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Injury to the central nervous system (CNS) provokes an inflammatory reaction and secondary damage that result in further tissue damage and destruction of neurons away from the injury site. Upon injury, expression of connexin 43 (Cx43), a gap junction protein, upregulates and is responsible for the spread and amplification of cell death signals through these gap junctions. In this study, we hypothesise that the downregulation of Cx43 by scaffold-mediated controlled delivery of antisense oligodeoxynucleotide (asODN), would minimise secondary injuries and cell death, and thereby support tissue regeneration after nerve injuries. Specifically, using spinal cord injury (SCI) as a proof-of-principle, we utilised a fibre-hydrogel scaffold for sustained delivery of Cx43asODN, while providing synergistic topographical cues to guide axonal ingrowth. Correspondingly, scaffolds loaded with Cx43asODN, in the presence of NT-3, suppressed Cx43 up-regulation after complete transection SCI in rats. These scaffolds facilitated the sustained release of Cx43asODN for up to 25 days. Importantly, asODN treatment preserved neurons around the injury site, promoted axonal extension, decreased glial scarring, and reduced microglial activation after SCI. Our results suggest that implantation of such scaffold-mediated asODN delivery platform could serve as an effective alternative SCI therapeutic approach. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Skin Research Institute of Singapore (SRIS) Published version The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The experiments in this work were supported by the Ministry of Education Tier 1 (RG38/19, RG37/20) and the National Research Foundation, Singapore, under its Intra-CREATE Thematic Grant (Award number: NRF2019-THE002-0001). We acknowledge the Agency for Science, Technology and Research (A*STAR) under its Industry Alignment Fund – Pre-Positioning Programme (IAF-PP) (Grant number H17/01/a0/0C9 and H1701a0004). We thank the Skin Research Institute of Singapore, Phase 2: SRIS@Novena for providing this work with the floor infrastructure and core equipment. JS Chin was also supported by IGS’s studentship.

SummaryThe transplantation of spinal cord progenitor cells (SCPCs) derived from human induced pluripotent stem cells (iPSCs) has beneficial effects on treating spinal cord injury (SCI). However, the presence of residual undifferentiated iPSCs amongst their differentiated progeny poses a high risk as it can develop teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs, no specific surface markers can identify them for subsequent removal. By profiling the size of SCPCs after a 10-day differentiation process, we found that the large-sized group contains significantly more cells expressing pluripotent markers. In this study, we employed a sized-based, label-free separation using an inertial microfluidic-based device to remove tumor-risk cells. The device can reduce the number of undifferentiated cells from an SCPC population with high throughput (i.e., > 3 million cells per minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining, flow cytometry analysis, and colony culture assay. We demonstrated the capabilities of our technology to reduce the percentage of OCT4-positive cells. Our technology has great potential for the ‘downstream processing’ of cell manufacturing workflow, ensuring better quality and safety of transplanted cells.

Central nervous system (CNS) pathologies, such as stroke and spinal cord injury, remain debilitating issues due to the inhibitory environment in the CNS. Many research works have focused on combinatorial therapeutic approaches, such as biomaterial scaffolding, cell transplantation and biomolecule delivery, in the hope of effectively improving functional recovery in vivo. Unfortunately, to date, there is still no effective treatment to regain mobility post-injury. In search of better therapeutic strategies, injectable hydrogels are becoming a popular treatment option for CNS diseases due to their tuneable mechanical properties and the minimally invasive nature of administration. Moreover, the ability to encapsulate exogenous cells and therapeutic molecules while providing an environment that is permissive to cells and promote cell survival incentivises the use of injectable hydrogels in CNS disease treatment. In this review, we will discuss the advances that have been achieved in the recent decade in injectable hydrogel systems for tissue regeneration after stroke and spinal cord injuries. In particular, we focus on the cellular response and tissue integration related to these hydrogel systems. We hope to provide useful insights on materials choices for future research work in injectable hydrogels for stroke and spinal cord regeneration.

Transverse Laser Modes In article number 2103550 by Changjin Huang, Yu‐Cheng Chen, and co‐workers, the key mechanism governing the frequency distributions of transverse modes in single cell biological lasers are revealed. The cover art illustrates the concept of distinctive laser fingerprints generated by cells, which could be employed to provide cellular information. Morphological information of cells could be interpreted through hyperspectral imaging of laser modes. Applications including cell classification and monitoring of cell adhesion are explored and demonstrated. [Image: see text]

Interlimb coordination is important for the enhancement of walking gait in spinal cord injured patients and many studies have recently attempted to dynamically map these movements for use in assistive devices. Nevertheless, there are many difficulties such as high variation of signal and lack of precise algorithms to extract continuous phases in real time. An improved phase learning to extract forelimb(s) and hindlimb phases from movements in real time is proposed. To quantify the performance of our proposed phase learning method, this phase learning is compared to Hilbert transform, a commonly used analytical method for offline process, with principal component analysis (PCA). The comparison between two methods demonstrated that a percentage of root mean square (RMS) time error between goal phase and output phase from our phase learning method is 7.94% as compared to that of Hilbert transform (7.44%). This phase learning that can extract phase in real time improves the analysis of interlimb coordination in robotic application.

In our previous studies, the initial robotic system could only provide passive rehabilitation for spinally injured rats. To provide a more physiological walking gait, the robotic system is improved by synchronizing the gait between fore-limbs and hindlimbs. An improved phase learning is used to extract phases from movements in real time. An improved phase synchronization algorithm is proposed to control a rat’s hindlimbs in synchronization with its forelimbs. To guarantee a phase synchronization performance of this robotic system, the RMS phase differences between the goal phase and the actual phase output from manipulators are quantified. Experimental results show that this approach has the potential to synchronize forelimbs movement with hindlimbs movement in real time as the RMS phase differences from our system are less than the variability of the walking phase difference from 14 healthy rats.

