Hongli Mo ,Li Yng ,Hofng Zhu ,Lihung Wu ,Peihong Ji,c ,Jiqun Yng ,Zhongwei Gu,b,c,d,*

a College of Materials Science and Engineering,Nanjing Tech University,Nanjing,211816,China

b Research Institute for Biomaterials,Tech Institute for Advanced Materials,Nanjing Tech University,Nanjing,210000,China

c NJTech-BARTY Joint Research Center for Innovative Medical Technology,Nanjing,210000,China

d Nanjing Industry Institute for Advanced Intelligent Equipment,Nanjing,210042,China

e Jiangsu Key Lab of 3D Printing Equipment and Manufacturing,Nanjing Normal University,Nanjing,210046,China

Keywords:3D bioprinting ECM-based materials Multi-material Composite and hybrid materials Stimuli-responsive materials

ABSTRACT 3D bioprinting has been developed as a promising technology in a wide variety of biomedical applications.However,the successful implementation of bioprinting is still heavily restricted by the relatively narrow range of printable and biocompatible materials.Recently,many comprehensive reviews of the main bioprinting methods,commonly used bioprinting materials and the trending bioprinting applications have been carried out.In this review,we focused on the trends in the development of materials for 3D bioprinting.The significance and recent advances of ECM-based materials,multi-material and stimuli-responsive materials for bioprinting were summarized successively.Moreover,the challenges in current studies and future perspectives of materials for 3D bioprinting were discussed.

1.Introduction

Three-dimensional (3D) printing,also known as additive manufacturing,have recently attracted significant interest in such fields as industry,agriculture,energy,aerospace,transportation,communications,culture,education,health care,national defense,military affairs and so on[1–8].In contrast to traditional manufacturing processes,3D printing enables to fabricate complex customized architectures through computer-aided design and spatial deposition of materials layer-bylayer.As an important branch of 3D printing,3D bioprinting is a stateof-the-art technology to fabricate biological constructs with specific geometries,hierarchical architectures and biological functions [9–11].Based on the principle of “additive manufacturing”,biomaterials,bioactive factors and even cells are integrally positioned with precise spatial control to reconstruct human tissues and organs that can mimic their native counterparts in terms of both structures and functionalities.In this way,3D bioprinting possesses a significant potential to address the unmet needs in the development of precision medicine and personalized treatments.And it is true that 3D bioprinting has been employed to help pharmaceutical and medical companies to create more specific drugs with less time and expense by providing printed organoid chips,enable a rapid production of patient-specific medical implants and surgical tools,change the way that doctors and surgeons plan procedures to optimize the surgical process and cost,and in the future,construct implantable artificial tissues or organs to reduce the waiting lists and increase the number of lives saved [12–19].

To finalize a construct,3D printing usually involves several technical steps which include developing the 3D geometry of the target anatomical area through CT/MRI scan,optimizing the printing file,selecting appropriate materials,printing,and analysis of the printed constructs [2,9].The material used for prototyping is an essential consideration for 3D printing [20–22].Initially,3D printing technologies were developed for nonbiological applications,and the typically 3D printers were designed for the deposition of metals,ceramics and thermoplastic polymers [23,24].The printing processes usually involved the use of organic solvents,high temperatures or crosslinking agents that are not compatible with living cells and biological materials.Therefore,one of the most important challenges in 3D bioprinting is to find suitable printing materials with excellent printability,biocompatibility,desired mechanical and degradation properties for tissue constructs [9,10,25].

Fig.1.The complexity of ECM.a).Main components of ECM[89].b).The unique composition of ECM in four different stem cell niches[88].The hemapoietic stemcell (HSC) niche consists of “endosteal niche” that is populated mainly by osteoblasts,and “vasculature niche” that is located in the perivascular space.In the hair follicle,multipotent stem cells are located in the bulge.In skeletal muscle,satellite cells reside in the niche between myofiber plasma membrane and basal lamina.In the adult brain,neural stem cells (NSCs) are intercalated into the ependymal layer and are also associated with the vasculature closely.

Currently,most of the materials used for 3D bioprinting are based on either natural or synthetic biomaterials.The main advantage of naturally derived materials (such as collagen,gelatin,chitosan,alginate,fibrin,hyaluronic acid) is their inherent bioactivity [26–41].They usually show high similarity to the extracellular matrix(ECM)and excellent biocompatibility.However,natural biomaterials are often weak in mechanical properties even after being cross-linked.Synthetic materials (such as PCL,PLA,PEG,PEEK,Pluronic) offer greater advantages over natural materials in that they can be tailored with specific physical properties and possess more uniformity than naturally derived materials [42–56].Challenges with the use of synthetic materials for 3D bioprinting include the poor biocompatibility,toxic degradation products and lack of bioactive ligands.Furthermore,human tissues are exquisite combinations of multiple cell types and well-defined gradient structures of ECM,each of which shows specific biological and mechanical effects on the cellular behaviors [57–61].It is believed that by replicating the native ECM of target tissues and organs in terms of composition and distribution,the desired microenvironment with predicted mechanical and functional properties will be reproduced in vitro (Fig.1a).However,it is impossible for any single component material to substitute all the functions of that the native ECM offered.The constructs printed with single material as standalone usually lack indispensable functionalities for functional systems.

To overcome the disadvantages mentioned above,a clearer understanding of the physiological properties of ECM and a greater capability to reproduce the complex ECM-mimetic 3D structures would be a significant advancement for 3D bioprinting.Besides,the development of composite/hybrid bioprinting materials and multi-material bioprinting technologies has become an irresistible trend [62–66].Meanwhile,living tissues and organs in the human body are existed in a dynamic biochemical environment.The artificial constructs printed with traditional materials are often failed to adapt themselves to match the development of tissues or organs in space or time.Therefore,the development of advanced stimuli-responsive biomaterials for 3D bioprinting of artificial biological constructs with the ability to adapt themselves to the complex dynamic physiological environment is of great significance[67–70].

