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Jianbo Gao, 1 Bing Xia, 1 Shengyou Li, 1 Liangliang Huang, 2 Teng Ma, 1 Xiaowei Shi, 1 Kai Luo, 3 Yujie Yang, 1 Laihe Zhao, 1 Hao Zhang, 4 Beier Luo, 5 Jinghui Huang 1 1Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, People’s Republic of China; 2Department of Orthopaedics, The General Hospital of Central Theater Command of People’s Liberation Army, Wuhan, People’s Republic of China; 3Department of Orthopaedics, The 985th Hospital of the PLA Joint Logistics Support Force, Taiyuan, People’s Republic of China; 4Department of Spinal Surgery, People’s Hospital of Longhua District, Shenzhen, People’s Republic of China; 5Department of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Beier LuoDepartment of Orthopaedics, Changhai Hospital, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of ChinaTel/Fax +862131161700Email bear_luo@126.comJinghui HuangDepartment of Orthopaedics, Xijing Hospital, Fourth Military Medical University, 169 Changle West Road, Xi’an, Shaanxi 710032, People’s Republic of ChinaTel/Fax +862984775275Email huangjh@fmmu.edu.cnPurpose: The clinical outcome of spinal cord injury is usually poor due to the lack of axonal regeneration and glia scar formation. As one of the most classical supporting cells in neural regeneration, Schwann cells (SCs) provide bioactive substrates for axonal migration and release molecules that regulate axonal growth. However, the effect of SC transplantation is limited by their poor migration capacity in the astrocyte-rich central nervous system.Methods: In this study, we first magnetofected SCs with chondroitinase ABC-polyethylenimine functionalized superparamagnetic iron oxide nanoparticles (ChABC/PEI-SPIONs) to induce overexpression of ChABC for the removal of chondroitin sulfate proteoglycans. These are inhibitory factors and forming a dense scar that acts as a barrier to the regenerating axons. In vitro, we observed the migration of SCs in the region of astrocytes after the application of a stable external magnetic field.Results: We found that magnetofection with ChABC/PEI-SPIONs significantly up-regulated the expression of ChABC in SCs. Under the driven effect of the directional magnetic field (MF), the migration of magnetofected SCs was enhanced in the direction of the magnetic force. The number of SCs with ChABC/PEI-SPIONs migrated and the distance of migration into the astrocyte region was significantly increased. The number of SCs with ChABC/PEI-SPIONs that migrated into the astrocyte region was 11.6- and 4.6-fold higher than those observed for the intact control and non-MF groups, respectively. Furthermore, it was found that SCs with ChABC/PEI-SPIONs were in close contact with astrocytes and no longer formed boundaries in the presence of MF.Conclusion: The mobility of the SCs with ChABC/PEI-SPIONs was enhanced along the axis of MF, holding the potential to promote nerve regeneration by providing a bioactive microenvironment and relieving glial obstruction to axonal regeneration in the treatment of spinal cord injury.Keywords: Schwann cells, astrocytes, magnetic field, superparamagnetic iron oxide nanoparticles, spinal cord injury, cell orientation

Introduction: Abnormal biomechanics plays a role in intervertebral disc degeneration. Adi- pose-derived stromal cells (ADSCs) have been implicated in disc integrity; however, their role in the setting of mechanical stimuli upon the disc's nucleus pulposus (NP) remains unknown. As such, the present study aimed to evaluate the influence of ADSCs upon NP cells in compressive load culture. Methods: Human NP cells were cultured in compressive load at 3.0MPa for 48 hours with or without ADSCs co-culture (the ratio was 50:50). We used flow cytometry, live/dead staining and scanning electron microscopy (SEM) to evaluate cell death, and determined the expression of specific apoptotic pathways by characterizing the expression of activated caspases-3, -8 and -9. We further used real-time (RT-) PCR and immunostaining to determine the expression of the ex- tracellular matrix (ECM), mediators of matrix degradation (e.g. MMPs, TIMPs and ADAMTSs), pro-inflammatory factors and NP cell phenotype markers. Results: ADSCs inhibited human NP cell apoptosis via suppression of activated caspase-9 and caspase-3. Furthermore, ADSCs protected NP cells from the degradative effects of compressive load by significantly up-regulating the expression of ECM genes (SOX9, COL2A1 and ACAN), tissue inhibitors of metalloproteinases (TIMPs) genes (TIMP-1 and TIMP-2) and cytokeratin 8 (CK8) protein expression. Alternatively, ADSCs showed protective effect by inhibiting com- pressive load mediated increase of matrix metalloproteinases (MMPs; MMP-3 and MMP-13), dis- integrin and metalloproteinase with thrombospondin motifs (ADAMTSs; ADAMTS-1 and 5), and pro-inflammatory factors (IL-1beta, IL-6, TGF-beta1 and TNF-alpha). Conclusions: Our study is the first in vitro study assessing the impact of ADSCs on NP cells in an un-physiological mechanical stimulation culture environment. Our study noted that ADSCs pro- tect compressive load induced NP cell death and degradation by inhibition of activated caspase-9 and -3 activity; regulating ECM and modulator genes, suppressing pro-inflammatory factors and preserving CK8. Consequently, the protective impact of ADSCs found in this study provides an essential understanding and expands our knowledge as to the utility of ADSCs therapy for in- tervertebral disc regeneration.

