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12 February 2016, Volume 20 Issue 7 Previous Issue    Next Issue

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Laquinimod inhibits the expression and function of hypoxia-inducible factor-2 alpha in osteoblasts Zhao Guang-zong, Fang Jun, Ding Gang, Zhang Long-qiang, Li Hua-zhuang, Gao Ke-hai 2016, 20 (7):  917-924.  doi: 10.3969/j.issn.2095-4344.2016.07.001 Abstract ( 334 )   PDF (531KB) ( 502 )   Save BACKGROUND: Fractures can induce bone cell hypoxia, and remarkably reduce the oxygen tension in cells. Hypoxia-inducible factor-2α is a key oxygen-dependent transcriptional activator to regulate the body function under hypoxia and mediate the release of various inflammatory factors after fractures. OBJECTIVE: To explore the role of Laquinimod in expression and function of hypoxia-inducible factor-2α in osteoblasts. METHODS: Mouse osteoblasts MC3T3-E1 (clone 14) were pretreated with Laquinimod at various concentrations(10-100 μmol/L) before hypoxia in the presence or absence of specific proteasome inhibitors MG132 or N-acetyl-leucyl-leucyl-norleucine. Then, the media were pre-conditioned in 1% or 21% oxygen tension for 1 to 24 hours. RESULTS AND CONCLUSION: Under hypoxia, the expression of hypoxia-inducible factor-2α in osteoblasts was increased remarkably, and Laquinimod could inhibit the expression of hypoxia-inducible factor-2α and its target genes in mouse MC3T3-E1 cells. Mechanistically, Laquinimod promoted hypoxia-inducible factor-2α degradation in a proteasome-dependent but von Hippel-Lindau protein-independent manner. Importantly, we found that Laquinimod disrupted the interaction between hypoxia-inducible factor-2α and its chaperone heat shock protein 90, but promoted the interaction between hypoxia-inducible factor-2α and the receptor of activated protein kinase C. These findings suggest that Laquinimod may promote the degradation of hypoxia-inducible factor-2α by affecting its folding and maturation. Laquinimod is a novel inhibitor of hypoxia-inducible factor-2α by changing its functional interaction with chaperone proteins heat shock protein 90 and receptor of activated protein kinase C.    中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Osx and Satb2 regulate osteoblast differentiation, bone formation and repair Hou Qiu-ke, Huang Yong-quan, Li Yun-jun, Chen Dong-feng 2016, 20 (7):  925-932.  doi: 10.3969/j.issn.2095-4344.2016.07.002 Abstract ( 445 )   PDF (569KB) ( 812 )   Save BACKGROUND: Osteoblasts occupy an important role in osteogenesis, which mainly come from bone marrow mesenchymal cells, and some transcription factors or local factors may promote the osteogenic differentiation of bone marrow stromal cells. OBJECTIVE: To study the role of Osx and Satb2 in C2C12 cells in the repair process of osteoporosis. METHODS: Twenty wild-type Sprague-Dawley rats were assigned into normal control group (n=10), sham group (n=5) and osteoporosis group (model group, n=5). Another 10 Osx-KO rats were enrolled in the study. Osteoporosis models were established by removal of both ovaries in the model group and Osx-KO group. In the sham group, bilateral ovaries were exposed but not removed. Changes in body mass and femoral bone density were detected in the four groups post operation. C2C12 cells were cultured in vitro, and siRNA-Satb2 and siRNA-Osx were designed. Expressions of Osx and Satb2 and their effects on osteoporosis were observed using cell experiments, gene silencing and western blot assay. RESULTS AND CONCLUSION: After 12 weeks, the body mass in the model and Osx-KO groups was significantly increased compared with the normal control and sham groups (P < 0.01); the bone density in the model and Osx-KO group was significantly decreased compared with the normal control and sham groups (P < 0.01). Satb2 and Osx were expressed in all the wild-type rats, but their expressions were decreased significantly in the Osx-KO rats (P < 0.001). Additionally, there was no difference in the Runx2 mRNA expression between the two kinds of rats. After silencing, the mRNA expressions of Satb2, Osx, Runx2 and ALP were all inhibited. These findings indicate that in the pathogenesis of osteoporosis, Osx and Satb2 may be protective molecules that have a regulatory role in the osteogenic differentiation, bone formation and repair.   中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Histological observation on new bone induced by platelet-rich fibrin Fu Dong-mei, Xiao Qiong, Yang Qin-qiu, Dong Lu, Chen Hong-liang, Sun Yong 2016, 20 (7):  933-939.  doi: 10.3969/j.issn.2095-4344.2016.07.003 Abstract ( 403 )   PDF (653KB) ( 597 )   Save BACKGROUND: In previous experiments, we have confirmed that platelet rich fibrin has the ability of osteoinduction, and have conducted a preliminary study on its microstructure and biomechanics. However, little is reported on its histology research. OBJECTIVE: To compare the histological changes after implanting platelet-rich fibrin, Bio-Oss and autologous bone and to analyze the pros and cons of platelet-rich fibrin implantation for repair of bone defects. METHODS: As previously reported, animal models of critical bone defects were established respectively on the bilateral femoral condyles of 12 beagle dogs. Then, platelet-rich fibrin, Bio-Oss+collagen membrane (Bio-Oss group) and autologous bone (autologous bone group)+collagen membrane were respectively implanted. At 3, 6, 8 and 12 months, one experimental dog from each group was killed, respectively, and histological observation was performed. Another beagle dog as blank control was enrolled to establish the animal model of critical bone