ORCID Profile
0000-0003-2030-8005
Current Organisations
Advanced Materials and BioEngineering Research (AMBER) Centre
,
Royal College of Surgeons in Ireland
,
Trinity College Dublin, Centre for Bioengineering
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Publisher: Elsevier BV
Date: 12-2010
DOI: 10.1016/J.BIOMATERIALS.2010.08.056
Abstract: One of the key challenges in tissue engineering is to understand the host response to scaffolds and engineered constructs. We present a study in which two collagen-based scaffolds developed for bone repair: a collagen-glycosaminoglycan (CG) and biomimetic collagen-calcium phosphate (CCP) scaffold, are evaluated in rat cranial defects, both cell-free and when cultured with MSCs prior to implantation. The results demonstrate that both cell-free scaffolds showed excellent healing relative to the empty defect controls and somewhat surprisingly, to the tissue engineered (MSC-seeded) constructs. Immunological analysis of the healing response showed higher M1 macrophage activity in the cell-seeded scaffolds. However, when the M2 macrophage response was analysed, both groups (MSC-seeded and non-seeded scaffolds) showed significant activity of these cells which are associated with an immunomodulatory and tissue remodelling response. Interestingly, the location of this response was confined to the construct periphery, where a capsule had formed, in the MSC-seeded groups as opposed to areas of new bone formation in the non-seeded groups. This suggests that matrix deposited by MSCs during in vitro culture may adversely affect healing by acting as a barrier to macrophage-led remodelling when implanted in vivo. This study thus improves our understanding of host response in bone tissue engineering.
Publisher: Elsevier BV
Date: 2010
DOI: 10.1016/J.BIOMATERIALS.2009.09.063
Abstract: In the literature there are conflicting reports on the optimal scaffold mean pore size required for successful bone tissue engineering. This study set out to investigate the effect of mean pore size, in a series of collagen-glycosaminoglycan (CG) scaffolds with mean pore sizes ranging from 85 microm to 325 microm, on osteoblast adhesion and early stage proliferation up to 7 days post-seeding. The results show that cell number was highest in scaffolds with the largest pore size of 325 microm. However, an early additional peak in cell number was also seen in scaffolds with a mean pore size of 120 microm at time points up to 48 h post-seeding. This is consistent with previous studies from our laboratory which suggest that scaffold specific surface area plays an important role on initial cell adhesion. This early peak disappears following cell proliferation indicating that while specific surface area may be important for initial cell adhesion, improved cell migration provided by scaffolds with pores above 300 microm overcomes this effect. An added advantage of the larger pores is a reduction in cell aggregations that develop along the edges of the scaffolds. Ultimately scaffolds with a mean pore size of 325 microm were deemed optimal for bone tissue engineering.
Publisher: Wiley
Date: 29-06-2020
Publisher: Elsevier BV
Date: 02-2021
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.BIOMATERIALS.2017.09.036
Abstract: The clinical translation of bioactive scaffolds for the treatment of large segmental bone defects has remained a challenge due to safety and efficacy concerns as well as prohibitive costs. The design of an implantable, biocompatible and resorbable device, which can fill the defect space, allow for cell infiltration, differentiation and neovascularisation, while also recapitulating the natural repair process and inducing cells to lay down new bone tissue, would alleviate the problems with existing treatments. We have developed a gene-activated scaffold platform using a bone-mimicking collagen hydroxyapatite scaffold loaded with chitosan nanoparticles carrying genes encoding osteogenic (BMP-2) and angiogenic (VEGF) proteins. With a single treatment, protein expression by mesenchymal stem cells (MSCs) seeded onto the scaffold is sustained for up to 28 days and is functional in inducing MSC osteogenesis. The in vivo safety and efficacy of this gene-activated scaffold platform was demonstrated resulting in the successful transfection of host cells, abrogating the requirement for multiple procedures to isolate cells or ex vivo cell culture. Furthermore, the level of bone formation at the exceptionally early time-point of 28 days was comparable to that achieved following recombinant BMP-2 protein delivery after 8 weeks in vivo, without the adverse side effects and at a fraction of the cost. This naturally derived cell-free gene-activated scaffold thus represents a new 'off-the-shelf' product capable of accelerating bone repair in critical-sized bone defects.
