An Amphiphilic Sulfonatocalix[5]arene as an Activator for Membrane Transport of Lysine-rich Peptides and Proteins.

Lysine (K) is an important target residue for protein and peptide delivery across membranes. K is the most frequently exposed residue in proteins, leading to high demand for the development of K-compatible transport activators. However, designing activators for K-rich peptides and proteins is more challenging than for arginine-rich species because of the kosmotropic nature of K and its recognition difficulty. In this study, we designed a new amphiphilic sulfonatocalix[5]arene (sCx5-6C) as a K-compatible transport activator. sCx5-6C was tailored with two key elements, recognition of K and the ability to embed into membranes. We measured the membrane transport efficiencies of α-poly-l-lysine, heptalysine, and histones across artificial membranes and of α-poly-l-lysine into live cells, activated by sCx5-6C. The results demonstrate that sCx5-6C acts as an efficient activator for translocating K-rich peptides and proteins, which cannot be achieved by known arginine-compatible activators.

Two-in-One Chemogene Assembled from Drug-Integrated Antisense Oligonucleotides To Reverse Chemoresistance.

Combinatorial chemo and gene therapy provides a promising way to cure drug-resistant cancer, since the codelivered functional nucleic acids can regulate drug resistance genes, thus restoring sensitivity of the cells to chemotherapeutics. However, the dramatic chemical and physical differences between chemotherapeutics and nucleic acids greatly hinder the design and construction of an ideal drug delivery system (DDS) to achieve synergistic antitumor effects. Herein, we report a novel approach to synthesize a nanosized DDS using drug-integrated DNA with antisense sequences (termed "chemogene") to treat drug-resistant cancer. As a proof of concept, floxuridine (F), a typical nucleoside analog antitumor drug, was incorporated in the antisense sequence in the place of thymine (T) based on their structural similarity. After conjugation with polycaprolactone, a spherical nucleic acid (SNA)-like two-in-one chemogene can be self-assembled, which possesses the capabilities of rapid cell entry without the need for a transfection agent, efficient downregulation of drug resistance genes, and chronic release of chemotherapeutics for treating the drug-resistant tumors in both subcutaneous and orthotopic liver transplantation mouse models.

A Crosslinked Nucleic Acid Nanogel for Effective siRNA Delivery and Antitumor Therapy.

Functional siRNAs are employed as cross-linkers to direct the self-assembly of DNA-grafted polycaprolactone (DNA-g-PCL) brushes to form spherical and nanosized hydrogels via nucleic acid hybridization in which small interfering RNAs (siRNAs) are fully embedded and protected for systemic delivery. Owing to the existence of multivalent mutual crosslinking events inside, the crosslinked nanogels with tunable size exhibit not only good thermostability, but also remarkable physiological stability that can resist the enzymatic degradation. As a novel siRNA delivery system with spherical nucleic acid (SNA) architecture, the crosslinked nanogels can assist the delivery of siRNAs into different cells without any transfection agents and achieve the gene silencing effectively both in vitro and in vivo, through which a significant inhibition of tumor growth is realized in the anticancer treatment.

Fluorescent strategy based on cationic conjugated polymer fluorescence resonance energy transfer for the quantification of 5-(hydroxymethyl)cytosine in genomic DNA.

DNA methylation is dynamically reprogrammed during early embryonic development in mammals. It can be explained partially by the discovery of 5-(hydroxymethyl)cytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC), which are identified as key players involved in both active and passive demethylation pathways. As one of the ten-eleven translocation oxidation products, 5-hmC was found relatively abundant in neuron cells and embryonic stem cells. Herein we report a new method for 5-hmC quantification in genomic DNA based on CCP-FRET (cationic conjugated polymers act as the energy donor and induce fluorescence resonance energy transfer) assay combined with KRuO4 oxidation. 5-hmC in genomic DNA can be selectively transformed into 5-fC by the oxidation of KRuO4 and then labeled with hydroxylamine-BODIPY (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore through the reaction between 5-fC and hydroxylamine-BODIPY. After the fluorescently labeled DNA was captured by CCP through electrostatic interactions, a significant FRET between CCP and hydroxylamine-BODIPY fluorophore was observed. This CCP-FRET-based assay benefits from light-harvesting, large Stokes shift, and optical signal amplification properties of the CCP. Furthermore, this CCP-FRET-based assay was quite successfully demonstrated for the 5-hmC quantification in three types of cells (mESc, HeLa, HEK 293T), providing a much more convenient choice for 5-hmC quantification in genomic DNA.

