Artificial nerve graft constructed by coculture of activated Schwann cells and human hair keratin for repair of peripheral nerve defects.

Studies have shown that human hair keratin (HHK) has no antigenicity and excellent mechanical properties. Schwann cells, as unique glial cells in the peripheral nervous system, can be induced by interleukin-1ß to secrete nerve growth factor, which promotes neural regeneration. Therefore, HHK with Schwann cells may be a more effective approach to repair nerve defects than HHK without Schwann cells. In this study, we established an artificial nerve graft by loading an HHK skeleton with activated Schwann cells. We found that the longitudinal HHK microfilament structure provided adhesion medium, space and direction for Schwann cells, and promoted Schwann cell growth and nerve fiber regeneration. In addition, interleukin-1ß not only activates Schwann cells, but also strengthens their activity and increases the expression of nerve growth factors. Activated Schwann cells activate macrophages, and activated macrophages secrete interleukin-1ß, which maintains the activity of Schwann cells. Thus, a beneficial cycle forms and promotes nerve repair. Furthermore, our studies have found that the newly constructed artificial nerve graft promotes the improvements in nerve conduction function and motor function in rats with sciatic nerve injury, and increases the expression of nerve injury repair factors fibroblast growth factor 2 and human transforming growth factor B receptor 2. These findings suggest that this artificial nerve graft effectively repairs peripheral nerve injury.

Low concentration of lipopolysaccharide acts on MC3T3-E1 osteoblasts and induces proliferation via the COX-2-independent NFkappaB pathway.

The translocations of lipopolysaccharide (LPS) from the gut and its effects on bone healing are usually of clinical interest during bone fracture. As already widely studied, Cyclooxygenase-2 (COX-2) is a key enzyme for prostaglandin E2 (PGE(2)) production, which induces the nuclear factor kappa B (NFkappaB) activation and is beneficial to fracture healing. In order to know their roles in skeletal regeneration, mouse MC3T3-E1 osteoblasts were treated with NFkappaB inhibitor BAY 11-7082 and sc791 (a selective COX-2 inhibitor), in the presence of LPS. Interestingly, LPS could induce osteoblasts proliferation through increasing NFkappaB activation and translocation. This induction was not related to COX-2 expression, suggesting that LPS-induced NFkappaB activation is independent of COX-2. It is possible that low concentration of LPS can act as a stimulating factor of the NFkappaB pathway in nonstimulated cells such as osteoblasts. COX-2 is not necessary for the NFkappaB pathway during LPS-induced proliferation of osteoblasts since sc791 had no effects on this induction. These studies provide insight into a potential mechanism by which LPS can affect bone tissue repair in the initial phase of inflammation.

Enhancement of Perovskite Solar Cells Efficiency using N-Doped TiO2 Nanorod Arrays as Electron Transfer Layer.

In this paper, N-doped TiO2 (N-TiO2) nanorod arrays were synthesized with hydrothermal method, and perovskite solar cells were fabricated using them as electron transfer layer. The solar cell performance was optimized by changing the N doping contents. The power conversion efficiency of solar cells based on N-TiO2 with the N doping content of 1% (N/Ti, atomic ratio) has been achieved 11.1%, which was 14.7% higher than that of solar cells based on un-doped TiO2. To get an insight into the improvement, some investigations were performed. The structure was examined with X-ray powder diffraction (XRD), and morphology was examined by scanning electron microscopy (SEM). Energy dispersive spectrometer (EDS) and Tauc plot spectra indicated the incorporation of N in TiO2 nanorods. Absorption spectra showed higher absorption of visible light for N-TiO2 than un-doped TiO2. The N doping reduced the energy band gap from 3.03 to 2.74 eV. The photoluminescence (PL) and time-resolved photoluminescence (TRPL) spectra displayed the faster electron transfer from perovskite layer to N-TiO2 than to un-doped TiO2. Electrochemical impedance spectroscopy (EIS) showed the smaller resistance of device based on N-TiO2 than that on un-doped TiO2.

A new mitochondrial RNA deletion fragment accelerated by oxidative stress in rat L6 cells.

RNA deletions may be easier to detect and more extensive than DNA deletions. Two large deletion fragments (1120 and 7811 bp) of mitochondrial RNA were observed in rat L6 muscle cells. At the site of the 1120 bp deletion, the remaining RNA fragment was re-linked by a short additional section (GGTATGAAGCT). These kinds of deletions were accelerated by oxidative stress and were not observed in mitochondrial DNA.

[Oxidative stress-induced differentiation of L6 myoblasts].

