Polymeric Materials for 3D Bioprinting

CHAPTER 4

Polymeric Materials for 3D Bioprinting PRIYA MUKHERJEE, MSC  • ANKITA RANI, M.TECH  •  PICHIAH SARAVANAN, PHD

INTRODUCTION Three-dimensional (3D) bioprinting is a printing technology in which organs and tissues are printed three dimensionally, through a layer-by-layer approach.1,2 The organs obtained through this technique find application in organ transplantion and for screening newly developed drugs.3 The demand on the organ transplantation and uncertainty over the evaluation of newly formulated drugs on animal organs necessitated the utilization of 3D printing technology in the field of medical science. The process of 3D bioprinting consists of three steps: prebioprinting, bioprinting, and postbioprinting. The first involves formation of organ blueprint and material selection with the utilization of bioimaging technologies for the capture of organ blueprint. These include computed tomography (CT), magnetic resonance imaging (MRI), and X-ray. The obtained images are converted into stereolithography (STL) files with an extension of .stl. These STL files provide the 3D representation of the image and the information is prototyped by the 3D bioprinter. Selection of material depends on the physiology of the organ to be synthesized and the type of printer adopted.3–5 The printing refers to the actual prototyping of the organ, in which foundation of the organ is formed first, then the print head moves above to form the next layer and in the same way the organ model is developed.4,6 The poststep involves conditioning of the developed prototype of the organ through the proliferation and differentiation process. This is achieved by incubating the organ at specific temperature for a specified period of time in a bioreactor.7,8 The essential steps involved in the bioprinting are shown in Fig. 4.1A, while Fig. 4.1B shows the sequential procedure of the bioprinting process. In general the printing adopts three general strategies: inkjet-based, extrusion-based, and laser-assisted bioprinting. The former utilizes current pulse or a heating element as liquefying and pressurizing agent for ink dispersion.5,9,10 This method dispenses the bioink in the form of droplets; therefore, it is classified as a discontinuous printing process. In the extrusion-based

printing, the bioink is extruded out from the printer, thus deposition occurs in continuous manner with a support of pneumatic or mechanical force.5,11 In the case of laser-based printing, the bioink is made in the form of ribbon and a pulsed laser is applied to push the ribbon at the specific location to print the desired biological components.5,11,12 The ink that is used for bioprinting is commonly referred as “Bioink”. This bioink in general consists of living cells while printing tissue or organ; whereas, in the case of printing scaffolds, they do not contain the living cells. Beside cells, the bioink consists of several polymer compositions in which the cells are suspended. Polymers form the basic framework of the organ nurturing cell adhesion, proliferation, and growth.2 They may be present in the form of individual hydrogels or polymers or as a mixture of both. Other materials that can be present in the bioink include the bioceramic compounds (e.g., hydroxyapatite, tricalcium phosphate). These bioceramics are primarily used for reinforcing the polymer matrix and for improving the efficiency of tissue formation.13–15 The common materials opted for the ink composition is schematically represented in Fig. 4.2. Hydrogels are made up of a network of same or different polymers, which are cross-linked with each other and having water molecules absorbed within their framework. The presence of water molecules in the cross-linked network allows easy exchange of nutrients throughout the framework. Structural similarity of hydrogels with the native extracellular matrix (ECM) employs their wide application as bioink. These hydrogels when mixed with the cells act as an encapsulating agent that preserves the cells from direct damage and provides microenvironment for their survival. Several widely used hydrogels include alginate, gelatin, and hyaluronic acid derived from natural polymers. Poly ethylene glycol (PEG) and PEG-derived hydrogels are of synthetic route.2,11 Natural polymers possess high resemblance with the ECM of tissues and results in good bioactivity. Alginate, gelatin, collagen, and hyaluronic acid are

3D Printing Technology in Nanomedicine. https://doi.org/10.1016/B978-0-12-815890-6.00004-9 Copyright © 2019 Elsevier Inc. All rights reserved.

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64

A

B FIG. 4.1  Essential steps and procedure in the 3D bioprinting process. (A) The sequential steps involved in

3D bioprinting process. (B) The processing procedure of bioprinting from the obtained blueprint. (The figure is adapted and reproduced with permission from Mironov V, Kasyanov V, Markwald RR. Organ printing: from bioprinter to organ biofabrication line. Curr Opin Biotechnol. 2011;22(5):667–673. Kang HW, Lee SJ, Ko IK, Kengla C, Yoo JJ, Atala A. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol. 2016;34(3):312–319.)

among the commonly used natural polymers. As they are of high molecular weight, they possess high viscosity and low solubility. The viscosity factor makes them suitable only for some specific printing strategies, whereas the solubility factor restricts the modification of their structural and functional properties.16,17 Synthetic polymers are manufactured either by selfassembly of monomers or polymers or by chemical modifications. As the design matrixes of these polymers are custom made, their structural and functional properties are easily controllable. Furthermore, they strengthen the mechanical integrity of the framework and eliminate the manufacturing limitations by offering architectural functionality and tunable viscosity.16 Poly(ethylene glycol), poly(lactic-co-glycolic acid) (PLGA), poly-caprolactone (PCL), and poly-l-lactic acid (PLLA) are among the widely used synthetic

polymers in the bioprinting field.2,16 The composite of natural and synthetic polymers offer tunable viscosity, with high functionality. Although the natural and synthetic polymers have equivalent potential as precursors, the present chapter aims in providing insight on the role of synthetic polymers in 3D bioprinting process. 

