Bioink properties before, during and after 3D bioprinting

Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction

Chemical insights into bioinks for 3D printing

International audienc

PHOTOCURABLE HYDROGELS FOR TISSUE ENGINERING APPLICATIONS

L'abstract è presente nell'allegato / the abstract is in the attachmen

Thermosensitive chitosan-based hydrogels for extrusion-based bioprinting and injectable scaffold for articular tissue engineering

La bio-impression est une forme avancée de fabrication additive qui permet de créer des structures 3D vivantes (contenant des cellules) et de créer des modèles 3D de tissus ou, à plus long terme, des tissus implantables pour remplacer les tissus ou organes malades ou endommagés. La bio-impression connaît une croissance rapide mais doit faire face à plusieurs défis. L'un d'entre eux consiste à trouver des matériaux extrudables contenant des cellules (appelée bioencres) qui combinent toutes les propriétés requises. Les hydrogels de chitosan thermosensibles qui forment des solutions à température ambiante mais gélifient rapidement à la température du corps sont d’intéressants candidats comme bioencre mais à ce jour il n'y a pas encore eu de résultats convaincants démontrant leur potentiel. De plus, les méthodes rhéologiques permettant de prédire leur imprimabilité font toujours défaut. L'objectif général de ce doctorat était d'étudier et optimiser les hydrogels thermosensibles à base de chitosan fabriqué avec un mélange de deux bases faibles, (bêta-glycérophosphate et hydrogénocarbonate de sodium) pour la bio-impression par extrusion, notamment pour l'ingénierie des tissus articulaires. Nous avons tout d’abord développé une approche rhéologique pour évaluer leur potentiel en tant que bioencres. Les cinétiques de gélification à température ambiante et du corps ont été caractérisées. Puis les essais de viscosité et de récupération ont été adaptés pour prendre en compte l’absence de stabilité des gels. La fidélité de forme et les propriétés mécaniques des structures imprimées ont également été caractérisées en fonction du taux de cisaillement appliqué et les résultats corrélés avec les données rhéologiques. Nous avons démontré qu'il était possible d'imprimer une structure avec une fidélité et une maniabilité adéquate; cependant, une concentration élevée de chitosan (3%p/v) est nécessaire, ce qui entraîne un taux de mortalité élevé des cellules, tandis que réduire la concentration à 2%p/v entraîne une très mauvaise fidélité de la forme. Nous avons surmonté ces limites en utilisant une approche basée sur la bio-impression FRESH (Freeform reversible embedding of suspended hydrogel). Un bain de support chaud a été conçu afin de soutenir les structures bioprintées et d'améliorer la thermoréticulation du chitosan pendant l'impression. Cette approche augmente drastiquement la fidélité et les propriétés mécaniques des structures imprimées avec une concentration de chitosane (2% p/v) adaptée à l'encapsulation de cellules. ii Enfin, nous avons étudié l'impact du chargement de particules de bioverre osteoconducteurs dans ces hydrogels thermosensibles, en vue de leur utilisation pour la fabrication de tissus osseux minéralisés. Les propriétés mécaniques et la cytocompatibilité in vitro étant affectées de manière négative par l'ajout de bioglass, notre stratégie a consisté à concentrer le bioverre sous forme de microbilles, puis incorporer ces microbilles dans l'hydrogel à base de chitosan chargé de cellules. Cette nouvelle stratégie a permis d'améliorer considérablement les propriétés mécaniques et la viabilité des cellules. Cet hydrogel bioactif hybride n’est pas utilisable comme bioencre, mais il est injectable et pourrait être utilisé comme matrice injectable pour la régénération de défauts osseux. Cependant, il reste encore beaucoup d’optimisation à faire pour la bio-impression de tissus de gradient complexes.Bioprinting is an advanced method that enables to engineer living 3D structures mimicking the tissue complexity found in-vivo. It allows to create 3D tissues to study drugs/biological mechanisms, also, in longer-term, implantable tissue to replace diseased/damaged body tissues/organs. Bioprinting is growing rapidly but faces several challenges. One of them is to find ideal bioinks which combine all the required properties. Hydrogels are generally used since cells require an aqueous environment. But it is very challenging to stack hydrogels into a 3D structure because hydrogels are weak by nature and cannot support the structure without collapsing. Among the potential candidates are thermosensitive chitosan hydrogels which form solutions at room temperature but rapidly gel at body temperature. However, their potential in bioprinting has not been yet studied. Moreover, comprehensive rheological methods to predict their printability are still missing. The general objective of this Ph.D. was to study and optimize the thermosensitive chitosan-based hydrogels for extrusion-based bioprinting and injectable scaffold for articular tissue engineering. The first objective was to develop a rheological approach to study printability of these time- and temperature-dependent hydrogels and assess their potential as bioinks. Chitosan-based physical hydrogels prepared by combining chitosan acidic solution with weak bases like beta-glycerophosphate and sodium-hydrogen-carbonate were studied. Gelation kinetics, shear-thinning viscosity as a function of shear rate corresponding to that applied during printing, and recovery tests were performed. The resolution and mechanical properties were characterized as a function of applied shear rate and results were correlated with rheological data. This work allowed us to determine the best chitosan hydrogel formulation for 3Dprinting and compare it with conventionally used bioink, alginate/gelatin. This methodology can also be useful for other temperature- and time-dependent materials. We demonstrated that printing structures with adequate fidelity and handability using chitosan-based hydrogels was feasible; however, a high concentration (3%w/v) was required, leading to high mortality rate of encapsulated cells. Decreasing chitosan concentration resulted in poor shape fidelity. The second objective was therefore to develop a method using Freeform reversible embedding of suspended hydrogel (FRESH) bioprinting to overcome these limitations. A warm support bath was designed to support chitosan-based bioprinted structures and enhance chitosan thermo-crosslinking during printing. This approach iv drastically increases the fidelity and mechanical properties of structures printed with low concentration chitosan (2%w/v) suitable for cell encapsulation. Lastly, we studied the impact of loading bioglass particles into such thermosensitive hydrogels for potential bone-mineralized tissue repair, which could promote bone ingrowth through osteoconductivity. The mechanical properties and in-vitro cytocompatibility are affected adversely by bioglass addition. A new strategy was implemented to encapsulate bioglass within chitosan-based microbeads, then incorporate these microbeads in the cell-laden chitosan-based hydrogel. This strategy improved mechanical properties and cell viability. This hybrid hydrogel could be used to form an injectable cell-loaded scaffold. The bioactive microbeads were freezable, increasing their potential for clinical applications. We demonstrated the potential of the thermosensitive chitosan-based hydrogels for bioprinting, especially with the FRESH approach. This opens interesting avenues toward tissue engineering. However, much works still remain to be done before bioprinting complex gradient tissues

