Network of Excellence for Functional Biomaterials (Scholarly Articles)

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  • Publication
    Sequestration of rhBMP-2 into Self-Assembled Polyelectrolyte Complexes Promotes Anatomic Localization of New Bone in a Porcine Model of Spinal Reconstructive Surgery.
    (Mary Ann Liebert, 2014-02-27) Abbah, Sunny-Akogwu; |~|
    Efficient and therapeutically safe delivery of recombinant human bone morphogenetic protein 2 (rhBMP-2) continues to be a central issue in bone tissue engineering. Recent evidence indicates that layer-by-layer self-assembly of polyelectrolyte complexes (PECs) can be used to recreate synthetic matrix environments that would act as tuneable reservoirs for delicate biomolecules and cells. Although preliminary in vitro as well as small-animal in vivo studies support this premise, translation into clinically relevant bone defect volumes in larger animal models remains unreported. Here we explored the use of native heparin-based PEC, deposited on a hydrated alginate gel template, to load bioactive rhBMP-2 and to facilitate lumbar interbody spinal fusion in pigs. We observed that triple PEC deposits with the highest protein sequestration efficiency and immobilization capacity promoted higher volume of new bone formation when compared with single PEC with low sequestration efficiency and immobilization capacity. This also resulted in a significantly enhanced biomechanical stability of the fused spinal segment when compared with PEC carriers with relatively low protein sequestration and immobilization capacities (p<0.05). Most importantly, PEC carriers showed a more orderly pattern of new bone deposition and superior containment of bone tissue within implant site when compared to collagen sponge carriers. We conclude that this growth factor sequestration platform is effective in the healing of clinically relevant bone defect volume and could overcome some of the safety concerns and limitations currently associated with rhBMP-2 therapy such as excessive heterotopic ossification.
  • Publication
    Preferential tendon stem cell response to growth factor supplementation.
    (Wiley, 2014) Holladay, Carolyn; Abbah, Sunny-Akogwu; |~|
    Tendon injuries are increasingly prevalent around the world, accounting for more than 100 000 new clinical cases/year in the USA alone. Cell-based therapies have been proposed as a therapeutic strategy, with recent data advocating the use of tendon stem cells (TSCs) as a potential cell source with clinical relevance for tendon regeneration. However, their in vitro expansion is problematic, as they lose their multipotency and change their protein expression profile in culture. Herein, we ventured to assess the influence of insulin-like growth factor 1 (IGF-1), growth and differentiation factor-5 (GDF-5) and transforming growth factor-β1 (TGFβ1) supplementation in TSC culture. IGF-1 preserved multipotency for up to 28 days. Upregulation of decorin and scleraxis expression was observed as compared to freshly isolated cells. GDF-5 treated cells exhibited reduced differentiation along adipogenic and chondrogenic pathways after 28 days, and decorin, scleraxis and collagen type I expression was increased. After 28 days, TGFβ1 supplementation led to increased scleraxis, osteonectin and collagen type II expression. The varied responses to each growth factor may reflect their role in tendon repair, suggesting that: GDF-5 promotes the transition of tendon stem cells towards tenocytes; TGFβ1 induces differentiation along several pathways, including a phenotype indicative of fibrocartilage or calcified tendon, common problems in tendon healing; and IGF-1 promotes proliferation and maintenance of TSC phenotypes, thereby creating a population sufficient to have a beneficial effect
  • Publication
    Nano-textured self-assembled aligned collagen hydrogels promote directional neurite guidance and overcome inhibition by myelin associated glycoprotein
    (Royal Society of Chemistry, 2011-01-11) Abu-Rub, Mohammad T.; Zeugolis, Dimitrios I.; McMahon, Siobhan; Pandit, Abhay; |~|
    The development of nerve guidance conduits is constantly evolving as the need arises for therapies for spinal cord injury. In addition to providing a path for regrowing axons to reconnect with their appropriate targets, the structural and biochemical cues provided by these conduits should be permissive for directional neurite outgrowth and be protective against inhibition in the vicinity of the injury site. Here, we adapted the use of iso-electric focusing to drive the alignment of supramolecular fibrils into self-assembled collagen hydrogels ([similar]300 µm diameter), and tested those hydrogels for the ability to direct and enhance the migration of neurites. Structural characterization revealed anisotropic alignment of nanofibrillar aggregates ([similar]20 nm diameter), arranged in micron-scale bundles ([similar]1 to 2 µm diameter) similar to the hierarchical size scales observed in native tissues. Neurite outgrowth extended bidirectionally along the axes of aligned hydrogels. Furthermore, it was shown that, as opposed to poly-D-lysine, neurite outgrowth on aligned hydrogels is not inhibited in the presence of myelin-associated glycoprotein (p > 0.05). These results highlight for the first time a structural and biochemical role for iso-electrically aligned collagen hydrogels in controlling neuronal growth, and indicate that the short-term signaling associated with these hydrogels can be used in adjunct therapy following injury to the spinal cord.
