A Histopathology-based Assessment of Biological Behavior in Oral Hyalinizing Extraosseous Lesions by Differential Stains.

AIM: The assessment of hyalinization to determine aggressive behavior in oral pathological lesions is a scarcely researched field that requires further exploration. The current study aims to predict the biological behavior of oral hyalinizing extraosseous lesions (OHEOL) by employing four differential stains with clinicopathologic correlation. MATERIALS AND METHODS: The study was performed on retrospectively diagnosed formalin-fixed paraffin-embedded cases of salivary gland tumors (SGTs) (n = 13), benign soft tissue (BST) lesions (n = 24), and oral submucous fibrosis (OSMF) (n = 53). The hematoxylin and eosin-stained sections were analyzed for the severity of hyalinization (SOH). Differential stains periodic acid Schiff (PAS), Alcian blue, safranin O, and picrosirius red with polarizing microscopy were used to assess the components of hyalinized tissue. The SOH was correlated with differential staining characteristics and clinicopathologic features to analyze possible correlation with aggressive potential in BST, advancement of disease in OSMF, and recurrence in SGT. RESULTS: Intensity of picrosirius red stain significantly correlated with SOH of SGTs (p = 0.044). The intensity of PAS stain (p = 0.040), picrosirius red polarizing greenish-yellow color (p = 0.002), and pattern of distribution of picrosirius red (p = 0.023) significantly correlated with recurrence of SGTs. The intensity of differential stains increased with the SOH in BST lesions indicating their correlation with SOH. The intensity (p = 0.008) and pattern (p = 0.010) of Alcian blue staining and intensity of safranin O stain (p = 0.003) significantly correlated with SOH in OSMF. Picrosirius red polarizing color reddish and yellowish red (p = 0.002) significantly correlated with SOH distinguishing early and advanced OSMF. CONCLUSION: Picrosirius red and PAS stains are reliable indicators of SOH and recurrence potential in SGT. Alcian blue, safranin O, and picrosirius red polarizing colors enable detection of SOH and accurately distinguish early from advanced OSMF. CLINICAL SIGNIFICANCE: SOH can be considered as a histological predictor of aggressive biologic behavior in OHEOL. These findings will result in appropriate management protocols.

A Histopathology-based Assessment of Biological Behavior in Oral Hyalinizing Odontogenic Tumors and Bone Lesions by Differential Stains.

AIM: Odontogenic tumors (OTs) and bone lesions of the oral cavity present diverse histological features and varying clinical behavior that makes predicting their biologic behavior difficult. The research undertaken in the current study aims to predict the biological behavior of oral hyalinizing odontogenic and bone lesions (OHO-BL) for the first time by employing four differential stains with clinicopathologic correlation. MATERIALS AND METHODS: The study was performed on retrospectively diagnosed formalin-fixed paraffin-embedded cases of OTs (n = 53) and bone lesions (n = 10). The severity of hyalinization (SOH) was assessed from stained tissue sections. Polarizing microscopy was used to analyze hyalinization in tissues stained with differential special stains, namely periodic acid-Schiff (PAS), Safranin-O, Alcian Blue, and Picrosirius red. SOH was also analyzed for possible correlation with recurrence and clinicopathologic correlation in OHO-BL. RESULTS: Intense staining was observed with PAS, Alcian Blue, and Safranin-O in OTs with increased SOH with a statistical significance. Polarizing greenish yellow color correlated significantly with the recurrence potential of the OT group. Recurrence in individual lesions of the OT group showed a statistically significant association with SOH. Such individual correlation was not observed in bone diseases. CONCLUSION: PAS, Alcian Blue, Safranin-O, and Picrosirius red are reliable stains to assess hyalinization in OHO-BL. Picrosirius red-polarizing microscopy is a dependable tool for identifying recurrent odontogenic lesions. CLINICAL SIGNIFICANCE: SOH can be considered a histological predictor of aggressive biologic behavior in oral hyalinizing odontogenic lesions that can enable the surgeon to arrive at an appropriate management protocol.

Rapid Nanogram Scale Screening Method of Microarrays to Evaluate Drug-Polymer Blends Using High-Throughput Printing Technology.

