finite element analysis of the biological samples can replicate experimental mode shape deflection. The use of polyurethane changes resonance character but results are also good approximations of the biological samples. CONCLUSIONS: A model of the bovine femur with consistent resonance behaviour is presented with alternatives (polyurethane and finite element analysis) that can serve in reducing the number of necessary biological samples. Future work will be to adapt results to human anatomy. Of clinical interest will be to influence bone pathologies such as post-surgical non-union, or bone functionality as part of haematopoiesis and endocrine secretion.

Chitosan-Coated Poly(lactic acid) Nanofibres Loaded with Essential Oils for Wound Healing.

Chronic skin wounds are characterised by a non-healing process that makes necessary the application of wound dressings on the damaged area to promote and facilitate the recovery of skin's physiological integrity. The aim of the present work is to develop a bioactive dressing that, once applied on the injured tissue, would exert antibacterial activity and promote adhesion and proliferation of fibroblasts. Nanofibres consisting of poly(lactic acid) (PLA) and essential oils (EOs) were electrospun and coated with a medium molecular weight chitosan (CS). Black pepper essential oil (BP-EO) or limonene (L), well-known for their antibacterial properties, were added to the PLA/acetone solution before electrospinning; phase separation phenomena occurred due to the poor solubility of the EOs in the PLA solution and led to fibres having surface nano-pores. The porous electrospun fibres were coated with CS to produce hydrophilic membranes that were easy to handle, biocompatible, and suited to promote cellular proliferation. The fibrous scaffolds were tested in terms of mechanical resistance, wettability, antibacterial activity, in-vitro cytotoxicity, and ability to promote fibroblasts' adhesion and proliferation. The results obtained proved that the CS coating improved the hydrophilicity of the fibrous mats, enhanced EO's antibacterial potential, and promoted cell adhesion and proliferation.

An In-Vitro Evaluation of the Characteristics of Zein-Based Films for the Release of Lactobionic Acid and the Effects of Oleic Acid.

Lactobionic acid (LBA) is widely used in different industrial sectors owing to its biocompatibility characteristics as well as antioxidant and antimicrobial properties. In this study, mixtures of the protein zein with LBA and with the addition of oleic acid (OA) as a ternary system were investigated as drug delivery films for the release of LBA. The chosen combinations exploit the vast difference in water solubility between LBA and the other two components (zein and OA). DSC thermograms and dynamic mechanical spectra, alongside electron microscopy images, were used to describe the microstructural features of the films and were found to provide insights for the release of LBA from the two examined zein-based films immersed in an aqueous physiological solution. For both film systems, a burst release behavior was observed, followed by a rapid and total extraction of LBA. The required immersion time for the total extraction of LBA was greatly reduced when oleic acid was added to the precursor solution mixture for producing the films. The LBA released from the zein-based films was found to exhibit both the expected antioxidant properties as well as exerting bacteriostatic effects towards Escherichia coli and Staphylococcus epidermidis.

Development of a hollow fibre-based renal module for active transport studies.

Understanding the active transport of substrates by the kidney in the renal proximal convoluted tubule is crucial for drug development and for studying kidney diseases. Currently, cell-based assays are applied for this this purpose, however, differences between assays and the body are common, indicating the importance of in vitro-in vivo discrepancies. Several studies have suggested that 3D cell cultures expose cells to a more physiological environments, thus, providing more accurate cell function results. To mimic the renal proximal tubule, we have developed a custom-made renal module (RM), containing a single polypropylene hollow fibre (Plasmaphan P1LX, 3M) that serves as a porous scaffold and compared to conventional Transwell cell-based bidirectional transport studies. In addition, a constant flow of media, exposed cells to a physiological shear stress of 0.2 dyne/cm2. MDCK-Mdr1a cells, overexpressing the rat Mdr1a (P-gp) transporter, were seeded onto the HF membrane surface coated with the basement membrane matrix Geltrex which facilitated cell adhesion and tight junction formation. Cells were then seeded into the HF lumen where attachment and tight junction formation were evaluated by fluorescence microscopy while epithelial barrier integrity under shear stress was shown to be achieved by day 7. qPCR results have shown significant changes in gene expression compared to cells grown on Transwells. Kidney injury marker such as KIM-1 and the hypoxia marker CA9 have been downregulated, while the CD133 (Prominin-1) microvilli marker has shown a fivefold upregulation. Furthermore, the renal transporter P-gp expression has been downregulated by 50%. Finally, bidirectional assays have shown that cells grown in the RM were able to reabsorb albumin with a higher efficiency compared to Transwell cell cultures while efflux of the P-gp-specific substrates Hoechst and Rhodamine 123 was decreased. These results further support the effect of the microenvironment and fluidic shear stress on cell function and gene expression. This can serve as the basis for the development of a microphysiological renal model for drug transport studies.

