Characterization of electrospun nanocomposite scaffolds and biocompatibility with adipose-derived human mesenchymal stem cells

Electrospun nanocomposite scaffolds were fabricated by encapsulating multi-walled carbon nanotubes (MWNT) in poly (lactic acid) (PLA) nanofibers. Scanning electron microscopy (SEM) confirmed the fabrication of nanofibers, and transmission electron microscopy identified the alignment and dispersion of MWNT along the axis of the fibers. Tensile testing showed an increase in the tensile modulus for a MWNT loading of 0.25 wt% compared with electrospun nanofibrous mats without MWNT reinforcement. Conductivity measurements indicated that the confined geometry of the fibrous system requires only minute doping to obtain significant enhancements at 0.32 wt%. Adipose-derived human mesenchymal stem cells (hMSCs) were seeded on electrospun scaffolds containing 1 wt% MWNT and 0 wt% MWNT, to determine the efficacy of the scaffolds for cell growth, and the effect of MWNT on hMSC viability and proliferation over two weeks in culture. Staining for live and dead cells and DNA quantification indicated that the hMSCs were alive and proliferating through day 14. SEM images of hMSCs at 14 days showed morphological differences, with hMSCs on PLA well spread and hMSCs on PLA with 1% MWNT closely packed and longitudinally aligned

Combinatorial scaffold morphologies for zonal articular cartilage engineering

AbstractArticular cartilage lesions are a particular challenge for regenerative medicine strategies as cartilage function stems from a complex depth-dependent organization. Tissue engineering scaffolds that vary in morphology and function offer a template for zone-specific cartilage extracellular matrix (ECM) production and mechanical properties. We fabricated multi-zone cartilage scaffolds by the electrostatic deposition of polymer microfibres onto particulate-templated scaffolds produced with 0.03 or 1.0mm3 porogens. The scaffolds allowed ample space for chondrocyte ECM production within the bulk while also mimicking the structural organization and functional interface of cartilage’s superficial zone. Addition of aligned fibre membranes enhanced the mechanical and surface properties of particulate-templated scaffolds. Zonal analysis of scaffolds demonstrated region-specific variations in chondrocyte number, sulfated GAG-rich ECM, and chondrocytic gene expression. Specifically, smaller porogens (0.03mm3) yielded significantly higher sGAG accumulation and aggrecan gene expression. Our results demonstrate that bilayered scaffolds mimic some key structural characteristics of native cartilage, support in vitro cartilage formation, and have superior features to homogeneous particulate-templated scaffolds. We propose that these scaffolds offer promise for regenerative medicine strategies to repair articular cartilage lesions

Application of Low-Frequency Alternating Current Electric Fields Via Interdigitated Electrodes: Effects on Cellular Viability, Cytoplasmic Calcium, and Osteogenic Differentiation of Human Adipose-Derived Stem Cells

Electric stimulation is known to initiate signaling pathways and provides a technique to enhance osteogenic differentiation of stem and/or progenitor cells. There are a variety of in vitro stimulation devices to apply electric fields to such cells. Herein, we describe and highlight the use of interdigitated electrodes to characterize signaling pathways and the effect of electric fields on the proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). The advantage of the interdigitated electrode configuration is that cells can be easily imaged during short-term (acute) stimulation, and this identical configuration can be utilized for long-term (chronic) studies. Acute exposure of hASCs to alternating current (AC) sinusoidal electric fields of 1 Hz induced a dose-dependent increase in cytoplasmic calcium in response to electric field magnitude, as observed by fluorescence microscopy. hASCs that were chronically exposed to AC electric field treatment of 1 V/cm (4 h/day for 14 days, cultured in the osteogenic differentiation medium containing dexamethasone, ascorbic acid, and β-glycerol phosphate) displayed a significant increase in mineral deposition relative to unstimulated controls. This is the first study to evaluate the effects of sinusoidal AC electric fields on hASCs and to demonstrate that acute and chronic electric field exposure can significantly increase intracellular calcium signaling and the deposition of accreted calcium under osteogenic stimulation, respectively

Morphological, electrical, and mechanical characterization of electrospun nanofiber mats containing multiwalled carbon nanotubes

ABSTRACT: This work focuses on the development of electrically conducting porous nanocomposite structures by the incorporation of multiwalled carbon nanotubes (MWNT) into electrospun poly(ethylene oxide) (PEO) nanofibers. Electron microscopy confirmed the presence of individual aligned MWNT encapsulated within the fibers and showed fiber morphologies with diameters of 100-200 nm. Electrical conductance measurements of the random nanofiber mats showed that by increasing the concentration of MWNT we were able to produce porous nanocomposite structures with dramatically improved electrical conductivity. Above a percolation threshold of 0.365 ( 0.09 MWNT weight percent (wt %) in PEO the conductance increased by a factor of 10 12 and then became approximately constant as the concentration of MWNT was further increased. Because of this percolation threshold, for a 1 wt % loading of MWNT, the conductivity is essentially maximized. Mechanical testing confirmed that the tensile strength did not change, and there was a 3-fold increase in the Young's modulus when comparing a 1 wt % MWNT loading to the pure electrospun PEO. Thus, the optimal MWNT concentration for PEO nanofiber mats with enhanced mechanical and electrical properties is ∼1 wt %

