Hydrogels for Bioprinting: A Systematic Review of Hydrogels Synthesis, Bioprinting Parameters, and Bioprinted Structures Behavior

Nowadays, bioprinting is rapidly evolving and hydrogels are a key component for its success. In this sense, synthesis of hydrogels, as well as bioprinting process, and cross-linking of bioinks represent different challenges for the scientific community. A set of unified criteria and a common framework are missing, so multidisciplinary research teams might not efficiently share the advances and limitations of bioprinting. Although multiple combinations of materials and proportions have been used for several applications, it is still unclear the relationship between good printability of hydrogels and better medical/clinical behavior of bioprinted structures. For this reason, a PRISMA methodology was conducted in this review. Thus, 1,774 papers were retrieved from PUBMED, WOS, and SCOPUS databases. After selection, 118 papers were analyzed to extract information about materials, hydrogel synthesis, bioprinting process, and tests performed on bioprinted structures. The aim of this systematic review is to analyze materials used and their influence on the bioprinting parameters that ultimately generate tridimensional structures. Furthermore, a comparison of mechanical and cellular behavior of those bioprinted structures is presented. Finally, some conclusions and recommendations are exposed to improve reproducibility and facilitate a fair comparison of results.


INTRODUCTION
Biofabrication can be defined as a multidisciplinary research field with a combination of principles, protocols, and fabrication techniques from engineering, electronics, material science, cell biology, and biomedicine (Silva, 2018). Bioprinting is a biofabrication technique that can control deposition of cells, extracellular matrix components, and biochemical factors layer by layer to create defined structures with several kinds of materials, bioactive molecules, and cells Eswaramoorthy et al., 2019). In this sense, bioprinting allows the generation of complex structures mimicking biological cues, which increases the possibilities of tissue creation by supporting and improving other traditional techniques of tissue engineering (Moldovan, 2019). Besides, all bioprinting techniques are also constantly and rapidly evolving thanks to the advances in technical processes and bioink (hydrogel with cells in culture medium) properties (Silva, 2018).
Synthesis, bioprinting, and cross-linking of bioinks have a great impact on the generation of biological structures and especially on its mechanical and cellular behavior. Therefore, bioink is one of the most critical components of 3D bioprinting and it is intimately related to the bioprinting technique and the selected cells (Kyle et al., 2017).
Although there are many bioprinting techniques, such as laser, inkjet, droplet, stereolithography, and electrospinning (Leberfinger et al., 2019), we have focused this review on extrusion-based bioprinting. This technique is widely used by researchers, mainly due to its low cost and versatility that allow mechanical modifications and a wide range of materials, but above all high cell densities (Kyle et al., 2017;Jovic et al., 2019;Leberfinger et al., 2019). It uses the widest range of biomaterials including hydrogels, biocompatible copolymers, and cell spheroids that have many different printability properties, such as viscosity, density, or shear thinning properties, among others.
Each bioprinting procedure needs a specific set of rheological and mechanical properties of the bioink to achieve a successful bioprinted structure. On the one hand, extrusion-based process must control the properties referred to shear thinning, like viscosity and shear stress, to mitigate cell damage. On the other hand, inkjet (droplet-based) process must control surface tension and viscosity of bioinks to get a proper droplet ejection (Leberfinger et al., 2019).
In this review, natural and synthetic materials used to produce hydrogels with different features and behaviors have been analyzed. According to the bioprinting process, those important parameters involved in the bioprinter setting have Post-printing FIGURE 1 | Schematic representation of three bioprinting stages: pre-printing (material selection, hydrogel synthesis, and bioink generation), extrusion-based bioprinting (parameters and cross-linking methods), and post-printing analysis (cellular and mechanical tests).
been exposed Sodupe-Ortega et al., 2018). Finally, tests for validation of bioprinted structures have been included and grouped into two blocks: cell and mechanical properties. Hence, the main goal of this systematic review is to analyze the impact of pre-printing stage (materials selection, hydrogel synthesis, and bioink generation) and extrusion-based bioprinting process (both bioprinting parameters and cross-linking methods) on post-printing results of the bioprinted structures (cell tests and mechanical properties) (Figure 1). their importance and/or functionalities, including concentration and viscosity; (3) printing settings gathers cartridge temperature, bed temperature, printing pressure, and printing speed; (4) crosslinking methods summarizes cross-linking process depending on its type and characteristics, and finally (5) validation tests registers types of cellular viability and mechanical tests. Specifically, main materials were classified as synthetic or natural, material name, cell-laden or post-printing cell addition, and according to its type of cross-linking (thermal, chemical, or physical). Furthermore, structural material was subdivided on material name and cross-linking type. Finally, sacrificial material was defined by material name, cross-linking type, and removal process. Papers were individually assigned to eight independent reviewers to be read in detail to extract available data.

Overall Findings
In all, 1,774 abstracts were found using the search string (Bioprinting AND Hydrogel) in three databases (PUBMED, WOS, and SCOPUS). Nine hundred and eleven papers were screened after removing duplicated, 783 of them excluded according to selection criteria, and 128 revised in full text. From those, 118 were finally considered for the review analysis (Figure 2). Other Alginate Gelatin GelMa Articles FIGURE 3 | Research trend in hydrogels for bioprinting using a bar chart (papers per year) combined with a stacked area chart (total uses of a material per year). The three most used materials (alginate, gelatin, and GelMA) are shown individually while the rest of materials are grouped in the category "other." It is important to note that some materials are used in more than one paper, so some stacked areas overtake its corresponding bar chart. Figure 3 shows an upward trend in the number of published papers during the last decade. There were no papers prior to 2009, only one published in this year, and an increase from 2015 onwards. However, 2017 meant a year of stagnation that could be due to an increase of research studies focused on the creation of more complex tissues, organoids, drug testing, and lab-on-a chip Ma X. et al., 2018;Reid et al., 2019), subjects that are out of the scope of this review. In 2018, the research community came back to the creation of new materials and structures. These studies could provide better results in terms of cellular viability, histo-differentiation of complex tissues and the formation of complex structures. On the other hand, a crucial point could rely on a higher accessibility to low-cost or home-made bioprinters (Ozbolat et al., 2017). Additionally, Figure 3 shows annual papers regarding the three most used materials as main component (alginate, gelatin, and GelMA) whereas Furthermore, the rest of materials was grouped in the category "other." Material trend is similar to year trend, with a few differences. In 2014 and 2017, total number of used materials is lower in comparison to previous years, which means that fewer papers used more than one material. On the other hand, papers published in the first trimester of 2019 showed a rising forecast for this year. It is difficult to make an approach to what kind of papers will be published in upcoming years, but everything indicates that new synthetic materials and mixtures of other complex materials could grow up (Ashammakhi et al., 2019).