Publisher: Wiley
Date: 25-11-2021
Abstract: After spinal cord injury (SCI), tissue engineering scaffolds offer a potential bridge for regeneration across the lesion and support repair through proregenerative signaling. Ideal biomaterial scaffolds that mimic the physicochemical properties of native tissue have the potential to provide innate trophic signaling while also minimizing damaging inflammation. To address this challenge, taking cues from the spinal cord's structure, the proregenerative signaling capabilities of native cord components are compared in vitro. A synergistic mix of collagen‐IV and fibronectin (Coll‐IV/Fn) is found to optimally enhance axonal extension from neuronal cell lines (SHSY‐5Y and NSC‐34) and induce morphological features typical of quiescent astrocytes. This optimal composition is incorporated into hyaluronic acid scaffolds with aligned pore architectures but varying stiffnesses (0.8–3 kPa). Scaffolds with biomimetic mechanical properties ( kPa), functionalized with Coll‐IV/Fn, not only modulate primary astrocyte behavior but also stimulate the production of anti‐inflammatory cytokine IL‐10 in a stiffness‐dependent manner. Seeded SHSY‐5Y neurons generate distributed neuronal networks, while softer biomimetic scaffolds promote axonal outgrowth in an ex vivo model of axonal regrowth. These results indicate that the interaction of stiffness and biomaterial composition plays an essential role in vitro in generating repair‐critical cellular responses and demonstrates the potential of biomimetic scaffold design.
Publisher: Wiley
Date: 10-2015
DOI: 10.1002/JBM.A.35567
Abstract: We have previously examined osteoblast behavior on porous collagen-glycosaminoglycan (CG) scaffolds with a range of mean pore sizes demonstrating superior cell attachment and migration in scaffolds with the largest pores (325 μm). Scaffolds provide a framework for construct development therefore, it is crucial to identify the optimal pore size for augmented tissue formation. Utilizing the same range of scaffolds (85 μm - 325 μm), this study aimed to examine the effects of mean pore size on subsequent osteoblast differentiation and matrix mineralization, and to understand the mechanism by which pore size influences behavior of different cell types. Consequently, primary mesenchymal stem cells (MSCs) were assessed and their behavior compared to osteoblasts. Results demonstrated that scaffolds with the largest pore size (325 μm) facilitated improved osteoblast infiltration, earlier expression of mature bone markers osteopontin (OPN) and osteocalcin (OCN), and increased mineralization. MSCs responded similarly to osteoblasts whereby cell attachment and scaffold infiltration improved with increasing pore size. However, MSCs showed reduced cell motility, proliferation, and scaffold infiltration compared to osteoblasts. This was associated with differences in the profile of integrin subunits (α2) and collagen receptors (CD44), indicating that osteoblasts have a stronger affinity for CG scaffolds compared to MSCs. In summary, these results reveal how larger pores promote improved cell infiltration, essential for construct development, however the optimal scaffold pore size can be cell type specific. As such, this study highlights a necessity to tailor both scaffold micro-architecture and cell-type when designing constructs for successful bone tissue engineering applications.
Publisher: MDPI AG
Date: 15-12-2021
DOI: 10.3390/PHARMACEUTICS13122161
Abstract: Injury to the peripheral or central nervous systems often results in extensive loss of motor and sensory function that can greatly diminish quality of life. In both cases, macrophage infiltration into the injury site plays an integral role in the host tissue inflammatory response. In particular, the temporally related transition of macrophage phenotype between the M1/M2 inflammatory/repair states is critical for successful tissue repair. In recent years, biomaterial implants have emerged as a novel approach to bridge lesion sites and provide a growth-inductive environment for regenerating axons. This has more recently seen these two areas of research increasingly intersecting in the creation of ‘immune-modulatory’ biomaterials. These synthetic or naturally derived materials are fabricated to drive macrophages towards a pro-repair phenotype. This review considers the macrophage-mediated inflammatory events that occur following nervous tissue injury and outlines the latest developments in biomaterial-based strategies to influence macrophage phenotype and enhance repair.
