Biodegradable Polymers and Stem Cells for Bioprinting

It is imperative to develop organ manufacturing technologies based on the high organ failure mortality and serious donor shortage problems. As an emerging and promising technology, bioprinting has attracted more and more attention with its super precision, easy reproduction, fast manipulation and advantages in many hot research areas, such as tissue engineering, organ manufacturing, and drug screening. Basically, bioprinting technology consists of inkjet bioprinting, laser-based bioprinting and extrusion-based bioprinting techniques. Biodegradable polymers and stem cells are common printing inks. In the printed constructs, biodegradable polymers are usually used as support scaffolds, while stem cells can be engaged to differentiate into different cell/tissue types. The integration of biodegradable polymers and stem cells with the bioprinting techniques has provided huge opportunities for modern science and technologies, including tissue repair, organ transplantation and energy metabolism.


Introduction
On the basis of statistical data, there were 25.7 million adults and 75,000 children suffering from cancer of stomach, liver, breast, lung, and so on during 1995-2009 [1]. There were 117,040 patients that needed organ transplantation, but only 28,053 suitable organs available in the USA in 2013 [2]. Besides huge economic costs and immunological rejections of allografts, organ shortage has become a great restriction in organ transplantation. For the shortcomings and limitations of the surgical transplantation, scientists have tried various new methods to increase man's life span. These new methods include biomaterial induction, cell therapy, tissue engineering and organ manufacturing approaches [3][4][5][6].
More and more evidence has shown that stem cells are the building blocks or backbones for regenerative medicine with their unique functions and advantages of self-renewal, high proliferation, and multiple-directional differentiation abilities [7,8]. The combination of biodegradable polymers with multinozzle 3D bioprinting technologies (i.e., cell assembling techniques) gives the hope to deposit various cells in place to mimic normal biological conditions in vitro and in vivo [9][10][11][12][13][14][15][16].
Recently, complex tissue constructs, such as vascularized liver, adipose, bone, cartilage and muscle tissues, have been fabricated by an integrated tissue-organ printer (ITOP) [17,18]. Bioprinting technology has been regarded as the most effective tool for regenerative medicine including tissue engineering and organ manufacturing. Some scientists have considered bioprinting technology as the conjunction of 3D printing and organ manufacturing techniques [19][20][21][22].
The main characteristic of bioprinting technology is printing cells and extracellular matrices (ECMs) layer by layer to form 3D tissue/organ-like constructs. Cells in bioprinting can be adult cells or stem cells extracted from the patient who requires organ transplantation, which solves the rejection problems that arise from the recipient's immune system. Excelling at tissue engineering, bioprinting can create customized structures with the computer-aided models (CAD) quickly, and print cells, cytokines, or ECMs automatically and precisely. Hence, this technology has been regarded as a forward-looking method to assemble cells and biomaterials rapidly and precisely [19][20][21][22].
However, organ manufacturing for organ replacement and reconstruction has not yet been completely applied to clinical treatment with reliable bioartificial organs [23,24]. Especially, functional branched vascular system manufacturing remains a technological barrier that needs to be broken through immediately. At present, most of the existing technologies are limited in simple tissue manufacturing and high-throughput drug screening areas [9][10][11][12][13][14][15][16]25]. Furthermore, 3D bioprinting involves in various aspects such as cell types, biomaterials, and growth factors, of which each factor plays a key role in determining the printing results. The selection of biomaterials is of fundamental importance in bioprinting, which contains a selection of adult or stem cells, synthetic or natural polymers, and growth factors.
3D bioprinting approaches include mechanical enhancement, biomimicry, autonomous self-assembly and mini-tissue formation stages. It brings hope to humanity of biomimetic structures with specially designed patterns, material composition and degradation kinetics, controllable mechanical properties and biological effects. Most importantly, it is possible to obtain complex tissue/organ structures with physical and chemical properties similar to their counterparts with the combination of various factors for tissue and organ regeneration [9][10][11][12][13][14][15][16]. Thus, there are four basic elements in bioprinting technologies: cells, growth factors, biodegradable polymers and bio-printer. Different organ manufacturing technologies differ in these four aspects. Generally, cells, as "bio-inks", are mixed in biodegradable polymer hydrogels before being printed [9][10][11][12][13][14][15][16].

