Spheroid construction strategies and application in 3D bioprinting

Tissue engineering has been striving toward designing and producing natural and functional human tissues.Cells are the fundamental building blocks of tissues.Compared with traditional two-dimensional cultured cells,cell spheres are threedimensional(3D)structures that can naturally form complex cell–cell and cell–matrix interactions.This structure is close to the natural environment of cells in living organisms.In addition to being used in disease modeling and drug screening,spheroids have significant potential in tissue regeneration.The 3D bioprinting is an advanced biofabrication technique.It accurately deposits bioinks into predesigned 3D shapes to create complex tissue structures.Although 3D bioprinting is efficient,the time required for cells to develop into complex tissue structures can be lengthy.The 3D bioprinting of spheroids significantly reduces the time required for their development into large tissues/organs during later cultivation stages by printing them with high cell density.Combining spheroid fabrication and bioprinting technology should provide a new solution to many problems in regenerative medicine.This paper systematically elaborates and analyzes the spheroid fabrication methods and 3D bioprinting strategies by introducing spheroids as building blocks.Finally,we present the primary challenges faced by spheroid fabrication and 3D bioprinting with future requirements and some recommendations.

3D bioprinting of in vitro porous hepatoma models:establishment,evaluation,and anticancer drug testing

Traditional tumor models do not tend to accurately simulate tumor growth in vitro or enable personalized treatment and are particularly unable to discover more beneficial targeted drugs.To address this,this study describes the use of threedimensional(3D)bioprinting technology to construct a 3D model with human hepatocarcinoma SMMC-7721 cells(3DP-7721)by combining gelatin methacrylate(GelMA)and poly(ethylene oxide)(PEO)as two immiscible aqueous phases to form a bioink and innovatively applying fluorescent carbon quantum dots for long-term tracking of cells.The GelMA(10%,mass fraction)and PEO(1.6%,mass fraction)hydrogel with 3:1 volume ratio offered distinct pore-forming characteristics,satisfactorymechanical properties,and biocompatibility for the creation of the 3DP-7721 model.Immunofluorescence analysis and quantitative real-time fluorescence polymerase chain reaction(PCR)were used to evaluate the biological properties of the model.Compared with the two-dimensional culture cell model(2D-7721)and the 3D mixed culture cell model(3DM-7721),3DP-7721 significantly improved the proliferation of cells and expression of tumor-related proteins and genes.Moreover,we evaluated the differences between the three culture models and the effectiveness of antitumor drugs in the three models and discovered that the efficacy of antitumor drugs varied because of significant differences in resistance proteins and genes between the three models.In addition,the comparison of tumor formation in the three models found that the cells cultured by the 3DP-7721 model had strong tumorigenicity in nude mice.Immunohistochemical evaluation of the levels of biochemical indicators related to the formation of solid tumors showed that the 3DP-7721 model group exhibited pathological characteristics of malignant tumors,the generated solid tumors were similar to actual tumors,and the deterioration was higher.This research therefore acts as a foundation for the application of 3DP-7721 models in drug development research.

Development of 3D bioprinting:From printing methods to biomedical applications

Biomanufacturing of tissues/organs in vitro is our big dream,driven by two needs:organ transplantation and accurate tissue models.Over the last decades,3D bioprinting has been widely applied in the construction of many tissues/organs such as skins,vessels,hearts,etc.,which can not only lay a foundation for the grand goal of organ replacement,but also be served as in vitro models committed to pharmacokinetics,drug screening and so on.As organs are so complicated,many bioprinting methods are exploited to figure out the challenges of different applications.So the question is how to choose the suitable bioprinting method?Herein,we systematically review the evolution,process and classification of 3D bioprinting with an emphasis on the fundamental printing principles and commercialized bioprinters.We summarize and classify extrusion-based,dropletbased,and photocuring-based bioprinting methods and give some advices for applications.Among them,coaxial and multi-material bioprinting are highlighted and basic principles of designing bioinks are also discussed.

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.

