Synthetic Polymers Derived Single-Network Inks/Bioinks for Extrusion-Based 3D Printing Towards Bioapplications

Three dimensional (3D) printing, also known as additive manufacturing technique has revolutionarized the field of manufacturing with a great impact as compared to the other traditional methods. This technique has shown steep rise over the past decade owing to their benchmark capabilities of fabricating new and complex 3D constructs, especially towards tissue engineering, and regenerative medicine. Among currently applied 3D printing techniques, extrusion-based 3D printing has gathered particular attention for the employment of ink/bioink materials to enable on-demand personalized fabrication due to its low cost, broad utility for various materials and ease of controlled printability. However, there still exist a lack in diversity of the ink materials with their optimized degradation rate, rheology and bioactivity for precisely fabricating complex and self-supported cell-laden 3D printed constructs. Therefore, to develop an array of such new materials is a major challenge for synthetic polymer chemists, material scientists and biomedical researchers for widening the future applicability of 3D (bio)printing. This review aims to summarize the recent advances in the rational designing and development of ink/bioink materials based on synthetic polymers as single network precursor due to their great opportunity to tune their physico-chemical 2 and mechanical properties in order to design and mimic in-human 3D tissue scaffolds with shape retention for both hard and soft tissues.


Introduction
3D printing based computer-aided additive manufacturing technique has emerged as a revolutionary manufacturing method and is currently being considered at the forefront research area in material sciences and biomedical engineering. 1,2 3D printing is a promising method for future era of personalized medicine via layer-by-layer fabricating patient-specific, customizable medical devices, organs, tissues and other bio-systems mimicking their native counterparts with complex and heterogeneous structures. 3,4 In the endeavour of such biofabrication, the selection of printing technology, design and materials are critically important in order to maintain the structureproperty-processing relationships in the 3D printed constructs. 5,6 To date there has been considerable development towards the engineering of different types of 3D printing devices, 7,8,9,10,11 and to increase their printing affordability and reliability such as with having tight control over the printing speed, higher resolutions, and multicomponents fabrications. 12,13 Further, development and optimization of the printable ink materials is highly required to achieve desired properties including physico-mechanical properties, rheological properties and biofunctionality to obtain self-supporting functional and high-strength products, depending on the applications. 14,15 Although there exist different ink materials ranging from polymers to composites and ceramics, 16 3 there is still a lack to achieve such diversity in 3D printable ink materials. On the other hand, the preparation of 3D printable cell-laden ink materials (i.e. bioinks) have attracted great deal of interest to the researchers in the recent years, 17 and have been considered as one of the most advanced tools to find new avenues in tissue engineering, 18 regenerative medicine, 19 drug delivery, 20 cell therapy, 21 etc. In the endeavor of mimicking the biocomplexity and heterogeneity of natural tissues in the 3D printed constructs at various scales, the development of such cellular embedded ink materials with "printability" (for e.g., with optimized printing process, speed, and resolution are critically important) 22,23 are pretty much challenging