Narayan C. Mishra

Fabrication and characterization of PCL/gelatin composite nanofibrous scaffold for tissue engineering applications by electrospinning method

In the present study, composite nanofibrous tissue engineering-scaffold consisting of polycaprolactone and gelatin, was fabricated by electrospinning method, using a new cost-effective solvent mixture: chloroform/methanol for polycaprolactone (PCL) and acetic acid for gelatin. The morphology of the nanofibrous scaffold was investigated by using field emission scanning electron microscopy (FE-SEM) which clearly indicates that the morphology of nanofibers was influenced by the weight ratio of PCL to gelatin in the solution. Uniform fibers were produced only when the weight ratio of PCL/gelatin is sufficiently high (10:1). The scaffold was further characterized by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric (TG) analysis, and X-ray diffraction (XRD). FT-IR and TG analysis indicated some interactions between PCL and gelatin molecules within the scaffold, while XRD results demonstrated crystalline nature of PCL/gelatin composite scaffold. Cytotoxicity effect of scaffold on L929 mouse fibroblast cells was evaluated by MTT assay and cell proliferation on the scaffold was confirmed by DNA quantification. Positive results of MTT assay and DNA quantification L929 mouse fibroblast cells indicated that the scaffold made from the combination of natural polymer (gelatin) and synthetic polymer (PCL) may serve as a good candidate for tissue engineering applications.

Surface modification of nanofibrous polycaprolactone/gelatin composite scaffold by collagen type I grafting for skin tissue engineering

In the present study, a tri-polymer polycaprolactone (PCL)/gelatin/collagen type I composite nanofibrous scaffold has been fabricated by electrospinning for skin tissue engineering and wound healing applications. Firstly, PCL/gelatin nanofibrous scaffold was fabricated by electrospinning using a low cost solvent mixture [chloroform/methanol for PCL and acetic acid (80% v/v) for gelatin], and then the nanofibrous PCL/gelatin scaffold was modified by collagen type I (0.2-1.5wt.%) grafting. Morphology of the collagen type I-modified PCL/gelatin composite scaffold that was analyzed by field emission scanning electron microscopy (FE-SEM), showed that the fiber diameter was increased and pore size was decreased by increasing the concentration of collagen type I. Fourier transform infrared (FT-IR) spectroscopy and thermogravimetric (TG) analysis indicated the surface modification of PCL/gelatin scaffold by collagen type I immobilization on the surface of the scaffold. MTT assay demonstrated the viability and high proliferation rate of L929 mouse fibroblast cells on the collagen type I-modified composite scaffold. FE-SEM analysis of cell-scaffold construct illustrated the cell adhesion of L929 mouse fibroblasts on the surface of scaffold. Characteristic cell morphology of L929 was also observed on the nanofiber mesh of the collagen type I-modified scaffold. Above results suggest that the collagen type I-modified PCL/gelatin scaffold was successful in maintaining characteristic shape of fibroblasts, besides good cell proliferation. Therefore, the fibroblast seeded PCL/gelatin/collagen type I composite nanofibrous scaffold might be a potential candidate for wound healing and skin tissue engineering applications.

Fabricating scaffolds by microfluidics

In this paper, we demonstrate for the first time the technique to using microfluidics to fabricate tissue engineering scaffolds with uniform pore sizes. We investigate both the bubble generation of the microfluidic device and the application of foam as a tissue engineering scaffold. Our microfluidic device consists of two concentric tapered channels, which are made by micropipettes. Nitrogen gas and aqueous alginate solution with Pluronic((R)) F127 surfactant are pumped through the inner and the outer channels, respectively. We observe rich dynamic patterns of bubbles encapsulated in the liquid droplets. The size of the bubble depends linearly on the gas pressure and inversely on the liquid flow rate. In addition, monodisperse bubbles self-assemble into crystalline structures. The liquid crystalline foams are further processed into open-cell solid foams. The novel foam gel was used as a scaffold to culture chondrocytes.

Fabrication and characterization of novel nano-biocomposite scaffold of chitosan-gelatin-alginate-hydroxyapatite for bone tissue engineering.

