Basement membrane properties and their recapitulation in organ-on-chip applications.

Drug discovery and toxicology is a complex process that involves considerable basic research and preclinical evaluation. These depend highly on animal testing which often fails to predict human trial outcomes due to species differences. Coupled with ethical concerns around animal testing, this leads to a high demand for improved in vitro cell culture platforms. Current research efforts, in this regard, however, are facing a challenge to provide physiologically relevant in vitro human organ models for a reliable assessment of the physiological responses of the body to drug compounds and toxins. The latest development in in vitro cell culture models, organ-on-chips (OOCs), seek to introduce more realistic models of organ function. Current OOCs often use commercial porous polymeric membranes as a barrier membrane for cell culture which is challenging due to the poor replication of the physiological architectures. Better recapitulation of the native basement membrane (BM) characteristics is desirable for modelling physical (e.g. intestine, skin and lung) and metabolic (e.g. liver) barrier models. In this review, the relevance of the physical and mechanical properties of the membrane to cell and system behaviour is elucidated. Key parameters for replicating the BM are also described. This review provides information for future development of barrier organ models focusing on BM-mimicking substrates as a core structure.

Physically Cross-Linked Gels of PVA with Natural Polymers as Matrices for Manuka Honey Release in Wound-Care Applications.

Manuka honey is a well-known natural material from New Zealand, considered to have properties beneficial for burn treatment. Gels created from polyvinyl alcohol (PVA) blended with natural polymers are potential burn-care dressings, combining biocompatibility with high fluid uptake. Controlled release of manuka honey from such materials is a possible strategy for improving burn healing. This work aimed to produce polyvinyl alcohol (PVA), PVA⁻sodium carboxymethylcellulose (PVA-CMC), PVA⁻gelatin (PVA-G), and PVA⁻starch (PVA-S) cryogels infused with honey and to characterize these materials physicochemically, morphologically, and thermally, followed by in vitro analysis of swelling capacity, degradation/weight loss, honey delivery kinetics, and possible activity against Staphylococcus aureus. The addition of honey to PVA led to many PVA crystals with defects, while PVA⁻starch⁻honey and PVA⁻sodium carboxymethylcellulose⁻honey (PVA-CMC-H) formed amorphous gels. PVA-CMC presented the highest swelling degree of all. PVA-CMC-H and PVA⁻gelatin⁻honey presented the highest swelling capacities of the honey-laden samples. Weight loss/degradation was significantly higher for samples containing honey. Layers submitted to more freeze⁻thawing cycles were less porous in SEM images. With the honey concentration used, samples did not inhibit S. aureus, but pure manuka honey was bactericidal and dilutions superior to 25% honey were bacteriostatic, indicating the need for higher concentrations to be more effective.

Towards functional 3D-stacked electrospun composite scaffolds of PHBV, silk fibroin and nanohydroxyapatite: Mechanical properties and surface osteogenic differentiation.

Bone tissue engineering scaffolds have two challenging functional tasks to fulfil: to encourage cell proliferation, differentiation and matrix synthesis and to provide suitable mechanical stability upon implantation. Composites of biopolymers and bioceramics combine the advantages of both types of materials, resulting in better processability and enhanced mechanical and biological properties through matrix reinforcement. In the present study, novel thick bone composite scaffolds were successfully fabricated using electrospun flat sheets of polyhydroxybutyrate-polyhydroxyvalerate/nanohydroxyapatite/silk fibroin essence (2% nanohydroxyapatite - 2% silk fibroin essence and 5% nanohydroxyapatite - 5% silk fibroin essence, respectively). Their potential asin vitrobone regeneration scaffolds was evaluated using mouse calvarian osteoblast cells (MC3T3-E1), in terms of morphology (scanning electron microscope), cell attachment, cell proliferation, Col type I, osteopontin and bone alkaline phosphatase activity (Quantitative Real Time Polymerase Chain Reaction [qRT-PCR], enzyme-linked immunosorbent assay, immunocytochemistry). Electrospun polyhydroxybutyrate-polyhydroxyvalerate scaffolds were used as reference constructs. The results showed that the compressive and tensile mechanical properties of the scaffolds are dependent on the change in their composition, and the treatment these underwent. Furthermore, methanol-treated and autoclaved (MA) P2 (2% nanohydroxyapatite, 2% silk fibroin essence) samples appeared to exhibit more promising tensile properties. Additionally, the compressive tests results confirmed that the methanol pre-treatment and the autoclaving step lead to an increase in the P2 secant modulus when compared to the non-methanol-treated ones, P2 and P5 (5% nanohydroxyapatite, 5% silk fibroin essence), respectively.Both formulations of polyhydroxybutyrate-polyhydroxyvalerate/nanohydroxyapatite/silk fibroin essence composite promoted greater cell adhesion and proliferation than the corresponding polyhydroxybutyrate-polyhydroxyvalerate control ones. Cells seeded on the composite fibrous scaffolds were extensively expanded and elongated on the fibre surface after one day in culture, whereas those seeded on the polyhydroxybutyrate-polyhydroxyvalerate scaffolds were not completely elongated. In addition, cells grown on P2 and P5 scaffolds had higher alkaline phosphatase activity when compared to those containing no nanohydroxyapatite/silk fibroin essence.

