Gellan-Xanthan Hydrogel Conduits with Intraluminal Electrospun Nanofibers as Physical,Chemical and Therapeutic Cues for Peripheral Nerve Repair

Optimal levels of functional recovery in peripheral nerve injuries remain elusive due to the architectural complexity of the neuronal environment. Commercial nerve repair conduits lack essential guidance cues for the regenerating axons. In this study, the regenerative potential of a biosimulated nerve repair system providing three types of regenerative cues was evaluated in a 10 mm sciatic nerve-gap model over 4 weeks. A thermo-ionically crosslinked gellan-xanthan hydrogel conduit loaded with electrospun PHBV-magnesium oleate-N-acetyl-cysteine (PHBV-MgOl-NAC) nanofibers was assessed for mechanical properties, nerve growth factor (NGF) release kinetics and PC12 viability. In vivo functional recovery was based on walking track analysis, gastrocnemius muscle mass and histological analysis. As an intraluminal filler, PHBV-MgOl-NAC nanofibers improved matrix resilience, deformation and fracture of the hydrogel conduit. NGF release was sustained over 4 weeks, governed by Fickian diffusion and Case-II relaxational release for the hollow conduit and the nanofiber-loaded conduit, respectively. The intraluminal fibers supported PC12 proliferation by 49% compared to the control, preserved up to 43% muscle mass and gradually improved functional recovery. The combined elements of physical guidance (nanofibrous scaffolding), chemical cues (N-acetyl-cysteine and magnesium oleate) and therapeutic cues (NGF and diclofenac sodium) offers a promising strategy for the regeneration of severed peripheral nerves.     

PVDF and P(VDF-TrFE) Electrospun Scaffolds for Nerve Graft Engineering: A Comparative Study on Piezoelectric and Structural Properties,and In Vitro Biocompatibility

Polyvinylidene fluoride (PVDF) and its copolymer with trifluoroethylene (P(VDF-TrFE)) are considered as promising biomaterials for supporting nerve regeneration because of their proven biocompatibility and piezoelectric properties that could stimulate cell ingrowth due to their electrical activity upon mechanical deformation. For the first time, this study reports on the comparative analysis of PVDF and P(VDF-TrFE) electrospun scaffolds in terms of structural and piezoelectric properties as well as their in vitro performance. A dynamic impact test machine was developed, validated, and utilised, to evaluate the generation of an electrical voltage upon the application of an impact load (varying load magnitude and frequency) onto the electrospun PVDF (15–20 wt%) and P(VDF-TrFE) (10–20 wt%) scaffolds. The cytotoxicity and in vitro performance of the scaffolds was evaluated with neonatal rat (nrSCs) and adult human Schwann cells (ahSCs). The neurite outgrowth behaviour from sensory rat dorsal root ganglion neurons cultured on the scaffolds was analysed qualitatively. The results showed (i) a significant increase of the β-phase content in the PVDF after electrospinning as well as a zeta potential similar to P(VDF-TrFE), (ii) a non-constant behaviour of the longitudinal piezoelectric strain constant d₃₃, depending on the load and the load frequency, and (iii) biocompatibility with cultured Schwann cells and guiding properties for sensory neurite outgrowth. In summary, the electrospun PVDF-based scaffolds, representing piezoelectric activity, can be considered as promising materials for the development of artificial nerve conduits for the peripheral nerve injury repair.     

Use of Natural Neural Scaffolds Consisting of Engineered Vascular Endothelial Growth Factor Immobilized on Ordered Collagen Fibers Filled in a Collagen Tube for Peripheral Nerve Regeneration in Rats

