Simple Technique for Microscopic Evaluation of Active Cellular Invasion into 3D Hydrogel Constructs.

Materials that are evaluated for bioengineering purposes are carefully tested to evaluate cellular interactions with respect to biocompatibility and in some cases cell differentiation. A key perspective that is often considered is the ability for decellularized synthetic or natural based matrices to facilitate cell migration or tissue ingrowth. Current methods of measuring cell migration range from simple scratch assays to Boyden chamber inserts and fluorescent imaging of seeded spheroids. Many of these methods require tissue processing for histological analysis and fixing and staining for imaging, which can be difficult and dependent on the stability of the hydrogel subject. Herein we present a simple platform that can be manufactured using 3D printing and easily applied to in vitro cell culture, allowing the researcher to image live cellular migration into a cellular materials. We found this to be an adaptable, cheap, and replicable technique to evaluate cellular interaction that has applications in the research and development of hydrogels for tissue engineering purposes.

Assessing the Effects of VEGF Releasing Microspheres on the Angiogenic and Foreign Body Response to a 3D Printed Silicone-Based Macroencapsulation Device.

Macroencapsulation systems have been developed to improve islet cell transplantation but can induce a foreign body response (FBR). The development of neovascularization adjacent to the device is vital for the survival of encapsulated islets and is a limitation for long-term device success. Previously we developed additive manufactured multi-scale porosity implants, which demonstrated a 2.5-fold increase in tissue vascularity and integration surrounding the implant when compared to a non-textured implant. In parallel to this, we have developed poly(ε-caprolactone-PEG-ε-caprolactone)-b-poly(L-lactide) multiblock copolymer microspheres containing VEGF, which exhibited continued release of bioactive VEGF for 4-weeks in vitro. In the present study, we describe the next step towards clinical implementation of an islet macroencapsulation device by combining a multi-scale porosity device with VEGF releasing microspheres in a rodent model to assess prevascularization over a 4-week period. An in vivo estimation of vascular volume showed a significant increase in vascularity (* p = 0.0132) surrounding the +VEGF vs. -VEGF devices, however, histological assessment of blood vessels per area revealed no significant difference. Further histological analysis revealed significant increases in blood vessel stability and maturity (** p = 0.0040) and vessel diameter size (*** p = 0.0002) surrounding the +VEGF devices. We also demonstrate that the addition of VEGF microspheres did not cause a heightened FBR. In conclusion, we demonstrate that the combination of VEGF microspheres with our multi-scale porous macroencapsulation device, can encourage the formation of significantly larger, stable, and mature blood vessels without exacerbating the FBR.

A Thermoresponsive Chitosan/ß-Glycerophosphate Hydrogel for Minimally Invasive Treatment of Critical Limb Ischaemia.

A reduction in blood supply to any limb causes ischaemia, pain and morbidity. Critical limb ischaemia is the most serious presentation of peripheral vascular disease. One in five patients with critical limb ischaemia will die within six months of diagnosis and one in three will require amputation in this time. Improving blood flow to the limb, via the administration of angiogenic agents, could relieve pain and avoid amputation. Herein, chitosan is combined with ß-glycerophosphate to form a thermoresponsive formulation (chitosan/ß-GP) that will flow through a syringe and needle at room temperature but will form a gel at body temperature. The chitosan/ß-GP hydrogel, with or without the angiogenic molecule desferrioxamine (DFO), was injected into the mouse hind limb, following vessel ligation, to test the ability of the formulations to induce angiogenesis. The effects of the formulations were measured using laser Doppler imaging to determine limb perfusion and CD31 staining to quantify the number of blood vessels. Twenty-eight days following induction of ischaemia, the chitosan/ß-GP and chitosan/ß-GP + 100 µM DFO formulations had significantly (p < 0.001 and p < 0.05, respectively) improved blood flow in the ischaemic limb compared with an untreated control. Chitosan/ß-GP increased vessel number by 1.7-fold in the thigh of the ischaemic limb compared with an untreated control, while chitosan/ß-GP + 100 µM DFO increased vessel number 1.8-fold. Chitosan/ß-GP represents a potential minimally invasive treatment for critical limb ischaemia.

