Alan M. Punnoose

Electrospun type 1 collagen matrices preserving native ultrastructure using benign binary solvent for cardiac tissue engineering.

Electrospinning is a well-established technique that uses a high electric field to fabricate ultrafine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers, due to its widespread occurrence in nature and its biocompatibility. Electrospinning of collagen alone has been reported, with fluoroalcohols such as hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE), but the resultant collagen lost its characteristic ultrastructural integrity of D-periodicity 67 nm banding, confirmed by transmission electron microscopy (TEM), and the fluoroalcohols used were toxic to the environment. In this study, we describe the use of glacial acetic acid and DMSO to dissolve collagen and generate electrospun nanofibers of collagen type 1, which is non-toxic and economical. TEM analysis revealed the characteristic feature of native collagen triple helical repeats, showing 67 nm D-periodicity banding pattern and confirming that the ultrastructural integrity of the collagen was maintained. Analysis by scanning electron microscopy (SEM) showed fiber diameters in the range of 200–1100 nm. Biocompatibility of the three-dimensional (3D) scaffolds was established by MTT assays using rat skeletal myoblasts (L6 cell line) and confocal microscopic analysis of immunofluorescent-stained sections of collagen scaffolds for muscle-specific markers such as desmin and actin. Primary neonatal rat ventricular cardiomyocytes (NRVCM) seeded onto the collagen scaffolds were able to maintain their contractile function for a period of 17 days and also expressed higher levels of desmin when compared with 2D cultures. We report for the first time that collagen type 1 can be electrospun without blending with copolymers using the novel benign solvent combination, and the method can be potentially explored for applications in tissue engineering.

Electrospun polycaprolactone matrices with tensile properties suitable for soft tissue engineering

The extracellular environment is a complex network of functional and structural components that impart chemical and mechanical stimuli that affect cellular function and fate. Cell differentiation on three dimensional scaffolds is also determined by the modulus of the substrate. Electrospun PCL nanofibers, which mimic the extra cellular matrix, have been developed with a wide variety of solvents and their combinations. The various studies have revealed that the solvents used influence the physical and mechanical properties, resulting in scaffolds with Youngs modulus in the range of 1.8–15.4 MPa, more suitable for engineering of hard tissue like bone. The current study describes the use of benign binary solvent-generated fibrous scaffolds with a Youngs modulus of 36.05 ± 13.08 kPa, which is almost 50 times lower than that of scaffolds derived from the commonly used solvents, characterized with myoblast, which can be further explored for applications in muscle and soft tissue engineering.

Gelatin electrospun nanofibrous matrices for cardiac tissue engineering applications

ABSTRACT The generation of in vitro tissue constructs using biomaterials and cardiac cells is a promising strategy for screening novel therapeutics and their effects on cardiac regeneration. Current cardiac mimetic tissue constructs are unable to stably maintain functional characteristics of cardiomyocytes for long-term cultures. The objective of our study was to fabricate and characterize nanofibrous matrices of gelatin for prolonged cultures of primary cardiomyocytes which previously has been used as copolymer or hydrogels. Gelatin nanofibrous matrices were successfully electrospun using a benign binary solvent, cross-linked without swelling and fusing and evaluated by scanning electron microscopy (SEM) and uniaxial tensile measurement. Scaffolds exhibited modulus 19.6 ± 3.6 kPa similar to native human myocardium tissue with fiber diameters of 200–600 nm and average porosity percentage of 49.9 ± 5.6. Myoblasts showed good cell adhesion and proliferation. Neonatal rat cardiomyocytes cultured on gelatin nanofibrous matrices showing synchronized contracting cardiomyocytes (beating) for 27 days were studied by video microscopy. Confocal microscopic analysis of immunofluorescence stained sections indicated the presence of cardiac specific Troponin T in long-term cultures. Semiquantitative RT-PCR analysis of 3D versus 2D cultures revealed enhanced expression of contractile protein desmin. Our studies show that the biophysical and mechanical properties of electrospun gelatin nanofibers are ideal for in vitro engineered cardiac constructs (ECC), to explore cardiac function in drug testing and tissue replacement. Together with stem cell techniques, they may be an ideal platform for prolongedin vitro studies in alternatives to animal usage for the pharmaceutical industry. GRAPHICAL ABSTRACT

Collagen 3 D fleece as scaffold for cardiac tissue engineering

BackgroundThe limited ability of cardiac muscle to regenerate after myocardial infarction motivates studies aimed at curative treatment options through cell engraftment. The purpose of this study was to test woven collagen fibres for possible use as 3 D scaffold for cardiac tissue engineering.MethodsNeonatal ventricular rat Cardiomyocytes were isolated and characterized using specific antibodies by immunocytochemistry and confocal microscopy following which they were seeded on collagen scaffolds.ResultsCollagen fleece fabricated out of woven collagen fibres harbored metabolically active cardiomyocytes. The video of the beating cells on the scaffold is substantial evidence that the collagen fleece could be used for the preparation of tailor made cardiac tissue construct.ConclusionCollagen fleece possesses the necessary tensile strength as well as the elasticity for maintaining pulsating cardiomyocytes. Their flexibility and compatibility project them as promising candidates for use in tailor made cardiac tissue constructs.

Antifibrotic effect of Centella asiatica Linn and asiatic acid on arecoline-induced fibrosis in human buccal fibroblasts.

