Section 3 3D Bioprinting

## **Chapter 8**

## Rheological Model of Materials for 3D Printing by Material Extrusion

*Jorge Mauricio Fuentes Fuentes*

## **Abstract**

In this chapter, the viscoelastic model of Maxwell and Kevin-Voigt and the rheological model are described. The operation and characteristic equations of a capillary rheometer are explained, as well as the Bagley and Rabinowitch corrections. Next, the method used to determine the viscosity of semicrystalline polymer is explained, using the capillary rheometer. Finally, the Rabinowitch is explained to define a rheological model that determines the viscosity of materials using a capillary rheometer.

**Keywords:** rheology, additive manufacturing, viscosity, manufacturing by extrusion, Bagley correction, Rabinowitch correction, cross WLF model

## **1. Introduction**

Semicrystalline materials, such as polypropylene, are difficult to print by MEX (material extrusion additive manufacturing), due to their lack of adhesion to the print bed and their high shrinkage [1]. The problem increases when the pieces have a large surface in relation to their height, so solutions such as cellophane adhesive tapes, adhesive sprays, and other types of solutions are used. The specimens suffer evident deformations (**Figure 1**), which might affect your application. This chapter shows how a rheological study of the material is carried out to select the optimal properties that would help to mitigate the warping problem.

In this chapter, an introduction to the viscoelasticity models and rheological models is made to later carry out the study of the rheological parameters of semicrystalline polymers, which would serve as a basis for carrying out similar studies on any other polymeric material for printing by MEX.

## **2. Viscoelasticity**

Viscoelasticity is the property that polymers must behave like an elastic solid and a viscous fluid [2]. Elastic deformation is instantaneous and independent of time. Viscous materials, such as water, resist shear flow, and they relax linearly with time when applying a strain. Elastic materials become taut when stretched and immediately return to their original state once the tension is removed. In a polymer, deformations occur with a delay in relation to the applied stresses. Polymers also have a plastic-type

#### **Figure 1.**

*a) PP specimens deformed after the printing process by MEX. b) Comparison of PP, PA, and PC specimens after the printing process by MEX.*

component related to non-recoverable deformation, linked to immediate permanent deformation.

In polymers, stress and time play important parameters in mechanical behavior because they help determine if it will be strong enough, if it will be tough to withstand blows without breaking, and how the polymer will deform under load.

The response of the material is conditioned by its viscoelastic nature. The analysis of the response of these materials can be carried out from mathematical models that try to explain the behaviors, among which are:


A fully viscous response is that of a Newtonian fluid, whose deformation is linear with time while stress is applied and is completely unrecoverable.

#### **2.1 Basic models of viscoelasticity**

When there is an elastic material, the deformation (ε) is instantaneous and proportional to the applied stress (*σ*), which is governed by Hooke's law, shown in Eq. (1), this behavior is represented by a spring [3, 4], which represents the stiffness modulus (ξÞ.

$$\mathbf{e} = \frac{\sigma}{\xi} \tag{1}$$

For a viscous fluid, the deformation is not instantaneous and will depend on time; and this deformation is not recoverable. This deformation is represented by a plunger or piston with a fluid inside, and its behavior is given by Newton's law, according to the Eq.(2). This equation indicates that the stress or stress applied is proportional to the strain rate, who's constant of proportionality is the viscous constant of the fluid (*η*)

$$
\sigma = \eta \frac{d\varepsilon}{dt} \tag{2}
$$

*Rheological Model of Materials for 3D Printing by Material Extrusion DOI: http://dx.doi.org/10.5772/intechopen.109630*

#### *2.1.1 Maxwell viscoelasticity model*

This model considers that the viscoelastic model of a polymer is given by the series union of a spring and a piston. An ideal elastic element is represented by a spring that obeys Hooke's law [5, 6], with a modulus of elasticity ξ.

Since there is a series coupling of both elements, the total deformation of the assembly will be the sum of the elastic deformation independent of time (ε1) and the viscous component (ε2) that is dependent on time, according to the Eq. (3), see **Figure 2a** and **b**.

$$
\mathfrak{e} = \mathfrak{e}\_1 + \mathfrak{e}\_2 \tag{3}
$$

On the other hand, the stresses when connected in series are equal according to the Eq. (4).

