Magnetically responsive tropoelastin hydrogel as a platform for soft tissue regeneration applications

Publicado em "European Cells and Materials. ISSN 1473-2262. Vol. 33, Suppl. 2, 2017 (0006)"The natural polymer tropoelastin is a structural protein of ECM of tissues requiring elasticity as part of their function, including ligaments and tendons. Tropoelastin has an innate capacity of self-assembly into high-order structures, and together with elastic resilience, structural stability and bioactivity bring forth pleasant singularities in adopting it as a building block to fabricate hydrogels. Moreover, easy tailoring of properties can be attained via incorporation of specific components into the polymeric network, including magnetic nanoparticles (MNPs), which are beneficial for on-demand therapies. Thus, the main goal of this work consisted in developing a magnetically responsive tropoelastin (MagTro) hydrogel as a platform to study the response of tendon cells to a mechanical stimulus induced by application of an external magnetic field (EMF). For this purpose, to first produce hydrogels, a solution of recombinant human tropoelastin was first freeze-dried overnight inside a mould and then chemically cross-linked inside an open desiccator via vapour glutaraldehyde. Thereafter, MagTro hydrogels were obtained through in situ precipitation of MNPs by immersing tropoelastin hydrogels in FeCl2 and FeCl3 solution overnight and secondly by soaking them in NaOH. Hydrogels were then analysed morphologically by Scanning Electron Microscopy (SEM and Cryo-SEM). Enzyme-triggered degradation was studied after 72h at 37oC in a human neutrophil elastase solution. Hydrogels exhibited a quick magnetic responsiveness to an EMF (Fig.1). Interestingly, MagTro hydrogels exhibited smaller pores as observed by Cryo-SEM. This feature can be tuned according to different soft tissue requirements by controlling different parameters of the fabrication process. Additionally, the release of tropoelastin into solution decreased, which suggests the formation of a surface coating of MNPs on tropoelastin network, protecting the hydrogel from a faster degradation. Preliminary results also indicate that cultured cells are viable and spread at the surface of the hydrogel. The application of an EMF to cell-laden MagTro hydrogels will be further investigated. Overall, the streamlined fabrication of MagTro hydrogels was successfully attained and the hydrogel formulation represents a promising potential platform for soft tissue regeneration.The authors acknowledge to BEAM-Master Joint Mobility Project an EU Australian cooperation in Biomedical Engineering Grant Agreement, 2014-1843/001 001-CPT EU-ICI-ECP and to FCT–Fundação para a Ciência e a Tecnologia in the framework of FCT-POPH-FSE, RC-A PhD grant SFRH/BD/96593/2013 and MEG grant IF/00685/2012.info:eu-repo/semantics/publishedVersio

A platelet lysate antibacterial bioactive patch for tendon repair

Platelet lysate (PL) is a class of platelet-rich hemoderivatives produced by cryogenic disruption of platelet concentrates, originating a pool of supra-physiological concentrations of growth factors (GFs) that is being widely explored in the medical field, namely in sports medicine and orthopaedics. In this concern, patch augmentation strategies have been receiving increased attention as the basis for the development of novel biomaterials aiming at tendon regeneration. In the present work, we assessed PL- membranes as prospective bioinstructive patches under the hypothesis that tendon cells positively respond to PL-derived biochemical signals. For this purpose, PL membranes were fabricated as previously described by Babo et al1 and characterized in terms of degradation, PL-derived proteins and GF release profiles. Cell behaviour was studied in terms of metabolic activity and proliferation, as well as extracellular matrix (ECM) production by culturing human tendon-derived cells (hTDCs) up to 21 days. In addition, the potential of PL membranes as antibacterial surfaces for biomedical implants was evaluated against Staphylococcus aureus ATCC 29213 by determining the number of viable counts, as well as biofilm formation and distribution up to 72h, using PDMS films as controls. Overall, our results showed that PL membranes remained stable for up to 30 days in PBS. In addition, PL-derived proteins, as well as specific GFs like basic fibroblast growth factor (bFGF) and platelet derived growth factor (PDGF)-BB followed a typical controlled release profile, as described by Babo et al1. Regarding the biological performance, PL-membranes were able to control the proliferation of seeded hTDCs, as demonstrated by maintenance of DNA content over 21 days of culture, in comparison to the controls in standard culture plastic. This result strongly suggests that PL-membranes can avoid an extensive proliferative phase, which in vivo is responsible for the formation of scar tissue, a major concern during tendon healing. These cells were metabolically active over time in culture and deposited tendon-related ECM proteins, including collagen types 1 and 3 and tenascin-C. Additionally, PL- membranes exhibited a significantly reduced number of viable counts of S. aureus, together with diminished bacteria adherence after 24h of incubation. No biofilm formation was observed in comparison to PDMS controls. Altogether, our results demonstrate that these PL-membranes can modulate cellular activity in situ, acting as a reservoir of bioactive molecules derived from PL, which supports their application as bioinstructive and protective patches for tendon regeneration. Finally, exploring the multitude of features of crosslinked PL proteins can potentially uncover uncharted prospective applications in regenerative medicine. References: 1. Babo P. Inflamm Regen. 2014; 34:33-44. Acknowledgements: The authors thank FCTâ Fundação para a Ciência e a Tecnologia in the framework of FCT-POPH-FSE, RC-A PhD grant SFRH/BD/96593/2013, ARF Post-Doc grant SFRH/BPD/100760/2014, and MEG grant IF/00685/2012. FCT–Fundação para a Ciência e a Tecnologia in the framework of FCT-POPH-FSE, RC-A PhDgrant SFRH/BD/96593/2013, ARF Post-Doc grant SFRH/BPD/100760/2014, and MEG grant F/00685/2info:eu-repo/semantics/publishedVersio

