OBJECTIVE:To evaluate the toxicity of retinoic acid in silicone oil to the retinal tissue.
METHOD:Twelve New Zealand white rabbits(24 eyes)were divided into three grorps at random. Three days after gas-compression vitrectomy,24 eyes were unedrgone gas/silicone oil exchange. The silicone oil 0.5 ml was injected intravitreally in 4 eyes as controls ,and 5mu;g/ml retinoic acid silicone oil 0.5ml in 10 eyes and 10 mu;g/ml retinoic acid silicone oil 0.5 ml in 10 eyes respectively as 2 study groups. After intravitrea[ injections, all the eyes were examined by ophthalmoscopy on the 1st, 3rd, 7th, 14th, 21st and 28th day. The retinas of the enucleated eyes on the 28th day were then examined by light microscopy and transmission electrone microscopy.
RESULT: No evidence of toxicity was found in retinas after intravitreal injections of silicone oil with 5 mu;g/ml or 10 mu;g/ml retinoic acid.
CONCLUSION :There was no toxic effect on the retinas by using 5 mu;g/ml or 10 mu;g/ml retinoic acid in intravitreal silicone oil tamponade operation.
（Chin J Ocul Fundus Dis,1997,13: 81-82）
ObjectiveTo observe the effect of epinephrine in intraocular irrigation solution on retinal vascular caliber and macular thickness.
MethodsA prospective control study. 32 eyes of 32 patients with macular hole who underwent vitrectomy were enrolled in this study. The patients including 14 males (14 eyes) and 27 females (18 eyes), with the average age of (64.0±4.5)years. Uncorrected visual acuity, corrected visual acuity, slit lamp biomicroscopy, indirect ophthalmoscopy, fundus color photography and optical coherence tomography were performed in all patients. Retinal vascular caliber located in 0.5-1.0 disc diameter from optic disk was measured from digital fundus photographs and summarized as central retinal artery (CRAE) and vein (CRVE) equivalents in all eyes at baseline and at the 1 month, 3 months follow-up visit. The macular thickness is the distance from retinal interface of inner plexiform layer to retinal pigment epithelium layer. The macula was divided into inner ring ( < 3 mm) and outer ring (3-6 mm) according to the distance from the fovea. The patients were divided into experiment group (include epinephrine in intraocular irrigation solution, 1:1000) and control group (without epinephrine in intraocular irrigation solution), 16 eyes in each group. The difference of CRAE and CRVE between two groups was not significant (P > 0.05). The difference of macular thickness between inner ring and outer ring was not significant (P > 0.05). The average follow-up was 3.5 months. CRAE, CRVE and macular thickness in inner ring and outer ring before and 1 month, 3 months after surgery were comparatively analyzed.
ResultsThe differences of CRAE and CRVE before and 1, 3 months after surgery both in experiment group (tCRAE=0.322, 0.148; tCRVE=0.317, 0.005) and control group (tCRAE=0.226, 0.137; tCRVE=0.284, 0.151) were not significant (P > 0.05). The differences of CRAE (t=0.624, 0.424) and CRVE (t=0.015, 0.041) between experiment group and control group also were not significant (P > 0.05). The differences of macular thickness in inner ring and outer ring before and 1, 3 months after surgery both in experiment group (tinner=0.322, 0.148;touter=0.317, 0.005) and control group (tinner=0.226, 0.137;touter=0.284, 0.151) were not significant (P > 0.05). The differences of macular thickness in inner ring (t=1.568, 0.373) and outer ring (t=-1.697, 0.536) between experiment group and control group also were not significant (P > 0.05).
ConclusionEpinephrine (1:1000) in intraocular irrigation solution has no effect on retinal vascular caliber and macular thickness in patients with macular hole.
