Deepti Singh

Synthesis of composite gelatin-hyaluronic acid-alginate porous scaffold and evaluation for in vitro stem cell growth and in vivo tissue integration

Engineering three-dimensional (3-D) porous scaffolds with precise bio-functional properties is one of the most important issues in tissue engineering. In the present study, a three-dimensional gelatin-hyaluronic acid-alginate (GHA) polymeric composite was synthesized by freeze-drying, which was followed by ionic crosslinking using CaCl2, and evaluated for its suitability in bone tissue engineering applications. The obtained matrix showed high porosity (85%), an interconnected pore morphology and a rapid swelling behavior. The rheological analysis of GHA showed a viscoelastic characteristic, which suggested a high load bearing capacity without fractural deformation. The influence of the GHA matrix on cell growth and on modulating the differentiation ability of mesenchymal stem cells was evaluated by different biochemical and immunostaining assays. The monitoring of cells over a period of four weeks showed increased cellular proliferation and osteogenic differentiation without external growth factors, compared with control (supplemented with osteogenic differentiation medium). The in vivo matrix implantation showed higher matrix-tissue integration and cell infiltration as the duration of the implant increased. These results suggest that a porous GHA matrix with suitable mechanical integrity and tissue compatibility is a promising substrate for the osteogenic differentiation of stem cells for bone tissue engineering applications.

Engineering three-dimensional macroporous hydroxyethyl methacrylate-alginate-gelatin cryogel for growth and proliferation of lung epithelial cells.

Three-dimensional (3D) growth of cell is of particular interest in the field of tissue engineering and regenerative medicine. Scaffolds used for this purpose are often tailor-made to mimic the microenvironment and the extracellular matrix of the tissue with defined role such as to provide appropriate structural, chemical, and mechanical support. The aim of the study was to design the macroporous matrix with potential in the field of tissue engineering especially for lung muscle regeneration. Blend of hydroxyethyl methacrylate-alginate-gelatin (HAG) cryogel scaffold was synthesized using cryogelation technique and this polymer material combination is being reported first time. The rheology study showed the elastic property of the material in wet state with no variation in storage modulus (G′), loss modulus (G″), and phase angle upon temperature variation. The microcomputer tomography (micro-CT) analysis confirmed the homogenous polymer structure with average pore diameter of 84 μm. Scaffold synthesized using polymer combinations which is mixture of polysaccharide (alginate) and protein (gelatin) provides supportive environment for human lung epithelial cell proliferation confirmed by cytoskeletal stain phalloidin and nuclei staining 4′,6-diamidino-2-phenylindole checked for over three weeks. The in vivo biocompatibility was further performed which showed integration of scaffold to the surrounding tissue with ability to recruit cells. However, at first week, small amount of infiltrating mast cells were found which subsequently diminished in following weeks. Immunohistochemistry for dendritic cells confirmed in vivo biocompatible nature of the HAG scaffold. The mechanical strength, stiffness, elastic measurements, in vivo compatibility, and in vitro lung cell proliferation show the potentiality of HAG materials for lung tissue engineering.

Effect of extracts of terminalia chebula on proliferation of keratinocytes and fibroblasts cells: An alternative approach for wound healing

Terminalia chebula is one of the traditional medicines used in the treatment of many diseases. In the present work, different concentrations of various organic and aqueous extracts (solvent-free) of T. chebula were tested on fibroblast (L929) and keratinocytes cells to evaluate its biocompatible concentration by using MTT and live-dead viability/cytotoxic assay. These extracts were found to be effective in decreasing the ammonia accumulation in the media, thereby reducing its toxic effect on cells. DPPH assay further confirmed the free-radical scavenging ability of the extracts which increased with the increase in concentration of each extract. Cell proliferation/apoptosis, cytoskeletal structure, and ECM production were further evaluated by live-dead assay and phalloidin/cytokeratin staining, respectively. The cytoskeletal structure and ECM secretion of the cells treated with extracts showed higher cellular activity in comparison to control. In conclusion, we have demonstrated the effect of these extracts of T. chebula on both types of skin cells and optimized concentration in which it could be used as a bioactive component for wound healing applications by increasing cell proliferation and decreasing free-radical production without affecting the normal cellular matrix. It can also find applications in other therapeutics applications where ammonia toxicity is a limiting factor.

