Polysaccharide‐based biomaterials in a journey from 3D to 4D printing

Abstract 3D printing is a state‐of‐the‐art technology for the fabrication of biomaterials with myriad applications in translational medicine. After stimuli‐responsive properties were introduced to 3D printing (known as 4D printing), intelligent biomaterials with shape configuration time‐dependent character have been developed. Polysaccharides are biodegradable polymers sensitive to several physical, chemical, and biological stimuli, suited for 3D and 4D printing. On the other hand, engineering of mechanical strength and printability of polysaccharide‐based scaffolds along with their aneural, avascular, and poor metabolic characteristics need to be optimized varying printing parameters. Multiple disciplines such as biomedicine, chemistry, materials, and computer sciences should be integrated to achieve multipurpose printable biomaterials. In this work, 3D and 4D printing technologies are briefly compared, summarizing the literature on biomaterials engineering though printing techniques, and highlighting different challenges associated with 3D/4D printing, as well as the role of polysaccharides in the technological shift from 3D to 4D printing for translational medicine.


| INTRODUCTION
Polysaccharides, mainly chitosan, 1 alginate, 2 agarose, 3 starch, 4 glycogen, 5 and cellulose, 6 as well as their blends 7 have been widely used for biomedical purposes, ranging from imaging and diagnostic to therapeutic, delivery and biosensing applications. 8,9 The bioactivity of polysaccharides gives reason for their usage in the treatment of diseases such as antitumor, antivirus, and immunoregulatory. 10,11 Although polysaccharides are best known for appropriate biocompatibility and nontoxic nature, they suffer from poor mechanical properties. Therefore, there have been several kinds of research in which surface-grafted 12 and crosslinked 13 polysaccharides have been employed for drug and gene delivery systems as well as electroconductive hydrogels. [14][15][16] Another limitation of polysaccharides could be difficulty of purification and extraction. There are also some reports emphasizing that the stability of polysaccharide-based scaffolds is limited in biological media, necessitating modification of polysaccharides and their extraction as well as processing circumstances. 17 Thus, there was a need for novel technologies manufacturing predesigned polysaccharide-based biomaterials, like electrospinning. 18 3D printing or additive manufacturing (AM) enables one to print a series of materials in a layer-by-layer manner, with the potential to control the shape and properties of each layer. The resultant structure from a 3D printer is usually a complex, customized, and solid one already formed as an image in a digital brain. In a brief classification, AM can be categorized into five main groups, including inject printing, binder jetting, extrusion-based printing, selective laser sintering (SLS), and stereolithography (SLA). 19,20 This modern technology enjoys several advantages in comparison with the classical processing methods.
For instance, AM has a great capability of reproducibility, appropriate control over the fabrication process, individualization of product series and facile modification of the final products. In the field of biomedical engineering, the ability to fabricate different shapes (meniscus, bone, nose, and ear) with excessive porosity is particularly underscored. For instance, the porous structure of 3D-printed scaffolds facilitates the delivery of nutrients to the cells, promotes cell viability, and provides the cell with a suitable media for the regeneration of organs or tissues. [21][22][23] The most outstanding applications of 3D printing technology are organ fabrication, precise printing of drugs, medical phantoms, and different aspects of cancer treatment ranging from diagnosis to drug delivery. 24,25 Although 3D printing of biopolymers is well-accepted among scientists, it generally suffers from some limitations, such as a lack of dynamism and responsiveness. Indeed, the final 3D-printed structure fails to follow a dynamic pattern of change in shape, swelling, self-repairing, self-assembly, multifunctionality, and shape-shifting properties as a function of time. On the other hand, lacking dynamism negatively affects and weakens biomimicry.
