Giles T. S. Kirby

Accelerating protein release from microparticles for regenerative medicine applications

There is a need to control the spatio-temporal release kinetics of growth factors in order to mitigate current usage of high doses. A novel delivery system, capable of providing both structural support and controlled release kinetics, has been developed from PLGA microparticles. The inclusion of a hydrophilic PLGA–PEG–PLGA triblock copolymer altered release kinetics such that they were decoupled from polymer degradation. A quasi zero order release profile over four weeks was produced using 10% w/w PLGA–PEG–PLGA with 50:50 PLGA whereas complete and sustained release was achieved over ten days using 30% w/w PLGA–PEG–PLGA with 85:15 PLGA and over four days using 30% w/w PLGA–PEG–PLGA with 50:50 PLGA. These three formulations are promising candidates for delivery of growth factors such as BMP-2, PDGF and VEGF. Release profiles were also modified by mixing microparticles of two different formulations providing another route, not previously reported, for controlling release kinetics. This system provides customisable, localised and controlled delivery with adjustable release profiles, which will improve the efficacy and safety of recombinant growth factor delivery.

PLGA/PEG‐hydrogel composite scaffolds with controllable mechanical properties

Biodegradable polymer scaffolds have great potential for regenerative medicine applications such as the repair of musculoskeletal tissues. Here, we describe the development of scaffolds that blend hydrogel components with thermoplastic materials, combining the unique properties of both components to create mouldable formulations. This study focuses on the structural and mechanical properties of the composite scaffolds, produced by combining temperature-sensitive poly(DL-lactic acid-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) particles with a hydrogel component [Pluronic F127, fibrin or hyaluronic acid (HyA)]. The composite formulations solidified over time at 37°C, with a significant increase (p ≤ 0.05) in compressive strength observed from 15 min to 2 h at this temperature. The maximum compressive strength was 1.2 MPa for PLGA/PEG-Pluronic F127 scaffolds, 2.4 MPa for PLGA/PEG-HyA scaffolds and 0.6 MPa for PLGA/PEG-fibrin scaffolds. Porosity for each of the PLGA/PEG-hydrogel formulations tested was between 50 and 51%. This study illustrates the ability to combine this thermoplastic PLGA/PEG system with hydrogels to fabricate composite scaffolds, and demonstrates that altering the particle to hydrogel ratio produces scaffolds with varying mechanical properties.

Stem Cells for Cutaneous Wound Healing.

Optimum healing of a cutaneous wound involves a well-orchestrated cascade of biological and molecular processes involving cell migration, proliferation, extracellular matrix deposition, and remodelling. When the normal biological process fails for any reason, this healing process can stall resulting in chronic wounds. Wounds are a growing clinical burden on healthcare systems and with an aging population as well as increasing incidences of obesity and diabetes, this problem is set to increase. Cell therapies may be the solution. A range of cell based approaches have begun to cross the rift from bench to bedside and the supporting data suggests that the appropriate administration of stem cells can accelerate wound healing. This review examines the main cell types explored for cutaneous wound healing with a focus on clinical use. The literature overwhelmingly suggests that cell therapies can help to heal cutaneous wounds when used appropriately but we are at risk of clinical use outpacing the evidence. There is a need, now more than ever, for standardised methods of cell characterisation and delivery, as well as randomised clinical trials.

Improved fabrication of melt electrospun tissue engineering scaffolds using direct writing and advanced electric field control

Direct writing melt electrospinning is an additive manufacturing technique capable of the layer-by-layer fabrication of highly ordered 3d tissue engineering scaffolds from micron-diameter fibers. The utility of these scaffolds, however, is limited by the maximum achievable height of controlled fiber deposition, beyond which the structure becomes increasingly disordered. A source of this disorder is charge build-up on the deposited polymer producing unwanted coulombic forces. In this study, the authors introduce a novel melt electrospinning platform with dual voltage power supplies to reduce undesirable charge effects and improve fiber deposition control. The authors produced and characterized several 90° cross-hatched fiber scaffolds using a range of needle/collector plate voltages. Fiber thickness was found to be sensitive only to overall potential and invariant to specific tip/collector voltage. The authors also produced ordered scaffolds up to 200 layers thick (fiber spacing 1 mm and diameter 40 μm) and characterized structure in terms of three distinct zones: ordered, semiordered, and disordered. Our in vitro analysis indicates successful cell attachment and distribution throughout the scaffolds, with little evidence of cell death after seven days. This study demonstrates the importance of electrostatic control for reducing destabilizing polymer charge effects and enabling the fabrication of morphologically suitable scaffolds for tissue engineering.