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

Clinically, rehabilitation is one of the most common treatment options for traumatic injuries. Despite that, recovery remains suboptimal and recent breakthroughs in regenerative approaches may potentially improve clinical outcomes. To date, there have been numerous studies on the utilization of either rehabilitative or regenerative strategies for traumatic injury treatment. However, studies that document the combined effects of rehabilitation and regenerative tissue engineering options remain scarce. Here, in the context of traumatic nerve injury treatment, we use a rat spinal cord injury (SCI) model as a proof of concept to evaluate the synergistic effects of regenerative tissue engineering and rehabilitation. Specifically, we implanted a pro-regenerative hybrid fiber–hydrogel scaffold and subjected SCI rats to intensive rehabilitation. Of note, the rehabilitation session was augmented by a novel customized training device that imparts normal hindlimb gait movements to rats. Morphologically, more regenerated axons were observed when rats received rehabilitation (∼2.5 times and ∼2 times enhancement after 4 and 12 weeks of recovery, respectively, p < 0.05). Besides that, we also observed a higher percentage of anti-inflammatory cells (36.1 ± 12.9% in rehab rats vs. 3.31 ± 1.48% in non-rehab rats, p < 0.05) and perineuronal net formation in rehab rats at Week 4. Physically, rehab animals were also able to exert higher ankle flexion force (∼0.779 N vs. ∼0.495 N at Week 4 and ∼1.36 N vs. ∼0.647 N at Week 12 for rehab vs. non-rehab rats, p < 0.001) and performed better than non-rehab rats in the open field test. Taken together, we conclude that coupling rehabilitation with regenerative scaffold implantation strategies can further promote functional recovery after traumatic nerve injuries. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Accepted version

Scaffolds can promote the healing of burns and chronic skin wounds but to date have suffered from issues with achieving full skin integration. Here, we characterise the wound response by both tissue integration and re-epithelialization to a scaffold using wet electrospinning to fabricate 3D fibrous structures. Two scaffold materials were investigated: poly(ε-caprolactone) (PCL) and PCL + 20% rat tail type 1 collagen (PCL/Coll). We assessed re-epithelisation, inflammatory responses, angiogenesis and the formation of new extracellular matrix (ECM) within the scaffolds in rat acute wounds. The 3D PCL/Coll scaffolds impeded wound re-epithelisation, inducing a thickening of wound-edge epidermis as opposed to a thin tongue of migratory keratinocytes as seen when 3D PCL scaffolds were implanted in the wounds. A significant inflammatory response was observed with 3D PCL/Coll scaffolds but not with 3D PCL scaffolds. Enhanced fibroblast migration and angiogenesis into 3D PCL scaffolds was observed with a significant deposition of new ECM. We observed that this deposition of new ECM within the scaffold was key to enabling re-epithelialization over the scaffold. Such scaffolds provide a biocompatible environment for cell integration to lay down new ECM and encourage re-epithelisation over the implanted scaffold. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University Published version This research is supported by the Agency for Science, Technology and Research (A*STAR) under its Industry Alignment Fund—Pre-Positioning Programme (IAF-PP) grant number H17/01/a0/0C9 as part of the Wound Care Innovation for the Tropics (WCIT) Programme and the Skin Research Institute of Singapore, Phase 2: SRIS@Novena grant number H17/01/a0/004. This work was also supported by A*STAR-NHG-NTU Skin Research Grant (SRG 15005), Ministry of Education Tier 1 (2017-T1-002-013) and Nanyang Technological University’s start-up grant. JS Chin is supported by IGS’s studentship.

Cellular lasers are cutting-edge technologies for biomedical applications. Due to the enhanced interactions between light and cells in microcavities, cellular properties and subtle changes of cells can be significantly reflected by the laser emission characteristics. In particular, transverse laser modes from single-cell lasers which utilize Fabry–Pérot cavities are highly correlated to the spatial biophysical properties of cells. However, the high chaotic and complex variation of laser modes limits their practical applications for cell detections. Deep learning technique has demonstrated its powerful capability in solving complex imaging problems, which is expected to be applied for cell detections based on laser mode imaging. In this study, deep learning technique was applied to analyze laser modes generated from single-cell lasers, in which a correlation between laser modes and physical properties of cells was built. As a proof-of-concept, we demonstrated the predictions of cell sizes using deep learning based on laser mode imaging. In the first part, bioinspired cell models were fabricated to systematically study how cell sizes affect the characteristics of laser modes. By training a convolutional neuron network (CNN) model with laser mode images, predictions of cell model diameters with a sub-wavelength accuracy were achieved. In the second part, deep learning was employed to study laser modes generated from biological cells. By training a CNN model with laser mode images acquired from astrocyte cells, predictions of cell sizes with a sub-wavelength accuracy were also achieved. The results show the great potential of laser mode imaging integrated with deep learning for cell analysis and biophysical studies.

Spinal cord injuries (SCIs) are followed by a complex series of events that contribute to the failure of regeneration. To date, there is no robust treatment that can restore the injury-induced loss of function. Since damaged spinal axons do not spontaneously regenerate in their native inhibitory microenvironment, a combined application of biomaterials and neurotrophic factors that induce nerve regeneration emerges as an attractive treatment for SCIs. In this study, we report the novel use of a three-dimensional (3D) hybrid scaffold to provide contact guidance for regrowth of axons in vivo. The scaffold comprises 3D aligned sparsely distributed poly(ε-caprolactone-co-ethyl ethylene phosphate) nanofibers that are supported and dispersed within a collagen hydrogel. Neurotrophin-3 was incorporated into the scaffold as an additional biochemical signal. To evaluate the efficacy of the scaffold in supporting nerve regeneration after SCIs, the construct was implanted into an incision injury, which was created at level C5 in the rat spinal cord. After 3 months of implantation, scaffolds with NT-3 incorporation showed the highest average neurite length (391.9 ± 12.9 μm, p ≤ 0.001) as compared to all the other experimental groups. In addition, these regenerated axons formed along the direction of the aligned nanofibers, regardless of their orientation. Moreover, the presence of the hybrid scaffolds did not affect tissue scarring and inflammatory reaction. Taken together, these findings demonstrate that our scaffold design can serve as a potential platform to support axonal regeneration following SCIs. NMRC (Natl Medical Research Council, S’pore) Accepted version

