Scaffolds For Neural Tissue Engineering Research Articles

Bioengineered Conductive Scaffolds for Neural Tissue Engineering

Bioengineered Conductive Scaffolds for Neural Tissue Engineering

Advanced approaches with combination of 2D nanomaterials and 3D printing for exquisite neural tissue engineering

The regeneration of neural tissue presents significant challenges due to the complexity of the nervous system, which can be damaged by traumatic and non-traumatic injuries. Traditional approaches to repairing nerve damage, such as transplantation of auto-, allo-, or xeno-grafts, have limitations. Recently, researchers have been studying the use of two-dimensional (2D) nanomaterials as novel therapeutic approaches for treating neurological disorders. This review first presents the classification of 2D nanoparticles for neural regeneration and their biocompatibility. Thereafter, we discuss the recent approaches combining 2D nanomaterials and three-dimensional (3D) printing to produce hydrogel-based scaffolds for exquisite neural tissue engineering. Furthermore, recent highlighted studies focusing on the combination of 2D nanomaterials and 3D bioprinting for neural tissue engineering are specially introduced. Through this review, we aim to contribute to crafting a range of strategies for the biomedical applications of 3D printing with diverse nanomaterials and hope to encourage further research on understanding the mechanisms of toxicity as well as the synergistic effects of 2D nanomaterials and hydrogel scaffolds for neural tissue engineering and regeneration.

Dual-Function Fibrous Co-Polypeptide Scaffolds for Neural Tissue Engineering.

This paper reports dual-function (high cell attachment and cell viability) fibrous scaffolds featuring aligned fibers, displaying good biocompatibility and no cytotoxicity. These scaffolds are fabricated through the electrospinning of a co-polypeptide comprising molar equivalents of N6 -carbobenzyloxy-l-lysine and γ-benzyl-l-glutamate, with the lysine moieties enhancing cell adhesion and the neural-stimulating glutamate moieties improving cell viability. These new scaffolds allow neural cells to attach and grow effectively without any special surface treatment or coating. Pheochromocytoma (PC-12) cells grown on these scaffolds exhibit better neuronal activity and longer neurite length, relative to those grown on scaffolds prepared from their respective homo-polypeptides. When the scaffolds are partially hydrolyzed such that they present net positive charge and increased hydrophilicity, the cell viability and neurite growth both increase further. Accordingly, these novel co-polypeptide fibrous scaffolds have potential applications in neural tissue engineering.

Recent advances in PLLA-based biomaterial scaffolds for neural tissue engineering: Fabrication, modification, and applications.

Repairing and regenerating injured neural tissue remains a worldwide challenge. Tissue engineering (TE) has been highlighted as a potential solution to provide functional substitutes for damaged organs or tissue. Among the biocompatible and biodegradable materials, poly-L-lactic-acid (PLLA) has been widely investigated in the TE field because of its tunable mechanical properties and tailorable surface functionalization. PLLA-based biomaterials can be engineered as scaffolds that mimic neural tissue extracellular matrix and modulate inflammatory responses. With technological advances, PLLA-based scaffolds can also have well-controlled three-dimensional sizes and structures to facilitate neurite extension. Furthermore, PLLA-based scaffolds have the potential to be used as drug-delivery carriers with controlled release. Moreover, owing to the good piezoelectric properties and capacity to carry conductive polymers, PLLA-based scaffolds can be combined with electrical stimulation to maintain stemness and promote axonal guidance. This mini-review summarizes and discusses the fabrication and modification techniques utilized in the PLLA-based biomaterial scaffolds for neural TE. Recent applications in peripheral nerve and spinal cord regeneration are also presented, and it is hoped that this will guide the future development of more effective and multifunctional PLLA-based nerve scaffolds.

Polymeric Fibers as Scaffolds for Spinal Cord Injury: A Systematic Review.

