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Neural tissue engineering holds great promise in repairing damaged nerve tissues. However, despite the promising results in regenerating the injured nervous system, tissue engineering approaches are still insufficient to result in full functional recovery in severe nerve damages. Majority of these approaches only focus on growth factors and cell-extracellular matrix (ECM) interactions. As another important component in nerve tissues, the potential of modulating cell-cell interactions as a strategy to promote regeneration has been overlooked. Within the central nervous system, there are considerably more cell-cell communications as compared to cell-ECM interactions, since the ECM only contributes 10%-20% of the total tissue volume. Therefore, modulating cell-cell interactions through cell adhesion molecules (CAMs) such as cadherins, neural cell adhesion molecules (NCAM) and L1, may be a potential alternative to improve nerve regeneration. This paper will begin by reviewing the CAMs that play important roles in neurogenic processes. Specifically, we focused on 3 areas, namely the roles of CAMs in neurite outgrowth and regeneration; remyelination; and neuronal differentiation. Following that, we will discuss existing tissue engineering approaches that utilize CAMs and biomaterials to control nerve regeneration. We will also suggest other potential methods that can deliver CAMs efficiently to injured nerve tissues. Overall, we propose that utilizing CAMs with biomaterials may be a promising therapeutic strategy for nerve regeneration. Accepted version

The loss of oligodendrocytes (OLs) and subsequently myelin sheaths following injuries or pathologies in the CNS leads to debilitating functional deficits. Unfortunately, effective methods of remyelination remain limited. Here, we present a scaffolding system that enables sustained non-viral delivery of microRNAs (miRs) to direct OL differentiation, maturation, and myelination. We show that miR-219/miR-338 promoted primary rat OL differentiation and myelination in vitro. Using spinal cord injury as a proof-of-concept, we further demonstrate that miR-219/miR-338 could also be delivered non-virally in vivo using an aligned fiber-hydrogel scaffold to enhance remyelination after a hemi-incision injury at C5 level of Sprague-Dawley rats. Specifically, miR-219/miR-338 mimics were incorporated as complexes with the carrier, TransIT-TKO (TKO), together with neurotrophin-3 (NT-3) within hybrid scaffolds that comprised poly(caprolactone-co-ethyl ethylene phosphate) (PCLEEP)-aligned fibers and collagen hydrogel. After 1, 2, and 4 weeks post-treatment, animals that received NT-3 and miR-219/miR-338 treatment preserved a higher number of Olig2+ oligodendroglial lineage cells as compared with those treated with NT-3 and negative scrambled miRs (Neg miRs; p < 0.001). Additionally, miR-219/miR-338 increased the rate and extent of differentiation of OLs. At the host-implant interface, more compact myelin sheaths were observed when animals received miR-219/miR-338. Similarly within the scaffolds, miR-219/miR-338 samples contained significantly more myelin basic protein (MBP) signals (p < 0.01) and higher myelination index (p < 0.05) than Neg miR samples. These findings highlight the potential of this platform to promote remyelination within the CNS. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Accepted version

Three-dimensional (3D) in vitro neural tissue models provide a better recapitulation of in vivo cell-cell and cell-extracellular matrix interactions than conventional two-dimensional (2D) cultures. Therefore, the former is believed to have great potential for both mechanistic and translational studies. In this paper, we review the recent developments in 3D in vitro neural tissue models, with a particular focus on the emerging bioprinted tissue structures. We draw on specific examples to describe the merits and limitations of each model, in terms of different applications. Bioprinting offers a revolutionary approach for constructing repeatable and controllable 3D in vitro neural tissues with diverse cell types, complex microscale features and tissue level responses. Further advances in bioprinting research would likely consolidate existing models and generate complex neural tissue structures bearing higher fidelity, which is ultimately useful for probing disease-specific mechanisms, facilitating development of novel therapeutics and promoting neural regeneration. NRF (Natl Research Foundation, S’pore) Accepted version

At present, the recovery prospect for patients with chronic neurodegenerative diseases or acute trauma in the central nervous system is sub-optimal. The controlled differentiation of neural stem/progenitor cells (NPCs) to functional neurons is a possible treatment strategy. In contrast to the classical approach of biochemicals supplementation for guided stem cell commitment, this study explores the feasibility of directing neuronal differentiation through synergistic integration of three-dimensional nanofibrous topographical cues and scaffold-mediated knockdown of RE-1 silencing transcription factor (REST) in mouse NPCs. Taking advantage of the strong adhesive property and latent reactivity of mussel-inspired polydopamine (PD) coating, electrospun polycaprolactone (PCL) nanofibers were successfully functionalized with REST siRNAs (denoted as siREST PD-fiber). Sustained REST knockdown in NPCs was achieved for up to five days in vitro and the silencing efficiency was significantly higher than that mediated through siRNA adsorption onto non-PD coated sample controls. The silencing of REST, together with nanofiber topographical effect, significantly enhanced NPC neuronal commitment (57.5% Map2(+) cells in siREST PD-fiber vs. 43.5% in siREST PD-film vs. 50% in PD-fiber controls, p 0.05) while reducing astrocytic and oligodendrocytic differentiation (10.7% O4(+) cells vs. ∼30% in siREST PD-film, p 0.01). Taken together, the synergistic effects of scaffold-mediated REST knockdown and topographical cues from PD-modified nanofibers may be a useful strategy for generating functional neurons for therapeutic purposes.

