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Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.
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Epigénesis Genética , Vaina de Mielina , Oligodendroglía , Remielinización , Animales , Vaina de Mielina/metabolismo , Humanos , Ratones , Remielinización/efectos de los fármacos , Oligodendroglía/metabolismo , Sistema Nervioso Central/metabolismo , Ratones Endogámicos C57BL , Rejuvenecimiento , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Organoides/metabolismo , Organoides/efectos de los fármacos , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/genética , Diferenciación Celular/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Masculino , Regeneración/efectos de los fármacos , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/genética , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patologíaRESUMEN
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, a biomimicking fiber platform that provides independent variation of fiber structural and intrinsic stiffness is reported. To demonstrate the functionality of these constructs as a mechanotransduction study platform, these substrates are utilized as artificial axons and the effects of axon structural versus intrinsic stiffness on CNS myelination are independently analyzed. 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, it is demonstrated that a stiff mechanical microenvironment impedes oligodendrocyte myelination, independently and distinctively from oligodendrocyte differentiation. Yes-associated protein is identified 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, the platform may have considerable utility as part of a drug discovery program in identifying molecules that promote both differentiation and myelination.
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Mecanotransducción Celular , Vaina de Mielina , Axones , Diferenciación Celular , OligodendroglíaRESUMEN
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.
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Sistema Nervioso Central/metabolismo , Portadores de Fármacos/química , MicroARNs/metabolismo , Remielinización/fisiología , Animales , Femenino , Hidrogeles/química , Inmunohistoquímica , MicroARNs/química , MicroARNs/genética , Microscopía Electrónica de Rastreo , Factores de Crecimiento Nervioso/metabolismo , Ratas , Ratas Sprague-Dawley , Remielinización/genéticaRESUMEN
RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(ß-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.
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Ésteres , Polímeros , Humanos , ARN Interferente Pequeño/genética , Células HeLa , Transfección , Silenciador del Gen , Técnicas de Transferencia de GenRESUMEN
The transplantation of spinal cord progenitor cells (SCPCs) derived from human-induced pluripotent stem cells (iPSCs) has beneficial effects in treating spinal cord injury (SCI). However, the presence of residual undifferentiated iPSCs among their differentiated progeny poses a high risk as these cells 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 used 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 (ie, >3 million cells/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.
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Células Madre Pluripotentes Inducidas , Células-Madre Neurales , Traumatismos de la Médula Espinal , Humanos , Médula Espinal/patología , Diferenciación Celular/fisiología , Traumatismos de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/patologíaRESUMEN
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.
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The neuroinflammatory response that is elicited after spinal cord injury contributes to both tissue damage and reparative processes. The complex and dynamic cellular and molecular changes within the spinal cord microenvironment result in a functional imbalance of immune cells and their modulatory factors. To facilitate wound healing and repair, it is necessary to manipulate the immunological pathways during neuroinflammation to achieve successful therapeutic interventions. In this review, recent advancements and fresh perspectives on the consequences of neuroinflammation after SCI and modulation of the inflammatory responses through the use of molecular-, cellular-, and biomaterial-based therapies to promote tissue regeneration and functional recovery will be discussed.
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To enhance the efficacy of photothermal therapy (PTT) at tumor sites, we designed a reactive oxygen species (ROS)-responsive gold nanoparticle (AuNP)-based nanosystem in which azide-decorated AuNPs (N3@AuNPs) and diselenide-coated alkyne-decorated AuNPs (Se/Ak@AuNPs) were separately prepared for selective clicking into nanoclusters when exposed to ROS. Se/Ak@AuNPs were dual-functionalized with alkyne moieties and diselenide linkers embedded in a long chain of polyethylene glycol (PEG) to enable the alkyne moieties of Se/Ak@AuNPs to be inaccessible to the azide moieties of N3@AuNPs owing to steric hindrance. At tumor sites where the ROS level is elevated due to the increased metabolic activity, cellular receptor signaling, mitochondrial dysfunction, and oncogene activity, the diselenide linkers were cleaved, leading to the liberation of the long PEG chains tethered to AuNPs, and the alkyne moieties could be recognized by the surrounding azide moieties to generate a click reaction. The clicked AuNPs formed clustered nanoparticles with increased size. Upon 808 nm laser irradiation, these large clusters of AuNPs significantly enhanced the photothermal conversion efficiency compared with that of isolated AuNPs. In vitro studies revealed that the AuNP clusters exhibited a noticeably higher apoptosis rate than AuNPs. Therefore, ROS-responsive clicked AuNP clusters can be a potential tool for PTT enhancement in cancer treatment.
