中药川芎对成骨细胞作用最佳浓度的体外实验

BACKGROUND: It has been proved that Rhizoma Chuanxiong can promote osteoblast proliferation and differentiation. OBJECTIVE: To further observe the effects of tetramethylpyrazine (Ligustrazine) extracted from Rhizoma Chuanxiong on osteoblast proliferation and type I collagen expression. METHODS: Mouse osteoblast cell line MC3T3-E1 was cultured in medium containing 1, 5, 10, 15 and   20 mg/L Ligustrazine, respectively. Osteoblast proliferation was detected by cell counting kit-8 assay, and alkaline phosphatase activity and type I collagen level measured using ELISA method. RESULTS AND CONCLUSION: Different concentrations of Ligustrazine all significantly promoted the osteoblast proliferation and type I collagen level compared with the blank control group (P < 0.005). The activity of alkaline phosphatase in all Ligustrazine groups except 15 and 20 mg/L groups was significantly higher than that in the blank control group (P < 0.005). These results show that Ligustrazine isolated from Rhizoma Chuanxiong can effectively promote the osteoblast proliferation and type I collagen expression, with the optimum concentration of 10 mg/L. 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     

新型壳聚糖基仿生网络复合膜诱导骨髓间充质干细胞定向成骨分化

Objective To study the osteogenic differentiation capacity of mesenchymal stem cells(MSCs)on chitosan based biomimetic network composites films.Methods The chitosan-based biomimetic network composites films were prepared using chitosan,gelatin and pectin in certain ratio by biomimetic approach.The experiment was designed as follow:experimental group 1 (composites films+routine medium),control group 1 (routine medium),experimental group 2(composites films+osteogenic medium),control group 2(osteogenic medium).The growth and proliferation of MSCs were observed by inverted phase contrast microscope and scanning electronmicroscopy(SEM) and detected through methyl thiazolyl tetrazolium(MTT) assay.Alkaline phosphatase(ALP) activity was used to investigate the osteogenic differentiation capacity.The formation of mineral was determined by special staining and the energy dispersive X-ray apparatus (EDX).Results MSCs could well adhere,grow and proliferate on the network composites films.The MTT assay demonstrated that there was no significant difference in cell viability between the experimental and control groups (P＞0.05).SEM observation showed that MSCs grew assembly on the surface of the films secreted a large number of extracelluar matrix,and formed scattered nodules.The cells on the experimental group 1 showed statistically higher ALP activity level than those cells on the other groups (P＜0.01).Moreover,the calcified nodules can be seen in the experimental groups by Alizarin red and Von Kossa staining.ALP staining showed blue-staining granules within the cytoplasm,and the deposition of Ca and P was detected on the films by SEM-EDX.Conclusion The novel chitosan-based biomimetic network composites films not only have good biocompatibility,but can also improve MSCs differentiation toward the osteogenic lineage.     

Sema7A干预钛微粒诱导鼠MC3T3-E1成骨细胞的凋亡

BACKGROUND: Semaphorin7A (Sema7A) is a kind of cell surface protein, which can promote the fusion of osteoclasts and the migration of osteoblasts at the same time, affecting the dynamic balance of the bone. It is speculated that Sema7A siRNA may inhibit osteoblast apoptosis induced by titanium particles.OBJECTIVE: To study the effect of Sema7A on the preosteoblast activity inhibited by titanium particles.METHODS:Mouse MC3T3-E1 preosteoblasts at passages 6 and 7 were divided into four groups: in blank control group, MC3T3-E1 cells were cultured alone; in standard control group, cell were cultured with titanium particles; in experimental groups 1 and 2, the cells were cultured with titanium particles+Sema7A overexpression plasmids and titanium particles+Sema7A siRNA, respectively. Apoptotic rate of MC3T3-E1 cells was detected by flow cytometry; the mRNA expression of bone sialoprotein, osteocalcin and type I collagen was detected by Q-PCR; western blot assay was adopted to detect the protein expression of bone sialoprotein, osteocalcin and type I collagen; alizarin red calcium nodule staining was taken to detect the degree of osteoblast mineralization.RESULTS AND CONCLUSION: The expressions of bone sialoprotein, osteocalcin and type I collagen were decreased in the standard control group and experimental group 1, but these expression were significantly increased in the experimental group 2 compared with the standard control group (P < 0.05). Flow cytometry results suggested that the apoptotic rate of osteoblasts in the experimental group 1 was significantly higher than that in the other groups (P < 0.05), and the apoptotic rate in the experimental group 2 was lower than that in the standard control group (P < 0.05). Alizarin red staining showed that there were no obvious mineralized nodules in the experimental group 1, but mineralized nodules formed in the experimental group 2. In brief, the genetic interference technique that inhibits the activity of Sema7A can interfere the process of mouse MC3T3-E1 preosteoblast differentiation inhibited by titanium particles, and thus provide a feasible way for the clinical treatment of wear particles-induced osteolysis using biotechnology.  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     

