DESIGN AND DEVELOPMENT OF NEW BIOINKS AND BIOPRINTING STRATEGIES TO PRODUCE SIMPLIFIED EQUIVALENTS OF HUMAN TISSUES, FOR EARTH AND SPACE EXPLORATION APPLICATIONS

[EN] Fifty years after the first human landed on the Moon mankind has started to plan the next steps for manned space exploration missions. When the time required to return to Earth exceeds 400-500 days (as in travels to Mars), the level of independence that the crew must possess is highly increased. To ensure the survival and good living conditions of this future explorers, new developments regarding medical infrastructure and resources must be carried out. In this work, the topic of this study was to investigate how 3D printing and bioprinting can be of use to improve the autonomy of the crew when facing the most probable clinical scenarios that can occur in long-term space exploratory missions. The printing parameters of thermoplastics, such as polycaprolactone (PCL), that can be used as a possible support material along bioprinting were studied, including the use or not of alternate layers or the printing starting point. For bioprinting, and taking bone constructs as the tissue of study, a new bioink based on human plasma (which potentially could be derived from the injured astronaut who needs the treatment), alginate, and methylcellulose (that can be grow and used in isolated areas) was designed. An inverted bioprinter was designed and built to be able to test the work capacity of this biomaterial in altered gravity conditions. As result, it was observed that the use of alternated layers involves the generation of more anisotropic structures, as the z-axis pore is limited to the layer height, while the x and y-pore size can be tailored to specific needs. The defects created because of the alignment, or the lack of it, at the new layer starting point, significantly affects the mechanical behavior of the scaffolds. ix The cooling down rate, or even printing speed, can be modified to obtain more chaotic micropatterns including nanofibers without the need of using further technologies. A plasma-based bioink was successfully designed and combined with hydroxyapatite-forming CPC which resembles the native mineral phase of bone. The results have shown that the plasma-based bioink and CPC form a synergistic system supporting adhesion, proliferation, and osteogenic differentiation of bone cells. Thanks to the inverted bioprinter, it has been demonstrated that the developed bioinks and support materials, like CPC, are suitable for extrusion (bio)printing. They are so adhesive that they can even be printed upside-down and therefore, against Earth’s gravity. Therefore, they could be used at altered gravity conditions on space exploration missions.[ES] Cincuenta años después de que el primer ser humano aterrizara en la Luna, la humanidad ha comenzado a planificar los próximos pasos para las misiones de exploración espacial tripuladas. Cuando el tiempo necesario para regresar a la Tierra supera los 400 - 500 días (como en los viajes a Marte), el nivel de independencia que debe poseer la tripulación aumenta considerablemente. Para garantizar la supervivencia y las buenas condiciones de vida de estos futuros exploradores, se deben realizar nuevos desarrollos en cuanto a infraestructuras y recursos médicos. En este trabajo, se estudió cómo la impresión 3D y la bioimpresión pueden ser de utilidad para mejorar la autonomía de la tripulación ante los escenarios clínicos más probables que pueden ocurrir en misiones de larga duración. Para ello se estudiaron algunos parámetros de impresión de termoplásticos, como la policaprolactona (PCL), que pueden utilizarse x como posible material de soporte a lo largo de la bioimpresión (ejemplo de parámetros: uso o no de capas alternas o la alineación del punto de partida de la impresión en cada capa). Para la bioimpresión, y tomando las construcciones óseas como tejido de estudio, se diseñó un nuevo material compuesto a base de plasma humano (que puede ser obtenido de los astronautas), alginato y metilcelulosa (que se puede obtener y utilizar en áreas aisladas). Se diseñó y construyó una bioimpresora invertida para poder probar la capacidad de trabajo de este biomaterial en condiciones de gravedad alterada. Como resultado se obtuvo que el uso de capas alternas implica la generación de más estructuras anisotrópicas, ya que normalmente el poro del eje z está limitado a la altura de la capa, mientras que el tamaño de los poros x e y se pueden adaptar a necesidades especıificas. Los defectos creados por la alineación, o la falta de ella, en el punto de inicio de la nueva capa, afectan significativamente al comportamiento mecánico de los andamios. La velocidad de enfriamiento, o incluso la velocidad de impresión, se puede modificar para obtener micropatrones más caóticos, incluso nanofibras, sin necesidad de utilizar tecnologías adicionales. Se diseñó con éxito una biotinta a base de plasma y se combinó con un cemento de fosfato cálcico formador de hidroxiapatita, que se asemeja al mineral nativo del hueso. Nuestros resultados mostraron que la deposición combinada de la biotinta basada en plasma y la pasta de CPC para dar lugar a constructos celularizados, forman un sistema sinérgico que apoya la adhesión, la proliferación y la diferenciación osteogénica de las células óseas. Gracias a la bioimpresora invertida desarrollada, se ha podido demostrar que la biotinta desarrollada y los materiales de soporte, como el cemento óseo de fosfato cálcico autoajustable, son aptos para la (bio)impresión extrusión. Son tan adhesivos que incluso se pueden imprimir al revés y, por lo tanto, contra la gravedad de la Tierra, pudiendo usarse en condiciones de gravedad alterada.DAAD Research Grant. Program: Short-Term Grants, 2018 (57378443). – Project "3D Printing of Living Tissue for Space Exploration" funded from the European Space Agency (ESA). Funds came them partially by the University of Dresden via a contract (No. 4000123640/17/NL/BJ/gp/TUD) of OHB System AG, Bremen and by OHB System AG itself, under a contract of the European Space Agency ESA (contract No. 4000123640/18/NL/BJ/gp). – "Low intensity ultrasounds for early detection and modulation of tumour and stroma". Convocatoria Proyecto Retos I+D+i 2017. AEI/FEDER: DPI2017-90147-R. Entidad financiadora: Agencia Estatal de Investigación, Ministerio de Economía, Industria y Competitividad, Gobierno de España.Peer reviewe