In this review,we firstly discuss the key requirements for ideal materials used in 3D bioprinting.Then we summarize the recent advances of ECM-based materials,multi-material and stimuli-responsive materials for bioprinting successively.Finally,the challenges in current studies and future perspectives of materials for 3D bioprinting are addressed.

2.Requirements for ideal 3D bioprinting materials

With the development of materials science,the varieties of materials for 3D bioprinting are expanding.To choose suitable materials that have desired functional and mechanical properties,and to achieve a successful printing procedure,several factors must be carefully considered.We will now discuss in more detail the key characteristics of ideal materials for 3D bioprinting.

Printability:Printability refers to the ability of materials to be accurately and controllably deposited in the specified space within a certain time [25,71,72].This performance is causally related to the accuracy of the structure and size of 3D printed product.For different printing technologies,the demand for printability of materials will also be different.For example,inkjet bioprinting has a great limitation on the viscosity of the material.The material needs to be capable of rapid cross-linking to ensure the layered formation of complex 3D structures.For micro-extrusion bioprinting,high viscosity materials can be used to maintain the initial 3D shape after printing,and the final stable structure can be formed by the cross-linking after printing.Therefore,it is required that the material should have a specific cross-linking mechanism or shear thinning properties.Currently,a large number of new materials have been reported for 3D bioprinting,but in the end these materials can only be limited to certain simple printing process,thus losing their practical application values.

Biocompatibility:With the deepening research,the understanding of biocompatibility has evolved from the initial requirement that materials can coexist with tissues and organs,and do not cause any adverse local or systemic effects,to the need for implanted materials to have positive interactions with the host tissues and/or immune systems,in order to achieve the purpose of regulating the activity and function of host cells,tissues and organs [73,74].At present,when 3D bioprinting is used in the development of medical models,rehabilitation aids,prostheses,surgical guides,etc.,the requirements for the biocompatibility of the materials are low.However,when 3D bioprinting is employed to prepare implantable medical devices (such as 3D printed joints,teeth,artificial eyes and other permanently implanted substitutes),tissue engineering scaffolds,and cell-laden tissue and organ substitutes,it is strictly required that the materials should have good biocompatibility.From the perspective of materials science,the biocompatibility is mainly affected by the chemical composition,structural morphology,surface characteristics,surface charge,and mechanical properties of the materials.Therefore,to improve the biocompatibility,the 3D bioprinting materials can be modified through:1).Surface modification,such as changing the materials topology,regulating the surface hydrophilicity and hydrophobicity,changing the surface charge strength,and surface coating with bioactive molecules;2).Compositing or hybridization of different types of materials,such as combining the advantages of both natural and synthetic materials to improve the mechanical properties and the biocompatibility of 3D bioprinting materials;3) Developing materials with the same or similar structure and performance as those existing in vivo through the principle of biomimicry.

Appropriate mechanical properties:The materials with certain mechanical strength that can resist the external force and maintain the printed initial shape and structure play an important role in the implementation of the functions of the printed constructs.According to the actual application goals,materials with different mechanical properties should be selected and designed [75–77].For example,to construct tissues and organs by 3D bioprinting,materials with corresponding mechanical properties should be selected according to the different mechanical environments required by the target tissues and organs.In addition,during the printing process,especially for the natural polymer materials with poor mechanical properties,sacrificial materials with better mechanical properties should be selected as a support [78,79].The support material should be easily removed from the printed constructs after printing completed.Meanwhile,the support material itself and the products during removal should have no negative effect on the structure and other properties of the printed constructs.

Biodegradability:After implantation in the body,3D bioprinting materials should be gradually degraded with the proliferation of cells and the production of ECM[80,81].The degradation rate should match the rate at which cells produce ECM to replace implanted materials,and the rate at which new tissue is generated.At the same time,the degradation products should also be non-toxic,easily metabolized and able to be excreted quickly.

Sterilization stability:Sterilization is a critical step in the biomedical applications of biomaterials [82–84].Usually,the sterilization of biomaterials is accomplished using physical or chemical processes,such as heat/steam treatment (autoclaving),e-beam or gamma irradiation,ethylene oxide treatment,incubating with ethanol and other techniques to inactivate all forms of microbial life.Each kind of sterilization method has its own advantages/disadvantages and applicable range.Therefore,the materials used for 3D bioprinting must be compatible with at least one kind of sterilization method.In other words,the materials must maintain its own characteristics or lose certain performance within acceptable range after being treated with appropriate sterilization method.

3.ECM-based materials for 3D bioprinting

The ECM is the natural 3D microenvironment in which cells reside[57,85].The ECM interacts with cells and provides cells with structural support and biochemical signals to facilitate a series of important cellular processes [61,86,87].Meanwhile,the ECM of each tissue and organ is unique in both physicochemical and biological properties that allow the cells to perform specific functions such as spreading,migration,proliferation,and differentiation (Fig.1b) [88].Therefore,it is extremely important for the researchers to recapitulate the native ECM in its components,structural organization and biomechanics for 3D bioprinting of tissues and organs.