Objective To investigate the feasibility of a new kind of porous β tricalcium phosphate (β-TCP) as a scaffold for the bone tissue engineering Methods The inverted phase contrast microscope was used to observe the growth of the marrow mesenchymal stem cells (MSCs) in the experimentalgroup and the control group at 10 days.In the experimental group, the MSCs were cultured with β-TCP(3 mm×3 mm×3 mm) in the 24-hole cultivation board, and in the control to control group, only MSCs were cultivated. The scanning electron microscope was used to observe growth of MSCs at 6 days. Cultivated with β-TCP at 3, 6, 9, 12 days, the MTT assay was used to judge the biocompatibility. The cytotoxicity was analyzed with the method that used the different density(100%， 50%， 10%， 1%，0%) leaching liquor gained from β-TCP to raise MSCs. MSCs were induced into the osteoblasts and were mixed with β-TCP, and the composite was used to repair a large radius bone defect in the rabbit. The specimens were made at 2,6,12 weeks. The histology imageology, and the radionuclide bone scan were used to analyze the bone formation. Results Some MSCs had a good adherence 4 hours after MSCs were inoculated and had a complete adherence at 12 hours. The cells were shaped like polyangle, spindle or converge monolayer after 8-10 days. The cells in the two groups had no difference. The cell adhesion was good, when observed by the inverted phase contrast microscope and the scanning electron microscope at 6 days. MTT showed that the absorbance (A)was not statistically different between the experimental group and the control group (P＞0.05); the different density leaching liquor had no cytotoxicity at the different time points. Histology, X-ray, and CT tomograph showed that itcould repair the large radius bone defect in the rabbit and its in vivo degradationrate was the same as the bone formation rate. Conclusion The new porous β-TCP has a unique three dimensional (3D) stereochemical structure and superordinary physicochemical property, and so it is a good scaffold for the bone tissue engineering.
Objective
To investigate the retinal toxicity and verify the safe dose of intravitreal injecting fluconazole.
Methods
Twelve healthy adult white rabbits were divided at random into 6 groups：a normal control group and 5 groups received intravitreal injection of a single dose of fluconazole ranging from 10 to 200 mu;g respectively.Retinal toxicity was examined by ophthalmoscopy, electroretinography, light and transmission electron microscopy (TEM) on the third and fourteenth day after injection.
Results
The ultrastructures of the retinal tissues of the normal control group and fluconazole 10～150 mu;g groups were normal on the third and fourteen day after injection.The light microscopy and TEM showed that cells of all the retinal layers in the 200 mu;g group revealed apparent degenerative changes on the fourteenth day after injection, and the light microscopic picture showed the vacuolar degeneration of outer segments of photoreceptors, the nuclei in outer nuclear layer drop out into inner segments, the vacuolar degeneration of nerve fiber layer, and the proliferation of pigment epithelium. TEM revealed expansion of paranucl eus space and karyopyknosis of the bipolar cells, the swelling of nerve fibers and disappearance of the synapses in the inner plexiform layer, the vacuolation and disappearance of microvilli of the pigment epithelium cells.
Conclusion
The safe dose of fluconazole injected intravitreally should be 100～150 mu;g.
(Chin J Ocul Fundus Dis，2000，16：139-212)
Three-dimensional (3D) printing, an emerging rapid prototyping technology, has been widely used in biomedical field. 3D printing was originally used to construct the visualization models and molds in medicine. With the development of 3D printing in biomedical field, the technology was gradually applied in complex tissue regeneration and organ reconstruction. Artificial tissues and organs obtained by 3D printing are expected to be used for organ transplantation, new drug development and drug toxicity evaluation in the field of medicine and health care research. This paper describes the individualized application of 3D printing technology in liver surgery and introduces the research progress of 3D bioprinting technology in liver transplantation, drug metabolism and hepatotoxicity evaluation, and prospects its future development trend to provide a reference for further study.
Objective
To analyze the glucolipotoxicity effects of glucose combined with free fatty acid (FFA) on ketone production and ultrastructure of skeletal muscle, by exogenous elevating circulating glucose and FFA concentration.
