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09 December 2016, Volume 20 Issue 51 Previous Issue    Next Issue

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Autologous iliac crest graft combined with autologous bone marrow stem cell transplantation for bone nonunion after fracture surgery Cheng Yin, Lu Xiao-bo, Zhang Yun, Ying Lv-fang 2016, 20 (51):  7605-7610.  doi: 10.3969/j.issn.2095-4344.2016.51.001 Abstract ( 363 )   PDF (872KB) ( 310 )   Save BACKGROUND: Bone nonunion is a common complication in the orthopedic treatment, and its morbidity reached 5%-10%, which results in the long-term functional disturbance of the limbs, and even disability. Autogenous iliac crest graft has been commonly used to treat bone nonunion, but some limitations still exist. OBJECTIVE: To investigate the treatment outcomes of autogenous iliac crest graft combined with autologous bone marrow stem cell transplantation for bone nonunion after fracture surgery. METHODS: Clinical and follow-up data from 69 patients with bone nonunion were analyzed retrospectively. All patients were allotted to combination (n=37) and iliac (n=32) groups, followed by treated with autologous iliac crest graft combined with autologous bone marrow stem cell transplantation or  autologous crest graft, respectively. Afterwards, the hospitalization time, fracture healing time, bone mineral density and Fereadez-Esteve callus scores were detected and compared between groups. RESULTS AND CONCLUSION: The hospitalization time did not differ significantly between groups (P > 0.05). The fracture healing time in the combination group was significantly shortened compared with the iliac group (P < 0.05). The bone mineral density and Fereadez-Esteve callus scores in the combination group were significantly higher than those in the iliac group at 3 and 6 months after surgery (P < 0.05). The excellent and good rate of the affected limb function in the combination group was significantly higher than that in the iliac group (P < 0.05). These results suggest that autogenous iliac crest graft combined with autologous bone marrow stem cell transplantation for bone nonunion can accelerate fracture healing, promote porosis and improve the functional recovery of affected limbs. 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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C- and N-terminal telopeptides of type I collagens, osteocalcin and bone-specific alkaline phosphatase used for the early diagnosis of bone nonunion Wang Qing-hua, Lin Jian-ping, Shen Ning-jiang, Shi Zhan-jun, Zhou Gang, Wu Duo-neng 2016, 20 (51):  7611-7621.  doi: 10.3969/j.issn.2095-4344.2016.51.002 Abstract ( 336 )   PDF (1122KB) ( 199 )   Save BACKGROUND: Detecting the serum levels of biochemical markers to assess bone fractures is simple, mini-invasive and specific. Thereafter, to predict bone nonunion by choosing an appropriate marker has become a hotspot. OBJECTIVE: To establish an animal model of bone nonunion and explore the changing rules of the biochemical markers during the process of nonunion. METHODS: Twenty New Zealand white rabbits aged 5-6 months were enrolled and divided into two groups. In bone defect group, a 15-mm length of bone (including the periosteum) was removed from the left mid-radius, and the medullary cavities were closed with bone wax. In bone fracture group, the mid-radius was fractured. X-ray examination was taken and blood samples were extracted preoperatively and at 2, 3, 4, 5, 6, 7, 8, 10, and 12 weeks after surgery. The serum levels of osteocalcin and bone-specific alkaline phosphatase (BSAP) as markers of bone formation, and C-terminal telopeptide of type I collagen (CTX), N-terminal telopeptide of type I collagen (NTX), and tartrate-resistant acid phosphatase 5b (TRACP 5b) as markers of bone resorption, were measured using biotin double-antibody sandwich ELISA. RESULTS AND CONCLUSION: In the bone defect group, the bone metabolism was at a high level, suggesting that the early diagnosis of bone nonunion depends on several biochemical indicators. In the bone defect group, the serum level of CTX peaked at 5 weeks, and the serum levels of osteocalcin, BSAP and NTX decreased obviously at 4 or 5 weeks, while the serum TRACP 5b concentration did not change significantly, indicating that all above markers except TRACP 5b sensitively reflect the bone turnover in vivo. Further studies are needed to determine whether systematic monitoring of the biochemical markers can reflect the bone turnover effectively and can be used for the early diagnosis of nonunion in the rabbit model. 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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Influence of mitogen-activated protein kinase inhibitor in the process of assemble flavone of rhizome drynaria promoting the osteogentic differentiation of myoblasts Zhang Li, Xu Bo-xing, Li Jie, Wang Wei 2016, 20 (51):  7622-7627.  doi: 10.3969/j.issn.2095-4344.2016.51.003 Abstract ( 327 )   PDF (945KB) ( 301 )   Save BACKGROUND: Previous studies have confirmed that drug-containing serum with low concentration of assemble flavone of rhizome drynaria (AFDR) promotes osteogentic differentiation of ciliary neurotrophic factor (CNTF)-modified myoblasts, but the underlying mechanism remains unclear. OBJECTIVE: To observe the influence of mitogen-activated protein kinase (MAPK) inhibitor in the process of AFDR promoting the osteogentic differentiation of myoblasts.    