Electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) scaffolds - a step towards ligament repair applications.

The incidence of anterior cruciate ligament (ACL) ruptures is approximately 50 per 100,000 people. ACL rupture repair methods that offer better biomechanics have the potential to reduce long term osteoarthritis. To improve ACL regeneration biomechanically similar, biocompatible and biodegradable tissue scaffolds are required. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with high 3-hydroxyvalerate (3HV) content, based scaffold materials have been developed, with the advantages of traditional tissue engineering scaffolds combined with attractive mechanical properties, e.g., elasticity and biodegradability. PHBV with 3HV fractions of 0 to 100 mol% were produced in a controlled manner allowing specific compositions to be targeted, giving control over material properties. In conjunction electrospinning conditions were altered, to manipulate the degree of fibre alignment, with increasing collector rotating speed used to obtain random and aligned PHBV fibres. The PHBV based materials produced were characterised, with mechanical properties, thermal properties and surface morphology being studied. An electrospun PHBV fibre mat with 50 mol% 3HV content shows a significant increase in elasticity compared to those with lower 3HV content and could be fabricated into aligned fibres. Biocompatibility testing with L929 fibroblasts demonstrates good cell viability, with the aligned fibre network promoting fibroblast alignment in the axial fibre direction, desirable for ACL repair applications. Dynamic load testing shows that the 50 mol% 3HV PHBV material produced can withstand cyclic loading with reasonable resilience. Electrospun PHBV can be produced with low batch variability and tailored, application specific properties, giving these biomaterials promise in tissue scaffold applications where aligned fibre networks are desired, such as ACL regeneration. .

X-ray computed tomography in life sciences.

Recent developments within micro-computed tomography (µCT) imaging have combined to extend our capacity to image tissue in three (3D) and four (4D) dimensions at micron and sub-micron spatial resolutions, opening the way for virtual histology, live cell imaging, subcellular imaging and correlative microscopy. Pivotal to this has been the development of methods to extend the contrast achievable for soft tissue. Herein, we review the new capabilities within the field of life sciences imaging, and consider how future developments in this field could further benefit the life sciences community.

A simple in vitro biomimetic perfusion system for mechanotransduction study.

In mechanotransduction studies, flow-induced shear stress (FSS) is often applied to two-dimensional (2D) cultured cells with a parallel-plate flow chamber (PPFC) due to its simple FSS estimation. However, cells behave differently under FSS inside a 3D scaffold (e.g. 10 mPa FSS was shown to induce osteogenesis of human mesenchymal stem cells (hMSC) in 3D but over 900 mPa was needed for 2D culture). Here, a simple in vitro biomimetic perfusion system using borosilicate glass capillary tubes has been developed to study the cellular behaviour under low-level FSS that mimics 3D culture. It has been shown that, compared to cells in the PPFC, hMSC in the capillary tubes had upregulated Runx-2 expression and osteogenic cytoskeleton actin network under 10 mPa FSS for 24 h. Also, an image analysis method based on Haralick texture measurement has been used to identify osteogenic actin network. The biomimetic perfusion system can be a valuable tool to study mechanotransduction in 3D for more clinical relevant tissue-engineering applications.

Direct electrical stimulation enhances osteogenesis by inducing Bmp2 and Spp1 expressions from macrophages and preosteoblasts.

The capability of electrical stimulation (ES) in promoting bone regeneration has already been addressed in clinical studies. However, its mechanism is still being investigated and discussed. This study aims to investigate the responses of macrophages (J774A.1) and preosteoblasts (MC3T3-E1) to ES and the faradic by-products from ES. It is found that pH of the culture media was not significantly changed, whereas the average hydrogen peroxide concentration was increased by 3.6 and 5.4 µM after 1 and 2 hr of ES, respectively. The upregulation of Bmp2 and Spp1 messenger RNAs was observed after 3 days of stimulation, which is consistent among two cell types. It is also found that Spp1 expression of macrophages was partially enhanced by faradic by-products. Osteogenic differentiation of preosteoblasts was not observed during the early stage of ES as the level of Runx2 expression remains unchanged. However, cell proliferation was impaired by the excessive current density from the electrodes, and also faradic by-products in the case of macrophages. This study shows that macrophages could respond to ES and potentially contribute to the bone formation alongside preosteoblasts. The upregulation of Bmp2 and Spp1 expressions induced by ES could be one of the mechanisms behind the electrically stimulated osteogenesis.

