Human tendon-derived cell sheets created by magnetic force-based tissue engineering hold tenogenic and immunomodulatory potential

Cell sheet technology and magnetic based tissue engineering hold the potential to become instrumen- tal in developing magnetically responsive living tissues analogues that can be potentially used both for modeling and therapeutical purposes. Cell sheet constructions more closely recreate physiological niches, through the preservation of contiguous cells and cell-ECM interactions, which assist the cellular guidance in regenerative processes. We herein propose to use magnetically assisted cell sheets (magCSs) constructed with human tendon- derived cells (hTDCs) and magnetic nanoparticles to study inflammation activity upon magCSs exposure to IL-1 β, anticipating its added value for tendon disease modeling. Our results show that IL-1 βinduces an inflammatory profile in magCSs, supporting its in vitro use to en- lighten inflammation mediated events in tendon cells. Moreover, the response of magCSs to IL-1 βis mod- ulated by pulsed electromagnetic field (PEMF) stimulation, favoring the expression of anti-inflammatory genes, which seems to be associated to MAPK(ERK1/2) pathway. The anti-inflammatory response to PEMF together with the immunomodulatory potential of magCSs opens new perspectives for their applicability on tendon regeneration that goes beyond advanced cell based modeling.This research was funded by the ERC CoG MagTendon (No. 772817), Fundação para a Ciência e Tecnologia (FCT) for the doctoral grant PD/BD/128089/2016 of A. Vinhas and the project MagTT PTDC/CTM-CTM/29930/2017 (POCI-01-0145-FEDER-29930), project Norte-01-0145-FEDER-02219015 supported by Norte Portugal Regional Operational Programme (NORTE 2020) and EC Twinning project Achilles (No. 810850)

Pulsed electromagnetic field modulates tendon cells response in il‐1β‐conditioned environment

Strategies aiming at controlling and modulating inflammatory cues may offer therapeutic solutions for improving tendon regeneration. This study aims to investigate the modulatory effect of pulsed electromagnetic field (PEMF) on the inflammatory profile of human tendonâ derived cells (hTDCs) after supplementation with interleukinâ 1β (ILâ 1β). ILâ 1β was used to artificially induce in-flammatory cues associated with injured tendon environments. The PEMF effect was investigated varying the frequency (5 or 17 Hz), intensity (1.5, 4, or 5 mT), and dutyâ cycle (10% or 50%) parameters to which ILâ 1βâ treated hTDCs were exposed to. A PEMF actuation with 4 mT, 5 Hz and a 50% duty cycle decreased the production of ILâ 6 and tumor necrosis factorâ α (TNFâ α), as well as the expression of TNFα, ILâ 6, ILâ 8, COXâ 2, MMPâ 1, MMPâ 2, and MMPâ 3, while ILâ 4, ILâ 10, and TIMPâ 1 expression increased. These results suggest that PEMF stimulation can modulate hTDCs response in an inflammatory environment holding therapeutic potential for tendon regenerative strategies.The authors thank Hospital da Prelada (Porto, Portugal) for providing tendon tissue and acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) for the doctoral grant PD/BD/128089/2016 and for MagTT project PTDC/CTM‐CTM/29930/2017 (POCI‐01‐0145‐FEDER‐29930), the project NORTE‐01‐0145‐FEDER‐000021 supported by Norte Portugal Regional Operational Programme (NORTE 2020), HORIZON 2020 under the TEAMING GRANT agreement No 739572—The Discoveries CTR and MagTendon No. 772817

Magnetic stimulation drives macrophage polarization in cell to–cell communication with IL-1β primed tendon cells