Synergistic use of biomaterials and licensed therapeutics to manipulate bone remodelling and promote non-union fracture repair.

Disrupted bone metabolism can lead to delayed fracture healing or non-union, often requiring intervention to correct. Although the current clinical gold standard bone graft implants and commercial bone graft substitutes are effective, they possess inherent drawbacks and are limited in their therapeutic capacity for delayed union and non-union repair. Research into advanced biomaterials and therapeutic biomolecules has shown great potential for driving bone regeneration, although few have achieved commercial success or clinical translation. There are a number of therapeutics, which influence bone remodelling, currently licensed for clinical use. Providing an alternative local delivery context for these therapies, can enhance their efficacy and is an emerging trend in bone regenerative therapeutic strategies. This review aims to provide an overview of how biomaterial design has advanced from currently available commercial bone graft substitutes to accommodate previously licensed therapeutics that target local bone restoration and healing in a synergistic manner, and the challenges faced in progressing this research towards clinical reality.

Evaluation of the activity of a chemo-ablative, thermoresponsive hydrogel in a murine xenograft model of lung cancer.

BACKGROUND: Minimally invasive intratumoural administration of thermoresponsive hydrogels, that transition from liquid to gel in response to temperature, has been proposed as a potential treatment modality for solid tumours. The aim of this study was to assess the inherent cytotoxicity of a poloxamer-based thermoresponsive hydrogel in a murine xenograft model of lung cancer. METHODS: In vitro viability assessment was carried out in a lung cancer (A549) and non-cancerous (Balb/c 3T3 clone A31) cell line. Following intratumoural administration of saline or the thermoresponsive hydrogel to an A549 xenograft model in female Athymic Nude-Foxn1nu mice (n = 6/group), localisation was confirmed using IVIS imaging. Tumour volume was assessed using callipers measurements over 14 days. Blood serum was analysed for liver and kidney damage and ex vivo tissue samples were histologically assessed. RESULTS: The thermoresponsive hydrogel demonstrated a dose-dependent cancer cell-specific toxicity in vitro and was retained in situ for at least 14 days in the xenograft model. Tumour volume increase was statistically significantly lower than saline treated control at day 14 (n = 6, p = 0.0001), with no associated damage of hepatic or renal tissue observed. CONCLUSIONS: Presented is a poloxamer-based thermoresponsive hydrogel, suitable for intratumoural administration and retention, which has demonstrated preliminary evidence of local tumour control, with minimal off-site toxicity.

A bioresorbable biomaterial carrier and passive stabilization device to improve heart function post-myocardial infarction.

The limited regenerative capacity of the heart after a myocardial infarct results in remodeling processes that can progress to congestive heart failure (CHF). Several strategies including mechanical stabilization of the weakened myocardium and regenerative approaches (specifically stem cell technologies) have evolved which aim to prevent CHF. However, their final performance remains limited motivating the need for an advanced strategy with enhanced efficacy and reduced deleterious effects. An epicardial carrier device enabling a targeted application of a biomaterial-based therapy to the infarcted ventricle wall could potentially overcome the therapy and application related issues. Such a device could play a synergistic role in heart regeneration, including the provision of mechanical support to the remodeling heart wall, as well as providing a suitable environment for in situ stem cell delivery potentially promoting heart regeneration. In this study, we have developed a novel, single-stage concept to support the weakened myocardial region post-MI by applying an elastic, biodegradable patch (SPREADS) via a minimal-invasive, closed chest intervention to the epicardial heart surface. We show a significant increase in %LVEF 14 days post-treatment when GS (clinical gold standard treatment) was compared to GS + SPREADS + Gel with and without cells (p ≤ 0.001). Furthermore, we did not find a significant difference in infarct quality or blood vessel density between any of the groups which suggests that neither infarct quality nor vascularization is the mechanism of action of SPREADS. The SPREADS device could potentially be used to deliver a range of new or previously developed biomaterial hydrogels, a remarkable potential to overcome the translational hurdles associated with hydrogel delivery to the heart.