Targeted drug delivery to specific neural cells within the central nervous system (CNS) plays important roles in treating neurological disorders, such as neurodegenerative (e.g. targeting neurons) and demyelinating diseases (e.g. targeting oligodendrocytes (OLs)). However, the presence of many other cell types within the CNS, such as microglial and astrocytes, may lead to non-specific uptake and subsequent side effects. As such, exploring an effective and targeted drug delivery system is of great necessity. Synthetic micro-/nano-particles that have been coated with biologically derived cellular membranes have emerged as a new class of drug delivery vehicles. However, the use of neural cell-derived membrane coatings remains unexplored. Here, we utilized this technique, and demonstrated the efficacy of targeted delivery by using four types of cell membranes that were derived from the CNS, namely microglial, astrocytes, oligodendrocyte progenitor cells (OPCs) and cortical neurons. Successful cell membrane coating over poly(-caprolactone) nanoparticles (NPs) was confirmed by dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM). Subsequently, an extensive screening of these cell membrane coated NPs was carried out on various CNS cells. Results suggested that microglial and OLs were the most sensitive cell types towards cell membrane coated NPs. Specifically, cell membrane coated NPs significantly enhanced the uptake efficiency of OLs (p < 0.001). Additionally, a temporal uptake study indicated that the OLs took up microglial membrane coated NPs (DPP-PCL-M Mem) most efficiently. Besides that, coating the NPs with four types of CNS cell membrane did not result in obvious specific uptake in microglial but reduced the activation of microglial, especially for DPP-PCL-M Mem (p < 0.01). Taken together, DPP-PCL-M Mem were uptaken most efficiently in OLs and did not induce significant microglial activation and may be most suitable for CNS drug delivery applications. Ministry of Education (MOE) National Research Foundation (NRF) Accepted version This work is supported by the National Research Foundation, Singapore, under its Intra-CREATE Thematic Grant Programme (NRF2019-THE002-0001) and the MOE Tier 1 grants (RG38/19 and RG37/20).

Current treatment approaches toward spinal cord injuries (SCI) have mainly focused on overcoming the inhibitory microenvironment that surrounds lesion sites. Unfortunately, the mere modulation of the cell/tissue microenvironment is often insufficient to achieve desired functional recovery. Therefore, stimulating the intrinsic growth ability of injured neurons becomes crucial. MicroRNAs (miRs) play significant roles during axon regeneration by regulating local protein synthesis at growth cones. However, one challenge of using miRs to treat SCI is the lack of efficient delivery approaches. Here, a 3D fiber‐hydrogel scaffold is introduced which can be directly implanted into a spinal cord transected rat. This 3D scaffold consists of aligned electrospun fibers which provide topographical cues to direct axon regeneration, and collagen matrix which enables a sustained delivery of miRs. Correspondingly, treatment with Axon miRs (i.e., a cocktail of miR‐132/miR‐222/miR‐431) significantly enhances axon regeneration. Moreover, administration of Axon miRs along with anti‐inflammatory drug, methylprednisolone, synergistically enhances functional recovery. Additionally, this combined treatment also decreases the expression of pro‐inflammatory genes and enhance gene expressions related to extracellular matrix deposition. Finally, increased Axon miRs dosage with methylprednisolone, significantly promotes functional recovery and remyelination. Altogether, scaffold‐mediated Axon miR treatment with methylprednisolone is a promising therapeutic approach for SCI., A novel approach of targeting axon local protein synthesis to promote the intrinsic growth ability of neurons by scaffold‐mediated microRNA delivery is introduced. The biofunctional 3D fiber‐hydrogel scaffold provides topographical cues that direct axon regrowth and a novel microRNA cocktail (miR‐132/miR‐222/miR‐431), which in the presence of methylprednisolone, enhances both nerve regeneration and functional recovery after spinal cord injury.

The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are delivered in the format of plasmids, mRNA, or ribonucleoprotein (RNP) complexes. In particular, non-viral approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local delivery and provide possible synergistic effects with other factors such as topographical cues that are co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be used to validate the efficiency of gene disruption.

The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are delivered in the format of plasmids, mRNA, or ribonucleoprotein (RNP) complexes. In particular, non-viral approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local delivery and provide possible synergistic effects with other factors such as topographical cues that are co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be used to validate the efficiency of gene disruption.

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.

Codelivery of CRISPR-Cas9 and chlorin e6 achieved spatially controlled tumor-specific gene editing with synergistic drug effects., Controlled release of CRISPR-Cas9 ribonucleoprotein (RNP) and codelivery with other drugs remain a challenge. We demonstrate controlled release of CRISPR-Cas9 RNP and codelivery with antitumor photosensitizer chlorin e6 (Ce6) using near-infrared (NIR)– and reducing agent–responsive nanoparticles in a mouse tumor model. Nitrilotriacetic acid–decorated micelles can bind His-tagged Cas9 RNP. Lysosomal escape of nanoparticles was triggered by NIR-induced reactive oxygen species (ROS) generation by Ce6 in tumor cells. Cytoplasmic release of Cas9/single-guide RNA (sgRNA) was achieved by reduction of disulfide bond. Cas9/sgRNA targeted the antioxidant regulator Nrf2, enhancing tumor cell sensitivity to ROS. Without NIR irradiation, Cas9 was degraded in lysosomes and gene editing failed in normal tissues. The synergistic effects of Ce6 photodynamic therapy and Nrf2 gene editing were confirmed in vivo. Controlled release of CRISPR-Cas9 RNP and codelivery with Ce6 using stimuli-responsive nanoparticles represent a versatile strategy for gene editing with potentially synergistic drug effects.

