Cellular response to cyclic compression of tissue engineered intervertebral disk constructs composed of electrospun polycaprolactone

There is lack of investigation capturing the complex mechanical interaction of tissue engineered IVD (intervertebral disc) constructs in physiologically-relevant environmental conditions. In this study, mechanical characterisation of anisotropic eletrospinning (ES) substrates made of polycaprolactone (PCL) was carried out in wet and dry conditions and viability of human bone marrow derived mesenchymal stem cells (hMSCs) seeded within double layers of ES PCL was also studied. Cyclic compression of IVD-like constructs composed of an agarose core confined by ES PCL double-layers was implemented using a bioreactor and the cellular response to the mechanical stimulation was evaluated. Tensile tests showed decrease of elastic modulus of ES PCL as the angle of stretching increased and at 60° stretching angle in wet, maximum ultimate tensile strength was observed. Based on the configuration of IVD-like constructs, the calculated circumferential stress experienced by the ES PCL double layers was 40 times of the vertical compressive stress. Confined compression of IVD-like constructs at 5% and 10% displacement dramatically reduced cell viability, particularly at 10%, although cell presence in small and isolated area can still be observed after mechanical conditioning. Hence, material mechanical properties of tissue-engineered scaffolds, composed of fibril structure of polymer with low melting point, are affected by the testing condition. Circumferential stress induced by axial compressive stimulation, conveyed to the ES PCL double-layer wrapped around an agarose core, can affect the viability of cells seeded at the interface, depending on the mechanical configuration and magnitude of the load

Microstructural and mechanical characteristics of PHEMA-based nanofibre-reinforced hydrogel under compression

Natural network-structured hydrogels (e.g. bacterial cellulose (BC)) can be synthesised with specific artificial hydrogels (e.g. poly(2-hydroxyethyl methacrylate)(PHEMA)) to form a tougher and stronger nanofibre-reinforced composite hydrogel, which possesses micro- and nano-porous structure. These synthetic hydrogels exhibit a number of advantages for biomedical applications, such as good biocompatibility and better permeability for molecules to pass through. In this paper, the mechanical properties of this nanofibre-reinforced hydrogel containing BC and PHEMA have been characterised in terms of their tangent modulus and fracture stress/strain by uniaxial compressive testing. Numerical simulations based on Mooney-Rivlin hyperelastic theory are also conducted to understand the internal stress distribution and possible failure of the nanofibre-reinforced hydrogel under compression. By comparing the mechanical characteristics of BC, PHEMA, and PHEMA-based nanofibre reinforced hydrogel (BC-PHEMA) under the compression, it is possible to develop a suitable scaffold for tissue engineering on the basis of fundamental understanding of mechanical and fracture behaviours of nanofibre-reinforced hydrogels

Three-dimensional hypoxic culture of human mesenchymal stem cells encapsulated in a photocurable, biodegradable polymer hydrogel: a potential injectable cellular product for nucleus pulposus regeneration

Nucleus pulposus (NP) tissue damage can induce detrimental mechanical stresses and strains on the intervertebral disc, leading to disc degeneration. This study demonstrates the potential of a novel, photo-curable, injectable, synthetic polymer hydrogel (pHEMA-co-APMA grafted with polyamidoamine (PAA)) to encapsulate and differentiate human mesenchymal stem cells (hMSC) towards a NP phenotype under hypoxic conditions which could be used to restore NP tissue function and mechanical properties. Encapsulated hMSC cultured in media (hMSC and chondrogenic) displayed good cell viability up to day 14. The genotoxicity effects of ultraviolet (UV) on hMSC activity confirmed the acceptability of 2.5min of UV light exposure to cells. Cytotoxicity investigations revealed that hMSC cultured in media containing p(HEMA-co-APMA) grafted with PAA degradation product (10% and 20%v/v concentration) for 14days significantly decreased the initial hMSC adhesion ability and proliferation rate from 24h to day 14. Successful differentiation of encapsulated hMSC within hydrogels towards chondrogenesis was observed with elevated expression levels of aggrecan and collagen II when cultured in chondrogenic media under hypoxic conditions, in comparison with culture in hMSC media for 14days. Characterization of the mechanical properties revealed a significant decrease in stiffness and modulus values of cellular hydrogels in comparison with acellular hydrogels at both day 7 and day 14. These results demonstrate the potential use of an in vivo photo-curable injectable, synthetic hydrogel with encapsulated hMSC for application in the repair and regeneration of NP tissue