Tissue Engineering: Understanding the Role of Biomaterials and Biophysical Forces on Cell Functionality Through Computational and Structural Biotechnology Analytical Methods.

Within the past 25 years, tissue engineering (TE) has grown enormously as a science and as an industry. Although classically concerned with the recapitulation of tissue and organ formation in our body for regenerative medicine, the evolution of TE research is intertwined with progress in other fields through the examination of cell function and behaviour in isolated biomimetic microenvironments. As such, TE applications now extend beyond the field of tissue regeneration research, operating as a platform for modifiable, physiologically-representative in vitro models with the potential to improve the translation of novel therapeutics into the clinic through a more informed understanding of the relevant molecular biology, structural biology, anatomy, and physiology. By virtue of their biomimicry, TE constructs incorporate features of extracellular macrostructure, molecular adhesive moieties, and biomechanical properties, converging with computational and structural biotechnology advances. Accordingly, this mini-review serves to contextualise TE for the computational and structural biotechnology reader and provides an outlook on how the disciplines overlap with respect to relevant advanced analytical applications.

Evaluation of the Immunomodulatory Effects of All-Trans Retinoic Acid Solid Lipid Nanoparticles and Human Mesenchymal Stem Cells in an A549 Epithelial Cell Line Model.

PURPOSE: To investigate two potential strategies aimed at targeting the inflammatory pathogenesis of COPD: a small molecule, all trans retinoic acid (atRA) and human mesenchymal stem cells (hMSCs). METHODS: atRA was formulated into solid lipid nanoparticles (SLNs) via the emulsification-ultrasonication method, and these SLNs were characterised physicochemically. Assessment of the immunomodulatory effects of atRA-SLNs on A549 cells in vitro was determined using ELISA. hMSCs were suspended in a previously developed methylcellulose, collagen and beta-glycerophosphate hydrogel prior to investigating their immunomodulatory effects in vitro. RESULTS: SLNs provided significant encapsulation of atRA and also sustained its release over 72 h. A549 cells were viable following the addition of atRA SLNs and showed a reduction in IL-6 and IL-8 levels. A549 cells also remained viable following addition of the hMSC/hydrogel formulation - however, this formulation resulted in increased levels of IL-6 and IL-8, indicating a potentially pro-inflammatory effect. CONCLUSION: Both atRA SLNs and hMSCs show potential for modulating the environment in inflammatory disease, though through different mechanisms and leading to different outcomes - despite both being explored as strategies for use in inflammatory disease. atRA shows promise by acting in a directly anti-inflammatory manner, whereas further research into the exact mechanisms and behaviours of hMSCs in inflammatory diseases is required.

A Custom Radiopaque Thermoresponsive Chemotherapy-Loaded Hydrogel for Intratumoural Injection: An In Vitro and Ex Vivo Assessment of Imaging Characteristics and Material Properties.

PURPOSE: Thermoresponsive hydrogels are gels which have different properties at varying temperatures. The objective of this study was to assess the material characteristics, imaging properties and chemotherapeutic drug release profile of a novel radiopaque thermoresponsive hydrogel in vitro, which is liquid at room temperature but solidifies at body temperature, to determine potential suitability for intratumoural delivery. MATERIALS AND METHODS: An iodinated radiopaque thermoresponsive hydrogel was formulated using iodixanol at a range of concentrations and assessed for sol-gel transition, radiopacity and imaging using CT and US. A lead formulation containing iodixanol at a concentration of 9.22% weight by weight (w/w, g of iodixanol per g of hydrogel) was evaluated in vitro for injectability, disintegration and dual drug release of cisplatin and paclitaxel from the hydrogel formulation. RESULTS: Radiopacity of the hydrogel increased in a concentration-dependent manner, but the highest concentration of iodixanol evaluated in this study (13.83% w/w) adversely affected the sol-gel transition of the hydrogel; therefore, 9.22% w/w iodixanol hydrogel was identified as the lead formulation. This formulation was readily visible on both CT and US. The formulation was hand injectable through a range of clinically relevant devices, had a sustained disintegration profile for up to 28 days and was able to deliver a sustained release of chemotherapeutic drug for up to 10 days. CONCLUSIONS: Favourable in vitro and ex vivo imaging and material characteristics of this thermoresponsive gel are demonstrated, suggesting potential interventional oncology applications for image-guided intratumoural delivery of sustained-release chemotherapy.