Spinal cord injury (SCI) is a complex neurological condition caused by trauma, inflammation, and other diseases, which often leads to permanent changes in strength and sensory function below the injured site. Changes in the microenvironment and secondary injuries continue to pose challenges for nerve repair and recovery after SCI. Recently, there has been progress in the treatment of SCI with the use of scaffolds for neural tissue engineering. Polymeric fibers fabricated by electrospinning have been increasingly used in SCI therapy owing to their biocompatibility, complex porous structure, high porosity, and large specific surface area. Polymer fibers simulate natural extracellular matrix of the nerve fiber and guide axon growth. Moreover, multiple channels of polymer fiber simulate the bundle of nerves. Polymer fibers with porous structure can be used as carriers loaded with drugs, nerve growth factors and cells. As conductive fibers, polymer fibers have electrical stimulation of nerve function. This paper reviews the fabrication, characterization, and application in SCI therapy of polymeric fibers, as well as potential challenges and future perspectives regarding their application.

Biocompatible chitosan/polyethylene glycol/multi-walled carbon nanotube composite scaffolds for neural tissue engineering.

Carbon nanotube (CNT) composite materials are very attractive for use in neural tissue engineering and biosensor coatings. CNT scaffolds are excellent mimics of extracellular matrix due to their hydrophilicity, viscosity, and biocompatibility. CNTs can also impart conductivity to other insulating materials, improve mechanical stability, guide neuronal cell behavior, and trigger axon regeneration. The performance of chitosan (CS)/polyethylene glycol (PEG) composite scaffolds could be optimized by introducing multi-walled CNTs (MWCNTs). CS/PEG/CNT composite scaffolds with CNT content of 1%, 3%, and 5% (1%=0.01 g/mL) were prepared by freeze-drying. Their physical and chemical properties and biocompatibility were evaluated. Scanning electron microscopy (SEM) showed that the composite scaffolds had a highly connected porous structure. Transmission electron microscope (TEM) and Raman spectroscopy proved that the CNTs were well dispersed in the CS/PEG matrix and combined with the CS/PEG nanofiber bundles. MWCNTs enhanced the elastic modulus of the scaffold. The porosity of the scaffolds ranged from 83% to 96%. They reached a stable water swelling state within 24 h, and swelling decreased with increasing MWCNT concentration. The electrical conductivity and cell adhesion rate of the scaffolds increased with increasing MWCNT content. Immunofluorescence showed that rat pheochromocytoma (PC12) cells grown in the scaffolds had characteristics similar to nerve cells. We measured changes in the expression of nerve cell markers by quantitative real-time polymerase chain reaction (qRT-PCR), and found that PC12 cells cultured in the scaffolds expressed growth-associated protein 43 (GAP43), nerve growth factor receptor (NGFR), and class III β‍-tubulin (TUBB3) proteins. Preliminary research showed that the prepared CS/PEG/CNT scaffold has good biocompatibility and can be further applied to neural tissue engineering research.

Pyrrole Plasma Polymer-Coated Electrospun Scaffolds for Neural Tissue Engineering.

Promising strategies for neural tissue engineering are based on the use of three-dimensional substrates for cell anchorage and tissue development. In this work, fibrillar scaffolds composed of electrospun randomly- and aligned-oriented fibers coated with plasma synthesized pyrrole polymer, doped and undoped with iodine, were fabricated and characterized. Infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction analysis revealed the functional groups and molecular integration of each scaffold, as well as the effect of plasma polymer synthesis on crystallinity. Scanning microscopy imaging demonstrated the porous fibrillar micrometric structure of the scaffolds, which afforded adhesion, infiltration, and survival for the neural cells. Orientation analysis of electron microscope images confirmed the elongation of neurite-like cell structures elicited by undoped plasma pyrrole polymer-coated aligned scaffolds, without any biochemical stimuli. The MTT colorimetric assay validated the biocompatibility of the fabricated composite materials, and further evidenced plasma pyrrole polymer-coated aligned scaffolds as permissive substrates for the support of neural cells. These results suggest plasma synthesized pyrrole polymer-coated aligned scaffolds are promising materials for tissue engineering applications.

