Enhancing Paper Packaging's Wet Strength Using the Synergy between Chitosan and Nanofibrillated Cellulose Additives.

The demand for eco-friendly packaging materials has urged researchers to look for alternatives to petroleum-based polymers. In this regard, paper-based products have turned out to be a promising choice; however, their weak resistance to water has limited their application. The use of various additives to enhance paper's moisture resistance is a common practice. However, considering the growing global agenda for sustainable development, the search for new bio-based paper additives has become increasingly important. This study investigated the potential synergistic impact of the addition of nanofibrillated cellulose (NFC) and chitosan additives (CHIT) to different fiber combinations to improve paper's properties, in particular, their wet strength. The efficacy of the additive application order was examined and was found to be crucial in achieving the desired outcomes. The results showed that incorporating CHIT after NFC enhanced the paper's tensile and burst indicators, as well as the paper stretch in the dry state, by 35-70%, 35-55%, and 20-35%, respectively. In addition, the tensile index and stretch in the wet state improved 9-13 times and 2.5-5.5 times over, respectively. The air permeability decreased 2.5-12 times over. These findings demonstrate that the sequential addition of the NFC and CHIT additives yield a greater enhancement of paper's properties than using each additive separately.

Neural cell membrane-coated DNA nanogels as a potential target-specific drug delivery tool for the central nervous system.

A major bottleneck in drug/gene delivery to enhance tissue regeneration after injuries is to achieve targeted delivery to the cells of interest. Unfortunately, we have not been able to attain effective targeted drug delivery in tissues due to the lack of efficient delivery platforms. Since specific cell-cell interactions exist to impart the unique structure and functionality of tissues and organs, we hypothesize that such specific cellular interactions may also be harnessed for drug delivery applications in the form of cell membrane coatings. Here, we employed neural cell-derived membrane coating technique on DNA nanogels to improve target specificity. The efficacy of neural cell membrane-coated DNA nanogels (NCM-nanogels) was demonstrated by using four types of cell membranes derived from the central nervous system (CNS), namely, astrocytes, microglia, cortical neurons, and oligodendrocyte progenitor cells (OPCs). A successful coating of NCMs over DNA nanogels was confirmed by dynamic light scattering, zeta potential measurements and transmission electron microscopy. Subsequently, an overall improvement in cellular uptake of NCM-nanogels over uncoated DNA nanogels (p < 0.005) was seen. Additionally, we observed a selective uptake of OPC membrane-coated DNA nanogels (NCM-O mem) by oligodendrocytes over other cell types both in vitro and in vivo. Our quantitative polymerase chain reaction (qPCR) results also showed selective and effective gene knockdown capacity of NCM-O mem for OPC transfection. The findings in this work may be beneficial for future drug delivery applications targeted at the CNS.

Targeting connexin 43 expression via scaffold mediated delivery of antisense oligodeoxynucleotide preserves neurons, enhances axonal extension, reduces astrocyte and microglial activation after spinal cord injury.

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.

A 3D Fiber-Hydrogel Based Non-Viral Gene Delivery Platform Reveals that microRNAs Promote Axon Regeneration and Enhance Functional Recovery Following Spinal Cord Injury.

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.

Biofunctional scaffolds with high packing density of aligned electrospun fibers support neural regeneration.

Neurons of the central nervous system do not regenerate spontaneously after injury. As such, biofunctional tissue scaffolds have been explored to provide a growth-promoting environment to enhance neural regeneration. In this regard, aligned electrospun fibers have proven invaluable for regeneration by offering guidance for axons to cross the injury site. However, a high fiber density could potentially limit axonal ingrowth into the scaffold. Here, we explore which fiber density provides the optimal environment for neurons to regenerate. By changing fiber electrospinning time, we generated scaffolds with different fiber densities and implanted these in a rat model of spinal cord injury (SCI). We found that neurons were able to grow efficiently into scaffolds with high fiber density, even if the gaps between fiber bundles were very small (<1 µm). Scaffolds with high fiber density showed good host-implant integration. Cell infiltration was not affected by fiber density. Efficient blood vessel ingrowth likely requires larger gaps between fibers or faster degrading fibers. We conclude that scaffolds with high fiber densities, and thus a large number of small gaps in between fiber bundles, provide the preferred environment for nerve regeneration after SCI.

Modulating Macrophage Phenotype by Sustained MicroRNA Delivery Improves Host-Implant Integration.

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.

Regenerative rehabilitation: exploring the synergistic effects of rehabilitation and implantation of a bio-functional scaffold in enhancing nerve regeneration.

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.

Biomimicking Fiber Scaffold as an Effective In Vitro and In Vivo MicroRNA Screening Platform for Directing 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.

Scaffold-Mediated Sustained, Non-viral Delivery of miR-219/miR-338 Promotes CNS Remyelination.

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.

Exploring new treatment for spinalized rats by synergising robotic rehabilitation system and regenerative medicine.

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.

