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.
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 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.
Objective To investigate the effects of celecoxib-poly lactide-co-glycolide microparticles (CEL-PLGA-MS) on rat retina after intravitreal injection. Methods A total of 32 male Brown Norway rats were randomly divided into CEL-PLGA-MS group and celecoxib group, 16 rats in each group. The rats in CEL-PLGA-MS group were divided into four dosage group, four rats in each group, which received intravitreal injection of PLGA with celecoxib at the concentration of 40, 80, 160, 320 mu;mol/L, respectively. The rats in celecoxib group were divided into four dosage group, four rats in each group, which received intravitreal injection of celecoxib at the concentration of 40, 80, 160, 320 mu;mol/L, respectively. Phosphate buffer solution (PBS) was injected in two rats as PBS control group. Two rats as normal control group received no treatment. The difference of retinal thickness among groups was measured by optical coherence tomography (OCT). The morphological and histological change of retina was evaluated under light microscope and transmission electron microscope. Results There was no difference of retinal thickness between normal control group and PBS control group (F=0.12,P＞0.05). At the first week after injection, the retinal thickness of CEL-PLGA-MS group and celecoxib group were thicker than that in normal control group and PBS control group (F=9.62, 46.13;P＜0.01). The retinal thickness of celecoxib group was thicker than that in CEL-PLGA-MS group (F=165.15,P＜0.01). The retinal thickness was estimated equal among 40, 80, 320 mu;mol/L dosage groups in CEL-PLGA-MS group (F=4.79,P＜0.01). The retinal thickness of 160, 320 mu;mol/L dosage group were thicker than that in 40, 80 mu;mol/L dosage group in celecoxib group (F=28.10,P＜0.01). At the second week after injection, there was no difference of retinal thickness between CEL-PLGA-MS and celecoxib group (F=3.79,P＞0.05); the retinal thickness of CEL-PLGA-MS and celecoxib group became thinner gradually compare to the first week after injection (F=7.28, 103.99; P＜0.01). At the fourth week after injection, the retinal thickness of celecoxib group was thicker than that in CEL-PLGA-MS group (F=19.11,P＜0.01). The retinal thickness of CEL-PLGA-MS group was approximately the same to normal control group and PBS control group (F=2.02,P＞0.05). The retinal thickness of celecoxib group was thicker than that in normal control group and PBS control group. No considerable abnormality of the retina was seen by light microscope and the retinal thickness corresponded with the values measured by OCT at the first week after injection. The abnormal structures of the retina were seen in 160, 320 mu;mol/L dosage group of celecoxib group and inner changed evidently by the transmission electron microscope. Disordered arrangement of microfilaments, dilated microtubule and some mitochondria vacuolation were observed in 320mu;mol/L dosage group of celecoxib group. Others changed slightly. Conclusions CEL-PLGA-MS has less toxicity on the retina than free-celecoxib after intravitreal injection. The safety of intravitreal injection with CEL-PLGA-MS is better than celecoxib.
Objective To evaluate the safety repeated intravitreal injection of bevacizumab (Avastin) with different dosage in rabbitsprime;eyes. Methods Fourteen chinchilla rabbits were randomly divided into 3 groups, including both eyes of 2 rabbits in the control group,the right eyes of the other 12 rabbits in the experimental group,and the left eyes of the 12 rabbits in the experimental control group. The eyes in the experimental group underwent intravitreal injection of bevacizumab with the dosage of 2.5 mg/0.1 ml and 5.0 mg/0.2 ml of bevacizumab; the eyes in the experimental control group underwent intravitreal injection of normal saline with the same dosage as in the experimental group. Injections were performed every two weeks and lasted six weeks. Clinical observation and retinal function tests were performed before and two days after every injection. The eyes were sacrificed 1 week and 4 weeks after last intravitreal injection respectively.Electron and optical microscope and TUNEL were performed.Results After intravitreal injection,no obvious anterior chamber flare, abnormal change of the ocular fundus, or vitreous opacity and hemorrhage was observed in all of the eyes.No change was found by indirect ophthalmoscope,Bultrasonic inspection, ultrasound biomicroscopy and optical coherence tomography. The number of anterior chamber flare before and after the injection with the dosage of 2.5 and 5.0 mg, the difference among the 3 groups didnprime;t differ much from each other (Pgt;0.05).Amplitude and pattern of ERG responses and flash VEP were similar between the control and experimental groups (Pgt;0.05). Some inflammatory cells were found in the some bevacizumabinjected eyes 1 week after injection, and vanished 3 weeks later. The histological configuration of the retina didnprime;t change in both experimental control and the control group. Electron microscopy showed that plasma cells were presented and vacuolelike change was observed in part of the photoreceptor cells in 5.0 mg experimental group 1 week after injections.Cellular apoptosis was observed in the photoreceptor cell layer. The number of apoptotic cells was more in 5.0 mg experimental group than that in the control and experimental control group 1 week after injections (Plt;0.01). Conclusion Multiintravitreal injection with 5.0 mg bevacizumab may have mild toxicity to the retina in the rabbits.
