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Drug deposition into the intrapericardial space is favourable for achieving localised effects and targeted cardiac delivery owing to its proximity to the myocardium as well as facilitating optimised pharmacokinetic profiles and a reduction in systemic side effects. Access to the pericardium requires invasive procedures but the risks associated with this have been reduced with technological advances, such as combining transatrial and subxiphoid access with different guidance methods. A variety of introducer devices, ranging from needles to loop-catheters, have also been developed and validated in pre-clinical studies investigating intrapericardial delivery of therapeutic agents. Access techniques are generally well-tolerated, self-limiting and safe, although some rare complications associated with certain approaches have been reported. This review covers these access techniques and how they have been applied to the delivery of drugs, cells, and biologicals, demonstrating the potential of intrapericardial delivery for treatments in cardiac arrhythmia, vascular damage, and myocardial infarction.

The recent rise of adoptive T cell therapy (ATCT) as a promising cancer immunotherapy has triggered increased interest in therapeutic T cell bioprocessing. T cell activation is a critical processing step and is known to be modulated by physical parameters, such as substrate stiffness. Nevertheless, relatively little is known about how biophysical factors regulate immune cells, such as T cells. Understanding how T cell activation is modulated by physical and biochemical cues may offer novel methods to control cell behavior for therapeutic cell processing. Inspired by T cell mechanosensitivity, we developed a multiwell, reusable, customizable, two-dimensional (2D) polyacrylamide (PA) hydrogel-integrated culture device to study the physicochemical stimulation of Jurkat T cells. Substrate stiffness and ligand density were tuned by concentrations of the hydrogel cross-linker and antibody in the coating solution, respectively. We cultured Jurkat T cells on 2D hydrogels of different stiffnesses that presented surface-immobilized stimulatory antibodies against CD3 and CD28 and demonstrated that Jurkat T cells stimulated by stiff hydrogels (50.6 ± 15.1 kPa) exhibited significantly higher interleukin-2 (IL-2) secretion, but lower proliferation, than those stimulated by softer hydrogels (7.1 ± 0.4 kPa). In addition, we found that increasing anti-CD3 concentration from 10 to 30 μg/mL led to a significant increase in IL-2 secretion from cells stimulated on 7.1 ± 0.4 and 9.3 ± 2.4 kPa gels. Simultaneous tuning of substrate stiffness and stimulatory ligand density showed that the two parameters synergize (two-way ANOVA interaction effect: p < 0.001) to enhance IL-2 secretion. Our results demonstrate the importance of physical parameters in immune cell stimulation and highlight the potential of designing future immunostimulatory biomaterials that are mechanically tailored to balance stimulatory strength and downstream proliferative capacity of therapeutic T cells.

Due to their homogeneity, tuneable properties, low cost and ease of manufacture, thermally induced phase separation (TIPS) polymeric microparticles are emerging as an exciting class of injectable device for the treatment of damaged tissue or complex diseases, such as cancer. However, relatively little work has explored enhancing surface functionalisation of this system. Herein, we present the functionalisation of TIPS microparticles with both small molecules and an antibody fragment of Herceptin™, via a heterobifunctional pyridazinedione linker capable of participating in SPAAC “click” chemistry, and compare it to the traditional method of preparing active-targeted microparticle systems, that is, physisorption of antibodies to the microparticle surface. Antigen-binding assays demonstrated that functionalisation of microparticles with Herceptin Fab, via a pyridazinedione linker, provided an enhanced avidity to HER2+ when compared to traditional physisorption methods.

Prostate cancer is a leading cause of death in men and despite improved surgical procedures that aid tumor resection, the risk of recurrence after surgery as a result of positive resection margins remains significant. Adjuvant chemotherapy is often required but this is associated with toxicity. Improved ways of delivering highly toxic chemotherapeutic drugs in a more controlled and targeted manner after the prostate has been removed during surgery could reduce the risk of recurrence and avoid systemic toxicity. The aim of this study was to develop a novel drug-device combination tissue scaffold that can be used to deliver the chemotherapeutic agent, docetaxel, into the tissue cavity that is created following radical prostatectomy. The device component investigated consisted of highly porous, poly(dl-lactide-co-glycolide) microparticles made using thermally induced phase separation. A facile method was established for loading docetaxel with high efficiency within one hour. Sustained drug release was observed from the microparticles when placed into a dynamic system simulating tissue perfusion. The drug released from the microparticles into perfusates collected at regular time intervals inhibited colony formation and exhibited sustained cytotoxicity against 3D spheroids of PC3 prostate cancer cells over 10 days. In conclusion, this study demonstrates the concept of combining docetaxel with the biodegradable microparticles at the point of care is technically feasible for achieving an effective drug-device combination tissue scaffold. This approach could provide an effective new approach for delivering adjuvant chemotherapy following radical prostatectomy.