due to the demand of various features depending on the application envisioned as discussed here. First, as cells are encapsulated within such ink materials, therefore all the components of the inks such as functionalized polymer based main ingredient along with other applied precursors for e.g. catalysts, cross-linker, drugs, bioactive molecules (like peptide sequences, 24 growth and differentiation factors, etc., 25,26 ) should be biocompatible in nature in order to maintain the cell survivability throughout the printing process as well as in the final printed article. Then, the ink materials should protect the cells and maintain their sensitivity to enable the survival of cells during the printing process for the biofabrication, and the desired cellular functions (for e.g., cell growth and proliferation, adhesion and differentiation) should be preserved in the fabricated structures. 27 Further, the post-extrusion structural and functional integrity in the product should be maintained with standardizing different parameters such as pressure, temperature, pH or light to avoid concerned cell resistance. 28,29 Finally, the materials should be biodegradable with controlled degradation kinetics and their waste compounds and intermediates should not have any toxic effect.
In the effort of developing ink materials for 3D printing with desired mechanical and biological properties, two major categories of material precursors have been adopted: (i) natural 4 polymers (biomaterials), and (ii) synthetic polymers. Natural polymers for e.g. polysaccharides and proteins, as typically extracted from plants, animals, bacteria, cells, etc., are currently widely applied materials as network precursor in bioink formation due to their greater biocompatible nature and higher cellular proliferation rates in comparison to synthetic polymers. 30 Note that in many reports of bioink formulation natural polymers were artificially modified with functional groups, for instance to enable network crosslinking, however such artificial biomaterials have been considered in this review in the category of natural polymers only. 31,32 Naturally derived polymers are often associated with some disadvantages which can arise challenges in their utility for biofabrication such as (1) variation in batch-to-batch which can led to the complication due to their variable printabilty and the issue of the reproducibility of constructs, and the cellular sensitivity to such variations, (2) tuning the structures, solubility, viscosity and other properties of biopolymers remains challenging as their functionalization are often more difficult, (3) fast biodegradability rate which often may not be suitable. On the other hand, synthetic polymers can overcome these limitations of natural polymers along with the opportunity of being tailored with specific physical, chemical and biological properties and can further lead to the synthesis of a broad library of new materials for (bio)inks. 33 Synthetic polymers further offer the ability to tune functional properties and rheological behaviour like printability and mechanical integrity of the inks, through monomer selection, architecture control, and post-polymerization functionalization opportunities. 34,35 Therefore, synthetic polymers hold great potential to be exploited as future ink materials of 3D printing for shaping into advanced and highly customisable architectures suitable for medical devices and tissue scaffolds including both hard and soft tissues. 36,37 However, a careful selection of synthetic polymer has to make in order to maintain biocompatibility and biodegradation issues.
For instance, poly(ethylene glycol) (PEG) corresponds to the synthetic polymer which has been fibroblast adhesion on the 3D-printed fiber surface of (RGDS(biotin))-PCL. 43

