labi Pal

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.

A Simple Approach for an Eggshell-Based 3D-Printed Osteoinductive Multiphasic Calcium Phosphate Scaffold

Natural origin bioceramics are widely used for bone grafts. In the present study, an eggshell-derived bioceramic scaffold is fabricated by 3D printing as a potential bone-graft analogue. The eggshell, a biological waste material, was mixed with a specific ratio of phosphoric acid and chitosan to form a precursor toward the fabrication of an osteoinductive multiphasic calcium phosphate scaffold via a coagulation-assisted extrusion and sintering for a multiscalar hierarchical porous structure with improved mechanical properties. Physicochemical characterization of the formed scaffolds was carried out for phase analysis, surface morphology, and mechanical properties. A similar scaffold was prepared using a chemically synthesized calcium phosphate powder that was compared with the natural origin one. The higher surface area associated with the interconnected porosity along with multiple phases of the natural origin scaffold facilitated higher cell adhesion and proliferation compared to the chemically synthesized one. Further, the natural origin scaffold displayed relatively higher cell differentiation activity, as is evident by protein and gene expression studies. On subcutaneous implantation for 30 days, promising vascular tissue in-growth was observed, circumventing a major foreign body response. Collagen-rich vascular extracellular matrix deposition and osteocalcin secretion indicated bonelike tissue formation. Finally, the eggshell-derived multiphasic calcium phosphate scaffold displayed improvement in the mechanical properties with higher porosity and osteoinductivity compared to the chemically derived apatite and unveiled a new paradigm for utilization of biological wastes in bone-graft application.

Accelerating full thickness wound healing using collagen sponge of mrigal fish (Cirrhinus cirrhosus) scale origin

The potentiality of collagen sponge as a skin substitute, derived from mrigal (Cirrhinus cirrhosus) scale has been explored in this study. Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the scale of mrigal were isolated and characterized. The yields of ASC and PSC were ∼3% and ∼7% based on the dry weight of scale while the hydroxyproline content was ∼90mg/g. Scanning electron microscope revealed progressive demineralization with EDTA on time dependent scale. Further, the D-Spacing in fibril bundles were calculated to be ∼67nm. Fourier transform infrared and circular dichroism spectra confirmed extracted protein to be collagen I, where both ASC and PSC comprised of two different α-chains (α1 and α2). The denaturation temperature (Td) of the collagen solution was 35°C closer to Td of mammalian collagen. In vitro cell culture studies on the extracted collagen sponge showed efficient cell growth and proliferation. Additionally, co-culture with fibroblast and keratinocyte cells showed development of stratified epidermal layer in vitro. Faster wound healing potential of the extracted collagen in a rat model proved its applicability as a dermal substitute.

Fabrication and characterization of polyvinyl alcohol/metal (Ca, Mg, Ti) doped zirconium phosphate nanocomposite films for scaffold-guided tissue engineering application

Nanocomposite films of polyvinyl alcohol (PVA) and zirconium phosphate (ZrP)/doped ZrP (doped with Ca, Mg, Ti) nanoparticles have been developed by solvent casting method to assess their potential as matrix material in scaffold-guided tissue engineering application. The prepared ZrP and doped ZrP nanoparticles as well as the nanocomposite films were characterized by various spectroscopic and microscopic techniques. Nanoindentation studies revealed improved nanomechanical properties in the PVA/doped ZrP nanocomposite films (highest for PVA/Ti doped ZrP: hardness=262.4MPa; elastic modulus=5800MPa) as compared to the PVA/ZrP and neat PVA films. In-vitro cell culture experiments carried out to access the cellular viability, attachment, proliferation, and migration on the substrates, using mouse fibroblast (3T3) cell lines, inferred enhanced bioactivity in the PVA/doped ZrP nanocomposite films (highest for PVA/Ca doped ZrP) in contrast to PVA/ZrP and neat PVA films. Controlled biodegradability as well as swelling behavior, superior bioactivity and improved mechanical properties of the PVA/doped ZrP nanocomposite films make them promising matrix materials for scaffold-guided tissue engineering application.

Single step synthesized sulfur and nitrogen doped carbon nanodots from whey protein: nanoprobes for longterm cell tracking crossing the barrier of photo-toxicity

Long-term cell tracking is a research interest for biological scientists across disciplines and applications. However, long-term cell tracking experiments are often limited due to photobleaching and phototoxicity. In the current study, a carbonaceous nanoprobe was developed using a single step microwave assisted degradation of whey protein in the aqueous phase. The CNDs were characterized via UV-Vis spectroscopy, fluorescence spectroscopy, HRTEM, DLS and FTIR. Due to choice of the precursor, the CNDs were observed to be doped with sulfur and nitrogen. The CNDs were capable of bioimaging. In a 2D cell culture system (culture flask), the cells retained fluorescence for up to five passages. In a 3D microenvironment, cell tracking was also successful for up to 10 days. The CNDs were observed to be capable of scavenging superoxides and hydroxyl radicals in vitro. The CNDs were also observed to save cells from phototoxicity and UV exposure via cytotoxicity, microscopy and nanoindentation analysis.