Publisher: Frontiers Media SA
Date: 03-12-2019
Publisher: Elsevier BV
Date: 05-2014
DOI: 10.1016/J.ACTBIO.2014.01.016
Abstract: An emerging paradigm in orthopedics is that a bone-healing outcome is the product of the anabolic (bone-forming) and catabolic (bone-resorbing) outcomes. Recently, surgical and tissue engineering strategies have emerged that combine recombinant human bone morphogenetic proteins (rhBMPs) and bisphosphonates (BPs) in order to maximize anabolism and minimize catabolism. Collagen-based scaffolds that are the current surgical standard can bind rhBMPs, but not BPs. We hypothesized that a biomimetic collagen-hydroxyapatite (CHA) scaffold would bind both agents and produce superior in vivo outcomes. Consistent with this concept, in vitro elution studies utilizing rhBMP-2 ELISA assays and scintillation counting of (14)C-radiolabeled zoledronic acid (ZA) confirmed delayed release of both agents from the CHA scaffold. Next, scaffolds were tested for their capacity to form ectopic bone after surgical implantation into the rat hind limb. Using CHA, a significant 6-fold increase in bone volume was seen in rhBMP-2/ZA groups compared to rhBMP-2 alone, confirming the ability of ZA to enhance rhBMP-2 bone formation. CHA scaffolds were found to be capable of generating mineralized tissue in the absence of rhBMP-2. This study has implications for future clinical treatments of critical bone defects. It demonstrates the relative advantages of co-delivering anabolic and anti-catabolic agents using a multicomponent scaffold system.
Publisher: Elsevier BV
Date: 08-2018
DOI: 10.1016/J.JCONREL.2018.05.022
Abstract: Gene-activated scaffolds have been shown to induce controlled, sustained release of functional transgene both in vitro and in vivo. Bone morphogenetic proteins (BMPs) are potent mediators of osteogenesis however we found that the delivery of plasmid BMP-2 (pBMP-2) alone was not sufficient to enhance bone formation. Therefore, the aim of this study was to assess if the use of a series of modified BMP-2 plasmids could enhance the functionality of a pBMP-2 gene-activated scaffold and ultimately improve bone regeneration when implanted into a critical sized bone defect in vivo. A multi-cistronic plasmid encoding both BMP-2 and BMP-7 (BMP-2/7) was employed as was a BMP-2-Advanced plasmid containing a highly truncated intron sequence. With both plasmids, the highly efficient cytomegalovirus (CMV) promoter sequence was used. However, as there have been reports that the elongated factor 1-α promoter is more efficient, particularly in stem cells, a BMP-2-Advanced plasmid containing the EF1α promoter was also tested. Chitosan nanoparticles (CS) were used to deliver each plasmid to MSCs and induced transient up-regulation of BMP-2 protein expression, in turn significantly enhancing MSC-mediated osteogenesis when compared to untreated controls (p < 0.001). When incorporated into a bone mimicking collagen-hydroxyapatite scaffold, the BMP-2-Advanced plasmid, under the control of the CMV promotor, induced MSCs to produce approximately 2500 μg of calcium per scaffold, significantly higher (p < 0.001) than all other groups. Just 4 weeks post-implantation in vivo, this cell-free gene-activated scaffold induced significantly more bone tissue formation compared to a pBMP-2 gene-activated scaffold (p < 0.001) as indicated by microCT and histomorphometry. Immunohistochemistry revealed that the BMP-2-Advanced plasmid accelerated differentiation of osteoprogenitor cells to mature osteoblasts, thus causing rapid healing of the bone defects. This study confirms that optimising the plasmid construct can enhance the functionality of gene-activated scaffolds and translate to accelerated bone formation in a critical sized defect.
Publisher: Elsevier BV
Date: 04-2018
DOI: 10.1016/J.ACTBIO.2018.02.004
Abstract: 3D scaffold-based in vitro cell culturing is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumours. The main challenge in treating neuroblastoma, a paediatric cancer of the sympathetic nervous system, is to combat tumour metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (Coll-GAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multi-functionality beyond this tumour type. Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.
Publisher: American Chemical Society (ACS)
Date: 28-03-2018
DOI: 10.1021/ACS.MOLPHARMACEUT.8B00044
Abstract: The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type often refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(l-lysine) polypeptide (star-PLL) with varying number and length of poly(l-lysine) arms as potential nonviral gene delivery vectors for MSCs. We demonstrate that star-PLL vectors are capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging, and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs to MSCs is a rapid process, which likely proceeds via a clathrin-independent mechanism. We identify a star-PLL composition with 64 poly(l-lysine) arms and five l-lysine subunits per arm as a particularly efficient vector that is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(l-lysine). Furthermore, it demonstrated comparable transgene expression to the widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL vectors to facilitate efficient, nontoxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.