Correction: On the quantification of local power densities in a new vibration bioreactor.

[This corrects the article DOI: 10.1371/journal.pone.0245768.].

Porous Optically Transparent Cellulose Acetate Scaffolds for Biomimetic Blood-Brain Barrierin vitro Models.

In vitro blood-brain barrier (BBB) models represent an efficient platform to conduct high-throughput quantitative investigations on BBB crossing ability of different drugs. Such models provide a closed system where different fundamental variables can be efficaciously tuned and monitored, and issues related to scarce accessibility of animal brains and ethics can be addressed. In this work, we propose the fabrication of cellulose acetate (CA) porous bio-scaffolds by exploiting both vapor-induced phase separation (VIPS) and electrospinning methods. Parameters of fabrication have been tuned in order to obtain porous and transparent scaffolds suitable for optical/confocal microscopy, where endothelial cell monolayers are allowed to growth thus obtaining biomimetic BBB in vitro models. Concerning VIPS-based approach, CA membranes fabricated using 25% H2O + 75% EtOH as non-solvent showed submicrometer-scale porosity and an optical transmittance comparable to that one of commercially available poly(ethylene terephthalate) membranes. CA membranes fabricated via VIPS have been exploited for obtaining multicellular BBB models through the double seeding of endothelial cells and astrocytes on the two surfaces of the membrane. Electrospun CA substrates, instead, were characterized by micrometer-sized pores, and were unsuitable for double seeding approach and long term studies. However, the potential exploitation of the electrospun CA substrates for modeling blood-brain-tumor barrier and studying cell invasiveness has been speculated. The features of the obtained models have been critically compared and discussed for future applications.

Damage in extrusion additive manufactured biomedical polymer: Effects of testing direction and environment during cyclic loading.

Although biodegradable polymers were widely researched, this is the first study considering the effect of combined testing environments and cyclic loading on the most important aspect related to additive manufacturing: the interfacial bond between deposited layers. Its results give confidence in applicability of the material extrusion additive manufacturing technology for biomedical fields, by demonstrating that the interface behaves in a manner similar to that of the bulk-polymer material. To do this, especially designed tensile specimens were used to analyse the degradation of 3D-printed polymers subjected to constant-amplitude and incremental cyclic loads when tested in air at room temperature (control) and submerged at 37 °C (close to in-vivo conditions). The mechanical properties of the interface between extruded filaments were compared against the bulk material, i.e. along filaments. In both cases, cyclic loading caused only a negligible detrimental effect compared to non-cyclic loading (less than 10 % difference in ultimate tensile strength), demonstrating the suitability of using 3D-printed components in biomedical applications, usually exposed to cyclic loading. For cyclic tests with a constant loading amplitude, larger residual deformation (>100 % greater) and energy dissipation (>15 % greater) were found when testing submerged in solution at 37 °C as opposed to in laboratory conditions (air at room temperature), as used by many studies. This difference may be due to plasticisation effects of water and temperature. For cyclic tests with incrementally increasing loading amplitudes, the vast majority of energy dissipation happened in the last two cycles prior to failure, when the polymer approached the yield point. The results demonstrate the importance of using an appropriate methodology for biomedical applications; otherwise, mechanical properties may be overestimated.