Advanced Material Catheter (AMCath), a minimally invasive endocardial catheter for the delivery of fast-gelling covalently cross-linked hyaluronic acid hydrogels.

Injectable hydrogels that aim to mechanically stabilise the weakened left ventricle wall to restore cardiac function or to deliver stem cells in cardiac regenerative therapy have shown promising data. However, the clinical translation of hydrogel-based therapies has been limited due to difficulties injecting them through catheters. We have engineered a novel catheter, Advanced Materials Catheter (AMCath), that overcomes translational hurdles associated with delivering fast-gelling covalently cross-linked hyaluronic acid hydrogels to the myocardium. We developed an experimental technique to measure the force required to inject such hydrogels and determined the mechanical/viscoelastic properties of the resulting hydrogels. The preliminary in vivo feasibility of delivering fast-gelling hydrogels through AMCath was demonstrated by accessing the porcine left ventricle and showing that the hydrogel was retained in the myocardium post-injection (three 200 µL injections delivered, 192, 204 and 183 µL measured). However, the mechanical properties of the hydrogels were reduced by passage through AMCath (≤20.62% reduction). We have also shown AMCath can be used to deliver cardiopoietic adipose-derived stem cell-loaded hydrogels without compromising the viability (80% viability) of the cells in vitro. Therefore, we show that hydrogel/catheter compatibility issues can be overcome as we have demonstrated the minimally invasive delivery of a fast-gelling covalently cross-linked hydrogel to the beating myocardium.

A collagen cardiac patch incorporating alginate microparticles permits the controlled release of hepatocyte growth factor and insulin-like growth factor-1 to enhance cardiac stem cell migration and proliferation.

Cardiac stem cells (CSCs) represent a logical cell type to exploit as a regenerative treatment option for tissue damage accrued as a result of a myocardial infarction. However, the isolation and expansion of CSCs prior to cell transplantation is time consuming, costly and invasive, and the reliability of cell expansion may also prove to be a major obstacle in the clinical application of CSC-based transplantation therapy after a myocardial infarction. In order to overcome this, we propose the incorporation of growth factor-eluting alginate microparticles into collagen-based scaffolds as an implantable biomaterial to promote the recruitment and expansion of CSCs in the myocardium. In order to obtain scaffolds able to enhance the motogenic and proliferative potential of CSCs, the aim of this work was to achieve a sustained delivery of both hepatocyte growth factor and insulin-like growth factor-1. Both proteins were initially encapsulated in alginate microparticles by spray drying and subsequently incorporated into a collagen scaffold. Microparticles were seen to homogeneously distribute through the interconnected scaffold pore structure. The resulting scaffolds were capable of extending the release of both proteins up to 15 days, a three-fold increase over non-encapsulated proteins embedded in the scaffolds. In vitro assays with isolated CSCs demonstrated that the sustained release of both bioactive proteins resulted in an increased motogenic and proliferative effect. As presently practiced, the isolation and expansion of CSCs for autologous cell transplantation is slow, expensive and difficult to attain. Thus, there is a need for strategies to specifically activate in situ the intrinsic cardiac regenerative potential represented by the CSCs using combinations of growth factors obviating the need for cell transplantation. By favouring the natural regenerative capability of CSCs, it is hypothesized that the cardiac patch presented here will result in positive therapeutic outcomes in MI and heart failure patients in the future. Copyright © 2016 John Wiley & Sons, Ltd.

A methylcellulose and collagen based temperature responsive hydrogel promotes encapsulated stem cell viability and proliferation in vitro.

