Somnath Maji

Gelatin/Carboxymethyl chitosan based scaffolds for dermal tissue engineering applications.

The present study delineates the preparation, characterization and application of gelatin-carboxymethyl chitosan scaffolds for dermal tissue engineering. The effect of carboxymethyl chitosan and gelatin ratio was evaluated for variations in their physico-chemical-biological characteristics and drug release kinetics. The scaffolds were prepared by freeze drying method and characterized by SEM and FTIR. The study revealed that the scaffolds were highly porous with pore size ranging between 90 and 170μm, had high water uptake (400-1100%) and water retention capacity (>300%). The collagenase mediated degradation of the scaffolds was dependent on the amount of gelatin present in the formulation. A slight yet significant variation in their biological characteristics was also observed. All the formulations supported adhesion, spreading, growth and proliferation of 3T3 mouse fibroblasts. The cells seeded on the scaffolds also demonstrated expression of collagen type I, HIF1α and VEGF, providing a clue regarding their growth and proliferation along with potential to support angiogenesis during wound healing. In addition, the scaffolds showed sustained ampicillin and bovine serum albumin release, confirming their suitability as a therapeutic delivery vehicle during wound healing. All together, the results suggest that gelatin-carboxymethyl chitosan based scaffolds could be a suitable matrix for dermal tissue engineering applications.

Nanocomposites of bio-based hyperbranched polyurethane/funtionalized MWCNT as non-immunogenic, osteoconductive, biodegradable and biocompatible scaffolds in bone tissue engineering

This study focused on the design of novel mechanically tough, biocompatible, osteoconductive and biodegradable scaffolds based on sunflower oil modified hyperbranched polyurethane (HBPU)/functionalized multi-walled carbon nanotube (f-MWCNT) nanocomposites (NCs), and the response of an animal model on their post-implantation. The NC was prepared by an in situ polymerization technique with different wt% of f-MWCNTs. The tensile strength of the NCs was enhanced to 36.98-47.6 MPa from 23.93 MPa (HBPU) and toughness from 12 767 to 18 427-19 440 due to the addition and efficient dispersion of the f-MWCNTs in the HBPU matrix. The post-60 days in vitro biodegraded NC retained sufficient strength (39 ± 1.65 MPa). The increase in wt% of f-MWCNTs had a significant effect on tailoring the physico-mechanical properties of the polymer. The hematological, histological and immunological indices of toxicity suggested the safety potential of the prepared systems within the tested animal model. Moreover, the cytokines (viz. IL-6 and TNF-α) detection, MTT assay and anti-hemolytic assay boosted the non-toxic behavior of the systems. The NC with interconnected pores size (200-330 μm) showed better proliferation and adherence of osteoblast (MG63) cells compared to the HBPU and the results were comparable with the control. Thus the findings confirmed the non-toxicity of f-MWCNTs in association with the polymer and thereby endorsed the NC as a potential biomimetic scaffold for bone tissue engineering.

Bio-functionalized MWCNT/hyperbranched polyurethane bionanocomposite for bone regeneration

The proper fabrication of biomaterials, particularly for purposes like bone regeneration, is of the utmost importance for the clinical success of materials that fulfill the design criteria at bio-interfacial milieu. Building on this aspect, a polyurethane nanocomposite (PNC) was fabricated by the combination of rapeseed protein functionalized multi-walled carbon nanotubes (MWCNTs) and vegetable-oil-based hyperbranched polyurethane. Biofunctionalized MWCNTs showed incredible biocompatibility compared to pristine MWCNTs as ascertained via in vitro and in vivo studies. PNC showed enhanced MG63 cell differentiation ability compared to the control and carboxyl functionalized MWCNT-based nanocomposite, as postulated by alkaline phosphatase activity together with better cellular adhesion, spreading and proliferation. Consequently, a critical-sized fracture gap (6 mm) bridged by the sticky PNC scaffold illustrated rapid bone neoformation within 30-45 d, with 90-93% of the defect area filling up. Histopathological studies demonstrated the reorganization of the normal tibial architecture and biodegradation of the implant. The subsequent toxicological study through cytokine expression, biochemical analysis and hematological studies suggested non-immunogenic and non-toxic effects of PNCs and their degraded/leached products. Their excellent bio-physiological features with high load-bearing ability (49-55.5 Mpa), ductility (675-790%) and biodegradability promote them as the best alternative biomaterials for bone regeneration in a comprehensive manner.