A novel nano-biocomposite scaffold was fabricated in bead form by applying simple foaming method, using a combination of natural polymers-chitosan, gelatin, alginate and a bioceramic-nano-hydroxyapatite (nHAp). This approach of combining nHAp with natural polymers to fabricate the composite scaffold, can provide good mechanical strength and biological property mimicking natural bone. Environmental scanning electron microscopy (ESEM) images of the nano-biocomposite scaffold revealed the presence of interconnected pores, mostly spread over the whole surface of the scaffold. The nHAp particulates have covered the surface of the composite matrix and made the surface of the scaffold rougher. The scaffold has a porosity of 82% with a mean pore size of 112±19.0μm. Swelling and degradation studies of the scaffold showed that the scaffold possesses excellent properties of hydrophilicity and biodegradability. Short term mechanical testing of the scaffold does not reveal any rupturing after agitation under physiological conditions, which is an indicative of good mechanical stability of the scaffold. In vitro cell culture studies by seeding osteoblast cells over the composite scaffold showed good cell viability, proliferation rate, adhesion and maintenance of osteoblastic phenotype as indicated by MTT assay, ESEM of cell-scaffold construct, histological staining and gene expression studies, respectively. Thus, it could be stated that the nano-biocomposite scaffold of chitosan-gelatin-alginate-nHAp has the paramount importance for applications in bone tissue-engineering in future regenerative therapies.

Fabrication and characterization of PCL/gelatin/chitosan ternary nanofibrous composite scaffold for tissue engineering applications

In the present study, we have fabricated a ternary composite nanofibrous scaffold from PCL/gelatin/chitosan, by electrospinning technique, using a solvent system—chloroform/methanol for polycaprolactone (PCL) and acetic acid for gelatin and chitosan, for tissue engineering applications. Field emission scanning electron microscopy (FE-SEM) was used to investigate the fiber morphology of the scaffold and it was found that the fiber morphology was influenced by the concentrations of PCL, gelatin, and chitosan in polymer solution during electrospinning. X-ray diffraction, Fourier transform infrared, and thermogravimetric (TG) analysis results showed some interactions among the molecules of PCL, gelatin, and chitosan within the scaffold. In-vitro cell culture studies were done by seeding L929 mouse fibroblasts on fabricated composite scaffold, which confirmed the cell viability, high cell proliferation rate, and cell adhesion on composite scaffold as indicated by MTT assay, DNA quantification, and FE-SEM analysis of cell-scaffold construct. Thus, the ternary composite scaffold made from the combination of PCL (synthetic polymer), gelatin, and chitosan (natural polymer) may find potential application in tissue engineering.

Antibiofilm activity of quercetin- encapsulated cytocompatible nanofibers against Candida albicans

In this study, nanofibers against pro dimorphic fungal sessile growth were developed. Quercetin was successfully encapsulated within poly(d,l-lactide-co-glycolide)–poly(ε-caprolactone) nanofibers using an electrospinning technique. Field emission scanning electron microscopy, fluorescent microscopy, and Fourier-transformed infrared spectrometer were used to confirm the formation as well as encapsulation of quercetin within the nanofibers. These fabricated nanofibers were further evaluated to determine the effectiveness of the antibiofilm activity against Candida albicans. The cytocompatibility of quercetin-encapsulated nanofibers was found to be similar to control and pure polymeric nanofibers based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay against human embryonic kidney (HEK-293) cell lines. These fabricated nanofibers potentially could be used as coatings on biomedical devices to inhibit microbial contaminations.

Fabrication and characterization of scaffold from cadaver goat-lung tissue for skin tissue engineering applications.

The present study aims to fabricate scaffold from cadaver goat-lung tissue and evaluate it for skin tissue engineering applications. Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue enzymatically, to have cell-free 3D-architecture of natural extracellular matrix. DNA quantification assay and Hematoxylin and eosin staining confirmed the absence of cellular material in the decellularized lung-tissue. SEM analysis of decellularized scaffold shows the intrinsic porous structure of lung tissue with well-preserved pore-to-pore interconnectivity. FTIR analysis confirmed non-denaturation and well maintainance of collagenous protein structure of decellularized scaffold. MTT assay, SEM analysis and H&E staining of human skin-derived Mesenchymal Stem cell, seeded over the decellularized scaffold, confirms stem cell attachment, viability, biocompatibility and proliferation over the decellularized scaffold. Expression of Keratin18 gene, along with CD105, CD73 and CD44, by human skin-derived Mesenchymal Stem cells over decellularized scaffold signifies that the cells are viable, proliferating and migrating, and have maintained their critical cellular functions in the presence of scaffold. Thus, overall study proves the applicability of the goat-lung tissue derived decellularized scaffold for skin tissue engineering applications.