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

Vascularization of tissue-engineered constructs is critical for proper cell and graft survival. In order to achieve this, pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), are often incorporated into scaffolds by methods that either involve multiple steps or risk compromising protein bioactivity. In this study, we demonstrate a simple approach to incorporate VEGF into polysaccharide electrospun fibers by taking advantage of the interactions between VEGF and sulfated polysaccharide, fucoidan. Pullulan/dextran (P/D) electrospun fibers (diameter ∼500 nm) incorporating fucoidan were fabricated by a one-step electrospinning process. Thereafter, VEGF was loaded onto the scaffolds. By varying the content of the chemical crosslinker, trisodium trimetaphosphate (STMP), from 10 to 12 and 16 wt% (denoted as STMP10, 12 and 16 respectively), the extent of fucoidan incorporation was significantly enhanced (

Assessment of intracellular mRNA expression is invaluable for understanding cellular signaling activities, identifying disease stages, and monitoring the gene expression pattern of therapeutic cells during their culture, expansion and/or differentiation process. Previous methods suffer from the need to disrupt the biological samples to perform polymerase chain reaction analysis which can be laborious, fragmented and destructive. Herein, we develop a mRNA nanosensor based on the sustained release of mRNA-specific molecular beacons (probes that fluoresce upon hybridization) from the biodegradable poly(d,l-lactide-co-glycolide) nanoparticles. Post cellular internalization, the particles gradually degrade and release the encapsulated probes which are initially weakly fluorescent. When the released probes meet and hybridize with target mRNA, they restore pre-quenched fluorescence. By virtue of quantifying the fluorescence intensity, we can estimate the cellular mRNA expression. As a case study, β-actin mRNA expression in mesenchymal stem cells cultured on a 3D matrix was monitored and compared with those cultured on a 2D plate for one week. Critically, the observed expression profile shows a great correlation with the established quantitative polymerase chain reaction analysis.

A proper protein orientation is often required in order to achieve specific protein-receptor interaction to elicit a desired biological response. Here, we present a Protein A-based biomimicking platform that is capable of efficiently orienting proteins for evaluating cellular behaviour. By absorbing Protein A onto aligned bio-mimicking polycaprolactone (PCL) fibers, we demonstrate that protein binding could be retained on these fibers for at least 7 days under physiologically relevant conditions. We further show that Protein A served as a molecular orientor to arrange the recombinant proteins in similar orientations. Such protein-orienting scaffolds were further verified to be biologically functional by using sensitive primary rat cortical neurons (CNs) and oligodendrocyte progenitor cells (OPCs), as model neural cells for a stringent proof of concept. Specifically, CNs that were seeded on fibers coated with Protein A and a known enhancer of neurite growth (L1 Cell Adhesion Molecular L1CAM) displayed the longest total neurite length (462.77 ± 100.79 μm, p < 0.001) as compared to the controls. Besides that, OPCs seeded on fibers coated with Protein A and Neuregulin-1 Type III (Nrg1 type III) (myelin enhancer) produced the longest myelin ensheathment length (19.8 ± 11.69 μm). These results demonstrate the efficacy of this platform for protein screening applications. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University National Medical Research Council (NMRC) National Research Foundation (NRF) Submitted/Accepted version This work is partially supported by the A*Star BMRC International Joint Grant - Singapore-China Joint Research Program (1610500024); the SingHealth-NTU-Research Collaborative Grant (SHS-NTU/038/ 206); the Singapore National Research Foundation under its NMRCCBRG grant (NMRC/CBRG/0096/2015); and the Ministry of Education (Singapore) Tier 1 grant, (RG38/19).

Synchronous forelimb-hindlimb gait pattern is important to facilitate natural walking behavior of an injured rat with total transection. Since our ultimate research goal is to build a rehabilitation robotic system to simulate the natural walking pattern for spinalized rats, this research aims to address an immediate goal of automating the inference of the rat’s hindlimb trajectory from its own forelimb movement. Our proposed method uses unsupervised learning to extract independent forelimb and hinblimb phases. From the phase information, a relationship between forelimb and hindlimb trajectory can then be calculated. Results show that the proposed method has the potential to be used in a rehabilitation robotic system.

Drivers of RNA interference are potent for manipulating gene and protein levels, which enable the restoration of dysregulated mRNA expression that is commonly associated with injuries and diseases. This review summarizes the potential of targeting neuroglial cells, using RNA interference, to treat nerve injuries sustained in the central nervous system. In addition, the various methods of delivering these RNA interference effectors will be discussed. Ministry of Education (MOE) Ministry of Health (MOH) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: SY Chew and J Lin would like to acknowledge partial funding support from the Singapore National Research Foundation under its National Medical Research Council Cooperative Basic Research Grant (NMRC-CBRG, NMRC/CBRG/0096/2015) and administered by the Singapore Ministry of Health’s National Medical Research Council; Ministry of Education Tier 1 grant (RG38/19). This research was supported in part by the National Research Foundation (NRF), Republic of Korea (2015K1A1A2032163, 2018K1A4A3A01064257, 2018R1A2B3003446).

Injuries within the peripheral nervous system (PNS) lead to sensory and motor deficits, as well as neuropathic pain, which strongly impair the life quality of patients. Although most current PNS injury treatment approaches focus on using growth factors/small molecules to stimulate the regrowth of the injured nerves, these methods neglect another important factor that strongly hinders axon regeneration - the presence of axonal inhibitory molecules. Therefore, this work sought to explore the potential of pathway inhibition in promoting sciatic nerve regeneration. Additionally, the therapeutic window for using pathway inhibitors was uncovered so as to achieve the desired regeneration outcomes. Specifically, we explored the role of Wnt signaling inhibition on PNS regeneration by delivering Wnt inhibitors, sFRP2 and WIF1, after sciatic nerve transection and sciatic nerve crush injuries. Our results demonstrate that WIF1 promoted nerve regeneration (p < 0.05) after sciatic nerve crush injury. More importantly, we revealed the therapeutic window for the treatment of Wnt inhibitors, which is one week post sciatic nerve crush when the non-canonical receptor tyrosine kinase (Ryk) is significantly upregulated. Ministry of Education (MOE) Nanyang Technological University Published version Financial support was received from SingHealth-NTU research collaborative grant (SHS-NTU/038/2016) and MOE AcRF Tier 1 grant (RG37/20).