N-acetyl-seryl-aspartyl-lysyl proline (Ac-SDKP) is a tetrapeptide possessing anti-fibrotic, angiogenic, anti-inflammatory, anti-apoptotic, and immunomodulatory properties. Currently, the main method to quantify the peptide is liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA), both of which are labour intensive and require expensive equipment and consumables. Furthermore, these techniques are generally utilised to detect very low or trace concentrations, such as in biological samples. The use of high concentrations of analyte might overload the extraction column or the separation column in LC-MS/MS or the ELISA plates, so the response could be a non-linear relationship at high analyte concentrations. Thus, they are not ideal for formulation development where detection of dose-equivalent concentrations is typically required. Therefore, a cost-effective, simple, and accurate quantification method for the peptide at a higher concentration needs to be developed. In this study, a simple and novel HPLC-UV method is proposed and validated using an Analytical Quality by Design (AQbD) approach. The method is first screened and optimised using chromatographic responses including capacity factor, resolution, tailing factor, and theoretical plate counts, fulfilling the International Council for Harmonisation (ICH) Q2 (R1) guidelines. The resultant optimised chromatography conditions utilised 10 mM phosphate buffer at pH 2.5 and acetonitrile as mobile phases, starting at 3% (v/v) acetonitrile and 97% (v/v) buffer and increasing to 9.7% (v/v) acetonitrile and 90.3% (v/v) buffer over 15 min at a flow rate of 1 mL/min at the column temperature of 25 °C. The injection volume is set at 10 μL and the VWD detector wavelength is 220 nm. The method established is suitable for detecting the peptide at a relatively high concentration, with a quantifiable range from 7.8 μg/mL to 2.0 mg/mL. In addition, the use of a relatively simple HPLC-UV approach could significantly reduce costs and allow easier access to quantify the peptide concentration. A limitation of this method is lower sensitivity compared with using LC-MS/MS and ELISA methods but running costs are lower and the methodology is simpler. The method is capable to quantify the peptide in various tested matrix solutions, with successful quantitation of the peptide in samples obtained from in vitro drug release study in PBS and from a chitosan-TPP nanogels formulation. Therefore, the method developed here offers a complementary approach to the existing quantification methods, quantifying this peptide at increased concentrations in simple to intermediately complex matrix solutions, such as HBSS, DMEM and FluoroBrite cell culture media.

Efficacy of cell therapies are often diminished by low cell retention. A method to visualise the location of grafted cells and biomaterials after delivery could be used to optimise therapies by verifying injection success, retention of the therapeutic and distribution of the product. To address these challenges an injectable cell-substrate consisting of highly porous microspheres containing the computed tomography (CT) contrast agent barium sulphate (BaSO4) was developed.Porous microspheres ( Conflict of Interest None

Introduction Cell therapy is a potential novel treatment for cardiac regeneration. Numerous studies have attempted to transplant cells to regenerate myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported in clinical trials. This is likely to arise from low cell retention following delivery and high cell death after transplantation. The current study aims to improve intramyocardial delivery of viable cells by using an injectable biodegradable substrate that supports cell attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). Methods Highly porous microcarriers Results TIPS microspheres exhibited a highly porous topography, with pore features ranging in shape and size (figure 1A). Pre-incubating the microcarriers with VTN-N; 0.5 μg/ml, resulted in an increase in iPSC attachment (93.4% vs 2.9% control; attached cells as % of seeded cells, n=5, P ≤ 0.0001) (figure 1B). The attached cells proliferated (doubling time≤24 hrs, n=3). iPSC attached to the microcarriers retained their pluripotent phenotype, demonstrated by positive expression of markers, SOX2, OCT4, TRA-1-60 and SSEA4. Pluripotency was further demonstrated by differentiation of TIPS-iPSC into a mixed cardiomyocyte-like population exhibiting a beating phenotype for up to 40 days in culture and positive expression of cardiac markers Troponin T and sarcomeric α-Actinin. Conclusion This work demonstrates that TIPS microcarriers offer a supporting matrix for culturing and differentiating iPSC and may provide an injectable biomaterial for cardiac regeneration. Conflict of Interest N/A

Glial cell alignment in tissue engineered constructs is essential for achieving functional outcomes in neural recovery. While gelatin methacrylate (GelMA) hydrogel offers superior biocompatibility along with permissive structure and tailorable mechanical properties, phosphate glass fibers (PGFs) can provide physical cues for directionality of neural growth. Aligned PGFs were fabricated by a melt quenching and fiber drawing method and utilized with synthesized GelMA hydrogel. The mechanical properties of GelMA and biocompatibility of the GelMA-PGFs composite were investigated in vitro using rat glial cells. GelMA with 86% methacrylation degree were photo-crosslinked using 0.1%wt photo-initiator (PI). Photocrosslinking under UV exposure for 60 s was used to produce hydrogels (GelMA-60). PGFs were introduced into the GelMA before crosslinking. Storage modulus and loss modulus of GelMA-60 was 24.73 ± 2.52 and 1.08 ± 0.23 kN/m2 , respectively. Increased cell alignment was observed in GelMA-PGFs compared with GelMA hydrogel alone. These findings suggest GelMA-PGFs can provide glial cells with physical cues necessary to achieve cell alignment. This approach could further be used to achieve glial cell alignment in bioengineered constructs designed to bridge damaged nerve tissue.

Recurrence of prostate cancer after radical prostatectomy is a consequence of incomplete tumor resection. Systemic chemotherapy after surgery is associated with significant toxicity. Improved delivery methods for toxic drugs capable of targeting positive resection margins can reduce tumor recurrence and avoid their known toxicity. This study evaluates the effectiveness and toxicity of docetaxel (DTX) release from highly porous biodegradable microparticles intended for delivery into the tissue cavity created during radical prostatectomy to target residual tumor cells. The microparticles, composed of poly(dl‐lactide‐co‐glycolide) (PLGA), are processed using thermally induced phase separation (TIPS) and loaded with DTX via antisolvent precipitation. Sustained drug release and effective toxicity in vitro are observed against PC3 human prostate cells. Peritumoral injection in a PC3 xenograft tumor model results in tumor growth inhibition equivalent to that achieved with intravenous delivery of DTX. Unlike intravenous delivery of DTX, implantation of DTX‐TIPS microparticles is not accompanied by toxicity or elevated systemic levels of DTX in organ tissues or plasma. DTX‐TIPS microparticles provide localized and sustained release of nontoxic therapeutic amounts of DTX. This may offer novel therapeutic strategies for improving management of patients with clinically localized high‐risk disease requiring radical prostatectomy and other solid cancers at high risk of positive resection margins., Docetaxel (DTX) release from highly porous biodegradable microparticles is investigated for the treatment of prostate cancer. Tumor growth inhibition is equivalent to that achieved with intravenous delivery of DTX while avoiding the toxicity associated with intravenous delivery. DTX thermally induced phase separation microparticles offer a novel therapeutic strategy for solid cancers associated with the risk of positive resection margins.

