Fabrication and characterization of bioresorbable, electroactive and highly regular nanomodulated cell interfaces.

Biomaterial-based implantable scaffolds capable of promoting physical and functional reconnection of injured spinal cord and nerves represent the latest frontier in neural tissue engineering. Here, we report the fabrication and characterization of self-standing, biocompatible and bioresorbable substrates endowed with both controlled nanotopography and electroactivity, intended for the design of transient implantable scaffolds for neural tissue engineering. In particular, we obtain conductive and nano-modulated poly(D,L-lactic acid) (PLA) and poly(lactic-co-glycolic acid) free-standing films by simply iterating a replica moulding process and coating the polymer with a thin layer of poly(3,4-ethylendioxythiophene) polystyrene sulfonate. The capability of the substrates to retain both surface patterning and electrical properties when exposed to a liquid environment has been evaluated by atomic force microscopy, electrochemical impedance spectroscopy and thermal characterizations. In particular, we show that PLA-based films maintain their surface nano-modulation for up to three weeks of exposure to a liquid environment, a time sufficient for promoting axonal anisotropic sprouting and growth during neuronal cell differentiation. In conclusion, the developed substrates represent a novel and easily-tunable platform to design bioresorbable implantable devices featuring both topographic and electrical cues.

Self-healing hydrogel as an injectable implant: translation in brain diseases.

Tissue engineering biomaterials are aimed to mimic natural tissue and promote new tissue formation for the treatment of impaired or diseased tissues. Highly porous biomaterial scaffolds are often used to carry cells or drugs to regenerate tissue-like structures. Meanwhile, self-healing hydrogel as a category of smart soft hydrogel with the ability to automatically repair its own structure after damage has been developed for various applications through designs of dynamic crosslinking networks. Due to flexibility, biocompatibility, and ease of functionalization, self-healing hydrogel has great potential in regenerative medicine, especially in restoring the structure and function of impaired neural tissue. Recent researchers have developed self-healing hydrogel as drug/cell carriers or tissue support matrices for targeted injection via minimally invasive surgery, which has become a promising strategy in treating brain diseases. In this review, the development history of self-healing hydrogel for biomedical applications and the design strategies according to different crosslinking (gel formation) mechanisms are summarized. The current therapeutic progress of self-healing hydrogels for brain diseases is described as well, with an emphasis on the potential therapeutic applications validated by in vivo experiments. The most recent aspect as well as the design rationale of self-healing hydrogel for different brain diseases is also addressed.

Apoptosis-Decellularized Peripheral Nerve Scaffold Allows Regeneration across Nerve Gap.

Peripheral nerve injury results in loss of motor and sensory function distal to the nerve injury and is often permanent in nerve gaps longer than 5 cm. Autologous nerve grafts (nerve autografts) utilize patients' own nerve tissue from another part of their body to repair the defect and are the gold standard in care. However, there is a limited autologous tissue supply, size mismatch between donor nerve and injured nerve, and morbidity at the site of nerve donation. Decellularized cadaveric nerve tissue alleviates some of these limitations and has demonstrated success clinically. We previously developed an alternative apoptosis-assisted decellularization process for nerve tissue. This new process may result in an ideal scaffold for peripheral nerve regeneration by gently removing cells and antigens while preserving delicate topographical cues. In addition, the apoptosis-assisted process requires less active processing time and is inexpensive. This study examines the utility of apoptosis-decellularized peripheral nerve scaffolds compared to detergent-decellularized peripheral nerve scaffolds and isograft controls in a rat nerve gap model. Results indicate that, at 8 weeks post-injury, apoptosis-decellularized peripheral nerve scaffolds perform similarly to detergent-decellularized and isograft controls in both functional (muscle weight recovery, gait analysis) and histological measures (neurofilament staining, macrophage infiltration). These new apoptosis-decellularized scaffolds hold great promise to provide a less expensive scaffold for nerve injury repair, with the potential to improve nerve regeneration and functional outcomes compared to current detergent-decellularized scaffolds.

Construction of brain-like spheroids containing endothelial tubular networks by an adhesive culture system.