Preparation and in vitro Evaluation of Injectable Alginate/Thiolated Chitosan Hydrogel Scaffold for Neural Tissue Engineering

Introduction: Spinal cord injuries are one of the main causes of disability with devastating neurological consequences and secondary conflicts in other organs. Tissue engineering and regenerative medicine have been recognized as novel, promising methods in the treatment of tissue injuries, especially in neurological damage in recent decades. Hydrogels have the advantage of compatibility with damaged tissue, and injectable hydrogels can be applied in minimally invasive surgeries. This study aimed to evaluate an injectable hydrogel-based scaffold consisting of thiolated chitosan and alginate for neural tissue regeneration. Materials and Methods: In the present study, an injectable hydrogel-based containing thiolated chitosan and alginate was prepared. Microbiology and pH tests were performed. Microstructural properties and porosity of scaffold were evaluated by scanning electron microscope (SEM). The swelling /shrinkage ratio and rates of biodegradation were also conducted. Finally, the viability of L929 cells on the scaffold was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Thiolated chitosan/ alginate hydrogel had low pH with no contamination. SEM showed hydrogel had a porous microstructure with a mean pore diameter of 21.89 ± 0.32 μm which is suitable for cell culture. Furthermore, according to MTT test results, this hydrogel was biocompatible. Conclusion: Thiolated chitosan/ alginate hydrogel is convenient for application in neural tissue engineering based on its structural properties and its ability to support cell proliferation. According to the in vitro analysis, it can also be used as a scaffold to create a suitable environment for increasing cell viability.

Fabrication and characterization of chitosan-based composite scaffolds for neural tissue engineering

With the development of peripheral nerve tissue engineering, study of biodegradable materials have extensive applied prospect. Chitosan (Cs) is a promising candidate for neural tissue engineering due to its similar chemical structure to glycosaminoglycan. However, due to its lack of elasticity and flexibility, Cs often needs to be used in combination with other materials. In this study, three composite scaffolds were prepared by freeze-drying method: Cs/hyaluronic acid (HA)/gelatin (Gel), Cs/collagen (Col), and Cs/polyethylene glycol (PEG). Mechanical properties, swelling behavior, porosity, and conductivity of these materials have been characterized. Compared with pure Cs, the addition of other materials reduced the average pore size, while improved the mechanical properties of the composite scaffold. Furthermore, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), live/dead staining, cell morphology, and immunofluorescence staining were performed to evaluate the cytotoxicity, biocompatibility, and differentiation of rat pheochromocytoma (PC12) cells on Cs/HA/Gel, Cs/Col, and Cs/PEG composite scaffolds. Our results indicated that all these Cs-based composite scaffolds had good biocompatibility without cytotoxicity, while Cs/PEG scaffolds possessed higher cell survival rate and could promote the adhesion, proliferation, and differentiation of PC12 cells. The Cs-based composite scaffolds developed in this study can serve as promising substitutes for neural tissue regeneration.

Strategy for Designing a Cell Scaffold to Enable Wireless Electrical Stimulation for Enhanced Neuronal Differentiation of Stem Cells.

Electrical stimulation (ES) offers significant advantages in modulating the behavior of stem cells on conductive scaffolds for neural tissue engineering. However, it is necessary to realize wireless ES to avoid the use of external wires in tissues. Thus, herein, a strategy is reported to develop a stem cell scaffold that allows wireless ES. A conductive annular graphene substrate is designed and grown by chemical vapor deposition; this substrate is used as a secondary coil to achieve wireless ES via electromagnetic induction in the presence of a primary coil. The substrate shows excellent biocompatibility for the culture of neural stem cells (NSCs). The results indicate that the applied wireless ES enhances neuronal differentiation, facilitates the formation of neurites, and does not substantially affect the viability and stemness maintenance of NSCs. Collectively, this system provides a strategy for achieving synergy between wireless ES and conductive scaffolds for neural regenerative medicine, which can be further utilized for the regeneration of other tissues.

