Application of 3-D Printing for Tissue Regeneration in Oral and Maxillofacial Surgery: What is Upcoming?

The ultimate goal of any surgical procedure is to improve perioperative form and function and to minimize operative and postoperative morbidity. In recent years, many exciting and novel technological advances have been introduced in the field of oral and maxillofacial surgery. One example of such technology that is continuing to increase in prevalence is the use of 3-dimensional (3-D) printing techniques with special properties, which seems hopeful for practitioners in the field of regenerative medicine. Tissue engineering is a critical and important area in biomedical engineering for creating biological alternatives for grafts, implants, and prostheses. One of the main triad bases for tissue engineering is scaffolds, which play a great role for determining growth directions of stem cells in a 3-dimensional aspect. Mechanical strength of these scaffolds is critical as well as interconnected channels and controlled porosity or pores distribution. However, existing 3-D scaffolds proved less than ideal for actual clinical applications. In this chapter, we review the application and advancement of rapid prototyping (RP) techniques in the design and creation of synthetic scaffolds for use in tissue engineering. Also, we survey through new and novel merging era of “bioprinting.”


Introduction
Three-dimensional printing-also known as rapid prototyping-was first introduced in 1980s; during past three decades, enormous changes and development have been performed by scientists through modifying this technology by uses, material, and also accuracy.
With increasing attention of scientific societies, recently, scientific literature bolded feasibility of 3-D-printed tissues and organs and its usage within laborious clinical situations. Also, this technology was used largely in accurate and highly customized devices, such as tracheobronchial splints, bionic ears, and even more. Within the field of craniofacial surgery, 3-D surgical models have been used as templates to create bone grafts, tailoring bioprosthetic implants, plate bending, cutting guides for osteotomies, and intraoperative oral splints. Using 3-D models and guides has been shown to shorten operative time and potentially reduce the complications associated with prolonged operative times.
The goal of surgical procedures for a clinician is to improve perioperative form, recovery of function, and also minimizing operative and postoperative morbidity. Many exciting and new technological advances have ushered in a new era in the field of oral and maxillofacial surgery over the last years, which within no exaggeration 3-D printing is the novelist and controversial one.
The aim of this chapter is to introduce 3-D printing method and its role in the contemporary oral and maxillofacial surgery and to review current advantages of its application in the field of regenerative medicine.

History of the technology
Three-dimensional (3-D) printing has been utilized in diverse aspects of manufacturing to produce different objects from guns, boats, and food to models of unborn babies. From over 1450 articles related to 3-D printing listed in PubMed, nearly a third of them were solely published in the last 2 years [1].
The developing agora for 3-D desktop printers encourages wide-ranging experimentations in that subject. Generally, medical indications of these printers are such as treatment planning, prosthesis, implant fabrications, medical training, and other usages [4].
Having being used in military, food industry, and art, rapid prototyping is receiving a lot of attention in the field of surgery in the last 10 years [6,14].
The pioneering usage of stereolithography in oral and maxillofacial surgery was by Brix and Lambrecht in 1985. Later this technique was used by them for treatment planning in craniofacial surgery [15].
In 1990, stereolithography was used by Mankovich et al. for treating patients having craniofacial deformities [16,17]. They used it to simulate bony anatomy of the cranium using computed tomography with complete internal components [17,18].
Today, maxillofacial surgery can benefit from additive manufacturing in various aspects and different clinical cases [22]. This technique can help with bending plates, manufacturing templates for bone grafts, tailoring implants, osteotomy guides, and intraoperative occlusal splints [23][24][25][26][27]. Rapid prototyping can shorten surgery duration and simplify pre and intraoperative decisions. It has enhanced efficacy and preciseness of surgeries [10].
There are many different 3-D printing techniques. Benefits and disadvantages are factors to differ each technology system [14]. Among this variety of different techniques, there is a huge discussion and usage in oral and maxillofacial region for SL, FDM, and PJ [1,28,30].
Each technology has its own characteristics, properties, and advantages which Table 1 summarizes some different three dimensional printing technologies.