Sustained delivery of siRNA/mesoporous silica nanoparticle complexes from nanofiber scaffolds for long-term gene silencing.

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. STATEMENT OF SIGNIFICANCE: 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.

Three-Dimensional Nanofiber Hybrid Scaffold Directs and Enhances Axonal Regeneration after Spinal Cord Injury.

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.

Nanofiber-mediated microRNA delivery to enhance differentiation and maturation of oligodendroglial precursor cells.

Remyelination in the central nervous system (CNS) is critical in the treatment of many neural pathological conditions. Unfortunately, the ability to direct and enhance oligodendrocyte (OL) differentiation and maturation remains limited. It is known that microenvironmental signals, such as substrate topography and biochemical signaling, regulate cell fate commitment. Therefore, in this study, we developed a nanofiber-mediated microRNA (miR) delivery method to control oligodendroglial precursor cell (OPC) differentiation through a combination of fiber topography and gene silencing. Using poly(ε-caprolactone) nanofibers, efficient knockdown of OL differentiation inhibitory regulators were achieved by either nanofiber alone (20-40%, p<0.05) or the synergistic integration with miR-219 and miR-338 (up to 60%, p<0.05). As compared to two-dimensional culture, nanofiber topography enhanced OPC differentiation by inducing 2-fold increase in RIP(+) cells (p<0.01) while the presence of miRs further enhanced the result to 3-fold (p<0.001). In addition, nanofiber-mediated delivery of miR-219 and miR-338 promoted OL maturation by increasing the number of MBP(+) cells significantly (p<0.01). Taken together, the results demonstrate the efficacy of nanofibers in providing topographical cues and microRNA reverse transfection to direct OPC differentiation. Such scaffolds may find useful applications in directing oligodendrocyte differentiation and myelination for treatment of CNS pathological conditions that require remyelination.

Astrocytic and vascular remodeling in the injured adult rat spinal cord after chondroitinase ABC treatment.

Upregulation of extracellular chondroitin sulfate proteoglycans (CSPG) is a primary cause for the failure of axons to regenerate after spinal cord injury (SCI), and the beneficial effect of their degradation by chondroitinase ABC (ChABC) is widely documented. Little is known, however, about the effect of ChABC treatment on astrogliosis and revascularization, two important factors influencing axon regrowth. This was investigated in the present study. Immediately after a spinal cord hemisection at thoracic level 8-9, we injected ChABC intrathecally at the sacral level, repeated three times until 10 days post-injury. Our results show an effective cleavage of CSPG glycosaminoglycan chains and stimulation of axonal remodeling within the injury site, accompanied by an extended period of astrocyte remodeling (up to 4 weeks). Interestingly, ChABC treatment favored an orientation of astrocytic processes directed toward the injury, in close association with axons at the lesion entry zone, suggesting a correlation between axon and astrocyte remodeling. Further, during the first weeks post-injury, ChABC treatment affected the morphology of laminin-positive blood vessel basement membranes and vessel-independent laminin deposits: hypertrophied blood vessels with detached or duplicated basement membrane were more numerous than in lesioned untreated animals. In contrast, at later time points, laminin expression increased and became more directly associated with newly formed blood vessels, the size of which tended to be closer to that found in intact tissue. Our data reinforce the idea that ChABC injection in combination with other synergistic treatments is a promising therapeutic strategy for SCI repair.

Convergent evidence identifying MAP/microtubule affinity-regulating kinase 1 (MARK1) as a susceptibility gene for autism.

Autism spectrum disorders (ASDs) are common, heritable, but genetically heterogeneous neurodevelopmental conditions. We recently defined a susceptibility locus for ASDs on chromosome 1q41-q42. High-resolution single-nucleotide polymorphisms (126 SNPs) genotyping across the chromosome 1q41-q42 region, followed by a MARK1 (microtubule affinity-regulating kinase 1)-tagged-SNP association study in 276 families with autism from the Autism Genetic Research Exchange, showed that several SNPs within the MARK1 gene were significantly associated with ASDs by transmission disequilibrium tests. Haplotype rs12740310*C-rs3737296*G-rs12410279*A was overtransmitted (P(corrected)= 0.0016), with a relative risk for autism of 1.8 in homozygous carriers. Furthermore, ASD-associated SNP rs12410279 modulates the level of transcription of MARK1. We found that MARK1 was overexpressed in the prefrontal cortex (BA46) but not in cerebellar granule cells, on postmortem brain tissues from patients. MARK1 displayed an accelerated evolution along the lineage leading to humans, suggesting possible involvement of this gene in cognition. MARK1 encodes a kinase-regulating microtubule-dependent transport in axons and dendrites. Both overexpression and silencing of MARK1 resulted in significantly shorter dendrite length in mouse neocortical neurons and modified dendritic transport speed. As expected for a gene encoding a key polarity determinant Par-1 protein kinase, MARK1 is involved in axon-dendrite specification. Thus, MARK1 overexpression in humans may be responsible for subtle changes in dendritic functioning.