PLA electrospun nanofibers modified with polypyrrole-grafted gelatin as bioactive electroconductive scaffold

Neural tissue engineering is a considerable strategy to generate new viable nerve tissue with the help of a scaffold with appropriate chemical, mechanical, and electrical properties. Surface modification of the electrospun nanofibers can be a suitable approach to increase the electroconductivity and cell attachment features of a scaffold. In this study, a neural tissue engineering scaffold containing polylactic acid (PLA) nanofibers, gelatin, and polypyrrole (PPy) was fabricated to combine electrospun nanofibers' topography benefits with the versatile advantages of gelatin and PPy. To this end, a conductive copolymer was chemically synthesized by grafting various weight ratios of pyrrole on gelatin chains, and then it was grafted on PLA nanofibers’ surface to improve its conductivity and bio interactions. The polypyrrole-grafted gelatin (GP) samples were characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, ultraviolet–visible (UV–vis) spectroscopy, and two-point probe conductivity test. The chemical structure, elemental composition, surface hydrophilicity, surface conductivity, and morphology of the modified scaffolds were evaluated by ATR-FTIR, energy-dispersive X-ray (EDX) spectroscopy, contact angle, two-point probe conductivity, and scanning electron microscopy (SEM) analyses, respectively. Likewise, In vitro studies including [(3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)] (MTT) and cell adhesion investigations were performed using pheochromocytoma (PC12) cells. The results revealed that scaffolds containing 15 and 20% polypyrrole could provide acceptable conditions for the adhesion and growth of nerve cells and can be introduced as a promising scaffold in nerve tissue engineering applications.

Design of peptide-PEG-Thiazole bound polypyrrole supramolecular assemblies for enhanced neuronal cell interactions

ABSTRACT Brain injuries, and neurodegenerative diseases can result in significant disability, lowered quality of life, and mortality. Current treatments including autologous nerve grafts are insufficient. This work presents the creation of new biomimetic supramolecular assemblies as a scaffold for potential applications in neural tissue regeneration. It is composed of polyethylene glycol amide core-conjugated with a thiazole-based building block and a peptide sequence (RVYNMGKGKGFCVPRPLVVYR). This peptide sequence is derived from laminin, a basal membrane protein, and a component of the extracellular matrix of neuronal cells. The product formed was allowed to self-assemble and bound to polypyrrole by layer-by-layer assembly to impart conductive properties (Lam-PEG-thiazole-PPy) resulting in the formation of fibrillar assemblies as indicated by electron microscopy studies. Rheological studies demonstrated that incorporation of PPy resulted in higher storage modulus indicative of higher ability to store deformation energy. Culture of rat olfactory-bulb derived neurons with the assemblies demonstrated that the Lam-PEG-thiazole-PPy assemblies promoted cell proliferation and were able to interact favorably with the cells. After 10 days of culture with the assemblies interconnected networks could be visualized. Furthermore, electrical stimulation resulted in a significant increase in axonal outgrowths. Thus, the assemblies presented herein exhibit potential for applications as a novel scaffold for neural tissue engineering.

Fabrication of nanochitosan incorporated polypyrrole/alginate conducting scaffold for neural tissue engineering

The utilization of conductive polymers for fabrication of neural scaffolds have attracted much interest because of providing a microenvironment which can imitate nerve tissues. In this study, polypyrrole (PPy)–alginate (Alg) composites were prepared using different percentages of alginate and pyrrole by oxidative polymerization method using FeCl3 as an oxidant and electrical conductivity of composites were measured by four probe method. In addition, chitosan-based nanoparticles were synthesized by ionic gelation method and after characterization merged into PPy–Alg composite in order to fabricate a conductive, hydrophilic, processable and stable scaffold. Physiochemical characterization of nanochitosan/PPy–Alg scaffold such as electrical conductivity, porosity, swelling and degradation was investigated. Moreover, cytotoxicity and proliferation were examined by culturing OLN-93 neural and human dermal fibroblasts cells on the Nanochitosan/PPy–Alg scaffold. Due to the high conductivity, the film with ratio 2:10 (PPy–Alg) was recognized more suitable for fabrication of the final scaffold. Results from FT-IR and SEM, evaluation of porosity, swelling and degradation, as well as viability and proliferation of OLN-93 neural and fibroblast cells confirmed cytocompatiblity of the Nanochitosan/PPy–Alg scaffold. Based on the features of the constructed scaffold, Nanochitosan/PPy–Alg scaffold can be a proper candidate for neural tissue engineering.