Poly(ethylene glycol) dicarboxylate/poly(ethylene oxide) hydrogel film co-crosslinked by electron beam irradiation as an anti-adhesion barrier

The cross-linked poly(ethylene glycol) dicarboxylate (PEGDC)/poly(ethylene oxide) (PEO) and poly(ethylene glycol) dimethacrylate (PEGDMA)/(PEO) hydrogels were developed for possible biomedical applications such as an anti-adhesion barrier. Various contents of PEGDC/PEO film were irradiated using an electron beam with various beam intensities in order to obtain various degrees of crosslinked hydrogels. The optimum dose (300 kGy) and total crosslinker content of 10% were used to prepare crosslinked hydrogel films with three different compositions (10% PEGDC, 10% PEGDMA, 5% PEGDC-5% PEGDMA). Among them, 10% PEGDC hydrogel film exhibited the highest elongation at break (69.33±6.87%) with high mechanical strength. 10% PEGDC hydrogel film showed the lowest hemolysis activity (6.03±0.01%) and the highest tissue adherence (75.67±1.15 cN). The result also indicated that the carboxyl groups in PEGDC affect the tissue adherence of hydrogel films via H-bonding interactions. In animal studies, 10% PEGDC anti-adhesion hydrogel film degraded within 3 weeks and demonstrated better anti-adhesive effect compared to Guardix-SG®.

Enhanced cell viability of hydroxyapatite nanowires by surfactant mediated synthesis and its growth mechanism

Hydroxyapatite (HA) nanowires were grown by a simple hydrothermal method using cetyl-trimethyl-ammonium-bromide (CTAB) as a surfactant. The growth of preferentially oriented nanowires were suggested to be formed by the orientation-dependent electrostatic interaction between the CTAB ions and HA crystals. We prepared HA nanowires with a diameter of 30 ± 7 nm and the length of few micrometers. The prolonged hydrothermal treatment with the ideal concentration of CTAB induce the preferential growth of HA nanowires in the c-axis direction. The pronounced peak broadening of mainly (002) planes with the increase of the length of nanowires can be attributed to the natural bending of nanowires with an extremely high aspect ratio. The in vitro cell cultural studies of the HA nanowires showed that the osteoblast cell response enhanced with the uniformity and the specific surface area of HA nanowires.

Hyperbranched poly(glycidol)/poly(ethylene oxide) crosslinked hydrogel for tissue engineering scaffold using e-beams.

A microporous hydrogel scaffold was developed from hyperbranched poly(glycidol) (HPG) and poly(ethylene oxide) (PEO) using electron beam (e-beam) induced cross-linking for tissue engineering applications. In this study, HPG was synthesized from glycidol using trimethylol propane as a core initiator and cross-linked hydrogels were made using 0, 10, 20, and 30% HPG with respect to PEO. The effects of %-HPG on the swelling ratio, cross-linking density, mechanical properties, morphology, degradation, and cytotoxicity of the hydrogel scaffolds were then investigated. Increasing the HPG content increased the pore size of the hydrogel scaffold, as well as the porosity, elongation at break, degree of degradation and swelling ratio. In contrast, the presence of HPG decreased the cross-linking density of the hydrogel. There was no significant difference in compressive modulus and tensile strength of all compositions. The pore size of hydrogel scaffolds could be easily tailored by controlling the content of HPG in the polymer blend. Evaluation of the cytotoxicity demonstrated that HPG/PEO hydrogel scaffold has potential for use as a matrix for cellular attachment and proliferation. These results indicate that cross-linked HPG/PEO hydrogel can function as a potential material for tissue engineering scaffolds. Moreover, a facile method to prepare hydrogel microporous scaffolds for tissue engineering by e-beam irradiation was developed.