Scaffold–cell bone engineering in a validated preclinical animal model: precursors vs differentiated cell source

The properties of osteoblasts (OBs) isolated from the axial skeleton (tOBs) differ from OBs of the orofacial skeleton (mOBs) due to the different embryological origins of the bones. The aim of the study was to assess and compare the regenerative potential of allogenic bone marrow‐derived mesenchymal progenitor cells with allogenic tOBs and allogenic mOBs in combination with a mPCL–TCP scaffold in critical‐sized segmental bone defects in sheep tibiae. After 6 months, the tibiae were explanted and underwent biomechanical testing, micro‐computed tomography (microCT) and histological and immunohistochemical analyses. Allogenic MPCs demonstrated a trend towards a better outcome in biomechanical testing and the mean values of newly formed bone. Biomechanical, microCT and histological analysis showed no significant differences in the bone regeneration potential of tOBs and mOBs in our in vitro study, as well as in the bone regeneration potential of different cell types in vivo. Copyright

Microparticles for Sustained Growth Factor Delivery in the Regeneration of Critically-Sized Segmental Tibial Bone Defects

This study trialled the controlled delivery of growth factors within a biodegradable scaffold in a large segmental bone defect model. We hypothesised that co-delivery of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) followed by bone morphogenetic protein-2 (BMP-2) could be more effective in stimulating bone repair than the delivery of BMP-2 alone. Poly(lactic-co-glycolic acid) (PLGA ) based microparticles were used as a delivery system to achieve a controlled release of growth factors within a medical-grade Polycaprolactone (PCL) scaffold. The scaffolds were assessed in a well-established preclinical ovine tibial segmental defect measuring 3 cm. After six months, mechanical properties and bone tissue regeneration were assessed. Mineralised bone bridging of the defect was enhanced in growth factor treated groups. The inclusion of VEGF and PDGF (with BMP-2) had no significant effect on the amount of bone regeneration at the six-month time point in comparison to BMP-2 alone. However, regions treated with VEGF and PDGF showed increased vascularity. This study demonstrates an effective method for the controlled delivery of therapeutic growth factors in vivo, using microparticles.

Improving Electrospun Fibre Stacking with Direct Writing for Developing Scaffolds for Tissue Engineering for Non-load Bearing Bone

Melt electrospinning can be used to produce fibres within the micro to nano scale with a deposition in a manner in-line with conventional 3D printing technology’s [1]. Technical issues such as charge build up in subsequent layers lead to limitations in the precision of fibre deposition as the number of layers increases.

Development of Advanced Dressings for the Delivery of Progenitor Cells

Culture surfaces that substantially reduce the degree of cell manipulation in the delivery of cell sheets to patients are described. These surfaces support the attachment, culture, and delivery of multipotent adult progenitor cells (MAPC). It was essential that the processes of attachment/detachment to the surface did not affect cell phenotype nor the function of the cultured cells. Both acid-based and amine-based surface coatings were generated from acrylic acid, propanoic acid, diaminopropane, and heptylamine precursors, respectively. While both functional groups supported cell attachment/detachment, amine coated surfaces gave optimal performance. X-ray photoelectron spectroscopy (XPS) showed that at a primary amine to carbon surface ratio of between 0.01 and 0.02, greater than 90% of attached cells were effectively transferred to a model wound bed. A dependence on primary amine concentration has not previously been reported. After 48 h of culture on the optimized amine surface, PCR, functional, and viability assays showed that MAPC retained their stem cell phenotype, full metabolic activity, and biological function. Consequently, in a proof of concept experiment, it was shown that this amine surface when coated onto a surgical dressing provides an effective and simple technology for the delivery of MAPC to murine dorsal excisional wounds, with MAPC delivery verified histologically. By optimizing for cell delivery using a combination of in vitro and in vivo techniques, we developed an effective surface for the delivery of MAPC in a clinically relevant format.

Cell sheets in cell therapies

This review aims to provide a broad introduction to the use of cell sheets and the role of materials in the delivery of cell sheets to patients within a clinical setting. Traditionally, cells sheets have been, and currently are, fabricated using established and accepted cell culture methods within standard formats (e.g., petri dishes) utilizing biological substrates. Synthetic surfaces provide a far more versatile system for culturing and delivering cell sheets. This has the potential to positively affect quality, and efficient, localized cell delivery has a significant impact on patient outcome and on the overall cost of goods. We highlight current applications of these advanced carriers and future applications of these surfaces and cell sheets with an emphasis both on clinical use and regulatory requirements.

Polycaprolactone-based Scaffolds Fabricated Using Fused Deposition Modelling or Melt Extrusion Techniques for Bone Tissue Engineering

Bone tissue engineering is one of the most exciting current research areas owing to advances in technologies able to produce three-dimensional scaffolds which can support tissue repair and regeneration. Bone repair is generally spontaneous but in some cases, involving large bone defects or trauma, healing does not occur. Autograft is still the most efficacious repair material but the drawbacks of this are well known; donor site morbidity, lack of material and post-operative complications. Tissue engineering approaches aim to address the shortfalls of existing graft alternatives. To do this they must be both structurally relevant, offering support and allowing tissue ingrowth as well as bioactive in order to compete with autograft efficacy. This chapter aims to outline and critically assess cutting edge approaches to scaffold fabrication for bone tissue engineering utilising specific additive manufacturing techniques with a focus on polycaprolactone constructs. Current methods of fused deposition modelling and melt extrusion will be described followed by the evolution of scaffolds with a focus on production, characterisation and in vivo assessment. Preclinical and clinical applications of these scaffolds will also be discussed with a view to future directions in this emerging field and progression towards the ultimate goal of an off-the-shelf graft alternative.