Complexing the Pre-assembled Brush-like siRNA with Poly(ß-amino ester) for Efficient Gene Silencing.

Small interfering RNA (siRNA) has been emerging as a highly selective and effective pharmaceutics for treating broad classes of diseases. However, the practical application of siRNA agent is often hampered by its poor crossing of the cellular membrane barrier and ineffective releasing from endosome to cytoplasm, leading to low gene silencing efficacy for clinical purposes. Thus far, cationic lipid and polymer-based vectors have been extensively explored for gene delivery. Yet condensing the rigid and highly negatively charged siRNA duplex to form a stable complex vehicle usually requires a large load of cationic carriers, prone to raising the toxicity issue for delivery. Herein, we develop a simple strategy that can efficiently condense the siRNAs into nanoparticle vehicles for target gene regulation. In this approach, we first employ a DNA-grafted polycaprolactone (DNA-g-PCL) brush as template to organize the small rigid siRNAs into a large brush-like structure (siRNA-brush) through nucleic acid hybridization. Then, the siRNA-brush assembly is condensed by an ionizable and biodegradable polymer (poly(ß-amino ester), PBAE) under acidic buffer condition to form a stable nanoparticle for siRNA delivery. Compared to the free siRNAs with poor complexing capability with PBAE, the large brush-like siRNA assemblies with more complicated topological architecture significantly promotes their electrostatic interaction with PBAE, enabling the formation of complexed nanoparticles at low weight ratio of polymer to siRNA. Additionally, PBAE/siRNA-brush complexes exhibit good biocompatibility and stability under physiological condition, as well as enhanced cellular internalization. When equipped with functional siRNAs, the obtained delivery system demonstrates excellent downregulation of target genes both in vitro and in vivo, through which the progression of hypertrophic scars can be retarded with negligible adverse effects in an xenografted mouse model.

A Virus-Mimicking Nucleic Acid Nanogel Reprograms Microglia and Macrophages for Glioblastoma Therapy.

Immunotherapy is recognized as one of the most promising approaches to treat cancers. However, its effect in glioblastoma (GBM) treatment is insufficient, which can in part be attributed to the immunosuppressive tumor microenvironment (TME). Microglia and macrophages are the main immune infiltrating cells in the TME of GBM. Unfortunately, instead of initiating the anti-tumor response, GBM-infiltrating microglia and macrophages switch to a tumor-promoting phenotype (M2), and support tumor growth, angiogenesis, and immunosuppression by the release of cytokines. In this work, a virus-mimicking membrane-coated nucleic acid nanogel Vir-Gel embedded with therapeutic miRNA is developed, which can reprogram microglia and macrophages from a pro-invasive M2 phenotype to an anti-tumor M1 phenotype. By mimicking the virus infection process, Vir-Gel significantly enhances the targetability and cell uptake efficiency of the miR155-bearing nucleic acid nanogel. In vivo evaluations demonstrate that Vir-Gel apparently prolongs the circulation lifetime of miR155 and endows it with an active tumor-targeting capability and excellent tumor inhibition efficacy. Owing to its noninvasive feature and effective delivery capability, the virus-mimicking nucleic acid nanogel provides a general and convenient platform that can successfully treat a wide range of diseases.

Nanobody-guided targeted delivery of microRNA via nucleic acid nanogel to inhibit the tumor growth.