3.1.ECM-derived biomaterials

The native ECM is complex in composition and the main components include collagen,non-collagenous glycoproteins (such as fibronectin,laminins,and tenascin),glycosaminoglycans (with predominant hyaluronic acid) and proteoaminoglycans (such asperlecan,and aggrecan) (Fig.1a) [89].To provide an environment that recapitulates the microenvironment in vivo,numerous isolated ECM components (collagen,fibronectin,etc.) and other nature or synthetic biomaterials have been explored for efficient 3D bioprinting[9,39–41,56,90].For example,Ng et al.developed a two-step bioprinting strategy to produce 3D in vitro pigmented human skin constructs with uniform skin pigmentation [91].The two-step bioprinting strategy involved the fabrication of hierarchical porous collagen-based structures and deposition of epidermal cells such as keratinocytes and melanocytes at pre-defined positions on top of the biomimetic dermal skin constructs.The 3D bioprinted pigmented skin constructs exhibited similar constitutive pigmentation as the skin donors.Since the gelation of collagen is typically achieved through the thermally driven self-assembly which is difficult to control,it is difficult to bioprint complex scaffolds using collagen in its native unmodified form.Researchers have developed some methods,including chemical modification of collagen to make it UV crosslinkable,pH,temperature and collagen concentration regulation to control the collagen gelation and print fidelity[92,93].However,the hydrogels obtained through these methods are usually incredibly soft and easy to collapse and it is difficult to print them with high fidelity at a height of more than several layers.To overcome this problem,Lee et al.presented a method to drive collagen self-assembly within a buffered support material through rapid pH change,which provides a 20-μm filament resolution [90].The chemically unmodified collagen was able to be directly bioprinted using freeform reversible embedding of suspended hydrogels to engineer components of the human heart at various scales with precise control of composition and microstructure.

Gelatin,which is derived from acidic or alkaline hydrolysis of collagen and possess better physical properties in aqueous solution,has been used extensively as an appealing alternative to collagen in 3D bioprinting for tissue engineering applications [94–97].To make gelatin printable and endow it with sufficient mechanical properties,chemical or enzymatic crosslinking is often required.During the past few years,research efforts focused on developing the gelatin methacryloyl (GelMA),a derivative of gelatin that can be synthesized via the chemical modification (methacrylation) of gelatin and crosslinked via free radical polymerization [26,35,37,98,99].GelMA shows good cell adhesion and migration ability due to the presence of Arg-Gly-Asp(RGD) sequences and matrix metalloproteinase (MMP) degradable motifs in the polymer chains.However,polymerization of GelMA requires the addition of a photoinitiator and ultraviolet (UV) irradiation.The cytotoxicity of these chemical photoinitiators and negative cellular effects of UV irradiation present a concern.Therefore,the development of alternative enzymatic crosslinking method without adding of initiators and novel biocompatibility photoinitiators that can be triggered by light with longer wavelength (such as blue light,or even visible light)has thus attract extensive attention.

Meanwhile,other natural-occurring biomaterials including hyaluronic acid,alginate,chitosan,silk,and so on have also received significant attention for different tissues bioprinting[29,31,32,36,100–104].To provide an in vitro environment that can more effectively replicate the in vivo microenvironment,biomaterials have been mixed with biochemical reagents such as growth factors and cytokines which provide biochemical cues that can induce cell differentiation or ECM production in other processes.The latest development of natural-occurring biomaterials and their applications in 3D bioprinting have been extensively reviewed elsewhere [12,25,105].

3.2.Decellularized ECM

Although the ECM-mimetic biomaterials showed great biocompatibility and printability,and have led to the development of 3D bioprinting,it is extremely difficult to precisely reproduce the complex composition and architecture of native ECM.One of the most promising strategy is the use of decellularized ECM (dECM) of the targeted tissue or organ (Fig.2a) [89,106,107].The dECM contains the proper proportion of proteins and polymers that makeup the ECM,as well as other biochemical cues from the original natural ECM.Bioprinting of dECM has a major advantage of the ability to modulate the behaviors of cells in the printed constructs appropriately,and the dECM also has the potential to meet all clinical performance requirements.Currently,dECM derived from numerous different organs including cartilage,heart,adipose,liver,skin,vascular tissue and bladder have been used for 3D bioprinting (Fig.2b and c).For example,Pati et al.prepared three kinds of dECM from adipose,cartilage and heart tissues with a combination of physical,chemical and enzymatic processes,and developed a method for the bioprinting of cell-laden constructs with the dECM bioinks [78].The printed cell-dECM constructs revealed higher levels of cell viability,differential lineage commitment and ECM formation.This research demonstrated that tissue printing with dECM is an attractive option that opens new avenues for tissue reconstruction with enhanced cells survival,differentiation and long-term functions.

Although many research groups showed 3D bioprinting of dECM from different organs with promising results,dECM-based tissue or organ bioprinting has not been well established.The main obstacle is the weak mechanical property of dECM.Usually,a frame printed using biomaterials with high mechanical property (e.g.PCL) is necessary to maintain the structure printed with dECM[78,108].Another commonly used effective strategy to make the dECM-based bioinks mechanically stronger is to combine the dECM with synthetic polymers or other active molecules.For example,PEG-based crosslinkers with varying molecular weights,geometries and functional groups was combined with dECM to make the dECM bioink mechanically stronger in the process and the final construct shear stiffness values ranging from approximately 100 Pa to 20 kPa[110].In another study,riboflavin was added to dECM to manipulate the mechanical properties of dECM via vitamin B2-induced photo-crosslinking [79].Polyethylene glycol diacrylate(PEGDA)has acrylate groups that are able to crosslink with each other,thereby trapping the dECM bioink,making the dECM bioink mechanically stronger in the process [110].

Fig.2.Processes and typical applications of dECM-based bioprinting.a)The manufacturing challenges of dECM-based bioprinting range from obtaining a consistent organ source to the issues in the final applications [106].b) Native tissues and dECM from cartilage,heart and adipose [78].c)Native tissues and dECM from liver[108] and skin [109].

For in vivo clinical applications of dECM-based bioprinting,challenges also include the prevention of transmission of infections and diseases.There is also a need to avoid an immune reaction to the presence of the allogeneic or xenogeneic dECM.Besides,the constituents of dECM vary depending on the source of tissues and organs.The inconsistent batch-to-batch reproducibility is another challenge that cannot be ignored.A typical example is Matrigel™which is a commercially available dECM cell culture matrix [111].It is extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma,a tumor rich in such ECM proteins as laminin,collagen IV,heparan sulfate proteoglycans and a number of growth factors.As a gelatinous ECM protein mixture,Matrigel™recapitulates the features of natural ECMs and is widely used as a substrate for cell culture.However,Matrigel™is not quantitatively and even qualitatively defined,and displays significant variability between batches.For safety and ethical reasons,it is very unlikely that such a material derivation from murine sarcoma cell lines could one day be suitable for clinical applications.Therefore,to make dECM a viable product for 3D bioprinting,further studies are needed.

