Delivery of definable number of drug or growth factor loaded poly(dl-lactic acid-co-glycolic acid) microparticles within human embryonic stem cell derived aggregates

Embryoid bodies (EBs) generated from embryonic stem cells are used to study processes of differentiation within a three dimensional (3D) cell environment. In many instances however, EBs are dispersed to single cell suspensions with a subsequent monolayer culture. Moreover, where the 3D integrity of an EB is maintained, cytokines or drugs of interest to stimulate differentiation are often added directly to the culture medium at fixed concentrations and effects are usually limited to the outer layers of the EB. The aim of this study was to create an EB model with localised drug and or growth factor delivery directly within the EB. Using poly(DL-lactic acid-co-glycolic acid) microparticles (MPs) with an average diameter of 13 μm, we have demonstrated controllable incorporation of defined numbers of MPs within human ES cell derived EBs, down to 1 MP per EB. This was achieved by coating MPs with human ES cell lysate and centrifugation of specific ratios of ES cells and MPs to form 3D aggregates. Using MPs loaded with simvastatin (pro or active drug) or BMP-2, we have demonstrated osteogenic differentiation within the 3D aggregates, maintained in culture for up to 21 days, and quantified by real time QPCR for osteocalcin. Immunostaining for RUNX2 and osteocalcin, and also histochemical staining with picrosirius red to demonstrate collage type 1 and Alizarin red to demonstrate calcium/mineralisation further demonstrated osteogenic differentiation and revealed regional staining associated with the locations of MPs within the aggregates. We also demonstrated endothelial differentiation within human ES cell-derived aggregates using VEGF loaded MPs. In conclusion, we demonstrate an effective and reliable approach for engineering stem aggregates with definable number of MPs within the 3D cellular structure. We also achieved localised osteogenic and endothelial differentiation associated with MPs releasing encapsulated drug molecules or cytokines directly within the cell aggregate. This provides a powerful tool for controlling and investigating differentiation within 3D cell cultures and has applications to drug delivery, drug discovery, stem cell biology, tissue engineering and regenerative medicine

Supercritical carbon dioxide: putting the fizz into biomaterials

This paper describes recent progress made in the use of high pressure or supercritical fluids to process polymers into three-dimensional tissue engineering scaffolds. Three current examples are highlighted: foaming of acrylates for use in cartilage tissue engineering; plasticization and encapsulation of bioactive species into biodegradable polyesters for bone tissue engineering; and a novel laser sintering process used to fabricate three-dimensional biodegradable polyester structures from particles prepared via a supercritical route

Thermoresponsive magnetic colloidal gels via surface-initiated polymerisation from functional microparticles

Novel magnetothermally responsive core-shell microparticles have been synthesized. The aqueous suspensions of these particles exhibit fast thermoreversible fluid-to-gel transitions and retain good magnetic properties. Rheological measurements demonstrated that the viscoelasticity of the prepared particle gels can be tuned, enabling these gels to have the mechanical properties that should facilitate their applications as 3D cell scaffolds for in vitro expansion of cells. Also, it was found that the responsive particles could be used in repeated heating-cooling cycles without marked changes in gel elasticity. Presto Blue viability assays of 3T3 fibroblasts and human mesenchymal sem cells cultured within the colloidal gel showed that the cells remained viable and proliferated, with significant increases in cell numbers over extended culture times. Confocal microscopy images of 3T3 cells cultured within the colloidal gel demonstrated that cells adhered, spread and retained their normal morphologies during proliferation.. Furthermore, magnetic separation allowed efficient recovery of cells after their expansion in vitro without need for enzyme-mediated release steps. Trypsin-free cell passages were performed allowing multiple growth, separation and reloading of cells within the colloidal gels. Overall, the results suggest this colloidal gel has potential as a 3D scaffold for in vitro expansion of a variety of cell types and for enzyme free cell harvesting

Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

Fundamental behaviour such as cell fate, growth and death are mediated through the control of key genetic transcriptional regulators. These regulators are activated or repressed by the integration of multiple signalling molecules in spatio-temporal gradients. Engineering these gradients is complex but considered key in controlling tissue formation in regenerative medicine approaches. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor complexity but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly using GAG-binding domains to promote cell targeting, and cell penetrating peptides (CPPs) to allow cell entry. Herein we demonstrate that GET can be used in a three dimensional (3D) hydrogel matrix to produce gradients of intracellular transduction of mammalian cells. Using a compartmentalised diffusion model with a source-gel-sink (So-G-Si) assembly, we created gradients of reporter proteins (mRFP1-tagged) and a transcription factor (TF, myogenic master regulator MyoD) and showed that GET can be used to deliver molecules into cells spatio-temporally by monitoring intracellular transduction and gene expression programming as a function of location and time. The ability to spatio-temporally control the intracellular delivery of functional proteins will allow the establishment of gradients of cell programming in hydrogels and approaches to direct cellular behaviour for many regenerative medicine applications

Targeted protein delivery: carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds.

Tissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen.This work was supported by the European Research Council [ERC Advanced Grant 320598 3D-E] and was also funded by a grant from the Medical Research Council, Arthritis Research UK, Reumafonds and the UKRM

The visualisation of vitreous using surface modified poly(lactic-co-glycolic acid) microparticles

This paper is freely available online under the BMJ Journals unlocked scheme, see http:// bjo.bmj.com/site/about/unlocked.xhtmAims To demonstrate the potential use of in vitro poly (lactic-co-glycolic acid) (PLGA) microparticles in comparison with triamcinolone suspension to aid visualisation of vitreous during anterior and posterior vitrectomy. Methods PLGA microparticles (diameter 10-60 mu m) were fabricated using single and/or double emulsion technique(s) and used untreated or following the surface adsorption of a protein (transglutaminase). Particle size, shape, morphology and surface topography were assessed using scanning electron microscopy (SEM) and compared with a standard triamcinolone suspension. The efficacy of these microparticles to enhance visualisation of vitreous against the triamcinolone suspension was assessed using an in vitro set-up exploiting porcine vitreous. Results Unmodified PLGA microparticles failed to adequately adhere to porcine vitreous and were readily washed out by irrigation. In contrast, modified transglutaminase-coated PLGA microparticles demonstrated a significant improvement in adhesiveness and were comparable to a triamcinolone suspension in their ability to enhance the visualisation of vitreous. This adhesive behaviour also demonstrated selectivity by not binding to the corneal endothelium. Conclusion The use of transglutaminase-modified biodegradable PLGA microparticles represents a novel method of visualising vitreous and aiding vitrectomy. This method may provide a distinct alternative for the visualisation of vitreous whilst eliminating the pharmacological effects of triamcinolone acetonide suspension.Peer reviewedFinal Published versio

Laminin and Fibronectin Treatment Leads to Generation of Dendritic Cells with Superior Endocytic Capacity

Copyright: 2010 Garcı´a-Nieto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Sampling the microenvironment at sites of microbial exposure by dendritic cells ( DC) and their subsequent interaction with T cells in the paracortical area of lymph nodes are key events for initiating immune responses. Most of our knowledge of such events in human is based on in vitro studies performed in the absence of extracellular matrix (ECM) proteins. ECM in basement membranes and interstitial spaces of different tissues, including lymphoid organs, plays an important role in controlling specific cellular functions such as migration, intracellular signalling and differentiation. The aim of this study was, therefore, to investigate the impact of two abundant ECM components, fibronectin and laminin, on the phenotypical and functional properties of DC and how that might influence DC induced T-cell differentiation. Methodology/Principal Findings: Human monocyte derived DC were treated with laminin and fibronectin for up to 48 hours and their morphology and phenotype was analyzed using scanning electron microscopy, flow cytometry and real time PCR. The endocytic ability of DC was determined using flow cytometry. Furthermore, co-culture of DC and T cells were established and T cell proliferation and cytokine profile was measured using H(3)-thymidine incorporation and ELISA respectively. Finally, we assessed formation of DC-T cell conjugates using different cell trackers and flow cytometry. Our data show that in the presence of ECM, DC maintain a 'more immature' phenotype and express higher levels of key endocytic receptors, and as a result become significantly better endocytic cells, but still fully able to mature in response to stimulation as evidenced by their superior ability to induce antigen-specific T cell differentiation. Conclusion: These studies underline the importance of including ECM components in in vitro studies investigating DC biology and DC-T cell interaction. Within the context of antigen specific DC induced T cell proliferation, inclusion of ECM proteins could lead to development of more sensitive assays.Peer reviewedFinal Published versio

Highly versatile cell-penetrating peptide loaded scaffold for efficient and localised gene delivery to multiple cell types: From development to application in tissue engineering

Gene therapy has recently come of age with seven viral vector-based therapies gaining regulatory approval in recent years. In tissue engineering, non-viral vectors are preferred over viral vectors, however, lower transfection efficiencies and difficulties with delivery remain major limitations hampering clinical translation. This study describes the development of a novel multi-domain cell-penetrating peptide, GET, designed to enhance cell interaction and intracellular translocation of nucleic acids; combined with a series of porous collagen-based scaffolds with proven regenerative potential for different indications. GET was capable of transfecting cell types from all three germ layers, including stem cells, with an efficiency comparable to Lipofectamine® 3000, without inducing cytotoxicity. When implanted in vivo, GET gene-activated scaffolds allowed for host cell infiltration, transfection localized to the implantation site and sustained, but transient, changes in gene expression – demonstrating both the efficacy and safety of the approach. Finally, GET carrying osteogenic (pBMP-2) and angiogenic (pVEGF) genes were incorporated into collagen-hydroxyapatite scaffolds and with a single 2μg dose of therapeutic pDNA, induced complete repair of critical-sized bone defects within 4 weeks. GET represents an exciting development in gene therapy and by combining it with a scaffold-based delivery system offers tissue engineering solutions for a myriad of regenerative indications

Direct three-dimensional printing of polymeric scaffolds with nanofibrous topography

Three-dimensional (3D) printing is a powerful manufacturing tool for making 3D structures with well-defined architectures for a wide range of applications. The field of tissue engineering has also adopted this technology to fabricate scaffolds for tissue regeneration. The ability to control architecture of scaffolds, e.g. matching anatomical shapes and having defined pore size, has since been improved significantly. However, the material surface of these scaffolds is smooth and does not resemble that found in natural extracellular matrix (ECM), in particular, the nanofibrous morphology of collagen. This natural nanoscale morphology plays a critical role in cell behaviour. Here, we have developed a new approach to directly fabricate polymeric scaffolds with an ECM-like nanofibrous topography and defined architectures using extrusion-based 3D printing. 3D printed tall scaffolds with interconnected pores were created with disparate features spanning from nanometres to centimetres. Our approach removes the need for a sacrificial mould and subsequent mould removal compared to previous methods. Moreover, the nanofibrous topography of the 3D printed scaffolds significantly enhanced protein absorption, cell adhesion and differentiation of human mesenchymal stem cells when compared to those with smooth material surfaces. These 3D printed scaffolds with both defined architectures and nanoscale ECM-mimicking morphologies have potential applications in cartilage and bone regeneration