ObjectiveTo interpret the mechanisms of vascular repair disorders in steroid-induced avascular necrosis of the femoral head (SANFH) via detection of the changes of proliferation, migration, and macrophage migration inhibitory factor (MIF)/vascular endothelial growth factor (VEGF) expressions of endothelial cells (ECs) under hypoxia/glucocorticoid. MethodsAccording to culture conditions, human umbilical vein ECs (HUVECs) at passage 3 were divided into group A (normal), group B (1.0×10-6 mol/L dexamethasone), group C (hypoxia), and group D (hypoxia+1.0×10-6 mol/L dexamethasone). The cell activity was detected by AlamarBlue; the number of viable cells was detected in live/dead cell staining; the cell morphology was observed after cytoskeleton staining; cell migration ability was compared by scratch test; and the levels of MIF and VEGF expressions were detected by ELISA. ResultsAt 24 hours after culture, the cell activity and the number of living cells in group C were significantly higher than those in the other 3 groups, showing significant difference between groups (P < 0.05), and group D had the worst cell activity and least living cells. Cytoskeleton staining showed that cells had normal morphology in groups A and B; cells had rich cytoskeleton and secretion granules in group C; cytoskeleton form disorder and nucleus pyknosis were observed in group D. Scratch test showed that the cell migration ability of group C was strongest while cell migration ability of group D was weakest. Accumulated concentration of MIF and VEGF in 4 groups significantly increased with time extending. Accumulated concentration of MIF in group C were significantly higher than that in other 3 groups at each time point (P < 0.05). Within 24 hours after intervention, stage concentration of MIF during 1-8 hours was significantly lower than that during 0-1 hour and 8-24 hours in every group (P < 0.05). Stage concentration of MIF in group C was significantly higher than other groups during 0-1 hour and 8-24 hours (P < 0.05). Within 2 hours after intervention, stage concentration of MIF in 4 groups during 0.5-1 hour was significantly higher than that during other stages (P < 0.05). Accumulated concentration of VEGF in group C was significantly higher than that in other groups at 8 and 24 hours (P < 0.05). The stage concentration of VEGF in groups C and D during 8-24 hours was significantly higher than that during 0-1 hour and 1-8 hours (P < 0.05). There was no significant difference in the stage concentration of VEGF within and among group A, B, C, and D at every stage within 2 hours after intervention (P > 0.05). ConclusionIn hypoxia environment, the proliferation and migration of ECs is enhanced, and the secretion of VEGF and MIF is increased. High concentration of dexamethasone will suppress the process above, which induces vascular repair disorders and aggravating SANFH.
Objective To investigate the effects of different temperatures on the system of in vitro physiological environment fostering limbs. Methods Twenty-four limbs were harvested from 6 adult Bama mini pigs and were randomly divided into 4 groups (n=6) according to different temperatures: limbs were placed in in vitro physiological environment foster-ing limbs at 26℃ (group A), 4℃ (group B), 10℃ (group C), and 18℃(group D). After 12 hours of perfusion, the morphology observation was done for the structure and ultrastructure changes of the skeletal muscle by light microscope and transmission electron microscope. The mRNA levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) were detected by real-time fluorescent quantitative PCR (RT-qPCR). Results Histological results showed that the skeletal muscle exhibited mild edema, integrity of the sarcolemma, and occasional perivascular inflammatory cell infiltration in groups B, C, and D, meanwhile, the cells of group C had normal morphology; however, muscle fibers degenerated, muscle cells were seriously damaged, a great number of inflammatory cells infiltrated in the fractured muscle fibers in group A. Transmission electron microscope results showed as follows: the muscle fibers arranged in disorder, and many focal solubility necrosis occurred in group A; the muscle fibers arranged in order relatively and sarcolemma was still intact, with mild swelling and flocculent degenerative mitochondria in group B; a large number of muscle fibers arranged in order and regularity with clear sarcomere in group C; and the muscle fibers arranged in disorder and irregularity and partly dissolved in group D. RT-qPCR results showed that the expressions of inflammatory factor TNF-α and IL-1β mRNA in group A were significantly higher than those in groups B, C, and D (P lt; 0.05); the expressions were significantly lower in groups B and C than in group D, and in group C than in group B (P lt; 0.05). Conclusion In the system of in vitro physiological environment fostering limbs, temperature plays an important role in the preservation of amputated limbs. It is suggested that 10℃ can significantly attenuate the reperfusion-induced skeletal muscle cell injuries in this system.
