Edible bird’s nests (EBN)—the nests of swiftlet birds harvested from the wild— are high-end healthcare food in East Asia, while their excessive harvesting poses increasing ecological, environmental, and food safety concerns. Here, we report for the first time a tissue-engineering (TE) approach for fabricating EBNs substitutes by integrating the technologies of three-dimensional (3D) printing and live cell culture. The engineered products, tissue-engineered edible bird’s nests (TeeBN), comprise two layers. The first is a feeding layer that encapsulates epithelial cells in 3D-printed biocompatible gelation scaffolds. These cells secrete bioactive ingredients, e.g., sialic acid and epidermal growth factors (EGF), recapitulating the natural production of these substances by birds. The second is a receiving layer, consisting of food-grade natural polymers, e.g., polysaccharides, which mimics the building blocks of natural EBNs while biologically stabilizing the factors released from the feeding layer. In vitro characterizations demonstrate that the feeding layer facilitates 3D cell growth and functions, and the receiving layer (as the end product) contains the necessary nutrients expected from natural EBNs—while without harmful substances commonly detected in natural EBNs. Further, in vivo metabolomics studies in mice indicate that TeeBN showed a similar profile of serum metabolites as natural EBN, reflecting comparable nutritional effects. In summary, we innovatively developed a tissue engineering-based substitute for EBNs with comparable metabolic functions and minimized safety risks, opening a new avenue for producing delicacy food from laboratorial cell culture with 3D printing technology.

 

Engineered vasculature is widely employed to maintain the cell viability within in vitro tissues. A variety of fabrication techniques for engineered vasculature have been explored, with combination of additive manufacturing with a sacrifice-based technique being the most common approach. However, the size deformation of vasculature caused by the swelling of sacrificial materials remains unaddressed. In this study, Pluronic F-127 (PF-127), the most widely used sacrificial material, was employed to study the deformation of the vasculature. Then, a thermoresponsive hydrogel comprising poly(N-isopropylacrylamide) (PNIPAM) and gelatin methacrylate (GelMA) was used to induce volume shrinkage at 37°C to compensate for the deformation of vasculature caused by the swelling of a three-dimensional (3D)- printed sacrificial template, and to generate vasculature of a smaller size than that after deformation. Our results showed that the vasculature diameter increased after the sacrificial template was removed, whereas it decreased to the designed diameter after the volume shrinkage. Human umbilical vein endothelial cells (HUVECs) formed an endothelial monolayer in the engineered vasculature. Osteosarcoma cells (OCs) were loaded into a hierarchical vasculature within the thermoresponsive hydrogel to investigate the interaction between HUVECs and OCs. New blood vessel infiltration was observed within the lumen of the engineered vasculature after in vivo subcutaneous implantation for 4 weeks. In addition, engineered vasculature was implanted in a rat ischemia model to further study the function of engineered vasculature for blood vessel infiltration. This study presents a small method aiming to accurately create engineered vasculature by additive manufacturing and a sacrifice-based technique.

Methacrylated gelatin (GelMA) has been intensively studied as a 3D printable scaffold material in tissue regeneration fields, which can be attributed to its well-known biological functions. However, the long-term stability of photo-crosslinked GelMA scaffolds is hampered by a combination of its fast degradation in the presence of collagenase and the loss of physical crosslinks at higher temperatures. To increase the longer-term shape stability of printed scaffolds, a mixture of GelMA and tyramine-conjugated 8-arm PEG (8PEGTA) was used to create filaments composed of an interpenetrating network (IPN). Photo-crosslinking during filament deposition of the GelMA and subsequent enzymatic crosslinking of the 8PEGTA were applied to the printed 3D scaffolds. Although both crosslinking mechanisms are radical based, they operate without interference of each other. Rheological data of bulk hydrogels showed that the IPN was an elastic hydrogel, having a storage modulus of 6 kPa, independent of temperature in the range of 10 – 40°C. Tensile and compression moduli were 110 kPa and 80 kPa, respectively. On enzymatic degradation in the presence of collagenase, the gelatin content of the IPN fully degraded in 7 days, leaving a stable secondary crosslinked 8PEGTA network. Using a BioMaker bioprinter, hydrogels without and with human osteosarcoma cells (hMG-63) were printed. On culturing for 21 days, hMG-63 in the GelMA/8PEGTA IPN showed a high cell viability (>90%). Thus, the presence of the photoinitiator, incubation with H2O2, and mechanical forces during printing did not hamper cell viability. This study shows that the GelMA/8PEGTA ink is a good candidate to generate cell-laden bioinks for extrusion-based printing of constructs for tissue engineering applications.

