Provas Banerjee

Quantitative Analysis of Histopathological Features of Precancerous Lesion and Condition Using Image Processing Technique

This paper aims at quantitative analysis of histopathological features of precancerous lesion and condition using image processing technique. The algorithm involves median and low pass filtering, segmentation by adaptive region growing, optimal and local thresholding, morphological operations such as opening and closing of gray scale and binary images and some numerical methods. Differentiation on the basis of type and level of precancerous type or condition is carried out based on image marker, defined as a vector of cancer related features viz. length and curvature of radius of rete-ridges and papillae, population density of cells within epithelium, etc. Implementation of presented algorithms is done in MATLAB. The results support quantitative analysis of pathological condition in respect with progression towards malignancy. This analysis may help in developing automated analysis tool

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.

Ex vivo bio-compatibility of honey-alginate fibrous matrix for HaCaT and 3T3 with prime molecular expressions

Honey’s inherent compositional diversity, bio-compatibility and time tested therapeutic efficacy, especially in tissue repair as a topical agent, attract researchers towards harnessing its biomaterial potential particularly in developing matrix for tissue engineering applications. Hence, this study fabricates fibrous mat from optimum honey-alginate formulation and alginate solution using wet spinning technology. The physical and morphological properties of the scaffolds are assessed and finally their comparative biological performances are evaluated through in vitro studies on adherence, viability and prime molecular expression of HaCaT and 3T3 cells. The honey-alginate scaffold demonstrates better performance than that of alginate in terms of cellular adherence, viability and proper expression of cell–cell adhesion molecule (E-cadherin) and prime molecules of extra cellular matrix (Collagen I and III) by HaCaT and 3T3 respectively.

Honey dilution impact on in vitro wound healing: Normoxic and hypoxic condition

Honey is known as a popular healing agent against tropical infections and wounds. However, the effects of honey dilutions on keratinocyte (HaCaT) wound healing under hypoxic condition is still not explored. In this study, we examined whether honey dilution have wound healing potential under hypoxic stress. The antioxidant potential and healing efficacy of honey dilution on in vitro wound of human epidermal keratinocyte (HaCaT cells) under hypoxia (3% O2), and normoxia is explored by nitro blue tetrazolium assay. The cell survival % quantified by MTT assay to select four honey dilutions like 10, 1, 0.1, and 0.01 v/v% and the changes in cellular function was observed microscopically. Further, the cell proliferation, migration, cell–cell adhesion, and relevant gene expression were studied by flow cytometry, migration/scratch assay, immunocytochemistry, and reverse transcription‐polymerase chain reaction, respectively. The expression pattern of cardinal molecular features viz. E‐cadherin, cytoskeletal protein F‐actin, p63, and hypoxia marker Hif 1α were examined. Honey dilution in 0.1% v/v combat wound healing limitations in vitro under normoxia and hypoxia (3%). Its wound healing potential was quantified by immunocytochemistry and real‐time PCR for the associated molecular features that were responsible for cell proliferation and migration. Our data showed that honey dilution can be effective in hypoxic wound healing. Additionally, it reduced superoxide generation and supplied favorable bioambience for cell proliferation, migration, and differentiation during hypoxic wound healing. These findings may reveal the importance of honey as an alternative and cost effective therapeutic natural product for wound healing in hypoxic condition.

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.

In situ histology of mice skin through transfer learning of tissue energy interaction in optical coherence tomography

Abstract. Tissue characterization method in optical coherence tomography (OCT) for in situ histology of soft tissues is presented and demonstrated for mice skin. OCT allows direct noninvasive visualization of subsurface anatomy. It is currently used for in situ investigation of lesions in skin, vessels, retinal layers, oral, and bronchial cavitities. Although OCT images present high resolution information about tissue morphology, reporting requires a reader experienced in interpretation of the images, viz., identification of anatomical layers and structures constituting the organ based on OCT speckle appearance. Our approach characterizes tissues through transfer learning of tissue energy interaction statistical physics models of ballistic and near-ballistic photons. The clinical information yield with our approach is comparable to conventional invasive histology. On cross evaluation with a mice model experiment, the epidermis, papillary dermis, dermis, and adipose tissue constituting the mice skin are identified with an accuracy of 99%, 95%, 99%, and 98%, respectively. This high accuracy of characterizing heterogeneous tissues using OCT justifies the ability of our computational approach to perform in situ histology and can be extended to regular clinical practice for diagnosis of vascular, retinal, or oral pathologies.

