Thermo-Responsive Nanocomposite Bioink with Growth-Factor Holding and its Application to Bone Regeneration

Three-dimensional (3D) bioprinting, which is being increasingly used in tissue engineering, requires bioinks with tunable mechanical properties, biological activities, and mechanical strength for in vivo implantation. Herein, a growth-factor-holding poly(organophosphazene)-based thermo-responsive nanocomposite (TNC) bioink system is developed. The mechanical properties of the TNC bioink are easily controlled within a moderate temperature range (5–37 °C). During printing, the mechanical properties of the TNC bioink, which determine the 3D printing resolution, can be tuned by varying the temperature (15–30 °C). After printing, TNC bioink scaffolds exhibit maximum stiffness at 37 °C. Additionally, because of its shear-thinning and self-healing properties, TNC bioinks can be extruded smoothly, demonstrating good printing outcomes. TNC bioink loaded with bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta1 (TGF-β1), key growth factors for osteogenesis, is used to print a scaffold that can stimulate biological activity. A biological scaffold printed using TNC bioink loaded with both growth factors and implanted on a rat calvarial defect model reveals significantly improved bone regenerative effects. The TNC bioink system is a promising next-generation bioink platform because its mechanical properties can be tuned easily for high-resolution 3D bioprinting with long-term stability and its growth-factor holding capability has strong clinical applicability.     

3D bioprinting of multicellular scaffolds for osteochondral regeneration

Regeneration of osteochondral tissue is of great potentialities in repairing severe osteochondral defects. However, anisotropic physiological characteristics and tissue linage difference make the regeneration of osteochondral tissue remain a huge challenge. Herein, a multicellular system based on a bilayered co-culture scaffold mimicking osteochondral tissues was successfully developed for an alternative of osteochondral regeneration via a 3D bioprinting strategy. The dual function of integrally repairing both cartilage and bone could be achieved by designing multiple-cells distribution and a cell-induced bioink containing bioceramic particles. As an important bioactive agent, the Li-Mg-Si bioceramics-containing bioink exhibited the function of simultaneously stimulating multiple cells for differentiation towards specific directions. The 3D bioprinted co-culture scaffolds showed the capacity for osteochondral tissue regeneration by inducing osteogenic and chondrogenic differentiation in vitro and accelerating the repair of severe osteochondral defects in vivo. This study offers a potential strategy for complex tissue reconstruction through bioprinting multiple tissue cells in combination of bioceramics-stimulating bioinks.     

Visible Light-Induced 3D Bioprinting Technologies and Corresponding Bioink Materials for Tissue Engineering: A Review

Three-dimensional (3D) bioprinting based on traditional 3D printing is an emerging technology that is used to precisely assemble biocompatible materials and cells or bioactive factors into advanced tissue engineering solutions. Similar technology, particularly photo-cured bioprinting strategies, plays an important role in the field of tissue engineering research. The successful implementation of 3D bioprinting is based on the properties of photopolymerized materials. Photocrosslinkable hydrogel is an attractive biomaterial that is polymerized rapidly and enables process control in space and time. Photopolymerization is frequently initiated by ultraviolet (UV) or visible light. However, UV light may cause cell damage and thereby, affect cell viability. Thus, visible light is considered to be more biocompatible than UV light for bioprinting. In this review, we provide an overview of photo curing-based bioprinting technologies, and describe a visible light crosslinkable bioink, including its crosslinking mechanisms, types of visible light initiator, and biomedical applications. We also discuss existing challenges and prospects of visible light-induced 3D bioprinting devices and hydrogels in biomedical areas.     

