24 research outputs found

    Improved production of acetate and propionate by Propionibacterium freudenreichii

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    Propionibacterium freudenreichii is a commercially important bacterium that is well-known for its role as ripening starter in the cheese industry and its probiotic potential. These bacteria may beneficially modulate the intestinal ecosystem and can exert anti-neoplastic effects via the production of short chain fatty acids (SCFAs), acetate and propionate. Several studies have demonstrated that the SCFA production by P. freudenreichii is responsible for its probiotic abilities. The aim of this work was to optimize the acetate and propionate production by P. freudenreichii towards its future use as a nutraceutical agent. In order to optimize the production of the abovementioned SCFAs in a minimal synthetic media the different composition of the several components were evaluated. Characterization of the acetate and propionate production in a medium mimicking the content of the human colon (MCHC) and a medium used by colorectal carcinoma cell lines (DMEM) was performed. The basal medium (BM) was found to be the most promising regarding the production of the SCFAs, showing 0.530 ¬Ī 0.011 g L-1 of biomass; high acetate and propionate yields (0.216 ¬Ī 0.001 g g-1 and 0.572 ¬Ī 0.002 g g-1, respectively), as well as high productivities (0.031 ¬Ī 0.000 g L-1 h-1 and 0.010 ¬Ī 0.000 g L-1 h-1, respectively). In the MCHC and DMEM media, it was possible to observe microbial growth (0.234 ¬Ī 0.006 g L-1 and 1.54 ¬Ī 0.00 g L-1, respectively); however the amounts of acetate and propionate were lower than the ones produced in BM medium. The results suggest that acetate and propionate production depends not only on the substrate type, but also on the medium constituents, being the simplest medium the one that show higher productivities as P. freudenreichii show low SCFA production when grown in MCHC and DMEM media. Future work will be conducted in order the increase bacteria growth and SCFA production in those media as this represents an essential feature for its use as a nutraceutical

    Chondrogenic differentiation induced by extracellular vesicles bound to a nanofibrous substrate

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    Extracellular vesicles (EVs) are being increasingly studied owing to its regenerative potential, namely EVs derived from human bone marrow mesenchymal stem cells (hBM-MSCs). Those can be used for controlling inflammation, repairing injury, and enhancing tissue regeneration. Differently, the potential of EVs derived from human articular chondrocytes (hACs) to promote cartilage regeneration has not been thoroughly investigated. This work aims to develop an EVs immobilization system capable of selectively bind EVs present in conditioned medium obtained from cultures of hACs or hBM-MSC. For that, an anti-CD63 antibody was immobilized at the surface of an activated and functionalized electrospun nanofibrous mesh. The chondrogenic potential of bound EVs was further assessed by culturing hBM-MSCs during 28 days under basal conditions. EVs derived from hACs cultured under differentiation medium or from chondrogenically committed hBM-MSCs induced a chondrogenic phenotype characterized by marked induction of SOX9, COMP, Aggrecan and Collagen type II, and matrix glycosaminoglycans synthesis. Indeed, both EVs immobilization systems outperformed the currently used chondroinductive strategies. These data show that naturally secreted EVs can guide the chondrogenic commitment of hBM-MSCs in the absence of any other chemical or genetic chondrogenic inductors based in medium supplementation.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD grant to M.R.C. (PD/BD/113797/2015) financed by the Doctoral Program on Advanced Therapies for Health (PATH) (FSE/POCH/PD/169/2013) and the project Cells4_IDs (PTDC/BTM-SAL/28882/2017). This work was also financed by the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125)

    The biomimetic surface topography of Rubus fruticosus leaves stimulate the induction of osteogenic differentiation of rBMSCs