With the number of stem cell-based therapies emerging on the increase, the need for novel and efficient delivery technologies to enable therapies to remain in damaged tissue and exert their therapeutic benefit for extended periods, has become a key requirement for their translation. Hydrogels, and in particular, thermoresponsive hydrogels, have the potential to act as such delivery systems. Thermoresponsive hydrogels, which are polymer solutions that transform into a gel upon a temperature increase, have a number of applications in the biomedical field due to their tendency to maintain a liquid state at room temperature, thereby enabling minimally invasive administration and a subsequent ability to form a robust gel upon heating to physiological temperature. However, various hurdles must be overcome to increase the clinical translation of hydrogels as a stem cell delivery system, with barriers including their low tensile strength and their inadequate support of cell viability and attachment. In order to address these issues, a methylcellulose based hydrogel was formulated in combination with collagen and beta glycerophosphate, and key development issues such as injectability and sterilisation processes were examined. The polymer solution underwent thermogelation at ~36 °C as determined by rheological analysis, and when gelled, was sufficiently robust to resist significant disintegration in the presence of phosphate buffered saline (PBS) while concomitantly allowing for diffusion of methylene blue dye solution into the gel. We demonstrate that human mesenchymal stem cells (hMSCs) encapsulated within the gel remained viable and showed raised levels of dsDNA at increasing time points, an indication of cell proliferation. Mechanical testing showed the "injectability", i.e. force required for delivery of the polymer solution through devices such as a syringe, needle or catheter. Sterilisation of the freeze-dried polymer wafer via gamma irradiation showed no adverse effects on the formed hydrogel characteristics. Taken together, these results indicate the potential of this gel as a clinically translatable delivery system for stem cells and therapeutic molecules in vivo.

A stimuli responsive liposome loaded hydrogel provides flexible on-demand release of therapeutic agents.

Lysolipid-based thermosensitive liposomes (LTSL) embedded in a chitosan-based thermoresponsive hydrogel matrix (denoted Lipogel) represents a novel approach for the spatiotemporal release of therapeutic agents. The entrapment of drug-loaded liposomes in an injectable hydrogel permits local liposome retention, thus providing a prolonged release in target tissues. Moreover, release can be controlled through the use of a minimally invasive external hyperthermic stimulus. Temporal control of release is particularly important for complex multi-step physiological processes, such as angiogenesis, in which different signals are required at different times in order to produce a robust vasculature. In the present work, we demonstrate the ability of Lipogel to provide a flexible, easily modifiable release platform. It is possible to tune the release kinetics of different drugs providing a passive release of one therapeutic agent loaded within the gel and activating the release of a second LTSL encapsulated agent via a hyperthermic stimulus. In addition, it was possible to modify the drug dosage within Lipogel by varying the duration of hyperthermia. This can allow for adaption of drug dosing in real time. As an in vitro proof of concept with this system, we investigated Lipogels ability to recruit stem cells and then elevate their production of vascular endothelial growth factor (VEGF) by controlling the release of a pro-angiogenic drug, desferroxamine (DFO) with an external hyperthermic stimulus. Initial cell recruitment was accomplished by the passive release of hepatocyte growth factor (HGF) from the hydrogel, inducing a migratory response in cells, followed by the delayed release of DFO from thermosensitive liposomes, resulting in a significant increase in VEGF expression. This delayed release could be controlled up to 14days. Moreover, by changing the duration of the hyperthermic pulse, a fine control over the amount of DFO released was achieved. The ability to trigger the release of therapeutic agents at a specific timepoint and control dosing level through changes in duration of hyperthermia enables sequential multi-dose profiles. STATEMENT OF SIGNIFICANCE: This paper details the development of a heat responsive liposome loaded hydrogel for the controlled release of pro-angiogenic therapeutics. Lysolipid-based thermosensitive liposomes (LTSLs) embedded in a chitosan-based thermoresponsive hydrogel matrix represents a novel approach for the spatiotemporal release of therapeutic agents. This hydrogel platform demonstrates remarkable flexibility in terms of drug scheduling and sequencing, enabling the release of multiple agents and the ability to control drug dosing in a minimally invasive fashion. The possibility to tune the release kinetics of different drugs independently represents an innovative platform to utilise for a variety of treatments. This approach allows a significant degree of flexibility in achieving a desired release profile via a minimally invasive stimulus, enabling treatments to be tuned in response to changing symptoms and complications.