defects, with no implantation. RESULTS AND CONCLUSION: At 3, 6, 8 and 12 months after implantation, there were significant differences in the new bone formation speed and amount between the platelet-rich fibrin group, Bio-Oss group and autologous bone group. These three kinds of bone grafts all had osteoinductive ability to different extents. In the platelet-rich fibrin group, the osteogenic effects were better at 3 and 6 months, and the new bone was similar to natural one; in the autologuos bone group, bone necrosis was noticeable at 3 and 6 months, but the osteogenic effects became better at 8 months, and the new bone was similar to natural one at 12 months; in the Bio-oss group, the osteogenic effects were similar to those in the platelet-rich fibrin group, but the residual of Bio-oss was visible at 12 months; in the blank control group, no bone formed at 3 months, indicating the animal model of critical bone defects was made successfully. In brief, the platelet-rich fibrin has good osteoinductive ability, with shorter time and better quality.   中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Differential expression of microRNAs in the intervertebral disc of hypoxia-inducible factor-1alpha deficient mice  Meng Xiang-chao, Liu Zhuo-chao, Wang Jun, Zhou Qi, Qi Jin, Zhang Xing-kai 2016, 20 (7):  940-946.  doi: 10.3969/j.issn.2095-4344.2016.07.004 Abstract ( 352 )   PDF (592KB) ( 638 )   Save BACKGROUND: It is confirmed that the absence of hypoxia-inducible factor-1α (HIF-1α) accelerates the degenerative process in the intervertebral discs, and microRNAs have an important role in degeneration of the intervertebral discs. OBJECTIVE: To evaluate the changes of microRNAs in the intervertebral discs of HIF-1α-deficient (HIF-1α-/-) mice which may mediate the signaling pathway of HIF-1α in the intervertebral discs. METHODS: As previously reported, HIF-1α-/- mice were established. HIF-1α-/- mice and HIF-1αflox/flox mice (control mice) aged 4 weeks were used. MRI and histological staining were used to evaluate the degeneration of the intervertebral discs. Total RNAs were extracted from the intervertebral discs tissues by Trizol, and the differential expression profile of microRNAs was harvested by significance analysis of microarrays and Cluster, based on microarray screening. Real-time quantitative reverse transcription-PCR was applied to verify the reliability of microRNA array results. RESULTS AND CONCLUSION: The number of nucleus pulposus cells in the intervertebral discs of HIF-1α-/- mice was decreased, the cells presented with small size and the color deepened in the cytoplasm. Finally, differential expression profile of microRNAs (n=10) was obtained, seven of which were upregulated and three were downregulated. In conclusion, the loss of HIF-1α may cause the imbalance of some important miRNAs, which may result in a large amount of dead nuclear pulposus cells and mediate disc degeneration in HIF-1α-/- mice.   中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Constructing and identifying a lentiviral vector of RNA interference targeting matrix metalloproteinases-3 gene in human degenerative nucleus pulposus cells Cao Jin, Fu Pei-rong, Fang Jing, Yang Jian-kun, Wei Hua-wei, Li Si-yuan, Gao Feng, Xi Yong-ming 2016, 20 (7):  947-956.  doi: 10.3969/j.issn.2095-4344.2016.07.005 Abstract ( 336 )   PDF (728KB) ( 403 )   Save BACKGROUND: Inhibiting the degradation of extracellular matrix in the intervertebral disc can delay the degenerative process of intervertebral disc. Matrix metalloproteinases-3 (MMP3) is considered as a key enzyme for degradation of extracellular matrix components such as type II collagen and aggrecan. OBJECTIVE: To construct the short hairpin RNA lentiviral vector targeting human MMP3 gene and to detect its efficiency of gene silence by infecting human degenerative nucleus pulposus cells. METHODS: According to the human MMP3 mRNA (NM_002422.4) sequence, four groups of the short hairpin RNA gene sequences targeting MMP3 were designed, synthesized and annealed to form double stranded DNA fragments, which were connected with the LV3 vectors digested by BamHI and EcoRI enzymes, and then transfected into the competent cells. The positive clones were identified by PCR, and analyzed by sequencing. The packaging and titer of lentivirus were determined after transfecting 293T cells. Human degenerative nucleus pulposus cells were infected with lentivirus vector, and the transfection efficiency of each group was observed under inverted fluorescence microscope. The interfering efficiency was detected by real time-PCR and western blot at 72 and 96 hours. RESULTS AND CONCLUSION: The ds-oligo DNA was successfully inserted into the lentiviral vector as confirmed by electrophoresis and sequence analysis. The recombinant lentivirus was harvested from 293T cells with a viral titer of 1-5 ×108 TU/mL. RNA interference targeting the GCC AGG CTT TCC CAA GCA AAT sequences with the highest interfering efficiency in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 mRNA expression by 98% and 72%, respectively; and at 96 hours, the interfering efficiency of protein expression was 57.2%. The recombinant lentivirus vector containing RNA interference targeting MMP3 gene is successfully constructed, which lays a foundation for further studies on the MMP3 function and gene therapy.  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程   Figures and Tables | References | Related Articles | Metrics

