Fundamentals of light-cell–polymer interactions in photo-cross-linking based bioprinting

APL Bioeng. 4, 041502 (2020); https://doi.org/10.1063/5.0022693 4, 041502 © 2020 Author(s). Fundamentals of light-cell–polymer interactions in photo-cross-linking based bioprinting Cite as: APL Bioeng. 4, 041502 (2020); https://doi.org/10.1063/5.0022693 Submitted: 23 July 2020 . Accepted: 21 September 2020 . Published Online: 12 October 2020 Daniel Nieto , Juan Antonio Marchal Corrales , Alberto Jorge de Mora , and Lorenzo Moroni COLLECTIONS Paper published as part of the special topic on Biophysics of Biofabrication This paper was selected as Featured This paper was selected as Scilight ARTICLES YOU MAY BE INTERESTED IN Illuminating interactions in light-based bioprinting Scilight 2020, 421101 (2020); https://doi.org/10.1063/10.0002283 Functional hydrogel bioink, a key challenge of 3D cellular bioprinting APL Bioengineering 4, 030401 (2020); https://doi.org/10.1063/5.0018548 Time dependent stress relaxation and recovery in mechanically strained 3D microtissues APL Bioengineering 4, 036107 (2020); https://doi.org/10.1063/5.0002898 Fundamentals of light-cell–polymer interactions in photo-cross-linking based bioprinting Cite as: APL Bioeng. 4, 041502 (2020); doi: 10.1063/5.0022693 Submitted: 23 July 2020 .Accepted: 21 September 2020 . Published Online: 12 October 2020 Daniel Nieto, 1,2,3,a) Juan Antonio Marchal Corrales, 3,4,5 Alberto Jorge de Mora, 6 and Lorenzo Moroni 2 AFFILIATIONS 1 Photonics4Life Research Group, Department of Applied Physics, Faculty of Physics, University of Santiago de Compostela, Santiago de Compostela 15782, Spain 2 Complex Tissue Regeneration Department, MERLN Institute for Technology Inspired Regenerative Medicine, Universiteitssingel 40, 6229ER Maastricht, The Netherlands 3 Department of Human Anatomy and Embryology, Institute of Biopathology and Regenerative Medicine, University of Granada, Granada 18016, Spain 4 Instituto de Investigaci on Biosanitaria de Granada (ibs.GRANADA), Granada 18012, Spain 5 Excellence Research Unit “Modeling Nature” (MNat), University of Granada, Granada 18016, Spain 6 SERGAS (Galician Health Service) and IDIS (Health Research Institute of Santiago de Compostela (IDIS), Orthopaedic Department, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain Note: This paper is part of the special issue on Biophysics of Biofabrication. a) Author to whom correspondence should be addressed: [email protected] ABSTRACT Biofabrication technologies that use light for polymerization of biomaterials have made significant progress in the quality, resolution, and generation of precise complex tissue structures. In recent years, the evolution of these technologies has been growing along with the development of new photocurable resins and photoinitiators that are biocompatible and biodegradable with bioactive properties. Such evolution has allowed the progress of a large number of tissue engineering applications. Flexibility in the design, scale, and resolution and wide applicability of technologies are strongly dependent on the understanding of the biophysics involved in the biofabrication process. In particular, understanding cell–light interactions is crucial when bioprinting using cell-laden biomaterials. Here, we summarize some theoretical mechanisms, which condition cell response during bioprinting using light based technologies. We take a brief look at the light–biomaterial interaction for a better understanding of how linear effects (refraction, reflection, absorption, emission, and scattering) and nonlinear effects (two-photon absorption) influence the biofabricated tissue structures and identify the different parameters essential for maintaining cell viability during and after bioprinting. V C2020 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http:// creativecommons.org/licenses/by/4.0/).https://doi.org/10.1063/5.0022693 I. INTRODUCTION Bioprinting is a fast emerging technique, which makes biofabricating artificial tissues and the microscale deposition of living cells possible. 1,2 The ultimate objective of bioprinting is to create 3D artificial tissues that mimic the natural biological microenvironments, where cells can function as well as they would in real tissues. The structural geometry and morphology of artificial structures should be controlled using bioprinting tools to maintain high functionality at various dimension scales to mimic the tissue complexity. 3,4 Light-based biofabrication methods have been developed and used to generate biological scaffolds and complex tissue structures. 