Aligned Fibroporous Matrix Generated from a Silver Ion and Graphene Oxide-Incorporated Ethylene Vinyl Alcohol Copolymer as a Potential Biomaterial for Peripheral Nerve Repair.

Developing an ideal nerve conduit for proper nerve regeneration still faces several challenges. The attempts to fabricate aligned substrates for neuronal growth have enhanced the hope of successful nerve regeneration. In this wok, we have attempted to generate an electrospun matrix with aligned fibers from a silver and graphene oxide-incorporated ethylene vinyl alcohol copolymer (EVAL). The presence of silver was analyzed using UV-visible spectra, XPS spectra, and ICP. Raman spectra and FTIR spectra confirmed the presence of GO. The complexation of Ag+ with - OH of EVAL enabled the generation of aligned fibers. The fiber diameter (>1 µm) provided sufficient space for forming focal adhesion by the neurites and filopodia of N2a and C6 cells, respectively. The fiber diameter enabled the neurites and filopodia of the cells to align on the fibers. The incorporation of GO has contributed to the cell-material interactions. The morphological and mechanical properties of fibers obtained in the study ensure that the EVAL-Ag-GO-0.01 matrix is a potential substrate for developing a nerve guidance conduit/nerve wrap (NGC/W).

In vitro Cytotoxicity Evaluation of Flowable Hyaluronic Acid-Acellular Stromal Vascular Fraction (HA-aSVF) Mixture for Tissue Engineering Applications.

Background: The stromal vascular fraction (SVF) is an aqueous fraction isolated from the adipose tissue that constitutes different kinds of cells and extracellular matrix components. Hyaluronic acid (HA) is a linear polysaccharide in vertebrate tissues and is considered a potential tissue engineering scaffold due to its biocompatible nature. In this study, we have evaluated the cytotoxicity of xenofree HA in combination with an acellular component of adipose SVF (HA-aSVF) to propose it as a candidate biomaterial for future applications. Materials and Methods: 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide assay of L-929 cells treated with HA-aSVF was used in our study. Data were normalized to cell control (untreated) and extracts of copper and ultra-high molecular weight polyethylene were used as positive (PC) and negative controls (NC). Results: Fibroblast cells retained the morphology after 24 h of treatment with HA-aSVF mixture and exhibited a similar percentage of cell activity compared to NC. PC showed a positive cytotoxic response as expected. The cells incubated with HA-aSVF showed a linear increase in cell activity indicating proliferation. Conclusion: The mixture of HA and acellular SVF in its flowable form is non-cytotoxic and showed improved cell proliferation. Hence the mixture can be proposed as a biomaterial and can be further explored for specific tissue engineering applications.

Graphene oxide decorated electrospun gelatin nanofibers: Fabrication, properties and applications.

Gelatin nanofiber fabricated by electrospinning process is found to mimic the complex structural and functional properties of natural extracellular matrix for tissue regeneration. In order to improve the physico-chemical and biological properties of the nanofibers, graphene oxide is incorporated in the gelatin to form graphene oxide decorated gelatin nanofibers. The current research effort is focussed on the fabrication and evaluation of physico-chemical and biological properties of graphene oxide-gelatin composite nanofibers. The presence of graphene oxide in the nanofibers was established by transmission electron microscopy (TEM). We report the effect of incorporation of graphene oxide on the mechanical, thermal and biological performance of the gelatin nanofibers. The tensile strength of gelatin nanofibers was increased from 8.29±0.53MPa to 21±2.03MPa after the incorporation of GO. In order to improve the water resistance of nanofibers, natural based cross-linking agent, namely, dextran aldehyde was employed. The cross-linked composite nanofibers showed further increase in the tensile strength up to 56.4±2.03MPa. Graphene oxide incorporated gelatin nanofibers are evaluated for bacterial activity against gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria and cyto compatibility using mouse fibroblast cells (L-929 cells). The results indicate that the graphene oxide incorporated gelatin nanofibers do not prevent bacterial growth, nevertheless support the L-929 cell adhesion and proliferation.

Galactosylated alginate-curcumin micelles for enhanced delivery of curcumin to hepatocytes.

Galactosylated alginate-curcumin conjugate (LANH2-Alg Ald-Cur) is synthesized for targeted delivery of curcumin to hepatocytes exploiting asialoglycoprotein receptor (ASGPR) on hepatocytes. The synthetic procedure includes oxidation of alginate (Alg), modification of lactobionic acid (LA), grafting of targeting group (modified lactobinic acid, LANH2) and conjugation of curcumin to alginate. Alginate-curcumin conjugate (Alg-Cur) without targeting group is also prepared for the comparison of properties. LANH2-Alg Ald-Cur self assembles to micelle with diameter of 235 ± 5 nm and zeta potential of -29 mV in water. Cytotoxicity analysis demonstrates enhanced toxicity of LANH2-Alg Ald-Cur over Alg-Cur on HepG2 cells. Cellular uptake studies confirm that LANH2-Alg Ald-Cur can selectively recognize HepG2 cells and shows higher internalization than Alg-Cur conjugate. Results indicate that LANH2-Alg Ald-Cur conjugate micelles are suitable candidates for targeted delivery of curcumin to HepG2 cells.

A Novel, Single Step, Highly Sensitive In-Vitro Cell-Based Metabolic Assay Using Honeycomb Microporous Polymer Membranes.