Human-Recombinant-Elastin-Based Bioinks for 3D Bioprinting of Vascularized Soft Tissues

Bioprinting has emerged as an advanced method for fabricating complex 3D tissues. Despite the tremendous potential of 3D bioprinting, there are several drawbacks of current bioinks and printing methodologies that limit  the ability to print elastic and highly vascularized tissues. In particular, fabrication of complex biomimetic structure that are entirely based on 3D bioprinting is still challenging primarily due to the lack of suitable bioinks with high printability, biocompatibility, biomimicry, and proper mechanical properties. To address these shortcomings, in this work the use of recombinant human tropoelastin as a highly biocompatible and elastic bioink for 3D printing of complex soft tissues is demonstrated. As proof of the concept, vascularized cardiac constructs are bioprinted and their functions are assessed in vitro and in vivo. The printed constructs demonstrate endothelium barrier function and spontaneous beating of cardiac muscle cells, which are important functions of cardiac tissue in vivo. Furthermore, the printed construct elicits minimal inflammatory responses, and is shown to be efficiently biodegraded in vivo when implanted subcutaneously in rats. Taken together, these results demonstrate the potential of the elastic bioink for printing 3D functional cardiac tissues, which can eventually be used for cardiac tissue replacement.     

Responsive biomaterials for 3D bioprinting: A review

Three-dimensional (3D) bioprinting enables a controlled deposition of cells, biomaterials, and biological compounds (i.e., bioinks) to build complex 3D biological models, biological living systems, and therapeutic products. Developing responsive biomaterials as novel bioinks has been a central focus of research in the field of bioprinting because of their controllable material properties in response to printing-induced external or internal stimuli. In this review, we highlight the most recent advances of responsive biomaterials for 3D bioprinting applications. We review commonly used stimuli-responsive biomaterials and strategies for utilizing multifunctional responsiveness to achieve desirable printability, structural formability, cell viability, and construct bioactivity for 3D bioprinting. We also summarize major bioink formulation strategies currently adopted in 3D bioprinting. We subsequently discuss several promising applications of 3D printing involving responsive biomaterials, such as bioprinting in a supporting bath, 4D bioprinting, and bioprinting new controlled drug delivery systems. Future perspectives on the design and development of novel multifunctional bioinks based on responsive biomaterials and technological innovations are also presented.     

Crosslinking Strategies for 3D Bioprinting of Polymeric Hydrogels

Three‐dimensional (3D) bioprinting has recently advanced as an important tool to produce viable constructs that can be used for regenerative purposes or as tissue models. To develop biomimetic and sustainable 3D constructs, several important processing aspects need to be considered, among which crosslinking is most important for achieving desirable biomechanical stability of printed structures, which is reflected in subsequent behavior and use of these constructs. In this work, crosslinking methods used in 3D bioprinting studies are reviewed, parameters that affect bioink chemistry are discussed, and the potential toward improving crosslinking outcomes and construct performance is highlighted. Furthermore, current challenges and future prospects are discussed. Due to the direct connection between crosslinking methods and properties of 3D bioprinted structures, this Review can provide a basis for developing necessary modifications to the design and manufacturing process of advanced tissue‐like constructs in future.     

The Next Frontier of 3D Bioprinting: Bioactive Materials Functionalized by Bacteria

3D bioprinting has become a flexible technical means used in many fields. Currently, research on 3D bioprinting is mainly focused on the use of mammalian cells to print organ and tissue models, which has greatly promoted progress in the fields of tissue engineering, regenerative medicine, and pharmaceuticals. In recent years, bacterial bioprinting has gradually become a rapidly developing research fields, with a wide range of potential applications in basic research, biomedicine, bioremediation, and other field. Here, this works reviews new research on bacterial bioprinting, and discuss its future research direction.     

Bioprinting of Multiscaled Hepatic Lobules within a Highly Vascularized Construct

Highly vascularized complex liver tissue is generally divided into lobes, lobules, hepatocytes, and sinusoids, which can be viewed under different types of lens from the micro‐ to macro‐scale. To engineer multiscaled heterogeneous tissues, a sophisticated and rapid tissue engineering approach is required, such as advanced 3D bioprinting. In this study, a preset extrusion bioprinting technique, which can create heterogeneous, multicellular, and multimaterial structures simultaneously, is utilized for creating a hepatic lobule (≈1 mm) array. The fabricated hepatic lobules include hepatic cells, endothelial cells, and a lumen. The endothelial cells surround the hepatic cells, the exterior of the lobules, the lumen, and finally, become interconnected with each other. Compared to hepatic cell/endothelial cell mixtures, the fabricated hepatic lobule shows higher albumin secretion, urea production, and albumin, MRP2, and CD31 protein levels, as well as, cytochrome P450 enzyme activity. It is found that each cell type with spatial cell patterning in bioink accelerates cellular organization, which could preserve structural integrity and improve cellular functions. In conclusion, preset extruded hepatic lobules within a highly vascularized construct are successfully constructed, enabling both micro‐ and macro‐scale tissue fabrication, which can support the creation of large 3D tissue constructs for multiscale tissue engineering.     