5,6 The optical nature of light (noncontact, optically selective, and precise processing) positions these technologies at the forefront of biofabrication techniques with the ability for high precision biofabrication at submicrometer resolution (<1lm). Most light based printers can be divided by the light source used for polymerization (using one photon or two photons), which is then projected over a bath filled with liquid photo to cross-linkable biomaterials or cell-laden hydrogels onto a moving stage. Most common lightbased technologies using photopolymerization include laser-based SLA, mask-based SLA, and digital light projection (DLP) using digital mirror devices (DMDs). These technologies are based on one photon polymerization, initially using UV light 7–10 and more recently visible light. 11–13 On the other hand, multiphoton polymerization-based 3D APL Bioeng. 4, 041502 (2020); doi: 10.1063/5.0022693 4, 041502-1 V CAuthor(s) 2020 APL Bioengineering REVIEW scitation.org/journal/apb laser lithography 14 is based on the absorption of two photons of near infrared light (NIR), to excite the same energy transition as ultraviolet (UV)one-photon absorption for cross-linking the biomaterial. A. SLA Conventional SLA was in fact the first technology introduced by Charles Hull in the 1980s for creating 3D constructs using UV light to polymerize materials. 15 This was followed by other studies discussing the kinetic modeling of linear, cross-linking photopolymerization and highlighting the opportunities as industrially cured coatings and dental fillings, and more generally three-dimensional rapid prototyping techniques. 16,17 SLA bioprinting presents some advantages in comparison with other bioprinting techniques, such as extrusion and inkjet systems. SLA bioprints photosensitive hydrogels in a layer by layer fashion rather than in struts or droplets. The bioprinting time for each layer is the same, but the total biofabrication time of the hole structure depends on the thickness. This bioprinting characteristic of SLA reduces the biofabrication time of the hole tissue structure. Moreover, SLA is a nozzle-free bioprinting technique, which avoids the inconvenience associated with nozzle-based bioprinting technologies, resulting in cell-laden structures with viability higher than 90%. 18,19 Commonly, SLA uses the light source that impinges into a bath filled with a photosensitive biomaterial, which is placed onto a Z moving stage. The Z stage moves down to a predefined distance and the material is polymerized. This distance determines the vertical resolution of the SLA printer. More recently, a top-down approach was used, where the light source that was placed below the bath containing the biomaterial, in this case, the Z platform, is moved up to a distance which determines the thickness of the layer and hence Z resolution. Maskbased SLA uses a mask and a light source to project the photomask [Fig. 1(d)]. Laser-based SLA uses laser, which is focused using a lens with XYZ movement to transfer the pattern. Bioprinting speeds using SLA are higher than other conventional methods. Nevertheless, this method presented poor biocompatibility with low resolution. 20 Although, initial studies have reported feature sixes of 150 lmpersingle layer and with axial resolution 250 lm, 21,22 with the development of biomaterials and hydrogels, the biocompatibility and resolution of FIG. 1. (a) A tumor angiogenesis model: (i) schematic showing the tumor angiogenesis model; (ii) schematic of the mask for printing; (iii) bioprinted microvasculature; and (iv) bioprinted tumor model. (b) A skeletal muscle model: (i) schematic showing the skeletal muscle tissue; (ii) schematic of the mask for printing; (iii) bioprinted skeletal tissue model; and (iv) PrestoBlue measurements of cell proliferation in the bioprinted structures. (c) A tendon-to-bone insertion model: (i) schematic of the tendon-to-bone insertion site; (ii) schematic of the mask for printing; (iii) bright-field optical image showing a bioprinted dye-laden GelMA structure; and (iv) bioprinted tendon-to-bone model. Reproduced with permission from Miri et al., Adv. Mater. 30, 1800242 (2018). Copyright 2018 John Wiley and Sons. (d) Schematics of stereolithographic bioprinting process: (i) laser-based and (ii) mask-based. (e) Schematic of the DLP bioprinting process: (i) gray scale digital mask and (ii) images of fluorescently labeled hiPSC-derived hepatic progenitor cells (hiPSC-HPCs). Reproduced with permission from Ma et al., Proc. Natl. Acad. Sci. U. S. A. 113, 2206–2211 (2016). Copyright 2016 PNAS. (f) The cross section of a TPP-bioprinted mouse paw bone imaged using scanning electron microscopy and the intricate contours within the structure that arose from the bioprinting process. (g) Schematic illustration of the experimental setup for two-photon bioprinting along with a zoomed description of focal plane and distribution of light intensity in the laser focus of a Gaussian beam is shown. Images (f) and (g) were reproduced with permission Miri et al., Lab Chip 19, 2019–2037 (2019). 50 Copyright 2019 Royal Society of Chemistry. APL Bioengineering REVIEW scitation.org/journal/apb APL Bioeng. 