Inflammation is part of the natural healing response, but it has been simultaneously associated with tendon disorders, as persistent inflammatory events contribute to physiological changes that compromise tendon functions. The cellular interactions within a niche are extremely important for healing. While human tendon cells (hTDCs) are responsible for the maintenance of tendon matrix and turnover, macrophages regulate healing switching their functional phenotype to environmental stimuli. Thus, insights on the hTDCs and macrophages interactions can provide fundamental contributions on tendon repair mechanisms and on the inflammatory inputs in tendon disorders. We explored the crosstalk between macrophages and hTDCs using co-culture approaches in which hTDCs were previously stimulated with IL-1β. The potential modulatory effect of the pulsed electromagnetic field (PEMF) in macrophage-hTDCs communication was also investigated using the magnetic parameters identified in a previous work. The PEMF influences a macrophage pro-regenerative phenotype and favors the synthesis of anti-inflammatory mediators. These outcomes observed in cell contact co-cultures may be mediated by FAK signaling. The impact of the PEMF overcomes the effect of IL-1β-treated-hTDCs, supporting PEMF immunomodulatory actions on macrophages. This work highlights the relevance of intercellular communication in tendon healing and the beneficial role of the PEMF in guiding inflammatory responses toward regenerative strategies.This research was funded by the ERC CoG grant MagTendon (No. 772817), FCT Project Mag TT PTDC/CTM-CTM/29930/2017, and project NORTE-01-0145-FEDER-000021 supported by Norte Portugal Regional Operational Programme (NORTE 2020). A. Vinhas is funded by Fundação para a Ciência e Tecnologia (FCT)-doctoral grant PD/BD/128089/2016

Contactless resolution of inflammatory signals in tailored macrophage-based cell therapeutics

In recent years, nanotechnology-based microRNA (miR) therapeutic platforms have shown great promise for immunotherapy and tissue regeneration, despite the unmet challenge of achieving efficient and safe delivery of miRs. The transport of miRs offers precision and regulatory value for a myriad of biological processes and pathways, including the control of macrophage (MÏ ) functions and, consequently, the inflammatory cascades MÏ are involved in. Thus, enforcement of MÏ can boost the regenerative process and provide new solutions for diverse chronic pathologies. In this study, we sought to develop a magnetically guided transporter to deliver an miR-155 antagonist to M1-primed MÏ . Furthermore, we determined its modulatory effect in reprogramming MÏ from inflammatory to pro-regenerative phenotypes, with the aim of tissue healing and regenerative medicine approaches. This strategy combines contactless and high-precision control of MÏ , anticipating new functional miR carriers for targeted strategies controlled by extracorporeal action. The magnetoplexes SPION@PEI-miR were efficiently delivered into MÏ without compromising cell viability and successfully induced miR-mediated gene silencing by enhancing the expression of anti-inflammatory markers (IL4 and IL10) and the production of M2Ï -related markers (CD206 and IL4). Given its multimodal features, SPION@PEI-miR represents a simple, safe, and nonviral theranostic platform that enables imaging, tracking, and miR delivery with modulatory effects on immune cells.This research was funded by the European Research Council, Consolidator Grant MagTendon, grant number 772817 and by the FCT - Fundação para a Ciência e a Tecnologia, scholarship number SFRD/BD/144816/2019

Controlling Macrophage Polarization to Modulate Inflammatory Cues Using Immune-Switch Nanoparticles

