Functional dyes in polymeric 3D printing: applications and perspectives

: Three-Dimensional Printing (3DP) is considered among the key-technologies for the next industrial revolution, with considerable effects on production processes, economy and society. In this context, the most relevant part of the market consists of polymeric 3D printing. The 3D printable liquids are composed of various components , among them dyes are usually underrated because they are introduced merely for aesthetical reasons or to enhance the objects’ resolution. In recent years, the capability of specific dyes to go beyond conventional use and to confer functional properties to 3D printed objects has become an emerging research area. Modifying elastic moduli upon light irradiation, inducing optical and emitting properties in the matrices or conferring temperature responsivity are just few examples of innovative stimuli-responsive materials that can be produced by combining well-designed dyes with the appropriate 3DP printed matrices. In this review, we discuss and critically analyze the most relevant recent results achieved in the use of smart dyes in the synthesis of stimuli responsive 3D printed polymers.


Introduction
Since the prehistoric era, colors have played an important role in the development of humankind. Humans learnt thousands of years ago how to modify the color of the materials by adding dyes and pigments for aesthetical purposes as well as for peculiar functions (e.g. camouflage, heat dissipation). Over the centuries, this knowledge has been adapted to various classes of materials including paintings, ceramics and textiles. Polymeric materials underwent similar processes since the XIX century and different approaches were developed for dispersing organic dyes and pigments in polymeric matrices. Recently the use of organic small chromophores in polymers has found new scopes, mainly addressed to transfer a plethora of features into the polymer libraries beyond the chromic tone. The preparation of materials capable to modulate color, mechanical, chemical, and optical properties under external stimuli are only a few examples of these appealing functional polymers. [1][2][3][4][5] Coumarins, azodyes, metal-organic complexes, spiropyran, and naphthalene diimides are some examples of the functional dye employed in these applications. [6][7][8][9][10] Conferring new functionalities to polymeric materials using dyes is particularly advantageous since a little amount of dyes could remarkably change the properties of the material while retaining the mechanical features of the matrix. Moreover, the possibility to variously decorate the chemical structures of the dyes allows insertion in the polymeric matrixes either by dispersion or by covalently link them to the polymeric backbones. In recent years, functional dyes have found a key-role also in the field of 3D printing. Recent improvements and innovations have encouraged the scientific and industrial research to enlarge the palette of available printable materials, towards new applications. 11,12 The first and more important use of dyes in 3D printing is for aesthetical purposes, and even today the main request of the market is related to the color/transparency of a component. 13,14 Additionally, in some 3D printing technology, dyes' role is intrinsically fundamental, determining printability and resolution. 15 On the other hand, fine-tuned chromophores are gaining an additional interest due to the possibility to translate their properties in the final 3D printed objects. This approach has led to a relatively unexplored research area, in which materials properties and 3D structuration are merged in new synergistic effects.
The scope of this review is to critically analyze the recent advancements in the use of dyes in polymeric 3D printing to create objects responsive to specific external stimuli such as light, pH, temperature, solvents. In the first part, the 3D printing techniques are described with a specific focus on the role of the dyes in each technology. Later, innovative uses of dyes in each 3D printing technique are discussed, highlighting the key role of those components. [16][17][18][19][20][21] Finally, a critical analysis is reported, to highlight the achieved results and future perspective.

Three-Dimensional Printing
The history of 3DP has begun in the 1980s, with the first patents of Stereoligraphic Apparatus (SLA), Fused Filament Fabrication (FFF) and Selective Laser Sintering (SLS). [22][23][24] The common first stage in all the 3DP techniques resides on the design of a Computer-Aided Design (CAD) file, a virtual model of the desired final product. The CAD file is then sectioned into 2D layers by dedicated software and sent to the printer, which will produce the part. 3DP allows the objects' construction by controlled adding of materials. Material saving, personalized production, on-demand fabrication, production of objects with architectures not achievable with other methods are some advantages among many of 3DP. Over the years, those technologies have increased their popularity due to the progressive printers' cost reduction, mainly by the cessation of many patents. In parallel, the development of the printers has led to increased printing speed and an improved resolution of the final objects, making these techniques attractive for many manufacturing fields. Metals, ceramics as well as biological materials can be 3D printed, but here we'll focus on polymeric 3D printing. [25][26][27][28][29] Polymers play a leading role in 3D printing market thanks to the possibility to modulate the final properties according to the expected applications. [30][31][32][33][34][35][36] 3DP has been investigated to overcome the limitation of conventional polymers production processes, becoming nowadays a consolidated alternative solution. The most common AM techniques are briefly described in the next paragraphs, with the related advantages and drawbacks. The description includes FFF, SLS, Direct Ink Writing (DIW), SLA, Digital Light Processing (DLP), and Two-Photon 3D printing (TPP) (Figure 1). Some of the most common materials employed in these techniques are also reported to facilitate the reading ( Figure 2). Fused Filament Fabrication FFF is the most common and widely used 3DP technique. It consists in a controlled deposition of melted or semi-melted polymers through a heated nozzle. An extruder is fed with a thin filament of thermoplastic polymer (usually of 1.75 mm or 3 mm), which deposits a layer of material according to the digital model. 37 At the end of a layer, the printing head lifts up on z-direction of one step, which corresponds to the slicing of the CAD file, and starts to print a new layer on the previous one. The possibility to print large dimension objects, the high user-friendly and the wide plethora of printable materials, make this technique the most diffused in the industry. The overall efficiency and quality of the printing process depends on many parameters that must be well-specified. 38,39 Among the many commercially available or synthetic thermoplastic polymers, acrylonitrile-butadiene-styrene copolymers (ABS), polyamides (PA), polycarbonate (PC) and polylactide (PLA) are the most used. 37 Fillers, like fibers or metallic particles, can be added to tune or confer specific properties to polymeric matrixes in various applications 40,41 Moreover, a further implementation resides in the usage of multiple printing nozzles which enables the printing of multi-material objects with different designs and structures. 42 FFF is a mature technology with few drawbacks, among which, the most important one is related to the limited resolution to about 50 μm. This hampers the printing of complex geometries (e.g. hanging parts) for which temporary supports, removed by chemical or mechanical treatments at the end, are needed during the printing process.