Electrospun nanofibre bundles and yarns for tissue engineering applications: A review.

Nanofibre membranes produced through the electrospinning process have been studied extensively over the past decade for a number of high demand applications including use as tissue engineered scaffolds. Despite possessing desirable properties including high surface area to volume ratios and enhanced mechanical properties, they ultimately suffer from a lack of cellular infiltration. Variations on the process include the production of highly aligned filaments of electrospun fibres referred to as bundles and yarns. Nanofibre bundle and yarn-based scaffolds have been shown to demonstrate superior cell infiltration rates compared to traditional electrospun nonwovens while also offering the capability to be incorporated into a wider array of post-processing technologies. In this review, fibre collection techniques currently employed within the literature for the fabrication of electrospun bundles and yarns along with their applications in the field of tissue engineering will be discussed.

Characterisation of debris from laser and mechanical cutting of bone.

Laser cutting of bones has been proposed as a technology in orthopaedic surgery. In this short study, the laser-bone interaction was examined using a pulsed erbium-doped yttrium aluminium garnet laser and compared to a conventional cutting technique. Microscopic analysis revealed the nature of waste debris and showed higher proportions of finer particles for conventional sagittal sawing compared to laser cutting.

Electrospun composites of PHBV, silk fibroin and nano-hydroxyapatite for bone tissue engineering.

Electrospinning of fibrous scaffolds containing nano-hydroxyapatite (nHAp) embedded in a matrix of functional biomacromolecules offers an attractive route to mimicking the natural bone tissue architecture. Functional fibrous substrates will support cell attachment, proliferation and differentiation, while the role of HAp is to induce cells to secrete extracellular matrix (ECM) for mineralization to form bone. Electrospinning of biomaterials composed of polyhydroxybutyrate-co-(3-hydroxyvalerate) with 2% valerate fraction (PHBV), nano-hydroxyapatite (nHAp), and Bombyx mori silk fibroin essence (SF), Mw=90KDa, has been achieved for nHAp and SF solution concentrations of 2 (w/vol) % each and 5 (w/vol) % each. The structure and properties of the nanocomposite fibrous membranes were investigated by means of Scanning Electron Microscopy in combination with Energy Dispersive X-Ray Analysis (SEM/EDX), Fourier Transformed Infrared Spectroscopy (FT-IR), uniaxial tensile and compressive mechanical testing, degradation tests and in vitro bioactivity tests. SEM images showed smooth, uniform and continuous fibre deposition with no bead formation, and fibre diameters of between 10 and 15 µm. EDX and FT-IR confirmed the presence of nHAp and SF. After one month in deionised water, tests showed less than 2% weight loss with the samples retaining their fibrous morphology, confirming that this material biodegrades slowly. After 28 days of immersion in Simulated Body Fluid (SBF) an apatite layer was visible on the surface of the fibres, proving their bioactivity. Preliminary in vitro biological assessment showed that after 1 and 3 days in culture, cells were attached to the fibres, retaining their morphology while presenting a flattened appearance and elongated shape on the surface of fibres. Young's modulus was found to increase from 0.7 kPa (±0.33 kPa) for electrospun samples of PHBV only to 1.4 kPa (±0.54 kPa) for samples with 2 (w/vol) % each of nHAp and SF. Samples prepared with 5 (w/vol) % each of nHAp and SF did not show a similar improvement.

Increased susceptibility of arterial tissue to wire perforation with the application of high-frequency mechanical vibrations.

