Review on the In Vitro Cytotoxicity Assessment in Accordance to the International Organization for Standardization (ISO)

@#Cytotoxicity is a predominant biological evaluation applied to search for a suitable and non-toxic bioactive compound and to determine the biocompatibility of medical devices-related human body. The broad usage of cytotoxicity tests leads to a robust establishment of cytotoxicity assays with high sensitivity and prompt results. In vitro assays are always prioritized over in vivo due to the reproducible data, reduce numbers of animal used and easily accessible material. Compounds concentration that execute 50% of cell population is determined by calculating the IC50. According to ISO10993, cytotoxicity tests must be performed to determine the biocompatibility of medical devices that has contact with human body. This is crucial to ensure the safety of research and its clinical use. Under the recommendation of ISO10995-Part 5, three categories of tests have been documented; extract elution, direct contact and indirect contact test. Each category plays significant role depending on the nature of experiment and sample used.

Effect of Ions Released and pH of Two Glass Ionomer Cements in Human Gingival Fibroblasts / Efecto de la liberación de iones y pH de dos cementos de ionómero de vidrio en fibroblastos gingivales humanos

Abstract 20. Conventional glass ionomer cements are used as dental provisional restorative materials, which present several advantages such as adhesion to the tooth mineral phase among others. On the other hand, the knowledge about biological property of glass ionomers shows various approaches and results. In this work, it was studied the in vitro biological response of human gingival fibroblasts in contact with commercial cements of glass ionomer: Mirafil® and Ionglass® and with their extracts, according to ISO 10993. The extracts of the cements, in which the cells were cultured, were adjusted at different concentrations ranging 0.1% to 100%. The cellular metabolic activity of gingival fibroblasts was measured using the Alamar Blue® reagent. The results showed a significant effect on the cellular metabolic activity correlated with the concentration of liberated ions (Al³+ and Ca²+) for both ionomers, as well as the pH variations of the culture media. This could mean that the cellular metabolic activity is substantially influenced by ions and pH of the cell culture.

Resumen 24. Los cementos de ionómero de vidrio convencionales se utilizan como materiales de restauración provisional para uso dental, los cuales presentan varias ventajas como la adhesión a la fase mineral de los dientes. Por otro lado, las propiedades biológicas de los ionómeros de vidrio muestran diversos enfoques y resultados. En éste trabajo se estudió la respuesta biológica in vitro de fibroblastos gingivales humanos en contacto con cementos comerciales de ionómero de vidrio: Mirafil® e Ionglass® y con sus respectivos extractos según la norma ISO 10993. Los extractos de los cementos en los que se cultivaron las células estaban en diferentes concentraciones: de 0.1% a 100%. La actividad metabólica celular se midió usando el reactivo Alamar Blue®. Los resultados mostraron un efecto significativo sobre la actividad metabólica celular correlacionada con la concentración de iones liberados (Al³+ y Ca²+) para ambos ionómeros, así como las variaciones de pH de los medios de cultivo. Ello podria explicar la influencia por los iones y el pH del cultivo celular en la actividad metabólica celular.

Analysis of toxin and multilocus sequence typing of Clostridium difficile strains isolated from China-Japan Friendship Hospital / 中华微生物学和免疫学杂志

Objective To analyze the characteristics of toxin, the PCR-ribotyping(RT) and the multilocus sequence typing(MLST) of Clostridium difficile strains isolated from China-Japan Friendship Hospital in order to provide a basis for monitoring the outbreak of nosocomial Clostridium difficile infection.Methods A total of 321 samples were collected from the patients with suspected Clostridium difficile infection(CDI) in China-Japan Friendship Hospital(CJFH) during 2012 to 2013.All Clostridium difficile strains were isolated and identified by the standard phenotypic culture method.Cytotoxicity test was performed to detect toxin B.Toxin genes (tcdA and tcdB) and binary toxin genes (cdtA and cdtB) harbored by those strains were analyzed.RT and MLST were used for homologous analysis.Clinical data of the patients were collected to analyze the isolation rate of Clostridium difficile in different populations.Results Forty-eight strains of Clostridium difficile were isolated from 46 patients with diarrhea and three of them were isolated from the same patient.The incidence of CDI among all patients, outpatients and inpatients were 14.3%(46/321), 12.8%(5/39) and 14.5%(41/282), respectively.Toxin B was detected in all of the strains as indicated by the cytotoxicity test.Strains of sequence type 1(ST1) showed the strongest cytotoxicity of all the isolated Clostridium difficile strains.Ten out of the 48 strains (20.8%) were tcdA(-)/tcdB(+) strains, which belonged to either ST37 or ST81.The results of RT and MLST were consistent in assigning the strains into nine types, in which the predominant type was ST1/RT027 accounting for 27.1% (13/48).All of the ST1/RT027 strains presented a toxin gene profile of tcdA(+)/tcdB(+) and cdtA(+)/cdtB(+).Most of the ST1/RT027 strains were isolated from the Traditional Chinese Medicine Department of Respiratory, where smallnosocomial outbreaks of ST1/RT027 strain infection might happen.Conclusion CDI diagnosed in CJFH mainly belongs to nosocomial infection.Most of the isolated strains harbor tcdA(+)/tcdB(+) genes.Surveillance for the outbreaks of CDI caused by ST1/RT027 strains over producing toxins A and B should be strengthened in hospitals.

