**4.4 Freeze-drying technique**

In this method a water-soluble polymer is frozen such that interpenetrating ice crystals are created which are later removed by sublimation, resulting in formation of porous scaffold [22].

## **4.5 Particle leaching and phase inversion**

In this process PLGA was dissolved in DMSO, and then the PLGA solution was thoroughly mixed with CaP particles in a ratio of 1:3 (w/w). This sticky mixture of CaP/PLGA was then poured into an aluminum foil mold filled with sugar crystals at a weight of 3 times the DMSO volume. The mixture diffuses throughout the mass of porogen crystals. After 2–3 minutes the mold was transferred to a refrigerator at −18°C for 1 h to set the mixture. The PLGA was precipitated and the sugar crystals were leached out of the precipitated CaP/PLGA mixture in ddH2O at room temperature (20°C) for 3 days, during which time the ddH2O was changed approximately 4 times each day. Every time a scaffold block was produced [58]. Thus, PLGA and two calcium phosphate phases (first is a particulate within the structure and second is a thin ubiquitous coating) get fabricated into a composite scaffold with a pore size and interconnecting macroporosity similar to that of human trabecular bone. The osteoconductive surface of calcium phosphate abrogates the putative foreign body giant cell response to the underlying polymer, whereas the internal calcium phosphate phase provides dimensional stability. The highly interconnected microporosity and the ability to wick up blood make the scaffold a clot-retention device and an osteoconductive support for growth of host bone. Such scaffold has been implemented in human patients for maintenance of alveolar bone height following tooth extraction. These scaffolds also augment alveolar bone height through standard sinus lift approaches. It was also observed that these scaffolds regenerated sufficient bone tissue in the wound site and provided good foundation for dental implant placement [16].

## **4.6 Phase separation**

It is a solvent based technique that employs change in temperature to induce phase separation of homogenous polymer-solvent solution through solid-liquid demixing or liquid-liquid phase separation [22, 47]. On this basis it is mainly divided into two types liquid-solid and liquid-liquid phase separation. The method is conducted by reducing the temperature of solution and extraction of solvent phase, till it reaches a porous polymer scaffold [47]. The phase separation majorly involves formation of a polymer-rich phase and polymer-poor phase upon rapid cooling of polymer-solvent solution by freeze-drying or freeze-extraction [4, 22]. As a result, the polymer-poor phase is eventually removed [22]. The solvent system utilized is usually a mixture of 1,4-dioxane and water and the temperature and

#### *Advances in Tissue Engineering Approaches for Craniomaxillofacial Bone Reconstruction DOI: http://dx.doi.org/10.5772/intechopen.94340*

time for the process is around 60°C for two hours. It has been observed that strong polymer-solvent interaction leads to solid-liquid phase separation, whereas weak polymer-solvent interactions results in liquid-liquid phase separation. The role of non-solvent such as water is to lower the degree of polymer-solvent interaction so as to induce liquid-liquid phase separation [4].

### **4.7 Computer-aided techniques**

The Computer-aided designing (CAD) is gaining popularity with respect to construction of model on the basis of constructive solid geometry or boundary representation principle. Models obtained by boundary representation require more storage space compared to constructive solid geometry. Therefore, as the model becomes larger or more detailed in internal structure the size of the file containing boundary-representation-derived-model drastically increases causing difficulty in operation. CAD methods are realized by utilizing various tools such as UG, CATIA and Pro/E. Some dedicated design software's such as Magics (3D printing preprocessing software developed by Belgium Materialize Company), have been recently developed in which the designer can directly instruct various integrated unit cells. MATERAILAS and computer-aided system for tissue scaffolds (CASTS) are some of the software and parametric library respectively that are used to design algorithms for minute detailing of the desired scaffold [52]. Implementation of CAD/ CAM softwares along with radiology procedures for easy acquisition and transfer of DICOM3 (Digital Imaging and Communications in Medicine) data allows the surgeon to perform three-dimensional measurements and reconstruct the deformed or missing anatomy by segmentation [59].