TYPES OF SYNTHETIC POLYMERS Polymeric materials have emerged as a highly efficient bioink material over prevailing ceramic and hydrogel in 3D bioprinting, owing to its higher degree of feasibility in fulfilling the needs of the medical science demand. These synthetic polymers endow the advantage of bulk handling with controllable degradability, microstructure, and strength which promote them as promising

CHAPTER 4  Polymeric Materials for 3D Bioprinting

65

FIG. 4.2  Schematic showing common materials opted for the ink composition.

material. Table 4.1 categorizes the synthetic polymers used in 3D bioprinting along with their molecular formula and structure.

Acrylonitrile Butadiene Styrene Acrylonitrile butadiene styrene (ABS) is a triblock copolymer of petrochemical origin. It belongs to the chemical family of styrene terpolymer32 with good strength and toughness. The low melting point (105°C) of this material further enhances its applicability. The ABS is made of three different monomers that provide various beneficial features, where heat tolerance is provided by acrylonitrile, robust impact strength is imparted by butadiene, and styrene impart rigidity.32 The fused deposition modeling (FDM) and selective laser sintering (SLS) printing technologies employ ABS as their precursor.33 Furthermore, it is used in cartilage engineering technologies.33 However, its deprived biodegradable nature and identical cell integration and processability similar to polylactic acid (PLA) restricts its applicability.33 

Eudragit Eudragit is a USFDA-approved methacrylic polymer in drug delivery system.34 For instance, Eudragit EPO is

used in theophylline tablet as carrier in drug delivery process.35,36 The performance of eudragit is appreciable for such drug delivery process that replaced PCL, a polymer widely used for similar application. Eudragit L 100-55, Eudragit 114 RL PO, Eudragit RS-100, and Eudragit EPO are some of the commercially available eudragit polymers manufactured by Evonik Nutrition & Care GmbH, Germany.37 Among these, Eudragit EPO possesses robust mechanical property, enhanced moisture protection, and taste masking making it as a predominant for drug carrier.35,38 Nevertheless, Eudragit RS-100 has shown potential in fabricating release of complex tablets with zero-order drug release.39 

Polybutylene Terephthalate Polybutylene terephthalate (PBT) is biocompatible thermoplastic polyester used in FDM printing technologies, as seen in ABS.33 PBT further exhibits dominant elasticity and easy processing ability in combination with strength and toughness.40,41 PBT is one among the common polymers that are widely used in biomedical field for in vivo and in vitro biocompatibility.42 It finds application in printing bone scaffolds of canine trabecular bones and in tissue regeneration.33,43 Moreover, it is also used as filler in orthopedic surgery.42 It possesses

66

TABLE 4.1

Synthetic Polymers Employed for 3D Bioprinting

1.

Synthetic Polymer With Its Molecular Formula

Structure

References

Acrylonitrile butadiene styrene (ABS) [(C3H3N·C4H6·C8H8)n]

18 n

C N 2.

Eudragit®

L-100 [(C9H14O4)n]

CH3

C

3.

Polybutylene terephthalate (PBT) [(C12H12O4)n]

C

O

O

O

C 2 H5

H

O

19

CH3

O n 20

O O

O

n

4.

Poly caprolactone (PCL) [(C6H10O2)n]

21

O O 5.

Poly-d,l-lactic acid (PDLLA) [(C3H6O3)n]

n 22

CH3 OH

HO O

n

     

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Sl. No

6.

Polyether ether ketone (PEEK) [(C19H12O3)n]

23

O O

C

O n

7.

Poly ethylene glycol (PEG) [(C2H4O)n·H2O,n ≥ 4]

24

O

H

O

H

8.

PEG-diacrylate (PEGDA) [CH2]COC(OCH2 CH2)nOCOC]CH2]

CH2

O

O

O Poly-glycolic acid (PGA) [(C2H2O2)n]

n

26

O O

10.

25

O

CH 2

9.

CHAPTER 4  Polymeric Materials for 3D Bioprinting

n

Polylactic acid (PLA) [(C3H4O2)n]

n

27

CH3

O O

n

Continued

67

     

68

TABLE 4.1

Synthetic Polymers Employed for 3D Bioprinting—cont’d

11.