Self-Assembly of a Multi-Functional Hydrogel from a Branched Peptide Amphiphile and Its Effects on Bone Marrow Mesenchymal Stem Cells

In this work, a branched peptide amphiphile (B-PA) presenting RGD and IKVAV motifs was fabricated by solid-phase peptide synthesis and self-assembled into a nanofiber hydrogel in which rabbit bone marrow mesenchymal stem cells (BMSCs) were seeded and cultured for seven days. Specifically, 1 wt% B-PA was self-assembled into a nanofiber hydrogel with the addition of culture medium and observed using transmission electron microscopy. The B-PA with a molecular weight of 2191.72 and a purity >95% self-assembled into nanofibers with diameters from 6 to 8 nm and lengths ranging from hundreds of nanometers to several micrometers. BMSCs were acquired from rabbits using differential adherence methods and identified by flow cytometry for cell phenotype. The cells were stained with calcein acetoxymethyl ester/propidium iodide to assess cell viability, CCK-8 to assess cell cytotoxicity and proliferation, and Hochest 33342 to assess cell adhesion. They were also immunofluorescently labeled with microtubule-associated protein-2 (MAP-2), neurofilament protein (NF), and glial fibrillary acidic protein (GFAP) to assess cell transdifferentiation. The B-PA hydrogel provided platform upon which the CD29+/44+ cells adhered and proliferated, and it induced the transdifferentiation of cells into neural cells expressing the markers MAP-2, NF, and GFAP. The hydrogel exhibited good cytocompatibility and multiple functions, and may therefore serve as a scaffold for neural tissue engineering.

Progress in the Applications of Smart Piezoelectric Materials for Medical Devices.

Smart piezoelectric materials are of great interest due to their unique properties. Piezoelectric materials can transform mechanical energy into electricity and vice versa. There are mono and polycrystals (piezoceramics), polymers, and composites in the group of piezoelectric materials. Recent years show progress in the applications of piezoelectric materials in biomedical devices due to their biocompatibility and biodegradability. Medical devices such as actuators and sensors, energy harvesting devices, and active scaffolds for neural tissue engineering are continually explored. Sensors and actuators from piezoelectric materials can convert flow rate, pressure, etc., to generate energy or consume it. This paper consists of using smart materials to design medical devices and provide a greater understanding of the piezoelectric effect in the medical industry presently. A greater understanding of piezoelectricity is necessary regarding the future development and industry challenges.

Modulation of Neuronal Cell Affinity on PEDOT-PSS Nonwoven Silk Scaffolds for Neural Tissue Engineering.

Peripheral nerve injury is a common consequence of trauma with low regenerative potential. Electroconductive scaffolds can provide appropriate cell growth microenvironments and synergistic cell guidance cues for nerve tissue engineering. In the present study, electrically conductive scaffolds were prepared by conjugating poly (3,4-ethylenedioxythiophene)-polystyrene sulfonate (PEDOT-PSS) or dimethyl sulfoxide (DMSO)-treated PEDOT-PSS on electrospun silk scaffolds. Conductance could be tuned by the coating concentration and was further boosted by DMSO treatment. Analogue NG108-15 neuronal cells were cultured on the scaffolds to evaluate neuronal cell growth, proliferation, and differentiation. Cellular viability was maintained on all scaffold groups while showing comparatively better metabolic activity and proliferation than neat silk. DMSO-treated PEDOT-PSS functionalized scaffolds partially outperformed their PEDOT-PSS counterparts. Differentiation assessments suggested that these PEDOT-PSS assembled silk scaffolds could support neurite sprouting, indicating that they show promise to be used as a future platform to restore electrochemical coupling at the site of injury and preserve normal nerve function.

