Interfacing of dextran coated ferrite nanomaterials with cellular system and delayed hypersensitivity on Guinea pigs.

The study focused on the interfacing of dextran coated ferrite nanomaterials (DFNM) with the cellular system and delayed hypersensitivity on Guinea pigs. In vitro study investigated the cytotoxic potential of DFNM on L929 cells, effect on antioxidant enzymes and Lipid peroxides (LPO) production on rat brain homogenates. DFNM was also repeatedly exposed topically to Guinea pigs for the evidence of skin sensitization and toxicity at the molecular level. Biochemical and hematological parameters were estimated. Liver and brain of Guinea pigs were homogenized and evaluated for the induction of LPO, glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD) and 8-hydroxyl-2-deoxyguanosine (8-OHdG). The results of the study demonstrated that there was no significant alternation in the level of antioxidant defense enzymes, LPO, hematological, biochemical or oxidative stress related DNA damage. Hence, it can be concluded that the synthesized DFNM was non-skin irritant or non-toxic at the molecular level under the laboratory conditions.

Analysis of IL-1 ß release from cryopreserved pooled lymphocytes in response to lipopolysaccharide and lipoteichoic acid.

Pyrogens are heterogeneous group of fever-inducing substances derived from Gram-positive and Gram-negative bacteria, fungi, and viruses. They incite immune response by producing endogenous pyrogens such as prostaglandins and other proinflammatory cytokines like IL-1ß, IL-6, and TNF-α. The present study was to analyze the influence of cryopreservation in IL-1ß release, a marker for inflammatory response from human lymphocytes, in response to exogenous pyrogenic stimulants. Lymphocytes isolated from pooled blood of multiple healthy individuals were cryopreserved in DMSO and glycerol for periods of 7, 14, 30, and 60 days and were challenged with LPS and LTA in vitro. The inflammatory cytokine, IL-1ß release, was measured by ELISA method. It was observed that the release of IL-1ß increases instantaneously after the initiation of incubation and reaches a maximum at 3 to 5 hours and then gradually decreases and gets stabilized for both pyrogens. Moreover it was also observed that the effect of cryoprotectants, DMSO (10%) and glycerol (10%), showed almost similar results for short-term storage, but DMSO-preserved lymphocytes yielded a better viability for long-term storage. Thus, the isolated cryopreserved lymphocytes system can be a promising approach for the total replacement/alteration to animal experimentation for pyrogenicity evaluation.

Cells-nano interactions and molecular toxicity after delayed hypersensitivity, in guinea pigs on exposure to hydroxyapatite nanoparticles.

The aim of the study was to evaluate the cells-nanoparticle interactions and molecular toxicity after delayed hypersensitivity in Guinea pigs, exposed to hydroxyapatite nanoparticles (HANP). The study focuses on synthesizing and characterizing HANPs and gaining an insight into the cytotoxicity, molecular toxicity, hypersensitivity and oxidative stress caused by them in vitro and in vivo. HANP was synthesized by chemical method and characterized by standard methods. Cytotoxicity was assessed on L929 cells by MTT assay and in vitro studies were carried out on rat liver homogenate. In vivo study was carried out by topical exposure of Guinea pigs with HANP, repeatedly, and evaluating the skin sensitization potential, blood parameters, oxidative stress in liver and brain and DNA damage (8-hydroxyl-2-deoxyguanosine: 8-OHdG) in liver. The results of the study indicated that there was no cytotoxicity (up to 600µg/mL) and oxidative damage (up to 100µg/mL), when exposed to HANPs. It was also evident that, there was no skin sensitization and oxidative damage when HANP were exposed to Guinea pigs.

Effect of fibrin glue on antioxidant defense mechanism, oxidative DNA damage and chromosomal aberrations.