MicroRNAs (miRNAs) play crucial roles in maintaining normal physiological processes by regulating gene expression network and thus the tumor-suppressive miRNA has emerged as a promising antitumor agent for cancer treatment. However, targeted delivery of miRNA remains a challenge owing to its intrinsic macromolecular and negatively-charged features. Herein, we first employ the miRNA as crosslinker to construct a nucleic acid nanogel, in which miRNA is embedded and protected inside the three-dimensional (3D) nanostructure. Thereafter, nanobody (Nb) conjugated DNA (Nb-DNA) strands are further loaded on nanogel surface through nucleic acid hybridization, to form a Nb-functionalized nanogel (Nb-nanogel) for tumor-targeted miRNA delivery and antitumor treatment. Both in vitro and in vivo experiments show that nanogel equipped with Nb targeting moieties can greatly promote the miRNA accumulation at the tumor site and cellular uptake efficiency, resulting in significant improvement of the miRNA-mediated antitumor efficacy. This research provides a new approach for targeted miRNA delivery and may pave a new avenue to realize efficient miRNA replacement therapy for cancer treatment.

Polydopamine-coated nucleic acid nanogel for siRNA-mediated low-temperature photothermal therapy.

Photothermal therapy (PTT) normally requires to maintain the temperature of tumor lesions above 50 °C, which potentially induces local inflammation and tumor metastasis. To avoid these side effects, it is vital to achieve effective antitumor efficacy at relatively low temperature (42-45 °C) during the PTT treatment. Herein, we design a polydopamine (PDA)-coated nucleic acid nanogel as a therapeutic complex for siRNA-mediated low-temperature PTT. First, siRNAs that target the heat-shock-protein 70 (Hsp70) serve as crosslinkers to guide the DNA-grafted polycaprolactone (DNA-g-PCL) assemble into nanosized hydrogel particles through nucleic acid hybridization. Thereafter, the obtained siRNA-embedded nanogels are further coated with a thin layer of polydopamine, which not only protects the nanogels against enzymatic degradation but also endows the nanogels with excellent photothermal conversion capacity under near infrared (NIR) light irradiation. After surface PEGylation, this triple shield siRNA delivery complex shows the capability of effective ablating the tumor under relatively mild condition.

In vitro biocompatibility of chitosan-based materials to primary culture of hippocampal neurons.

The natural biomaterial chitosan has been widely used as a promising nerve guidance conduit material for peripheral nerve repair. This study aimed to investigate in vitro biocompatibility of chitosan to primarily cultured hippocampal neurons, one type of central nervous system (CNS) cells. The substrate made up of chitosan fibers or membranes was found to support the survival and growth of the attached hippocampal neurons by using light and electron microscopy as well as immunocytochemistry for neurofilament 200, growth-associated protein-43, microtubule-associated protein 2, beta-tubulin III and synaptophysin. MTT assay indicated that the cell viability of hippocampal neurons in chitosan fiber or membrane extract was not significantly different from that in hydroxyapatite extract or plain neuronal medium, but significantly higher than that in organotin extract after culture for different times. Western analysis revealed that no significant difference in the protein level of growth-associated protein-43 and beta-tubulin III was detected between hippocampal neurons cultured in chitosan extract and in plain neuronal culture medium. The results collectively demonstrate that chitosan is biocompatible to primary culture of hippocampal neurons without cytotoxic effects on cell phenotype and functions, raising a potential possibility of using chitosan for CNS therapy.

A non-cationic nucleic acid nanogel for the delivery of the CRISPR/Cas9 gene editing tool.