3D bioprinting of functional human skin: production and in vivo analysis

Significant progress has been made over the past 25 years in the development of in vitro-engineered substitutes that mimic human skin, either to be used as grafts for the replacement of lost skin, or for the establishment of in vitro human skin models. In this sense, laboratory-grown skin substitutes containing dermal and epidermal components offer a promising approach to skin engineering. In particular, a human plasma-based bilayered skin generated by our group, has been applied successfully to treat burns as well as traumatic and surgical wounds in a large number of patients in Spain. There are some aspects requiring improvements in the production process of this skin; for example, the relatively long time (three weeks) needed to produce the surface required to cover an extensive burn or a large wound, and the necessity to automatize and standardize a process currently performed manually.This work was partially supported by grant DPI2014-61887-EXP from the Spanish Ministerio de Economía y Competitividad

DESIGN AND DEVELOPMENT OF NEW BIOINKS AND BIOPRINTING STRATEGIES TO PRODUCE SIMPLIFIED EQUIVALENTS OF HUMAN TISSUES, FOR EARTH AND SPACE EXPLORATION APPLICATIONS

[EN] Fifty years after the first human landed on the Moon mankind has started to plan the next steps for manned space exploration missions. When the time required to return to Earth exceeds 400-500 days (as in travels to Mars), the level of independence that the crew must possess is highly increased. To ensure the survival and good living conditions of this future explorers, new developments regarding medical infrastructure and resources must be carried out. In this work, the topic of this study was to investigate how 3D printing and bioprinting can be of use to improve the autonomy of the crew when facing the most probable clinical scenarios that can occur in long-term space exploratory missions. The printing parameters of thermoplastics, such as polycaprolactone (PCL), that can be used as a possible support material along bioprinting were studied, including the use or not of alternate layers or the printing starting point. For bioprinting, and taking bone constructs as the tissue of study, a new bioink based on human plasma (which potentially could be derived from the injured astronaut who needs the treatment), alginate, and methylcellulose (that can be grow and used in isolated areas) was designed. An inverted bioprinter was designed and built to be able to test the work capacity of this biomaterial in altered gravity conditions. As result, it was observed that the use of alternated layers involves the generation of more anisotropic structures, as the z-axis pore is limited to the layer height, while the x and y-pore size can be tailored to specific needs. The defects created because of the alignment, or the lack of it, at the new layer starting point, significantly affects the mechanical behavior of the scaffolds. ix The cooling down rate, or even printing speed, can be modified to obtain more chaotic micropatterns including nanofibers without the need of using further technologies. A plasma-based bioink was successfully designed and combined with hydroxyapatite-forming CPC which resembles the native mineral phase of bone. The results have shown that the plasma-based bioink and CPC form a synergistic system supporting adhesion, proliferation, and osteogenic differentiation of bone cells. Thanks to the inverted bioprinter, it has been demonstrated that the developed bioinks and support materials, like CPC, are suitable for extrusion (bio)printing. They are so adhesive that they can even be printed upside-down and therefore, against Earth’s gravity. Therefore, they could be used at altered gravity conditions on space exploration missions.[ES] Cincuenta años después de que el primer ser humano aterrizara en la Luna, la humanidad ha comenzado a planificar los próximos pasos para las misiones de exploración espacial tripuladas. Cuando el tiempo necesario para regresar a la Tierra supera los 400 - 500 días (como en los viajes a Marte), el nivel de independencia que debe poseer la tripulación aumenta considerablemente. Para garantizar la supervivencia y las buenas condiciones de vida de estos futuros exploradores, se deben realizar nuevos desarrollos en cuanto a infraestructuras y recursos médicos. En este trabajo, se estudió cómo la impresión 3D y la bioimpresión pueden ser de utilidad para mejorar la autonomía de la tripulación ante los escenarios clínicos más probables que pueden ocurrir en misiones de larga duración. Para ello se estudiaron algunos parámetros de impresión de