Basic principles of emulsion templating and its use as an emerging manufacturing method of tissue engineering scaffolds

Tissue engineering (TE) aims to regenerate critical size defects, which cannot heal naturally, by using highly porous matrices called TE scaffolds made of biocompatible and biodegradable materials. There are various manufacturing techniques commonly used to fabricate TE scaffolds. However, in most cases, they do not provide materials with a highly interconnected pore design. Thus, emulsion templating is a promising and convenient route for the fabrication of matrices with up to 99% porosity and high interconnectivity. These matrices have been used for various application areas for decades. Although this polymer structuring technique is older than TE itself, the use of polymerised internal phase emulsions (PolyHIPEs) in TE is relatively new compared to other scaffold manufacturing techniques. It is likely because it requires a multidisciplinary background including materials science, chemistry and TE although producing emulsion templated scaffolds is practically simple. To date, a number of excellent reviews on emulsion templating have been published by the pioneers in this field in order to explain the chemistry behind this technique and potential areas of use of the emulsion templated structures. This particular review focusses on the key points of how emulsion templated scaffolds can be fabricated for different TE applications. Accordingly, we first explain the basics of emulsion templating and characteristics of PolyHIPE scaffolds. Then, we discuss the role of each ingredient in the emulsion and the impact of the compositional changes and process conditions on the characteristics of PolyHIPEs. Afterward, current fabrication methods of biocompatible PolyHIPE scaffolds and polymerisation routes are detailed, and the functionalisation strategies that can be used to improve the biological activity of PolyHIPE scaffolds are discussed. Finally, the applications of PolyHIPEs on soft and hard TE as well as in vitro models and drug delivery in the literature are summarised

Laser direct writing (LDW) of magnetic structures

Laser direct writing (LDW) has been used to pattern 90nm thick permalloy (Ni 81 Fe 19 ) into 1-D and 2-D microstructures with strong shape anisotropy. Sub-nanosecond laser pulses were focused with a 0.75 NA lens to a 1.85μm diameter spot, to achieve a fluence of approximately 350 mJ.cm -2 and ablate the permalloy film. Computer-controlled sample scanning then allowed structures to be defined. Scan speeds were controlled to give 30% overlap between successive laser pulses and reduce the extent of width modulation in the final structures. Continuous magnetic wires that adjoined the rest of the film were fabricated with widths from 650 nm - 6.75μm and magneto-optical measurements showed coercivity reducing across this width range from 47 Oe to 11 Oe. Attempts to fabricate wires narrower than 650nm resulted in discontinuities in the wires and a marked decrease in coercivity. This approach is extremely rapid and was carried out in air, at room temperature and with no chemical processing. The 6-kHz laser pulse repetition rate allowed wire arrays across an area of 4 mm x 0.18 mm to be patterned in 85 s

Boosting the osteogenic and angiogenic performance of multiscale porous polycaprolactone scaffolds by In vitro generated extracellular matrix decoration

Tissue engineering (TE)-based bone grafts are favorable alternatives to autografts and allografts. Both biochemical properties and the architectural features of TE scaffolds are crucial in their design process. Synthetic polymers are attractive biomaterials to be used in the manufacturing of TE scaffolds, due to various advantages, such as being relatively inexpensive, enabling precise reproducibility, possessing tunable mechanical/chemical properties, and ease of processing. However, such scaffolds need modifications to improve their limited interaction with biological tissues. Structurally, multiscale porosity is advantageous over single-scale porosity; therefore, in this study, we have considered two key points in the design of a bone repair material; (i) manufacture of multiscale porous scaffolds made of photocurable polycaprolactone (PCL) by a combination of emulsion templating and three-dimensional (3D) printing and (ii) decoration of these scaffolds with the in vitro generated bone-like extracellular matrix (ECM) to create biohybrid scaffolds that have improved biological performance compared to PCL-only scaffolds. Multiscale porous scaffolds were fabricated, bone cells were cultured on them, and then they were decellularized. The biological performance of these constructs was tested in vitro and in vivo. Mesenchymal progenitors were seeded on PCL-only and biohybrid scaffolds. Cells not only showed improved attachment on biohybrid scaffolds but also exhibited a significantly higher rate of cell growth and osteogenic activity. The chick chorioallantoic membrane (CAM) assay was used to explore the angiogenic potential of the biohybrid scaffolds. The CAM assay indicated that the presence of the in vitro generated ECM on polymeric scaffolds resulted in higher angiogenic potential and a high degree of tissue infiltration. This study demonstrated that multiscale porous biohybrid scaffolds present a promising approach to improve bioactivity, encourage precursors to differentiate into mature bones, and to induce angiogenesis