Simulated hypergravity induces changes in human tendon-derived cells: from cell morphology to gene expression

Gravity influences physical and biological processes, having an impact on development, as well as homeostasis of living systems. The musculoskeletal system is comprised of several mechano- responsive tissues and altered gravitational forces are known to influence distinct properties, including bone mineral density and skeletal muscle mass. This is particularly relevant in a near- weightlessness (microgravity) environment, which is found during spaceflight and, not less importantly, during bed resting. Over the years, several studies have been conducted under simulated conditions of altered gravity owing to the advances on ground-based facilities, such as bioreactors for microgravity / hypo-gravity (1g) studies. Interestingly, microgravity-induced alterations are comparable to tissue degeneration caused by disuse and ageing. In turn, exposing musculoskeletal tissues to hypergravity may constitute a way of simulating (over)loading or, eventually, to be used as a measure to rescue cell phenotype after exposure to near-weightlessness conditions. Different studies have focused on bone, cartilage and skeletal muscle, but effects on tendons and ligaments have been underappreciated. Therefore, we evaluated the influence of increasing g-levels (5g, 10g, 15g and 20g) and different hypergravity exposure periods (4 and 16 h) on the behaviour of human tendon- derived cells (hTDCs). For this purpose, hTDCs were exposed to simulated hypergravity conditions using the Large Diameter Centrifuge (LDC) from the European Space Research and Technology Centre (ESTEC, ESA, The Netherlands). Human TDCs cultured under standard conditions (1g, normogravity, Earth gravity force) were used as controls. The effects of hypergravity on the viability of hTDCs, as well as on the expression of tendon related markers at the gene level were evaluated. Simulated hypergravity resulted in a reduced cell content after 16 h independently of g-level, as determined by DNA quantification. Additionally, the different g-levels studied led to changes in cell and cytoskeleton morphology. Strikingly, a 16-hour period of exposure resulted in alterations of gene expression profiles. Overall, gene expression of tendon-related markers, including collagen types I (col1a1) and III (col3a1), scleraxis (scx), tenomodulin (tnmd), decorin (dcn) and tenascin (tnc), seemed to be increased upon hypergravity stimulation and in comparison to cells cultured under control conditions. Altogether, these results highlight that altered gravity, particularly simulated hypergravity, has an influence on the phenotype of tendon cells, opening new avenues for research focused on using altered gravity as a model for overloading-induced tendon tissue injury or as measure to rescue the phenotype of degenerated tendon cells. Acknowledgements The authors would like to thank ESA Education Office for Spin Your Thesis! 2016 programme. R.C-A acknowledges the PhD grant SFRH/BD/96593/2013 from FCT â Fundação para a Ciência e a Tecnologia. SFRH/BD/96593/2013 from FCT –Fundação para a Ciência e a Tecnologiainfo:eu-repo/semantics/publishedVersio

Bioinstructive Layer-by-Layer-Coated Customizable 3D Printed Perfusable Microchannels Embedded in Photocrosslinkable Hydrogels for Vascular Tissue Engineering

The development of complex and large 3D vascularized tissue constructs remains the major goal of tissue engineering and regenerative medicine (TERM). To date, several strategies have been proposed to build functional and perfusable vascular networks in 3D tissue-engineered constructs to ensure the long-term cell survival and the functionality of the assembled tissues after implantation. However, none of them have been entirely successful in attaining a fully functional vascular network. Herein, we report an alternative approach to bioengineer 3D vascularized constructs by embedding bioinstructive 3D multilayered microchannels, developed by combining 3D printing with the layer-by-layer (LbL) assembly technology, in photopolymerizable hydrogels. Alginate (ALG) was chosen as the ink to produce customizable 3D sacrificial microstructures owing to its biocompatibility and structural similarity to the extracellular matrices of native tissues. ALG structures were further LbL coated with bioinstructive chitosan and arginine–glycine–aspartic acid-coupled ALG multilayers, embedded in shear-thinning photocrosslinkable xanthan gum hydrogels and exposed to a calcium-chelating solution to form perfusable multilayered microchannels, mimicking the biological barriers, such as the basement membrane, in which the endothelial cells were seeded, denoting an enhanced cell adhesion. The 3D constructs hold great promise for engineering a wide array of large-scale 3D vascularized tissue constructs for modular TERM strategies.publishe