A Smart Magnetically Active Nanovehicle for on-Demand Targeted Drug Delivery: Where van der Waals Force Balances the Magnetic Interaction

The magnetic field is a promising external stimulus for controlled and targeted delivery of therapeutic agents. Here, we focused on the preparation of a novel magnetically active polymeric micelle (MAPM) for magnetically targeted controlled drug delivery. To accomplish this, a number of superparamagnetic as well as biocompatible hybrid micelles were prepared by grafting four armed pentaerythretol poly(ε-caprolactone) (PE-PCL) onto the surface of Fe3O4 magnetic nanoparticles (MNPs) of two different ranges of size (∼5 nm and ∼15 nm). PE-PCL (four-armed) was synthesized by ring-opening polymerization, and it has been subsequently grafted onto the surface of modified MNP through urethane (-NHCO-) linkage. Polymer-immobilized MNP (5 and 15 nm) showed peculiar dispersion behavior. One displayed uniform dispersion of MNP (5 nm), while the other (15 nm) revealed associated structure. This type of size dependent contradictory dispersion behavior was realized by taking the van der Waals force as well as magnetic dipole-dipole force into consideration. The uniformly dispersed polymer immobilized MNP (5 nm) was used for the preparation of MAPM. The hydrodynamic size and bulk morphology of MAPM were studied by dynamic light scattering and high-resolution transmission electron microscopy. The anticancer drug (DOX) was encapsulated into the MAPM. The magnetic field triggers cell uptake of MAPM micelles preferentially toward targeted cells compare to untargeted ones. The cell viabilities of MAMP, DOX-encapsulated MAPM, and free DOX were studied against HeLa cell by MTT assay. In vitro release profile displayed about 51.5% release of DOX from MAPM (just after 1 h) under the influence of high frequency alternating magnetic field (HFAMF; prepared in-house device). The DOX release rate has also been tailored by on-demand application of HFAMF.

Nano-Bio Engineered Carbon Dot-Peptide Functionalized Water Dispersible Hyperbranched Polyurethane for Bone Tissue Regeneration

The present study delves into a combined bio-nano-macromolecular approach for bone tissue engineering. This approach relies on the properties of an ideal scaffold material imbued with all the chemical premises required for fostering cellular growth and differentiation. A tannic acid based water dispersible hyperbranched polyurethane is fabricated with bio-nanohybrids of carbon dot and four different peptides (viz. SVVYGLR, PRGDSGYRGDS, IPP, and CGGKVGKACCVPTKLSPISVLYK) to impart target specific in vivo bone healing ability. This polymeric bio-nanocomposite is blended with 10 wt% of gelatin and examined as a non-invasive delivery vehicle. In vitro assessment of the developed polymeric system reveals good osteoblast adhesion, proliferation, and differentiation. Aided by this panel of peptides, the polymeric bio-nanocomposite exhibits in vivo ectopic bone formation ability. The study on in vivo mineralization and vascularization reveals the occurrence of calcification and blood vessel formation. Thus, the study demonstrates carbon dot/peptide functionalized hyperbranched polyurethane gel for bone tissue engineering application.

PAMAM (generation 4) incorporated gelatin 3D matrix as an improved dermal substitute for skin tissue engineering

The study explored the prospects of PAMAM (generation 4) applicability in gelatin based scaffolds for skin tissue engineering. The effect of PAMAM on physico-chemical and biological characteristics of gelatin scaffolds was evaluated. Gelatin scaffolds (with/without PAMAM) were prepared by lyophilization, chemically crosslinked by glutaraldehyde and characterized for their morphology (pore size), chemical features (bond nature), water adsorption, biodegradation and biological compatibility. The study demonstrated that addition of PAMAM did not significantly alter the pore size distribution or porosity of the scaffolds. However, water adsorption potential and collagenase mediated degradation significantly enhanced over period of the study. Both the scaffolds (with/without PAMAM) were highly biocompatible and hemocompatible. PAMAM (G4) blended scaffolds showed relatively higher cellular adhesion and proliferation of both keratinocytes and fibroblasts with an improved gene expression profile of native collagen type I of fibroblasts. Moreover, expression of angiogenesis inducing genes, HIF1α and VEGF were also higher in PAMAM blended gelatin matrix. Also, PAMAM incorporated gelatin matrix showed a slower rate of drug release which confirms its suitability for therapeutic delivery during wound healing. These results clearly suggest that blending PAMAM (G4) into the matrix could provide an additional support to scaffold assisted wound healing.

Development of gelatin/carboxymethyl chitosan/nano-hydroxyapatite composite 3D macroporous scaffold for bone tissue engineering applications

The present study delineates a relatively simpler approach for fabrication of a macroporous three-dimensional scaffold for bone tissue engineering. The novelty of the work is to obtain a scaffold with macroporosity (interconnected networks) through a combined approach of high stirring induced foaming of the gelatin/carboxymethyl chitosan (CMC)/nano-hydroxyapatite (nHAp) matrix followed by freeze drying. The fabricated macroporous (SGC) scaffold had a greater pore size, higher porosity, higher water retention capacity, slow and sustained enzymatic degradation rate along with higher compressive strength compared to that of non-macroporous (NGC, prepared by conventional freeze drying methodology) scaffold. The biological studies revealed the increased percentage of viability, proliferation, and differentiation as well as higher mineralization of differentiated human Whartons jelly MSC microtissue (wjhMSC-MT) on SGC as compared to NGC scaffold. RT-PCR also showed enhanced expression level of collagen type I, osteocalcin and Runx2 when seeded on SGC. μCT and histological analysis further revealed a penetration of cellular spheroid to a greater depth in SGC scaffold than NGC scaffold. Furthermore, the effect of cryopreservation on microtissue survival on the three-dimensional construct revealed significant higher viability upon revival in macroporous SGC scaffolds. These results together suggest that high stirring based macroporous scaffolds could have a potential application in bone tissue engineering.