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    The interaction between cells and biomaterials is essential for the success of biomedical applications in which the implantation of biomaterials in the human body is necessary. It has been demonstrated that material's chemical, mechanical, and structural properties can influence cell behaviour. The surface topography of biomaterials is a physical property that can have a major role in mediating cell√Ę material interactions. This interaction can lead to different cell responses regarding cell motility, proliferation, migration, and even differentiation. The combination of biomaterials with mesenchymal stem cells (MSCs) for bone regeneration is a promising strategy to avoid the need for autologous transplant of bone. Surface topography was also associated with the capacity to control MSCs differentiation. Most of the topographies studied so far involve machine-generated surface topographies. Herein, our strategy differentiates from the above mentioned since we selected natural surface topographies that can modulate cell functions for regenerative medicine strategies.√ā¬†Rubus fruticosus√ā¬†leaf was the selected topography to be replicated in polycaprolactone (PCL) membranes through polydimethylsiloxane moulding and using soft lithography. Afterwards, rat bone marrow stem cells (rBMSCs) were seeded at the surface of the imprinted PCL membranes to characterize the bioactive potential of our biomimetic surface topography to drive rBMSCs differentiation into the osteogenic lineage. The selected surface topography in combination with the osteogenic inductive medium reveals having a synergistic effect promoting osteogenic differentiation.This work is a result of the project FROnTHERA (NORTE-01-0145-FEDER-000023), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and Portuguese Foundation for Science and Technology under the doctoral programme in Tissue Engineering, Regenerative Medicine and Stem Cells (PD/59/2013), (PD/BD/128087/2016) (COVID/BD/151599/2020) and by the project Cells4_IDs (PTDC/BTM-SAL/28882/2017)

    Biomimetic surface topography as a potential modulator of macrophages inflammatory response to biomaterials

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    The implantation of biomaterial devices can negatively impact the local microenvironment through several processes including the injury incurred during the implantation process and the associated host inflammatory response. Immune cell responses to implantable biomaterial devices mediate host-material interactions. Indeed, the immune system plays a central role in several biological processes required for the integration of biomaterials such as wound healing, tissue integration, inflammation, and foreign body reactions. The implant physicochemical properties such as size, shape, surface area, topography, and chemistry have been shown to provide cues to the immune system. Its induced immune-modulatory responses towards inflammatory or wound healing phenotypes can determine the success of the implant. In this work, we aim to evaluate the impact of some biomimetic surface topographies on macrophages' acute inflammatory response. For that, we selected 4 different biological surfaces to replicate through soft lithography on spin casting PCL membranes. Those topographies were: the surface of E. coli, S.eppidermidis and L929 cells cultured in polystyrene tissue culture disks, and an Eggshell membrane. We selected a model based on THP-1-derived macrophages to study the analysis of the expression of both pro-inflammatory and anti-inflammatory markers. Our results revealed that depending on the surface where these cells are seeded, they present different phenotypes. Macrophages present a M1-like phenotype when they are cultured on top of PCL membranes with the surface topography of E. coli and S. epidermidis. When cultured on membranes with L929 monolayers or Eggshell membrane surface topography, the macrophages present a M2-like phenotype. These results can be a significant advance in the development of new implantable biomaterial devices since they can help to modulate the inflammatory responses to implanted biomaterials by controlling their surface topography.FCT -Fundação para a Ciência e a Tecnologia(PD/59/2013

    Desenvolvimento de uma Propionibacterium melhorada para potencial uso como um nutracêutico para a prevenção - tratamento do cancro colorectal