4.Multi-material 3D bioprinting

Single material-based 3D bioprinting usually leads to the limited variation in the physico-chemical properties of printed objects and other concerns for medical applications.To address this issue,there is a rapid increasing interest in the modification and diversification of generic printing materials via blending other materials with unique properties(such as combining a bioink with a polymeric scaffold),or by the inclusion of fillers and additives with distinct properties (such as nanoparticles,nanoplatelets and fibers) to generate 3D printable composite with high performance,including optimal mechanical properties,desired biocompatibility,improved biomimicking of tissues structures,and good fidelity [112–116].In this part,we present the newest progress in the development of 3D multi-material printing technology and printable composite/hybrid materials,as well as their potential use in the field of tissue engineering and other biomedical applications.

Fig.3.Printing of composite scaffolds through multi-head printing which containing a plastic support ink(a[117–119],b[78,120–122]and c[79,123]),and which containing at least two non-plastic (bio)inks (d [124,125] and e [126–128]).

4.1.Multi-material printing technology

Modern multi-material 3D printers can fabricate blended composite materials procedurally or simultaneously with precise control of material composition and properties through a multi-head printer,and from which objects with various and gradient functionality are created(Fig.3).

Blended composite scaffolds with multi-layered structure mostly composed of one plastic support ink and another non-plastic bioink(Fig.3a–c).The support ink of these scaffolds is commonly made from polycaprolactone (PCL),polylactic acid (PLA) or copolymers of them printed by Fused Deposition Modeling (FDM) or regular extrusion,coupled with heating at glass transition or melting temperature.This plastic is the main ingredient in the 3D printable composites,which enables the structure integrity of the composite materials and provides consistency during the printing process [116].It acts as a mold surrounding the bioink to prevent its overflow into the same layer(Fig.3a and b),or as a rigid individual layer to separate the two bioink layers(Fig.3c).This procedure has been widely proved to be useful as bone or cartilage grafts where PCL or PLA layers are combined with alginate,agarose,gelatin or Matrigel™bioinks[119,121,122,129–131].They are also used to manufacture robust cell culture platforms [132].For example,Shim et al.successfully carried out the 3D bioprinting of a multilayered and cell-rich cytocompatible composite material with a multihead deposition system using PCL and chondrocyte cell-encapsulated alginate hydrogel in different layers,which resulted in outstanding reconstruction of mice cartilage (Fig.4) [121].The printing process of cell-encapsulated alginate hydrogels showed negligible effects on the viability of the chondrocytes.The system simultaneously facilitated scaffold construction and deposited controllable amounts of cells and growth factors with spatial positioning to form well-defined pre-tissue constructs.Histochemical analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in printed PCL-alginate hybrid scaffold without an adverse tissue response.This 3D printing method for the formation of multilayered constructs may have extensive applications in tissue engineering.

Heterogeneous layer-by-layer printing is another way of achieving the desired properties of composites with multi-layered successive alternating structure,containing at least two different non-plastic bioinks(Fig.3d and e).Each bioink can encapsulate specific cells or network precursors,such as cross-linkers,photo-initiators or ions.For example,Li et al.reported a robust alginate/methylcellulose (Alg/MC) blend hydrogel for 3D bioprinting for the first time,with a strategy to improve adhesion between printed layers(Fig.5)[125].The optimized Alg/MC blend hydrogel exhibited a highly thixotropic property,great extrudability and stackability.With treatment by a trisodium citrate (TSC)solution,the interfacial bonding between the printed layers was significantly improved.The TSC solution acted as a chelating agent to remove the superficial calcium ions at each layer.Post-crosslinking in a CaCl2bath after 3D printing further enhanced the adhesion strength between the layers.The Alg/MC hydrogel with the aid of TSC demonstrated superior printability,high stackability (150 layers could be printed) and high shape fidelity.A good cell viability of ＞95% was obtained for a freshly 3D bioprinted Alg/MC construct.

Fig.4.3D bioprinting of a multi-layered PCL and alginate hydrogel[121].a)Schematics of the fabrication process of cell-printed 3D PCL–alginate gel hybrid scaffold using a multi-head deposition system.Photo-images of:b)fabricated porous 3D PCL scaffold,c)chondrocyte-printed 3D PCL–alginate gel hybrid scaffold for in vivo experiments,and d) simplified 2D hybrid scaffold for in vitro experiments.

It is noteworthy that in heterogeneous layer-by-layer printing,one of the printing materials can be used as a temporary sacrificial template that will be simply removed after printing without causing the collapse of structure (Fig.3e).A bio-blood-vessel (BBV) has been developed using a coaxial cell printing technique via the extrusion of a pluronic(CPF-127) (inside) and a hybrid bioink composed of vascular-tissuederived dECM (VdECM) and alginate (outside) (Fig.6) [128].This printable composite material not only offered a desired environment to promote cell proliferation,differentiation and neovascularization,but also enables a direct fabrication of tubular BBVs.Moreover,by tuning the printing parameters,the BBVs can be fabricated in varied desired dimensions.