4, 041502 (2020); doi: 10.1063/5.0022693 4, 041502-2 V CAuthor(s) 2020 SLA improved to 50 lm. 18 SLAs have been commonly used in manufacturing industries and more recently used for tissue engineering applications. Initially, SLA has been used for bone tissue models. Catros et al. used an SLA bioprinter for patterning nanohydroxyapatite (nHA) and osteoblastic cells in 2D and adapted to the biofabrication of 3D composite materials toward healing bone defects. 23 Wang used an SLA bioprinting system in combination with visible photosensitive bioinks [poly(ethylene glycol) diacrylate (PEGDA), gelatin methacryloyl (GelMA), and eosin Y based photoinitiator], resulting in NIH 3T3 cell bioprinting with 50 lm resolution and high cell viability. 24 B. Digital light projection The printing speed of SLA can be significantly improved by using mask-less DMD-based bioprinting. The DMD-based bioprinting uses an array of micromirrors (the dimension of each micromirror can be in the order of 5–10 lm) to selectively switch the light intensity of each micromirror (each individual mirror can be controlled on two positions, being either 0-dark or 1-light reflecting and with speeds on the order of kilohertz) and project it over light-sensitive biopolymers that polymerize the preselected light patterns transferred by the DMD in a layer by layer fashion [Fig. 1(e)]. DMD technology has arisen as an alternative for high-throughput DLP printing, resulting in good biocompatibility for seeding cells. 25 Zhu et al. used a DMD bioprinter for generating prevascularized tissue models with complex geometries (widths 50 lm and heights ffi50 lm) using a bioink of endothelial cells, GelMA, and glycidal methacrylate–- hyaluronic acid. 26 Miri et al. were able to generate biological tissue structures such as tumor angiogenesis [Fig. 1(a)], muscle strips [Fig. 1(b)], and musculoskeletal junctions [Fig. 1(c)]with printing resolutions on the order of 10 lmbyusingaDMD bioprinter working at 365 nm, in combination with microfluidics. 8 Ma et al. have used DMD to fabricate hexagonal lobule structures of GelMA (15% w/v) seeded with HUVECs (Human Umbilical Vein Endothelial Cells) (with a resolution 50 lm) that where incorporated on a liver-on-a-chip. 27 These studies highlight the high speed of bioprinting associated with DMD (under 1 min), accuracy (10–50 lm), and versatility (from biocompatible scaffolds to cell-laden structures with different geometries) of the mask-less methods. C. Multiphoton polymerization-based 3D laser lithography The challenge associated with 3D biofabrication using singlephoton photopolymerization is to avoid the off-focal photopolymerization that may ultimately cure undesirable parts of the designed construct. 28,29 Biofabrication using multiphoton polymerization benefits from the high resolution inherent in the two-photon polymerization (TPP) process, which can generate 3D structures with micro/nanoscale resolution [Fig. 1(f)]. 30 The nonlinear optical phenomenon associated with TPP occurs when irradiating using a focused femtosecond laser beam at infrared wavelength, by simultaneous absorption of multiple photons, which induces photopolymerization of a small area (100 nm), based on the radical generation due to the interaction between the used photoinitiator and the femtosecond laser beam. This interaction allows the generation of 3D tissue structures with ultra-high-resolution (from lmto nm) that cannot be achieved by other conventional photolithographic methods. 31 Ovsianikov et al. used TPP to fabricate biodegradable tissue scaffolds using gelatin modified with methacrylamide (GelMod), which were seeding with adipose-derived stem cells, presenting good adhesion and resulting in proliferation and differentiation to adipocytes. 32 Koroleva et al. demonstrated that hybrid Zr–Si porous scaffolds were fabricated using TPP promoted mesenchymal stem cells (hMSCs) to differentiate toward the osteogenic lineage. 33 Commonly used TPP systems include two X–Y galvanometric scanners to move the laser focus in the X–Y coordinates and a high resolution Z stage that performs axial scanning [Fig. 1(g)]. Due to the coherent properties of the laser beam with optimal focusing capabilities, the TPP process can generate high-resolution 3D features. Nevertheless, the throughput is restricted by the sequential laser scanning process. This limitation is further enhanced when printing complex hollow structures or large volume structures. Different optical solutions have been proposed, which include microlens arrays, spatial light modulators, and diffractive optical elements, most of them based on splitting the laser into multiple foci. Geng et al. have used TPP in combination with a DMD scanner for generating tens of laser foci that can be controlled individually by achieving diffraction-limited resolution (500 nm–1600 nm) and a processing speed of 22.7 kHz. 34 These studies highlighted the opportunities of TPP associated with high resolution features. Nevertheless, several key challenges still remain, which include the failure of biofabricating cell-laden constructs with