Correlating optical biopsy with histopathology of wounds under topical intervention with honey

Non-invasive visualization of wound bed with depth information is important to understand the tissue repair under therapeutic interventions. In this context, present study performs skin swept source-optical coherence tomography (i.e. optical biopsy/OCT) and correlates findings with histo-pathological observations on different types of traumatic non-healing wounds under honey dressing. Correlation between OCT and microscopic observations were explored especially in respect to progressive growth and maturation of the epithelial and sub-epithelial components. Characteristic transition of uniform hypo-lucid band for de-pigmented zone to wavy highly lucid band for the pigmented zone in the OCT images could be directly correlated with the microscopic findings on transformation of flattened pre-mature epithelium of de-pigmented area to thicker and matured epithelium with rete-pegs in pigmented zone. This correlated evaluation of multimodal images demonstrates the applicability of SS-OCT in wound research and importance of integrated approach in validation of newer technology.

Honey Extracted Polyphenolics Reduce Experimental Hypoxia in Human Keratinocytes Culture

Hypoxic assault affects fundamental cellular processes and generates oxidative stress on healthy cells/molecules. Honey extracted polyphenolics (HEP) as a natural antioxidant reduced hypoxic cytotoxicity in this study. Different honey samples were physicochemically characterized to identify preferred (jamun) honey [pH 3.55 ± 0.04, conductivity (μs/cm) = 6.66 ± 0.14, water content % (w/w) = 14.70 ± 0.35, total solid content % (w/w) = 85.30 ± 0.35, phenol content (mg GAE/100 g) = 403.55 ± 0.35, flavonoid content (mg QE/100 g) = 276.76 ± 4.10, radical scavenging activity (% 500 μL) = 147.75 ± 3.13, catalase activity (absorbance at 620 nm) = 0.226 ± 0.01]. HEP was tested in different doses on hypoxic and normoxic cells (HaCaT) using viability and antioxidant assays. Cardinal molecular expressions such as cadherin-catenin-cytoskeleton complex (namely, E-cadherin, β-catenin, and F-actin), hypoxia marker (Hif 1 α), proliferation marker (Ki67), and epithelial master regulator (p63) were studied by immuno-cytochemisty (ICC) and qRT-PCR. The 0.063 mg/mL HEP demonstrated better vitality and functionality of HaCaT cells as per viability assay (*, P < 0.01) even under hypoxia. ICC and qRT-PCR observations indicated restoration of cellular survival and homeostasis under 0.063 mg/mL HEP after hypoxic assault. Furthermore, major spectral changes for nucleic acid and membrane phospholipid reorganizations by Fourier transform infrared spectroscopy illustrated a positive impact of 0.063 mg/mL HEP on hypoxic cells considering proliferation and cellular integrity. It was concluded that a specific dose of jamun HEP reduces hypoxic cytotoxicity.

Honey based fibrous scaffold for tissue engineering application

Natural product like honey is well known for its compositional diversity, medicinal properties including anti-inflammatory role, wound repair efficacy and nutritional competence. This multi-dimensional character of honey attracts regenerative medicine researchers. However, flow behavior of honey restricts its applications as a sustainable biomaterial matrix. In this juncture this study develops honey-biomaterial formulations and fabricates fibrous scaffold through wet spinning technique towards regenerative medicine applications. The cell culture study demonstrates the bio-compatibility of the fabricated constructs for fibroblasts (3T3) and keratinocyte (HaCaT) through MTT assay and by scanning electron microscopy regarding adhesion and penetration of cells on the scaffold. Better performance has been exhibited by the honey-biomaterial based scaffolds in comparison to alginate fibrous construct. This study also shows better expression of prime molecules like collagen I and III in 3T3 and p63 and E-cadherin in HaCaT on honey based matrix than that of alginate matrix or control group. Thus present work develops a honey-based fibrous matrix with biocompatible interface for plausible tissue engineering applications.