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Orientadores: Anderson de Oliveira Lobo, Marcus Alexandre Finzi Corat Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Os meniscos do joelho são estruturas fibrocartilaginosas intra-articulares, de complexa organização da matriz extracelular (MEC), que desempenham um papel essencial na função da articulação. A baixa capacidade regenerativa intrínseca do tecido, associada a elevada incidência de lesões e a falta de um tratamento consensualmente eficaz disponível, evidencia a urgente necessidade do desenvolvimento de novas terapias para lesões meniscais, no qual a engenharia de tecidos tem emergido como uma das mais promissoras abordagens. O sucesso desta estratégia terapêutica para os meniscos está diretamente associado ao desenvolvimento scaffolds ideias capazes de reproduzir tanto a microestrutura da MEC como o formato anatômico tridimensional (3D) do tecido nativo, além de possuírem propriedades mecânicas adequadas. Nesse sentido, o principal objetivo deste trabalho foi construir um scaffold meniscal apto a cumprir com esses critérios através de uma nova estratégia de biofabricação elaborada a partir da bioimpressão 3D de hidrogel reforçado com nanofibras poliméricas biomimeticamente alinhadas. Dividido em duas partes, a primeira etapa se concentrou na otimização mecânica das membranas nanofibrosas. Para isso, membranas constituídas por fibras de policaprolactona (PCL) alinhadas radialmente e circunferencialmente incorporadas com duas diferentes concentrações de nanotubos de carbono (CNT): 0,5 e 1,0 mg.mL-1 foram produzidas a partir de um sistema específico de eletrofiação. As imagens obtidas por microscopia eletrônica de varredura (MEV) demonstraram a distribuição espacial e o alinhamento das nanofibras, bem como a diminuição no tamanho médio das fibras com a adição de CNT, que teve sua presença confirmada por imagens de microscopia eletrônica de transmissão (MET). A análise mecânica por ensaio de tração e os ensaios biológicos in vitro mostraram uma melhora nas propriedades mecânicas relacionadas ao aumento do teor de CNT sem influenciar negativamente na sobrevivência das células-tronco mesenquimais (CTM) cultivadas sobre as membranas. Já na segunda etapa, os primeiros objetivos específicos foram utilizar essas membranas de nanocompósitos à base de PCL/CNT para reforçar mecanicamente hidrogeis bioimpressos à base de colágeno tipo 1. Para isso, as membranas de nanofibras foram inseridas entre as camadas de hidrogel durante o processo de bioimpressão em diferentes proporções: Hidrogel:nanofibras 1:0, 4:1 e 2:1. Os testes de compressão mecânica uniaxial demonstraram que a presença de camadas de nanofibras melhorou significativamente as propriedades mecânicas do hidrogel bioimpresso sem afetar de forma negativa a viabilidade das CTM que permaneceram viáveis mesmo após 7 dias de cultivo estático dentro do scaffold. Além disso, os resultados mostraram que a incorporação de nanofibras melhorou a integridade estrutural e a fidelidade da forma do scaffold em comparação com o hidrogel sem membranas nanofibrosas. A partir desses achados, nosso próximo passo foi utilizar essa estratégia de biofabricação para construir um scaffold meniscal com formato 3D e dimensões personalizadas. Para tanto, um modelo 3D digital do menisco medial do joelho humano foi reconstruído a partir de imagens obtidas por ressonância magnética nuclear (RMN) utilizando um software livre e de código aberto. Então, membranas com nanofibras biomimeticamente alinhadas puderam ser produzidas com tamanho bidimensional compatível com o menisco medial humano e, finalmente, o scaffold biomimético meniscal 3D personalizado pode ser fabricado com sucesso através da atual abordagem de biofabricação. Por fim, as descobertas do presente estudo sugerem uma potencial aplicabilidade deste método para a engenharia de tecidos do menisco do joelho Abstract: Knee menisci are intra-articular fibrocartilaginous structures with a complex extracellular matrix (ECM) organization that play an essential role in joint function. The low intrinsic regenerative capacity of the tissue, associated with a high incidence of injuries and the lack of consensually effective treatment available, highlights the urgent need to develop new therapies for meniscal injuries, in which tissue engineering has emerged as one of the most promising approaches. The success of this therapeutic strategy for menisci is strongly associated with the development of ideal scaffolds able to reproduce both the ECM microstructure and the three-dimensional (3D) anatomical shape of the native tissue, in addition to having good mechanical properties. In this sense, the main objective of this work was to build a meniscal scaffold able to fulfill these criteria through a new biofabrication strategy elaborated from the 3D bioprinting of a hydrogel reinforced with biomimetically aligned polymeric nanofibers. Divided into two parts, the first step focused on the mechanical optimization of nanofibrous membranes. For this, mats