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.

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）.

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.

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.

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.

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.

Revolutionizing preclinical research for pancreatic cancer:the potential of 3D bioprinting technology for personalized therapy

Pancreatic cancer(PC)is a prevalent digestive malignancy worldwide and ranks as the fourth leading cause of cancer-related deaths globally.The incidence and mortality rates have been increasing annually,and due to its insidious onset and high malignancy,most patients are diagnosed at an advanced stage,with a 5-year survival rate of less than 8%(1).PC can be classified into endocrine and exocrine tumors,with over 95% of pancreatic malignant tumors originating from the exocrine portion of the pancreas.

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.

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.

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.

Additive-lathe 3D bioprinting of bilayered nerve conduits incorporated with supportive cells

Nerve conduits have been identified as one of the most promising treatments for peripheral nerve injuries,yet it remains unsolved how to develop ideal nerve conduits with both appropriate biological and mechanical properties.Existing nerve conduits must make trade-offs between mechanical strength and biocompatibility.Here,we propose a multi-nozzle additive-lathe 3D bioprinting technology to fabricate a bilayered nerve conduit.The materials for printing consisted of gelatin methacrylate(GelMA)-based inner layer,which was cellularized with bone marrow mesenchymal stem cells(BMSCs)and GelMA/poly(ethylene glycol)diacrylate(PEGDA)-based outer layer.The high viability and extensive morphological spreading of BMSCs encapsulated in the inner layer was achieved by adjusting the degree of methacryloyl substitution and the concentration of GelMA.Strong mechanical performance of the outer layer was obtained by the addition of PEGDA.The performance of the bilayered nerve conduits was assessed using in vitro culture of PC12 cells.The cell density of PC12 cells attached to cellularized bilayered nerve conduits was more than 4 times of that on acellular bilayered nerve conduits.The proliferation rate of PC12 cells attached to cellularized bilayered nerve conduits was over 9 times higher than that on acellular bilayered nerve conduits.These results demonstrate the additive-lathe 3D bioprinting of BMSCs embedded bilayered nerve conduits holds great potential in facilitating peripheral nerve repair.

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

It is a severe challenge to construct 3D scaf- folds which hold controllable pore structure and similar morphology of the natural extracellular matrix （ECM）. In this study, a compound technology is proposed by com- bining the 3D bioprinting and electrospinning process to fabricate 3D scaffolds, which are composed by orthogonal array gel microfibers in a grid-like arrangement and inter- calated by a nonwoven structure with randomly distributed polycaprolactone （PCL） nanofibers. Human adipose- derived 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 demon- strates 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 per- formance in tissue engineering applications.

Printability during projection-based 3D bioprinting

Since projection-based 3D bioprinting(PBP)could provide high resolution,it is well suited for printing delicate structures for tissue regeneration.However,the low crosslinking density and low photo-crosslinking rate of photocurable bioink make it difficult to print fine structures.Currently,an in-depth understanding of the is lacking.Here,a research framework is established for the analysis of printability during PBP.The gelatin methacryloyl(GelMA)-based bioink is used as an example,and the printability is systematically investigated.We analyze the photo-crosslinking reactions during the PBP process and summarize the specific requirements of bioinks for PBP.Two standard quantized models are established to evaluate 2D and 3D printing errors.Finally,the better strategies for bioprinting five typical structures,including solid organs,vascular structures,nerve conduits,thin-wall scaffolds,and micro needles,are presented.

Inkjet 3D bioprinting for tissue engineering and pharmaceutics

3D bioprinting has the capability to create 3D cellular constructs with the desired shape using a layer-by-layer approach.Inkjet 3D bioprinting,as a key component of 3D bioprinting,relies on the deposition of cell-laden droplets to create native-like tissues/organs which are envisioned to be transplantable into human body for replacing damaged ones.Benefiting from its superiorities such as high printing resolution and deposition accuracy,inkjet 3D bioprinting has been widely applied to various areas,including,but not limited to,tissue engineering and drug screening in pharmaceutics.Even though inkjet 3D bioprinting has proved its feasibility and versatility in various fields,the current applications of inkjet 3D bioprinting are still limited by the printing technique and material selection.This review,which specifically focuses on inkjet 3D bioprinting,firstly summarizes the techniques,materials,and applications of inkjet 3D bioprinting in tissue engineering and drug screening,subsequently discusses the major challenges that inkjet 3D bioprinting is facing,and lastly summarizes potential solutions to those challenges.