  • Publication
    Thermosensitive hydrogel for prolonged delivery of lentiviral vector expressing neurotrophin-3 in vitro
    (Wiley, 2011-11-01) McMahon, Siobhan; Nikolskaya, Natalia; Ní Choileain, Siobhan; Hennessy, Niamh; O'Brien, Timothy; Rochev, Yury; |~|
    Background The development of tissue engineering scaffolds for gene delivery has the potential to enhance gene transfer efficiency and safety via controlled temporal and spatial delivery. Lentiviral delivery can be carried out using the natural biopolymer thermoresponsive gel, chitosan/b-glycerol phosphate (b-GP) as a carrier.Methods Three chitosan/b-GP scaffolds were prepared with varying concentrations of chitosan and b-GP to obtain a pH and gelation temperature suitable for in situ delivery. A lentiviral vector expressing either green fluorescent protein (Lenti GFP) or neurotrophin-3 (Lenti NT-3) was incorporated into the chitosan/ b-GP scaffolds and also into collagen 0.1% w/v (control). Viral elution medium was removed at various timepoints and added to the culture medium of preseeded HeLa or primary dorsal root ganglia (DRG) cells, respectively. GFP gene expression was quantified using fluorescence-activated cell sorting analysis. The effect of Lenti NT-3 was analyzed by measuring DRG neurite outgrowth.Results Collagen displayed its most significant elution of virus on day 1 and chitosan/b-GP (with a final concentration of 2.17% chitosan) on day 3.Conclusions The system shows promise for the in situ, thermoresponsive delivery of lentiviral vectors providing long-term gene expression for therapeutic factors to treat conditions such as injury to the nervous system. Copyright (C) 2011 John Wiley & Sons, Ltd.
  • Publication
    Controlling dispersion of axonal regeneration using a multichannel collagen nerve conduit
    (Elsevier, 2010-03-21) Yao, Li; Pandit, Abhay; Enterprise Ireland - Proof of Concept Grant (PC/2007/027); Science Foundation Ireland - Research Frontiers Program, under Grant Number (08/RFP/ENM1218); Science Foundation Ireland - The Short Term Travel Fellowship (STTF) supplement; Leiden University Fund (LUF)/ Slingelands
    Single channel conduits are used clinically in nerve repair as an alternative to the autologous nerve graft. Axons regenerating across single channel tubes, however, may disperse resulting in inappropriate target reinnervation. This dispersion may be limited by multichannel nerve conduits as they resemble the structure of nerve multiple basal lamina tubes. In this study, we investigated the influence of channel number on the axonal regeneration using a series of 1-, 2-, 4-, and 7-channel collagen conduits and commercial (NeuraGen®) single channel conduits. Nerve conduits were implanted in rats with a 1 cm gap of sciatic nerve. After four months, quantitative results of regeneration were evaluated with nerve morphometry and the accuracy of regeneration was assessed using retrograde tracing: two tracers being applied simultaneously to tibial and peroneal nerves to determine the percentage of motor neurons with double projections. Recovery of function was investigated with compound muscle action potential recordings and ankle motion analysis. We showed that the fabricated 1-channel and 4-channel conduits are superior to other types of conduits in axonal regeneration. Simultaneous tracing showed a significantly lower percentage of motor neurons with double projections after 2- and 4-channel compared with 1-channel conduit repair. This study shows the potential influence of multichannel guidance on limiting dispersion without decreasing quantitative results of regeneration.