Biomaterials available for 3-D printing
As researchers aim to investigate new materials for 3-D printing in last decade, it is obvious to see variety of biomaterials with different properties and also different applications. As Table 2 summarizes all biomaterials used within studies all over the world for generating scaffolds for bone tissue engineering, it has to be noticed that from this large spectrum of biomaterials Tenacious support material can be difficult to remove completely. Support material may cause skin irritation.
Cannot be heat sterilized. High cost materials.

DLP (digital light processing)
Liquid resin is cured layer by layer by a projector light source. The object is built upside down on an incrementally elevating platform.
Good accuracy, smooth surfaces, relatively fast.
Lower cost technology.
Light curable liquid polymers and wax-like materials for casting. Support materials must be removed. Resin is messy and can cause skin sensitization, and may be irritant by contact Limited shelf life and vat life. Cannot be heat sterilized. Higher cost materials.

Powder binder
Plaster or cementaceous material set by drops of (colored) water from 'inkjet' print head. Object built layer by layer in a powder bed, on an incrementally descending platform.
Lower cost materials and technology.
Can print in color. Un-set material provides support Relatively fast process. Safe materials.
Low resolution. Messy powder. Low strength. Cannot be soaked or heat sterilized.
Object built layer by layer in powder bed. Heated build chamber raises temperature of material to just below melting point. Scanning laser then sinters powder layer by layer in a descending bed.
Range of polymeric materials including nylon, elastomers, and composites. Strong and accurate parts.
Self-supported process.
Polymeric materials-commonly nylon may be autoclaved. Printed object may have full mechanical functionality. Lower cost materials if used in large volume. Rough surface. Less post-processing required. Lower resolution.

Advantages Disadvantages
Thermoplastic Fused deposition modeling (FDM) First 3-DP technology, most used in 'home' printers. Thermoplastic material extruded through nozzle onto build platform.
High porosity. Variable mechanical strength. Low-to mid-range cost materials and equipment. Low accuracy in low cost equipment. Some materials may be heat sterilized.
Low cost but limited materialsonly thermoplastics.
Limited shape complexity for biological materials.
Support material must be removed. Hyaluronic acid based hydrogel TCP, tri-calcium phosphate; HA, hydroxyapatite; DBM, demineralized bone matrix; PLGA, poly(lactic-co-glycolic acid); PLA, poly(d,l-lactic acid); PGA, poly(glycolic acid); PLLA, poly(l-lactic acid); PVDC, polyvinylidene chloride; PEG, polyethylene glycol; DA, diacrylate; MMP, matrix metalloproteinases; ABB, anorganic bovine bone; Puramatrix, a selfassembling peptide nanomaterial. just a whole bit of them are available for application in 3-D printing. As follows, we discuss four large categories of materials for 3-D printing of scaffolds and craniofacial tissues, which researches still aim to determine these materials complete properties and advantages.

Polymers and hydrogels
Polymer hydrogels are ideal candidates for the development of printable materials for tissue engineering. Hydrogels are known for remarkable tunability of rheological also presenting great mechanical, chemical, and biological properties; high biocompatibility; and similarity to native extracellular matrix (ECM) [32]. For three-dimensional printing of polymers and hydrogels, the use of materials with controlled viscosity should been noticed. This defines the range of printability of the ink. Polymer inks, which are typically printed in the prepolymer phase, need enough viscosity allowing structural support of subsequent printed layers, also enough fluidity to prevent nozzle clogging. For avoiding these difficulties, alginate hydrogels have been cross-linked with calcium ions immediately before the ink leaves the printing head or just after extrusions [33].
In recent researches, for providing suitable ink for bioprinting applications, prepolymerized cell-laden methacrylated gelatin hydrogels have been used successfully [34,35]. Synthetic hydrogels used for cell encapsulation may limit cell-cell interactions. These interactions are critical for efficient cell proliferation, differentiation, and finally, tissue development. This can represent one of the limitations of bioprinting cell-laden hydrogels which is not present in 3-D printed scaffolds with cells seeded onto or in bioprinting of dense cell aggregates, which will discuss as follow. Hence, the requirement for the development of ECM-derived hydrogels that have tunable physical and chemical properties, are compatible with high cell viability, and provide the adequate binding sites (RGDs) for cell attachment and matrix remodeling during their early proliferative stage [32].
Synthetic polymers are most commonly used materials for 3-D printing in biomedical applications [36,37]. However, since high temperature is usually involved during the printing of these materials, the direct incorporation of cells or growth factors in the polymer mixture is generally avoided as the cell viability or bioactivity [37] cannot be maintained throughout the manufacturing process.
Although hydrogels provide great advantages for tissue engineering applications, such as the ability of exposing cells to highly hydrated 3-D microenvironments that is similar to the natural ECM [32]. In contrast, they generally present very low stiffness (in the kPa range) compared with the majority of load-bearing tissues in the craniofacial complex (in the GPa range). Therefore, reconstruction of tissues subjected to higher mechanical loads, such as bones and teeth, usually requires the use of ceramic materials or composite scaffolds which provide more mechanical advantages, where polymers are commonly combined with inorganic fillers to increase scaffold stiffness [38].