Classification of Bioprinting Techniques
Based on the working principles, bioprinting technologies have been divided into four classes: inkjet bioprinting, extrusion bioprinting, laser-assisted bioprinting, and ultrasonic bioprinting ( Figure 1) [9][10][11][12][13][14][15][16]. Among them, the former three are commonly employed in modern tissue engineering and organ manufacturing areas. In detail, inkjet bioprinting technology is based on simple home printing techniques. Cells and biomaterials such as hydrogels are printed separately layer by layer to form an object using thermal or acoustic methods [26]. Tissues/Organs can be gradually matured when the cells communicate and connect to each other after printing ( Figure 1A). During thermal inkjet printing, heat is generated at the printer head and the cells and biomaterials are forced out of the nozzle through pressure pulses. The system temperature can rise 4-10˝C with no obvious detrimental effect on cell viability [27,28].
In laser-assisted bioprinting or laser bioprinting, a bubble between a solution and a piece of glass is usually created through a vapor pressure (i.e., laser pulse). This pressure shoots a small drop of the solution, including cells, towards the collector substrate. Drop by drop, a tissue-like structure can be produced through the repeated processes. Laser bioprinting has the advantage of high resolution in living cell arrangement. In the meantime, cells undergo thermal and mechanical deformation from the laser during the printing processes. Usually, the damage can be decreased to some degree through incorporating biodegradable polymers in the "bio-inks". Especially, with scaffold biomaterials employed, elegant 3D structures can easily be printed [29,30]. Laser bioprinting can also be employed for multiple cell printing using different cell types in the substrate, which is a vital element for tissue engineering and organ manufacturing ( Figure 1F).
In general, different bioprinting techniques have dissimilar printing themes and limitations [31][32][33][34][35]. For instance, diversity and affordability are the prominent features of an inkjet bioprinting technique. Different cells and ECMs can be deposited together or separately. When cells are printed in situ (e.g., in patient body), there is no need to have a flat surface or platform to support the outcomes [33]. However, only liquids with low viscosities and low cell numbers can be printed to avoid clogging in the nozzle and to reduce shear stress on the cells. In extrusion bioprinting, the structural integrity of cells and ECMs obviously precedes those in inkjet bioprinting. Multiple cell types with high cell densities can be printed simultaneously using multinozzle printers. It is possible to directly print organs with a branched vascular system because of cells encapsulated in the intrinsic connectible ECMs or scaffold materials [34,35]. Nevertheless, the limited ECM selection and short storage time period of biodegradable polymer hydrogels have an impact on the technical development and practice [9][10][11][12][13][14][15][16][31][32][33][34][35][36]. In laser-assisted bioprinting, a broad range of biomaterials with various viscosities can be printed without clogging problems. However, the cell printing efficiency needs to be improved [37].
Currently, some achievements have been attained with various cells and biodegradable polymers. For example, adipose-derived stem cells (ADSCs) and mesenchymal stem cells (MSCs) were printed using a laser-assisted bioprinting (LaBP) technique [38,39]. Around five to seven living  [9]; (B) A robotic printing platform and its crescent construct [9]; (C) A direct-write system and its preliminary 3D figures [9]; (D) A modular tissue printing platform with four "cartridges" to load cell suspensions or hydrogels developed in Brigham and Women's Hospital, Harvard Medical School, Prof. Yoo's group [9]; (E) A bioprinting tubular structure with cellular cylinders developed in University of Missouri, Columbia, USA, Prof. Forgacs' group [9]; (F) A laser-guided direct writing (LGDW) system and its patterned factor-linked beads on a stem cell monolayer with micrometer accuracy (Bar = 200 µm) developed in University of Minnesota, Prof. Odde's group [9].
