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Liu G.,Shanghai JiaoTong University | Liu G.,Tongji University | Zhang Y.,Shanghai JiaoTong University | Liu B.,Shanghai JiaoTong University | And 3 more authors.
Biomaterials | Year: 2013

Adipose tissue derived stem cells (ASCs) based therapies for the repair and regeneration of various tissues have been widely investigated recently because of their multilineage potential and self-renewal capability. Our previous study demonstrated that autologous ASCs loaded onto natural coral scaffolds could repair cranial critical-sized defects (CSDs) in a canine model. The objective of this study was to determine whether the use of allogeneic ASCs could heal the same defect without the use of immunosuppressive therapy. The pedigree mismatch, mixed lymphocyte reaction assays (MLRs) and allogeneic skin graft experiments were performed to confirm unrelated ASC donors and recipients. A total of 12 adult Beagle dogs were enrolled in this study and divided into two groups. Bilateral cranial CSDs were created in each animal. The right-side defect was treated with allogeneic ASCs delivered onto a coral scaffold, and the left defect was either filled with an autologous ASC/coral composite (Group 1, n = 5) or with one coral scaffold alone (Group 2, n = 5). The systematic immune response and bone healing were evaluated postoperatively. The results showed that allogeneic ASC transplantation did not induce a systemic immune response by the hosts, and allogeneic ASCs could repair the cranial CSDs in an analogous way to that of the autologous cells. Moreover, both the green fluorescently labeled allogeneic and autologous ASCs were detected within the lacunae of newly formed bone in the defect site at 24 weeks, illustrating that the grafted ASCs contributed directly to bone regeneration in vivo. Thus, we concluded that allogeneic ASCs have the capacity to regenerate bone within craniofacial defects, providing an alternative source of seed cells for bone tissue engineering. © 2013 Elsevier Ltd.


Ma K.,Luoyang Orthopaedic Traumatological Hospital | Ma K.,Henan University | Zhang C.,Luoyang Orthopaedic Traumatological Hospital | Huang M.-Y.,Luoyang Orthopaedic Traumatological Hospital | And 2 more authors.
Oncology Reports | Year: 2016

The main objective of this study was to explore whether autophagy could be triggered by cinobufagin, and to clarify the role of autophagy in the antitumor effects of cinobufagin on U2OS cells and the underlying mechanisms. U2OS cells were exposed to 15, 30, 60 and 120 mg/l cinobufagin for 0, 12, 24 and 48 h. An MTT assay was used to measure cell viability. FITC-Annexin V/PI staining and flow cytometry were used to analyze the apoptotic ratio, while apoptotic morphological changes were assessed by PI and Hoechst 33258 viable cell staining. The effects of autophagy on the cells were investigated with GFP-LC3b green fluorescence plasmid transfection and transmission electron microscopy. The levels of caspase-3, -8, - 9, cleaved PARP, LC3-II/LC3-I, p62 and the activation of JNK/p-38 were detected by western blot analysis. Reactive oxygen species (ROS) fluorescence intensity was examined under fluorescence microscopy with an analysis software system. Cell proliferation was obviously inhibited by cinobufagin in a dose- and time-dependent manner. The apoptosis ratio was gradually increased with treatment time as evidenced by flow cytometric analysis and Hoechst 33258 staining. Exposure to cinobufagin resulted in the activation of caspase-3, -8, -9, as well as cleaved PARP which indicated that cinobufagin induced caspase-dependent apoptosis. Autophagy was confirmed in the cinobufagin-treated cells as evidenced by formation of autophagosomes, accumulation of GFP-LC3 fluorescence particles as well as the upregulation of LC3-II/LC3-I levels. Inhibition of autophagy diminished apoptosis as detected by the MTT assays. Moreover the percentage of apoptotic cells decreased following pretreatment with 3-MA, CQ and si-beclin-1. Cinobufagin also induced phosphorylation of the JNK and p38 signaling pathway as well as ROS generation. The JNK and p38 inhibitors significantly attenuated coexistence of apoptosis and autophagy-related proteins. The ROS scavenger also prevented phosphorylation of the JNK and p38 signaling pathway. Our research proved that cinobufagin triggered apoptosis and autophagic cell death via activation of the ROS/JNK/p-38 axis. © 2016, Spandidos Publications. All rights reserved.


