Indirect Cytotoxicity Test Research Articles

PH Evolution and Cytotoxicity of [Alpha]-Tricalcium Phosphate Cement with Three Different Additives

An application of calcium phosphates is as bone cements, among which the system based on alpha-tricalcium phosphate (α-TCP) exhibits excellent properties. The aim of this study is to analyze pH evolution and cytotoxicity of α-TCP cement with three different additives. Changes on the pH were measured at intervals of 12h during seven days. But initial measurements were executed at each 15 minutes. Indirect cytotoxicity test was performed according to ISO (10993-5, 1992) employing CHO-k1 cells and RPMI 1640 as culture medium. It was used a colorimetric method which uses the tetrazolium compound. The additives used on the liquid phase were disodium hydrogen phosphate (Na2HPO4) and/or citric acid (C6H8O7) and/or tannic acid (C76H52O46). The results indicate that the cement without additives does not have requirements to be applied like bone cement, while the other cements composition exhibit different responses in the pH and the cytotoxicity test. In conclusion, due to the presence of additives it was possible to control pH evolution during setting and cytotoxic response. However, further investigation is necessary in order to determine the influence of these additives, mainly tannic acid, on the in vivo behavior of these bone cements.

Investigation of PLA-based scaffolds fabricated via SVM rapid prototyping

Poly-lactic acid (PLA) is a biodegradable polymer that has been well accepted as a tissue engineering scaffold material. Recently, PLA has been applied in selective vacuum manufacturing (SVM), a new RP technique being developed, for fabricating scaffold. For this RP technique to be accepted for this purpose, its fabricated scaffolds must be tested for their properties. This paper presents an investigation of the properties of scaffolds fabricated from SVM technique. The results illustrated that the fabricated PLA scaffolds had porous structure. The porosity was about 71.65% with pore size ranged from 20 to 90 μm. The compressive modulus of elasticity was 2.07 ± 0.25 MPa, lying within the lower range of mechanical properties reported for soft tissue application. An indirect cytotoxicity test showed the cell viability of 75.92% which means that the specimens posed no threat to the cells and could be used as scaffolds for mammalian tissue culture.

The in vitro indirect cytotoxicity test and in vivo interface bioactivity evaluation of biodegradable FHA coated Mg–Zn alloys

Abstract A kind of biodegradable fluoridated hydroxyapatite (FHA) coating was prepared on Mg–Zn alloy to improve the interface bioactivity in bone healing via electrodeposition method. The in vitro cytotoxicity evaluation of the ions released during degradation was taken. No toxicity was shown and even higher cells’ viability appeared on the 7th day compared with the normal culture case (negative control). In vivo implantation was carried out in the femoral condyle of adult New Zealand rabbits. The cross section showed by Micro-CT scan confirmed that the better interface contacts happened in the coated group after one month implantation. Also the coating left can still be normally observed by scanning electron microscope (SEM) with a little degradation. As a result, the FHA coating may be a promising candidate to enhance interface bioactivity for biodegradable Mg alloys in orthopaedics.

Degradation, cytotoxicity, and biocompatibility of NIPAAm‐based thermosensitive, injectable, and bioresorbable polymer hydrogels

A thermosensitive, injectable, and bioresorbable polymer hydrogel, poly(N-isopropylacrylamide-co-dimethyl-γ-butyrolactone acrylate-co-acrylic acid) [poly(NDBA)], was synthesized by radical copolymerization with 7.00 mol % dimethyl-γ-butyrolactone acrylate in tetrahydrofuran. The chemical composition was determined by acid titration in conjunction with (1) H NMR quantification. The molecular weight and polydispersity were determined by gel permeation chromatography in conjunction with static light scattering. The degradation properties of the polymer hydrogel were characterized using differential scanning calorimetry, percentage mass loss, cloud point test, and swelling ratio over time. It was found that the initial lower critical solution temperature (LCST) of the polymer is between room temperature and body temperature and that it takes about 2 weeks for the LCST to surpass body temperature under physiological conditions. An indirect cytotoxicity test indicated that this copolymer has relatively low cytotoxicity as seen with 3T3 fibroblast cells. The in vivo-gelation and degradation study showed good agreement with in vitro-degradation findings, and no detrimental effects to adjacent tissues were observed after the complete dissolution of the polymer. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

Electrospun Scaffolds of a Polyhydroxyalkanoate Consisting of ω-Hydroxylpentadecanoate Repeat Units: Fabrication and In Vitro Biocompatibility Studies