Extrusion-Based 3D Printing Technique
Extrusion-based 3D printing is the commercially available and most common rapid prototyping technique due to its affordability, versatility, and compatibility with a wide selection of ink materials usable for biofabrication and tissue regeneration from small vessels to large constructs/organs. 45,46 Here, the inks are extruded through a micronozzle of extrusion head and controlled by either an endless screw, pneumatic pressure or a mechanical piston ( Figure 1A). 47,48 The 3D objects are constructed via the blueprints from a computer-aided design (CAD) file by direct-ink-writing in a layer-by-layer fashion and controlling an xyz stage of the deposition path of the print-heads. 49 Extrusion-based 3D printers can be successfully engineered under sterile conditions with nozzle diameters and multiple extrusion heads providing the unique advantage of depositing different types of layers in the final printed article via delivering multiple (bio)ink materials with varying components, cells and cellular density ( Figure 1B). 8,50 To successfully enable the "printability" of an ink, a suitable viscosity should be achieved typically by inducing either external shear force (i.e. shear-thinning property) or pre-crosslinking to enable its extrusion from the nozzle, however as the external mechanical stress is removed post-extrusion a rapid filament shape retention with efficient recovery of mechanical properties (i.e. self-healing property) is desired to keep an integer and self-supported 3D printed structures with no structural collapsing. 51 For instance, UV irradiation (or heating) can be applied for such shape retention to the tip of the printing nozzle or directly over the printed structure on the deposition plate. Burdick 8 and coworkers developed a general extrusion-based bioprinting method of "in-situ crosslinking" for photo-crosslinkable non-viscous hydrogel inks (such as from 5 wt% methacrylated hyaluronic acid (MeHA)) via introducing light through a photopermeable capillary which enables their simultaneous extrusion and crosslinking, prior to deposition ( Figure 1C and D). 52 The printed constructs depicted the uniform filament formation along with the high viability of the encapsulated cells. Another fascinating approach has gained significant interest recently based on extrusion-based 3D bioprinting using suspension baths, illustrating their ability to suspend and completely encapsulate the extruded filament materials. 53 Such secondary support provides the flow restriction of bioink and trigger the cross-linking (physically or chemically) immediately after the deposition, and enables the printed construct with improved resolution and controlled heterogeneity. 54 Feinberg and co-workers presented such approach as Freeform Reversible Embedding of Suspended Hydrogels (FRESH) printing where a thermoreversible support bath (CaCl 2 ) was utilized to embed soft hydrogel (based on alginate) to exemplify the construction of a bioprinted full-size human heart model. 55 Recently, a co-axial extrusion-based 3D bioprinting approach has been also introduced to enable simultaneous printing of bioink along with crosslinker solutions in a coaxial system to promote gelation during extrusion. 56 Khademhosseini, Dentini and co-workers demonstrated a coaxial needle extrusion system where the internal needle carried the flow of cell-laden bioink materials (based on alginate and gelatin methacroyl) and the external needle was designed to the simultaneous flow of ionic crosslinking solution (CaCl 2 ) to generate the ionically crosslinked hydrogel microfibers at the tip of dispensing system, which was further secondary crosslinked covalently via exposure of UV light. 57 9 Hydrogels are highly hydrated three-dimensional polymeric networks and have been exploited for various biomedical applications, for e.g., as a scaffold for tissue engineering, and drug delivery. 58,59 Hydrogel based ink materials can provide a perfect soft material for soft tissue engineering as to create a soft tissue is more challenging due to the demand of features like high elasticity, flexibility, viscosity and inter-layer adhesion. 60,61 From a biological point of view, hydrogels having a high water content can be considered as an ideal candidate for cell-laden bioink materials for extrusionbased 3D printing because they can closely simulate the native extracellular matrix (ECM) microenvironments with having aqueous 3D environment, and provides cells survivability with retained rounded morphology and homogeneous encapsulation during the extrusion process and there after promote new tissue formation and functioning in 3D space. 18,62 However, fabrication of hydrogel based scaffolds or cell-laden constructs with shape fidelity remains challenging, and there exist a current demand of optimization in the mechanical properties of the hydrogel materials within a biofabrication window ( Figure 1B). 47 On one hand, the soft hydrogels which are well suited for cells are disadvantageous for fabrication process to achieve the constructs with maintained predesigned structure fidelity, and on the other hand the stiff hydrogels which can typically provide the shape fidelity can affects the cellular functioning by limiting the migration and proliferation of the cells within the dense   can offers the tailoring into its mechanics by maintaining an adequate viscoelastic characteristics and integrity, 63,64 including self-healing and shear-thinning properties, and also provides optimization opportunity into its functionality, and degradation kinetics of the printed products. 65,66 In general, hydrogel ink formulation requires a specific viscous polymeric solution that can be immediately form high networking post-printing either by physically or chemically cross-linking of polymers. 67 Prestwich and coworkers described the formation of extrudable hydrogels with suitable rheological properties for bioprinting of vessel-like constructs derived from tetrahedral polyethylene glycol tetracrylates via co-crosslinking thiolated hyaluronic acid and gelatin derivatives. 68   interactions. 85 The prepared hydrogels exhibited thermoplasticity, self-repairability, and reprocessability for various 3D printed constructs over a lower temperature range. By tuning the feed ratios and concentration of monomer in the copolymer, the effective mechanical properties of the formed supramolecular hydrogels were obtained with high tensile strength upto 1.3 MPa, large stretchability upto more than 1300% and increased compressibility to 11 MPa at swelling equilibrium state. Moreover, these supramolecular hydrogels biomaterials depicted medicinally desirable antibacterial, anti-inflammatory activities along with the biocompatible properties.

