Natural materials

The use of naturally occurring materials as scaffolds to support cell growth and proliferation significantly impacted the origin and progress of tissue engineering and regenerative medicine. However, the majority of these materials failed to provide adequate cues to guide cell differentiation toward the formation of new tissues. Over the past decade, a new generation of multifunctional and smart natural-based materials has been developed to provide biophysical and biochemical cues intended to specifically guide cell behavior. In this chapter, the use of extracellular matrix proteins and blood-derivatives intrinsic capacity to mimic the biophysical and biological characteristics of native tissues is reviewed. Furthermore, the design of a variety of nanostructures using the well-explored characteristics of nucleic acids is summarized. In the second section, the exploitation of supramolecular chemistry to create new dynamic functional hydrogels that mimic the extracellular matrix structure and/or composition is surveyed. Then, the incorporation of nanoelements in polymeric networks for the design of smart nanocomposite materials with tailored functionalities to guide cell behavior is introduced. Finally, the future perspectives in the development of new biomaterials for tissue engineering and regenerative medicine are presented.Te authors acknowledge the fnancial support of the European Union Framework Programme for Research and Innovation Horizon 2020, under the TEAMING grant agreement No 739572 – Te Discoveries CTR, Marie Skłodowska-Curie grant agreement No 706996 and European Research Council grant agreement No 726178; FCT (Fundação para a Ciência e a Tecnologia) and the Fundo Social Europeu através do Programa Operacional do Capital Humano (FSE/POCH) in the framework of Ph.D. grants PD/BD/113807/2015 (BBM) and PD/BD/129403/2017 (SMB), Post-Doc grant SFRH/ BPD/112459/2015 (RMD) and project SmarTendon (PTDC/NAN-MAT/30595/2017); Project NORTE01-0145-FEDER-000021 supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF

Writing 3D In vitro models of human tendon within a biomimetic fibrillar support platform

Tendinopathies are poorly understood diseases for which treatment remains challenging. Relevant in vitro models to study human tendon physiology and pathophysiology are therefore highly needed. Here we propose the automated 3D writing of tendon microphysiological systems (MPSs) embedded in a biomimetic fibrillar support platform based on cellulose nanocrystals (CNCs) self-assembly. Tendon decellularized extracellular matrix (dECM) was used to formulate bioinks that closely recapitulate the biochemical signature of tendon niche. A monoculture system recreating the cellular patterns and phenotype of the tendon core was first developed and characterized. This system was then incorporated with a vascular compartment to study the crosstalk between the two cell populations. The combined biophysical and biochemical cues of the printed pattern and dECM hydrogel were revealed to be effective in inducing human-adipose-derived stem cells (hASCs) differentiation toward the tenogenic lineage. In the multicellular system, chemotactic effects promoted endothelial cells migration toward the direction of the tendon core compartment, while the established cellular crosstalk boosted hASCs tenogenesis, emulating the tendon development stages. Overall, the proposed concept is a promising strategy for the automated fabrication of humanized organotypic tendon-on-chip models that will be a valuable new tool for the study of tendon physiology and pathogenesis mechanisms and for testing new tendinopathy treatments.The authors thank Hospital da Prelada (Porto, Portugal) for providing adipose tissue samples. The authors acknowledge the financial support from Project NORTE-01-0145-FEDER 000021 supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), the European Union Framework Program for Research and Innovation HORIZON 2020, under the Twinning Grant Agreement 810850-Achilles, and European Research Council Grant Agreement 772817 and 101069302, Fundação para a Ciência e a Tecnologia for the Ph.D. grant PD/BD/129403/2017 (to S.M.B.) financed through the doctoral program in Tissue Engineering, Regenerative Medicine and Stem Cells (TERM&SC), for Contract 2020.03410.CEECIND and 2022.05526.PTDC (to R.M.A.D.). The authors acknowledge Doctor Alberto Pardo for performing the rheology measurements of the PL bioink. The schematics in Figures 1, 2A, 4A, and 6A and Table of Contents graphic were created with BioRender.com