In general, different bioprinting techniques have dissimilar printing themes and limitations [31][32][33][34][35]. For instance, diversity and affordability are the prominent features of an inkjet bioprinting technique. Different cells and ECMs can be deposited together or separately. When cells are printed in situ (e.g., in patient body), there is no need to have a flat surface or platform to support the outcomes [33]. However, only liquids with low viscosities and low cell numbers can be printed to avoid clogging in the nozzle and to reduce shear stress on the cells. In extrusion bioprinting, the structural integrity of cells and ECMs obviously precedes those in inkjet bioprinting. Multiple cell types with high cell densities can be printed simultaneously using multinozzle printers. It is possible to directly print organs with a branched vascular system because of cells encapsulated in the intrinsic connectible ECMs or scaffold materials [34,35]. Nevertheless, the limited ECM selection and short storage time period of biodegradable polymer hydrogels have an impact on the technical development and practice [9][10][11][12][13][14][15][16][31][32][33][34][35][36]. In laser-assisted bioprinting, a broad range of biomaterials with various viscosities can be printed without clogging problems. However, the cell printing efficiency needs to be improved [37].
Currently, some achievements have been attained with various cells and biodegradable polymers. For example, adipose-derived stem cells (ADSCs) and mesenchymal stem cells (MSCs) were printed using a laser-assisted bioprinting (LaBP) technique [38,39]. Around five to seven living cells were printed using a laser-induced forward transfer technique with high precision at micron scales [40].
Most of the natural biodegradable polymers can be dissolved in cell friendly inorganic solvents to form solutions or hydrogels with good biocompatibility and biodegradability. The solution or hydrogel states of the biodegradable polymers have certain fluidity during bioprinting [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58]. The mobility of the polymer solutions or hydrogels can be modified in rapid prototyping (RP) devices, and can be manipulated with high resolution and mature accuracy. This is vital important for the biological property retention and tissue/organ recreation during and after cell printing processes [70,71]. These polymers are mainly applied to temporary prosthesis, scaffold formation and drug delivery fields. Several relevant parameters of biodegradable polymers, such as extreme temperatures, organic solvents, and water deficiencies can negatively influence cell viabilities.
Evidently, the number of biomaterials that can be simultaneously printed depends mainly on the nozzle numbers of an extrusion-based bioprinter. Different material systems can be integrated into a construct by choosing proper printing parameters. For example, in our group, we have developed various multinozzle bioprinters for different organ manufacturing. Especially, two robotic arm controlled injectable nozzles and two extrusion-based syringes have been combined for solid organs, such as the breast, liver and heart, manufacturing [91]. We have assembled various cells, such as ADSCs and hepatocytes, using our homemade multiple nozzle cell 3D printers, to produce complex organ precursors with branching vascular systems inside, and to convert ADSCs into vascular endothelial cells and smooth muscle cells in the 3D constructs [22][23][24]. Figure 2 is an illustration of a two-syringe RP technique [10,48]. Figure 3 is an illustration of liver manufacturing with a four-nozzle bioprinter and multiple biomaterials. Four different biomaterials can be simultaneously printed together.Thereby, it can meet the requirements for stable vascular system formation within a hybrid hierarchical polyurethane-cell/hydrogel construct [10,11]. Correspondingly, it was the first time some new words, such as "inner scaffold" and "outer scaffold", have appeared in our manuscripts and cover letters. With the similar integrations of extrusion nozzles and biomaterials, Kang and coworkers have produced human scale bone, cartilage and muscle tissues with a composite gelatin/fibrinogen/hyaluronan/glycerol hydrogel [17]. The combination of different biodegradable polymers in one hydrogel has been proved to be an effective way in 3D bioprinting techniques, especially for complex organ manufacturing.
Molecules 2016, 21, 539 6 of 13 extrusion nozzles and biomaterials, Kang and coworkers have produced human scale bone, cartilage and muscle tissues with a composite gelatin/fibrinogen/hyaluronan/glycerol hydrogel [17]. The combination of different biodegradable polymers in one hydrogel has been proved to be an effective way in 3D bioprinting techniques, especially for complex organ manufacturing.