The search for effective strategies for peripheral nerve regeneration has attracted much attention in recent years. In this study, ordered collagen fibers were used as intraluminal fibers after nerve injury in rats. Vascular endothelial growth factor (VEGF) plays an important role in nerve regeneration, but its very fast initial burst of activity within a short time has largely limited its clinical use. For the stable binding of VEGF to ordered collagen fibers, we fused a collagen-binding domain (CBD) to VEGF through recombinant DNA technology. Then, we filled the ordered collagen fibers-CBD-VEGF targeting delivery system in a collagen tube to construct natural neural scaffolds, which were then used to bridge transected nerve stumps in a rat sciatic nerve transection model. After transplantation, the natural neural scaffolds showed minimal foreign body reactions and good integration into the host tissue. Oriented collagen fibers in the collagen tube could guide regenerating axons in an oriented manner to the distal, degenerating nerve segment, maximizing the chance of target reinnervation. Functional and histological analyses indicated that the recovery of nerve function in the natural neural scaffolds-treated group was superior to the other grafted groups. The guiding of oriented axonal regeneration and effective delivery systems surmounting the otherwise rapid and short-lived diffusion of growth factors in body fluids are two important strategies in promoting peripheral nerve regeneration. The natural neural scaffolds described take advantage of these two aspects and may produce synergistic effects. These properties qualified the artificial nerve conduits as a putative candidate system for the fabrication of peripheral nerve reconstruction devices.     

Peripheral Nerve Regeneration and Muscle Reinnervation

Injured peripheral nerves but not central nerves have the capacity to regenerate and reinnervate their target organs. After the two most severe peripheral nerve injuries of six types, crush and transection injuries, nerve fibers distal to the injury site undergo Wallerian degeneration. The denervated Schwann cells (SCs) proliferate, elongate and line the endoneurial tubes to guide and support regenerating axons. The axons emerge from the stump of the viable nerve attached to the neuronal soma. The SCs downregulate myelin-associated genes and concurrently, upregulate growth-associated genes that include neurotrophic factors as do the injured neurons. However, the gene expression is transient and progressively fails to support axon regeneration within the SC-containing endoneurial tubes. Moreover, despite some preference of regenerating motor and sensory axons to “find” their appropriate pathways, the axons fail to enter their original endoneurial tubes and to reinnervate original target organs, obstacles to functional recovery that confront nerve surgeons. Several surgical manipulations in clinical use, including nerve and tendon transfers, the potential for brief low-frequency electrical stimulation proximal to nerve repair, and local FK506 application to accelerate axon outgrowth, are encouraging as is the continuing research to elucidate the molecular basis of nerve regeneration.     

Nanohydroxyapatite-coated hydroxyethyl cellulose/poly (vinyl) alcohol electrospun scaffolds and their cellular response

The present study focused on the preparation of nanohydroxyapatite (nHA)-coated hydroxyethyl cellulose/polyvinyl alcohol (HEC/PVA) nanofibrous scaffolds for bone tissue engineering application. The electrospun HEC/PVA scaffolds were mineralized via alternate soaking process. FESEM revealed that the nHA was formed uniformly over the nanofibers. The nHA mineralization enhanced the tensile strength and reduced the elongation at breakage of scaffolds. The wettability of the nanofibrous scaffolds was significantly improved. The in vitro biocompatibility of scaffolds was evaluated with human osteosarcoma cells. nHA-coated scaffolds had a favorable effect on the proliferation and differentiation of osteosarcoma cell and could be a potential candidate for bone regeneration.     

New Frontiers in Peripheral Nerve Regeneration: Concerns and Remedies

Topical advances in studying molecular and cellular mechanisms responsible for regeneration in the peripheral nervous system have highlighted the ability of the nervous system to repair itself. Still, serious injuries represent a challenge for the morphological and functional regeneration of peripheral nerves, calling for new treatment strategies that maximize nerve regeneration and recovery. This review presents the canonical view of the basic mechanisms of nerve regeneration and novel data on the role of exosomes and their transferred microRNAs in intracellular communication, regulation of axonal growth, Schwann cell migration and proliferation, and stromal cell functioning. An integrated comprehensive understanding of the current mechanistic underpinnings will open the venue for developing new clinical strategies to ensure full regeneration in the peripheral nervous system.     