Three-dimensional hypoxic culture of human mesenchymal stem cells encapsulated in a photocurable, biodegradable polymer hydrogel: a potential injectable cellular product for nucleus pulposus regeneration

Nucleus pulposus (NP) tissue damage can induce detrimental mechanical stresses and strains on the intervertebral disc, leading to disc degeneration. This study demonstrates the potential of a novel, photo-curable, injectable, synthetic polymer hydrogel (pHEMA-co-APMA grafted with polyamidoamine (PAA)) to encapsulate and differentiate human mesenchymal stem cells (hMSC) towards a NP phenotype under hypoxic conditions which could be used to restore NP tissue function and mechanical properties. Encapsulated hMSC cultured in media (hMSC and chondrogenic) displayed good cell viability up to day 14. The genotoxicity effects of ultraviolet (UV) on hMSC activity confirmed the acceptability of 2.5min of UV light exposure to cells. Cytotoxicity investigations revealed that hMSC cultured in media containing p(HEMA-co-APMA) grafted with PAA degradation product (10% and 20%v/v concentration) for 14days significantly decreased the initial hMSC adhesion ability and proliferation rate from 24h to day 14. Successful differentiation of encapsulated hMSC within hydrogels towards chondrogenesis was observed with elevated expression levels of aggrecan and collagen II when cultured in chondrogenic media under hypoxic conditions, in comparison with culture in hMSC media for 14days. Characterization of the mechanical properties revealed a significant decrease in stiffness and modulus values of cellular hydrogels in comparison with acellular hydrogels at both day 7 and day 14. These results demonstrate the potential use of an in vivo photo-curable injectable, synthetic hydrogel with encapsulated hMSC for application in the repair and regeneration of NP tissue

Electrospun polycaprolactone nano-fibers supports growth of human mesenchymal stem cells

Electrospun polycaprolactone (PCL) layers with sub-micron fibres arranged in random, semi-aligned and aligned manners were prepared and their anisotropic mechanical property were further characterised at various stretching angle. Proof of principle study was carried out to assess biocompatibility of electrospun layers with human mesenchymal stem cells (hMSCs). Cell adhesion, viability and orientation outcomes showed that aligned PCL nano-fibers can be applied as a template to guide the alignment of hMSCs and could be a promising scaffold for tissue engineering of anisotropic annulus fibrosus in spinal inter-vertebral disc

Calcium Stearate as an Effective Alternative to Poly(vinyl alcohol) in Poly-Lactic-<i>co</i>-Glycolic Acid Nanoparticles Synthesis

Poly­(d,l-lactic-<i>co</i>-glycolic acid) (PLGA) nanoparticles (NPs) are among the most studied systems for drug and gene targeting. So far, the synthesis of stable and uniform PLGA NPs has involved the use of a large excess of polyvinyl surfactants such as poly­(vinyl alcohol) (PVA) and polyvinylpyrrolidone (PVP), whose removal requires multistep purification procedures of high ecological and economic impact. Hence the development of environment-friendly and cost-effective synthetic procedures for the synthesis of PLGA NPs would effectively boost their use in clinics. This work aims to address this issue by investigating more efficacious alternatives to the so far employed polyvinyl surfactants. More specifically, we developed an innovative synthetic process to achieve stable and uniformly distributed PLGA NPs that involves the use of calcium stearate (CSt), gaining benefits of its high biocompatibility and efficacy at low concentrations and avoiding consequently expensive purification steps. With the help of minimum quantities of polysorbate 60 and sorbitane monostearate, CSt-stabilized PLGA NPs with different sizes and structures were synthesized. The influence of CSt on the encapsulation efficiency of bioactive molecules has been also investigated. The effective encapsulation of both hydrophobic (curcumin) and hydrophilic (fibrinogen labeled with Alexa647) biomolecules into NPs was demonstrated by confocal microscopy, and their release quantified by spectrofluorimetric analyses. Finally, degradation and cytotoxicity studies showed that CSt stabilized NPs were stable under physiological conditions and with good biocompatibility, thus looking promising for further investigation as controlled release devices