METHODS: Transfected-CNTF myoblasts were preprocessed prior to osteogentic induction. Changes of alkaline phosphatase specific activities were detected in blank serum, AFDR drug serum and p38 pathway inhibitor SB203580 groups. mRNA and protein expression levels of core binding factor a1 and alkaline phosphatase were detected by real-time PCR and western blot, respectively. RESULTS AND CONCLUSION: Real-time PCR showed that the mRNA expression levels of core binding factor a1 and alkaline phosphatase in the AFDR group were the highest; while p38 inhibitor SB203580 significantly downregulated the above levels. Western blot findings showed that a significant reduction in the protein expression levels of core binding factor a1 and alkaline phosphatase after p38 inhibitor SB203580 addition. These results suggest that p38 pathway inhibitor can downregulate osteogenesis-related gene and protein expressions, and thus AFDR promoting the osteogentic differentiation of CNTF-modified myoblasts probably through activating p38 MAPK signaling pathway. 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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Growth differentiation factor 5 induces adipose-derived stem cells differentiating into chondrocytes Li Ming-hui, Liu Yang, Sun Kai, Tan Jun-feng, Zhang Mi 2016, 20 (51):  7628-7633.  doi: 10.3969/j.issn.2095-4344.2016.51.004 Abstract ( 281 )   PDF (868KB) ( 263 )   Save BACKGROUND: Growth differentiation factor 5 (GDF5) has been shown to play a crucial role in the development of chondrocytes via different regulatory mechanisms. OBJECTIVE: To explore the effect of GDF5 on the chondrogenic differentiation of adipose-derived stem cells (ADSCs). METHODS: ADSCs in passage 4 isolated from Japanese White rabbits were cultured in the medium containing 0 (blank control group), 10, 50, 100, 150 and 200 μg/L, respectively. The morphological changes were observed, and the optimal concentration of GDF-5 was screened, and its round coverslips at 1, 2 and 3 weeks of culture were selected to undergo immunocytochemistry, toluidine blue staining and immunofluorescent staining. RESULTS AND CONCLUSION: The growth of ADSCs was stable in the medium containing 10 and 50 μg/L GDF5, while apoptotic cells appeared after induction with 200 μg/L GDF5. In the medium containing 100 and 150 μg/L GDF5, some spindle-shaped cells changed into irregular shape, and became round or oval after 7-10 days of culture. The optimal concentration of GDF5 was 100 μg/L. The cells transfected by 100 μg/L GDF5 were positive for type II collagen obviously and with blue-stained nucleus. The transfected cells were positive for toluidine blue, and metachromatic granules were visible in the cytoplasm. The proteoglycan mRNA expression of the transfected cells was significantly increased, and reached the highest at 3 weeks. These results suggest that GDF5 promotes the chondrocytic differentiation of ADSCs. 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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Tendon-bone healing after acute anterior cruciate ligament reconstruction with versus without remnant preservation Zhang Lei, Yue Na, Zhang Tai-liang, Xie Chong-xin, Bai Jing-ping 2016, 20 (51):  7634-7641.  doi: 10.3969/j.issn.2095-4344.2016.51.005 Abstract ( 310 )   PDF (2299KB) ( 203 )   Save BACKGROUND: Reconstruction with remnant preservation can enhance tendon-bone healing. However, the study limits on the histological level, and there is a lack of research based on the modular biological level. OBJECTIVE: To investigate whether anterior cruciate ligament reconstruction with remnant preservation can enhance tendon-bone healing. METHODS: Seventy-two New Zealand rabbits were randomly allocated to three groups (n=24 per group), followed by cruciate ligament reconstruction without remnant (group A), with remnant preservation (femoral tensioning and augmented suture) (group B) and with remnant preservation (graft passing remnant anterior cruciate ligament sheath) (group C), respectively. RESULTS AND CONCLUSION: Hematoxylin-eosin staining showed that the tendon-bone healing in the groups B and C surpassed that in the group A, and group B was better than group C. Real-time PCR revealed that the expression level of osteoprotegrin mRNA and the osteoprotegrin/receptor activator of nuclear factor-κB ligand (RANKL) ratio were greater in the groups B and C than in the group A, and highest in the group C, while the expression levels of RANKL mRNA in the groups B and C were lower than that in the group A. In conclusion, these two kinds of anterior cruciate ligament reconstruction methods with remnant preservation can enhance tendon-bone healing, which have obtained most obvious achievements in the anterior cruciate ligament reconstruction in the graft passing anterior cruciate ligament remnant sheath that may be related to the up-regulation of osteoprotegrin mRNA and down-regulation of RANKL mRNA. 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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Chemical extraction method versus detergent-enzymatic method for the preparation of tissue-engineered trachea matrix Jiang Yuan, Xu Yan-fei, Zhang Si-quan, Shi Hong-can 2016, 20 (51):  7642-7647.  