Spinal cord injury (SCI) leads to the impairment of impulse conduction and subsequently to an abnormality of limbs function. To regain locomotor performance in SCI cases, we establish a robust combinatorial regenerative and rehabilitative approach to enhance axonal regeneration in the Sprague-Dawley rat with complete thoracic SCI. This system consists of a body weight support system, five-bar linkage for driving the rat's ankles, and treadmill for training motor functions. This system is tested in a rat which is totally transected at T9 and T10 of the spinal cord. A nanofiber scaffold is implanted in a gap between T9 and T10 of the spinal cord in a spinalized rat for stimulating axonal regrowth. The position errors are quantified under five static load conditions (no load, 10, 30, 60, and 100 g) and dynamic load condition. Average root mean square (RMS) position errors in x - and y- axes of the manipulator are 2.1% and 5.3%, respectively. According to a preliminary test, this system can provide the constant force to support the body weight and can drive the rat's hindlimbs without inducing anxiety or irritation. From our experiment, average RMS position errors in x - and y- axes of the manipulator are 10% and 11.7%, respectively. The contribution of this research is the developmental rehabilitation robotic system for a rat with complete thoracic SCI in quadruped posture which can provide more natural walking posture. The scope of this letter is a developmental rehabilitation robotic system.

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

Biological lasers which utilize Fabry–Pérot (FP) cavities have attracted tremendous interest due to their potential in amplifying subtle biological changes. Transverse laser modes generated from cells serve as distinct fingerprints of individual cells; however, most lasing signals lack the ability to provide key information about the cell due to high complexity of transverse modes. The missing key, therefore, hinders it from practical applications in biomedicine. This study reveals the key mechanism governing the frequency distributions of transverse modes in cellular lasers. Spatial information of cells including curvature can be interpreted through spectral information of transverse modes by means of hyperspectral imaging. Theoretical studies are conducted to explore the correlation between the cross‐sectional morphology of a cell and lasing frequencies of transverse modes. Experimentally, the spectral characteristics of transverse modes are investigated in live and fixed cells with different morphological features. By extracting laser modes in frequency domain, the proposed concept is applied for studying cell adhesion process and cell classification from rat cortices. This study expands a new analytical dimension of cell lasers, opening an avenue for subcellular analysis in biophotonic applications., This study reveals the key mechanism governing the frequency distributions of transverse modes in single cell biological lasers. Morphological information of cells can be interpreted through hyperspectral imaging of laser modes. Applications including cell classification and monitoring of cell adhesion are explored and demonstrated.

Single cellular lasers have recently attracted tremendous research due to their outstanding lasing characteristics for cell sensing and tracking. Thanks to enhanced light−cell interactions in Fabry–Pérot microcavities, transverse laser modes from cellular lasers are highly correlated to the spatial biophysical properties of cells. However, the huge complexity and randomness of laser modes set a critical challenge towards practical applications in cell analysis. In this study, deep learning is applied to unravel the complex laser modes generated from single-cell lasers by establishing the correlation between laser modes and cellular physical properties. Primary cells extracted from rat brains and cell-like droplets are investigated and trained through a convolutional neuron network based on laser mode images. Detailed simulations and experiments are conducted to study the effect of cell size on laser modes. Predictions of cell diameters with a sub-micron accuracy are achieved with deep learning. Finally, the potential application of using deep-learning-enhanced laser modes for cell classification is demonstrated. Neuron and glial cells extracted from rat brains are classified through hyperspectral images of laser modes. The results demonstrate that deep learning has the potential to enable laser modes with biological significance and functions, offering new possibilities for biophotonic applications. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) This research was supported by A*STAR under its AME YIRG Grant (Project No. A2084c0063). Funding support by the Ministry of Education Academic Research Tier 1 grant (RG38/19) was acknowledged by S.Y.C. and N.Z.

Glial cells are mechanosensitive, and thus, engineered systems have taken a step forward to design mechanotransduction platforms in order to impart diverse mechanical stresses to cells. Mechanical strain encountered in the central nervous system can arise from diverse mechanisms, such as tissue reorganization, fluid flow, and axon growth, as well as pathological events including axon swelling or mechanical trauma. Biomechanical relevance of the in vitro mechanical testing requires to be placed in line with the physiological and mechanical changes in central nervous tissues that occur during the progression of neurodegenerative diseases. Mechanotransduction signaling utilized by glial cells and the recent approaches intended to model altered microenvironment adapted to pathological context are discussed in this review. New insights in systems merging substrate's stiffness and topography should be considered for further glial mechanotransduction studies, while testing platforms for drug discoveries promise great advancements in pharmacotherapy. Potential leads and strategies for clinical outcomes are expected to be developed following the exploration of these glial mechanosensitive signaling pathways.

Spinal cord injuries (SCI) often lead to persistent neurological dysfunction due to failure in axon regeneration. Unfortunately, currently established treatments, such as direct drug administration, do not effectively treat SCI due to rapid drug clearance from our bodies. Here, we introduce a three-dimensional aligned nanofibers-hydrogel scaffold as a bio-functionalized platform to provide sustained non-viral delivery of proteins and nucleic acid therapeutics (small non-coding RNAs), along with synergistic contact guidance for nerve injury treatment. A hemi-incision model at cervical level 5 in the rat spinal cord was chosen to evaluate the efficacy of this scaffold design. Specifically, aligned axon regeneration was observed as early as one week post-injury. In addition, no excessive inflammatory response and scar tissue formation was triggered. Taken together, our results demonstrate the potential of our scaffold for neural tissue engineering applications.