Leveraging three-dimensional (3D) bioprinting in the fields of tissue engineering and regenerative medicine has rapidly accelerated progress toward the development of living tissue constructs and biomedical devices. Ongoing vigorous research has pursued the development of 3D in vitro tissue models to replicate the key aspects of human physiology by incorporating relevant cell populations and adequate environmental cues. Given their advantages of being able to intimately mimic the heterogeneity and complexity of their native counterparts, 3D in vitro models hold promise as alternatives to conventional cell cultures or animal models for translational application to model human physiology/pathology and drug screening. Research has highlighted the importance of in vitro models, and a sophisticated biomanufacturing strategy is vitally required. In particular, vascularization is critical for the prolonged survival and functional maturation of the engineered tissues, which has remained one of the major challenges in the establishment of physiologically relevant 3D in vitro models. To this end, 3D bioprinting can efficiently generate solid and reproducible vascularized tissue models with high architectural and compositional similarity to the native tissues, leading to improve the structural maturation and tissue-specific functionality. Multiple bioprinting strategies have been developed to vascularize in vitro tissues by spatially controlled patterning of vascular precursors or generating readily perfusable vascular structures. This review presents an overview of the advanced 3D bioprinting strategies for vascularized tissue model development. We present the key elements for rebuilding functional vasculature in 3D-bioprinted tissue models and discuss the recent achievements in the engineering of 3D vascularized in vitro models using 3D bioprinting. Finally, we delineate the current challenges and future outlooks of 3D bioprinting-based vascularized tissue models.

 

Large bone defects such as those that occur after trauma or resections due to cancer still are a challenge for surgeons. Main challenge in this area is to find a suitable alternative to the gold-standard therapy, which is highly risky, and a promising option is to use biomaterials manufactured by 3D printing. In former studies, we demonstrated that the combination of polylactic acid (PLA) and bioglass (BG) resulted in a stable 3D-printable material, and porous and finely structured scaffolds were printed. These scaffolds exhibited osteogenic and anti-inflammatory properties. This 3D-printed material fulfills most of the requirements described in the diamond concept of bone healing. However, the question remains as to whether it also meets the requirements concerning angiogenesis. Therefore, the aim of this study was to analyze the effects of the 3D-printed PLA-BG composite material on angiogenesis. In vitro analyses with human umbilical vein endothelial cells (HUVECs) showed a positive effect of increasing BG content on viability and gene expression of endothelial markers. This positive effect was confirmed by an enhanced vascular formation analyzed by Matrigel assay and chicken chorioallantoic membrane (CAM) assay. In this work, we demonstrated the angiogenic efficiency of a 3D-printed PLA–BG composite material. Recalling the osteogenic potential of this material demonstrated in former work, we manufactured a mechanically stable, 3D-printable, osteogenic and angiogenic material, which could be used for bone tissue engineering.

 