Mesenchymal stromal cells (MSC) hold significant potential for tissue engineering applications. Modular tissue engineering involves the use of cellularized “building blocks” that can be assembled via a bottom-up approach into larger tissue-like constructs. This approach emulates more closely the complexity associated hierarchical tissues compared with conventional top-down tissue engineering strategies. The current study describes the combination of biodegradable porous poly(DL-lactide-co-glycolide) (PLGA) TIPS microcarriers with canine adipose-derived MSC (cAdMSC) for use as implantable conformable building blocks in modular tissue engineering applications. Optimal conditions were identified for the attachment and proliferation of cAdMSC on the surface of the microcarriers. Culture of the cellularized microcarriers for 21 days in transwell insert plates under conditions used to induce either chondrogenic or osteogenic differentiation resulted in self-assembly of solid 3D tissue constructs. The tissue constructs exhibited phenotypic characteristics indicative of successful osteogenic or chondrogenic differentiation, as well as viscoelastic mechanical properties. This strategy paves the way to create in situ tissue engineered constructs via modular tissue engineering for therapeutic applications.

Peripheral arterial disease and critical limb ischemia are common symptoms of cardiovascular disease. Vascular surgery is used to create a bypass around occluded blood vessels to improve blood flow to ischemic muscle, thus avoiding the need for amputation. Attempts to vascularize tissues by therapeutic angiogenesis using delivery of exogenous angiogenic agents are underwhelming. A material‐based approach that provides an endogenous stimulus capable of promoting angiogenesis and increased tissue perfusion would provide a paradigm shift in treatment options available. It is reported here that microporous biodegradable films produced using thermally induced phase separation provide a localized biophysical stimulus of proangiogenic genes in vivo that is associated with increased blood vessel density and restoration of blood flow to ischemic tissue. These findings show, for the first time, that acellular, nonfunctionalized biodegradable biomaterials can provide an innovative, material‐based approach for therapeutic angiogenesis to enhance tissue reperfusion in vivo., Microporous biodegradable film produced using thermally induced phase separation is developed to promote therapeutic angiogenesis for cardiovascular disease. The film stimulates restoration of vascular perfusion in ischemic tissue via upregulation of proangiogenic growth genes that result in increased vessel in density. The new material has potential transform treatment of cardiovascular disease.

Adipose‐derived mesenchymal stromal cells (AdMSC) release numerous soluble factors capable of stimulating angiogenesis. Improved methods for delivering these cells to maximize their potency are now sought that ideally they retain viable cells in the target tissue while promoting the secretion of angiogenic factors. Substrate surface topography is a parameter that can be used to manipulate the behavior of AdMSC but challenges exist with translating this parameter into materials compatible with minimally invasive delivery into tissues for in situ delivery of the angiogenic secretome. The current study investigates three compositions of hierarchically structured, porous biodegradable microcarriers for the culture of AdMSC and the influence of their surface topographies on the angiogenic secretome. All three compositions perform well as cell microcarriers in xeno‐free conditions. The attached AdMSC retain their capacity for subsequent trilineage differentiation. The secretome of AdMSC attached to the microcarriers consists of multiple proangiogenic factors, including significantly elevated levels of vascular endothelial growth factor, which stimulates angiogenesis in vitro. The unique properties of hierarchically structured, porous biodegradable microcarriers investigated in this study offer a radically transformative approach for achieving targeted in vivo delivery of AdMSC and enhancing the potency of their proangiogenic activity to induce neovascularization in ischemic tissue., This study investigates highly textured microspheres designed for use as implantable cell microcarriers that are shown to stimulate the secretion of proangiogenic factors from attached mesenchymal stromal cells. These findings suggest that the technology can provide a feasible and transformative new approach for treating cardiovascular disease through the targeted in situ delivery of factors that will promote new blood vessel growth.

The meteoric rise of cancer immunotherapy in the past decade has led to promising treatments for a number of hard-to-treat malignancies. In particular, adoptive T cell therapy has recently reached a major milestone with two products approved by the US FDA. However, the inherent complexity of cell-based immunotherapies means that their manufacturing time, cost, and controllability limit their effectiveness and geographic reach. One way to address these issues may lie in complementing the dominant, reductionistic mentality in modern medicine with complex systems thinking. In this opinion article, we identify key concepts from complexity theory to address manufacturing challenges in cell-based immunotherapies and raise the possibility of a unifying framework upon which future bioprocessing strategies may be designed.

Advances in healthcare technology for continence have historically been limited compared to other areas of medicine, reflecting the complexities of the condition and social stigma which act as a barrier to participation. This whitepaper has been developed to inspire and direct the engineering science community towards research opportunities that exist for continence technologies that address unmet needs in diagnosis, treatment and long-term management. Our aim is to pinpoint key challenges and highlight related research opportunities for novel technological advances. To do so, we draw on experience and expertise from academics, clinicians, patients and patient groups linked to continence healthcare. This is presented in four areas of consideration: the clinical pathway, patient perspective, research challenges and effective innovation. In each we introduce seminal research, background information and demonstrative case-studies, before discussing their relevance to engineering science researchers who are interested in approaching this overlooked but vital area of healthcare.