Publisher: Mary Ann Liebert Inc
Date: 10-2010
Publisher: Mary Ann Liebert Inc
Date: 05-2011
Publisher: Wiley
Date: 14-09-2022
Abstract: Diabetic foot ulcers (DFUs) are chronic wounds sustained by pathological fibroblasts and aberrant extracellular matrix (ECM). Porous collagen‐based scaffolds (CS) have shown clinical promise for treating DFUs but may benefit from functional enhancements. Our previous work showed fibroblasts differentiated from induced pluripotent stem cells are an effective source of new ECM mimicking fetal matrix, which notably promotes scar‐free healing. Likewise, functionalizing CS with this rejuvenated ECM shows potential for DFU healing. Herein, an approach to DFU healing is demonstrated for the first time using biopsied cells from DFU patients, reprogramming those cells, and functionalizing CS with patient‐specific ECM as a personalized acellular tissue‐engineered scaffold. A two‐pronged approach is taken: 1) direct ECM blending into scaffold fabrication, and 2) seeding scaffolds with reprogrammed fibroblasts for ECM deposition followed by decellularization. The decellularization approach reduces cell number requirements and maintains naturally deposited ECM proteins. Both approaches show enhanced ECM deposition from DFU fibroblasts. Decellularized scaffolds additionally enhance glycosaminoglycan deposition and subsequent vascularization. Finally, reprogrammed ECM scaffolds from patient‐matched DFU fibroblasts outperform those from healthy fibroblasts in several metrics, suggesting ECM is in fact able to redirect resident pathological fibroblasts in DFUs toward healing, and a patient‐specific ECM signature may be beneficial.
Publisher: Elsevier BV
Date: 04-2016
DOI: 10.1016/J.BIOMATERIALS.2016.01.065
Abstract: Today, chronic respiratory disease is one of the leading causes of mortality globally. Epithelial dysfunction can play a central role in its pathophysiology. The development of physiologically-representative in vitro model systems using tissue-engineered constructs might improve our understanding of epithelial tissue and disease. This study sought to engineer a bilayered collagen-hyaluronate (CHyA-B) scaffold for the development of a physiologically-representative 3D in vitro tracheobronchial epithelial co-culture model. CHyA-B scaffolds were fabricated by integrating a thin film top-layer into a porous sub-layer with lyophilisation. The film layer firmly connected to the sub-layer with delamination occurring at stresses of 12-15 kPa. Crosslinked scaffolds had a compressive modulus of 1.9 kPa and mean pore diameters of 70 μm and 80 μm, depending on the freezing temperature. Histological analysis showed that the Calu-3 bronchial epithelial cell line attached and grew on CHyA-B with adoption of an epithelial monolayer on the film layer. Immunofluorescence and qRT-PCR studies demonstrated that the CHyA-B scaffolds facilitated Calu-3 cell differentiation, with enhanced mucin expression, increased ciliation and the formation of intercellular tight junctions. Co-culture of Calu-3 cells with Wi38 lung fibroblasts was achieved on the scaffold to create a submucosal tissue analogue of the upper respiratory tract, validating CHyA-B as a platform to support co-culture and cellular organisation reminiscent of in vivo tissue architecture. In summary, this study has demonstrated that CHyA-B is a promising tool for the development of novel 3D tracheobronchial co-culture in vitro models with the potential to unravel new pathways in drug discovery and drug delivery.
Publisher: Elsevier BV
Date: 09-2020
Publisher: MDPI AG
Date: 30-07-2021
Abstract: Impaired skin wound healing due to severe injury often leads to dysfunctional scar tissue formation as a result of excessive and persistent myofibroblast activation, characterised by the increased expression of α-smooth muscle actin (αSMA) and extracellular matrix (ECM) proteins. Yet, despite extensive research on impaired wound healing and the advancement in tissue-engineered skin substitutes, scar formation remains a significant clinical challenge. This study aimed to first investigate the effect of methacrylate gelatin (GelMA) biomaterial stiffness on human dermal fibroblast behaviour in order to then design a range of 3D-printed GelMA scaffolds with tuneable structural and mechanical properties and understand whether the introduction of pores and porosity would support fibroblast activity, while inhibiting myofibroblast-related gene and protein expression. Results demonstrated that increasing GelMA stiffness promotes myofibroblast activation through increased fibrosis-related gene and protein expression. However, the introduction of a porous architecture by 3D printing facilitated healthy fibroblast activity, while inhibiting myofibroblast activation. A significant reduction was observed in the gene and protein production of αSMA and the expression of ECM-related proteins, including fibronectin I and collagen III, across the range of porous 3D-printed GelMA scaffolds. These results show that the 3D-printed GelMA scaffolds have the potential to improve dermal skin healing, whilst inhibiting fibrosis and scar formation, therefore potentially offering a new treatment for skin repair.
