*4.7.2 4D bioprinting*

The director of the Self-Assembly lab at the Massachusetts Institute of Technology (MIT), Skylar Tibbits, demonstrated the concept of 4D printing for the first time in 2014 [64]. 4D printing is an invasive and robust [42] technique that involves the development of raw printing materials and design of the mechanism and multilayer architecture of printed structures that directly incorporate a preprogrammed transformation [50]. With the rapid progress of nanotechnology 4D bioprinting has been developed to incorporate time as the fourth dimension in combination to the 3D bioprinting strategies, to bring about changes in confirmation, shape and functionalities (shape, property, self-assembly or self-repair) of the printed objects [22, 50]. So, basically 4D printing can be defined as the ability of 3D printed material to actuate when an external stimulus is applied [50]. The types of stimuli can be physical (e.g. temperature, pressure, electricity, light and magnetic field), chemical (e.g. humidity and pH) [42] and biological (cell traction force-CTF). Mechanisms of CTF have been utilized for cell origami technology in which 3D constructs of cell are developed by folding two-dimensional elements into predefined shapes. Currently the stimuli-responsive biomaterials have largely replaced CTF-based and manual folding approaches for 4D printing [64].

A case study: Maxillo-orbital surgery for placement of titanium implants to anatomically reduce bone fracture presents several challenges. In many cases deep insertion of titanium mesh implant to the orbital floor may result in damage to optic nerve and vision loss. However, if the mesh is not inserted deep enough, the reconstruction of orbital floor will not deplete and the eye will lack support. Therefore, titanium mesh implants must be inserted into the orbital wall and should tightly fit the surface of orbital floor. Under certain circumstances the titanium mesh may deviate from its position, thus increasing or decreasing the volume of orbital cavity, and as a result symptom such as diplopia, exophthalmos and enophthalmos are not relieved [23]. 4D printing has revolutionized our approach to address such complicated issues by empowering the surgeons to place and modify of scaffold in real-time during the surgical procedure. This technique enables actuation of a 3D material by application of external stimulus and can be performed after the scaffold has been placed at the site of injury/defect.

A 3D printed product should exhibit smart behavior such as "Shape memory" or "Self-actuation", to be considered for 4D printing [42]. A variety of materials have been developed for this purpose such as shape memory polymer (SMP), electroactive polymer (EAP) and hygromorph composites (moisture induced morphine – that utilizes moisture induced anisotropic swelling of natural fibers to drive actuation in development of hygromorph composites) [50].

Shape memory is defined as ability of materials to "remember" and recover their original shape. This suggests that the original shape of a material can be deformed to fix into a secondary form, by application of an external physical force. The material retains this new shape until a specific stimulus (e.g. temperature, ultraviolet light, humidity, electric and magnetic field) is applied that triggers the transformation of matter and the original shape is regained. Humidity is utilized as one such stimulus for shape changing materials, and this increases the utility of hydrogels for 4D printing. Since, hydrogels have relatively low stiffness, natural fibers are preferred for fabrication of scaffolds. Hygromorphs biocomposites are the natural fiber biocomposites, making their mark as the new class of smart materials and can be used in 4D printing [50].

Currently two design strategies are employed for generating actuation with 4D printed hygromorph biocomposites: mono-material printing and multi-material printing deposition. The mono-material printing approach presumes that a given material possesses different mechanical properties and different coefficients of expansions in different directions (anisotropy), induced by the orientation of the fibers within the filament and the printing process itself [50].

Scientists have synthesized renewable bioscaffolds by utilizing PCL and crosslinkers with castor oil which displayed both shape memory and shape recovery at physiological temperatures. Additionally, scaffolds made from epoxidized acrylate material based on renewable soyabean oil, using 3D laser printing, have been shown to express temperature-responsive shape memory. The major disadvantage of solid-state SMP in terms of 4D bioprinting is that the cells can only be seeded on the surface but cannot be uniformly dispersed within it. Moreover, the incitation mechanisms utilized to trigger deformation procedures also pose substantial restrictions. For example, dramatic changes in physical and chemical parameters such as UV and pH may have possible negative effects on cell viability but variation in temperature (between 4 and 40°C) and Ca2+ concentration does not have any detrimental effect on living cells [64].

The main factors influencing the process of 4D printing are: (a) type of additive manufacturing process utilized; (b) the nature of the responsive material; (c) type of stimulus; (d) mechanism of interaction between the material and stimulus; and (e) mathematical modeling of the material transformation [42]. The stimuli-responsive biomaterials have made it possible to realize spatio-temporal distributions and release of bioactive cues and cells for heterogenous tissue regeneration. The Project Cyborg software designed by MIT is a platform that offers abilities to simulate selfassembly and optimization of design constructs of programmable materials [64].

#### *4.7.3 Reverse modeling*

Reverse modeling design is an image-based technique that reconstructs bone tissue microstructure based on its CT or MRI image. This technique employs binary value method to analyze slice information, where element "1" represents the solid and "0" represents the void. The 2D model thus created is transformed into STL (standard tessellation language) files and transmitted to AM equipment to construct 2D layer. Layer-by-layer method is then used to obtain the 3D structure. This method combines advanced medical imaging system, powerful image analysis software and rapid AM technique to mimic microarchitecture of bone tissue [52].

Cutting et al. in 1986 elaborated the use of 3D computed tomography (CT) images in planning virtual surgeries, and these principles have now been extrapolated to develop customized 3D scaffolds for craniofacial reconstruction [14].

#### *4.7.4 Mathematical modeling*

This method mainly utilizes shape functions to construct porous scaffold with implicit function surfaces or irregular polygonal models. Triply periodic minimal surface (TPMS) method uses trigonometric functions to derive complex porous structure with minimal surface, in which the curvature at any point is zero. It is similar to the natural surface geometries of beetle shells, butterfly wings and crustacean bones, where periodicity exists in three independent directions and no sealed cavities are present in the geometry. TPMS based method has been used for designing tissue scaffolds and a simple primitive (P-type) unit. Other types of TPMS units such as diamond (D-type) and gyroid (G-type) have also been proposed for bone scaffold designing. Capfer et al. studied two types of TPMS-based structures

including network solids and sheet solids. (a) In network solids the minimal surface makes the solid void interface, whereas in (b) sheet solids minimal surfaces to sheets with predefined thickness are inflated to construct porous solids. The latter was found to possess considerably higher mechanical stiffness and Poisson's ratio [52].