Magnetic Patterning by Electron Beam-Assisted Carbon Lithography

We report on the proof of principle of a scalable method for writing the magnetic state by electron-stimulated molecular dissociative adsorption on ultrathin Co on Re(0001). Intense microfocused low-energy electron beams are used to promote the formation of surface carbides and graphitic carbon through the fragmentation of carbon monoxide. Upon annealing at the CO desorption temperature, carbon persists in the irradiated areas, whereas the clean surface is recovered elsewhere, giving origin to chemical patterns with nanometer-sharp edges. The accumulation of carbon is found to induce an in-plane to out-of-plane spin reorientation transition in Co, manifested by the appearance of striped magnetic domains. Irradiation at doses in excess of 1000 L of CO followed by ultrahigh vacuum annealing at 380 °C determines the formation of a graphitic overlayer in the irradiated areas, under which Co exhibits out-of-plane magnetic anisotropy. Domains with opposite magnetization are separated here by chiral Neél walls. Our fabrication protocol adds lateral control to spin reorientation transitions, permitting to tune the magnetic anisotropy within arbitrary regions of mesoscopic size. We envisage applications in the nano-engineering of graphene-spaced stacks exhibiting the desired magnetic state and properties

An Efficient Cu_<i>x</i>O Photocathode for Hydrogen Production at Neutral pH: New Insights from Combined Spectroscopy and Electrochemistry

Light-driven water splitting is one of the most promising approaches for using solar energy in light of more sustainable development. In this paper, a highly efficient p-type copper­(II) oxide photocathode is studied. The material, prepared by thermal treatment of CuI nanoparticles, is initially partially reduced upon working conditions and soon reaches a stable form. Upon visible-light illumination, the material yields a photocurrent of 1.3 mA cm^–2 at a potential of 0.2 V vs a reversible hydrogen electrode at mild pH under illumination by AM 1.5 G and retains 30% of its photoactivity after 6 h. This represents an unprecedented result for a nonprotected Cu oxide photocathode at neutral pH. The photocurrent efficiency as a function of the applied potential was determined using scanning electrochemical microscopy. The material was characterized in terms of photoelectrochemical features; X-ray photoelectron spectroscopy, X-ray absorption near-edge structure, fixed-energy X-ray absorption voltammetry, and extended X-ray absorption fine structure analyses were carried out on pristine and used samples, which were used to explain the photoelectrochemical behavior. The optical features of the oxide are evidenced by direct reflectance spectroscopy and fluorescence spectroscopy, and Mott–Schottky analysis at different pH values explains the exceptional activity at neutral pH

Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors

<div><p>Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.</p></div

Additional file 1: Table S1. of Tissue distribution and acute toxicity of silver after single intravenous administration in mice: nano-specific and size-dependent effects

Body weight gain and relative organ weight (%) after IV administration of differently coated AgNPs. Table S2. Silver tissue concentration determined by ICP-MS after IV administration of AgNPs and AgAc. Figure S1. Silver tissue concentration after IV administration of differently coated AgNPs and AgAc. Figure S2. Principal component analysis of silver tissue concentration data. Figure S3. Histology of spleen and liver after IV administration of 10 mg Ag/Kg in mice. Figure S4. Histology of liver from mice treated with 10 nm AgNPs. (PDF 728 kb