Research on the Cytotoxicity of the Heterogeneous Acellular Corneal Stromal Leaching Solution on the CHL Cells / 现代检验医学杂志

Objective To select the largest non-toxic leaching solution concentration through the experimental observation of the cytotoxicity of the ostrich acellular corneal stromal leaching solution to the Chinese hamster lung fibroblasts cells(CHL) for the further chromosome distortion experiment.Methods The leaching solution made from the ostrich acellular corneal stromal material was diluted with concentrate of 1 ∶ 2,1 ∶ 4 and the original concentration were used to culture with the CHL cells,the negative and positive control were also set up at the same time,to evaluate the impact on cell growth after 24 hour by MTT colorimetric method.Results The leaching solution diluted with 1∶4 was non-toxic,and could promote the growth of the cells.Conclusion Combined with the results of classification and cell morphological features,this cytotoxicity test can be used to screen the best benchmark non-toxic concentrations for the chromosome aberration test of the CHL cells.

Biocompatibility study of acellular bladder matrix patch / 重庆医学

Objective To evaluate the biocompatibility of acellular bladder matrix patch as the pelvic floor repair alternative ma-terial .Methods The combination of in vivo and in vitro biological experiments including cytotoxicity ,haemolysis and vaginal im-plant tests were employed to evaluate the biocompatibility of this material .Results In the cytotoxicity test ,the material toxicity was the grade 0-1 .The hemolysis rate of the material extract liquid was 1 .5% .The general and histological observation showed that the material did not cause the host immunologic rejection and led to slight foreign body reaction after implanting ,which was ba-sically degraded in 12 weeks after transplantation .Conclusion Acellular bladder matrix exhibits better biocompatibility ,but needs to adjust its degradation rate in order to adapt to the requirements of pelvic floor repair operation .

Production and cytotoxicity studies of lovastatin from aspergillus niger PN2 an endophytic fungi isolated from Taxus baccata.

Aspergillus niger PN2 an endophytic fungus, was isolated from the healthy tissues of Taxus baccata. The fungus was screened for the production of lovastatin on a solid state fermentation with wheat bran as a substrate. The fungal species were identified by their characteristic cultural morphology and molecular analysis. The presence of lovastatin was confirmed by spectroscopic method, nuclear magnetic resonance (NMR), thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods of analysis. The lovastatin production was quantified by ultraviolet (UV) analysis. The maximum amount of lovastatin production was recorded as 1.5 mg/g substrate. The extracted fungal lovastatin demonstrate a strong cytotoxic activity in in vitro culture of tested human cancer cells (HeLa and HepG2) by apoptotic assay. These results designate that the fungus, A. niger PN2 is an excellent candidate for lovastatin production and can serve as a potential organism for genetic engineering to enhance the production of lovastatin to a higher level.

Alterations in NK Cell Cytotoxicity Induced by Peripheral Nerve Injury in Mice / 대한마취과학회지

BACKGROUND: Chronic pain is often associated with changes in the immune responses, which highlights the need for the aggressive pain control to obtain a better prognosis. This study examined splenic NK cell cytotoxicity in an attempt to assess the possible changes in the immune function under chronic neuropathic pain after a partial transsection of the sciatic nerve. METHODS: After confirming tactile allodynia in response to the von Frey filament, a modified lactate dehydrogenase (LDH) release assay was used to determine the cytotoxic activity of splenic NK cells on the YAC-1 cell line in C3H/HeN (H-2k) mice (n = 6). NK cells as effector cells were mixed with YAC-1 cells as target cells (1 x 10(4)/100microliter), resulting in an effector-target ratio of 1 : 25, 1 : 50, 1 : 100 in the culture medium. RESULTS: At 1 and 2 weeks after the nerve injury, all the subjects showed significant mechanical sensitivity compared with those observed before surgery. The percentage of NK cell cytotoxicity of the neuropathic mice increased significantly 1 week after the nerve injury but decreased within 2 weeks compared with the normal mice. CONCLUSIONS: In terms of the altered NK cell cytotoxicity, neuropathic pain can cause changes in the normal performance of the immune function.

Change of the Invasiveness with Selective Cox-2 Inhibition in an Oral Squamous Cell Carcinoma Cell Line, KB; Preliminary in Vitro Study

Cyclooxygenase-2 (Cox-2) is known as one of the critical factor in carcinomas of various organs. However, the importance of Cox-2 in oral squamous cell carcinoma has not been fully described yet. The purpose of this study is to evaluate the anti-cancer effect of selective cox-2 inhibitor, celecoxib in an oral squamous cell carcinoma cell line, KB with respect to cytotoxicity test, in vitro invasion and MMP-2 expression. In cytotoxicity test, celecoxib treated group showed definitely concentration dependent cytotoxicity. In addition, administration of celecoxib reduced the invasive potential of KB cell line significantly in invasion assay. However, there was no remarkable difference of the MMP-2 expression between the celecoxib treated group and the control group. Considering these data, celecoxib had a potential cytotoxic agent to oral squamous cell carcinoma cells. Also, it had anti-invasive property without acting on the MMP-2 expression mechanism. Therefore, it was postulated that celecoxib had the possibility of anti-cancer agent in treatment strategies of oral squamous cell carcinoma.