Although multi-material 3D printing combines different properties into one project and provides indispensable strength and functionality for functional products,however,the interfacial performance between multiple materials with vastly different properties would limit the structural reliability of the composites.Abundant reactive groups on the polymer chains make it possible to achieve strong bonding between multiple materials interfaces and solve the compatibility problems.Wirthl et al.developed a class of adhesives that can undergo instant and tough bonding between hydrogels and antagonistic materials [133].Tough hydrogels strongly attach,within seconds,to plastics,elastomers,leather,bone,and metals,reaching unprecedented interfacial toughness exceeding 2000 J/m2.Soft and transparent multi-layered hybrids of elastomers and ionic hydrogels endure biaxial strain with more than 2000% increase in area,facilitating soft transducers,generators,and adaptive lenses.These adhesives are applicable in rapid prototyping and in delicate environments that can print multi-material structures with strong interfacial adhesion.

4.2.Composite/hybrid materials for 3D biopringting

As abovementioned,3D biopringting of multiple materials is commonly achieved by switchable nozzles or core–shell/coaxial printing to integrate materials into one project with precise distribution in different layers or regions,enriching the functionality of printing system.In this case,each printer head contains a single material and the printing materials are isolated before printing.Another type of multimaterial printing is achieved by the introduction of appropriate doping agents into generic printable materials and/or pre-blending of other bioactive components before printing,endowing variable physico-chemical and biological characteristics to the resultant composites.The difference between the two approaches of multi-material printing is the compositional path of materials:the former method utilizes multi-head printer to combine materials and the pure materials are isolated before printing,while the latter combines and hybridizes materials ahead of printing,in which usually a single-head printer is utilized.

Fig.5.Schematic illustration of the extrusion-based bioprinting process with the Alg/MC hydrogel and cells-TSC solution[125].The construct is built layer-by-layer,wherein each layer is formed by extruding the Alg/MC hydrogel from syringe 1,followed by extruding a cells-TSC solution from syringe 2.The construct is post-crosslinked in a CaCl2 solution before culturing at 37 °C in a cell culture media.

Fig.6.Tissue engineered bio-blood-vessels (BBV) constructed using a tissue-specific bioink and 3D coaxial cell printing technique [128].a) A hybrid bioink was prepared by mixing VdECM and sodium alginate,which was used to encapsulate the atorvastatin/PLGA microspheres and endothelial progenitor cells.b) A 3D coaxial printing technique was applied to construct the cell/drug-laden BBV.c) Schematic depiction of the BBV fabrication process.The ionic gelation of alginate realized BBV printing (i),the thermal crosslinking of collagen fibers was induced by incubation at 37 °C (ii),medium immersion dissolved and removed CPF127 to obtain BBV with a hollow tubular shape (iii).

Fig.7.Tough,elastic,and highly printable NICE bioinks [136].a)The NICE bioinks use nanosilicates to reinforce an ionic-covalent entanglement hydrogel made from GelMA and κCA,creating a dually reinforced hydrogel network.b) The NICE bioinks print mechanical stiffand elastomeric hydrogel structures with a high aspect-ratios and high print fidelity(scale bar=1 mm).c)The 3D printed structures from NICE bioink are mechanically(film)and physiologically(bifurcated vessel)stable and have high structural fidelity (3D printed ear).

Hybrid constructs can be obtained by doping nano-objects into the generic printable materials.For example,specific additives such as nanoparticles and fibers were included to give toughness and improve mechanical strength of the composite material[134–136].As shown in Fig.7,Gaharwar et al.presented an enhanced nano-engineered ioniccovalent entanglement (NICE) bioink for the fabrication of advanced cell-laden 3D composite with mechanical stiffness,high elasticity and superior printability [136].The NICE bioinks use nanosilicates to reinforce an ionic-covalent entanglement (ICE) hydrogel made from bioactive GelMA with kappa-carrageenan (κCA).The NICE bioink can be used to print freestanding,high aspect-ratio structures over 3 cm and 150 layers tall with excellent shape fidelity,and the structures become tough and elastic after cross-linking,which can support more than 50-times their own weight.Meanwhile,cells within the 3D-printed structure maintained high viability and proliferating throughout the 120-day period,which is vital for long-term tissue regeneration.NICE bioinks can be used to bioprint complex,large-scale,cell-laden constructs for tissue engineering with high structural fidelity and mechanical stiffness for applications in custom bioprinted scaffolds and tissue engineered implants.

Gold nanorods (GNR) were added to give electrical properties and to improve cell signaling between cardiac cells [137].As shown in Fig.8,a GNR-incorporated GelMA-based bioink is developed for printing 3D functional cardiac tissue constructs.The GNR concentration is adjusted to create a proper microenvironment for the spreading and organization of cardiac cells.At optimized concentrations of GNR,the nanocomposite bioink has a low viscosity,similar to pristine inks,which allows for the easy integration of cells at high densities.In the printed GNR constructs,cardiac cells show improved cell adhesion and organization when compared to the constructs without GNRs.Furthermore,the incorporated GNRs bridge the electrically resistant pore walls of polymers,improve the cell-to-cell coupling and promote synchronized contraction of the bioprinted constructs.Additionally,micro carriers can also be introduced into the matrix to achieve the local delivery of drugs and bioactive agents,as depicted in Fig.6 [128].

Apart from nano-objects abovementioned,it is noted that addition of appropriate chemicals or bioactive molecules into the printed subjects can tune their properties as well [138].In these cases,the composite can be easily fabricated by physical blending the modifiers with bioinks.For example,growth factors can be included to the matrix material via simple mixing to induce cell differentiation and enhance biological properties of the bioink [79,121].Vascular endothelial growth factor (VEGF) is an angiogenic cytokine and it relates to the proliferation and differentiation of endothelial cells.Studies have demonstrated the VEGF encapsulated in the heart tissue derived dECM(hdECM) bioinks played an important role in promoting strong vascularization and tissue matrix formation in vivo (Fig.9).The patterned patch also exhibited enhanced cardiac functions,including reduced cardiac remodeling and fibrosis,along with improvements in cardiomyogenesis and neovascularization post myocardial infarction.

