Bioprinting has been widely investigated for tissue engineering and regenerative medicine applications.However,it is still difficult to reconstruct the complex native cell arrangement due to the limited printing resol...Bioprinting has been widely investigated for tissue engineering and regenerative medicine applications.However,it is still difficult to reconstruct the complex native cell arrangement due to the limited printing resolution of conventional bioprinting techniques such as extrusion-and inkjet-based printing.Recently,an electrohydrodynamic(EHD)bioprinting strategy was reported for the precise deposition of well-organized cell-laden constructs with microscale filament size,whereas few studies have been devoted to developing bioinks that can be applied for EHD bioprinting and simultaneously support cell spreading.This study describes functionalized alginate-based bioinks for microscale EHD bioprinting using peptide grafting and fibrin incorporation,which leads to high cell viability(>90%)and cell spreading.The printed filaments can be further refined to as small as 30μm by incorporating polyoxyethylene and remained stable over one week when exposed to an aqueous environment.By utilizing the presented alginate-based bioinks,layer-specific cell alignment along the printing struts could be observed inside the EHD-printed microscale filaments,which allows fabricating living constructs with cell-scale filament resolution for guided cellular orientation.展开更多
Three-dimensional(3D)bioprinting has revolutionized tissue engineering by enabling precise fabrication with bioinks.Among these techniques,digital light processing(DLP)stands out due to its exceptional resolution,spee...Three-dimensional(3D)bioprinting has revolutionized tissue engineering by enabling precise fabrication with bioinks.Among these techniques,digital light processing(DLP)stands out due to its exceptional resolution,speed,and biocompatibility.However,the progress of DLP is hindered by the limited availability of suitable bioinks.Currently,some studies involve simple mixing of different materials,resulting in bioinks that lack uniformity and photopolymerization characteristics.To address this challenge,we present an innovative one-pot synthesis method for bioinks based on methacrylated gelatin/alginate with hydroxyapatite(HAP).This approach offers significant advantages in terms of efficiency and uniformity.The synthesized bioinks demonstrate excellent printability,stability,and notably enhanced mechanical properties,facilitating optimal in vitro compatibility.Additionally,the HAP-hybrid bioinks printed scaffolds demonstrated impressive bone repair capabilities in vivo compared with pure organic bioinks.In conclusion,the Gel/Alg/HAP bioinks presented herein offer an innovative solution for DLP bioprinting within the field of bone tissue engineering.Their multifaceted advantages help overcome the limitations of restricted bioink choices,pushing forward the boundaries of bioprinting technology and contributing to the progress of regenerative medicine and tissue engineering.展开更多
Cryobioprinting has tremendous potential to solve problems to do with lack of shelf availability in traditional bioprinting by combining extrusion bioprinting and cryopreservation.In order to ensure the viability of c...Cryobioprinting has tremendous potential to solve problems to do with lack of shelf availability in traditional bioprinting by combining extrusion bioprinting and cryopreservation.In order to ensure the viability of cells in the frozen state and avoid the possible toxicity of dimethyl sulfoxide(DMSO),DMSO-free bioink design is critical for achieving successful cryobioprinting.A nontoxic gelatin methacryloyl-based bioink used in cryobioprinting is composed of cryoprotective agents(CPAs)and a buffer solution.The selection and ratio of CPAs in the bioink directly affect the survival of cells in the frozen state.However,the development of universal and efficient cryoprotective bioinks requires extensive experimentation.We first compared two commonly used CPA formulations via experiments in this study.Results show that the effect of using ethylene glycol as the permeable CPA was 6.07%better than that of glycerol.Two datasets were obtained and four machinelearning models were established to predict experimental outcomes.The predictive powers of multiple linear regression(MLR),decision tree(DT),random forest(RF),and artificial neural network(ANN)approaches were compared,suggesting an order of ANN>RF>DT>MLR.The final selected ANN model was then applied to another dataset.Results reveal that this machine-learning method can accurately predict the effects of cryoprotective bioinks composed of different CPAs.Outcomes also suggest that the formulations presented here have universality.Our findings are likely to greatly accelerate research and development on the use of bioinks for cryobioprinting.展开更多
Geometric and structural integrity often deteriorate in 3D printed cell-laden constructs over time due to cellular compaction and hydrogel shrinkage.This study introduces a new approach that synergizes the advantages ...Geometric and structural integrity often deteriorate in 3D printed cell-laden constructs over time due to cellular compaction and hydrogel shrinkage.This study introduces a new approach that synergizes the advantages of cell compatibility of biological hydrogels and mechanical stability of elastomeric polymers for structure fidelity maintenance upon stereolithography and extrusion 3D printing.Enabling this advance is the composite bioink,formulated by integrating elastomeric microparticles from poly(octamethylene maleate(anhydride)citrate)(POMaC)into biologically derived hydrogels(fibrin,gelatin methacryloyl(GelMA),and alginate).The composite bioink enhanced the elasticity and plasticity of the 3D printed constructs,effectively mitigating tissue compaction and swelling.It exhibited a low shear modulus and a rapid crosslinking time,along with a high ultimate compressive strength and resistance to deformation from cellular forces and physical handling;this was attributed to packing and stress dissipation of elastomeric particles,which was confirmed via mathematical modelling.Enhanced functional assembly and stability of human iPSC-derived cardiac tissues and primary vasculature proved the utility of the composite bioink in tissue engineering.In vivo implantation studies revealed that constructs containing POMaC particles exhibited improved resilience against host tissue stress,enhanced angiogenesis,and infiltration of pro-reparative macrophages.展开更多
