Various methods of 3D and Bio-printing

Summary There is growing need for synthetic tissue replacement materials designed in a way that mimic complex structure of tissues and organs. Among various methods for fabrication of implants (scaffolds), 3D printing is very powerful technique because it enables creation of scaffolds with complex internal structures and high resolution, based on medical data sets. This method allows fabrication of scaffolds with desired macro- and micro-porosity and fully interconnected pore network. Rapid development of 3D printing technologies has enabled various applications from the creation of anatomical training models for complex surgical procedures to the printing of tissue engineering constructs. The aim of current investigations was to develop compatible printers and materials (bioinks) to obtain biomimetic scaffolds, which allow printing of living cells without significant loss of cell viability. The advanced level of such printing assumes “in situ” printing, i.e. printing cells and biomaterials directly onto or in a patient that will reduce recovery time.


INTRODUCTION
Tissue engineering strategy typically involves combination of cells and bioactive factors with a 3D scaffold to form useful construct for implantation [1,2]. Ideally, scaffold should be completely resorbed during tissue integration. Biomaterial scaffolds should mimic important aspects of targeted tissue, restoring their function and providing an environment suitable for cell differentiation and proliferation [3,4]. Traditional techniques used for production of such scaffolds are gas foaming, solvent casting, fiber bonding, phase separation, particulate leaching, and freeze drying techniques that provide macroscale scaffold features but often lack the complexity of native tissue [5].
Fabrication methods that enable production of complex geometries have significant advantages since they provide production of scaffolds of irregular shape that can perfectly fit the defect geometry. Besides, they can also mimic tissue complexity through precise positioning of multiple materials and cell types. As it is known, scaffolds should provide not only macroscale structural design, but also microscale features necessary for cellular sophisticated control over fabrication of a new tissue. Recently, 3D fabrication or rapid-prototyping technology has become popular and accessible, allowing everyday investigations of potential new fabrication techniques with better geometric accuracy on the macro and micro scale level [6,7]. Those investigations have opened door to innumerable approaches of scaffolds engineering such as high-resolution imaging and 3D printing technology known as laser sintering which was successfully used to create functional jawbone replacement [8]. This method has enabled creation of articulated joints, cavities that promote muscle attachment, and grooves to guide nerve and vein regrowth, and also reduced surgical preparation and accelerated recovery. In addition, designed vasculature may enable creation of larger constructs useful for nutrient transport for tissue growth. Functional tissue constructs could also be applied as a diagnostic tool for drug testing or other therapeutic procedures.

ACellUlAR SCAffOlD fABRICATION
Rapid prototyping techniques use multi-axis positioning systems and one of various methods to generate a 3D construct through subsequent layer fabrication (extrusion, deposition, solidification, polymerization, sintering or binding using many other methods) [8,9]. First step is creating a model in a computer-aided design (CAD) program and export it into the file format that describes the volume or surface mesh in 3D space such as *.stl (stereolithography), *.obj (object), or *.amf (additive manufacturing file). Second step is translation of the 3D data into slices to be patterned by the printer program using the program generally known as a 'slicer' . These techniques enable user to configure algorithm that determines pattern used to fill the layers and then the program calculates necessary parameters such as extrusion speed, cure time, or laser speed to accurately fill the pattern.
Previously, these techniques were adapted to mold casting, but recent rapid development increased their versatility and precision. Nowadays techniques are able to create scaffolds that fully mimic macroscale organs geometry and print layers with thickness less than 20 μm allowing complete reproduction of the tissue microarchitecture. Techniques with higher precision are currently under investigation to enable reproduction of smaller tissue features such as hepatic lobules and kidney nephrons.

