Current advances and future perspectives in extrusion-based bioprinting

4 - Extrusion-Based Bioprinting

Three-dimensional (3D) extrusion-based bioprinting is the most widely used bioprinting technology to fabricate bionic tissue or organ constructs by combining biomaterial ink and living cells for tissue engineering and regenerative medicine. One critical issue of this technique is the selection of suitable biomaterial ink to simulate extracellular matrix (ECM) that provides mechanical support for cells and regulates their physiological activities. Previous studies have demonstrated that it is an enormous challenge to form and maintain reproducible 3D constructs and eventually achieve the balance among biocompatibility, mechanical properties, and printability. This review highlights the properties of extrusion-based biomaterial inks and recent developments as well as details various biomaterial inks classified by their function. Key approaches related to their modification methods according to the functional requirements are also discussed, along with the selection strategies by varying extrusion paths and methods in extrusion-based bioprinting. This systematical review will assist researchers in identifying the most suitable extrusion-based biomaterial inks based on their requirements, as well as in elaborating current challenges and prospects of extrudable biomaterial inks in the field of bioprinting of in vitro tissue models.

Extrusion-based bioprinting (EBB) is a rapidly developing technique that has made substantial progress in the fabrication of constructs for cartilage tissue engineering (CTE) over the past decade. With this technique, cell-laden hydrogels or bio-inks have been extruded onto printing stages, layer-by-layer, to form three-dimensional (3D) constructs with varying sizes, shapes, and resolutions. This paper reviews the cell sources and hydrogels that can be used for bio-ink formulations in CTE application. Additionally, this paper discusses the important properties of bio-inks to be applied in the EBB technique, including biocompatibility, printability, as well as mechanical properties. The printability of a bio-ink is associated with the formation of first layer, ink rheological properties, and crosslinking mechanisms. Further, this paper discusses two bioprinting approaches to build up cartilage constructs, i.e., self-supporting hydrogel bioprinting and hybrid bioprinting, along with their applications in fabricating chondral, osteochondral, and zonally organized cartilage regenerative constructs. Lastly, current limitations and future opportunities of EBB in printing cartilage regenerative constructs are reviewed.

This article begins with a description of extrusion-based bioprinting for tissue scaffold fabrication. It also examines various extrusion-based bioprinting processes and related tissue scaffolding strategies, presents the selection criteria of various bioinks with various polymers and their printed scaffolds for applications in tissue engineering and regenerative medicines, and provides future research recommendations to address the shortcomings and issues found in current extrusion-based bioprinting processes.

In the last decade, bioprinting has emerged as a facile technique for fabricating tissues constructs mimicking the architectural complexity and compositional heterogeneity of native tissues. Amongst different bioprinting modalities, extrusion-based bioprinting (EBB) is the most widely used technique. Coaxial bioprinting, a type of EBB, enables fabrication of concentric cell-material layers and enlarges the scope of EBB to mimic several key aspects of native tissues. Over the period of development of bioprinting, tissue constructs integrated with vascular networks, have been one of the major achievements made possible largely by coaxial bioprinting. In this review, current advancements in biofabrication of constructs with coaxial bioprinting are discussed with a focus on different bioinks that are particularly suitable for this modality. This review also expounds the properties of different bioinks suitable for coaxial bioprinting and then analyses the key achievements made by the application of coaxial bioprinting in tissue engineering, drug delivery and in-vitro disease modelling. The major limitations and future perspectives on the critical factors that will determine the ultimate clinical translation of the versatile technique are also presented to the reader.