Modification of Decellularized Goat-Lung Scaffold with Chitosan/Nanohydroxyapatite Composite for Bone Tissue Engineering Applications

Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue, and the scaffold was modified with chitosan/nanohydroxyapatite composite for the purpose of bone tissue engineering applications. MTT assay with osteoblasts, seeded over the chitosan/nanohydroxyapatite-modified decellularized scaffold, demonstrated significantly higher cell growth as compared to the decellularized scaffold without modification. SEM analysis of cell-seeded scaffold, after incubation for 7 days, represented a good cell adhesion, and the cells spread over the chitosan/nanohydroxyapatite-modified decellularized scaffold. Expression of bone-tissue-specific osteocalcin gene in the osteoblast cells grown over the chitosan/nanohydroxyapatite-modified decellularized scaffold clearly signifies that the cells maintained their osteoblastic phenotype with the chitosan/nanohydroxyapatite-modified decellularized scaffold. Therefore, it can be concluded that the decellularized goat-lung scaffold-modified with chitosan/nanohydroxyapatite composite, may provide enhanced osteogenic potential when used as a scaffold for bone tissue engineering.

Influence of quercetin and nanohydroxyapatite modifications of decellularized goat-lung scaffold for bone regeneration

In the present study, goat-lung scaffold was fabricated by decellularization of lung tissue and verified for complete cell removal by DNA quantification, DAPI and H&E staining. The scaffold was then modified by crosslinking with quercetin and nanohydroxyapatite (nHAp), and characterized to evaluate the suitability of quercetin-crosslinked nHAp-modified scaffold for regeneration of bone tissue. The crosslinking chemistry between quercetin and decellularized scaffold was established theoretically by AutoDock Vina program (in silico docking study), which predicted multiple intermolecular hydrogen bonding interactions between quercetin and decellularized scaffold, and FTIR spectroscopy analysis also proved the same. From MTT assay and SEM studies, it was found that the quercetin-crosslinked nHAp-modified decellularized scaffold encouraged better growth and proliferation of bone-marrow derived mesenchymal stem cells (BMMSCs) in comparison to unmodified decellularized scaffold, quercetin-crosslinked decellularized scaffold and nHAp-modified decellularized scaffold. Alkaline Phosphatase (ALP) assay results showed highest expression of ALP over quercetin-crosslinked nHAp-modified scaffold among all the tested scaffolds (unmodified decellularized scaffold, quercetin-crosslinked decellularized scaffold and nHAp-modified decellularized scaffold) indicating that quercetin and nHAp is very much efficient in stimulating the differentiation of BMMSCs into osteoblast cells. Alizarin red test quantified in vitro mineralization (calcium deposits), and increased expression of alizarin red over quercetin-crosslinked nHAp-modified scaffold indicating better stimulation of osteogenesis in BMMSCs. The above findings suggest that quercetin-crosslinked nHAp-modified decellularized goat-lung scaffold provides biomimetic bone-like microenvironment for BMMSCs to differentiate into osteoblast and could be applied as a potential promising biomaterial for bone regeneration.

Tooth Tissue Engineering: Potential and Pitfalls

All over the world a large number of people suffer from tooth diseases like dental caries, tooth abscess, and plaques. Tooth loss or damage, which occurs frequently in our society are generally repaired by applying several conventional methods, such as root-canal treatment, direct pulp capping and dental implants. These methods are quite painful, create damage to the surrounding tooth tissues and also may at times have adverse side-effects. The limitations of the conventional methods can be overcome by applying the concept of tooth tissue engineering. Tooth tissue engineering is the application of biosciences and engineering to regenerate a biofunctional tooth, which can be used to replace the missing tooth or repair the damaged tooth. Tissue engineering involves three key elements - cell, scaffold and growth factors, which interact with each other to regenerate a specific tissue. The success of tissue engineering depends on the proper selection of these three key elements and understanding the interactions among them. To bring us close to the realization of a tissue-engineered tooth, immense progress is going on in understanding how tooth is first developed, and there is a good advancement in tooth regeneration. In this review, “tooth tissue engineering” will be discussed, along with the recent advancements and challenges in bring a biofunctional tooth from laboratory out into clinical use.