Selective Laser Sintering
In SLS a high-power laser is used to sinter metals, glasses, ceramics or polymer powders. The laser scans the powder bed in XY directions to sinter the desired points, following the CAD project. Once a layer is completed, the building stage lowers down by one thickness step and a new layer of powder is deposited and scanned by the laser. The process is repeated till the printed object is fabricated. 43 The most used polymers in SLS are polyamide 12 (PA12), polystyrene (PS) and polycarbonate (PC); nevertheless, the limited availability of materials is the main limitation in this technique. 44,45 The SLS processing temperature must be precisely controlled to obtain an optimal adhesion between layers and good resolution. In semi-crystalline polymers, the processing temperature must be fixed in between the melting and the crystallization values 44 Other key-factors to take into account are the powder capability to absorb the laser wavelength, the laser irradiation intensity, the melted material viscosity, the shape and size of the powder which should be preferentially spherical and in a range between 20 and 80 µm. 44 Moreover, the chamber must be kept in an inert atmosphere to prevent any oxidative process during printing. Differently from FFF, supports are not needed for hanging parts since the non-sintered powder acts as a support for top layers. Furthermore, un-sintered materials are also recovered and reused in the following processes. This makes SLS a widely diffused technique for industrial applications, especially for structural components.

Direct Ink Writing
The working principle of DIW is very similar to FFF with the only difference that this technique enables the printing of inks with high viscosity or eventually shear-thinning. 32 A syringe is fed under a pneumatical compression or a screw, extruding the material at a fine-controlled temperature, pressure and viscosity. This process allows the polymer to be deposited according to a CAD file on a building stage. 46  DIW is widely used in printing biological materials, using various materials such as polymers, ceramics and composites are suitable for DIW. Nevertheless, each material requires peculiar extrusion speed to obtain a homogenous and well-defined final product. 34

Photopolymerization techniques
In 1986 Charles Hull patented the first 3D printer based on photopolymerization. 22 Photopolymerization is a wellknown polymerization process induced by light irradiation, presenting several intriguing advantages (e.g. high rate and stereospecificity) which made it appealing for applications in coating industries, electronic and inks technology. The photopolymerization process takes place upon irradiation with light in the ultraviolet, visible or nearinfrared region of the spectra. [55][56][57][58][59] Photoinitiators absorb radiation and generate reactive species (e.g. radicals or cations) that react with the functional moieties present on the monomers, that propagate leading to the polymerization. The photocurable formulations, including 3D printable formulations, are usually liquid and are composed of several ingredients that will be introduced afterward according to 3D printing technologies. SLA, DLP and TPP are the three main techniques based on photopolymerization each one presenting advantages and drawbacks. The main difference in comparison to the above-discussed techniques resided on the fact that the photopolymerization-based techniques produce thermosets polymers (e.g. polymers with three dimensional crosslinked networks). Here we'll use the term vat polymerization (VP) techniques for all the photo-induced 3D printing technologies.