With donor organs not readily available, the need for a tissue-engineered oesophagus remains high, particularly for congenital childhood conditions such as atresia. Previous attempts have not been successful, and challenges remain. Small intestine submucosa (SIS) is an acellular matrix material with good biological properties; however, as is common with these types of materials, they demonstrate poor mechanical properties. In this work, electrospinning was performed to mechanically reinforce tubular SIS with polylactic-co-glycolic acid (PLGA) nanofibres. It was hypothesised that if attachment could be achieved between the two materials, then this would (i) improve the SIS mechanical properties, (ii) facilitate smooth muscle cell alignment to support directional growth of muscle cells and (iii) allow for the delivery of bioactive molecules (VEGF in this instance). Through a relatively simple multistage process, adhesion between the layers was achieved without chemically altering the SIS. It was also found that altering mandrel rotation speed affected the alignment of the PLGA nanofibres. SIS-PLGA scaffolds performed mechanically better than SIS alone; yield stress improvement was 200% and 400% along the longitudinal and circumferential directions, respectively. Smooth muscle cells cultured on the aligned fibres showed resultant unidirectional alignment. In vivo the SIS-PLGA scaffolds demonstrated limited foreign body reaction judged by the type and proportion of immune cells present and lack of fibrous encapsulation. The scaffolds remained intact at 4 weeks in vivo, and good cellular infiltration was observed. The incorporation of VEGF within SIS-PLGA scaffolds increased the blood vessel density of the surrounding tissues, highlighting the possible stimulation of endothelialisation by angiogenic factor delivery. Overall, the designed SIS-PLGA-VEGF hybrid scaffolds might be used as a potential matrix platform for oesophageal tissue engineering. In addition to this, achieving improved attachment between layers of acellular matrix materials and electrospun fibre layers offers the potential utility in other applications. STATEMENT OF SIGNIFICANCE: Because of its multi-layered nature and complex structure, the oesophagus tissue poses several challenges for successful clinical grafting. Therefore, it is promising to utilise tissue engineering strategies to mimic and form structural compartments for its recovery. In this context, we investigated the use of tubular small intestine submucosa (SIS) reinforced with polylactic-co-glycolic acid (PLGA) nanofibres by using electrospinning and also, amongst other parameters, the integrity of the bilayered structure created. This was carried out to facilitate smooth muscle cell alignment, support directional growth of muscle cells and allow the delivery of bioactive molecules (VEGF in this study). We evaluated this approach by using in vitro and in vivo models to determine the efficacy of this new system.

With increasing global energy demands, unconventional formations, such as shale rocks, are becoming an important source of natural gas. Extensive efforts focus on understanding the complex behavior of fluids (including their transport in the sub-surface) to maximize natural gas yields. Shale rocks are mudstones made up of organic and inorganic constituents of varying pore sizes (1-500 nm). With cutting-edge imaging technologies, detailed structural and chemical description of shale rocks can be obtained at different length scales. Using this knowledge to assess macroscopic properties, such as fluid permeability, remains challenging. Direct experimental measurements of permeability supply answers, but at elevated costs of time and resources. To complement these, computer simulations are widely available; however, they employ significant approximations, and a reliable methodology to estimate permeability in heterogeneous pore networks remains elusive. For this study, permeability predictions obtained by implementing two deterministic methods and one stochastic approach, using a kinetic Monte Carlo algorithm, are compared. This analysis focuses on the effects resulting from pore size distribution, the impact of micro- and macropores, and the effects of anisotropy (induced or naturally occurring) on the predicted matrix permeability. While considering multiple case studies, recommendations are provided on the optimal conditions under which each method can be used. Finally, a stochastic analysis is performed to estimate the permeability of an Eagle Ford shale sample using the kinetic Monte Carlo algorithm. Successful comparisons against experimental data demonstrate the appeal of the stochastic approach proposed.

This paper identifies restimulation opportunities in existing multistage completed horizontal wells with plans for a customized refracturing solution applying breakthrough stimulation and diversion processes to increase oil production in a tight carbonate formation, offshore Black Sea. Because operators are shifting strategy in a low oil price environment from new well drilling toward well interventions, refracturing is gaining more focus, particularly for tight and less conventional reservoirs. Many potential candidates also have suboptimal completions for refracturing, so the challenge for operators is selecting the best candidates and designing a refracturing treatment for improved well performance in these complex situations. This paper describes the well screening and selection process for the restimulation of existing horizontal wells with multistage openhole completions. During Phase 1 of the project, pilot candidates were ranked using a weighted average score of specific decision criteria for evaluating the refracturing potential. The goal of the screening process was to identify wellbores with the most bypassed reserves and to determine the root cause. Top candidates demonstrated bypassed reserve potential because of large completion spacing and lower average permeability than was originally estimated. The design process emphasized identifying areas where incremental oil could be accessed by substantially increasing total exposed conductive surface area and placing new fractures between existing using novel approaches to refracturing incorporating flow diverting technology. The application of an engineered pressure-managed design approach optimized proppant cycles, and flow diverting refracturing methods were a fundamental component in recognizing that the restimulation pilot was realistic, achievable, and justified economically. By dynamically managing and adapting proppant schedules, diverter volume fractions, and total materials pumped over time, new induced fracture surface areas can be reliably created in the most economic manner. Phase 2 consisted of executing the refracturing operation on the selected pilot well, which had been originally hydraulically fractured in 2009. A repressurization procedure of the reservoir was performed before the main treatment to equalize pressure depletion along the lateral and ultimately enhance the coverage of newly fractured zones along the wellbore. The refracturing treatment on the pilot well consisted of four proppant cycles with application of engineered pressure management to improve fracture initiations and flow distribution. A degradable particulate diverter technology was used as primary isolation of each fracturing cycle. Restimulation results of the pilot well demonstrated technical and production success, with huge potential to implement this technology during the next phase of field revitalization (Phase 3). This pilot project has proved that the combination of a well selection process aimed at finding unstimulated and bypassed reservoir volume and the application of customized technical solutions for refracturing can be successfully applied to increase recovery factors and identify new opportunities in mature field redevelopment.