doi: 10.3969/j.issn.2095-4344.2016.51.006 Abstract ( 314 )   PDF (1737KB) ( 239 )   Save BACKGROUND: Acellular tracheal matrix is similar to the native trachea with structure and biological performance preserved after decellularization, and it is an important aim in tissue engineering to find an effective method of decellularization. OBJECTIVE: To select the optimal decellularization method through comparing chemical extraction method with detergent-enzymatic method for preparing rabbit tissue engineering trachea matrix. METHODS: Thirty tracheas from New Zealand white rabbits were randomly divide into three groups. Twenty of rabbit tracheas were subjected to decellularization using 2% TritonX-100 combined with deoxyribonuclease I and ribonuclease (chemical extraction method group), and sodium deoxycholate combined with deoxyribonuclease I (detergent-enzymatic group), respectively. The other ten were given no intervention as controls. Samples were collected and observed by hematoxylin-eosin staining, Masson-trichrome staining, safranin O staining, DAPI staining and scanning electronic microscope. RESULTS AND CONCLUSION: Hematoxylin-eosin staining demonstrated that compared with the control group, almost all cellular components of the mucosal epithelium were removed in the detergent-enzymatic and chemical extraction groups, and there were few remnant chondrocytes. Masson-trichrome staining indicated that compared with the control group, components of the mucosal layer chondrocytes in the chemical extraction and detergent-enzymatic groups were completely removed, with only part of remained chondrocytes in the cartilage zone. Glycosaminoglycan was slightly decreased both in the chemical extraction and detergent-enzymatic groups shown by Safranin O staining, but more reduction was found in the chemical extraction group. DAPI staining reveled that only a small amount of cartilage cells remained in the dense layer of cartilage and lacuna both in this two methods. Scanning electronic microscope showed that using the detergent-enzymatic method there were the hierarchical structures of native trachea, but slight disruption using the chemical extraction method. In conclusion, decellularized rabbit trachea matrix obtained by detergent-enzymatic method is better, with little disruption to the matrix. 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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Validity of kinetic factors on evaluating the vertical jumping ability after anterior cruciate ligament reconstruction Xie Di, Chen Hui-fang, Qi Jian-hong, Liu Hai-bin, Gao Feng, Zhu Zhen-hua, Yang Wen-ning 2016, 20 (51):  7648-7653.  doi: 10.3969/j.issn.2095-4344.2016.51.007 Abstract ( 232 )   PDF (893KB) ( 302 )   Save BACKGROUND: Evaluation of vertical jumping ability is usually only limited to height measurements. The measurements of parameters that describe kinetic factors may provide a better assessment of a patient’s jumping ability. OBJECTIVE: To determine the deficit in one-legged vertical jumping ability and to clarify the relationships between the maximum jumping height and the maximum power, force and velocity during one-legged vertical jumps after anterior cruciate ligament reconstruction. METHODS: Twenty-five healthy subjects (10 males and 15 females) and 25 anterior cruciate ligament reconstructed patients (10 males and 15 females) participated in this study. The isokinetic quadriceps femoris strength and one-legged vertical jumping ability were evaluated by the height, power, force and velocity in all subjects. RESULTS AND CONCLUSION: (1) The maximum height of the one-legged vertical jumps was only significantly correlated with the maximum force in the healthy subjects (P < 0.05). (2) However, for the reconstructed and unreconstructed legs in anterior cruciate ligament reconstructed patients, the maximum jumping height was significantly correlated with the maximum power, force and velocity during one-legged vertical jumps (P < 0.05). (3) These findings suggest the importance of a knee strategy during one-legged vertical jumps for rehabilitation after anterior cruciate ligament reconstruction. Assessment of the jumping ability after anterior cruciate ligament reconstruction may be determined by the maximum power instead of the maximum jumping height. 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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A new ligament cross-sectional area measuring instrument: design and application Zhu Jian-fei, Cheng Yong-zhong, Hou Wang-yang, Cheng Hao, Cheng Ling, Wen Jian-min, 2016, 20 (51):  7654-7659.  doi: 10.3969/j.issn.2095-4344.2016.51.008 Abstract ( 278 )   PDF (951KB) ( 281 )   Save BACKGROUND: There is a lack of study on material properties and parameters of foot finite element models in China. Vernier caliper is a common method for measuring the width and thickness of ligaments and tendons to calculate the cross-sectional area. OBJECTIVE: To design a new ligament cross-sectional area measuring instrument to improve the measurement accuracy. METHODS: The cross-sectional area of the five fresh cadaver ankle ligaments was respectively measured using the new instrument and vernier caliper, and then a comparative analysis of the two measurement methods was performend. RESULTS AND CONCLUSION: The cross-sectional area of anterior talofibular ligament, calcaneofibular ligament, tibionavicular ligament and calcaneotibial ligament was (20.61±7.52), (22.38±11. 49), (33.09±9.91) and (28.20±10.88) mm2, respectively measured by the vernier caliper, and (17.59±4.03), (20.77±7.91), (28.08±8.14) and (30.39±7.98) mm2 by the new ligament cross-sectional area measuring instrument. These results suggest that this new measuring instrument is accurate, reliable and easy to operate, which can be used as a special instrument to measure ligament cross-sectional area, but further studies will be necessary. 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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Phrenic nerve transfer versus intercostal nerve transfer for the repair of brachial plexus root avulsion injuries Abdixbir Abra, Li Ping, Ilhamjan Usman, Exmetjan Yüsup 2016, 20 (51):  7660-7665.  