Hence, 4D printing was introduced and progressed to mimic natureinspired structures. 26 4D printing assists in promoting the structural configuration of printed materials as a function of time. 4D printing makes good use of biomedical, chemistry, materials, and computer science to develop advanced materials. Biomaterials with sensitivity to particular stimuli are the building blocks of 4D printing technology, which can be classified as physical, chemical, and biological stimuli-responsive materials. 27 Physically responsive materials are sensitive to temperature, light, humidity, electricity, and magnetic field, while chemical ones take action when a change in pH and ion concentration exists. More intricately, biological stimuli materials are responsive to cell traction forces, glucose, and enzymes. 25,28,29 Similar to 3D printing, 4D printing is based on a powerful mathematical model of the desired structure/image. Likewise, 4D printing can be classified into four main technologies, including SLA, fused-deposition modeling, powder bed, and inkjet head 3D printing. The strategy to be selected is dependent on the mechanical properties of the used biomaterials and their flexibility as well as printability. 30 Very recently, a new class of 3D printers has been introduced, called the 5D printer. On this note, the 5D printing technique is not the next generation of 4D printing. It allows one to print curved layers by two additional axes, leading to a higher degree of freedom. This printer can move the print bed and printing head in two more angles. 31 Besides the capability of printing complicated curved layers, 5D printers can create scaffolds possessing adequately high mechanical properties. For instance, 5D-printed scaffold can tolerate a pressure four times, on average, higher than that tolerated in 3D-printed scaffold. Therefore, hard and complex tissues, like bone and teeth parts can be printed accordingly. 32 On the other hand, this class of bioprinters fails to print smart materials that reveal shape change over time. Forecasts suggest that 5D printing can support the development of surgical tools and prosthetics. Although 5D printing seems promising, there are some important blind spots and unanswered questions that need further investigations. To name but a few, the following questions still remain open: • Does 5D printing process, itself, leave any trace on the response of the ink to the environmental clues or the seeded cells' biological functionality?
• Does the dynamics of 5D bioprinting disrupt the cells' metabolic activities seeded within the scaffold?
• Is it applicable to print scaffolds that are implantable in the body by inducing the capability of size alteration?
• Is it possible to have a 5D-printed scaffold undergone reaction when surrounded with immune cells or under pathological circumstances?
By and large, although bioprinted scaffolds have been repetitively reported as efficient and have experienced an exemplary progression, the complexity of innate multicellular tissues jeopardizes the accuracy and biological dynamicity. Thus, and unfortunately indeed, recapitulating the real features of native tissue is the main concern.
Figure 1a shows the history of progression from 3D to 4D printing in the development of biomaterials for medical applications. It is evident from the timeline graph that applications of 3D and 4D printing are becoming more and more delicate, critically viable, and targeted. Moreover, attempts in using 3D and 4D printing techniques to shape polysaccharides follow an ascending trend (Figure 1b). Although progressing very fast, a long way should be paved for the appropriate selection of printable polysaccharides. Besides highly printable character, the chosen candidates should have high sensitivity to possible stimuli, great biological features, good mechanical properties, sustainability, and recyclability. In this review article, we wrote a short introduction of 3D and 4D printing concepts, followed by clarification of the need for a shift from 3D to 4D printing while considering the polysaccharides' role, and challenges associated with the application of 4D printing to polysaccharides. We have also proposed possible solutions to existing challenges.

| OVERVIEW OF 3D AND 4D PRINTING TECHNOLOGIES
"3D printing is actually 2D printing over and over again," told by Prof. According to ISO/ASTM52900-15, AM can also be divided into seven categories: material extrusion, vat photopolymerization, powder bed fusion, material jetting, binder jetting, sheet lamination, and directed energy deposition. 34,35 This new technology is considered invaluable due to addressing three critical concerns. First, it can provide complex geometries that are not achievable by traditional routes. Second, it can print different kinds of biopolymers simultaneously, without the need for toxic chemical reagents and solvents. Third, it leaves no waste. 36 However, when using 3D printers, there is no opportunity to deform the scaffold. The fabricated scaffolds are nondynamic and there is a need to mimic the nature-inspired structures using smarter materials. To resolve this situation, 4D printing is introduced utilizing advanced and smart materials showing stimuli-responsiveness behavior. In this regard, polysaccharides received popularity in 4D printing due to their multidimensional responsiveness. 37,38 Basically, 4D printing can be classified to three main categories: liquid solidification, powder solidification, and direct material extrusion. 39,40 Shape memory polymers (SMPs), alloys (SMAs), and composites (SMCs) are smart materials suited for 4D printing. 41 Nevertheless, not only is there more rigor in choosing materials for 4D printing, but also the expectations are very high.