Objective To analyze published literature about the clinical studies on elective single versus double embryo transfer using meta-analysis, so as to provide more convincing evidence for the clinical application of elective single embryo transfer. Methods We electronically searched foreign and domestic biomedical databases including PubMed, Ovid, EMbase, MEDLINE and CENTRAL, to collect randomized controlled trials (RCTs) on elective single versus double embryo transfer. According to Cochrane systematic review method, two reviewers independently screened studies according to inclusion and exclusion criteria, extracted data, and assessed methodological quality of the included studies. Then, meta-analysis was performed using RevMan 5.1 software. Results A total of 9 RCTs involving 2 784 cases were included, of which, 1 452 were in the trial group while the other 1 332 were in the control group. The results of meta-analysis indicated that, compared with elective double embryo transfer, during each transfer period elective single embryo transfer reduced the live birth rate (RR=0.66, 95%CI 0.59 to 0.73, Plt;0.000 01), multiple pregnancy rate (RR=0.05 95%CI 0.02 to 0.11, Plt;0.000 01), preterm birth rate (RR=0.39, 95%CI 0.26 to 0.60, Plt;0.000 1), and low birth weight rate (RR=0.25, 95%CI 0.15 to 0.44, Plt;0.000 01). However, it had no effect on the ectopic pregnancy rate (RR=0.55, 95%CI 0.11 to 2.77, P=0.47), miscarriage rate (RR=1.33, 95%CI 0.92 to 1.91, P=0.13), and neonatal mortality rate (RR=0.31, 95%CI 0.03 to 2.76, P=0.29). Conclusion Compared with elective double embryo transfer, during each transfer period elective single embryo transfer reduces the live birth rate, multiple pregnancy rate, preterm birth rate, and low birth weight rate. No significant difference was found between the two groups in the other indicators.
Objective To establish a rapid, simple, and economic method to prepare osteoporosis (OP) in vitro model. Methods Eighty pairs of fresh goat femur were collected from 18-month-old female goats and were randomly divided into 4 groups (20 pairs in each group). The femur was immersed decalcifying solution (18% EDTA) for 1-5 days (group B), 6-10 days (group C), and 11-15 days (group D), while group A had no treatment as control. Four pairs of femur were taken out every day. Quantitative computed tomography was used to scan the medial and lateral femoral condyles, and the bone mineral density (BMD) was calculated. Electronic universal testing machine was used to do three-point bending test and compress and tensile ultimate strenght test, and the mechanical parameters for femur were calculated. Results With demineralized time passing, BMD of the medial and lateral femoral condyles were downtrend in groups A, B, C, and D, showing significant differences among 4 groups (P lt; 0.05); BMD of the lateral femoral condyle was significantly higher than that of the medial femoral condyle in each group (P lt; 0.05). The three-point bending test showed that broken load, ultimate strength, and elastic modulus of groups A and B were significantly higher than those of groups C and D (P lt; 0.05); but no significant difference was found between groups A and B, and between groups C and D (P gt; 0.05). Compress and tensile ultimate strength test showed that the compress and tensile ultimate strengths were significantly higher in group A than in groups C and D (P lt; 0.05), and in group B than in group D (P lt; 0.05), but no significant difference was found between groups A and B, between groups B and C, and between groups C and D (P gt; 0.05). Conclusion The 18% EDTA immersing for 6-15 days is a fast, simple, economical method to prepare an OP in vitro model of goat femur.