**Figure 2.** *(a) Diagram of Maxwell's viscoelastic model. (b) Series coupling of the elastic element (spring) and viscous element (plunger).*

$$
\sigma = \sigma\_1 = \sigma\_2 \tag{4}
$$

If the time variable for the deformations is considered, the Eqs. (3) and (4) become:

$$\frac{\text{de}\_1}{\text{dt}} = \frac{1}{\xi} \frac{\text{d}\sigma\_1}{\text{dt}} \tag{5}$$

$$\frac{d\varepsilon\_2}{dt} = \frac{1}{\eta}\sigma\_2\tag{6}$$

Deriving the Eq.(3) respect to time:

$$\frac{\text{de}}{\text{dt}} = \frac{\text{de}\_1}{\text{dt}} + \frac{\text{de}\_2}{\text{dt}} \tag{7}$$

$$\frac{\text{de}}{\text{dt}} = \frac{1}{\xi} \frac{\text{d}\sigma\_1}{\text{dt}} + \frac{1}{\eta} \sigma\_2 = \frac{\text{de}}{\text{dt}} = \frac{1}{\xi} \frac{\text{d}\sigma}{\text{dt}} + \frac{\sigma}{\eta} \tag{8}$$

If it is considered that a constant effort is applied, the expression is as indicated in Eq. (9):

$$\frac{d\varepsilon}{dt} = \frac{\sigma\_0}{\eta} \tag{9}$$

Integrating the previous Eq. (9), considering that the immediate response in the elastic spring corresponds to the constant tension, we have:

$$\mathbf{e} = \frac{\sigma\_0}{\eta} \mathbf{t} + \frac{\sigma\_0}{\xi} \tag{10}$$

It can be seen in **Figure 3** that a viscoelastic element that works at constant tension will have an immediate deformation due to its elastic component and an increasing linear deformation, due to the viscous response of the polymer.

The Maxwell model is useful when it comes to predicting instantaneous elastic deformation; however, when it comes to viscous deformation over time, it does not fit reality, since this curve is not linear.

#### *2.1.2 Kelvin-Voigt model*

In this model of viscoelasticity, the behavior of a polymer is considered as the parallel union of a piston and a spring [3, 4], as shown in the **Figure 4**.

In this model, when applying the tension, part of the energy will be stored by the spring and the rest will be slowly dissipated when the viscous element (piston) moves, resulting in a total deformation that depends on time [5, 6]. When the load is no longer applied, the original shape of the spring will recover, but not the piston, see diagram of **Figure 5**.

According to this model, the total tension applied will be equal to the sum of the tensions of the spring and that of the piston, see Eq. (11). The total deformation will be equal to the deformation of the spring and that of the piston, see Eq. (12), since they are in parallel.

*Rheological Model of Materials for 3D Printing by Material Extrusion DOI: http://dx.doi.org/10.5772/intechopen.109630*

*Representation of the deformation with respect to time according to the Maxwell model under the action of a constant stress.*

**Figure 4.**

*Diagram of the kelvin-Voigt viscoelastic model in which there is a parallel coupling of the elastic element and the viscous element.*

**Figure 5.** *Diagram of the elongation that a viscoelastic element undergoes over time that complies with the kelvin-Voigt model.*

$$
\sigma = \sigma\_1 + \sigma\_2 \tag{11}
$$

$$
\mathfrak{e} = \mathfrak{e}\_1 = \mathfrak{e}\_2 \tag{12}
$$

Considering the addition of stresses, we have the general expression of the Kelvin-Voigt model:

$$\mathbf{e} = \frac{\sigma\_0}{\eta} \mathbf{t} + \frac{\sigma\_0}{\xi} \tag{13}$$

$$
\sigma = \xi e\_1 + \eta \frac{d e\_2}{d t} = \xi e + \frac{d e}{d t} \tag{14}
$$

When dealing with a long-term phenomenon, we have that: *σ* ¼ *σ*1*σ*0, and the solution of the differential Eq. is given by the form:

$$\mathbf{e} = \frac{\sigma\_0}{\xi} \left[ \mathbf{1} - \mathbf{e}^{\frac{-\xi}{\eta}} \right] \tag{15}$$

The behavior of the polymer applying this model is shown in the **Figure 6**, in this it is observed that when a constant tension is applied to the material, it experiences a

**Figure 6.**

*Representation of the exponentially deformation with respect to time according to Maxwell model.*

progressive elongation exponentially. However, this does not square with reality, since at time 0, there is an instantaneous deformation.