Ceramics
Ceramic scaffolds are usually composed of calcium and phosphate mineral phases, such as hydroxyapatite [39] or b-tricalcium phosphate [40]. The noticeable ability of these scaffolds to upregulate osteogenesis due to inherent properties of the formation of a bioactive ion-rich cellular microenvironment, also as mentioned before their ability to mechanically provide space maintenance, makes these materials interesting choice for 3-D scaffold fabrication for craniofacial applications. In contrast, ceramic scaffolds are not compatible with cell encapsulation for bioprinting. In 3-D printed ceramic scaffolds, cells quickly populate the scaffold surface, which establishing close cell-cell interactions lead to promotion of cell proliferation and differentiation. On the other hand, ceramics with properties lead to lower rates of degradation than hydrogels, which aids in prolonged guided tissue remodeling and structural support. In contrast, ceramic scaffolds are too brittle for implantation in load-bearing defect sites. Ideal scaffolds would combine the high calcium content of calcium and phosphate ceramics with the outstanding toughness of natural bone, which perhaps can only be obtained by creating scaffolds that are biomimetically mineralized and hierarchically structured, as recent researches demonstrated that in [41].
Fused deposition of ceramics (FDC) in a direct printing mode generally consists of extruding a slurry including a high content (>50% w/v) of inorganic components [42]. The manufacturing of such scaffolds follows 3 steps: 1. Mixture phase, which involves the preparation of the slurry. The bioceramic particles are mixed in a solvent (aqueous or nonaqueous) with a low concentration of organic polymers/surfactants, called the binder, to obtain adequate flowability.

2.
Green ceramic and binder burnout phase involving the deposition of filaments of slurry following a predetermined pattern prior to drying and exposure to high temperature to burn out the organic component of the mixture.

3.
Sintering phase, which involves the exposure of the green form to elevated temperature (above 1000°C) to initiate the migration of atoms between adjacent ceramic particles, hence creating physical bonds called "necks." It is critical for reproducible manufacturing of 3-D rapid prototyped bioceramics to have shape retention, a challenge that can be reached by adjusting the viscosity of the slurry and the evaporation rate of the solvent [43].

Composite materials
Printable composites, which are usually in the form of copolymers, polymer-polymer mixtures, or polymer-ceramic mixtures [44], allow ability for the combination of variety of advantageous properties of their included components, which provide a remarkable candidate as "bioink". Considering the advantages of polymer composite hydrogels, such as interpenetrating polymer networks (IPNs) or hybrid hydrogels [45], the incorporation of synthetic fillers to printable materials recently discussed in researches [33]. The addition of silicate fillers [38] and a range of nanoparticles have been used to synthesize different types of composite scaffolds [46] to promote greater control over viscosity and stiffness of polymer hydrogels.
In addition, several of silica-containing hydrogels with higher expression of genes encoding morphogenetic cytokines, such as bone morphogenetic proteins (BMPs) seems promising [47]. The combination and manufacturing mixture of hydrogels with filler materials and/or natural peptides with morphogenetic capacity demonstrate great future for application in 3-D printing in aim to reach ultimate goal in regenerative craniofacial repair.