Osteochondral tissue coculture: An in vitro and in silico approach.

Osteochondral tissue engineering aims to regenerate functional tissue-mimicking physiological properties of injured cartilage and its subchondral bone. Given the distinct structural and biochemical difference between bone and cartilage, bilayered scaffolds, and bioreactors are commonly employed. We present an osteochondral culture system which cocultured ATDC5 and MC3T3-E1 cells on an additive manufactured bilayered scaffold in a dual-chamber perfusion bioreactor. Also, finite element models (FEM) based on the microcomputed tomography image of the manufactured scaffold as well as on the computer-aided design (CAD) were constructed; the microenvironment inside the two FEM was studied and compared. In vitro results showed that the coculture system supported osteochondral tissue growth in terms of cell viability, proliferation, distribution, and attachment. In silico results showed that the CAD and the actual manufactured scaffold had significant differences in the flow velocity, differentiation media mixing in the bioreactor and fluid-induced shear stress experienced by the cells. This system was shown to have the desired microenvironment for osteochondral tissue engineering and it can potentially be used as an inexpensive tool for testing newly developed pharmaceutical products for osteochondral defects.

Fabrication and Characterisation of Stimuli Responsive Piezoelectric PVDF and Hydroxyapatite-Filled PVDF Fibrous Membranes.

Poly(vinylidene fluoride) has attracted interest from the biomaterials community owing to its stimuli responsive piezoelectric property and promising results for application in the field of tissue engineering. Here, solution blow spinning and electrospinning were employed to fabricate PVDF fibres and the variation in resultant fibre properties assessed. The proportion of piezoelectric ß-phase in the solution blow spun fibres was higher than electrospun fibres. Fibre production rate was circa three times higher for solution blow spinning compared to electrospinning for the conditions explored. However, the solution blow spinning method resulted in higher fibre variability between fabricated batches. Fibrous membranes are capable of generating different cellular response depending on fibre diameter. For this reason, electrospun fibres with micron and sub-micron diameters were fabricated, along with successful inclusion of hydroxyapatite particles to fabricate stimuli responsive bioactive fibres.

Three-dimensional visualisation of soft biological structures by X-ray computed micro-tomography.

Whereas the two-dimensional (2D) visualisation of biological samples is routine, three-dimensional (3D) imaging remains a time-consuming and relatively specialised pursuit. Current commonly adopted techniques for characterising the 3D structure of non-calcified tissues and biomaterials include optical and electron microscopy of serial sections and sectioned block faces, and the visualisation of intact samples by confocal microscopy or electron tomography. As an alternative to these approaches, X-ray computed micro-tomography (microCT) can both rapidly image the internal 3D structure of macroscopic volumes at sub-micron resolutions and visualise dynamic changes in living tissues at a microsecond scale. In this Commentary, we discuss the history and current capabilities of microCT. To that end, we present four case studies to illustrate the ability of microCT to visualise and quantify: (1) pressure-induced changes in the internal structure of unstained rat arteries, (2) the differential morphology of stained collagen fascicles in tendon and ligament, (3) the development of Vanessa cardui chrysalises, and (4) the distribution of cells within a tissue-engineering construct. Future developments in detector design and the use of synchrotron X-ray sources might enable real-time 3D imaging of dynamically remodelling biological samples.

The Effect of Branching (Star Architecture) on Poly(d,l-lactide) (PDLLA) Degradation and Drug Delivery.

This study focuses on the comparative evaluation of star (branched) and linear poly(l,d-lactic acid) (PDLLA) as degradable materials employed in controlled release. The polymers were prepared via ring-opening polymerization initiated by decanol (linear), pentaerythritol (4-armed star) and dipentaerythritol (6-armed star), and processed both in the form of films and nanoparticles. Independent of the length or number of their arms, star polymers degrade slower than linear polymers, possibly through a surface (vs bulk) mechanism. Further, the release of a model drug (atorvastatin) followed zero-order-like kinetics for the branched polymers, and first-order kinetics for linear PDLLA. Using NHOst osteoblastic cells, both linear and star polymers were devoid of any significant toxicity and released atorvastatin in a bioavailable form; cell adhesion was considerably lower on star polymer films, and the slower release from their nanoparticles appeared to be beneficial to avoid atorvastatin overdosing.