Sacrificial microgel‑laden bioink‑enabled 3D bioprinting of mesoscale pore networks

Three-dimensional(3D)bioprinting is a powerful approach that enables the fabrication of 3D tissue constructs that retain complex biological functions.However,the dense hydrogel networks that form after the gelation of bioinks often restrict the migration and proliferation of encapsulated cells.Herein,a sacrificial microgel-laden bioink strategy was designed for directly bioprinting constructs with mesoscale pore networks(MPNs)for enhancing nutrient delivery and cell growth.The sacrificial microgel-laden bioink,which contains cell/gelatin methacryloyl(GelMA)mixture and gelled gelatin microgel,is first thermo-crosslinked to fabricate temporary predesigned cell-laden constructs by extrusion bioprinting onto a cold platform.Then,the construct is permanently stabilized through photo-crosslinking of GelMA.The MPNs inside the printed constructs are formed after subsequent dissolution of the gelatin microgel.These MPNs allowed for effective oxygen/nutrient diffusion,facilitating the generation of bioactive tissues.Specifically,osteoblast and human umbilical vein endothelial cells encapsulated in the bioprinted large-scale constructs(≥1 cm)with MPNs showed enhanced bioactivity during culture.The 3D bioprinting strategy based on the sacrificial microgel-laden bioink provided a facile method to facilitate formation of complex tissue constructs with MPNs and set a foundation for future optimization of MPN-based tissue constructs with applications in diverse areas of tissue engineering.

3D bioprinting:current status and trends-a guide to the literature and industrial practice

The multidisciplinary research field of bioprinting combines additive manufacturing,biology and material sciences to cre-ate bioconstructs with three-dimensional architectures mimicking natural living tissues.The high interest in the possibility of reproducing biological tissues and organs is further boosted by the ever-increasing need for personalized medicine,thus allowing bioprinting to establish itself in the field of biomedical research,and attracting extensive research efforts from companies,universities,and research institutes alike.In this context,this paper proposes a scientometric analysis and critical review of the current literature and the industrial landscape of bioprinting to provide a clear overview of its fast-changing and complex position.The scientific literature and patenting results for 2000-2020 are reviewed and critically analyzed by retrieving 9314 scientific papers and 309 international patents in order to draw a picture of the scientific and industrial landscape in terms of top research countries,institutions,journals,authors and topics,and identifying the technology hubs worldwide.This review paper thus offers a guide to researchers interested in this field or to those who simply want to under-stand the emerging trends in additive manufacturing and 3D bioprinting.

The construction of in vitro tumormodels based on 3D bioprinting

Cancer is characterized by a high fatality rate,complex molecular mechanism,and costly therapies.The microenvironment of a tumor consists of multiple biochemical cues and the interaction between tumor cells,stromal cells,and extracellular matrix plays a key role in tumor initiation,development,angiogenesis,invasion and metastasis.To better understand the biological features of tumor and reveal the critical factors of therapeutic treatments against cancer,it is of great significance to build in vitro tumor models that could recapitulate the stages of tumor progression and mimic tumor behaviors in vivo for efficient and patient-specific drug screening and biological studies.Since conventional tissue engineering methods of constructing tumor models always fail to simulate the later stages of tumor development due to the lack of ability to build complex structures and angiogenesis potential,three-dimensional(3D)bioprinting techniques have gradually found its applications in tumor microenvironment modeling with accurate composition and well-organized spatial distribution of tumor-related cells and extracellular components in the past decades.The capabilities of building tumor models with a large range of scale,complex structures,multiple biomaterials and vascular network with high resolution and throughput make 3D bioprinting become a versatile platform in bio-manufacturing aswell as inmedical research.In this review,wewill focus on 3D bioprinting strategies,design of bioinks,current 3D bioprinted tumor models in vitro classified with their structures and propose future perspectives.

Using 3D bioprinting to produce mini-brain

Recent progresses in three-dimensional （3D） bioprinting technology accelerate the coming of the era of personalized medicine. With vari- ous printing approaches and materials developed, 3D bioprinting may have a broad range of medical applications, including the fabrication of delicate tissues/organs/or the clinical use in the future or for the es- tablishment of tissues in disease models. The principal advantages of 3D bioprinting are personalized design and precise fabrication, which are of critical importance for tissue engineering. To date, several types of biomimetic tissues, such as cartilage, skin, and vascular tissues have been fabricated by 3D bioprinting （Liaw and Guvendiren, 2017）.

Liquid-phase 3D bioprinting of gelatin alginate hydrogels:influence of printing parameters on hydrogel line width and layer height

Extrusion-based 3D bioprinting is a direct deposition approach used to create three-dimensional(3D)tissue scaffolds typically comprising hydrogels.Hydrogels are hydrated polymer networks that are chemically or physically cross-linked.Often,3D bioprinting is performed in air,despite the hydrated nature of hydrogels and the potential advantage of using a liquid phase to provide cross-linking and otherwise functionalize the hydrogel.In this work,we print gelatin alginate hydrogels directly into a cross-linking solution of calcium chloride and investigate the influence of nozzle diameter,distance between nozzle and surface,calcium chloride concentration,and extrusion rate on the dimensions of the printed hydrogel.The hydrogel layer height was generally found to increase with increasing extrusion rate and nozzle distance,according to the increased volume extruded and the available space,respectively.In addition,the hydrogel width was generally found to increase with decreasing nozzle distance and cross-linking concentration corresponding to confinement-induced spreading and low crosslinking regimes,respectively.Width/height ratios of^1 were generally achieved when the nozzle diameter and distance were comparable above a certain cross-linking concentration.Using these relationships,biocompatible 3D multilayer structures were successfully printed directly into calcium chloride cross-linking solution.