Increased neuritogenesis on ternary nanofiber matrices of PLCL and laminin decorated with black phosphorus

Recently, the composites of varying biomaterials and two-dimensional (2D) nanomaterials have been increasingly developed in the field of tissue engineering and regeneration owing to their excellent physicochemical and mechanical properties. In particular, the combination of biocompatible polymers and 2D nanomaterials can endow the nanocomposite with novel biofunctional properties while maintaining the inherent character of every single material. These nanocomposites are known to have the potential for improvement of cellular behaviors such as cell adhesion, proliferation, and differentiation. In this study, poly(l-lactide-co-ε-caprolactone) (PLCL) and laminin (Lam) nanofibers functionalized with black phosphorus (BP) were successfully fabricated to increase the neuritogenesis of HT22 hippocampal neuronal cells using an electrospinning process. The ternary nanofiber matrices of PLCL/Lam/BP were found to be suitable for supporting the attachment and proliferation of hippocampal neurons. Furthermore, these PLCL/Lam/BP nanofiber matrices could not only promote neurite outgrowth and alignment but also increase the neuritogenesis of hippocampal neurons by providing optimal microenvironments for neuronal differentiation. The underlying mechanism of this phenomenon was confirmed by determining the expression levels of genes related to neurogenesis. In conclusion, these results suggest that BP-functionalized composite nanofibers could be a promising candidate as a scaffold for neural tissue engineering and regeneration.

An Electroactive Oligo-EDOT Platform for Neural Tissue Engineering.

The unique electrochemical properties of the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) make it an attractive material for use in neural tissue engineering applications. However, inadequate mechanical properties, and difficulties in processing and lack of biodegradability have hindered progress in this field. Here, the functionality of PEDOT:PSS for neural tissue engineering is improved by incorporating 3,4-ethylenedioxythiophene (EDOT) oligomers, synthesized using a novel end-capping strategy, into block co-polymers. By exploiting end-functionalized oligoEDOT constructs as macroinitiators for the polymerization of poly(caprolactone), a block co-polymer is produced that is electroactive, processable, and bio-compatible. By combining these properties, electroactive fibrous mats are produced for neuronal culture via solution electrospinning and melt electrospinning writing. Importantly, it is also shown that neurite length and branching of neural stem cells can be enhanced on the materials under electrical stimulation, demonstrating the promise of these scaffolds for neural tissue engineering.

Double coating of graphene oxide–polypyrrole on silk fibroin scaffolds for neural tissue engineering

The desired scaffolds for neural tissue engineering need to have electrical conductivity. In this study, we doubly coated graphene oxide and polypyrrole on silk fibroin scaffolds (SF@GO-PPY) by a facile method to improve its electrical conductivity. The graphene oxide–polypyrrole double coating was distributed homogeneously on silk fibroin scaffolds. Compared with silk fibroin scaffolds, the SF@GO-PPY scaffold showed higher electrical conductivity, electrochemical property, mechanical property, and thermal stability. The π–π stacking interaction between polypyrrole and graphene oxide might contribute to the superior conductive and electrochemical property of the SF@GO-PPY scaffold. Moreover, in vitro cell experiment carried out on SH-SY5Y cells showed no cytotoxicity of all the scaffolds. Thus, the results indicated that the SF@GO-PPY scaffold might be a suitable candidate for the application in neural regeneration field.

Nebulized jet-based printing of bio-electrical scaffolds for neural tissue engineering: a feasibility study

In this paper we investigate the application of a direct writing technique for printing conductive patterns onto a biocompatible electrospun-pyrolysed carbon-fibre-based substrate. The result is a first study towards the production of bio-electrical scaffolds that could be used to enhance the promotion of efficient connections among neurons for in vitro studies in the field of neural tissue engineering. An electrospinning process is employed for production of the materials derived from the precursor polyacrylonitrile, in which the embedding of carbon nanotubes (CNTs) is also investigated. Subsequently, the methodology of research into suitable parameters for the printed electronics, using a commercial silver nanoparticle (Øavg,particle size ∼ 100 nm) ink, is described. The results show values of 2 Ω cm for the resistivity of the carbon-fibre materials and conductive printed lines of resistance ∼50 Ω on glass and less than ∼140 Ω on carbon-fibre samples. Biocompatibility results demonstrate the possibility of using electrospun-pyrolysed mats, also with embedded CNTs, as potential neural substrates for spatially localized electrical stimulation across a tissue. In addition, the data concerning the potential toxicity of silver suspensions are in accordance with the literature, showing a dose-dependent behaviour. This work is a pioneering feasibility study of the use of the flexible and versatile printed electronic approach, combined with engineered biocompatible substrates, to realize integrated bio-electrical scaffolds for in vitro neural tissue engineering applications.