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    Disserta√ß√£o de mestrado em Gen√©tica MolecularPropionibacterium freudenreichii is a commercially important bacterium that is well-known for its role as ripening starter in the cheese industry and its probiotic potential. These bacteria may beneficially modulate the intestinal ecosystem and can exert anti-neoplastic effects, particularly against colorectal cancer (CRC), via the production of short chain fatty acids (SCFAs), namely acetate and propionate. Thus, propionibacteria can be envisaged as a potential nutraceutical towards the prevention/treatment of CRC. In that sense, the aim of this thesis was to develop strategies to enhance the production of SCFA by P. freudenreichii under the simulation of human colon environment, as well as to evaluate its effects on CRC cells. In order to optimize and characterize the production of SCFAs, acetate and propionate by P. freudenreichii, different culture media and different fermentation conditions were evaluated. Moreover, the SCFAs toxic concentrations for the bacterium were determined. Additionally, a digestive stress challenge and random mutagenesis of P. freudenreichii DSM 20271 were performed. Finally, the effect of the propionibacteria fermentation broth on CRC cells and the CRC cells conditioned medium on the growth and biotransformation performance of the bacteria were studied. The basal medium (BM) was found to be the best to produce SCFA by P. freudenreichii with high amounts of acetate and propionate being produced, mainly when supplemented with glycerol. However, the results obtained with the medium ‚Äúmimicking the content of the human colon‚ÄĚ (MCHC) were not favorable regarding SCFAs production. The adapted P. freudenreichii to digestive stress lost the ability to produce high amounts of SCFAs in yeast extract-lactate (YEL) and BM media, in particular propionate. Moreover, partial inhibition of the bacteria growth and SCFAs production occurred at the following concentrations of pure SCFAs: 4 g L-1 acetate; 3 g L-1 propionate; 6 g L-1 propionate; 1 g L-1 acetate and 3 g L-1 propionate. Pure acetate and propionate, as well as the bacterial fermentation broth inhibited the CRC cells RKO proliferation and promoted their accumulation in the sub-G1 phase of the cell cycle. In conclusion, the results gathered in this work suggest that the coculture of P. freudenreichii and CRC cells was found to be possible and favorable for the bacteria and that P. freudenreichii could potentially be used in the CRC prevention/treatment via their ability to produce SCFAs.A Propionibacterium freudenreichii √© uma bact√©ria comercialmente importante, conhecida pela sua utiliza√ß√£o como cultura de arranque na produ√ß√£o de queijo, bem como pelo seu potencial probi√≥tico. Estas bact√©rias podem modular beneficamente o ecossistema intestinal e exercer os efeitos antineopl√°sicos, em particular contra o cancro colorectal (CRC), atrav√©s da produ√ß√£o de √°cidos gordos de cadeia curta (AGCC), nomeadamente acetato e propionato. Assim, as propionibact√©rias podem ser vistas como potenciais nutrac√™uticos para a preven√ß√£o/tratamento do CRC. Nesse sentido, o objectivo desta tese foi desenvolver estrat√©gias para aumentar a produ√ß√£o de AGCC pela P. freudenreichii em condi√ß√Ķes que simulam o c√≥lon humano, bem como avaliar o seu efeito nas c√©lulas de CRC. A fim de otimizar e caracterizar a produ√ß√£o dos AGCC, acetato e propionato, diferentes meios de cultura e diferentes condi√ß√Ķes foram avaliados. Al√©m disso, as concentra√ß√Ķes de AGCC t√≥xicas para a bact√©ria foram determinadas. Adicionalmente, realizaram-se ensaios de adapta√ß√£o ao stresse digestivo e de muta√ß√£o aleat√≥ria na P. freudenreichii DSM 20271. Finalmente, foi estudado o efeito do meio de fermenta√ß√£o da bact√©ria nas c√©lulas de CRC, bem como o efeito do meio condicionado das c√©lulas de CRC no crescimento e produ√ß√£o de AGCC pela bact√©ria. O meio basal (BM) demonstrou ser o melhor para produzir AGCC pela P. freudenreichii, obtendo-se grandes quantidades de acetato e propionato, principalmente no meio BM suplementado com glicerol. No entanto, os resultados obtidos com o meio que "imita o conte√ļdo do c√≥lon humano" (MCHC) n√£o foram favor√°veis relativamente √† produ√ß√£o de AGCC. A P. freudenreichii adaptada ao stresse digestivo perdeu a capacidade de produzir grandes quantidades de AGCC, particularmente propionato, nos meios de extrato de levedura-lactato (YEL) e BM. Adicionalmente, observou-se uma inibi√ß√£o parcial do crescimento bacteriano e produ√ß√£o de AGCC para as seguintes concentra√ß√Ķes de AGCC puros: 4 g L-1 acetato; 3 g L-1 propionato; 6 g L-1 propionato; 1 g L-1 acetato e 3 g L-1 propionato. O acetato e o propionato, bem como o meio de fermenta√ß√£o da bact√©ria inibiram a prolifera√ß√£o das c√©lulas de CRC RKO e induziram um aumento de c√©lulas na fase sub-G1 do ciclo celular. Em conclus√£o, os resultados deste trabalho sugerem que a co-cultura entre c√©lulas de CRC e P. freudenreichii √© poss√≠vel e favor√°vel para a bact√©ria e que a P. freudenreichii poder√° potencialmente ser usada na preven√ß√£o/tratamento de CRC atrav√©s da sua capacidade de produzir AGCC