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Oro , Nanopartículas del Metal , Oro/farmacología , Especies Reactivas de Oxígeno , Terapia Fototérmica , AzidasRESUMEN
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.
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Sistema Nervioso Central , Sistemas de Liberación de Medicamentos , Nanogeles , Sistemas de Liberación de Medicamentos/métodos , Neuronas , Membrana Celular , ADN , Portadores de Fármacos/químicaRESUMEN
Transplanting human induced pluripotent stem cells (iPSCs)-derived spinal cord progenitor cells (SCPCs) is a promising approach to treat spinal cord injuries. However, stem cell therapies face challenges in cell survival, cell localization to the targeted site, and the control of cell differentiation. Here, we encapsulated SCPCs in thiol-modified hyaluronan-gelatin hydrogels and optimized scaffold mechanical properties and cell encapsulation density to promote cell viability and neuronal differentiation in vitro and in vivo. Different compositions of hyaluronan-gelatin hydrogels formulated by varying concentrations of poly(ethylene glycol) diacrylate were mechanically characterized by using atomic force microscopy. In vitro SCPC encapsulation study showed higher cell viability and proliferation with lower substrate Young's modulus (200 Pa vs 580 Pa) and cell density. Moreover, the soft hydrogels facilitated a higher degree of neuronal differentiation with extended filament structures in contrast to clumped cellular morphologies obtained in stiff hydrogels (p < 0.01). When transplanted in vivo, the optimized SCPC-encapsulated hydrogels resulted in higher cell survival and localization at the transplanted region as compared to cell delivery without hydrogel encapsulation at 2 weeks postimplantation within the rat spinal cord (p < 0.01). Notably, immunostaining demonstrated that the hydrogel-encapsulated SCPCs differentiated along the neuronal and oligodendroglial lineages in vivo. The lack of pluripotency and proliferation also supported the safety of the SCPC transplantation approach. Overall, the injectable hyaluronan-gelatin hydrogel shows promise in supporting the survival and neural differentiation of human SCPCs after transplantation into the spinal cord.
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Scaffolds can promote the healing of burns and chronic skin wounds but to date have suffered from issues with achieving full skin integration. Here, we characterise the wound response by both tissue integration and re-epithelialization to a scaffold using wet electrospinning to fabricate 3D fibrous structures. Two scaffold materials were investigated: poly(ε-caprolactone) (PCL) and PCL + 20% rat tail type 1 collagen (PCL/Coll). We assessed re-epithelisation, inflammatory responses, angiogenesis and the formation of new extracellular matrix (ECM) within the scaffolds in rat acute wounds. The 3D PCL/Coll scaffolds impeded wound re-epithelisation, inducing a thickening of wound-edge epidermis as opposed to a thin tongue of migratory keratinocytes as seen when 3D PCL scaffolds were implanted in the wounds. A significant inflammatory response was observed with 3D PCL/Coll scaffolds but not with 3D PCL scaffolds. Enhanced fibroblast migration and angiogenesis into 3D PCL scaffolds was observed with a significant deposition of new ECM. We observed that this deposition of new ECM within the scaffold was key to enabling re-epithelialization over the scaffold. Such scaffolds provide a biocompatible environment for cell integration to lay down new ECM and encourage re-epithelisation over the implanted scaffold.
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Biological lasers which utilize Fabry-Pérot (FP) cavities have attracted tremendous interest due to their potential in amplifying subtle biological changes. Transverse laser modes generated from cells serve as distinct fingerprints of individual cells; however, most lasing signals lack the ability to provide key information about the cell due to high complexity of transverse modes. The missing key, therefore, hinders it from practical applications in biomedicine. This study reveals the key mechanism governing the frequency distributions of transverse modes in cellular lasers. Spatial information of cells including curvature can be interpreted through spectral information of transverse modes by means of hyperspectral imaging. Theoretical studies are conducted to explore the correlation between the cross-sectional morphology of a cell and lasing frequencies of transverse modes. Experimentally, the spectral characteristics of transverse modes are investigated in live and fixed cells with different morphological features. By extracting laser modes in frequency domain, the proposed concept is applied for studying cell adhesion process and cell classification from rat cortices. This study expands a new analytical dimension of cell lasers, opening an avenue for subcellular analysis in biophotonic applications.