Stereolithographic apparatus
In the SLA approach/method a reservoir (or vat) is filled with a photocurable formulation and a UV laser (e.g. 355 nm) scans the surface, according to a CAD project, inducing the photopolymerization process and curing the monomers. 55 The XY plane resolution and the penetration depth along Z axis depend on many factors, including the instrumental parameters such as the exposure time, the scan speed and the intensity of the light source, as well as chemical variables as the presence of additives and fillers. 42 When a layer is built, the building platform is lowered in the vat, new liquid resin covers the cured layers and another slice can be polymerized in the same way. Once the process is done, a post-curing stage in a UV oven is often needed to complete the polymerization and confer enhanced mechanical properties to the final object. SLA allows printing materials with different properties by varying the photocurable formulation of the operating liquid resins. On the other hand, due to the instrument configuration, multimaterial printing is not possible on XY plane while it is possible to change materials along Z adjusting the photocurable formulation during the process. Furthermore, if compared with FFF, the printing rate is slower. Digital light processing DLP is an evolution of SLA where a projector (e.g. emission wavelength at 405 nm) equipped with a Digital Mirror Device (DMD) is located under the vat and projects the image of each cross-section into the liquid resin. The projection occurs through the transparent bottom of the vat and follows the CAD project. DLP presents some advantages compared to SLA in terms of shorter production times due to the simultaneous polymerization of an entire layer, usage of a lower volume of resin per produced object, lack of restriction in height of the printed parts, minimal cleaning steps of the final products and high resolution (30-50 µm). 42 Similarly to SLA, a post-curing step with UV light is needed to complete the polymerization of all the reactive groups.
Moreover, an LCD screen can be used instead of a DMD device to assemble 3D printers, saving costs but sacrificing the overall printing resolution. As SLA evolution, DLP also suffers from similar drawbacks like the production of multi-material objects, reduced printing dimension on XY plane which makes this technology less appealing for some industrial application where huge printing section is necessary. 42 Two-Photon 3D printing TPP offers the possibility to print objects with a resolution at the micrometer and nanometer scale. TPP is based on femtosecond pulsed laser, highly focused on a liquid resin, generating a high photon absorption probability on the focus area while keeping it very low outside. 60 As a result, a very tiny voxel is polymerized, allowing the fabrication of objects with high resolution, below 100 nm. 61 The low printing speed, the lack of efficient photoinitiators, and the reduced dimension of the printed objects relegate TPP mainly to academic research, especially on biological, micromechanics, and optics field. 62 3D printable VP formulations To explain better the importance of dyes in VP 3D printing, here we briefly describe the components of photocurable formulations. Those can be adapted to all the abovementioned 3D printing technologies based on photopolymerization, slightly modifying the ingredients. Each formulation is mainly composed of a monomer, a photoinitiator, and a dye. Many monomers are commercially available and in recent years many other resins have been developed and reported and reviewed. 37,63 The most common monomers in VP configuration are acrylates or methacrylates, with two or more polymerizable groups. Monomers with only one polymerizable group, such as acrylic or methacrylic acid, can also be used by adding a cross-linker. Unfortunately, these kinds of resins suffer from volume shrinkage during the polymerization process. Most of the acrylate systems can show strong internal stresses and deformations responsible for the reduced resolution or breakage of the final objects. 55 On the other hand, methacrylate monomers are less subject to shrinkage, but their curing rate is slower than acylates. 15 A rapid curing process along with low final distortion of objects can be obtained by mixing acrylate and methacrylate monomers. 37 As an alternative, aromatic or high molecular weight (meth)acrylates which present less volume shrinkage but higher viscosity, can be used. 64,65 Nevertheless, in these cases the printing parameters must be well-controlled and heating of the formulation during the printing process should also be considered. 25 Epoxy resins and thiol-ene systems (i.e. thiol monomers that react with alkene or alkynes) can also be used in VP as long as the monomers present at least two reactive groups, to produce thermosets. These formulations show a reduced shrinkage, fewer stresses, and higher conversion in comparison to acrylate monomers. 66,67 For thiol-ene systems, the weakening of the mechanical properties due to the presence of -C-S-C-bonds can be reduced by the addition of allyl ethers to the thiol/methacrylate mixtures. 68 Finally, polymers with higher T g can be obtained replacing alkene with alkyne in mixture with thiols monomers. 69 At last, it is worthy to mention that while the reactive groups of the monomers control the kinetics of the reaction, the backbone of the monomers has more influence on the physicochemical characteristics (e.g. strength, hydrophilicity, brittleness), so both the aspect should be taken into account while formulating a 3D printable material. The first step in a photopolymerization process is the generation of reactive species. This step requires a photoinitiator, able to absorb the irradiation wavelength and generate radicals or cationic species that can further react with monomers. The photoinitiator determines the polymerization rate, the conversion of the functional groups, the final resolution, and the threshold of polymerization. 62,70 The penetration of light into the resin and the curing depth are two important parameters that control the final resolution in VP and are both depending on the photoinitiator. The curing depth (Cp) is defined as follows: 71 = ln ( ) where E and E c are the laser exposure on the resin surface and the critical exposure of the resin under a specific laser irradiation wavelength and D p is the penetration depth: 71 is the concentration of the photoinitiator and ε is its molar extinction coefficient. It is possible to regulate the resolution of the product to obtain the desired properties of the final object by adjusting the quantity and type of the photoinitiator, the exposure time for each layer, as well as the energy of the light source. Other important parameters that can affect the C p are the light density, the monomer reactivity, the temperature and the exposure time. 72 The photoinitiator can be radical or cationic species, according to the mechanism used to start the polymerization.