A miniaturized, high-throughput assay was optimized to screen polymer-drug solid dispersions using a 2-D Inkjet printer. By simply printing nanoliter amounts of polymer and drug solutions onto an inert surface, drug/polymer microdots of tunable composition were produced in an easily addressable microarray format. The amount of material printed for each dried spot ranged from 25 ng to 650 ng. These arrays were used to assess the stability of drug/polymer dispersions with respect to recrystallization, using polarized light microscopy. One array with a panel of 6 drugs formulated at different ratios with a poly(vinylpyrrolidone-vinyl acetate) (PVPVA) copolymer was developed to estimate a possible bulk (gram-scale) approximation threshold from the final printed nanoamount of formulation. Another array was printed at a fixed final amount of material to establish a literature comparison of one drug formulated with different commercial polymers for validation. This new approach may offer significant efficiency in pharmaceutical formulation screening, with each experiment in the nanomicro-array format requiring from 3 up to 6 orders of magnitude lower amounts of sample than conventional screening methods.

A Comparative Evaluation of Collagen in Ameloblastoma and Oral Squamous Cell Carcinoma using Picrosirius Red Staining with Polarizing Microscopy and CD44v6 Immunoreactivity.

Background: Solid multicystic ameloblastoma (SMA) is a locally aggressive, benign odontogenic tumor of odontogenic origin with greater rate of recurrence. Epithelial-mesenchymal interaction plays an important role in tooth morphogenesis that shows complete differentiation of epithelial and ectomesenchymal components to the level of tooth formation. Tumor stroma in ameloblastoma is normal mature collagen that prevents differentiation to the level of tooth formation. Current study evaluates the role of stromal elements in aggressive behavior of SMA using picrosirius red staining with polarizing microscopy and CD44v6 immunohistochemistry (IHC). Objectives: To compare nature of collagen using picrosirius red staining under polarized microscope and IHC expression of CD44v6 marker in SMA and oral squamous cell carcinoma (OSCC). Methods: Thirty blocks were retrieved from departmental archives and subjected to picrosirius red staining and CD44v6 IHC staining. Slides stained with picrosirius red were observed under polarized microscope to report the birefringence pattern. IHC slides were annotated for intensity of staining of tumor cells. Results: In contrast to OSCC's 40% red, 40% yellowish-red, and 20% greenish-yellow birefringence, SMA displayed 87% red, 13% yellowish-red, and 0% greenish-yellow. Compared to OSCC, which had tumor cells stained 9% strongly, 64% moderately, 27% mildly, and 0% negatively, SMA revealed 0% strong, 10% moderate, 60% weak, and 30% negative staining. Conclusion: As opposed to OSCC, which exhibited a greater quantity of greenish-yellow birefringence of immature collagen, SMA showed predominantly red birefringence, which is suggestive of mature collagen with a lack of metastasis. Comparing SMA to OSCC, the lack of significant CD44v6 positivity suggests that there has not been perineural invasion or regional metastases in SMA.

Detection of amyloid in abdominal fat pad aspirates in early amyloidosis: Role of electron microscopy and Congo red stained cell block sections.

BACKGROUND: Fine-needle aspiration biopsy (FNA) of the abdominal fat pad is a minimally invasive procedure to demonstrate tissue deposits of amyloid. However, protocols to evaluate amyloid in fat pad aspirates are not standardized, especially for detecting scant amyloid in early disease. MATERIALS AND METHODS: We studied abdominal fat pad aspirates from 33 randomly selected patients in whom subsequent tissue biopsy, autopsy, and/or medical history for confirmation of amyloidosis (AL) were also available. All these cases were suspected to have early AL, but had negative results on abdominal fat pad aspirates evaluated by polarizing microscopy of Congo Red stained sections (CRPM). The results with CRPM between four reviewers were compared in 12 cases for studying inter observer reproducibility. 24 cases were also evaluated by ultrastructural study with electron microscopy (EM). RESULTS: Nine of thirty-three (27%) cases reported negative by polarizing microscopy had amyloidosis. Reanalysis of 12 mixed positive-negative cases, showed considerable inter-observer variability with frequent lack of agreement between four observers by CRPM alone (Cohen's Kappa index of 0.1, 95% CI -0.1 to 0.36). EM showed amyloid in the walls of small blood vessels in fibroadipose tissue in four out of nine cases (44%) with amyloidosis. CONCLUSION: In addition to poor inter-observer reproducibility, CRPM alone in cases with scant amyloid led to frequent false negative results (9 out of 9, 100%). For improved detection of AL, routine ultrastructural evaluation with EM of fat pad aspirates by evaluating at least 15 small blood vessels in the aspirated fibroadipose tissue is recommended. Given the high false negative rate for CRPM alone in early disease, routine reflex evaluation with EM is highly recommended to avert the invasive option of biopsying various organs in cases with high clinical suspicion for AL.