Fig.8.A gold nanorod (GNR)-incorporated GelMA-based bioink for 3D bioprinting of functional cardiac tissue constructs [137].a) Schematic of coating GelMA molecules on commercial cetyltrimethylammonium bromide(CTAB)-coated GNRs(C-GNRs).b)Schematic of preparation for G-GNR/GelMA prepolymer solution.c)Schematic of bioprinting process using the G-GNR nanocomposite bioink.The inset showed the bioprinted 30-layered construct.d) G-GNR nanocomposite bioinkprinted constructs expressed higher levels of gap junction protein Cx-43 on day 14 than pristine GelMA/alginate bioink-printed ones.

On the other hand,the hydrogel bioink can also be modified covalently by grafting bioactive compounds.By chemical conjugation of drugs,RGD peptide ligands and growth factors,the bioink is able to deliver and release fragile therapeutic compounds in a spatiotemporal manner[48,119,122,128,139–144].Studies carried out in this area will undoubtedly cross-fertilize the field of bioprinting.

As aforementioned,all the trials to construct bioprintable composites with the inclusion of appropriate nano additives and bioactive molecules have made it possible to design various bioinks with improved performances,including mechanical and biological properties,defining printability and biocompatibility of the bioink.Whereas,it is noteworthy that particle fillers can be used in almost all 3D bioprinting technologies due to their uniform shape and good dispersibility [20].On the contrary,fiber fillers are not proper for many printing methods,such as selective laser sintering,vat photopolymerization and inkjet printing.It is difficult to make a smooth layer of powder-fiber mixture for selective laser sintering.The high aspect ratio structure of fiber fillers would affect the transparency of bioinks in vat photopolymerization and make the printer easily jam in inkjet printing,respectively.However,the shear and extensional flow field in the nozzle during extrusion can cause the fiber fillers’ orientation,because of which,damage-tolerant composites with anisotropic structures and programmable mechanics can be easily constructed without adding any other assistant materials can be fabricated [145].

5.Stimuli-responsive materials for bioprinting

Despite these remarkable advances in the replication of ECM structure for tissue and organ regeneration,general 3D bioprinted constructs might be insufficient to adapt the dynamic physiological environment due to their static nature after printing.Natural human tissues play their unique and complex functions by constant undergoing physiological dynamic changes introduced by external stimuli.Therefore,it is essential to build structures mimicking the physiological behaviors of natural human tissues under the varying external stimuli.The emergence of using stimuli-responsive materials that can transform the shape or function of printed 3D constructs over time in 3D bioprinting technology has provided a solution to this issue.This novel technique is now interpreted as ‘4D bioprinting’ because of the successful integration of ‘time’ as the fourth dimension within the existed 3D bioprinting [70,146].

Advanced 4D bioprinted constructs are fabricated by employing similar procedures as 3D bioprinting technology.The used materials capable of reshaping their structure in response to external stimuli(such as temperature,water,pH,ionic concentrations,light,electric and magnetic field) offer an opportunity of the continuing evolution and maturation of bioprinted living cellular constructs in a programmed manner [147].

By far,many pioneering works have demonstrated the successful incorporation of stimuli-responsive materials in printing process.Transformation of 4D bioprinted constructs has also been achieved following the programmed route [70,146,147].However,4D bioprinting technology in mimicking the complex native tissues remains challenging due to the limited choices and insufficient functionalities of materials to some extent[70,148,149].To meet the requirements of 4D bioprinting,it is prior to develop stimuli-responsive materials with good printability,excellent biocompatibility,gentle responsiveness,and proper mechanical supporting ability [147].Therefore,several categories of stimuli-responsive materials with great application potential in 4D bioprinting are introduced in the following section,along with their practical biomedical applications.

Fig.9.Stem cell-laden dECM bioinks encapsulated with VEGF for 3D bioprinting of complex tissue construct[79].a)Illustration of pre-vascularized stem cell patch including multiple cell-laden bioinks and supporting PCL polymer.b) Schematics of the experimental groups.c) Histological analysis and immunofluorescence staining results of the experimental groups four weeks after implantation(Scale bar 200 mm;BV:blood vessel).d)Quantitative analysis of blood vessel diameter and number of blood vessels in the confined area four weeks after implantation.

5.1.Temperature-responsive materials

Temperature-responsive materials can achieve shape transformation under external temperature stimulation,which are probably the most widely used materials in 4D bioprinting to date due to the obvious difference between 3D constructs fabrication and cell culture environment.Typical temperature-responsive materials used in 4D bioprinting process include shape memory polymers (SMPs) and temperatureresponsive hydrogels [147].

Fig.10.4D programmable culture substrate for neural cell differentiation modulation [151].a) Fabrication of micro-well arrays and the time-dependent transformation.b) Illustration of cell patterning process.c) Optical images and 3D surface plots of 4D substrate.d) Time-dependent differentiation behavior of NSCs.

Temperature-responsive SMPs possess intrinsic shape recovery capability,which enables the shape transformation and recovery under the stimulation of temperature.Miao et al.developed a smart and biocompatible scaffold based on soybean oil epoxidized acrylate,which can be fixed with a temporary shape at -18 °C and recovered to its original shape at 37 °C [150].High attachment and proliferation of multipotent human bone marrow mesenchymal stem cells (hMSCs) on the printed scaffold demonstrated its great potential for 4D bioprintingbased tissue engineering.The same group has recently developed a 4D programmable culture substrate using temperature-responsive SMPs(Fig.10).Results in this work have shown great potential in enhancing dynamic growth of neural stem cells (NSCs) and neural differentiation along with axonal alignment(Fig.10d).A major shortcoming is that the solid structure of temperature-responsive SMPs makes it impossible to incorporate cells in them:cells can only be attach on the scaffold after printing process.