Electrospinning was used to fabricate fibrous scaffolds of lipase-catalyzed poly(ω-pentadecalactone) (PPDL). The slow resorbability of this biomaterial is expected to be valuable for tissue-engineering applications requiring long healing times. The effect of solvent systems and instrumental parameters on fiber morphology was investigated. PPDL electrospinning was optimized and defect-free fibers (diameter 410 ± 150 nm) were obtained by using a mixed three-solvent system. Scaffolds were characterized by scanning electron microscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXS). TGA showed no residual solvent in the scaffolds. DSC and WAXS results indicated that electrospun PPDL is semicrystalline. Biocompatibility of PPDL scaffolds was evaluated through indirect cytotoxicity tests using embryonic rat cardiac H9c2 cells. The ability of PPDL electrospun mats to support cell growth was verified by culturing H9c2 cells onto the scaffold. Cell adhesion, proliferation and morphology were evaluated. The results indicated that PPDL mats are not cytotoxic and they support proliferation of H9c2 cells. The cumulative results of this study suggest further exploration of PPDL fibrous mats as scaffolds for tissue-engineered constructs.

Manipulating Degradation Time in a N-isopropylacrylamide-Based Co-polymer with Hydrolysis-Dependent LCST

A thermosensitive, bioresorbable and in situ gelling co-polymer, poly(N-isopropylacrylamide-co-dimethyl-γ-butyrolactone acrylate-co-acrylic acid), was synthesized by radical co-polymerization with varying dimethyl-γ-butyrolactone acrylate (DBA) content. The materials properties were characterized using differential scanning calorimetry, gel-permeation chromatography in conjunction with static light scattering, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR) and acid titration. The initial lower critical solution temperature (LCST) of the synthesized co-polymer is between room temperature and body temperature. With the increase of DBA content, the LCST decreases, but then increases after the ring-opening hydrolysis of the DBA side-group. The FT-IR and NMR spectra show the co-polymerization of three monomers, as well as the hydrolysis-dependent ring-opening of the DBA side-group. The addition of acrylic acid increases the initial LCST and accelerates the degradation rate of the co-polymer. An indirect cytotoxicity test indicated that this co-polymer has relatively low cytotoxicity as seen with 3T3 fibroblast cells.

Bioactive Magnetoelastic Materials as Coatings for Implantable Biomaterials

Enhanced fibroblast activity at the soft tissue-implant interface can dramatically decrease the stability, function, and lifespan of biomedical implants such as bone anchored prostheses. Although bone anchoring systems dramatically improve prosthetic limb mechanical stability, uncontrolled fibrosis at the soft tissue-mounting post interface is a significant problem. The aberrant cell growth leads to irregular skin folds that prevent proper sealing to the bone anchoring post and also serves as a site for opportunistic infection and failure of the prosthetic system. We are developing a bioactive vibrational coating to control fibrous tissue overgrowth. The coating is based on a magnetoelastic (ME) material that can be set to vibrate at a predetermined amplitude and frequency using a controlled magnetic field. We hypothesize that small local vibrations can be used to selectively control cell adhesion and gene expression to promote and maintain functional stability at the implant-tissue interface. For bone anchored prostheses, the ME coating would be applied around the mounting post at the soft tissue interface. The specific aims of this work were to (1) modify the coating for use in contact with a biologic environment and (2) determine if local vibrational strain can efficiently control cell attachment to the coating without significantly influencing viability. First, two common biocompatible polymers, polyurethane and chitosan, were deposited as thin films on the ME coating to allow for its use in tissue culture. An indirect cytotoxicity test was used to determine fibroblast (L929) viability in media conditioned for 24 and 48 hours with uncoated, chitosan coated, and polyurethane coated ME materials. Results demonstrated that both polymer coatings returned cell survival to levels statistically indistinguishable from controls (cells cultured on tissue cultured polystyrene, TCP) with cell viability over 96% under all coating conditions. Second, the affect of local vibrations on cell adhesion was tested in vitro. A cell viability assay (Calcein-AM) followed by fluorescent imaging was used to quantify attachment and viability of fibroblasts cultured directly on the bioactive ME material. Results clearly indicated that controlled local vibrations can induce complete cell detachment from the ME material compared with non-vibrated controls at up to 72 hours post-seeding. Further, cells detached via applied vibrations showed no significant decrease in viability compared to adherent controls. These results suggest the potential for this novel coating to effectively control fibrous tissue overgrowth using the mild application of tunable local vibrations, a significant and cost-effective approach that could improve the stability, function, and lifespan of biomedical implants and reduce the need for surgical revision.