Spheroids which are composed of several types of cells have been widely studied in the pharmaceutical field as their structure and functions are similar to human organs. Three-dimensional brain-like tissues are one of the most important tissues for the development of medicines to treat brain diseases and for in vitro brain models. In this study, spheroids mainly containing neurons, astrocytes, and endothelial cells were fabricated using a novel 3D culture plate, "MicoCell™" to construct a brain mimicking tissue. Due to the multicavity structures of MicoCell, ∼102 of attached spheroids were fabricated in a single plate. Spheroids in MicoCell were attached onto a mild cell adhesive surface, allowing for easy immunostaining and microscopic observation. Spheroid formation was improved by adding a Rho-Kinase inhibitor during cultivation. Endothelial cells formed vascular network structures in spheroids and some parts of the vascular structures attached onto the bottom of a culture plate. Co-culture of multiple cell types required optimization of the culture medium during spheroid formation. The mixture of neural stem cell medium and endothelial growth medium showed good spheroid formation and a vascular network. These results indicated that our culture plates and brain mimicking spheroids would be a suitable candidate for pharmaceutical applications such as drug screening and for in vitro brain models.

Cold atmospheric plasma modification and electrical conductivity induction in gelatin/polyvinylidene fluoride nanofibers for neural tissue engineering.

BACKGROUND: This research follows some investigations through neural tissue engineering, including fabrication, surface treatment, and evaluation of novel self-stimuli conductive biocompatible and degradable nanocomposite scaffolds. METHODS: Gelatin as a biobased material and polyvinylidene fluoride (PVDF) as a mechanical, electrical, and piezoelectric improvement agent were co-electrospun. In addition, polyaniline/graphene (PAG) nanoparticles were synthesized and added to gelatin solutions in different percentages to induce electrical conductivity. After obtaining optimum PAG percentage, cold atmospheric plasma (CAP) treatment was applied over the best samples by different plasma variable parameters. Finally, the biocompatibility of the scaffolds was analyzed and approved by in vitro tests using two different PC12 and C6 cell lines. In the present study the morphology, FTIR, dynamic light scattering, mechanical properties, wettability, contact angle tests, differential scanning calorimetric, rate of degradation, conductivity, biocompatibility, gene expression, DAPI staining, and cell proliferation were investigated. RESULTS: The PAG percentage optimization results revealed fiber diameter reduction, conductivity enhancement, young's modulus improvement, hydrophilicity devaluation, water uptake decrement, and degradability reduction in electrospun nanofibers by increasing the PAG concentration. Furthermore, ATR-FTIR, FE-SEM, AFM, and contact angle tests revealed that helium CAP treatment improves scaffold characterizations for 90 s in duration time. Furthermore, the results of the MTT assay, FE-SEM, DAPI staining, and RT-PCR revealed that samples containing 2.5% w/w of PAG are the most biocompatible, and CAP treatment increases cell proliferation and improves neural gene expression in the differentiation medium. CONCLUSIONS: According to the results, the samples with the 2.5% w/w of PAG could provide a suitable matrix for neural tissue engineering in terms of physicochemical and biological.

Local delivery of fingolimod through PLGA nanoparticles and PuraMatrix-embedded neural precursor cells promote motor function recovery and tissue repair in spinal cord injury.

Spinal cord injury (SCI) is a devastating clinical problem that can lead to permanent motor dysfunction. Fingolimod (FTY720) is a sphingosine structural analogue, and recently, its therapeutic benefits in SCI have been reported. The present study aimed to evaluate the therapeutic efficacy of fingolimod-incorporated poly lactic-co-glycolic acid (PLGA) nanoparticles (nanofingolimod) delivered locally together with neural stem/progenitor cells (NS/PCs) transplantation in a mouse model of contusive acute SCI. Fingolimod was encapsulated in PLGA nanoparticles by the emulsion-evaporation method. Mouse NS/PCs were harvested and cultured from embryonic Day 14 (E14) ganglionic eminences. Induction of SCI was followed by the intrathecal delivery of nanofingolimod with and without intralesional transplantation of PuraMatrix-encapsulated NS/PCs. Functional recovery, injury size and the fate of the transplanted cells were evaluated after 28 days. The nanofingolimod particles represented spherical morphology. The entrapment efficiency determined by UV-visible spectroscopy was approximately 90%, and the drug content of fingolimod loaded nanoparticles was 13%. About 68% of encapsulated fingolimod was slowly released within 10 days. Local delivery of nanofingolimod in combination with NS/PCs transplantation led to a stronger improvement in neurological functions and minimized tissue damage. Furthermore, co-administration of nanofingolimod and NS/PCs not only increased the survival of transplanted cells but also promoted their fate towards more oligodendrocytic phenotype. Our data suggest that local release of nanofingolimod in combination with three-dimensional (3D) transplantation of NS/PCs in the acute phase of SCI could be a promising approach to restore the damaged tissues and improve neurological functions.