Stereolithography (SlA)
SLA techniques use deflected laser beam or projected light source to cure and harden given areas of photopolymer at the surface of some material ( Figure 1) [8,10]. Various photopolymers with suitable viscosity and ability to harden can be used in construct creation with SLA. Cooke used SLA to fabricate 3D scaffolds for bone tissue engineering using biodegradable polymers, like diethyl fumarate and polypropylene fumarate [10]. Also, photo-curable ceramic acrylate suspension was used to form a construct of cancellous bone and bone scaffolds using hydroxyapatite [7].
The disadvantage of SLA methods is limited resolution by the diameter of laser beam (about 250 μm), although small-spot laser systems and digital light processing projection produced features of about 70 μm. These techniques can also be used to design hydrogel scaffolds from natural and synthetic polymers that expand in water and are significantly less rigid than traditional SLA constructs. Hydrogels have become popular as tissue engineering biomaterials due to their high water content and mechanics similar to soft tissue. Some researchers use this technique for creation of 2-hydroxyethyl methacrylate scaffolds using photolithography for formation of patterns from non-swollen prepolymer, which were then hydrated and seeded with cells [11]. SLA has also been used to make molds that are used to cast negative replicas of the printed molds. Chu et al. made printed mold of a mandible generated using CAD program and data from computed tomography imaging. The mold was filled with a hydroxyapatite/acrylate mixture and heated to cure the scaffold [12].
Accordingly, SLA seems to be a versatile and attractive technique for creating tissue-engineering scaffolds because of its precision and increasing availability of biologically relevant photopolymers.

Powder-fusion printing (PfP)
PFP uses granular materials (plastic, resin, or metal) for printing that are selectively bound together ( Figure 2) [8,13]. In selective laser sintering-melting (SLS/SLM), plastic or metal granules are sintered together by a laser beam that is directed across the powder bed, to increase local temperature influencing particle fusion in the heated area along the laser path. 3D scaffolds are generated by layer-by-layer deposition of the powder. After fabrication, unfused powder is removed and the resulting part is mechanically strong construct with carefully designed geometry and porosity. As in SLA, the resolution of SLS printing depends on the spot size of the laser beam and the size of powder particles. Typical laser-based systems have minimum features of about 400 μm, with minimum void size of about 50 μm. SLS techniques have also been developed to fabricate constructs with various biopolymers used in a wide variety of medical implants.
Scaffolds can also be made from granular material by binding the particles with solvents or adhesives whereby figure 2. Powder Bed Fusion printer: A layer, typically 0.1mm thick material is spread over the built platform, then laser fuses the first layer or first cross section of the model. A new layer of powder is spread across the previous layer using a roller. Further layers or cross sections are fused and added. The process repeats until the entire model is created. Loose, un-fused powder remains in position but is removed during post processing. Slika 2. Štampač praškastog fuzionog sloja: sloj materijala, tipično debljine 0,1 mm, nanosi se preko platforme, i laserski lepi prvi sloj ili prvi presek modela. Novi sloj praha se širi preko prethodnog sloja pomoću valjka. Dalji slojevi ili preseci se spajaju i dodaju. Proces se ponavlja sve dok se ne kreira ceo model. Neiskorišćeni prah ostaje na poziciji, ali se uklanja kasnije tokom obrade. figure 1. SLA 3D printer used for printing resin-based photopolymers Liquid material solidifies by a high-powered laser or light source, "activating" the photopolymerization reaction. If laser is used to "draw" the object' s layers, this method is known as pure SLA. If the method is based on te digital light projection of entire slice of the object this process is known as digital light processing (DLP). Slika 1. 3D štampač SLA koji se koristi za štampanje fotopolimera na bazi smola Tečni materijal se očvršćava pomoću laserskog ili svetlosnog izvora velike snage, aktiviranjem reakcije fotopolimerizacije. Ako se koristi laser za "crtanje" slojeva objekta, ova metoda je poznata kao čista stereolitografija. Ako je metoda zasnovana na projekciji digitalne projekcije čitavog objekta, ovaj proces je poznat kao proces digitalne obrade svetlosti (DLP).
they are built layer-by-layer. Also, scaffolds can be fabricated from natural biopolymers and polysaccharides like gelatin, dextran, and starch. Microporous structures can be achieved with the addition of porogens and particulate leaching. For example, Simpson et al. fabricated porous poly (lactic-co-glycolic) acid scaffold using PFP and precisely reproduced the shape of an entire human finger phalanx [14]. These porous structures were also investigated from the aspect of cell attachment, growth, and matrix deposition.
Although PFP is limited to powdered materials, its advantage is capability to fabricate scaffolds from several materials such as titanium and magnesium that are not readily printable with other techniques. PFP is particularly suitable for bone and other rigid tissues scaffolds because bound or fused material creates constructs of superior mechanical properties. In addition, some materials naturally found in bone such as tricalcium phosphate can also be printed using PFP techniques, allowing creation of complex scaffold shapes, including in advance designed interconnected porosity. The resolution and minimum pore size are limited by the powder characteristics, and additional sintering is necessary to solidify parts that contain cracks and other damages. The focus of current research is on developing new materials for PFP and refinement of printing parameters to improve scaffold surface design.