  • Publication
    A Highly Effective Gene Delivery Vector
    (The Royal Society of Chemistry, 2010-06) Newland, Ben; Tai, Hongyun; Zheng, Yu; Velasco, Diego; Di Luca, Andrea; Howdle, Steven M.; Alexander, Cameron; Wang, Wenxin; Pandit, Abhay
    A hyperbranched 2-(dimethylamino)ethyl methacrylate (DMAEMA) based polymer has been synthesised by a one-pot in situ deactivation enhanced atom transfer radical polymerisation (DE-ATRP); it exhibits much higher transfection ability than linear poly(DMAEMA) and is comparable to the well known branched poly(ethylene imine) (PEI) and the SuperFect dendrimer but with lower cytotoxicity.
  • Publication
    The influence of size and charge of chitosan/polyglutamic acid hollow spheres on cellular internalization, viability and blood compatibility
    (Elsevier, 2010-11) Dash, Biraja C.; Réthoré, Gildas; Monaghan, Michael; Pandit, Abhay
    Polymeric hollow spheres can be tailored as efficient carriers of various therapeutic molecules due to their tunable properties. However, the entry of these synthetic vehicles into cells, their cell viability and blood compatibility depend on their physical and chemical properties e.g. size, surface charge. Herein, we report the effect of size and surface charge on cell viability and cellular internalization behaviour and their effect on various blood components using chitosan/polyglutamic acid hollow spheres as a model system. Negatively charged chitosan/polyglutamic acid hollow spheres of various sizes 100, 300, 500 and 1000 nm were fabricated using a template based method and covalently surface modified using linear polyethylene glycol and methoxyethanol amine to create a gradient of surface charge from negative to neutrally charged spheres respectively. The results here suggest that both size and surface charge have a significant influence on the sphere¿s behaviour, most prominently on haemolysis, platelet activation, plasma recalcification time, cell viability and internalization over time. Additionally, cellular internalization behaviour and viability was found to vary with different cell types. These results are in agreement with those of inorganic spheres and liposomes, and can serve as guidelines for tailoring polymeric solid spheres for specific desired applications in biological and pharmaceutical fields, including the design of nanometer to submicron-sized delivery vehicles.
  • Publication
    Non-viral gene therapy for myocardial engineering
    (John Wiley & Sons, Ltd., 2010-05) Holladay, Carolyn A.; O'Brien, Timothy; Pandit, Abhay; Science Foundation Ireland
    Despite significant advances in surgical and pharmacological techniques, myocardial infarction (MI) remains the main cause of morbidity in the developed world because no remedy has been found for the regeneration of infarcted myocardium. Once the blood supply to the area in question is interrupted, the inflammatory cascade, among other mechanisms, results in the damaged tissue becoming a scar. The goals of cardiac gene therapy are essentially to minimize damage, to promote regeneration, or some combination thereof. While the vector is, in theory, less important than the gene being delivered, the choice of vector can have a significant impact. Viral therapies can have very high transfection efficiencies, but disadvantages include immunogenicity, retroviral-mediated insertional mutagenesis, and the expense and difficulty of manufacture. For these reasons, researchers have focused on non-viral gene therapy as an alternative. In this review, naked plasmid delivery, or the delivery of complexed plasmids, and cell-mediated gene delivery to the myocardium will be reviewed. Pre-clinical and clinical trials in the cardiac tissue will form the core of the discussion. While unmodified stem cells are sometimes considered therapeutic vectors on the basis of paracrine mechanisms of action basic understanding is limited. Thus, only genetically modified cells will be discussed as cell-mediated gene therapy. Copyright © 2009 John Wiley & Sons, Inc.