Publisher: Elsevier BV
Date: 04-2017
DOI: 10.1016/J.ACTBIO.2017.02.031
Abstract: In order to identify the mechanisms by which skeletal maturity alters the mechanosensitivity of mesenchymal stromal cells (MSCs) and, the implications for osteogenesis and angiogenesis during bone formation, we compared the response of MSCs derived from children and skeletally-mature healthy adults cultured on soft and stiff collagen-coated polyacrylamide substrates. MSCs from children were more mechanosensitive, showing enhanced angiogenesis and osteogenesis on stiff substrates as indicated by increased endothelial tubule formation, PGF production, nuclear-translocation of YAP, ALP activity and mineralisation. To examine these mechanisms in more detail, a customised PCR array identified an age-dependent, stiffness-induced upregulation of NOX1, VEGFR1, VEGFR2, WIF1 and, of particular interest, JNK3 in cells from children compared to adults. When JNK3 activity was inhibited, a reduction in stiffness-induced driven osteogenesis was observed - suggesting that JNK3 might serve as a novel target for recapitulating the enhanced regenerative potential of children in adults suffering from bone degeneration. We investigated the age-associated changes in the capacity of MSCs for bone regeneration involving the mechanosensitive signalling pathways, which reduce the ability of adult cells to respond to biophysical cues in comparison to cells from children, who are still undergoing bone development. Our results offer new insights into the mechanobiology of MSCs and sheds new light on age-altered mechanosensitivity and, on why children have such an immense capacity to regenerate their skeletal system. We have identified the mechanisms by which skeletal maturity alters the mechanosensitivity of mesenchymal stromal cells and an age-dependent, stiffness-induced upregulation of a number of prominent genes including, most notably, JNK3 in children cells, thus suggesting its potential to promote enhanced bone repair.
Publisher: Informa UK Limited
Date: 07-2010
Publisher: Elsevier BV
Date: 07-2012
DOI: 10.1016/J.JMBBM.2011.11.009
Abstract: In stem cell biology, focus has recently turned to the influence of the intrinsic properties of the extracellular matrix (ECM), such as structural, composition and elasticity, on stem cell differentiation. Utilising collagen-glycosaminoglycan (CG) scaffolds as an analogue of the ECM, this study set out to determine the effect of scaffold stiffness and composition on naive mesenchymal stem cell (MSC) differentiation in the absence of differentiation supplements. Dehydrothermal (DHT) and 1-ethyl-3-3-dimethyl aminopropyl carbodiimide (EDAC) crosslinking treatments were used to produce three homogeneous CG scaffolds with the same composition but different stiffness values: 0.5, 1 and 1.5 kPa. In addition, the effect of scaffold composition on MSC differentiation was investigated by utilising two glycosaminoglycan (GAG) types: chondroitin sulphate (CS) and hyaluronic acid (HyA). Results demonstrated that scaffolds with the lowest stiffness (0.5 kPa) facilitated a significant up-regulation in SOX9 expression indicating that MSCs are directed towards a chondrogenic lineage in more compliant scaffolds. In contrast, the greatest level of RUNX2 expression was found in the stiffest scaffolds (1.5 kPa) indicating that MSCs are directed towards an osteogenic lineage in stiffer scaffolds. Furthermore, results demonstrated that the level of up-regulation of SOX9 was higher within the CHyA scaffolds in comparison to the CCS scaffolds indicating that hyaluronic acid further influences chondrogenic differentiation. In contrast, enhanced RUNX2 expression was observed in the CCS scaffolds in comparison to the CHyA scaffolds suggesting an osteogenic influence of chondroitin sulphate on MSC differentiation. In summary, this study demonstrates that, even in the absence of differentiation supplements, scaffold stiffness can direct the fate of MSCs, an effect that is further enhanced by the GAG type used within the CG scaffolds. These results have significant implications for the therapeutic uses of stem cells and enhance our understanding of the physical effects of the in vivo microenvironment on stem cell behaviour.
Publisher: Elsevier BV
Date: 2021
Publisher: Elsevier BV
Date: 2021
Location: Ireland
Location: Ireland
No related grants have been discovered for Fergal O'Brien.