脐血间充质干细胞移植联合紫杉醇对卵巢癌A2780细胞增殖、 凋亡及侵袭的影响

BACKGROUND:An intensive study on ovarian cancer A2780 cells contributes to modify treatment strategies targeting ovarian cancer stem cells and enhance survival rate of ovarian cancer patients. OBJECTIVE: To investigate the effects of umbilical cord blood mesenchymal stem cells (UC-MSCs) transplantation combined with paclitaxel on proliferation, apoptosis, and invasion of A2780 human ovarian cancer cells in vitro. METHODS: A2780 human ovarian cancer cells were assigned into three groups: blank control group (routine cell culture), UC-MSCs group (addition of suspension containing 1×106 UC-MSCs) and combined treatment group (combined addition of suspension containing 1×106 UC-MSCs and 250 μL of 1 μmol/L paclitaxel solution). CD133+ antigen expression, proliferation, apoptosis, and invasion were determined by immunofluorescent staining, flow cytometry, TUNEL staining, and Transwell chamber invasion assay, respectively. RESULTS AND CONCLUSON: CD133+ antigen expression in the UC-MSCs group and combined treatment group was significantly decreased compared with the blank control group (P < 0.05). Proliferation and invasion abilities were significantly decreased, while apoptosis cell number was significantly increased in the combined treatment group compared with the other two groups (P < 0.05). These findings indicate that the combined treatment of UC-MSCs transplantation and paclitaxel can inhibit proliferation and invasion and promote apoptosis of A2780 human ovarian cancer 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     

富血小板纤维蛋白诱导口腔缺损软组织的修复与再生

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     

Estrogen regulates osteogenic differentiation of human periodontal ligament stem cells via Wnt/beta-catenin signaling pathway北大核心

BACKGROUND: Estrogen can promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), but the molecular mechanism is unclear. OBJECTIVE: To study the regulatory effect of estrogen on the osteogenic differentiation of hPDLSCs via Wnt/β-catenin signaling pathway. METHODS: The hPDLSCs were isolated and purified by digestion method combined with limited dilution clone method. Three experimental groups were set as follows: osteogenic induction only (control group); 1×10-7 mol/L estrogen with osteogenic induction (estrogen group); and 100 µg/L Wnt3a protein with osteogenic induction (Wnt3a group). Alkaline phosphatase activity was detected at 1, 3, 5, 7 days of osteogenic induction. Western blot was used to detect the expression of Wnt/β-catenin signaling pathway related proteins β-catenin, P-GSK-3β, GSK-3β, CyclinD1 and osteoblast-related proteins Runx2 and OCN after 7 days of osteogenic induction. RESULTS AND CONCLUSION: The activity of ALP in all groups increased with time. The expression level of ALP in the estrogen group and Wnt3a group was higher than that in the control group at 1, 3, 5 and 7 days of induction (P < 0.05), while there was no significant difference between the former two groups (P > 0.05). The western blot results showed that the expression levels of β-catenin, P-GSK-3β, CyclinD1, Runx2 and OCN in the estrogen group and Wnt3a group were higher than those in the control group (P < 0.05), while the expression of GSK-3β was lower than that in the control group (P < 0.05). But there were no differences in the expression of Wnt/β-catenin signaling pathway related proteins and mid-late osteogenic markers between estrogen group and Wnt3a group (P > 0.05). To conclude, estrogen can enhance the osteogenic differentiation of hPDLSCs, and the underlying mechanism is likely to activate the Wnt/β-catenin signaling pathway in activated hPDLSCs exposed to estrogen. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