The persistence of inflammatory mediators in tissue niches significantly impacts regenerative outcomes and contributes to chronic diseases. Interleukin-4 (IL4) boosts pro-healing phenotypes in macrophages (MÏ ) and triggers the activation of signal transducer and activator of transcription 6 (STAT6). Since the IL4/STAT6 pathway reduces MÏ responsiveness to inflammation in a targeted and precise manner, IL4 delivery offers personalized possibilities to overcome inflammatory events. Despite its therapeutic potential, the limited success of IL4-targeted delivery is hampered by inefficient vehicles. Magnetically assisted technologies offer precise and tunable nanodevices for the delivery of cytokines by combining contactless modulation, high tissue penetration, imaging features, and low interference with the biological environment. Although superparamagnetic iron oxide nanoparticles (SPION) have shown clinical applicability in imaging, SPION-based approaches have rarely been explored for targeted delivery and cell programming. Herein, we hypothesized that SPION-based carriers assist in efficient IL4 delivery to MÏ , favoring a pro-regenerative phenotype (M2Ï ). Our results confirmed the efficiency of SPION-IL4 and MÏ responsiveness to SPION-IL4 with evidence of STAT6-mediated polarization. SPION-IL4-treated MÏ showed increased expression of M2Ï associated-mediators (IL10, ARG1, CCL2, IL1Ra) when compared to the well-established soluble IL4. The ability of SPION-IL4 to direct MÏ polarization using sophisticated magnetic nanotools is valuable for resolving inflammation and assisting innovative strategies for chronic inflammatory conditions.This research was funded by the European Research Council, Consolidator Grant Magtendon, grant number 772817. FCT-Fundação para a Ciência e a Tecnologia, grant number SFDH/BD/144816/2019. FCT-Fundação para a Ciência e a Tecnologia under the Scientific Employment Stimulus—Individual Call: 2020.01157.CEECIND

Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts

During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and -smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca2+]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca2+]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca2+ in the extracellular fluid. The biphasic [Ca2+]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca2+]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling

Terrestrial behavior in titi monkeys (Callicebus, Cheracebus, and Plecturocebus) : potential correlates, patterns, and differences between genera

For arboreal primates, ground use may increase dispersal opportunities, tolerance to habitat change, access to ground-based resources, and resilience to human disturbances, and so has conservation implications. We collated published and unpublished data from 86 studies across 65 localities to assess titi monkey (Callicebinae) terrestriality. We examined whether the frequency of terrestrial activity correlated with study duration (a proxy for sampling effort), rainfall level (a proxy for food availability seasonality), and forest height (a proxy for vertical niche dimension). Terrestrial activity was recorded frequently for Callicebus and Plecturocebus spp., but rarely for Cheracebus spp. Terrestrial resting, anti-predator behavior, geophagy, and playing frequencies in Callicebus and Plecturocebus spp., but feeding and moving differed. Callicebus spp. often ate or searched for new leaves terrestrially. Plecturocebus spp. descended primarily to ingest terrestrial invertebrates and soil. Study duration correlated positively and rainfall level negatively with terrestrial activity. Though differences in sampling effort and methods limited comparisons and interpretation, overall, titi monkeys commonly engaged in a variety of terrestrial activities. Terrestrial behavior in Callicebus and Plecturocebus capacities may bolster resistance to habitat fragmentation. However, it is uncertain if the low frequency of terrestriality recorded for Cheracebus spp. is a genus-specific trait associated with a more basal phylogenetic position, or because studies of this genus occurred in pristine habitats. Observations of terrestrial behavior increased with increasing sampling effort and decreasing food availability. Overall, we found a high frequency of terrestrial behavior in titi monkeys, unlike that observed in other pitheciids

Estímulo magnético e materiais magneticamente responsivos para modular a inflamação e o seu impacto no comportamento celular para a regeneração do tendão