Herbally derived polymeric nanofibrous scaffolds for bone tissue regeneration

Hydroxyapatite (HA), the bone mineral and Cissus quadrangularis (CQ), a medicinal plant with osteogenic activity, are attaining increasing interest as a potential therapeutic agent for enhanced bone tissue regeneration. In the present study a synergistic effect of these two agents were analyzed by fabricating PCL‐CQ‐HA nanofibrous scaffolds by electrospinning and compared with PCL‐CQ and PCL (control) nanofibrous scaffolds. Morphology, composition, hydrophilicity, and mechanical properties of the electrospun PCL, PCL‐CQ, PCL‐CQ‐HA nanofibrous scaffolds were examined by Field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), Contact angle and Tensile tests, respectively. The response of human foetal osteoblast cells on these scaffolds were evaluated using MTS assay, alkaline phosphatase activity, alizarin red staining, and osteocalcin expression for bone tissue regeneration. While the observed cellular response to both groups of scaffolds was better than for the control PCL scaffold, the PCL‐CQ‐HA nanofibrous scaffolds provided the most favorable substrate for cell proliferation and mineralization. The results showed that PCL‐CQ‐HA nanofibrous scaffolds had appropriate surface roughness for the osteoblast adhesion, proliferation, and mineralization comparing with other scaffolds. The observed investigation of physicochemical and biological properties suggests that the CQ‐HA loaded PCL nanofibrous scaffolds serve as a potential biocomposite material for bone tissue engineering. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 39835.     

Application of low loading of collagen in electrospun poly[(l‐lactide)‐co‐(ε‐caprolactone)] nanofibrous scaffolds to promote cellular biocompatibility

Electrospinning of various polymers has been used to produce nanofibrous scaffolds that mimic the extracellular matrix and support cell attachment for the potential repair and engineering of nerve tissue. In the study reported here, an electrospun copolymer of l ‐lactide and ε‐caprolactone (67:33 mol%) resulted in a nanofibrous scaffold with average fibre diameter and pore size of 476 ± 88 and 253 ± 17 nm, respectively. Blending with low loadings of collagen (<2.5% w/w) significantly reduced the average diameter and pore size. The uniformity of fibre diameter distributions was supported with increasing collagen loadings. The nanofibrous scaffolds significantly promoted the attachment and proliferation of olfactory ensheathing cells compared to cells exhibiting asynchronous growth. Furthermore, analysis of cell health through mitochondrial activity, membrane leakage, cell cycle progression and apoptotic indices showed that the nanofibrous membranes promoted cell vigour, reducing necrosis. The study suggests that the use of more cost‐effective, low loadings of collagen supports morphological changes in electrospun poly[(l ‐lactide)‐co‐(ε‐caprolactone)] nanofibrous scaffolds, which also support attachment and proliferation of olfactory ensheathing cells while promoting cell health. The results here support further investigation of the electrospinning of these polymer blends as conduits for nerve repair. © 2013 Society of Chemical Industry     

Bio-Scaffolds as Cell or Exosome Carriers for Nerve Injury Repair

Central and peripheral nerve injuries can lead to permanent paralysis and organ dysfunction. In recent years, many cell and exosome implantation techniques have been developed in an attempt to restore function after nerve injury with promising but generally unsatisfactory clinical results. Clinical outcome may be enhanced by bio-scaffolds specifically fabricated to provide the appropriate three-dimensional (3D) conduit, growth-permissive substrate, and trophic factor support required for cell survival and regeneration. In rodents, these scaffolds have been shown to promote axonal regrowth and restore limb motor function following experimental spinal cord or sciatic nerve injury. Combining the appropriate cell/exosome and scaffold type may thus achieve tissue repair and regeneration with safety and efficacy sufficient for routine clinical application. In this review, we describe the efficacies of bio-scaffolds composed of various natural polysaccharides (alginate, chitin, chitosan, and hyaluronic acid), protein polymers (gelatin, collagen, silk fibroin, fibrin, and keratin), and self-assembling peptides for repair of nerve injury. In addition, we review the capacities of these constructs for supporting in vitro cell-adhesion, mechano-transduction, proliferation, and differentiation as well as the in vivo properties critical for a successful clinical outcome, including controlled degradation and re-absorption. Finally, we describe recent advances in 3D bio-printing for nerve regeneration.     