3D Bioprinting for Tissue and Organ Fabrication

The field of regenerative medicine has progressed tremendously over the past few decades in its ability to fabricate functional tissue substitutes. Conventional approaches based on scaffolding and microengineering are limited in their capacity of producing tissue constructs with precise biomimetic properties. Three-dimensional (3D) bioprinting technology, on the other hand, promises to bridge the divergence between artificially engineered tissue constructs and native tissues. In a sense, 3D bioprinting offers unprecedented versatility to co-deliver cells and biomaterials with precise control over their compositions, spatial distributions, and architectural accuracy, therefore achieving detailed or even personalized recapitulation of the fine shape, structure, and architecture of target tissues and organs. Here we briefly describe recent progresses of 3D bioprinting technology and associated bioinks suitable for the printing process. We then focus on the applications of this technology in fabrication of biomimetic constructs of several representative tissues and organs, including blood vessel, heart, liver, and cartilage. We finally conclude with future challenges in 3D bioprinting as well as potential solutions for further development.United States. Office of Naval Research. Young Investigator ProgramNational Institutes of Health (U.S.) (Grants EB012597, AR057837, DE021468, HL099073 and R56AI105024)Presidential Early Career Award for Scientists and Engineer

Innovative approaches to the use of nanotools for creating tendon tissue-mimetic constructs and disease models

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisThe intricate composition and architecture of living tissues determine their functionality, resulting from the complex interplay between cells and the extracellular matrix (ECM). Properly addressing these characteristics is crucial for the development of 3D in vitro advanced platforms that have the potential to enhance the efficacy of knowledge-based therapy development, while significantly reducing the need for animal experimentation. This Thesis aimed at addressing specific research challenges for the development of effective tendon-mimetic constructs for tissue regeneration and disease modeling. These challenges include i) recreating the complex hierarchical and fibrillar architecture of tendon extracellular matrix; ii) the ability to remotely actuate mechanotransduction mechanisms in tissue engineered construct; iii) delivering biochemical cues involved in tendon tissue development and healing and iv) incorporating multiple cell types to replicate the complex biological processes that occur in native tissue. To address these challenges, we used state-of-the-art technologies such as 3D bioprinting and organ-on-chip technology and biomaterials, in particular nanoparticles, with unique (bio) functional properties. Building on these concepts, In the first experimental chapter (Chapter 3), cellulose nanocrystals (CNCs) surface charge chemistry was exploited to induce their ion-mediated self-assembly to recreate the unique biophysical cues provided by native tissue fibrillar Extracellular Matrix (ECMs) while allowing the design of embedded bioengineered constructs with arbitrary geometries. This system was further explored in Chapter 4, by combining magnetically- and matrix assisted 3D bioprinting for creating anisotropic microstructures. The topographical and biochemical cues of this biomimetic microstructure were combined with its magneto-mechanical stimulation during in vitro maturation, to boost stem cells mechanosignaling and to promote their commitment toward tenogenic lineage. Resourcing zinc-doped iron oxide magnetic nanoparticles incorporated into electrospun fibers (sMRF) specifically designed for this purpose. In Chapter 5 sMRF served as inspiration for developing a compartmentalized tendon-on-chip (3D-TenoC) model to study crosstalk and biochemical signaling in tendon physiology and pathophysiology. This 3D-TenoC model faithfully recreated essential characteristics of human tendons, including anisotropy and spatiotemporal distribution of cells. Overall, this thesis showcases that by combining specific bionanomaterials and advanced 3D bioprinting technologies for the construction of 3D models with an unprecedented ability to mimic native tendon tissue ECM anisotropy, physical stimulus, and customizable biochemical cues, while accommodating multiple cell types. These strategies have the potential to play a significant role in generating valuable biological data and integrating ongoing advancements in tendon tissue engineering.A intrincada composição e arquitetura dos tecidos vivos determinam sua funcionalidade, resultante da complexa interação entre as células e a matriz extracelular (ECM). A abordagem adequada dessas características é crucial para o desenvolvimento de plataformas 3D in vitro avançadas que têm o potencial de aumentar a eficácia do desenvolvimento de terapias baseadas em conhecimento, contribuído em simultâneo para reduzir significativamente a necessidade de experimentação animal. Esta Tese focou-se em desafios de investigação específicos para o desenvolvimento de construções miméticas de tendão eficazes para regeneração deste tecido e modelação in vitro das suas patologias. Esses desafios incluem i) recriar a complexa arquitetura fibrilar hierárquica da matriz extracelular do tendão; ii) incorporar a capacidade de acionar remotamente mecanismos de mecanotransdução nas construções produzidas por engenharia de tecidos; iii) incorporar sinais bioquímicos envolvidos no desenvolvimento e cicatrização do tecido do tendão; e iv) incorporar diferentes tipos de células de forma a replicar os processos biológicos complexos que ocorrem no tecido nativo. Para enfrentar esses desafios, usamos tecnologias de ponta, como bioimpressão 3D e tecnologia organ-on-chip, e biomateriais, em particular nanopartículas, com (bio)funcionalidades únicas. Com base nesses conceitos, no primeiro capítulo experimental (Capítulo 3), a química de superfície de nanocristais de celulose (CNCs) foi explorada para induzir a sua automontagem mediada por iões, permitindo simultaneamente recriar os sinais biofísicos únicos providenciados pela ECM fibrilar de tecido nativo e o design de construções de bioengenharia com geometrias arbitrárias. Este sistema foi depois explorado no Capítulo 4, no qual a bioimpressão 3D assistida por sistemas magnéticos e matriz de suporte foi explorada para criar microestruturas anisotrópicas. Os sinais topográficos e bioquímicos desta microestrutura biomimética foram combinados com estimulação magneto-mecânica durante sua a maturação in vitro para acionar a mecanosinalização de células estaminais e promover seu o seu direcionamento para a linhagem tenogénica. Recorrendo a nanopartículas magnéticas de óxido de ferro dopadas com zinco incorporadas em fibras electrofiadas (sMRF) especificamente concebidas para o efeito, no Capítulo 5, as sMRF serviram de inspiração para o desenvolvimento de um modelo de tendão-em-chip compartimentado (3D-TenoC) para estudar a comunicação e sinalização bioquímica que ocorre na fisiologia e patofisiologia do tendão. Este 3D TenoC permitiu recriar características essenciais dos tendões humanos, incluindo anisotropia e distribuição espácio-temporal das suas células. No geral, esta tese demostra como bionanomateriais específicos e tecnologias de bioimpressão 3D podem ser combinados para contruir modelos 3D com capacidade impar de recriar a anisotropia da ECM do tendão nativo, providenciar estímulos físico e sinais bioquímicos personalizáveis, e acomodar simultaneamente vários tipos de células. Estas estratégias demostram potencial para desempenhar um papel relevante na geração de dados biológicos valiosos assim como para integrarem avanços em continuo desenvolvimento na engenharia de tecidos de tendão.Fundação para a Ciência e a Tecnologia (FCT) - PD/BD/129403/201