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisTendon pathologies are a significant cause for disability in an increasingly active and ageing population. There is a substantial gap on the understanding of molecular and cellular responses leading to the onset and evolution of tendinopathic conditions but growing evidence suggests that persistent inflammatory events play a key role and may contribute to either triggering tendon injuries and/or prevent regenerative processes. Therefore, inflammatory events are can be the ultimate target for assisting new therapeutic strategies. In this thesis, we investigated magnetic stimuli applying pulsed electromagnetic field (PEMF) in combination with magnetically responsive materials using complementary cell based approaches for guiding and stimulating tendon regeneration through modulation of the inflammatory response. We firstly explored the signature features of tendon- and ligament- derived cells as endogenous living agents with predictive tenogenic behavior, and the influence of cryopreservation on their functional behavior (Chapter IV). Subsequently, (Chapter V), we selected IL-1b, a powerful pro-inflammatory mediator, to artificially induce an inflammatory profile in human tendon-derived cells (hTDCs) in culture. With these studies, we established the exogenous supplementation of IL-1b as an in vitro inflammation model for tendon tissue which was used in further studies to assess PEMF role in controlling the inflammatory response of hTDCs (Chapter V), envisioning the design of non-invasive and remote-actuated systems to modulate pathophysiological responses. Macrophages have a pivotal role in healing and in the orchestration of the inflammation process. As macrophage communication with hTDCs may provide insights in tendon repair mechanisms, co-cultures of IL-1b-treated-hTDCs and macrophages were further investigated in Chapter VI. Taking advantage of a potential synergistic effect of PEMF action over magnetic nanoparticles, the response of IL-1b-treated-hTDCs was further assessed in different magnetic based strategies envisioning translational approaches. These included magnetic cell sheet constructions (Chapter VII), which provide more complex and dynamic cell to cell and cell-matrix interactions, and magnetic responsive polymeric membranes (Chapter VIII) to externally trigger and guide tendon derived cell responses. The achievements from this Thesis support the emergent role of magnetic based strategies in general and of PEMF in particular, in the modulation of tendon cell responses, antagonizing the effect of inflammatory mediators and consequently, stimulating tendon regeneration.As patologias de tendão constituem uma causa significativa de incapacidade numa população cada vez mais ativa e envelhecida. Há uma lacuna substancial na compreensão das respostas moleculares e celulares que despoletam e/ou permitem a evolução das condições tendinopáticas, embora estudos recentes sugiram que a presença de eventos inflamatórios persistentes contribua para o avanço das lesões tendinosas e, portanto, podem auxiliar em novas estratégias terapêuticas. Nesta tese, investigamos a influência de estímulos magnéticos na resposta celular, aplicando um campo eletromagnético pulsado (CEMP) ou um CEMP combinado com materiais magneticamente responsivos usando abordagens baseadas em células estaminais para orientar e estimular a reparação do tendão. Explorámos inicialmente as características das células derivadas de tendões e ligamentos como agentes endógenos com comportamento tenogénico preditivo e a influência da criopreservação na manutenção do seu fenótipo no Capítulo IV. Em seguida, no Capítulo V, selecionamos um mediador pro inflamatório, IL-1b para induzir um perfil inflamatório, in vitro, em células derivadas de tendão humano (hCDTs). Estabelecemos a suplementação exógena de IL-1b como um modelo de inflamação in vitro para abordagens de tendão que foi posteriormente usado para avaliar o papel do CEMP no controlo da resposta inflamatória de hCDTs (Capítulo V), prevendo a utilização de métodos não invasivos e sistemas remotamente ativados para modular as respostas fisiopatológicas. Os macrófagos têm um papel fundamental na cicatrização e na orquestração do processo inflamatório. A comunicação de macrófagos com hCDTs pode fornecer informações sobre os mecanismos de reparação do tendão, pelo que co culturas de hCDTs tratadas com IL-1b e macrófagos foram investigadas no Capítulo VI. Tirando vantagem de um efeito combinado do CEMP sobre nanopartículas magnéticas, a resposta de hCDTs tratadas com IL-1b foi ainda avaliada baseada em diferentes estratégias magnéticas que preveem abordagens translacionais. Estes incluíram matrizes celulares magnéticas (Capítulo VII), que fornecem interações célula-célula e célula-matriz mais complexas, e membranas poliméricas magnéticas (Capítulo VIII) para guiar as repostas celulares de células derivadas de tendão. Os resultados desta tese fornecem informações sobre o papel emergente das estratégias magnéticas em geral e do CEMP em particular, na modulação das respostas celulares do tendão, antagonizando o efeito dos mediadores inflamatórios e, consequentemente, estimulando a regeneração do tendão

O papel do "purinoma" na atividade dos miofibroblastos cardíacos: interação complexa entre transportadores, enzimas e recetores purinérgicos