clinically relevant dimensions. TPP systems that are commercially available are very expensive and are difficult to adapt to the particular application. The dearth of biomaterials (biocompatible and biodegradables) for TPP is another inconvenience for covering different biological applications. The dearth of water soluble PIs limits the uses of photopolymers with high water contents. Although TPP is very precise, it is a relatively slow process, which results in small scaffolds of structures difficult to handle in tissue engineering. Biological experiments need statistical experiments with a huge amount of identical structures. In this sense, efforts need to be taken to develop novel photopolymers and photoinitiators for TPP aimed at increasing the fabrication speed and reducing cytotoxic effects. Table I shows the characteristics of the most common light based technologies using photopolymerization. D. Basic light interaction process during bioprinting During photolithographic biofabrication, the primary light interactions can be caused mainly by linear phenomena of refraction, reflection, absorption, emission, and scattering and nonlinear effects as in the case of multiphoton polymerization. 35 Reflection occurs when light reflects on the surface of the biomaterial without penetrating it. Refraction occurs as a consequence of the change in the propagation angle of light when it passes from air to the biomaterial during bioprinting. Absorption and dispersion that occur both in a biological APL Bioengineering REVIEW scitation.org/journal/apb APL Bioeng. 4, 041502 (2020); doi: 10.1063/5.0022693 4, 041502-3 V CAuthor(s) 2020 biomaterial and in cells are dependent on the wavelength of light, which is complete through intracellular and extracellular constituents. Emission is a phenomenon that consists of an energy transmission to the atoms of the biological component, in which their electrons are promoted to higher levels of energy. While the energy of photons used to irradiate cells has a dramatic impact on phototoxicity, it can be expected that the bioprinting limitations are mostly related to the photon energy and the wavelength of the light source used to photo-crosslink the biomaterials. Although cells exhibit a very distinct irradiation sensitivity, the phototoxicity increases dramatically with decreasing irradiation wavelength. 36 Infrared light, as long as we do not pass the cellular thermal threshold through which apoptosis and destruction are induced, is safe. Nevertheless, ultraviolet light is mostly used for light-curing photopolymerization on stereolithographic bioprinting and has been reported to damage the DNA of cells. 37,38 Following light absorption, in certain circumstances, cells can undergo a wide variety of photochemical and photophysical processes, which include fluorescence, thermal effects, photoablation effects, plasma-induced ablation, and photodisruption. 39 These effects must be avoided during bioprinting. Photoablation occurs due to the action TABLE I. Most common light based bioprinting technologies using photopolymerization. Bioprinting technology Advantages Common advantages Disadvantages Common disadvantages References Digital light projection (DMD) High cell viability Direct incorporation of cells during bioprinting Dynamic bioprinting High resolution Noncontact biofabrication systems (No shear, mechanical and thermal stress, nor clogging during bioprinting) High resolution and density; additive operation Photopolymerization is cell friendly (pH, temperature) Customized systems/ required skills Moderate cost for high resolution systems UV light can damage micromirrors Require photocurable bioink (limited biomaterials) Monomer toxicity (biomaterial reactions during bioprinting) Custom made equipment (require technical staff) 8 9 27 Laser-based SLA High resolution (1 50 lm) Bioprinting of high viscosity Selective exposure of bioinks Medium speed Limited scalability Laser source might have an adverse effect on the cellular genetic material Moderate cost for high resolution systems 6 12 27 Mask-based SLA High cell viability Easy control of matrix properties Low cost technology Fast speed Monomer toxicity and use of ultraviolet radiation Require a mask pattern Multiple step processes 3 25 27 Multiphoton bioprinting Ultra-high resolution (nm to few micrometers) High penetration depth No UV light required High water content bioinks Limited by the speed of printing for highthroughput screening High cost technology Require optimization of photocurable bioink 11 24 25 27 APL Bioengineering REVIEW scitation.org/journal/apb APL Bioeng. 4, 041502 (2020); doi: 10.1063/5.0022693 4, 041502-4 V CAuthor(s) 2020 of an intense ultraviolet (UV) laser pulse that photochemically decomposes various cellular and extracellular components. Plasma-induced ablation and photodisruption occur as a consequence of exposing the biological material to a power density above 1011 W/cm 2 . 