On the other hand,temperature-responsive hydrogels with switchable swelling property might be preferable to 4D bioprinting because of the high water content in bioprinted constructs,which makes them a better accommodation for living cells.Hydrogels based on the classical poly (N-isopropylacrylamide) (PNIPAM) have been used in 4D bioprinting due to its proper low critical solution temperature (LCST).Although PNIPAM hydrogels offer an opportunity for bioprinting,the lack of biodegradability,bioactivity and lowered biocompatibility upon LCST could be limitations for their development.More promising temperature-responsive hydrogels are urgently needed for 4D bioprinting [152,153].

5.2.Humidity-responsive materials

Humidity-responsive materials can be swollen or shrunken with the variation of surrounding humidity,resulting the change of their size,shape and structure.Humidity,in other word,water provides a facile and completely safe condition for the transformation of these materials,as well as a potential application for the living cell constructs[147,154].Kirillova et al.printed a photoinitiator-loaded methacrylated alginate or hyaluronic acid solution with or without cells onto the surface of glass or polystyrene substrate [68].After photoinduced crosslinking following slight drying,the crosslinking density of top and bottom printed layers varied,resulting an immediate self-folding after being immersed in aqueous solution(Fig.11a).In addition,adding and removal of Ca2+allows the reversible shape transformation,leading to unfolding and refolding of printed constructs,respectively (Fig.11b).This approach provides a facile way for creation of cell-laden hollow tubes with the internal diameters reaching to 20 μm,which may make a difference in biofabrication of small blood vessels such as venules or capillaries.In another remarkable work,Gladman et al.printed biomimetic composite hydrogel architectures with rigid cellulose fibrils embedded in a soft acrylamide matrix [67].The printed hydrogel architectures perfectly mimic the composition of native plant cell walls and can change shape once immersed in water induced by the alignment of rigid cellulose fibrils (Fig.11c).The authors designed and showed a successful deformation process of calla lily flower and orchid Dendrobium helix (Fig.11d),demonstrating the great biomimicking ability of the bioprinted composite hydrogels architectures.Despite a lack of study on living cell-laden architectures in this work,it provides a new pathway for tissue engineering and biomedical device fabrication.

Fig.11.4D bioprinting of humidity-responsive materials-based architectures [67].a) 4D biofabrication of cell-laden self-folding hydrogel tubes.b) Ca2+ ionsresponsiveness of methacrylated alginate hydrogel tube.c)4D printing of biomimetic complex flower morphologies based on composite hydrogel.d) Predictive 4D printing and shape transformation of biomimetic architectures inspired by calla lily flower and orchid Dendrobium helix.

5.3.Magnetic-responsive materials

Magnetic field is widely used in biomedical applications.Commonly,magnetic-responsive materials are fabricated by the incorporation of magnetic nanoparticles,such as Fe,Fe2O3,Fe3O4,and NdFeB [155–158].Some of them have already been used for bioprinting.For example,streptavidin-coated iron nanoparticles are embedded in bioinks composed of agarose and type I collagen[155].Using this strategy,Betsch et al.aligned collagen fibers by applying a straightforward magnetic field during the bioprinting process.The study on chondrocyte-loaded hydrogel constructs demonstrated a markedly increased expression of cartilage specific ECM components such as collagen type II.Kim et al.developed a direct writing ink by mixing uncured elastomer matrix with NdFeB and silica nanoparticles[156].With the applying a magnetic field during the extrusion of ink,the authors showed a complex shape control and the derived diverse functions of printed soft robot.It suggested a highly promising method for 4D bioprinting in the application of flexible electronics,biomedical devices and soft robotics.

5.4.Photo-responsive materials

Light is also a common-used strategy in 4D bioprinting due to its great space-time controllability with high resolution.Photo-responsive materials are capable of altering their polarity and conformation activated by a broad range of light from ultraviolet (UV) to infrared (IR)bands,resulting in shape and structure transformation for bioprinted constructs.Among them,near infrared(NIR)and IR light are preferable because of the relatively lower cell toxicity and higher penetration.According to polymer types,the underlying mechanisms differ,mainly including photothermal effect and photodegradation.

Adding biocompatible photothermal particle in polymer matrix,such as graphene,carbon nanotube and polydopamine nanoparticles can be helpful to fabrication of photo-responsive materials and 4D bioprinted constructs [159–161].The materials include temperatureresponsive property because the transformation effect of the constructs is in fact induced by produced heat when exposed to radiation.Besides,photo-responsive materials with photodegradation property are usually made by decoration of photodegradable moieties (such as coumarin or o-nitrobenzyl ether groups) on polymeric chains [162–164].The photodegradation-induced deformation can be modulated by the number of photodegradable moieties and dose of radiation.Thus,4D bioinks based on these materials are capable of fabricating cell culture matrices,further mimicking dynamic degradation nature of ECM.Arakawa et al.developed biomimetic hierarchical vascular networks in a cell-laden photodegradable hydrogel [165].The results presented in this work showed a delicate creation of microchannel networks mimicking native vasculature by using multi-photon lithography.Therefore,this photodegradable material-based strategy exhibits great potential in fabrication of complex multi-cellular tissues with programmed vascular networks.

5.5.Outlook of stimuli-responsive bioprinting materials

Although great efforts have been made in this area,stimuli-responsive materials suit for 4D bioprinting remain limited.Materials in many researches undergo transformation in harsh condition,which may end up severe cell damage.The proliferation and differentiation potential of cells in these printed constructs requires further evaluation.Thus,biocompatible materials with gentle responsiveness are still the prerequisite of 4D bioprinting,in which living cells are involved especially.Besides,current studies on 4D bioprinting of multi-responsive architectures remain rare.Due to the complexity and variability of environment in vivo,the 4D bioprinting architectures aim to not only reproduce the dynamic growth process of native tissues,but also adapt themselves to the multiple biological responses during implantation and culture process.Therefore,the stimulation parameters should be diverse and safe to the living cells and surrounding tissues.Materials capable of being responsive to multiple stimuli are required to fabricate 4D printed constructs with shape transformation and self-maturation abilities.