In addition to the parameters mentioned in Table 1, there are some major and basic requirements for both 3D and 4D bioprinting (i) Printing parameters such as printing speed, extrusion rates, nozzle There are also some major differences between 3D and 4D: (i) materials for 4D printing are smarter, advanced, designed, or selfassembled, while thermoplastics, metals, and ceramics are the common materials for 3D printing; (ii) 4D printing device is a multimaterial 3D printer (Figure 2) 58 ; and (iii) the final scaffold achieved by 3D printing remains unchanged by the time (after applying stimuli), while in 4D printing it does change. 60 Table 2 summarizes the comparison between 3D and 4D printing technologies.

| CHALLENGES IN BIOMATERIALS DEVELOPMENT BY 3D AND 4D PRINTING
There exist some challenges in the printing of biopolymers, originating from material defects. Printability, biocompatibility, biomimicry, degradation pattern, and degradation byproducts are the main limitations. [69][70][71] Fortunately, there are also several possible resorts for the addressed issues. For instance, modifying commercial printers, material modifications, devising state-of-the-art solvent systems, incorporation of polysaccharides with other bioactive materials, and developing some postprocessing techniques such as surface coating and plasma radiation can be counted. [72][73][74] Due to the considerable biological features of polysaccharides, they can be of great interest as inks. However, their poor mechanical properties must be considered T A B L E 1 Key parameters included in stimuli-responsiveness and applications of bioprinting technologies.  T A B L E 2 The comparison of 3D and 4D printing technologies in a brief view. It can be seen that printing variables and properties of the printed articles must be matched for a target application. F I G U R E 3 (a) Co-printing of the vasculature, cells, and extra cellular matrix (ECM) to improve vascularization in a printed cell-laden tissue construct. 75 (b) A suggested 3D bioprinting strategy to fabricate vascularized tissue using the combination of 3D extrusion printing with cell-directing materials is a multiscaled approach for printing vascularized tissue in a layer-by-layer manner.
T A B L E 3 Different steps of designing polysaccharide-based inks/applications of polysaccharides and polysaccharide-based inks in 3D and 4D printing, their benefits and their challenges.   (Table 3 and Figure 4), some reports have indicated that their inappropriate shape-morphing ability is a serious limitation associated with the 4D printing of polysaccharides. However, other excellent properties of these biomaterials such as biocompatibility, nontoxicity, and abundance cannot be ignored. Hence, scientists have suggested overcoming their shape-morphing issues by blending with other biopolymers. 103 For instance, alginate's undesired shape-morphing ability can be resolved when it is mixed with methylcellulose or dopamine.
The resulting hydrogel has great rheological properties, shape-morphing ability, and extrudability. 104,105 Another example of improving shape morphing capability of polysaccharides via blending is the addition of multiwalled carbon nanotubes, which brings not only an efficient photothermal conversion capability (a photo-responsive shape-changing composite) but also stronger mechanical properties. 106  4D printing is capable to meet this requirement because the printed scaffolds can change their shape and structure as the organ grows. In this regard, with the help of scanning technologies such as computed tomography (CT) and magnetic resonance imaging (MRI), the growth pattern of each patient would be captured and the shape configuration of the 4D-printed scaffold could be tuned. [108][109][110][111][112] This technology is also able to innovate new routes for more advanced research, 98 helpful for analyzing body defects and regeneration, 113  The picture reveals the schematic illustration of the necessary steps to successfully design bioinks for bioprinting. Accordingly, we need to designate the molecular weight (MW), viscosity, and concentration of the used biomaterials based on the application (Step 1). Then, the crosslinker and its concentration must be defined in order to improve the properties of the bioink (Step 2). The additives are usually added in an optimized manner in order to add a special characteristic to the final printed scaffold (Step 3). Then, standard tests will be performed to determine the optimized concentration in which the platform has the best properties (Step 4). Finally, biological agents (e.g., drug, growth factors, macromolecules) would be incorporated, if needed.
