Yumin Yang

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.

Chitosan/silk fibroin-based, Schwann cell-derived extracellular matrix-modified scaffolds for bridging rat sciatic nerve gaps

Extracellular matrix (ECM) plays a prominent role in establishing and maintaining an ideal microenvironment for tissue regeneration, and ECM scaffolds are used as a feasible alternative to cellular and molecular therapy in the fields of tissue engineering. Because of their advantages over tissue-derived ECM scaffolds, cultured cell-derived ECM scaffolds are beginning to attract attention, but they have been scarcely studied for peripheral nerve repair. Here we aimed to develop a tissue engineered nerve scaffold by reconstituting nerve cell-derived ECM with natural biomaterials. A protocol was adopted to prepare and characterize the cultured Schwann cell (SC)-derived ECM. A chitosan conduit and silk fibroin (SF) fibers were prepared, cultured with SCs for ECM deposition, and subjected to decellularization, followed by assembly into a chitosan/SF-based, SC-derived ECM-modified scaffold, which was used to bridge a 10 mm rat sciatic nerve gap. The results from morphological analysis as well as electrophysiological examination indicated that regenerative outcomes achieved by our developed scaffold were similar to those by an acellular nerve graft (namely a nerve tissue-derived ECM scaffold), but superior to those by a plain chitosan/SF scaffold. Moreover, blood and histopathological parameters confirmed the safety of scaffold modification by SC-derived ECM. Therefore, a hybrid scaffold based on joint use of acellular and classical biomaterials represents a promising approach to nerve tissue engineering.

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.

Long-term outcome of the repair of 50 mm long median nerve defects in rhesus monkeys with marrow mesenchymal stem cells-containing, chitosan-based tissue engineered nerve grafts

Despite great progress in the fields of tissue engineering and stem cell therapy, the translational and preclinical studies are required to accelerate the clinical application of tissue engineered nerve grafts, as an alternative to autologous nerve grafts, for peripheral nerve repair. Rhesus monkeys (non-human primates) are more clinically relevant and more suitable for scaling up to humans as compared to other mammalians. Based on this premise, and considering a striking similarity in the anatomy and function between human and monkey hands, here we used chitosan/PLGA-based, autologous marrow mesenchymal stem cells (MSCs)-containing tissue engineered nerve grafts (TENGs) for bridging a 50-mm long median nerve defect in rhesus monkeys. At 12 months after grafting, locomotive activity observation, electrophysiological assessments, and FG retrograde tracing tests indicated that the recovery of nerve function by TENGs was more efficient than that by chitosan/PLGA scaffolds alone; histological and morphometric analyses of regenerated nerves further confirmed that the morphological reconstruction by TENGs was close to that by autografts and superior to that by chitosan/PLGA scaffolds alone. In addition, blood test and histopathological examination demonstrated that TENGs featured by addition of autologous MSCs could be safely used in the primate body. These findings suggest the efficacy of our developed TENGs for peripheral nerve regeneration and their promising perspective for clinical applications.

Chitosan/polyglycolic acid nerve grafts for axon regeneration from prolonged axotomized neurons to chronically denervated segments

Peripheral nerve regeneration for long-term delayed injuries is usually unsatisfied. Here we attempted to use a chitosan/polyglycolic acid (PGA) artificial nerve graft to bridge a long-term delayed 10-mm defect in SD rats based on the previous studies on the graft used for immediate repair of 30-mm-long dog sciatic nerve defects and for clinical treatment of a 35-mm-long median nerve defect at elbow of a human patient. In this study, for experimental groups, the rat sciatic nerve had been transected leaving a 10-mm defect, which was maintained for 3 or 6 months before implantation with the chitosan/PGA artificial nerve graft. The animals non-grafted or grafted with autograft served as negative or positive control group. In experiment groups, nerve regeneration with functional recovery was achieved as measured by electrophysiological and histological techniques, although differences in the quantity and the quality of the regenerated nerve were observed between the 3- and 6-month delayed subgroups. The results showed that: (1) a few denervated Schwann cells survived and sustained their ability to myelinate axons at least 6 months, and (2) the atrophic denervated muscle could be reinnervated by regenerated axons through new muscle-nerve connections. These observations provide the possibility of guiding regenerated axons from survived axotomized neurons to distal nerve stump by the chitosan/PGA artificial nerve graft.

Nerve conduits based on immobilization of nerve growth factor onto modified chitosan by using genipin as a crosslinking agent.