Objective Titania and Ag containing nano-hydroxyapatite/polyamide 66 (TiO2-Ag-nHA/PA66) composite bone fill ing material has good biocompatibil ity and biological safety. To investigate the antibacterial effect and Ag+ release characteristics of TiO2-Ag-nHA/PA66 composite bone fill ing material containing different concentrations of Ag+ in vitro. Methods The n-HA/PA66 composite bone fill ing material A1 (material A1) was prepared by co-polymerization method, and TiO2-Ag-nHA/PA66 composite bone fill ing materials A2 and A3 (materials A2 and A3) were prepared by thesame way containing Ag+ of 0.22wt% and 0.64wt%, respectively, and the TiO2 content was 2.35wt%. The materials A2 and A3 were respectively immersed in 50 mL simulated body fluid (SBF), and Ag+ concentration was measured by atomic absorption spectrometry at 1, 3, 7, 14, 21, and 49 days. The inhibition ring test and colony count method were used to evaluate antibiotic effect against Staphylococcus aureus and Escherichia coli, the anti-adhesion capacity of Staphylococcus aureus and Escherichia coli was observed by scanning electron microscope (SEM). Results There was no significant difference in the Ag+ concentration between materials A2 and A3 at 1 day and 3 days (P gt; 0.05); and there were significant differences in the Ag+ concentration between materials A2 and A3 after 7 days (P lt; 0.05). The inhibition ring diameters of materials A2 and A3 to Staphylococcus aureus and Escherichia coli reached the maximum at 1 day, which were (13.40 ± 2.88), (9.40 ± 1.14) mm and (23.60 ± 1.14), (18.80 ± 0.84) mm, showing significant difference (P lt; 0.05) between materials A2 and A3 respectively; and then, the diameter of inhibition ring reduced with the time. The antibacterial effect of materials A2 and A3 against Staphylococcus aureus and Escherichia coli lasted 15, 33 days and 9, 24 days, respectively. No inhibition ring was observed around material A1 all the time. And the inhibitory rates of materials A2 and A3 were 89.74% ± 3.62%, 94.18% ± 2.05% and 78.65% ± 5.64%, 85.96% ± 2.50%; showing significant differences (P lt; 0.05) among materials A1, A2, and A3. SEM showed that bacterial adhesion of materials A2 and A3 was obviously fewer than that of material A1. Conclusion TiO2-Ag-nHA/PA66 composite bone fill ing material has antibacterial property against Staphylococcus aureus and Escherichia coli, and it has a good release effect in SBF.
ObjectiveTo systematically review the clinical effects of short-term and conventional fertilization for vitro fertilization-embryo transfer (IVF-ET). MethodsRandomized controlled trials (RCTs) about the clinical effects of short-term fertilization versus conventional fertilization for IVF-ET were searched in PubMed, The Cochrane Library (Issue 8, 2014), CBM, CNKI, WanFang Data and VIP from inception to August 2014. Two reviewers independently screened literature according to the inclusion and exclusion criteria, extracted data, and assessed methodological quality of included studies. Then meta-analysis was performed using RevMan 5.2 software. ResultsA total of six RCTs involving 1 373 patients were finally included. The results of meta-analysis indicated that:short-term fertilization was superior to conventional fertilization in increasing high quality embryo rates (OR=1.42, 95%CI 1.18 to 1.70, P=0.000 2) as well as clinical pregnancy rates (OR=1.67, 95%CI 1.33 to 2.09, P < 0.000 01). However, the two groups were alike in fertilization rates, polyspermy rates, and miscarriage rates. ConclusionCurrent evidence indicates that short-term fertilization is superior to conventional fertilization in increasing high quality embryo rates as well as clinical pregnancy rates. Due to limited quality and quantity of the included studies, the above conclusion should be verified by conducting more large-scale, high quality RCTs with long-term follow-up.
Objective To observe the morphology and growing status of mesenchymal stem cells(MSCs) of human bone marrow in vitro, in order to confirm that MSCs of human bone marrow are ideal seed cells and provide basic theory for further MSCs research. Methods The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration were used to isolate and purify MSCs of human bone marrow. We observed the cellular growth status and morphology of the primary MSCs and the surface antigens of second passage MSCs were tested. Results The primary culture cells fused into monolayer after 14-16 d. The passage cells kept the same morphological characteristics of primary culture cells. Ultrastructure of the second passage MSCs showed that the shape of nuclei was irregular, there were multiple nucleoli in some of the nuclei, and morphological differentiation of intracytoplasm organelles was immature. The growth curve of the first, fifth and tenth passage cells showed a logarithmic growth at day 3, a peak growth at day 5, and no clones occurred after tenth passage. Cloning efficiency of first passage, fifth passage and tenth passage was respectively 25.83%±2.93%, 14.67%±1.63% and 4.67%±0.52%. Test of MSCs phenotypic characteristics showed a high homogeneity among the cells and surface antigen profiles were positive for CD29, CD44 and negative for CD34, CD45. Conclusion The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration are simple, economic and efficient to isolate and purify MSCs from human bone marrow. With a high proliferating ability in vitro, MSCs from human bone marrow are ideal seed cells for tissue engineers.
Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo. This novel cell culture model has been proved to be close to the natural system in vivo. In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.
