Composite Inks for Extrusion Printing of Biological and Biomedical Constructs

Abstract

Extrusion-based three-dimensional (3D) printing is an emerging technology for the fabrication of complex structures with various biological and biomedical applications. The method is based on the layer-by-layer construction of the product using a printable ink. The material used as the ink should possess proper rheological properties and desirable performances. Composite materials, which are extensively used in 3D printing applications, can improve the printability and offer superior performances for the printed constructs. Herein, we review composite inks with a focus on composite hydrogels. The properties of different additives including fibers and nanoparticles are discussed. The performances of various composite inks in biological and biomedical systems are delineated through analyzing the synergistic effects between the composite ink components. Different applications, including tissue engineering, tissue model engineering, soft robotics, and four-dimensional printing, are selected to demonstrate how 3D-printable composite inks are exploited to achieve various desired functionality. The review finally presents an outlook of future perspectives on the design of composite inks.

Graphical Abstract

1. Introduction

Three-dimensional (3D) printing has attracted tremendous attention due to its high versatility and functionality. This technology rapidly found its place in numerous biological and biomedical applications, including but not limited to, inert implants and tissue-engineered implants,^1–4 microfluidic devices and bioelectronics,^5–7 organoid models and organ-on-a-chip platforms,^8,9 and drug-screening systems.¹⁰ A typical 3D printing instrument uses a printable material, termed the “ink”, to fabricate prescribed constructs in a layer-by-layer fashion. The incorporation of cells in the ink creates what is often called a “bioink,” although this term is sometimes used for acellular materials for biomedical applications. The fabrication of cell-laden constructs is often designated as “bioprinting”. Single-phase inks have been widely used for various applications in bioprinting and general printing. However, their uses have been long-bottlenecked by the limited choice of materials, low printability, insufficient mechanical performances, poor biomimicry, and lack of spatiotemporal controls.¹¹ The escalating need for printable materials with specific performances has spurred recent efforts on designing functional 3D printing inks that are functional over a broader range of applications.

Composite inks offer increased versatility to attain the desired functions. By incorporating at least one type of insoluble additives, such as fibers, nanotubes, or nanoparticles, into a single-phase matrix, composite inks leverage the advantages of each individual component to achieve printed structures with improved properties.¹² The fibrous structure of several human native tissues, from soft tissues such as vocal fold lamina propria¹³ to hard tissues such as musculoskeletal system,^14,15 motivates the use of composite inks in the field of tissue engineering. Composite inks enable four-dimensional (4D) printing via the addition of controllable shape-changing features.^16–18 Broader mechanical, electrical, and physicochemical tunability can be achieved through the use of composite inks.^19,20 For example, the addition of nanoclays²¹ can improve the printability of acrylamide and agarose inks via enhancing their thixotropy. The incorporation of nanotubes in composite hydrogel networks can provide additional focal adhesion sites²² and promote cell adhesion and migration within bioprinted scaffolds.

Rapid advancements in the field of composite inks are apparent through the significant increase in the number of publications in the field (Figure 1). In the present review, state-of-the-art composite inks for extrusion-based printing are discussed with a focus on biological and biomedical applications. Chemical and biological properties, including surface-functionalization, chemical composition, electrostatic charge, and biological activity of different composites are described and compared. The advantages and limitations of different composite ink additives are reviewed. The performances of composite inks are also evaluated by elucidating how they may enhance the properties of printed structures. Finally, a perspective view of the latest progress and related applications of composite inks in biological and biomedical systems is presented. Figure 2 shows a graphical summary of the present review.

Figure 1-.

Increase in the number of publications in the field of (a) 3D printing and bioprinting, and (b) composite inks with different applications. The data are extracted from Scopus^®.

Figure 2-.

Main applications and performance targets of composite inks for extrusion (bio)printing.

2. Concept of composite inks

Composite materials are mixtures of two or more materials of different phases with significantly different properties.¹² Based on this definition, a single-phase mixture of non-fibrous precursors is not categorized as a composite ink, although it could be a multicomponent ink.²³ As such, monomer solutions, such as collagen or fibrin solutions, are not considered as composite inks. If these materials are mixed with a base ink to form a fibrous multi-phase material prior to printing, the resulting ink can be categorized as a composite ink. Emulsions, which are composed of insoluble droplets of a liquid dispersed in another liquid, are, however, considered composite materials as they form multi-phase materials.²⁴ In comparison with single-phase inks, composite systems ideally have an improved/extended range of properties. Matrigel, a commercial composite hydrogel rich in fibrous proteins such as collagens and laminin, is one of the well-known bioinks that is used in combination with other printable materials to achieve enhanced properties.^25,26 For example, Matrigel-agarose hydrogel can provide both proper fidelity and an excellent microenvironment for cell growth²⁷ due to the existence of agarose and fibrous proteins, such as collagen and laminin.

3. Additives used in composite inks

Depending on the matrix and the additive, desirable enhancements in mechanical stability and printability^28,29 may be achieved. Different types of fibers, nanotubes, or nanoparticles can be incorporated in soft hydrogel systems to fabricate composite hydrogels (Table 1). In this section, the most common additives used in fabricating composite inks are reviewed, with a focus on composite hydrogels.

Table 1-.

Characteristics of different additives used in the fabrication of composite inks.

Additive	Source	Structure	Diameter	Young’s Modulus	Cytotoxicity	Biodegradability	Conductivity	Main advantage	Refs.
Collagen	Natural	Fiber	30~300 nm	1~2 GPa (Dry condition)	No	Enzymatic, controllable	No	Abundant cell-binding ligands	31,206
Fibrin	Natural	Fiber	40~250 nm	1~28 MPa	No	Enzymatic, controllable	No	High extensibility (>330%)	43,45,207
HAp	Natural/ Synthetic	Particle-/ Needle-shaped	0.1~70 μm	40~150 GPa	No	Degradable by osteoclasts	No	Bone inducibility	208–212
CNT	Synthetic	Nanotube	1~2 nm (SWCNT), 5~100 nm (MWCNT)	As high as 1.8 TPa	Low (concentrations <1 mg mL−1)	Possibly slow enzymatic degradation by macrophages	Yes (armchair type)	High stiffness, antimicrobial	19,213,214
CNC	Natural	Nanocrystal	2~30 nm	20~50 GPa	Low	Non-biodegradable in the human body	No	Tunable surface chemistry	86,215–217
CNF	Natural	Fiber	10~100 nm	20~50 GPa	Low (concentrations <0.1 mg mL−1)	Non-biodegradable in the human body	No	Shear-thinning properties	86,215,216,218–220
Nanoclay	Natural/ Synthetic	Nanoparticle	50~200 nm	-	No	-	Yes	Electrostatic charge	102,221