Bioinks are formulations of biomaterials and living cells, sometimes with growth factors or other biomolecules, while extrusion bioprinting is an emerging technique to apply or deposit these bioinks or biomaterial sol...Bioinks are formulations of biomaterials and living cells, sometimes with growth factors or other biomolecules, while extrusion bioprinting is an emerging technique to apply or deposit these bioinks or biomaterial solutions to create three-dimensional (3D) constructs with architectures and mechanical/biological properties that mimic those of native human tissue or organs. Printed constructs have found wide applications in tissue engineering for repairing or treating tissue/organ injuries, as well as in vitro tissue modelling for testing or validating newly developed therapeutics and vaccines prior to their use in humans. Successful printing of constructs and their subsequent applications rely on the properties of the formulated bioinks, including the rheological, mechanical, and biological properties, as well as the printing process. This article critically reviews the latest developments in bioinks and biomaterial solutions for extrusion bioprinting, focusing on bioink synthesis and characterization, as well as the influence of bioink properties on the printing process. Key issues and challenges are also discussed along with recommendations for future research.展开更多
Engineering hydrogels that resemble biological tissues of various lengths via conventional fabrication techniques remains challenging.Three-dimensional(3D)bioprinting has emerged as an advanced approach for constructi...Engineering hydrogels that resemble biological tissues of various lengths via conventional fabrication techniques remains challenging.Three-dimensional(3D)bioprinting has emerged as an advanced approach for constructing complex biomimetic 3D architectures,which are currently restricted by the limited number of available bioinks with high printability,biomimicry,biocompatibility,and proper mechanical properties.Inspired by ubiquitous coacervation phenomena in biology,we present a unique mineral-biopolymer coacervation strategy that enables the hierarchical assembly of nanoclay and recombinant human collagen(RHC).This system was observed to undergo a coacervation transition(liquid‒liquid phase separation)spontaneously.The formed dense phase separated from its supernatant is the coacervate of clay-RHC-rich complexes,where polymer chains are sandwiched between silicate layers.Molecular dynamics simulation was first used to verify and explore the coacervation process.Then,the coacervates were demonstrated to be potential bioinks that exhibited excellent self-supporting and shear-thinning viscoelastic properties.Through extrusion-based printing,the versatility of the bioink was demonstrated by reconstructing the key features of several biological tissues,including multilayered lattice,vascular,nose,and ear-like structures,without the need for precrosslinking operations or support baths.Furthermore,the printed scaffolds were cytocompatible,elicited minimal inflammatory responses,and promoted bone regeneration in calvarial defects.展开更多
The printability of bioink and post-printing cell viability is crucial for extrusion-based bioprinting.A proper bioink not only provides mechanical support for structural fidelity,but also serves as suitable three-dim...The printability of bioink and post-printing cell viability is crucial for extrusion-based bioprinting.A proper bioink not only provides mechanical support for structural fidelity,but also serves as suitable three-dimensional(3D)microenvironment for cell encapsulation and protection.In this study,a hydrogel-based composite bioink was developed consisting of gelatin methacryloyl(GelMA)as the continuous phase and decellularised extracellular matrix microgels(DMs)as the discrete phase.A flow-focusing microfluidic system was employed for the fabrication of cell-laden DMs in a high-throughput manner.After gentle mixing of the DMs and GelMA,both rheological characterisations and 3D printing tests showed that the resulting DM-GelMA hydrogel preserved the shear-thinning nature,mechanical properties,and good printability from GelMA.The integration of DMs not only provided an extracellular matrix-like microenvironment for cell encapsulation,but also considerable shear-resistance for high post-printing cell viability.The DM sizes and inner diameters of the 3D printer needles were correlated and optimised for nozzle-based extrusion.Furthermore,a proof-of-concept bioink composedg of RSC96 Schwann cells encapsulated DMs and human umbilical vein endothelial cell-laden GelMA was successfully bioprinted into 3D constructs,resulting in a modular co-culture system with distinct cells/materials distribution.Overall,the modular DM-GelMA bioink provides a springboard for future precision biofabrication and will serve in numerous biomedical applications such as tissue engineering and drug screening.展开更多
Extrusion-based bioprinting (EBB) holds potential for regenerative medicine. However, the widely-used bioinks of EBB exhibit some limitations for skin regeneration, such as unsatisfactory bio-physical (i.e., mechanica...Extrusion-based bioprinting (EBB) holds potential for regenerative medicine. However, the widely-used bioinks of EBB exhibit some limitations for skin regeneration, such as unsatisfactory bio-physical (i.e., mechanical, structural, biodegradable) properties and compromised cellular compatibilities, and the EBB-based bioinks with therapeutic effects targeting cutaneous wounds still remain largely undiscussed. In this review, the printability considerations for skin bioprinting were discussed. Then, current strategies for improving the physical properties of bioinks and for reinforcing bioinks in EBB approaches were introduced, respectively. Notably, we highlighted the applications and effects of current EBB-based bioinks on wound healing, wound scar formation, vasculari-zation and the regeneration of skin appendages (i.e., sweat glands and hair follicles) and discussed the challenges and future perspectives. This review aims to provide an overall view of the applications, challenges and promising solutions about the EBB-based bioinks for cutaneous wound healing and skin regeneration.展开更多