Synthetic Polymer With Its Molecular Formula

Structure

Polylactic-co-glycolic acid (PLGA) {[(C3H4O2)n·(C2H2O2)m]OH}, n:m = 50:50

References 28

O

Poly propylene fumarate (PPF) [C3H6OH(C7H8O4)n OH]

O

n

CH3 12.

H

O OH

m

O 29

CH3

O

CH3

O

OH

OH

O O n

13.

14.

Polyurethane (PU) [RONH(CO)R′]n

30

O

O

N

N

H

H

Poly-vinyl alcohol (PVA) [(C2H4O)n]

O

O

n

31

OH

     

n

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CHAPTER 4  Polymeric Materials for 3D Bioprinting physical and chemical properties similar to PCL or PLA and does not have any distinct advantages.33 PBTs undergo degradation in aqueous media through hydrolysis or oxidation route as in most polymers.42 The higher melting point (225°C) and nonbiodegradable nature leading to the formation of crystalline remnants in in vivo application limit its applicability.33,44,45 

Poly Caprolactone PCL is economically cheaper and possesses great bioink features including stiffness, biocompatibility, and degradability.33 Primarily, it is used as an FDM and stereolithography (SLA) polymeric material in hard tissue engineering.46,47 Its quite low melting point (60°C) with commanding rheological and viscoelastic properties facilitates thermal treatment of PCL for applicability in melt-based extrusion printing.33,48 PCL is one of the nontoxic polymers that bear notable stability. In general the stability lasts for a period of 6 months with biological half-life of 3 years.33 SLS-printed PCL scaffolds exhibit characteristics like poriferous structure having interconnectedness, rough surface, and comparable compactness to that of bone leading to bone regeneration and cell ingrowth capability.33,47 However, its longer biological half-life develops secondary problem in scaffolds made for applications other than bone tissue engineering.48 Furthermore, its higher hydrophobicity characteristics lead to low bioactivity, that is, slow cell growth and tissue adhesion.49 

Poly-d,l-Lactic Acid Poly-d,l-Lactic acid (PDLLA) is a lactic acid-derived polymer with amorphous structure. It is hydrophobic in nature possessing convenient biocompatibility with durable mechanical features finding its way in biomedical applications especially in SLA techniques.33,50,51 It is one among the widely used polymers in forming biocompatible and porous scaffolds and hence applied in resorbable devices of orthopedic rehabilitation and tissue engineering.51,52 PDLLA being hydrophobic does not allow water dissemination in its matrix and prolongs the decaying process; however, inclusion of hydrophilic groups enhances the water uptake and consequently hinders the decaying.50,52–54 However, such inclusion of hydrophilic groups might degrade the polymer to monomer by lowering the pH and trigger obnoxious allergic and inflammatory reactions in the body.50,52,55,56 

Polyether Ether Ketone Polyether ether ketone (PEEK) is an expensive, nonbiodegradable polymer possessing superior

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biocompatibility, low heat conductivity and radiolucent with comparable bioinertness.33,57,58 The semicrystalline nature of the material allows good heat resistance and chemical stability.59 It possesses strength and elasticity as that of cortical bone that reduces the chance of osteopenia after implementation by minimizing the stress shielding.60,61 It is mostly used in FDM and SLS technologies as bioinks in prototyping craniofacial implants and bone replacement.33 The unique radiolucent property provides advantage in orthopedic applications by permitting radiographic assessment.60,62 The submissive bioinert nature suppresses osteointegrative properties and hence restricts its applicability for tissue engineering.60 It further has the potential to prompt reactions such as dislodging, encapsulation, and extrusion in the body affecting the body tissues.33 Moreover, it has a very high melting point of 350°C further retarding its usage in SLS technique.33,63 

Poly Ethylene Glycol Poly ethylene glycol (PEG) is a good hydrophilic polymer formed by radical polymerization reaction.64 It has a linear or branched structure containing disymmetric or asymmetric hydroxyl ion as tail groups.65 The hydroxyl group can also be replaced by functional groups such as acetylene, acrylate, amine, azide, carboxyl, methoxyl, thiol, and vinyl chloride.65,66 The robust biocompatibility promoted PEG as widely used material in drug delivery system, tissue engineering scaffold formation, and surface modifications for producing amphiphilic block copolymers and ionomers.65,67 It is inherently resistant to protein adsorption and cell adhesion and predominantly forms hydrogel.67 They are nonbiodegradable and have low mechanical strength and their nonbiodegradability was attributed to the presence of C–C polymer backbone.64 However, degradability of PEG can be triggered by hydrolytic and enzymatic degradation.64,68 

PEG-Diacrylate PEG polymerized with acrylate generates PEG-diacrylate (PEGDA). The photoinitiator characteristic of the material induces the bioactivity through a simple UV exposure.69 It is a hydrophilic photoresist polymer, used primarily in direct laser writing (DLW) and SLA70 technique. Its properties such as biocompatibility, high water content, and tunable mechanical properties promote it as a good candidate.71 Application of PEGDA includes cell encapsulation, delivery, and cross-linking with other polymers. Similar to other polymers, it is also used for scaffold formation in tissue engineering system72 and for hydrogel generation.73 Partial diminution