Decellularized Caprine liver extracellular matrix as a 2D substrate coating and 3D hydrogel platform for vascularized liver tissue engineering

Development of a vascularized liver tissue construct is a need of an hour to circumvent the current demand of liver transplantation in health care sector. An appropriate matrix must support liver cell viability, functionality, and development of microvasculature. With this perspective, here, we report the use of decellularized caprine liver extracellular matrix (CLECM) derived hydrogel for tissue engineering applications. First, CLECM was used as a substrate coating material for 2D hepatocyte culture. HepG2 cells cultured on CLECM‐coated surface showed higher albumin, urea, glycogen, and GAGs synthesis in comparison with collagen‐coated surface (taken as control for the study). Thereafter, the cells were encapsulated in CLECM hydrogels for 3D culture. In CLECM hydrogels, HepG2 cells showed highly differentiated and polarized phenotype with the appearance of bile canaliculi‐like structures and enhanced expression of mature hepatocyte markers. We further showed that CLECM hydrogels also supported the development of microvasculature in vitro, thus making it a suitable candidate for development of a prevascularized liver tissue construct. In conclusion, we proved the superiority of CLECM over collagen for 2D/3D human hepatocyte and endothelial cell culture. CLECM could serve as an efficient biomaterial platform in the development of a liver tissue construct for application in tissue engineering.

Ectopic vascularized bone formation by human mesenchymal stem cell microtissues in a biocomposite scaffold

Three-dimensional multicellular human bone marrow mesenchymal stem cells (hBM-MSCs) are showing a great promise in the repair of bone tissue due to its osteogenic differentiation potential, mimicking in vivo microenvironment and immunomodulatory property. In the present study, the potential of hBM-MSC microtissues (MTs) in combination with a biocomposite material to form vascularized bone-like tissue at an ectopic site in an immunocompromised mouse was evaluated. The scaffold was fabricated using gelatin, carboxymethyl cellulose, polyvinyl alcohol and nano-hydroxyapatite (GCnHP) by the freeze-drying method. The physico-chemico-biological characteristics were compared with control scaffold devoid of polyvinyl alcohol (GCnH). The scaffolds (GCnH and GCnHP) were highly porous and had interconnected pores. GCnHP showed higher mechanical strength, higher water adsorption and a lower rate of collagenase-mediated degradation in comparison to GCnH. The scaffolds also supported growth and proliferation of hBM-MSCs MTs and subsequent differentiation into osteoblast-like cells. The differentiated cells showed matrix mineralization and high expression of runX2, alkaline phosphatase, collagen type 1 and osteocalcin genes. A high expression of VEGF was also observed suggesting the potential of hBM-MSC MTs to induce angiogenesis. H&E and Massons trichrome staining of the 4-weeks in vivo implanted scaffold revealed the presence of newly synthesized collagen and infiltration of host vasculature. IHC assessment showed expression of osteocalcin and osterix. These results demonstrate the efficacy of the combination of hBM-MSC MTs and biocomposite material as a promising approach for in vivo non-load bearing bone tissue repair for future clinical and various regenerative medicine applications.

Molecular Mechanisms Associated with Particulate and Soluble Heteroglycan Mediated Immune Response

Immune responses are outcomes of complex molecular machinery which occur inside the cells. Unravelling the cellular mechanisms induced by immune stimulating molecules such as glycans and determining their structure‐function relationship are therefore important factors to be assessed. With this viewpoint, the present study identifies the functional receptor binding unit of a well characterized heteroglycan and also delineates the cellular and molecular processes that are induced upon heteroglycan binding to specific cell surface receptors in immune cells. The heteroglycan was acid hydrolysed and it was revealed that 10–30 kDa fractions served as the functional receptor binding unit of the molecule. Increasing the size of 10–30 kDa heteroglycan showed prominent immune activity. The whole soluble heteroglycan was also conjugated with hyperbranched dendrimers so as to generate a particulate form of the molecule. Dectin‐1 and TLR2 were identified as the major receptors in macrophages that bind to particulate as well as soluble form of the heteroglycan and subsequently caused downstream signaling molecules such as NF‐κβ and MAPK to get activated. High levels of 1L‐1β and IL‐10 mRNA were observed in particulate heteroglycan treated macrophages, signifying that increasing the size and availability of the heteroglycan to its specific receptors is pertinent to its biological functioning. Upregulated expression of PKC and iNOS were also noted in particulate heteroglycan treated RAW 264.7 cells than the soluble forms. Taken together, our results indicate that biological functions of immunomodulatory heteroglycan are dependent on their size and molecular weight. J. Cell. Biochem. 117: 1580–1593, 2016.