Fabrication and characterization of chitosan/kefiran electrospun nanofibers for tissue engineering applications

Chitosan (CS)-based nanofibrous scaffolds are very promising in tissue engineering applications. However, electrospinning of CS is not possible unless using toxic solvents such as trifluoroacetic acid or by blending with other polymers. In the present study, we investigated CS-based nanofibers' fabrication by blending it with kefiran as a natural polysaccharide. A series of solutions with various CS to kefiran ratios were prepared and underwent electrospinning. The effects of main process parameters, including applied voltage and needle tip-to-collector distance on nanofibers' diameter and morphology, were also studied. Nanofibers containing 80% CS and 20% Kefiran with an average diameter of 81 ± 17 nm were successfully electrospun. Thermogravimetric analysis indicated the presence of both polymers in blend nanofibers. The diameter of CS/kefiran nanofibers increased with enhanced applied voltage, while needle tip-to-collector distance did not significantly affect the mean diameters. Appropriate viability of l929 cells on the obtained scaffolds was demonstrated utilizing Alamar blue assay. Also, cell attachment onto the fiber surface was confirmed by scanning electron microscopy. Results indicated that CS/kefiran nanofibrous scaffolds would be promising for tissue engineering applications.     

Development of poly (L-lactide-co-caprolactone) multichannel nerve conduit with aligned electrospun nanofibers for Schwann cell proliferation

Biomaterials are playing a significant role in understanding and promoting the plasticity and repair of the nervous system. Biomimetic nanofibrous scaffolds mimicking important features of the native extracellular matrix provide a promising strategy to restore functions or achieve favorable responses for tissue regeneration and autograft nerve conduit is one of the most promising nerve regeneration strategies. The present study is based on novel fabrication method by using a special collector for 3D multichannel nerve conduit, longitudinally oriented with aligned electrospun nanofibers. The conduit contained a high number of channels (varying from 7 to 19) and each channel showed a separate morphology. Nerve channels were fabricated with the varying length ranging from 4 to 9 cm and total diameter ranging from 2200 ± 40 µm to 3951 ± 196 µm, while the channel diameter ranging from 350 ± 86 µm to 780 ± 20 µm. It has been clearly shown that the average porosity of nerve conduits has reached almost 89%. In this study, we optimized the parameters to control the structural stability, including the size and the number of channels in the nerve conduit. We also checked in vitro cell biocompatibility of multichannel nerve conduit and demonstrated that Schwann cells have the tendency to grow along the direction of nanofibers and high cell growth was observed in high number of channels compared to low number of channels. These results elaborated the potential use of this biocompatible multichannel nerve conduit for further in vivo testing.     

Poly(l-lactide-co-?-caprolactone) electrospun nanofibers for encapsulating and sustained releasing proteins

The aim of this study was to investigate coaxial electrospun poly(l-lactide-co-?-caprolactone) [PLLACL] nanofibers for the application in nerve tissue engineering. The hypothesis was that the nanofibrous mats fabricated by coaxial electrospun PLLACL could be effective scaffolds for releasing proteins, such as Bovine Serum Albumin (BSA) or/and Nerve Growth Factor (NGF), in a sustained manner. To test the hypothesis, the coaxial electrospun nanofibers with PLLACL as the shell and BSA/NGF as the core were characterized. Morphologies and mechanical properties of nanofibrous mats were examined. BSA released behavior was studied. The results demonstrated that BSA could be sustainedly released from coaxial electrospun PLLACL nanofibers, however, BSA released from mix electrospun nanofibers present the burst release behavior. Bioactivity of released NGF from coaxial electrospun nanofibers was verified by testing the differentiation of rat pheochromocytoma cells (PC12).     

