Organisation and dynamics of well-defined graft copolymers at the air-water interface

Novel amphiphilic graft copolymers with a backbone of poly(norbornene) (PNB) with poly(ethylene oxide) (PEO) grafts have been synthesised by a combination of ring opening metathesis and anionic polymerisation methods. The polymer has been prepared with hydrogenous and deuterated grafts and with grafts of different degrees of polymerisation. These graft copolymers spread at the air-water and air-PEO solution interface forming thin films and their organisation and dynamic behaviour is discussed. Monolayer behaviour was characterised from surface pressure isotherms and it was demonstrated that the shape of the isotherm is dependent on graft length and on the concentration of PEO in the subphase. Using neutron reflectometry the organisation of such spread films at the air-water interface have been obtained over a range of surface concentrations for each length of PEO graft. Data were analysed by both exact calculation methods and the partial kinematic approximation and the models adopted were verified by applying the model independent Bayesian analysis. All yield the same description i.e. the hydrophobic backbone remains at the uppermost surface while the PEO grafts penetrate the subphase. The PEO layer increases in thickness with increased surface concentration and graft length. In each case the rate of increase with surface concentration was initially rapid but above a critical concentration, a slower rate was observed. In this latter regime the variation of the tethered layer height scales with surface density (ơ) and degree of polymerisation of the graft (N) as, r(_s) = N(^1.06)ơ(^0.33),which agrees well with scaling and self consistent field theory of polymer brushes. The dynamic behaviour of each copolymer film spread on water has been studied using surface quasi-elastic light scattering. A resonance between the capillary and dilational waves is observed at identical surface concentrations for each copolymer film. The viscoelastic behaviour of the dilational mode is reminiscent of Kramers-Kronig relations. The phenomenon of resistive mode mixing was observed in frequency dependency studies

Human osteoblasts within soft peptide hydrogels promote mineralisation in vitro

Biomaterials that provide three-dimensional support networks for the culture of cells are being developed for a wide range of tissue engineering applications including the regeneration of bone. This study explores the potential of the versatile ionic-complementary peptide, FEFEFKFK, for such a purpose as this peptide spontaneously self-assembles into β-sheet-rich fibres that subsequently self-associate to form self-supporting hydrogels. Via simple live/dead cell assays, we demonstrated that 3 wt% hydrogels were optimal for the support of osteoblast cells. We went on to show that these cells are not only viable within the three-dimensional hydrogel but they also proliferate and produce osteogenic key proteins, that is, they behave like in vivo bone cells, over the 14-day period explored here. The gel elasticity increased over time when cells were present – in comparison to a decrease in control samples – indicating the deposition of matrix throughout the peptide scaffold. Moreover, significant quantities of calcium phosphate were deposited. Collectively, these data demonstrate that ionic-complementary octapeptides offer a suitable three-dimensional environment for osteoblastic cell function

A Systematic Comparative Assessment of the Response of Ovarian Cancer Cells to the Chemotherapeutic Cisplatin in 3D Models of Various Structural and Biochemical Configurations-Does One Model Type Fit All?

Epithelial Ovarian Cancer (EOC) is a silent, deadly and aggressive gynaecological disease with a relatively low survival rate. This has been attributed, to some extent, to EOC's high recurrence rate and resistance to currently available platinum-based chemotherapeutic treatment methods. Multiple groups have studied and reported the effect of chemotherapeutic agents on various EOC 3D in vitro models. However, there are very few studies wherein a direct comparative study has been carried out between the different in vitro 3D models of EOC and the effect of chemotherapy within them. Herein, we report, for the first time, a direct comprehensive systematic comparative study of three different 3D in vitro platforms, namely (i) spheroids, (ii) synthetic PeptiGels/hydrogels of various chemical configurations and (iii) polymeric scaffolds with coatings of various extracellular matrices (ECMs) on the cell growth and response to the chemotherapeutic (Cisplatin) for ovary-derived (A2780) and metastatic (SK-OV-3) EOC cell lines. We report that all three 3D models are able to support the growth of EOC, but for different time periods (varying from 7 days to 4 weeks). We have also reported that chemoresistance to Cisplatin, in vitro, observed especially for metastatic EOC cells, is platform-dependent, in terms of both the structural and biochemical composition of the model/platform. Our study highlights the importance of selecting an appropriate 3D platform for in vitro tumour model development. We have demonstrated that the selection of the best platform for producing in vitro tumour models depends on the cancer/cell type, the experimental time period and the application for which the model is intended