#### *2.1.3 Combined model (burgers)*

In general terms, the Kelvin-Voight model Eq. satisfactorily explains a real behavior such as creep, which the Maxwell model does not consider. On the other hand, the Kelvin-Voight model does not explain instantaneous deformation (**Figure 7**), which the Maxwell model does and with a good approximation.

A good, more correct approximation that has been obtained to simulate the viscoelastic behavior of polymers is to use a combined model that considers the Kelvin-Voigt and Maxwell models and adjusts more to the real behavior, which is applicable from the point of view engineering [7]. In this model, the two models are coupled in series, according to the **Figure 8**, where the first component corresponds to the Maxwell model and the second component corresponds to the Kelvin-Voigt model.

Identifying the subscripts M to the Maxwell model, KV to the Kelvin-Voigt model, E to the elastic component of the spring, and V to the viscous component of the piston, the above equations become:

Maxwell's equation:

$$\mathbf{e}\_{\mathbf{M}} = \frac{\sigma\_0}{\eta\_{\mathbf{M}-\mathbf{V}}} \mathbf{t} + \frac{\sigma\_0}{\xi\_{\mathbf{M}-\mathbf{E}}} \tag{16}$$

Kelvin-Voigt equation:

$$
\varepsilon\_{\rm KV} = \frac{\sigma\_0}{\xi\_{\rm KV-E}} \left[ 1 - \mathbf{e}^{\frac{-\rm kV-E}{\eta\_{\rm KV-V}} \cdot t} \right] \tag{17}
$$

If the two models are considered in series, the total elongation of the system is equal to the sum of each of the Maxwell and Kelvin-Voigt systems individually. Therefore, the expression that defines the yield in the combined model is:

**Figure 7.**

*Schematic representation of the actual deformation that a polymer undergoes, in which the instantaneous deformation, the deformation and the viscous recovery of the material are observed.*

**Figure 8.**

*Scheme of the combined viscoelastic model (Burguers).*

$$
\varepsilon\_{\text{TOTAL}} = \varepsilon\_{\text{M}} + \varepsilon\_{\text{KV}} \tag{18}
$$

$$\mathbf{e}\_{\text{TOTAL}} = \frac{\sigma\_0}{\eta\_{\text{M}-\text{V}}} \mathbf{t} + \frac{\sigma\_0}{\xi\_{\text{M}-\text{E}}} + \frac{\sigma\_0}{\xi\_{\text{KV}-\text{E}}} \left[ \mathbf{1} - \mathbf{e}^{\frac{-\xi\_{\text{KV}-\text{E}}}{\eta\_{\text{KV}-\text{V}}}} \right] \tag{19}$$

Each of the terms is defined as:

<sup>ξ</sup>*<sup>M</sup>*�*<sup>E</sup>*: Elastic constant of the spring in Maxwell's element.

<sup>η</sup>*<sup>M</sup>*�*<sup>V</sup>*: Viscous constant of the piston in Maxwell's element.

ξ*KV*�*<sup>E</sup>*: Elastic constant of the spring in the Kelvin-Voigt element.

η*KV*�*<sup>V</sup>*: Viscous constant of the piston in the Kelvin-Voigt element.

See **Table 1**.

The combination of simple moldings such as Maxwell and Kelvin-Voigt allows to obtain models that are more adjusted to the real viscoelastic behavior of polymeric materials.

$$\varepsilon\_{\rm M} = \frac{\sigma\_0}{\xi\_{\rm M-E}} + \frac{\sigma\_0 \cdot \mathbf{t}}{\eta\_{\rm M-V}} \tag{20}$$

$$
\varepsilon\_{\rm KV} = \frac{\sigma\_0}{\xi\_{\rm KV-E}} \left[ \mathbf{1} - \mathbf{e}^{\frac{-\xi\_{\rm KV-E}}{\eta\_{\rm KV-V}}} \right] \tag{21}
$$

## **3. Polymer rheology**

To carry out the correct modeling and simulation of the extrusion process through a nozzle for printing by MEX, it is necessary to precisely know the viscosity of the material, which depends on the physical parameters to which it is subjected [8, 9].