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Cell therapy is an emerging option for regenerating skeletal muscle. Improved delivery methods for anchorage-dependent myoblasts are likely to improve integration and function of transplanted muscle cells. Highly porous microspheres, produced using thermally induced phase separation (TIPS), have features ideally suited for minimally invasive cell delivery. The purpose of this study was to investigate, for the first time, the use of TIPS microspheres as highly porous microcarriers for manipulation of human skeletal muscle myoblasts (HSMM) under defined culture conditions. HSMM cells readily attached to the surface of poly (DL-lactide-co-glycolide) (PLGA) TIPS microcarriers, where they were induced to continue proliferating or to be driven towards differentiation whilst under static-dynamic culture conditions for 7 days. Switching from proliferation medium to differentiation medium for 7 days, resulted in increased protein expression of skeletal muscle cell contractile apparatus components, MyoD and skeletal muscle myosin heavy chain, compared with cells cultured on conventional culture plasticware for the same duration (p < 0.001). Growth of myoblasts on the surface of the microcarriers and their migration following simulated delivery, caused no change to the proliferative capacity of cells over 7 days. Results from this study demonstrate that TIPS microspheres provide an ideal vehicle for the expansion and delivery of myoblasts for therapeutic applications. Transplantation of myoblasts anchored to a substrate, rather than in suspension, will reduce the amount of ex vivo manipulation required during preparation of the product and allows cells to be delivered in a more natural state. This will improve the ability to control cell dosage and increase the likelihood of efficacy.

In recent years, the thermally induced phase-separation process has been used extensively to form porous materials, such as scaffolds and drug delivery devices with pore sizes ranging from the nano- to the micro-scale. This chapter discusses the methods that are used for creating these tissue-engineering scaffolds. Porous tissue scaffolds offer several benefits for the engineering of soft tissues. These most notably include infiltration of cells and tissue infiltration together with vascularization to support perfusion of oxygen and nutrients. Phase separation technologies provide an almost unrivaled platform for the manufacture of 3D tissue-engineering scaffolds. The manufacturing processes involved are often relatively low cost, scalable, and compatible with a wide range of materials. The structural features attained can be designed to resemble native extracellular matrix or provide novel substrates capable of stimulating specific cellular responses. These attributes are likely to result in the technology being increasingly utilized as the field of tissue engineering and regenerative medicine grows.

The permeability of shales is important, because it controls where oil and gas resources can migrate to and where in the Earth hydrocarbons are ultimately stored. Shales have a well-known anisotropic directional permeability that is inherited from the depositional layering of sedimentary laminations, where the highest permeability is measured parallel to laminations and the lowest permeability is perpendicular to laminations. We combine state of the art laboratory permeability experiments with high-resolution X-ray computed tomography and for the first time can quantify the three-dimensional interconnected pathways through a rock that define the anisotropic behaviour of shales. Experiments record a physical anisotropy in permeability of one to two orders of magnitude. Two- and three-dimensional analyses of micro- and nano-scale X-ray computed tomography illuminate the interconnected pathways through the porous/permeable phases in shales. The tortuosity factor quantifies the apparent decrease in diffusive transport resulting from convolutions of the flow paths through porous media and predicts that the directional anisotropy is fundamentally controlled by the bulk rock mineral geometry. Understanding the mineral-scale control on permeability will allow for better estimations of the extent of recoverable reserves in shale gas plays globally.

The electrohydrodynamic (EHD) technique has traditionally been confined to the bench-top. Recently, a portable EHD device was constructed that allows the process to be applied in a much more flexible and versatile manner. In this article, the effect of electrode and collector height on the fibers generated by EHD and wound-dressing forming capabilities of the device tested on different types of simulated wound morphologies were investigated. Dressings were also fabricated on living tissue to investigate jet behavior and product morphology. The EHD device was found to be capable of controlled deposition of micrometer-sized poly(D,L-lactide-co-glycolide) acid fibers that can form an opaque, waterproof wound dressing on human skin within a few minutes with excellent adherence.

Vascular occlusion can result in fatal myocardial infarction, stroke or loss of limb in peripheral arterial disease. Interventional balloon angioplasty is a common first line procedure for vascular disease treatment, but long term success is limited by restenosis and neointimal hyperplasia. Cellular therapies have been proposed to mitigate these issues; however efficacy is low, in part due to poor cell retention. We show that magnetic targeting of mesenchymal stem cells gives rise to a 6-fold increase in cell retention following balloon angioplasty in a rabbit model using a clinically applicable permanent magnet. Cells labelled with superparamagnetic iron oxide nanoparticles exhibit no negative effects on cell viability, differentiation or secretion patterns. The increase in stem cell retention leads to a reduction in restenosis three weeks after cell delivery.