This study describes a novel, simple and versatile system for cell-based assays at the bench-top. The system consists of Polyurethane (PU) based honeycomb membrane with the active compounds/assay reagents dispensed on its pore linings. Membranes with functionalized pores were thus created and used for conducting cell based assays. As proof-of-concept Flourocein acetate (FDA) and Propidium iodide (PI) were embedded on the pore linings and live/dead assays were performed on L929 and Hacat cell lines. The results proved the sensitivity of the membrane based cell assay. To ensure the capacity of this system for high throughput applications, membrane based live/dead assay was performed on L929 cells with varying levels of viability. The results from this experiment were quantified by microscopic and spectrofluourimetric techniques both of which were found to correlate well. It was concluded that this simple membrane based cell assay is highly versatile and enables multiple compounds to be tested on the same cell/tissue. Furthermore, this method requires low volumes of assay reagents and eliminates many of the wet techniques that are involved in a conventional assay, without compromising on the sensitivity. It is anticipated that this functionalized membrane system could be easily adapted for both manual and automated high content screening experiments including in vitro biomaterial evaluation as well as cytotoxicity of nanomaterials.

Gum arabic-curcumin conjugate micelles with enhanced loading for curcumin delivery to hepatocarcinoma cells.

Curcumin is conjugated to gum arabic, a highly water soluble polysaccharide to enhance the solubility and stability of curcumin. Conjugation of curcumin to gum arabic is confirmed by (1)H NMR, fluorescence and UV spectroscopy studies. The conjugate self assembles to spherical nano-micelles (270 ± 5 nm) spontaneously, when dispersed in aqueous medium. Spherical morphology of the self assembled conjugate is evidenced by field emission scanning electron microscopy and transmission electron microscopy. The self assembly of the amphiphilic conjugate into micelle in aqueous medium significantly enhances the solubility (900 fold of that of free curcumin) and stability of curcumin in physiological pH. The anticancer activity of the conjugate micelles is found to be higher in human hepatocellular carcinoma (HepG2) cells than in human breast carcinoma (MCF-7) cells. The conjugate exhibits enhanced accumulation and toxicity in HepG2 cells due to the targeting efficiency of the galactose groups present in gum arabic.

Modified dextran cross-linked electrospun gelatin nanofibres for biomedical applications.

Electrospun gelatin nanofibres attract attention of bioengineering arena because of its excellent biocompatibility and structural resemblance with native extracellular matrix. In this study, we have developed gelatin nanofibres using an innovative cross-linking approach to minimize cytotoxic effects. Gelatin was dissolved in water:acetic acid (8:2, v/v) solution and electrospun to form nanofibres with diameter in the range of 156 ± 30 nm. The nanofibres were cross-linked with a modified polysaccharide, namely, dextran aldehyde (DA). Cross-linking with DA could be achieved without compromising the fibrous architecture. DA cross-linked gelatin nanofibres maintained the fibrous morphology in aqueous medium. These mats exhibit improved mechanical properties and gradual degradation behaviour. The nanofibres were evaluated for cytotoxicity, cell adhesion, viability, morphology and proliferation using L-929 fibroblast cells. The results confirmed that DA cross-linked mats were non cytotoxic towards L-929 cells with good cell adhesion, spreading and proliferation.

Intelligent thermoresponsive substrate from modified overhead projection sheet as a tool for construction and support of cell sheets in vitro.

Cell sheet engineering using thermoresponsive culture dishes allows harvesting of intact in vitro cell sheet. In this study, commercially available polyethylene terephthalate-based overhead projection transparency sheet (OHPS) was identified as a substrate for coating thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate) (NGMA) copolymer having lower critical solution temperature of 28°C. Since OHPS is highly hydrophobic and rigid, the surface was modified by alkali treatment (OHPS-M) to functionalize the surface with carboxyl and hydroxyl groups so as to make it more suitable for efficient coating of NGMA copolymer and cell culture. To impart thermoresponsiveness, OHPS-M was coated with NGMA (OHPS-MC). Surface morphology, surface chemistry, and thermoresponsive coating were analyzed by profilometry, scanning electron microscopy, water contact angle, and Fourier transform infrared spectroscopy. The cytotoxicity, cell adhesion, and proliferation on OHPS-M and OHPS-MC were analyzed using L929 cells. Specific cytocompatibility analysis was done using SIRC (Rabbit corneal) cells. Data revealed cytocompatible nature of OHPS-M and OHPS-MC. Suitability of OHPS-MC for cell sheet harvest and transfer efficiency was assessed using primary corneal cells. Corneal cell sheet constructs retrieved by temperature variation was characterized by reverse transcriptase polymerase chain reaction for markers specific to differentiated corneal cells (keratin 3 and keratin 12) and proliferating cell nuclear antigen, and assessed for viability using fluorescein diacetate staining, tissue architecture by scanning electron microscopy, and cell-cell contacts by connexin-43 staining. The retrieved cell sheets retained corneal epithelial characteristics such as keratin 3/12, viability, and tissue architecture with intact cell-cell contacts as in native tissue. The results proved that surface modification and coating with NGMA on OHPS offer a novel biocompatible thermosensitive cell culture substrate for generating cell sheet constructs. In addition, OHPS-M can also serve as an efficient carrier tool for retrieved cell sheet.