Biocompatibility of various root canal filling materials ex vivo

To evaluate the biocompatibility of a resin-based endodontic filler (RealSeal) using the indirect cytotoxicity test. Human gingival fibroblasts were cultured ex vivo. Pellets of the materials to be tested were incubated for 24, 48, and 72 h at 37 degrees C under sterile conditions to obtain their eluates. The fibroblasts were exposed to either diluted (50%) or undiluted eluates for 24 h. A culture medium with foetal calf serum was added to the control wells. Cell viability was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. The data concerning cell viability were statistically analyzed using one-way anova test and Bonferroni multiple comparisons test. Eluates obtained after 24 h of incubation with the resin filler did not reduce cellular viability. An increase in cellular viability, as compared with control cells, was observed in the gutta-percha group. The undiluted eluate from the polyether material was cytotoxic, causing an 82 +/- 4% decrease in cellular viability. Eluates obtained after 48 h of incubation with the resin filler increased cellular viability, whereas the polyether significantly reduced viability. Gutta-percha did not cause any detectable change. After 72 h of incubation the eluate of the resin filler caused an increase in cellular viability, as did gutta-percha, whereas polyether caused a significant decrease. RealSeal resin filler was nontoxic in this laboratory model. Further investigations are necessary to verify its usefulness in clinical applications.

Avaliação do possível efeito tóxico de um alcano semifluorinado de uso oftalmológico sobre cultura de células Vero

PURPOSE: Perfluorocarbon liquids (PFCLs) are used in vitreoretinal surgery. PFCLs may cause inflammatory reactions, cellular injury and destruction of the normal retinal architecture. In order to avoid these effects, semifluorinated alkanes (SFA) were developed. We assessed the potential use of an SFA known as perfluorohexiloctano (F6H8) as long-term vitreous replacement under controlled cell culture conditions. METHODS: We analyzed indirect cytotoxicity, where the cells only come into contact with soluble elements that can be eliminated by perfluorohexiloctano. We therefore analyzed direct toxicity (contact toxicity) of perfluorohexiloctano by means of scanning electronic microscopy and immunocytochemistry reagents for actin. Cells embedded in a treatment-free culture medium were used as control, a positive control for toxicity with an undeniably toxic effect on cells, and a weight control that produced a mechanical compression similar to the amount of perfluorohexiloctano used in the experiment. RESULTS: The indirect cytotoxicity test showed that F6H8 did not affect cell growth. Our direct toxicity tests showed that cellular alterations caused by perfluorohexiloctano were similar to those produced by the weight control and different from toxicity control. CONCLUSION: Perfluorohexiloctano does not present indirect toxicity and this product has a compressive rather than a toxic effect on cultured cells.

In vitro cytotoxicity of fibre-polymethyl methacrylate composite used in dentures.

The aim of this study was to determine the in vitro cytotoxicity of fibre-polymethyl methacrylate (PMMA) composite used in denture bases. Both heat-cured and autopolymerized PMMA were tested as the matrix of the fibre composite or as a plain denture polymer. The fibres tested included unsized E-glass and silica fibres, silica fibres which were sized with a silane coupling agent only, as well as commercially epoxy-silane or methacrylate-chrome complex sized E-glass fibres. The indirect contact cytotoxicity test by agar diffusion was carried out according to the international standard ISO 10993-5 after storing the test specimens in water for 24 h. PVC plastic was used as a positive control and polyethylene plastic was used as a negative control. Within the limitations of the agar diffusion test, it can be concluded that neither the unreinforced PMMA nor the fibre composite made from PMMA and fibres was cytotoxic.

A rapid method for detection of mycoplasmas in mammalian cell cultures and comparison with other routine techniques

A simple and rapid method for the detection of mycoplasmas in mammalian cell cultures has been developed on the basis of the enzymatic activity of adenosine phosphorylase. This enzyme, which has been found in high activity in mycoplasmas in contrast to mammalian cells, catalyzes the transformation of 6-methylpurine deoxyriboside (6-MPDR) into the two cytotoxic products 6-methylpurine and 6-methylpurine riboside and is the basis of an established indirect cytotoxicity test for mycoplasmas. In this study estimation of parasitic incidence relies on the direct visualization of the enzymatic conversion of 6-MPDR by isocratic ion-pair reversed-phase high-performance liquid chromatography. The final result of the test is available after 3 to 24 h of incubation of the cells together with the indicator metabolite 6-MPDR and depends on the adenosine phosphorylase activity of the respective mycoplasma species. The direct detection of enzymatic activity results in a high sensitivity, allowing the observation of infections that could not be found by the indirect cytotoxicity test. Comparison of the direct adenosine phosphorylase activity test with other commonly used methods reveals distinct cost and time advantages.