    Biofuncionalização da superfície de membranas fibrosas de policaprolactona para terapias avançadas de tecido esquelético e neural

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    Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e C√©lulas EstaminaisDamage of the skeletal and neural tissues has a significant impact over the quality-of-life of patients and high socio-economical costs. Current treatment options are not effective in long term, due to the suboptimal integration with the host tissue and limited bioactivity of implantable biomaterials. The immobilization of biomolecules at the surface of biomedical devices has attracted increasing interest, allowing for their local bioavailability avoiding systemic side effects and longer half-life. Envisioning the development of advanced therapies, the electrospun nanofibrous meshes (NFMs) were used as a substrate due to their fibrous structure mimic the extracellular matrix (ECM) of many tissues, allowing cell-cell and cell-biomaterial interactions. For that, the surface of polycaprolactone NFMs was activated and functionalized with amine groups allow for covalent immobilization of defined antibodies, with the capacity to selectively bind autologous biomolecules. Different biofunctional substrates with chondrogenic inductive properties were developed through the surface biofunctionalization of NFM with endogenous human fibronectin, extracellular vesicles or the combination of endogenous Transforming Growth Factor-133 and Insulin-like Growth Factor-I. All these biofunctional substrates successfully induced the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) under basal culture conditions. Blood-derived Nerve Growth Factor bound to the surface of NFMs remains bioactive, being an effective inducer of the neurogenic differentiation of a relevant cell line. Additionally, we developed a biofunctional system able to mimic the vasculature of bone tissue, comprising Bone Morphogenetic Protein 2 and Vascular Endothelial Growth Factor in a parallel pattern design. This biofunctional system enabled a spatially defined osteogenic and angiogenic differentiation of hBM-MSCs. The surface biofunctionalization of biomaterial substrates enables developing biofunctional systems envisioning patient-specific devices promoting skeletal and neural tissue regeneration that can maximize and extend the local efficacy and minimize the side effects of the use of biologic based therapies in patients.A deteriora√ß√£o dos tecidos esquel√©tico a neural t√™m um impacto significativo na qualidade de vida dos pacientes e um elevado custo socioecon√≥mico. Os tratamentos atualmente dispon√≠veis n√£o s√£o eficazes a longo termo, devido √† inadequada integra√ß√£o com o tecido hospedeiro e √† baixa bioatividade dos biomateriais implantados. A imobiliza√ß√£o de biomol√©culas constitui uma estrat√©gia alternativa, permitindo a biodisponibilidade local das biomol√©culas evitando efeitos colaterais sist√™micos. Ambicionando desenvolver terapias avan√ßadas, malhas fibrosas produzidas por "electrospinning" (NFMs) foram usadas como substratos polim√©ricos devido √† sua estrutura fibrosa similar a matriz extracelular (ECM) de muitos tecidos, promovendo as intera√ß√Ķes c√©lula-c√©lula e c√©lula-biomaterial. Para isso, NFMs de policaprolactona foram ativadas e funcionalizadas com grupos amina, permitindo a imobiliza√ß√£o covalente de anticorpos pr√©-definidos, com capacidade de ligar seletivamente biomol√©culas aut√≥logas. Foram desenvolvidos diferentes substratos biofuncionais, com propriedades indutoras de diferencia√ß√£o condrog√©nica, mediante liga√ß√£o de fibronectina humana, ves√≠culas extracelulares ou a combina√ß√£o do fator de transforma√ß√£o do crescimento beta 3 com o fator de crescimento semelhante √† insulina tipo I. Todos estes substratos biofuncionalizados foram capazes de induzir a diferencia√ß√£o condrog√©nica de c√©lulas estaminais mesenquimais derivadas de medula √≥ssea humana (hBM-MSCs) sendo cultivadas em condi√ß√Ķes basais. O fator de crescimento nervoso ligado √† superf√≠cie das NFMs permanece bioativo, sendo um indutor eficaz da diferencia√ß√£o neurog√©nica de uma linha celular relevante. Numa outra abordagem, foi desenvolvido um sistema biofuncional capaz de mimetizar a vasculatura de um tecido √≥sseo, ligando paralelamente a prote√≠na morfogen√©tica √≥ssea 2 e o fator de crescimento do endot√©lio vascular sobre uma mesma NFM. Este sistema biofuncional permitiu a diferencia√ß√£o osteog√©nica e angiog√©nica de hBM-MSCs espacialmente definida. Concluindo, a bioftmcionaliza√ß√£o de substratos produzidos por "electrospinning" permite o desenvolvimento de dispositivos biom√©dicos personalizados, capazes de promover a regenera√ß√£o do tecido esquel√©tico e neural, maximizando a efic√°cia local e minimizando os efeitos colaterais do uso de terapias biol√≥gicas em pacientes.To the financial support of the Portuguese Fotmdatron for Science and Technology to maize possible this PhD by awarded me with a PhD scholarship (PD/BD/113797/2015) under the Doctoral Program on Advanced Therapies for Health (FSE/POCK/PD/169/2013). The experimental work was funded by the projects SPARTAN (PTDC/CTM-BIO/4388/2014) and FRONthera (NORTE-01-0145-FEDER-0000232)