Cell aggregates and spheroids
Over recent years, many of researches aimed to evaluate and study cell aggregates and spheroids for use in tissue engineering and regenerative medicine [48]. As this method cited correctly and appropriately as "scaffold-free printing," in fact small quantities of hydrogel are used to facilitate cell aggregation. In this method for 3-D printing, or in an appropriate way called "bioprinting," multicellular spheroids are deposited using extrusion printers and allowed to self-assemble into the desired 3-D structure (Figure 1). As it is clear, these systems allow direct fabrication of tissue constructs which in contrast to other methods have extremely high cell densities. Although in load-bearing tissues with high amount of mineral components and noticeable mechanical properties use of this methods still looks uncertain, the ability to position aggregates of heterotypic cells with microscale precision (Figure 2) seems promising as an excellent alternative to bioprint complex tissues consisting variety of cells [49].

Manufacturing of scaffolds with 3-D printing technology
Researches aimed to investigate novel technologies for 3-D printing and introduced some novel methods including phase-separation, self-assembly, electrospinning, freeze drying, solvent casting/particulate leaching, gas foaming, and melt molding [52]. Using scaffolds, the architecture of native extracellular matrices can be mimicked at the nanoscale level and therefore provide the primary base for the regeneration of new tissue [53]. Originally, a "top-down" approach was used as a tissue engineering method for scaffold fabrication. In this method, cells are seeded onto a biodegradable and biocompatible scaffold and are predicted to migrate and fill the scaffold hence creating their own matrix. By using this technique, several avascular tissues such as bladder [54] and skin [55] have been engineered effectively. However, due to the limited diffusion properties of these scaffolds, this technique faces several challenges for fabrication of more complex tissues such as heart and liver [56]. Therefore, "bottom-up" methods have been developed to overcome this problem [57]. Bottom-up approaches include cell-encapsulation with microscale hydrogels, cell aggregation by self-assembly, generation of cell sheets, and direct printing of cells [58]. These complex tissue blocks can be assembled using various methods including microfluidics [59], magnetic fields [60], acoustic fields [61], and surface tension [62]. These methods are relatively easy and have provided a solid foundation for the fabrication of scaffolds. However, as mentioned previously, these conventional methods suffer from several limitations including inadequate control over scaffold properties such as pore size, pore geometry, distribution of high levels of interconnectivity, and mechanical strength. As such, it is necessary to develop technologies with sufficient control so as to design more intricate tissue-specific scaffolds. In addition, scaffolds can be coated using surface modification techniques (such as introducing functional groups) to enhance cell migration, attachment and proliferation. Three-dimensional printing allows scaffolds to become more precisely fabricated (similar to that of the computer-aided design (CAD)) with higher flexibility in the type of materials used to make such scaffolds. Three-dimensional printing uses an additive manufacturing process where a structure is fabricated using a layerby-layer process. Materials deposited for the formation of the scaffold may be cross-linked or polymerized through heat, ultraviolet light, or binder solutions. Using this technology, 3-D printed scaffolds can be prepared for optimized tissue engineering [52].
For appropriate formation of tissue architecture, the seeding cells (often stem cells) require a 3-D environment/matrix similar to that of the ECM. The ECM acts as a medium to provide proteins and proteoglycans among other nutrients for cellular growth. The ECM also provides structural support to allow for cellular functionality such as regulating cellular communication, growth, and assembly [63]. With this in mind, scientists and engineers originally attempted to replicate the ECM through conventional techniques, which consequently established a framework for using more advanced techniques, such as 3-D printing, to yield higher quality scaffolds. The 3-D printing technique can create defined scaffold structures with controlled pore size and interconnectivity and the ability to support cell growth and tissue formation [64][65][66]. The current methods for 3-D printing involve a CAD, which is then relayed to each 3-D printing system to "print" the desired scaffold structure. Through various 3-D printing technologies, discussed below, researchers are trying to fabricate biocompatible scaffolds that efficiently support tissue formation ( Table 3).