Journals Analysis
In this section, a classification of journals has been made to analyze what type of publications deal with our topics. Four main categories and other four subcategories from JCR or SJR were used to group journals. The main categories are: (1) material, journals of chemical/material-centered issues; (2) cellular, journals focused on the cellular/histological/biochemical topic; (3) engineering, journals focused on the technical and/or mechanical issues, and (4) multidisciplinary, journals that allow multiple topics. Additionally, the four combined subcategories are: (1) engineering/material, (2) cellular/material, (3) engineering/cellular, and (4) engineering/material/cellular. Figure 4 shows distribution of all 50 journals. Material contains the highest number of journals with 17 (29 papers). "Applied Materials & Interfaces" and "Biomaterials Science & Engineering" are the two most common journals in this category with 6 and 4 papers, respectively. All combinations of the material category reach 33 journals (86 papers). The remaining categories contain fewer journals. Cellular and engineering categories includ only four and seven journals (5 and 8 papers), respectively. "Scientific Reports" and "Plos One" are two journals associated to multidisciplinary category with 8 and 3 papers, respectively. "Journal of nanotechnology in Engineering and Medicine" is the only journal associated to the engineering/material/cellular category with one paper. Finally, the material/engineering subcategory is by far the most common with 12 journals (50 papers). In this subcategory, most papers are published by "Biofabrication" (23 papers) and "Acta Biomateralia" (8 papers).

Natural vs. Synthetic
Natural and synthetic polymers can be considered as a broad cataloging of materials to synthesize hydrogels. In this sense, natural polymers are defined as bio-derived materials present in nature that can be extracted using physical or chemical methods (e.g., gelatin, alginate, or chitosan). On the other hand, those human-made polymers are named synthetic and are usually classified into plastics, elastomers, and synthetic fibers (Ouellette and Rawn, 2015). In general, authors use natural materials more than synthetic ( Figure 5) due to their better biological properties (Silva, 2018) at the expense of the best mechanical properties of synthetic materials (Abelardo, 2018). A chronological classification of papers show few studies between 2009 and 2014 (10 out of 118) followed by a huge increment in the use of natural materials in 2015 (89% of all papers in this year). After that, natural materials clearly have a downward trend in favor of synthetic materials that reached 36, 42, 48, and 85% throughout 2016, 2017, 2018, and 2019, respectively. Maybe this trend is due to high biocompatibility and affordable price of natural materials during the first years of bioprinting. However, rheological properties of natural materials are not the best for printability, and mechanical properties of the bioprinted structures are only appropriate for some applications. For this reason, once these natural materials reached their technical and biological limitations, the use of synthetic materials began to rise in order to solve these former problems.