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Platelet-rich fibrin for repair of oral soft tissue defects Wang Tuo, Yang Qin-qiu, Dong Lu, Xiao Qiong, Chen Hong-liang, Sun Yong, Zhong Ke 2016, 20 (7):  957-965.  doi: 10.3969/j.issn.2095-4344.2016.07.006 Abstract ( 491 )   PDF (1004KB) ( 653 )   Save BACKGROUND: Insufficient oral soft tissues in the implant zone may have a negative effect on the wound healing and the aesthetic restoration in the late stage. Platelet-rich fibrin can promote the wound healing of soft tissue defects. But there is still a lack of in-depth studies on the promotion of oral soft tissue defects in animal experiments. OBJECTIVE: To compare the repairing effects of platelet-rich fibrin and collagen membrane on soft tissue defects of the hard palate in New Zealand rabbits. METHODS: Fifty-four New Zealand rabbits were randomly divided into three groups (n=14 per group): platelet-rich fibrin group, collagen membrane group and blank control group. A 5 mm-diameter circular full-thickness soft tissue defect was made in the front of the hard palate, 2 mm distant to the rear maxillary incisors and mucosal edge of the bilateral hard palates. Autologous platelet-rich fibrin membrane or collagen membrane were implanted into the defect in the platelet-rich fibrin group and collagen membrane group, respectively. No treatment was given in the blank control group. General observation of the wound and wound healing analysis were performed at days 3, 7, 14, 21, 28, 56 post operation. Hematoxylin-eosin staining, CD31 immunohistochemical staining and Masson staining were used to observe inflammatory reaction, angiogenesis and collagen formation in the surgical site. RESULTS AND CONCLUSION: The wound healing rate was fastest in the platelet-rich fibrin group, and no obvious scar formed. At 3 days post operation, there was no difference in the wound healing rates among the three groups; at 7 days, the wound healing rate in the platelet-rich fibrin group was significantly higher than that in the collagen membrane group and blank control group (P < 0.05). At 3 and 7 days after operation, the inflammatory reaction in the platelet-rich fibrin group was less than that in the collagen membrane and blank control groups (P < 0.05); at 14, 21, 28 and 56 days, there was no significant difference between the three groups. At 7, 14, 21 days after operation, the average absorbance value of CD31 in the platelet-rich fibrin group was significantly higher than that in the collagen membrane and blank control groups (P < 0.05). The average absorbance value of collagen formation in the platelet-rich fibrin group was significantly higher than that in the collagen membrane and blank control groups at 7 days after operation (P < 0.05), significantly higher than that in the blank control group at 14 days (P < 0.05), but lower than that in the collagen membrane and blank control groups at 21, 28 and 56 days after operation (P < 0.05). These findings show that platelet-rich fibrin can reduce inflammatory reactions in the process of wound healing, accelerate the angiogenesis, regulate the metabolism of collagen, reduce the formation of scar and improve the quality of wound healing, thereby promoting the repair of oral soft tissue defects. 中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Allogeneic versus heterogeneous bone marrow mesenchymal stem cells for laryngeal cartilage repair Liu Yi-chang, Zhou Jing 2016, 20 (7):  966-971.  doi: 10.3969/j.issn.2095-4344.2016.07.007 Abstract ( 261 )   PDF (485KB) ( 457 )   Save BACKGROUND: Because chondrocytes have no regeneration ability, to select suitable seed cells is the primary problem to repair cartilage defects. OBJECTIVE: To investigate the effect of allogeneic versus heterologous bone marrow mesenchymal stem cells (BMSCs) in repairing laryngeal cartilage defects after chondrogenic induction. METHODS: BMSCs from human and rabbits were isolated and cultured. Passage 3 cells were cultured in chondrogenic induction medium containing transforming transforming growth factor beta 1 and bone morphogenetic protein, and then were dropped onto a poly(lactic-co-glycolic acid) (PLGA) scaffold. Thirty New Zealand rabbits were randomly assigned into three groups: blank control group, human BMSCs group, rabbit BMSCs group. Animal models of laryngeal cartilage defects were made in the three groups. After modeling, saline-soaked PLGA scaffold, PLAG scaffold with human BMSCs or with rabbit BMSCs were implanted respectively into the rabbits in the normal blank, human BMSCs and rabbit BMSCs groups. The expression of type II collagen in the larynx and its surrounding tissues was detected by immunohistochemistry at 4 and 8 weeks postoperatively. RESULTS AND CONCLUSION: The animals in each group breathed normally with no presence of wheezing, and their eating and activity were good. Moreover, there was no purulency or infection in the three groups. At 4 and 8 weeks after operation, the positive rates of type II collagen in the two BMSCs groups were significantly higher than that in the blank control group (P < 0.05). There was no significant difference between two BMSCs groups (P > 0.05). These results show that both allogeneic and heterologous BMSCs have good therapeutic effects on the repair of laryngeal cartilage defects in rabbits.  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程   Figures and Tables | References | Related Articles | Metrics