constituted by polycaprolactone (PCL) fibers aligned radially and circumferentially incorporated with two different concentrations of carbon nanotubes (CNT): 0.5 and 1.0 mg.mL-1 were produced by a specific electrospinning system. The images obtained by scanning electron microscopy (SEM) demonstrated the spatial distribution and alignment of the nanofibers, as well as the decrease in the average fiber size with the addition of CNT, which had its presence confirmed by transmission electron microscopy (TEM) images. Mechanical analysis by tensile test and in vitro biological assays showed an improvement in mechanical properties related to the increase in CNT content without negatively influencing the survival of mesenchymal stem cells (MSCs) seeded on the membranes. In the second stage, the first specific objectives were to use these nanocomposite membranes based on PCL/CNT to mechanically reinforce bioprinted type 1 collagen hydrogels. Thus, the nanofibrous mats were inserted between the hydrogel layers during the bioprinting process in different ratios: hydrogel:nanofibers 1:0, 4:1, and 2:1. Uniaxial mechanical compression tests demonstrated that the presence of nanofiber layers significantly improved the mechanical properties of the bioprinted hydrogel without negatively affecting the viability of MSCs that remained viable even after 7 days of culture within the scaffold. Furthermore, the results evidenced that the incorporation of nanofibers enhanced the scaffold's structural integrity and shape fidelity compared to the nanofiber-free hydrogel. Based on these findings, our next step was to use this biofabrication strategy to create a meniscal scaffold with a custom shape and 3D dimensions. For this, a 3D digital model of the medial meniscus of the human knee was reconstructed from images obtained by nuclear magnetic resonance (NMR) using free and open-source software. Then, biomimetically aligned nanofibers mats could be produced with a two-dimensional size compatible with the human medial meniscus and, finally, the patient-specific 3D meniscal scaffold was successfully fabricated using the current biofabrication approach. Lastly, the findings of the present study suggest a potential applicability of this method for knee meniscus tissue engineering Doutorado Clínica Médica Doutor em Ciências CAPES 001

Alan IS Morais, 1,* Xichi Wang, 2–4,* Ewerton G Vieira, 1 Bartolomeu C Viana, 1, 5 Edson C Silva-Filho, 1 Josy A Osajima, 1 Samson Afewerki, 3, 4 Marcus AF Corat, 6 Heurison S Silva, 5 Fernanda R Marciano, 5 Guillermo U Ruiz-Esparza, 3, 4 Thiago D Stocco, 6, 7 Mirian MM de Paula, 6 Anderson O Lobo 1 1LIMAV-Interdisciplinary Laboratory for Advanced Materials, Materials Science and Engineering Graduate Program, UFPI-Federal University of Piauí, Teresina, PI CEP 64049-550, Brazil; 2Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People’s Republic of China; 3Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham & Women´s Hospital, Cambridge, MA 02139, USA; 4Harvard-MIT Division of Health Science and Technology, Massachusetts Institute of Technology, MIT, Cambridge, MA 02139, USA; 5Department of Physics, UFPI-Federal University of Piauí, Teresina, PI CEP 64049-550, Brazil; 6Multidisciplinary Center for Biological Research, University of Campinas (UNICAMP), Campinas 13083-877, Brazil; 7Faculty of Physiotherapy, Santo Amaro University, São Paulo 04829-300, Brazil*These authors contributed equally to this workCorrespondence: Anderson O Lobo Email lobo@ufpi.edu.brBackground: The facile preparation of oxygen-generating microparticles (M) consisting of Polycaprolactone (PCL), Pluronic F-127, and calcium peroxide (CPO) (PCL-F-CPO-M) fabricated through an electrospraying process is disclosed. The biological study confirmed the positive impact from the oxygen-generating microparticles on the cell growth with high viability. The presented technology could work as a prominent tool for various tissue engineering and biomedical applications.Methods: The oxygen-generated microparticles fabricated through electrospraying processes were thoroughly characterization through various methods such as X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) analysis, and scanning electron microscopy (SEM)/SEM-Energy DispersiveSpectroscopy (EDS) analysis.Results: The analyses confirmed the presence of the various components and the porous structure of the microparticles. Spherical shape with spongy characteristic microparticles were obtained with negative charge surface (ζ = – 16.9) and a size of 17.00 ± 0.34 μm. Furthermore, the biological study performed on rat chondrocytes demonstrated good cell viability and the positive impact of increasing the amount of CPO in the PCL-F-CPO-M.Conclusion: This technological platform could work as an important tool for tissue engineering due to the ability of the microparticles to release oxygen in a sustained manner for up to 7 days with high cell viability.Keywords: oxygen-generating-microparticles, electrospraying, tissue engineering, calcium peroxide, cartilage