3D bioprinting of conductive hydrogel for enhanced myogenic differentiation

Recently,hydrogels have gained enormous interest in three-dimensional(3D)bioprinting toward developing functional substitutes for tissue remolding.However,it is highly challenging to transmit electrical signals to cells due to the limited electrical conductivity of the bioprinted hydrogels.Herein,we demonstrate the 3D bioprinting-assisted fabrication of a conductive hydrogel scaffold based on poly-3,4-ethylene dioxythiophene(PEDOT)nanoparticles(NPs)deposited in gelatin methacryloyl(GelMA)for enhanced myogenic differentiation of mouse myoblasts(C2C12 cells).Initially,PEDOT NPs are dispersed in the hydrogel uniformly to enhance the conductive property of the hydrogel scaffold.Notably,the incorporated PEDOT NPs showed minimal influence on the printing ability of GelMA.Then,C2C12 cells are successfully encapsulated within GelMA/PEDOT conductive hydrogels using 3D extrusion bioprinting.Furthermore,the proliferation,migration and differentiation efficacies of C2C12 cells in the highly conductive GelMA/PEDOT composite scaffolds are demonstrated using various in vitro investigations of live/dead staining,F-actin staining,desmin and myogenin immunofluorescence staining.Finally,the effects of electrical signals on the stimulation of the scaffolds are investigated toward the myogenic differentiation of C2C12 cells and the formation of myotubes in vitro.Collectively,our findings demonstrate that the fabrication of the conductive hydrogels provides a feasible approach for the encapsulation of cells and the regeneration of the muscle tissue.

An Overview on Materials and Techniques in 3D Bioprinting Toward Biomedical Application

Three-dimensional(3D)bioprinting,an additive manufacturing based technique of biomaterials fabrication,is an innovative and auspicious strategy in medical and pharmaceutical fields.The ability of producing regenerative tissues and organs has made this technology a pioneer to the creation of artificial multi-cellular tissues/organs.A broad variety of biomaterials is currently being utilized in 3D bioprinting as well as multiple techniques employed by researchers.In this review,we demonstrate the most common and novel biomaterials in 3D bioprinting technology further with introducing the related techniques that are commonly taking into account by researchers.In addition,an attempt has been accomplished to hand over the most relevant application of 3D bioprinting techniques such as tissue regeneration,cancer investigations,etc.by presenting the most important works.The main aim of this review paper is to emphasis on strengths and limitations of existence biomaterials and 3D bioprinting techniques in order to carry out a comparison through them.

3D bioprinting of cell-laden constructs for regenerative medicine

Human tissue consists of various tissue-specific cells,extracellular matrix components and microstructures,and growth factors.With promising multi-cell and multi-material integration manufacturing feature,3D extrusion bioprinting has shown outstanding application potential in the field of regenerative medicine.For functional tissue regeneration,bioprinted constructs not only play the role of a cell-delivery system,but also serve as an important host niche for cell proliferation and work.In order to meet the specific requirements of different tissue regeneration,development of bio-inks that provide tissue-specific biophysical cues and biochemical microenvironments is an important research topic.Furthermore,reconstruction of tissues with anisotropic structure,such as articular cartilage and meniscus,largely depend on the design of 3D bioprinting path for accurate arrangement of specific bio-inks.This review summarizes the advanced designs of tissue-specific 3D bioprinting of cell-laden constructs for functional regeneration of skeletal and locomotor systems such as bone,cartilage,skeletal muscle,and blood vessels via the collaboration of bio-ink and printing processes.It may provide a basis for synergistic design for functional regenerative constructs bioprinting in the future.

Development of a 3D subcutaneous construct containing insulin-producing beta cells using bioprinting

Type 1 diabetes is caused by insulin deficiency due to the loss of beta cells in the islets of Langerhans.In severe cases,islet transplantation into the portal vein is performed.However,due to the loss of transplanted islets and the failure of islet function,the 5-year insulin independence rate of these patients is<50%.In this study,we developed a long-term,insulin-secreting,3 Dbioprinted construct implanted subcutaneously with the aim of preventing islet loss.The bioprinted construct was fabricated by the multi-layer bioprinting of beta-cell(mouse insulinoma-6:MIN-6)-encapsulated alginate bioink and poly(caprolactone)biodegradable polymer.A glucose response assay revealed that the bioprinted constructs proliferated and released insulin normally during the 4-week in vitro period.Bioprinted MIN-6 generated clusters with a diameter of 100-200μm,similar to the original pancreatic islets in the construct.In an in vivo study using type 1 diabetes mice,animals implanted with bioprinted constructs showed three times higher insulin secretion and controlled glucose levels at 8 weeks after implantation.Because the implanted,bioprinted constructs had a positive effect on insulin secretion in the experimental animals,the survival rate of the implanted group(75%)was three times higher than that of the non-implanted group(25%).The results suggest that the proposed,3 D-bioprinted,subcutaneous construct can be a better alternative to portal vein islet transplantation.