Toluidine Blue with a Synergistic Effect in Morphological Assessment of Oral Cytosmears.

OBJECTIVES: One percent toluidine blue being the most effective adjunct is often used to detect dysplasia. Not much attention has been given to the effect of toluidine blue in enhancement of cytological smears. The present study assessed the smears before and after application of toluidine blue in smokers against non-smokers using three different stains [Papanicolaou (PAP), Hematoxylin and Eosin (H and E), and Giemsa]. STUDY DESIGN: Twenty-five individuals from each group participated in the study. The oral cytosmears were obtained before and after application of toluidine blue and assessed for clumping of squamous cells, cellular and nuclear pleomorphism, micronuclei, binucleation, bacterial colony units, and keratin flakes. RESULTS: In smokers, the maximum enhancement in cytological smears post-toluidine blue application was shown by Giemsa stain than PAP and H and E stains. Among the individual parameters, nuclear pleomorphism exhibited greatest significant difference between smokers and non-smokers. CONCLUSION: Toluidine blue enhanced the staining characteristics both in terms of sensitivity and specificity and thereby was found to be synergistic in assessment of cytosmears. The cellular alterations noticed in the smears of smokers with clinically normal buccal mucosa can be used as a means of education tool in counselling for smoking cessation.

Glutaraldehyde treatment elicits toxic response compared to decellularization in bovine pericardium.

Glutaraldehyde-stabilized bovine pericardium is used for clinical application since 1970s because of its desirable features such as less immunogenicity and acceptable durability. However, a propensity for calcification is reported on account of glutaraldehyde treatment. In this study, commercially available glutaraldehyde cross-linked bovine pericardium was evaluated for its in vitro cytotoxic effect, macrophage activation, and in vivo toxic response in comparison to decellularized bovine pericardium. Glutaraldehyde-treated bovine pericardium and its extract were observed to be cytotoxic and it also caused significant inflammatory cytokine release from activated macrophages. Significant antibody response, calcification response, necrotic, and inflammatory response were noticed in glutaraldehyde-treated bovine pericardium in comparison to decellularized bovine pericardium in a rat subcutaneous implantation model. Glutaraldehyde-treated bovine pericardium also failed in acute systemic toxicity testing and intracutaneous irritation testing as per ISO 10993. With respect to healing and implant remodeling, total lack of host tissue incorporation and angiogenesis was noticed in glutaraldehyde-treated bovine pericardium compared to excellent host fibroblast incorporation and angiogenesis within the implant in decellularized bovine pericardium. In conclusion, using in vitro and in vivo techniques, this study has demonstrated that glutaraldehyde-treated bovine pericardium elicits toxic response compared to decellularized bovine pericardium which is not congenial for long-term implant performance.

Short duration gluteraldehyde cross linking of decellularized bovine pericardium improves biological response.

Gluteraldehyde stabilized bovine pericardium is used for clinical application since 1970s because of its desirable features such as less immunogenicity and acceptable durability. However, a propensity for calcification and long term implant failure is reported because of gluteraldehyde treatment. There is also failure of implant to integrate into host tissue because of its resistance to tissue remodeling. Decellularized bovine pericardium, a potential alternative allows tissue remodeling but it has problems such as immunogenicity and chronic inflammatory response. In this study, decellularized bovine pericardium was subjected to short duration, low concentration gluteraldehyde cross-linking at two levels and its biological response (both in vitro and in vivo) was compared with un-crosslinked decellularized bovine pericardium and fully crosslinked normal bovine pericardium. It was observed that both un-crosslinked and partially crosslinked decellularized bovine pericardium to be non-cytotoxic and it caused significantly less inflammatory cytokine release such as TNF alpha and IL1beta from activated macrophages. Among all groups, short duration 0.2% Gluteraldehyde treated decellularized bovine pericardium showed significantly less antibody response and inflammatory response compared to un-crosslinked decellularized pericardium, short duration 0.6% gluteraldehyde treated decellularized bovine pericardium or completely cross linked bovine pericardium in juvenile rat subcutaneous implantation model. Moreover, short duration 0.2% gluteraldehyde crosslinked decellularized bovine pericardium showed minimum calcification, better host fibroblast incorporation, new collagen deposition and angiogenesis within the implant. These attributes may finally lead to better implant remodeling and sustained implant function during clinical use.

Surface reactivity of calcium phosphate based ceramics in a cell culture system.