Pyrazolobenzothiazine-based carbothioamides as new structural leads for the inhibition of monoamine oxidases:design, synthesis, in vitro bioevaluation and molecular docking studies

Binding mode of potent inhibitor (green) &amp; cognate ligand (pink) in the active site of MAO-B.</p

Magnetically-assisted 3D bioprinting of anisotropic tissue-mimetic constructs

Recreating the extracellular matrix organization and cellular patterns of aniso-tropic tissues in bioengineered constructs remains a significant biofabrication challenge. Magnetically-assisted 3D bioprinting strategies can be exploited to fabricate biomimetic scaffolding systems, but they fail to provide control over the distribution of magnetic materials incorporated in the bioinks while pre-serving the fidelity of the designed composites. To overcome this dichotomy, the concepts of magnetically- and matrix-assisted 3D bioprinting are combined here. By allowing low viscosity bioinks to remain uncrosslinked after printing, this approach enables the arrangement of incorporated magnetically-responsive microfibers without compromising the resolution of printed structures before inducing their solidification. Moreover, the fine design of these magnetic microfillers allows the use of low inorganic contents and weak magnetic field strengths, minimizing the potentially associated risks. This strategy is evalu-ated for tendon tissue engineering purposes, demonstrating that the synergy between the biochemical and biophysical cues stemming from a tendon-like anisotropic fibrous microstructure, combined with remote magneto-mechanical stimulation during in vitro maturation, is effective on directing the fate of the encapsulated human adipose-derived stem cells toward tenogenic pheno-type. In summary, the developed strategy allows the fabrication of anisotropic high-resolution magnetic composites with remote stimulation functionalities, opening new horizons for tissue engineering applications.The authors acknowledge the financial support from project NORTE-01-0145-FEDER 000021 supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); the European Union Framework Program for Research and Innovation HORIZON 2020, under the Twinning grant agreement no. 810850-Achilles, European Research Council grant agreement no. 772817, Fundação para a Ciência e a Tecnologia for the PhD grants PD/BD/129403/2017 (S.M.B.) and PD/BD/143039/2018 (S.P.B.T.) financed through doctoral the program in Tissue Engineering, Regenerative Medicine and Stem Cells (TERM&SC), for 2020.03410. CEECIND (R.M.A.D.) and project PTDC/NAN-MAT/30595/2017. Xunta de Galicia and Ministerio de Universidades (Spain) for postdoctoral grants ED481B2019/025 (A.P.) and Margarita Salas (R.R.), respectively. Schematics in Figures 1 and 5 were created with BioRender.com