Novel alginate-gelatin hybrid nanoparticle for drug delivery and tissue engineering applications

Novel alginate-gelatin hybrid nanoparticles were fabricated using single oil in water (O/W) emulsification techniques. Physicochemical property of the particle was characterized using scanning electron microscopy and Fouriers transmission infrared spectroscopy. Particle size was determined using zeta potential metastasize analyzer and was found to be in range of 400-600 nm. AGNPs were used for culturing human keratinocytes for two weeks to check biocompatibility of synthesized AGNPs. 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed increased metabolic activity of cells cultured on AGNPs in comparison to two-dimensional (2D) system (control). Cellular attachment on nanoparticle was further confirmed using SEM and 4′,6-diamidino-2-phenylindole staining. The drug release profile shows possible electrostatic bond between alginate and gelatin resulting in controlled release of drug from AGNPs. For the first time alginate-gelatin hybrid nanosystem has been fabricated and all results showed it can be used as potential system for delivery of drug and therapeutical agents to cells and can also be used for regenerative medicine applications.

Enhanced proliferation and growth of human lung epithelial cells on gelatin microparticle loaded with ephedra extracts

The objective of this work was to evaluate the effect of extracts of Ephedra gerardiana loaded onto gelatin particles on human lung epithelial cells. Particles were synthesized using oil-water emulsification technique and were further stabilized by glutaraldehyde. Particle size was evaluated using SEM and zeta potential analyzer and was found to be in the range of 600 nm-1.32 µm. Drug release profile showed controlled and constant release of extract over the period of 5 days. In vitro biocompatibility of gelatin particles loaded with solvent-free extract of Ephedra gerardiana was tested with human lung epithelial cells. Gelatin particle acted not only as scaffold for cellular adhesion but also as carrier matrix for controlled release of extracts. The cell viability was significantly high when cultured in the presence of Ephedra extract in comparison to cells without Ephedra and 2D systemas seen in MTT, SEM, and live/dead staining assay. It is concluded that gelatin microparticle functions both as drug delivery systemand scaffold; however, the main finding was the effect of Ephedra extract on human lung cells resulting in enhanced proliferation and consequent promotion of ECM production indicating that extract could be a bioactive component that can be utilized in tissue engineering and regenerative medicine.

Effect of cross-linkers in fabrication of carrageenan-alginate matrices for tissue engineering application.

The three‐dimensional (3D) scaffold serves as a structural substrate and as a niche for cell proliferation to ensure tissue regeneration. Ideal scaffolds should have porous structures with high pore interconnectivity to allow cell adherence, differentiation, and proliferation while ensuring suitable mechanical strength and biodegradability without inflicting any immune response. Cross‐linker is one of the major factors that affect the mechanical and biological properties of scaffolds. In this study, different chemical cross‐linker effects on scaffold architecture were examined. Porous 3D scaffolds based on carrageenan and alginate (CA) were successfully fabricated by a freeze‐drying technique and using various cross‐linkers like glutaraldehyde (GA), genipin, and ethyl (dimethylamino propyl) carbodiimide/N‐hydroxysuccinimide (EDC/NHS). The chemical cross‐linker effects on the CA scaffold were characterized by scanning electron microscopy, differential scanning calorimetry, and thermogravimetry. A human fibroblast cell line (L929) was seeded into the fabricated scaffold, and cell proliferation was assessed by MTT and live/dead assay. Overall results suggested the potential cross‐linkers for ideal CA biomaterial could be EDC/NHS among other agents tested as the scaffold CAEN was found to be porous, interconnected, and physically and mechanically stable. When compared to matrices with other cross‐linkers, higher cell attachment, better cellular response, and higher metabolic activity could be observed in the scaffold synthesized using EDC/NHS as cross‐linker.

Functionalizing cellulose scaffold prepared by ionic liquid with bovine serum albumin for biomedical application

The present study reports the preparation of a cellulose scaffold for tissue engineering directly from cellulose fiber using ionic liquid (IL) by the NaCl leaching method with bovine serum albumin (BSA), which is well known protein utilized for biomedical applications like degradation of polymer, cell attachment and proliferation on scaffold. The 1-n-allyl-3-methylimidazolium chloride (AmimCl) IL was used as a solvent for cellulose. The morphology of the scaffold was studied by scanning electron microscopy (SEM) and the images showed that the pore sizes of the scaffolds were about 200 µm. In addition, the water uptake (WU) and degree of degradation of the cellulose scaffold were measured. Meanwhile, the biocompatibility and bioactivity of the scaffold were determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) assay and the Live/Dead viability test. The various results demonstrated the ability of the Mesenchymal stem cells (MSC) to attach to the surface of the scaffolds amplified as percentage of BSA increased in cellulose scaffold.