Raunak Kumar Das

Amelioration of excision wounds by topical application of green synthesized, formulated silver and gold nanoparticles in albino Wistar rats.

Wound healing, a complex biological process, has attained a lot of attention as dermatologists are primarily interested in stimulated wound closure without formation of scar or a faint scar. The recent upsurgence of nanotechnology has provided novel therapeutic materials in the form of silver and gold nanoparticles which accelerate the wound healing process. The effect of formulated nanoparticles using Coleus forskohlii root extract (green synthesized) has been tried out for ameliorating full thickness excision wounds in albino Wistar male rats. The evaluation of in vivo activity of nanoparticles in wound healing was carried out on open wounds made by excision on the dorsal sides of albino Wistar rats under anesthesia, and the healing of the wounds was assessed. Histological aspects of the healing process were studied by a HE (Hematoxylin and Eosin) staining method to assess various degrees of re-epithelialization and the linear alignment of the granulation tissue whereas Van Giesons histochemical staining was performed to observe collagen fibers. The healing action shown by the formulated nanoparticles was remarkable during the early stages of wound healing, which resulted in the substantial reduction of the whole healing period. Topical application of formulated gold nanoparticles was found to be more effective in suppressing inflammation and stimulating re-epithelialization compared to silver nanoparticles during the healing process. The results throw light on the amelioration of excision wounds using nanoparticles which could be a novel therapeutic way of improving wound healing in clinical practice. The mechanism of advanced healing action of both types of nanoparticles could be due to their antimicrobial, antioxidant and anti-inflammatory properties.

Assessment of malignant potential of oral submucous fibrosis through evaluation of p63, E-cadherin and CD105 expression

Background The assessment of malignant potential of oral submucous fibrosis grades vis-à-vis their progression towards malignancy is associated with expression of possible multiple molecular markers. Aims To analyse p63, E-cadherin and CD105 expression in this premalignant pathosis with a view to unravel and understand the expression of these molecules as markers. Methods The oral mucosal biopsies (normal, oral submucous fibrosis with and without dysplasia) were studied with routine H&E, and by immunohistochemistry for p63, E-cadherin and CD105 expression. p63 was assessed as percentage of positive nuclei. E-cadherin expression was estimated through (i) distance between basement membrane and E-cadherin expression initiation point, (ii) ratio between epithelial thickness and epithelial thickness displaying E-cadherin, and (iii) E-cadherin intensity variation along the expression path. CD105 expression was assessed qualitatively. Results The p63+ cells were highest in severely dysplastic tissues followed by other dysplastic grades, normal oral mucosa and non-dysplastic conditions. However, the p63+ cells displayed the feature of progressive maturation only in normal mucosa. There was a loss of membranous E-cadherin in basal layers of all diseased conditions; it was highest in severe dysplasia. There was significant variation (p<0.0001) in E-cadherin intensity within and between the tissues (normal and diseased). CD105 expression increased abruptly in dysplasia. Conclusions The malignant potential of this pre-cancerous condition is likely to be correlated with an increase in p63 and CD105 expression and a concomitant loss of membranous E-cadherin. This may lead to marker identification through greater validation.

Epithelio-mesenchymal transitional attributes in oral sub-mucous fibrosis.

Evaluating molecular attributes in association with its epithelial and sub-epithelial changes of oral sub-mucous fibrosis is meaningful in exploring the plausibility of an epithelio-mesenchymal transition (EMT) and malignant potentiality of this pathosis. In this study histopathological and histochemical attributes for basement membrane and connective tissue in biopsies of oral sub-mucous fibrosis (n = 55) and normal oral mucosa (n = 16) were assessed and expressions of p63, E-cadherin, β-catenin, N-cadherin and TWIST were analyzed immunohistochemically. The p63 and its isoforms (TA and ∆N), PARD3, E-cadherin and β-catenin were also assessed transcriptomically by q-PCR and EMT players like TWIST1, ZEB1, MMP9 and micro-RNA 205 were searched in gene expression microarrays. Oral epithelium demonstrating impairment in progressive maturation in oral sub-mucous fibrosis concomitantly experienced an increase in basement membrane thickness and collagen deposition along with alteration in target molecular expressions. In comparison to non-dysplastic conditions dysplastic stages exhibited significant increase in p63 and p63∆N expressions whereas, E-cadherin and β-catenin exhibited loss from the membrane with concurrent increase in cytoplasm. Further the N-cadherin and TWIST were gained remarkably along with the appearance of nuclear accumulation features of β-catenin. The microarray search had noticed the up-regulation of TWIST1, ZEB1 and MMP9 along with down regulation of micro-RNA 205. The simultaneous increase in basement membrane thickness and sub-epithelial collagen deposition were the plausible indicators for increased matrix stiffness with expected impact on oral epithelial functional homoeostasis. This was corroborated with the increase in expressions of epithelial master regulator p63 and its oncogenic isoform (∆N) along with membranous loss of E-cadherin (EMT hallmark) and its associate β-catein and gain of mesenchymal markers like N-cadherin and TWIST. These also became indicative for the induction of epithelial to mesenchymal transitional mechanism in oral sub-mucous fibrosis when connoted here with the relevant modulation in expressions of EMT regulators.