termoplásticos, como la policaprolactona (PCL), que pueden utilizarse x como posible material de soporte a lo largo de la bioimpresión (ejemplo de parámetros: uso o no de capas alternas o la alineación del punto de partida de la impresión en cada capa). Para la bioimpresión, y tomando las construcciones óseas como tejido de estudio, se diseñó un nuevo material compuesto a base de plasma humano (que puede ser obtenido de los astronautas), alginato y metilcelulosa (que se puede obtener y utilizar en áreas aisladas). Se diseñó y construyó una bioimpresora invertida para poder probar la capacidad de trabajo de este biomaterial en condiciones de gravedad alterada. Como resultado se obtuvo que el uso de capas alternas implica la generación de más estructuras anisotrópicas, ya que normalmente el poro del eje z está limitado a la altura de la capa, mientras que el tamaño de los poros x e y se pueden adaptar a necesidades especıificas. Los defectos creados por la alineación, o la falta de ella, en el punto de inicio de la nueva capa, afectan significativamente al comportamiento mecánico de los andamios. La velocidad de enfriamiento, o incluso la velocidad de impresión, se puede modificar para obtener micropatrones más caóticos, incluso nanofibras, sin necesidad de utilizar tecnologías adicionales. Se diseñó con éxito una biotinta a base de plasma y se combinó con un cemento de fosfato cálcico formador de hidroxiapatita, que se asemeja al mineral nativo del hueso. Nuestros resultados mostraron que la deposición combinada de la biotinta basada en plasma y la pasta de CPC para dar lugar a constructos celularizados, forman un sistema sinérgico que apoya la adhesión, la proliferación y la diferenciación osteogénica de las células óseas. Gracias a la bioimpresora invertida desarrollada, se ha podido demostrar que la biotinta desarrollada y los materiales de soporte, como el cemento óseo de fosfato cálcico autoajustable, son aptos para la (bio)impresión extrusión. Son tan adhesivos que incluso se pueden imprimir al revés y, por lo tanto, contra la gravedad de la Tierra, pudiendo usarse en condiciones de gravedad alterada.DAAD Research Grant. Program: Short-Term Grants, 2018 (57378443). – Project "3D Printing of Living Tissue for Space Exploration" funded from the European Space Agency (ESA). Funds came them partially by the University of Dresden via a contract (No. 4000123640/17/NL/BJ/gp/TUD) of OHB System AG, Bremen and by OHB System AG itself, under a contract of the European Space Agency ESA (contract No. 4000123640/18/NL/BJ/gp). – "Low intensity ultrasounds for early detection and modulation of tumour and stroma". Convocatoria Proyecto Retos I+D+i 2017. AEI/FEDER: DPI2017-90147-R. Entidad financiadora: Agencia Estatal de Investigación, Ministerio de Economía, Industria y Competitividad, Gobierno de España.Peer reviewe

Design of Thermoplastic 3D-Printed Scaffolds for Bone Tissue Engineering: Influence of Parameters of “Hidden” Importance in the Physical Properties of Scaffolds

Additive manufacturing (AM) techniques are becoming the approaches of choice for the construction of scaffolds in tissue engineering. However, the development of 3D printing in this field brings unique challenges, which must be accounted for in the design of experiments. The common printing process parameters must be considered as important factors in the design and quality of final 3D-printed products. In this work, we study the influence of some parameters in the design and fabrication of PCL scaffolds, such as the number and orientation of layers, but also others of “hidden” importance, such as the cooling down rate while printing, or the position of the starting point in each layer. These factors can have an important impact oin the final porosity and mechanical performance of the scaffolds. A pure polycaprolactone filament was used. Three different configurations were selected for the design of the internal structure of the scaffolds: a solid one with alternate layers (solid) (0 , 90 ), a porous one with 30% infill and alternate layers (ALT) (0 , 90 ) and a non-alternated configuration consisting in printing three piled layers before changing the orientation (n-ALT) (0 , 0 , 0 , 90 , 90 , 90 ). The nozzle temperature was set to 172 C for printing and the build plate to 40 C. Strand diameters of 361 26 m for room temperature cooling down and of 290 30 m for forced cooling down, were obtained. A compression elastic modulus of 2.12 0.31 MPa for n-ALT and 8.58 0.14 MPa for ALT sca olds were obtained. The cooling down rate has been observed as an important parameter for the final characteristics of the scaffold.AEI/FEDER, UE project: DPI2017-90147-R.Peer reviewe