Additive manufacturing has shown promising results in reconstructing three-dimensional (3D) living tissues for various applications, including tissue engineering, regenerative medicine, drug discovery, and high-throughput drug screening. In extrusion-based bioprinters, stable formation of filaments and high-fidelity deposition of bioinks are the primary challenges in fabrication of physiologically relevant tissue constructs. Among various bioinks, gelatin methacryloyl (GelMA) is known as a photocurable and physicochemically tunable hydrogel with a demonstrated biocompatibility and tunable biodegradation properties. The two-step crosslinking of GelMA (reversible thermal gelation and permanent photo-crosslinking) has attracted researchers to make complex tissue constructs. Despite promising results in filament formation and printability of this hydrogel, the effect of temperature on physicochemical properties, cytocompatibility, and biodegradation of the hydrogel are to be investigated. This work studies the effect of thermoreversible, physical crosslinking on printability of GelMA. The results of 3D printing of GelMA at different temperatures followed by irreversible chemical photo-crosslinking show that the decrease in temperature improves the filament formation and shape fidelity of the deposited hydrogel, particularly at the temperatures around 15 °C. Time dependant mechanical testing of the printed samples revealed that decreasing the extruding temperature increases the elastic properties of the extruded filaments. Furthermore, our novel approach in minimizing the slippage effect during rheological study enabled to measure changes in linear and non-linear viscoelastic properties of the printed samples at different temperatures. A considerable increase in storage modulus of the extruded samples printed at lower temperatures confirms their higher solid behavior. Scanning electron microscopy revealed a remarkable decrease in porosity of the extruded hydrogels by decreasing the temperature. Chemical analysis by Fourier-transform infrared spectroscopy and circular dichroism showed a direct relationship between the coil-helix transition in hydrogel macromers and its physical alterations. Finally, biodegradation and cytocompatibility of the extruded hydrogels decreased at lower extruding temperatures.

3D bio-printer is a rapidly developing technology that has made great innovations in tradition manufacture process during the last decade. Due to the different printing forming methods, various 3D bio-printers are divided into inkjet-based bio-printer, nozzle-based bio-printer and laser-based bio-printer, which have widespread application in basic research and pharmaceutics to clinics. However, the most important challenge is to produce the uniform droplets and realize the function of printing on-demand. In this paper, we discuss the current advantages in printing on-demand technology and design a novel nozzle with the functions of printing on-demand and continuous extrusion. The nozzle control mechanism is detailed described in the process of droplet-generation and experiment analysis has been designed to verify the feasibility of nozzle control process. These experiment results show that the coaxial focusing nozzle with self-filling function has great application prospect to generate viable end products for tissue engineering and regenerative medicine.

Investigation of Hydrogel and Gelatin Bath Formulations for Extrusion-Based 3D Bioprinting using Deep Learning

Towards preserving post-printing cell viability and improving the resolution: Past, present, and future of 3D bioprinting theory

From Tissue Engineering to Organ Engineering

Printability–A key issue in extrusion-based bioprinting

Extrusion-based 3D bioprinting is a widely used technique for fabricating cell-laden constructs in tissue engineering and regenerative medicine. However, the mechanical stresses experienced by cells during the printing process can negatively impact their viability. This study examines the influence of nozzle geometry—specifically contrac- tion angle and outlet diameter—on stress distribution and its effects on cell survival. Through a combination of experimental analysis and theoretical modeling, we explore how nozzle design affects the balance between shear and extensional stresses during bioprinting. The findings highlight the importance of optimizing nozzle parameters to minimize mechanical damage and improve post-printing cell viability. The pro- posed model provides a framework for guiding nozzle design, offering insights for the development of customized bioprinting strategies that enhance construct fidelity and biological functionality. These results contribute to advancing bioprinting techniques for applications in tissue engineering and regenerative medicine.

Editorial: Tissue Engineering: Perspectives, Challenges, and Future Directions

Hydrogels for extrusion-based bioprinting: General considerations

In this review, we propose a comprehensive overview of additive manufacturing (AM) technologies and design possibilities in manufacturing metamaterials for various applications in the biomedical field, of which many are inspired by nature itself. It describes how new AM technologies (e.g. continuous liquid interface production and multiphoton polymerization, etc) and recent developments in more mature AM technologies (e.g. powder bed fusion, stereolithography, and extrusion-based bioprinting (EBB), etc) lead to more precise, efficient, and personalized biomedical components. EBB is a revolutionary topic creating intricate models with remarkable mechanical compatibility of metamaterials, for instance, stress elimination for tissue engineering and regenerative medicine, negative or zero Poisson’s ratio. By exploiting the designs of porous structures (e.g. truss, triply periodic minimal surface, plant/animal-inspired, and functionally graded lattices, etc), AM-made bioactive bone implants, artificial tissues, and organs are made for tissue replacement. The material palette of the AM metamaterials has high diversity nowadays, ranging from alloys and metals (e.g. cobalt–chromium alloys and titanium, etc) to polymers (e.g. biodegradable polycaprolactone and polymethyl methacrylate, etc), which could be even integrated within bioactive ceramics. These advancements are driving the progress of the biomedical field, improving human health and quality of life.