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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]

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

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.

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 (

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

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).

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

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.

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.

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.

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.

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