Biomedical implant failure due to the host's response remains a challenging problem. In particular, the formation of the fibrous capsule is a common barrier for the normal function of implants. Currently, there is mounting evidence indicating that the polarization state of macrophages plays an important role in effecting the foreign body reaction (FBR). This opens up a potential avenue for improving host‐implant integration. Here, electrospun poly(caprolactone‐co ‐ethyl ethylene phosphate) nanofiber scaffolds are utilized to deliver microRNAs (miRs) to induce macrophage polarization and modulate FBR. Specifically, C57BL/6 mice that are treated with M2‐inducing miRs, Let‐7c and miR‐124, display relatively thinner fibrous capsule formation around the scaffolds at both Week 2 and 4, as compared to treatment with M1‐inducing miR, Anti‐Let‐7c. Histological analysis shows that the density of blood vessels in the scaffolds are the highest in miR‐124 treatment group, followed by Anti‐Let‐7c and Let‐7c treatment groups. Based on immunohistochemical quantifications, these miR‐encapsulated nanofiber scaffolds are useful for localized and sustained delivery of functional miRs and are able to modulate macrophage polarization during the first 2 weeks of implantation to result in significant alteration in host‐implant integration at longer time points. 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

To date, the aim of spinal cord injury (SCI) researches in animals is to find the most effective treatment method which can lead to faster recovery. In order to evaluate if the method is effective, robust functional assessments are crucial. From the past to present, indicators to observe the recovery of the motor function in rodent SCI models are using human observance or the Basso, Beattie, and Bresnahan score (BBB score), force detection, and imaging approaches. Nevertheless, these indicators do not meet some requirements for a severe full transection injury case. The goal of this project is to develop a novel force sensing system for measuring the ground reaction force of rats with severe SCI. In total, this system was tested with 12 spinalized rats. Following a full transection at the T9-T10 level of the spinal cord in rats with a 2mm gap, a nanofiber scaffold containing Neurotrophin-3 (NT-3), as previously described, was implanted [1]. After 12 weeks of rehabilitative training, results showed that rats that underwent rehabilitation were able to gradually exert more force as compared to rats that did not undergo rehabilitation. At Week 6, the ground reaction force recorded in rats with rehabilitation was 0.8 ± 0.1 N in left limb and 0.75 ± 0.14 N in right limb. On the other hand, rats without rehabilitation exerted 0.52 ± 0.06 N in left limb and 0.47 ± 0.09 N in right limb. At Week 12, the force recorded in rehabilitated rats increased to 1.43 ± 0.13 N in left limb and 1.28 ± 0.17 N in right limb whereas in rats without rehabilitation, the force recorded was only 0.74 ± 0.12 N in left limb and 0.54 ± 0.11 N in right limb. These results not only showed that rehabilitation enhanced recovery of motor function, but also demonstrated the viability of measuring the ground reaction force applied by the rats as an assessment for a full spinal cord transection injury model.

Genome editing, especially via the simple and versatile type II CRISPR/Cas9 system, offers an effective avenue to precisely control cell fate, an important aspect of tissue regeneration. Unfortunately, most CRISPR/Cas9 non-viral delivery strategies only utilise micro-/nano-particle delivery methods. While these approaches provide reasonable genomic editing efficiencies, their systemic delivery may lead to undesirable off-target effects. For in vivo applications, a more localized and sustained delivery approach may be useful, particularly in the context of tissue regeneration. Here, we developed a scaffold that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and non-viral manner. Specifically, using mussel-inspired bioadhesive coating, polyDOPA-melanin (pDOPA), we adsorbed Cas9:sgRNA lipofectamine complexes onto bio-mimicking fiber scaffolds. To evaluate the genome-editing efficiency of this platform, U2OS.EGFP cells were used as the model cell type. pDOPA coating was essential in allowing Cas9:sgRNA lipofectamine complexes to adhere onto the scaffolds with a higher loading efficiency, while laminin coating was necessary for maintaining cell viability and proliferation on the pDOPA-coated fibers for effective gene editing (21.5% editing efficiency, p 0.001). Importantly, U2OS.EGFP cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds via reverse transfection. Overall, we demonstrate the efficacy of such fiber scaffolds in providing localized, sustained and non-viral delivery of Cas9:sgRNA complexes. Such genome editing scaffolds may find useful applications in tissue regeneration. STATEMENT OF SIGNIFICANCE: Currently, there is a lack of effective non-viral means to deliver CRISPR/Cas9 components for genome editing. Most existing approaches only utilize micro-/nano-particles by injection or systemic delivery, which may lead to undesirable off-target effects. Here, we report a platform that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and sustained manner. We used mussel-inspired bioadhesive coating to functionalize the bio-mimicking fiber scaffolds with Cas9:sgRNA lipofectamine complexes, to allow effective gene editing for the cells seeded on the scaffolds. Importantly, the cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds. Such genome editing scaffolds may find useful applications in tissue regeneration.

MicroRNAs effectively modulate protein expression and cellular response. Unfortunately, the lack of robust nonviral delivery platforms has limited the therapeutic application of microRNAs. Additionally, there is a shortage of drug‐screening platforms that are directly translatable from in vitro to in vivo. Here, a fiber substrate that provides nonviral delivery of microRNAs for in vitro and in vivo microRNA screening is introduced. As a proof of concept, difficult‐to‐transfect primary neurons are targeted and the efficacy of this system is evaluated in a rat spinal cord injury model. With this platform, enhanced gene‐silencing is achieved in neurons as compared to conventional bolus delivery (p < 0.05). Thereafter, four well‐recognized microRNAs (miR‐21, miR‐222, miR‐132, and miR‐431) and their cocktails are screened systematically. Regardless of age and origin of the neurons, similar trends are observed. Next, this fiber substrate is translated into a 3D system for direct in vivo microRNA screening. Robust nerve ingrowth is observed as early as two weeks after scaffold implantation. Nerve regeneration in response to the microRNA cocktails is similar to in vitro experiments. Altogether, the potential of the fiber platform is demonstrated in providing effective microRNA screening and direct translation into in vivo applications. MOH (Min. of Health, S’pore) NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Published version