3D bioprinting for cell culture and tissue fabrication

Three-dimensional （3D） bioprinting is a computer-assisted technology which precisely controls spatial position of biomaterials, growth factors and living cells, offering unprecedented possibility to bridge the gap between structurally mimic tissue constructs and functional tissues or organoids. We briefly focus on diverse bioinks used in the recent progresses of biofabrication and 3D bioprinting of various tissue architectures including blood vessel, bone, cartilage, skin, heart, liver and nerve systems. This paper provides readers a guideline with the conjunction between bioinks and the targeted tissue or organ types in structuration and final functionalization of these tissue analogues. The challenges and perspectives in 3D bioprinting field are also illustrated.

Advanced biomedical and electronic dual-function skin patch created through microfluidic-regulated 3D bioprinting

Artificial skin involves multidisciplinary efforts,including materials science,biology,medicine,and tissue engineering.Recent studies have aimed at creating skins that are multifunctional,intelligent,and capable of regenerating tissue.In this work,we present a specialized 3D printing ink composed of polyurethane and bioactive glass(PU-BG)and prepare dual-function skin patch by microfluidic-regulated 3D bioprinting(MRBP)technique.The MRBP endows the skin patch with a highly controlled microstructure and superior strength.Besides,an asymmetric tri-layer is further constructed,which promotes cell attachment and growth through a dual transport mechanism based on hydrogen bonds and gradient structure from hydrophilic to superhydrophilic.More importantly,by combining the features of biomedical skin with electronic skin(e-skin),we achieved a biomedical and electronic dual-function skin patch.In vivo experiments have shown that this skin patch can enhance hemostasis,resist bacterial growth,stimulate the regeneration of blood vessels,and accelerate the healing process.Meanwhile,it also mimics the sensory functions of natural skin to realize signal detection,where the sensitivity reached up to 5.87 kPa􀀀1,as well as cyclic stability(over 500 cycles),a wide detection range of 0–150 kPa,high pressure resolution of 0.1%under the pressure of 100 kPa.This work offers a versatile and effective method for creating dual-function skin patches and provide new insights into wound healing and tissue repair,which have significant implications for clinical applications.

Fabrication of a 3D bioprinting model for posterior capsule opacification using GelMA and PLMA hydrogel-coated resin

Posterior capsule opacification(PCO)remains the predominant complication following cataract surgery,significantly impairing visual function restoration.In this study,we developed a PCO model that closely mimics the anatomical structure of the crystalline lens capsule post-surgery.The model incorporated a threaded structure for accurate positioning and observation,allowing for opening and closing.Utilizing 3D printing technology,a stable external support system was created using resin material consisting of a rigid,hollow base and cover.To replicate the lens capsule structure,a thin hydrogel coating was applied to the resin scaffold.The biocompatibility and impact on cellular functionality of various hydrogel compositions were assessed through an array of staining techniques,including calcein-AM/PI staining,rhodamine staining,BODIPY-C11 staining and EdU staining in conjunction with transwell assays.Additionally,the PCO model was utilized to investigate the effects of eight drugs with anti-inflammatory and anti-proliferative properties,including 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR),THZ1,sorbinil,4-octyl itaconate(4-OI),xanthohumol,zebularine,rapamycin and caffeic acid phenethyl ester,on human lens epithelial cells(HLECs).Confocal microscopy facilitated comprehensive imaging of the PCO model.The results demonstrated that the GelMA 605%þPLMA 2%composite hydrogel exhibited superior biocompatibility and minimal lipid peroxidation levels among the tested hydrogels.Moreover,compared to using hydrogel as the material for 3D printing the entire model,applying surface hydrogel spin coating with parameters of 2000 rpm�2 on the resin-based 3D printed base yielded a more uniform cell distribution and reduced apoptosis.Furthermore,rapamycin,4-OI and AICAR demonstrated potent antiproliferative effects in the drug intervention study.Confocal microscopy imaging revealed a uniform distribution of HLECs along the anatomical structure of the crystalline lens capsule within the PCO model,showcasing robust cell viability and regular morphology.In conclusion,the PCO model provides a valuable experimental platform for studying PCO pathogenesis and exploring potential therapeutic interventions.