Materials
The selection of materials is one of the most important decisions for the hydrogel generation. They have a great impact on biocompatibility and cellular viability as well as the mechanical behavior of the bioprinted structures, what is mandatory for a good bioprinting result. In this sense, all 118 papers used 34 different materials, although some chemical modifications were performed in some of them (e.g., alginate with norbornene) that are not considered as different material in this review. Specifically, the most common materials was alginate appearing in 58 papers followed by gelatin (26), GelMA (25), hyaluronic acid (16), and polyethylene glycol (PEG) with its chemical modifications (16). 1 2* 1 FIGURE 6 | Combination of the 10 most used materials in hydrogels among them. The materials are shown in the external ring (total of papers and name). The middle ring segments represent one-material hydrogels and hydrogels, marked with (*), that are a combination of this material with non-selected materials (one-material papers mixed with non-selected materials papers). Inner lines represent hydrogels with two of the selected materials, but in some case other non-selected materials can be included (number of papers next to each inner line).
Although complex tissues and organs generation are out of the scope of this review, we consider interesting to include some information about those papers that define its biological purpose (61 out of 118). Most of them have a low frequency or a generic soft tissue use, but cartilage (22 papers) and vascular (9) usually use alginate (12 and 6 papers) and GelMA (9 and 2 papers), respectively.
In this review, the 10 most used materials were selected for a detailed analysis. Figure 6 shows the combination of these 10 materials in different hydrogels (103 papers). However, in order to make clear this figure, those papers that use hydrogels with more than two of these selected materials (12) and papers that use materials different of these ten (3) were excluded from this figure and analyzed later in this section.
Alginate is the most used material in bioprinting appearing in a total of 58 papers. It is used with the other selected materials in pairs in 28 papers, with more than two selected materials in 10 papers, alone in 10 papers and with other chemical modifications in other 10 papers. Some of these interesting chemical modifications that improve its characteristics are: oxidized alginate (ox-alg)  which gives alginate a faster degradation and more reactive groups (Boontheekul et al., 2005), methacrylated alginate (MeAlg/AlgMA) which allows photo-polymerization thanks to its methacryloyl groups (García-Lizarribar et al., 2018;Ji et al., 2019), both oxidized and methacrylated together (ox-MeAlg) , and alginate with norbornene (alg-norb)  which provides alginate an ultrafast light-triggered cross-linking. Firstly, gelatin is the hydrogel which appears more times with alginate in just two materials combinations He et al., 2016;Wang et al., 2016;Ding et al., 2017Ding et al., , 2018Giuseppe et al., 2017;Aljohani et al., 2018;Berg et al., 2018;Gao et al., 2018;Li et al., 2018c). It is important to note that alginate/gelatin combination allows hydrogel to have good rheological properties (alginate) with proper thermoresponsive behavior (gelatin). Secondly, different types of cellulose have been used in combination with alginate: cellulose nanocrystals (CNC) , methylcellulose Schütz et al., 2017;Ahlfeld et al., 2018;Gonzalez-Fernandez et al., 2019), and nanofibrillated cellulose (NFC) Apelgren et al., 2017). Gelatin and cellulose are followed by GelMA Zhang X. et al., 2018;Kosik-Kozioł et al., 2019;Krishnamoorthy et al., 2019), PEGderived Yu et al., 2018), agarose López-Marcial et al., 2018), hyaluronic acid , and collagen  to produce hydrogels of two materials. The rest of studies uses three and four-materials hydrogels with 7 and 3 papers, respectively. It is particularly interesting that 5 out of these 10 papers utilized alginate and GelMA with other different components: hyaluronic acid methacrylated , PEG , PEGDA , PEDGA/cellulose (García-Lizarribar et al., 2018), and PEGMA/agarose . In the same way, four hydrogels are composed by alginate and collagen with different components: gelatin , chitosan , agarose , and gelatin/chitosan . Finally, other hydrogel is composed by alginate, hyaluronic acid, and cellulose . Despite the lack of good rheological properties of natural materials, alginate is one of the best natural polymer in rheology. Additionally, good biocompatibility and its facility to form reticular structures using Ca 2+ ions have popularized its use in bioprinting (Lee and Mooney, 2012).
Gelatin is the second most used material with 26 papers. It is a component of hydrogels with other selected materials (17 papers) and with other combinations (7) mainly due to its poor rheological properties. Maybe for this reason, it is used alone in just two papers Tijore et al., 2018b). In other studies, several modifications have been performed to enhance its properties. So, furfuryl-gelatin (f-gelatin) allows cross-linking with visible light (AnilKumar et al., 2019), but other studies combined with hyaluronic acid Shin et al., 2018;AnilKumar et al., 2019), GelMA (McBeth et al., 2017), cellulose , PEGDA , and PEG . Additionally, it is also used in threematerials Haring et al., 2019) and four-materials  hydrogels. Specifically, two important features of gelatin can be remarked: cellular adhesion, mainly due to presence of RGD (arginine-glycine-aspartate) sequences, and thermoresponsive behavior that sustains the use of gelatin as supporting material.
GelMA is the third material and the first synthetic one, compose by gelatin with methacrylated groups . In general, GelMA has excellent rheological properties that improve printability, shape fidelity, and stability of the hydrogel due to UV photopolymerization of its methacrylated groups . For this reason, modifications of this material are unusual and only Haring et al. (2019) modifieds GelMA with a dopamine molecule. Hence, it appears in 25 papers, it is combined with other selected material in 8 papers, used alone in othersix Billiet et al., 2014;Ersumo et al., 2016;Gu et al., 2018;Pepelanova et al., 2018;Zhou et al., 2019), with more than two of the selected materials, and used with other different materials in 5 papers. Alginate is the most common combination of GelMA appearing in 4, 3, and 2 papers for two-, three-, and four-material hydrogels, respectively (further details in the alginate section). Other common materials used with GelMA are hyaluronic acid Noh et al., 2019), gelatin (McBeth et al., 2017, and collagen . The rest of papers combines GelMA with pluronic Suntornnond et al., 2017), carrageenan (Li et al., 2018b,c), gellan gum , and polyisocyanide (PIC) (Celikkin et al., 2018).
Hyaluronic acid (HA) and its derived materials are quite used being the fourth material in the list with 16 papers. It is an anionic, non-sulfated glycosaminoglycan that is present in connective and neural tissues as well as it is one of the major components of the skin. For this reason, HA is mostly used in skin tissue engineering. It appears alone in five papers Stichler et al., 2017;Lee et al., 2018;Wang et al., 2018;Kiyotake et al., 2019). Furthermore, there are three studies that use HA combined with gelatin Shin et al., 2018;AnilKumar et al., 2019) but other authors use GelMA Noh et al., 2019), alginate/cellulose , and chitosan . This material is modified in half of the papers, obtaining: hyaluronic acid methacrylated (HAMA) Mouser et al., 2017), acrylated-HA and tyramine-conjugated HA , dopamine-conjugated HA , and thiol functionalized HA . Concretely, HAMA provides great tunability for specific uses at different methacrylation degrees (Xia et al., 2017).
Cellulose is the following material with 15 papers. Cellulose fibers are obtained from natural resources and are widely used in bioprinting to improve mechanical properties of hydrogels. Depending on the polymerization degree, the length of its polymeric chain, hydrogels with cellulose can have from high tensile strength (long chains) to solubility properties (short chains). It is usually modified replacing some hydroxyl groups with methoxy groups forming methylcellulose. Specifically, cellulose is used alone in three papers (Béduer et al., 2018;Cochis et al., 2018;Contessi Negrini et al., 2018). Additionally, it is combined with alginate (detailed above), gelatin , or hyaluronic acid , being the two remaining papers combinations of alginate/cellulose with hyaluronic acid  and GelMA/PEGDA (García-Lizarribar et al., 2018).
Collagen is other popular material in bioprinting appearing in 12 papers that is the main component of the extracellular matrix, e.g., connective tissues as cartilage. In this review, collagen is used alone in four papers Kim et al., 2016;Ren et al., 2016;Ahn et al., 2017)and appears in combination with alginate , GelMA , and agarose (Köpf et al., 2016). The rest of combinations with more material was described in previous sections.
Polyethylene glycol (PEG) and its derivatives PEG diacrylated (PEGDA) and PEG methacrylated (PEGMA) are used in the included studies in 6, 8, and 2 papers, respectively. PEG is a synthetic material formed by polymerization of ethylene oxide, highly valuable for its hydrophilicity that facilitates exchange of cell's nutrients and waste. Despite the fact that PEG is used alone in one paper with norbornene groups  and combined with gelatin , and alginate/GelMA  in other studies, It also appears with silk fibroin (Zheng et al., 2018), poly(N -(2-hydroxypropyl) methacrylamide lactate) methacrylated (pHPMA-lactate) , and polycaprolactone-diacrylated (PCL-DA) . It is important to note that PEG-derived materials allow hydrogel to be photo-crosslinked, which provides better mechanical stability after bioprinting. Specifically, PEGDA presents high hydrophilicity, a bioinert structure, and lack of toxic or immunogenic responses (Zalipsky and Harris, 1997). PEGDA is used alone (Schmieg et al., 2018) and combined with alginate  and gelatin . It also appears with alginate/GelMA  and with alginate/GelMA/cellulose (García-Lizarribar et al., 2018). Additionally, it is combined with gellan gum , carbomer hydrogel , and laponite (Peak et al., 2018). Finally, PEGMA is used in two papers, one of them with alginate and a modification of PEGMA that includes a fibrinogen molecule  and the other one with alginate/GelMA/agarose .
Chitosan is the last material included in this detailed analysis with six papers. It is a natural-obtained and biodegradable polymer very similar to other extracellular matrix components that provides great cellular viability. However, its low mechanical properties and its slow gelation make chitosan a material rarely used alone. For this reason, to solve these poor mechanical properties it is usually combined with other materials as hyaluronic acid , alginate/collagen , collagen/agarose , and alginate/gelatin/collagen . Chitosan also appears combined with silk  and modified with hydroxybutil groups to improve its water solubility or with oxidized chondroitin sulfate to improve its mechanical properties .