OBJECTIVE: To explore the relationship between the differentiation of L6 myoblasts and oxidative stress. METHODS: MTT assay was used to determine the viability of L6 myoblasts, from which the total RNA was extracted for amplification of the myogenin gene fragment by RT-PCR. H(2)O(2)-induced morphological changes of the cells were observed. RESULTS: The myoblasts treated with low concentration of reactive oxygen (50 micromol/L H(2)O(2)) for 1 h exhibited accelerated cell growth (P<0.05), and treatment with 50 and 150 micromol/L H(2)O(2) induced the gene expression of myogenin, a molecular marker for differentiation of myoblasts. Morphological study revealed myotube formation and accelerated differentiation of the myoblasts induced by H(2)O(2). CONCLUSION: The reactive oxygen may serve as the intracellular signal molecules to induce the growth and differentiation of the myoblasts.

[FK506 promotes apoptosis of macrophages activated by homogenate of allogenic nerve].

OBJECTIVE: To study the effect of FK506 in promoting apoptosis of peripheral blood-derived macrophages activated by homogenate of allogenic nerve tissues. METHODS: Homogenate of the allogenic nerve tissues was prepared using the sciatic nerve and injected in one-month-old SD rats, from which the macrophages activated by the homogenate were collected from the abdominal cavity and cultured in vitro. The cells were divided into 4 groups according to different concentrations of FK506 for treatment, namely 0 (group A, control group), 0.25 ng/ml (group B), 0.5 ng/ml (group C), and 1.0 ng/ml (group D). The cells of the 4 groups were inoculated into 96-well plate respectively for detecting the viability of the macrophages by MTT assay and for morphological evaluation of the cell apoptosis by transmission electron microscopy and fluorescence microscopy. RESULTS: The cells in groups B and C exhibited reduced viability and signs of apoptosis, and necrosis was observed in group D. Transmission electron microscopy and fluorescence microscopy identified early apoptotic changes and the presence of apoptotic body in the macrophages. The apoptotic rates of groups B and C were much higher than that in group A found by flow cytometry. CONCLUSION: FK506 can promote the apoptosis of macrophage activated by allogenic nerve homogenate and reduce macrophage-mediated immunological rejection of peripheral nerve allograft.

[Construction of artificial nerve bridge by three-dimensional culture of Schwann cells with human hair keratins].

OBJECTIVE: To culture Schwann cells (SCs) and human hair keratins (HHKs) for artificial nerve bridge construction. METHODS: SCs were purified by primary culture and labeled with BrdU, which were then cultured with HHKs decorated by ECM. The artificial nerve bridge was implanted into the defect of sciatic nerve, beneath the skin, and in the skeletal muscles of SD rat, respectively. The morphology of the SCs cultured with HHKs was monitored by inverted microscope and evaluated by immunocytochemical staining. Growth of BrdU-labeled SCs in vivo was observed by immunocytochemical staining on paraffin sections. RESULTS: In vitro cultured SCs were capable of adhering to HHKs and grew well four weeks after implantation. The HHK component in the artificial nerve bridge underwent degradation in the defect of the sciatic nerve, beneath the skin, and in the skeletal muscles of SD rat, and SC survival and proliferation were verified. CONCLUSION: SCs can survive in three-dimensional culture with HHKs for construction of artificial nerve bridge to repair nerve defects.

Changes in content of macrophage migration inhibitory factor secreted by Schwann cells after peripheral nerve injury.

OBJECTIVE: To investigate whether Schwann cells can secrete macrophage migration inhibitory factor (MIF) after peripheral nerve injury. METHODS: Two kinds of infant rat Schwann cells(which were derived from intact and injured nerves respectively) were cultured in 10% newborn calf serum (NCS) DMEM/F12 medium for 72 h. Then the level of MIF in the conditioned media was determined by an enzyme-linked immunoadsordent assay (ELISA). As control,MIF level was also determined in 10% NCS DMEM/F12 medium without any cells. RESULTS: The concentration of MIF in the conditioned medium of Schwann cells derived from injured nerves was significantly higher than that of control samples (P<0.05), while the concentration of MIF in the conditioned medium of Schwann cells from intact nerves was not elevated. CONCLUSION: After peripheral nerve injury, Schwann cells can secrete MIF which may play an important role as an immunomodulatory cytokine for macrophage activation, inflammatory reactions and immune responses.

In vitro culture and study of the biological characteristics of rabbit keratocytes.

OBJECTIVE: To improve the technique for culturing rabbit keratocytes in vitro and investigate the biological characteristics of these cells. METHODS: Fresh rabbit corneas were obtained and the epithelial and endothelial cells were removed after digestion with trypsin. The stroma was rinsed, minced, and incubated in DMEM/F12 medium supplemented with 10% fetal bovine serum and the biological characteristics of the keratocytes were observed with MTT assay and compared with those of the cells in serum-free culture media. RESULT: On about the third day of incubation, some keratocytes germinated from the stromal tissues and migrated onto the flask surface presenting fibroblast-like arrangement with spindle-shaped appearance. The keratocytes became confluent after 10 day's incubation and the peak of cell proliferation occurred on day 8 in the presence of serum in the media, while in the absence of serum, the peak took place on day 10. CONCLUSION: The method improved for in vitro keratocyte culture is convenient and effective, and the presence of serum in the media may to some degree affect the growth of the keratocytes.