Methods
Fifty Wistar rats were divided into high-fat-feed induced obesity group (OB group, n=40) and ordinary feed as normal control group (NC group, n=10). Circulating glucose and FFA levels were increased by infusion in high-fat-fed obese rats. The levels of serum lipid, plasma FFA and beta-hydroxybutyric acid were detected by the horizontal colorimetry, and the microstructure of skeletal muscle was observed by transmission electron microscopy, especially the changes of the mitochondrial structure. Euglycemic-hyperinsulinemic clamp with tracer infusion was performed to assess peripheral insulin sensitivity.
Results
The average weight and body fat ratio in the OB group was higher than that in the NC group (P<0.05). Insulin clamp test to assess peripheral insulin sensitivity showed that the steady-state glucose Infusion rate in the OB group during clamp test was significantly lower than that in the NC group [OB: (19.26±1.84) mg/(kg·min)vs. NC: (28.82±1.69) mg/(kg·min), P<0.05]. The mitochondrial denaturation of skeletal muscle in the OB group of rats was observed, and the swelling and crest permutation, the accumulation of lipid droplets and cavitation were formed, and hypertrophy of mitochondria were also seen after intralipid and glucose infusion, which was obvious in the combined infusion group.
Conclusions
By exogenous elevating circulating glucose and FFA concentration, the products of ketone body increases. The mitochondrial damage of skeletal muscle suggests that mitochondrial may be the potential target of glucoxicity and lipotocicity.
Objective To optimize the in vitro culture system of C57/BL6 marrow mesenchymal stem cells (MSCs) and to investigate the effect of alcohol and acetaldehyde on MSCs. Methods The MSCs were isolated from the femur marrow of C57/BL6 mice and were cultured in the optimized system, so that highlypurified MSCs were harvested and identified by immunohistochemistry. Then, MSCs were cultured in the medium containing alcohol or its metabolic product acetaldehyde, with the following concentration groups: alcohol 5.7，17.0，50.0，100.0 and 150.0 mmol/L; acetaldehyde 4.5, 0.9, 0.18, 0.036, 0.007 2, 0.001 44 , 0.000 28 mmol/L. MSCs were cultured with α-MEM as the control group. After 3 days, their proliferation activity was measured by the MTT method. Results MSCs within 6 passages had a good stability and a high proliferation activity. They were identified to express CD90 but no CD34. The MTT assay showed that alcohol at the concentration greater than 100.0 mmol/L and acetaldehyde at the concentration greater than 4.5 mmol/L could inhibit proliferation of MSCs(P＜0.05) . But the proliferation activity might rise with an increase in the acetaldehyde concentration smaller than 0.18 mmol/L(P＜0.05) . Conclusion Theoptimized culture system can effectively isolate and culture MSCs. Both alcoholand acetaldehyde can inhibit proliferation of MSCs but toxicity of acetaldehydeis more serious.
ObjectiveTo study the effect of three-dimensional (3D) printed β-tricalcium phosphate (β-TCP) scaffold loaded poly (lactide-co-glycolide) (PLGA) anti-tuberculosis drug sustained release microspheres on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its cytotoxicity.MethodsIsoniazid and rifampicin/PLGA sustained release microspheres were prepared by W/O/W multiple emulsion method. The β-TCP scaffolds were prepared by 3D printing technique. The microspheres were loaded on the scaffolds by centrifugal oscillation method to prepare composite materials. The BMSCs of Sprague Dawley rat were isolated and cultured by whole bone marrow adherent method, and the third generation cells were used for the following experiments. BMSCs were co-cultured with osteogenic induction medium (group A), PLGA anti-tuberculosis drug sustained release microsphere extract (group B), 3D printed β-TCP scaffold extract (group C), and 3D printed β-TCP scaffold loaded PLGA anti-tuberculosis drug sustained release microsphere composite extract (group D), respectively. Cytotoxicity was detected by cell counting kit 8 (CCK-8) method; the calcium deposition was observed by alizarin red staining; and the mRNA expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were detected by real-time fluorescence quantitative PCR (RT-qPCR).ResultsCCK-8 assay showed that the absorbance (A) value of groups A, B, C, and D increased gradually with the culture time prolonging. After cultured for 24, 48, and 72 hours, the A value decreased in the order of groups A, C, B, and D. There was no significant difference between groups B and D (P>0.05), but there were significant differences between other groups (P<0.05). The cytotoxicity was evaluated as grade 0-2, and the toxicity test was qualified. Alizarin red staining showed that red mineralized nodules were formed in all groups at 21 days after osteogenic induction, but the number of mineralized nodules decreased sequentially in groups C, D, A, and B. RT-qPCR test results showed that the relative expressions of OCN and BSP genes in groups A, B, C, and D increased gradually with the culture time prolonging. The relative expression of ALP gene increased at 7 and 14 days, and decreased at 21 days. After cultured for 7, 14, and 21 days, the relative expressions of ALP, OCN, and BSP genes decreased sequentially in groups C, D, A, and B; the differences were significant between groups at different time points (P<0.05).Conclusion3D printed β-TCP loaded PLGA anti-tuberculosis drug sustained release microsphere composites have no obvious cytotoxicity to BMSCs, and can promote BMSCs to differentiate into osteoblasts to a certain extent.