Cationic photopolymerization, or eventually hybrid cationic and radical, is more suitable for SLA because a 355 nm UV laser is optimal to interact with those photoinitiators. 55 At 405 nm, the typical wavelength of DLP printers, there is a higher availability of radical photoinitiators, so that systems are preferred. 55 At last, dyes are typically organic or organometallic molecules that can be inserted in a polymer either by dispersion or by covalent linkage to the chains through appropriate functional groups. The introduction of dyes in polymers allows obtaining light-emitting devices, electroluminescent polymers, temperature sensors, mechanochromic materials, or for liquid crystals dispersed in polymers. [73][74][75][76][77][78] In VP, dyes are added into the formulation to improve the features of the objects, such as resolution or strength, while dyes absorb in the UV or visible range, while not affecting the polymerization process itself. 37 Figure 2. Chemical structure of commercially available polymers and monomer for application in 3D printing. Similarly, the cure depth is dependent on the light absorber concentration and its molar extinction coefficient. Dyes are able to spatially confine the polymerization process into the irradiated areas, according to the CAD file, while avoiding over-curing phenomena both in width and depth. 72,79 The amount of dyes loaded in 3DP matrices must be evaluated considering the broadness of the absorption spectrum and the dye molar extinction coefficient to minimize competitive photon absorption processes with other components like photo-initiators or photo-crosslinkers. An overall good resolution and fast polymerization are thus related to a well-evaluated balance between the dyes and the photoinitiator percentage in the matrix. In summary, In most of the applications, dyes do not seem to lend other important functionalities to the formulation other than conferring color to the final product. 79 This effect could be obtained using dyes in very low concentration as reported for solving the yellowing of the resin during the printing process by the addition of ultramarine dye (0.01% wt). 80 As an example, Sudan I 1 (Figure 3) is a dye that has been widely used to limit the curing depth and obtain better zresolution. 81 For the same purpose, 1 was also mixed with diacrylate and thiol monomers showing a slower conversion reaction due to the competitive absorption among 1 and the photoinitiator. 15 This evidences that an optimal compromise should be carefully evaluated between curing rate and z-resolution.  Figure 3) has been used to generate silver nanoparticle in situ using a formulation of PEGDA and AgNO 3 . 14 The role of 2 was to avoid the curing out of the irradiated area and to control the thickness of each layer. Finally, 2 could also be degraded by UV light irradiation to obtain a decolorized final product. The evolution in the use of dyes in 3DP is the goal of this review and it will be discussed in the following section, with particular focus on the synthesis of stimuli-responsive and functional polymers. 82 As last ingredient of photocurable formulations, fillers can be added to synthesize (nano)composites and to impart some specific properties such as electrical and thermal conductivity, luminescence, tunable stiffness to the polymers. This is the most common strategy to overcome polymeric materials limitations. In this regard, 3D printing of composites is largely investigated and recently reviewed. 41,[83][84][85][86] Functional dyes in 3D printing In this section, recent advances in using functional dyes in 3D printing are discussed, while extensive reviews of chromophore molecules are reported elsewhere. [87][88][89][90][91][92][93][94][95] The dyes employed in each technology are introduced with a particular focus on the compromises needed to merge specific dyes and process peculiarities.
-Organic molecules and dyes in FFF Few functional dyes were employed in FFF 3D printing, due to the unfortunate possibility of thermal degradation during the extrusion. Mechanocrhomic dyes are a class of chromophores known to change color when subjected to mechanical stress. 77  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 Dispersed aggrega-chromic dyes can modify their optical properties when disaggregating after the exposure to a mechanical force, or due to the mechanically-induced chemical rearrangement of the chromophores covalently connected to the polymeric chains. 96 Mechanochromic spiropyranes in FFF have been recently introduced showing a color change from colorless to purple upon mechanical elongation due to the reversible switch between the spiropyran and merocyanine isomers (Figure 4a). 97 Three different spiropyranes 3-5 ( Figure 4b) were copolymerized via ring opening polymerization with PCL to obtain two matrices bearing the dyes inside the polymeric chain, in which the elongation stress was focused mainly on Cspiro-O bond, and one sample with a spiropyran as chain-end, in which the force can't trigger the isomerization reaction. The polymers were printed by extrusion at 110°C and without evidence of thermal degradation during the process, indicating the compatibility of these dye with FFF technology. Moreover, the purple color of the merocyanine form was not observed, suggesting that the stress during the printing process did not promote the chemical rearrangement of the dyes. The reversible color changes were finally evaluated as a response to external mechanical stress or to UV irradiation, suggesting potential applications in various devices (Figure 4c and 4d). An inexpensive, biodegradable and biocompatible colorful polymer printed in FFF was obtained by mixing a PLA matrix with crystals of a thermochromic triclinic polymorph of alkoxyphenyl N-substituted naphthalene diimides 6-11 (Figure 5a). 98,99 The first sample composed of PLA and 4 (in 1:19 and 1:9 ratios) was melted at 200°C without indication of thermal degradation and showing a reversible negative thermochromism, which was enhanced at higher concentration of 4. Similar but less evident positive thermochromism was obtained with 6 combined with PLA in a 1:9 ratio. The activation of the thermochromism at different temperature can be controlled by modulating the alkoxyphenyl chain length, making these materials suitable for thermal sensors (Figure  5b and 5c). Photonic crystals are dielectrics materials in which an inner propagation of some light frequencies is forbidden providing a color proper of the crystal nanostructure. These materials can also be introduced in formulations as an alternative to dyes and pigments as in the case of the colorless dendritic block copolymer with a molecular weight of 484 kDa 12 (Figure 6a). 100 During the FFF printing process at 200°C, 12 rapidly selfassembles and produces a nanostructured material that can reflect violet and transmit yellow light (Figure 6b-d). The reflected light wavelength can be modulated within defined limits related to the processability, by increasing the molecular weight of 12. Not only, violet, orange and green light-reflected materials have been produced, but also a sample able to transmit light was obtained with further use as an optical filter.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 FFF can be used to process colorful functional materials without occurring in degradation despite the temperature and mechanical stresses generated during the printing process. This led to easy and low-cost production of functional materials with excellent resolution and complex geometries, making easy even-home production of components with high added value.
