Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies

Skin fibrosis still constitutes an unmet clinical need. Although pharmacological strategies are at the forefront of scientific and technological research and innovation, their clinical translation is hindered by the poor predictive capacity of the currently available in vitro fibrosis models. Indeed, customarily utilised in vitro scarring models are conducted in a low extracellular matrix milieu, which constitutes an oxymoron for the in-hand pathophysiology. Herein, we coupled macromolecular crowding (enhances and accelerates extracellular matrix deposition) with transforming growth factor beta 1 (TGF beta 1; induces trans-differentiation of fibroblasts to myofibroblasts) in human dermal fibroblast cultures to develop a skin fibrosis in vitro model and to screen a range of anti-fibrotic families (corticosteroids, inhibitors of histone deacetylases, inhibitors of collagen crosslinking, inhibitors of TGF beta 1 and pleiotropic inhibitors of fibrotic activation). Data obtained demonstrated that macromolecular crowding combined with TGF beta 1 significantly enhanced collagen deposition and myofibroblast transformation. Among the anti-fibrotic compounds assessed, trichostatin A (inhibitors of histone deacetylases); serelaxin and pirfenidone (pleiotropic inhibitors of fibrotic activation); and soluble TGF beta receptor trap (inhibitor of TGF beta signalling) resulted in the highest decrease of collagen type I deposition (even higher than triamcinolone acetonide, the gold standard in clinical practice). This study further advocates the potential of macromolecular crowding in the development of in vitro pathophysiology models.Peer reviewe

The influence of animal species, gender and tissue on the structural, biophysical, biochemical and biological properties of collagen sponges

peer-reviewedAlthough collagen type I is extensively used in biomedicine, no study to-date has assessed how the properties of the produced scaffolds are affected as a function of species, gender and tissue from which the collagen was extracted. Herein, we extracted and characterised collagen from porcine and bovine, male and female and skin and tendon tissues and we subsequently fabricated and assessed the structural, biophysical, biochemical and biological properties of collagen sponges. All collagen preparations were of similar purity and free-amine content (p > 0.05). In general, the porcine groups yielded more collagen; had higher (p < 0.05) denaturation temperature and resistance to enzymatic degradation; and lower (p < 0.05) swelling ratio and compression stress and modulus than the bovine groups of the same gender and tissue. All collagen preparations supported growth of human dermal fibroblasts and exhibited similar biological response to human THP-1 monocytes. These results further illustrate the need for standardisation of collagen preparations for the development of reproducible collagen-based devices

Development and characterisation of cytocompatible polyester substrates with tunable mechanical properties and degradation rate

Although it has been repeatedly indicated the importance to develop implantable devices and cell culture substrates with tissue-specific rigidity, current commercially available products, in particular cell culture substrates, have rigidity values well above most tissues in the body. Herein, six resorbable polyester films were fabricated using compression moulding with a thermal presser into films with tailored stiffness by appropriately selecting the ratio of their building up monomers (e.g. lactide, glycolide, trimethylene carbonate, dioxanone, ε-caprolactone). Typical NMR and FTIR spectra were obtained, suggesting that the fabrication process did not have a negative effect on the conformation of the polymers. Surface roughness analysis revealed no apparent differences between the films as a function of polymer composition. Subject to polymer composition, polymeric films were obtained with glass transition temperatures from -52 °C to 61 °C; contact angles in water from 81 ° to 94 °; storage modulus from 108 MPa to 2,756 MPa and loss modulus from 8 MPa to 507 MPa (both in wet state, at 1 Hz frequency and at 37 °C); ultimate tensile strength from 8 MPa to 62 MPa, toughness from 23 MJ/m3 to 287 MJ/m3, strain at break from 3 % to 278 %, macro-scale Young's modulus from 110 MPa to 2,184 MPa (all in wet state); and nano-scale Young's modulus from 6 kPa to 15,019 kPa (in wet state). With respect to in vitro degradation in phosphate buffered saline at 37 °C, some polymeric films [e.g. poly(glycolide-lactide) 30 / 70] started degrading from day 7 (shortest timepoint assessed), whilst others [e.g. poly(glycolide-co-ε-caprolactone) 10 / 90] were more resilient to degradation up to day 21 (longest timepoint assessed). In vitro biological analysis using human dermal fibroblasts and a human monocyte cell line (THP-1) showed the potential of the polymeric films to support cell growth and controlled immune response. Evidently, the selected polymers exhibited properties suitable for a range of clinical indications.This work has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie, grant agreement no. 676338; the Widespread: Twinning, grant agreement no. 810850; and the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, grant agreement no. 866126. This work was also supported by Science Foundation Ireland, Career Development Award, grant agreement no. 15/CDA/3629 and Science Foundation Ireland/European Regional Development Fund, grant agreement no. 13/RC/2073. We would also like to thank Darlene Nebinger, Danielle Lord and Oswaldo Fabian from Medtronic North Haven, USA, for all their technical/experimental support