In the field of regenerative medicine,the importance of 3D bioprinting is self-evident and nonnegligible.However,3D bioprinting technology also requires bioink with excellent performance as support material to fabrica...In the field of regenerative medicine,the importance of 3D bioprinting is self-evident and nonnegligible.However,3D bioprinting technology also requires bioink with excellent performance as support material to fabricate a multi-functional bioinspired scaffold.Collagen-based bioink is regarded as an ideal 3D bioprinting ink for its excellent biocompatibility,controllable printability and cell loading property.It is an important breakthrough in regenerative medicine with the progress of collagen-based bioink,which fabricates bioinspired scaffolds with different functions and is applied in different repair scenarios.This review summarizes the different applications of collagen-based bioink and classifies them as soft tissue and hard tissue according to the target region.The applications of target region in soft tissues include skin,cartilage,heart and blood vessels,while in hard tissues include femur,skull,teeth and spine.When the collagen-based bioink is applied in repairing soft tissue,the requirements of function are higher,while the mechanical properties must be further improved in repairing hard tissue.We further summarize the characteristics of collagen-based bioink and point out the most important properties that should be considered in different repair scenarios,which can provide reference for the preparation of bioinks with different functions.Finally,we point out the main challenges faced by collagen-based bioink and prospect the future research directions.展开更多
Osteosarcoma,an aggressive bone cancer found most often in children and adolescents,remains difficult to treat,and little improvement in survival rate has been observed over recent decades.The tumor microenvironment(T...Osteosarcoma,an aggressive bone cancer found most often in children and adolescents,remains difficult to treat,and little improvement in survival rate has been observed over recent decades.The tumor microenvironment(TME),especially the extracellular matrix(ECM),is a critical factor determining cancer progression and chemotherapy resistance,yet traditional 2D models generally fail to replicate its properties.Recent development of 3D-bioprinted tumor models has facilitated improved simulation of the complexity of the TME,but specific models involving bioinks tailored to osteosarcoma remain underdeveloped.Gelatin methacryloyl(GelMA)is a common bioink that can rapidly gel and contains Arg-Gly-Asp(RGD)sequences.However,it lacks collagen’s triple-helix structure that is essential for ECM-cell communication.Hyaluronic acid(HA)is a macromolecule that is aberrantly expressed in osteosarcoma by mechanisms that remain largely unexplored.In this study,we developed a composite bioink containing GelMA,collagen,and HA,and applied it to 3D bioprint an in vitro osteosarcoma model.We found that HA significantly enhanced osteosarcoma cell proliferation and chemoresistance,as well as the expression of epithelial-mesenchymal transition and cancer stem cell markers.Furthermore,we found that HA abundance was positively correlated with hypoxia and angiogenesis signaling pathways,and this occurred mainly via upregulation of hypoxia-inducible factor-1α(HIF-1α)and vascular endothelial growth factor A(VEGFA)expression,thereby contributing to increased chemoresistance.Overall,our study provides a protocol for building in vitro realistic 3D-bioprinted models for studying osteosarcoma,highlights the role of HA in osteosarcoma progression,and offers a platform for developing new chemotherapy treatments.展开更多
Chronic kidney diseases affect a significant portion of the global population and their prevalence is expected to increase in the coming years.Advanced in vitro models are crucial for understanding disease onset and f...Chronic kidney diseases affect a significant portion of the global population and their prevalence is expected to increase in the coming years.Advanced in vitro models are crucial for understanding disease onset and for improving drug testing.Emerging strategies have enhanced the accuracy of these models by incorporating 3D culture and perfusion systems.Notably,efforts have focused on modeling the nephron,particularly endothelialized and epithelialized tubular structures,with perfusion to simulate toxin exchange for nephrotoxicity testing.New approaches combining biomaterials with patient-derived kidney epithelial cells show promise for high-throughput personalized drug screening.However,these methods often rely on decellularized extracellular matrix materials,such as Matrigel®and collagen,which suffer from batch-to-batch variability.To address reproducibility issues,we used norbornene-functionalized alginate to produce peptide-functionalized thiol-ene crosslinked hydrogels.By varying the composition of crosslinkers and peptide functionalization,we tuned the cell interaction with the hydrogels.The rapid reaction kinetics enabled the bioprinting of cell-laden tubular structures using microfluidic bioprinting,without the need for ionic crosslinking,by adapting the printer with UV irradiation at the nozzle.The bioprinted fibers successfully formed monolayers,indicating their potential for creating advanced kidney in vitro models.Thiol-ene crosslinked hydrogels proved to be highly tunable and adaptable to microfluidic bioprinting,demonstrating significant promise for further application to create kidney in vitro models.展开更多
Articular cartilage damage caused by trauma or degenerative pathologies such as osteoarthritis can result in significant pain,mobility issues,and disability.Current surgical treatments have a limited capacity for effi...Articular cartilage damage caused by trauma or degenerative pathologies such as osteoarthritis can result in significant pain,mobility issues,and disability.Current surgical treatments have a limited capacity for efficacious cartilage repair,and long-term patient outcomes are not satisfying.Three-dimensional bioprinting has been used to fabricate biochemical and biophysical environments that aim to recapitulate the native microenvironment and promote tissue regeneration.However,conventional in vitro bioprinting has limitations due to the challenges associated with the fabrication and implantation of bioprinted constructs and their integration with the native cartilage tissue.In situ bioprinting is a novel strategy to directly deliver bioinks to the desired anatomical site and has the potential to overcome major shortcomings associated with conventional bioprinting.In this review,we focus on the new frontier of robotic-assisted in situ bioprinting surgical systems for cartilage regeneration.We outline existing clinical approaches and the utilization of robotic-assisted surgical systems.Handheld and robotic-assisted in situ bioprinting techniques including minimally invasive and non-invasive approaches are defined and presented.Finally,we discuss the challenges and potential future perspectives of in situ bioprinting for cartilage applications.展开更多