40 Fluorescence originates from the transition from an excited singlet state to a ground state vibrational mode. Thermal effects are the result of the conversion of absorbed light energy into heat. The abovementioned mechanisms need to be considered carefully to increase cell viability. Light power, selected wavelengths, and exposure time are perhaps the main determinants that dominate the process during light based biofabrication. It is essential to adapt the light beam and optimize the light parameter to minimize cell damage without losing its ability to light cure or achieve cross-linking in the medium. Most Stereolithography (SLA) printers can be divided by the light source used for polymerization (using one photon or two photons), which is then projected over a bath filled with liquid photo to cross-linkable biomaterials or cell-laden hydrogels onto a moving stage. Although photo-cross-linking is typically associated with SLA bioprinting, other biofabrication technologies, such as extrusion, may use photocross-linking as a secondary process. Bram et al. have used extrusion bioprinting based on a two-step cross-linking approach. Secondary photo-cross-linking was applied for shape maintenance. This two-step cross-linking methodology can be used with a broad window of extrusion biofabrication parameters that allow printing at a low viscosity (4 mPa s) to maintain high cell viability (>80%)andwithgoodshapefidelity.This eliminates the problems associated with low viscosity bioinks (complex chemical modifications, multiple initiation systems, and viscosity enhancers). 59 There is a vast amount of reviews covering the advantage and disadvantages of different bioprinting technologies and more recently the fundamentals and practical aspects of light based bioprinting, 41,42 but few of them covering the primary cell–light interactions. In this article, we consider the impact and behavior of light during bioprinting, focusing on high resolution structures with high cell viability. Section II is related to polymer–light interactions centered on high resolution structures. Section III is devoted to biophysical principles at that cell–light interaction level and parameters involved to maintain high cell viability, and Sec. IV presents a future outlook and conclusions. II. POLYMER–LIGHT INTERACTIONS Photopolymerization comprises the reaction of monomers that form large networks when irradiated with light (by the single-photon or two-photon absorption). This absorption can be promoted by the reactant monomer or by the transfer of energy absorbed by a photoinitiator. 43 Photoinitiators used for photopolymerization generate free radicals when they are exposed to light and react with monomers and/ or oligomers for initiating polymer chain reactions and growth. A. Photopolymerization mechanism (single-photon vs two-photon) During photopolymerization, the photon interactions at the initial step differ from the ordinary thermal polymerization, but the following steps being propagation, termination, and chain transfer remain the same. Under such premises, the photopolymerization bioprinting process can be classified into two categories: single-photon photopolymerization and multiphoton photopolymerization (Fig. 2). In single-photon SLA, the polymerization process is originated via linear single-photon absorption [Fig. 3(a)]. 44 The energy of the FIG. 2. Diagram of stereolithographic processes based on the exciton radiation form and energy (single photon and multi-photon). APL Bioengineering REVIEW scitation.org/journal/apb APL Bioeng. 4, 041502 (2020); doi: 10.1063/5.0022693 4, 041502-5 V CAuthor(s) 2020 photon, E p , is equivalent or superior than the material bandgap E g . With Ep¼hv¼hc=kbeing the governing law of this process, it means that high energy photons and short wavelengths are required. In most cases, UV wavelengths shorter than 365 nm are selected. One photon absorption with Ep>Egmotivates the electron to move from the valence band to the conduction band. This process alters the chemical bond inducing polymerization process, which is responsible for biomaterial cross-linking. Noncoherent light sources at low power levels can also induce linear absorption. Nevertheless, as the light intensity increases, typically using coherence laser sources, nonlinear absorption can take place. The UV (k UV ) photosensible biomaterial can be also photopolymerized by infrared (IR) wavelengths of nearly double wavelength (k IR ¼2k UV ). 