Enzymatically degradable, starch-based layer-by-layer films: application to cytocompatible single-cell nanoencapsulation

The build-up and degradation of cytocompatible nanofilms in a controlled fashion have great potential in biomedical and nanomedicinal fields, including single-cell nanoencapsulation (SCNE). Herein, we report the fabrication of biodegradable films of cationic starch (c-ST) and anionic alginate (ALG) by electrostatically driven layer-by-layer (LbL) assembly technology and its application to the SCNE. The [c-ST/ALG] multilayer nanofilms, assembled either on individual Saccharomyces cerevisiae or on the 2D flat gold surface, degrade on demand, in a cytocompatible fashion, via treatment with α-amylase. Their degradation profiles are investigated, while systematically changing the α-amylase concentration, by several surface characterization techniques, including quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. DNA incorporation in the LbL nanofilms and its controlled release, upon exposure of the nanofilms to an aqueous α-amylase solution, are demonstrated. The highly cytocompatible nature of the film-forming and -degrading conditions is assessed in the c-ST/ALG-shell formation and degradation of S. cerevisiae. We envisage that the cytocompatible, enzymatic degradation of c-ST-based nanofilms paves the way for developing advanced biomedical devices with programmed dissolution in vivo.publishe

In vitro and in vivo assessment of magnetically actuated biomaterials and prospects in tendon healing

Aim: To expand our understanding on the effect of magnetically actuated biomaterials in stem cells, inflammation and fibrous tissue growth. Materials & methods: Magnetic biomaterials were obtained by doping iron oxide particles into starch poly-ϵ-caprolactone (SPCL) to create two formulations, magSPCL-1.8 and 3.6. Stem cell behavior was assessed in vitro and the inflammatory response, subcutaneously in Wistar rats. Results: Metabolic activity and proliferation increased significantly overtime in SPCL and magSPCL-1.8. Electromagnetic fields attenuated the presence of mast cells and macrophages in tissues surrounding SPCL and magSPCL-1.8, between weeks 1 and 9. Macrophage reduction was more pronounced for magSPCL-1.8, which could explain why this material prevented growth of fibrous tissue overtime. Conclusion: Magnetically actuated biomaterials have potential to modulate inflammation and the growth of fibrous tissue.This work was supported by POLARIS funded under FP7-REPGOT and Incentivo/SAU/LA0026/2014 from the Foundation for Science and Technology. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.info:eu-repo/semantics/publishedVersio

The effects of platelet lysate patches on the activity of tendon-derived cells

Platelet-derived biomaterials are widely explored as cost-effective sources of therapeutic factors, holding a strong potential for endogenous regenerative medicine. Particularly for tendon repair, treatment approaches that shift the injury environment are explored to accelerate tendon regeneration. Herein, genipin-crosslinked platelet lysate (PL) patches are proposed for the delivery of human-derived therapeutic factors in patch augmentation strategies aiming at tendon repair. Developed PL patches exhibited a controlled release profile of PL proteins, including bFGF and PDGF-BB. Additionally, PL patches exhibited an antibacterial effect by preventing the adhesion, proliferation and biofilm formation by S. aureus, a common pathogen in orthopaedic surgical site infections. Furthermore, these patches supported the activity of human tendon-derived cells (hTDCs). Cells were able to proliferate over time and an up-regulation of tenogenic genes (SCX, COL1A1 and TNC) was observed, suggesting that PL patches may modify the behavior of hTDCs. Accordingly, hTDCs deposited tendon-related extracellular matrix proteins, namely collagen type I and tenascin C. In summary, PL patches can act as a reservoir of biomolecules derived from PL and support the activity of native tendon cells, being proposed as bioinstructive patches for tendon regeneration.Authors also acknowledge Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia in the framework of FCT-POPH-FSE, the PhD grant SFRH/BD/96593/2013 of R.C-A, the postdoctoral grant SFRH/BPD/100760/2014 of A.R.F. and the consolidator grant IF/00593/2015 of M.E.G.; to the project RL3-TECT-NORTE-07-0124-FEDER000020 cofinanced by ON.2 under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF) and the European Research Council for the project ComplexiTE grant agreement ERC-2012- ADG 20120216-321266.info:eu-repo/semantics/publishedVersio