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of the 3D geometry of the scaffold promoted by the water binding property of PEGDA is seen as major limitation of this polymer.74 However, the diminution can be overhauled by combining it with pentaerythritoltetraacrylate (PETTA) or pentaerythritoltriacrylate.74 

Poly-Glycolic Acid Poly-glycolic acid (PGA) is a major synthetic polymer in 3D scaffold architecture due to its chemical versatility, ease of processing along with good biocompatibility, and biological performances.75 The biodegradation of PGA yields glycolic acid monomer, which is easily removed from the body by certain catabolic pathways in the form of carbon dioxide and water.76 Moreover, the physical and mechanical properties of PGA can be regulated by its copolymers.75 PGA is used in bone internal fixation devices and in preparation of resorbable sutures.75 In comparison to PDLLA the degradation product of PGA is nontoxic.76 The seeding density and spreading of the cells can be catalyzed by surface functionalization of PGA through hydrolysis of ester bonds.76 Although the hydrolysis technique generates beneficial features, it can be limited by its restructuring surface morphology and mechanical strength.76 It is susceptible to bulk erosion resulting in scaffold collapsing thereby liberating acidic degradation products affecting the body.75–78 

Polylactic Acid PLA is hydrolytically degradable aliphatic polyester and possesses properties such as biocompatibility, degradability, and printing ability making it as an eminent polymeric bioink.33,79–81 It is a prominent polymer used as precursor in the FDM technique. It is capable of generating filaments at its melting point (175°C) and hence predominantly used in musculoskeletal tissue engineering for substituting ligaments and nonbiodegradable fibers.33,75 The degradation of PLA releases acidic by-product and tampers its long-term biocompatibility through prompting tissue inflammation and cell demise.33,82 Moreover, its brittleness makes its comprehensive strength lower than that of bone restricting its usage.33 The limitation can be overcome by combining it with low-cost ceramic materials such as calcium phosphate by forming scaffolds of improved bone strength and reduced acid generation.33,83,84 

Polylactic-Co-Glycolic Acid Polylactic-co-glycolic acid (PLGA) is a classification of polymers with good biodegradable nature and pronounced cytocompatibility properties.85–87 PLA and PGA combine to form copolymer PLGA that aids in

cell proliferation and differentiation along with hosting tissues.75,88 It further presents mechanical features equivalent to that of human calcareous bone and is osteoconductive.89 PLGA is sometimes used in bone regeneration animal model and in many tissue-restoring systems; however, the hydrophobic feature defers its usage.89,90 Furthermore, its linear structures results in low mechanical stiffness, promotes high degradation rate, and thereby restricts its usage as scaffold material.85,90 The broken PLGA debris in in vivo tests is susceptible to promote inflammatory reaction, and this fracturing can be restricted by mixing it with PCL.90 

Poly Propylene Fumarate Poly propylene fumarate (PPF) is another classification of polymers with linear monomers structure possessing biodegradable characteristics and widely used in SLA as a base polymer for cross-linking.91 It is made from fumaric acid, which is composed of unsaturated C–C bonds as building blocks for the cross-linkable networks.92 Owing to the presence of fumaric acid as monomer the degradation yields harmless end products such as poly(acrylic acid-co-fumaric acid), fumaric acid, and propylene glycol.93 Its biocompatibility spans between soft and hard tissues, while its tensile properties are analogous to that of trabecular bone and this makes them as appropriate polymer for bone cement.94–96 

Polyurethane Polyurethane (PU) is also a classification of polymers but bears thermosetting nature. It is considered as good biodegradable elastomer having excellent biocompatibility and mechanical strength.97 It is further differentiated by the use of solvent as organic solvent–based traditional PU and water-based PU; the former adopts volatile organics, while water is used as solvent in the latter.98 PU finds its application in SLA and DLP printing techniques, and its degradation can be compared with PLA.97,98 High printing resolution and good cytocompatibility further add to characteristics strength in 3D bioprinting.98 It is preferred for the chondrocytes generation in cartilage tissue engineering, bone fabrication, and construction of muscle and nerve scaffolds.97,98 It exhibits a distinct ideal elastomeric property that undergoes repetitive contraction and relaxation and makes perfect choice for muscle generation.99 

Poly-Vinyl Alcohol Poly-vinyl alcohol (PVA) are synthesized in the presence of vinyl alcohol and acetate, and these monomers