Three-dimensional(3D)bioprinting is widely used in ophthalmic clinic,including in diagnosis,surgery,prosthetics,medications,drug development and delivery,and medical education.Articles published in 2011–2022 into bio...Three-dimensional(3D)bioprinting is widely used in ophthalmic clinic,including in diagnosis,surgery,prosthetics,medications,drug development and delivery,and medical education.Articles published in 2011–2022 into bioinks,printing technologies,and bioprinting applications in ophthalmology were reviewed and the strengths and limitations of bioprinting in ophthalmology highlighted.The review highlighted the trade-offs of printing technologies and bioinks in respect to,among others,material type cost,throughput,gelation technique,cell density,cell viability,resolution,and printing speed.There is already widespread ophthalmological application of bioprinting outside clinical settings,including in educational modelling,retinal imaging/visualization techniques and drug design/testing.In clinical settings,bioprinting has already found application in pre-operatory planning.Even so,the findings showed that even with its immense promise,actual translation to clinical applications remains distant,but relatively closer for the corneal(except stromal)tissues,epithelium,endothelium,and conjunctiva,than it was for the retina.This review similarly reflected on the critical on the technical,practical,ethical,and cost barrier to rapid progress of bioprinting in ophthalmology,including accessibility to the most sophisticated bioprinting technologies,choice,and suitability of bioinks,tissue viability and storage conditions.The extant research is encouraging,but more work is clearly required for the push towards clinical translation of research.展开更多
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 man...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.展开更多
The three-dimensional (3D)bioprinting technology has progressed tremendously over the past decade.By controlling the size, shape,and architecture of the bioprinted constructs,3D bioprinting allows for the fabrication ...The three-dimensional (3D)bioprinting technology has progressed tremendously over the past decade.By controlling the size, shape,and architecture of the bioprinted constructs,3D bioprinting allows for the fabrication of tissue/organ-like constructs with strong structural-functional similarity with their in vivo counterparts at high fidelity.The bioink,a blend of biomaterials and living cells possessing both high biocompatibility and printability,is a critical component of bioprinting.In particular, gelatin methacryloyl (GelMA)has shown its potential as a viable bioink material due to its suitable biocompatibility and readily tunable physicochemical properties.Current GelMA-based bioinks and relevant bioprinting strategies for GelMA bioprinting are briefly reviewed.展开更多
Three-dimensional(3D)organoids derived from pluripotent or adult tissue stem cells seem to possess excellent potential for studying development and disease mechanisms alongside having a myriad of applications in regen...Three-dimensional(3D)organoids derived from pluripotent or adult tissue stem cells seem to possess excellent potential for studying development and disease mechanisms alongside having a myriad of applications in regenerative therapies.However,lack of precise architectures and large-scale tissue sizes are some of the key limitations of current organoid technologies.3D bioprinting of organoids has recently emerged to address some of these impediments.In this review,we discuss 3D bioprinting with respect to the use of bioinks and bioprinting methods and highlight recent studies that have shown success in bioprinting of stem cells and organoids.We also summarize the use of several vascularization strategies for the bioprinted organoids,that are critical for a complex tissue organization.To fully realize the translational applications of organoids in disease modeling and regenerative medicine,these areas in 3D bioprinting need to be appropriately harnessed and channelized.展开更多
Three-dimensional(3D)extrusion-based bioprinting is widely used in tissue engineering and regenerative medicine to create cell-incorporated constructs or scaffolds based on the extrusion technique.One critical issue i...Three-dimensional(3D)extrusion-based bioprinting is widely used in tissue engineering and regenerative medicine to create cell-incorporated constructs or scaffolds based on the extrusion technique.One critical issue in 3D extrusion-based bioprinting is printability or the capability to form and maintain reproducible 3D scaffolds from bioink(a mixture of biomaterials and cells).Research shows that printability can be affected by many factors or parameters,including those associated with the bioink,printing process,and scaffold design,but these are far from certain.This review highlights recent developments in the printability assessment of extrusion-based bioprinting with a focus on the definition of printability,printability measurements and characterization,and printability-affecting factors.Key issues and challenges related to printability are also identified and discussed,along with approaches or strategies for improving printability in extrusion-based bioprinting.展开更多
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 so...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.展开更多
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...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.展开更多
The significance of bioink suitability for the extrusion bioprinting of tissue-like constructs cannot be overemphasized.Gelatin,derived from the hydrolysis of collagen,not only can mimic the extracellular matrix to imm...The significance of bioink suitability for the extrusion bioprinting of tissue-like constructs cannot be overemphasized.Gelatin,derived from the hydrolysis of collagen,not only can mimic the extracellular matrix to immensely support cell function,but also is suitable for extrusion under certain conditions.Thus,gelatin has been recognized as a promising bioink for extrusion bioprinting.However,the development of a gelatin-based bioink with satisfactory printability and bioactivity to fabricate complex tissue-like constructs with the desired physicochemical properties and biofunctions for a specific biomedical application is still in its infancy.Therefore,in this review,we aim to comprehensively summarize the state-of-the-art methods of gelatin-based bioink application for extrusion bioprinting.Wefirstly outline the properties and requirements of gelatin-based bioinks for extrusion bioprinting,highlighting the strategies to overcome their main limitations in terms of printability,structural stability and cell viability.Then,the challenges and prospects are further discussed regarding the development of ideal gelatin-based bioinks for extrusion bioprinting to create complex tissue-like constructs with preferable physicochemical properties and biofunctions.展开更多
基金This work was financially supported by the National Key Research and Development Program of China(No.2018YFA0703003)the National Natural Science Foundation of China(No.52125501)+1 种基金the Key Research Project of Shaanxi Province(Nos.2021LLRH-08,2020GXLH-Y-021,and 2021GXLH-Z-028)the Youth InnovationTeam of Shaanxi Universities and the Fundamental Research Funds for the Central Universities.