3.1. Collagen

Collagen is the most abundant protein in the body. Over 30 different types of collagens can be distinguished, many of which comprising a long fibrous structure.³⁰ Collagen fibers are made of aggregations of fibrils, which have a triple-helix structure consisting of two alpha-1 and one alpha-2 polypeptide chains.³¹ The most observable amino acid sequences in collagen are glycine-proline-X and glycine-X-hydroxyproline, where X can be any other type of amino acid.³² Glycine, which has only one carboxylic group and one amine group without any side chain, is the main amino acid available in the chemical composition of collagen. Although collagen fibrils possess a larger stiffness than many other fibrous proteins (1~2 GPa),³³ their mechanical properties deteriorate at temperatures above 40 $°$C.³⁴ Collagen is the main protein in the extracellular matrix (ECM). It is a biocompatible and biofunctional material, containing numerous cell-adhesion ligands.³⁵ Owing to these properties, collagen fibers have been extensively used in various composite hydrogels to enhance their intrinsic properties.^36,37 For example, collagen/Pluronic composite hydrogels have Young’s modulus that is 3 to 9 times greater than that of the pure hydrogel.³⁸ Based on the rule of mixture, this enhancement is due to the presence of the stiff collagen fibers in the matrix. It is also reported that cell proliferation and adhesion in agarose/collagen composites are significantly higher than in the corresponding single-phase hydrogel.³⁹ Rapid biodegradation is detrimental to tissue-engineering applications of collagen. A vapor-phase titanium-infiltration method has recently been reported to prolong the biodegradation of collagen fibers.⁴⁰

3.2. Fibrin

Fibrin, known as a natural fibrous coagulation protein, plays an important role in blood-clotting. It is obtained from the enzymatic transformation of fibrinogen. The physical and mechanical properties of the fibers, such as their density and Young’s modulus, highly depend on diameter.⁴¹ The fibrin fiber diameter varies within the approximate range from 40 to 250 nm. Chloride ions can modulate the structure of fibrin fibers by inhibiting the formation of thicker fibers.⁴² Thin fibrin fibers have a much greater Young’s modulus than thick fibers, as the composition of protofibrils in the cross-section of thin fibers is denser. As a result, thick fibrin fibers are easier to be dissolved⁴³ and are more suitable for preparing composite inks. A unique characteristic of fibrin fiber is its outstanding extensibility, over 330%,⁴⁴ which is attributed to the fact that the fiber becomes stiffer as mechanical strain is increased.^45,46 The cytocompatibility and tunability of fibrin make it suitable for biological functions, ^47,48 for example to promote cell adhesion^45,49 or to induce angiogenesis.⁵⁰

3.3. Carbon nanotubes (CNTs)

Discovered in 1991 by Ijima,⁵¹ CNTs are known as one of the stiffest materials available, with Young’s modulus of 1.8 TPa.⁵² These nanomaterials are hollow tubes with high specific surface areas, solely composed of carbon atoms. Depending on the number of concentric tubes, CNTs are classified as single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). The SWCNTs have diameters of approximately 1~2 nm, while MWCNTs have a larger diameter of 5~100 nm.⁵³ Both types of CNTs have been broadly used for reinforcing composite inks.^54–56 For tissue-engineering applications, MWCNTs are more common as their dimensions are similar to those of fibrous proteins in the ECM.^22,57 Unlike MWCNTs, SWCNTs easily penetrate the cell membrane and, therefore, are often used for drug-delivery.^58,59 In another classification, CNTs are divided into three major groups based on the orientation of the hexagonal arrangement of the sidewall; armchair, zigzag, and chiral.⁶⁰ The electrical properties of armchair CNTs are similar to those of metals, while zigzag and chiral CNTs behave as semiconductors.⁶⁰ These electrical characteristics have led to CNT usage for the fabrication of conductive 3D-printing inks.^61–63

Pristine CNTs are hydrophobic and aggregate into rope-shaped structures by means of weak Van der Waals forces. The sidewall of the CNTs consists of sp² carbons that can be hybridized by means of reactive double bonds. Hence, CNTs are very potent to be functionalized, either chemically or physically, with other moieties in a stable manner.⁶⁴ Functionalization with hydrophilic groups can increase the dispersibility of CNTs in aqueous media,⁶⁵ which is essential in preparing CNT-based composite inks. Surface-functionalization may remarkably enhance the electrical conductance of these nanomaterials.⁶⁶ The biocompatibility of CNTs has been controversial for several years, and there has been no consensus on it.^67,68 However, recent studies report that the functionalized MWCNTs in concentrations lower than 750 μg mL⁻¹ do not cause a significant reduction in cell viability.¹⁹ Since CNT is a synthetic material, its biodegradation is of high importance, especially in tissue-engineering applications. It has been reported that the CNTs can be degraded by macrophages,^69,70 as well as different enzymes and chemical compounds, such as horseradish peroxidase^71,72 and sodium hypochlorite.⁷³