A correlative imaging based methodology for accurate quantitative assessment of bone formation in additive manufactured implants.

A correlative imaging methodology was developed to accurately quantify bone formation in the complex lattice structure of additive manufactured implants. Micro computed tomography (µCT) and histomorphometry were combined, integrating the best features from both, while demonstrating the limitations of each imaging modality. This semi-automatic methodology registered each modality using a coarse graining technique to speed the registration of 2D histology sections to high resolution 3D µCT datasets. Once registered, histomorphometric qualitative and quantitative bone descriptors were directly correlated to 3D quantitative bone descriptors, such as bone ingrowth and bone contact. The correlative imaging allowed the significant volumetric shrinkage of histology sections to be quantified for the first time (~15 %). This technique demonstrated the importance of location of the histological section, demonstrating that up to a 30 % offset can be introduced. The results were used to quantitatively demonstrate the effectiveness of 3D printed titanium lattice implants.

Current clinical opinion on surgical approaches and rehabilitation of hand flexor tendon injury-a questionnaire study.

The management of flexor tendon injury has seen many iterations over the years, but more substantial innovations in practice have been sadly lacking. The aim of this study was to investigate the current practice of flexor tendon injury management, and variation in practice from the previous reports, most troublesome complications, and whether there was a clinical interest in potential innovative tendon repair technologies. An online survey was distributed via the British Society for Surgery of the Hand (BSSH) and a total of 132 responses were collected anonymously. Results showed that although most surgeons followed the current medical recommendation based on the literature, a significant number of surgeons still employed more conventional treatments in clinic, such as general anesthesia, ineffective tendon retrieval techniques, and passive rehabilitation. Complications including adhesion formation and re-rupture remained persistent. The interest in new approaches such as use of minimally invasive instruments, biodegradable materials and additive manufactured devices was not strong, however the surgeons were potentially open to more effective and economic solutions.

Automated fabrication of a scalable heart-on-a-chip device by 3D printing of thermoplastic elastomer nanocomposite and hot embossing.

The successful translation of organ-on-a-chip devices requires the development of an automated workflow for device fabrication, which is challenged by the need for precise deposition of multiple classes of materials in micro-meter scaled configurations. Many current heart-on-a-chip devices are produced manually, requiring the expertise and dexterity of skilled operators. Here, we devised an automated and scalable fabrication method to engineer a Biowire II multiwell platform to generate human iPSC-derived cardiac tissues. This high-throughput heart-on-a-chip platform incorporated fluorescent nanocomposite microwires as force sensors, produced from quantum dots and thermoplastic elastomer, and 3D printed on top of a polystyrene tissue culture base patterned by hot embossing. An array of built-in carbon electrodes was embedded in a single step into the base, flanking the microwells on both sides. The facile and rapid 3D printing approach efficiently and seamlessly scaled up the Biowire II system from an 8-well chip to a 24-well and a 96-well format, resulting in an increase of platform fabrication efficiency by 17,5000-69,000% per well. The device's compatibility with long-term electrical stimulation in each well facilitated the targeted generation of mature human iPSC-derived cardiac tissues, evident through a positive force-frequency relationship, post-rest potentiation, and well-aligned sarcomeric apparatus. This system's ease of use and its capacity to gauge drug responses in matured cardiac tissue make it a powerful and reliable platform for rapid preclinical drug screening and development.

State of the art composites comprising electrospun fibres coupled with hydrogels: a review.

Research into scaffolds tailored for specific tissue engineering and biomaterial applications continues to develop as these structures are commonly impeded by their limitations. For example, electrospun fibres and hydrogels are commonly exploited because of their ability to mimic natural tissues; however, their clinical use remains restricted due to negligible cellular infiltration and poor mechanical properties, respectively. A small number of research groups are beginning to investigate composite scaffolds based on electrospun fibres and hydrogels in an attempt to overcome their individual shortcomings. This review paper discusses the various methodologies and approaches currently undertaken to create these novel composite structures and their intended applications. The combination of these two commonly used scaffold architectures to create synergistically superior structures is showing potential with regards to therapeutic use within the tissue engineering community. FROM THE CLINICAL EDITOR: This review discusses methodologies to create novel electrospun nanofibers and hydrogels, and their intended applications. The combination of these two scaffold architectures has important future clinical applications, although their use is currently limited to the experimental tissue engineering community.