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Effects of cyclic tensile strain on actin cytoskeleton rearrangement in annulus fibrosus cells Zhang De-hong, Fang Peng-fei, Wang Xing-sheng, Zhao Ji-rong, Li Xiao-na 2016, 20 (7):  972-980.  doi: 10.3969/j.issn.2095-4344.2016.07.008 Abstract ( 416 )   PDF (597KB) ( 594 )   Save BACKGROUND: When the intervertebral disc is under stress, the hydraulic pressure generated inside the nucleus pulposus makes the annulus fibrosus extend outward and expand, and the annulus collagen fibers are stretched so that the extracellular matrix of annulus fibrosus cells is also under the pressure. In the intervertebral disc, aggrecan is the main component of proteoglycans, matrix metalloproteinase-2 is a major enzyme for extracellular matrix degradation, and tissue inhibitor of metalloproteinase is a multifunctional specific inhibition factor for matrix metalloproteinase activity. There is a mutual regulation between the latter two to keep the homeostasis between them. OBJECTIVE: To investigate the mechanism of cyclic tensile strain in the metabolism of intervertebral disc annulus matrix. METHODS: Rat anulus fibrosus cells were subjected to 2% or 10% cyclic tensile strain at 1.0 Hz for 2 and 12 hours using Flexcell4000 tension system. Then cells were collected and cultured in conditioned medium for gene and protein detection. Real-time quantitative PCR was used to detect mRNA expression of aggrecan, matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2. Gelatin zymography was used to detect matrix metalloproteinases-2 activity. RESULTS AND CONCLUSION: The use of 2% cyclic tensile strain had no obvious effect on the stress fiber of actin cytoskeleton, whereas actin cytoskeleton was depolymerized in response to 10% cyclic tensile strain. The 2% cyclic tensile strain raised the expression of Aggrecan at 12 hours; whereas raised the matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2 at 2 hours, both of which were in homeostasis; matrix metalloproteinases-2 activity had no significant changes. 10% cyclic tensile strain had no effect on the mRNA expression of Aggrecan. No matter stretching 2 or 12 hours, the matrix metalloproteinases-2 was up-regulated, and the tissue inhibitor of metalloproteinase-2 was down-regulated, both of which were not in balance. Moreover, the matrix metalloproteinases-2 activity was not significantly changed. These findings indicate that the mRNA expressions of Aggrecan, matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2 alter in response to cyclic tensile strain in rat anulus fibrosus cells, and the tensile strain induces different mechano-responses in the actin cytoskeleton.  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Effect of simvastatin on bone mineral density and biomechanical properties of ovariectomized rats Zhang Yan, Liu Hao, Xing Lei, Zhang Guo-bin, Tian Fa-ming 2016, 20 (7):  981-986.  doi: 10.3969/j.issn.2095-4344.2016.07.009 Abstract ( 353 )   PDF (421KB) ( 368 )   Save BACKGROUND: Osteoporosis and its complications severely threaten the elder’s health. Simvastatin, widely accepted as a lipid-lowering drug, is reported to potentially promote bone formation, but it is in debate when orally administered, and there is no evidence to support whether this is due to the region difference. OBJECTIVE: To investigate the effect of orally administered simvastatin on bone mass and biomechanical properties of the femur and vertebrae in osteopenia rats induced by ovariectomy (OVX). METHODS: Twenty-four 6-month-old female Sprague-Dawley rats were subjected to OVX+orally administered saline vehicle (OVX group, n=8), OVX+orally administered simvastatin (5 mg/kg/d; intervention group, n=8) or sham surgery (sham group, n=8). After 8 weeks of treatment, all rats were sacrificed and the level of procollagen type I N-terminal propeptide in blood serum was assessed by ELISA. Bone mineral density was determined in the L5 vertebra and left femur using dual-energy X-rays. Furthermore, the biomechanical properties of the L4 vertebra and right femur, including maximum load and elastic modulus, were detected by compression testing and three-point bending test, respectively. RESULTS AND CONCLUSION: The serum level of procollagen type I N-terminal propeptide in the sham group was significantly lower than that in the other two groups. OVX rats showed significantly lower bone mineral density in both the L5 vertebra and left femur than sham rats (P < 0.05). Rats in the intervention group showed higher bone mineral density than those in the OVX group, with statistically significant difference in the L5 vertebra (P < 0.05), but insignificant difference in the femur. Maximum load and elastic modulus of the L4 vertebra in the OVX group were significantly lower than those in the sham and intervention group. Markedly lower elastic modulus of the femur was found in the OVX group than the sham and intervention groups. These findings demonstrate that simvastatin treatment can partially prevent bone loss in OVX rats with more notable effect on the vertebrae than the femur, and for this model, the vertebra is superior to the femur used in biomechanical test. 中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Digital rapid prototyping implant template assists anterior tooth restoration in maxillary esthetic zone: a follow-up of 12 months Yuan Zhi, Chen Yi-hui 2016, 20 (7):  987-992.  