    Stimulation of neurite outgrowth using autologous NGF bound at the surface of a fibrous substrate

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    Peripheral nerve injury still remains a major clinical challenge, since the available solutions lead to dysfunctional nerve regeneration. Even though autologous nerve grafts are the gold standard, tissue engineered nerve guidance grafts are valid alternatives. Nerve growth factor (NGF) is the most potent neurotrophic factor. The development of a nerve guidance graft able to locally potentiate the interaction between injured neurons and autologous NGF would be a safer and more effective alternative to grafts that just release NGF. Herein, a biofunctional electrospun fibrous mesh (eFM) was developed through the selective retrieval of NGF from rat blood plasma. The neurite outgrowth induced by the eFM-NGF systems was assessed by culturing rat pheochromocytoma (PC12) cells for 7 days, without medium supplementation. The biological results showed that this NGF delivery system stimulates neuronal differentiation, enhancing the neurite growth more than the control condition.Portuguese Foundation for Science and Technology (FCT), grant numbers PTDC/BTM-SAL/28882/2017 (Cells4_Ids) and PTDC/CTM-BIO/4388/2014 (SPARTAN). The PhD grant of M.R.C. (PD/BD/113797/2015) was funded by the FCT Doctoral Program on Advanced Therapies for Health (PATH) (FSE/POCH/PD/169/2013) and the A.M. was financed by the IF grant IF/00376/201

    Fibronectin bound to a fibrous substrate has chondrogenic induction properties

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    Articular cartilage is an avascular tissue characterized by a dense and specific extracellular matrix (ECM). Fibronectin (FN) is a key constituent of the pericellular ECM, assembled into a fibrillar matrix through a cell-mediated process, being implicated in chondrogenic events. In this study, we evaluate the chondrogenic potential of FN bound to the surface of an electrospun nanofibrous mesh (NFM). For that, an√ā¬†anti-FN antibody was immobilized at the surface of NFMs, rendering them capable of selectively binding endogenous FN (eFN) from blood plasma. The chondrogenic potential of bound eFN was further assessed by culturing human bone marrow-derived mesenchymal stem cells (hBM-MSCs) for 28 days, in a basal growth medium. The biological results indicate that NFMs√ā¬†functionalized with eFN were able to successfully induce the chondrogenesis of hBM-MSCs,√ā¬†as demonstrated√ā¬†by the high expression of√ā¬†SOX9,√ā¬†Aggrecan, and√ā¬†Collagen type II. Therefore, biofunctionalized nanofibrous substrates comprising eFN significantly enhance the efficacy of a cartilage tissue-engineering strategy.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the Ph.D. grant of M.R.C. (PD/BD/113797/2015) financed by the FCT Doctoral Program on Advanced Therapies for Health (PATH) (FSE/POCH/PD/169/2013), the IF grant of A.M. (IF/00376/2014), and the project SPARTAN (PTDC/CTMBIO/4388/2014