In vitro biocompatibility of electrospun silk fibroin mats with Schwann cells

In this study, electrospinning was used to fabricate silk‐fibroin (SF)‐based mats, which served as substrates for the culturing of rat Schwann cells. Microscopic observation and physical parameter measurements revealed that the electrospun SF mats had a nanofibrous structure with favorable physical properties. Fourier transform infrared analysis provided chemical characterization of the molecular confirmation of the SF proteins in the mats. The morphology and immunocytochemistry showed that the mats supported the survival and growth of the cultured Schwann cells, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide analysis indicated that the electrospun SF mat extract had no cytotoxic effects on Schwann cell proliferation. Collectively, all of the results suggest that the electrospun SF mats might become a candidate scaffold for tissue‐engineered nerve grafts to promote peripheral nerve regeneration. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

The Effect of Electrospun Gelatin Fibers Alignment on Schwann Cell and Axon Behavior and Organization in the Perspective of Artificial Nerve Design

Electrospun fibrous substrates mimicking extracellular matrices can be prepared by electrospinning, yielding aligned fibrous matrices as internal fillers to manufacture artificial nerves. Gelatin aligned nano-fibers were prepared by electrospinning after tuning the collector rotation speed. The effect of alignment on cell adhesion and proliferation was tested in vitro using primary cultures, the Schwann cell line, RT4-D6P2T, and the sensory neuron-like cell line, 50B11. Cell adhesion and proliferation were assessed by quantifying at several time-points. Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers. Schwann cell morphology and organization were investigated by immunostaining of the cytoskeleton. Cells were elongated with their longitudinal body parallel to the aligned fibers. B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers. The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.     

The Role of Dietary Nutrients in Peripheral Nerve Regeneration

Peripheral nerves are highly susceptible to injuries induced from everyday activities such as falling or work and sport accidents as well as more severe incidents such as car and motorcycle accidents. Many efforts have been made to improve nerve regeneration, but a satisfactory outcome is still unachieved, highlighting the need for easy to apply supportive strategies for stimulating nerve growth and functional recovery. Recent focus has been made on the effect of the consumed diet and its relation to healthy and well-functioning body systems. Normally, a balanced, healthy daily diet should provide our body with all the needed nutritional elements for maintaining correct function. The health of the central and peripheral nervous system is largely dependent on balanced nutrients supply. While already addressed in many reviews with different focus, we comprehensively review here the possible role of different nutrients in maintaining a healthy peripheral nervous system and their possible role in supporting the process of peripheral nerve regeneration. In fact, many dietary supplements have already demonstrated an important role in peripheral nerve development and regeneration; thus, a tailored dietary plan supplied to a patient following nerve injury could play a non-negotiable role in accelerating and promoting the process of nerve regeneration.     

Fabrication of althea officinalis loaded electrospun nanofibrous scaffold for potential application of skin tissue engineering

The Althea Officinalis (AO) extract is well known as a traditional herbal drug for its wound healing ability owing to the anti-inflammatory and antimicrobial properties. Furthermore, its mucilaginous properties provide moisturizing and nutritional effects on skin cell proliferation. Therefore, AO extract can be applied in the temporary skin substitute for the ability to expedite the therapy duration. In this study, different concentrations of AO extract (0, 5, 10, 15, and 20 wt %) were incorporated into the nanofibrous scaffolds to study their potential for the skin tissue repairing. The desired scaffolds were prepared by electrospinning the blend of poly(ε -caprolactone) and gelatin as a synthesized and natural polymer. The electrospun nanofibers were characterized by SEM, FTIR, DSC, TGA, tensile, AO extract release, and cellular culture tests. This study proposed incorporating the AO extract into the nanofibrous scaffolds for accelerating the skin tissue repairing and the optimized amount of AO extract as about 15% was introduced for offering the most desirable electrospun scaffolds for this application. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48587.     

Influence of Post-Treatment with 75% (v/v) Ethanol Vapor on the Properties of SF/P(LLA-CL) Nanofibrous Scaffolds

In order to improve the water-resistant ability of silk fibroin (SF) and SF/P(LLA-CL) blended nanofibrous scaffolds for tissue engineering applications, 75% (v/v) ethanol vapor was used to post-treat electrospun nanofibers. SEM indicated that the treated SF and SF/P(LLA-CL) nanofibrous scaffolds maintained a nanofibrous structure and possessed good water-resistant ability. Characterization of (13)C CP-MAS NMR clarified that 75% (v/v) ethanol vapor could induce SF conformation from random coil or α-helix to β-sheet. Although the water contact showed that treated SF/P(LLA-CL) blended nanofibrous scaffolds were hydrophobic, the water uptake demonstrated that their hydrophilicity was greatly superior to those of pure P(LLA-CL) nanofibrous scaffolds. Furthermore, the treated SF/P(LLA-CL) nanofibrous scaffolds, both in dry state and wet state, could retain good mechanical properties. Therefore, 75% (v/v) ethanol vapor treatment might be an ideal method to treat SF and SF/P(LLA-CL) nanofibrous scaffolds for biomedical applications.     