Organ regeneration: integration application of cell encapsulation and 3D bioprinting

3D bioprinting has shown great promise in the field of tissue engineering, which offers a vital and significant platform for organ regeneration. Therefore, an increasing focus on 3D bioprinting, coupled with a growing knowledge of cell–cell interaction and cell encapsulation, has driven researchers to discover the preferable biomaterials that enable the greatest possible to reconstruct artificial organs with the different cells and hydrogel. One important challenge is to adapt materials selected to improve the cell survival rate. In this paper, we firstly summarise the fundamentals and the latest application of biomaterials that have significant characteristics such as porous, biodegradability, biological compatibility, and adaptability, and further describe formation mechanisms of droplet under the different cell encapsulation technologies, and finally highlight integration application of cell encapsulation and 3D bioprinting.     

Hydrogel Bioink Reinforcement for Additive Manufacturing: A Focused Review of Emerging Strategies

Bioprinting is an emerging approach for fabricating cell-laden 3D scaffolds via robotic deposition of cells and biomaterials into custom shapes and patterns to replicate complex tissue architectures. Bioprinting uses hydrogel solutions called bioinks as both cell carriers and structural components, requiring bioinks to be highly printable while providing a robust and cell-friendly microenvironment. Unfortunately, conventional hydrogel bioinks have not been able to meet these requirements and are mechanically weak due to their heterogeneously crosslinked networks and lack of energy dissipation mechanisms. Advanced bioink designs using various methods of dissipating mechanical energy are aimed at developing next-generation cellularized 3D scaffolds to mimic anatomical size, tissue architecture, and tissue-specific functions. These next-generation bioinks need to have high print fidelity and should provide a biocompatible microenvironment along with improved mechanical properties. To design these advanced bioink formulations, it is important to understand the structure–property–function relationships of hydrogel networks. By specifically leveraging biophysical and biochemical characteristics of hydrogel networks, high performance bioinks can be designed to control and direct cell functions. In this review article, current and emerging approaches in hydrogel design and bioink reinforcement techniques are critically evaluated. This bottom-up perspective provides a materials-centric approach to bioink design for 3D bioprinting.     

3D Bioprinting: from Benches to Translational Applications

Over the last decades, the fabrication of 3D tissues has become commonplace in tissue engineering and regenerative medicine. However, conventional 3D biofabrication techniques such as scaffolding, microengineering, and fiber and cell sheet engineering are limited in their capacity to fabricate complex tissue constructs with the required precision and controllability that is needed to replicate biologically relevant tissues. To this end, 3D bioprinting offers great versatility to fabricate biomimetic, volumetric tissues that are structurally and functionally relevant. It enables precise control of the composition, spatial distribution, and architecture of resulting constructs facilitating the recapitulation of the delicate shapes and structures of targeted organs and tissues. This Review systematically covers the history of bioprinting and the most recent advances in instrumentation and methods. It then focuses on the requirements for bioinks and cells to achieve optimal fabrication of biomimetic constructs. Next, emerging evolutions and future directions of bioprinting are discussed, such as freeform, high‐resolution, multimaterial, and 4D bioprinting. Finally, the translational potential of bioprinting and bioprinted tissues of various categories are presented and the Review is concluded by exemplifying commercially available bioprinting platforms.     