3D bioprinting of collagen-based materials for oral medicine

Oral diseases have emerged as one of the leading public health challenges globally.Although the existing clinical modalities for restoration of dental tissue loss and craniomaxillofacial injuries can achieve satisfactory therapeutic results,they cannot fully restore the original complex anatomical structure and physiological function of the tissue.3D printing of biological tissues has gained growing interest in the field of oral medicine with the ability to control the bioink component and printing structure for spatially heterogeneous repairing constructs,holding enormous promise for the precise treatment of oral disease.Particularly,collagen-based materials have been recognized as promising biogenic bioinks for the regeneration of several tissues with high cell-activating and biocompatible properties.In this review,we summarize 3D printing methods for collagen-based biomaterials and their mechanisms.Additionally,we highlight the animal sources of collagen and their characteristics,as well as the methods of collagen extraction.Furthermore,this review provides an overview of the 3D bioprinting technology for the regeneration of the pulpal nerve and blood vessels,cartilage,and periodontal tissue.We envision that this technique opens up immense opportunities over the conventional ones,with high replicability and customized function,which can ultimately promote effective oral tissue regeneration.

Simulating the in vivo tumor microenvironment-advances in building a vascularized model of hepatocellular carcinoma through 3D bioprinting

Over the past decade,there has been notable progress in the systemic treatment of liver cancer.However,despite the emergence of new therapeutic strategies,they have not universally achieved success,with patients afflicted by liver diseases frequently displaying resistance to these treatments(1).Consequently,liver cancer remains a global health challenge,and hepatocellular carcinoma(HCC)stands as the fourth most common cause of cancer-related deaths globally,constituting 80-90%of primary liver cancer cases(2,3).This poses a substantial threat to both the survival and overall well-being of individuals.

Innovations in 3D bioprinting and biomaterials for liver tissue engineering:Paving the way for tissue-engineered liver

The liver is a pivotal organ that maintains internal homeostasis and actively participates in multiple physiological processes.Liver tissue engineering(LTE),by which in vitro biomimetic liver models are constructed,serves as a platform for disease research,drug screening,and cell replacement therapies.3D bioprinting is used in tissue engineering to create microenvironments that closely mimic authentic tissues with carefully selected functional biomaterials.Ideal functional biomaterials exhibit characteristics such as high biocompatibility,mechanical strength,flexibility,processability,and tunable degradability.Biomaterials can be categorized into natural and synthetic biomaterials,each with its own advantages and limitations,and their combinations serve as a primary source of 3D bioprinting materials.It is noteworthy that the liver decellularized extracellular matrix(dECM),obtained by removing cellular components from tissues,possesses traits such as bioactivity,biocompatibility,and non-immunogenicity,making it a common choice among functional biomaterials.Furthermore,crosslinking of biomaterials significantly impacts the mechanical strength,physicochemical properties,and cellular behavior of the printed structures.This review covers the current utilization of biomaterials in LTE,focusing on natural and synthetic biomaterials as well as the selection and application of crosslinking methods.The aim is to enhance the fidelity of in vitro liver tissue models by providing a comprehensive coverage of functional biomaterials,thereby establishing a versatile platform for tissue-engineered livers.

Integrated 3D bioprinting-based geometry-control strategy for fabricating corneal substitutes

Background:The shortage of donor corneas is a severe global issue,and hence the development of corneal alternatives is imperative and urgent.Although attempts to produce artificial cornea substitutes by tissue engineering have made some positive progress,many problems remain that hamper their clinical application worldwide.For example,the curvature of tissue-engineered cornea substitutes cannot be designed to fit the bulbus oculi of patients.Objective:To overcome these limitations,in this paper,we present a novel integrated three-dimensional(3 D) bioprintingbased cornea substitute fabrication strategy to realize design,customized fabrication,and evaluation of multi-layer hollow structures with complicated surfaces.Methods:The key rationale for this method is to combine digital light processing(DLP) and extrusion bioprinting into an integrated 3 D cornea bioprinting system.A designable and personalized corneal substitute was designed based on mathematical modelling and a computer tomography scan of a natural cornea.The printed corneal substitute was evaluated based on biomechanical analysis,weight,structural integrity,and fit.Results:The results revealed that the fabrication of high water content and highly transparent curved films with geometric features designed according to the natural human cornea can be achieved using a rapid,simple,and low-cost manufacturing process with a high repetition rate and quality.Conclusions:This study demonstrated the feasibility of customized design,analysis,and fabrication of a corneal substitute.The programmability of this method opens up the possibility of producing substitutes for other cornea-like shell structures with different scale and geometry features,such as the glomerulus,atrium,and oophoron.