【Abstract】 Objective To observe the effect of cytokines and combinations in the inducement of human umbil icalcord blood-derived CD34+ cells into hepatocyte-l ike cells. Methods The mononuclear cells (MNCs) were derived by density gradient centrifugation and the CD34+ cells were sperated from MNCs. The human umbil ical cord blood-derived CD34+ cells were cultivated through 49 different combinations of cytokines including leukemia inhibitor factor (LIF), oncostatin M, bFGF, aFGF, hepatocyte growth factor, EGF and stem cell factor for 28 days, and the concentrations of the cytokines were 10, 10, 10, 10, 20, 20 and 50 ng/mL, respectively. The mRNAs of cytokeratin 19 (CK-19), CK-18, glutamine synthetase (GS), human albumin (ALB) and α-fetoprotein (AFP) were detected every seven days. The ALB secretion abil ity, detoxification abil ity and hepatin synthesis abil ity of the induced cells were detected by immunofluorescence assay, indocyanine green (ICG) and periodic acid-schiff assay, respectively. The fresh umbil ical cord blood-derived CD34+ cells were detected at the same time as a control. Results The mRNAs of CK-19, CK-18 and GS could be transcribed in all the induced cells, but the transcription of the mRNAs of ALB and AFP which was the special mark of mature hepatocyte and l iver stem cell, respectively, was not found. All the mRNAs could not be found in freshly isolated umbil ical cord blood-derived CD34+ cells. All the cells induced in vitro could not release ALB, and not help the detoxification of ICG which was the fundamental function of mature l iver cells. These results were the same in the control group. The hepatin synthesis abil ity of all the induced cells increased by comparison to the fresh ones. Conclusion Though some mRNAs of proteins which are transcribed in hepatocytes can be found in the induced cells, umbil ical cord blood-derived CD34+ cells could not be transdifferentiated into hepatocyte-l ike cells through cytokines in vitro.
Objective To investigate the feasibil ity of replacing urinary epithel ial cells with oral mucosa cell to reconstruct tissue engineered urethra by being seeded on bladder acellular matrix graft (BAMG). Methods Eighteen male New Zealand rabbits, aged 10 weeks, weighing 0.3-0.5 kg, were used in this study. Oral mucosa cell of 12 rabbits were isolated and seeded onto a culture dish with a feeder layer of 3T3 and a culture dish without 3T3, respectively. The morphologic change and growth condition of oral mucosa cells were observed by inverted phase contrast microscope after 2 days of seeding. The quantity of oral mucosa cells was counted using cell counting meter; the cell growth curve was drawn and the immunofluorescence staining with broad-spectrum keratin antibody was carried out. The bladders taken from the rest 6 rabbits were decelluled to make BAMG and the tissue of 1 cm × 1 cm was randomly selected to observe the effect of acellularization. The second passage oral mucosa cells cultured with 3T3 were appl ied to steril ized BAMG to obtain a issueengineered mucosa. The tissue-engineered mucosa was assessed using HE staining and scanning electron microscope after being cultured for 1 week. Results Oral mucosa cells seeded onto a feeder layer of 3T3 could be passaged for 7 or 8 generations with homogeneous forms and full function. Oral mucosa cells cultured without 3T3 could only be subcultured for 2 generations before aging and had multiple shapes and different sizes. Oral mucosa cells cultured by the two methods both started logarithmic growth on the 8th day and reached the peak value on the 14th day, which was indicated by the cell growth curve. However, more cells could be obtained through oral mucosa cells cultured with 3T3 than those cultured without 3T3. Oral mucosa cells manifestated green colour fluorescence cultured with or without 3T3. After the cells were removed, the BAMG presented as a porous membrane. The HE staining showed that the effect of acellularization was good and there were no cells at BAMG. The second passage oral mucosa cells cultured with 3T3 were expanded and seeded onto steril ized BAMG to obtain a tissue-engineered mucosa. Good compatibil ity of the compound graft was assessed using HE staining and scanning electron microscope. HE staining and scanning electron microscope showed that oral mucosa cells had good biocompatibil ity with BAMG after the tissue engineered mucosa was cultured for 1 week. Conclusion Oral mucosa cells of rabbit can be cultured in vitro and attain magnitude quantities. Oral mucosa cell also have good biocompatibil ity with BAMG and the compound graft could be a new material for urethral reconstruction.