Designing responsive dressings for inflammatory skin disorders; encapsulating antioxidant nanoparticles into biocompatible electrospun fibres.

Inflammatory skin disorders are highly prevalent and current treatments are marred by side-effects. Here, we have designed anti-inflammatory fibrous sheets with the potential to treat low exudate inflammatory skin disorders such as psoriasis or atopic dermatitis. Antioxidant and anti-inflammatory nanoparticles composed of crosslinked poly(propylene sulfide) (PPS) were encapsulated in poly(ethylene oxide) (PEO) fibres via electrospinning from an aqueous suspension. The loading of nanoparticles did not adversely effect the homogenous nature of the electrospun fibres; furthermore, nanoparticles retained their morphology, size and anti-inflammatory character after electrospinning. The PPS-nanoparticle-loaded nanofibres were found to be highly cytocompatible when tested on human dermal fibroblasts. These findings suggest they have significant potential to topically treat inflamed tissues that are characterized by high reactive oxygen species (ROS) levels.

On the quantification of local power densities in a new vibration bioreactor.

We investigate the power densities which are obtainable locally in a vibration bioreactor. These reactor systems are of great relevance for research about oncological or antibacterial therapies. Our focus lies on the local liquid pressure caused by resonance vibration in the fluid contained by the reactor's petri dish. We use for the excitation one piezoelectric patch which offer advantages concerning controllability and reproducibility, when compared to ultrasound. The experimental work is extended by finite element analyses of bioreactor details. The peaks of the vibration response for water, sodium chloride (0.1N Standard solution), and McCoy's 5A culture medium are in good alignment. Several natural frequencies can be observed. Local power density can reach multiple times the magnitude used in ultrasound studies. Based on the observed local power densities, we are planning future work for the exposure of cell cultures to mechanical vibration.

Printability and mechanical performance of biomedical PDMS-PEEK composites developed for material extrusion.

Polydimethylsiloxane (PDMS) materials are widely adopted in the manufacture of facial prostheses, lab-on-chip devices and scaffolds for soft-tissue engineering applications; however, their processing by additive manufacturing (AM) has proved challenging. Liquid silicone rubbers (LSRs) are favoured for their high shape fidelity when cast, but their low viscosity and surface tension often prevent self-support, post-extrusion. Poly(ether) ether ketone (PEEK) particle reinforcement through interfacial bonding has proven to enhance key properties of PDMS, expanding their end-use functionality. Still, the impact of such particles on the printability of LSR-PDMS is not explored. In this study, for the first time, solvent-free biocompatible PDMS-PEEK composites (up to 30 wt% PEEK) were successfully characterised for material extrusion (ME) printing. Rheological analysis confirmed shear-thinning of all PDMS-PEEK composites under applied load (within the tolerances of the printer) and dominant storage moduli at rest (i.e. prints can self-support), considered highly desirable for ME-based printing. Attained rheological datasets were used to guide initial printability studies, which revealed finer track fidelity with rising fractional content of PEEK, at comparable print speed and displacement values. Composites with higher PEEK content demonstrated significant increases in Shore A hardness and stiffness (in tension and compression) in bulk form. Last but not least, enhanced shape fidelity (thanks to PEEK reinforcement) and geometrical autonomy further expanded the manufacturing freedom of PDMS, whereby infill density could be controlled in order to increase the range of mechanical performance, previously unachievable with conventional casting fabrication. Fundamentally, this could lead to the manufacture of bespoke spatially graded multi-material structures and devices that could be used to replicate the heterogenous properties of soft human tissues and in other advanced material applications.

Dry vs. wet: Properties and performance of collagen films. Part II. Cyclic and time-dependent behaviours.