45 TPP is based on this mechanism and comprises the absorption of two photons simultaneously through a virtual state for molecule excitation [Fig. 3(a)]. The virtual levels (fs) have a particularly short lifetime, which results in the instantaneous absorption of almost two photons. This excitation process depends quadratically on the incident light intensity 46 and the TPP requires high light intensities (>GW/cm 2 ). The TPP process can be described as follows: 47 a molecule in a ground energy state Gs is excited to an excited state Es. In this process, the molecules absorb two single photons (Gs and Es are separated with an energy difference of Ef above the Gs) as shown in Fig. 3(a). A virtual state Vs is created by the absorption of the two photons [both photons having the same energy levels ¼E1 (degenerate, Ef ¼2E1)]. With further excitation, they lose energy and move to another state R, where vibrational relaxation is induced by the lowest vibrational level of the lowest-energy Es [Fig. 3(a), dashed arrow], and then return to the ground state by a pathway that can be radiative or nonradiative. The TPP process is originated precisely at the focal volume of the laser beam [Fig. 3(b)], which facilitates the generation of precise and high-resolution 3D structures. The biggest limiting factor in the extensive use of TPP for biological applications is the slow manufacturing time that accompanies high-resolution structuring which can compromise cell viability. This can be overcome by using optical systems to modulate the behavior of light. Gittard et al. have demonstrated TPP using a multiple spotlight approach by generating microstructure arrays for tissue engineering. Computer-generated hologram patterns were used to generate multiple spotlights from one laser beam, significantly reducing the manufacturing time. These multiple foci were used to simultaneously produce multiple tissue scaffolds by TPP. 48 Atry et al. demonstrated the applicability of diffractive optical elements for fabricating large scaffolds at rates several times faster than by single spotlight. 49 The two key properties that conditioned the resolution of laser direct write bioprinting, mask based SLA, and DMD-based bioprinting processes, which are mainly determined by the thickness of the photosensitive resin, are the directionality of the FIG. 3. (a) Single-photon and two-photon absorption processes, (b) Gaussian beam profile of a laser beam, and (c) single-photon and two photon absorption features on the biomaterial. APL Bioengineering REVIEW scitation.org/journal/apb APL Bioeng. 4, 041502 (2020); doi: 10.1063/5.0022693 4, 041502-6 V CAuthor(s) 2020 impinging light and the lower scattering of light (perpendicular to the laser). The thickness (z resolution) can be controlled by adjusting the laser characteristics (pulse width, light wavelength, power, repetition rate, and size of the beam) and the properties of the resin (including viscosity and superficial stress). 50 According to the polymerization kinetics of the photo-cross-linking mechanisms, we can assume the following relationship to define the thickness of the light-cured material: Cd¼Dpln Ei Ec  ; where C d is the depth of curing (lm), D p is the depth of penetration (lm), E i is the irradiation of light (mJ/cm 2 ), and E c is the threshold value of energy of the gelation point for the liquid resin (mJ/cm 2 ). As E i approaches E c , the layer is cured, and the resin is solidified. Because of the nonlinear nature of two photon polymerization and the threshold behavior, high-quality and high-resolution features can be obtained. By adjusting different laser parameters (pulse energy and pulse repetition rate), the printing resolution (<100 nm) can be increased by overcoming the diffraction limit. The two-photon absorption mechanism happens in a resin that initially does not absorb the selected wavelength of the laser light, allowing its penetration in the material [Fig. 3(c)]. The bioprinting resolution of TPP is related to the incident laser light and the square of its intensity. A high magnification focal lens focuses laser energy at a small focal point where the highest amount of absorption takes place. Assuming a Gaussian laser beam profile with an intensity distribution I(r, z) at distances (z in the direction of propagation and r along the cross section) from the center can be defined as Ir;z ðÞ ¼I0 w2 0 wz ðÞ 2 "# e 2r2 wz ðÞ 2; where I 0 ,x 0 ,andx(z) are the intensity at the center of the Gaussian beam (r ¼0, z ¼0), the waist of the beam, and the radius of the beam in the plane with a distance of z, respectively. The average intensity at the focus plane can be defined as Ifocus ¼w pw021fht; where W is the power average, 1is the width of the selected pulse, f is the repetition rate, h is the Planck constant, and tis the frequency of light. The photon-polymerization is initiated when the density of radicals P (r, z) surpasses the threshold Pth [P (r, z) Pth]. The intensity at the focal plane (z ¼0) reaches the threshold, where Ir;z ðÞ ¼Ir;0 ðÞ ¼I0exp 2r2 w02  : All the aforementioned interactions and effects have influence over the bioprinting process. B. Photoinitiators The polymerization efficiency of the developed bioinks depends strongly on the selection of the photoinitiator. The photoinitiator should be efficient in free radical generation with low toxicity. For engineering of living tissues, photoinitiators sensitive to UV are the most