-Organic molecules and dyes in DIW DIW is a very good choice for the development of functional materials since it operates at room temperature while using liquids and pastes. One of the most important and exploited uses of DIW is to print hydrogels with cells that can mimic the extracellular matrix. Thanks to the low viscosity, hydrogels can be easily printed through an extrusion and the shape can be retained after the printing process by a rapid sol-gel transition or by a secondary cross-linking procedure that can be used to stabilize the final construct. 101 This technique is also known as bioprinting and was largely combined with the use of functional dyes. A multifunctional 3DP hydrogel composed of sodium alginate, sodium polyacrylate and layered rare-earth hydroxide, consisting mainly of lanthanide ions of Eu 3+ and Tb 3+ 13-16 (Figure 7a) was recently reported. 102 These ions, other than as co-cross linkers, ionic carriers and rheology modifiers, could emit light and their photoluminescence could be tuned, by modulating the concentration of the cation in the hydrogel matrix composed of 17, sodium alginate and sodium polyacrylate matrix (Figure 7b). The emission, under 254 nm UV light, shifted from yellow to red and green according to the different ratios between the Eu 3+ and Tb 3+ . These final properties, along with the highresolution printability and the tunable stiffness has permitted the creation of skin like sensors capable to detect human motion. Photoluminescent 3D printed hydrogels were also used to evaluate the O 2 concentration available for cells. 103 A O 2sensitive luminescent platinum (II) meso(2,3,4,5,6pentafluoro)phenyl porphyrin 18 and a coumarin derivative 19 as a fluorescence standard (Figure 7c) were dispersed in an alginate-based bio-ink with cytocompatible nanoparticles, made of styrene-maleic anhydride copolymer. The platinum complex has conferred a red emission upon excitation with blue wavelength and it could change its emission as a function of O 2 concentration. This probe was successfully used to map the oxygen concentration in printed objects containing green microalgae (Chlorella Sorokiniana) and human mesenchymal stem (hTERT-MSC) cells (Figure 7d). Similarly, transition metals ions were employed to print hydrogels by co-assembling with the peptide hydrogelators. 104 The covalent bonds between the ligands and the peptides were designed to prevent aggregation with consequent self-quenching of the chromophores. 2,2'dipyridine (bpy) as a ligand was used to realize the emitting complexes 20-22 with Eu 3+ , Ru 2+ and Ir 3+ cations ( Figure  7e). Different emission patterns, as well as, a white fluorescent hydrogel could be generated by playing with the relative ratios of the metals' complexes ( Figure 7f). The final hydrogels were proven to be mechanically stable in water and suitable for application in soft electronics. An interesting application of organic fluorescent dyes in the polymeric matrix demonstrated that not only the photophysical properties but also the mechanical behavior of well-known and discussed hydrogels could be modulated by the presence of a chromophore (Figure 8a). 46,105 In a PAS matrix loaded with tetra-(4-pyridylphenyl)ethylene 23 (Figure 8b), pH variations were shown to induce (dis)aggregation phenomena of the dye which were depicted by a change in the monolayer emission (Figure 8c). At the same time, in an acidic environment, due to the increasing protonation of the dye and its consequent solubilization, the brightness decreased, and a material deformation occurred, due to the electrostatic interaction between the protonated 23 and PAS. Both the dyes' concentration and the time needed to reach the equilibrium were investigated in detail. Another type of material able to change the shape through pH variation in a totally reversible and rapid way was designed by mixing PEG-(NH-MA) 2 with α-cyclodextrin (α-CD), creating a polypseudorotaxane 3D printed hydrogel. 106 The material was not only responsive to pH variations, due to the deprotonation of a hydroxyl groups of α-CD, but, when mixed with polyacrylate, it was also sensitive to the variations in the aqueous ionic strength. α-CD has also been reported in combination with Pluronic F-127 to created reversible shape 3D printed hydrogel in response to solvent exchange between water and DMSO. It is worth noting how this effect could be used to convert solvent chemical energy into a mechanical one. 107 The mentioned spiropyran can be also used in DIW in combination with commercial silicone inks composed of PDMS microbeads in which 24 was incorporated via platinum-catalyzed hydrosilation in 0.25 wt% ( Figure  9a). 108 The switch between spiropyran 24 and the merocyanine 25 was shown to be activated either by compression or elongation and to be completely reversible. The decoration of the 24 scaffold with a nitro group was translated in longer time needed to reverse the equilibrium from 25 in comparison to the unsubstituted dye (Figure 9b). Reversible deformation of printed polymers could also be achieved by using the trans/cis isomerization of azobenzene dyes 26, 27 (Figure 9c). 