3DICE coding matrix multidirectional macro-architecture modulates cell organization, shape, and co-cultures endothelization network

Natural extracellular matrix governs cells providing biomechanical and biofunctional outstanding properties, despite being porous and mostly made of soft materials. Among organs, specific tissues present specialized macro-architectures. For instance, hepatic lobules present radial organization, while vascular sinusoids are branched from vertical veins, providing specific biofunctional features. Therefore, it is imperative to mimic such structures while modeling tissues. So far, there is limited capability of coupling oriented macro-structures with interconnected micro-channels in programmable long-range vertical and radial sequential orientations. Herein, a three-directional ice crystal elongation (3DICE) system is presented to code geometries in cryogels. Using 3DICE, guided ice crystals growth templates vertical and radial pores through bulky cryogels. Translucent isotropic and anisotropic architectures of radial or vertical pores are fabricated with tunable mechanical response. Furthermore, 3D combinations of vertical and radial pore orientations are coded at the centimeter scale. Cell morphological response to macro-architectures is demonstrated. The formation of endothelial segments, CYP450 activity, and osteopontin expression, as liver fibrosis biomarkers, present direct response and specific cellular organization within radial, linear, and random architectures. These results unlock the potential of ice-templating demonstrating the relevance of macro-architectures to model tissues, and broad possibilities for drug testing, tissue engineering, and regenerative medicine.The authors are grateful for the Portuguese Foundation for Science and Technology (FCT) distinction attributed to R. F. Canadas (SFRH/ BD/92565/2013), and to J. M. Oliveira (IF/00423/2012, IF/01285/ 2015). R. F. Canadas is also thankful to FCT, Fundo Europeu de Desenvolvimento Regional (FEDER), and Programa Operacional Competitividade e Internacionalizaç˜ao (POCI) for funding the B-Liver Project (PTDC/EMD-EMD/29139/2017). The authors are also thankful to FCT for supporting the project Hierarchitech (M-ERA-NET/0001/2014) and for the funds provided under the 3 BioMeD project (JICAM/0001/2017). The authors acknowledge that this material and collaboration is based in part upon work supported by Luso-American Development Foundation (FLAD), 2016/CON15/CAN6). U. Demirci is also grateful for the Canary Center at Stanford for Cancer Early Detection Seed Award. The authors are also grateful for the support provided by Diana Bicho and Nicolas Cristini on scaffold characterization and cell culture, respectively

Estratégias avançadas de engenharia para bioimpressão de cartilagem específica para cada paciente

