Yahong Zhao

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.

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.

Chitosan Degradation Products Promote Nerve Regeneration by Stimulating Schwann Cell Proliferation via miR-27a/FOXO1 Axis

Natural polysaccharides are biomaterials widely used for constructing scaffolds in tissue engineering. While natural polysaccharides have been shown to robustly promote tissue regeneration, the underlying molecular mechanism remains largely unknown. Here, we show that chitooligosaccharides (COS), the intermediate products of chitosan degradation, stimulate peripheral nerve regeneration in rats. Our experiment also shows that COS stimulate the proliferation of Schwann cells (SCs) during nerve regeneration. By analyzing the transcriptome and gene regulatory network, we identified the miR-27a/FOXO1 axis as the main signaling pathway for mediating the proliferative effects of COS on SCs. COS increase the expression level of miR-27a and cause a reduction of FOXO1, which subsequently accelerates the cell cycle and stimulates SC proliferation to stimulate nerve regeneration. These findings define a basic pathway for oligosaccharides-mediated cell proliferation and reveal a novel aspect of polysaccharide biomaterials in tissue engineering.

Synthesis and protective effects of aralkyl alcoholic 2-acetamido-2-deoxy-β-D-pyranosides on hypoglycemia and serum limitation induced apoptosis in PC12 cell.

Neuroprotective agents have been in the focus of attention in the treatment of ischemic stroke. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., possessed a wide range of biological activities, especially neuroprotection. In an attempt to improve neuroprotective effects of new salidroside analogs for ischemic stroke, a series of novel aralkyl alcoholic 2-acetamido-2-deoxy-β-d-pyranosides were synthesized and their protective activities against the hypoglycemia and serum limitation induced cell death in rat pheochromocytoma cells (PC12 cells) were studied. Most compounds showed strong neuroprotective effects, especially for 4g and 4h, which exhibited a great potency superior to salidroside. MTT assay, Hoechst 33342 staining, and flow cytometry with annexin V/PI staining collectively showed that pretreatment with 4g and 4h attenuated cell viability loss and apoptotic cell death in cultured PC12 cells. Caspase-3 colorimetric assay and Rhodamine 123 staining revealed the changes in expression levels of caspase-3 and mitochondrial membrane potential in PC12 cells on exposure to hypoglycemia and serum limitation with and without 4g and 4h pretreatment, respectively. All the results suggested that 4g and 4h protects the PC12 cells against hypoglycemia and serum limitation induced apoptosis possibly by modulation of apoptosis-related gene expression and restoration of the mitochondrial membrane potential. Therefore, these novel findings may provided a new framework for the design of new aralkyl alcoholic 2-acetamido-2-deoxy-β-d-pyranosides as neuroprotective agents for treating cerebral ischemic stroke and neurodegenerative diseases.

Chitosan degradation products facilitate peripheral nerve regeneration by improving macrophage-constructed microenvironments.

Chitosan-based artificial nerve grafts have been widely employed to repair peripheral nerve defects. Our previous study has shown that chitosan constructed nerve graft not only provides suitable scaffolds for nerve regeneration, its degradation products, chitooligosaccharides (COS), also promote nerve repair. However, the involved mechanisms are still not fully elucidated. In the present study, we observed that pro-inflammatory cytokines, as well as macrophage infiltration, were transiently up-regulated in the injured sciatic nerves which were bridged with silicon tubes filled with COS. Based upon transcriptome analysis, the axis of miR-327/CCL2 in Schwann cells (SCs) was identified as a potential target of COS. The following experiments have confirmed that COS stimulate CCL2 expression by down-regulating miR-327 in SCs. Consequently, the resulting CCL2 induces macrophage migration at injury sites to re-construct microenvironments and thus facilitates nerve regeneration. Collectively, our data provide a theoretical basis for the clinical application of chitosan-based grafts in peripheral nerve regeneration.

Nanoparticle mediated controlled delivery of dual growth factors

Peripheral nerve functional recovery after nerve injury generally requires multiple growth factors by synergistic effect. However, the optical combination of multiple synergistic growth factors for axonal regeneration has been scarcely considered up to now. Meanwhile, the use of growth factors in promoting nerve regeneration was limited by its short biological half-life in vivo, its vulnerability to structure disruption or hydrolyzation, leading to loss of bioactivity. Herein, a novel polymeric nanoparticle delivery system composed of heparin and ɛ-poly-L-lysine (PL) was prepared for control release of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF). The nanoparticles were synthesized by polyelectrolyte complexation in aqueous solution at room temperature, followed by cross-linking with biological genipin. The obtained nanoparticles had a spherical shape, with a mean diameter of about 246 nm, and high growth factors encapsulation efficiency as well as good stability. NGF and bFGF were encapsulated in the nanoparticles and showed a continuous and slow release behavior in vitro. The bioactivities of the released growth factors were evaluated, and exhibited the synergistic effect. The controlled release of the dual synergistic growth factors would improve the treatment of peripheral nerve injury to mimic the natural cellular microenvironments.