Corticosteroids have long been used to treat intraocular inflammation by intravitreal injection. We describe dexamethasone loaded poly-DL-lactide-co-glycolide (PLGA) microparticles that were fabricated by thermally induced phase separation (TIPS). The dexamethasone loaded microparticles were evaluated using a two-compartment, in vitro aqueous outflow model of the eye (PK-Eye) that estimates drug clearance time from the back of the eye via aqueous outflow by the anterior route. A dexamethasone dose of 0.20±0.02mg in a 50μL volume of TIPS microparticles resulted in a clearance t1/2 of 9.6±0.3days using simulated vitreous in the PK-Eye. Since corticosteroids can also clear through the retina, it is necessary to account for clearance through the back of the eye. Retinal permeability data, published human ocular pharmacokinetics (PK) and the PK-Eye clearance times were then used to establish in vitro in vivo correlations (IVIVCs) for intraocular clearance times of corticosteroid formulations. A t1/2 of 48h was estimated for the dexamethasone-TIPS microparticles, which is almost 9 times longer than that reported for dexamethasone suspension in humans. The prediction of human clearance times of permeable molecules from the vitreous compartment can be determined by accounting for drug retinal permeation and determining the experimental clearance via the anterior aqueous outflow pathway using the PK-Eye.

Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.

Tissue engineering is a rapidly advancing field that is likely to transform how medicine is practised in the near future. For hollow organs such as those found in the cardiovascular and respiratory systems or gastrointestinal tract, tissue engineering can provide replacement of the entire organ or provide restoration of function to specific regions. Larger tissue-engineered constructs often require biomaterial-based scaffold structures to provide support and structure for new tissue growth. Consideration must be given to the choice of material and manufacturing process to ensure the de novo tissue closely matches the mechanical and physiological properties of the native tissue. This review will discuss some of the approaches taken to date for fabricating hollow organ scaffolds and the selection of appropriate biomaterials.

Delivery of cells into tubular tissue constructs with large diameters poses significant spatial and temporal challenges. This study describes preliminary findings for a novel process for rapid and uniform seeding of cells onto the luminal surface of large tubular constructs. Fibroblasts, tagged with superparamagnetic iron oxide nanoparticles (SPION), were directed onto the luminal surface of tubular constructs by a magnetic field generated by a k4-type Halbach cylinder device. The spatial distribution of attached cells, as measured by the mean number of cells, was compared with a conventional, dynamic, rotational cell-delivery technique. Cell loading onto the constructs was measured by microscopy and magnetic resonance imaging. The different seeding techniques employed had a significant effect on the spatial distribution of the cells ( p < 0.0001). The number of attached cells at defined positions within the same construct was significantly different for the dynamic rotation technique ( p < 0.05). In contrast, no significant differences in the number of cells attached to the luminal surface were found between the defined positions on the construct loaded with the Halbach cylinder. The technique described overcomes limitations associated with existing cell-delivery techniques and is amenable to a variety of tubular organs where rapid loading and uniform distribution of cells for therapeutic applications are required.

Microcarriers are widely used for the expansion of cells in vitro, but also offer an approach for combining cell transplantation and tissue bulking for regenerative medicine in a minimally invasive manner. This could be beneficial in conditions associated with muscle damage or atrophy, such as faecal incontinence, where the use of bulking materials or cell transplantation alone has proven to be ineffective. Microcarriers currently available have not been designed for this purpose and are likely to be suboptimal due to their physical and biochemical properties. The aim of this study was to investigate macroporous microspheres of polylactide-co-glycolide (PLGA), prepared using a thermally induced phase separation technique, for their suitability as cell microcarriers for the transplantation of smooth muscle cells. Cell attachment, growth and migration were studied and compared with commercially available porcine gelatin microcarriers (Cultispher-S) in suspension culture. Smooth muscle cells attached more rapidly to the PLGA microcarriers, which also significantly enhanced the rate of cell growth compared with Cultispher-S microcarriers. The majority of smooth muscle cells attached to the PLGA microcarriers in suspension culture were able to migrate away over a 15 day period of static culture, unlike Cultispher-S microcarriers which retained the majority of cells. The ability of PLGA microcarriers to enhance cell growth combined with their capacity to release cells at the sites of delivery are features that make them ideally suited for use as a cell transplantation delivery device in tissue engineering and regenerative medicine.

Antibodies to tumor necrosis factor alpha (TNF-α) have been successful in treating perianal fistulae in Crohn's disease, but current modes of delivery are limited. Microspheres are currently being assessed as scaffolds for tissue engineering and drug delivery devices. The aim of this study was to produce anti-TNF-α antibody-encapsulated microspheres using thermally induced phase separation (TIPS) and to characterize their behavior.Anti-TNF-α antibody was encapsulated into the microspheres (100 mg infliximab/g poly[lactide-co-glycolide] w/w) using a novel technique combining a vibration encapsulator unit with a TIPS process, using either lyophilized particulate antibody or an aqueous solution of antibody. Microspheres were incubated in phosphate-buffered saline for collection of supernatant and assessment of degradation. The amount and biological activity of the encapsulated antibody released from the microspheres was assessed by enzyme-linked immunosorbent assay and its ability to neutralize recombinant human (rh)TNF-α in vitro with a cytotoxicity assay. An in vitro wound scratch assay was used to assess the effect of released antibody on fibroblast migration. Ultrastructural characteristics of the different microspheres were characterized by scanning electron microscopy.Highly porous microspheres released anti-TNF-α antibody under zero-order kinetics and inhibited the cytotoxic activity of rhTNF-α, producing a significant increase in cell viability compared with cells treated with rhTNF-α alone. This effect was most pronounced with microspheres fabricated by blending lyophilized particulate anti-TNF-α antibody into the polymer solution, which also significantly reduced the release of lactate dehydrogenase.Anti-TNF-α antibody encapsulated into highly porous microspheres was released in a controlled manner and exhibited biological activity against TNF-α. The technique used to produce TIPS microspheres is rapid and provides high encapsulation efficiency. This technique could also be applied to other therapeutic peptides where rapid fabrication and high yields are required.