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisOrgan shortage and transplantation needs have led to congestion in healthcare systems resulting in a huge socioeconomic impact. Tissue Engineering has been revolutionizing the engineering of functional tissues, making them great alternatives to achieve a better, faster and effective worldwide patient care. Fibrocartilage is an avascular and aneural tissue characterized by the reduced number of cells and can be found in different tissues, such as intervertebral disc (IVD) and meniscus. These tissues own poor regenerative properties where a massive number of individuals have been affected by their degeneration. The current available treatments have shown poor clinical outcomes and none of them can be consensually designated as the “gold” standard treatment. Tissue engineers have been trying to overcome all the current challenges by developing novel approaches where different biomaterials have been explored to achieve a suitable implant (Chap. I and II). However, the pursuit for the “perfect” biomimetic implant is still a big challenge. Therefore, the combination of high-resolution imaging techniques (magnetic resonance imaging and micro-computed tomography) with 3D printing can be a powerful tool to closely mimic the fibrocartilaginous native tissue. This approach can provide reproducibility of the produced scaffolds and allows the production of patient-specific implants, helping to improve patient recovery time and biofunctionality reestablishment (Chap. III). The concept of patientspecificity is explored in this thesis using natural-based materials, where silk fibroin (SF) plays the central role due to its high processing versatility and remarkable mechanical properties. In the first work, indirect printed patient-specific hierarchical scaffolds were produced combining SF with ionicdoped β-tricalcium phosphates (Chap. V). Furthermore, using a 3D printing extrusion-based technology, an innovative SF-based bioink was developed (Chap. VI). Using the previously developed horseradish peroxidase-mediated crosslinking system, 3D patient-specific memory-shape implants were produced (Chap. VII). As third work, a step forward in terms of mimicking the IVD native tissue was given, where the previously developed SF bioink was combined with elastin (Chap. VIII). Finally, an extrusion-based 3D printing hybrid system comprising a gellan gum/fibrinogen cell-laden bioink and a SF methacrylated bioink was developed to produce cell-laden patient-specific implants (Chap. IX). In summary, the proposed novel 3D printing approaches revealed to be promising alternatives for the production of patient-specific implants for fibrocartilage regeneration.A escassez de órgãos e a necessidade de transplantação levaram ao congestionamento dos sistemas de saúde, resultando num enorme impacto socioeconómico. Engenharia de Tecidos tem revolucionado a fabricação de tecidos, tornando-se uma ótima alternativa para criar um melhor atendimento ao paciente. Fiibrocartilagem é um tecido avascular e aneural caracterizado pelo reduzido numero de células e pode ser encontrado em diferentes tecidos, como o disco intervertebral (DIV) e o menisco. Estes tecidos possuem fracas propriedades regenerativas, contribuindo para um elevado número de indivíduos afetado pela sua degeneração. Os tratamentos atualmente disponíveis revelam resultados inadequados e nenhum é consensualmente designado como o tratamento padrão. Engenheiros têm tentado superar os desafios encontrados, utilizando diferentes biomateriais para desenvolver novas estratégias para produzir implantes adequados (Cap. I e II). No entanto, a procura por um implante biomimético “perfeito” permanece um grande desafio. A combinação de técnicas de imagem de alta resolução (ressonância magnética e tomografia micro-computadorizada) com a impressão 3D pode ser uma ferramenta poderosa para mimetizar o tecido fibrocartilaginoso. Esta abordagem promove a produção de implantes reprodutiveis e específicos para cada paciente, ajudando a melhorar o tempo de recuperação e o restabelecimento da biofuncionalidade do tecido (Cap. III). O conceito de implantes específicos para cada paciente é explorado nesta tese usando materiais de origem natural, onde a fibroína de seda (SF) desempenha um papel central devido à sua elevada versatilidade de processamento e notáveis propriedades mecânicas. No primeiro trabalho, foram produzidos implantes hierárquicos específicos para cada paciente, impressos indiretamente, combinando SF com fosfatos de β-tricálcio dopados com iões (Cap. V). Para além disso, usando uma tecnologia de impressão 3D, desenvolveu-se uma “bioink” de SF usando um processamento rápido (Cap. VI). Utilizando um sistema de reticulao com base na enzima peroxidase, foram produzidos implantes 3D específicos para cada paciente (Cap. VII). No terceiro trabalho, foi feita uma melhoria em termos de mimetização do DIV cojungando elastina com a “bioink” de SF (Cap. VIII). Finalmente, foi desenvolvido um sistema híbrido de impressão 3D baseado em extrusão usando uma “bioink” de goma gelana/fibrinogénio com células encapsuladas e uma “bioink” de SF metacrilada (Cap. IX). Em resumo, estas novas abordagens de impressão 3D revelaram ser alternativas promissoras para a produção de implantes específicos para cada paciente visando a regeneração de fibrocartilagem

Deep learning in bioengineering and biofabrication: a powerful technology boosting translation from research to clinics

Bioengineering has been revolutionizing the production of biofunctional tissues for tackling unmet clinical needs. Bioengineers have been focusing their research in biofabrication, especially 3D bioprinting, providing cutting-edge approaches and biomimetic solutions with more reliability and cost–effectiveness. However, these emerging technologies are still far from the clinical setting and deep learning, as a subset of artificial intelligence, can be widely explored to close this gap. Thus, deep-learning technology is capable to autonomously deal with massive datasets and produce valuable outputs. The application of deep learning in bioengineering and how the synergy of this technology with biofabrication can help (more efficiently) bring 3D bioprinting to clinics, are overviewed herein.The authors acknowledge financial support provided through projects B-FABULUS (PTDC/BBB-ECT/2690/2014), Fun4TE (PTDC/EMD-EMD/31367/2017) and JUSThera (ref: NORTE-01-0145-FEDER-000055), financed by the Portuguese Foundation for Science and Technology (FCT) and co-financed by European Regional Development Fund (FEDER) and Operational Program for Competitiveness and Internationalization (POCI). JB Costa acknowledges the Junior Researcher contract (POCI-01-0145-FEDER 031367) attributed by FCT to Fun4TE. The FCT distinction attributed to J Silva-Correia (IF/00115/2015) under the Investigator FCT program is also greatly acknowledged

Carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds for bone tissue engineering applications