Morphology-induced physico-mechanical and biological characteristics of TPU-PDMS blend scaffolds for skin tissue engineering applications: Morphology-induced physico-mechanical and biological characteristics of TPU-PDMS blend scaffolds for skin tissue engineering applications

Composition and architecture of scaffolds are the most important factors determining the performance of skin substitutes. In this work, morphology induced unique physical and biological characteristics of compatibilized TPU-PDMS blend scaffolds at 90:10, 80:20, and 70:30 blend ratios of TPU and PDMS was studied. The fiber morphology, porosity, surface wettability, and mechanical properties of electrospun scaffolds were distinctly influenced by the presence of PDMS. Interestingly, the scaffold architecture varied from electrospun fibers to porous fibers and finally occurrence of unique porous beads noticed at 30% PDMS in the microstructure which was confirmed using FESEM. Micro-CT analysis revealed that the porosity of electrospun scaffolds was enhanced from 61% to 79% with 30 parts of PDMS addition. Moreover, MTT assay and cell proliferation were studied using human skin fibroblast cells and found to be significantly enhanced with the PDMS percentage. TPU-PDMS blends offer better overall performance at 70:30 blend ratio of TPU and PDMS (T70P30). Only 4% of hemolysis was observed for T70P30 blends, which establishes the hemocompatibility of the material. In comparison, the results reveal the potential of the cytocompatible T70P30 scaffold for the fabrication of skin substitutes for tissue engineering applications.

Carbon nanodot impregnated fluorescent nanofibers for in vivo monitoring and accelerating full-thickness wound healing

Semiconductor quantum dots are overwhelmingly used for in situ monitoring and imaging of cell-scaffold interactions. However, quantum dots suffer from oxidative biodegradation in biological systems, besides being toxic due to the presence of heavy metals. In this study, we report the development of an intrinsically fluorescent nanofibrous scaffold of polycaprolactone-gelatin for skin tissue regeneration and noninvasive monitoring of scaffold activity in vivo. The presence of the incorporated carbon nanodots played a critical role in imparting the scaffold with these novel characteristics. The developed scaffold was uniform and bead free with fiber diameter of 698 ± 420 nm and pore diameter of 2.93 ± 1.13 μm. Inclusion of carbon nanodots not only bestowed uniform fluorescence of the scaffold but also promoted fibroblast cell adhesion, migration and proliferation. Co-culture of fibroblast and keratinocyte cells on the scaffold surface also enabled the development of a stratified epithelial layer. The scaffold exhibited antioxidant properties by scavenging free radicals and reducing the expression of antioxidative enzymes. Upon implantation in a full-thickness excision wound, the scaffold accelerated the progression of healing and the regenerated skin exhibited a stratified epithelial layer with mature dermal tissue. The scaffold enabled noninvasive monitoring of the wound healing kinetics in vivo through two-photon microscopy. With excellent photoluminescence, biocompatibility, and photo stability, the scaffold can suitably be used for prolonged monitoring of cell-scaffold interactions and further efficiently reduce the oxidative stress during continuous imaging. Additionally, being synthesized from inexpensive precursors employing a simple procedure, carbon nanodot production is cost-effective and the developed scaffold would be an off-the-shelf, readily available economical product.

Effect of Vitamin E and a Long-Chain Alcohol n-Octanol on the Carbohydrate-Based Nonionic Amphiphile Sucrose Monolaurate—Formulation of Newly Developed Niosomes and Application in Cell Imaging

We have introduced new niosome formulations using sucrose monolaurate, vitamin E and n-octanol as independent additives. Detailed characterization techniques including turbidity, dynamic light scattering, transmission electron microscopy, ξ potential, and proton nuclear magnetic resonance measurements have been introduced to monitor the morphological transition of the carbohydrate-based micellar assembly into niosomal aggregates. Moreover, microheterogeneity of these niosomal aggregates has been investigated through different fluorescence spectroscopic techniques using a hydrophobic probe molecule coumarin 153 (C153). Further, it has been observed that vitamin E and octanol have an opposing effect on the rotational motion of C153 in the respective niosome assemblies. The time-resolved anisotropy studies suggest that incorporation of vitamin E and octanol into the surfactant aggregates results in slower and faster rotational motion of C153, respectively, compared to the micellar assemblies. Moreover, the ability to entrap a probe molecule by these niosomes is utilized to encapsulate and deliver the anticancer drug doxorubicin inside the mammalian cells which is monitored through fluorescence microscopic images. Interestingly, the niosome composed of vitamin E demonstrated better cytocompatibility toward primary chondrocyte cell lines compared to the octanol-forming niosome.