3.4. Cellulose

Cellulose is the most abundant renewable organic polymer, which can be naturally produced or chemically synthesized. It is made of glucose units that form straight chains through hydrogen bonds.⁷⁴ The chemical composition of cellulose is (C₆H₁₀O₅)_n, where n denotes the degree of polymerization and therefore, implies the number of glucose units. In contrast to pristine CNT (contact angle=150~160$°$),⁷⁵ cellulose is considered a hydrophilic material with a contact angle of 20~30$°$.⁷⁶ The most popular derivative of cellulose for the fabrication of composite hydrogels is the cellulose nanocrystal (CNC). It is extracted by hydrolyzing the amorphous region of cellulose. These tubular structures have a diameter in the range of 2~20 nm.⁷⁷ The method of extraction leaves a sulfate half-ester group on CNC’s surface⁷⁸ and makes it potent for surface-functionalization. As a result, functionalized CNCs are commonly utilized for enhancing cell-material interactions in composite hydrogels.⁷⁹ The hydrogen bonds within the chemical structure of CNCs increase their mechanical properties, including their tensile strength and their stiffness (Young’s modulus of 167.5 GPa).⁸⁰ Consequently, CNCs are commonly used to mechanically reinforce hydrogels.^81–83 It has been reported that CNCs can also enhance the printability of alginate sulfate hydrogels.⁸⁴ Unique features of CNC-loaded composite hydrogels include temperature- and pH-dependent swelling,^85,86 which is helpful in fabricating stimuli-responsive composite inks. Among the additives described in this section, cellulose-based fibers, including CNCs and cellulose nanofibrils (CNFs), appear to be the most promising fiber additive for multiple functions in 3D-printing applications.^87,88

3.5. Nanoparticles (NPs)

Composite inks made of NPs can work synergistically to obtain unique characteristics that are not achievable using single-phase inks. Depending on material requirements, various types of NPs can be incorporated. The following section explicates different types of NP-added composite inks and their properties.

3.5.1. Metal NPs

Physical and chemical properties of metal NPs, including size distribution, particle morphology, surface chemistry, surface coating, and cytotoxicity are critical to their interactions with the composite matrix. Depending on the target properties, metal and metal oxide nanoparticles, such as silver, gold, iron, manganese, and titanium can be added to hydrogels to prepare inks for specific targeted properties.^89–92 In general, previous studies have demonstrated that metal NPs with larger diameters are less cytotoxic.⁹³ In addition, positively charged NPs show dose-dependent hemolytic activity and cytotoxicity, while their negative surface charge yields higher cytocompatibility⁹⁴. The shape and structure of NPs (nanospheres, nanorods, nanocubes, nanodisks, etc.) are important factors that affect their intrinsic properties.^95,96

3.5.2. Non-Metal NPs

Mineral nanoparticles such as hydroxyapatite (HAp, Ca₁₀(PO₄)₆(OH)₂) and nanoclays are examples of non-metal nanomaterials. As the main inorganic component of the bone, HAp has a morphology, composition, crystal form, and crystallinity similar to those of native bone minerals. They, therefore, exhibit good osteoinductivity and osteoconductivity.⁹⁷ Nanoclays are the building blocks of clay minerals. Depending on their chemical composition and structures, nanoclays can be categorized into one of several types, including montmorillonite, bentonite, kaolinite, saponite, and hectorite.⁹⁸ One of the most widely used types of nanoclays is the synthetic hectorite Laponite [Na^+0.7(Si₈Mg_5.5Li_0.3)O₂₀(OH)₄)^−0.7]. Laponite consists of disk-shaped particles of approximately 25 nm in diameter and 1 nm in thickness.⁹⁹ The disk surface possesses a permanent negative charge due to isomorphic substitutions in the crystal structure. Its edge possesses a pH-dependent charge from unsatisfied valences in the disrupted crystal lattice.⁹⁸ The addition of Laponite to water forms a strong thixotropic fluid. It can increase the viscosity of polymer solutions to meet requirements for direct ink-writing.^100–102 Nanoclays have also been found to possess hemostatic properties through the absorption and activation of blood coagulation factor XII and plasma proteins.^103,104 Due to their excellent biocompatibility and degradability, nanoclays have been explored as vehicles for drug-delivery.^105,106 Promotion of cell adhesion and proliferation constitutes another benefit of nanoclays’ incorporation for regenerative medicine.^107,108

4. Performances of composite inks

The performances of composite inks can be significantly improved compared to single-phase inks by leveraging the unique properties of additives. In this section, we will brief on how the composites improve the printability of the inks and the mechanical behaviors of the printed structures. The enhancement of biological and biomedical performance will be discussed. Some distinct benefits enabled by the composites will also be presented. All the composite bioinks that we have discussed throughout this review along with their effects on the performances of their base bioinks are briefed in Table 2.

Table 2-.

Summary of the composite inks with their principal performances reviewed in the present article.

Base ink	Composite ink (base-additive)	Additive concentration	Target performance	Other improvements	Ref.
Gelatin	Alginate/gelatin-CNCs	3.76 wt%	Printability	Cell interaction	79
	Gelatin-Laponite XLG	6% (w/v)	Printability	-	101
	Gelatin-Laponite XLG and RD	6% (w/v)	Self-supporting ability	Mechanical properties	102
	Gelatin-Laponite XLG	Up to 1.2 wt%	Rheological properties	Biological properties/drug-delivery	222
	Gelatin-gas	-	Pore size and porosity		141
GelMA	GelMA-CNTs	0.3% (w/v)	Flexible conductivity	-	62
	GelMA-AuNRs	Up to 0.5 mg mL−1	Conductivity	Cell adhesion, cell-to-cell coupling	91
	GelMA-strontium NPs	0.5–5 mg mL−1	Printability	Osteogenic capacity	222
	GelMA-Laponite XLG	0.5–1.2 wt%	Printability	Porosity	223
	GelMA-PEO	Up to 1.6% (w/v)	Pore size and porosity	-	138
	GelMA-HAp	50 wt%	Stiffness	Osteogenic capacity	170
	GelMA-PLGA	-	Stiffness	-	178
Alginate	Alginate-CNFs	0.5– 1.5 wt%	Printability	Mechanical Properties	81
	Alginate-CNFs	20 wt%	Stiffness	Mechanical Properties	2
	Alginate sulfate-CNCs	1.36 wt%	Printability	Cell spreading	84
	Alginate-Laponite XLG and RD	6% (w/v)	Self-supporting ability	Mechanical properties	102
	Alginate-PEGDA-Laponite XLG	5 wt%	Printability	Mechanical properties	125
	Alginate-collagen	15 mg mL−1	Stiffness	Mechanical properties	158
	Alginate-gelatin microparticles	-	Drug-release control	Osteogenic properties	177
	Alginate-Pluronic	13%	Porosity	Printability	136
Agarose	Agarose-collagen	0–0.3% (w/v)	Anisotropy	Biological properties	152
	Agarose-Matrigel	-	Printability and cell alignment	-	27
Matrigel	Matrigel-collagen	0.8 mg mL−1	Cell alignment	-	25
	Matrigel-melt electrowriting-based PCL	-	3D electrophysiology	-	26
PCL	PCL-CNTs	Up to 5% (w/v)	Mechanical and chemical properties	-	57
	PCL-CNTs	5 mg mL−1	Mechanical properties	Bioactuation	183
	PCL-PPSu-AgNPs	5% (wt%)	Anti-microbial properties	Enhanced degradation	89
	PCL-HAp	Up to 20 wt%	Mechanical properties	Increased degradation	108
Other polymers	Polylactic acid (PLA)-CNTs	-	Mechanical properties	Unaffected cell viability	54
	Polybutylene terephthalate (PBT)-CNTs	Up to 4% (w/v)	Electrical properties	Mechanical properties	63
	Epoxy diacrylate-CNTs	-	Impact resistance in meniscus implants	-	56
	PVA-cellulose nanowhiskers (CNWs)	1–7 wt%	Controlling pore morphology	Increased thermal stability and mechanical properties	83
	Polyacrylamide (PAM)/hydroxypropyl methylcellulose (HPMC)-AgNP	Up to 2 wt%	Anti-microbial properties	Increased water-uptake, increased wound-closure	90
	Gellan gum-Laponite	0.5–1% (w/v)	Printability	Localized drug delivery	224
	NIPAM-Laponite XLG	6% (w/v)	Printability	-	29
	Hyaluronic acid methacrylate-AgNPs	-	Electrical properties	-	146
	PEGDA-Laponite XLG and RD	6% (w/v)	Self-supporting properties	Mechanical properties	102
	PEGDMA-HAp	2% w/v	Osteogenesis	-	156
	MeHA-Ga-AgNPs	-	Conductivity	-	146
	N,N-dimethylacrylamide- (Nanoclays and CNFs)	-	Mechanical properties	-	17
	Silicone rubber-magnetizable microparticles	-	Mechanical properties	-	187