Thiol–Ene Clickable Gelatin: A Platform Bioink for Multiple 3D Biofabrication Technologies

Bioprinting can be defined as the art of combining materials and cells to fabricate designed, hierarchical 3D hybrid constructs. Suitable materials, so called bioinks, have to comply with challenging rheological processing demands and rapidly form a stable hydrogel postprinting in a cytocompatible manner. Gelatin is often adopted for this purpose, usually modified with (meth‐)acryloyl functionalities for postfabrication curing by free radical photopolymerization, resulting in a hydrogel that is cross‐linked via nondegradable polymer chains of uncontrolled length. The application of allylated gelatin (GelAGE) as a thiol–ene clickable bioink for distinct biofabrication applications is reported. Curing of this system occurs via dimerization and yields a network with flexible properties that offer a wider biofabrication window than (meth‐)acryloyl chemistry, and without additional nondegradable components. An in‐depth analysis of GelAGE synthesis is conducted, and standard UV‐initiation is further compared with a recently described visible‐light‐initiator system for GelAGE hydrogel formation. It is demonstrated that GelAGE may serve as a platform bioink for several biofabrication technologies by fabricating constructs with high shape fidelity via lithography‐based (digital light processing) 3D printing and extrusion‐based 3D bioprinting, the latter supporting long‐term viability postprinting of encapsulated chondrocytes.     

Design,physical prototyping and initial characterisation of ‘lockyballs’

Directed tissue self-assembly or bottom-up modular approach in tissue biofabrication is an attractive and potentially superior alternative to a classic top-down solid scaffold-based approach in tissue engineering. For example, rapidly emerging organ printing technology using self-assembling tissue spheroids as building blocks is enabling computer-aided robotic bioprinting of three-dimensional (3D) tissue constructs. However, achieving proper material properties while maintaining desirable geometry and shape of 3D bioprinted tissue engineered constructs using directed tissue self-assembly, is still a challenge. Proponents of directed tissue self-assembly see the solution of this problem in developing methods of accelerated tissue maturation and/or using sacrificial temporal supporting of removable hydrogels. In the meantime, there is a growing consensus that a third strategy based on the integration of a directed tissue self-assembly approach with a conventional solid scaffold-based approach could be a potential optimal solution. We hypothesise that tissue spheroids with ‘velcro®-like’ interlockable solid microscaffolds or simply ‘lockyballs’ could enable the rapid in vivo biofabrication of 3D tissue constructs at desirable material properties and high initial cell density. Recently, biocompatible and biodegradable photo-sensitive biomaterials could be fabricated at nanoscale resolution using two-photon polymerisation (2PP), a development rendering this technique with high potential to fabricate ‘velcro®-like’ interlockable microscaffolds. Here we report design studies, physical prototyping using 2PP and initial functional characterisation of interlockable solid microscaffolds or so-called ‘lockyballs’. 2PP was used as a novel enabling platform technology for rapid bottom-up modular tissue biofabrication of interlockable constructs. The principle of lockable tissue spheroids fabricated using the described lockyballs as solid microscaffolds is characterised by attractive new functionalities such as lockability and tunable material properties of the engineered constructs. It is reasonable to predict that these building blocks create the basis for a development of a clinical in vivo rapid biofabrication approach and form part of recent promising emerging bioprinting technologies.     

A Review on the 3D Printing of Functional Structures for Medical Phantoms and Regenerated Tissue and Organ Applications

Medical models, or “phantoms,” have been widely used for medical training and for doctor-patient interactions. They are increasingly used for surgical planning, medical computational models, algorithm verification and validation, and medical devices development. Such new applications demand high-fidelity, patient-specific, tissue-mimicking medical phantoms that can not only closely emulate the geometric structures of human organs, but also possess the properties and functions of the organ structure. With the rapid advancement of three-dimensional (3D) printing and 3D bioprinting technologies, many researchers have explored the use of these additive manufacturing techniques to fabricate functional medical phantoms for various applications. This paper reviews the applications of these 3D printing and 3D bioprinting technologies for the fabrication of functional medical phantoms and bio-structures. This review specifically discusses the state of the art along with new developments and trends in 3D printed functional medical phantoms (i.e., tissue-mimicking medical phantoms, radiologically relevant medical phantoms, and physiological medical phantoms) and 3D bio-printed structures (i.e., hybrid scaffolding materials, convertible scaffolds, and integrated sensors) for regenerated tissues and organs.     

