Guicai Li

Porous chitosan scaffolds with surface micropatterning and inner porosity and their effects on Schwann cells.

Chitosan is found to promote the regeneration of peripheral nerve system in our previous studies, whereas the regeneration speed is not satisfied with clinical request. Micropatterning could promote cell orientation and growth, however, the effect of porous chitosan micropatterning on nerve regeneration is rarely reported. In this study, the porous chitosan micropatterning with surface ridge/groove and inner porosity structure was fabricated using a combination of micromodeling and lyophilization method. The morphology and stability of the prepared chitosan micropatterning were evaluated, the regulation of Schwann cells behavior by chitosan micropatterning was evaluated. The results showed that the chitosan micropatterning displayed stripe-like structure with a clear and complete edge. The micropatterning with 30/30 μm was more stable than 20/20 μm sample. Schwann cells on chitosan micropatterning showed orientation adhesion and began to grow along a certain direction after culture for 2 h, and displayed the minimal orientation angle and the largest length/width ratio on 30/30 μm micropatterning after further culture for 3 d and 5 d, indicating the most obvious cell orientation. Moreover, the secretion of nerve growth factor (NGF) demonstrated that the micropatterned chitosan had no negative influence on the physiological function of Schwann cells. Thus, the results indicate that the porous chitosan micropatterning can regulate Schwann cell growth well, which may have potential application in nerve regeneration. The study provides an important basis for constructing porous nerve conduit with micropatterning structure in the inner wall.

Effect of silanization on chitosan porous scaffolds for peripheral nerve regeneration.

The aim of this study was to evaluate the feasibility of using 3-aminopropyltriethoxysilane (APTE) silanization treatment for modification and biocompatibility of lyophilized chitosan porous scaffolds. The process is beneficial for biomaterial development due to its low toxicity and simplicity. The silanization treatment with low APTE concentration showed no significant influence on the morphology of chitosan scaffolds, while a skin-like surface was observed for the silanized scaffolds treated with high APTE concentration. The porosity and surface amino densities were increased after silanization whereas the swelling ratio was reduced, and the degradation ratio in PBS and anti-acid degradation properties of the silanized chitosan scaffolds were significantly improved. The in vitro Schwann cells culture demonstrated that the silanized scaffolds with 8% APTE could obviously facilitate the attachment and proliferation of Schwann cells, indicating great potential for the application in peripheral nerve regeneration.

Preparation of graphene oxide/polyacrylamide composite hydrogel and its effect on Schwann cells attachment and proliferation

Various hydrogel materials have been developed for improving the regeneration of peripheral nerve. Among which the graphene related hydrogels with excellent mechanical properties have attracted great attention. However, the effect of these hydrogels on peripheral nerve regeneration is still unclear. In the present study, the graphene oxide/polyacrylamide (GO/PAM) composite hydrogels were fabricated by in-situ free radical polymerization. The morphology, wettability, composition, swelling ratio, mechanical property and degradation behavior of the prepared GO/PAM composite hydrogels were separately characterized. The effect of GO/PAM hydrogel on the attachment and proliferation of Schwann cells was evaluated. Moreover, the release of biofactors by Schwann cells and adsorption of matrix proteins were further measured. The results showed that the color of the hydrogel became darker with the increased GO concentration, while the surface pore structure also displayed large variation when GO concentration was increased. The hydrophobicity and mechanical properties of hydrogel were increased with the ascending GO concentration. In addition, the variation of GO concentration displayed no obvious influence on the degradation of the composite hydrogel in different medium. The GO/PAM composite hydrogel with 0.4% GO (G0.4) could effectively enhance the attachment and proliferation of Schwann cells. Furthermore, the cells on G0.4 hydrogel displayed higher biofactors release and larger matrix adsorption than other samples. The results demonstrated that GO with suitable concentration in PAM hydrogel could effectively promote Schwann cell growth. The study may provide an important experimental basis for the design and development of new nerve grafts with potential application for peripheral nerve regeneration.

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.

Neuroprotective effect of schizandrin A on oxygen and glucose deprivation/reperfusion-induced cell injury in primary culture of rat cortical neurons

Brain ischemia appears to be associated with innate immunity. Recent reports showed that C3a and C5a, as potent targets, might protect against ischemia induced cell death. In traditional Chinese medicine, the fruit of Schizandra chinesis Baill (Fructus schizandrae) has been widely used as a tonic. In the present study, we sought to evaluate the neuroprotective effects of schizandrin A, a composition of S. chinesis Baill, against oxygen and glucose deprivation followed by reperfusion (OGD/R)-induced cell death in primary culture of rat cortical neurons, and to test whether C3a and C5a affected cortical neuron recovery from ischemic injury after schizandrin A treatment. The results showed that schizandrin A significantly reduced cell apoptosis and necrosis, increased cell survival, and decreased intracellular calcium concentration ([Ca2+]i) and lactate dehydrogenase (LDH) release in primary culture of rat cortical neurons after OGD/R. Mechanism studies suggested that the modulation of extracellular-regulated kinase (ERK), c-Jun NH2-terminal kinases (JNK), and p38, as well as caspase-3 activity played an important role on the progress of neuronal apoptosis. C5aR participated in the neuroprotective effect of schizandrin A in primary culture of rat cortical neurons after OGD/R. Our findings suggested that schizandrin A might act as a candidate therapeutic target drug used for brain ischemia and related diseases.