文摘Bioprinting has been widely investigated for tissue engineering and regenerative medicine applications.However,it is still difficult to reconstruct the complex native cell arrangement due to the limited printing resolution of conventional bioprinting techniques such as extrusion-and inkjet-based printing.Recently,an electrohydrodynamic(EHD)bioprinting strategy was reported for the precise deposition of well-organized cell-laden constructs with microscale filament size,whereas few studies have been devoted to developing bioinks that can be applied for EHD bioprinting and simultaneously support cell spreading.This study describes functionalized alginate-based bioinks for microscale EHD bioprinting using peptide grafting and fibrin incorporation,which leads to high cell viability(>90%)and cell spreading.The printed filaments can be further refined to as small as 30μm by incorporating polyoxyethylene and remained stable over one week when exposed to an aqueous environment.By utilizing the presented alginate-based bioinks,layer-specific cell alignment along the printing struts could be observed inside the EHD-printed microscale filaments,which allows fabricating living constructs with cell-scale filament resolution for guided cellular orientation.
基金financial support from the National Natural Science Foundation of China(Nos.82202335,82230071,and 82172098)the Shanghai Sailing Program(No.22YF1414000).
文摘Three-dimensional(3D)bioprinting has revolutionized tissue engineering by enabling precise fabrication with bioinks.Among these techniques,digital light processing(DLP)stands out due to its exceptional resolution,speed,and biocompatibility.However,the progress of DLP is hindered by the limited availability of suitable bioinks.Currently,some studies involve simple mixing of different materials,resulting in bioinks that lack uniformity and photopolymerization characteristics.To address this challenge,we present an innovative one-pot synthesis method for bioinks based on methacrylated gelatin/alginate with hydroxyapatite(HAP).This approach offers significant advantages in terms of efficiency and uniformity.The synthesized bioinks demonstrate excellent printability,stability,and notably enhanced mechanical properties,facilitating optimal in vitro compatibility.Additionally,the HAP-hybrid bioinks printed scaffolds demonstrated impressive bone repair capabilities in vivo compared with pure organic bioinks.In conclusion,the Gel/Alg/HAP bioinks presented herein offer an innovative solution for DLP bioprinting within the field of bone tissue engineering.Their multifaceted advantages help overcome the limitations of restricted bioink choices,pushing forward the boundaries of bioprinting technology and contributing to the progress of regenerative medicine and tissue engineering.
基金supported by the Major Science and Technology Special Project of Henan Province,China(No.171100210600)the Program of China Scholarship Council(No.201807045057)+2 种基金the High Level Talent Internationalization Training Program of Henan Province,China(No.2019004)the Scientific and Technological Research Project of Henan Province,China(Nos.212102310854 and 222102310526)the Open Foundation of the State Key Laboratory of Fluid Power and Mechatronic Systems(No.GZKF-202105)。
文摘Cryobioprinting has tremendous potential to solve problems to do with lack of shelf availability in traditional bioprinting by combining extrusion bioprinting and cryopreservation.In order to ensure the viability of cells in the frozen state and avoid the possible toxicity of dimethyl sulfoxide(DMSO),DMSO-free bioink design is critical for achieving successful cryobioprinting.A nontoxic gelatin methacryloyl-based bioink used in cryobioprinting is composed of cryoprotective agents(CPAs)and a buffer solution.The selection and ratio of CPAs in the bioink directly affect the survival of cells in the frozen state.However,the development of universal and efficient cryoprotective bioinks requires extensive experimentation.We first compared two commonly used CPA formulations via experiments in this study.Results show that the effect of using ethylene glycol as the permeable CPA was 6.07%better than that of glycerol.Two datasets were obtained and four machinelearning models were established to predict experimental outcomes.The predictive powers of multiple linear regression(MLR),decision tree(DT),random forest(RF),and artificial neural network(ANN)approaches were compared,suggesting an order of ANN>RF>DT>MLR.The final selected ANN model was then applied to another dataset.Results reveal that this machine-learning method can accurately predict the effects of cryoprotective bioinks composed of different CPAs.Outcomes also suggest that the formulations presented here have universality.Our findings are likely to greatly accelerate research and development on the use of bioinks for cryobioprinting.
文摘Geometric and structural integrity often deteriorate in 3D printed cell-laden constructs over time due to cellular compaction and hydrogel shrinkage.This study introduces a new approach that synergizes the advantages of cell compatibility of biological hydrogels and mechanical stability of elastomeric polymers for structure fidelity maintenance upon stereolithography and extrusion 3D printing.Enabling this advance is the composite bioink,formulated by integrating elastomeric microparticles from poly(octamethylene maleate(anhydride)citrate)(POMaC)into biologically derived hydrogels(fibrin,gelatin methacryloyl(GelMA),and alginate).The composite bioink enhanced the elasticity and plasticity of the 3D printed constructs,effectively mitigating tissue compaction and swelling.It exhibited a low shear modulus and a rapid crosslinking time,along with a high ultimate compressive strength and resistance to deformation from cellular forces and physical handling;this was attributed to packing and stress dissipation of elastomeric particles,which was confirmed via mathematical modelling.Enhanced functional assembly and stability of human iPSC-derived cardiac tissues and primary vasculature proved the utility of the composite bioink in tissue engineering.In vivo implantation studies revealed that constructs containing POMaC particles exhibited improved resilience against host tissue stress,enhanced angiogenesis,and infiltration of pro-reparative macrophages.
文摘Bioinks are formulations of biomaterials and living cells, sometimes with growth factors or other biomolecules, while extrusion bioprinting is an emerging technique to apply or deposit these bioinks or biomaterial solutions to create three-dimensional (3D) constructs with architectures and mechanical/biological properties that mimic those of native human tissue or organs. Printed constructs have found wide applications in tissue engineering for repairing or treating tissue/organ injuries, as well as in vitro tissue modelling for testing or validating newly developed therapeutics and vaccines prior to their use in humans. Successful printing of constructs and their subsequent applications rely on the properties of the formulated bioinks, including the rheological, mechanical, and biological properties, as well as the printing process. This article critically reviews the latest developments in bioinks and biomaterial solutions for extrusion bioprinting, focusing on bioink synthesis and characterization, as well as the influence of bioink properties on the printing process. Key issues and challenges are also discussed along with recommendations for future research.