4.1. Improvement in printability

Extrusion 3D printing accommodates the printing of a wide range of materials.¹⁰⁹ The major physicochemical parameters determining the printability of ink are its rheological properties and crosslinking mechanisms.¹¹⁰ An ideal ink should have a sufficient viscosity and high yield stress post-printing and before crosslinking.¹¹¹ Under most scenarios, it is preferred to be shear-thinning to facilitate the extrusion process¹¹² (Figure 3a–b). Tuning rheological properties are especially critical but challenging for tasks when live cells are encapsulated within the bioinks: high yield stress and viscosity benefit the printing fidelity but generate large shear stresses in the printing nozzle and impede cell viability. For bioprinting, low-viscosity bioinks are usually preferred.¹¹⁰ Many novel setups have been developed to enable the printing of low-viscosity bioinks employing the strategy of crosslinking at the printing nozzle during a printing task. Those developments include an in situ UV-curing system in conjunction with transparent printing nozzles,¹¹³ diffusion-based coaxial nozzles,¹¹⁴ and microfluidics-based extruders with micromixer at the nozzle exit.¹¹⁵ While those recent advancements in 3D printing hardware also provide solutions to print low viscous inks, they usually require additional accessories and may not be readily accessible.

Figure 3-.

(a) Typical 3D printing setup for direct writing of composite inks. Adapted with permission from ref ²⁹. Copyright 2018 American Chemical Society. (b) Important rheological indicators for good printability, including shear-shinning, yield strength, and thixotropy. (c) Nanomposite inks enable the direct printing of high-aspect-ratio tubes with different inclination angles. Reproduced with permission from ref ¹⁰². Copyright 2017 American Chemical Society. (d) Printed lattice, ear, and nose structures of PEGDA-alginate-Laponite composite hydrogels with high fidelity. Reproduced with permission from ref ¹²⁵. Copyright 2015 Wiley-VCH Verlag.

Various composites have been employed to provide suitable printability for low-viscosity inks through different strategies. One strategy is to introduce additives of a solid phase into the inks to improve the printability of the target inks. CNFs as a biocompatible and low-cost material is shear-thinning inside an aqueous environment. Due to the entangled network inside the suspension, it can form a stable physical hydrogel at as low as 2 wt% of concentration before and after extrusion printing.¹¹⁶ It has been demonstrated that CNFs can significantly improve the post-printing fidelity of alginate hydrogels while maintaining the high viability of embedded cells.¹¹⁷ Furthermore, by varying the concentrations of the added composite contents, the rheological behaviors of the composite inks can be tailored to suit different applications. For example, addition of Pluronic F127 micelles into gelatin methacrylate (GelMA) solutions equipped the composite bioinks to be directly printable at physiological temperature.¹¹⁸ The viscosity is also tunable through varying the micelles to GelMA ratio.

Another strategy to improve the printability of low-viscosity inks relies on the interactions between the composite materials and the ink matrices. When composites are added to the low-viscosity inks, they can interact with the polymeric ink matrices by forming reversible bonding, such as dynamic covalent bonds, hydrogen bonds, electrostatic interactions, and Van der Waals interactions.^82,119,120 Those reversible bonds can weakly crosslink the polymeric inks and provide desirable rheological properties for the printing. During extrusion, the weakly crosslinked composite inks can flow through the printing nozzle by rupturing the bonds. The bonds can rapidly reform after the extrusion and mechanically support the printed structures. For example, silica nanoparticles can bond with polymer chains to form a hydrogel network.¹²¹ The formed hydrogel is shear-thinning and self-healing. Strong adhesion can also be achieved between the printed layers. This strategy usually ends with a secondary crosslinking, such as irreversible covalent crosslinking, to permanently stabilize the printed structures.^122,123

One model system using this strategy to improve ink printability is by incorporating nanoclays to form composite inks. Laponite, a member of the smectite mineral family, has been widely used in the bioprinting field to enable the direct extrusion of hydrogel composites in the air.^29,124 The unique anisotropic electrostatic charges allow the laponites to form anionic, cationic, and physical interactions with various types of polymers. When they are blended with a hydrogel-precursor, not only can they form ionic bonds and hydrogen bonds with the hydrogel polymers, but also can bond with each other and form a “house-of-cards” arrangement.¹⁰² The interactions with polymers and themselves can be easily broken by applying shear forces and reform when stresses are relaxed. Such properties enabled their omnipotent ability to improve the printability of various polymer inks and bioinks, such as alginate,¹²⁵ GelMA,¹²⁶ poly(ethylene glycol) diacrylate (PEGDA),¹⁰² and N-isopropylacrylamide (NIPAm),¹²⁷ among others (Figure 3c–d). For example, the addition of Laponite improved the rheological properties of PEGDA-alginate inks and enabled the direct extrusion of double-network hydrogels.¹²⁵