Computer assisted textile-based technologies constitute an attractive route to strategize scaffolding (including stitching, braided, woven, non-woven and knitted) of more complex fibrous 3D scaffolds suitable for engineering of soft tissue such as ligament in the craniofacial regions [28].

With advancements in our knowledge about materials along with a boon in computer-aided technologies, several methods have evolved recently that increases the precision and accessibility of craniomaxillofacial bone reconstruction (**Figure 3**).

Recently rapid prototyping has emerged as an effective tool for 3D printing of porous scaffolds with interconnected porous network [42], complex geometries, well defined and reproducible architectures [22]. The basic concept of rapid prototyping involves presentation of cycles of cross-sectional sheets from where the data is exerted into the solid free form fabrication machine to produce the physical model. As the layers are built from bottom to top, each newly manufactured layer sticks to the previous. Several techniques originate from this principle of rapid prototyping. We have discussed some of the major types of this technique in the following sections [42].

### *4.7.1 3D printing*

3D printing has emerged as a promising tool of additive manufacturing (AM) that enables us to optimize the processes of preoperative planning, develop intraoperative guidance tools, reduce operative time and improve bifunctional and esthetic outcome [12, 60]. Liu et.al fabricated Al2O3 scaffolds with a through-hole structure using 3D printing and sol-gel technology. Alumina (Al2O3) is a bioinert ceramic and exhibit negligible tissue reaction and therefore several researchers incorporate other components into Al2O3 to enhance the mechanical strength of the scaffold. Fabrication of Al2O3 / borosilicate glass scaffolds using urea- formaldehyde resin as in-powder adhesive by 3D printing has been demonstrated to maximize the tensile

**Figure 3.**

*Computer-assisted scaffold modeling for fabrication of implants.*

strength [40]. In the last decade, investigators have reported 3D-printed prostheses of nose, ears, eyes, face, and hand [12]. With the help of direct writing technology tricalcium phosphate scaffolds have been fabricated and used to repair rabbit trephine defects [61].

Significant improvements in clinical imaging and user-friendly 3D software with progression of open source platforms, associated with recent hardware developments have enabled 3D printer to build layers as small as 16 μm thickness for stereolithography (Polyjet, Stratasys); 178 μm thickness for fused deposition modeling (Fortus 900 mc, Stratasys); 80 μm thickness for selective laser sintering (sPro 230 HS, 3D systems) and 75 μm resolution for stereolithography (3D systems) [12, 62]. These 3D printing techniques including stereolithography, multi jet modeling, selective laser sintering, binder jet technique and fused deposition modeling provide appreciation of visuospatial relationship between the anatomical structures created and craniomaxillofacial reconstructive surgery [12].


powder bed. These droplets are dispensed and driven either by thermal or piezoelectric processes into a powder bed. The thermal inkjet printing employs temperature between 100 and 300°C to nucleate a bubble and eject the droplets, which produces shear and thermal stress on natural polymer inks, resulting in inconsistent droplet volume. Piezoelectric technology utilizes pressure or acoustic waves produced via piezoelectric actuator to generate the drops and therefore can be used with a range of polar and non-polar solvents [52].

A wide range of powder materials such as polymers, ceramics, proteins and cells can be processed using this technique. However, the ink's viscosity is limited to 5 to 20 Pa.s to avoid high ejection pressure or continuous flow of material [3].