Collagen is an abundant protein found in the extracellular matrix of many tissues. Due to its biocompatibility, it is a potentially ideal biomaterial for many tissue engineering applications. However, harvested collagen often requires restructuring into a three-dimensional matrix to facilitate applications such as implantation into poorly accessible tissue cavities. The aim of the current study was to produce a conformable collagen-based scaffold material capable of supporting tissue regeneration for use in wound repair applications. Microporous collagen spheres were prepared using a thermally induced phase separation (TIPS) technique and their biocompatibility was assessed. The collagen spheres were successfully cross-linked with glutaraldehyde vapour, rendering them mechanically more stable. When cultured with myofibroblasts the collagen spheres stimulated a prolonged significant increase in secretion of the angiogenic growth factor, vascular endothelial growth factor (VEGF), compared with cells alone. Control polycaprolactone (PCL) spheres failed to stimulate a similar prolonged increase in VEGF secretion. An enhanced angiogenic effect was also seen in vivo using the chick embryo chorioallantoic membrane assay, where a significant increase in the number of blood vessels converging towards collagen spheres was observed compared with control PCL spheres. The results from this study indicate that microporous collagen spheres produced using TIPS are biologically active and could offer a novel conformable scaffold for tissue regeneration in poorly accessible wounds.

Conformable scaffold materials capable of rapid vascularization and tissue infiltration would be of value in the therapy of inaccessible wounds. Microporous spheres of poly(D,L-lactide-co-glycolide) (PLGA) containing bioactive glass (BG) were prepared using a thermally induced phase separation (TIPS) technique, and the bioactivity, in vitro degradation, and tissue integration of the microporous spheres were assessed. Microporous spheres containing 10% (w/w) BG stimulated a significant increase in vascular endothelial growth factor secretion from myofibroblasts consistently over a 10-day period (p < 0.01) compared with the neat PLGA microporous spheres. The microporous spheres degraded steadily in vitro over a 16-week period, with the neat PLGA microporous spheres retaining 82% of their original weight and microporous spheres containing 10% (w/w) BG retaining 77%. Both types of microporous spheres followed a similar pattern of size reduction throughout the degradation study, resulting in a 23% and 20% reduction after 16 weeks for the neat PLGA microporous spheres and PLGA microporous spheres containing 10% (w/w) BG, respectively (p < 0.01). After in vivo implantation into a subcutaneous wound model, the TIPS microporous spheres became rapidly integrated (interspherically and intraspherically) with host tissue, including vascularization of voids inside the microporous sphere. The unique properties of TIPS microporous spheres make them ideally suited for regenerative medicine applications where tissue augmentation is required.

Repair of superficial injury to the gastrointestinal mucosa involves the process of restitution, the rapid migration of epithelial cells across damaged areas. The effect of 45S5 bioactive glass on epithelial restitution was assessed using a novel co-culture model incorporating wounded intestinal epithelial cell monolayers and sub-epithelial myofibroblasts to simulate in vivo conditions that occur during superficial mucosal ulceration. Epithelial wound healing was not increased by culture medium conditioned with bioactive glass, with 1% (w/v) bioactive glass inhibiting cell migration. Epithelial wounds co-cultured with myofibroblasts grown on surfaces coated with 0.1% (w/v) bioactive glass increased wound healing compared with co-cultures containing no bioactive glass. Myofibroblasts grown on surfaces coated with bioactive glass secreted significantly increased amounts of basic fibroblast growth factor but did not increase epithelial cell proliferation, indicating the wound healing observed was due to restitution. These data from a model of superficial mucosal injury suggest bioactive glass may function as a stimulant of paracrine mucosal signaling networks that promotes rapid epithelial repair.

BAckgroundNone of the proposed materials tested for the management of perianal fistulae has proven to be a definitive treatment.AimTo assess a new repair scaffold and drug delivery device conceived to target perianal fistula repair.MethodsPoly(d,l-lactide-co-glycolide) porous microspheres containing either antibacterial silver-releasing degradable phosphate glass or metronidazole were prepared using thermally induced phase separation.ResultsIon- and drug-release profiling of the microspheres revealed continued release of silver ions from microspheres filled with silver-doped phosphate glass and high encapsulation efficiency for metronidazole [78% and 82% for microspheres loaded with 2.5% and 1.3% (w/w), respectively]. Microbicidal activity was confirmed by growth inhibition of bacterial species (Staphylococcus aureus, Escherichia coli and Bacteroides fragilis), which characteristically dominate the colonization of perianal fistula tracts. Microspheres containing > 3 mol% silver or metronidazole resulted in strong bacterial inhibition/kill against B. fragilis; the presence of one sphere containing > 3 mol% silver had a potent inhibitory effect against all the microbes studied. Microspheres became rapidly integrated with host tissue following subcutaneous implantation into a rodent wound model.ConclusionsThe study demonstrates a novel scaffold for guided tissue regeneration providing local release of antimicrobial agents sufficient to counter bacterial colonization and warrants further investigation.

Microsphere-based controlled release technologies have been utilized for the long-term delivery of proteins, peptides and antibiotics, although their synthesis poses substantial challenges owing to formulation complexities, lack of scalability, and cost. To address these shortcomings, we used the electrospray process as a reproducible, synthesis technique to manufacture highly porous (94%) microspheres while maintaining control over particle structure and size. Here we report a successful formulation recipe used to generate spherical poly(lactic-co-glycolic) acid (PLGA) microspheres using the electrospray (ES) coupled with a novel thermally induced phase separation (TIPS) process with a tailored Liquid Nitrogen (LN2) collection scheme. We show how size, shape and porosity of resulting microspheres can be controlled by judiciously varying electrospray processing parameters and we demonstrate examples in which the particle size (and porosity) affect release kinetics. The effect of electrospray treatment on the particles and their physicochemical properties are characterized by scanning electron microscopy, confocal Raman microscopy, thermogravimetric analysis and mercury intrusion porosimetry. The microspheres manufactured here have successfully demonstrated long-term delivery (i.e. 1week) of an active agent, enabling sustained release of a dye with minimal physical degradation and have verified the potential of scalable electrospray technologies for an innovative TIPS-based microsphere production protocol.