109 PHMS with molecular weight of 2100-2400 g/mol were initially functionalized with 26 and 27 using platinum-catalyzed hydrosilylation reactions. The obtained material was subsequently printed via a syringe on a film of a commercial polyimide, which confers flexibility and excellent resistance against organic solvents. Under 442 nm irradiation, both materials have shown a deformation that could be totally recovered, in the second's timescale, once the light was switched off. After ten cycles the materials did not show fatigue (Figure 9d). Furthermore, relative contributions of Young's moduli and thicknesses of both layers could be used to modulate the motion of the materials. Coumarin can be used to reversibly modulate the mechanical properties of polymers by UV light irradiation to generate a polyester, derived from bio-renewable resources. 110 Coumarin appendage 28 reacts via a [2 + 2] cycloaddition to form 29 under UV light (Figure 10a). Thanks to the reactivity of the coumarin, a 3D printable and biodegradable polyester were prepared without the need of solvents and photoinitiators. The so obtained viscoelastic liquids change their properties to generate an elastomeric product by photo-cross linking reaction upon irradiation at 350 nm (Figure 10b). This result highlighted the possibility to produce tunable bio-based polymers with minimal toxicity, for biomedical purposes. The same approach was used to fabricate free-standing photo-cross-linked hydrogels exploiting the reversible [2 + 2] cycloaddition of coumarin 30. 111 Irradiation at 254 nm of the prepared hydrogel was depicted by complete reversible degradation into water-soluble copolymer 31 which have shown low acute cytotoxicity suggesting application as a cell-culture scaffold (Figure 10c) A similar concept has been applied to ruthenium (II) trisbipyridyl dication 32 and sodium ammonium persulfate to mediate the cross-linking of a 3D printed hyaluronic acid and gelatine hydrogels with phenolic hydroxyl moieties through visible light (Figure 10d). 112 The mechanical properties of the final polymer can be tuned varying the ruthenium concentration demonstrating the potential use in regenerative medicine. Not only hydrogels can be printed in DIW, but also liquid crystal elastomers (LCE), as reported for 33 (Figure 10e) when mixed with n-butylamine as a chain extender and a photoinitiator. 113 In the printing process, LCE were aligned along the printing pathway and the material showed 40% of reversible contraction from room temperature up to 200°C, only along the printing direction. The DIW printed structure was then photo cross-linked under UV-light. These smart devices have been reported suitable for medical functions.
The reported examples show also that DIW can be a promising 3D printing technique in which organic dyes can be successfully introduced in polymeric matrices. Lightemitting materials, reversible chemical reactions, or  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59 controlled shape deformations are the most promising properties conferred to objects obtained by merging dyes with DIW technique. -Organic molecules and dyes in SLA/DLP SLA and DLP are the most suitable techniques for taking advantage of the use of the functional dyes since they work at room temperature and employing liquid formulations. Organic molecules and dyes can be introduced as additives in liquid formulations, conferring specific and useful functionalities or simply improving the control over the polymerization process and objects' final resolution. In the context of SLA and DLP techniques, the collection of the available dyes and polymeric matrices is wide and varied. A variation on the Young's modulus have been reported by using well-known azodyes 34 and 35 (Figure 11a). Those were used in two different polymeric matrices with similar chemical structures but different glass transition temperatures, namely BEDA with molecular weight of 572 g/mol and BEMA with molecular weight of 1700g/mol. 79 Under laser irradiation at 532 nm, the Young's moduli of the two polymers showed remarkable changes depending on the polymers' T g (BEMA has a T g of -38°C, while BEDA 46°C). BEMA with 35 increased its elastic modulus by 18% and BEMA with 34 by 58% at 25°C. On the other hand, at the same temperature, BEDA reduced the elastic modulus by 27% containing 35, and with 34 by 75%. Furthermore, when the temperature was 48°C, BEDA containing 34 increased Young's modulus by 620%, showing an opposite behavior. These experimental data were applied to microcantilevers, to locally control changes in mechanical properties by laser irradiation. The same azo dyes were employed in a following work to exploit the trans/cis switching of azobenzene dyes under visible light for 3D printed devices with photo-activated gas permeability. 114 The formulations were printed with DLP then CO 2 and O 2 permeability analyses were carried out on the resulting membranes under Nd:YAG laser beam irradiation at 532 nm and at different humidity conditions. Under laser irradiation, the CO 2 permeability was increased by 14% for PEGDA containing 35 and 70% for PEGDA doped with 34 at 25°C and 0% of humidity. The processes were reported as completely reversible showing a carbon dioxide permeability increase when the laser was switched off. On the other hand, the CO 2 permeability was thirty times higher than O 2 permeability, and the laser irradiation seemed not to have an effect. Finally, a 3D prototype gas separator was printed and tested in CO 2 permeability, with a matrix assembled with 532 nm transparent polymer and an azo-based membrane (Figure 11b). CO 2 was flowed through the azobenzene-based membrane and during laser irradiation, different CO 2 permeability was detected by monitoring the pH of the aqueous solution where the CO 2 gurgles, forming carbonic acid. Environmentally sensitive materials, able to respond to variations in physical parameters such as light and temperature has been also reported using anthracene 36 in different polymeric matrices. 