Hydrogel Properties Concentration
Maybe, concentration is the most important parameter of hydrogels for to reasons: to assure reproducibility of the experiment, and to increase printability of the hydrogel. The importance of this parameter is clear when 89.3% of all papers define the amount of each material present in the hydrogel mixture accurately. Most papers reveal researchers are trying to develop new materials/mixtures or modifying former hydrogels to get new specific properties.
In the material section, three mains polymers stand out over the rest: alginate, gelatin and GelMA (Table 1). Alginate is the most used component in hydrogel mixtures with 122 different concentrations in 52 different papers. In general, the most used concentration range is 2-4% w/v (35 papers). Specifically, the frequency of use for 2, 3, and 4% w/v of alginate is 15, 14, and 12 papers, respectively. The rest of concentration varies between 1% w/v (10 papers) and 5% w/v (7 papers). Although standard concentrations of alginate are up to 5% w/v, Markstedt et al. (2015) and Nguyen et al. (2017) use 10, 20, 30 and 40% w/v of alginate mixed with NFC and Aljohani et al. (2018) uses 18% w/v of alginate mixed with 4% w/v of gelatin and 12% w/v of agar. In summary, the range of concentration 2-4% gives alginate its better viscosity for bioprinting as it will be seen in the next section .
Gelatin is the second material with 45 concentrations in 22 papers, but with heterogeneity of values. Concentrations are distributed in a range of 1-20%, being 5% w/v the most common value (5 papers) and 10% w/v (4 papers), or 15% w/v (2 papers) other common values. It is noted that gelatin provides good thermoresponsive properties to hydrogel, but its concentration is highly dependent on the bioink application.

Viscosity
This parameter can be considered an important factor for hydrogel printability. As it is known, viscosity indicates fluidity Dots shows concentration of these materials which applies in each reference.
and for this reason it is very important for hydrogel extrusion. So, the more the viscous, the more the inner pressure of hydrogel during the extrusion process and increased cell damage. Pepelanova et al. (2018) proposes shear-thinning hydrogels to get an easy filament deposition during the printing process and a high shape fidelity after printing (low shear stress). However, only 12.1% of analyzed papers details viscosity or perform rheological tests of hydrogels. He et al. (2016) performs tests with a mixture of alginate/gelatin to established a "300-30,000 cps" as the optimum range of viscosities for this kind of hydrogels. Other tests performed by Campbell et al. (2015) with a mixture of collagen/alginate recommend a viscosity higher than "2,000 cps" to maintain shape fidelity. Raddatz et al. (2018) studies some alginate concentrations and their viscosities which vary from 13.5 mPa·s (0.5% w/v) to 2,156 mPa·s (4% w/v). As said before, these viscosities are obtained with the most used concentration of alginate. Hence, according to these range of concentrations that change viscosity of hydrogel, stiffness can be modified for a proper balance between good shape fidelity (harder hydrogels) and better printability (softer hydrogels). Finally, other authors show results of hydrogel behavior in graphics, but do not provide specific values of viscosity obtained from a rheological study of a non-Newtonian fluid Das et al., 2015;Pepelanova et al., 2018;Aydogdu et al., 2019;Jeon et al., 2019).

Bioprinting Parameters
Bioprinting parameters can be defined as those bioprinter settings (firmware inputs) needed to properly produce bioprinted structures. In this sense, only a specific range of values are adequate for bioprinting and its selection is a key factor to obtain viable bioprinted structures. However, these values are highly dependent on the hydrogel composition, so they should be carefully selected in each case. An important feature of hydrogel is printability that was no analyzed in this review because it is rarely used Gao et al., 2018). It is defined in three levels of meaning according to viscosity (shear thinning property), curation (crosslinking), and biofabrication window (range of bioprinting parameters) . Some objective metrics to measure printability and the printability window are Pr , extrudability, extrusion uniformity, and structural integrity , but only few papers used them. For this reason, the most relevant bioprinting parameters have been selected to be analyzed in this review: cartridge temperature, bed temperature, printing pressure and printing speed.