In this study, we have developed 50- to 100-µm-sized titanium phosphate glass microcarriers (denoted as Ti5) that show enhanced proliferation of human mesenchymal stem cells and MG63 osteosarcoma cells, as well as enhanced human mesenchymal stem cell expression of bone differentiation markers, in comparison with commercially available glass microspheres at all time points. We also demonstrate that these microcarriers provide superior human mesenchymal stem cell proliferation with conventional Dulbecco’s Modified Eagle medium than with a specially developed commercial stem cell medium. The microcarrier proliferative capacity is revealed by a 24-fold increase in MG63 cell numbers in spinner flask bioreactor studies performed over a 7-day period, versus only a 6-fold increase in control microspheres under the same conditions; the corresponding values of Ti5 and control microspheres under static culture are 8-fold and 7-fold, respectively. The capability of guided osteogenic differentiation is confirmed by ELISAs for bone morphogenetic protein-2 and osteopontin, which reveal significantly greater expression of these markers, especially osteopontin, by human mesenchymal stem cells on the Ti5 microspheres than on the control. Scanning electron microscopy and confocal laser scanning microscopy images reveal favorable MG63 and human mesenchymal stem cell adhesion on the Ti5 microsphere surfaces. Thus, the results demonstrate the suitability of the developed microspheres for use as microcarriers in bone tissue engineering applications.

Summary Background Defective neutrophil recruitment has been described as a primary pathogenic abnormality in Crohn’s disease. Cantharidin-induced blisters provide a novel investigative tool to assess cellular influx and inflammatory mediator production during acute inflammation and allows the effects of therapy on these parameters to be measured. Aims To determine whether reduced neutrophil tissue penetration in Crohn's disease relates to impaired production of inflammatory mediators, and whether it can be reversed by granulocyte-colony stimulating factor (G-CSF). Methods Neutrophil and monocyte/macrophage populations and inflammatory mediators were measured in cantharidin blisters at 24 h. Neutrophil chemotaxis was assessed in vitro using blister fluid as the chemoattractant. The effect of s.c. G-CSF on blister phenotype was determined. Results Significantly fewer neutrophils migrated into blisters in Crohn's patients. The production of neutrophil chemokines, but not other inflammatory mediators, was reduced. This significantly correlated with reduced chemotaxis in vitro. Differences were unrelated to caspase-recruitment domain 15 genotype. G-CSF significantly increased blister neutrophil concentrations in control subjects and Crohn's patients. Conclusions Reduced neutrophil migration during acute inflammation in Crohn's disease is associated with impaired production of appropriate chemoattractants. G-CSF therapy increases neutrophil tissue migration, which may partially account for its observed therapeutic effect.

Attempts to stimulate therapeutic angiogenesis using gene therapy or delivery of recombinant growth factors, such as vascular endothelial growth factor (VEGF), have failed to demonstrate unequivocal efficacy in human trials. Bioactive glass stimulates fibroblasts to secrete significantly increased amounts of angiogenic growth factors and therefore has a number of potential applications in therapeutic angiogenesis. The aim of this study was to assess whether it is possible to encapsulate specific quantities of bioactive glass and fibroblasts into alginate beads, which will secrete growth factors capable of stimulating angiogenesis. Human fibroblasts (CCD-18Co) were encapsulated in alginate beads with specific quantities of 45S5 bioactive glass and incubated in culture medium (0-17 days). The conditioned medium was collected and assayed for VEGF or used to assess its ability to stimulate angiogenesis by measuring the proliferation of human dermal microvascular endothelial cells. At 17 days the beads were lysed and the amount of VEGF retained by the beads measured. Fibroblasts encapsulated in alginate beads containing 0.01% and 0.1% (w/v) 45S5 bioactive glass particles secreted increased quantities of VEGF compared with cells encapsulated with 0% or 1% (w/v) 45S5 bioactive glass particles. Lysed alginate beads containing 0.01% and 0.1% (w/v) 45S5 bioactive glass contained significantly more VEGF (p

Neovascularization of tissue-engineered constructs remains a limiting factor for the engineering of larger tissue constructs. Attempts to stimulate neovascularization, using recombinant protein or gene transfer of angiogenic growth factors, have been proposed; however, these approaches have been associated with problems regarding the delivery and duration of exposure of the growth factor. This study was performed to determine the ability of biologically active glass to stimulate the secretion of angiogenic growth factors from human stromal cells and subsequent angiogenesis. CCD18Co human fibroblasts were cultured on tissue culture surfaces coated with specific quantities of 45S5 Bioglass particles. At 24-, 48-, and 72-h intervals the gene expression of vascular endothelial growth factor (VEGF) and the protein secretion of VEGF and basic fibroblast growth factor (bFGF) from fibroblasts were measured. The effect of conditioned medium collected from Bioglass-stimulated fibroblasts on human dermal microvascular endothelial cells was assessed using in vitro angiogenesis assays. Results showed that surfaces coated with Bioglass produced a significant increase in the secretion of VEGF and bFGF. Conditioned medium from stimulated fibroblasts significantly increased the proliferation of human dermal microvascular endothelial cells and induced a significant increase in the formation of anastomosed networks of human endothelial cell tubules. It is concluded that the ability of 45S5 Bioglass to stimulate the release of angiogenic growth factors and to promote angiogenesis provides a novel alternative approach for stimulating neovascularization of tissue-engineered constructs.