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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

Cells can sense mechanical signals through cytoskeleton reorganization. We previously discovered the formation of omni-directional actin protrusions upon compression loading, namely compression-induced actin protrusions (CAPs), in human mesenchymal stem cells (MSCs) in 3D micro-tissues. Here, the regulatory roles of three RhoGTPases (CDC42, Rac1 and RhoA) in the formation of CAPs were investigated. Upon compression loading, extensive formation of CAPs was found, significantly associated with an upregulated mRNA expression of Rac1 only, but not CDC42, nor RhoA. Upon chemical inhibition of these RhoGTPase activity during compression, only Rac1 activity was significantly suppressed, associating with the reduced CAP formation. Silencing the upstream regulators of these RhoGTPase pathways including Rac1 by specific siRNA dramatically disrupted actin cytoskeleton, distorted cell morphology and aborted CAP formation. Silencing cortactin (CTTN), a downstream effector of the Rac1 pathway, induced a compensatory upregulation of Rac1, enabling the MSCs to respond to the compression loading stimulus in terms of CAP formation, although at a reduced number. The importance of Rac1 signalling in CAP formation and the corresponding upregulation of lamellipodial markers also suggest that these CAPs are lamellipodia in nature. This study delineates the mechanism of compression-induced cytoskeleton reorganization, contributing to rationalizing mechanical loading regimes for functional tissue engineering.

The clustered regularly interspaced short palindromic repeat (CRISPR) systems have a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or interference (CRISPRi). However, the safety concerns associated with viral vectors and the possible off-target issues of systemic administration remain huge concerns to be safe delivery methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide (SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 systems. Specifically, an amphiphilic negatively charged SAP− is first coated onto PCL nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively charged SAP+-RGD are then absorbed via electrostatic interactions. The SAP-coated scaffolds facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF expression in mammalian cells, and the secreted GDNF subsequently promotes neurite outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide coated nanofibers can be a new route to establish a bioactive interface, which provides a simple and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) 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.

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.

Effective remyelination in the central nervous system (CNS) facilitates the reversal of disability in patients with demyelinating diseases such as multiple sclerosis. Unfortunately until now, effective strategies of controlling oligodendrocyte (OL) differentiation and maturation remain limited. It is well known that topographical and biochemical signals play crucial roles in modulating cell fate commitment. Therefore, in this study, we explored the combined effects of scaffold topography and sustained gene silencing on oligodendroglial precursor cell (OPC) development. Specifically, microRNAs (miRs) were incorporated onto electrospun polycaprolactone (PCL) fiber scaffolds with different fiber diameters and orientations. Regardless of fiber diameter and orientation, efficient knockdown of differentiation inhibitory factors were achieved by either topography alone (up to 70%) or fibers integrated with miR-219 and miR-338 (up to 80%, p < 0.05). Small fiber promoted OPC differentiation by inducing more RIP+ cells (p < 0.05) while large fiber promoted OL maturation by inducing more MBP+ cells (p < 0.05). Random fiber enhanced more RIP+ cells than aligned fibers (p < 0.05), regardless of fiber diameter. Upon miR-219/miR-338 incorporation, 2 μm aligned fibers supported the most MBP+ cells (∼17%). These findings indicated that the coupling of substrate topographic cues with efficient gene silencing by sustained microRNA delivery is a promising way for directing OPC maturation in neural tissue engineering and controlling remyelination in the CNS. NMRC (Natl Medical Research Council, S’pore) Accepted version

While substrate topography influences cell behavior, RNA interference (RNAi) has also emerged as a potent method for understanding and directing cell fate. However, the effects of substrate topography on RNAi remain poorly understood. Here, we report the influence of nanofiber architecture on siRNA-mediated gene-silencing in human somatic and stem cells. The respective model cells, human dermal fibroblasts (HDFs) and mesenchymal stem cells (MSCs), were cultured onto aligned or randomly oriented electrospun poly(e-caprolactone) fibers of different average diameters (300 nm, 700 nm and 1.3 μm). In HDFs, decreasing fiber diameter from 1.3 μm to 300 nm improved Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Collagen-I silencing efficiencies by ∼ 3.8 and ∼4.4 folds respectively (p < 0.05) while the effective siRNA uptake pathway was altered from clathrin-dependent endocytosis to macropinocytosis. In MSCs, aligned fibers generated significantly higher level of gene silencing of RE-1 silencing transcription factor (REST) and green fluorescent protein (GFP) (∼1.6 and ∼1.5 folds respectively, p < 0.05), than randomly-oriented fibers. Aligned fiber topography facilitated functional siRNA uptake through clathrin-mediated endocytosis and membrane fusion. Taken together, our results demonstrated a promising role of three-dimensional fibrous scaffolds in modulating siRNA-mediated gene-silencing and established the critical synergistic role of these substrates in modulating cellular behavior by RNAi.

Herein we combine cell sheet technology and electrospun scaffolding to rapidly generate circumferentially aligned tubular constructs of human aortic smooth muscles cells with contractile gene expression for use as tissue engineered blood vessel media. Smooth muscle cells cultured on micropatterned and N-isopropylacrylamide-grafted (pNIPAm) polydimethylsiloxane (PDMS), a small portion of which was covered by aligned electrospun scaffolding, resulted in a single sheet of unidirectionally aligned cells. Upon cooling to room temperature, the scaffold, its adherent cells, and the remaining cell sheet detached and were collected on a mandrel to generating tubular constructs with circumferentially aligned smooth muscle cells which possess contractile gene expression and a single layer of electrospun scaffold as an analogue to a small diameter blood vessel's internal elastic lamina (IEL). This method improves cell sheet handling, results in rapid circumferential alignment of smooth muscle cells which immediately express contractile genes, and introduction of an analogue to small diameter blood vessel IEL.