  • Publication
    A reliable method for detecting complexed DNA in vitro
    (The Royal Society of Chemistry, 2010-09-08) Holladay, C.; Keeney, M.; Mathew, B; Wang, W.; Pandit, Abhay; Science Foundation Ireland
    Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique hypothesized to allow quantification of both bound and unbound DNA. A comparison of the two methods was performed by quantifying the elution of six different varieties of DNA complexes from a model biomaterial (collagen) scaffold. After seven days of elution, the PicoGreen® assay only allowed detection of three types of complexes (those formed using Lipofectin¿ and two synthesised copolymers). However, the Cy5 fluorescent labeling technique enabled detection of all six varieties including those formed via common transfection agents poly(ethylene imine), poly-l-lysine and SuperFect¿. This allowed reliable quantification of the elution of all these complexes from the collagen scaffold. Thus, while intercalating dyes may be effective and reliable for detecting double-stranded, unbound DNA, the technique described in this work allowed reliable quantification of DNA independent of complexation state.
  • Publication
    Spinal cord injury in vitro: Modelling axon growth inhibition
    (Elsevier, 2010-06) Abu-Rub, Mohammad; McMahon, Siobhan; Zeugolis, Dimitrios I.; Pandit, Abhay
    Over the past three decades, tremendous progress has been made in elucidating mechanisms underlying regenerative failure after spinal cord injury and in devising therapeutic approaches to promote functional nerve regeneration. Various in vitro assays have been developed using brain and/or spinal cord neuronal cells to study axon growth in conditions that represent the post-injury environment. This review outlines the current models used to dissect, analyze and manipulate specific aspects of spinal cord injury leading to axon growth inhibition.
  • Publication
    Liposomal gene delivery mediated by tissue-engineered scaffolds
    (Elsevier, 2010-01-01) Kulkarni, Mangesh; Griser, Udo; O'Brien, Timothy; Pandit, Abhay; Science Foundation Ireland
    In the absence of any ideal gene delivery carrier despite the recent explosion of novel carrier systems, the current trend is to explore the complementary synergy promised by a combination of delivery systems such as liposomes, which are the most widely researched versatile non-viral carriers, and tissue-engineered scaffolds with macrostructures of defined architecture comprised of natural or synthetic macromolecules. Here, we discuss the recent advances in liposomal gene delivery and the possible benefits of a combined liposome¿scaffold approach, such as long-term expression, enhanced stability, reduction in toxicity and ability to produce spatio-temporal expression patterns. This approach is generating significant impact in the field as a result of its potential for extended localised gene delivery for applications in a variety of clinical conditions.
  • Publication
    Type II collagen-hyaluronan hydrogel - a step towards a scaffold for intervertebral disc tissue engineering
    (European Cells & Materials Ltd, 2010-09-06) Calderon, Laura; Collin, Estelle; Murphy, Mary; O'Halloran, Damien; Pandit, Abhay; Science Foundation of Ireland, Research Frontiers Programme
    Intervertebral disc regeneration strategies based on stem cell differentiation in combination with the design of functional scaffolds is an attractive approach towards repairing/regenerating the nucleus pulposus. The specific aim of this study was to optimise a composite hydrogel composed of type II collagen and hyaluronic acid (HA) as a carrier for mesenchymal stem cells. Hydrogel stabilisation was achieved by means of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking. Optimal hydrogel properties were determined by investigating different concentrations of EDC (8 mM, 24 mM and 48 mM). Stable hydrogels were obtained independent of the concentration of carbodiimide used. The hydrogels cross-linked by the lowest concentration of EDC (8 mM) demonstrated high swelling properties. Additionally, improved proliferation of seeded rat mesenchymal stem cells (rMSCs) and hydrogel stability levels in culture were observed with this 8 mM cross-linked hydrogel. Results from this study indicate that EDC/NHS (8 mM) cross-linked type II collagen/HA hydrogel was capable of supporting viability of rMSCs, and furthermore their differentiation into a chondrogenic lineage. Further investigations should be conducted to determine its potential as scaffold for nucleus pulposus regeneration/repair.