体外静水压环境下细胞因子诱导骨髓间充质干细胞向髓核样细胞分化

BACKGROUND: Differentiation of bone marrow mesenchymal stem cells is induced by integrated factors. In vitro interaction of cytokine complex and certain cell mechanical stimulation is carried out to further improve the efficiency of bone marrow mesenchymal stem cells differentiating into nucleus pulposus-like cells.OBJECTIVE: To investigate the differentiation of bone marrow mesenchymal stem cells into nucleus pulposus-like cells induced by transforming growth factor-β1 and insulin-like growth factor-1 under hydrostatic pressure.METHODS:Bone marrow mesenchymal stem cells from adult rats were separated, cultured and purified in vitro. Passage 3 cells were induced in vitro with transforming growth factor-β1 and insulin-like growth factor-1 under hydrostatic pressure (hydrostatic pressure group), with transforming growth factor-β1 and insulin-like growth factor-1 under normal pressure (drug group), or with normal culture medium under normal pressure (blank control group).RESULTS AND CONCLUSION: At day 14 after culture, polygonal nucleus pulposus-like cells were observed in the hydrostatic pressure group, but irregular cells in the drug group. There was no obvious change in the blank control group. Levels of collagen type II and DNA were higher in the hydrostatic pressure group than the other two groups. These findings indicate that the combination of transforming growth factor-β1 and insulin-like growth factor-1 can successfully induce the differentiation of bone marrow mesenchymal stem cells into nucleus pulposus-like cells under hydrostatic pressure, and the differentiation efficiency is higher under hydrostatic pressure than under normal pressure.  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     

透明质酸在去细胞异体神经移植中对瘢痕的影响

BACKGROUND:In terms of the histocompatibility, immune rejection and scar formation after repair, acellular nerve allograft is closer to autologous nerve cells. At present, hyaluronic acid has been applied for autologous peripheral nerve repair; however, research on the nerve allograft is rarely reported. OBJECTIVE: To explore the effect of hyaluronic acid on the anastomotic scar in acellular nerve allograft repair of rat sciatic nerve defect. METHODS: Thirty-six Sprague-Dawley rats were randomly divided into three groups (n=12 per group). The rat model of nerve defect of 10 mm was established by cutting the sciatic nerve of the left hind leg and then given nerve allograft combined with the injection of hyaluronic acid at anastomosis (experimental group), only nerve allograft (control group) and autologous nerve graft (nerve autograft group), respectively. Afterwards, the healing of the proximal anastomosis was observed and scar components were assessed. RESULTS AND CONCLUSION:Gross observations showed that the rat skin and muscle fascia had no significant differences in healing among groups, while the surrounding tissue adhesion in the experimental group was milder than that in the control group (P < 0.05). Masson staining found that collagen deposition in the epinerium could be observed in each group. In the experimental group, a small amount of collagen fibers arranged orderly in the epineurium; in the control group numerous collagen fibers accumulated and arranged irregularly; in the nerve autograft group, sparse epineurial collagen fibers appeared in an order arrangement. The gray value of collagen type I in the experimental group was higher than that in the control group (P < 0.05), while the gray value of collagen type III was lower than that in the control group (P < 0.05). No significant differences were found in the sum gray values of collagen type I and III among groups (P > 0.05). These findings indicate that in the peripheral nerve repair, hyaluronic acid abrogates the scar formation by increasing the deposition of collagen type III and reducing the deposition of collagen type I. 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     