doi: 10.3969/j.issn.2095-4344.2016.07.010 Abstract ( 432 )   PDF (536KB) ( 614 )   Save BACKGROUND: Dental implantation in maxillary esthetic zone is difficult to achieve desired outcomes. OBJECTIVE: To observe the feasibility of digital rapid prototyping implant template for anterior implantation. METHODS: Eighty patients scheduled to receive anterior implant treatment were enrolled. According to the wishes of patients, they were divided into two groups, with 40 cases in each group, and were given routine implant treatment (control group) and digital rapid prototyping template-aided dental restoration (observation group). After implantation, the X-axis, Y-axis and Z-axis errors of the two groups were measured, and the patients were followed up for 12 months. RESULTS AND CONCLUSION: After implantation, the errors of X, Y and Z axes in the observation group was significantly lower than those in the control group (P < 0.05); at 24 hours after implantation, patients in the two groups showed good appearance of the anterior tooth with no dental prosthesis loosing. Imaging examinations in the two groups showed no obvious bone resorption. These findings indicate that the aid therapy using digital rapid prototyping can obtain satisfactory therapeutic effect on anterior tooth restoration in the maxillary esthetic zone and precisely localize the dental implantation site.  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程   Figures and Tables | References | Related Articles | Metrics

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Human bone marrow mesenchymal stem cells promote epithelial mesenchymal  transition in lung cancer cells Wu Jia-bin, Wang Tao, Yang Wei-lin, Wang Jun-jie, Xiao Jie-fei, Wang Ru-chen, Chen Zhen-guang 2016, 20 (7):  993-999.  doi: 10.3969/j.issn.2095-4344.2016.07.011 Abstract ( 273 )   PDF (626KB) ( 616 )   Save BACKGROUND: The complex relationship between bone marrow mesenchymal stem cells and cancers severely limit the clinical application of mesenchymal stem cells. So it is urgent to study the role of mesenchymal stem cells in tumor growth and metastasis. OBJECTIVE: To explore the effect of human bone marrow mesenchymal stem cells on epithelial mesenchymal transition in non-small cell lung cancer A549 and PAa cells. METHODS: The A549 and PAa cells were cultured with mesenchymal stem cell supernatant (mesenchymal stem cell conditioned medium, MSCs-CM). The cellular morphology was observed under a microscope. The mRNA and protein expression of E-cadherin, N-cadherin, Vimentin, Slug, Snail, and Twist were determined by RT-PCR and western blot. Transwell and wound healing assay were used to detect the change of migration and metastatic ability. RESULTS AND CONCLUSION: Compared with the control group, the cellular morphology of experimental group showed mesenchymal-like changes. In response to MSCs-CM, there was decreased E-cadherin but increased N-cadherin, Vimentin and Slug, Snail, Twist at mRNA and protein levels compared with the control group (P < 0.05). The migration and metastatic abilities of the experimental group were also increased. So, human bone marrow mesenchymal stem cells can promote epithelial mesenchymal transition in A549 and PAa cells, and enhance the migration and metastatic abilities of A549 and PAa cells.   中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Inhibitory effects of extracellular cholesterol and lipopolysaccharide on cellular cholesterol efflus Liu Jie, Zheng Yun-mei, Tian Zhi-hui, Chang Guang-ming, Li Hai-dong 2016, 20 (7):  1000-1005.  doi: 10.3969/j.issn.2095-4344.2016.07.012 Abstract ( 429 )   PDF (400KB) ( 487 )   Save BACKGROUND: Cholesterol is closely linked to the occurrence and progression of atherosclerosis. Current approaches to study cellular cholesterol dynamics have their own limitations. OBJECTIVE: To measure the cholesterol efflux rate of RAW 264.7 mouse macrophages by BODIPY-Cholesterol labeling and to explore the effects of extracellular cholesterol and lipopolysaccharide on the cholesterol efflux rate. METHODS: RAW 264.7 cells were cultured in vitro with DMEM containing 10% fetal bovine serum, and labeled with BODIPY-Cholesterol for 1, 2, 4, 8 hours. Then, the cells were rinsed with serum-free DMEM and inoculated for 6, 12, 24, 48, 96 hours to optimize the labeling time and incubation time. We measured and compared the cholesterol efflux rates after cultured cells were treated with cholesterol, lipopolysaccharide, human sera with high cholesterol or human sera with normal cholesterol. RESULTS AND CONCLUSION: The best labeling time for BODIPY-Cholesterol was 2-8 hours. Cholesterol efflux rates were gradually decreased after the cells that were labeled for 2 hours were incubated with increasing concentrations of cholesterol (0.1, 0.5, 2.5 mmol/L, P < 0.01). Treating cells with lipopolysaccharide also decreased the cholesterol efflux rate (P < 0.05). Furthermore, the cholesterol efflux rate was decreased after cells were treated with human sera with high cholesterol (P < 0.05). These findings indicate that BODIPY-Cholesterol can be used to measure cellular cholesterol efflux rate and to study the effects of extracellular cholesterol and lipopolysaccharide on the cholesterol efflux rate.  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Preparation and properties of a new human acellular dermal matrix Jiang Tao, Zhang Ai-jun, Li Xue-yang, Ma Zhi-bing, Shen Cai-qi, Jin Pei-sheng 2016, 20 (7):  1006-1012.  