## *Rheological Model of Materials for 3D Printing by Material Extrusion DOI: http://dx.doi.org/10.5772/intechopen.109630*

**Table 1.** *the*

 *curve of the material.*

#### **Figure 9.**

*Diagram of the relationship between dynamic viscosity and kinematic viscosity.*

There are three types of viscosity: dynamic viscosity, kinematic viscosity, and apparent viscosity. The dynamic or absolute viscosity (μ) represents the internal resistance between the molecules of a moving fluid and determines the forces that move and deform it. The kinematic viscosity (ν) relates the dynamic viscosity to the density of the fluid used and represents the resistance of a fluid to sliding. Instead, the apparent viscosity "η" is defined as the ratio between the shear stress (τ) and the strain rate (γ) for fluids with nonlinear behavior (non-Newtonian). If the fluidity curve is plotted (shear force vs. strain rate), it is also defined as the slope at each point on the curve, see **Figure 9**.

In **Figure 10**, the required shear stress is represented as a function of the shear speed to be reached for different liquids. In a Newtonian fluid, the slope of the curve (n) is 1, while in a dilatant fluid (n > 1), the viscosity increases with the shear strain rate. For some fluids such as polymer melts, some paints, and fluids with suspended particles, the viscosity decreases with increasing shear stress (n < 1), these fluids are called pseudoplastic or shear-thinning.

Thermoplastic polymers under low or no shear conditions behave like a Newtonian fluid, while under high shear conditions they behave like a pseudoplastic, decreasing rapidly with increasing shear rate [10], as can be seen in the example of polypropylene shown in **Figure 11**.

#### **3.1 Rheology for 3D printing**

It is necessary to know if the designed plastic will be extrudable by the MEX method, to avoid performing many time-consuming and expensive empirical tests [12], as well as avoiding nozzle clogging, setting the parameters of the 3D printing machine, as well as avoiding phenomena such as contraction and adhesion to the printing table.

To simulate plastic extrusion through the die, you can use the rheology data and compare it with a plastic that extrudes correctly and compare the rheological curves, which will show, for example, shear rate and shear stress [13, 14].

*Rheological Model of Materials for 3D Printing by Material Extrusion DOI: http://dx.doi.org/10.5772/intechopen.109630*

**Figure 10.** *Power law schematic showing logτ versus log(dγ=dτ) for different types of fluids.*

#### **Figure 11.**

*Viscosity graph of PP, POLYFORT® FIPP 30 T K1005 at 3 temperatures [11].*

The cutting rate can be determined by:

• making an initial shear rate measurement of the melt viscosity to obtain the power law index.


### **3.2 Viscosity measurement**

One of the most important characteristics to predict the behavior of an extrusion or injection process is the viscosity of the melted material. There are several methods for obtaining viscosity as a function of shear rate [15]. Instruments used to measure shear rates must shear the fluid at measurable rates, and the stress developed must be known, for which a rotational viscometer or capillary rheometer can be used [16]. For the tests of this work, a capillary rheometer was used. Extrusion pressure or volumetric flow rate can be controlled as the independent variable, and the other is the measured dependent variable [17] .

## *3.2.1 Rotational rheometer*

In this rheometer, the fluid is sheared at a given temperature between an annular space due to the rotation of a coaxial internal cylinder inside another cylinder or by the rotation of a conical plate on a stationary plate or vice versa [18]. Rotational viscometers using two coaxial cylinders measure low viscosities of liquids, see **Figure 12a**. In a plate-and-cone viscometer, the polymer is contained between the lower plate and the cone, which rotates at a constant speed (Ω), see **Figure 12b**. These viscometers are used for viscosities less than 10 s�<sup>1</sup> . This viscometer is expensive equipment, it gives information at the molecular level, and it only serves to give information about the fluid in the Newtonian regime.

**Figure 12.** *a) Diagram of a coaxial cylinder viscometer. b) Diagram of a plate and cone viscometer.*

## *3.2.2 Capillary tube rheometer*

There are three main reasons why the capillary rheometer is widely used in the plastics industry [19]:

the shear rate and flow geometry in the capillary rheometer are very similar to the conditions actually encountered in extrusion and injection modeling;

a capillary rheometer typically covers the wider shear rate ranges (10 � <sup>6</sup> s � <sup>1</sup> –10 <sup>6</sup> s � <sup>1</sup> );

a capillary rheometer provides good practical data and information on matrix swelling, melt instability, and extrudate defects.

