Santanu Dhara

Isolation and characterization of fish scale collagen of higher thermal stability

Collagen is the most abundant protein found in animal body and widely used for biomedical and pharmaceutical applications. However, its applicability is severely limited due to high cost. Fish processing waste, which otherwise cause serious environmental pollution, is a promising cost effective collagen source. In the present study, collagen was isolated from scales of Labeo rohita (Rohu) and Catla catla (Catla). It is first time that these species are used as sources of collagen. Thermo-gravimetric analysis (TGA) revealed maximum demineralization achieved after 48h of EDTA treatment of intact scale. The isolated protein was confirmed as collagen by different physico-chemical techniques like FTIR, SDS-PAGE, and CD. Further amino acid analysis corroborates isolation of type I collagen. A major characteristic of obtained collagen was found to have denaturation temperature (T(d)) of 36.5 degrees C, which is promising as an advantage for biomedical application due to closeness in T(d) to mammalian collagen.

Dextrin cross linked with poly(HEMA): a novel hydrogel for colon specific delivery of ornidazole

We report on the synthesis and characterization of a novel hydrogel based on dextrin grafted with poly(2-hydroxyethyl methacrylate) by embedding N,N′-methylene bis acrylamide (MBA) as cross linker, into a polymeric network in the presence of potassium persulphate (KPS) initiator for colon specific delivery of ornidazole. Various grades of hydrogels [Dxt-g-p(HEMA)] have been synthesized by altering the reaction parameters and the best one optimized. The developed hydrogel has been characterized using FTIR spectra, 13C NMR spectra, elemental analysis, XRD study, SEM analysis, TGA analysis, swelling study and cell viability study. The equilibrium swelling ratio of the hydrogels has been recorded in different media and found to be at a maximum at pH 7.4. A cell viability study indicates that the hydrogel is non-cytotoxic in nature. The drug delivery results demonstrate that Dxt-g-p(HEMA) delivers ornidazole successfully in the colonic region in a controlled way and is a good candidate for an orally administered drug delivery system. The release mechanism and kinetics of ornidazole from various hydrogels have been determined using different linear and nonlinear mathematical models, which confirm that ornidazole release from hydrogel follows first order kinetics and a non-Fickian diffusion mechanism.

Collagen scaffolds derived from fresh water fish origin and their biocompatibility

Collagen, a major component of native extracellular matrix, has diverse biomedical applications. However, its application is limited due to lack of cost-effective production and risk of disease transmission from bovine sources currently utilized. This study describes fabrication and characterization of nano/micro fibrous scaffolds utilizing collagen extracted from fresh water fish origin. This is the first time collagen extracted from fresh water fish origin was studied for their biocompatibility and immunogenicity. The nano/micro fibrous collagen scaffolds were fabricated through self-assembly owing to its amphiphilic nature and were subsequently cross-linked. In vitro degradation study revealed higher stability of the cross-linked scaffolds with only ~50% reduction of mass in 30 days, while the uncross-linked one degraded completely in 4 days. Further, minimal inflammatory response was observed when collagen solution was injected in mice with or without adjuvant, without significant dilution of sera. The fish collagen scaffolds exhibited considerable cell viability and were comparable with that of bovine collagen. SEM and fluorescence microscopic analysis revealed significant proliferation rate of cells on the scaffolds and within 5 days the cells were fully confluent. These findings indicated that fish collagen scaffolds derived from fresh water origin were highly biocompatible in nature.