Regulating the production of brain-derived neurotrophic factor (BDNF) in Schwann cells (SCs) is critical for their application in traumatic nerve injury, neurodegenerative disorders, and demyelination disease in both central and peripheral nervous systems. The present study investigated the possibility of using electrical stimulation (ES) to activate SCs to release BDNF. We found that short-term ES was capable of promoting BDNF production from SCs, and the maximal BDNF release was achieved by ES at 6 V (3 Hz, 30 min). We further examined the involvement of intracellular calcium ions ([Ca2+]i) in the ES-induced BDNF production in SCs by pharmacological studies. We found that the ES-induced BDNF release required calcium influx through T-type voltage-gated calcium channel (VGCC) and calcium mobilization from internal calcium stores, including inositol triphosphate-sensitive stores and caffeine/ryanodine-sensitive stores. In addition, calcium-calmodulin dependent protein kinase IV (CaMK IV), mitogen-activated protein kinase (MAPK), and cAMP response element-binding protein (CREB) were found to play important roles in the ES-induced BDNF release from SCs. In conclusion, ES is capable of activating SCs to secrete BDNF, which requires the involvement of calcium influx through T-type VGCC and calcium mobilization from internal calcium stores. In addition, activation of CaMK IV, MAPK, and CREB were also involved in the ES-induced BDNF release. The findings indicate that ES can improve the neurotrophic ability in SCs and raise the possibility of developing electrically stimulated SCs as a source of cell therapy for nerve injury in both peripheral and central nervous systems.

Various scaffolds have been attempted for intervertebral disc regeneration, but their effectiveness was limited by loss of nutrients within the scaffolds. It has been suggested that the disc is not severely hypoxic and limited availability of oxygen results in disc degeneration. Therefore, a certain oxygen level might be beneficial for disc regeneration, which has not been given enough attention in previous studies. Here, we used perfluorotributylamine (PFTBA) for the first time as an oxygen regulator in alginate scaffold for disc regeneration in vitro and in vivo. We found that the characteristics of alginate were not affected by PFTBA and the oxygen level of the scaffold was regulated. Then, human nucleus pulposus (NP) cells were cultured in the PFTBA-enriched alginates. It was found that PFTBA could promote NP cell survival and proliferation. In addition, 2.5% PFTBA was capable of regulating extracellular matrix (ECM) to a disc-like tissue graft with little effect on the expression of NP cell markers. Finally, 2.5% PFTBA-enriched alginate was found to restore the disc height and the ECM in a mouse disc degeneration model, indicating its beneficial effect on alleviating disc degeneration. These findings highlight the promising application of PFTBA in further intervertebral disc regeneration.

Zhongyang Liu,1,* Liangliang Huang,1,* Liang Liu,1,* Beier Luo,2,* Miaomiao Liang,3 Zhen Sun,1 Shu Zhu,1 Xin Quan,1 Yafeng Yang,1 Teng Ma,1 Jinghui Huang,1 Zhuojing Luo1 1Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, 2Department of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai, 3Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi’an, People’s Republic of China *These authors contributed equally to this work Abstract: Schwann cells (SCs) are attractive seed cells in neural tissue engineering, but their application is limited by attenuated biological activities and impaired functions with aging. Therefore, it is important to explore an approach to enhance the viability and biological properties of SCs. In the present study, a magnetic composite made of magnetically responsive magnetic nanoparticles (MNPs) and a biodegradable chitosan–glycerophosphate polymer were prepared and characterized. It was further explored whether such magnetic nanocomposites via applied magnetic fields would regulate SC biological activities. The magnetization of the magnetic nanocomposite was measured by a vibrating sample magnetometer. The compositional characterization of the magnetic nanocomposite was examined by Fourier-transform infrared and X-ray diffraction. The tolerance of SCs to the magnetic fields was tested by flow-cytometry assay. The proliferation of cells was examined by a 5-ethynyl-2-deoxyuridine-labeling assay, a PrestoBlue assay, and a Live/Dead assay. Messenger ribonucleic acid of BDNF, GDNF, NT-3, and VEGF in SCs was assayed by quantitative real-time polymerase chain reaction. The amount of BDNF, GDNF, NT-3, and VEGF secreted from SCs was determined by enzyme-linked immunosorbent assay. It was found that magnetic nanocomposites containing 10% MNPs showed a cross-section diameter of 32.33±1.81 µm, porosity of 80.41%±0.72%, and magnetization of 5.691 emu/g at 8 kOe. The 10% MNP magnetic nanocomposites were able to support cell adhesion and spreading and further promote proliferation of SCs under magnetic field exposure. Interestingly, a magnetic field applied through the 10% MNP magnetic scaffold significantly increased the gene expression and protein secretion of BDNF, GDNF, NT-3, and VEGF. This work is the first stage in our understanding of how to precisely regulate the viability and biological properties of SCs in tissue-engineering grafts, which combined with additional molecular factors may lead to the development of new nerve grafts. Keywords: Schwann cell, magnetic field, nanocomposite, cell proliferation