Bioprinting: From a DIY Revolution to Patients

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Design of Thermoplastic 3D-Printed Scaffolds for Bone Tissue Engineering: Influence of Parameters of ¿Hidden¿ Importance in the Physical Properties of Scaffolds

Additive manufacturing (AM) techniques are becoming the approaches of choice for the construction of scaolds in tissue engineering. However, the development of 3D printing in this field brings unique challenges, which must be accounted for in the design of experiments. The common printing process parameters must be considered as important factors in the design and quality of final 3D-printed products. In this work, we study the influence of some parameters in the design and fabrication of PCL scaolds, such as the number and orientation of layers, but also others of ¿hidden¿ importance, such as the cooling down rate while printing, or the position of the starting point in each layer. These factors can have an important impact oin the final porosity and mechanical performance of the scaolds. A pure polycaprolactone filament was used. Three dierent configurations were selected for the design of the internal structure of the scaolds: a solid one with alternate layers (solid) (0, 90), a porous one with 30% infill and alternate layers (ALT) (0, 90) and a non-alternated configuration consisting in printing three piled layers before changing the orientation (n-ALT) (0, 0, 0, 90, 90, 90). The nozzle temperature was set to 172 C for printing and the build plate to 40 C. Strand diameters of 361 26 m for room temperature cooling down and of 290 30 m for forced cooling down, were obtained. A compression elastic modulus of 2.12 0.31 MPa for n-ALT and 8.58 0.14 MPa for ALT scaolds were obtained. The cooling down rate has been observed as an important parameter for the final characteristics of the scaold.This project was funded by AEI/FEDER, UE project: DPI2017-90147-R

Development of a hierarchical clustering method for anomaly identification and labelling of marine machinery data

The application of artificial intelligence models for the fault diagnosis of marine machinery increased expeditiously within the shipping industry. This relates to the effectiveness of artificial intelligence in capturing fault patterns in marine systems that are becoming more complex and where the application of traditional methods is becoming unfeasible. However, despite these advances, the lack of fault labelling data is still a major concern due to confidentiality issues, and lack of appropriate data, for instance. In this study, a method based on histogram similarity and hierarchical clustering is proposed as an attempt to label the distinct anomalies and faults that occur in the dataset so that supervised learning can then be implemented. To validate the proposed methodology, a case study on a main engine of a tanker vessel is considered. The results indicate that the method can be a preliminary option to classify and label distinct types of faults and anomalies that may appear in the dataset, as the model achieved an accuracy of approximately 95% for the case study presented

A Systematic Review on the Generation of Organic Structures through Additive Manufacturing Techniques

Additive manufacturing (AM) has emerged as a transformative technology in the fabrication of intricate structures, offering unparalleled adaptability in crafting complex geometries. Particularly noteworthy is its burgeoning significance within the realm of medical prosthetics, owing to its capacity to seamlessly replicate anatomical forms utilizing biocompatible materials. Notably, the fabrication of porous architectures stands as a cornerstone in orthopaedic prosthetic development and bone tissue engineering. Porous constructs crafted via AM exhibit meticulously adjustable pore dimensions, shapes, and porosity levels, thus rendering AM indispensable in their production. This systematic review ventures to furnish a comprehensive examination of extant research endeavours centred on the generation of porous scaffolds through additive manufacturing modalities. Its primary aim is to delineate variances among distinct techniques, materials, and structural typologies employed, with the overarching objective of scrutinizing the cutting-edge methodologies in engineering self-supported stochastic printable porous frameworks via AM, specifically for bone scaffold fabrication. Findings show that most of the structures analysed correspond to lattice structures. However, there is a strong tendency to use organic structures generated by mathematical models and printed using powder bed fusion techniques. However, no work has been found that proposes a self-supporting design for organic structures

Generation of Controlled Micrometric Fibers inside Printed Scaffolds Using Standard FDM 3D Printers

New additive manufacturing techniques, such as melting electro-writing (MEW) or near-field electrospinning (NFES), are now used to include microfibers inside 3D printed scaffolds as FDM printers present a limited resolution in the XY axis, not making it easy to go under 100 µm without dealing with nozzle troubles. This work studies the possibility of creating reproducible microscopic internal fibers inside scaffolds printed by standard 3D printing. For this purpose, novel algorithms generating deposition routines (G-code) based on primitive geometrical figures were created by python scripts, modifying basic deposition conditions such as temperature, speed, or material flow. To evaluate the influence of these printing conditions on the creation of internal patterns at the microscopic level, an optical analysis of the printed scaffolds was carried out using a digital microscope and subsequent image analysis with ImageJ software. To conclude, the formation of heterogeneously shaped microfilaments (48 ± 12 µm, mean ± S.D.) was achieved in a standard FDM 3D Printer with the strategies developed in this work, and it was found that the optimum conditions for obtaining such microfibers were high speeds and a reduced extrusion multiplier