Objective To evaluate the toxic effects of staphylococcus aureus exotoxins and neutrophils on retinal pigment epithelium (RPE) cells (RPEC). Methods An in-vitro model of bacteroidal endophthalmitis was established by co-culturing of human RPE cell line D407 and human peripheral blood neutrophils in the present of staphylococcus aureus exotoxins ATCC29213. The level of lactate dehydrogenase hydroxide（LDH）in the cuture supernant was measured, and the viability of RPE was evlauated by flow cytometry and Hoechst 33342/Propidium Iodide（PI）staining. Results When RPE cells were cultured with the exotoxin ATCC29213, the LDH level and necrotic RPE cells were positive proportional to the dosage of exotoxin, but only 250mu;l or 500mu;l of ATCC29213 had a statistical significant effect. When RPE cells were co-cultured with neutrophils in the present of ATCC29213 for 6 hours, 100mu;l of ATCC29213 already had a statistical significant effect on LDH level and necrotic RPEC, and the effect was proportional to the amount of neutrophils in the culture. Conclusion Both staphylococcus aureus exotoxins and neutrophils can damage the RPEC by inducing necrosis, and their function had synergetic effect.
Objective To observe the regulation effect of transforming growth factor alpha (TGFalpha;) on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muuml;ller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7～10 day, and then cultured Muuml;ller cells according to literature. The 3～4 generation cultured cells of the same primary cell were divided into two groups at random: ① TGFalpha; group: maintained in different concentrations of TGFalpha; as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;② Control group: cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFalpha; on GLAST activity in Muuml;ller cells were observed by L-3H-glutamate uptake detection; the expression of GLAST mRNA in Muuml;ller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFalpha; concentration, both L3H glutamate uptake and GLAST mRNA expression were increased. The L-3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFalpha; on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant (Plt;0.05). Conclusion TGF-alpha; can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.
Objective To observe the retinal toxicity of intravitreal injection of Bevacizumab (Avastin) in albino rabbit eyes at different doses. Methods Sixteen New Zealand albino rabbits，thirty-two eyes were divided into four groups at random. Three groups were prepared for Avastin experiment, named A, B, C. Each group received intravitreal injection of Avastin at dose 1.25 mg/0.05ml，2.5 mg/0.1ml and 6.25 mg/0.25 ml respectively. The other group named D served as a control, and accepted intravitreal injection of 0.9% normal saline 0.1 ml. Then test it by electroretinagram (ERG) after 1, 2 and 4 weeks. In addition, each group was removing two rabbitprime;s eyes to observe the retinal morphology and ultra structure by light microscope and transmission electron microscopy after intravitreal injection avastin 1, 2 and 4 weeks. Results The ERG pattern and amplitude of each group were normal after intravitreal injection Avastin 1, 2 and 4 weeks. （P＞0.05）Between study and control groups, there was no significant difference in retinal morphology which was observed by light microscope at any stage of the study. By electron microscopic observation, retinal ultramicrostructure was no evident retinal toxicity being tested both at group A and B (1.25 mg/0.05 ml and 2.5 mg/0.1 ml). But at group C (6.25 mg/0.25 ml), significant mitochondrial swelling and hydropic changes were seen in the inner segments of photoreceptors. And there was no improvement of the pathological changes in four weeks. Conclusion It is safe that intravitreal injection of Avastin in rabbitprime;s eyes at dose 1.25 mg or 2.5 mg at single time. （Chin J Ocul Fundus Dis，2008，24：193-196）
Objective To observe the effects of structure and function of cornea, chamber angle and retina of varying doses of Bevacizumab which was injected intravitreally in rabbits. Methods Twenty-four New Zealand albino rabbits were divided into three groups randomly, the right eyes in three groups received int ravitreal injection Avastin at dose 1.25 mg，2.5 mg and 5 mg respectively as experimental eye, the left eyes accepted intravitreal injection 0.9% normal saline at the same volume as a control eye. The anterior segment of eye and ocular fundus were examined and intraocular pressure was measured by slit-lamp microscope and direct ophthalmoscope before and after injection. It was tested by Electroretino gram (ERG) before and after injection 1, 4, 8 weeks. At the 8th week, it carried out corneal endothelium counting; then enucleated eyes to observe by the light microscope and transmission electron microscope. Results No statistically significant difference regard to IOP,corneal endothelium counting, a-and b-waves of ERG at any stage of study in every group（P＞0.05）. No obvious change at cornea, chamber angle, retinal structure and retinal ultrastructure in every group under light microscope. Conclusion This study indicated that there is no obvio us toxicity of intravitreal injection with Avastin 1.25~5.0 mg in normal rabbit eyes. （Chin J Ocul Fundus Dis，2008，24：189-192）