Cartridge temperature
In this review, cartridge temperature is defined as the internal temperature of the cartridge/printhead and it is inversely related to hydrogel viscosity. Thus, the higher the temperature, the lower the viscosity, inducing a less shear stress decreasing cell damage. Although we are using this terminology for better understanding, printing temperature it is commonly used in many papers to refer the same concept. Only 45% of all the analyzed papers indicate their cartridge temperature. In this sense, up to 65 papers lack this critical parameter in its methodology. We grouped papers in five different ranges: below 20, 20-30, 30-40, above 40 • C, and room temperature.
Other authors define cartridge temperature as room temperature. In our opinion, this definition can lead to misunderstandings due to the existence of different regulatory frameworks for this concept (e.g., 20-25 • C for the USP-NF or 15-25 • C for the European Pharmacopeia). In this review, there are 6 papers that use room temperature for their cartridge temperature Kundu et al., 2015;Li et al., 2017;Zheng et al., 2018;AnilKumar et al., 2019;Ji et al., 2019). Hence, this indistinct setting of temperatures makes reproducibility of their experiments difficult, since small variations of this parameter can significantly modify the hydrogel behavior during the extrusion process as commented by Mouser et al. (2016).
Some other studies use more than one temperature or an extra wide range of temperatures that do not fit in our selected ranges. First, Mouser et al. (2016)
In order to understand the importance of heating systems, Ahn et al. (2017) perform some interesting experiments that include not only a heated bed, but even an upper heated system over the nozzle. Specifically, they use a 23 • C non-heated bed, a 30 • C heated bed, a 32 • C upper heating system, and a 36 • C heated bed with upper heating system. These results are quite interesting, mainly because they enhance the importance of bed temperature, but additionally they propose a broad heated printing volume that can be controlled using a closed chamber or a specific heating system. They conclude that nonheated bed obtains the worst shape fidelity, but a combination of heated bed with upper heated system improved the shape fidelity of the bioprinted structures and got the best results in its experiments.

Printing pressure
Many authors (55) do not inform about printing pressure used during their experiments or use the indistinct term low pressure . From our point of view, this parameter is critical for a proper management of live cells during bioprinting. Additionally, it is important to note that there is no pressure unit defined for bioprinting. Although most studies use Pa, other studies use bar, psi, N/mm 2  or mTorr  units. In order to compare all the papers, all units were converted to SI (kPa) in this review.
Finally, Aljohani et al. (2018) use 0.5 and 1 Pa for a gelatin/alginate/agar hydrogel. This is a very low pressure compared with the rest of papers (>5 kPa). On the other hand, Narayanan et al. (2016) use a cell-laden alginate/PLA hydrogel at 2,000 kPa, while Wei et al. (2019) use 2.2, 2.8, 4.3, and 4.7·10 5 kPa to print an alginate hydrogel with post-printing cell addition. It is important to note that those ranges of pressure are far away from commonly used with alginate hydrogels (10-300 kPa). However, printing pressure is no longer a critical parameter of bioprinting using post-printing cell addition. In cell laden bioprinting the pressure ranges are in accordance to cellular viability, where the data obtained corroborate this affirmation. In all papers in which there are values of printing pressure and viability, 75.3% of pressure values have a viability over 80%.

Printing speed
Printing speed (X-Y movement) is important because it is directly related with the total bioprinting time. Aditionally, extrusionbased controls the hydrogel flow (filament width) using mainly printing speed and printing pressure. So, printing speed appears in 65 papers with 87 different velocities that vary from 0.2 to 150 mm/s. By taking a closer look, 91% of values are in the range of 1-30 mm/s where 57% of speeds are below 10 mm/s. In fact, the most used speed is 10 mm/s (13 entries) Wang et al., 2016;Nguyen et al., 2017;Ahlfeld et al., 2018; TABLE 2 | Pressure ranges for all papers that studied this setting parameter. There is a printing speed that stands out because of its high value. It is used by Gao et al. (2018) with gelatin/alginate hydrogels at 150 mm/s.