used. Photoinitiators can be separated into two categories (in relationship with the radical generation mechanisms): (1) Type-I photoinitiators (cleavable photoinitiators) and (2) Type-II (bimolecular photoinitiating). During bioprinting, for initiating polymerization, Type-I photoinitiators generate two radicals. The starting process of Type-II (e.g., benzophenone/tertiary amine) presents more complexity. For example, benzophenone is excited and promotes fast electron transfer (from the lone pair of tertiary amine), which is followed by the proton transfer process; this process provides the radical (Hdonor) that initiates photopolymerization. To avoid the UV light damaging effects, including DNA damage and cancer effects, 51 some visible light photoinitiators have been investigated and demonstrated to be useful for bioprinting with cells. LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) is a UV photosensible photoinitiator, which has also been demonstrated to be sensitive to blue light (near UV). 52 Eosin Y(2 0,40,50,70-tetrabromofluorescein disodium salt) is sensitive around 514 nm. Hydrogels developed for working with Eosin Y maintain cell function and present less toxicity than Irgacure 2959 (1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propanone). 53–55 Natural photoinitiators such as Riboflavin (FR) and Vitamin B2, were demonstrated to induce photo-cross-linking on alginate hydrogels 56 and were used as visible light photoinitiators in the thiol–ene polymerization of polyethylene glycol (PEG)-based hydrogels. 57 A photoinitiator based on a ruthenium complex [tris-bipyridyl-ruthenium (II) hexahydrate] and sodium persulfate (SPS) was used to initiate visible light crosslinking of hyaluronic acid/gelatin-based bioinks. 58 Soliman et al. have used ruthenium (Ru) and sodium persulfate (SPS) cross-linkers in combination with allyl-functionalized gelatin (Gel-AGE) bioink for extrusion bioprinting based on the dual-step cross-linking approach, where a primary (partial) cross-linking in the absence of light is performed to alter the bioink’s rheological properties with subsequent secondary post-printing cross-linking for shape maintenance. 59 Lim et al. have used a Vis þRu/SPS system, demonstrating better cell cytocompatibility than the commonly used UV þI2959 system. Encapsulated cells remained >85% viable even when using high Ru/SPS concentrations, visible-light intensities, and longtime exposure times (21days), which highlight the potential Vis þRu/SPS system to avoid the cell damage associated with UV light and for maintaining high cell viability, shape fidelity, and metabolic activity. 60 More recently, poly-aketoester based photoinitiators have demonstrated good cell viability in combination with methacrylates and polyethylene glycol (PEG) diacrylate-based hydrogels. 61 It appears, therefore, clear that visible light sensible materials are emerging as optimal PI for cell-laden bioinks for bioprinting. A list of the commonly used PIs and their light absorbing peaks are showed in Table II. III. CELL–LIGHT INTERACTIONS Human cells vary in size in a range from 5 lm of erythrocytes (red blood cells), 20 lm of leukocytes, to tens of centimeters of neuronal axons. 62 They can therefore be either larger or smaller than the light wavelength used for bioprinting. The interaction of cells and cellladen biomaterials with light can be caused mainly by linear phenomena of refraction, reflection, absorption, emission, and scattering. 63 During bioprinting, the light refracts when it travels from the light source (by air at a particular angle) and reaches a substance (biomaterial) which presents another refractive index. The refractive index APL Bioengineering REVIEW scitation.org/journal/apb APL Bioeng. 4, 041502 (2020); doi: 10.1063/5.0022693 4, 041502-7 V CAuthor(s) 2020 determines the phase and speed of light propagation. Reflection occurs when light reflects on the surface of the biomaterial without penetrating in it, due to the difference between the refractive index of air and biomaterial. Reflection at the microscopic level will depend on the cell surface morphology and because of it will not be uniform. Light absorption is complete through intracellular and extracellular constituents and occurs as a consequence of the transition from a grown state (low energy state) to an excited state (high energy state) of a molecule. 