Fabrication of hierarchical polycaprolactone/gel scaffolds via combined 3D bioprinting and electrospinning for tissue engineering

It is a severe challenge to construct 3D scaffolds which hold controllable pore structure and similar morphology of the natural extracellular matrix(ECM).In this study,a compound technology is proposed by combining the 3D bioprinting and electrospinning process to fabricate 3D scaffolds,which are composed by orthogonal array gel microfibers in a grid-like arrangement and intercalated by a nonwoven structure with randomly distributed polycaprolactone(PCL) nanofibers.Human adiposederived stem cells(hASCs) are seeded on the hierarchical scaffold and cultured 21 d for in vitro study.The results of cells culturing show that the microfibers structure with controlled pores can allow the easy entrance of cells and the efficient diffusion of nutrients,and the nanofiber webs layered in the scaffold can significantly improve initial cell attachment and proliferation.The present work demonstrates that the hierarchical PCL/gel scaffolds consisting of controllable 3D architecture with interconnected pores and biomimetic nanofiber structures resembling the ECM can be designed and fabricated by the combination of 3D bioprinting and electrospinning to improve biological performance in tissue engineering applications.     

Sociotechnical alignment in biomedicine: The 3D bioprinting market beyond technology convergence

The nature of hybrid technologies has been frequently interpreted with the concept of technology convergence. However, this concept tends to highlight only technical aspects of technology and market evolution. In order to provide a more comprehensive picture, the concept of sociotechnical alignment is explored here.The field of 3D bioprinting (the production of biological structures with automated, computer-controlled bioprinters) is focused on here. In the emergent global bioprinting market, companies have relied on three core technologies (tissue engineering, additive manufacturing, and software development) and continue to receive inputs from other technologies.On the biological side, bioprinting has benefited from new approaches such as the use of induced pluripotent stem cells. On the engineering side, it has been possible to use relatively cheap technologies such as open-source processing Arduino boards. On the software side, the proliferation of open source packages has strengthened the possibilities of bioprinting. The combination between these and other technology fringes involves a process of sociotechnical alignment whereby technical, scientific, and political issues are always at play.As a result, different companies have been able to realize different market strategies, having varied geographical reach. However, the first movements towards extensive globalization can also be noticed. In this way, the current diversity of the bioprinting market may be jeopardized in the years to come.     

Designer ‘blueprint’ for vascular trees: morphology evolution of vascular tissue constructs

Organ printing is a variant of the biomedical application of rapid prototyping technology or layer-by-layer additive biofabrication of 3D tissue and organ constructs using self-assembled tissue spheroids as building blocks. Bioengineering of perfusable intraorgan branched vascular trees incorporated into 3D tissue constructs is essential for the survival of bioprinted thick 3D tissues and organs. In order to design the optimal ‘blueprint’ for digital bioprinting of intraorgan branched vascular trees, the coefficients of tissue retraction associated with post-printing vascular tissue spheroid fusion and remodelling must be determined and incorporated into the original CAD. Using living tissue spheroids assembled into ring-like and tube-like vascular tissue constructs, the coefficient of tissue retraction has been experimentally evaluated. It has been shown that the internal diameter of ring-like and the height of tubular-like tissue constructs are significantly reduced during tissue spheroid fusion. During the tissue fusion process, the individual tissue spheroids also change their shape from ball-like to a conus-like form. A simple formula for the calculation of the necessary number of tissue spheroids for biofabrication of ring-like structures of desirable diameter has been deduced. These data provide sufficient information to design optimal CAD for bioprinted branched vascular trees of desirable final geometry and size.     

生物医用材料的3D打印技术与发展

近年来,由于3D打印技术的迅猛发展,有关3D打印技术的应用受到科学界广泛关注,特别是生物医学领域。3D打印材料的瓶颈制约着3D打印技术的发展,特别是用于生物医学领域的打印材料,只有非常局限的几种,但是研究可打印生物医用材料意义重大,经济效益显著。简述了3D打印技术在生物医学工程上的应用。重点综述了用于3D打印的生物医用材料,主要涉及金属、陶瓷、高聚物、复合材料以及生物墨水等,并且阐述了3D打印材料的发展前景。