Hybrid manufacturing strategies for tissue engineering scaffolds using methacrylate functionalised poly(glycerol sebacate)

Poly(glycerol sebacate) is an attractive biomaterial for tissue engineering due to its biocompatibility, elasticity and rapid degradation rate. However, poly(glycerol sebacate) requires harsh processing conditions, involving high temperatures and vacuum for extended periods, to produce an insoluble polymer matrix. These conditions make generating accurate and intricate geometries from poly(glycerol sebacate), such as those required for tissue engineering scaffolds, difficult. Functionalising poly(glycerol sebacate) with methacrylate groups produces a photocurable polymer, poly(glycerol sebacate)-methacrylate, which can be rapidly crosslinked into an insoluble matrix. Capitalising on these improved processing capabilities, here, we present a variety of approaches for fabricating porous tissue engineering scaffolds from poly(glycerol sebacate)-methacrylate using sucrose porogen leaching combined with other manufacturing methods. Mould-based techniques were used to produce porous disk-shaped and tubular scaffolds. Porogen size was shown to influence scaffold porosity and mechanical performance, and the porous poly(glycerol sebacate)-methacrylate scaffolds supported the proliferation of primary fibroblasts in vitro. Additionally, scaffolds with spatially variable mechanical properties were generated by combining variants of poly(glycerol sebacate)-methacrylate with different stiffness. Finally, subtractive and additive manufacturing methods were developed with the capabilities to generate porous poly(glycerol sebacate)-methacrylate scaffolds from digital designs. These hybrid manufacturing strategies offer the ability to produce accurate macroscale poly(glycerol sebacate)-methacrylate scaffolds with tailored microscale porosity and spatially resolved mechanical properties suitable for a broad range of applications across tissue engineering

Comparison of the anabolic effects of reported osteogenic compounds on human mesenchymal progenitor-derived osteoblasts

There is variability in the reported effects of compounds on osteoblasts arising from differences in experimental design and choice of cell type/origin. This makes it difficult to discern a compound’s action outside its original study and compare efficacy between compounds. Here, we investigated five compounds frequently reported as anabolic for osteoblasts (17β-estradiol (oestrogen), icariin, lactoferrin, lithium chloride, and menaquinone-4 (MK-4)) on human mesenchymal progenitors to assess their potential for bone tissue engineering with the aim of identifying a potential alternative to expensive recombinant growth factors such as bone morphogenetic protein 2 (BMP-2). Experiments were performed using the same culture conditions to allow direct comparison. The concentrations of compounds spanned two orders of magnitude to encompass the reported efficacious range and were applied continuously for 22 days. The effects on the proliferation (resazurin reduction and DNA quantification), osteogenic differentiation (alkaline phosphatase (ALP) activity), and mineralised matrix deposition (calcium and collagen quantification) were assessed. Of these compounds, only 10 µM MK-4 stimulated a significant anabolic response with 50% greater calcium deposition. Oestrogen and icariin had no significant effects, with the exception of 1 µM icariin, which increased the metabolic activity on days 8 and 22. 1000 µg/mL of lactoferrin and 10 mM lithium chloride both significantly reduced the mineralised matrix deposition in comparison to the vehicle control, despite the ALP activity being higher in lithium chloride-treated cells at day 15. This demonstrates that MK-4 is the most powerful stimulant of bone formation in hES-MPs of the compounds investigated, highlighting its potential in bone tissue engineering as a method of promoting bone formation, as well as its prospective use as an osteoporosis treatment

Considerations using additive manufacture of emulsion inks to produce respiratory protective filters against viral respiratory tract infections such as the COVID-19 virus

This review paper explores the potential of combining emulsion-based inks with additive manufacturing (AM) to produce filters for respiratory protective equipment (RPE) in the fight against viral and bacterial infections of the respiratory tract. The value of these filters has been highlighted by the current severe acute respiratory syndrome coronavirus-2 crisis where the importance of protective equipment for health care workers cannot be overstated. Three-dimensional (3D) printing of emulsions is an emerging technology built on a well-established field of emulsion templating to produce porous materials such as polymerized high internal phase emulsions (polyHIPEs). PolyHIPE-based porous polymers have tailorable porosity from the submicron to 100 s of μm. Advances in 3D printing technology enables the control of the bulk shape while a micron porosity is controlled independently by the emulsion-based ink. Herein, we present an overview of the current polyHIPE-based filter applications. Then, we discuss the current use of emulsion templating combined with stereolithography and extrusion-based AM technologies. The benefits and limitation of various AM techniques are discussed, as well as considerations for a scalable manufacture of a polyHIPE-based RPE