Applications of Alginate-Based Bioinks in 3D Bioprinting.

Three-dimensional (3D) bioprinting is on the cusp of permitting the direct fabrication of artificial living tissue. Multicellular building blocks (bioinks) are dispensed layer by layer and scaled for the target construct. However, only a few materials are able to fulfill the considerable requirements for suitable bioink formulation, a critical component of efficient 3D bioprinting. Alginate, a naturally occurring polysaccharide, is clearly the most commonly employed material in current bioinks. Here, we discuss the benefits and disadvantages of the use of alginate in 3D bioprinting by summarizing the most recent studies that used alginate for printing vascular tissue, bone and cartilage. In addition, other breakthroughs in the use of alginate in bioprinting are discussed, including strategies to improve its structural and degradation characteristics. In this review, we organize the available literature in order to inspire and accelerate novel alginate-based bioink formulations with enhanced properties for future applications in basic research, drug screening and regenerative medicine.no funding source acknowledge

Bioprinting of Chondrocyte-Laden Hydrogel Constructs and their In-Vitro Characterization for Cartilage Tissue Engineering

Abstract Articular cartilage lines the ends of bones, provides low friction and load bearing, and allows for efficient joint movement. Once damaged, articular cartilage has difficulty of repairing itself due to lack of blood and nerve supply. Cartilage tissue engineering (CTE) aims to provide solutions to cartilage defects and involves the use of cells, scaffolds, and stimulating factors, alone or in combination. Hydrogel, a crosslinked polymeric network containing large amounts of water, is regarded as the ideal scaffolding material for CTE due to its structural similarity to native cartilage. Encapsulating chondrocytes in hydrogels is a promising approach to provide high cell seeding density, uniform cell distribution and a suitable microenvironment for encapsulated chondrocytes. However, fabrication of hydrogel scaffolds with desired microstructure/ internal structure and living cells is the key issue, which limits hydrogel’s applications in cartilage tissue engineering. To address these issues, this thesis aimed to bioprint cartilage constructs that incorporate living cells and characterize them in vitro for CTE. This aim was achieved via pursuing the following three specific objectives. The first objective was to fabricate CTE scaffolds based on the bioprinting technique and to study the influence of scaffold design on the mechanical performance. Gelatin and alginate mixtures were synthesized and printed into porous hydrogel scaffolds with the help of thermal/submerged ionic crosslinking process. The scaffold geometries, including stand orientation and the spacing between them, were adjusted for bioprinting and their influence on the scaffold mechanical properties were investigated. Results showed that there was a significant influence of internal design on the mechanical performance of printed hydrogel scaffolds and porosity, contact area between strands and spacing variation were three key factors that influence the mechanical performance of scaffolds. The second objective was to develop a 3D Bioplotting technique or process supplemented with the submerged cross-linking mechanism to fabricate alginate hydrogel constructs with living cells. In vitro biological performance of the printed alginate constructs was evaluated in terms of cell viability, proliferation and secretion of sulfated glycosaminoglycan (GAG) and Collagen type II. Chondrocytes were homogeneously distributed in the bioprinted hydrogels and cell viabilities were around 80%. Cartilage extracellular matrix (ECM) including glycosaminoglycan (GAG) and Collagen type II were synthesized by embedded chondrocytes, demonstrating the promising biocompatibility of this bioprinting technique. The third objective was to test the hypothesis that homogeneously dispersed hydroxyapatite in alginate hydrogel promotes the formation of calcified cartilage matrix. Cell growth, extracellular matrix (ECM) production, and mineralization potential were evaluated in the presence or absence of hydroxyapatite particles for comparison. The hydroxyapatite (HAP) phase was evenly dispersed into alginate hydrogel with the addition of a surfactant-sodium citrate (SC). Chondrocytes embedded in this composite hydrogel demonstrated expression of alkaline phosphatase (ALP) after 14 days of culture. Characteristic ECM in calcified cartilage such as minerals and Collagen type X showed a significantly higher synthesis in composite hydrogels with pre-incorporated HAP than that of alginate hydrogels. These results provided researchers with a facile technique to bioprint porous chondrocyte-laden hydrogel constructs for application in CTE and demonstrated a technique of inducing chondrocytes to synthesize calcified cartilage matrix by simply mixing HAP into hydrogel. Taken all together, this thesis presented the techniques/methods developed to bioprint cartilage constructs with living cells and would bring forward the fabrication of constructs for the repair of cartilage defects