Although the development of three-dimensional (3D) printing technology is growing rapidly in the biomedical field, it remains a challenge to achieve arbitrary 3D structures with high resolution and high efficiency. Protein hydrogels fabricated by two-photon polymerization (TPP) have excellent mechanical properties, high precision, and 3D architecture. However, a large number of the amino acid group in bovine serum albumin (BSA) would be consumed when the protein-based hydrogels use dyes of free radical type II photoinitiators. In this study, we use glycidyl methacrylate (GMA) to modify BSA molecules to obtain a series of BSA-GMA materials, allowing the protein material to be two-photon polymerized with a water-soluble free radical type I photoinitiator. The precisely controllable 3D structure of the BSA-GMA hydrogel was fabricated by adjusting the concentration of the precursor solution, the degree of methacrylation, and the processing parameters of the TPP technique. Importantly, BSA-GMA materials are free of acidic hazardous substances. Meanwhile, the water-soluble initiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphite (LAP) allows TPP on the vinyl group of the GMA chain and thus without consuming its amino acid group. The as-prepared BSA-GMA hydrogel structure exhibits excellent autofluorescence imaging, pH responsiveness, and biocompatibility, which would provide new avenues for potential applications in tissue engineering and biomedical fields to meet specific biological requirements.

Intramembranous ossification (IMO) and endochondral ossification (ECO) are two pathways of bone regeneration. The regeneration of most bone, such as limb bone, trunk bone, and skull base bone, mainly occurs in the form of endochondral ossification, which has also become one of the effective ways for bone tissue engineering. In this work, we prepared a well-structured and biocompatible methacrylated gelatin/polymethacrylic acid (GelMA/PMAA) hydrogel by digital light processing (DLP) printing technology, which could effectively chelate iron ions and continuously activate the hypoxia-inducible factor-1 alpha (HIF-1α) signaling pathway to promote the process of endochondral ossification and angiogenesis. The incorporation of PMAA endowed the hydrogel with remarkable viscoelasticity and high efficacy in chelation of iron ions, giving rise to the activation of HIF-1α signaling pathway, improving chondrogenic differentiation in the early stage, and facilitating vascularization in the later stage and bone remodeling. Therefore, the findings have significant implications on DLP printing technology of endochondral osteogenesis induced by the iron-chelating property of biological scaffold, which will provide an effective way in the development of novel bone regeneration.

Three-dimensional (3D) bioprinting technology is one of the most advanced techniques currently applied in tissue engineering and regenerative medicine and has developed rapidly in the past few years. Despite many breakthroughs, there are still several challenges of 3D bioprinting technology awaiting to be addressed, and one of them is the urgency of optimizing bioinks (natural or synthetic hydrogel), which are critical elements in 3D bioprinting, for specific properties. Different from traditional hydrogels, microgels, which are a new type of bioink, are micron-sized gels with excellent mechanical and biological properties, which make them great candidates for applications in 3D bioprinting. Different from the dense and limited pore size of traditional hydrogels, the pore structure of microgel is adjustable, enabling better cell loading before 3D bioprinting, and the printed pores are conducive to the exchange of metabolic substances and cell migration. The “bottom-up” modular microgel has stronger customizable characteristics, and it can freely adjust its mechanical properties, such as hardness, toughness, and rheological properties. In this review, we review the application of microgels in the field of biomedicine and discuss the future development of microgels in 3D bioprinting.

Surgeons use different medical devices in the surgery, such as patient-specific anatomical models, cutting and positioning guides, or implants. These devices must be sterilized before being used in the operation room. There are many sterilization processes available, with autoclave, hydrogen peroxide, and ethylene oxide being the most common in hospital settings. Each method has both advantages and disadvantages in terms of mechanics, chemical interaction, and post-treatment accuracy. The aim of the present study is to evaluate the dimensional and mechanical effect of the most commonly used sterilization techniques available in clinical settings, i.e., Autoclave 121, Autoclave 134, and hydrogen peroxide (HPO), on 11 of the most used 3D-printed materials fabricated using additive manufacturing technologies. The results showed that the temperature (depending on the sterilization method) and the exposure time to that temperature influence not only the mechanical behavior but also the original dimensioning planned on the 3D model. Therefore, HPO is a better overall option for most of the materials evaluated. Finally, based on the results of the study, a recommendation guide on sterilization methods per material, technology, and clinical application is presented.