基金the projects of National Natural Science Foundation of China(No.32301209)Key R&D Projects of Henan Province(Nos.231111312400 and 241111220400).
文摘Engineering hydrogels that resemble biological tissues of various lengths via conventional fabrication techniques remains challenging.Three-dimensional(3D)bioprinting has emerged as an advanced approach for constructing complex biomimetic 3D architectures,which are currently restricted by the limited number of available bioinks with high printability,biomimicry,biocompatibility,and proper mechanical properties.Inspired by ubiquitous coacervation phenomena in biology,we present a unique mineral-biopolymer coacervation strategy that enables the hierarchical assembly of nanoclay and recombinant human collagen(RHC).This system was observed to undergo a coacervation transition(liquid‒liquid phase separation)spontaneously.The formed dense phase separated from its supernatant is the coacervate of clay-RHC-rich complexes,where polymer chains are sandwiched between silicate layers.Molecular dynamics simulation was first used to verify and explore the coacervation process.Then,the coacervates were demonstrated to be potential bioinks that exhibited excellent self-supporting and shear-thinning viscoelastic properties.Through extrusion-based printing,the versatility of the bioink was demonstrated by reconstructing the key features of several biological tissues,including multilayered lattice,vascular,nose,and ear-like structures,without the need for precrosslinking operations or support baths.Furthermore,the printed scaffolds were cytocompatible,elicited minimal inflammatory responses,and promoted bone regeneration in calvarial defects.
基金This work was supported by National Natural Science Foundation of China,Nos.32171353,52073314Guangdong Key Areas Research and Development Program,No.2020B1111150003+1 种基金Guangdong Basic and Applied Basic Research Foundation,No.2022A1515011388Science and Technology Projects of Guangzhou,No.202002020078。
文摘The printability of bioink and post-printing cell viability is crucial for extrusion-based bioprinting.A proper bioink not only provides mechanical support for structural fidelity,but also serves as suitable three-dimensional(3D)microenvironment for cell encapsulation and protection.In this study,a hydrogel-based composite bioink was developed consisting of gelatin methacryloyl(GelMA)as the continuous phase and decellularised extracellular matrix microgels(DMs)as the discrete phase.A flow-focusing microfluidic system was employed for the fabrication of cell-laden DMs in a high-throughput manner.After gentle mixing of the DMs and GelMA,both rheological characterisations and 3D printing tests showed that the resulting DM-GelMA hydrogel preserved the shear-thinning nature,mechanical properties,and good printability from GelMA.The integration of DMs not only provided an extracellular matrix-like microenvironment for cell encapsulation,but also considerable shear-resistance for high post-printing cell viability.The DM sizes and inner diameters of the 3D printer needles were correlated and optimised for nozzle-based extrusion.Furthermore,a proof-of-concept bioink composedg of RSC96 Schwann cells encapsulated DMs and human umbilical vein endothelial cell-laden GelMA was successfully bioprinted into 3D constructs,resulting in a modular co-culture system with distinct cells/materials distribution.Overall,the modular DM-GelMA bioink provides a springboard for future precision biofabrication and will serve in numerous biomedical applications such as tissue engineering and drug screening.
基金This study was supported by the Beijing Natural Science Foundation(7204306)National Natural Science Foundation of China(82002056,81830064,81721092,32000969,81974019)+9 种基金National Key Research and Development Program of China(2018YFA0108700,2017YFA0105602)NSFC Projects of International Cooperation and Exchanges(81720108004)China Postdoctoral Science Foundation(2020M673672)Key Support Program for Growth Factor Research(SZYZ-TR-03)Chinese PLA General Hospital for Military Medical Innovation Research Project(CX19026)the CAMS Innovation Fund for Medical Sciences(CIFMS,2019-I2M-5-059)the Military Medical Research and Development Projects(AWS17J005)the Research Team Project of Natural Science Foundation of Guangdong Province of China(2017A030312007)the key program of guangzhou science research plan(201904020047)The Special Project of Dengfeng Program of Guangdong Provincial People’s Hospital(DFJH201812,KJ012019119,KJ012019423).
文摘Extrusion-based bioprinting (EBB) holds potential for regenerative medicine. However, the widely-used bioinks of EBB exhibit some limitations for skin regeneration, such as unsatisfactory bio-physical (i.e., mechanical, structural, biodegradable) properties and compromised cellular compatibilities, and the EBB-based bioinks with therapeutic effects targeting cutaneous wounds still remain largely undiscussed. In this review, the printability considerations for skin bioprinting were discussed. Then, current strategies for improving the physical properties of bioinks and for reinforcing bioinks in EBB approaches were introduced, respectively. Notably, we highlighted the applications and effects of current EBB-based bioinks on wound healing, wound scar formation, vasculari-zation and the regeneration of skin appendages (i.e., sweat glands and hair follicles) and discussed the challenges and future perspectives. This review aims to provide an overall view of the applications, challenges and promising solutions about the EBB-based bioinks for cutaneous wound healing and skin regeneration.
基金the financial support from the National Natural Science Foundation of China(No.32122046,12272032,82072082,32101102)the National Key R&D Program of China(No.2020YFC0122204,2018YFA0703100)+2 种基金the Youth Innovation Promotion Association of CAS[No.2019350]the Shenzhen Fundamental Research Foundation[No.JCYJ20210324115814040]the 111 Project(No.B13003).