A cytotoxicity assay utilizing a fluorescent dye that determines accurate surviving fractions of cells

A cytotoxicity assay has been developed based on the measurement of the proliferative activity of surviving cells as quantified by a cell-incorporated fluorescent dye, 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The BCECF proliferative assay is fast (the results are obtained within 3–4 days depending on the cell line), accurate, not labor-intensive, does not require the use of radioisotopes or toxic compounds, and is amenable to automation. The BCECF proliferative assay was compared with two other indirect cytotoxicity tests, a tryptan blue exclusion test and a BCECF viability test. Neither of these two latter assays reflected in any way the killing of cells by ricin. In contrast, using the BCECF proliferation assay, an optimal period of cell culturing after exposure to a toxin could be found so that the cytotoxicity values produced agreed with the surviving fractions of cells measured in a direct cytotoxicity assay. Under non-optimal conditions, the assay reflected the cell kill only qualitatively. Although it is common practice to conduct indirect cytotoxicity tests without validating them with a direct assay, our experiments demonstrate that the values obtained in such non-optimized indirect cytotoxicity tests may not parallel the cell kill and may, therefore, be meaningless.

Variant embryonal carcinoma cells lacking SSEA-1 and forsmann antigens remain developmentally pluripotent

Six embryonal carcinoma (EC) cell lines that are resistant to the cytotoxic, galactose-specific lectin abrin were isolated from mutagenized populations of either PSA-1 or F9 cells. The LD 10 for each of the variant lines was at least 150-fold greater than that for parental cells. Indirect cytotoxicity tests demonstrated that all of the variant cell lines lacked both Stage Specific Embryonic Antigen-1 (SSEA-1, less than 1% of wild-type levels) and Forsmann antigen (less than 5% of wild-type levels). When abrin-resistant cells were fused to previously isolated SSEA-1-negative cells ( M. J. Rosenstraus (1983), Dev. Biol. 99, 318–323) that express Forsmann antigen, the resulting hybrids expressed SSEA-1. This implies the mutation conferring abrin resistance is in a different gene than that defined by the previously isolated mutation. Thus, we have identified two genes that are required for SSEA-1 expression, one of which also appears to be required for Forsmann antigen expression. The F9-derived variants differentiated into visceral-like or parietal-like endoderm when treated with retinoic acid in the absence or presence of 8-bromo-cAMP, respectively. PSA-1-derived variants formed differentiated teratocarcinomas containing derivatives of all three germ layers. Thus the SSEA-1 and Forsmann haptenic determinants are not required for EC cells to differentiate into a broad spectrum of cell types; nor do they appear to be involved in the cell-cell interactions that are postulated to regulate visceral versus parietal endoderm differentiation.

Isolation and characterization of an embryonal carcinoma cell line lacking SSEA-1 antigen.

Stage-specific embryonic antigen 1 (SSEA-1) is a glycoconjugate that is expressed on embryonal carcinoma cells but not on their differentiated derivatives. To determine if SSEA-1 is required for the expression of the EC cell phenotype, a variant cell line lacking this antigen was isolated by exposing mutagenized OTF9-63 (an F9 subclone) cells to anti-SSEA-1 antiserum plus complement. Indirect cytotoxicity tests demonstrated that the variant cell line exhibits at least a 500-fold reduction in SSEA-1 expression. Somatic cell hybrids constructed between variant and wild-type cells expressed SSEA-1, suggesting that the lack of SSEA-1 expression in the mutant is probably due to loss, rather than masking, of the antigenic site. The variant cell line was tumorigenic in syngeneic mice, forming teratocarcinomas composed exclusively of embryonal carcinoma cells. Monolayer cultures of the variant cell line formed parietal endodermal (laminin-positive) cells following exposure to retinoic acid whereas aggregates grown in suspension culture formed visceral endoderm. Also, like their parental cells, the variant cells sorted out to form simple embryoid bodies when mixed in suspension with parietal-like endodermal cells. Thus, the expression of a number of aspects of the embryonal carcinoma phenotype is not dependent on the presence of SSEA-1.

Isolation and characterization of peanut agglutinin-resistant embryonal carcinoma cell-surface variants.