doi: 10.3969/j.issn.2095-4344.2016.51.009 Abstract ( 335 )   PDF (812KB) ( 341 )   Save BACKGROUND: Phrenic nerve transfer and intercostal nerve transfer are the accepted surgery strategies for the treatment of brachial plexus root avulsion injuries; however, which surgery is more suitable for the repair remains inconclusive. OBJECTIVE: To observe the treatment outcomes of brachial plexus root avulsion injuries by transferring the phrenic nerve to the anterior division of the upper trunk of brachial plexus and the intercostal nerve to the musculocutaneous nerve. METHODS: Twenty patients with brachial plexus root avulsion injuries were included. Among them, 9 were treated with phrenic nerve transfer to the anterior division of the upper trunk of brachial plexus (phrenic nerve transfer group), and 11 were treated with intercostal nerve transfer to the musculocutaneous nerve (intercostal nerve transfer group). Postoperative follow-up ranged from 15 to 36 months. Incision length, blood loss, and operation time were recorded. Muscle strength of the biceps and elbow flexion angle were evaluated. The repair outcome was evaluated by assessing the functional recovery of musculocutaneous nerve according to the criteria issued by the Branch of Hand Surgery of Chinese Medicine Association, and the excellent and good rate was calculated. RESULTS AND CONCLUSION: The excellent and good rate was 66.7% and 63.6%, respectively, in phrenic nerve transfer group and intercostal nerve transfer group, which is not significantly different between both groups (P > 0.05). Smaller length of operation incision, reduced blood loss, and shorter operation time were found in the phrenic nerve transfer group. Two and three patients in bad recovery were in phrenic nerve transfer and intercostal nerve transfer groups, respectively. These findings suggest that the two kinds of surgery strategies for the repair of brachial plexus root avulsion injuries can obtain good results in the functional recovery of elbow flexion. Phrenic nerve transfer exerts superiorities in operation incision, blood loss and operation time. 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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Isolation and culture of primary microvascular endothelial cells from mouse brains Zhang Zuo-hui, Chen Chen, Chen Hao, Cui Gui-yun, Hua Fang 2016, 20 (51):  7666-7671.  doi: 10.3969/j.issn.2095-4344.2016.51.010 Abstract ( 453 )   PDF (1442KB) ( 503 )   Save BACKGROUND: Brain microvascular endothelial cells (BMECs) are important tools in the field of neuroscience research; therefore, how to obtain highly purified BMECs is a key and difficulty in vitro. OBJECTIVE: To develop a simple method of isolating and culturing highly purified BMECs. METHODS: C57BL/6 mice aged 6-8 weeks old were selected, and microvessels were obtained using enzyme digestion and gradient centrifugation. Further, endothelia cells were purified by certain drugs, followed by identified by CD31 and GFAP immunofluorescence staining. The expression of Claudin-5 was detected using immunofluorescence staining with anti-Claudin-5 antibody. RESULTS AND CONCLUSION: Mouse BMECs grew and arranged in spiral or cobblestone-like. Immunofluorescence staining showed that the purity of BMECs reached above 99% and Claudin-5 was highly expressed. In conclusion, a simple method of easy accessibility is developed to obtain highly purified primary mouse BMECs. 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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A culture method for cortical neurons derived from neonatal Sprague-Dawley rats Wang Dong-yan, Yang Jin-wei, Cheng Jing-ru, Ma Wei, Li Xing-tong, Guo Jian-hui, Li Li-yan 2016, 20 (51):  7672-7677.  doi: 10.3969/j.issn.2095-4344.2016.51.011 Abstract ( 276 )   PDF (1893KB) ( 326 )   Save BACKGROUND: Primary culture in vitro of neurons plays an important role in the development, regeneration, signal transduction mechanisms, neuropharmacology and gene expressions of the nervous system. OBJECTIVE: To establish a simple method for primary culture of high-purity cortical neurons in neonatal Sprague-Dawley rats. METHODS: Cortical tissues were acquired from neonatal Sprague-Dawley rats born 1 day. In traditional experimental group, the whole cortex was removed; in improved experimental group, the cortical tissues, 2-3 mm thick on the brain surface were removed. Single cell suspensions were prepared after papain digestion and centrifugation and were then seeded onto 24-well culture plates containing neuron solutions for primary culture (1×105 per well). Cells were identified by neuronal specific markers MAP-2 and Tuj1 after 3-day culture. The number of neurons and neurite length were observed under inverted phase contrast microscope and recorded at 6, 24, 48 and 72 hours, 5 and 7 days of culture, resprctively. RESULTS AND CONCLUSION: The cultured cells expressing MAP-2 and Tuj1 were neurons that could be used in the following experiments. The purity of neurons in the improved experimental group was 92% at 3 days, while only 51% in the traditional experimental group. Cells in both two groups had attached to the wall presenting with small processes at 6 hours, and a simple neural network formed at the 3rd day until dense neural networks could be found at the 5th day. To conclude, our culture method herein is simple and convenient, and can be used to produce neurons with high purity, which will be helpful for the experimental studies on cortical neurons from Sprague-Dawley rats. 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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Isolation, cultivation and identification of human skin microvascular endothelial cells Wang Guang-yu, Wang Yu, Zhu Yan-ping, Kang Yu-dong, Wang Fu-sheng, Ding Yi, Dong Yu, Xu Xu-ying 2016, 20 (51):  7678-7683.  