低强度脉冲式超声对关节软骨的修复

BACKGROUND: Articular cartilage injuries can result from a variety of causes. Conventional therapy cannot obtain the optimal clinical results. Low-intensity pulsed ultrasound has been shown to promote the repair of injured articular cartilage. OBJECTIVE: To investigate the effects of low-intensity pulsed ultrasound on the repair of injured articular cartilage. METHODS: Twenty New Zealand white rabbits were used to establish knee arthritis models and equally randomized into study and control groups, respectively. Rabbits in the study group received low-intensity pulsed ultrasound treatment, and sham low-intensity pulsed ultrasound treatment was given in the control group. At 8 weeks after treatment, pathological change and histological scores in articular cartilage tissue collected from both groups were determined. Moreover, the ultrastructure and type II collagen expression of chondrocytes were determined. Matrix metalloproteinase-13 mRNA expression was detected by quantitative real-time PCR. RESULTS AND CONCLUSION: At 8 weeks after treatment, toluidine blue staining showed a disordered arrangement of cells, decreased number of cartilage cells in each layer and cluster in the control group. Light disordered arrangement of cells, decreased appearance of the superficial layer cells and the cluster phenomenon were observed in the study group. Articular cartilage tissue scores were significantly decreased in the study group compared with the control group (P < 0.05). The chondrocytes were small, enlarged intracellular mitochondria and rough endoplasmic reticulum, cytoplasmic swelling, collagen fibrils coarse, well developed Golgi apparatus, and nuclear fragmentation were observed in the control group. In addition, the normal structure of organelles disappeared and cell degeneration was observed in the control group. In the study group, the size of chondrocytes and the Golgi complex and other organelles were normal, and the protein polysaccharide granules were observed in the cytoplasm and membrane. The mRNA expression of matrix metalloproteinase-13 in the study group was significantly lower than that in the control group (P < 0.05). Type II collagen immunoreactivity in the study group was stronger than that in the control group. No incision infection, suppuration, red swelling appeared in all rabbits. Our results suggest that low-intensity pulsed ultrasound can be used for the treatment of articular cartilage injury by alleviating the degradation of collagen type II and inhibiting the expression of matrix metalloproteinase-13. 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     

Proliferation and differentiation of periosteum cells induced by icariin北大核心

BACKGROUND: Icariin has a broad prospect for promoting cell proliferation. Differentiation direction of periosteal cells is uncertain, but the cells are easy to be induced by ultrasound, oxygen or bone morphogenetic protein 7 (BMP7). Periosteal cells have been applied in bone tissue engineering; however, icariin effects on the proliferation and differentiation of periosteal cells is little reported. OBJECTIVE: To investigate the effect of icariin on the proliferation and differentiation of human periosteal cells, thus providing theoretical basis for icariin applied in bone tissue engineering. METHODS: The human periosteum was obtained and the primary cells were isolated in vitro. After culture and expansion, periosteal cells were cultured in 24-well plates, and induced by 0.001, 0.01 and 0.1 mg/L icariin and 50 µg/L BMP7, respectively. The corresponding avsorbance values of different groups were detected. The levels of alkaline phosphatase and calcium nodules in periosteal cells were measured at 1, 3, 5 and 7 days, and the mRNA levels of osteocalcin, osteopontin and Runx2 were detected at 3, 5 and 7 days. RESULTS AND CONCLUSION: The periosteal cells proliferated well after induction with icariin, and could proliferate well in different concentrations of icariin and the positive control group (P < 0.05). Compared with the control group, the periosteal cells induced by icariin were able to produce more alkaline phosphatase and calcium nodules (P < 0.05). The mRNA expression of osteocalcin, osteopontin and Runx2 in periosteal cells could be up-regulated by icariin (P < 0.05). These findings imply that icariin can promote proliferation and differentiate of periosteal cells into osteoblasts, and it can be used as an inducer for the preparation of seed cells in bone tissue engineering. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