115 Anthracene can undergo [4+4] cycloaddition generating 37 under UV light with λ > 300 nm and the almost complete reversible reaction occurs under irradiation with λ < 300 nm (Figure 12a). The printed material, a linear 37-containing poly(D,L-lactide)poly(tetramethylene oxide) glycol copolymers (AN-PDLLAPTMEG) with different content of PDLLA, showed excellent recovery of the initial shape after deformation in the dynamic mechanical analysis (Figure 12b). Furthermore, the possibility to 3D print this material using SLA or DLP configurations without the need of a photoinitiator may ensure the biosafety and the possible use in biomedical applications. A 37-containing copolymers PLA and PEG exhibited dual stimuli responsivity to light and heat to deform and recover the initial shape. Moreover, the  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 anthracene change in fluorescence before and after the irradiation at 365 nm could be exploited and used for information storage and simple anticounterfeiting.   (Figure 13c). 116 The resins' printability, the intrinsic emission and the dependence on the dye concentration were investigated to show an inverse relationship between the material transmissivity and the dye concentration, probably due to the reabsorption processes ( Figure 13d). Additionally, the printed device has shown potential application as a polarity sensor providing different emission upon interaction with various organic solvents. Coordination polymers represent an alternative to conventional matrices to create interesting materials for chemosensing applications, due to their high flexibility, dynamic structures, and possibility to create stimuliresponsive devices. An example of such materials was introduced by employing the commercially available 4-4'bipyridine 42 as metal coordinator in combination with copper (II) 1D coordination polymer inserted with thymine as a terminal ligand (Figure 14a and b). 83 The polymer was characterized by both coordinated and solvation water molecules that were partially released by heating, causing a color shift from blue to violet. It is worth noting that the dehydration process was proven to be completely reversible, making this material suitable as a moisture sensor, also in organic solvents. More complex dyes bearing reactive azide groups on a squaraine backbone as 43 was also used to react via click chemistry reaction with alkyne moieties showing high selectivity in a dedicated post-printing functionalization process (Figure 14c). 117 Two different formulations were prepared with the former containing and excess of reactive thiol and the latter characterized by an excess of alkyne moieties able to react with the 43 to provide selective staining on the printed object (Figure 14d). The same copper-mediated azide-alkyne cycloaddition approach was used to functionalize complex polyesters with a modified coumarine 44 to confer emission properties to the polymer (Figure 14c). 118 The prepared copolymer, 3D printed with continuous DLP (cDLP), was further treated with 44 to quickly create a bright printed object (Figure 14e). It is worth mentioning that these copolymers were employed for regenerative medicine, thanks to the biocompatibility and degradation into safe by-products. A clear example of such applications was recently reported 3D printing with cDLP discs of PPF functionalized with propargyl alcohol moieties, which could further react with 44 by azide-alkyne click mechanism (Figure 14f). 119 The reactivity of the printed discs toward azides was visually confirmed by the color of the final objects. The same reaction was then applied to functionalize the PPF disc with N 3 -GRGDS peptide to prepare biocompatible scaffolds for application in biomedical treatments. Mechanophores polymeric matrices represent an interesting alternative for the preparation of material responsive to external stimuli, as reported for HEA containing oxanorbornadiene 45 (0.99 wt%) and Irgacure 819 46 as photoinitiator (0.49 wt%). 120 The samples were prepared by DLP under visible light irradiation to avoid degradation of 45, while providing high efficiency of the crosslinking. Subsequent compression of the sample for one minute was shown to prompt the formation of the 47 and 48, which are the molecular constituents of 45 ( Figure  15b),. Furthermore, complex shape objects containing 0.01%wt Nile Red 49 were additionally printed to demonstrate the excellent final resolution and the possibility to reversibly compress these products ( Figure  15c). Natural or biocompatible materials were as well employed for VP polymerization. For instance, Chitosan, a biocompatible and biodegradable polymer, is very attractive for biomedical applications. A bio-ink was obtained straightforwardly by functionalizing chitosan with methacrylic anhydride and finally mixed with LAP 50 (Figure 15c) to start the polymerization using 405 nm light source which is less harmful to cell lines compared to UV radiation. 121 The resulting polymer has shown high fidelity and resolution, along with good biocompatibility. PGS is another example of a biocompatible polymer that could undergo photo cross-linkage when mixed with an acrylate moiety 52. 