doi: 10.3969/j.issn.2095-4344.2016.51.012 Abstract ( 466 )   PDF (2242KB) ( 332 )   Save BACKGROUND: Currently, the enzymatic digestion combined with magnetic activated cell sorting for isolating microvascular endothelial cells are cumbersome and do harm to cells. Therefore, how to simplify the isolation and culture of human dermal microvascular endothelial cells to obtain highly purified endothelial cells in vitro becomes a hotspot. OBJECTIVE: To explore a simple and effective cultivation method of microvascular endothelial cells from diabetic patient skins in vitro, and to detect the cell growth. METHODS: Diabetic patients with chronic foot wounds after amputation were enrolled to collect the limb proximal skin and topical skin around the wound superficial dermal tissue. Human dermal microvascular endothelial cells were obtained using adherent method and trypsin method, folloewd by purified utilizing trypsin digestion and repeated attachment method when passage culture. RESULTS AND CONCLUSION: Human dermal microvascular endothelial cells were obtained successfully, Primary cultured endothelial cells completely adhered to the wall at 24 hours, entered the logarithmic phase at the 10th day, and the cell concentration reached 80% at the 12th-13th day. While the passage cells grew more actively than primary cells, and fully covered the bottom in a “cobblestone” arrangement after 5-7 days of culture. Immunohistochemical staining showed that cultured cells were positive for FVIII and CD31-associated antigens with 100% positive rate. MTT assay showed that cell growth curves of 2, 4, and 5 generations of dermal microvascular endothelial presented the inverted "S" shape. These results suggest that abundant highly purified human dermal microvascular endothelial cells can be obtained through the adherent method and a small amount of short-term trypsin method. 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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Isolation, identification and culture of porcine heart valve myofibroblasts Liu Feng-dan, Hu Wei-lin, Chen Zheng-ping, Li Yong-sheng 2016, 20 (51):  7684-7689.  doi: 10.3969/j.issn.2095-4344.2016.51.013 Abstract ( 242 )   PDF (856KB) ( 714 )   Save BACKGROUND: Valvular interstitial cells are the main components of the heart valves. Myofibroblasts, as a kind of valvular interstitial cells, can express alpha-smooth muscle actin and type I collagen fiber, and hold differentiation potential. These cells cannot only play a support role in the valve structure, but also play a regulatory role in the process of the valve normal physiological and pathological responses. OBJECTIVE: To obtain a reliable method of separation, primary culture and identification of myofibroblasts laying a foundation for further study on the cardiac valvular calcification. METHODS: Aortic valve myofibroblas extracted from porcine hearts were primary cultured by trypsin and collagenase combined digestive method, common enzyme-digestion method and tissue-culture method, respectively. The myofibroblast activity and morphology were observed using microscope, and myofibroblasts were identified using light microscope and immunocytochemistrial method. RESULTS AND CONCLUSION: Myofibroblasts had a higher activity and purity cultured by trypsin combined with collagenase II digestion method. Aortic valve myofibroblasts were positive for alpha-smooth muscle actin and negative for von Willebrand factor under fluorescence microscope, suggesting that myofibroblasts were successfully obtained. 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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Expression of growth hormone and insulin-like growth factor-1 in the temporal cortex of a Lewis dwarf model rat Cai Yi-qi, Wang Kai-fei, Wang Ying-ying, Zhang Su-hua 2016, 20 (51):  7690-7696.  doi: 10.3969/j.issn.2095-4344.2016.51.014 Abstract ( 250 )   PDF (992KB) ( 222 )   Save BACKGROUND: Insulin-like growth factor-1(IGF-1), a main active factor in growth hormone (GH), plays various biological functions, such as improving cognitive ability and anti-apoptotic action. OBJECTIVE: To detect the expressions of GH and IGF-1 in the temporal cortex of Lewis dwarf rats, and to explore the effect of different concentrations of GH on the differentiation of hippocampal nerve stem cells (NSCs). METHODS: Lewis dwarf rats aged 11(adult) and 20 (senile) month olds and normal wild-type rats were euthanized by decapitation, underwent the craniotomy quickly, and the temporal cortex in the cold saline was extracted. GH and IGF-1 levels were detected using western blotting. After isolation, purification and identification of the rat hippocampal NSCs, the effect of GH in different concentrations (10, 30, 90 μg/L) on the NSCs differentiation was determined at 96 hours after culture. RESULTS AND CONCLUSION: The GH level in the temporal cortex did not differ significantly among rats (P > 0.05). While the IGF-I level in the temporal cortex of Lewis dwarf rats was significantly higher than that of the wild-type rats (P < 0.05). The GH level in the temporal cortex of adult female Lewis dwarf rats was significantly lower than that of the male rats (P < 0.05). Immunofluorescence showed that the proportion of β III-tubulin-positive neurons was significantly higher than that in the control group (P < 0.05) after the hippocampal NSCs and precursor cells cultured for 96 hours with GH (30 μg/L), but there was no significant difference between the control group and treatment group with GH of 10 or 90 μg/L. These results suggest that GH and IGF-I are expressed in the temporal cortex of both Lewis dwarf and wild-type rats which are independent from pituitary GH and the peripheral circulating IGF-1. Additionally, GH can promote the differentiation of hippocampal NSCs and precursor cells into neurons. 