Synthesis and Properties of Thermotropic Copolyesters Based on Poly(ethylene terephthalate) and 4'-Acetoxy-4-biphenyl-carboxylic Acid.

A series of novel copolyesters based on polyethylene terephthalate (PET) and 4'-hydroxy-biphenyl-4-carboxylic acid (HBCA) was obtained by melt polycondensation of bis(2-hydroxyethyl) terephthalate and 4'-acetoxybiphenyl-4-carboxylic acid (ABCA) as co-monomers with Sb2O3 as a catalyst. Using this synthetic procedure, a set of copolymers containing 20-80 mol% of HBCA units was prepared. According to NMR spectroscopy, the copolymers were of random composition. Copolyesters comprising 60-80 mol% of HBCA possessed increased heat resistance and formed nematic melts at 270 °C and higher. The liquid crystal (LC) phase formation was accompanied by transition to non-Newtonian characteristics of the melt flow, as well as an equalization of storage and loss moduli values. According to XRD and polarizing microscopy, the LC glassy phase of the copolyesters coexists with crystalline regions of poly-(4'-hydroxy-4-biphenylcarboxylate), non-melting up to 400 °C and above. The mechanical characteristics of these LC copolyesters showed similar or better values than those of well-known LC polymers. These novel copolyesters can be useful in obtaining heat-resistant materials with an ordered structure and, as a consequence, improved performance.

A quantitative and qualitative comparative analysis of collagen fibers to determine the role of connective tissue stroma in oral squamous cell carcinoma using special stains and polarized microscopy.

BACKGROUND: Solid tumors such as oral squamous cell carcinoma (OSCC) are composed of malignant epithelial cells and the stroma in which these cells are dispersed. As the tumor progresses, the extracellular matrix undergoes dramatic morphological and architectural changes. Special stains make analysis easy and less erroneous by highlighting the area of interest and can be used to study these changes. AIM: The aim of the study was to analyze morphological changes in collagen fibers in various histological grades of OSCC using Masson's trichrome (MT) and Picrosirius red (PSR). STUDY DESIGN: The study comprised 74 tissue samples, divided into two groups: Group I consisted of 63 cases of histologically proven OSCC (39 cases of well-differentiated squamous cell carcinoma [WDSCC], 17 moderately differentiated squamous cell carcinoma [MDSCC] and 7 poorly differentiated squamous cell carcinoma [PDSCC]) and Group II consisted of 11 cases of normal mucosa as controls. MATERIALS AND METHODS: Sections were stained with hematoxylin and eosin, MT and PSR and observed under light and polarizing microscope, respectively. STATISTICAL ANALYSIS: ANOVA, Tukey's honestly significant difference post hoc multiple comparison test, Chi-square test and paired t-test were used for the statistical analysis. RESULTS: As the grade of OSCC progressed, collagen fibers became thin, loosely packed and haphazard. The mean area fraction also decreased. They exhibited orange-red hue and strong birefringence in WDSCC, yellowish-orange hue and strong birefringence in MDSCC and greenish-yellow hue and weak birefringence in PDSCC. CONCLUSION: Initially, there is a reorganization of the collagen fibers in an attempt to prevent the invasion of tumor cells, but as cancer progresses, the stromal change enhances movement of the tumor cells within it, leading to metastasis.

Updates in processing of anterior fat pad aspirate for amyloid (with video and sketches).

Fat pad aspiration is a commonly used method for detecting amyloid in tissue. Amyloid is detected in the small blood vessels of the aspirated adipose tissue. Optimum evaluation of amyloid with electron microscopy requires at least 15 blood vessels in the fat pad aspirate. The presence of a significant proportion of adipocytes in the aspirate dilutes the fibrovascular portion. This may compromise the evaluation for amyloid with electron microscopy and in FFPE with proteomic studies by mass spectroscopy for confirmation of the amyloid subtype. This video article describes the updated protocol for processing the anterior fat pad aspirate. It demonstrates how to remove the interference of blood and fatty component in the fat pad aspirate performed by the previously reported procedure.