122 The final DLP printed samples have shown excellent mechanical properties and good biocompatibility while being characterized by elastic modulus values as a function of the cross-linker concentration and the exposure time during the printing process. Those biomaterials were as well used with functional dyes as it has also been reported using a rosin derived monomer 52 and HEA in a mixture of methacrylated ethyl cellulose macromonomer 53, hexamethylene diisocyanate 54 as cross-linkers. 123 The formulation was printed in SLA with 55 as photoinitiator and, subsequently, the thermoset was heated to promote a secondary cross-linking between 54 and free hydroxy moieties to improve the mechanical and thermal properties of the polymer (Figure 15d). The obtained sample was also characterized by good selfhealing by exposing the "broken moieties" to UV curing and heating, shape-memory behavior, and aggregation-induced emission. Moreover, the fluorescence, mainly due to the rosin, was shifted from 467 to 498 upon curing/heating treatment making this sample suitable to applications as sensor, organic light-emitting diodes and in biology. -Organic molecules and dyes in TPP In TPP field, very few works report the use of dyes, due to the lower impact of such molecules to improve the printing performances. In fact, in TPP, the ultra-focalized irradiation and the multiple photons polymerization mechanisms are sufficient to ensure the micrometer o nanometer resolution of the structures. Nevertheless, azodyes as 56-58 have been employed to evaluate the trans/cis photoisomerization and their applicability in TPP (Figure 16a). 124 Alkyl and bulky groups  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 were introduced on the dyes' scaffolds to improve the solubility and the dispersion while avoiding the formation of hydrogel with consequent dyes aggregation while printing photonic crystals (Figure 16b). A similar azobenzene dye 35 was embedded in a polymeric matrix using BEDA 2EO as a crosslinking monomer, 46 as a photoinitiator in a 1:97:2 ratio to obtain a rapid yet reversible deformation of printed nanometer membranes (Figure 16c). 125 The formulation was printed to form a square membrane of 120 µm dimension which, under 532 nm laser irradiation, has shown a volume expansion of 28% and a decrease in the refractive index of about 0.16. The reversibility of the process was demonstrated by observing the deformation and recovery by switching on and off the laser several times. Nevertheless, the weakness of the system was related to the possible photobleaching of the azodye due to the energetic laser irradiation. Also coumarins 59 and 60 have found applications in TPP, to reduce the amount of photoinitiator needed to start the polymerization, as well as, of the sub-products residue left at the end of the polymerization. 126 Novel coumarin derivatives were prepared with substituent groups to expand the conjugation, covering the visible spectra, to enhance the photosensitizer effect with a concurrent reduction of the photoinitiator concentration from 1-2% to 0.4%. At the same time, a good printing resolution was achieved, as demonstrated by the 3D printed giraffe ( Figure  16d). Coumarin and other commercial dyes 61-64, as fluorescein, were also used to create 3D printed objects able to emit light without reducing the printing resolution and enabling the internal structure verification by optical microscopy. 127 Coumarin-based ketones 65-68 were instead used as initiators in TPP, evaluating their photochemical and photophysical properties by varying the side chains as well as the central ketone ring. 128 Similarly coumarins 69-71 bearing oxime-esters groups were able to start the polymerization process upon irradiation in the 400-460 nm spectral window with low-intensity laser. 62

Conclusion and perspectives
In 3DP field, dyes have conventional uses mainly for aesthetical porpuses (i.e. desired colors) or, in the best case, as elements to enhance the resolution of the printed structures. In this review we have reported recent advances on the use of dyes for imparting functional properties to materials: dyes can confer specific properties such as modification of mechanical behavior, light emission, tunable permeability, controlled wettability, or mechanochromic features. Furthermore, these investigations can spread all over the most common 3DP technologies by adapting the chromophores' functionalization to the techniques' peculiarities. Functional devices such as waveguides, sensors for humidity and solvents, biosubstrates, and artificial membranes can be produced. Future applications can be based on inclusion of other responsive dyes, such as derivatives of pyrene or benzoxadiazole to evaluate changes in pH, temperature or the hydrophilic/hydrophobic behavior of the polymeric matrixes. 8 BODIPY, porphyrin, rhodamine and carbazole can find applications in temperature sensing by the changes of the emission properties and quantum yield. 5 The resulting devices can be used as reversible sensors, potential biomedical drug delivery systems and actuators. 129,130 The innovations presented here demonstrates that this area is still growing, and this will enlarge the applications of 3D printing. The wide variety of possible functionalities provided by dyes to common polymeric matrices will prompt both academic and industrial researchers to expand even more the progress in this field . 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60