doi: 10.3969/j.issn.2095-4344.2016.07.013 Abstract ( 430 )   PDF (758KB) ( 748 )   Save BACKGROUND: Early vascularization is crucial for wound healing. A high-porosity, macrovoid allogeneic skin leads to the rapid vascularization and cellular infiltration. OBJECTIVE: To obtain a new allogeneic skin product with high porosity, good cell permeability and good histocompatibility using an improved preparation method of human acellular dermal matrix. METHODS: Cell components of healthy human skins were removed by the improved method and the traditional method, respectively. The improved method was to remove the subcutaneous fat, eliminate the epidermis (1 mol/L NaCl solution at 37 ℃ for 24 hours) followed by shaking processing (2% NaOH at 45 ℃ for 4 hours), and then, the solution was neutralized with PBS rinsing, dried and stored at 4 ℃ for standby. We detected the porosity and degradation time in vitro of the acellular dermal matrices prepared by two methods and the cytotoxicity of the material infiltration liquid on the adipose-derived stem cells. Hematoxylin-eosin staining was used for the detection of the cell residual, the integrity of collagen and cell biocompatibility. Scanning electron microscopy was used to detect the pore size. RESULTS AND CONCLUSION: Both the two methods could completely remove the cell components, and maintain the integrity of the collagen scaffold. The porosity of acellular dermal matrix with the improved method was (93.22±0.99)%, which was significantly higher than that with the traditional method [(74.28±2.06)%; P < 0.001]. However, there was no significant difference in in vitro degradation time between the two kinds of acellular dermal matrices (P > 0.05). No obvious cytotoxicity of the acellular dermal matrix prepared with the improved method was detected. At 3-7 days of co-culture, the adipose-derived stem cells cultured on the acellular dermal matrix prepared with the improved method could penetrate the basement membrane to the deep dermis, while there was no obvious cell invasion and growth in the deep dermis prepared by the traditional method. Compared with the traditional method, the improved method is more suitable for cell infiltration and growth with higher porosity and larger pore size.   中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Regulation of endometrial stromal cells through the Wnt/beta-catenin signaling pathway Chen Man-ru, Liao Zhi 2016, 20 (7):  1013-1018.  doi: 10.3969/j.issn.2095-4344.2016.07.014 Abstract ( 467 )   PDF (495KB) ( 505 )   Save BACKGROUND: Increasing number of genes and signaling pathways are involved in regulating stem cells periodically, and wherein Wnt/β-catenin signaling pathway is an important pathway of stem cells. OBJECTIVE: To investigate the effects of Wnt/β-catenin signal pathway on mouse endometrial stromal cells. METHODS: After injection of Wnt/β-catenin pathway inhibitor or activator, endometrial tissues from Balb/c mice were obtained, some of which were used for detection of invasion of endometrial stromal cells and western blot detection, and the rest of which were used for preparing animal models of endometriosis followed by immunohistochemical detection. RESULTS AND CONCLUSION: The expression of β-catenin and GSK3β proteins was significantly higher in the activator group than the inhibitor group (P < 0.05). The number of transmembrane cells was significantly higher in the activator group than the inhibitor and control groups (P < 0.05). Immunohistochemical findings showed positive expression of E-cadherin in ectopic endometrial tissues of the inhibitory group, and strongly positive expression of vascular endothelial growth factor in ectopic endometrial tissues of the activator group. These findings indicate that Wnt/β-catenin signaling pathway may cause endometriosis by strengthening ectopic endometrial implantation, invasion and metastasis.   中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Improved extraction of primary vascular endothelial cells from the rabbit aorta Xiao Zi-chun, Tan Jin-hai, Zhou Yi 2016, 20 (7):  1019-1024.  doi: 10.3969/j.issn.2095-4344.2016.07.015 Abstract ( 466 )   PDF (518KB) ( 625 )   Save BACKGROUND: Primary vascular endothelial cells are mostly harvested through aorta endothelial cell cultures and micro-artery endothelial cell cultures using enzyme digestion and tissue adhesion methods, and the quality and purity of harvested cells cannot meet the need for current scientific research. OBJECTIVE: To investigate an improved extraction of primary vascular endothelial cells and the relevant identification method. METHODS: A segment of rabbit aorta was cut to culture vascular endothelial cells using the improved extraction method in group A or using adhesion method in group B. In the group A, the vascular intima was striped out with microsurgical instruments, and digested enzymatically to acquire single primary cells followed by culture in endothelial cell culture medium. In the group B, the whole vascular intima was adhered to the culture dish that was incubated in a 5%CO2, 37 ℃ incubator for 1 hour. Cell pellets in the two groups were cultured in vitro. Cell morphology was observed using a microscope; immunohistochemical staining was used to detect CD31, VIII factor and Vimentin protein for identification of vascular endothelial cells. RESULTS AND CONCLUSION: The purity and number of vascular endothelial cells extracted by the improved method were higher than those by the adhesion method. Immunohistochemical findings showed positive expression of CD31 and VIII factor, but negative expression of Vimentin. These findings indicate that the improved extraction method can obtain more vascular endothelial cells with higher purity, which is of strong operability and practicality.   中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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6-Hydroxydopamine up-regulates divalent metal transporter-1 and ferroportin-1 in C6 glioma cell lines Xu Man-man, Xu Ling-yu, Yang Xiao, Du Xin-xing 2016, 20 (7):  1025-1030.  