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Increasing evidence indicates superiority of three-dimensional (3D) in vitro cell culture systems over conventional two-dimensional (2D) monolayer cultures in mimicking native in vivo microenvironments. Tissue-engineered 3D culture models combined with stem cell technologies have advanced Alzheimer's disease (AD) pathogenesis studies. However, existing 3D neuronal models of AD overexpress mutant genes or have heterogeneities in composition, biological properties and cell differentiation stages. Here, we encapsulate patient induced pluripotent stem cell (iPSC) derived neural progenitor cells (NPC) in poly(lactic-co-glycolic acid) (PLGA) microtopographic scaffolds fabricated via wet electrospinning to develop a novel 3D culture model of AD. First, we enhanced cellular infiltration and distribution inside the scaffold by optimizing various process parameters such as fiber diameter, pore size, porosity and hydrophilicity. Next, we compared key neural stem cell features including viability, proliferation and differentiation in 3D culture with 2D monolayer controls. The 3D microfibrous substrate reduces cell proliferation and significantly accelerates neuronal differentiation within seven days of culture. Furthermore, 3D culture spontaneously enhanced pathogenic amyloid-beta 42 (Aβ42) and phospho-tau levels in differentiated neurons carrying familial AD (FAD) mutations, compared with age-matched healthy controls. Overall, our tunable scaffold-based 3D neuronal culture platform serves as a suitable in vitro model that robustly recapitulates and accelerates the pathogenic characteristics of FAD-iPSC derived neurons.

In recent years, there has been a marked increase in tissue engineering research, particularly in the development of electrospun fiber-based substrates as in vitro cell culture platforms. Many scaffolds fabricated via electrospinning have focused on two-dimensional (2D) mats composed of aligned, random or core-shell nanofibers depending on the application and type of extracellular matrix present inside native tissue. In this study, a novel coaxial electrospinning process with a mixture of polylactic-co-glycolic acid (PLGA) and polyethylene oxide (PEO) for sheaths along with polyvinyl alcohol (PVA) for cores was used to produce hollow microfibers. Subsequent removal of PVA and PEO by dissolution resulted in the formation of hollow fibers containing pores inside the shell. The influence of key electrospinning parameters on fiber diameter and pore size was analyzed and optimized via application of design of experiments (DOE). In addition, a regression model was built to show the mathematical relationships and interdependence between process variables and response. Next, the feasibility of this platform for in vitro cell culture was verified by successfully encapsulating and culturing pheochromocytoma 12 (PC12) cells inside the hollow microfibers, where surface perforations could facilitate nutrient and oxygen diffusion and waste removal. After three days of cultivation, it was found that hollow fibers could provide both guidance and support for adherence and proliferation of PC12 cells. This technique based on coaxial cell electrospinning paves the way for development of controllable, tunable and low-cost platforms facilitating guided and constrained culture of cells inside microfibers, thus highlighting its potential as a tool for designing new types of biohybrid materials.

Rapid surface functionalization of nanomaterials using covalent linkage following "green chemistry" remains challenging, and the quest for developing simple protocols is persisting. We report a nanomechanical microfluidic approach for the coupling of allenamide functionalized organic derivatives on the surface of thiol-modified silica nanoparticles using allenamide-thiol chemistry. The coupling principle involves the use of a microfluidic surface acoustic wave device that generates acoustic streaming-based chaotic fluid micromixing that enables mixing of laterally flowing fluids containing active components. This approach was used to demonstrate the direct surface labeling of thiol-modified silica nanoparticles using a selected group of modified fluorescent tags containing allenamide handles and achieved a total labeling efficiency of 83-90%. This green approach enabled a highly efficient surface functionalization under aqueous conditions, with tunable control over the conjugation process via the applied field. The dye-labeled silica particles were characterized using various analytical techniques and found to be biocompatible with potential in live cell bioimaging. It is envisaged that this bioconjugation strategy will find numerous applications in the field of bioimaging and drug delivery. S.S. and C.T.L. acknowledge support from the Biomedical Institute for Global Health Research & Technology (BIGHEART), National University of Singapore.

Electrical responsive scaffolds are important for regulating adhesion, mitigation, and proliferation of electroactive cells in tissue engineering. Conventional approaches to fabricate suitable scaffolds with enhanced electrical conductivity require multiple steps including the surface coating of conductive polymers or the encapsulation of conductive fillers. In this work, a new type of three-dimensional porous carbon fiber sponge-based electrical conductive scaffold with a fiber diameter of 8.1 (±2.1) μm was fabricated by one-step high-temperature carbonization at 800 °C from an abundantly available biomaterial - cotton. The as-carbonized cotton has a suitable pore size from several dozens to hundreds of micrometers, low toxicity, and good biocompatibility. Its hydrophilicity and biocompatibility were further modified by surface oxidation and polydopamine coating. The topological feature, surface functional groups, hydrophilicity, and electrical conductivity of the carbonized cotton were studied. The as-carbonized, oxidized, and polydopamine-coated scaffolds have electrical conductivities of 32.6, 44.9 and 128.2 S/m, respectively. The cytocompatibility and the effect of electrical stimulation on cell behavior were evaluated by cell culture with and without electrical stimulation. It was observed that the polydopamine coating significantly improved the hydrophilicity and cytocompatibility. The electrical stimulation accelerated cell proliferation and differentiation of nerve cells. This work demonstrated that carbonized biomaterial represents a promising material category that can be used for tissue engineering especially in electroactive tissues.