Ma K.,Luoyang Orthopaedic Traumatological Hospital | Ma K.,Henan University | Huang M.-Y.,Luoyang Orthopaedic Traumatological Hospital | Guo Y.-X.,Luoyang Orthopaedic Traumatological Hospital | Hu G.-Q.,Henan University
Oncology Letters | Year: 2016

The aim of the present study was to observe whether autophagy was induced by matrine, and to investigate the role of autophagy in the antitumor effects of matrine on human osteosarcoma MG-63 cells and its underlying mechanism. MG-63 cells were cultured in vitro in matrine at a concentration of 0.6, 0.8, 1.0 and 1.2 g/l for 0, 24, 48 and 72 h. A MTT assay was used to evaluate the proliferation inhibition of MG-63 cells by matrine, and Annexin V-fluorescein isothiocyanate/propidum iodide (PI) staining flow cytometry was used to analyze the apoptotic rate. Alterations in cell morphology was assessed by PI and Hoechst 33258 cell staining. Matrine-induced autophagy in MG-63 cells was confirmed by green fluorescent protein-microtubule-associated protein 1-light chain 3 (LC3) b transfection and fluorescence microscopy, and cell viability was investigated by MTT assay following inhibition of autophagy by chloroquine (CQ) pretreatment. The expression level of apoptosis-associated proteins B-cell lymphoma-2 (Bcl-2) and Bcl-2-like protein 4 (Bax), autophagy-associated LC3II protein, and the activa- tion of extracellular signal-regulated kinase (ERK) was detected by western blotting. Cell proliferation was clearly inhibited by matrine in a dose- and time-dependent manner. Flow cytometry and Hoechst 33258/PI staining verified that matrine induced apoptosis in a time-dependent manner when cells were exposed to 1.1 g/l matrine; fluorescence microscopy demonstrated that green fluorescence puncta were enhanced with prolonged time of matrine incubation. Western blotting confirmed that the expression of pro-apoptosis-associated proteins Bax and LC3II, and phosphorylated-ERK were upregulated, and anti-apoptosis protein Bcl-2 was downregulated in a time-dependent manner following treatment with matrine. The cell viability of the matrine + CQ group was increased compared with the matrine group alone, which revealed that matrine treatment alone induced protective autophagy in MG-63 cells. In addiiton, expression of LC3II/LC3I decreased and the expression of BAX/Bcl-2 increased in the matrine + U0126 group compared with the matrine alone group. The present study demonstrated, to the best of our knowledge, for the first time that matrine induced protective autophagy via ERK activation in MG-63 cells, and matrine combined treatment with CQ or U0126 led to an increase in apoptosis in osteosarcoma cells. © 2016, Spandidos Publications. All rights reserved.


Rao Y.,Luoyang Orthopaedic Traumatological Hospital | Zhu W.,Luoyang Orthopaedic Traumatological Hospital | Liu H.,Luoyang Orthopaedic Traumatological Hospital | Jia C.,Luoyang Orthopaedic Traumatological Hospital | And 2 more authors.
Journal of International Medical Research | Year: 2013

Objectives: To investigate the safety and therapeutic efficacy of autologous olfactory ensheathing cell (OEC) transplantation in cervical spinal cord injury (SCI). Methods: Patients with cervical SCI of >6 months' duration were treated with autologous OECs, injected into the area surrounding the SCI under magnetic resonance imaging guidance, twice a week for 4 weeks. Patients were evaluated before treatment and at 3, 6, 12 and 24 months posttreatment, using the American Spinal Injury Association (ASIA) Impairment Scale, the ASIA sensory and motor score and the Functional Independence Measure (FIM) score. Results: Eight patients were recruited to the study. Three months after treatment, ASIA and FIM scores had improved significantly compared with pretreatment, though by 1 year no further significant improvements in the ASIA score were seen. The return of substantial sensation and motor activity in various muscles below the injury level was observed in three patients during follow-up. In addition, bladder function was restored in two patients. There were no serious complications postoperatively or during the follow-up period. Conclusions: This study provides preliminary evidence of the safety and possible efficacy of autologous OEC transplantation. © The Author(s) 2013.

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