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

Influence of the Thermodynamic and Kinetic Control of Self‐Assembly on the Microstructure Evolution of Silk‐Elastin‐Like Recombinamer Hydrogels

Complex recombinant biomaterials that merge the self-assembling properties of different (poly)peptides provide a powerful tool for the achievement of specific structures, such as hydrogel networks, by tuning the thermodynamics and kinetics of the system through a tailored molecular design. In this work, elastin-like (EL) and silk-like (SL) polypeptides are combined to obtain a silk-elastin-like recombinamer (SELR) with dual selfassembly. First, EL domains force the molecule to undergo a phase transition above a precise temperature, which is driven by entropy and occurs very fast. Then, SL motifs interact through the slow formation of β-sheets, stabilized by H-bonds, creating an energy barrier that opposes phase separation. Both events lead to the development of a dynamic microstructure that evolves over time (until a pore size of 49.9 ± 12.7 µm) and to a delayed hydrogel formation (obtained after 2.6 h). Eventually, the network is arrested due to an increase in β-sheet secondary structures (up to 71.8 ± 0.8%) within SL motifs. This gives a high bond strength that prevents the complete segregation of the SELR from water, which results in a fixed metastable microarchitecture. These porous hydrogels are preliminarily tested as biomimetic niches for the isolation of cells in 3D cultures.Este trabajo forma parte de los proyectos de investigación MAT2016-78903-R, MAT2016-79435-R, RTI2018-096320-B-C22 y DTS19/00162 del Ministerio de Ciencia e Innovación, del proyecto VA317P18 de la Junta de Castilla y León, del proyecto 0624_2IQBIONEURO_6_E del programa Interreg V A España Portugal POCTEP y del Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

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

10.1038/srep08729Scientific Reports

Alternative uses for co-products: Harnessing the potential of valuable compounds from meat processing chains

peer-reviewedOpportunities for exploiting the inherent value of protein-rich meat processing co-products, in the context of increased global demand for protein and for sustainable processing systems, are discussed. While direct consumption maybe the most profitable route for some, this approach is influenced greatly by local and cultural traditions. A more profitable and sustainable approach may be found in recognizing this readily available and under-utilised resource can provide high value components, such as proteins, with targeted high value functionality of relevance to a variety of sectors. Applications in food & beverages, petfood biomedical and nutrition arenas are discussed. Utilization of the raw material in its entirety is a necessary underlying principle in this approach to help maintain minimum waste generation. Understanding consumer attitudes to these products, in particular when used in food or beverage systems, is critical in optimizing commercialization strategies.This work forms part of the ReValueProtein Research Project (Grant Award No. 11/F/043) which is supported by the Irish Department of Agriculture, Food and the Marine (DAFM) and the Food Institutional Research Measure (FIRM) both funded by the Irish Government under the National Development Plan 2007–2013.Department of Agriculture, Food and the Marin

Hypoxia preconditioning of bone marrow mesenchymal stem cells before implantation in orthopaedics

[No abstract available]This work is supported by the: European Commission, Horizon 2020, Marie Skłodowska-Curie Actions, Innovative Training Networks (ITN) Programme Tendon Therapy Train, under the grant agreement number 676338; Science Foundation Ireland, Career Development Award Programme, under the grant agreement number 15/CDA/3629; and Science Foundation Ireland and the European Regional Development Fund, under the grant agreement number 13/RC/2073

Hypoxia preconditioning of bone marrow mesenchymal stem cells before implantation in orthopaedics

[No abstract available]This work is supported by the: European Commission, Horizon 2020, Marie Skłodowska-Curie Actions, Innovative Training Networks (ITN) Programme Tendon Therapy Train, under the grant agreement number 676338; Science Foundation Ireland, Career Development Award Programme, under the grant agreement number 15/CDA/3629; and Science Foundation Ireland and the European Regional Development Fund, under the grant agreement number 13/RC/2073.peer-reviewe

Design and characterization of a three-layer collagen-based scaffold to modulate BMSC behaviour for enthesis regeneration

The enthesis is a specialised tissue interface between tendon and bone, essential for adequate force transmission. Following injuries and surgical repair, the enthesis is often not re-established and traditionally used tissue substitutes have lacked to reproduce the complexity of the native tissue. We hypothesised that a collagen-based three-layer scaffold that mimic the composition of the enthesis, in combination with bioactive molecules, will enhance the functional regeneration of the tissue.Financial support was received from the European Union, Horizon 2020 Programme, Grant Agreement 676338