High-frequency mechanical vibrations (20-50 kHz), delivered via small diameter flexible wire waveguides represent a minimally invasive technology for the treatment of chronic total occlusions and in other tissue ablation applications. Tissue disruption is reported to be caused by repetitive mechanical contact and cavitation. This work focuses on the effects of vibrating wire waveguides in contact with arterial tissue. An apparatus with clinically relevant parameters was used, characterized as operating at 22.5 kHz and delivering amplitudes of vibration of 17.8-34.3 µm (acoustic intensity, I(SATA): 1.03-3.83 W/cm(2)) via 1.0-mm diameter waveguides. Inertial cavitation (in water at 37 °C) was determined to occur above amplitudes of vibration greater than 31.4 µm (I(SATA) = 3.21 W/cm(2)). The energized waveguides were advanced through tissue samples (porcine aorta) and the force profiles were measured for a range of acoustic intensities. The results show that the tissue perforation initiation force, perforation initiation energy, and total energy required to perforate the tissue reduces with increasing acoustic intensity. No significant reduction in perforation force or energy was observed in the inertial cavitation region. Multistage perforation was evident through the force profile and histological examination of the tissue samples post wire waveguide perforation.

Cell encapsulation and cryostorage in PVA-gelatin cryogels: incorporation of carboxylated ε-poly-L-lysine as cryoprotectant.

It is desirable to produce cryopreservable cell-laden tissue-engineering scaffolds whose final properties can be adjusted during the thawing process immediately prior to use. Polyvinyl alcohol (PVA)-based solutions provide platforms in which cryoprotected cell suspensions can be turned into a ready-to-use, cell-laden scaffold by a process of cryogelation. In this study, such a PVA system, with DMSO as the cryoprotectant, was successfully developed. Vascular smooth muscle cell (vSMC)-encapsulated cryogels were investigated under conditions of cyclic strain and in co-culture with vascular endothelial cells to mimic the environment these cells experience in vivo in a vascular tissue-engineering setting. In view of the cytotoxicity DMSO imposes with respect to the production procedure, carboxylated poly-L-lysine (COOH-PLL) was substituted as a non-cytotoxic cryoprotectant to allow longer, slower thawing periods to generate more stable cryogels. Encapsulated vSMC with DMSO as a cryoprotectant responded to 10% cyclic strain with increased alignment and proliferation. Cells were stored frozen for 1 month without loss of viability compared to immediate thawing. SMC-encapsulated cryogels also successfully supported functional endothelial cell co-culture. Substitution of COOH-PLL in place of DMSO resulted in a significant increase in cell viability in encapsulated cryogels for a range of thawing periods. We conclude that incorporation of COOH-PLL during cryogelation preserved cell functionality while retaining fundamental cryogel physical properties, thereby making it a promising platform for tissue-engineering scaffolds, particularly for vascular tissue engineering, or cell preservation within microgels.

High power, low frequency ultrasound: meniscal tissue interaction and ablation characteristics.

This study evaluates high power low frequency ultrasound transmitted via a flat vibrating probe tip as an alternative technology for meniscal debridement in the bovine knee. An experimental force controlled testing rig was constructed using a 20 kHz ultrasonic probe suspended vertically from a load cell. Effect of variation in amplitude of distal tip displacement (242-494 µm peak-peak) settings and force (2.5-4.5 N) on tissue removal rate (TRR) and penetration rate (PR) for 52 bovine meniscus samples was analyzed. Temperature elevation in residual meniscus was measured by embedded thermocouples and histologic analysis. As amplitude or force increases, there is a linear increase in TRR (Mean: 0.9 to 11.2 mg/s) and PR (Mean: 0.08 to 0.73 mm/s). Maximum mean temperatures of 84.6°C and 52.3°C were recorded in residual tissue at 2 mm and 4 mm from the ultrasound probe-tissue interface. There is an inverse relationship between both amplitude and force, and temperature elevation, with higher settings resulting in less thermal damage.

Endothelialization of PVA/gelatin cryogels for vascular tissue engineering: effect of disturbed shear stress conditions.