文摘In the field of regenerative medicine,the importance of 3D bioprinting is self-evident and nonnegligible.However,3D bioprinting technology also requires bioink with excellent performance as support material to fabricate a multi-functional bioinspired scaffold.Collagen-based bioink is regarded as an ideal 3D bioprinting ink for its excellent biocompatibility,controllable printability and cell loading property.It is an important breakthrough in regenerative medicine with the progress of collagen-based bioink,which fabricates bioinspired scaffolds with different functions and is applied in different repair scenarios.This review summarizes the different applications of collagen-based bioink and classifies them as soft tissue and hard tissue according to the target region.The applications of target region in soft tissues include skin,cartilage,heart and blood vessels,while in hard tissues include femur,skull,teeth and spine.When the collagen-based bioink is applied in repairing soft tissue,the requirements of function are higher,while the mechanical properties must be further improved in repairing hard tissue.We further summarize the characteristics of collagen-based bioink and point out the most important properties that should be considered in different repair scenarios,which can provide reference for the preparation of bioinks with different functions.Finally,we point out the main challenges faced by collagen-based bioink and prospect the future research directions.
基金supported by the Natural Science Foundation of Zhejiang Province(No.TGY24E050004)the Ningbo Major Research and Development Plan Project(No.2024Z208)+2 种基金the National Natural Science Foundation of China(No.12202387)the Ningbo Health Technology Project(No.2023Y16)the Natural Science Foundation of Ningbo(No.2022J212).
文摘Osteosarcoma,an aggressive bone cancer found most often in children and adolescents,remains difficult to treat,and little improvement in survival rate has been observed over recent decades.The tumor microenvironment(TME),especially the extracellular matrix(ECM),is a critical factor determining cancer progression and chemotherapy resistance,yet traditional 2D models generally fail to replicate its properties.Recent development of 3D-bioprinted tumor models has facilitated improved simulation of the complexity of the TME,but specific models involving bioinks tailored to osteosarcoma remain underdeveloped.Gelatin methacryloyl(GelMA)is a common bioink that can rapidly gel and contains Arg-Gly-Asp(RGD)sequences.However,it lacks collagen’s triple-helix structure that is essential for ECM-cell communication.Hyaluronic acid(HA)is a macromolecule that is aberrantly expressed in osteosarcoma by mechanisms that remain largely unexplored.In this study,we developed a composite bioink containing GelMA,collagen,and HA,and applied it to 3D bioprint an in vitro osteosarcoma model.We found that HA significantly enhanced osteosarcoma cell proliferation and chemoresistance,as well as the expression of epithelial-mesenchymal transition and cancer stem cell markers.Furthermore,we found that HA abundance was positively correlated with hypoxia and angiogenesis signaling pathways,and this occurred mainly via upregulation of hypoxia-inducible factor-1α(HIF-1α)and vascular endothelial growth factor A(VEGFA)expression,thereby contributing to increased chemoresistance.Overall,our study provides a protocol for building in vitro realistic 3D-bioprinted models for studying osteosarcoma,highlights the role of HA in osteosarcoma progression,and offers a platform for developing new chemotherapy treatments.
基金funding from the European Union’s Hori-zon 2020 FET Open programme under grant agreement No.964452.
文摘Chronic kidney diseases affect a significant portion of the global population and their prevalence is expected to increase in the coming years.Advanced in vitro models are crucial for understanding disease onset and for improving drug testing.Emerging strategies have enhanced the accuracy of these models by incorporating 3D culture and perfusion systems.Notably,efforts have focused on modeling the nephron,particularly endothelialized and epithelialized tubular structures,with perfusion to simulate toxin exchange for nephrotoxicity testing.New approaches combining biomaterials with patient-derived kidney epithelial cells show promise for high-throughput personalized drug screening.However,these methods often rely on decellularized extracellular matrix materials,such as Matrigel®and collagen,which suffer from batch-to-batch variability.To address reproducibility issues,we used norbornene-functionalized alginate to produce peptide-functionalized thiol-ene crosslinked hydrogels.By varying the composition of crosslinkers and peptide functionalization,we tuned the cell interaction with the hydrogels.The rapid reaction kinetics enabled the bioprinting of cell-laden tubular structures using microfluidic bioprinting,without the need for ionic crosslinking,by adapting the printer with UV irradiation at the nozzle.The bioprinted fibers successfully formed monolayers,indicating their potential for creating advanced kidney in vitro models.Thiol-ene crosslinked hydrogels proved to be highly tunable and adaptable to microfluidic bioprinting,demonstrating significant promise for further application to create kidney in vitro models.
基金the funding provided by the United Kingdom(UK)Engineering and Physical Sciences Research Council(EPSRC)Doctoral Prize Fellowship(EP/R513131/1)。
文摘Articular cartilage damage caused by trauma or degenerative pathologies such as osteoarthritis can result in significant pain,mobility issues,and disability.Current surgical treatments have a limited capacity for efficacious cartilage repair,and long-term patient outcomes are not satisfying.Three-dimensional bioprinting has been used to fabricate biochemical and biophysical environments that aim to recapitulate the native microenvironment and promote tissue regeneration.However,conventional in vitro bioprinting has limitations due to the challenges associated with the fabrication and implantation of bioprinted constructs and their integration with the native cartilage tissue.In situ bioprinting is a novel strategy to directly deliver bioinks to the desired anatomical site and has the potential to overcome major shortcomings associated with conventional bioprinting.In this review,we focus on the new frontier of robotic-assisted in situ bioprinting surgical systems for cartilage regeneration.We outline existing clinical approaches and the utilization of robotic-assisted surgical systems.Handheld and robotic-assisted in situ bioprinting techniques including minimally invasive and non-invasive approaches are defined and presented.Finally,we discuss the challenges and potential future perspectives of in situ bioprinting for cartilage applications.