Modification of β-Sheet Forming Peptide Hydrophobic Face: Effect on Self-Assembly and Gelation

β-Sheet forming peptides have attracted significant interest for the design of hydrogels for biomedical applications. One of the main challenges is the control and understanding of the correlations between peptide molecular structure, the morphology, and topology of the fiber and network formed as well as the macroscopic properties of the hydrogel obtained. In this work, we have investigated the effect that functionalizing these peptides through their hydrophobic face has on their self-assembly and gelation. Our results show that the modification of the hydrophobic face results in a partial loss of the extended β-sheet conformation of the peptide and a significant change in fiber morphology from straight to kinked. As a consequence, the ability of these fibers to associate along their length and form large bundles is reduced. These structural changes (fiber structure and network topology) significantly affect the mechanical properties of the hydrogels (shear modulus and elasticity)

Biofabrication of the osteochondral unit and its applications: Current and future directions for 3D bioprinting

Multiple prevalent diseases, such as osteoarthritis (OA), for which there is no cure or full understanding, affect the osteochondral unit; a complex interface tissue whose architecture, mechanical nature and physiological characteristics are still yet to be successfully reproduced in vitro. Although there have been multiple tissue engineering-based approaches to recapitulate the three dimensional (3D) structural complexity of the osteochondral unit, there are various aspects that still need to be improved. This review presents the different pre-requisites necessary to develop a human osteochondral unit construct and focuses on 3D bioprinting as a promising manufacturing technique. Examples of 3D bioprinted osteochondral tissues are reviewed, focusing on the most used bioinks, chosen cell types and growth factors. Further information regarding the applications of these 3D bioprinted tissues in the fields of disease modelling, drug testing and implantation is presented. Finally, special attention is given to the limitations that currently hold back these 3D bioprinted tissues from being used as models to investigate diseases such as OA. Information regarding improvements needed in bioink development, bioreactor use, vascularisation and inclusion of additional tissues to further complete an OA disease model, are presented. Overall, this review gives an overview of the evolution in 3D bioprinting of the osteochondral unit and its applications, as well as further illustrating limitations and improvements that could be performed explicitly for disease modelling

Evaluation of a synthetic peptide-based bioink (PeptiInk Alpha 1) for in vitro 3D bioprinting of cartilage tissue models

Cartilage pathology in human disease is poorly understood and requires further research. Various attempts have been made to study cartilage pathologies using in vitro human cartilage models as an alternative for preclinical research. Three-dimensional (3D) bioprinting is a technique that has been used to 3D-bioprint cartilage tissue models in vitro using animal-derived materials such as gelatine or hyaluronan, which present challenges in terms of scalability, reproducibility, and ethical concerns. We present an assessment of synthetic self-assembling peptides as bioinks for bioprinted human in vitro cartilage models. Primary human chondrocytes were mixed with PeptiInk Alpha 1, 3D-bioprinted and cultured for 14 days, and compared with 3D chondrocyte pellet controls. Cell viability was assessed through LIVE/DEAD assays and DNA quantification. High cell viability was observed in the PeptiInk culture, while a fast decrease in DNA levels was observed in the 3D pellet control. Histological evaluation using hematoxylin and eosin staining and immunofluorescence labeling for SOX-9, collagen type II, and aggrecan showed a homogeneous cell distribution in the 3D-bioprinted PeptiInks as well as high expression of chondrogenic markers in both control and PeptiInk cultures. mRNA expression levels assessed by - qRT-PCR (quantitative real time-polymerase chain reaction) confirmed chondrogenic cell behavior. These data showed promise in the potential use of PeptiInk Alpha 1 as a bioprintable manufacturing material for human cartilage in vitro models

Trends in qualitative research in language teaching since 2000

This paper reviews developments in qualitative research in language teaching since the year 2000, focusing on its contributions to the field and identifying issues that emerge. Its aims are to identify those areas in language teaching where qualitative research has the greatest potential and indicate what needs to be done to further improve the quality of its contribution. The paper begins by highlighting current trends and debates in the general area of qualitative research and offering a working definition of the term. At its core is an overview of developments in the new millennium based on the analysis of papers published in 15 journals related to the field of language teaching and a more detailed description, drawn from a range of sources, of exemplary contributions during that period. Issues of quality are also considered, using illustrative cases to point to aspects of published research that deserve closer attention in future work, and key publications on qualitative research practice are reviewed

Functionalised peptide hydrogel for the delivery of cardiac progenitor cells.