Objective To evaluate the cytotoxicity of microdosis peracetic acid (PAA) so as to provide the evidence for making residual l imit of PAA steril ization. Methods Mouse fibroblasts (L929 cell l ine) cultured in vitro were observed to evaluate the influence of microdosis PAA including 1 × 10-6, 2 × 10-6, 3 × 10-6, 4 × 10-6, 5 × 10-6, and 10 × 10-6 (V/V). Theproliferation of cells was determined by MTT assay at 2, 4, and 7 days of culture. The growth curve and the relative growth rate (RGR) were obtained. The cytotoxicity of PAA at different concentrations was evaluated according to RGR. Results At 2, 4, and 7 days after culture, fibroblasts of 1 × 10-6 group grew with normal morphology analogous to control group, while the cell growth of other groups were poor. With the increase of PAA concentration, the absorbance (A) values decreased, which suggested that there was a significant negative correlation between cell prol iferation and PAA concentration. And the correlation coefficient was — 1.000 at 2 and 4 days, — 0.964 at 7 days. There was no significant difference in A value between 1 × 10-6 group and the control group (P gt; 0.05), while there were significant differences in A value between the control group and other concentration groups (P lt; 0.05). The growth curve of 1 × 10-6 group was similar to that of the control group, both had obvious phase of exponential growth. The growth curves of other groups had no obvious phase of exponential growth. The cytotoxicity of 1 × 10-6 group was classified as level 1, 2 × 10-6 group as level 2, 3 × 10-6 group as level 3, 4 × 10-6 group as level 3-4, 5 × 10-6 group and 10 × 10-6 group as level 4. Conclusion PAA of 1 × 10-6 had no obvious cytotoxicity. The residual l imit of PAA less than 1 × 10-6 was recommended.
The objective of the study is to analyze the biological characteristics and stability of the linear derivative Bac2a from bactenecin, compared with the control peptide melittin. The secondary structure, antibacterial activity, hemolytic activity, cell toxicity and stability of the Bac2a were determined by circular dichroism spectroscopy, broth micro-dilution method and MTT assay. The results showed that Bac2a was a nonregular curl in aqueous solution, however, it was an α-helix structure in the hydrophobic environment. The minimal inhibitory concentration (MIC) of Bac2a ranged from 2 to 32 μmol/L, so the bacteriostatic activity of Bac2a was strong. The hemolytic rate was only 14.81% when the concentration of Bac2a was 64 μmol/L, which showed that the hemolytic rate of Bac2a was low. The therapy index of Bac2a was 3.26, and the cytotoxicity was relatively low, thus the cell selectivity was relatively high. In addition, with the heating treatment of 100℃ for 1 h, Bac2a still possessed rather a high antibacterial activity and showed a good heating stability. In a word, Bac2a has good application prospects in food, medicine and other fields, and is expected as a substitute for traditional antibiotics.