Chitosan–collagen scaffolds with nano/microfibrous architecture for skin tissue engineering

In this study, a hierarchical nano/microfibrous chitosan/collagen scaffold that approximates structural and functional attributes of native extracellular matrix has been developed for applicability in skin tissue engineering. Scaffolds were produced by electrospinning of chitosan followed by imbibing of collagen solution, freeze-drying, and subsequent cross-linking of two polymers. Scanning electron microscopy showed formation of layered scaffolds with nano/microfibrous architechture. Physicochemical properties of scaffolds including tensile strength, swelling behavior, and biodegradability were found satisfactory for intended application. 3T3 fibroblasts and HaCaT keratinocytes showed good in vitro cellular response on scaffolds thereby indicating the matrices, cytocompatible nature. Scaffolds tested in an ex vivo human skin equivalent wound model, as a preliminary alternative to animal testing, showed keratinocyte migration and wound re-epithelization-a prerequisite for healing and regeneration. Taken together, the herein proposed chitosan/collagen scaffold, shows good potential for skin tissue engineering.

Enhanced Redifferentiation of Chondrocytes on Microperiodic Silk/ Gelatin Scaffolds: Toward Tailor-Made Tissue Engineering

Direct-write assembly allows rapid fabrication of complex three-dimensional (3D) architectures, such as scaffolds simulating anatomical shapes, avoiding the need for expensive lithographic masks. However, proper selection of polymeric ink composition and tailor-made viscoelastic properties are critically important for smooth deposition of ink and shape retention. Deposition of only silk solution leads to frequent clogging due to shear-induced β-sheet crystallization, whereas optimized viscoelastic property of silk-gelatin blends facilitate the flow of these blends through microcapillary nozzles of varying diameter. This study demonstrates that induction of controlled changes in scaffold surface chemistry, by optimizing silk-gelatin ratio, can govern cell proliferation and maintenance of chondrocyte morphology. Microperiodic silk-gelatin scaffolds can influence postexpansion redifferentiation of goat chondrocytes by enhancing Sox-9 gene expression, aggregation, and driving cartilage matrix production, as evidenced by upregulation of collagen type II and aggrecan expression. The strategy for optimizing redifferentiation of chondrocytes can offer valuable consideration in scaffold-based cartilage repair strategies.

Development of chitosan-tripolyphosphate fibers through pH dependent ionotropic gelation

Incorporation of phosphate groups into a material may be of particular interest as they act as templates for hydroxyapatite growth through complexation with Ca(2+) and thus improve the osteoconduction property. The phosphate groups can be incorporated into chitosan through ionotropic gelation with tripolyphosphate (TPP). Interestingly, the ion pairs formed through negatively charged phosphate groups with protonated amine functionality of chitosan in ionotropic gelation are expected to provide chitosan with an amphoteric character, which may facilitate protein adhesion following enhanced attachment of anchorage dependant cells than chitosan, which shows poor cell adhesion properties. In this study, chitosan-tripolyphosphate (TPP) fibers with varying phosphate contents were prepared through wet spinning in STPP baths of different pH. Gelation kinetics and gel strength of chitosan with STPP solutions of three different pH were evaluated and compared with that of NaOH solution for evaluation of their influence on nature of gelation. The solution pH of STPP baths was found to have significant control on the extent of ionic cross-linking and physico-chemical properties of the fibers. Moreover, this kinetically driven ionotropic gelation of chitosan by TPP results in low degree of crystallinity of chitosan-TPP fibers and consequently their lower thermal stability than chitosan fibers.

Carbon nanodots from date molasses: new nanolights for the in vitro scavenging of reactive oxygen species