Analysis of collagen fibers in keratocystic odontogenic tumor and ameloblastoma: A polarizing microscopic study.

INTRODUCTION: Collagen forms an integral part of connective tissue and maintains its structural integrity. It has natural birefringence which is attributed to the arrangement of its fibers and is enhanced by special stains such as picrosirius red through polarizing microscopy. The polarization colors differ according to the fiber thickness and pattern of arrangement which in turn related to aggressiveness. Hence, the present study was conducted to evaluate collagen fibers in keratocystic odontogenic tumor (KCOT) and ameloblastoma using polarizing microscopy. AIM: This study aims to compare and correlate different types and patterns of collagen fibers in KCOT and ameloblastoma using picrosirius red stain under polarizing microscopy to delineate their aggressiveness. MATERIALS AND METHODS: The color, thickness, and orientation of collagen fibers in the KCOTs (n = 15) and ameloblastomas (n = 15) were studied histochemically by staining the sections with picrosirius red and examined under polarizing microscope using image analyzer software. RESULTS: When collagen fiber bundles in KCOT and ameloblastoma were compared, significant difference was noted between yellowish-orange collagen fiber bundles, but no significant difference was observed between greenish-yellow and orange-red collagen bundles. With respect to orientation and organization, the results are statistically significant (P < 0.05). CONCLUSION: The connective tissue stroma of KCOT could be regarded not just as a structural support but as a functional part of the lesion. In KCOT, the thin, parallel, and loosely arranged greenish-yellow collagen fibers may be attributed to its high recurrence rate and biological aggressiveness.

Natural enamel caries in quinoline: Volumetric data and the pattern of infiltration.

This study aimed to test the hypotheses that (i) a parameter related to permeability, αd (ratio of squared water volume by the nonmineral volume) is, among all major component volumes (mineral, water, and organic volumes) the best predictor of quinoline infiltration in natural enamel caries (NEC), and (ii) the pore volume fraction infiltrated by quinoline (Vqui ) in NEC is much lower than previous estimates that neglected water and organic enamel volumes. Mineral and nonmineral volumes and αd were measured at 341 histological points (from 20 approximal NEC lesions), and transport of quinoline was tracked by orientation-independent polarizing microscopy. R2 values of Vqui were 0.596 (αd ), 0.033 (mineral volume), 0.474 (water volume), and 0.011 (organic volume). Vqui values were 23% (body of the lesion), 7% (dark zone), and 9% (translucent zone), lower than previous estimates (with high effect size). Transport of quinoline occurred both parallelly and perpendicularly to prism paths, and dark zones were seen where only transport parallel to prisms occurred. In conclusion, αd was the main predictor of quinoline infiltration, but it differed from the water volume with a small effect size, and the pore volume fraction with quinoline was much lower than previous estimates.

Evaluation of Collagen in Leukoplakia, Oral Submucous Fibrosis and Oral Squamous Cell Carcinomas Using Polarizing Microscopy and Immunohistochemistry

Objectives: Picrosirius red and MMP are capable of degrading extracellular matrix proteins, expressed in lesions such as squamous cell carcinomas. The present study was undertaken with an aim to analyze and compare changes in collagen using Picrosirius red staining under polarizing microscopy and immunohistochemical staining using anti MMP-13 in samples of oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. Materials and Methods: A total of 70 slides were prepared and divided into 3 groups. Group I comprised 10 slides of normal gingival tissue, Group II 40 slides of potentially malignant disorders and Group III 20 slides of well differentiated oral squamous cell carcinoma. Half the slides for each group were stained with Picrosirius red stain and the remainder with antibodies to MMP-13. Rerults: In Group II, MMP-13 connective tissue expression was greater in OSMF as compared to leukoplakia. Group III showed elevated expression among 70% of cases. Picrosirius red staining in Group II cases, showed higher staining Yellow-Orange andGreen-Yellow mature fibers in OSMF than leukoplakia cases while in Group III, 50% OSCC cases showed Green-yellow stained immature thin fibers. Conclusion: In future, therapeutic measures targeted against MMP-13 may inhibit collagenolysis to some extent and delay spread of tumors. An easy and reliable method to determine the state of the stroma in such cases may be Picrosirius red staining with polarizing microscopy.