UV radiation is the most common curating agent in SLA. When a two-dimensional layer of gelatin methacrylate (GelMA) and different concentration of photoinitiator were cured with UV exposure, it was found that the concentration of photoinitiator affected the porosity of GelMA hydrogel by polymerizationinduced phase separation. Similarly, fabrication of poly(propylene fumarate) (PPF) was carried out by embedding PPF/diethyl fumarate photopolymer with PLGA microspheres loaded with bone morphogenetic proteins (BMPs). PPF is known to form cross linked polymer network when combined with photoinitiator bisacrylphosphrine oxide and exposed to UV light [9]. Cha et al. utilized nano-SLA to print micropillar and microridge patterns on the scaffold and investigated the effect of these patterns on cell adhesion, proliferation and osteogentic differentiation [36]. PolyHIPEs (poly high internal phase emulsion) are the class of material where porosity is created due to phase separation between two immiscible liquids in presence an emulsifier. 2-ethylhexyl acrylates (EHA) and isobornyl acrylate (IBOA), when mixed together and combined with the photoinitiators (diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide/2-hydroxy-2-methylpropiophenone) formed a porous structure in presence of water, upon curation and photopolymerization by laser. In this method the curation is carried out by laser instead of UV radiation. Similarly methacrylated poly(D,L-lactide) (PDLLA) scaffolds were prepared using Irgacure 2959 as photoinitiator [9].

Ceramics are known to be non-photocurable and therefore, they require photocurable resin to bind the particles together [9]. Ceramic materials are primarily made up of metals with inorganic calcium or phosphate salts and are generally osteoconductive and osteoinductive [3]. The scientists investigated that bioceramic slurry of HA and TCP, mixed with photocurable FA1260T resin, cured with SLA and sintered at 1400°C (to remove the solidified photocurable resin and fuse the bioceramic particles together), resulted in fabrication of biocompatible osteoinductive scaffold. In one of the studies, researchers investigated the utility of thiol-ene reactions to produce photopolymer networks, as an alternative to the use of photoinitiators. These reactions occur between alkene and thiolmonomers to form an alkyl sulfide group that is regarded to photo-trigger the chemical reaction, thus eliminating the need of photoinitiator. A 1:1 ratio of thiol (pentaerythritol tetrakis(3-mercaptopropionate) (PETMP)) and alkene (poly (ethylene glycol)divinyl ether (PEG-DVE)), has been shown to crosslink without the presence of photoinitiator and was also biocompatible [9].

5.Laser-assisted 3D printing also known as laser-assisted bioprinting (LAB) basically has three main components (a) a pulsed laser source, (b) a transparent glass slide or ribbon, as a target, serving as a support for the printing material and (c) a receiving substrate to collect the materials. While printing, a focused laser pulse stimulates a small area of the target which is mainly made up of an energy absorbing layer on the surface and bioink solution underlay. Evaporation of a portion of the energy absorbing layer results in formation of a droplet that is collected by the receiving substrate and crosslinked [3]. Pure calcium silicate and dilute magnesium doped scaffolds of different layer thickness and macropore sizes, prepared by varying the layer deposition mode from single-layer printing to double layer printing, have been demonstrated to improve bone tissue ingrowth in craniomaxillofacial bone defect treatment [37]. Varying the layer configuration from single to double layer printed versions has been shown to significantly enhance side-wall pore size and strut thickness [37].

As, LABs are not equipped with nozzle and obviate direct contact between dispenser and bioink, they minimize the problem of material or cell clogging [3, 42].

6.Fused deposition modeling (FDM) was developed and patented by Scott Crump and is one of the most popular rapid prototyping technique [42]. It is the most widely used extrusion based additive manufacturing technique that fabricates scaffolds without the use of toxic organic solvents [22]. Extrusion based printing uses pneumatic, piston or screw driven system to create

pressure and push out the suspension, solution or emulsion [1]. FDM is a heat utilizing technique, where the thermoplastic filament is guided into a liquefier for melting through rollers and extruded from the computer-controlled nozzle in a layer-by-layer manner to create a scaffold [22, 25]. Thus, the fabrication process involves movement of computer-controlled nozzle in X-Y plane in order to create the desired pattern after which the nozzle move upward along the Z axis to a predefined distance, to print the next layer [42]. The thermoplastic polymers used for fabrication of scaffolds are called as bioinks [1]. To ensure good interlayer adhesion, the previously formed layer is kept at temperatures just below the solidification peak of thermoplastic material [34]. The process temperature depends on the melting temperature of the building material, which is generally very high for biological molecules [25].