64,65 Emission is a phenomenon that consists of energy transmission to the atoms of the biological component, in which their electrons are promoted to higher levels of energy, in some circumstances unstable, leaving holes under them. When electrons return to these holes with less energy, the excess of energy is returned as light, with different characteristics from the incident ray. Scattering occurs due to loss of directionality of light and spread of the light beam spot. This phenomenon is what regulates the light intensity distribution in the cell-laden biomaterials. Light scattering in tissues is dominated by Mie scattering. This type of scattering occurs when particles are the same size as the wavelength of light (when particles are much smaller than the wavelength, Rayleigh scattering predominates). Cells, nuclei, and organelles all fall into this classification. In addition, the lipid membranes that enclose these structures have a different refractive index than the surrounding medium (around 1.5). The dispersion of light into the tissue is motivated by the differences of the refractive index. When the difference between the medium and the cells increases, the dispersion also increases. The relationship with the wavelength can be complex, but in general, the longer the wavelength, the lower the scattering, which indeed is one of the benefits of TPP. 66 In Rayleigh scattering, subcellular components such as organelles can be a scattering component. This type of scattering depends mostly on the following parameters: the dimensions of the scattering compounds (cells or organelles in bioprinting), scattering centers and the surrounding medium refractive index variations, and the light wavelength [see Fig. 4(b)]. 67 We can consider that Rayleigh scattering is inversely proportional to the square of the wavelength of light. That means that a short wavelength (UV) will be more scattered than a long wavelength (IR). By taking scattering individually as a mechanism for optical loss during bioprinting, we can assume that the longer the wavelength is, the deeper will the light penetrate into a cell laden biomaterial sample [Fig. 4(a)]. Absorption is controlled by Beer’s law, especially when working with monochromatic beams. 68 It establishes empirically an absorption coefficient in the matter, and corelates this absorption to the wavelength of the incident light. In studies mainly on human skin tissues, due to a high interest in being an area permanently exposed to radiation, an increase in absorption and less penetration of light with shorter wavelengths (in studies with wavelengths from 300 to 800 nm) has been demonstrated. 69 This law is valid in liquid media such as cellular and intracellular interstitial fluids, and establishes an increase in absorption with the concentration of solute in the medium in cellladen biomaterials. Let us consider a sample which is in a solution, contained in a box which is transparent to the radiation of interest (monochromatic) and with uniform thickness. With I 0 being the intensity of the radiation that enters the sample and Ibeing the intensity of the radiation that goes across the sample, the transmittance T is given by T ¼I/I 0 . Beer’s law can be expressed as log10 I I0 ¼abc; where bis the thickness of the box, cis the concentration of the sample in the solution, and ais the capacity of the sample to absorb radiation. Beer’s law can be simplified as A ¼abc, with A being the absorbance, and is expressed as A¼log10 I I0  : Beer’s law says that the concentration and the absorbance are linearly proportional (when the cell thickness and the radiation wavelength remain constant). Therefore, both the absorption and the dispersion that occur both in a biological biomaterial and in cells are conditioned by the wavelength and increased in the blue region of the electromagnetic spectrum compared to the red and infrared regions. 69 Following light absorption, cells undergo a wide variety of photochemical and photophysical processes. Some cellular elements generate fluorescence (emissivity) as they are excited directly or when they get energy from another cellular element. This is defined as autofluorescence and the constituent it emits is called fluorochrome. Fluorescence, which has a half-life between 1 and 10 ns, originates from the energy transition (excited singlet state to a ground state vibrational mode). 70 Other processes that can be observed in light– biological matter interactions, apart from the autofluorescence discussed above and photochemical processes, are thermal effects, photoablation effects, photodisruption, and plasma-induced ablation. 40 Thermal effects can be considered as the result of the conversion of absorbed light energy into heat. They can be produced by pulsed and continuous wave (CW) lamps and lasers. They are nonspecific, TABLE II. Common PIs used in light-based bioprinting. Name (chemical) Abbreviation Absorbing peak (nm) Sources 20,40,50,70-Tetrabromofluorescein disodium salt Eosin Y 514 54,55 2,20-Azobis[2-methyl-n-(2-hydroxyethyl)propionamide] VA-086 385 53 Lithium phenyl-2,4,6-trimethylbenzoylphosphinate LAP 375 52 1-[4–(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propanone Irgacure 2959 257 8 Riboflavin (Vitamin B2) RF 220–240 56,57 Ruthenium with a reagent (sodium persulfate) Ru (SPS) 400–450 58–60 Poly-a-ketoester based photoinitiators Poly-a-ketoesters 330 61 APL Bioengineering REVIEW scitation.org/journal/apb APL Bioeng. 4, 041502 (2020); doi: 10.1063/5.0022693 4, 041502-8 V CAuthor(s) 2020