doi: 10.3969/j.issn.2095-4344.2016.07.016 Abstract ( 260 )   PDF (364KB) ( 351 )   Save BACKGROUND: Previous studies have confirmed that 6-hydroxydopamine is capable to increase the expression of divalent metal transporter-1 and reduce the expression of ferroportin-1 in the neurons and microglia, which may lead to iron deposition in the substantia nigra after Parkinson’s disease. However, it is unclear whether 6-hydroxydopamine can play diverse roles in astrocytes. OBJECTIVE: To observe the effects of 6-hydroxydopamine on the expression of divalent metal transporter-1 and ferroportin-1 in rat C6 glioma cell lines. METHODS: C6 glioma cell lines from rats were cultured in 10 μmol/L 6-hydroxydopamine for 24 hours. Then, protein expressions of divalent metal transporter-1 and ferroportiner-1 were measured by western blot method. RESULTS AND CONCLUSION: The protein expressions of divalent metal transporter-1 and ferroportin-1 in C6 glioma cell lines were increased by 2.5 times (P < 0.01) and 1 time (P < 0.05), respectively, after treatment with 6-hydroxydopamine. These findings indicate that 6-hydroxydopamine can promote iron transport rate in astrocytes by increasing both divalent metal transporter-1 and ferroportin-1 expressions, and astrocytes has a different response to 6-hydroxydopamine from neurons and microglia.   中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Inflammation induced by degenerative lumbar scoliosis Meng Ning-bo 2016, 20 (7):  1031-1036.  doi: 10.3969/j.issn.2095-4344.2016.07.017 Abstract ( 309 )   PDF (481KB) ( 316 )   Save BACKGROUND: Degenerative lumbar scoliosis patients will appear to have abnormal changes in various cytokines in the serum of the body, which is closely related to patient’s pathogenesis. OBJECTIVE: To monitor the changes in the serum levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β during the onset of degenerative lumbar scoliosis. METHODS: Sixty-six patients with degenerative lumbar scoliosis were enrolled as observation group, and according to Schwab evaluation system, there were 16 cases of type I, 32 of type II, and 18 of type III. Additionally, 60 healthy persons undergoing homochronous physical examination were extracted as control group. Blood samples were taken at preoperative 1, 2, 3 weeks and at postoperative 1, 2, 3, 4, 5, 6 weeks to determine serum levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β as well as to observe the relevant changes. RESULTS AND CONCLUSION: In the observation group, the levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β were increased gradually at 1-3 weeks before treatment, and then showed a decrease trend at 6 weeks after treatment. Compared with the control group, in the observation group, the levels of tumor necrosis factor-α were significantly higher at 1-4 weeks after admission (P < 0.05); the levels of interleukin-6 and interleukin-1β were significantly higher at 1-5 weeks after admission (P < 0.05). These findings indicate that the levels of tumor necrosis factor-α, interleukin-6 and interleukin-1 reached the peak at 1 week before treatment, and then gradually decreased. Changes in the levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β have an important role to assess disease development, which can be used to determine the severity of inflammation based on which we can select the appropriate treatment.  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Tissue-engineered cartilage for repair of sports-induced cartilage injury Wang Da-peng, Zhang Lan, Zhao Na 2016, 20 (7):  1037-1043.  doi: 10.3969/j.issn.2095-4344.2016.07.018 Abstract ( 434 )   PDF (565KB) ( 451 )   Save BACKGROUND: Sports-induced cartilage injury is very common; due to the poor self-healing capacity of the cartilage, cartilage repair has always been a difficult problem. OBJECTIVE: To review the features of different seed cells in tissue-engineered cartilage construction and to explore the application of tissue-engineered cartilage construction in the repair of sports-induced cartilage injury in vitro. METHODS: We searched PubMed database, Wanfang database and CNKI database for articles related to tissue-engineered cartilage repair of sports-induced cartilage injuries, as well as stem cells and scaffold materials used in tissue-engineered cartilage construction. Totally 190 articles were retrieved, and finally 47 articles were included in result analysis after repetitive studies were excluded. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells under different conditions can differentiate into chondrocytes, and have better potential of chondrogenic differentiation compared with adipose-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells. But, their safety still needs to be further studied. Good scaffolds cannot only induce stem cell differentiation, but also be the key to cartilage construction. Composite materials are the future direction of the scaffold research.  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics

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Peripheral nerve repair: theory and technology application He Xin-ze, Wang Wei, Hu Tie-min, Ma Jian-jun,Yu Chang-yu, Gao Yun-feng, Cheng Xing-long, Wang Pei 2016, 20 (7):  1044-1050.  doi: 10.3969/j.issn.2095-4344.2016.07.019 Abstract ( 388 )   PDF (685KB) ( 1789 )   Save BACKGROUND: Recovery of motor and sensory function from peripheral nerve injury is relatively slow and incomplete. It is a difficult problem for orthopedic surgeons that mainly leads to the decline in the quality of life in patients. OBJECTIVE: To conclude the methods and corresponding outcomes in peripheral nerve regeneration by analyzing the new treatment means for peripheral nerve injury. METHODS: PubMed, Wanfang, CNKI databases were retrieved for relevant articles using key words of “nerve injury, regeneration”, and then retrieval data were sorted and analyzed. RESULTS AND CONCLUSION: In recent years, in-depth studies on peripheral nerve repair have been made in the following aspects: surgical mode, drug, cytokine, gene transfer and biomaterials as well as traditional Chinese medicine. If the detect size is four times longer than the diameter of nerves, the nerve regeneration chamber can achieve good outcomes. The methods of restoring nerve continuity following nerve injury are developed from surgical anastomosis to photochemohistological method, thermal laser welding, plastic repair and other emerging technologies. Studies have found that plasminogen activator, nerve growth factor, neurotrophic factor, recombinant erythropoietin, human tissue kallikrein, B vitamins and their derivatives, herbal preparations, immunosuppressive agents all can promote nerve regeneration.  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程   References | Related Articles | Metrics