F I G U R E 5 (a) The basic process depicting the 3D printing of polysaccharides-based skin scaffold under the sufficient condition to achieve implantable mature skin. 86 (b) Utilization of alginate-based 3D-printed scaffold for plant cell culturing. 121 (c) Different applications of bioprinted polysaccharides in tissue engineering.
(d) Illustration of surgical procedure for implanting the printed scaffold. (e) Histological assessment of wound healing process after 1, 2, and 10 weeks.
(f) Quantitative diagram of regenerated adipocyte area 1 week after the implantation. 85,122 biomaterials for 3D and 4D bioprinting. 117 In the form of a hydrogel, they can be easily utilized in pressure-assisted micro-syringe and inkjet techniques, such that the final scaffold reveals high porosity and interconnectivity, particularly the ability to cell culture and drug loading 118-120 ( Figure 5). 121

| Polysaccharides in 3D bioprinting
Although several polysaccharides have been examined for printability potential, only a few of them reveal thermal stability in terms of melt strength or viscosity in printing. The overall strategy is to blend them Polysaccharide-based 3D printed scaffolds can support the homogeneous distribution of functional chondrocytes in addition to the retention of chondrocyte phenotype. 157 Hence, they seem to be a potent option for clinical uses. The possibility of nanofibers fabrication from cellulose acetate and chitosan can endorse exploiting them for regulation of morphology and tuning the release profile of the printed scaffold. 158,159 Of particular note, chitosan is well known in the biomedical engineering and bioprinting industry due to its great ability to mimic the heart, bone, cartilage, vascular, skin, and neuronal extracellular matrix 160-165 (see Figure 6 166,167 ). It also enjoys repairability due to its ability to cell attachment and cell differentiation. However, its mechanical properties and printability pose a limitation on its usage in digital printing. In addition, printing accuracy and resolution of the ultimate bioprinted scaffold must be carefully supervised.
Thus, chitosan should be modified with other polysaccharides to resolve its poor printability. The addition of polyethylene glycol (PEG), gelatin, and pectin can guarantee the facile extrusion of chitosan by controlling its viscosity. [168][169][170][171] From this perspective, some believe that chitosan is a modifier rather than a continuous phase in the formulation of polysaccharide-based biomaterials for 3D printing. Moreover, the presence of imine bonds between oxidized hyaluronate and glycol chitosan as well as the acyl hydrazone bonds between oxidized hyaluronate and adipic acid, the dihydrazide can result in the development of a highly printable chitosan-based platform with self-healing capability. 172,173 Neat polysaccharides, particularly cellulose and lignin, severely boost the mechanical strength of chitosan inks in F I G U R E 6 A schematic illustration of (a) 3D-printed heart model reported by several papers in literature, 162,166 (b) 3D-printed bone model, both the computer design before printing and the actual printed model. 167 comparison to proteins such as gelatin. 174,175 Unlike chitosan with limited printability potential, alginate has attracted a great deal of attention because of its excellent printability. Moreover, biocompatibility, low cost, low toxicity, and fast gelation (when Ca 2+ exists as a cross-linker 176,177 ) are other characteristics of alginate. This is the reason for the diversity of investigations carried out to print alginatebased inks and their rapid bioprinting progression. 121 great bio-interaction and integration with the native tissue. 184,185 Besides alginate, chitosan, and their blends, some other polysaccharides are occasionally applied in 3D printing. Pectin provides the user with a great media for cell attachment, and cell organization as well as primary human cells, mesenchymal stem cells, fibroblasts, and osteoblasts growth. However, weak shear-thinning properties can limit its practicality for 3D printing. The addition of other biopolymers to pectin was accordingly examined. The incorporation of carboxylated cellulose nanofibrils into pectin not only enhanced its viscoelastic behaviors but also its printability and shear-thinning properties. 186,187 Similarly, methylcellulose can intelligently be utilized to strengthen teins can also bring about appropriate cell adhesion, especially adhesion to mesenchymal and epithelial cells. [190][191][192]

| Polysaccharides in 4D bioprinting
To be used in 4D printing, materials must own sensitiveness to a particular stimulus (or multistimuli), as mentioned earlier. These stimuli can be chemical, physical, or even biological. However, they have to The Schematic illustration of the chemical crosslinking of alginate with PEG via exposing them to CaCl 2 solution and UV light. The presence of PEG activates temperature and salt concentration responsiveness which is a key factor in 4D bioprinting, (b) Crosslinking mechanism after material being surrounded by ca 2+ ions and exposure to UV light, (c) Diameter of the 4D bioprinter and the pattern with which the target scaffold is printed. 200 provide shape change as a function of time, after applying the motives.