Bioprinting advantages aiming for clinical use
The goal of tissue engineering is to create functional tissues and organs for regenerative therapies and ultimately organ transplantation/replacement. Trial and error was the long and tedious process mainly used to advance the field of regenerative medicine by clarifying the success of techniques.
Researchers needed to come up with a list of requirements in order to measure their successes or failures in tissue fabrication [48,67]. This list was generated from the observations of natural human tissue.
As gold standard of fabricated tissues is to be as similar as possible to natural tissues in the human body in different parameters, then these fabricated tissues must:

2.
Be capable of essential functions in vivo [48].
Also, the printers used for tissue fabrication required standardization as well [67,69].

1.
The bioprinting machines required set extreme sterilization methods to eliminate unwarranted contamination with previously used materials or foreign matter from the environment.

2.
The conditions for printing must be ideal for tissue fabrication, so factors such as humidity and temperature must be closely monitored.

3.
Nozzle size and methods of delivery affect the viability of the materials being printed; therefore, there must be set ideals for delivery methods in relation to the various printing materials.  Table 3. Preclinical researches on various 3-D printing techniques for manufacturing scaffolds for tissue engineering [52].
As a result, researchers created a few methods of printing with the goal of finding a solution to the given problems for optimal tissue biofabrication [48,68,69]. Thermal inkjet bioprinting with bioink and direct-write bioprinting both make use of modified inkjet printers but with varied application techniques. Organ printing with tissue spheroids is the recent achievement of researches which seems promising to fabricate tissues directly. Table 4 review advantages and disadvantages of all three common methods "Thermal Inkjet Bioprinting," "Direct-Write Bioprinting," and "Spheroid Organ Printing." Organ printing, otherwise known as the biomedical

• Thin
• Only nourishable via diffusion "Bioink," which is a water-based liquid consisting of proteins, enzymes, and cells suspended in a media or saline.

Direct-write bioprinting
• Digital control of print.
• Several printing units simultaneously.
• Application of variety of materials simultaneously.
• Faster turnaround time for printed products.
Possibility of printing tissues with different compositions.
• The bioink of direct-write printers may consist of hydrogels of varying consistencies that are composed of agarose, alginate, collagen type I, and Pluronic F127.
• This method categorized in pneumatic, mechanical, and a pneumatic-mechanical hybrid. It was concluded that the pneumatic systems work better with high viscosity materials, while mechanical systems are better suited in working with materials of low viscosity.

Spheroid organ printing
Spheroids are punched into "biopaper" which is a sprayed layer of hydrogel. Each spheroid is made of living cells, thereby creating a ball of "living materials" capable of selfassembly and self-fusion. Alternatively, the spheroids can be digitally placed, undergo selfassembly, and fuse without the use of hydrogel.
• Self-organization is defined as, "a process in which patterning at the global level of a system emerges solely from numerous interactions among the lower-level components of the system." • Self-assembly is defined to be, "the autonomous organization of components into patterns or structures without human intervention." Researchers fabricated three types of spheroids to create a vascular tree: solid or nonlumenized spheroids, spheroids with one big lumen (mono-lumenized spheroids), and microvascularized tissue spheroids. application of rapid prototyping, may be defined as additive layer-by-layer biomanufacturing of cells. Advantages of organ printing include its automated approach offering a pathway for a scalable and reproducible mass production of tissue-engineered products. This also allows the precise simultaneous 3-D positioning of several cell types, hence enabling the creation of tissue with a high level of cell density. Organ printing may be used to solve the problem of vascularization in thick tissue constructs, and moreover, this technology may be done in situ. Therefore, this emerging transforming technology has potential for surpassing traditional solid scaffold-based tissue engineering [72].