Biodegradable and biocompatible graphene-based scaffolds for functional neural tissue engineering: A strategy approach using dental pulp stem cells and biomaterials.

Neural tissue engineering aims to restore the function of nervous system tissues using biocompatible cell-seeded scaffolds. Graphene-based scaffolds combined with stem cells deserve special attention to enhance tissue regeneration in a controlled manner. However, it is believed that minor changes in scaffold biomaterial composition, internal porous structure, and physicochemical properties can impact cellular growth and adhesion. The current work aims to investigate in vitro biological effects of three-dimensional (3D) graphene oxide (GO)/sodium alginate (GOSA) and reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of cell viability and cytotoxicity. Herein, the effects of the 3D scaffolds, coating conditions, and serum supplementation on DPSCs functions are explored extensively. Biodegradation analysis revealed that the addition of GO enhanced the degradation rate of composite scaffolds. Compared to the 2D surface, the cell viability of 3D scaffolds was higher (p < 0.0001), highlighting the optimal initial cell adhesion to the scaffold surface and cell migration through pores. Moreover, the cytotoxicity study indicated that the incorporation of graphene supported higher DPSCs viability. It is also shown that when the mean pore size of the scaffold increases, DPSCs activity decreases. In terms of coating conditions, poly- l-lysine was the most robust coating reagent that improved cell-scaffold adherence and DPSCs metabolism activity. The cytotoxicity of GO-based scaffolds showed that DPSCs can be seeded in serum-free media without cytotoxic effects. This is critical for human translation as cellular transplants are typically serum-free. These findings suggest that proposed 3D GO-based scaffolds have favorable effects on the biological responses of DPSCs.

Defining the role of 17ß-estradiol in human endometrial stem cells differentiation into neuron-like cells.

Human endometrial stem cells (hEnSCs) that can be differentiated into various neural cell types have been regarded as a suitable cell population for neural tissue engineering and regenerative medicine. Considering different interactions between hormones, growth factors, and other factors in the neural system, several differentiation protocols have been proposed to direct hEnSCs towards specific neural cells. The 17ß-estradiol plays important roles in the processes of development, maturation, and function of nervous system. In the present research, the impact of 17ß-estradiol (estrogen, E2) on the neural differentiation of hEnSCs was examined for the first time, based on the expression levels of neural genes and proteins. In this regard, hEnSCs were differentiated into neuron-like cells after exposure to retinoic acid (RA), epidermal growth factor (EGF), and also fibroblast growth factor-2 (FGF2) in the absence or presence of 17ß-estradiol. The majority of cells showed a multipolar morphology. In all groups, the expression levels of nestin, Tuj-1 and NF-H (neurofilament heavy polypeptide) (as neural-specific markers) increased during 14 days. According to the outcomes of immunofluorescence (IF) and real-time PCR analyses, the neuron-specific markers were more expressed in the estrogen-treated groups, in comparison with the estrogen-free ones. These findings suggest that 17ß-estradiol along with other growth factors can stimulate and upregulate the expression of neural markers during the neuronal differentiation of hEnSCs. Moreover, our findings confirm that hEnSCs can be an appropriate cell source for cell therapy of neurodegenerative diseases and neural tissue engineering.

Biomimicking Fiber Platform with Tunable Stiffness to Study Mechanotransduction Reveals Stiffness Enhances Oligodendrocyte Differentiation but Impedes Myelination through YAP-Dependent Regulation.

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.

Three-dimensional imaging and quantification of real-time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy.