Stem Cells in Bioprinting
There is a current trend to combine stem cells and growth factors with ECMs in 3D milieus to mimic organ development [92][93][94][95][96][97][98][99][100][101][102][103][104][105]. In fact, with the proper use of growth factors, stem cells can be extrusion nozzles and biomaterials, Kang and coworkers have produced human scale bone, cartilage and muscle tissues with a composite gelatin/fibrinogen/hyaluronan/glycerol hydrogel [17]. The combination of different biodegradable polymers in one hydrogel has been proved to be an effective way in 3D bioprinting techniques, especially for complex organ manufacturing.
Especially, embryonic stem cells (ESCs) have been used for developmental biology, regenerative medicine, cell replacement therapy and drug discovery with high differentiation potentialities [106]. However, in view of the moral issue, the applications of ESCs in 3D bioprinting techniques have been greatly influenced. Currently, some somatic stem cells, such as adipose-derived stem cells (ADSCs) and induced pluripotent stem cells (iPSCs), are becoming increasingly popular and attractive [107][108][109][110][111][112].
iPSC is a perfect cell type for its feature of overcoming the difficulty of the limitations associated with the current cell sources and has developed very quickly. iPSCs have the potential to differentiate into almost all kinds of cells for different tissues, such as nerve, blood, bone, and heart, under appropriate conditions. Currently, iPSC lines of rats [122], pigs [123], humans [124], monkeys [125] and rabbits [126] have been set up successfully. Some researchers have chosen to focus on iPSCs for disease model establishment, drug discovery template, energy metabolic research, cell therapy and organ manufacturing. In detail, the methods of generating integration-free iPSCs are as followings: non-integrating DNA methods, RNA-based methods, proteins-based methods [127][128][129]. Similar to other cell printing techniques, 3D stem cell printing can provide stem cells with complex 3D cellular microenvironments similar to their native counterparts. Patient self-derived stem cells can solve the immune rejection issues.

Future Outlook
Bioprinting technology has obtained growing popularity for its favorable meanings. Some groups have made great progress with the pertinent techniques in tissue engineering, organ manufacturing and drug screening areas. Biodegradable polymers are usually used as the cell printing ECMs or accommodations, while stem cell techniques are especially helpful in complex organ manufacturing. The development and future of bioprinting techniques are promising: (1) it can solve the donor shortage problems of organ transplantation; (2) it can overcome the immune rejection symptoms caused by allograft tissue/organ transplantation; (3) it can reduce the high cost and heavy burden of the patients with reverse engineering and personal manufacturing techniques; (4) it can establish a platform for high-throughput drug screening and organ manufacturing; and (5) it can be easily accepted by researchers and doctors with those CAD tissue/organ models.
Nevertheless, the development of bioprinting is still imperative and challenging. In particular, several aspects should be further addressed: (1) lack of design principles for bioprinting despite an emergency in additive manufacturing areas [131][132][133]; (2) further 4D, 5D even 6D development based on the 2D and 3D printing techniques [134,135]; (3) optimal stem cells induction approaches before, during or after the printing processes; (4) multiple cell type or biomaterial system incorporation protocols for complex tissue/organ manufacturing; (5) special cell culture incubators to acquire large amount of cells in a short time period; (6) biological functionality realization within the 3D printed substitutions; (7) harsh environment factor reduction (such as temperature and pressure) for biological property maintenance of the integrated cells and growth factors; and (8) typical zone or layer designs for complex organ products. Further multidisciplinary research and cooperation are still urgently needed relating to biology, chemistry, medicine, materials, mechanics, and pathophysiology sciences and technologies. More and more high-throughput manufacturing products, such as the liver, heart, kidney, skin, cartilage and bones, will become possible with promising 3D bioprinting technologies. We are herein expecting a bright and fruitful future for the modern multinozzle 3D bioprinting techniques.