Surface reactivity of Calcium Phosphate materials--Hydroxyapatite (HA), Tricalcium Phosphate (beta-TCP), Hydroxyapatite-Tricalcium Phosphate (HA-TCP) were elucidated in a cell culture system. MG-63 osteoblast-like cells were seeded onto the ceramic discs to evaluate changes in the cell morphology and functionality with respect to the different substrates. The dissolution and re-precipitation of calcium phosphate phases on the surface of the discs in the culture medium was found to be prominent on beta-TCP when compared with HA. Low calcium (Ca), magnesium (Mg) and alkaline phosphatase (ALP) levels and high phosphorous (P) levels in the medium of beta-TCP were observed. This indicated that P must have leached out into the medium from beta-TCP and Ca in turn deposited from the medium onto beta-TCP resulting in the apatite phase transformation. The low ALP activity in beta-TCP medium is however an indication of low osteoblastic activity. Under the phase contrast microscope, the osteoblast cells around HA material were found to be confluent and viable, while in the vicinity of beta-TCP only cellular debris was observed. In the case of HA-TCP, only a few viable cells surrounded the material amidst the debris. Scanning electron microscopy revealed numerous cells on the surface of HA showing different cell behaviour like anchorage, attachment, adhesion and spreading in the early time period as the surface was only slightly disturbed with re-crystallisation. But with time the entire surface of HA had changed due to precipitation and re-crystallization which did not support cell behaviour while the cells surrounding the material showed normal growth. On the contrary, cells were scarcely observed on the entirely changed surface of beta-TCP and HA-TCP even from the earlier days of the culture and the morphology of cells surrounding the material too started changing. These results establish that HA promoted the activity of osteoblast cells. HA surface remained unaltered for some time, while the surface of beta-TCP underwent dissolution of surface ions and resulted in the re-crystallization of apatite over the surface. The resulting changes in the surrounding milieu of beta-TCP with high phosphate and low Ca levels probably was responsible for the death of the cells.

In vitro cytocompatibility studies of Diamond Like Carbon coatings on titanium.

Diamond like carbon (DLC) films were deposited on to titanium (Ti) substrates by Plasma Enhanced Chemical Vapour Deposition (PECVD) process. The quality of the films were checked by Raman spectra and nano-hardness tests. The cytocompatibility of titanium and DLC coated titanium were studied using continuous cell lines of mouse fibroblast cells ( L-929), Human Osteoblast cells (HOS) and primary human umbilical cord vein endothelial cells (HUVEC). The cellular responses to the materials were assessed both quantitatively and qualitatively. The adhesion and spreading of cells on materials were compared using Ti as a control. Present study indicates an improved cytocompatibility of DLC coated Ti in comparison to bare Ti.

In vitro studies on the effect of physical cross-linking on the biological performance of aliphatic poly(urethane urea) for blood contact applications.

The effect of physical cross-linking in candidate cycloaliphatic and hydrophobic poly(urethane urea) (4,4'-methylenebis(cyclohexylisocyanate), H(12)MDI/hydroxy-terminated polybutadiene, HTPBD/hexamethylenediamine, HDA) and poly(ether urethane urea)s (H(12)MDI/HTPBD-PTMG/HDA) on the in vitro calcification and blood-material interaction was studied. All the candidate poly(urethane urea)s and poly(ether urethane urea)s elicit acceptable hemolytic activity, cytocompatibility, calcification, and blood compatibility in vitro. The studies on blood-material interaction reveal that the present poly(urethane urea)s are superior to polystyrene microtiter plates which were used for the studies on blood-material interaction. The present investigation reveals the influence of physical cross-link density on biological interaction differently with poly(urethane urea) and poly(ether urethane urea)s. The higher the physical cross-link density in the poly(urethane urea)s, the higher the calcification and consumption of WBC in whole blood. On the other hand, the higher the physical cross-link density in the poly(ether urethane urea)s, the lesser the calcification and consumption of WBC in whole blood. However a reverse of the above trend has been observed with the platelet consumption in the poly(urethane urea)s and poly(ether urethane urea)s.

Endomyocardial fibrosis is associated with selective deposition of type I collagen.

BACKGROUND: Endomyocardial fibrosis is a distinct form of heart disease leading to restrictive ventricular filling and cardiac failure. The disease is characterized by a marked thickening of the endocardium due to the deposition of dense fibrous tissue composed of wavy bundles of collagen. Changes in collagen composition and an abnormal increase in its concentration result in a stiffer myocardium and ventricular diastolic dysfunction. The nature of cardiac collagens and the relative proportions of collagen types in endomyocardial fibrosis have not been documented in the literature. METHODS AND RESULTS: This study analyzed collagen composition in the cardiac tissues of 13 patients with endomyocardial fibrosis and 6 individuals who were the victims of traffic accidents or suicidal deaths and did not have any heart disease. We estimated the relative proportions of types I and III collagen after pepsin digestion of the tissue and separation of the emerging peptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The mean type I:III collagen ratio was 0.51+/-0.06 in normal individuals, and 0.93+/-0.43 in patients with endomyocardial fibrosis (p<0.05). The alteration in the type I:III collagen ratio was due to a disproportionate increase in type I collagen. CONCLUSIONS: The results indicate that a selective increase in type I collagen may contribute to the impaired diastolic distension of the ventricles in patients with endomyocardial fibrosis.

Lipoprotein adsorption onto modified chitosan beads: preliminary study.

Among the currently used LDL-apheresis techniques selective removal seems to be a better option. We have made a preliminary study on adsorption of LDL onto modified and bare chitosan and PVA beads. It seems that bare chitosan itself maybe an excellent adsorbent system for LDL. Further detailed studies are required to examine the specificity and selectivity in adsorption.