Herein, we report a non-cationic DNA-crosslinked nanogel for intracellular delivery of a Cas9 and single guide RNA (Cas9/sgRNA) complex. A DNA-grafted polycaprolactone brush (DNA-g-PCL) is first loaded with the Cas9/sgRNA complex and then crosslinked by DNA linkers via nucleic acid hybridization to form a nanosized hydrogel, in which the gene editing tools are embedded and protected inside. With compact architecture, the Cas9/sgRNA complex-containing nanogel exhibited excellent physiological stability against nuclease digestion and enhanced cellular uptake efficiency, making the delivery system a promising tool for target genome editing.

pH-Responsive and Gemcitabine-Containing DNA Nanogel To Facilitate the Chemodrug Delivery.

Herein, we construct a structure-switchable gemcitabine (Ge)-containing DNA nanogel that can respond to the intracellular acidic environment, subsequently facilitating the chemodrug release inside the cells. Based on the structural similarity between Ge and deoxycytidine (dC), dC nucleotides in the component DNA strands used for nanogel assembly are fully replaced by Ge during their synthesis. By changing the designed sequences, two Ge-containing Y-shaped motifs with different sticky ends are first assembled and then associated together to form nanogel by sticky-end hybridizations. In particular, one of the sticky-end sequences is arbitrarily designed to be rich of Ge and the other is designed to be partially complementary to the first Ge-rich sticky end. At the neutral or basic condition, the Ge-rich sticky ends hybridize with the partially complementary sticky ends on the second Y motifs, keeping the assembled nanogel stable. Upon being exposed to the acidic condition, Ge-rich sticky ends intend to form intramolecular i-motif-like quadruplex structures, resulting in the disassembly of the nanogel. On the one hand, the nanosized feature enables the Ge-containing nanogel with rapid cellular uptake behavior. On the other hand, the pH-responsive feature endows the rapid disassembly of the nanogel to facilitate the enzymatic drug release inside the cell, resulting in the enhanced anticancer activity of the DNA-based drug delivery system.

Promoting the Delivery of Nanoparticles to Atherosclerotic Plaques by DNA Coating.

Many nanoparticle-based carriers to atherosclerotic plaques contain peptides, lipoproteins, and sugars, yet the application of DNA-based nanostructures for targeting plaques remains infrequent. In this work, we demonstrate that DNA-coated superparamagnetic iron oxide nanoparticles (DNA-SPIONs), prepared by attaching DNA oligonucleotides to poly(ethylene glycol)-coated SPIONs (PEG-SPIONs), effectively accumulate in the macrophages of atherosclerotic plaques following an intravenous injection into apolipoprotein E knockout (ApoE-/-) mice. DNA-SPIONs enter RAW 264.7 macrophages faster and more abundantly than PEG-SPIONs. DNA-SPIONs mostly enter RAW 264.7 cells by engaging Class A scavenger receptors (SR-A) and lipid rafts and traffic inside the cell along the endolysosomal pathway. ABS-SPIONs, nanoparticles with a similarly polyanionic surface charge as DNA-SPIONs but bearing abasic oligonucleotides also effectively bind to SR-A and enter RAW 264.7 cells. Near-infrared fluorescence imaging reveals evident localization of DNA-SPIONs in the heart and aorta 30 min post-injection. Aortic iron content for DNA-SPIONs climbs to the peak (∼60% ID/g) 2 h post-injection (accompanied by profuse accumulation in the aortic root), but it takes 8 h for PEG-SPIONs to reach the peak aortic amount (∼44% ID/g). ABS-SPIONs do not appreciably accumulate in the aorta or aortic root, suggesting that the DNA coating (not the surface charge) dictates in vivo plaque accumulation. Flow cytometry analysis reveals more pronounced uptake of DNA-SPIONs by hepatic endothelial cells, splenic macrophages and dendritic cells, and aortic M2 macrophages (the cell type with the highest uptake in the aorta) than PEG-SPIONs. In summary, coating nanoparticles with DNA is an effective strategy of promoting their systemic delivery to atherosclerotic plaques.

Repairing a 35-mm-long median nerve defect with a chitosan/PGA artificial nerve graft in the human: a case study.

