A self-assembly based supramolecular bioink with hierarchical control As a new bioprinting tool

Tissue engineering aims to capture details of the extracellular matrix (ECM) that stimulate cell growth and tissue regeneration. Molecularly complex materials or advanced additive fabrication techniques are often used to capture aspects of the ECM. Promising biofabrication techniques often lack nano and molecular scale control, as well as materials that can recreate the natural ECM or selectively guide cell behaviour. On the other hand, complex biomaterials based on molecular self-assembly tend to lack reproducibility and order beyond the nanoscale. We propose a new material fabrication platform that integrates the benefits of bioprinting and molecular self-assembly to overcome the current major limitations. Our approach relies on the co-assembly of peptide amphiphiles (PAs) with biomolecules and/or proteins found in the ECM, whilst exploiting the droplet-on-demand (DoD) printing process. Taking advantage of the interfacial fluid forces during printing, it is possible to guide the self-assembly into aligned or disordered nanofibers, hydrogel structures of different geometries and sizes, surface topographies and higher-ordered structures made from multiple hydrogels. The co-assembly process can be performed during printing and in cell-friendly conditions, whilst exhibiting high cell viability (\u3e 88 %). Moreover, multiple cell types can be spatially distributed on the outside or embedded within the tuneable biomimetic scaffolds. The combination of self-assembly with 3D-bioprinting, provides a basis for a new biofabrication platform to create hydrogels of complex geometry, structural hierarchy and tuneable chemical composition. Please click Additional Files below to see the full abstract

Interfacial Self-Assembly to Spatially Organize Graphene Oxide Into Hierarchical and Bioactive Structures

Multicomponent self-assembly holds great promise for the generation of complex and functional biomaterials with hierarchical microstructure. Here, we describe the use of supramolecular co-assembly between an elastin-like recombinamer (ELR5) and a peptide amphiphile (PA) to organise graphene oxide (GO) flakes into bioactive structures across multiple scales. The process takes advantage of a reaction – diffusion mechanism to enable the incorporation and spatial organization of GO within multiple ELR5/PA layers. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and ImageJ software were used to demonstrate the hierarchical organisation of GO flakes within the ELR5/PA layers and the distribution profiles of GO throughout the ELR5/PA membranes. Furthermore,atomic force microscopy (AFM) revealed improved Young’s moduli of the ELR5/PA/GOmembranes compared to the ELR5/PA membranes. Lastly, we investigated biocompatibility of the ELR5/PA/GO membrane via various cell culture methods

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex-vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro

Bioengineered 3D models of human pancreatic cancer recapitulate in vivo tumour biology

Patient-derived in vivo models of human cancer have become a reality, yet their turnaround time is inadequate for clinical applications. Therefore, tailored ex vivo models that faithfully recapitulate in vivo tumour biology are urgently needed. These may especially benefit the management of pancreatic ductal adenocarcinoma (PDAC), where therapy failure has been ascribed to its high cancer stem cell (CSC) content and high density of stromal cells and extracellular matrix (ECM). To date, these features are only partially reproduced ex vivo using organoid and sphere cultures. We have now developed a more comprehensive and highly tuneable ex vivo model of PDAC based on the 3D co-assembly of peptide amphiphiles (PAs) with custom ECM components (PA-ECM). These cultures maintain patient-specific transcriptional profiles and exhibit CSC functionality, including strong in vivo tumourigenicity. User-defined modification of the system enables control over niche-dependent phenotypes such as epithelial-to-mesenchymal transition and matrix deposition. Indeed, proteomic analysis of these cultures reveals improved matrisome recapitulation compared to organoids. Most importantly, patient-specific in vivo drug responses are better reproduced in self-assembled cultures than in other models. These findings support the use of tuneable self-assembling platforms in cancer research and pave the way for future precision medicine approaches

Cross-linking of a biopolymer-peptide co-assembling system

Producción CientíficaThe ability to guide molecular self-assembly at the nanoscale into complex macroscopic structures could enable the development of functional synthetic materials that exhibit properties of natural tissues such as hierarchy, adaptability, and self-healing. However, the stability and structural integrity of these kinds of materials remains a challenge for many practical applications. We have recently developed a dynamic biopolymer-peptide co-assembly system with the capacity to grow and undergo morphogenesis into complex shapes. Here we explored the potential of different synthetic (succinimidyl carboxymethyl ester, poly (ethylene glycol) ether tetrasuccinimidyl glutarate and glutaraldehyde) and natural (genipin) cross-linking agents to stabilize membranes made from these biopolymer-peptide co-assemblies. We investigated the cross-linking efficiency, resistance to enzymatic degradation, and mechanical properties of the different cross-linked membranes. We also compared their biocompatibility by assessing the metabolic activity and morphology of adipose-derived stem cells (ADSC) cultured on the different membranes. While all cross-linkers successfully stabilized the system under physiological conditions, membranes cross-linked with genipin exhibited better resistance in physiological environments, improved stability under enzymatic degradation, and a higher degree of in vitro cytocompatibility compared to the other cross-linking agents. The results demonstrated that genipin is an attractive candidate to provide functional structural stability to complex self-assembling structures for potential tissue engineering or in vitro model applications.Ministerio de Economía, Industria y Competitividad (Project MAT2013-42473-R and MAT2015-68901R)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13, VA313U14 and VA015U