Heart failure (HF) remains one of the leading causes of death worldwide; most commonly developing after myocardial infarction (MI). Since adult cardiomyocytes characteristically do not proliferate, cells lost during MI are not replaced. As a result, the heart has a limited regenerative capacity. There is, therefore, a need to develop novel cell-based therapies to promote the regeneration of the heart after MI. The delivery and retention of cells at the injury site remains a significant challenge. In this context, we explored the potential of using an injectable, RGDSP-functionalised self-assembling peptide - FEFEFKFK - hydrogel as scaffold for the delivery and retention of rat cardiac progenitor cells (CPCs) into the heart. Our results show that culturing CPCs in vitro within the hydrogel for one-week promoted their spontaneous differentiation towards adult cardiac phenotypes. Injection of the hydrogel on its own, or loaded with CPCs, into the rat after injury resulted in a significant reduction in myocardial damage and left ventricular dilation

ISO-1 Binding to the Tautomerase Active Site of MIF Inhibits Its Pro-inflammatory Activity and Increases Survival in Severe Sepsis

MIF is a proinflammatory cytokine that has been implicated in the pathogenesis of sepsis, arthritis, and other inflammatory diseases. Antibodies against MIF are effective in experimental models of inflammation, and there is interest in strategies to inhibit its deleterious cytokine activities. Here we identify a mechanism of inhibiting MIF pro-inflammatory activities by targeting MIF tautomerase activity. We designed small molecules to inhibit this tautomerase activity; a lead molecule, "ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)," significantly inhibits the cytokine activity in vitro. Moreover, ISO-1 inhibits tumor necrosis factor release from macrophages isolated from LPStreated wild type mice but has no effect on cytokine release from MIFdeficient macrophages. The therapeutic importance of the MIF inhibition by ISO-1 is demonstrated by the significant protection from sepsis, induced by cecal ligation and puncture in a clinically relevant time frame. These results identify ISO-1 as the first small molecule inhibitor of MIF proinflammatory activities with therapeutic implications and indicate the potential of the MIF active site as a novel target for therapeutic interventions in human sepsis

Designing Peptide/Graphene Hybrid Hydrogels through Fine-Tuning of Molecular Interactions

A recent strategy that has emerged for the design of increasingly functional hydrogels is the incorporation of nanofillers in order to exploit their specific properties to either modify the performance of the hydrogel or add functionality. The emergence of carbon nanomaterials in particular has provided great opportunity for the use of graphene derivatives (GDs) in biomedical applications. The key challenge when designing hybrid materials is the understanding of the molecular interactions between the matrix (peptide nanofibers) and the nanofiller (here GDs) and how these affect the final properties of the bulk material. For the purpose of this work, three gelling β-sheet-forming, self-assembling peptides with varying physiochemical properties and five GDs with varying surface chemistries were chosen to formulate novel hybrid hydrogels. First the peptide hydrogels and the GDs were characterized; subsequently, the molecular interaction between peptides nanofibers and GDs were probed before formulating and mechanically characterizing the hybrid hydrogels. We show how the interplay between electrostatic interactions, which can be attractive or repulsive, and hydrophobic (and π–π in the case of peptide containing phenylalanine) interactions, which are always attractive, play a key role on the final properties of the hybrid hydrogels. The shear modulus of the hydrid hydrogels is shown to be related to the strength of fiber adhesion to the flakes, the overall hydrophobicity of the peptides, as well as the type of fibrillar network formed. Finally, the cytotoxicity of the hybrid hydrogel formed at pH 6 was also investigated by encapsulating and culturing human mesemchymal stem cells (hMSC) over 14 days. This work clearly shows how interactions between peptides and GDs can be used to tailor the mechanical properties of the resulting hydrogels, allowing the incorporation of GD nanofillers in a controlled way and opening the possibility to exploit their intrinsic properties to design novel hybrid peptide hydrogels for biomedical applications