 

Increasing evidence indicates that macrophages play an important role in angiogenesis and bone regeneration. Because the phenotypic polarization of macrophage is extremely sensitive to the pore size of materials, poly(ether-ether-ketone) (PEEK) scaffolds with pore sizes of 0, 200, and 400 μm were prepared, and the influence of pore size-mediated macrophage polarization on subsequent angiogenesis and osteogenesis was examined. The interaction results of macrophages and scaffolds indicated that macrophages were responsive to the pore size of three-dimensional (3D)-printed PEEK scaffolds, and large pore size scaffolds showed greater potential in inducing M1 to M2 transition of macrophage and enhanced macrophage secretion of high concentrations of osteogenesis-related and angiogenesis-related cytokines. When human umbilical vein endothelial cells (HUVECs) and bone marrow mesenchymal stem cells (BMSCs) were cultured in the conditioned medium derived from co-culture of macrophages and scaffolds, HUVECs showed good angiogenic responses in terms of cell migration and angiogenic gene expression, while BMSCs showed good osteogenic differentiation effect in in vitro mineralization and osteogenesis-related gene expression. The results of bone defect repair showed that the bone volume/total volume ratio and trabecular thickness of the large pore size PEEK scaffold were significantly higher, and it had better biomechanical properties and achieved a better osseointegration effect. Our data demonstrate that large-pore PEEK scaffolds promote angiogenesis and osteogenic differentiation in vitro and osseointegration in vivo, most likely because scaffolds with larger pore size are able to mediate a higher degree of M1 to M2 transition in macrophages.

 The skin plays an important role in vitamin D synthesis, humoral balance, temperature regulation, and waste excretion. Due to the complexity of the skin, fluids loss, bacterial infection, and other life-threatening secondary complications caused by skin defects often lead to the damage of skin functions. 3D bioprinting technology, as a customized and precise biomanufacturing platform, can manufacture dressings and tissue engineering scaffolds that accurately simulate tissue structure, which is more conducive to wound healing. In recent years, with the development of emerging technologies, an increasing number of 3D-bioprinted wound dressings and skin tissue engineering scaffolds with multiple functions, such as antibacterial, anti-inflammatory, antioxidant, hemostatic, and antitumor properties, have significantly improved wound healing and skin treatment. In this article, we review the process of wound healing and summarize the classification of 3D bioprinting technology. Following this, we shift our focus on the functional materials for wound dressing and skin tissue engineering, and also highlight the research progress and development direction of 3D-bioprinted multifunctional wound healing materials.

In the inkjet printing process, the droplet experience two phases, namely the jetting and the impacting phases. In this review article, we aim to understand the physics of a jetted ink, which begins during the droplet formation process. Following which, we highlight the different impacts during which the droplet lands on varying substrates such as solid, liquid, and less commonly known viscoelastic material. Next, the article states important process-specific considerations in determining the success of inkjet bioprinted constructs. Techniques to reduce cell deformation throughout the inkjet printing process are highlighted. Modifying postimpact events, such as spreading, evaporation, and absorption, improves cell viability of printed droplet. Last, applications that leverage on the advantage of pixelation in inkjet printing technology have been shown for drug screening and cell–material interaction studies. It is noteworthy that inkjet bioprinting technology has been integrated with other processing technologies to improve the structural integrity and biofunctionality of bioprinted construct.

 

Three-dimensional (3D) bioprinting is a promising and innovative biomanufacturing technology, which can achieve precise position controlling of cells and extracellular matrix components, and further create complex and functional multi-cellular tissues or organs in a 3D environment. Bioink in the form of the cell-loaded hydrogel is most commonly used in bioprinting, and it is vital to the process of bioprinting. The bionic scaffold should possess suitable mechanical strength, biocompatibility, cell proliferation, survival, and other biological characteristics. The disadvantages of natural polymer hydrogel materials include poor mechanical properties as well as low printing performance and shape fidelity. Over the past years, a series of synthetic, modified, and nanocomposite hydrogels have been developed, which can interact through physical interactions, chemical covalent bond crosslinking, and bioconjugation reactions to change the characteristics to satisfy the requirements. In this review, a comprehensive summary is provided on recent research regarding the unique properties of hydrogel bioinks for bioprinting, with optimized methods and technologies highlighted, which have both high-value research significance and potential clinical applications. A critical analysis of the strengths and weaknesses of each hydrogel-based biomaterial ink is presented at the beginning or end of each section, alongside the latest improvement strategies employed by current researchers to address their respective shortcomings. Furthermore, we propose potential repair sites for each hydrogel-based ink based on their distinctive repair features, while reflecting on current research limitations. Finally, we synthesize and analyze expert opinions on the future of these hydrogel-based bioinks in the broader context of tissue engineering and regenerative medicine, offering valuable insights for future investigations.