Deposition of a high entropy thin film by aerosol-assisted chemical vapor deposition.

Herein we report for the first time the synthesis of a high entropy (CuZnCoInGa)S metal sulfide thin film deposited by AACVD using molecular precursors.

Synthesis of High Entropy and Entropy-Stabilized Metal Sulfides and Their Evaluation as Hydrogen Evolution Electrocatalysts.

High entropy metal chalcogenides are materials containing five or more elements within a disordered sublattice. These materials exploit a high configurational entropy to stabilize their crystal structure and have recently become an area of significant interest for renewable energy applications such as electrocatalysis and thermoelectrics. Herein, we report the synthesis of bulk particulate HE zinc sulfide analogues containing four, five, and seven metals. This was achieved using a molecular precursor cocktail approach with both transition and main group metal dithiocarbamate complexes which are decomposed simultaneously in a rapid (1 h) and low-temperature (500 °C) thermolysis reaction to yield high entropy and entropy-stabilized metal sulfides. The resulting materials were characterized by powder XRD, SEM, and TEM, alongside EDX spectroscopy at both the micro- and nano-scales. The entropy-stabilized (CuAgZnCoMnInGa)S material was demonstrated to be an excellent electrocatalyst for the hydrogen evolution reaction when combined with conducting carbon black, achieving a low onset overpotential of (∼80 mV) and η10 of (∼255 mV).

Modeling and design of optimal flow perfusion bioreactors for tissue engineering applications.

Perfusion bioreactors have been used in different tissue engineering applications because of their consistent distribution of nutrients and flow-induced shear stress within the tissue-engineering scaffold. A widely used configuration uses a scaffold with a circular cross-section enclosed within a cylindrical chamber and inlet and outlet pipes which are connected to the chamber on either side through which media is continuously circulated. However, fluid-flow experiments and simulations have shown that the majority of the flow perfuses through the center. This pattern creates stagnant zones in the peripheral regions as well as in those of high flow rate near the inlet and outlet. This non-uniformity of flow and shear stress, owing to a circular design, results in limited cell proliferation and differentiation in these areas. The focus of this communication is to design an optimized perfusion system using computational fluid dynamics as a mathematical tool to overcome the time-consuming trial and error experimental method. We compared the flow within a circular and a rectangular bioreactor system. Flow simulations within the rectangular bioreactor are shown to overcome the limitations in the circular design. This communication challenges the circular cross-section bioreactor configuration paradigm and provides proof of the advantages of the new design over the existing one.

Status and challenges of electrical stimulation use in chronic wound healing.

Non-healing wounds have led to a soaring clinical and socioeconomical need for advanced wound-care techniques. Electrical stimulation is an emerging therapy inspired by the wound's endogenous electric field. Promising results of clinical trials have encouraged efforts to create wearable stimulation devices, uncover multiple cellular targets, and optimize stimulation regimes. However, the field faces a translational bottleneck. This review aims to highlight the gaps between in vivo treatments and in vitro associated experiments by discussing the current knowledge of the generation, characterization, and targets of electrical stimuli. It becomes clear that enabling the translation of this technology will require increasing the complexity of the current models for skin endogenous and controlled ion transport, and investigating which stimulus has an optimum effect on cells derived from chronic wound-prone patients.

Electrical stimulation of titanium to promote stem cell orientation, elongation and osteogenesis.