Cross-Linking Methods
Cross-linking is usually a post-printing procedure that consists of the modification of the internal structure of the printed hydrogel to harden it and to achieve the expected mechanical properties of the bioprinted structure. It can be performed in three different ways depending on its reaction trigger: thermal (controlled by temperatures changes), chemical (controlled by the addition of reacting agents), or physical (triggered by physical procedures, usually UV light). In this sense, hydrogel composition determines the cross-linking type to use.  Figure 7 shows all cross-linking methods of the three most used materials: alginate, gelatin, and GelMA with its combinations. Additionally, Table 3 summarizes all analyzed studies with these three materials. Thermal cross-linking is commonly used in gelatin or agarose hydrogels (16 papers). From those 16 papers, six of them perform cross-linking at 37 • C McBeth et al., 2017;Law et al., 2018;Tijore et al., 2018a,b;Zheng et al., 2018) and three use room temperature (Köpf et al., 2016;Lin et al., 2016;Berg et al., 2018). The rest use thermal cross-linking without specifying temperature Giuseppe et al., 2017;Bandyopadhyay et al., 2018;Chen et al., 2018;Cochis et al., 2018;Xu X. et al., 2018). On the other hand, in alginate/gelatin hydrogels Berg et al. (2018) uses room temperature, He et al. (2016) uses a cool substrate, and Bandyopadhyay et al. (2018) and Giuseppe et al. (2017) use thermal cross-linking but without specific temperature. For gelatin hydrogels Blaeser et al. (2013) and Tijore et al. (2018a) use 37 • C for thermal cross-linking and Xu X. et al. (2018) do not give any detail on how the thermal cross-linking performs.
Chemical cross-linking is commonly used to harden alginate, chitosan, or gelatin, but it is used with other materials too (69 papers). In general, solution with Ca 2+ cations are used to trigger the cross-linking reaction. In this sense, 49 out of 69 papers utilize different concentrations of CaCl 2 solution to perform the chemical cross-linking. Concentrations vary from 10 mM to 0.5 M or from 1 to 10% w/v. However, other Ca 2+ solutes are used to perform chemical cross-linking. Specifically, Gao et al. (2018) uses CaSO 4 or Wei et al. (2019), and Kundu et al. (2015) use NaCl 2 , also Freeman and Kelly (2017) uses CaCO 3 and CaSO 4 . Although exposition time of the crosslinking agent is quite relevant, its definition is infrequent and, in some cases, highly different. So, Ahlfeld et al. (2018) uses 10 min while Raddatz et al. (2018) uses a 30 s mist. In order to clarify this issue for alginate Naghieh et al. (2018) performs an analysis of the cross-linking effect of CaCl 2 at 0, 2, 4, and 24 h of exposure time. Chemical cross-linking is mostly done to alginate (52 papers) with CaCl 2 solution (46 papers), and detailed concentrations can be seen in Figure 6. Other (non-Ca 2+ ) solutions are used in two papers Aydogdu et al., 2019) which use hydrazine and NaOH, respectively, and other two studies do not provide information Yoon et al., 2019). Other specific cross-linking agents are genipin Kim et al., 2016), mTgase (Tijore et al., 2018b) or 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) , and MAL-PEG-MAL  for gelatin, or different solute for other materials like thrombin , mushroom tyrosinase , and oxidative reactions .
Finally, physical cross-linking is usually performed with the exposure of the bioprinted structures to UV light. In this sense, GelMA is the most used material with this kind of crosslinking, but all materials modified with methacrylated groups can be photo-crosslinked, such as HAMA, AlgMA or PEGMA. Among all papers (41) with this kind of cross-linking, only two of them do not use UV: AnilKumar et al. (2019) that use visible light with Rose Bengal and Riboflavin as photoinitiatorand Das et al. (2015) that use sonication procedures. In general, physical cross-linking needs a photoinitiator that triggers the reaction and some usually agents are Irgacure D-2959 or Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The most used UV wavelength is 365 nm (18 papers) followed by 405 nm (3 papers) and 312 nm (1 paper). On the other hand, Mouser et al. (2017) and Bertassoni et al. (2014) use 300-600 and 360-480 nm wavelength ranges instead of specific values, respectively. Also, Celikkin et al. (2018) use physical crosslinking without giving any kind of information. An important caution with physical cross-linking is the UV radiation effects on cells that depends mainly on wavelength and exposure time. In this sense, in 14 papers there is not information of the used wavelength. Table 3 shows that most of physical cross-linking is made to GelMA (24 papers), three papers perform physical cross-linking to alginate Jeon et al., 2019;Ji et al., 2019) and one paper use it with gelatin (AnilKumar et al., 2019). Finally, UV light power is used in a range from 2 mW to 6 W, while the exposure time varies from 10 s to 30 min.

Cellular Tests
Currently, cellular viability is one of the most common features to assess bioprinted structures that must be used on patients or drug testing. Here, post-printing analysis are focused on cellular and mechanical tests, but several biological measures appear during this review: gene expression (25 papers) that is usually related to cellular differentiation, and cell morphology (51 papers) that controls qualitatively cell shape or clustering. FIGURE 7 | Cross-linking used for the three most common materials and its combinations. Blue, yellow, and red circles represent chemical, physical and thermal cross-linking, respectively. Superposed circles indicate that two cross-linking classifications have been combined. Each circle contains a classification codification (upper code) and the number of papers for the corresponding code combination (lower number). Chemical cross-linking has been chosen as primary classification, therefore papers not using it have been codified under C7 for graphical representation purposes and no papers are allocated to this code alone, e.g., alginate C2/13 -P4/1 (blue and yellow circles superposed) indicates that 13 papers used C2 ([100-500] mM Ca 2+ solution) to cross-link alginate and another paper used C2 and P4 (UV light-no data of wavelength) to perform the cross-linking. 3 | The three most used materials (alginate, gelatin, GelMA, and its combinations in pairs) with its different cross-linking methods.