CHAPTER 4  Polymeric Materials for 3D Bioprinting were responsible for the biocompatible, biodegradable, bioinert, and semicrystalline nature. Similar to PEG, PVA is also a water-soluble polymer and used in SLS printing technique. The tensile potency of PVA resembles to that of human articular cartilage. PVA can further form complex with appropriate adhesive and provide a suitable matrix for bone cell ingrowth.100 Its superior hydrophilicity and chemical stability allows withstanding in extreme pH and temperature while its semicrystalline structure allows efficient oxygen and nutrients passage to the cell (i.e., vital elements for cell survival and allows elimination of waste easily through the cells).101 It is widely beneficial in various loads bearing treatments such as craniofacial defect treatment and bone tissue engineering applications.100–103 PVA being water-soluble readily swells in the presence of water and possesses difficulty in handling.100 

FACTORS AFFECTING BIOPRINTED CONSTRUCTS Polymeric biomaterial characteristics such as porosity, surface area, biocompatibility, biodegradability, and mechanical strength are among the most important factors that decide the properties of constructed scaffolds. Porosity is a foremost factor that affects cell growth, removal of degraded compounds, and vascularization of the printed scaffold.104–106 The nutrient transport and mechanical properties are controlled by the porous nature of the material.104 The features such as ECM production and organization are again dependent on the porosity.104 In general, a pore size of 100 and 150 μm is required for bone formation although a size greater than 300 μm intensifies the bone formation and vascularization process.104,105,107,108 Materials having a pore size of 250 μm and greater are suitable for blood vessel formation as compared to those of smaller size.109 In case of tissue engineering, both narrow and broader pore size is preferred, for example, ∼5 μm is required for neovascularization, 5–15 μm for fibroblast ingrowth, 20 μm for hepatocytes ingrowth, 20–125 μm for adult mammalian skin ingrowth, and 200–350 μm for osteoconduction.110–115 The next comes is surface area an important factor for cell anchorage and proliferation. It is well known that larger and accessible surface area favors cell and tissue attachment in scaffold.110,116 It is vital in case of tissue or organ’s function restoration or replacement, as high surface to volume ratio facilitates accommodation of larger number of cells.110 The biocompatibility nature follows the surface area and refers to the material ability to act as a substrate for supporting the system’s

71

cellular activity and perform a specific function without creating any unwanted reaction.110,117 Thus, it is determined by the dependent of chemistry (monomers, copolymers, functional group etc.), morphology, and structure (linear branched and cross-linked) of the chosen polymers.110 For example, polymers such as PLA, PLGA, and PGA exhibit good biocompatibility.110,118 The biodegradable tendency of a polymer is very important because they allow natural degradation of scaffold after a specified time replacing themselves with the newly grown tissues. The drug delivery system and tissue engineering system are highly revolutionized by the invention of these biodegradable polymers.110 The polymer biodegradation is facilitated by the cleavage of the sensitive hydrolytic or enzymatic bonds present in the polymer.110,119 This biodegradability is an intrinsic property of the polymers and is influenced by chemical structure, molecular weight, crystallinity, glass transition temperature (Tg), and the range of hydrophilicity/ hydrophobicity.110,120 The nonbiodegradable polymer imparts immutable support to the patient and over a time attains durability.110 Mechanical property also is an equally important factor of polymers because the regeneration potential of hard tissues depends on it.110 As the scaffold needs to withstand the load and stress for a successful growth of the new tissues, the rheological properties of polymers such as maximum strain, elastic modulus, and tensile strength play a crucial role. The associated factors including pore connectivity, orientation, shape, size, and volume have influence on scaffold’s mechanical property and structural integrity.110 Table 4.2 presents a list of physical properties of some widely used polymers in 3D bioprinting. 

APPLICATION OF SYNTHETIC POLYMERS IN 3D BIOPRINTING Scaffold Printing for Bone Tissue A scaffold is a basic structural unit playing a vital role in tissue engineering as supporting material where they act as a framework for an organ. These scaffolds are followed by seeding of cells, which grow on the surface provided by them and they take a shape similar to that of organ and degrades once the organ is fully developed. There are several traditional methods adopted for scaffold formation, which includes gas foaming, melt molding, phase separation, and solvent casting and particulate leaching. However, there are many drawbacks associated with these techniques: uncontrollable pore size, shape, and geometry, undesired spatial distribution of cells, and interconnectivity.12,122

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TABLE 4.2

Physical Properties of Some Synthetic Polymer S.No.

Polymer

Crystallinity %

Density (g/cm3)

Degradability

References

1.

PGA

50(46–52)

1.6

Biodegradable

113, 121

2.

PLA

0–42

1.24–1.26

Biodegradable

113, 121

3.

PCL

55

1.11

Biodegradable

113, 121

4.

PVA

40–50

1.25–1.32

Biodegradable

121

5.

PLGA

Amorphous

1.27–1.34

Biodegradable

121

6.