The development of a minimally invasive, robust, and inexpensive technique that permits real-time monitoring of cell responses on biomaterial scaffolds can improve the eventual outcomes of scaffold-based tissue engineering strategies. Towards establishing correlations between in situ biological activity and cell fate, we have developed a comprehensive workflow for real-time volumetric imaging of spatiotemporally varying cytosolic calcium oscillations in pure microglial cells cultured on electrospun meshes. Live HMC3 cells on randomly oriented electrospun fibers were stained with a fluorescent dye and imaged using a laser scanning confocal microscope. Resonance scanning provided high-resolution in obtaining the time-course of intracellular calcium levels without compromising spatial and temporal resolution. Three-dimensional reconstruction and depth-coding enabled the visualization of cell location and intracellular calcium levels as a function of sample thickness. Importantly, changes in cell morphology and in situ calcium spiking were quantified in response to a soluble biochemical cue and varying matrix architectures (i.e., randomly oriented and aligned fibers). Importantly, raster plots generated from spiking data revealed calcium signatures specific to culture conditions. In the future, our approach can be used to elucidate correlations between calcium signatures and cell phenotype/activation, and facilitate the rational design of scaffolds for biomedical applications.

Graphene-Based Nanocomposites for Neural Tissue Engineering.

Graphene has made significant contributions to neural tissue engineering due to its electrical conductivity, biocompatibility, mechanical strength, and high surface area. However, it demonstrates a lack of biological and chemical cues. Also, it may cause potential damage to the host body, limiting its achievement of efficient construction of neural tissues. Recently, there has been an increasing number of studies showing that combining graphene with other materials to form nano-composites can provide exceptional platforms for both stimulating neural stem cell adhesion, proliferation, differentiation and neural regeneration. This suggests that graphene nanocomposites are greatly beneficial in neural regenerative medicine. In this mini review, we will discuss the application of graphene nanocomposites in neural tissue engineering and their limitations, through their effect on neural stem cell differentiation and constructs for neural regeneration.

Integration of Phase-Change Materials with Electrospun Fibers for Promoting Neurite Outgrowth under Controlled Release.

We report a temperature-regulated system for the controlled release of nerve growth factor (NGF) to promote neurite outgrowth. The system is based upon microparticles fabricated using coaxial electrospray, with the outer solution containing a phase-change material (PCM) and the inner solution encompassing payload(s). When the temperature is kept below the melting point of the PCM, there is no release due to the extremely slow diffusion through a solid matrix. Upon increasing the temperature to slightly pass the melting point, the encapsulated payload(s) can be readily released from the melted PCM. By leveraging the reversibility of the phase transition, the payload(s) can be released in a pulsatile mode through on/off heating cycles. The controlled release system is evaluated for potential use in neural tissue engineering by sandwiching the microparticles, co-loaded with NGF and a near-infrared dye, between two layers of electrospun fibers to form a tri-layer construct. Upon photothermal heating with a near-infrared laser, the NGF is released with well-preserved bioactivity to promote neurite outgrowth. By choosing different combinations of PCM, biological effector, and scaffolding material, this controlled release system can be applied to a wide variety of biomedical applications.

Current and novel polymeric biomaterials for neural tissue engineering.

The nervous system is a crucial component of the body and damages to this system, either by of injury or disease, can result in serious or potentially lethal consequences. Restoring the damaged nervous system is a great challenge due to the complex physiology system and limited regenerative capacity.Polymers, either synthetic or natural in origin, have been extensively evaluated as a solution for restoring functions in damaged neural tissues. Polymers offer a wide range of versatility, in particular regarding shape and mechanical characteristics, and their biocompatibility is unmatched by other biomaterials, such as metals and ceramics. Several studies have shown that polymers can be shaped into suitable support structures, including nerve conduits, scaffolds, and electrospun matrices, capable of improving the regeneration of damaged neural tissues. In general, natural polymers offer the advantage of better biocompatibility and bioactivity, while synthetic or non-natural polymers have better mechanical properties and structural stability. Often, combinations of the two allow for the development of polymeric conduits able to mimic the native physiological environment of healthy neural tissues and, consequently, regulate cell behaviour and support the regeneration of injured nervous tissues.Currently, most of neural tissue engineering applications are in pre-clinical study, in particular for use in the central nervous system, however collagen polymer conduits aimed at regeneration of peripheral nerves have already been successfully tested in clinical trials.This review highlights different types of natural and synthetic polymers used in neural tissue engineering and their advantages and disadvantages for neural regeneration.

Nanopatterned Scaffolds for Neural Tissue Engineering and Regenerative Medicine.