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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Construction of a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene and its transfection into vaginal epithelial cells Shen Fu-jin, Xu Xue-xian, Zhou Li-mei 2016, 20 (51):  7697-7702.  doi: 10.3969/j.issn.2095-4344.2016.51.015 Abstract ( 232 )   PDF (1643KB) ( 306 )   Save BACKGROUND: LL37, the only antimicrobial peptide of the cathelicidin family identified from the human, not only promotes the proliferation of endothelial cells, but also plays an important role in angiogenesis and re-epithelialization. OBJECTIVE: To construct a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene, and to detect the expression and secretion of LL37 after transfected into the canine vaginal epithelial cells. METHODS: The cDNA encoding LL37 was amplified by PCR. Recombinant adenovirus expression plasmid encoding LL37 and green fluorescent protein (EGFP) was constructed, and identified using restriction endonuclease technology and DNA sequencing method. Adenovirus particles were generated by cotransfecting the 293-packaging cell line. The adenovirus were collected, amplified and concentrated, and viral titers were determined by end-point dilution assay by applying serial dilutions of the purified viruses to 293 cells. Primary cultured canine vaginal epithelial cells were transfected by the recombinant adenovirus. The transfection efficacy was observed by fluorescence microscope, and the cultured supernatant was collected to determine the expression of LL37 by ELISA method at 1, 2, 3, 5 and 7 days after transfection. RESULTS AND CONCLUSION: The adenovirus vector GV314-LL37 with the titer of 3×109 pfu/mL was successfully constructed and identified by DNA sequencing methods. Canine vaginal epithelial cells were successfully isolated and cultured and grew stably. After transfection, vaginal epithelial cells could express the EGFP and LL37 efficiently in a time-dependent manner detected by fluorescence microscope and ELISA method. The transfection efficacy of EGFP reached to 89% at 72 hours. The level of LL37 in the cell culture supernatant in the transfection group was significantly higher than that in the control group, the highest expression of LL37 was found at 3 days that lasting for 7 days. In conclusion, the recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene is successfully constructed, which can express and secrete LL37 after transfected into canine vaginal epithelial cells, providing a foundation for constructing the tissue-engineered vagina possessing anti-infection and neovascularization. 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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Root resorption and interleukin-17 expression in a rat model of kidney deficiency uring orthodontic treatment Yu Yan-heng, Huang Xin-yan, Zheng Rong, Li Chao, Dong Yue, Gao Xu-guang, Wu Li-peng 2016, 20 (51):  7703-7709.  doi: 10.3969/j.issn.2095-4344.2016.51.016 Abstract ( 250 )   PDF (2082KB) ( 198 )   Save BACKGROUND: The mechanism underlying orthodontic-induced external root resorption is not yet clear, and it differs individually. Kidney deficiency has been proved to be related to bone diseases which mediated by different cytokines. Interleukin-17 is an important cytokine involved in external root resorption. So figuring out whether kidney deficiency and interleukin-17 are related to root resorption will be helpful for etiological research. OBJECTIVE: To explore the relationship between kidney deficiency physique, interleukin-17 and root resorption during orthodontic treatment in rats. METHODS: Thirty-six Wistar rats were selected and equivalently randomized into two groups, followed by modeled into kidney deficiency (kidney deficiency group) or injected with normal saline (control group), respectively. Afterwards, the right maxillary of each rat served as an orthodontic force model, and the left maxillary as a non-orthodontic force model. All rats were respectively sacrificed under general anesthesia at the 3, 7 and 14 days after given orthodontic force. Then, the mesial surface of the root of maxillary first molars and the expression level of interleukin-17 were observed through hematoxylin-eosin staining and immunohistochemical method. RESULTS AND CONCLUSION: Histological observation showed that significantly increasing root resorption in a time-dependent manner could be observed, and there were various absorbed lacunae of osteoclasts on the enamel in the kidney deficiency orthodontic force group. The alveolar bone resorption and widened periodontal membrane appeared in the control orthodontic force group. While no remarkable root and alveolar bone resorptions were found in the other two non-orthodontic force groups. The expression level of interleukin-17 in the kidney deficiency orthodontic force group was higher than that in the control orthodontic force group; the expression level of interleukin-17 in the kidney deficiency non-orthodontic force group was higher than that in the control non-orthodontic force group. In conclusion, kidney deficiency patients are easy to develop root resorption, the mechanism of which is maybe relevant to the upregulation of interleukin-17. 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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Neuroprotective effect of tamoxifen in a model rat with aucte spinal cord injury Huang Wei 2016, 20 (51):  7710-7716.  