Fabrication of biodegradable synthetic vascular networks and their use as a model of angiogenesis

One of the greatest challenges currently faced in tissue engineering is the incorporation of vascular networks within tissue-engineered constructs. The aim of this study was to develop a technique for producing a perfusable, three-dimensional cell friendly model of vascular structures that could be used to study the factors affecting angiogenesis and vascular biology in engineered systems in more detail. Initially, biodegradable synthetic pseudo-vascular networks were produced via the combination of robocasting and electrospinning techniques. The internal surfaces of the vascular channels were then recellularized with human dermal microvascular endothelial cells (HDMECs) with and without the presence of human dermal fibroblasts (HDFs) on the outer surface of the scaffold. After 7 days in culture, channels that had been reseeded with HDMECs alone, demonstrated irregular cell coverage. However when using a co-culture of HDMECs inside and HDFs outside the vascular channels, coverage was found to be continuous throughout the internal channel. Using this cell combination, collagen gels loaded with vascular endothelial growth factor were deposited onto the outer surface of the scaffold and cultured for a further 7 days after which endothelial cell (EC) outgrowth from within the channels into the collagen gel was observed showing the engineered vasculature maintains its capacity for angiogenesis. Furthermore the HDMECs appeared to have formed perfusable tubules within the gel. These results show promising steps towards the development of an in vitro platform upon which to study angiogenesis and vascular biology in a tissue-engineering context

Manufacturing of PolyHIPE-based Porous Microparticles for Bone Tissue Engineering

Particle-based systems have great potential as scaffolds for tissue engineering, since they are injectable, avoiding the need for open surgery. When a bone cancer is removed, the void that’s formed requires filling with a biomaterial to encourage bone regrowth. Two main methods of filling these voids include using autograft material or bone ceramics. Injectable cell-scaffolds allow keyhole surgery which would be impossible with current methods of treatment. Here we are investigating a method for production of spherical microporous particles of 100-800 µm. The microporous material is constructed from a HIPE (High Internal Phase Emulsion) via photocuring. The stir-emulsion method produces a wide range of particle sizes and the T-junction fluidic device produces a very narrow range of particle sizes. With both methods it is possible to change the mean particle size and the particle size distribution. The porosity of the particles can be altered independently by the use of temperature during the initial polyHIPE formation. Mesenchymal hES-MPs cells were cultured on particles which had been coated with acrylic acid via plasma deposition. The cells enable the agglomeration of particles into 3D structures with cell growth both into and between particles

Unidirectional Neuronal Cell growth and Differentiation on Aligned Polyhydroxyalkanoate Blend Microfibres with Varying Diameters

Polyhydroxyalkanoates (PHAs) are a family of prokaryotic-derived biodegradable and biocompatible natural polymers known to exhibit neuroregenerative properties. In this work, poly(3-hydroxybutyrate), P(3HB) and poly(3-hydroxyoctanoate), P(3HO), have been combined to form blend fibres for directional guidance of neuronal cell growth and differentiation. A 25:75 P(3HO)/P(3HB) blend (PHA blend) was used for the manufacturing of electrospun fibres as resorbable scaffolds to be used as internal guidance lumen structures in nerve conduits. The biocompatibility of these fibres was studied using neuronal and Schwann cells. Highly aligned and uniform fibres with varying diameters were fabricated by controlling electrospinning parameters. The resulting fibre diameters were 2.4 ± 0.3 µm, 3.7 ± 0.3 µm and 13.5 ± 2.3 µm for small, medium and large diameter fibres respectively. The cell response to these electrospun fibres was investigated with respect to growth and differentiation. Cell migration observed on the electrospun fibres showed topographical guidance in accordance with the direction of the fibres. The correlation between fibre diameter and neuronal growth under two conditions; individually and in co-culture with Schwann cells was evaluated. Results obtained from both assays revealed that all PHA blend fibre groups were able to support growth and guide aligned distribution of neuronal cells and there was a direct correlation between the fibre diameter and neuronal growth and differentiation. This work has led to the development of a family of unique biodegradable and highly biocompatible 3D substrates capable of guiding and facilitating the growth, proliferation and differentiation of neuronal cells as internal structures within nerve conduits

Erratum: Wally, Z.J.; van Grunsven, W.; Claeyssens, F.; Goodall, R.; Reilly, G.C. Porous Titanium for Dental Implant Applications. Metals 2015, 5, 1902–1920.

The authors wish to make the following corrections to the citations in the published paper [1].[...