The isolation and characterization of variant embryonal carcinoma (EC) cells possessing altered cell-surface structures is described. The lectin peanut agglutinin (PNA), which binds to EC cells but not their differentiated derivatives, was used to select the variants. Clones resistant to the cytotoxic effect of PNA were isolated at a frequency of 4 X 10(-5) following mutagenesis. The resistant phenotype was stable in the absence of selection in all eight clones tested. The increased frequency of resistant clones following mutagenesis and the stability of the phenotype suggests a mutational origin. Somatic cell hybrids constructed between wild-type cells and two different PNA-resistant cell lines were sensitive to PNA; this suggests that the resistant phenotype is recessive. Binding assays demonstrated that resistant cells exhibited a twofold to fourfold reduction in the total amount of PNA bound. Together with the recessive behavior of the phenotype, this suggests that resistant cells are deficient for PNA receptors. The PNA-resistant cells also showed reduced binding of monoclonal antibody against stage-specific embryonic antigen 1 (SSEA-1) in indirect cytotoxicity tests. All eight PNA-resistant lines isolated were tumorigenic in syngeneic mice and gave rise to well-differentiated teratocarcinomas. The PNA-resistant cells behaved like their wild-type parents in a cell recognition assay; when incubated in suspension with endodermal cells, they sorted out to form simple embryoid bodies (a core of EC cells surrounded by an endodermal rind). Thus, EC cells can form tumors, differentiate, and recognize differentiated cells in a sorting assay despite a reduction in expression of the embryo-specific cell surface structures (s) that bind PNA and anti-SSEA-1 antibody.

Presence of common surface antigens(s) on endodermal tumors and embryonal tissues of rats, hamsters and mice.

Antiserum against yolk-sac carcinoma of rat was prepared in rabbits. After appropriate absorption in vitro or in vivo this antiserum was examined on different tumors and normal tissues or rat, hamster and mouse. The methods used were indirect immunofluorescence and indirect immunoperoxidase staining and cytotoxicity tests. The immune serum was found to react with the cell membrane of different rat and hamster yolk-sac carcinomas. It reacted also with the cell surface of rat hepatoma cells. By absorption on hyalin and blocking with amniotic fluid it was shown that the antigen was neither a basement membrane component nor alpha-fetoprotein. The antiserum was cytotoxic to yolk-sac carcinoma and hepatoma cells. The immune reaction was limited to the cell membrane, as observed in immunofluorescence and in immunoperoxidase staining. The specificity of the antiserum was proved by cross-absorptions with various tumor lines and by removing its activity with the soluble fraction of yolk-sac carcinoma cells. Non-endodermal rat and hamster tumor lines did not react with the anti-yolk-sac carcinoma immune serum. Most normal adult tissues, including spermatozoa, were negative, but a positive reaction was observed in ovaries and on glandular cells of the uterus. In embryonal tissues this surface antigen(s) was detected in the endoderm of 8-day-old rat embryos 7-day-old mouse embryos and in yolk-sac endoderm of both species. The data indicate that the antigen(s) is associated with endodermal differentiation.

A tumor-specific cytotoxic and neutralizing factor in rats immunized with ascites hepatoma induced by azo dyes.

Transplantation immunity of Donryu rats against ascites hepatoma AH-64A induced by azo dye was demonstrated by intraperitoneal injection of tumor cells pretreated with heteroantibodies in vitro. Hyper-immunity was induced by successive challenges with fresh tumor cells. The cytotoxic effect of the serum of resistant rats (RRS) against AH-64A tumor cells was not reduced after absorption with normal rat liver cells, but was slightly reduced after absorption with normal rat spleen cells. The cytotoxicity was absorbed completely with 5 times 10(6) AH-64A tumor cells. AH-64A, -B, -C, and -D are ascites hepatoma cell lines originating from a single Donryu rat. AH-64A and AH-64B cross-reacted with RRS while AH-64C and AH-64D, chemically induced DBLA-6 leukemia cells and normal lymph node cells of rats, did not react with RRS in indirect immunofluorescence and cytotoxicity tests. A neutralization test was carried out by treating 2 times 10(5) tumor cell with either RRS or immune spleen cell in vitro and then injecting them subcutaneously into irradiated rats (400 R). It was found that 1:20 dilution of RRS protected the rats against AH-64A tumor cell growth while 1:40 and 1:80 dilutions of RRS caused some protection. A subcutaneous tumor mass developed after transplantation of tumor cells treated with RRS, but after about 2 weeks this began to decrease in size and disappeared completely within 6 weeks after transplantation. Treatment of AH-64A tumor cells with immune spleen cells at cell-to-cell ratios of 1:200 and 1:100 caused complete neutralization while normal spleen cells at a ratio of 1:200 had slight effect. Treat;ent with immune spleen cells prevented tumor growth from t;e start. Most of the surviving animals were resistant to c,allenge with 1 times 10(5) fresh AH-64A cells. RRS was fractionated by cellulose acetate membrane electrophoresis and the amounts of beta1- and gamma-globulin fractions were found to be 48 and 42% more than in normal rat serum. The immunoelectrophoretic pattern of resistant rat serum showed a stronger IgM precipitin line than that of normal rat serum.