Most of the nanoimaging tools like quantum dots and metallic nanoparticles are shown to have different levels of cytotoxicity via various mechanisms. However carbon nanodots (CNDs) are a new group of ultra small nano structures (average 4-6 nm) which is potential candidate of next generation optical imaging. Being carbonaceous in origin, CNDs possess excellent luminescence and photostability with significantly less cytotoxicity. In present study, we have synthesized carbon nano-dots from date molasses by microwave irradiation at ∼pH 11. The synthesized carbon nanodots were characterized using UV-Vis spectroscopy, fluorescence spectroscopy, TEM, XRD analysis, FTIR study and Zeta potential measurement. The average sizes of the dots were found to be 5-7 nm. A clear band emission was visible around 480 nm when an excitation beam of 415 nm was incident. For biological applicability, MTT assay and hemocompatibility studies were performed. The results exhibited the material to be highly cytocompatible within the application limit. Upon immediate exposure to CNDs, no significant changes to cellular surface morphology were observed via AFM imaging. Significant hemolysis or blood cell aggregation was not observed after incubation of CNDs with blood. After labelling with CNDs, MG-63 cells were found to be unbleached up to several hours even on exposure to light. We are reporting first time in this study the free radical scavenging property of CNDs in ex vivo and in vitro models. Antioxidant activity was measured ex vivo via potassium permanganate assay and DPPH assay. In vitro superoxide inhibition activity was measured both by spectroscopy and under microscope by NBT reduction assay. Hydroxyl free radical inhibition activity was measured via DCFH-DA Assay. The results were comparable with scavenging activity of standard antioxidant molecules (BHT and l-ascorbic acid). A novel assay for quantitative analysis of cellular oxidative stress was also proposed. Therefore, this material could be useful for long-term live cell imaging and cell tracking in a scaffold with minimal cytotoxicity and oxidative stress.

Shape forming of ceramics via gelcasting of aqueous particulate slurries

Gelcasting is a promising technique for shape forming of bulk dense or porous ceramic, metal structures. The process offers a number of advantages over processes such as slip casting, injection molding in forming complex ceramic shapes. It is shown here that the optimization of slurry rheology, choice of mold material, mold design and the drying conditions have a significant role in the overall success of the process. In this process, components of simple or complex shapes can be produced to near net shape by direct casting. If required complex shapes can also be produced by machining the green gelcast bodies. The process of gelcasting also has a lot of potential in forming highly porous ceramic shapes.

One pot synthesis of intriguing fluorescent carbon dots for sensing and live cell imaging.

We report a simple one-pot synthesis of highly fluorescent carbon dots (CDs) via modified hydrothermal (MHT) treatment of alkaline solution of dopamine and cysteine. These CDs (λex=320 nm, λem=390 nm, and quantum yield ∼ 5.1%) are of ∼ 2-3 nm in diameter. Further attempt of synthesizing CDs in some common water-miscible solvents ends up the fact that the MHT product from acetone medium is nonfluorescent. However, CDs, produced in aqueous medium, are so stable that they can be dried as a deliverable solid (WCD) without any alteration of fluorescing property if reversibly dispersed in water. Fluorescence of WCD is quenched selectively in acetone. Quenching occurs presumably due to the disruption of radiative recombination along with the hindrance in quantum confinement of the emissive energy traps to the particle surface. Successive quenching of fluorescence of WCD in different acetone concentration admixed in water paves the way to selective acetone sensing (LOD=8.75 × 10(-7) M). The synthesized CDs (in aqueous medium) are cytocompatible and are efficient fluorescent probe for cell imaging. Only living cells are recognized exclusively from fluorescence imaging leaving aside dead cells, while cells are treated with CDs.

In vitro cytocompatibility and blood compatibility of polysulfone blend, surface-modified polysulfone and polyacrylonitrile membranes for hemodialysis

The fabrication of dialysis membranes with significant biocompatibility is an active area of research. In this context, three types of asymmetric flat sheet membranes were fabricated and compared for potential use as hemodialysis membranes. A polysulfone–polyvinylpyrrolidone and polyethylene glycol-based polymer blend membrane, a polysulfone membrane surface-modified with trimesoyl chloride and m-phenylene diamine, and a polyacrylonitrile membrane were synthesized. All three types of membrane were characterized in terms of their surface morphology, permeability, hydrophilicity, surface charge, porosity and mechanical strength. They were then subjected to comprehensive cytocompatibility and hemocompatibility tests as well as analysing the transport of uremic toxins. On the basis of protein adsorption, oxidative stress, cell proliferation and adhesion, all three membranes were comparable. However, the blend and surface-modified membranes showed excellent results for hemolysis, platelet adhesion, blood cell aggregation and degree of thrombus formation. All these results indicated the suitability of the blend and surface-modified membranes for possible dialysis applications.