Development of collagen-hydroxyapatite scaffolds incorporating PLGA and alginate microparticles for the controlled delivery of rhBMP-2 for bone tissue engineering.

The spatiotemporally controlled delivery of the pro-osteogenic factor rhBMP-2 would overcome most of the severe secondary effects linked to the products delivering this protein for bone regeneration. With this in mind, the aim of the present work was to develop a controlled rhBMP-2 release system using collagen-hydroxyapatite (CHA) scaffolds, which had been previously optimized for bone regeneration, as delivery platforms to produce a device with enhanced capacity for bone repair. Spray-drying and emulsion techniques were used to encapsulate bioactive rhBMP-2 in alginate and PLGA microparticles, with a high encapsulation efficiency. After incorporation of these microparticles into the scaffolds, rhBMP-2 was delivered in a sustained fashion for up to 28days. When tested in vitro with osteoblasts, these eluting materials showed an enhanced pro-osteogenic effect. From these results, an optimal rhBMP-2 eluting scaffold composition was selected and implanted in critical-sized calvarial defects in a rat model, where it demonstrated an excellent healing capacity in vivo. These platforms have an immense potential in the field of tissue regeneration; by tuning the specific therapeutic molecule to the tissue of interest and by utilizing different collagen-based scaffolds, similar systems can be developed for enhancing the healing of a diverse range of tissues and organs.

Supramolecular hydrogels with reverse thermal gelation properties from (oligo)tyrosine containing block copolymers.

Novel block copolymers comprising poly(ethylene glycol) (PEG) and an oligo(tyrosine) block were synthesized in different compositions by N-carboxyanhydride (NCA) polymerization. It was shown that PEG2000-Tyr(6) undergoes thermoresponsive hydrogelation at a low concentration range of 0.25-3.0 wt % within a temperature range of 25-50 °C. Cryogenic transmission electron microscopy (Cryo-TEM) revealed a continuous network of fibers throughout the hydrogel sample, even at concentrations as low as 0.25 wt %. Circular dichroism (CD) results suggest that better packing of the ß-sheet tyrosine block at increasing temperature induces the reverse thermogelation. A preliminary assessment of the potential of the hydrogel for in vitro application confirmed the hydrogel is not cytotoxic, is biodegradable, and produced a sustained release of a small-molecule drug.

Development of a thermoresponsive chitosan gel combined with human mesenchymal stem cells and desferrioxamine as a multimodal pro-angiogenic therapeutic for the treatment of critical limb ischaemia.

Critical limb ischaemia (CLI) is a debilitating ischaemic disease caused by vascular occlusion. Pro-angiogenic therapeutics have the potential to produce collateral vasculature, delaying or negating the need for amputation or invasive revascularisation. Thermoresponsive hydrogels can provide an in situ depot for the sustained release of drugs and provide protection and cohesion for encapsulated cells. Human mesenchymal stem cells (hMSCs) have demonstrated strong angiogenic potential in vitro and angiogenic efficacy in vivo. Desferrioxamine (DFO), a pharmacological activator of the pro-angiogenic hypoxia inducible factor-1α pathway, has shown pro-angiogenic efficacy in vivo. This study combined hMSCs and DFO with a thermoresponsive chitosan/ß-glycerophosphate (ß-GP) gel, to function as an injectable, multimodal, pro-angiogenic therapeutic for the treatment of CLI. This gel underwent a thermogelation beginning at 33°C, and provided a sustained, biologically active release of DFO over the space of seven days, whilst permitting the survival, proliferation and migration of encapsulated hMSCs. hMSCs encapsulated in gel containing a 100µM concentration of DFO displayed an upregulation in VEGF expression. The combination of hMSCs and DFO within the gel resulted in a synergistic enhancement in bioactivity, as measured by increased VEGF expression in gel-exposed human umbilical vein endothelial cells. This formulation displays significant potential as an injectable pro-angiogenic therapeutic for the treatment of CLI.