Papilloma virus in cervical carcinoma: detection of viral antigen in cancer cells.

Paraffin sections of biopsies from histopathologically confirmed cases of uterine cervical carcinoma, cervical scrapings from dysplasia, chronic cervicitis, tumour cells from carcinoma of the oral cavity and normal tissues from healthy normal cervix and oral cavity scrapings were examined for the presence of Human papilloma virus antigens. The techniques adopted were the Indirect Immunofluorescence Stainig and the Peroxide-Anti-Peroxidase techniques. The HPV-antigen was present in 38 percent and 41 percent of invasive carcinoma cervix, by PAP and IIF methods respectively. In cervical dysplasia 8-13% revealed HPV antigen while oral carcinoma cells and normal tissue samples were totally negative.

Detection of herpes simplex virus type-2 DNA and human papilloma virus DNA sequences in cervical carcinoma tissue by molecular hybridization.

Biopsy samples from one hundred and two patients with squamous cell carcinoma of the uterine cervix and tissues from twelve healthy normal cervical tissues (post hysterectomy) were examined for HSV-type 2 and HPV-11 DNA sequences by molecular hybridization technique. In the carcinoma tissue extracts 53% contained HSV-2 DNA, 27% -HSV-1-DNA and 36% showed HPV-11 gene sequences while 5.7% were found to contain both HPV and HSV-2 DNA. Biopsies from healthy cervix were completely negative for HSV-2 and HPV-11 DNA sequences.

In vivo biocompatibility of an aliphatic crosslinked polyurethane in rabbit.

A cage implantation technique has been adopted in a rabbit animal model to investigate the biocompatibility of an aliphatic crosslinked polyurethane based on hexamethylene diisocyanate (HDI). In this study, four cages are represented; the cage containing the candidate polyurethane material, biomedical grade polyurethane (Tecoflex), the commercial grade polyvinylchloride (PVC), and an empty cage which were implanted subcutaneously in rabbits. Exudates were aspirated from these cages at 4, 7, 14, and 21 days postimplantation. Exudates were analyzed for variations in proteins, cell counts, and extracellular enzymes. Results with the four types of cage implants showed that the candidate polyurethane caused an inflammatory response comparable to that caused by medical-grade Tecoflex and the empty cage control.

Pulmonary vasospasm in rabbits infused with stroma free haemoglobin solution.

Effect of infusion of stroma free haemoglobin solution into mice and rabbits was studied. Though no abnormalities were noted in mice, pulmonary vasospasm and hepatocytic necrosis were seen in all rabbits. In addition to reports that low adenosine deaminase levels in the solution could be a cause of vasoconstriction, it is proposed on the basis of present results that either denaturation of haemoglobin molecule itself or release of super oxide anion during oxygenation of haemoglobin, could cause cellular damage leading to toxicity.

Demonstration of HSV-I antigen in patients with oral cancer by immunofluorescence and immunoperoxidase techniques.

Biopsies from one hundred and seventy five patients suffering from carcinoma of the oral cavity were examined for the presence of Herpes simplex virus Type 1 (HSV-1) antigen, using Immunofluorescence (Direct and indirect) and immunoperoxidase techniques. Seventy-seven percent of the biopsies showed significant fluorescence, while 71% showed positivity with immunoperoxidase staining. There was no correlation either in the degree of fluorescence or in the intensity of peroxidase staining with clinical stage of the disease. Normal oral biopsies showed 30% and 20% positivity in immunofluorescence and immunoperoxidase respectively. In the case of oral cancer cells, the fluorescence was very much diminished when HSV-1 antiserum was absorbed with specific homologous virus-infected Vero cells.

Detection of herpes simplex virus type-2 antigen(s) in biopsies from carcinoma of the uterine cervix.

Indirect immunofluorescence technique was employed to detect herpes simplex virus type-2 (HSV-2) antigens in tumor biopsies from 215 patients with carcinoma of the uterine cervix. A total of 169 samples (79%) revealed brilliant nuclear fluorescence. Inflammatory cells infiltrating the tumor mass were positive to 60 of the 215 patients (28%). Samples showed no significant variation in the degree of fluorescence or proportion of cells binding HSV antibody with advancement in the clinical stage of the disease. Fluorescence was totally abolished when incubated with HSV-2 antiserum absorbed with a specific homologous virus. Among controls, there was fluorescence in 27% of cervical scrapings from normal women and 34% (42/124) among patients with gynecological disorders other than cervical malignancy. In cervical dysplasia 23 out of 40 patients (58%) expressed herpes virus-associated antigens. There was membrane fluorescence in live malignant cell preparations in 3 of 28 patients (11%). Normal cervix tissue from hysterectomy specimens and breast cancer cells were negative for herpes simplex virus-related antigens. Pre-immune serum and PBS showed nonspecific fluorescence in 25% and 23% of sera, respectively.