The application of three-dimensional (3D) bioprinting has increased in the biomedical field. The lack of bioinks with both biocompatibility and printability is still a problem to be solved. Silk fibroin materials have good biocompatibility and have a broad application prospect in the field of biomedical materials. At present, most research usually involves Bombyx mori silk fibroin (BSF). However, BSF has low cell adhesion. Compared with BSF, Antheraea pernyi silk fibroin (ASF) isolated from typical non-mulberry silk exhibits a unique arginine-glycine-aspartate (RGD) sequence with good cell adhesion enhancement. In this study, we developed a bioink based on ASF for digital light processing (DLP) 3D bioprinting. The ASF-based bioinks (ASF-MA) were produced by a methacryloylation process using methacrylic anhydride (MA) to achieve the properties of photopolymerization reaction. The ASF-MA hydrogel has mechanical properties, biocompatibility, and especially cell adhesion. Meanwhile, we found that the ASF-MA hydrogels promoted the adhesion, migration, and proliferation of S16 cells. Hence, the ASF-MA hydrogels had the potential applications in biomedical fields.

 

Temporomandibular joint (TMJ) osteoarthritis causes fibrocartilage damage to the TMJ disc and mandibular condyle, resulting in local pain and functional impairment that further reduces patients’ quality of life. Tissue engineering offers a potential treatment for fibrocartilage regeneration of the TMJ disc and mandibular condyle. However, the heterogeneous structure of TMJ fibrocartilage tissue poses significant challenges for the fabrication of biomimetic scaffolds. Over the past two decades, some researchers have attempted to adopt three-dimensional (3D) printing techniques to fabricate biomimetic scaffolds for TMJ fibrocartilage regeneration, but publications on such attempts are limited and rarely report satisfactory results, indicating an urgent need for further development. This review outlines several popular 3D printing techniques and the significant elements of tissue-engineered scaffolds: seed cells, scaffold materials, and bioactive factors. Current research progress on 3D-printed scaffolds for fibrocartilage regeneration of the TMJ disc and mandibular condyle is reviewed. The current challenges in TMJ tissue engineering are mentioned along with some emerging tissue-engineering strategies, such as machine learning, stimuli-responsive delivery systems, and extracellular vesicles, which are considered as potential approaches to improve the performance of 3D-printed scaffolds for TMJ fibrocartilage regeneration. This review is expected to inspire the further development of 3D printing techniques for TMJ fibrocartilage regeneration.

This article provides an overview of the different types of blood-derived biomaterials that can be used as solvent additives in the formulation of inks/bioinks for use in solvent extrusion printing/bioprinting. We discuss the properties of various blood sub-products obtained after blood fractionation in terms of their use in tailoring ink/bioink to produce functional constructs designed to improve tissue repair. Blood-derived additives include platelets and/or their secretome, including signaling proteins and microvesicles, which can drive cell migration, inflammation, angiogenesis, and synthesis of extracellular matrix proteins. The contribution of plasma to ink/bioink functionalization relies not only on growth factors, such as hepatocyte growth factor and insulin growth factors, but also on adhesive proteins, such as fibrinogen/fibrin, vitronectin, and fibronectin. We review the current developments and progress in solvent-based extrusion printing/bioprinting with inks/bioinks functionalized with different blood-derived products, leading toward the development of more advanced patient-specific 3D constructs in multiple medical fields, including but not limited to oral tissues and cartilage, bone, skin, liver, and neural tissues. This information will assist researchers in identifying the most suitable blood-derived product for their ink/bioink formulation based on the intended regenerative functionality of the target tissue.