6.Perspective and challenges

For 3D bioprinting of tissues and organs,the materials play multiple functions such as protecting,supporting and maintaining the activity of cells,promoting cellular proliferation and differentiation,guiding tissue regeneration and promoting functional maturity.Since human tissues and organs are extremely complex both structurally and functionally,it is impossible for the traditional materials or any single component material to have all the characteristics needed to reconstruct the tissue functions.The development of novel materials that support high precision and rapid prototyping,possess specific biological functions,and have better compatibility with printers and printing software has become an inevitable trend.

It is believed that an important development direction is to deepen the understanding of natural ECM in the future.By simulating the complex components and gradient distribution of ECM,bioactive materials with specific functions can be developed.At the same time,3D bioprinting technology should be used to precisely regulate the presentation of functional materials to achieve replication of specific ECM and construct ECM-like microenvironment in vitro,creating favorable conditions for the survival,proliferation,differentiation and function maintenance of cells.Another important research direction based on biomimicry is the development of functional adaptive materials,that is,materials can spontaneously reprogram their shapes,properties or functions according to external stimuli.For example,in the early stage of printing,the material can not only play a role of protection and support,but also enable cells to produce and deposit ECM,and spontaneously construct a microenvironment suitable for the growth of cells.With the continuous proliferation of cells,the material should be degraded to give space for the growth of cells,and the degradation products should have no side effects.

Besides,the development of advanced printers and printing processes also cannot be ignored.Kang et al.developed an integrated tissue-organ printer (ITOP) system [166].Based on gelatin,fibrinogen,hyaluronic acid and glycerol,they prepared different bioinks for printing of bone,cartilage and skeletal muscle by regulating the ratio of each components.Meanwhile,the mandible,cartilage(ear)and skeletal muscle were successfully constructed with Pluronic F-127 as outer layer sacrifice and PCL as mechanical reinforcement material.Liu et al.reported a bionic printing technology of multi-cellular/material system to construct artificial tissues and organs in vitro [65].By using the highly automatic multi-material printer,rapid printing of seven different kinds of bioinks was achieved,and different organoids such as brain,lung,heart,kidney,pancreas,stomach,small intestine,large intestine,bladder and prostate other organs were constructed.Meanwhile,owing to its inherent advantages such as using the body as a bioreactor and eliminating the risks associated with manual implantation of pre-fabricated constructs,new attempts are consistently being made to advance the in situ bioprinting technology [19,167–169].For example,Albanna et al.developed a mobile skin bioprinting system that provides rapid on-site management of extensive wounds [168].The in situ printed cell (dermal fibroblasts and epidermal keratinocyte)-laden hydrogel (fibrin and collagen) showed rapid wound closure,reduced contraction and accelerated re-epithelialization.It can be assumed that with the development of more advanced biomaterials,higher resolution of bioprinting methods,high-end integrated printers with scanning,online monitoring and inspection systems,the in situ bioprinting showed great potential in the construction of tissues directly on the intended anatomical location of the living body.

The 3D bioprinting also faces other challenges common to tissue engineering and regenerative medicine.For example,the issues of internal vascularization in the bioprinted constructs.Some studies have demonstrated the feasibility of constructing vascular networks in organs (Fig.12) [127,170–175].One of the challenges in printing vascular network is the compatibility of materials and cells with the printer and printing processes.In addition,it may take longer for vascular networks to develop and mature throughout the tissue structure than for cells to survive.Bioreactor can help maintain the activity of tissue builders and buy time for tissue fusion,remodeling,and maturation in post-processing.In addition,the natural vascular network is a nonuniform network in size that changes gradually from large to small.One possible solution in the future is to use self-sacrificing materials to build a network of interconnected large-diameter blood vessels in the process of 3D printing.After implantation,the medium-and small-diameter blood vessels will be constructed by the spontaneous growth of the host vascular network around the implants,and the capillaries will be developed by inducing the differentiation of stem cells in the printed implants.

7.Concluding remarks

In this review,we briefly introduced the requirements for ideal 3D bioprinting materials and summarized the recent advances of ECMbased materials,multi-material and stimuli-responsive materials for bioprinting.Meanwhile,the challenges and future perspectives of materials for 3D bioprinting were addressed.In the future,the research focuses will include:development of novel 3D bioprinting materials with excellent biocompatibility,better printabilities and appropriate mechanical properties;development of heterogeneous and gradient composite materials;development of biomaterials that are suitable for in situ bioprinting;improvement of the biological characteristics of materials by means of biomimetics or recombination with bioactive factors;improvement of the survival and functions maintenance of print cells and tissues or organs by designing and controlling the degradation performance and vascularization-promoting abilities of printing materials;establishment the standards of 3D bioprinting materials and the production process specifications,etc.We believe that with the expansion of printing materials and the improvement of their performance,3D bioprinting will eventually bring about revolutionary changes in the application of health care.

Fig.12.3D bioprinting of vascular structures.a)Schematic of the co-bioprinting of vasculature,cells,and ECM,and the heterogeneous vascularized tissue constructs by adopting Pluronic F-127 as the sacrificial template [170].b) Schematic and confocal microscope images of the bioprinted thick vascularized tissue constructs[127].c) Schematic and photographs of the bioprinted GelMA hydrogel with microchannels via the micromolding of agarose template microfibers [171].d)Hierarchically branched tubular networks [172],microvascular network fabricated via omnidirectional printing of a fugitive ink [173],stereolithographic printed hydrogels with entangled vascular networks [174],and coaxially printed branched vasculature [175].

Declaration of competing interest

No conflict of interest exits in the submission of this manuscript,and manuscript is approved by all authors for publication.

Acknowledgements

This work was supported by the Key R&D Plan of Jiangsu Province(BE2018010-3),the National Natural Science Foundation of China(31800803 and 31771067),and the Scientific Research Foundation for Talent Introduction of Nanjing Tech University (39803129 and 39803130).