    Chondrogenesis-inductive nanofibrous substrate using both biological fluids and mesenchymal stem cells from an autologous source

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    During the last decade, many cartilage tissue engineering strategies have been developed, being the stem cell-based approach one of the most promising. Transforming Growth Factor-√é¬≤3 (TGF-√é¬≤3) and Insulin-like Growth Factor-I (IGF-I) are key proteins involved in the regulation of chondrogenic differentiation. Therefore, these two growth factors (GFs) were immobilized at the surface of a single electrospun nanofibrous mesh (NFM) aiming to differentiate human Bone Marrow-derived Mesenchymal Stem Cells (hBM-MSCs). The immobilization of defined antibodies (i.e. anti-TGF-√é¬≤3 and anti-IGF-I) allows the selective retrieval of the abovementioned GFs from human platelet lysates (PL). Biochemical assays, involving hBM-MSCs cultured on biofunctional nanofibrous substrates under basal culture medium during 28√Ę ¬Įdays, confirm the biological activity of bound TGF-√é¬≤3 and IGF-I. Specifically, the typical spherical morphology of chondrocytes and the immunolocalization of collagen type II confirmed the formation of a cartilaginous ECM. Therefore, the proposed biofunctional nanofibrous substrate is able to promote chondrogenesis.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD grant of MRC (PD/BD/113797/2015) financed by the FCT Doctoral Program on Advanced Therapies for Health (PATH) (FSE/POCH/PD/169/2013), the Post-doc fellowships of MAS and ARP (SFRH/BPD/73322/2010 and SFRH/BPD/90332/2012), the IF grant of AM (IF/00376/2014), and the projects SPARTAN (PTDC/CTM-BIO/4388/2014) and FRONthera (NORTE-01-0145-FEDER-0000232).info:eu-repo/semantics/publishedVersio

    Spatial immobilization of endogenous growth factors to control vascularization in bone tissue engineering

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    The intimate crosstalk between endothelial and bony cells is essential for the reconstruction of bone tissue defects. Indeed, a successful bone repair is greatly dependent on the formation of new blood vessels, to ensure the supply of nutrients and gases, as well as the removal of metabolites. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) are involved on cells differentiation and bone vascularization aiming to develop viable bone tissue. Herein it is hypothesized that endogenous BMP-2 and VEGF bound in a parallel arrangement over a single nanofibrous substrate (NFM) can lead to a successful osteogenic and angiogenic differentiation of mesenchymal stem cells. For that, an engineered biofunctional system was developed comprising anti-BMP-2 and anti-VEGF antibodies, immobilized over an electrospun NFMs in a parallel pattern design, with the attempt to recreate the vasculature of bone tissue. The osteogenic and angiogenic potential of this engineered biofunctional system was demonstrated by culturing human bone marrow-derived mesenchymal stem cells (hBM-MSCs) during 21 days without exogenous induction. A chick chorioallantoic membrane (CAM) assay showed that the engineered biofunctional system, comprising bound endogenous BMP-2 and VEGF, is able to induce an increased angiogenic response. The angiogenic ability of this system, together with the osteogenic inductor BMP-2, enable obtaining an effective vascularized bone tissue engineering approach.The authors would like to acknowledge Norte 2020, for financing the PhD scholarship of C. O. (Norte-08-5369-000037) and Portuguese Foundation for Science and Technology (FCT) for the PhD grant of M. R. C. (PD/BD/113797/2015) financed by the FCT Doctoral Program on Advanced Therapies for Health(PATH) (FSE/POCH/PD/169/2013), the post-doctoral grant of E. M. F (SFRH/BPD/96197/2013), the IF grant of A. M. (IF/00376/2014), and the projects SPARTAN (PTDC/CTM-BIO/4388/2014) and FRONthera (NORTE-01-0145-FEDER-000023). Thiswork was developed under the framework of the CooperationAgreement established with the Serviço de Imuno-Hemoterapia do Centro Hospitalar de S. João, EPE
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