Biologically inspired approaches employing nanoengineering techniques have been influential in the progress of neural tissue repair and regeneration. Neural tissues are exposed to complex nanoscale environments such as nanofibrils. In this chapter, we summarize representative nanotechniques, such as electrospinning, lithography, and 3D bioprinting, and their use in the design and fabrication of nanopatterned scaffolds for neural tissue engineering and regenerative medicine. Nanotopographical cues in combination with other cues (e.g., chemical cues) are crucial to neural tissue repair and regeneration using cells, including various types of stem cells. Production of biologically inspired nanopatterned scaffolds may encourage the next revolution for studies aiming to advance neural tissue engineering and regenerative medicine.

Highly aligned nanocomposite scaffolds by electrospinning and electrospraying for neural tissue regeneration.

Neural tissue engineering offers a promising avenue for repairing neural injuries. Advancement in nanotechnology and neural scaffold manufacturing strategies has shed light on this field into a new era. In this study, a novel tissue engineered scaffold, which possesses highly aligned poly-ε-caprolactone microfibrous framework and adjustable bioactive factor embedded poly (d, l-lactide-co-glycolide) core-shell nanospheres, was fabricated by combining electrospinning and electrospraying techniques. The fabricated nanocomposite scaffold has cell favorable nanostructured feature and improved hydrophilic surface property. More importantly, by incorporating core-shell nanospheres into microfibrous scaffold, a sustained bioactive factor release was achieved. Results show rat pheochromocytoma (PC-12) cell proliferation was significantly promoted on the nanocomposite scaffold. In addition, confocal microscope images illustrated that the highly aligned scaffold increased length of neurites and directed neurites extension along the fibers in both PC-12 and astrocyte cell lines, which indicates that the scaffold is promising for guiding neural tissue growth and regeneration. From the clinical editor: In an attempt to direct neural cell growth, biomimetic neural scaffold was produced by electrospinning integrated with co-axial electrospraying techniques. In-vitro data provided a framework for future designs for neuronal regeneration.

In vitro model of neurotrauma using the chick embryo to test regenerative bioimplantation.

Effective repair of spinal cord injury sites remains a major clinical challenge. One promising strategy is the implantation of multifunctional bioscaffolds to enhance nerve fibre growth, guide regenerating tissue and modulate scarring/inflammation processes. Given their multifunctional nature, such implants require testing in models which replicate the complex neuropathological responses of spinal injury sites. This is often achieved using live, adult animal models of spinal injury. However, these have substantial drawbacks for developmental testing, including the requirement for large numbers of animals, costly infrastructure, high levels of expertise and complex ethical processes. As an alternative, we show that organotypic spinal cord slices can be derived from the E14 chick embryo and cultured with high viability for at least 24 days, with major neural cell types detected. A transecting injury could be reproducibly introduced into the slices and characteristic neuropathological responses similar to those in adult spinal cord injury observed at the lesion margin. This included aligned astrocyte morphologies and upregulation of glial fibrillary acidic protein in astrocytes, microglial infiltration into the injury cavity and limited nerve fibre outgrowth. Bioimplantation of a clinical grade scaffold biomaterial was able to modulate these responses, disrupting the astrocyte barrier, enhancing nerve fibre growth and supporting immune cell invasion. Chick embryos are inexpensive and simple, requiring facile methods to generate the neurotrauma model. Our data show the chick embryo spinal cord slice system could be a replacement spinal injury model for laboratories developing new tissue engineering solutions.

Incorporation of montmorillonite into microfluidics-generated chitosan microfibers enhances neuron-like PC12 cells for application in neural tissue engineering.

The complexity in structure and function of the nervous system, as well as its slow rate of regeneration, makes it more difficult to treat it compared to other tissues. Neural tissue engineering aims to create an appropriate environment for nerve cell proliferation and differentiation. Fibrous scaffolds with suitable morphology and topography and better mimicry of the extracellular matrix have been promising for the alignment and migration of neural cells. On this premise, to improve the properties of the scaffold, we combined montmorillonite (MMT) with chitosan (CS) polymer and created microfibers with variable diameters and varied concentrations of MMT using microfluidic technology and tested its suitability for the rat pheochromocytoma cell line (PC12). According to the findings, CS/MMT 0.1 % compared to CS/MMT 0 % microfibers showed a 201 MPa increase in Young's modulus, a 68 mS/m increase in conductivity, and a 1.4-fold increase in output voltage. Analysis of cell mitochondrial activity verified the non-toxicity, resulting in good cell morphology with orientation along the microfiber. Overall, the results of this project showed that with a low concentration of MMT, the properties of microfibers can be significantly improved and a suitable scaffold can be designed for neural tissue engineering.