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Adhesión Celular/fisiología , Rayos Láser , Óptica y Fotónica/instrumentación , Óptica y Fotónica/métodos , Animales , Diseño de Equipo , Luz , Modelos Animales , Modelos Teóricos , RatasRESUMEN
Injuries within the peripheral nervous system (PNS) lead to sensory and motor deficits, as well as neuropathic pain, which strongly impair the life quality of patients. Although most current PNS injury treatment approaches focus on using growth factors/small molecules to stimulate the regrowth of the injured nerves, these methods neglect another important factor that strongly hinders axon regeneration-the presence of axonal inhibitory molecules. Therefore, this work sought to explore the potential of pathway inhibition in promoting sciatic nerve regeneration. Additionally, the therapeutic window for using pathway inhibitors was uncovered so as to achieve the desired regeneration outcomes. Specifically, we explored the role of Wnt signaling inhibition on PNS regeneration by delivering Wnt inhibitors, sFRP2 and WIF1, after sciatic nerve transection and sciatic nerve crush injuries. Our results demonstrate that WIF1 promoted nerve regeneration (p < 0.05) after sciatic nerve crush injury. More importantly, we revealed the therapeutic window for the treatment of Wnt inhibitors, which is 1 week post sciatic nerve crush when the non-canonical receptor tyrosine kinase (Ryk) is significantly upregulated.
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Glial cells are mechanosensitive, and thus, engineered systems have taken a step forward to design mechanotransduction platforms in order to impart diverse mechanical stresses to cells. Mechanical strain encountered in the central nervous system can arise from diverse mechanisms, such as tissue reorganization, fluid flow, and axon growth, as well as pathological events including axon swelling or mechanical trauma. Biomechanical relevance of the in vitro mechanical testing requires to be placed in line with the physiological and mechanical changes in central nervous tissues that occur during the progression of neurodegenerative diseases. Mechanotransduction signaling utilized by glial cells and the recent approaches intended to model altered microenvironment adapted to pathological context are discussed in this review. New insights in systems merging substrate's stiffness and topography should be considered for further glial mechanotransduction studies, while testing platforms for drug discoveries promise great advancements in pharmacotherapy. Potential leads and strategies for clinical outcomes are expected to be developed following the exploration of these glial mechanosensitive signaling pathways.
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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 nonspecific uptake and subsequent side effects. As such, exploring an effective and targeted drug delivery system is of great necessity. Synthetic micro-/nanoparticles 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. A successful cell membrane coating over poly(ε-caprolactone) nanoparticles (NPs) was confirmed using dynamic light scattering, zeta potential measurements, and transmission electron microscopy. 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 toward 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 the 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.
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Membrana Celular/metabolismo , Sistema Nervioso Central/metabolismo , Materiales Biocompatibles Revestidos/metabolismo , Nanopartículas/metabolismo , Neuronas/citología , Animales , Membrana Celular/química , Células Cultivadas , Sistema Nervioso Central/química , Materiales Biocompatibles Revestidos/química , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Tamaño de la Partícula , Ratas , Propiedades de SuperficieRESUMEN
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.
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Sistemas CRISPR-Cas , Edición Génica , Ribonucleoproteínas/metabolismo , Andamios del Tejido , Adhesivos/química , Animales , Proteína 9 Asociada a CRISPR/química , Línea Celular , Expresión Génica , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Lípidos/química , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Nanofibras/química , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , Ribonucleoproteínas/genética , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
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.
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Actinas , Células Madre Mesenquimatosas , Actinas/metabolismo , Colágeno , Humanos , Células Madre Mesenquimatosas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
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.
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Neuritas , Neuronas , Animales , Células Cultivadas , RatasRESUMEN
Although cell membrane-coated fiber scaffolds can be useful for regenerative medicine by presenting both cell surface antigens and topographical cues, it remains unknown whether changes in cellular behavior on cell membrane-coated scaffolds are due to specific cell-cell interactions. In this work, the effects of scaffold fiber diameters and surface charges on the cell membrane coating efficiency were explored. Furthermore, fibroblast membrane-coated scaffolds improved the growth of human keratinocytes as compared to red blood cell membrane-coated and plain scaffolds. These results suggest the biofunctionality of cell membrane-coated scaffolds and the specific cell-cell interactions that are preserved to modulate cellular response.
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Membrana Celular/química , Materiales Biocompatibles Revestidos/química , Queratinocitos/química , Ingeniería de Tejidos , Humanos , Ensayo de Materiales , Tamaño de la Partícula , Andamios del Tejido/químicaRESUMEN
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.