fused deposition modeling (fDM)
FDM techniques enable useful platform for scaffolds creation by using precise xyz positioning system to direct the position of a nozzle during material deposition [8,15]. The material is deposited in layers and solidified into a previously defined shape. Traditional SFF printers are frequently used for rapid prototyping by using a small diameter polymer feedstock of acrylonitrile butadiene styrene which is forced through the nozzle heated to temperatures higher than 200°C.
Biodegradable polymers used in tissue engineering typically melt at lower temperatures and can be printed at more moderate temperatures (60-100 °C). Using this method it is possible to produce precise lattice structure, if temperature is precisely controlled and optimized with speed parameters during generation of filament with required accuracy. Newer generations of FDM systems use heated reservoir for extrusion of polymer pellets rather than fibers. Scaffolds produced by this technique from alginate and PCL implanted in mice have shown enhanced cartilage and collagen formation over a 4-week implantation [8].
Decreasing nozzle size and layer height increases x-y and z resolution, leading to significantly slower extrusion rates. Theoretical resolution is limited by the precision of the linear motion system (motors, gears, timing belts, and leadscrews) and retention properties of extruded material. Although FDM techniques enable the achievement of high degree of positional accuracy in the xy plane, their substantial limitation is in disability to print overhanging or unsupported parts because there is no supporting material from previous layers. Therefore, hardening during cooling or cross-linking after extrusion is essential for satisfied support of subsequent layers. Also, this drawback can be solved with introducing filament of support material during the process of printing, usually through additional extruder ( Figure 3).
Recent improvements in hydrogel rheological properties enable printing of these materials using FDM. For example, Hong et al. created printable hydrogel using a network of PEG and alginate with silicate nano-platelets [16]. These gels possessed zero-shear viscosity above 10 kPa·s, enabling shape retention after printing and a shear thinning that facilitated extrusion. The size and accuracy of printed hydrogel construct are dependent on the volume contained in the syringe and rheological properties of the hydrogel. Viscosity plays a key role in construct accuracy, because high-viscosity materials possess structural rigidity that is important for support of extruded successive layers, and secondary cross-linking step is typically used to lock the printed shape and improve mechanical properties of these constructs.
Extrusion-based printers typically use pneumatic pressure or a motor actuated plunger for material deposition. Pneumatic systems simplify control of the applied force to extruded material. The system should be calibrated for each material with adjustments of the nozzle size, nozzle geometry (tapered tip, cylindrical needle, and length), and gas pressure.
FDM seems to be one of the most versatile printing techniques for creation of biomimetic scaffolds due to its ability to make multilayered constructs built from various materials and print soft biomaterials like hydrogels ( Figure 4). Scaffolds printed by this technique may exhibit anisotropic mechanical properties that can be useful for creating scaffolds with intended alignment such as ligament or tendon.