4.2. Improvement in mechanical properties

Besides printability, another important reason for using composite inks is to obtain properties and performances that are not achievable with single-phase materials. At the beginning of bioprinting development, the focus was emphasizing on matching the stiffness of the bioprinted scaffolds with targeted tissues.⁶ However, stiffness alone does not represent how real biological tissues behave. Soft tissues are viscoelastic, porous, tough, and highly stretchable. The intrinsic toughness of most single-phase polymer scaffolds is at least one or two orders of magnitude lower than that of tissues, which significantly limits their applications in load-bearing activities or mechanically active jobs.^128,129 Through synergizing the interactions between additives and polymers inside the composite inks, the mechanical properties of the printed structures can be significantly improved to be more biomimetic.

Enhancement in mechanical performances can be realized through the arrangement of materials inside the printed composite structures. A common arrangement is a programmed distribution of a stiff and a soft element. In terms of the elastic behavior, the resulting composite would have stiffness in between those of the two elements due to the rule of mixtures. However, the elongation and toughness can be greatly improved by designing the interfaces between the two elements. For example, a printed composite network structure consisting of interconnected segments of poly(glycerol sebacate) acrylate (PGSA) with different mechanical stiffness was created.¹³⁰ The composite network was demonstrated to sustain 50% more strain before failure compared to mesh networks composed of single PGSA segments. The energy absorption rate was also improved by 100% through the rupturing of the softer segments during straining to effectively dissipate the energy. 3D-printed poly(N-acryloyl glycinamide) (PNAGA)-clay composite hydrogels also showed significantly improved toughness and stretchability compared to those of the single-network PNAGA hydrogels. The composite structures can withstand more than 550% of tensile strain and 90% compressive strain without rupture due to their dissipative sacrificial network (Figure 4a).¹³¹

Figure 4-.

(a) Photos showing printed PNAGA-clay scaffolds and their ability to resist large compressive strain. Reproduced with permission from ref ¹³¹. Copyright 2017 American Chemical Society. (b) Printed biomimetic microporous structures using bioinks containing two aqueous emulsion composites (GelMA and PEO). The size of micropores between 1–100 µm was created (i-iv). The growths of endothelial cells and fibroblasts in hydrogels printed using single-phase bioinks (v-vi) were significantly lower than those of the porous hydrogels printed using composite bioinks (vii-viii). Reproduced with permission from ref ¹³⁸. Copyright 2018 Wiley-VCH Verlag. (c) Schematic showing aligned glass fibers embedded within the extruded elastomeric matrix. (d) Anisotropic elastic modulus and coefficient of thermal expansion of printed filaments enabled by the extrusion of composite inks allow precise control of extrinsic curvature after a temperature change. Reproduced with permission from ref ¹⁵³. Copyright 2019 National Academy of Sciences.

4.3. Structural enhancement

The structural resemblance is also important to ensure proper biological functions. Studies have shown that pore sizes of 5–20 µm are desirable for fibroblast and hepatocyte ingrowth, 20–120 µm for wound healing, and >90 µm for vascularization.^132,133 However, the intrinsic pore sizes for most hydrogels are in the nanometer-scale while the bioprintable pore size (defined by the spacing between extruded filaments) is usually greater than 100 µm.^134,135 Composite bioinks have been explored to bridge this gap. One type of composite bioink yielding micropores is termed the micelle-laden template porous ink, where Pluronic block copolymers of poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) (F-127) are used as biocompatible fugitive spacers within a bioink.¹³⁶ Pluronic F127 as a non-ionic surfactant reduces the critical micelle concentration and increases its volume fraction with an elevation of temperature. It undergoes phase-transitions and self-assemble to micelles, resulting in micellar crystallization and formation of a self-supporting gel phase.^136,137 When it is blended with bioink, the micelles inside the printed scaffolds can be flushed away by lowering the temperature and thereby leaving empty spaces as micropores (~6–8 µm). Another type of porous composite ink exploits the benefit of liquid-liquid emulsion which overcomes the pore size limitation. Instead of using micelles, this type of ink uses an emulsion containing two immiscible aqueous polymers emulsions. GelMA and poly(ethylene oxide) (PEO) solution blend can form emulsion bioinks induced by phase separation. After the bioprinting, the GelMA phase can be crosslinked through free radical polymerization while the PEO phase is unaffected. The uncrosslinked aqueous PEO phase can then be washed away in the cell culture medium and leave interconnected pores (25–53 µm) inside the GelMA scaffolds (Figure 4b).^138–140 Although the current configuration has a limited printing time window for approximately 30 minutes due to the stability of the emulsion, it shows the novel printing modes enabled by composite inks and their potentials to improve the performance of existing 3D-printing technologies. Gas-liquid composite inks have also been explored as a pore-forming 3D-printing technique. A study utilized a microfluidic microdroplet-generator to generate air bubbles, instead of conventional liquid droplets, within a gelatin liquid ink.¹⁴¹ By changing the air pressure and orifice design of the microdroplet-generator, pore size could be varied within 80–800 µm. The dynamically adjustable air pressure also enabled the fabrication of layered and smoothly graded gradient porous structures to mimic the structural complexity of bones. It is worth-noting that although the printed final constructs are not necessarily composites (such as when the F-127 micelles are leached out leaving on the porous hydrogel constructs), the inks used contained immiscible phases and are considered to be composite inks.