PCL-PEG-PCL
Other notable system include a 3D printed drug delivery implant developed by Roberts, Hayes and coworkers based on thermo-responsive supramolecular polyurethane (SPU), able to form self-assemble polymer network via hydrogen bonding and N2N stacking ( Figure 6). 86 The material displayed suitable mechanical properties (with self-supporting and stiff, yet flexible) for hot-melt extrusion-based approach at a relatively low processing temperature (100 °C). Further to establish the potential application of SPU for 3D printing of a biomedical device, the biocompatibility of the polymeric films of polyurethane was determined by MTT assay using mouse fibroblasts (L929) which illustrated their non-cytotoxicity with more than 94% of cell viability. A multicomponent synthetic material SPU-PEG-paracetamol was prepared based on SPU co-formulated with PEG (4 wt% and 8 wt%) and incorporating paracetamol drug (16% w/w) to afford 3D printed robust implant constructs which exhibited appropriated mechanical performance, and prolonged drug release over a time period of 5 to 8.5 months. Such release rates

Stimuli-responsive Polymers
Stimuli-responsive polymers are smart or intelligent materials which can undergo alteration in their chemical, physical, morphological or mechanical properties upon interactions with their surrounding environment. 88,89 In the past decade these materials have been increasingly applied to a wide range applications, including controlled and triggered drug delivery, (bio)sensing, actuators, coatings, diagnostics, tissue engineering and biomedical devices. 90

Nanoengineered Polymers
Nanoengineered polymers are hybrid materials in which polymeric matrix are dispersed with one or more type of inorganic or organic nanofillers such as silica or metallic nanoparticles, nanoclay, nanorods, carbon nanotubes and nanofibers, graphene, metallic nanowires, quantum dots, etc. 98,99 Due with shape-fidelity and enhanced electrical conductivities >800 S/m for electronic applications. 105 The printed 3D scaffold further revealed their potential biomedical application towards nerve tissue engineering and regeneration by exhibiting their significant cellular response (attachment, proliferation, and neurogenic differentiation) to human mesenchymal stem cells (hMSCs) and viability to multiple, distinct cell types, including hMSCs.
Polymer/clay nanocomposite ink materials based on PEG precursor colloidal solutions incorporated with biocompatible and bioactive disk-shaped 2D laponite (XLG) nanoclay were investigated by Gaharwar group for 3D bioprinting ( Figure 11). 106 Such hydrogels based on diacrylated PEG-laponite demonstrated shear-thinning and self-healing properties due to rapid internal phase rearrangement, thus enabling to print a range of complex 3D structures upon UV based photo-crosslinking. The rheological characteristic of the nanocomposite hydrogels was appeared to be independent of addition of PEG and dominated by the behaviour of laponite network, whereas their self-recovery time was found to be controlled by the ratio of PEG:laponite.

Conclusions and Future Perspectives
The field of 3D printing based additive manufacturing is burgeoning enormously over the past few years and a range of affordable 3D printers are accessible for laboratories to date. A lot of efforts have been devoted to this field, essentially towards (i) engineering and optimization of the printing techniques for enabling the creation of the complex and highly organised structures with controlled processing and higher resolution, and (ii) developing the ink materials with suitable rheological properties and functionality. The particular challenge is the development of (bio)ink materials for 3D printing of biologically relevant scaffolds/tissue replacement, which allows the cells to adhere, differentiate and proliferate, and preserves the bioactivity of their other embedded compounds such as growth factors, signalling peptides, drugs, etc. Currently, researchers are focusing on the designing of (bio)ink materials mainly based on natural polymers or combination of natural and 30 for advancing the field of 3D printing to the next frontier with the envision of creating synthetic and in-human 3D printed tissues. The fundamental understanding of engineering 3D printing technique with controlled processing parameters, evaluating new and advanced polymers-based ink/bioink materials, and designing the biomimetic hard and soft tissue scaffolds with controlled structural integrity and functioning, are critically important to fabricate the targeted tissue/organ, and therefore will drive further innovation in the field of tissue engineering and regenerative medicines.

Conflicts of interest
The authors declare no conflict of interest.