Towards an optimal microenvironment for nucleus pulposus regeneration: a glycobiology approach

Neck and low back pain are the two highest causes of job-related disability in the UK and the USA. These pathologies are strongly related to intervertebral disc degeneration (IVD). Disc degeneration diseases (DDD) are characterised by changes in extracellular matrix (ECM) composition which lead to an important disorganisation of IVD tissue. Better knowledge of IVD biology and DDD in the last decades has promoted the development of new tissue engineering approaches to restore the disc function from a biological viewpoint. The ultimate objective of this thesis was to develop an optimal functionalized cell delivery system using an ECMmimicking injectable hydrogel that enhances the production and the deposition of newly synthesised ECM to aid the regeneration of NP tissue. It was hypothesised that the modulation of the glycoenvironment of NP cells will promote the maintenance of their phenotype. In the first phase of this thesis, an injectable type II collagen hydrogel stabilised with poly(ethylene glycol)ether tetrasuccinimidyl glutarate and supplemented with hyaluronic acid was successfully developed. The hydrogel system was stable in culture and had the capability to support cell growth. In addition, NP cells maintained a low type I collagen expression and their cell morphology after culture in the hydrogel. These characteristics, in addition to its injectable properties, make this hydrogel a promising candidate as a carrier of cells for future translation in vivo. The results obtained in this study highlighted the importance of ECM composition on NP cell behaviour. Highly glycosylated, the ECM of IVD tissue plays a crucial role on cell behaviour and IVD biology. Therefore, as a step forward, the glycoenvironment of the IVD was mapped in an effort to understand IVD glycoenvironment and its impact on IVD biology. A subset of specific and selective histological markers to distinguish the cell and ECM phenotypes of NP, AF and cartilage tissue and their stage of maturation was identified. The detailed CS composition and quantity of chondroitin sulfates (CS) revealed a change in sulfation pattern of CS with maturity. The depletion of CS has been shown to greatly affect IVD biology of the intervertebral disc and CS were chosen for the investigations conducted in the last part of this thesis. Therefore, the behaviour of GAGs, specifically of CS, and xylosyltransferase I (XT-I) and glucuronyltransferase I (GTI), two key enzymes involved at crucial points of CS synthesis, was evaluated in a bovine ageing IVD model. Important changes in GAGs composition during disc ageing were highlighted in this study. CS, specifically, were affected at a structural and quantitative levels with important changes in sulfated disaccharide composition upon ageing. A correlation between the expressions of XT-I and GT-I and CS content was shown in this study. The delivery via electroporation restored the expression of both enzymes at a protein level. A trend, although not significant, towards the increase of CS production after delivery of XT-I and GT-I was seen. In accord with the results of this study, the best therapeutic approach to modulate the expression of GAGs might be a dual delivery of XT-I and GT-I or in combination with aggrecan protein core up-regulation. Glycans were shown in this thesis to be essential to IVD biology. A better understanding of their effects on cell behaviour will promote the development of new biological tissue engineering approaches for IVD regeneration

Hyperbranched PEGmethacrylate linear pDMAEMA block copolymer as an efficient non-viral gene delivery vector

A unique hyperbranched polymeric system with a linear poly-2-dimethylaminoethyl methacrylate (pDMAEMA) block and a hyperbranched polyethylene glycol methyl ether methacrylate (PEGMEMA) and ethylene dimethacrylate (EGDMA) block was designed and synthesized via deactivation enhanced atom transfer radical polymerisation (DE-ATRP) for efficient gene delivery. Using this unique structure, with a linear pDMAEMA block, which efficiently binds to plasmid DNA (pDNA) and hyperbranched polyethylene glycol (PEG) based block as a protective shell, we were able to maintain high transfection levels without sacrificing cellular viability even at high doses. The transfection capability and cytotoxicity of the polymers over a range of pDNA concentration were analysed and the results were compared to commercially available transfection vectors such as polyethylene imine (branched PEI, 25 kDa), partially degraded poly(amido amine) dendrimer (dPAMAM; commercial name: SuperFect (R)) in fibroblasts and adipose tissue derived stem cells (ADSCs). (c) 2012 Elsevier B.V. All rights reserved.peer-reviewe

Daily variations in dietary lysine content alter the expression of MurF-1 and atrogin-1 in chicken muscle

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AMPK regulates the S6K1 pathway and protein synthesis in avian QM7 myoblasts

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