We have developed a chitosan/polyglycolic acid (PGA) artificial nerve graft which was previously used for bridge implantation of dog sciatic nerves across 30-mm long defects. Here we describe a clinical trial of this graft for repairing a 35-mm-long median nerve defect at elbow of a human patient. During the 3-year follow-up period, functional recovery of the injured median nerve was assessed by pinch gauge test, hydraulic hand dynamometry, static two-point discrimination and touch test with monofilaments, in couple with electrophysiological examinations. The motor and sensory function of the median nerve demonstrated an ongoing recovery postimplantation, reaching M4 and S3+ levels during the follow-up period. The results indicate that the chitosan/PGA artificial nerve graft could be used for surgical repair of larger defects in major peripheral nerves at a higher level in the human.

Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro.

Silk-based materials have been used in the field of bone or ligament tissue engineering. In order to explore the feasibility of using purified silk fibroin to construct artificial nerve grafts, it is necessary to evaluate the biocompatibility of silk fibroin material with peripheral nerve tissues and cells. We cultured rat dorsal root ganglia (DRG) on the substrate made up of silk fibroin fibers and observed the cell outgrowth from DRG during culture by using light and electron microscopy coupled with immunocytochemistry. On the other hand, we cultured Schwann cells from rat sciatic nerves in the silk fibroin extract fluid and examined the changes of Schwann cells after different times of culture. The results of light microscopy, MTT test and cell cycle analysis showed that Schwann cells cultured in the silk fibroin extract fluid showed no significant difference in their morphology, cell viability and proliferation as compared to that in plain L15 medium. Furthermore, no significant difference was found in expression of the factors secreted by Schwann cells, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and S-100, between Schwann cells cultured in the silk fibroin extraction fluid and in plain L15 medium by the aid of immunocytochemistry, RT-PCR and Western analysis. Collectively, these data indicate that silk fibroin has good biocompatibility with DRG and is also beneficial to the survival of Schwann cells without exerting any significant cytotoxic effects on their phenotype or functions, thus providing an experimental foundation for the development of silk fibroin as a candidate material for nerve tissue engineering applications.

Influence of Poly(L-Lactic Acid) Aligned Nanofibers on PC12 Differentiation.

The aim of this study was to unveil the mechanism by which aligned nanofibers influence neuronal differentiation. PC12 cells were seeded on three different poly(L-lactic acid) (PLLA) substrates (PLLA films (control), electrospun PLLA random nanofibers (RF) and electrospun PLLA aligned nanofibers (AF)). Subsequently, cellular experiments, cDNA microarrays and molecular biological approaches were employed to investigate the impacts of the different PLLA substrates on PC12 cell differentiation. Scanning electron microscope observation revealed that neurite outgrowth in the AF group was parallel to the direction of nanofiber alignment and that the filopodias at the neurite tips spread along the aligned nanofiber axis. Meanwhile, both neurite length and the expression of GAP43 (a neuronal differentiation marker gene) were higher in the AF group than those in the control and RF groups. These results suggested that the PLLA aligned nanofibers enhanced PC12 cell differentiation. cDNA microarray experiment revealed that 876 and 1937 genes had significantly changed expression in the RF and AF groups, respectively. Based on gene ontology analysis, 493 and 1193 differentially expressed genes involved in neuronal differentiation were found in the RF and AF groups, respectively. Pathway analysis showed that the PLLA aligned nanofibers mainly mediated their effects via integrin-mediated pathways. qRT-PCR and western blotting assays further confirmed that gene and protein expression levels in the integrin-mediated FAK-MEK-ERK pathway (e.g., Tln1, Mapk6, phosphorylated-ERK1/2) were enhanced by the PLLA aligned nanofibers. Both PC12 cell differentiation and the expressions of genes and proteins in the integrin-mediated FAK-MEK-ERK pathway were inhibited when integrins were blocked by the pentapeptide GRGDS. In addition, the Pafah1b-1 gene was found to be involved in PLLA aligned nanofibers' promotion of PC12 cell differentiation. Taken together, the results suggested that PLLA aligned nanofibers might cooperate with nerve growth factor (NGF) to induce PC12 cell differentiation by activating the integrin-mediated FAK-MEK-ERK pathway and the Pafah1b1 gene.

microRNA regulatory mechanism by which PLLA aligned nanofibers influence PC12 cell differentiation.