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Clinical anatomy of the mesorectum Li Wen-rui, Zhou Le-qun, Zhang Wei-guang 2016, 20 (7):  1051-1056.  doi: 10.3969/j.issn.2095-4344.2016.07.020 Abstract ( 373 )   PDF (511KB) ( 630 )   Save BACKGROUND: Currently, it is still controversial about the border, surrounding fascia, space of pelvic cavity, distribution of nerves and lymph nodes of the mesorectum, and the development of new technologies makes a progress in related anatomic research. OBJECTIVE: To summarize the previous studies so as to describe clearly the progress of mesorectal anatomy and to discuss its clinical value. METHODS: Using “rectum; mesentery; fascia; space; nerve; lymph node; total mesorectal excision (TME); clinical anatomy” as key words, a computer-based search of PubMed was done for articles related to the mesorectum and surrounding fasciae, space of pelvic cavity, distribution of nerves and lymph nodes. RESULTS AND CONCLUSION: Fresh or frozen specimens are often used for studying the mesenterium, fascia, nerves and lymph nodes by using traditional pelvic and perineum anatomical methods. Computer-assisted anatomical dissection can combine immunostaining with computer imaging. A three-dimensional model can well reflect the relationship among the different anatomical structures, as well as nerve traveling and spatial location. Mesorectum is located behind the denonvilliers and in the front of the sacral fascia of the rectum. Pelvic splanchnic nerve of the mesorectum is derived from the anterior sacral nerve root, runs through the presacral fascia, and enters into the neuro-fascial layer via the pesacral space, which is divided into the upper and lower parts according to the peritoneum. There are more folds in the rear of lymph nodes within the mesorectum within and near the peritoneum. There are still a lot of controversies about anatomical relationship between the mesorectum and surrounding structures, and to elaborate these issues can provide an objective basis for guiding clinical work.  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 References | Related Articles | Metrics

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A randomized control clinical study on small-needle-knife therapy combined with exercise therapy for knee osteoarthritis: 3-month follow-up visit Zhao Ming-lei, Bai Yue-hong, Zhang Ying, Shi Wen-min 2016, 20 (7):  1057-1064.  doi: 10.3969/j.issn.2095-4344.2016.07.021 Abstract ( 673 )   PDF (541KB) ( 750 )   Save BACKGROUND: Small-needle-knife therapy for knee osteoarthritis has no uniform location, operation and mechanisms of action. Studies have proved that exercise therapy can enhance muscle strength, increase stability of the knee, improve joint range of motion, and effectively relieve pain. OBJECTIVE: To observe the clinical effect of small-needle-knife therapy combined with exercise therapy for treatment of knee osteoarthritis via a randomized controlled clinical trial. METHODS: 122 patients were randomly divided into treatment group (n=61; small-needle-knife therapy combined with exercise therapy) and control group (n=61; low-frequency therapy combined with exercise therapy). Then, clinical efficacy in the two groups were assessed by statistical analysis of visual analog scale, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), swelling degree of the knee joint, quadriceps circumference, flexion and extension of the knee joint before and after treatment. Meanwhile, adverse reactions in patients were recorded for safety evaluation. RESULTS AND CONCLUSION: (1) The visual analog scale and WOMAC scores in the two groups were both significantly improved at 2 weeks after treatment (P < 0.05). Moreover, these scores in the treatment group were significantly lower than those in the control group (P < 0.05). (2) At 12 weeks after treatment, the WOMAC score in the treatment group was better than that in the control group (P < 0.05), and the range of motion of the knee joint was also better in the treatment group than the control group (P < 0.05). (3) According to the full analysis set and per protocol set, the total efficiency rats in the treatment group were both superior to those in the control group (P < 0.001). (4) In the treatment group, there were four cases of surgery, four cases lost to follow-up, and two cases of mild adverse reactions; in the control group, there were six cases of surgery, three cases lost to follow-up, and no adverse reaction. Taken together, small-needle-knife therapy and physiotherapy both have certain clinical effects on knee osteoarthritis. Small-needle-knife therapy combined with exercise therapy is superior to physiotherapy combined with exercise therapy in the total efficiency. Follow-up results of 3 months have been confirmed, but long-term effects need further exploration.   中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程 Figures and Tables | References | Related Articles | Metrics