Material Chemical Physical Thermal References
Alginate C1 Campbell et al., 2015;Markstedt et al., 2015;Narayanan et al., 2016;Freeman and Kelly, 2017;Naghieh et al., 2018; C1/C2 Khalil and Sun, 2009 C1/C5 Kundu et al., 2015C2 Jia et al., 2014Izadifar et al., 2016;Apelgren et al., 2017;Kosik-Kozioł et al., 2017;Nguyen et al., 2017;Polley et al., 2017;Schütz et al., 2017;Ahlfeld et al., 2018;Habib et al., 2018;He et al., 2018;Maiullari et al., 2018;Yu et al., 2018;   On the one hand, the most of gene expression studies used osteogenic-and chondrogenic-related markers such as cartilage formation genes (12 papers): ACAN (aggrecan), COL1, COL2, COL10 (collagen type I, II, X), or SOX-9. On the other hand, 37 papers conclude that there no morphological differences after bioprinting in comparison to a 2D culture and 19 papers clarify that the increasing of the stiffness, due to the increasing of the viscosity and concentration of the material or modifications of crosslinking parameters, tend cells to adopt a round shape losing its functionality (Prasad and Alizadeh, 2019). In this sense, two kinds of tests are commonly performed to determine the live/dead proportion of cells (viability tests) and its metabolic activity (metabolic tests). Table 4 compiles cellular tests grouped in these two categories, including reagents and techniques. In total, 19 Live/Dead assay kits to measure the viability of bioprinted structures have been used, but none of these kits mention its composition. So, we could not perform any kind of analysis in this category. On the other hand, calcein AM has been used in 60 papers for staining alive cells and in combination with two complementary compounds: Ethidium homodimer (37 papers) or propidium iodide (23 papers) as an orange-red and red stain for dead cells, respectively (see Table 4). Additionally, propidium iodide also appears alone as viability cell marker in 10 papers, combined with fluorescein diacetate in four papers Campbell et al., 2015;Campos et al., 2015;Jeon et al., 2019), combined with acridine orange (2 papers) (Lin et al., 2016;Kosik-Kozioł et al., 2017), and combined with other unspecified agents (4 papers) (Duarte Köpf et al., 2016;Giuseppe et al., 2017;Law et al., 2018). Alamar blue is a cellular viability reagent used in eight papers for staining living cells in blue color with metabolic reduction (O'Brien et al., 2000). Another test used is the trypan blue exclusion test (Béduer et al., 2018) that only stain cells with altered cell membrane, marking dead cells. It is measured as the ratio of non-stained to total cells by optical microscopy. Finally, Hoechst 33342 is a fluorescence probe that binds to the nucleus of alive cells (AnilKumar et al., 2019).
Although several studies present results of specific gene expression, cellular differentiation, or morphology, we have focused our interest in viability results. To do this, five different groups according to periods of viability measurements are established: at 0-(just after printing), 1-, 3-, 7-, and 21-days. However, data heterogeneity does not allow a statistical inference. It is important to note that comparison among studies could be relatively unfair due to many different conditions, such as: variations in bioprinted structures (grid, tubular scaffolds, discs,. . . ), different cell lines survival characteristics (fibroblast vs. HUVECs), different measure periods (e.g., 2, 11, or 28 days), or different assay kits. Table 5 shows papers (97) that study cellular viability including material, cell type and viability. According to our analysis, the total number of papers for each group is the following: 36 papers for 0-day, 37 papers for 1-day, 25 papers for 3-days, 35 papers for 7-days, and 24 papers for 21-days. However, some papers' time points did not fit in our five groups, and they have been grouped in the closet category as follows: 0-day and 1day groups fit perfectly but in 3-, 7-, and 21-day are counted from 2 to 4, 5 to 11 and 12 to 28 days, respectively. In this sense, 0-, 1-, and 7-days groups are the most used by authors, although only few of them use all groups in their studies Li et al., 2018c).
As mentioned before, heterogeneity of studies (different materials, cross-linking methods, temperatures, and cell lines, among others) diminishes importance of mean viability (82.70%) obtained at 0-day. Maybe, a detailed analysis of viability by materials or cell lines could be significant, reducing the variability. For this reason, the three most used materials (alginate, gelatin, and GelMA) and two of their combinations (alginate with GelMA, and alginate with gelatin) have been selected in Figure 8 to compare their viabilities. Results show that most of the mean cellular viabilities are up to 80% with a 0-day mean viability over 83%. This could indicate that cellular viability just after printing, has been partly sorted out. After that, 1-day viability decreases in most cases, being more accused in the alginate-GelMA combination (58.50% from 83.05%) with the most important exception of gelatin that increases (94.90% from 74.60%). Maybe, this decreasing trend could be due to nutritional or environmental conditions of cells during this first stage. In this sense, during these first hours after the bioprinting process, cells must adapt to a new environment which in some cases, stops their growth while other provoke their death. After this stressing period, 3-days group usually increases its cell viability, showing an adaptation to the new material in which they are embedded. Moreover, similar trend is found in 7-days group. Finally, after 21-days every material behaves in its own way, in GelMA and alginate/gelatin cellular viability continue its increase, while in alginate, gelatin and alginate/GelMA decrease. On the contrary, gelatin startes with a very high viability at 0-and 1-day, but after 3-days decreases, reaching its minimal 21-days later. It is noticeable that GelMA shows the best cellular viability, despite the fact that it is supposed to have the worst one even though it is synthetic (Abelardo, 2018).
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org  Wu Y. et al., 2018 *Khalil andJia et al., 2014;Nguyen et al., 2017;Ahlfeld et al., 2018;Hafeez et al., 2018;Raddatz et al., 2018;Jeon et al., 2019;Yoon et al., Ahn et al., 2017;Stichler et al., 2017;Cochis et al., 2018;Xu C. et al., 2018;Zheng et al., 2018;Li et al., 2019 All studies are included in the closest period when they used a different timing. References marked with (*) make viability assay but use multiple cell lines or conditions, or not provide any value. decisive too. In general, multipotent cells (e.g., Mesenchymal Stem Cells) are selected due to their differentiation potential. A total of 46 different cell lines are used in these papers. It is important to note that many papers are only qualifying cell viability using a fuzzy scale, such as good, regular or bad survival and other measures not related to viability, such as cell distribution. Figure 9 shows that Mesenchymal Stem Cells (MSCs) are the most used cells (16 papers), but only some of these papers provide quantitative cell viability (9) (Duarte Campos et al., 2013; Campos et al., 2015;Daly et al., 2016a;Freeman and Kelly, 2017;Giuseppe et al., 2017;Schütz et al., 2017;Gonzalez-Fernandez et al., 2019;Ji et al., 2019;Xin et al., 2019) and the remaining reports provided a qualitative value (6) Stichler et al., 2017;Ahlfeld et al., 2018;Tijore et al., 2018b;Zheng et al., 2018;Jeon et al., 2019) with the only exception of AnilKumar et al. (2019) that does not perform any kind of test. The widely use of MSCs could be due to their ability to be differentiated into bone, cartilage, muscle, marrow, ligament and connective tissue cells (Caplan, 2011).
Ultimate tensile strength (UTS) is the maximum stress that a material can withstand while being stretched or pulled before breaking. This property appears in 12 papers and nine of them use alginate in their hydrogel. In this case, when alginate is used (mixed or not with another component) UTS values are within 40-500 kPa range with concentrations of 2-5%w/v.
Finally, yield stress is little studied (3 papers) and most of them provide graphical results. Bandyopadhyay et al. (2018) obtain a yield stress of 3,350 kPa using an alginate/gelatin/collagen hydrogel. Lastly, Li et al. (2018b) study the ultimate shear stress (12 kPa) in a GelMA/Carrageenan hydrogel.
As exposed before, all these properties are highly dependent of pre-and printing process. In this sense, concentration, and crosslinking parameters, are the parameters that affect the most to mechanical stability of bioprinted structure. This is evident when the main modifications in studies relating mechanical properties are made with changes in these parameters. For example, changes in concentration to analyze how it affects the mechanical properties are made in 11 papers, 4 of them young modulus is observed Mouser et al., 2016;Naghieh et al., 2018;Krishnamoorthy et al., 2019), in 4 papers UTS Yang et al., 2017;Bandyopadhyay et al., 2018;He et al., 2018), and in 3 compressive modulus Schuurman et al., 2013;Giuseppe et al., 2017). All of them obtain the same conclusion: mechanical stability rises when the concentration increases. Mechanical properties are also influenced by crosslinking parameters. In this sense Giuseppe et al. (2017) proposed a 15 min time of exposition of Ca 2+ for alginate/gelatin blend after measuring different time point and analyze it compressive modulus, noting that with higher time of exposition modulus increases. Also, Kang et al. (2017) made modifications in their photoinitiator, its concentration and power of UV irradiated. In the same way as before, the higher the concentration and the irradiation power the higher the stiffness.