4.4. Biological resemblance

For tissue-engineering applications, the printed scaffolds need to have the biological resemblance of the tissues as well. Naturally derived fibers, such as collagen, fibrin, and decellularized ECM (dECM), have been employed with various bioinks to provide cell-binding ligands and tissue-specific microenvironments to obtain better biological performances.^142–144 Graphene nanocomposite inks have been demonstrated to improve the conductivity of the printed scaffolds for the regeneration of neural tissues.¹⁴⁵ Silver nanoparticles (AgNPs) have been explored to improve the conductivity of the bioinks for 3D bioprinting of cardiac tissue constructs.¹⁴⁶ Similarly, by mixing gold nanorods (AuNRs) into GelMA bioinks, conductive functional heart tissues were bioprinted.⁹¹ Nanocomposite inks have also shown great support for cell viability, proliferation, and differentiation.¹⁴⁷ For example, the addition of dECM with pre-existing fibrous contents to the basic bioink matrices constitutes composite bioinks. Such composite bioinks provide tissue-specific growth and differentiation factors that can improve and regulate cellular functions of cells.¹⁴⁸ Other additives, such as nanoclay, can slowly release bioactive substances, such as magnesium and silicon ions, to promote differentiation and osteogenesis of osteoblasts.¹⁴⁹ In addition, some composite bioinks, such as AgNP-incorporated bioinks, have shown desirable resistance to bacterial growth and have the potential to be used as open wound-dressings.⁹⁰

4.5. Anisotropy

Composite inks can also enable the creation of structures with mechanical and physical anisotropies, which are difficult to access using single-phase inks. Many biological tissues are anisotropic mainly due to the highly orientated collagen fibers to maximize the load-bearing abilities.¹⁵⁰ Collagen orientation and anisotropy also have great influences on cell proliferation and migration.¹⁵¹ When collagen fibers are incorporated inside bioinks, bioprinting can align the collagen fibers either with shear forces when passing through the printing nozzle or with a magnetic field, assisted by iron NPs during the printing. An agarose-collagen-iron NP composite bioink has been used to study the effect of collagen alignment on cartilage tissue engineering.¹⁵² Human knee articular chondrocytes were demonstrated to secrete markedly more collagen II and aggrecan contents in bioprinted (aligned collagen) constructs compared to cast (random collagen) samples. The compressive moduli were also strengthened with the collagen alignment. Anisotropy in elastic moduli, swelling ratio, and thermal expansion can bring advanced features such as shape-morphing. Recent progress exploited elastomeric inks with anisotropic fillers and enabled precise control of elastic modulus and coefficient of thermal expansion of the printed filaments. Due to the anisotropic thermal expansion differences, the printed planar structures can be programmed to form different out-of-plane shapes with temperature change (Figure 4c–d).¹⁵³

5. Applications of composite inks

5.1. Tissue engineering

Three-dimensional bioprinting shows great application prospects in tissue engineering due to its capability of shape-control, decent resolution, and high accuracy.¹⁵⁴ Composite bioinks combine the advantages of each component and potentially improve the performances of the components in terms of mechanical strength, printability, gelation kinetics, and bioactivity. Composite bioinks can more accurately simulate the strength and elasticity of tissues, to achieve the purpose of precise tissue repair.¹⁵⁵

5.1.1. Bone and cartilage

3D printing of scaffolds can yield an accurate repair for bone and cartilage.^2,14,156 HAp can be compounded with other materials such as hydrogels to prepare composite bioinks. Besides the chemical properties of HAp, which is similar to those of the bone tissue, HAp can also promote the vitality and proliferation of bone cells by increasing the surface characteristics and roughness of the bioink, which is beneficial to repair of the bone defects.¹⁵⁷ In a previous study, human mesenchymal stem cells (hMSCs) were bioprinted in poly(ethylene glycol)-dimethacrylate (PEGDMA) hydrogels encapsulated with nanoparticles of bioactive glass (BG) or HAp.¹⁵⁶ It was reported that HAp was more effective compared to BG for hMSCs osteogenesis in bioprinted bone constructs.

Various types of composite bioinks have been utilized for cartilage-regeneration.^2,84,117,158 Researchers used a CNF-enabled composite bioink co-printed with human induced pluripotent stem cells (hiPSCs) to fabricate cartilage tissue constructs.² Two bioinks were investigated in this study: CNFs with alginate (CNF/alginate) and CNFs with hyaluronic acid (CNF/hyaluronic acid). The authors reported that the CNF/alginate bioink was suitable for bioprinting of hiPSCs to support cartilage-generation in co-culture with chondrocytes.² In another study, collagen type I and agarose were separately used to mix with sodium alginate to serve as the bioinks.¹⁵⁸ The results of this research indicated that the alginate/collagen composite bioink effectively constructed in vitro 3D-printed cartilage tissues.

5.1.2. Skin

Skin is the largest organ of the human body, with a complex multi-layer structure, and serves as an immune barrier to protect the underlying muscles, bones, ligaments, and internal organs.¹⁵⁹ Three-dimensional bioprinting of the skin is currently a trending research area. As such, different types of bioprinting methods and bioinks are being further studied.^159–162 Collagen, gelatin, and chitosan are the most widely used natural polymer materials as the bioinks for bioprinting the skin.¹⁵⁹ In addition, there are also many bioinks made of polymer materials to 3D-bioprint the skin structure.^89,163 In a recent study, AgNPs were combined with polycaprolactone-block-poly(1,3-propylene succinate)‎ (PCL-PPSu) to make the antibacterial bioink for bioprinting the skin. AgNPs can reduce the adhesion of microorganisms to the constructs, without compromising the cell activity.⁸⁹

5.1.3. Cardiac tissue

The cardiac system is essential for the survival of organs and tissues.^164,165 The conductivity of the heart muscle is the key for the heart to be able to beat rhythmically.¹⁶⁶ In a recent study, an AuNR-incorporated GelMA-based bioink was developed to fabricate conductive cardiac tissues (Figure 5a).⁹¹ Immunostaining showed the sarcomeric α-actinin and gap junction protein connexin 43 (Cx-43) expressions increased in the AuNR/GelMA hydrogel when compared to the pristine GelMA hydrogel (Figure 5b). In another study, Shin et al. developed a conductive bioink with granular microgels.¹⁴⁶ The microgels of HA modified with both methacrylate and gallol groups (MeHA-Ga) were fabricated through a water-in-oil emulsion and subsequently underwent in situ metal-reduction with AgNPs to provide electrical conductivity (Figure 5c). The granular hydrogels had a higher surface area than traditional hydrogels and therefore possessed higher electrical conductivity (Figure 5d).

Figure 5-.