3D bioprinting of a biomimetic meniscal scaffold for application in tissue engineering

Appropriate biomimetic scaffolds created via 3D bioprinting are promising methods for treating damaged menisci.However,given the unique anatomical structure and complex stress environment of the meniscus,many studies have adopted various techniques to take full advantage of different materials,such as the printing combined with infusion,or electrospining,to chase the biomimetic meniscus,which makes the process complicated to some extent.Some researchers have tried to tackle the challenges only by 3D biopringting,while its alternative materials and models have been constrained.In this study,based on a multilayer biomimetic strategy,we optimized the preparation of meniscus-derived bioink,gelatin methacrylate(GelMA)/meniscal extracellular matrix(MECM),to take printability and cytocompatibility into account together.Subsequently,a customized 3D bioprinting system featuring a dual nozzle+multitemperature printing was used to integrate the advantages of polycaprolactone(PCL)and meniscal fibrocartilage chondrocytes(MFCs)-laden GelMA/MECM bioink to complete the biomimetic meniscal scaffold,which had the best biomimetic features in terms of morphology and components.Furthermore,cell viability,mechanics,biodegradation and tissue formation in vivo were performed to ensure that the scaffold had sufficient feasibility and functionality,thereby providing a reliable basis for its application in tissue engineering.

3D bioprinting of integral ADSCs-NO hydrogel scaffolds to promote severe burn wound healing

Severe burns are challenging to heal and result in significant death throughout the world.Adiposederived mesenchymal stem cells(ADSCs)have emerged as a promising treatment for fullthickness burn healing but are impeded by their low viability and efficiency after grafting in vivo.Nitric oxide(NO)is beneficial in promoting stem cell bioactivity,but whether it can function effectively in vivo is still largely unknown.In this study,we bioprinted an efficient biological scaffold loaded with ADSCs and NO(3D-ADSCs/NO)to evaluate its biological efficacy in promoting severe burn wound healing.The integral 3D-ADSCs/NO hydrogel scaffolds were constructed via 3D bioprinting.Our results shown that 3D-ADSCs/NO can enhance the migration and angiogenesis of Human Umbilical Vein Endothelial Cells(HUVECs).Burn wound healing experiments in mice revealed that 3D-ADSCs/NO accelerated the wound healing by promoting faster epithelialization and collagen deposition.Notably,immunohistochemistry of CD31 suggested an increase in neovascularization,supported by the upregulation of vascular endothelial growth factor(VEGF)mRNA in ADSCs in the 3D biosystem.These findings indicated that 3D-ADSC/NO hydrogel scaffold can promote severe burn wound healing through increased neovascularization via the VEGF signalling pathway.This scaffold may be considered a promising strategy for healing severe burns.

Coaxial 3D bioprinting of organ prototyps from nutrients delivery to vascularization

Vascular networks inside organs provide the means for metabolic exchange and adequate nutrition.Similarly,vascular or nutrient networks are needed when building tissue constructs>500μm in vitro due to the hydrogel compact pore size of bioinks.As the hydrogel used in bioinks is rather soft,it is a great challenge to reconstruct effective vascular networks.Recently,coaxial 3 D bioprinting was developed to print tissue constructs directly using hollow hydrogel fibers,which can be treated as built-in microchannels for nutrient delivery.Furthermore,vascular networks could be printed directly through coaxial 3 D bioprinting.This review summarizes recent advances in coaxial bioprinting for the fabrication of complex vascularized tissue constructs including methods,the effectiveness of varying strategies,and the use of sacrificial bioink.The limitations and challenges of coaxial 3 D bioprinting are also summarized.

Advances in 3D bioprinting technology for cardiac tissue engineering and regeneration

Cardiovascular disease is still one of the leading causes of death in the world,and heart transplantation is the current major treatment for end-stage cardiovascular diseases.However,because of the shortage of heart donors,new sources of cardiac regenerative medicine are greatly needed.The prominent development of tissue engineering using bioactive materials has creatively laid a direct promising foundation.Whereas,how to precisely pattern a cardiac structure with complete biological function still requires technological breakthroughs.Recently,the emerging three-dimensional(3D)bioprinting technology for tissue engineering has shown great advantages in generating micro-scale cardiac tissues,which has established its impressive potential as a novel foundation for cardiovascular regeneration.Whether 3D bioprinted hearts can replace traditional heart transplantation as a novel strategy for treating cardiovascular diseases in the future is a frontier issue.In this review article,we emphasize the current knowledge and future perspectives regarding available bioinks,bioprinting strategies and the latest outcome progress in cardiac 3D bioprinting to move this promising medical approach towards potential clinical implementation.