胰酶联合Ⅱ型胶原酶分离培养髓核细胞：培养基中葡萄糖浓度的选择

BACKGROUND: Intervertebral disc degeneration is the pathological basis of degenerative spinal diseases. Studies on the influential factors of intervertebral disc degeneration contribute to the prevention and treatment of degenerative spinal disease. OBJECTIVE: To observe the growth and proliferation of nucleus pulposus cells isolated by trypsin plus type II collagenase digestion in complete medium with different glucose concentrations, exploring the optimal glucose concentration for growth of nucleus pulposus cells. METHODS: Nucleus pulposus cells isolated and cultured by trypsin plus type II collagenase digestion method were observed under an inverted microscope, and the cell number was counted. Morphology of nucleus pulposus cells was observed after hematoxylin-eosin staining and toluidine blue staining. Collagen type II immunoreactivity was detected by immunohistochemical staining combined with immunofluorescent staining. Nucleus pulposus cells were incubated in complete medium containing various glucose concentrations (0, 6.25, 12.5, 17.5, and 25 mmol/L) for 24 hours, and then cell proliferation and apoptosis were determined. RESULTS AND CONCLUSION: The stained nucleus pulposus cells showed polygonal and short spindle, with one or two nuclei. Cellular pseudopod appeared gradually and then became slim with increased passage numbers. The isolated and cultured nucleus pulposus cells positively expressed collagen type II and aggrecan Proliferative activity of nucleus pulposus cells cultured in medium with 17.5 mmol/L glucose was significantly higher than that in medium with 0 and 25 mmol/L glucose (P < 0.05 or P < 0.01). There was no significant difference in cell apoptosis between these groups except for 0 mmol/L glucose (P < 0.05). These results confirm that a large number of nucleus pulposus cells can be harvested by trypsin plus type II collagenase digestion and the optimal glucose concentration is 17.5 mmol/L. 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     

人类脂肪来源成体干细胞体外定向成软骨的诱导实验

Objective To investigate the feasibility of inducing human adipose derived adult stem cells (hADASc) into chondrocytes by gene transfection.Methods hADASc were cultured in vitro, and transfected with gene PGL3-TGF-pl, whose Luceferase activity (RUL value) was measured.PT-PCR was used to monitor the expression of tranfected hADASc.Anti collagen II immunohistochemical staining was performed in the experimental group (transfected hADASc) , active control group (untransfected hADASc cultured in chondrocytes induction medium) and negative control group (untransfected hADASc cultured in conventional medium).The immuno-staining image was analyzed with an automated imaging analysis system and computed for PU values.Results Nucleated tissue cells with features of stem cells were isolated from in vitro culture of human subcutaneous adipose tissue.The RUL value of the experimental group (9 212.583±315.240) was higher than that of the control group(317.000?0.710, P＜0.01).RT-PCR revealed that the transfected hADASc could express the TGF-β1.Anti collagen Ⅱ immunohistochemical staining was positive in experimental group and active control group, with PU values (13.864±2.416 and 13.637±2.548,respectively) statistically different from that of the negative control group (6.013 ±0.827, P＜0.05).Conclusions Nucleated tissue cells with features of stem cells can be isolated from culture of human subcutaneous adipose tissue in vitro.The PGL3-TGF-β1-transfected hADASc may express TGF-β1.The endogenous TGF-β1-induced hADASc produces collagen Ⅱ , with similar actions of exogenous TGF-β1.     

差速贴壁法原代培养椎间盘髓核细胞中的应用优势

BACKGROUND: As the main function cell of intervertebral disc, nucleus pulposus cells are the focus of studying the degenerative mechanism; thereby, it is crucial to maintaining the physiological function of nucleus pulposus cells in vitro and the stability of the cell phenotype. OBJECTIVE: To study the excellence of differential velocity adherent procedure in primary culture of nucleus pulposus cells of rat intervertebral disc through comparison. METHODS: Twenty male Wistar rats aged 4 weeks were enrolled, and then nucleus pulposus cells of intervertebral disc were isolated and cultured in vitro; cell passage culture was performed in different groups when the primary cells were merged to 90%. Differential velocity adherent group cells adhered for 30 minutes, and non-adherent cells were aspirated and transferred to new culture dish after readjusting the concentration; the controls received no intervention. Passages 1 and 2 cells in the differential velocity adherent group were isolated and purified by the same procedure. The morphology of three generations of cells in the two groups was compared, the purity of the identification was detected by immunohistochemistry, the cell viability was detected by cell counting kit-8 and the cell growth curve was drawn. RESULTS AND CONCLUSION: Inverted phase contrast microscope and hematoxylin-eosin staining revealed that the cell homogeneity of the differential velocity adherent group was significantly higher than that of the control group, and there were more kinds of fibroblast-like cells in nucleus pulposus cells in the control group. Identification and purity analysis of collagen type II showed that the cytoplasm of two groups were both stained brown, indicating that they were the nucleus pulposus chrondrocytes. The positive rate of differential velocity adherent group was significantly higher than that of the control group (P < 0.05). The cell growth curve of cell counting kit-8 showed cells in the two groups all passed by the latency phase within 2 days, then to the logarithmic phase of 3 days, and entered the lag phase, while the growth rate of the control group was more rapid during the latency and the early logarithmic phases. These findings suggest that differential velocity adherence method is a practical and effective procedure for the isolation and purification of primary cultured rat nucleus pulposus cells. Through the primary culture, twice differential velocity adherence procedure, the passage 3 rat nucleus pulposus cells are metabolic exuberant, consistent with the phenotype, the cell purity is higher, and the logarithmic growth phase can be used as the optimal time for studying the mechanism of intervertebral disc 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     