Embryonic Stem Cell Differentiation to Cardiomyocytes on Nanostructured Scaffolds for Myocardial Tissue Regeneration

With recent advances in developmental and stem cell biology, the application of stem cells in tissue engineering has received great attention and designing of suitable scaffolds to support cell growth, differentiation, and functional tissue organization are advancing toward effective tissue regeneration. Regeneration of the infarct myocardium after myocardial infarction (MI), which is caused by the abrupt occlusion of one or more of the coronary arteries in the heart is one of the most demanding aspects in tissue engineering. Embryonic stem cells (ESCs) can differentiate into many cell types and has been considered as a cell source for cardiac regeneration. In this regard, nanofibrous scaffolds received great attention in tissue engineering field due to their similarity in morphology to native extracellular matrix (ECM) and various scaffolds have been studied as cardiac patches over the previous years. In this study poly (ε-caprolactone) (PCL)/gelatin nanofibrous scaffolds were fabricated by electrospinning and embroyonic bodies (EBs) were formed using ESCs seeded on the nanofibrous scaffolds. SEM images revealed cell outgrowth from EBs and the spreading of cells over the nanofibrous scaffolds were observed. Immunocytochemistry results showed the cellular expression of cardiac proteins, namely α-actinin and connexin 43 on the nanofibrous scaffolds indicating the differentiation of EBs to cardiomyocytes. Results of our study showed that PCL/gelatin nanofibrous scaffolds can act as a promising substrate for differentiation of EBs to cardiomyocytes and could be applied for cardiac tissue engineering.     

Enhanced chondrogenic differentiation of human bone marrow mesenchymal stem cells on PCL/PLGA electrospun with different alignments and compositions

The simultaneous effect of electrospun scaffold alignment and polymer composition on chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSC) is investigated. Aligned and randomly oriented polycaprolactone/poly(lactic-co-glycolic acid) (PLGA) hybrid electrospun scaffolds with two different ratios are fabricated by electrospinning. It is found that aligned nanofibrous scaffolds support higher chondrogenic differentiation of hBMMSCs compared to random ones. The aligned scaffolds show a higher expression level of chondrogenic markers such as type II collagen and aggrecan. It is concluded that the aligned nanofibrous scaffold with higher PLGA ratio could significantly enhance hBMMSC proliferation and differentiation to chondrocytes.     

Preparation of electrospun nanofibrous polycaprolactone scaffolds using nontoxic ethylene carbonate and glacial acetic acid solvent system

In recent years, solution electrospinning has attracted the interest of researchers due to the possibility to design nanofibrous scaffolds with large surface area to volume ratios. Polycaprolactone (PCL), because of its biocompatibility and easy processability, has been widely used to develop electrospun structures for tissue engineering. However, the use of organic solvents and the poor PCL solution stability still hinder the development of the solution electrospinning process. The relatively benign glacial acetic acid (GAC) as a solvent of PCL was used to fabricate microfibrous fibers or beaded fibers. Thus, ethylene carbonate (EC) as a nontoxic assistant solvent was added to the PCL/GAC solution to successfully fabricate electrospun nanofibrous PCL scaffolds. The stability of the PCL/GAC/EC solution system was demonstrated as the viscosity, which showed no significant change during 48 h. The ultrafine PCL fiber diameter decreased as EC concentration was increased from 0 to 9 vol% and started to slightly increase when EC concentration increased beyond 9 vol%. MTT assay evidenced that MC3T3-E1 cells on the nanofibrous PCL scaffolds exhibited a better enhancement on cell proliferation. In summary, EC was added in PCL/GAC to establish a stable and low toxic solution electrospinning system, which provides promising strategy in tissue engineering field. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48387.