OBJECTIVE: Aligned nanofibers (AFs) are regarded as promising biomaterials in nerve tissue engineering. However, a full understanding of the biocompatibility of AFs at the molecular level is still challenging. Therefore, the present study focused on identifying the microRNA (miRNA)-mediated regulatory mechanism by which poly-L-lactic acid (PLLA) AFs influence PC12 cell differentiation. APPROACH: Firstly, the effects of PLLA random nanofibers (RFs)/AFs and PLLA films (control) on the biological responses of PC12 cells that are associated with neuronal differentiation were examined. Then, SOLiD sequencing and cDNA microarray were employed to profile the expressions of miRNAs and mRNAs. The target genes of the misregulated miRNAs were predicted and compared with the mRNA profile data. Functions of the matched target genes (the intersection between the predicted target genes and the experimentally-determined, misregulated genes) were analyzed. MAIN RESULTS: The results revealed that neurites spread in various directions in control and RF groups. In the AF group, most neurites extended in parallel with each other. The glucose consumption and lactic acid production in the RF and AF groups were higher than those in the control group. Compared with the control group, 42 and 94 miRNAs were significantly dysregulated in the RF and AF groups, respectively. By comparing the predicted target genes with the mRNA profile data, five and 87 matched target genes were found in the RF and AF groups, respectively. Three of the matched target genes in the AF group were found to be associated with neuronal differentiation, whereas none had this association in the RF group. The PLLA AFs induced the dysregulation of miRNAs that regulate many biological functions, including axonal guidance, lipid metabolism and long-term potentiation. In particular, two miRNA-matched target gene-biological function modules associated with neuronal differentiation were identified as follows: (1) miR-23b, miR-18a, miR-107 and miR-103 regulate the Rras2 and Nf1 gene and thereby, affect cytoskeleton regulation and MAPK pathway; (2) miR-92a, miR-339-5p, miR-25, miR-125a-5p, miR-351 and miR-19b co-regulate the Pafah1b1 gene, affecting PC12 cell migration and differentiation. SIGNIFICANCE: This work demonstrates a bioinformatic approach to accomplish miRNA-mRNA profile integrative analysis and provides more insights for understanding the regulatory mechanism of miRNA in AFs affecting neuronal differentiation. These findings will be greatly beneficial for the application and design of AFs in nerve tissue engineering.

Neural tissue engineering options for peripheral nerve regeneration.

Tissue engineered nerve grafts (TENGs) have emerged as a potential alternative to autologous nerve grafts, the gold standard for peripheral nerve repair. Typically, TENGs are composed of a biomaterial-based template that incorporates biochemical cues. A number of TENGs have been used experimentally to bridge long peripheral nerve gaps in various animal models, where the desired outcome is nerve tissue regeneration and functional recovery. So far, the translation of TENGs to the clinic for use in humans has met with a certain degree of success. In order to optimize the TENG design and further approach the matching of TENGs with autologous nerve grafts, many new cues, beyond the traditional ones, will have to be integrated into TENGs. Furthermore, there is a strong requirement for monitoring the real-time dynamic information related to the construction of TENGs. The aim of this opinion paper is to specifically and critically describe the latest advances in the field of neural tissue engineering for peripheral nerve regeneration. Here we delineate new attempts in the design of template (or scaffold) materials, especially in the context of biocompatibility, the choice and handling of support cells, and growth factor release systems. We further discuss the significance of RNAi for peripheral nerve regeneration, anticipate the potential application of RNAi reagents for TENGs, and speculate on the possible contributions of additional elements, including angiogenesis, electrical stimulation, molecular inflammatory mediators, bioactive peptides, antioxidant reagents, and cultured biological constructs, to TENGs. Finally, we consider that a diverse array of physicochemical and biological cues must be orchestrated within a TENG to create a self-consistent coordinated system with a close proximity to the regenerative microenvironment of the peripheral nervous system.