Directing stem cell fate remains a major area of interest and also a hurdle to many, particularly in the field of regenerative medicine. Unfortunately, conventional methods of over-expressing inductive factors through the use of biochemical induction cocktails have led to sub-optimal outcomes. A potential alternative may be to adopt the opposite by selectively silencing genes or pathways that are pivotal to stem cell differentiation. Indeed, over recent years, there have been an increasing number of studies on directing stem cell fate through gene knockdown via RNA interference (RNAi). While the effectiveness of RNAi in controlling stem cell differentiation is evident from the myriad of studies, a chaotically vast collection of gene silencing targets have also been identified. Meanwhile, variations in methods of transfecting stem cells have also affected silencing efficiencies and the subsequent extent of stem cell differentiation. This review serves to unite the pioneers who have ventured into the emerging field of RNAi-enhanced stem cell differentiation by summarizing and evaluating the current approaches adopted in utilizing gene silencing to direct stem cell fate and their corresponding outcomes.

SiRNA delivery has found useful applications particularly as therapeutic agents against genetic diseases. Currently, the delivery of siRNA typically takes the form of nanoparticles. In order to expand the applications of these potent but labile molecules for long-term use required by tissue engineering and regenerative medicine, alternative delivery vehicles are required. This work presents a scaffold-mediated approach to siRNA delivery. By encapsulating siRNA within polycaprolactone (PCL) nanofibers (300-400nm in diameter) controlled release of intact siRNA could be achieved for at least 28days under physiological conditions. The successful transfection of HEK 293 cells with GAPDH siRNA released from fibrous scaffolds at day 5, 15 and 30 demonstrated that the encapsulated molecules remained bioactive throughout the period of sustained release, providing silencing efficiency of 61-81% that was comparable to conventional siRNA transfection. Direct seeding of cells on these biofunctional scaffolds, with and without transfection reagent, demonstrated enhanced cellular uptake and efficient GAPDH gene-silencing. This work demonstrates the potential of nanofibrous scaffold-mediated siRNA delivery for long-term gene-silencing applications. The combination of topographical features provided by nanofibrous scaffolds may provide synergistic contact guidance and biochemical signals to mediate and support cellular development in regenerative medicine.

Artificial nerve guide conduits have the advantage over autografts in terms of their availability and ease of fabrication. However, clinical outcomes associated with the use of artificial nerve conduits are often inferior to that of autografts, particularly over long lesion gaps. There have been significant advances in the designs of artificial nerve conduits over the years. In terms of materials selection and design, a wide variety of new synthetic polymers and biopolymers have been evaluated. The inclusion of nerve conduit lumen fillers has also been demonstrated as essential to enable nerve regeneration across large defect gaps. These lumen filler designs have involved the integration of physical cues for contact guidance and biochemical signals to control cellular function and differentiation. Novel conduit architectural designs using porous and fibrous substrates have also been developed. This review highlights the recent advances in synthetic nerve guide designs for peripheral nerve regeneration, and the in vivo applicability and future prospects of these nerve guide conduits.

Peripheral nerve regeneration can be enhanced by the stimulation of formation of bands of Büngner prior to implantation. Aligned electrospun poly(epsilon-caprolactone) (PCL) fibers were fabricated to test their potential to provide contact guidance to human Schwann cells. After 7 days of culture, cell cytoskeleton and nuclei were observed to align and elongate along the fiber axes, emulating the structure of bands of Büngner. Microarray analysis revealed a general down-regulation in expression of neurotrophin and neurotrophic receptors in aligned cells as compared to cells seeded on two-dimensional PCL film. Real-time-PCR analyses confirmed the up-regulation of early myelination marker, myelin-associated glycoprotein (MAG), and the down-regulation of NCAM-1, a marker of immature Schwann cells. Similar gene expression changes were also observed on cells cultured on randomly oriented PCL electrospun fibers. However, up-regulation of the myelin-specific gene, P0, was observed only on aligned electrospun fibers, suggesting the propensity of aligned fibers in promoting Schwann cell maturation.

This paper reports on mathematical and finite element modeling of the effects of secondary phase (Y2O3) volume and location on the thermal conductivity of aluminum nitride (AlN) substrates. Finite element analysis results were also compared with experimental results. From finite element analysis, thermal conductivity of AlN(Y2O3) substrates with secondary phase located at the grain boundaries was in agreement with Buhr’s model. Maxwell’s equation was found to give a good estimate of the thermal conductivity of AlN(Y2O3) substrates with the secondary phase located at the triple points, with a varying gradient depending on the thermal conductivity of the individual components. With increasing secondary phase volume, thermal conductivity was higher when the secondary phase was located at the triple points as compared with grain boundaries. © 2002 Elsevier Science B.V. All rights reserved.

MicroRNA-based therapeutics have rapidly emerged in recent years as promising platforms for the treatment of genetic disorders and cancer. Perhaps less widely explored is the application of microRNAs to regenerative medicine and tissue engineering. However, microRNAs hold tremendous potential in these fields. This lack of advancement is probably triggered by the conventional mindset adopted in the field, which is to up-regulate factors in favor of regeneration as opposed to an atypical gene silencing approach. Compounded upon this may also be the fact that most studies on microRNAs have focused on the biological aspects of profiling and identifying microRNA involvement and signaling pathways. The translational use of this knowledge to engineering approaches is not directly straightforward in most cases. In this theme issue, we aim to highlight the potential of microRNA gene silencing in tissue engineering and regenerative medicine. The unique requirements of tissue regeneration prompt for special considerations in gene silencing. Most obvious may be the involvement of tissue scaffolds in regenerative medicine vs. typical systemic delivery of microRNAs for genetic disorders and cancer therapies. NMRC (Natl Medical Research Council, S’pore) Accepted version