制备小鼠去细胞拟胚体对Lewis肺癌细胞的促分化作用

BACKGROUND: Co-culture with embryonic stem cells or embryonic tissues can induce differentiation of carcinoma cells into normal epithelial cells or decrease malignancy of carcinoma cells. Acellular embryoid bodies retain the structure and important cytokines of embryonic tissues. OBJECTIVE:To prepare acellular embryoid bodies from mouse embryonic stem cells and to investigate their effects on differentiation of mouse Lewis lung carcinoma cells at three-dimensional culture in vitro. METHODS: Mouse embryonic stem cells (D3) were dynamically cultured for 7 days to produce embryoid bodies followed by decellularization with 0.1% sodium dodecyl sulfate. Mouse Lewis lung carcinoma cells were co-cultured with acellular embryoid bodies as test group or cultured in three-dimensional matrigel medium for 7 days as control group, respectively. Cell proliferation and expression of E-cadherin were detected by immunohistochemical staining and western blot assay, respectively. In addition, mRNA expressions of Slug and E-cadherin were observed using RT-PCR technology. RESULTS AND CONCLUSION: Uniform mouse embryoid bodies were successfully prepared, and were completely decellularized with sodium dodecyl sulfate. After 7-day three-dimensional matrigel culture, in the control group, multicellular tumor spheroids were formed, accompanied by a higher Ki67 positive rate; Lewis lung carcinoma cells in the test group were repopulated in the acellular embryoid bodies showing significantly lower Ki67 positive rate. Compared with the control group, the absorbance of Paxillin in the test group was significantly smaller, and the absorbance of E-cadherin was significantly higher (P < 0.05). Besides, mRNA expressions of Slug and E-cadherin were significantly decreased and increased in the test group compared with the control group, respectively (P < 0.05). These findings indicate that the acellular embryoid bodies can promote differentiation of mouse Lewis lung carcinoma cells in three-dimensional culture in vitro. 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; ORCID: 组织工程0000-0003-2685-4218(吕卫东)     

20 W微波照射骨骼肌卫星细胞的增殖与分化：兔骨折内固定模型研究

BACKGROUND:Low power microwave irradiation has been shown to promote the healing of fractures with internal fixation; however, its action mechanisms on the skeletal muscle around the fracture site are unclear. OBJECTIVE:To study the effects of low power microwave irradiation (20 W) on the proliferation ability of skeletal muscle satellite cells in a rabbit model of femoral fracture with internal fixation. METHODS:Forty male New Zealand rabbits were used to establish femoral fracture followed by internal fixation models, and then were equally randomized into spontaneous recovery and microwave treatment groups. Low power microwave irradiation (20 W) was given for 30 consecutive days in the microwave treatment group on day 4 after modeling, while no microwave irradiation was given in the spontaneous recovery group. Rabbit thigh muscles adjacent to the implant were obtained to isolate skeletal muscle satellite cells. Immunohistochemical staining, hematoxylin-eosin staining and quantitative RT-PCR were used to evaluate the ability of the proliferation and differentiation of skeletal muscle satellite cells. RESULTS AND CONCLUSON: Hematoxylin-eosin staining showed that there was no significant difference in the morphology and histology of skeletal muscle tissues between the spontaneous recovery and microwave treatment groups. However, the relative mRNA expression of MyoG in the cultured skeletal muscle satellite cells in vitro and the number of α-sarcometric actin-postive cells in the microwave treatment group were significantly increased compared with the spontaneous recovery group (P < 0.05). The proliferative ability of skeletal muscle satellite cells was inhibited at the early stage, but not at the later stage. Our results suggest that low power microwave irradiation (20 W) can promote the proliferation and differentiation of skeletal muscle satellite cells around the implant in a rabbit model of femoral fracture with internal fixation, and thereby confirm the efficacy and safety of low power microwave irradiation for the internal fixation of fractures. 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     