Biocompatibility evaluation of electrospun silk fibroin nanofibrous mats with primarily cultured rat hippocampal neurons.

In this study, electrospinning was performed to fabricate silk fibroin (SF) nanofibrous mats, which were used as substrates for in vitro culture of rat hippocampal neurons. The light and electron micrographs demonstrated that the electrospun SF nanofibrous mat supported the survival and growth of the attached hippocampal neurons. MTT assay and immunocytochemistry in couple with Western blot analysis respectively indicated there was no significant difference in both the cell viability and expression levels of some proteins, including GAP-43, MAP-2, NF, and β-tubulin, between hippocampal neurons cultured in the electrospun SF nanofibrous mat extract and in plain neuronal medium. Our results indicated that electrospun SF nanofibrous mats were biocompatible to primary culture of hippocampal neurons without cytotoxic effects on the cell phenotype and functions, raising a potential possibility of using these mats for CNS therapeutic applications.

Fabrication and characterization of polyacrylamide/silk fibroin hydrogels for peripheral nerve regeneration

Various hydrogels have been used for repairing peripheral nerve injury; however, the silk fibroin (SF)-based hydrogels in peripheral nerve regeneration are still rarely reported. In this study, the SF/pAM hydrogels with different SF concentrations and ethanol treatment time were developed by solution blending and in situ radical polymerization. The physiochemical properties of composite hydrogels were measured, the cytotoxicity of hydrogels was evaluated by L929 fibroblasts, and the effect on peripheral nerve regeneration was evaluated via Schwann cells culture in vitro. The results showed that the physiochemical properties of SF/pAM hydrogels could be changed by varying SF concentration and ethanol treatment time, and the mechanical property was enhanced with increasing SF concentration, while the presence of SF in pAM hydrogels and ethanol treatment does not affect hydrogels structure in per se. All the composite hydrogels displayed no obvious cytotoxicity, while the SF/pAM composite hydrogels with 10% SF and 60-min ethanol treatment could obviously accelerate the attachment and proliferation of Schwann cells. Therefore, the SF/pAM composite hydrogels possessed the beneficial properties required for in situ cell scaffolding and may have potential application in peripheral nerve regeneration.

Electrospun silk fibroin-based neural scaffold for bridging a long sciatic nerve gap in dogs

Silk fibroin (SF)‐derived silkworms represent a type of highly biocompatible biomaterial for tissue engineering. We have previously investigated biocompatibility of SF with neural cells isolated from the central nervous system or peripheral nerve system in vitro, and also developed a SF‐based nerve graft conduit or tissue‐engineered nerve grafts by introducing bone marrow mesenchymal stem cells, as support cells, into SF‐based scaffold and evaluated the outcomes of peripheral nerve repair in a rat model. As an extension of the previous study, the electrospun technique was performed here to fabricate SF‐based neural scaffold inserted with silk fibres for bridging a 30‐mm‐long sciatic nerve gap in dogs. Assessments including functional, histological and morphometrical analyses were applied 12 months after surgery. All the results indicated that the SF‐based neural scaffold group achieved satisfactory regenerative outcomes, which were close to those achieved by autologous nerve grafts as the golden‐standard for peripheral nerve repair. Overall, our results raise a potential possibility for the translation of SF‐based electrospun neural scaffolds as an alternative to nerve autografts into the clinic.

Nerve growth factor loaded heparin/chitosan scaffolds for accelerating peripheral nerve regeneration

Artificial chitosan scaffolds have been widely investigated for peripheral nerve regeneration. However, the effect was not as good as that of autologous grafts and therefore could not meet the clinical requirement. In the present study, the nerve growth factor (NGF) loaded heparin/chitosan scaffolds were fabricated via electrostatic interaction for further improving nerve regeneration. The physicochemical properties including morphology, wettability and composition were measured. The heparin immobilization, NGF loading and release were quantitatively and qualitatively characterized, respectively. The effect of NGF loaded heparin/chitosan scaffolds on nerve regeneration was evaluated by Schwann cells culture for different periods. The results showed that the heparin immobilization and NGF loading did not cause the change of bulk properties of chitosan scaffolds except for morphology and wettability. The pre-immobilization of heparin in chitosan scaffolds could enhance the stability of subsequently loaded NGF. The NGF loaded heparin/chitosan scaffolds could obviously improve the attachment and proliferation of Schwann cells in vitro. More importantly, the NGF loaded heparin/chitosan scaffolds could effectively promote the morphology development of Schwann cells. The study may provide a useful experimental basis to design and develop artificial implants for peripheral nerve regeneration and other tissue regeneration.