Dissertação de mestrado em Bioquímica AplicadaOs fibroblastos cardíacos (FC) desempenham um papel crucial na remodelação cardíaca, nomeadamente nas alterações eletrofisiológicas e hemodinâmicas que seguem o enfarte do miocárdio. Durante a isquemia e reperfusão miocárdica são libertadas grandes quantidades de ATP e UTP, levando à ativação de diversos subtipos de recetores P2Y (metabotrópicos) e P2X (ionotrópicos). Desta forma, os nucleótidos de adenina e uracilo poderão desempenhar um papel crucial como mediadores da sinalização autócrina e/ou parácrina entre FC e entre estes e os cardiomiócitos. O fenótipo e a expressão de recetores purinérgicos P2 em culturas de FC de ratos Wistar adultos foram confirmados por imunofluorescência aplicada à microscopia confocal. As oscilações do cálcio intracelular ([Ca2+]i) foram monitorizadas usando um leitor de microplacas (Synergy HT, BioTek) após incubação das células com uma sonda fluorescente, Fluo-4NW (2.5 mM, 45 min a 37 ºC). A proliferação/viabilidade dos FC em cultura foi monitorizada utilizando o ensaio MTT. A produção colagénio do tipo I foi avaliada por histoquímica enzimática através da marcação com Sirius Red. Os FC de ratos Wistar adultos em cultura exibiram um fenótipo de miofibroblastos caraterizado pela imunorreatividade positiva contra o recetor do domínio discoide 2 (DDR2) e a proteína contrátil do músculo liso (α-SMA). Estas células apresentaram imunorreatividade contra os recetores P2Y1, P2Y2, P2Y4, P2Y11, P2Y12 e P2Y13. Os nucleótidos de adenina e uracilo (100 μM) aumentaram os níveis de [Ca2+]i com a seguinte potência: UTP (51±1,6%, n=11) ≈ ATP (48±1,2%, n=16) >> ADP (31±1,2%, n=15) ≈ UDP (28±1,8%, n=7). A incubação das culturas de FC com ADP (3-300 μM, n=2-15) causou um aumento dos níveis [Ca2+]i dependente da concentração, não sendo observado nenhum efeito atenuante na presença dos antagonistas dos recetores sensíveis ao ADP, P2Y1, P2Y12 e P2Y13, respetivamente MRS 2179 (10 μM, n=4), AR-C 66096 (0,1 μM, n=3) e MRS 2211 (10 μM, n=3).No entanto o efeito produzido pelo ADP (100 μM, n=13) foi significativamente (p<0,05) atenuado pelos antagonistas não-seletivos P2, suramina (100 μM, n=4) e RB-2 (100 μM, n=3). O envolvimento de recetores sensíveis ao UTP, P2Y2 e P2Y4, foi avaliado usando agonistas seletivos destes recetores, respetivamente MRS 2768 (10 μM, n=5) e MRS 4062 (10 μM, n=7); enquanto o último mimetizou o efeito do UTP, o agonista seletivo P2Y2 foi ineficaz. O envolvimento do recetor P2Y4 no controlo dos níveis de [Ca2+]i nos FC foi confirmado pela atenuação do efeito dos nucleótidos de uracilo (e.g. UTP) e adenina (e.g. ATP, ADP) na presença de RB-2 (100 μM, n=7). O ADP (30 μM, n=5) e o seu análogo estável, ADPßS (30 μM, n=8), aumentaram a proliferação e a produção de colagénio do tipo I das culturas de FC. A proliferação induzida pelo ADPßS foi atenuada (p<0,05) pelo antagonista seletivo do recetor P2Y1, MRS 2179 (300 nM, n=6), mas não pelos antagonistas dos recetores P2Y12 e P2Y13, AR-C 66096 (0,1 μM, n=6) e MRS 2211 (10 μM, n=6), respetivamente. Os dados sugerem que os recetores P2Y1, P2Y4 e P2Y11 podem afetar de forma diferencial os transientes de [Ca2+]i e a proliferação de FC. O recetor P2Y4 