Oxidative stress is involved in diverse biological phenomenon, and is caused by the imbalance between reactive oxygen species (ROS) and antioxidant defense system. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is the most critical biomarker in the estimation of ROS-induced DNA damage. This investigation focuses on the effect of fibrin glue on lipid peroxidation (LPO), antioxidant enzymes and oxidative DNA damage (both in vitro and in vivo). The blood biochemical parameters of the implanted animals and in vitro chromosomal aberrations were also studied. Fibrin glue was applied on the calvarial defect made on the anesthetized rats for an observation period of 4, 12, 26 and 52 weeks. At the end of the observation period, animals were anesthetized; blood was collected for serum analysis and was sacrificed. Brain was collected for the detection of 8-OHdG using competitive ELISA and liver was collected for analyzing the antioxidant enzymes and LPO. The results of this study suggest that the effect of fibrin glue on rat brain (in vivo and in vitro) and mice liver (in vitro) did not make any significant influence on LPO and antioxidant defense system. Similarly, there was no change observed in the expression of 8-OHdG. Serum constituents of implanted rats were observed to be within the normal range. The normal karyotype obtained indicates that the physiological saline extract of fibrin glue does not induce any chromosomal anomalies. Hence, it was concluded that the fibrin glue material does not have any potential to produce oxidative stress, alterations in the C-8 position of guanine and chromosomal anomalies.

Analysis of mitochondrial DNA damage using 8-hydroxy 2'deoxyguanosine on in vitro and in vivo exposure of biomaterials.

The present study was carried out to evaluate the effect of HAP-EVA, fibrin glue, HA-BG, Latex and Dental material on oxidative stress related mtDNA damage by in vitro and in vivo methods. In vivo studies of these biomaterials were carried out by implanting biomaterials (five materials) on animals for period of 1, 4, 12, 26 and 52 weeks. At the end of observations, animals were anesthetized, sacrificed and tissues surrounding the implanted materials were collected. Brain, bone and muscles were used for the extraction of mtDNA. Similarly mtDNA was extracted from the homogenate of fresh brain, bone and muscles on exposure to the physiological saline extract of all the above five biomaterials (In vitro). The extracted mtDNA were subjected to analyse the presence of 8-OHdG. The results of study indicated that there was no significant increase in the level of 8-OHdG and thereby does not influence on the GC-TA transversions.

Assessment of oxidative stress and chromosomal aberration inducing potential of three medical grade silicone polymer materials.

Medical expenditures for devices are increasing along with the ageing of human population and the synthesis of materials such as silicone polymers is on upsurge for manufacturing these devices. The International Organization for Standardization (ISO) emphasizes a battery of tests for preclinical assessment of biocompatibility of medical devices. Genotoxicity assays have become an integral component of these test procedures and it employs a set of in vitro and in vivo experiments to detect mutagens. Hence, this study was performed with an intention to investigate the genotoxic potential of the physiological saline extracts of three medical grade silicone polymer materials by the in vitro chromosomal aberration assay using human peripheral blood lymphocytes. Further, the oxidative stress inducing potential of the material extracts was investigated in vivo in mice liver homogenates using cyclophosphamide as positive control. The investigation revealed that none of the three materials were able to produce marked human lymphocyte chromosomal aberration, suggesting the absence of mutagens. The materials also showed negative results in their oxidative stress inducing potential, which was revealed by the normal levels of lipid peroxidation and unaltered levels of glutathione and its metabolizing enzymes in the mice liver tissue homogenates. It was interesting to observe a significant correlation between the genotoxic and antioxidant parameters investigated. Hence, it is suggested that the estimation of antioxidant status would serve as a better preliminary testing procedure prior to evaluating the genetic and molecular toxicity mechanisms of medical devices and/or materials intended for manufacture of such devices.

Amelioration of melatonin on oxidative stress and genotoxic effects induced by cisplatin in vitro.