doi: 10.3969/j.issn.2095-4344.2016.51.017 Abstract ( 268 )   PDF (1903KB) ( 322 )   Save BACKGROUND: Tamoxifen has been found to exert neuroprotection by reducing cerebral hemorrhage and edema surrounding the injured site of the spinal cord. OBJECTIVE: To investigate the neuroprotective effect of tamoxifen on rat acute spinal cord injury and the underlying mechanism. METHODS: Sixty Sprague-Dawley rats were equivalently randomized into five groups, and modeled into spinal cord injury at T10 level using modified Allen’s weight-drop method (70 g/cm), except those in sham operation group. At 30 minutes after modeling, all rats were given the intraperitoneal injection of 2.5, 5.0 and 10 mg/kg tamoxifen or same amount of normal saline, once daily. Basso, Beattie, Bresnahan (BBB) scores were recorded at 24, 48 and 72 hours after surgery. The injured spinal cord was removed at 72 hours to observe its edema. Meanwhile, the levels of interleukin-1β, interleukin-10 and tumor necrosis factor-α, as well as Caspase-3 activity were detected by ELISA; the protein levels of nuclear factor-κB p65, phosphorylated I-κBα and Caspase-3 were detected by western blot assay. RESULTS AND CONCLUSION: Compared with the model group, the hind limb function in the tamoxifen groups was significantly improved. Tamoxifen significantly decreased the water content in the rat spinal cord and inhibited spinal cord edema at 72 hours after surgery. ELISA results showed that tamoxifen significantly reduced the activity of interleukin-1β, interleukin-10, tumor necrosis factor-α and Caspase-3 (P < 0.05). Western blot assay revealed that tamoxifen significantly downregulated the expression levels of nuclear factor-κB p65, phosphorylated I-κBα and Caspase-3. These results suggest that tamoxifen protects against spinal cord injury via suppressing inflammatory response and apoptosis-associated proteins. 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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Bone marrow mesenchymal stem cells differentiate into cartilage and bone: roles of Wnt5a/PCP signaling pathways Peng Xu, Zhang Xiao-mei, Wei Shi-hang, Liu Yan1, He Xue-ling 2016, 20 (51):  7717-7723.  doi: 10.3969/j.issn.2095-4344.2016.51.018 Abstract ( 618 )   PDF (783KB) ( 412 )   Save BACKGROUND: Wnt5a is able to inhibit canonical Wnt signaling and activate non-canonical Wnt signaling pathway. In recent years, it has been found that non-classical Wnt5a/PCP signaling pathway mediated by Wnt5a plays an important role in the process of bone marrow mesenchymal stem cell proliferation and differentiation, but the underlying mechanism is unclear. OBJECTIVE: To summarize the progress in downstream effector molecules related to Wnt5a/PCP signaling pathway, and its roles in the chondrogenic and osteogenic differentiation of bone marrow mesenchymal stem cells. METHODS: A computer-based online search of CqVip, CNKI and PubMed databases between January 2000 and February 2016 was performed using the Chinese keywords of “BMSCs, Wnt signaling pathways, chondrogenic differentiation, osteogenic differentiation” and English keywords of “BMSCs, chondrogenic differentiation, osteogenic differentiation, Wnt, Fzd, Ror2, RhoA, ROCK, JNK”, respectively. Literatures related to bone marrow mesenchymal stem cell chondrogenic and osteogenic differentiation were selected. Finally, 43 eligible articles were included for analysis through excluding the old and repeated research. RESULTS AND CONCLUSION: Wnt5a, a representative protein in non-canonical Wnt signaling pathway, paticipates in the cytoskeleton, cell migration and cell polarization and other activities by mediating its downstream signaling molecules such as Fzd, Ror, RhoA, ROCK, JNK, thereby regulating its proliferation and differentiation. But it is unclear how Wnt5a/PCP participates in the bone marrow mesenchymal stem cell chondrogenic and osteogenic differentiation and how the downstream effector molecules interact or function independently, which requires further studies. 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 meniscus: seed cells and physicochemical factors Han Chang-xu, Ma Li-bo, Ren Yi-zhong 2016, 20 (51):  7724-7730.  doi: 10.3969/j.issn.2095-4344.2016.51.019 Abstract ( 266 )   PDF (687KB) ( 221 )   Save BACKGROUND: As meniscectomy may result in various adverse reactions, tissue-engineered meniscus is expected to be used for meniscus repair. Seed cell selection and optimal physicochemical stimuli are crucial for the construction of tissue-engineered meniscus. OBJECTIVE: To overview the seed cells for the tissue-engineered meniscus construction and the research progress of physicochemical factors. METHODS: The first author retrieved the CNKI and Medline databases using the keywords of “meniscus, tissue engineering, seed cells, physical and chemical factors” in English and Chinese, respectively, to retrieve articles related to the seed cells and physicochemical factors of the tissue-engineered meniscus construction. Irrelative and repetitive articles were excluded, and 49 eligible articles were enrolled for analysis. RESULTS AND CONCLUSION: The seed cells must maintain their phenotype and synthetic ability after physicochemical stimulation in combination with scaffolds, to achieve the reproducibility of tissue-engineered meniscus. Most of cytokines can be used for promoting the proliferation and differentiation of chondrocytes, but the underlying mechanisms were little known. Their application in the meniscus tissue engineering needs to be studied in depth. Currently it is urgent to improve physicochemical stimuli in order to construct the tissue-engineered meniscus. The shear force does harm to chondrocyte phenotype, and dynamic compression loading has been proved to enhance Ca+ and glycosaminoglycan release. The fibrous cartilage stimulated by shear force and other factors may be helpful for constructing the tissue-engineered meniscus. An elaborated randomized controlled trial and the long-term quantitative analysis are of importance to assess the research results. 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 | Related Articles | Metrics