Mechanically, poly(vinyl alcohol) (PVA)-based cryogels are extremely well suited for vascular tissue engineering applications. However, their surface properties lead to a slow rate of endothelialization, and the mode of cell attachment leaves the endothelium susceptible to removal under physiological shear stress conditions. In this study, abrupt and ramped disturbed shear stress conditions created by a turbulent orbital flow were used to examine endothelialization on PVA/gelatin cryogels. Cell proliferation rate and apoptosis were evaluated by fluorescent activated cell sorter (FACS) analysis, and the expression of cell-adhesion molecules was used to evaluate the response of cells on cryogels to static and shear conditions by real-time polymerase chain reaction (RT-PCR). Application of a ramped shear stress had a profound effect on endothelial cell proliferation (22.30 +/- 0.20-fold increase), necrosis (eliminated), apoptosis (1.04 +/- 0.18 increase), and overall facilitation of endothelialization while concomitantly increasing nitric oxide (NO) synthesis levels. Ramped shear stress was also effective in helping the retention of the endothelial cells on the cryogel surface, whereas abrupt application caused widespread removal. Under static conditions, Selectin-P expression decreased, whereas both inter-cellular adhesion molecule (ICAM) and platelet endothelial cell adhesion molecule (PECAM)-I expression increased on cryogels over a 10-day culture period. Under both shear stress conditions, Selectin-P expression was decreased both on cryogels and tissue culture polystyrene (TCPS). Controlled application of disturbed shear stress shortens endothelialization times on cryogel surfaces, in contrast to the established antiproliferative effect of shear stress caused by laminar flow, without compromising their functionality. This demonstrates how such mechanical stimuli can be exploited to alter cellular behavior and facilitate the required outcomes for tissue engineering applications.

Thermal behavior and mechanical properties of physically crosslinked PVA/Gelatin hydrogels.

Poly (vinyl alcohol)/Gelatin hydrogels are under active investigation as potential vascular cell culture biomaterials, tissue models and vascular implants. The PVA/Gelatin hydrogels are physically crosslinked by the freeze-thaw technique, which is followed by a coagulation bath treatment. In this study, the thermal behavior of the gels was examined by differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis (DMTA). Rheological measurement and uniaxial tensile tests revealed key mechanical properties. The role of polymer fraction in relation to these mechanical properties is explored. Gelatin has no significant effect on the thermal behavior of PVA, which indicates that no substantial change occurs in the PVA crystallite due to the presence of gelatin. The glass transition temperature, melting temperature, degree of crystallinity, polymer fraction, storage modulus (G') and ultimate strength of one freeze-thaw cycle (1FT) hydrogels are inferior to those of 3FT hydrogels. With coagulation, both 1FT and 3FT hydrogels shifted to a lower value of T(g), melting temperature and polymer fraction are further increased and the degree of crystallinity is depressed. The mechanical properties of 1FT, but not 3FT, were strengthened with coagulation treatment. This study gives a detailed investigation of the microstructure formation of PVA/Gelatin hydrogel in each stage of physical treatments which helps us to explain the role of physical treatments in tuning their physical properties for biomechanical applications.

Therapeutic ultrasound angioplasty: the risk of arterial perforation. An in vitro study.

The use of therapeutic ultrasound delivered via small diameter wire waveguides may represent an emerging minimally invasive approach in the treatment of chronic total occlusions (CTOs), calcified and fibrous plaques. The distal-tip mechanical vibrations (typically 0-210 microm peak-to-peak) have been reported to debulk rigid calcified and fibrous tissues while healthy elastic arterial tissue remains largely unaffected. The risk of arterial (healthy tissue) perforation with energized waveguides is not fully understood. An ultrasonic apparatus capable of delivering a range of wire waveguide distal-tip displacements, up to 80 microm peak-to-peak (p-p), at an operational frequency of 22.5 KHz (+/- 6%) has been developed. For three distal-tip displacement settings (32, 50 and 80 microm p-p) with 1.0 mm diameter waveguides, the force required to perforate healthy porcine aortic tissue was experimentally determined. The results show a distinct two stage perforation, thought to be the result of different mechanical properties of the layers in the arterial wall. The average maximum force (N) required to cause perforation with the 1.0 mm diameter ultrasonic waveguide activated at the three settings was experimentally determined to be 2.7 N (32 microm p-p), 2.6 N (50 microm p-p) and 2 N (80 microm p-p). The force required to cause perforation of the tissue with no ultrasound was found to be approximately 4 N. These results highlight that when ultrasound energy is applied to the waveguide, less force is required to perforate healthy arterial tissue. This reduction in perforation force is more pronounced at higher ultrasonic displacements, similar to those reported in clinical studies for the effective removal of diseased calcified and fibrous plaques.