BIOPRINTINg
Bioprinting belongs to additive manufacturing techniques for creation of the cell-based scaffolds [17]. These techniques are presumably adapted for printing with cells at the same time as material, since they have minimal impact on the cell viability and function. Biological materials used for printing should match natural environment of the host tissue to support function of those cells. Additionally, cells should be able to overcome shear stress during the printing process and survive in real non-physiological conditions of the printing regime [8].
Bioprinting techniques are classified into the three categories: microextrusion, lasser-assisted bioprinting (LAB) and inkjet-based bioprinting. Among them, inkjet bioprinting is the most promising for the creation of complex architectures, successfully mimicking native tissue and organs. In inkjet-bioprinting, bioink droplets are deposited onto the substrate that gels to form polymeric structures, while microextrusion bioprinting uses mechanical extruder to deposit bioink. Additionally, extrusion-bioprinting is useful for high cell density, due to its easier processing, but it is a slower than drop-based bioprinting. LAB requests a picoliter (pL) resolution through which cells and liquid materials can be printed. This printing method is rapidly growing and it is promising for the fabrication of tissue-like constructs.

extrusion bioprinting
Extrusion-bioprinting is one of the most economical techniques for rapid prototyping ( Figure 5) [8,17]. It typically includes pressure or screw/plunger-actuated dispensing of a fluid containing cells and/or biomaterials. It should provide shear thinning enabling minimal resistance under flow and quick chemical or physical cross-link after extrusion to support successive layers. This technique allows accurate deposition of the material and fabrication of complex patterned structures, including the use of multiple cell types, enabling accelerated growth and new tissue formation. Increasing print resolution and print speed are desirable in extrusion-bioprinting. Additionally, by the modification of printing mechanics, printing time can be diminished and coextrusion of multiple materials can be permitted. The main disadvantage is relatively long fabrication time to achieve high resolution in complex structures. This method enables successful fabrication of clinically relevant scaffolds for tissue engineering, because it is ideally adjusted for biological materials due to its ability to deposit multiple materials with wide-ranging properties. Extrusion bioprinted scaffolds are typically soft, due to their high water content that makes them limited to soft tissues application.

laser-assisted bioprinting (lAB)
LAB, or biological laser printing, is a group of laser techniques that use laser energy to facilitate densification of scaffold materials ( Figure 6) [8,17]. One type of LAB uses laser pulse (laser based direct writing (LDW)) for local heating a slide with an energy-absorbing layer and solution of cells. The laser pulse induces sublimation or evaporation of material, expelling the solution of cells on the opposite side and precisely depositing them on the substrate. This method includes laser-induced forward transfer and matrix-assisted pulsed laser evaporation, which can be used for deposition of fibroblasts, keratinocytes, human mesenchymal stem cells, various cancer cells and biopolymers.
As lasers technique allows high precision, this method is suitable for bioprinting of the smallest details of native tissues and organs. This technique allows direct printing of cells, but with several limitations, like detrimental effect on cell survival and their long-term behavior.