PDLLA

Amorphous

1.25

Biodegradable

113, 121

A

B FIG. 4.3  Sequential procedure involved in scaffold printing using specific polymer (A) represents the

complete process flow, showing the printing technique for a PCL framework (B) image of fabricated PCL layer. (The figure is adapted and reproduced with permission from Kang HW, Lee SJ, Ko IK, Kengla C, Yoo JJ, Atala A. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol. 2016;34(3):312–319.)

The innovation of bioprinting defeated the aforesaid limitations, through connectivity between scaffold image obtained from the medical imaging techniques and printing process. Furthermore, the discovery of new generation printers capable to deliver the prototype of the obtained scaffold image resolved the issues encountered.122 In general, the bone tissue printing is always preceded by a scaffold fabrication process. Common steps included in bone tissue printing are sequenced as follows: combined selection of polymeric materials and required cells for scaffold and bone printing, scaffold

fabrication and characterization, cell culture, seeding of cells in scaffold by the bioprinting process, evaluation of cell viability and proliferation on the scaffold, and finally experimenting with an animal model. A hydrated environment is important for the cell survival and is provided by dissolving the cell culture either in enzymatic solution or in some hydrogel solutions.123,124 PCL, PLA, PLGA, PEEK, and PBT are some of the synthetic polymers frequently used in scaffold printing for bone tissue. Fig. 4.3 depicts the step-bystep procedure involved in the cartilage printing using specific polymer.

CHAPTER 4  Polymeric Materials for 3D Bioprinting

73

TABLE 4.3

Polymers in Scaffold Printing for Bone Tissue Engineering Polymer

Application

Bioprinting Type

Significant Characteristics

References

PCL/Bioactive Borate glass composite

Established angiogenesis in scaffold interior (bone tissue engineering)

Extrusion

High elastic modulus, bioactivity enhanced by Borate glass

48

PCL/HA filled with CNT

Effective in stimulation of bone cell regeneration

Extrusion

HA resulted in increased bioactivity

49

PLA/HA

self-fitting implant (shape memory effect)

Extrusion (fused filament ­fabrication)

High elastic modulus, Mixing of HA resulted in increased mechanical strength

89

PLGA

Bone tissue regeneration (periosteum and bicortical) in rabbit model

NM

Biocompatible, highly osteoconductive

125

TiO2 coated PEEK

Better cervical fusion of bone tissue

NM

Biocompatible, TiO2 increased bioactivity

126

PEEK

Frontal bone defect reconstruction

NM

Biocompatible, widely used for craniofacial implants

127

CaP coated PBT

Increased bone formation

NM

Highly biocompatible, major polymer for articular cartilage repair

128

PCL/β-TCP

Increased Cell proliferation

Extrusion

Biocompatibility increased by β-TCP addition.

129

PEG hydrogel coated PCL

Controlled craniofacial tissue mineralization

Precision ­extrusion

PEG resulted higher cell growth, inhibited cell differentiation and tissue mineralization

130

NM, not mentioned; TCP, tricalcium phosphate.

Table 4.3 provides complete list of synthetic polymers used for scaffold printing with their prospective application in bone tissue engineering. Least bioactive synthetic polymers are often mixed with some bioactive compounds to promote the biocompatibility of the scaffold surface. For instance, Goncalves and coworkers in 2016 mixed PCL with silicon doped hydroxyapatite (HA) and carbon nanotubes (CNTs). The CNTs enhanced the scaffold’s surface electrical conductivity thereby stimulating higher cell growth on the other hand silicon increased the bioactivity.49 Similarly, a study Murphy and coworkers in 2017 mixed PCL with bioactive borate glass, which resulted in enhanced angiogenesis property on the surface of the scaffold.48 In recent years, sol-gel-derived TiO2 coating on scaffold surface is also preferred for increasing the bioactivity of the inert polymers.126 The advantage of sol-gel-derived TiO2 coating over HA coating is its nondegradable property.131,132 These bioceramic compounds are well known for their reinforcing property,

and they are mainly used in hard tissue engineering as a constituent of polymer matrix in scaffold synthesis. As discussed earlier in the polymer section, PLA being brittle in nature is generally mixed with ceramic compounds for enhanced strength. The PLA/HA scaffold’s shape memory effect and mechanical properties were studied by Senatov and coworkers in 2015, and they observed that the mixing of HA into PLA scaffold restricted appearance of cracks during the compression heating cycle and also decreased the molecular mobility of the PLA molecular chains.89 

Muscle Tissue Printing The development on application of polymers for muscle tissues printing is still in cradle stage as compared to cartilage. The presence of highly vascular network within the muscle hinders its development to the next stage. In addition, the use of polymers interferes with new tissue growth, and cytotoxic behavior leads to the reduction of cell-cell contact