Electrical stimulation of cells allows exogenous electric signals as stimuli to manipulate cell growth, preferential orientation and bone remodelling. In this study, commercially pure titanium discs were utilised in combination with a custom-built bioreactor to investigate the cellular responses of human mesenchymal stem cells via in-vitro functional assays. Finite element analysis revealed the homogeneous delivery of electric field in the bioreactor chamber with no detection of current density fluctuation in the proposed model. The custom-built bioreactor with capacitive stimulation delivery system features long-term stimulation with homogeneous electric field, biocompatible, sterilisable, scalable design and cost-effective in the manufacturing process. Using a continuous stimulation regime of 100 and 200 mV/mm on cp Ti discs, viability tests revealed up to an approximately 5-fold increase of cell proliferation rate as compared to non-stimulated controls. The human mesenchymal stem cells showed more elongated and differentiated morphology under this regime, with evidence of nuclear elongation and cytoskeletal orientation perpendicular to the direction of electric field. The continuous stimulation did not cause pH fluctuations and hydrogen peroxide production caused by Faradic reactions, signifying the suitability for long-term toxic free stimulation as opposed to the commonly used direct stimulation regime. An approximate of 4-fold increase in alkaline phosphatase production and approximately 9-fold increase of calcium deposition were observed on 200 mV/mm exposed samples relative to non-stimulated controls. It is worth noting that early stem cell differentiation and matrix production were observed under the said electric field even without the presence of chemical inductive growth factors. STATEMENT OF SIGNIFICANCE: This manuscript presents a study on combining pure titanium (primarily preferred as medical implant materials) and electrical stimulation in a purpose-built bioreactor with capacitive stimulation delivery system. A continuous capacitive stimulation regime on titanium disc has resulted in enhanced stem cell orientation, nuclei elongation, proliferation and differentiation as compared to non-stimulated controls. We believe that this manuscript creates a paradigm for future studies on the evolution of healthcare treatments in the area of targeted therapy on implantable and wearable medical devices through tailored innovative electrical stimulation approach, thereby influencing therapeutic conductive and electroactive biomaterials research prospects and development.

Capacitive electrical stimulation of a conducting polymeric thin film induces human mesenchymal stem cell osteogenesis.

Electroactive materials based on conductive polymers are promising options for tissue engineering and regenerative medicine applications. In the present work, the conducting copolymers of poly (3,4-ethylenedioxythiophene) and poly (d, l-lactic acid) (PEDOT-co-PDLLA) with PEDOT:PDLLA molar ratios of 1:50, 1:25, and 1:5 were synthesized and compared to the insulating macromonomer of EDOT-PDLLA as an experimental control. Bone marrow-derived human mesenchymal stem cells (hMSC-BM) were cultured on the copolymers and the macromonomer thin films inside a bioreactor that induced a capacitive electrical stimulation (CES) with an electric field of 100 mV/mm for 2 h per day for 21 days. Under CES, the copolymers exhibited good cell viability and promoted the differentiation from hMSC-BM to osteogenic lineages, revealed by higher mineralization mainly when the contents of conducting segments of PEDOT (i.e., copolymer with 1:25 and 1:5 PEDOT:PDLLA ratios) were increased. The results indicate that the intrinsic electrical conductivity of the substrates is an important key point for the effectiveness of the electric field generated by the CES, intending to promote the differentiation effect for bone cells.

Instructive electroactive electrospun silk fibroin-based biomaterials for peripheral nerve tissue engineering.

Aligned sub-micron fibres are an outstanding surface for orienting and promoting neurite outgrowth; therefore, attractive features to include in peripheral nerve tissue scaffolds. A new generation of peripheral nerve tissue scaffolds is under development incorporating electroactive materials and electrical regimes as instructive cues in order to facilitate fully functional regeneration. Herein, electroactive fibres composed of silk fibroin (SF) and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) were developed as a novel peripheral nerve tissue scaffold. Mats of SF with sub-micron fibre diameters of 190 ± 50 nm were fabricated by double layer electrospinning with thicknesses of ∼100 µm (∼70-80 µm random fibres and ∼20-30 µm aligned fibres). Electrospun SF mats were modified with interpenetrating polymer networks (IPN) of PEDOT:PSS in various ratios of PSS/EDOT (α) and the polymerisation was assessed by hard X-ray photoelectron spectroscopy (HAXPES). The mechanical properties of electrospun SF and IPNs mats were characterised in the wet state tensile and the electrical properties were examined by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The cytotoxicity and biocompatibility of the optimal IPNs (α = 2.3 and 3.3) mats were ascertained via the growth and neurite extension of mouse neuroblastoma x rat glioma hybrid cells (NG108-15) for 7 days. The longest neurite outgrowth of 300 µm was observed in the parallel direction of fibre alignment on laminin-coated electrospun SF and IPN (α = 2.3) mats which is the material with the lowest electron transfer resistance (Ret, ca. 330 Ω). These electrically conductive composites with aligned sub-micron fibres exhibit promise for axon guidance and also have the potential to be combined with electrical stimulation treatment as a further step for the effective regeneration of nerves.

Finite Element Modelling of a Cellular Electric Microenvironment.

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).