Collagen constitutes one-third of human-body proteins, providing mechanical strength and structural stability. Films of collagen are widely used in tissue engineering as scaffolds for wound healing and corneal implants, among other applications, presupposing the investigation of their mechanical properties and performance under various loading and environmental conditions. Part I of this research (Bose et al., 2020) demonstrated a drastic change in the mechanical response of collagen films under in-aqua conditions when compared to dry specimens. It was also observed that collagen films exhibited a strain-rate-dependent hardening behaviour with a strain-rate-sensitivity exponent ranging from 0.02 to 0.2. In Part II, the cyclic and time-dependent behaviours of collagen films were analysed under different loading and environmental conditions. Strain ratchetting was observed for collagen subjected to cyclic loading under various stress levels and environmental (in-air and in-aqua) conditions, while the in-aqua samples demonstrated an increase in the stiffness (50% in the first cycle), which may be referred to as cyclic stiffening. In contrast, the dry samples showed a drop in the modulus after the first cycle, without any subsequent changes. Additionally, time-dependent viscoelastic properties were analysed, using dynamic mechanical analysis as well as creep and stress-relaxation techniques. Tan Î´ values for dry samples ranged from 0.05 to 0.075, while for hydrated ones it varied from 0.12 to 0.24. Collagen films exhibited primary and secondary creep stages, while the initial stress-relaxation was fast followed by a monotonous decay. The stress-strain-time data obtained from experiments were fitted in Prony series to estimate the relaxation moduli and times.

Cell marbles: A novel cell encapsulation technology by wrapping cell suspension droplets using electrospun nanofibers for developmental engineering.

Developmental Engineering aims to imitate natural tissue regeneration processes via an additive manufacturing approach. This research developes a technology to fabricate ready-made cell marbles (CMs) by wrapping cell suspension droplets of (3-15 µl) with electrospun hydrophobic nanofibers, as modular building blocks for developmental engineering. Human dermal fibroblasts and/or immortalised keratinocytes were suspended in the culture media cores of the CMs. The encapsulated cells were observed to precipitate at bottoms or up-inclined inner surfaces of the fibrous shells within 10 min. The CMs were mechanically strong enough to be handled as soft solids, thus easily and accurately delivered using forceps into three distinct culture systems, including tissue culture plastics, cellulosic scaffolds and in vitro fibrin wound models. The release of the cells, culture media and nanofibers into specific delivery points within the investigated culture systems was achieved via the controlled rupture of the CMs triggered by the simple hydrophobic-hydrophilic interaction between the nanofibers and the aqueous surroundings. Further cell and tissue culture studies indicated that the prominent traits of the skin cells were well preserved during cell encapsulation and delivery processes, suggesting the great potential of the CMs for additive tissue manufacturing in developmental engineering.

Dry vs. wet: Properties and performance of collagen films. Part I. Mechanical behaviour and strain-rate effect.

Collagen forms one-third of the body proteome and has emerged as an important biomaterial for tissue engineering and wound healing. Collagen films are used in tissue regeneration, wound treatment, dural substitute etc. as well as in flexible electronics. Thus, the mechanical behaviour of collagen should be studied under different environmental conditions and strain rates relevant for potential applications. This study's aim is to assess the mechanical behaviour of collagen films under different environmental conditions (hydration, submersion and physiological temperature (37 °C)) and strain rates. The combination of all three environment factors (hydration, submersion and physiological temperature (37 °C)) resulted in a drop of tensile strength of the collagen film by some 90% compared to that of dry samples, while the strain at failure increased to about 145%. For the first time, collagen films were subjected to different strain rates ranging from quasi-static (0.0001 s-1) to intermediate (0.001 s-1, 0.01 s-1) to dynamic (0.1 s-1, 1 s-1) conditions, with the strain-rate-sensitivity exponent (m) reported. It was found that collagen exhibited a strain-rate-sensitive hardening behaviour with increasing strain rate. The exponent m ranged from 0.02-0.2, with a tendency to approach zero at intermediate strain rate (0.01 s-1), indicating that collagen may be strain-rate insensitive in this regime. From the identification of hyperelastic parameter of collagen film, it was found that the Ogden Model provides realistic results for future simulations.