The stimuli responsiveness of polysaccharides will provide us with the opportunity to utilize them in 4D printing technology. They can easily respond to physical stimuli like temperature, light, electricity, magnetic field, or even pressure, chemical species such as reactive oxygen species (ROS), redox species (e.g., glutathione), glucose, enzymes, and some ions (e.g., calcium). 193,194 For example, chitosan is responsive to glucose, 195 pH (under acidic conditions, due to the presence of basic amine groups), 127 or even an electric field. 196 Moreover, reports have indicated that agarose, sodium alginate, and hyaluronic acid respond to temperature deviation, chitosan and agarose react to voltage changes, alginate glycerin arouses in response to PH, and hyaluronic acid is affected when tension is applied 197 (see Table 4). Additionally, a combination of cellulose, dextran, and graphene reveals pH and near-infrared (NIR) sensitive properties. 198 Noteworthily, some of them have multiresponsiveness to more than one stimulus. 199 There are methods to modify polysaccharides preparing them as 4D bioprinting's inks. The can utilize different methods such as blending with other biopolymers, dispersing some additives to increase the printability as well as using chemical crosslinking strategies. 205 A holistic understanding of the required printing factors is essential to overcome the barriers related to printability, precision, and accuracy. 206 Additionally, there exist some other challenges related to both 3D and 4D printing of polysaccharides.
For instance, intemperate interconnectivity, thick structure as well as very low viscosity are the problems that some polysaccharide-based inks are suffering from. 92,207 There exist two possible clarifications in this direction. First, advanced material design has to be contemplated to improve printability, mechanical properties, and biological features. Second, the advanced digital simulation needs to be mature and enhanced, leading to the fabrication of smarter materials. Although plenty of efforts should be integrated into a protocol to resolve 4D printing challenges, it is believed that 4D printing technology would find amazing applications in the near future. For instance, it would become a unique method of surgery to implant medical devices more efficiently. Using the state-of-the-art 4D printing technique, we would be able to provide the surgeon with all the needed data about blood loss, blood clots, as well as breathing difficulties. Moreover, smart devices could prepare detailed information about the anatomies of the individual patient (at anytime and anywhere after the surgery), as an impossible task in the past. 98 Considerably, we believe that 5D printing will also have a bright future, especially in cancer treatment. The ability to monitor the distortion of the tumor's anatomy, the possibility of tumor invasion to the surrounding structure, and monitoring the possible changes occurring after neoadjuvant treatments are the important factors that would help complicated surgical planning using 5D bioprinters, an interesting subject that has been recently studied by a group of scientists. 208 What we may need to take huger steps is a deeper understanding of the interaction of the printed organ with the host tissue and the native microenvironment, the possible response of the printed organ to the body's immune system, and the pathological conditions. 209 By far, many studies have to be conducted and plenty of challenges must be resolved to reach such a stage of bioprinting knowledge.

CONFLICT OF INTEREST STATEMENT
The authors have no conflict of interest to declare.

DATA AVAILABILITY STATEMENT
The authors confirm that the data supporting the findings of this study are available within the article.