CONCLUSIONS
This article is a systematic review of hydrogel implications during bioprinting process, including a descriptive statistical analysis of materials, bioprinting parameters, mechanical tests, and viability assays.
Maybe, the omission of relevant bioprinting parameters is one of the most important drawbacks detected in most of the papers, making the reproducibility of their results difficult. Obviously, many research fields are involved in bioprinting, so it is possible that authors focused their interest on those parameters directly related to its scope, playing down the rest of essential information. For this reason, we propose some suggestions to solve this problem in section "Recommendations and future works." First, alginate is the most commonly used material followed by gelatin and GelMA. For this reason, the concentration and crosslinking analysis are highly influenced by these three materials.
Here, we show that the most used concentrations are 2-4, 5, and 10% w/v for alginate, gelatin, and GelMA, respectively. Likewise, most cross-linking methods for alginate are chemical and based on Ca 2+ cations, while 37 • C is the most common temperature for thermal cross-linking of gelatin, and UV light is the standard physical cross-linking of GelMA.
Secondly, cell-laden hydrogels are the most used. Consequently, cartridge temperature is usually defined in the range of 30-40 • C (allowing cell survival) and the printing pressure at 100-200 kPa (reducing cell stress). Obviously, the addition of cells after hydrogel bioprinting minimize the importance of these parameters.
Finally, MSCs are the cell line most used in combination with hydrogels. In general, good viability results are obtained with all cell lines. Regarding mechanical tests, the Young modulus is widely used in bioprinting, although there is no consensus on the most important mechanical property of each bioprinted structure.

RECOMMENDATIONS AND FUTURE WORKS
In our opinion, those missed bioprinting parameters are usually related to a poor reproducibility. Moreover, inappropriate evaluation tests may cause an unfair comparison of results. For this reason, some guidelines and recommendations are detailed below. Additionally, in order to facilitate reading and understanding for future papers, the International Systems of Units (SI) must be used.
Hence, we strongly recommend defining the following parameters in all studies. Concentration of materials and protocols to prepare hydrogels should be fully detailed and could be complemented with its viscosity. Although cartridge temperature and printing pressure are two essential parameters needed to set the bioprinter, bed temperature and printing speed will increase the reproducibility of the study. A quantitative measure of printability or the hydrogel printability window will facilitate its practical use. Additionally, in the cross-linking step, concentration for chemical-based cross-linking, temperature for thermal-based cross-linking, light wavelength/power for physicalbased cross-linking must be defined. Furthermore, the exposition time must be defined for all three cross-linking types. On the other hand, cellular tests must include the identification of cell line and assay-kit information with quantifications at different time points (0, 1, 3, 7, and 21-days). And finally, those studies whose bioprinted structures have a specific clinical application must perform mechanical tests to mimic the tissue/organ properties (e.g., compressive stress for cartilage tissue).
In summary, due to time and space restrictions, this review could not analyze all the information available in the selected papers. Thus, future works could focus on comparing results of commercial vs. homemade bioprinters (cell viability, mechanical behavior), analyzing other rheological properties (swelling ratio, surface tension), printability vs. precision, or degradation speed for different hydrogels.

AUTHOR CONTRIBUTIONS
EM performed literature search, screening, data extraction, preparation of graphics, tables, and manuscript. JG-B contributed to screening, data extraction, preparation of graphics, tables, and manuscript. JP contributed to screening, data extraction, and preparation of manuscript. EL, JC, AM-G, MD, JC-A, and DT contributed to screening, data extraction, and revision work to the manuscript. FS-M performed revision work to the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This study has been co-funded by Consejería de Economía, Ciencia y Agenda Digital, Junta de Extremadura, through Grants IB16200 (PI: JP) and PD16067 to JG-B. Co-funded by Unión Europea/FEDER and FSE. Supported by competitive grant 'Miguel Servet I' from Instituto de Salud Carlos III to JC (CP17/00021 and CD17/00021 co-financed by FEDER and FSE) and Sara Borrell grant from Instituto de Salud Carlos III to EL (CD19/00048 co-financed by FSE).