Surface characteristics and biocompatibility of bioinert nitrides ion plated titanium implant / 대한치주과학회지

Even though titanium(Ti) and its alloys are the most used dental implant materials, there are some problems that Ti wears easily and interferes normal osteogenesis due to the metal ions. Ti coated with bioactive ceramics such as hydroxyapatite has also such problems as the exfoliation or resorption of the coated layer. Recent studies on implant materials have been proceeding to improve physical properties of the implant substrate and bio Compatibility of the implant surfaces. The purpose of the present study was to examine the physical property and bone tissue compatibility of bioinert nitrides ion plated Ti. Button type specimens(l4mm in diameter, 2.32mm in height) for the abrasion test and cytotoxicity test and thread type implants(3.75mm in diameter, 6mm in length) for the animal experiments were made from Ti(grade 2) and 316LVM stainless steel. Ti specimens were ion plated with TiN, ZrN by the low temperature arc vapor deposition, and the depth profile of the TiN/Ti, ZrN/Ti ion plated surface was examined by Auger Electron Spectroscopy. Three kind of button type specimens of TiN/Ti, ZrN/Ti and Ti were used for abrasion test, and HEPAlClC7 cells and CCD cells were cultivated for 4 days with the specimens for cytotoxicity test. Thread type implants of TiN, ZrN/Ti, Ti, 316LVM were implanted on the femur of 6 adult dogs weighing l0kg-l3kg. Two dogs were sacrified for histological examination after 45 days and 90 days, and four dogs were sacrificed for the removal torque test of the implants after 90 days. The removal torque force was measured by Autograph (Shimadzu Co., AGS-l000D series, Japan). Abrasion resistance of TiN/Ti was the highest, and that of ZrN/Ti and Ti were Followed. The bioinert nitride ion plated Ti had much better abrasion resistance, compared with Ti. In the cytotoxicity test, the number of both cells were increased in all specimens, and there were no significant difference in cytotoxic reaction among all groups (p>0.1). In histological examination, 316LVM showed the soft tissue engagement in interface between the implant and bone, but the other materials after 45 day5 noted immature new bone formation in the medullary portion along the implant surface, and those after 90 days showed implant support by new bone formation in both the cortical and the medullary portion. The removal torque force of TiN/Ti showed significantly higher than that of Ti(p0.05), and that of 316LVM was lowest among all groups(p<0.05). These results suggest that bioinert nitrides ion plated Ti can resolve the existing problems of Ti and bioactive ceramics, and it may be clinically applicable to human.

The study of biocompatibility of polyurethane sponges made in China / 医疗卫生装备

Objective To evaluate the biocompatibility of four kinds of polyurethane sponges made in China. Methods According to ASTM Standards for Medical Devices of America, polyurethane sponges are used to perform such biological tests as cytotoxicity test, acute toxicity test, pyrogenic reactions test, stimulation test of conjunctiva and cornea, sensitization test. The data are analyzed and evaluated according to the criterion. Results Reaction scales of these polyurethane sponges in cytotoxicity are 0 or 1 level. No toxicity effects and pyrogenic reactions are observed in vivo test. No conjunctiva and cornea irritation reactions and no sensitization reactions are found. Conclusions The four kinds of polyurethane sponges have high biocompatibility and can become ideal dressings of Vacuum-Assisted Closure.

Study of non-linearity relation of MTS cytotoxicity assay / 中国药理学通报

AIM We found in experiment that the relation of cell number-OD in MTS cytotoxicity assay is non-linearity. In this paper we probe into the relations of cell number-OD and drug dose-inhibition rate. METHODS Cells HL-60 and Raji were used in this experiment. Assay on the relation of cell number-OD: Cells of different concentrations were seeded into 96 well plates. OD was read at 490 nm after incubating with MTS for 1～5 hour. The regression curves were estimated from the data with the software SPSS 11.0. Cytotoxicity assay: Cells were seeded into 96 well plate and incubated with drug of different doses for 72 hours. OD was read at 490 nm after incubating with MTS for 3 hour. The regression curves were estimated from the data with the software SPSS 11.0. RESULTS The cubic curves fit well the relations of cell number-OD of the 2 strains of cells at 5 time points and the coefficient R2 of these cubic curves is 0.997～1.000, greater than the R2 0.938～0.993 of linear model. For the relations of dose-inhibition rate, the cubic curves also have best fitness and their R2 is 0.998～1.000 greater than the R2 0.948～0.987 of linear model of logarithm dose-probit. CONCLUSION For the relations of cell number-OD in MTS cytotoxicity assay, the fitness of cubic curves are better than the linear model. A more accurate IC 50 would be obtained by the cubic curve equation than by probit regression that is in common use.