Accepted ManuscriptIn bone tissue engineering, the development of advanced biomimetic scaffolds has led to the quest for biomotifs in scaffold design that better recreate bone matrix structure and composition and hierarchy at different length scales. In this study, an advanced hierarchical scaffold consisting of silk fibroin combined with decellularized cell-derived extracellular matrix and reinforced with carbon nanotubes was developed. The goal of the carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds is to harvest the individual properties of its constituents to introduce hierarchical capacity in order to improve standard silk fibroin scaffolds. The scaffolds were fabricated using enzymatic cross-linking, freeze modeling, and decellularization methods. The developed scaffolds were assessed for pore structure and mechanical properties showing satisfying results to be used in bone regeneration. The developed carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds showed to be bioactive in vitro and expressed no hemolytic effect. Furthermore, cellular in vitro studies on human adipose-derived stem cells (hASCs) showed that scaffolds supported cell proliferation. The hASCs seeded onto these scaffolds evidenced similar metabolic activity to standard silk fibroin scaffolds but increased ALP activity. The histological stainings showed cells infiltration into the scaffolds and visible collagen production. The expression of several osteogenic markers was investigated, further supporting the osteogenic potential of the developed carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds. The hemolytic assay demonstrated the hemocompatibility of the hierarchical scaffolds. Overall, the carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds presented the required architecture for bone tissue engineering applications.Research and Innovation Staff Exchanges (RISE) action (H2020 Marie Skłodowska-Curie actions) for funds obtained through the BAMOS project (H2020-MSCA-RISE-2016-73415) and the R&D Project KOAT PTDC/BTMMAT/29760/2017 (POCI-01-0145- FEDER-029760), financed by Fundação para a Ciência e a Tecnologia (FCT) and co-financed by FEDER and POCI. F.R.M. acknowledges FCT for her contract under the Transitional Rule DL 57/2016 (CTTI-57/18-I3BS(5)). V.P.R. acknowledges the Junior Researcher contracts (POCI-01-0145-FEDER-031367; POCI-01-0145-FEDER-029139) attributed by the FCT under the projects Fun4TE project (PTDC/EMD-EMD/31367/2017) and BLiver (PTDC/EMD-EMD/29139/2017). J.B.C. acknowledges the Junior Researcher contract (POCI-01-0145-FEDER-031367) attributed by FCT to the Fun4TE project (PTDC/EMDEMD/31367/2017)

3D Bioprinted Highly Elastic Hybrid Constructs for Advanced Fibrocartilaginous Tissue Regeneration

Advanced strategies to bioengineer a fibrocartilaginous tissue to restore the function of the meniscus are necessary. Currently, 3D bioprinting technologies have been employed to fabricate clinically relevant patient-specific complex constructs to address unmet clinical needs. In this study, a highly elastic hybrid construct for fibrocartilaginous regeneration is produced by coprinting a cell-laden gellan gum/fibrinogen (GG/FB) composite bioink together with a silk fibroin methacrylate (Sil-MA) bioink in an interleaved crosshatch pattern. We characterize each bioink formulation by measuring the rheological properties, swelling ratio, and compressive mechanical behavior. For in vitro biological evaluations, porcine primary meniscus cells (pMCs) are isolated and suspended in the GG/FB bioink for the printing process. The results show that the GG/FB bioink provides a proper cellular microenvironment for maintaining the cell viability and proliferation capacity, as well as the maturation of the pMCs in the bioprinted constructs, while the Sil-MA bioink offers excellent biomechanical behavior and structural integrity. More importantly, this bioprinted hybrid system shows the fibrocartilaginous tissue formation without a dimensional change in a mouse subcutaneous implantation model during the 10-week postimplantation. Especially, the alignment of collagen fibers is achieved in the bioprinted hybrid constructs. The results demonstrate this bioprinted mechanically reinforced hybrid construct offers a versatile and promising alternative for the production of advanced fibrocartilaginous tissue.United States National Institutes of Health (1P41EB023833-346 01) and the Portuguese Foundation for Science and Technology (PTDC/BBB-ECT/2690/2014 and PTDC/EMD-EMD/ 31367/2017). FCT/MCTES is acknowledged for the Ph.D. scholarship attributed to J.B.C. (PD/BD/113803/2015) and the financial support provided to J.S.-C. (IF/00115/2015) and J.M.O. (IF/01285/2015) under the program “Investigador FCT