4D Printing: The Development of Responsive Materials Using 3D-Printing Technology

Additive manufacturing, widely known as 3D printing, has revolutionized the production of biomaterials. While conventional 3D-printed structures are perceived as static, 4D printing introduces the ability to fabricate materials capable of self-transforming their configuration or function over time in response to external stimuli such as temperature, light, or electric field. This transformative technology has garnered significant attention in the field of biomedical engineering due to its potential to address limitations associated with traditional therapies. Here, we delve into an in-depth review of 4D-printing systems, exploring their diverse biomedical applications and meticulously evaluating their advantages and disadvantages. We emphasize the novelty of this review paper by highlighting the latest advancements and emerging trends in 4D-printing technology, particularly in the context of biomedical applications.The authors would like to acknowledge grants from the Universidad de Buenos Aires, UBACYT 20020150100056BA and PIDAE 2022 (Martín F. Desimone), and from CONICET PIP 0826 (Martín F. Desimone), and PIBAA 28720210100962CO (Sofia Municoy), which supported this work

Co-axial printing of growth factor-laden microspheres for pancreatic islet transplantation

Type I diabetes is an autoimmune disease affecting millions of people in the world. It occurs when the pancreas cannot produce insulin, resulting in episodes of hyperglycaemia that can lead to heart attacks, renal failure, or death. The main cause is the auto destruction of beta cells that produce insulin, located in the pancreatic islets (or islets of Langerhans). Current treatments include insulin injections that decrease the blood glucose level. However, it can sometimes generate hypoglycaemia or insulin resistance on the patients. Bioprinting allows controlled engineering of pancreatic islets with hydrogel scaffolds and transplanting them into the patients. Nevertheless, immunotolerance of the grafted constructs has yet to be achieved. Currently, the islets are implanted together with immunosuppressors to avoid the rejection, but these affect the functionality of the beta cells. Co-transplanting regulatory T cells (Tregs) that regulate the autoimmune response could be the solution to immune rejection. Thus, co-axial extrusion printing is a promising approach, as it allows printing two types of bioinks. Pancreatic islets can be printed in the core of the structure and Tregs in the shell, protecting the islets. This project was mainly focused on the development of the bioink for the shell. The ink consists of a hydrogel that promotes cell growth and allows bioprinting (2% alginate/7.5% gelatin methacrylolyl (GelMA)/3.5% gelatin), and growth factors for Treg functionality (IL-2). The growth factors were encapsulated in GelMA microspheres for a sustained release inside the ink. The release rate of IL-2 was studied, as well as the ink properties and printability

Additive manufacturing of bioactive glass biomaterials

Tissue engineering (TE) and regenerative medicine have held great promises for the repair and regeneration of damaged tissues and organs. Additive manufacturing has recently appeared as a versatile technology in TE strategies that enables the production of objects through layered printing. By applying 3D printing and bioprinting, it is now possible to make tissue-engineered constructs according to desired thickness, shape, and size that resemble the native structure of lost tissues. Up to now, several organic and inorganic materials were used as raw materials for 3D printing; bioactive glasses (BGs) are among the most hopeful substances regarding their excellent properties (e.g., bioactivity and biocompatibility). In addition, the reported studies have confirmed that BG-reinforced constructs can improve osteogenic, angiogenic, and antibacterial activities. This review aims to provide an up-to-date report on the development of BG-containing raw biomaterials that are currently being employed for the fabrication of 3D printed scaffolds used in tissue regeneration applications with a focus on their advantages and remaining challenges