Annulus fibrosus (AF) impairment is associated with reherniation, discogenic pain, and disc degeneration after surgical partial discectomy. Due to a limited intrinsic healing capacity, defects in the AF persist over time and it is hence necessary to adopt an appropriate strategy to close and repair the damaged AF. In this study, a cell-free biodegradable scaffold made of polycaprolactone (PCL), electrospun, aligned microfibers exhibited high levels of cell colonization, alignment, and AF-like extracellular matrix deposition when evaluated in an explant culture model. The biomimetic multilayer fibrous scaffold was then assessed in an ovine model of AF impairment. After 4 weeks, no dislocation of the implants was detected, and only one sample out of six showed a partial delamination. Histological and immunohistochemical analyses revealed integration of the implant with the surrounding tissue as well as homogeneously aligned collagen fiber organization within each lamella compared to the disorganized and scarcer fibrous tissue in a randomly organized control fibrous scaffold. In conclusion, this biomimetic electrospun implant exhibited promising properties in terms of AF defect closure, with AF-like neotissue formation that fully integrated with the surrounding ovine tissue. Accepted version

Acute wound healing is an orderly process of four overlapping events: haemostasis, inflammation, proliferation and remodelling. A drug delivery system with a temporal control of release could promote each of these events sequentially. However, acute wound healing normally proceeds very well in healthy individuals and there is little need to promote it. In the elderly and diabetics however, healing is often slow and wounds can become chronic and we need to promote their healing. Targeting the events of acute wound healing would not be appropriate for a chronic wound, which have stalled in the proinflammatory phase. They also have many additional problems such as poor circulation, low oxygen, high levels of leukocytes, high reactive oxygen species, high levels of proteolytic enzymes, high levels of proinflammatory cytokines, bacterial infection and high pH. The future challenge will be to tackle each of these negative factors to create a wound environment conducive to healing. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University Supported by a SUG from NTU. MOE T1 grant T1-002-098, T1-002- 013. BMRC IAFPP Wound Care Innovation in the Tropics. A*Star-SHGNTU Skin Research Grant 15005. JSC holds an NTU IGS studentship.

Neural tissue regeneration following traumatic injuries is often subpar. As a result, the field of neural tissue engineering has evolved to find therapeutic interventions and has seen promising outcomes. However, robust nerve and myelin regeneration remain elusive. One possible reason may be the fact that tissue regeneration often follows a complex sequence of events in a temporally-controlled manner. Although several other fields of tissue engineering have begun to recognise the importance of delivering two or more biomolecules sequentially for more complete tissue regeneration, such serial delivery of biomolecules in neural tissue engineering remains limited. This review aims to highlight the need for sequential delivery to enhance nerve regeneration and remyelination after traumatic injuries in the central nervous system, using spinal cord injuries as an example. In addition, possible methods to attain temporally-controlled drug/gene delivery are also discussed for effective neural tissue regeneration. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Accepted version

Highly fluorescent polymeric nanofibers fabricated via electrospinning of PCL-DPP-PCL (photostable polycaprolactones–di(thiophene-2-yl)-diketopyrrolopyrrole–photostable polycaprolactones) and commercial PCL mixture show superior photostability and cytocompatibility for long-term tracking of cell–substrate interaction. As a proof of concept, these PCL-DPP-PCL nanofibers enable clear visualization of intricate cell–substrate interactions such as oligodendrocyte myelination. MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Accepted version

Reductionist approaches based on 3D fibers reveal that single-cell migration along fibers is driven by lateral actin-based waves for various cell types., In vivo, cells migrate on complex three-dimensional (3D) fibrous matrices, which has made investigation of the key molecular and physical mechanisms that drive cell migration difficult. Using reductionist approaches based on 3D electrospun fibers, we report for various cell types that single-cell migration along fibronectin-coated nanofibers is associated with lateral actin-based waves. These cyclical waves have a fin-like shape and propagate up to several hundred micrometers from the cell body, extending the leading edge and promoting highly persistent directional movement. Cells generate these waves through balanced activation of the Rac1/N-WASP/Arp2/3 and Rho/formins pathways. The waves originate from one major adhesion site at leading end of the cell body, which is linked through actomyosin contractility to another site at the back of the cell, allowing force generation, matrix deformation and cell translocation. By combining experimental and modeling data, we demonstrate that cell migration in a fibrous environment requires the formation and propagation of dynamic, actin based fin-like protrusions.

A key hallmark of many diseases, especially those in the central nervous system (CNS), is the change in tissue stiffness due to inflammation and scarring. However, how such changes in microenvironment affect the regenerative process remains poorly understood. Here, we report a biomimicking fiber platform that provides independent variation of fiber structural and intrinsic stiffness. To demonstrate the functionality of these constructs as a mechanotransduction study platform, we utilized these substrates as artificial axons and independently analysed the effects of axon structural vs. intrinsic stiffness on CNS myelination. While studies have shown that substrate stiffness affects oligodendrocyte differentiation, the effects of mechanical stiffness on the final functional state of oligodendrocyte (i.e. myelination) has not been shown prior to this. Here, we demonstrate that a stiff mechanical microenvironment impedes oligodendrocyte myelination, independently and distinctively from oligodendrocyte differentiation. We identified YAP to be involved in influencing oligodendrocyte myelination through mechanotransduction. The opposing effects on oligodendrocyte differentiation and myelination provide important implications for current work screening for promyelinating drugs, since these efforts have focused mainly on promoting oligodendrocyte differentiation. Thus, our novel platform may have considerable utility as part of a drug discovery programme in identifying molecules that promote both differentiation and myelination. Ministry of Education (MOE) Ministry of Health (MOH) National Medical Research Council (NMRC) National Research Foundation (NRF) Accepted version This work was supported partially by the Singapore National Research Foundation under its NMRC-CBRG grant (NMRC/CBRG/0096/2015), administered by the Singapore Ministry of Health’s National Medical Research Council, and also by the MOE Tier 1 grant (RG38/19). The NTU Research Scholarship supporting W. Ong and J. Lin is also acknowledged.