The efficacy of FDM largely depends on the parameters such as nozzle temperature, nozzle diameter, extrusion speed, layer thickness and raster angle [22]. Its solvent free technology cost effectiveness and high speed renders some advantages to this technique [42]. Since this process is carried out at high temperature incorporation of biological molecules becomes impossible [34]. Furthermore, this technique faces a limitation in terms of availability of a medical grade biocompatible thermoplastic material with viscosity that is adequately low for extrusion but at the same time high enough for scaffolding [42]. PCL/HA bone scaffolds fabricated using CT-guided FDM have been found to exhibit cortical bone like features, displayed close mechanics to that of natural bone and integrated tightly with the surrounded tissue [52]. Scientists have developed computer-aided low-temperature deposition manufacturing system that has been successfully demonstrated to fabricate 3D scaffolds identical to the patient-specific alveolar bone defects. But as the resulting bone substitutes are in the form of blocks or granules, they face limitations in clinical applications requiring restoration of complex structure in craniomaxillofacial region [16].

7.Fused filament fabrication (FFF): Similar to other 3D printing techniques, the FFF also involves layer-by-layer deposition of thermoplastic material through a heated nozzle onto the platform or previously printed layers. FFF has certain advantages that include (a) minimizing the cost of production runs (b) reducing the production waste (c) shortening the design manufacture cycle (d) ability to build intricate geometries and (e) ability to tailor microstructure and properties in each layer. The mechanical performance of FFF is controlled by slicing and printing parameters. The slicing parameters include raster angle (in-plane angle), the inter-filament distance, layer height, filament orientation, nozzle diameter, filling pattern (e.g. honeycomb, hexagonal, triangular) and the build orientation (out-of-plane). The printing parameters include nozzle geometry, nozzle temperature, printing speed, printing trajectory, bed temperature and calibration. Though development of fiber-reinforced composites developed using FFF have enhanced mechanical properties, the major limitation of these printed composites are inherent extrusion-induced defects, such as porosity (due to poor interfacial bonding between the fibers and the polymer and between the printed beads or the printed layers) [50].

No matter which specific technique is used to produce the 3D biomodel, the following are proven advantages of using 3D printing for reconstructive surgery [12]:

a. 3D printing involves direct visualization of anatomic structures and their spatial relationships, thus improving the understanding of complex underlying

conditions which significantly enhance the quality of diagnosis and treatment planning [12].


It has been a general observation that 3D printed biocomposites have low fiber content (< 30 wf%) and a very low aspect ratio (L/d), that reduces their overall viscosity and improves printability. In addition, discontinuous or short fiber-reinforcement exhibit high porosity of biocomposite because of low pressure applied during printing. Therefore, the future trends in 3D printing are expected to deliver higher mechanical properties with improved material selection. The use of continuous natural fiber for biocomposites could bring about drastic improvements in mechanical performance due to high fiber content and better control of anisotropy by fiber orientation [50].

With advent of 3D printing technology, it is now possible to fabricate cellbased 3D scaffolds. The use of stem cells for clinical applications must fulfill Good Manufacturing Practice (GMP) requirements to ensure safety and quality of the treatment [63]. Bone marrow stromal and adipose derived stem cells find preferred applicability for orthopedic and maxillofacial tissue regeneration [61]. Hamlet and colleagues investigated a cell-based approach for alveolar bone regeneration using

hydrogel as bioink for cell delivery. Bioprinting of periodontal ligament cells has also been performed to create a 3D hydrogel microarray. The process of bioprinting the cells using a pressure-assisted valve-based bioprinting system is carried out within a sterile hood and controlled by a computer [3].