[Amplification of brain-derived neurotrophic factor receptor trkB gene and construction of its eukaryotic expression vector].

OBJECTIVE: To construct a recombinant eukaryotic expression vector of rat brain-derived neurotrophic factor receptor trkB gene. METHODS: Using the total RNA extracted from rat brain tissue as the template, the trkB gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers containing the restriction sites of EcoRI and BamHI. The amplified fragment of trkB gene was digested with EcoRI and BamHI, and then subcloned into cloning vector pMD18-T and then expression vector pEGFP-C2. The recombinant plasmid was identified by restriction endonuclease analysis and PCR. RESULTS: The amplified DNA fragment was about 1 461 bp in length, and enzyme digestion and PCR analysis showed that trkB gene had been successfully cloned into the vectors pMD18-T and pEGFP-C2. CONCLUSION: The trkB gene of rat has been successfully amplified and cloned into the eukaryotic expression vector pEGFP-C2.

The possible influence of varying diameter of aligned electrospun fibers on Schwann cells maturation in culture.

The development of Schwann cells, the principal glial cell in the peripheral nervous system, occurs through a series of transitional embryonic and postnatal phases, which are tightly regulated by a number of axonal signals. During the axon ensheathment and myelin growth, the diameter of the axon play an important role in the maturation of Schwann cells. Because of electrospun fibers similar to protein fibers within the native extracellular matrix, the scaffolds are being developed as neural tissue engineering scaffolds. Until now, the correlation between varying diameter of aligned electrospun fibers and Schwann cells maturation has not been investigated. We hypothesize that the different diameter of aligned electrospun fibers may influence the maturation of Schwann cells and may help improve the outcome of cell-based approaches to cure demyelinated lesions or peripheral nerve regeneration.

[Distribution of neuronal nitric oxide synthase-immunopositive neurons in rat corpus striatum and their ultrastructures].

OBJECTIVE: To observe the distribution of neuronal nitric oxide synthase (nNOS)-immunopositive neurons in rat corpus striatum and their ultrastructural features. METHODS: Brain tissue specimens were obtained from normal SD rats, in which nNOS-immunopositive neurons were visualized by ABC immunocytochemistry and observed under immunoelectron microscope with pre-embedding staining. RESULTS: Under light microscope, nNOS-immunopositive neurons appeared brown with distinct profiles of the cell body and processes. These neurons, mostly medium-sized and small cells, were located mainly in the lateral region of the corpus striatum. Only a few immunopositive neurons were detected in the medial region of the corpus striatum. Immunohistochemistry and transmission electron microscopy identified the nNOS-immunopositive neurons as interneurons possessing large nuclei with small amount of cytoplasma. The immunopositive granules were visualized as black plaques, and the larger ones distributed mainly in the cell bodies, some with monolayer membrane encapsulation. The small granules did not have the encapsulation, scattering in perinuclear regions and under the cell membrane, but not in the cell body. The immunopositive granules were also found in the axons and dendrites, but not in the vesicles of the synapses. In addition, many immunopositive terminals were found close to the blood vessels. CONCLUSIONS: nNOS-immunopositive neurons in rat corpus striatum are mainly medium-sized and small cells as is typical of the interneurons. The immunopositive granules locate in the cytoplasma, axons and dendrites, and larger granules have membrane coating while small ones do not, possibly in relation to their functions.

[Construction of artificial nerve bridge by three-dimensional culture of interleukin-1beta- activated Schwann cells with human hair keratins].

OBJECTIVE: To culture interleukin-1beta (IL-1beta)-activated Schwann cells (SCs) with human hair keratins (HHKs) for artificial nerve bridge construction. METHODS: SCs purified by primary culture with or without IL-1beta activation were cultured with HHKs decorated by extracellular matrix (ECM), and the artificial nerve bridge was implanted into the defect of rat sciatic nerve. The morphology of the SCs cultured with HHKs was monitored by inverted microscope, scanning electron microscope and evaluated by immunocytochemical staining, and the expression of nerve growth factor (NGF) in the sciatic nerve was observed by in situ hybridization. RESULTS: Activated SCs showed better ability to adhere to the HHKs and grew well. The HHKs component in the artificial nerve bridge underwent degradation in the sciatic nerve defect after 3 to 4 weeks, and IL-1beta activation resulted in enhanced NGF expression in the SCs. CONCLUSION: The constructed artificial nerve bridge by three-dimensional culture of IL-1beta-activiated SCs with HHKs decorated by ECM promotes the repair of sciatic nerve defects and accelerates sciatic nerve regeneration.