Spinal cord injury (SCI) is a traumatic event which leads to the loss of sensory and motor functions of the body. Complete recovery of these functions are usually limited due to the inability of the damaged axons within the central nervous system (CNS) to regenerate autonomously. Here, a combinatorial regenerative and rehabilitative approach to regrow damaged axons was proposed. Sprague-Dawley rats were subjected to a severe T9-T10 full tranection injury with a 2mm gap. Neurotrophin-3 (NT-3) loaded fibrous scaffold was implanted within the gap to provide topographical guidance for the axons to cross the injured region. To study the effect of rehabilitation, the rats were separated into 2 groups; those that undergo rehabilitation (trained, N=4) and those that do not undergo rehabilitation (untrained, N=3). In order to rehabilitate the rats, a rehabilitation robotic system consisting of a body weight support, hindlimb manipulator, and treadmill was developed. Preliminary results showed that rats which underwent rehabilitation had more robust axonal regeneration within the scaffold after 1 month. However, the Basso, Beattie, and Bresnahan (BBB) score, which is an indicator of locomotor recovery, do not show much significance between trained and untrained rats.

Our ability to rescue functional deficits after demyelinating diseases or spinal cord injuries is limited by our lack of understanding of the complex remyelination process, which is crucial to functional recovery. In this study, we developed an electrospun suspended poly(e-caprolactone) microfiber platform to enable the screening of therapeutics for remyelination. As a proof of concept, this platform employed scaffold-mediated non-viral delivery of a microRNA (miR) cocktail to promote oligodendrocyte precursor cells (OPCs) differentiation and myelination. We observed enhanced OPCs differentiation when the cells were transfected with miR-219 and miR-338 on the microfiber substrates. Moreover, miRs promoted the formation of MBP+ tubular extensions around the suspended fibers, which was indicative of myelination, instead of flat myelin membranes on 2D substrates. In addition, OPCs that were transfected with the cocktail of miRs formed significantly longer and larger amounts of MBP+ extensions. Taken together, these results demonstrate the efficacy of this functional screening platform for understanding myelination. Statement of Significance The lack of understanding of the complex myelination process has hindered the discovery of effective therapeutic treatments for demyelinating diseases. Hence, in vitro models that enable systematic understanding, visualization and quantification of myelination are valuable. Unfortunately, achieving reproducible in vitro myelination by oligodendrocytes (OLs) remains highly challenging. Here, we engineered a suspended microfiber platform that enables sustained non-viral drug/gene delivery to study OL differentiation and myelination. Sustained drug delivery permits the investigation of OL development, which spans several weeks. We show that promyelinogenic microRNAs promoted OL differentiation and myelination on this platform. Our engineered microfiber substrate could serve as a drug/gene screening platform and facilitate future translation into direct implantable devices for in vivo remyelination purposes.

The use of human induced pluripotent stem cell-derived neural progenitor cells (hiPSC-NPCs) is an attractive therapeutic option for damaged nerve tissues. To direct neuronal differentiation of stem cells, we have previously developed an electrospun polycaprolactone nanofiber scaffold that was functionalized with siRNA targeting Re-1 silencing transcription factor (REST), by mussel-inspired bioadhesive coating. However, the efficacy of nanofiber-mediated RNA interference on hiPSC-NPCs differentiation remains unknown. Furthermore, interaction between such cell-seeded scaffolds with injured tissues has not been tested. In this study, scaffolds were optimized for REST knockdown in hiPSC-NPCs to enhance neuronal differentiation. Specifically, the effects of two different mussel-inspired bioadhesives and transfection reagents were analyzed. Scaffolds functionalized with RNAiMAX Lipofectamine-siREST complexes enhanced the differentiation of hiPSC-NPCs into TUJ1+ cells (60% as compared to 22% in controls with scrambled siNEG after 9 days) without inducing high cytotoxicity. When cell-seeded scaffolds were transplanted to transected spinal cord organotypic slices, similar efficiency in neuronal differentiation was observed. The scaffolds also supported the migration of cells and neurite outgrowth from the spinal cord slices. Taken together, the results suggest that this scaffold can be effective in enhancing hiPSC-NPC neuronal commitment by gene-silencing for the treatment of injured spinal cords. ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Accepted version

A low toxicity and efficient delivery system is needed to deliver small interfering RNAs (siRNA) in vitro and in vivo. The use of mesoporous silica nanoparticles (MSN) is becoming increasingly common due to its biocompatibility, tunable pore size and customizable properties. However, bolus delivery of siRNA/MSN complexes remains suboptimal, especially when a sustained and long-term administration is required. Here, we utilized electrospun scaffolds for sustained delivery of siRNA/MSN-PEI through surface adsorption and nanofiber encapsulation. As a proof-of-concept, we targeted collagen type I expression to modulate fibrous capsule formation. Surface adsorption of siRNA/MSN-PEI provided sustained availability of siRNA for at least 30 days in vitro. As compared to conventional bolus delivery, such scaffold-mediated transfection provided more effective gene silencing (p 0.05). On the contrary, a longer sustained release was attained (at least 5 months) when siRNA/MSN-PEI complexes were encapsulated within the electrospun fibers. In vivo subcutaneous implantation and biodistribution analysis of these scaffolds revealed that siRNA remained localized up to ∼290 μm from the implants. Finally, a fibrous capsule reduction of ∼45.8% was observed after 4 weeks in vivo as compared to negative scrambled siRNA treatment. Taken together, these results demonstrate the efficacy of scaffold-mediated sustained delivery of siRNA/MSN-PEI for long-term non-viral gene silencing applications.The bolus delivery of siRNA/mesoporous silica nanoparticles (MSN) complexes shows high efficiency to silence protein agonists of tumoral processes as cancer treatments. However, in tissue engineering area, scaffold mediated delivery is desired to achieve a local and sustained release of therapeutics. We showed the feasibility and the efficacy of siRNA/MSN delivered from electrospun scaffolds through surface adsorption and nanofiber encapsulation. We showed that this method enhances siRNA transfection efficiency and sustained targeted proteins silencing in vitro and in vivo. As a proof of concept, in this study, we targeted collagen type I expression to modulate fibrous capsule formation. However this platform can be applied to the release and transfection of siRNA or miRNA in cancer and tissue engineering applications.