In this study, we made an effort to evaluate the possible protective actions of melatonin on cisplatin-induced oxidative damage in mice brain homogenate and genotoxic effects in human lymphocytes under in vitro conditions. The tissue homogenate was divided into three parts. The first portion was kept as control treated with dimethyl sulphoxide (DMSO) (group 1) while the second and third portion were treated with 24 µg/g tissue cisplatin alone (group 2) and 24 µg/g tissue cisplatin in combination with 3 mM melatonin (group 3), respectively. We measured the oxidative stress biomarkers such as lipid peroxidation, 8-hydroxy 2' deoxyguanosine (8-OHdG) and antioxidant parameters such as reduced glutathione, superoxide dismutase, glutathione peroxidase, and glutathione reductase in brain homogenate. Likewise peripheral venous blood was collected from healthy donors and human lymphocyte culture was done using karyotyping medium. Cultures were divided into three groups. Group 1 was the control i.e. lymphocytes treated with DMSO 5 µg/mL. In group 2, lymphocytes were treated with 2 µg/mL cisplatin and group 3 with a combination of 2 µg/mL cisplatin and 0.3 mM melatonin. Incubation of tissue homogenates with cisplatin elevated the malondialdehyde and 8-OHdG levels which were then reversed by melatonin. Reduction in antioxidant parameters with respect to corresponding controls were also restored by melatonin treatment. Furthermore, supplementation of melatonin was found to modulate the chromosome damage elicited by cisplatin which was determined using Giemsa (GTG) banding and karyotyping. These findings suggest that melatonin improves the cellular function and helps them to survive in the belligerent environment created by free radicals.

Pre-clinical evaluation of titanium nitride coated titanium material.

The titanium nitride coated titanium is a material intended for the fabrication of left ventricular assist device. As per International standards, a material is subjected to pre-clinical evaluation before fabrication of a device. Toxicity/biocompatibility studies such as acute systemic toxicity, intracutaneous irritation, in vitro haemolysis and implantation in muscle were studied as per international standards for the titanium nitride coated titanium. Acute systemic toxicity was studied in mice using physiological saline and cotton seed oil extracts of the material. Intracutaneous irritation was done by injecting the extracts of the test material and control intradermally into rabbits. Grading of erythema and oedema of test and control animals were recorded at 24, 48 and 72 h. In vitro haemolysis was carried out with material and extract of the material with rabbit blood. The muscle implantation was carried out in nine anesthetized rabbits. The implanted animals were sacrificed at the end of 1, 4 and 12 weeks, the tissue with the implanted materials were collected and subjected to histopathological analysis. The results of the study did not show any significant irritation or systemic toxicity with physiological saline and cotton seed oil extracts of the material. The percentage of hemolysis induced by the material and extract was under acceptable range. Results of the histopathological evaluation suggest that the test material did not produce any cellular changes. Hence the present study concluded that the test material is non-toxic, non-irritant, non-haemolytic and biocompatible.

Detection of pyrogenicity on medical grade polymer materials using rabbit pyrogen, LAL and ELISA method.

The objective of the study is to detect the pyrogenicity of five medical grade gelatinous polymer materials, intended for the manufacturing of capsule for pharmaceutical applications, by an indigenously developed ELISA, LAL and rabbit pyrogen assays. The ELISA methodology includes the incubation of the sample extract with blood from a healthy donor at 37°C. Any pyrogen present in the extract induces the IL-1ß which can be determined by ELISA. The rabbit pyrogen and LAL assays were performed as per standards. The result of the ELISA method indicated that all the materials extract induced high level of IL-1ß as a marker for pyrogenicity. The rise in temperature of rabbit pyrogen was above 0.5°C in all materials extract. LAL assay induced an endotoxin level above 0.5EU. All the five polymer materials were found pyrogenic in all the assays. The ELISA method is very sensitive because the lowest limit of detection was 10pg/ml endotoxin. Hence it can be concluded that the ELISA method will be an added advantage for the quality control release of a batch of medical products and improving the existing methodologies in the context of reduction and replacement in the use of animal models.