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within the tissue.133 Owing to this reason the focus of tissue engineering has diverted toward the formation of scaffold-free organs that rarely employs synthetic polymers. However, there were some attempts reported by the researchers on the use of polymers toward muscle bioprinting. Merceron and his coworkers in 2015 developed Musculotendon unit (MTU) utilizing PU and PCL as a structural unit.99 The significant elastic property of the PU over PCL motivated them to use it for muscle tissue printing. The PU possesses very good elastic property with low young modulus as compared to PCL and thereby making suitable candidate for muscle printing. Their study explicitly revealed the rheological behavior of the PU and PCL experimentally.99 In 2016 the team led by Anthony Atala of Wake Forest School of Medicine, Medical Center Boulevard, Winston–Salem, North Carolina, USA prototyped PCL-based human-scale tissue constructs of any shape with the support of novel integrated tissueorgan printer (ITOP).124 Such construction of muscle tissue without disturbing the vascular network by the polymers is seen as alternative approach for utilizing polymers (see Fig. 4.1B). 

Cartilage Printing Cartilage tissues are vital for reducing the friction between the successive bones and they are responsible for osteoarthritis, relapsing polychondritis, etc.134 In general, joint replacement surgery and cell transplantation are some of the familiar therapeutic procedures practiced for such diseases.135–137 In most cases, failure of these therapeutic procedures occurs when the implant is rejected by the host, leading to the death of cells present in the tissue due to necrosis and the unmatched biomechanical properties between the original tissue and the newly formed tissue.137,138 Polymers used for cartilage printing generally include PEG or PEG-derived hydrogels, ABS, PLA, PEGDMA, and PGA. Modifications are preferred in some instance for decreasing the viscosity and for enhancing the biocompatibility of the polymer. Gao et al.138 showed such modification for decreasing the viscosity of PEGDMA to enhance the inkjet printing of cartilage tissue. This was achieved by conjugating PEGDMA with peptide chain that enhanced the growth of chondrocytes encapsulated within the hydrogel, thus resulting in ease of bioprinting.139 Table 4.4 consolidates the polymers that are commonly chosen for cartilage bioprinting. 

TABLE 4.4

Synthetic Polymers Employed in Cartilage Printing Polymer/Polymer Composite

Application

Printing Strategy

Significant Characteristics

References

ABS

Nucleus pulposus tissue regeneration

FDM

Biocompatible, low melting point (105°C) prompting application for FDM, widely used for cartilage tissue engineering.

140

PLA

Nucleus pulposus tissue regeneration

FDM

Biocompatible, hydrophilic, suitable melting point (175°C) for application in FDM, major polymer in cartilage tissue engineering.

140

PEGDMA (derived from PEG) and PEGDA

Utilized as bioink containing chondrocyte for direct cartilage repair and neocartilage generation

Inkjet

Elasticity modulus comparable to cartilage tissue, Photocross linkable, water-soluble, low viscosity employs application in inkjet bioprinters

134,141

Acrylated peptidePEG hydrogel

Good chondrogenic differentiation

Inkjet

Tunable mechanical property, able to form hydrogel, conjugation of acrylated peptides increased the biocompatibility and reduced the viscosity of the hydrogel.

139

PCL/ alginate/β(TGF)

Cartilage regeneration

Extrusion

PCL maintained the 3D structure of scaffold carrying alginate hydrogel encapsulated cells and TGF

142

PGA scaffold

Cartilage regeneration

Extrusion

Biocompatible, flexible mechanical property, nontoxic

143

PEGDMA, poly(ethylene glycol) dimethacrylate; TGF, transforming growth factor.

CHAPTER 4  Polymeric Materials for 3D Bioprinting

Organ and Tissue Printing In general, accidents and deformities result in organ loss or damage. The traditional treatments involve the use of prosthesis or medicine that may not regenerate the organ to its original state. The ultimate demand in the 3D printing is prototyping a full organ hoping for a solution in the organ transplantation. They are capable of delivering various organs such as human external ear, eye, kidney, and liver. However, there are many obstacles in adopting these after implantation.5 In the field of organ printing, majority of researchers use sacrificial layer in which polymers such as PCL and PEG are deployed. These layers provide considerable mechanical strength during the printing process and are removed subsequently by immersing the construct in an aqueous solution without disrupting cells viability.144,145 For instance, relevant experiment was reported by Lee and group in 2014 for ear generation by 3D bioprinting, where PEG was deposited as a sacrificial layer.144 Table 4.5 lists down the recent developments of organs and tissues printing through various polymeric precursors. The combined organ and tissue printing finds an importance in the dentistry. Studies have proved that bioceramic mixed with PCL as additive for increasing the biocompatibility and for reducing the hydrophobic behavior of PCL. The presence of bioceramics enhances the bioactivity of PCL resulting in better cell adhesion for promoting the growth of human dental pulp cells (hDPCs) on the scaffold surface.84 G5 glass, a type of calcium phosphate (CaP), has also been used for such

applications along with increasing mechanical strength of scaffold. In a study by Navarro and group (2012), it is found that the uniform distribution of G5 glasses on the PCL/PEG scaffold surface increased the modulus of compression. Moreover, it made the surface more rough and hydrophilic making suitable for the growth of mesenchymal cells.147 