文摘Three-dimensional(3D)bioprinting is widely used in ophthalmic clinic,including in diagnosis,surgery,prosthetics,medications,drug development and delivery,and medical education.Articles published in 2011–2022 into bioinks,printing technologies,and bioprinting applications in ophthalmology were reviewed and the strengths and limitations of bioprinting in ophthalmology highlighted.The review highlighted the trade-offs of printing technologies and bioinks in respect to,among others,material type cost,throughput,gelation technique,cell density,cell viability,resolution,and printing speed.There is already widespread ophthalmological application of bioprinting outside clinical settings,including in educational modelling,retinal imaging/visualization techniques and drug design/testing.In clinical settings,bioprinting has already found application in pre-operatory planning.Even so,the findings showed that even with its immense promise,actual translation to clinical applications remains distant,but relatively closer for the corneal(except stromal)tissues,epithelium,endothelium,and conjunctiva,than it was for the retina.This review similarly reflected on the critical on the technical,practical,ethical,and cost barrier to rapid progress of bioprinting in ophthalmology,including accessibility to the most sophisticated bioprinting technologies,choice,and suitability of bioinks,tissue viability and storage conditions.The extant research is encouraging,but more work is clearly required for the push towards clinical translation of research.
基金sponsored by the National Natural Science Foundation of China(No.U1609207)。
文摘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.
基金the National Institutes of Health (K99CA201603,R21EB025270, R21EB026175)Doctoral New Investigator Grant from American Chemical Society Petroleum Research Fund (56840-DNI7).G.L. Y.acknowledges Natural and Science Foundation of Hubei Province (2014CFB778).
文摘The three-dimensional (3D)bioprinting technology has progressed tremendously over the past decade.By controlling the size, shape,and architecture of the bioprinted constructs,3D bioprinting allows for the fabrication of tissue/organ-like constructs with strong structural-functional similarity with their in vivo counterparts at high fidelity.The bioink,a blend of biomaterials and living cells possessing both high biocompatibility and printability,is a critical component of bioprinting.In particular, gelatin methacryloyl (GelMA)has shown its potential as a viable bioink material due to its suitable biocompatibility and readily tunable physicochemical properties.Current GelMA-based bioinks and relevant bioprinting strategies for GelMA bioprinting are briefly reviewed.
文摘Three-dimensional(3D)organoids derived from pluripotent or adult tissue stem cells seem to possess excellent potential for studying development and disease mechanisms alongside having a myriad of applications in regenerative therapies.However,lack of precise architectures and large-scale tissue sizes are some of the key limitations of current organoid technologies.3D bioprinting of organoids has recently emerged to address some of these impediments.In this review,we discuss 3D bioprinting with respect to the use of bioinks and bioprinting methods and highlight recent studies that have shown success in bioprinting of stem cells and organoids.We also summarize the use of several vascularization strategies for the bioprinted organoids,that are critical for a complex tissue organization.To fully realize the translational applications of organoids in disease modeling and regenerative medicine,these areas in 3D bioprinting need to be appropriately harnessed and channelized.
基金The authors acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada(NSERC,Grant No.:RGPIN-2014-05648).
文摘Three-dimensional(3D)extrusion-based bioprinting is widely used in tissue engineering and regenerative medicine to create cell-incorporated constructs or scaffolds based on the extrusion technique.One critical issue in 3D extrusion-based bioprinting is printability or the capability to form and maintain reproducible 3D scaffolds from bioink(a mixture of biomaterials and cells).Research shows that printability can be affected by many factors or parameters,including those associated with the bioink,printing process,and scaffold design,but these are far from certain.This review highlights recent developments in the printability assessment of extrusion-based bioprinting with a focus on the definition of printability,printability measurements and characterization,and printability-affecting factors.Key issues and challenges related to printability are also identified and discussed,along with approaches or strategies for improving printability in extrusion-based bioprinting.
基金supported by the Key-Area Research and Development Program of Guangdong Province(2019B010941001)the Shenzhen Double Chain Project for Innovation and Development Industry supported by the Bureau of Industry and Information Technology of Shenzhen(201908141541)Shenzhen Fundamental Research Foundation(GJHZ20170314154845576 and GJHS20170314161106706).
文摘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.
基金the National Key Research and Development Program of China(No.2018YFA0703000)the Science Fund for Creative Research Groups of the National Natural Science Foundation of China(No.51521064)。
文摘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.
基金support for this work from the National Key R&D Program of China(No.2018YFA0703100)the National Natural Sci-ence Foundation of China(Nos.32122046,82072082,and 32000959)+2 种基金the Youth Innovation Promotion Association of CAS(No.2019350)the Guangdong Natural Science Foundation(No.2019A1515111197)the Shenzhen Fundamental Research Foun-dation(Nos.JCYJ20190812162809131,JCYJ20200109114006014,JCYJ20210324113001005,and JCYJ20210324115814040).
文摘The significance of bioink suitability for the extrusion bioprinting of tissue-like constructs cannot be overemphasized.Gelatin,derived from the hydrolysis of collagen,not only can mimic the extracellular matrix to immensely support cell function,but also is suitable for extrusion under certain conditions.Thus,gelatin has been recognized as a promising bioink for extrusion bioprinting.However,the development of a gelatin-based bioink with satisfactory printability and bioactivity to fabricate complex tissue-like constructs with the desired physicochemical properties and biofunctions for a specific biomedical application is still in its infancy.Therefore,in this review,we aim to comprehensively summarize the state-of-the-art methods of gelatin-based bioink application for extrusion bioprinting.Wefirstly outline the properties and requirements of gelatin-based bioinks for extrusion bioprinting,highlighting the strategies to overcome their main limitations in terms of printability,structural stability and cell viability.Then,the challenges and prospects are further discussed regarding the development of ideal gelatin-based bioinks for extrusion bioprinting to create complex tissue-like constructs with preferable physicochemical properties and biofunctions.