é o principal responsável pela elevação rápida dos níveis de [Ca2+]i induzida pelo UTP, ATP e ADP. Além disso, os resultados mostram que a ativação de recetores P2Y1 sensíveis ao ADP possui um efeito proliferativo (pró-fibrótico).Cardiac fibroblasts (CF) play crucial roles in cardiac remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. ATP and UTP are released in huge amounts during heart ischemia and reperfusion, strenghtening the activation of many subtypes of metabotropic P2Y and ionotropic P2X receptors. The adenine and uracil nucleotides may play a key role of autocrine/paracrine mediators. The phenotype and the expression of P2 purinoceptors in cultured CF from adult Wistar rats were confirmed by immunofluorescence confocal microscopy. Intracellular Ca2+ ([Ca2+]i) oscillations were monitored in the time-lapse mode using a microplate reader (Sinergy HT, BioTek) after loading the cells with the fluorescent Ca2+ dye, Fluo- 4NW (2.5 μM, 45 min at 37ºC). CF proliferation/viability was monitored using the MTT assay. Type I collagen production was evaluated using the Sirius Red assay. Cultured CF from adult rats exhibit a myofibroblast phenotype characterized by positive immunoreactivity against discoidin domain receptor 2 (DDR2) and α-smooth muscle actin (α-SMA). These cells exhibit immunoreactivity against P2Y1, P2Y2, P2Y4, P2Y11 P2Y12 and P2Y13 receptors. Adenine and uracil nucleotides increased [Ca2+]i oscillations. The rank potency order of the nucleotides (100 μM) was the following: UTP (51±8, n=11) ≈ ATP (48±9, n=16) >> ADP (31±10, n=15) ≈ UDP (28±7, n=7). Incubation of CF cultures with ADP (3-300 μM, n=2-15) concentration-dependently increased [Ca2+]I, not observed any effect in the presence of antagonists ADP-sensitive P2Y1, P2Y12 e P2Y13 receptors, respectively, MRS 2179 (10 μM, n=4), AR-C 66096 (0,1 μM, n=3) and MRS 2211 (10 μM, n=3). However the effect of ADP (100 μM, n=13) was significantly (p<0,05) attenuated by suramin (100 μM, n=4) and Reactive Blue-2 (100 μM, n=3). Involvement of UTP-sensitive P2Y2 and P2Y4 receptors were evaluated using MRS 2768 (10 μM, n=5) and MRS 4062 (10 μM, n=7), respectively; while the latter mimicked the effect of UTP, the P2Y2 agonist was ineffective. Involvement of the P2Y4 receptor was confirmed, because antagonism by Reactive Blue-2 (RB-2, 100 μM, n=7) was more effective than by Suramin (100 μM, n=10). UTP-induced [Ca2+]i oscillations were also attenuated by the P2Y11 receptor antagonist, NF340 (10 μM, n=12). ADP (30 μM, n=5) and ADPßS (30 μM, n=8) increased cells growth and type I collagen production in CF culture. ADPßS-induced proliferation was attenuated (p<0,05) by selective P2Y1, MRS 2179 (300 nM, n=6), but not P2Y12 and P2Y13 receptors with ARC 66096 (0,1 μM, n=6) and MRS 2211 (10 μM, n=6), respectively. Data suggest that P2Y1, P2Y4 and P2Y11 receptors differentially affect [Ca2+]i transients and proliferation of rat CF. P2Y4 receptor is the major responsible for the quick elevation of [Ca2+]i levels induced by UTP, ATP and ADP. Furthermore, results show that activation of P2Y1 possess a proliferative effect.Fundação para a Ciência e Tecnologia (FCT) - projetos PTDC/DTP-FTO/0802/2012 e PEst-OE/SAU/UI0215/201