Hybrid gelation processes in enzymatically gelled gelatin:impact on nanostructure, macroscopic properties and cellular response

Physical, chemical and hybrid tilapia fish gelatin hydrogels were investigated by small-angle neutron scattering (SANS), molecular dynamic simulations and their biological effect in cell cultures studied; results from the different experimental techniques were then correlated and linked to the rheological properties of the gels (F. Bode et al., Biomacromolecules, 2011, 12, 3741–3752). Hydrogels were obtained by cross-linking with the microbial enzyme transglutaminase (mTGase) under two conditions: above and below gelatin physical gelation temperature (ca. 23 °C). Hydrogels cross-linked at 37 °C, from the sol-state, are referred to as ‘chemical’ gels (C); hydrogels cross-linked at 21 °C, thus with concurrent physical gelation, are referred to as ‘physical-co-chemical’ gels (PC). The SANS data were appropriately described by a combination of a Lorentzian and a power law model. For physical gels, the correlation length (ξ) obtained from the fits decreased linearly with gelatin concentration, from 42 to 26 Å for 3.5 to 10% w/w gelatin, respectively. Independently of gelation temperature, all physical gels at a given concentration showed a similar correlation length ξ (26 ± 2 Å), with no significant difference with the sol-state (23 ± 2 Å). In both C and PC gels, ξ increased with mTGase concentration over the range studied: 40 to 167 Å for 10 and 40 U mTGase per g gelatin in C gels (after 120 min cross-linking) and 40 to 82 Å for 10 and 40 U mTGase per g gelatin for PC gels. ξ reached a plateau at the highest mTGase concentration studied for both types of gels. In addition, kinetic studies on C gels revealed that ξ increased linearly with time in the first two hours and grew faster with increasing mTGase concentration. ξ values in the PC gels were smaller than in the corresponding C gels. Cell proliferation studies showed that the gels were compatible with cell growth and indicated no statistically relevant dependence on mTGase concentration for C gels. For PC gels, cell proliferation decreased with increases in mTGase concentration, by approximately 80% from 10 to 40 U mTGase per g gelatin. With the exception of the highest mTGase concentration studied, PC gels overall showed a slightly (but statistically significant) higher cell proliferation than the corresponding chemical gels

Anisotropic Fibrous Scaffolds for Articular Cartilage Regeneration

Articular cartilage lesions, which can progress to osteoarthritis, are a particular challenge for regenerative medicine strategies, as cartilage function stems from its complex depth-dependent microstructural organization, mechanical properties, and biochemical composition. Fibrous scaffolds offer a template for cartilage extracellular matrix production; however, the success of homogeneous scaffolds is limited by their inability to mimic the cartilage's zone-specific organization and properties. We fabricated trilaminar scaffolds by sequential electrospinning and varying fiber size and orientation in a continuous construct, to create scaffolds that mimicked the structural organization and mechanical properties of cartilage's collagen fibrillar network. Trilaminar composite scaffolds were then compared to homogeneous aligned or randomly oriented fiber scaffolds to assess in vitro cartilage formation. Bovine chondrocytes proliferated and produced a type II collagen and a sulfated glycosaminoglycan-rich extracellular matrix on all scaffolds. Furthermore, all scaffolds promoted significant upregulation of aggrecan and type II collagen gene expression while downregulating that of type I collagen. Compressive testing at physiological strain levels further demonstrated that the mechanical properties of trilaminar composite scaffolds approached those of native cartilage. Our results demonstrate that trilaminar composite scaffolds mimic key organizational characteristics of native cartilage, support in vitro cartilage formation, and have superior mechanical properties to homogenous scaffolds. We propose that these scaffolds offer promise in regenerative medicine strategies to repair articular cartilage lesions

Application of the Denovo Discrete Ordinates Radiation Transport Code to Large-Scale Fusion Neutronics

Fusion energy systems pose unique challenges to the modeling and simulation community. These challenges must be met to ensure the success of the ITER experimental fusion reactor. ITER's complex systems require detailed modeling that goes beyond the scale of comparable simulations to date. In this work, the Denovo radiation transport code was used to calculate neutron fluence and kerma for the JET streaming benchmark. This work was performed on the Titan supercomputer at the Oak Ridge Leadership Computing Facility. Denovo is a novel three-dimensional discrete ordinates transport code designed to be highly scalable. Sensitivity studies have been completed to examine the impact of several deterministic parameters. Results were compared against experiment as well as the MCNP and Shift Monte Carlo codes