Evaluation of the Nature of Collagen Fibers in KCOT, Dentigerous Cyst and Ameloblastoma using Picrosirius Red Stain - A Comparative Study.

BACKGROUND: Reciprocal interaction between dental epithelium and mesenchyme is thought to be crucial for normal odontogenesis. Thus, the mesenchymal influence of the fibrous capsules may play an important role in the maintenance of epithelial expression. Collagen is the major component of the extracellular matrix and possibly there is an alteration in the nature and structure of collagen in various pathological conditions. Studies by polarizing microscopy have also shown that there is a difference in collagen and probably these differences may play a role in their biologic behaviour. AIM: The purpose of this study was to evaluate the nature of collagen fibers in keratocystic odontogenic tumour (KCOT), dentigerous cyst (DC), unicystic ameloblastoma (UA) and solid/multicystic ameloblastoma (SMA) and correlating this with their biological behaviour. MATERIALS AND METHODS: Five diagnosed cases each of UA, SMA, KCOT and DC were taken and stained using Picrosirius red stain kit and evaluated using a polarizing microscope. STATISTICAL ANALYSIS: Chi-square test was used to analyse the results. RESULTS AND CONCLUSION: Collagen fibers in dentigerous cysts showed predominant yellowish-red birefringence and fibers in KCOT and ameloblastomas showed a predominantly greenish-yellow birefringence. Hence, our study suggests that the nature and character of collagen fibers may influence the clinical behaviour of the lesion. Since ours is a pilot study, to corroborate our view, studies with larger sample size are required to substantiate the results.

Collagen in histologic stages of oral submucous fibrosis: A polarizing microscopic study.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous condition in which there is excessive deposition of collagen in connective tissue. The purpose of present study was to compare changes in birefringence of collagen fibers in histopathological stages of OSF. MATERIALS AND METHODS: Collagen in connective tissue of 91 cases of OSF was studied by staining 5 µm thick sections with picrosirius red. The polarization colors of thin (0.8 µm or less) and thick (1.6-2 µm) collagen fibers were recorded. RESULTS: The birefringence of thin collagen fibers showed no difference in both histopathological connective tissue stages and degrees of epithelial dysplasia. The polarization colors of thick collagen fibers showed a gradual change from predominantly yellow-orange to greenish-yellow in advancing connective tissue stages and degrees of epithelial dysplasia. CONCLUSION: The results of present study show a significant change in birefringence of collagen between connective tissue stages and between mild, moderate to severe degree of epithelial dysplasia. This change in birefringence colors and arrangement of collagen fibers may give an implication of impending neoplastic change in OSF.

The effect of lactic acid concentration and ph of lactic acid buffer solutions on enamel remineralization / 대한치과보존학회지

There are considerable in vitro and in vivo evidences for remineralization and demineralization occurring simultaneously in incipient enamel caries. In order to "heal"the incipient dental caries, many experiments have been carried out to determine the optimal conditions for remineralization. It was shown that remineralization is affected by different pH, lactic acid concentrations, chemical composition of the enamel, fluoride concentrations, etc. Eighty specimens from sound permanent teeth without demineralization or cracks, 0.15 mm in thickness, were immersed in lactic acid buffered demineralization solutions for 3 days. Dental caries with a surface zone and subsurface lesion were artificially produced. Groups of 10 specimens were immersed for 10 or 12 days in lactic acid buffered remineralization solutions consisting of pH 4.3 or pH 6.0, and 100, 50, 25, or 10 mM lactic acid. After demineralization and remineralization, images were taken by polarizing microscopy (x100) and micro-computed tomography. The results were obtained by observing images of the specimens and the density of the caries lesions was determined. 1. As the lactic acid concentration of the remineralization solutions with pH 4.3 was higher, the surface zone of the carious enamel increased and an isotropic zone of the subsurface lesion was found. However, the total decalcification depth increased at the same time. 2. In the remineralization solutions with pH 6.0, only the surface zone increased slightly but there was no significant change in the total decalcification depth and subsurface zone. In the lactic acid buffer solutions with the lower pH and higher lactic acid concentration, there were dynamic changes at the deep area of the dental carious lesion.