Incorporation of nerve growth factor (NGF) into a nerve conduit can improve peripheral nerve regeneration. Here, genipin, a natural and low toxic agent, was used to crosslink chitosan, a natural polysaccharide, and concurrently to immobilize NGF onto modified chitosan, followed by fabrication of chitosan (CS)-genipin (GP)-NGF nerve conduits. MTT test showed that the cell viability of Schwann cells cultured in the conduit extract was not significantly different from that in plain medium. The neurite outgrowth measurement and immunocytochemistry with anti-growth-associated protein-43 and anti-neurofilament indicated that NGF released from CS-GP-NGF nerve conduits retained the bioactivity of stimulating neuronal differentiation of PC12 cells. Fracture strength measurements and vitamin B12 release analysis confirmed that CS-GP-NGF nerve conduits possessed good mechanical properties and adequate permeability. We also investigated the in vitro release kinetics of NGF from CS-GP-NGF nerve conduits by ELISA. The continuous release profile of NGF, within a 60-day time span, consisted of an initial burst that was controlled by a concentration gradient-driven diffusion, followed by a zero-order release that was controlled by a degradation of chitosan matrix. Collectively, CS-GP-NGF nerve conduits had an integrated system for continuous release of NGF, thus holding promise for peripheral nerve repair applications.

Evaluation on in vitro biocompatibility of silk fibroin-based biomaterials with primarily cultured hippocampal neurons.

Silk fibroin-based biomaterials have recently found increasing applications in the tissue-engineering field including the generation of artificial nerve guides for peripheral nerve repair. The aim of this study was to investigate the suitability of silk fibroin as a candidate biomaterial for central nervous system (CNS) therapy. We found that substrates made up of silk fibroin fibers supported the survival and growth of the attached hippocampal neurons by using morphological observation. We also cultured the hippocampal neurons in silk fibroin extract for different times, and observed no significant difference occurring in their morphology, cell viability for these cultured hippocampal neurons as compared to those cultured in plain neuronal culture medium. Moreover, immunocytochemistry, RT-PCR, and Western blot analysis revealed that no significant difference was found in mRNA or protein levels of the growth-associated molecules, such as brain-derived neurotrophic factor, growth-associated protein-43, neurofilament, nerve growth factor, and nerve growth factor-receptor P75, between the hippocampal neurons cultured in the silk fibroin extract and in plain neuronal culture medium. Taken together, all the results demonstrate that silk fibroin has good biocompatibility with primarily cultured hippocampal neurons without any significant cytotoxic effects on their cell phenotype and functions, suggesting a potential possible use of silk fibroin for preparing the tissue-engineered nerve guides or drug delivery vehicles to treat CNS injuries or diseases.

Nanoparticle-mediated delivery of TGF-β1 miRNA plasmid for preventing flexor tendon adhesion formation

Treatment of the disrupted digital flexor tendon is troublesome because of the lack of sufficient healing capacity and the formation of adhesions. Sustained gene delivery may be a promising approach of modulating gene expression in enhancing tendon healing and decreasing adhesions. In this study, a microRNA-based RNAi plasmid was used to specifically silence the expression of TGF-β1 gene associated with scar and adhesion formation in the flexor tendons. The miRNA plasmids were complexed with polylactic-co-glycolic acid (PLGA) nanoparticles to form nanoparticle/TGF-β1 miRNA plasmid (nanoparticle/plasmid) complexes. In vitro and in vivo transfection efficiencies experiments against tenocytes revealed that nanoparticle/plasmid complexes have significantly superior transfection efficiency over the lipofectamine/plasmid complexes. The gene and protein expression associated with adhesion of tendon treated with nanoparticle/plasmid complexes were evaluated by real-time PCR and immunoblotting. The grading of adhesions for tendons treated with nanoparticle/plasmid complexes was less severe than that treated with the nanoparticle/mock plasmid complexes. However, the ultimate strength of repaired tendons treated with nanoparticle/plasmid complexes was significantly lower than that of tendons treated with the nanoparticle/mock plasmid complexes.

Effect of chitooligosaccharide on neuronal differentiation of PC-12 cells

Chitosan is now being widely used biomaterial in the tissue engineering field, and has great potential as a candidate material for preparing nerve guidance conduits due to its various favorable properties, especially that of good nerve cell affinity. Chitosan can be degraded in vivo into chitooligosaccharide. We have investigated the in vitro effects of chitooligosaccharide on neuronal differentiation of PC‐12 cells to see what effects chitooligosaccharide have on certain functions in the regenerating neurons. The morphologic observation and assessment using the specific reagent of tetrazolium salt WST‐8 indicated that neurite outgrowths from PC‐12 cells and the viability of PC‐12 cells were enhanced by treatment of chitooligosaccharide. The real‐time quantitative RT‐PCR and Western blot analysis revealed showed that chitooligosaccharide could upregulate the expression of neurofilament‐H mRNA or protein and N‐cadherin protein in PC‐12 cells. The maximum effect of 0.1 mg/ml chitooligosaccharide was obtained after 2 week culture. All the data suggest that chitooligosaccharide possesses good nerve cell affinity by supporting nerve cell adhesion and promoting neuronal differentiation and neurite outgrowth.

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.