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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.

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 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

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.

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

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.

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

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.

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. 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) Published version

Correction for ‘Scaffold mediated gene knockdown for neuronal differentiation of human neural progenitor cells’ by Wai Hon Chooi et al., Biomater. Sci., 2018, 6, 3019–3029.