Vascularization
In order to create a complete and functional organ, the researchers must be able to create thick complex tissues with full vascularization containing lumens of various sizes, large vascular structures to microstructures, in order to sustain the surrounding organ tissue. The best way to achieve this type of vascularization is to fabricate the vascular system and tissue simultaneously, of which is easier said than done [48]. Thorough vascularization remains a common theme for current bioprinting limitations. Without a functional circulatory system, tissue constructs are limited to a means of diffusion for nutrition, which in itself is limited to just a few hundred microns [69].
Current methods of vascularization call for the infiltration of host microvessels into an implanted construct [67,73,74].
Yet, this strategy is lacking in control and specificity for the developing microvessels. The invading microvessels have a limited penetration depth which prevents the successful incorporation of the microvessels into larger layered constructs. Additionally, the penetration of the vascular system itself may result in a distortion of the region penetrated or in the destruction of the fabricated tissue altogether. For these reasons, it would be ideal to construct tissues with direct vessel in-growth, or vascularization created within the tissue itself, all before implantation.

Tissue components and costs
In addition to vascularization, native tissues contain unique cellular combinations and organizations. There is a need to develop techniques that mimic the complexity of native tissues in order to drive tissue recovery and replacement for medical applications [69]. With the production of organs such as kidneys, for example, at least one million glomeruli and nephrons would need to be generated. Not only would the fabrication be a massive undertaking but also the fabricated tissue would need to be scalable. Scalability of biofabricated tissues is not presently a reality. Yet, spheroids have shown promise toward being scalable with further development. Finally, another major limitation for the development of natural-like, fully functioning fabricated human tissue is economic [68]. This challenge must definitely be overcome if biofabrication technology is to allow the creation of a functional living human organ.

Future aspects of 3-D printing for regenerative medicine
In this chapter, we have illustrated current guiding principles for 3-D bioprinting in tissue fabrication, as well as recent advances and technological developments. The speed at which our knowledge has advanced with additive manufacturing and automated printing systems shows a promise to expand our basic science and engineering capabilities toward addressing health care problems. One of the significant developments in 3-D bioprinting is to manufacture cell microenvironments from molecular to macroscopic scales, which are requested and suitable for tissue engineering and regenerative medicine. As novel methods and technologies introduced in recent years for 3-D printing of biomaterials, promising overview of future appears to manufacture scaffolds for tissue engineering that reach the gold standards and also better comprehensions of stem cells microenvironments and interactions. By aid of various novel technologies, such as microfluidic systems [75,76], biopatterning [77], and layer-by-layer assembly [76,78], researchers are now able to biomanufacture microtissue constructs within scaffolds and even also within scaffold-free environments. Considering the great and enormous improvements of biomaterial for tissue engineering, in contrast, there are still certain challenges and difficulties that need more attention. Vascularization is one of the limitations which receive most of attentions [79,80] due to the fact that this challenge leads to hypoxia, apoptosis, and immediate cell death. For resolving this issue and providing sufficient space for vascularization, researchers attempts to fabricate porous scaffolds [81], to provide sufficient space for vascularization. However, this approach cannot overcome the vascularization challenge completely due to the diffusion of cells and other materials into these porous structures [82]. Forming interconnected, well-defined vascular structures during biomanufacturing process seems to lead to resolving this difficulty and providing better results during process. Other issues that have to be noticed are mechanical strength and stability in 3-D tissue engineering which is one of the key requirements [83]. To be clear in regeneration of hard (e.g., bone) and soft (e.g., vascular grafts) tissues, modulus of elasticity is a crucial parameter that desires improvement [84][85][86]. Furthermore, the development of a totally closed bioprinting system that integrates printing and post-printing processes such as in-vitro culture and maturation of tissue constructs continues to be a challenge.
With advances in near future, which help finding solutions for the challenges mentioned above, bioprinting technologies will potentially help improvements of rapid clinical solutions and advances in medical implants. Further, we envision that the integration of cells and biomaterials through bioprinting with microfluidic technologies are likely to create unique microenvironments for various applications in cancer biology, tissue engineering, and regenerative medicine [87][88][89][90][91]. Additionally, developments on high-throughput biomanufacturing of 3-D architectures will pave the way for further advancements of in vitro screening and diagnostic applications, potentially enabling complex organ constructs. In the meantime, it is only the effective interplay of engineering concepts in combination with the well-established fundamentals of biology that will realize the true potential of this exciting area.