UVA1 irradiation inhibits fibroblast proliferation and alleviates pathological changes of scleroderma in a mouse model

The purpose of the present study was to compare the effects of different doses of ultraviolet radiation A1 (UVA1) on human fibroblast proliferation and collagen level in a mouse model of scleroderma,so as to identify appropriate irradiation doses for clinical treatment of scleroderma.Monolayer from human fibroblasts was cultured in vitro,and a mouse model of scleroderma was established by subcutaneous injection of 100 μL of 400 μg/mL bleomycin into the back of BALB/c mice for 4 weeks.The mouse models and human fibroblasts were divided into UVA1exposed (100,60 and 20 J/cm 2) and UVA-unexposed groups.At 0,24 and 48 h after exposure,cell proliferation and levels of hydroxyproline and collagen were detected.UVA1 irradiation was performed 3 times weekly for 10 weeks,and the pathological changes of skin tissues,skin thickness and collagen level were observed after phototherapy.Cell proliferation and the levels of hydroxyproline and collagen were inhibited after phototherapy,and there was a significant difference between the UVA1-exposed cells and UVA1-unexposed cells (P < 0.001).In addition,UVA1 phototherapy improved dermal sclerosis and softened the skin,and there were significant differences between the high-dose UVA1 group and the model group,and the negative group (P < 0.05).It is concluded that UVA1 radiation can reduce cell proliferation,and decrease hydroxyproline and collagen levels in a dose-dependent manner in vitro.High-dose UVA1 phototherapy has marked therapeutic effect on scleroderma in the mouse model.Decreased collagen level may be related to the reduced number and activity of cells,as well as inhibition of collagen synthesis.     

生长分化因子5诱导脂肪干细胞向软骨细胞的转化

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     

Atorvastatin effects on proliferation and apoptosis of rat bone marrow-derived endothelial progenitor cells in vitro北大核心

BACKGROUND: Atorvastatin has a cardiovascular protective effect that significantly improves endothelial function and promotes the mobilization, migration, and differentiation of endothelial progenitor cells. However, the screening of atorvastatin concentration for in vitro cell culture is not well documented. OBJECTIVE: To investigate the effects of different concentrations of atorvastatin on rat bone marrow-derived EPCs growth characteristics. METHODS: Bone marrow mononuclear cells from Sprague-Dawley rats were induced in selective culture fluid to culture EPCs. Immunofluorescence staining was used to identify cell surface markers. Harvested EPCs were divided into control group and atorvastatin groups with four different concentrations (0.01, 0.1, 1, and 10 µmol/L) for culture. The growth and proliferation of EPCs were observed under light microscope and MTT assay. Flow cytometry was used to detect apoptosis in EPCs. Nitric oxide and endothelial nitric oxide synthase levels in the culture fluid were measured by nitrate reductase method. RESULTS AND CONCLUSION: The number of cells tended to increase in the control and atorvastatin groups, and it was highest in the 1 µmol/L atorvastatin group. The cell number in the 10 µmol/L atorvastatin group began to decrease at 7 days of culture. Among the five groups, the apoptotic rate of cells was lowest in the 1 µmol/L atorvastatin group and highest in the 10 µmol/L atorvastatin group. The levels of nitric oxide and endothelial nitric oxide synthase were significantly higher in the 0.01, 0.1 and 1.0 µmol/L atorvastatin groups compared with the control group (P < 0.01), but lower in the 10 µmol/L atorvastatin group compare with the other groups (P < 0.01). Overall, atorvastatin can promote the proliferation of endothelial progenitor cells and reduce apoptosis by increasing the production of endothelial nitric oxide synthase and nitric oxide, and 1 µmol/L atorvastatin is most suitable for the EPCs culture. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.