Assessment of molecular events during in vitro re-epithelialization under honey-alginate matrix ambience.

Re-epithelialization is one of the most important stages of cutaneous regeneration and its success requires supportive micro-ambience which may be provided with suitable bio-matrix. Biocompatibility and efficacy of such bio-matrix in re-epithelialization could be explored by multimodal analysis of structural and functional attributes of in vitro wound healing model including evaluation of prime molecular expressions of the epithelial cells during repair. Present study examines the influence of honey-alginate and alginate matrices on re-epithelialization in keratinocyte (HaCaT) population in a 2-D wound model. Cellular viability, proliferation and cell-cell adhesion status were assessed during wound closure using live/dead cell assay and by evaluating expressions of Ki67, p63 and E-cadherin along-with % change in cellular electrical impedance. Efficacy of honey-alginate matrix in comparison to only alginate one was demonstrated by a quicker reduction in wound gap, improved cellular viability, enhanced expressions of Ki67, p63 and its isoforms (TAp63, ΔNp63) as well as E-cadherin. Faster restoration of electrical attribute (% of impedance change) after wounding also indicated better impact of honey-alginate matrix in re-epithelialization.

Swept-source optical coherence tomography of lower limb wound healing with histopathological correlation

Direct noninvasive visualization of wound bed with depth information is important to understand the tissue repair. We correlate skin swept-source-optical coherence tomography (OCT) with histopathological and immunohistochemical evaluation on traumatic lower limb wounds under honey dressing to compare and assess the tissue repair features acquired noninvasively and invasively. Analysis of optical biopsy identifies an uppermost brighter band for stratum corneum with region specific thickness (p < 0.0001) and gray-level intensity (p < 0.0001) variation. Below the stratum corneum, variation in optical intensities is remarkable in different regions of the wound bed. Correlation between OCT and microscopic observations are explored especially in respect to progressive growth and maturation of the epithelial and subepithelial components. Characteristic transition of uniform hypolucid band in OCT image for depigmented zone to wavy highly lucid band in the pigmented zone could be directly correlated with the microscopic findings. The transformation of prematured epithelium of depigmented area, with low expression of E-cadherin, to matured epithelium with higher E-cadherin expression in pigmented zone, implicated plausible change in their optical properties as depicted in OCT. This correlated evaluation of multimodal images demonstrates applicability of swept-source-OCT in wound research and importance of integrated approach in validation of new technology.

Computer-aided molecular pathology interpretation in exploring prospective markers for oral submucous fibrosis progression

Evaluation of molecular pathology markers using a computer‐aided quantitative assessment framework would help to assess the altered states of cellular proliferation, hypoxia, and neoangiogenesis in oral submucous fibrosis and could improve diagnostic interpretation in gauging its malignant potentiality.

Computational analysis of p63+ nuclei distribution pattern by graph theoretic approach in an oral pre-cancer (sub-mucous fibrosis)

Background: Oral submucous fibrosis (OSF) is a pre-cancerous condition with features of chronic, inflammatory and progressive sub-epithelial fibrotic disorder of the buccal mucosa. In this study, malignant potentiality of OSF has been assessed by quantification of immunohistochemical expression of epithelial prime regulator-p63 molecule in correlation to its malignant (oral squamous cell carcinoma [OSCC] and normal counterpart [normal oral mucosa [NOM]). Attributes of spatial extent and distribution of p63 + expression in the epithelium have been investigated. Further, a correlated assessment of histopathological attributes inferred from H&E staining and their mathematical counterparts (molecular pathology of p63) have been proposed. The suggested analytical framework envisaged standardization of the immunohistochemistry evaluation procedure for the molecular marker, using computer-aided image analysis, toward enhancing its prognostic value. Subjects and Methods: In histopathologically confirmed OSF, OSCC and NOM tissue sections, p63 + nuclei were localized and segmented by identifying regional maxima in plateau-like intensity spatial profiles of nuclei. The clustered nuclei were localized and segmented by identifying concave points in the morphometry and by marker-controlled watersheds. Voronoi tessellations were constructed around nuclei centroids and mean values of spatial-relation metrics such as tessellation area, tessellation perimeter, roundness factor and disorder of the area were extracted. Morphology and extent of expression are characterized by area, diameter, perimeter, compactness, eccentricity and density, fraction of p63 + expression and expression distance of p63 + nuclei. Results: Correlative framework between histopathological features characterizing malignant potentiality and their quantitative p63 counterparts was developed. Statistical analyses of mathematical trends were evaluated between different biologically relevant combinations: (i) NOM to oral submucous fibrosis without dysplasia (OSFWT) (ii) NOM to oral submucous fibrosis with dysplasia (OSFWD) (iii) OSFWT-OSFWD (iv) OSFWD-OSCC. Significant histopathogical correlates and their corroborative mathematical features, inferred from p63 staining, were also investigated into. Conclusion: Quantitative assessment and correlative analysis identified mathematical features related to hyperplasia, cellular stratification, differentiation and maturation, shape and size, nuclear crowding and nucleocytoplasmic ratio. It is envisaged that this approach for analyzing the p63 expression and its distribution pattern may help to establish it as a quantitative bio-marker to predict the malignant potentiality and progression. The proposed work would be a value addition to the gold standard by incorporating an observer-independent framework for the associated molecular pathology.