We report multiscale structured fibers and patterned films based on a semiconducting polymer, poly(3-hexylthiophene) (P3HT), as photoconductive biointerfaces to promote neuronal stimulation upon light irradiation. The micro/nanoscale structures of P3HT used for neuronal interfacing and stimulation include nanofibers with an average diameter of 100 nm, microfibers with an average diameter of about 1 μm, and lithographically patterned stripes with width of 3, 25, and 50 μm, respectively. The photoconductive effect of P3HT upon light irradiation provides electrical stimulation for neuronal differentiation and directed growth. Our results demonstrate that neurons on P3HT nanofibers showed a significantly higher total number of branches, while neurons grown on P3HT microfibers had longer and thinner neurites. Such a combination strategy of topographical and photoconductive stimulation can be applied to further enhance neuronal differentiation and directed growth. These photoconductive polymeric micro/nanostructures demonstrated their great potential for neural engineering and development of novel neural regenerative devices. Ministry of Education (MOE) Nanyang Technological University M.W. is grateful to the funding support by a start-up grant of Nanyang Assistant Professorship (M4080992) from Nanyang Technological University and AcRF Tier 2 (ARC 36/13: M4020172) from the Ministry of Education, Singapore.

Electroconductive and injectable hydrogels are attracting increasing attention owing to the needs of electrically induced regulation of cell behavior, tissue engineering of electroactive tissues, and achieving minimum invasiveness during tissue repair. In this study, a novel in situ formed 3D conductive and cell-laden hydrogel is developed, which can be broadly used in bioprinting, tissue engineering, neuroengineering etc. An instantaneous, uniform spatial distribution and encapsulation of cells can be achieved as a result of hydrogen bonding induced hydrogel formation. Particularly, the cell-laden hydrogel can be easily obtained by simply mixing and shaking the polydopamine (PDA) functionalized rGO (rGO-PDA) with polyvinyl alcohol (PVA) solution containing cells. Graphene oxide is reduced and functionalized by dopamine to restore the electrical conductivity, while simultaneously enhancing both hydrophilicity and biocompatibility of reduced graphene oxide. In vitro culture of PC12 cells within the cell-laden hydrogel demonstrates its biocompatibility, noncytotoxicity as well as the ability to support long-term cell growth and proliferation. Enhanced neuronal differentiation is also observed, both with and without electrical stimulation. Overall, this 3D conductive, cell-laden hydrogel holds great promise as potential platform for tissue engineering of electroactive tissues.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by progressive memory loss, impairment of other cognitive functions, and inability to perform activities of daily life. The key to understanding AD aetiology lies in the development of effective disease models, which should ideally recapitulate all aspects pertaining to the disease. A plethora of techniques including in vivo, in vitro, and in silico platforms have been utilized in developing disease models of AD over the years. Each of these approaches has revealed certain essential characteristics of AD; however, none have managed to fully mimic the pathological hallmarks observed in the AD human brain. In this review, we will provide details into the genesis, evolution, and significance of the principal methods currently employed in modelling AD, the advantages and limitations faced in their application, including the headways made by each approach. This review will focus primarily on two-dimensional and three-dimensional in vitro modelling of AD, which during the last few years has made significant breakthroughs in the areas of AD pathology and therapeutic screening. In addition, a glimpse into state-of-the-art neural tissue engineering techniques incorporating biomaterials and microfluidics technologies is provided, which could pave the way for the development of more accurate and comprehensive AD models in the future.

Micro/nanofiber-based scaffolds are widely used as a physical graft for axon growth in vitro for nerve tissue engineering. In this study, the fabrication of hollow coaxial microfibers with a 3D structure for directional neuronal cell culture is reported. The coaxial microfibers with poly-ε-caprolactone (PCL) sheath and polyvinyl alcohol (PVA) mixed with PC 12 cells as core are prepared using a coaxial double cylinder based on the liquid-liquid coflowing electrospinning method. By optimizing the electrospinning conditions, uniform microfibers with an average diameter of 54.6 µm are obtained using a PCL concentration of 20% and a tip-to-collector distance of 6 cm with a voltage of 6 kV. After selectively dissolving the PVA core, pheochromocytoma 12 (PC12) cells are successfully cultured inside these fibers. It is experimentally confirmed that such 3D hollow fibers can support and guide PC12 cells to grow, proliferate, and differentiate inside the hollow fibers and form 3D-connected neuronal networks with axons extended along the hollow fibers. This implies that complex nerve connection could be constructed using the low cost and powerful liquid-liquid coflowing electrospinning method, which open the door to culture well-controlled nerve connections as in vitro models for neural tissue regeneration, brain disease models, and so on.