Mulberroside a protects against ischemic impairment in primary culture of rat cortical neurons after oxygen–glucose deprivation followed by reperfusion

Mulberroside A is a natural polyhydroxylated stilbene compound present at relatively high abundance in the roots and twigs of Morus alba L. It is known for its nephroprotective, hypoglycemic, and antidiabetic effects. Because its metabolite, oxyresveratrol, possessed purported anti‐inflammatory and neuroprotective effects, we proposed that mulberroside A may elicit neuroprotective effects that can be used in the treatment of brain ischemic injury. Therefore, we decided to investigate the pharmacological properties of mulberroside A in primary culture of rat cortical neurons after oxygen–glucose deprivation followed by reperfusion (OGD/R), evaluating its ability to counteract the hypoxia–ischemia impairment. The results showed that mulberroside A elicited neuroprotective effects comparable to nimodipine. The mechanistic studies showed that mulberroside A decreased the expressions of tumor necrosis factor‐α (TNF‐α), interleukin (IL)−1β, and IL‐6 and inhibited the activation of NALP3, caspase‐1, and nuclear factor‐κB and the phosphorylation of extracellular signal‐regulated protein kinases, the c‐Jun N‐terminal kinase, and p38, exhibiting anti‐inflammatory antiapoptotic effects. Our results also further demonstrate that the proinflammatory cytokines of IL‐1β, IL‐6, and TNF‐α are promising targets for treatment of cerebral ischemic injury. Although further investigation is required for its development, all of these findings led us to speculate that mulberroside A is a candidate for the treatment of ischemic stroke, which would act as a multifactorial neuroprotectant.

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.

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.

Regulating Schwann Cells Growth by Chitosan Micropatterning for Peripheral Nerve Regeneration In Vitro

To address the effect of chitosan micropatterning on nerve regeneration, two sizes of parallel microstripes of chitosan are fabricated on the surface of coverslips using a micromodeling method. The morphology of the prepared polydimethylsiloxane stamps and chitosan micropatterning is observed by scanning electron microscopy and the wettability of the prepared micropatterning is evaluated using water contact-angle measurements. Schwann cell (SC) culture is used to evaluate the effect of chitosan micropatterning on cell behavior. The results show that the stripe-like chitosan micropatterning can be successfully fabricated on coverslip surfaces. SCs on 30/30 μm chitosan micropatterning shows the most obvious cell orientation. Moreover, the secretion of nerve growth factor by SCs indicate that the chitosan micropatterning has no negative influence on the normal physiological function of the cells. Thus, the study suggests that chitosan micropatterning can induce and regulate the growth of SCs well, which may have potential application in peripheral nerve regeneration.

Tailoring of chitosan scaffolds with heparin and γ-aminopropyltriethoxysilane for promoting peripheral nerve regeneration

Chitosan has been well known for promoting peripheral nerve regeneration, however, its effect is still not as good as that of autografts. In this study, the feasibility of using negatively charged heparin and positively charged γ-aminopropyltriethoxysilane (APTE) treatment as biocompatible modification of lyophilized porous chitosanscaffolds was evaluated. The morphology of the prepared chitosan scaffolds as a function of treatment with different charged molecules showed no significant differences, while a skin-like surface was observed for the scaffolds modified with high APTE concentration and heparin. The quantitative and qualitative characterization of heparin and amino densities by Toluidine Blue O (TBO) and Acid Orange (AO) assays confirmed the successful immobilization of heparin and APTE on the chitosan scaffolds. The measurement of surface charge densities indicated that the scaffolds treated with APTE showed increased charge densities while heparin decreased the cationic charge density. Moreover, the fabricated charge processed chitosan scaffolds were stable after immersion in phosphate buffer saline for more than ten days. Further on, the chitosan scaffolds processed with 2 mg/mL heparin did facilitate the attachment, proliferation and maintain the biological function of Schwann cells in vitro. The study demonstrates that chitosan scaffolds treated with suitable heparin concentration provides an effective selection for biomaterials surface modification and shows great potential for the application in peripheral nerve regeneration.