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Dopamine and 5-hydroxytryptamine signaling pathways in the pathogenesis of spinal cord injury-induced osteoporosis: new theory and novel therapeutic strategies Zhang Miao, Zhang Xu-ran, Li Rui, Sun Yu 2016, 20 (51):  7731-7737.  doi: 10.3969/j.issn.2095-4344.2016.51.020 Abstract ( 312 )   PDF (446KB) ( 253 )   Save BACKGROUND: Current basic and clinical research have showed that increases in bone resorption and bone loss accur earlier after spinal cord injury (SCI) than disuse atrophy, revealing that other mechanisms are involved in the pathogenesis of the SCI-induced osteoporosis (SIO). OBJECTIVE: To introduce the current lab and clinical research progress in SIO focusing on the functional changes of two major neurotransmitters in the spinal cord, including dopamine (DA), 5-hydroxytryptamine (5-HT) and their receptors, as well as their regulatory functions on bone metabolism, aiming at finding a new treatment strategy for SIO. METHODS: A computer-based online search in PubMed and Embase databases was conducted for clinical and basic research related to SIO published from January 1967 to August 2016, using the keywords of “spinal cord injury; osteoporosis; dopamine; serotonin; 5-hydroxytryptamine” in English. Irrelevant, poorly related and repetitive studies were excluded, and finally 41 eligible articles were included. RESULTS AND CONCLUSION: DA and 5-HT are major neurotransmitters in the central nervous system, both involving in the regulation of bone remodeling. After SCI, loss of innervation and descending neurotransmitters especially DA, 5-HT and subsequent deregulation of their receptors are responsible for the onset of post-traumatic bone loss. The above research progress, in combination with the emerging clinical and lab investigations targeting 5-HT, DA and their receptors for improving neural functions after SCI, provides possible therapeutic pathways for SIO. 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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Can exosomal micro-RNAs be as biomarkers of diseases? Li Yi, Tang Pei-fu 2016, 20 (51):  7738-7745.  doi: 10.3969/j.issn.2095-4344.2016.51.021 Abstract ( 387 )   PDF (857KB) ( 1168 )   Save BACKGROUND: Exosome, a kind of cystic vesicle with bilayer structure, is widely distributed in the body fluids. Exosomes are involved in various cellular communications, and its contents including proteins, short chain peptides, DNA, RNA, phospholipids, and miRNA are resistant to degradation. OBJECTIVE: To clarify the characters of exosomes, and to investigate the possibility of exosomal miRNAs as biomarkers for different diseases to provide a new strategy for clinical diagnosis. METHODS: A computer-based search of CNKI and PubMed databases was performed by the first author for articles related to exosomal miRNAs. The keywords were “exosome, microvesicles, extracellular vesicles, miRNA, biomarker, early diagnosis, progrosis” in Chinese and English, respectively. Totally 50 eligible articles were included in result analysis. RESULTS AND CONCLUSION: After reviewing researches of exosomes in different diseases, we can confirm that exosomes broadly participant in physiological and pathological process of various system diseases. The abnormal expression of exosomal micro-RNAs has been identified in many studies, indicating the exosomal micro-RNAs have a great potential to be biomarkers for disease diagnosis. Further studies should focus on extracting the contents of exosomes, the pathogenesis of exosomes is involved in and screening the appropriate exosomal miRNAs for early diagnosis. 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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Periodontitis and p38 mitogen activated protein kinase pathway: research and development Tang Ya-ping, Liu Qi, Guo Cheng-wei, Xie Rui-di 2016, 20 (51):  7746-7752.  doi: 10.3969/j.issn.2095-4344.2016.51.022 Abstract ( 207 )   PDF (735KB) ( 237 )   Save BACKGROUND: A series of inflammatory signal pathways are activated accompanied by increasing expressions of inflammatory factors after periodontal tissues being stimulated by exogenous substances like bacterium. Inflammation is closely related to periodontal tissue repair and regeneration. OBJECTIVE: To illustrate the correlation of immune response of periodontitis and periodontitis-related inflammatory cytokines to p38 mitogen activated protein kinase (p38MAPK) pathway, and to provide a new idea and method for the treatment of periodontitis in the future. METHODS: The related literatures of periodontitis and p38MAPK pathway published from January 1990 to January 2016 were retrieved from the databases of CBM, CNKI, SWIC and PubMed. The research and progress of periodontal tissue repair and regeneration after periodontitis were analyzed. RESULTS AND CONCLUSION: Totally 36 literatures were enrolled finally. Inflammation-regulated and inflammation-associated signaling pathways as well as subsequent expression of inflammatory mediators can partly control the excessive disease-induced inflammation and immune responses of the host, among which p38MAPK signaling pathway may be involved in the occurrence and development of periodontitis. More studies, however, are needed to validate these findings. The regulation of p38MAPK signaling pathway is still of great significance in the treatment of periodontal disease, and we hope to provide a new insight and basis for clinical diagnosis and treatment of periodontitis. 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