Cardiac tissue engineering using composite bioinks. (a) Schematic of preparation for AuNR/GelMA prepolymer solution with AuNRs; (b) Expression of F-actin, sarcomeric α-actinin, Cx-43 increase in AuNR/GelMA hydrogel group. Reproduced with permission from ref ⁹¹. Copyright 2017 Wiley-VCH Verlag. (c) Schematic of conductive granular hydrogels with microgels jamming through vacuum filtration; (d) i) bulk hydrogels with AgNPs, ii) granular hydrogels without AgNPs, iii) granular hydrogels with AgNPs pre-embedded during microgel fabrication (“pre-emb”), or iv) granular hydrogels with AgNPs through the “in situ” process. The brown line describes the proposed electron. Reproduced with permission from ref ¹⁴⁶. Copyright 2019 Wiley-VCH Verlag.

5.2. Cancer modeling

Three-dimensionally bioprinted tissue models can accurately emulate the tissue pathophysiological microenvironments, thus providing in vitro platforms for cancer research.^167–169 Composite hydrogels can both emulate the tumor microenvironments and regulate cell migration in in vitro models.^170–172 Using composite hydrogels for 3D printing breast tumor models could promote the understanding of the underlying mechanism in bone metastasis of breast cancer cells.^172,173 Several studies have combined HAp with PEGDA, GelMA, and poly(ethylene glycol) (PEG) to fabricate breast cancer-bone metastasis models.^170–173 Cui et al. developed a breast cancer metastasis model with a composite bioink containing HAp, PEGDA, and GelMA, which composed of bone matrix, vascular channel, and breast cancer cells.¹⁷⁰ This model took control of spatial distribution to study the pathophysiology of breast cancer metastasis and improved the understanding of the interaction between osteoblasts, blood vessels, and tumors.

5.3. Drug-delivery

To control drug-delivery, various types of nanoparticles, such as ceramic nanoparticles, metal nanoparticles, and carbon-based nanomaterials combined with polymer networks to form composite hydrogels with controlled drug release.^58,174–176 In a previous study, bone morphogenetic protein 2 (BMP-2) was loaded on gelatin microparticles (GMPs) dispersed in the bioink supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study the bone-formation.¹⁷⁷ The results of immunohistochemistry showed that osteocalcin expression was higher in the BMP-2 sustained-release group. Similarly, in another study, transforming growth factor-beta 1 (TGF-β1)-embedded poly(lactic-co-glycolic acid) (PLGA) nanospheres were loaded into the GelMA and PEGDA bioink to fabricate engineering cartilage.¹⁷⁸ Nanospheres could effectively prolong the release of TGF-β1 and induce the differentiation of MSC into chondrocytes.

5.4. Other biomedical applications

5.4.1. 4D printing

Biological organs and tissues often change shapes over time or with different functions. In recent years, 4D printing has attracted great attention because it enables specific changes in the shape or the physical and/or chemical characteristics of 3D-printed structures over time or under different stimuli.¹⁷⁹ As an example of a conductive bioink with tunable stiffness, toughness, and extensibility, sulfonate-modified silica nanoparticles (SiO₂-SO₃-Na⁺) have been used in combination with hydrogel to prepare a composite hydrogel system (Figure 6a).¹⁸⁰ The sulfonate groups on the nanoparticles interacted with the quaternary ammonium groups (RN (CH₃)₃⁺Cl⁻) on the polymer network and enhanced the material dynamic and reversible properties.¹⁸⁰ Hydrogel composite inks composed of stiff CNFs embedded in a soft N-isopropylacrylamide (NIPAM) matrix, which was inspired by botanical systems, have been also utilized (Figure 6b).¹⁷ The structures 3D-printed using the above composite inks could be controlled by increasing the temperature to produce morphological changes. Agarose nanofibers and polyacrylamide have been used to fabricated a composite hydrogel.²¹ The experimental results demonstrated that this composite hydrogel could construct a whale, which was able to open the mouth and cock the tail by cooling treatment.²¹

Figure 6-.

Biomedical applications of composite bioinks. (a) Highly elastic, transparent, and conductive composite hydrogels allowed (i) resilience on a printed Eiffel tower (ii) in which the repeatable extensive deformation (iii) was recovered. Multi-armed gripper, (iv) as designed, and printed to rapidly swell from initially flat and open to curved and enclosed within 10 min. Reproduced with permission from ref ¹⁸⁰. Copyright 2017 Wiley-VCH Verlag. (b) Predictive 4D printing of biomimetic architectures as a native calla lily flower (scale bars, 5 mm). Reproduced with permission from ref ¹⁷. Copyright 2016 Nature Publishing Group. (c) A deformable 3D printed structure was prepared by mixing magnetizable nanoparticles and elastomer ink. The researchers also printed a hexagonal structure that could wrap the pill and use a rolling motion to carry the pill under the rotating magnetic field generated by the permanent magnet. Reproduced with permission from ref ¹⁸⁷. Copyright 2018 Nature Publishing Group. (d) Electricity caused contraction of the 3D-printed stingray structure containing CNTs/GelMA seeded with cardiomyocytes. Reproduced with permission from ref ¹⁸¹. Copyright 2018 Wiley-VCH Verlag.

5.4.2. Actuators, soft robotics, and artificial muscles

Soft composite hydrogels with excellent cytocompatibility are widely used for the fabrication of bioactuators,^181–183 soft robots,¹⁸⁴ artificial muscles,¹⁸⁵ and smart sensors.¹⁰⁵ Currently, soft robots and artificial muscles that rely on the development of actuators can be designed for various applications.¹⁸⁶ Composite bioinks often combine multiple material characteristics to prepare materials with excellent mechanical properties and good biocompatibility. In a recent study, the researchers printed deformable structures by mixing magnetizable nanoparticles and elastomer inks.¹⁸⁷ They demonstrated the printing of a hexagonal structure that could wrap the pill and use a rolling motion to carry the pill under a rotating magnetic field (Figure 6c). The cell-mediated traction can promote the deformation of the hydrogel. The bioactuator functions using the traction of the cells through delicate designs. One study has integrated self-actuating cardiac muscles to flexible gold microelectrodes to create actuators with life-like movements (Figure 6d).^181–183 Skeletal muscle cells and cardiomyocytes are often used in bioactuators because of their ability of spontaneous contraction. For example, neonatal rat cardiomyocytes could be combined with CNT-incorporated GelMA hydrogels to produce functional cardiac patches.¹⁸³ These patches could form 3D biohybrid actuators that show controllable linear cyclic contraction/extension, pumping, and swimming actions.¹⁸³