Immunohistochemical Evaluation of p63, E-Cadherin, Collagen I and III Expression in Lower Limb Wound Healing under Honey

Honey is recognized traditionally for its medicinal properties and also appreciated as a topical healing agent for infected and noninfected wounds. This study evaluates impact of honey-based occlusive dressing on nonhealing (nonresponding to conventional antibiotics) traumatic lower limb wounds (n = 34) through clinicopathological and immunohistochemical (e.g., expression of p63, E-cadherin, and Collagen I and III) evaluations to enrich the scientific validation. Clinical findings noted the nonadherence of honey dressing with remarkable chemical debridement and healing progression within 11–15 days of postintervention. Histopathologically, in comparison to preintervention biopsies, the postintervention tissues of wound peripheries demonstrated gradual normalization of epithelial and connective tissue features with significant changes in p63+ epithelial cell population, reappearance of membranous E-cadherin (P < .0001), and optimum deposition of collagen I and III (P < .0001). Thus, the present study for the first time reports the impact of honey on vital protein expressions in epithelial and connective tissues during repair of nonhealing lower limb wounds.

Correlated analysis of semi-quantitative immunohistochemical features of E-cadherin, VEGF and CD105 in assessing malignant potentiality of oral submucous fibrosis.

Oral submucous fibrosis, a potentially premalignant condition for oral squamous cell carcinoma, manifests both non-dysplastic and dysplastic grades. Early and specific identification of its malignant potentiality suffers from diagnostic limitations that may be addressed by correlated molecular pathology attributes having histopathological backdrop. Present study correlates expressional alteration in prime epithelial marker E-cadherin, with neo-angiogenic molecules viz. VEGF and CD105 for elucidation of malignant potentiality in different stages of oral submucous fibrosis. Sixty-eight incision biopsies from normal oral mucosa (n = 10), non-dysplastic (n = 18) and different dysplastic grades (n = 40) of oral submucous fibrosis were semi-quantitatively analyzed for immunohistochemical expressions of E-cadherin (membranous and cytoplasmic), VEGF and CD105 which were further statistically correlated. The loss of membranous E-cadherin with increase in cytoplasmic accumulation in differentiative layers of epithelium through the progression of dysplasia was noted along with up-regulation in VEGF expressions. The number of CD105(+) blood vessels and their major axis also showed significant increase from non-dysplasia toward higher grades of dysplasia. The positive correlation between deregulated expression of epithelial cell-cell adhesion molecule and increase in neo-angiogenic attributes of oral submucous fibrosis with increase in dysplastic grades indicated elucidatory potential of molecular expression features in assessment of malignant potentiality in oral submucous fibrosis.

Finding an optimum immuno-histochemical feature set to distinguish benign phyllodes from fibroadenoma.

PURPOSE Benign phyllodes and fibroadenoma are two well-known breast tumors with remarkable diagnostic ambiguity. The present study is aimed at determining an optimum set of immuno-histochemical features to distinguish them by analyzing important observations on expressions of important genes in fibro-glandular tissue. METHODS Immuno-histochemically, the expressions of p63 and α-SMA in myoepithelial cells and collagen I, III and CD105 in stroma of tumors and their normal counterpart were studied. Semi-quantified features were analyzed primarily by ANOVA and ranked through F-scores for understanding relative importance of group of features in discriminating three classes followed by reduction in F-score arranged feature space dimension and application of inter-class Bhattacharyya distances to distinguish tumors with an optimum set of features. RESULTS Among thirteen studied features except one all differed significantly in three study classes. F-Ranking of features revealed highest discriminative potential of collagen III (initial region). F-Score arranged feature space dimension and application of Bhattacharyya distance gave rise to a feature set of lower dimension which can discriminate benign phyllodes and fibroadenoma effectively. CONCLUSIONS The work definitely separated normal breast, fibroadenoma and benign phyllodes, through an optimal set of immuno-histochemical features which are not only useful to address diagnostic ambiguity of the tumors but also to spell about malignant potentiality.