The influence of substrate stiffness on the behavior and functions of Schwann cells in culture.

Solid tissues in the body possess a range of stiffness and provide cells with an instructive microenvironment. Scaffolds in tissue engineering should be rationally designed to become an adhesion substrate friendly to cells. Schwann cells are the principal glial cell in the peripheral nervous system and used as support cells for generating tissue-engineered nerve grafts. Although an important mechanical cue, substrate stiffness, has been documented to make significant effects on many types of cells cultured on the substrate, such a study for Schwann cells is still lacking. In this study, we investigated cell adhesion, survival, proliferation, migration, cytoskeleton, and neurotrophic actions of Schwann cells cultured on polyacrylamide gel substrates with different stiffness, and determined an optimal elastic modulus value for these substrates. Our data not only highlight the importance of substrate stiffness in the crosstalk between Schwann cells and surrounding microenvironment, but also introduce a new parameter, in addition to biocompatibility, biodegradability, and neuroaffinity, for designing scaffolds in nerve tissue engineering.

Joint use of a chitosan/PLGA scaffold and MSCs to bridge an extra large gap in dog sciatic nerve.

BACKGROUND: Tissue-engineered nerve grafts (TENGs) constitute a promising alternative to nerve autografts that are recognized as the gold standard for surgical repair of peripheral nerve gaps. OBJECTIVE: To investigate the feasibility of using TENGs for bridging extra large peripheral nerve gaps in large animals. METHODS: TENGs were constructed by incorporating autologous bone marrow mesenchymal stem cells (MSCs) into a neural scaffold that consisted of a chitosan conduit inserted with poly(lactic-co-glycolic acid) (PLGA) fibers. A 60-mm-long sciatic nerve gap in dogs was bridged by TENGs, chitosan/PLGA scaffolds, or nerve autografts. At 12 months postsurgery, behavioral analysis, electrophysiology, retrograde fluorogold tracing, and histological examination were performed. RESULTS: The outcomes of TENGs were similar to those of autografts and better than those of scaffolds alone. CONCLUSION: Introduction of autologous MSCs to a chitosan/PLGA scaffold improved the repair and rehabilitation of a large gap after peripheral nerve injury in dogs. Autologous MSCs may be a source of support cells for neural tissue engineering.

Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration.

Surgical repair of severe peripheral nerve injuries represents not only a pressing medical need, but also a great clinical challenge. Autologous nerve grafting remains a golden standard for bridging an extended gap in transected nerves. The formidable limitations related to this approach, however, have evoked the development of tissue engineered nerve grafts as a promising alternative to autologous nerve grafts. A tissue engineered nerve graft is typically constructed through a combination of a neural scaffold and a variety of cellular and molecular components. The initial and basic structure of the neural scaffold that serves to provide mechanical guidance and optimal environment for nerve regeneration was a single hollow nerve guidance conduit. Later there have been several improvements to the basic structure, especially introduction of physical fillers into the lumen of a hollow nerve guidance conduit. Up to now, a diverse array of biomaterials, either of natural or of synthetic origin, together with well-defined fabrication techniques, has been employed to prepare neural scaffolds with different structures and properties. Meanwhile different types of support cells and/or growth factors have been incorporated into the neural scaffold, producing unique biochemical effects on nerve regeneration and function restoration. This review attempts to summarize different nerve grafts used for peripheral nerve repair, to highlight various basic components of tissue engineered nerve grafts in terms of their structures, features, and nerve regeneration-promoting actions, and finally to discuss current clinical applications and future perspectives of tissue engineered nerve grafts.