Others Other applications of these synthetic polymers in 3D bioprinting lies in pharmacology field focusing on targeted drug release system.5 They are necessary for the controlled delivery of drugs, and hence they are chosen as a carrier compound. Mostly the drugs are delivered by encapsulating the targeted compound in the polymer matrix. Polymers having slow hydrolytic degradation rate are applied for covering the drug, which act as barriers for the drug diffusion. Furthermore, the thickness of polymer layer controls the drug delivery rate and time. PCL, PGA, PEO (Poly ethyleneoxide), PLA, and Eudragit are commonly used as carriers.148,149 

LIMITATION AND FUTURE PERSPECTIVE 3D bioprinting has been used in versatile fields varying from integration of live cells to biosensors and from stem cell fabrication to artificial organ generation suggesting potential futuristic applications. It has shown commanding bioactivity in tissue engineering applications emerging as a strong fabrication tool for generating intricate of microscale and macroscale. However,

TABLE 4.5

List of Organs Printed by 3D Bioprinting Technology Organ/Tissue

Polymers/­ Hydrogel Used

Technique

Biomaterial Role

References

Ear

PCL/PEG/­ alginate

MtoBS

PCL and alginate were the constituent biomaterials of scaffold, PEG was the sacrificial material

144

Cardiac tissue

PCL/CNT (­carbon ­nanotube)

Extrusion

PCL was used because of its appreciable mechanical, biocompatible, biodegradable, and nontoxic property, CNT was used for reducing the hydrophobicity of PCL scaffold surface making it bioactive

146

Mesenchymal tissue

PLA/PEG/G5 (CaP glass)

Extrusion

G5 glasses and PEG improved bioactivity and mechanical strength of PLA

147

Dental tissue

PCL/Biodentine

Extrusion

Biodentine increased the bioactivity by ­reducing the hydrophobicity of PCL surface

84

MtoBS, multihead tissue/organ building system.

75

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3D bioprinting is still in its infant stage and its further growth and dispersion is limited by various factors. The layer-by-layer printing technique used in 3D bioprinting presents difficulty in printing complex hollow 3D structures.150 Although this problem is solved by incorporating polymers as sacrificial materials, they further prolong the problems in process complexity.150–152 Moreover, the sacrificial materials must possess features such as compatibility with the biomaterials along with cytocompatibility that further limits the development of new sacrificial materials.150 Inefficiency in printing prevascularized tissues is also a major hurdle in 3D bioprinting while cell culturing and biomaterial synthesis in bioink preparation is a time-consuming process.150,152 Hence, faster and high resolution bioprinters along with new advanced biomaterials and sacrificial agents are necessary for 3D bioprinting. When considering the biomimetic materials, they lack in mechanical strength and hence composites are to be used as bioinks for supporting printed tissues. The design of tunable composite mixture with desired structures and features can be an alternative approach for overcoming the setbacks of printing. Therefore, it is mandatory to consider factors such as biocompatibility, degradation kinetics, cytocompatibility, mechanical properties, and by-product generation before developing biomaterials. Thereby PCL and PLA have emerged as low cost biocompatible polymers with good mechanical properties and prompted them as a prominent in 3D bioprinting. Hence the newly developed biomaterials should undertake these properties while dealing with issues such as fragility, cytocompatibilty, and degradability. On the other hand, skin tissue bioprinting techniques require suppressed growth and development of in vivo skin architectures, regeneration of skin appendages like epidermis, skin follicles, and sebaceous glands while the multiple organ system development faces challenges such as scalability, vascularization, and viability. Thus, it is evident that the demands of the 3D printings are very specific to the application and new synthetic polymers should be developed for fulfilling the need. Although the 3D bioprinting incurs various setbacks, it is foreseen as a promising technique providing tremendous prospective in the area of medical science and engineering. 

CONCLUSION The combination of the medical imaging technology with the printing process has led to fabrication of natural organ’s artificial prototype possessing identical characteristics of the original. The polymers have extended

the ability to cater the demand on any type of organ needed for transplantation. The discussion has clearly advocated the application of synthetic polymer on the synthesis of organs with high mechanical strength, high functionality, and tunable viscosity. It is evident that synthetic polymers have emerged as a highly efficient bioink that can successfully deliver the necessary biological matters. Therefore, one can conclude that 3D bioprinting with the support of synthetic polymers has emerged as an appropriate technology that has softened the persisting hurdles encountered during the implementation. Thus the innovation of 3D bioprinting is seen as a breakthrough in medical science and speculated as an end solution for numerous mysterious diseases.

ACKNOWLEDGMENT The corresponding author is grateful to Science and Engineering Research Board [SERB], Department of science and Technology for the financial support received under Early Career Research Award with grant code ECR/2016/001400.

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