Inkjet bioprinting
Inkjet bioprinting enables precious deposition of cells and biomaterials, using some advances of 2D inkjet printing to create 3D scaffolds [8,18]. In this method a limited volume of fluid is falling into the precise pattern specified by the corresponding software. One of the most important  (C) For support bath hydrogel 3DP, biomaterial is extruded into the support hydrogel material. At 22°C, the hydrogel bath is stable enough to support the extruded print material, but at 37°C, the hydrogel bath transitions into the more liquid state to release the 3D printed object. The support bath allows formation of complex structures. advantages of this technique is the speed at which it can construct scaffolds with complex 3D architecture. This high speed limits the number of polymeric materials that can be used for bio-printing, since their gelation time has to be greater or equal to the drop deposition time.
Inkjet bio-printers can be adjusted to print materials at increased resolutions and speeds. They use thermal or piezoelectric energy to deposit droplets of solution into desired patterns and consist of one or many ink chambers with multiple nozzles with corresponding piezoelectric or heating components. A short pulse of current is used to actuate the component and eject a droplet of ink. In thermal bio-printers, there is often a strong increase of temperature in local spots, inducing formation of vapor bubbles and collapsing, leading to ejecting ink droplets onto the substrate. In piezoelectric inkjet printing, piezocrystals induce pressure increase, which further influence the droplet ejection. Deposition from the nozzle onto the printing surface happens when an electric charge induces vibration in the crystals, and vibration propagates to printing surface. It has been shown that heat and mechanical stresses during thermal bioprinting cause decrease of cell viability. Some researchers use this method for printing of retinal ganglion and glia cells isolated from adult central nervous system without causing an adverse effect on cell viability, while some of them succeeded in use of thermo-sensitive gels by modifying cartridge of commercially available inkjet printer to create multilayered scaffolds [8].
The main disadvantage of inkjet printing is request for biological agents to be in a liquid state, to allow deposition. Deposited droplets then solidify into the required geometry, through cross-linking based on physical, chemical, pH, or ultraviolet methods. Due to chemical cross-linking, many natural materials frequently change their chemical properties. In addition, some cross-linking mechanisms induce decrease of cell viability and functionality (Figure 7).
Although inkjet bioprinting enables encapsulation of live cells, their concentration has to be relatively low in order to form cohesive droplets and prevent clogging of the nozzle. Despite numerous disadvantages, this method has a great potential due to its low cost, high resolution, and high compatibility with many biomaterials. Additionally, these printers enable accurate deposition of fine droplets with precise volume to create high-resolution scaffolds with cells intact. Droplet size can be modulated from 1 to 300 pL with deposition rates from 1 to 10,000 droplets per second. Therefore, this method enables scaffolds creation with accuracy within 100 μm, which is very promising for creating complex scaffolds. Although it cannot produce very tall structures, influenced by the typical mechanical properties of the gel inks, due to its ability to print multiple structures and cell types it is very convenient for printing complex tissues with great accuracy.

INSTeAD Of CONClUSION: exPeCTeD fATe Of TheSe MeThODS IN The fUTURe
Adaptation of current 3D printing methods for biological applications has enormous importance for future fabrication of tissue grafts and artificial organs. Besides tissue engineering, 3D printing is also used in the area of drug delivery, analysis of chemical and biological agents and organ-on-a-chip devices [19].
Despite its huge potential in regenerative strategies, the main challenges are related to necessity of improved resolution, increased speed and printing that enables cells survival [18]. Current efforts in improvement of printing resolution in lithography assume the development of methods like electron beam lithography and multi-photon absorption polymerization, because these methods are suitable for creation of scaffolds with extremely precise feature sizes, of the order of only of tens of nanometers [20].
Materials used for 3D bioprinting must meet the following criteria: should be biocompatible, support cell growth and differentiation and retain its shape long enough to preserve scaffold integrity until solidification locks in scaffold geometry. The most commonly used materials for such purposes are collagen, gelatin, hyaluronic acid, alginate, modified copolymers, and photo-polymerizablemacromers [21].
For design of complex scaffolds that mimic tissue, additional research is necessary for accurate mapping of complex tissues to be able to make well-reproduced scaffolds with required structures and biological properties. One of the main challenges in future in 3D printing is figure 7. A binder is firstly jetted and selectively sprayed into a powder bed. When one layer is printed, the powder bed drops incrementally and a roller or blade applies and flattens the powder over the surface of the bed, prior to the next pass of the jet heads, with the binder for the subsequent layer to be formed and fused with the previous layer. Slika 7. Vezivno sredstvo se prvo izbacuje kroz mlaznicu i selektivno prska na rezervoar praha. Kada se odštampa jedan sloj, rezervoar praha postepeno opada, a valjak ili sečivo poravnava prah pre sledećeg prolaza mlaznih glava, sa vezivnim sredstvom za sledeći sloj koji se formira i kondenzuje sa prethodnim slojem. direct "in situ" bioprinting, or printing cells and biomaterials directly onto or in a patient. Some recent research showed capabilities of bioprinting directly into wounds or burn defects [22]. Further improvements of the printing speed and resolution are needed for "in situ" printing that will enhance tissue regeneration and reduce patients recovery time.