Human induced pluripotent stem cell-derived planar neural organoids assembled on synthetic hydrogels.

The tailorable properties of synthetic polyethylene glycol (PEG) hydrogels make them an attractive substrate for human organoid assembly. Here, we formed human neural organoids from iPSC-derived progenitor cells in two distinct formats: (i) cells seeded on a Matrigel surface; and (ii) cells seeded on a synthetic PEG hydrogel surface. Tissue assembly on synthetic PEG hydrogels resulted in three dimensional (3D) planar neural organoids with greater neuronal diversity, greater expression of neurovascular and neuroinflammatory genes, and reduced variability when compared with tissues assembled upon Matrigel. Further, our 3D human tissue assembly approach occurred in an open cell culture format and created a tissue that was sufficiently translucent to allow for continuous imaging. Planar neural organoids formed on PEG hydrogels also showed higher expression of neural, vascular, and neuroinflammatory genes when compared to traditional brain organoids grown in Matrigel suspensions. Further, planar neural organoids contained functional microglia that responded to pro-inflammatory stimuli, and were responsive to anti-inflammatory drugs. These results demonstrate that the PEG hydrogel neural organoids can be used as a physiologically relevant in vitro model of neuro-inflammation.

Hydrodynamic Assembly of Astrocyte Cells in Conductive Hollow Microfibers.

The manufacturing of 3D cell scaffoldings provides advantages for modeling diseases and injuries as it enables the creation of physiologically relevant platforms. A triple-flow microfluidic device is developed to rapidly fabricate alginate/graphene hollow microfibers based on the gelation of alginate induced with CaCl2 . This five-channel microdevice actualizes continuous mild fabrication of hollow fibers under an optimized flow rate ratio of 300:200:100 µL min-1 . The polymer solution is 2.5% alginate in 0.1% graphene and a 30% polyethylene glycol solution is used as the sheath and core solutions. The biocompatibility of these conductive microfibers by encapsulating mouse astrocyte cells (C8D1A) within the scaffolds is investigated. The cells can successfully survive both the manufacturing process and prolonged encapsulation for up to 8 days, where there is between 18-53% of live cells on both the alginate microfibers and alginate/graphene microfibers. These unique 3D hollow scaffolds can significantly enhance the available surface area for nutrient transport to the cells. In addition, these conductive hollow scaffolds illustrate unique advantages such as 0.728 cm3  gr-1 porosity and two times more electrical conductivity in comparison to alginate scaffolds. The results confirm the potential of these scaffolds as a microenvironment that supports cell growth.

Hesperidin guided injured spinal cord neural regeneration with a combination of MWCNT-collagen-hyaluronic acid composite: In-vitro analysis.

Restoring the lost bioelectrical signal transmission along with the appropriate microenvironment is one of the major clinical challenges in spinal cord regeneration. In the current research, we developed a polysaccharide-based protein composite Multiwalled Carbon Nanotubes (MWCNTs)/ Collagen (Col)/ Hyaluronic acid (HA) composite with Hesperidin (Hes) natural compound to investigate its combined therapeutic effect along with biocompatibility, antioxidant activity, and electrical conductivity. The multifunctional composites were characterized via FT-IR, XRD, SEM, HR-TEM, BET, C.V, and EIS techniques. The electrical conductivity and modulus of the MWCNT-Col-HA-Hes were 0.06 S/cm and 12.3 kPa, similar to the native spinal cord. The in-vitro Cytotoxicity, cell viability, antioxidant property, and cell migration ability of the prepared composites were investigated with a PC-12 cell line. In-vitro studies revealed that the multifunctional composites show higher cell viability, antioxidant, and cell migration properties than the control cells. Reduction of ROS level indicates that the Hes presence in the composite could reduce the cell stress by protecting it from oxidative damage and promoting cell migration towards the lesion site. The developed multifunctional composite can provide the antioxidant microenvironment with compatibility and mimic the native spinal cord by providing appropriate conductivity and mechanical strength for spinal cord tissue regeneration.