AIM The aim of the present study was to investigate the in vitro antifibrogenic effects of Centella asiatica Linn (CA) and its bioactive triterpene aglycone asiatic acid (AA) on arecoline-induced fibrosis in primary human buccal fibroblasts (HBF). METHODS An ethanolic extract of CA was prepared, and AA was purchased commercially. High-performance thin-layer chromatography (HPTLC) was performed to quantify AA in the CA extract; colorimetric assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was performed to determine an half-maximal inhibitory concentration. HBF were cultured and stimulated with arecoline. The inhibitory effects of CA and AA at different concentrations were assessed using gene-expression studies on fibrosis-related markers: transforming growth factor-β1, collagen 1 type 2, and collagen 3 type 1. The stimulatory effect of arecoline and the inhibitory effect of AA on fibroblast morphology and extracellular matrix were assessed qualitatively using Masson trichrome stain. RESULTS The HPTLC analysis determined 1.2% AA per 100 g of CA extract. Arecoline produced a concentration-dependent increase in the fibrotic markers, treatment with CA significantly downregulated fibrotic markers at higher concentrations, and AA downregulated at lower concentrations. Arecoline altered fibroblast morphology and stained strongly positive for collagen, and AA treatment regained fibroblast morphology with faint collagen staining. CONCLUSION CA and AA can be used as antifibrotic agents.

Electrospun Type 1 Collagen Matrices Using a Novel Benign Solvent for Cardiac Tissue Engineering

Electrospinning is a well-established technique that uses a high electric field to fabricate ultra fine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers due to its biocompatibility and widespread occurrence in nature. Electrospinning of Collagen alone has been reported with fluoroalcohols such as Hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE), which are toxic to the environment. In this study we describe the use of a novel benign binary solvent to generate nanofibers of Collagen type 1, which is non-toxic and economical. Transmission electron microscopy (TEM) analysis revealed the characteristic feature of native collagen namely the 67 nm banding pattern, confirming that the triple helical structure was maintained. Scanning Electron Microscopy (SEM) analysis showed the fiber diameters to be in the 200-800 nm range. Biocompatibility of the three dimensional (3D) scaffolds was established by MTT assays using skeletal myoblasts and Confocal Microscopic analysis of immunofluorescent stained sections for muscle specific markers such as Desmin and Actin. Primary neonatal rat ventricular cardiomyocytes seeded onto the scaffolds were able to maintain their contractile function for a period of 17 days. Our work provides evidence that Collagen 1 can be electrospun without combining with other polymers using a novel benign solvent and we are currently exploring the potential of this approach for cardiac and skeletal muscle tissue engineering. This article is protected by copyright. All rights reserved.

Electrospun cellulose acetate phthalate nanofibrous scaffolds fabricated using novel solvent combinations biocompatible for primary chondrocytes and neurons

Electrospun nanofibres have been shown to exhibit extracellular matrix (ECM)-like characteristics required for tissue engineering in terms of porosity, flexibility, fibre organization and strength. This study focuses on developing novel cellulose acetate phthalate (CAP) scaffolds by electrospinning for establishing 3-D chondrocyte and neuronal cultures. Five solvent combinations were employed in fabricating the fibres, namely, acetone/ethanol (9:1), dimethylformamide/tetrahydrofuran/acetone (3:3:4), tetrahydrofuran/acetone (1:1), tetrahydrofuran/ethanol (1:1) and chloroform/methanol (1:1). The electrospun fibres were characterized by scanning electron microscopy (SEM) analysis and confirmed to be within the nanometre range. Based on the morphology of the fibers from SEM results, two solvent combinations such as acetone/ethanol and dimethylformamide/tetrahydrofuran/acetone were selected for stabilization as CAP exhibits a pH dependent solubility. Fourier-Transform Infrared (FTIR) analysis revealed the hydrolysis of CAP which was overcome by EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] and EDC/NHS (N-hydroxysuccinimide) cross-linking resulting in its stability (pH of 7.2) for three months. MTT [3-(4, 5-dimethylthiazol-2-yl)-1, 5-diphenyltetrazolium bromide] assay performed using L6 myoblast confirmed the biocompatibility of the scaffolds. 3-D primary chondrocyte and neuronal cultures were established on the scaffolds and maintained for a period of 10 days. H&E staining and SEM analysis showed the attachment of the chondrocytes and neurons on CAP scaffolds prepared using dimethylformamide/tetrahydrofuran/acetone and acetone/ethanol respectively.

Standardization of Human Corneal Endothelial Cell Isolation and the Use of Denuded Human Amniotic Membrane as a Scaffold for Human Corneal Endothelial cells

Objectives: To standardize the isolation of human corneal endothelial cells (HCECs) and to use the denuded human amniotic membrane (HAM) as a scaffold for isolated HCECs. Methods: Human amniotic membrane was denuded using 1.2 units/ml of Dispase II at 37°C for 60 minutes followed by mechanical scraping. Corneal endothelial and Descemet’s membrane sheets were peeled from human donor cadaveric eyes unfit for surgical use and enzymatically digested with 2 mg/ml of collagenase II solution at 37°C and 5% CO2 for 2 hrs. Isolated cells were resuspended in culture medium with supplements and plated onto uncoated cultureware for four hours to eliminate fibroblasts which adhere more rapidly than endothelial cells. After preplating, the non-adherent cells were seeded onto gelatin coated dishes or onto denuded amniotic membrane in OptiMEM media supplemented with growth factors. The cells were analyzed by microscopy for adherence and polygonal morphology. Results: Microscopy of the denuded amniotic membrane showed no epithelial cell remnants. Enzymatic digestion of cornea left behind acellular Descemet’s sheets with the endothelial cells floating individually or in clusters with preplating aiding in a more fibroblast free endothelial cell isolation. Few isolated cells managed to scaffold onto the amniotic membrane and retain that adhesion during subsequent media replacements. Conclusion: Prolonged Treatment of HAM using the mild enzyme Dispase-II results in denuding the membrane, which serves as a successful scaffold for harvested corneal endothelial cells. This approach may be further explored as a cost effective alternative for endothelial cell proliferation and as an in vitro model for corneal tissue engineering studies.