Soft robots can deform responsively by actuation to mimic the complex movements of animals. Using the suitable composite bioinks with precisely defined configuration can significantly enhance the functions of soft robots.^184,188 Artificial muscle is a biomedical device that can contract, expand, or rotate under the action of external stimuli (such as electricity, pH, pressure, magnetic field, or temperature). Soft actuators allow artificial muscles to perform the actions required for their function.¹⁸⁵ Similarly, the composite hydrogel materials controlled by the electric field used to create soft robots can be used to produce artificial muscles.¹⁸⁹

6. Summary and Outlook

The extrusion 3D printing method can accommodate a wide range of composite inks due to its easy-to-use nature and excellent compatibility with inks of various viscosities.^190,191 The shear-induced alignment of certain composite additives also enabled new stimuli-responsiveness modes in the printed constructs, such as mechanical, physical, and electrical anisotropies.^17,152 Many additives tend to change the rheological properties of the basic ink matrices, which results in higher viscosity or yield stress values and therefore inducing higher shear stresses during fabrication. Such induction particularly impedes the results of biofabrication because excessive shear stress decreases cell viability.¹⁹² The size of additives or aggregations also tends to clot the printing needle, which may require nozzles with a larger size.¹³⁸ Therefore, the ratios of the additives to the inks and the additive sizes are extremely important to ensure smooth and trouble-free printing. Furthermore, the tradeoff between the printing speed and resolution needs to be balanced to fit the target applications for single-nozzle extrusion. The speed can be improved by utilizing multimaterial extrusion printers with rapid material switch ability while keeping the resolution the same.^193,194 Although most composite ink systems mentioned in this review are for extrusion printing, they can be generalized to other 3D-printing technologies depending on the ink viscosity, transparency, crosslinking mechanism, and gelation kinetics.

In terms of the printing materials themselves, tremendous achievements have been made in the past few years regarding the development of composite inks. With designer characteristics and flexibility, composite inks are showing great potentials for clinically repairing/replacing biological tissues. For example, a bioprinted functional full-scale composite ear combining thermoplastics and cell-laden hydrogels is concrete proof.¹⁹⁵ However, long-term stability should be emphasized when developing composite inks. Despite the tremendous efforts that have been made to improve the toughness of composite materials, few of them enhance the fatigue threshold. For example, the threshold of nature rubber and hydrogels is within 1–100 J m⁻², while their intrinsic toughness is over 1,000 J m⁻².^196,197 It is critically needed to improve the threshold of printed composite inks when repairing mechanically active tissues, such as tendon and cardiac tissues where cyclic loadings are experienced. A potential solution to improve the toughness and threshold of the printed composite structures is to use a reversible chemical crosslinking mechanism. Commonly used reversible bonds include dynamic covalent bonds, ionic bonds, hydrogen bonds, hydrophobic interactions, dipole-dipole interactions, and host-guest interactions. Some of these bonds can reform quickly upon rupture while others may require outside stimuli, such as elevated temperature, pH change, presence of enzymes, or light exposure.^198–200 Improving the fatigue threshold may be realized through the interactions between the matrices and the composite materials. A recent effort in using polyvinyl alcohol (PVA) with crystallinity achieved a fatigue threshold of over 1,000 J m⁻².¹⁹⁶ The coating of ordered nanocrystalline PVA over porcine cartilage also obtained a high interfacial threshold. The adhesion is robust with over 5,000 cycles of reciprocating sliding.²⁰¹ However, the current configuration is only demonstrated on limited composite materials. It is necessary to propose new strategies or generalize the existing ones to other material systems.

For tissue-engineering applications, composite bioinks can simulate tissue characteristics. For example, the bioink can simulate the antibacterial properties of the skin by adding AgNPs.⁸⁹ Adding AuNRs to the bioink to achieve a conductive scaffold for repairing muscle defects could be a perspective in extending the applications of composite bioinks.⁹¹ Composite bioinks can accurately simulate certain complicated properties of tissues, to achieve the purpose of precise tissue-repair.¹⁵⁵ However, at present, it is extremely difficult to reconstruct some highly heterogeneous tissues with complicated properties such as the neuronal tissues and osteochondral tissue. Therefore, the development of new composite bioinks and designing new scaffolds to emulate specific tissues are currently the main focus in the field.

The rapidly evolving 4D printing technology is driving the advanced design of stimuli-triggered soft robotics. Common strategies utilize the anisotropy or gradient material properties created by the precision positioning of composite inks to sense the external triggers.^11,202,203 Shape-changing hydrogels and hydrogel actuators are especially interesting for applications as artificial muscles and cell-manipulators. In spite of the sensitivity to various environmental changes, such as temperature, humidity, and light, the responses of most current 4D printing technologies are much slower compared to traditional robotics. The slow real-time responses of hydrogel actuators may be due to the slow water diffusion-induced hydrogel volume transition and the hysteresis behavior caused by reversible chemical bonds and intermolecular interactions.²⁰⁴ To drive the field forward, composite inks yielding hydrogels with fast responses to external stimuli need to be developed. Methods such as designing hydrogels without mechanical hysteresis may provide a solution to this challenge.^131,205 The limited sensing ability for each type of hydrogels also restricts the ability of 4D-printed devices. Developing multi-responsive and biocompatible triggers is also critical to ensuring the implementation of this technology in biological environments.

In recognition of the recent development in composite inks and their related applications for 3D printing, we have provided a review covering the commonly used composite materials, their performances, design principles, and latest advances in this field. We hope this review could provide insights and initiate new ideas towards the design and applications of composite inks. We also wish the design principle discussed in this review could minimize the trial-and-errors in the research path. Despite the rapid advances of 3D printing in the past decade, there are still many opportunities for composite inks remained to be explored, especially for composite bioinks and its related biomedical applications. We believe composite inks will open new arenas towards the design of next-generation regenerative medicines and biomedical devices with the collaborations of researchers from various fields.