In this equipment, the fluid is forced to pass from a container through a small diameter hole or capillary in a nozzle, by mechanical or pneumatic actuators or pistons [12]. The fluid is kept at a constant temperature due to the use of electric heating resistances (**Figure 13**).

Under constant flow and isothermal conditions for an incompressible fluid, the viscous force resisting the movement of a column of fluid in the capillary is equal to the applied force tending to move the column in the direction of flow, then:

**Figure 13.** *Diagram of a capillary rheometer.*

where,

R is the radius of the column.

Lis the length of the column.

Δ*P* is the pressure drop across the capillary.

*τ* is the shear stress.

According to the above, the shear stress *τ* is maximum on the cylinder walls and is zero in the center. For the calculations the maximum shear stress will be used.

In normal capillary rheometry, the molten material vents to the atmosphere, and the driving static pressure in the reservoir is taken as Δ*P*. In such cases, end effects involving viscous and elastic deformations at the capillary inlet and outlet must be considered when calculating the actual shear stress in the capillary wall, particularly if the ratio of capillary length to radius (L/R) is small.

For a fluid that exhibits Newtonian behavior, the shear rate at the wall is given by:*γ*\_

$$
\dot{\chi} = \frac{4\,\mathrm{Q}}{\pi \mathrm{R}^3} \tag{23}
$$

where Q is the volumetric flow rate through the capillary due to pressure Δ*P*, then the viscosity of the melt can be expressed as:

$$
\eta = \frac{\pi}{\dot{\lambda}} = \frac{\pi \mathbf{R}^4 \Delta \mathbf{P}}{\mathbf{8LQ}} \tag{24}
$$

Values measured by capillary rheometers are often presented as plots of shear stress versus shear rate at certain temperatures. These values are called the apparent shear stress and the apparent shear rate at the tube wall.

## **3.3 Bagley's correction**

This correction is applied due to the overpressure that occurs when going from a large cylinder (container or charge cylinder) to a small one (nozzle). The Bagley method is considered in which the overpressure is evaluated by relating it to an apparent increase in the length of the nozzle [20]. The total pressure drop ΔPtot is given by the sum of the pressure drop in the reservoir, at the inlet ΔPe, in the capillary Δ*P*, and at the outlet, due to the swelling of the polymer at the outlet. The pressure drop in the capillary is required to make the calculations and the pressure drop in the reservoir and the pressure drop at the outlet due to swelling can be neglected [21].

$$
\Delta \mathbf{P\_{tot}} = \Delta \mathbf{P\_e} + \Delta P \tag{25}
$$

For two or three capillaries, measurements are made of the total pressure drop as a function of the volumetric flow, for which the piston moves at different speeds, so that the flow varies, with the corresponding pressure variation, see **Figure 14**. As the flow increases, the cutting speed increases. With greater capillary length, there is a greater pressure drop.

For the same flow, the pressure drop for the three capillaries is taken. For each flow value, a minimum of 2 L/D points are obtained, and straight lines are made for each flow. In our case we have three capillaries (**Figure 15**). In total, seven flow data were taken as a function of piston speed (**Figure 16**).

**Figure 14.** *Pressure drop curve (*Δ*PTot) versus volume flow (Q) for three nozzles (3 L/D ratios).*

The determination of the correction value is carried out with a test in which at least three nozzles with different values of the L/D ratio are used.

$$\tau\_{\text{corr}} = \frac{\Delta \mathbf{P}}{2(\frac{\text{L}}{\text{R}} + \text{e})} \tag{26}$$

where,

*τcorr* is the corrected shear stress in [Pa].

*e* is the additional apparent length of capillary measured at a given shear rate measured in [mm].

In order to determine, which is an empirical constant that tries to correct the effects of exit and entrance of material in the molten state in the capillary, it is extrapolated to Δ*P* ¼ 0, from the representation of Δ*P* versus L/D at constant shear rate and for capillaries of different lengths.

Extrapolating means that the L/D ratio is 0, that is, there is no change in diameter. When it intercepts in Δ*P*, the value of pressure drops at the inlet (Δ*Pe*) is obtained, then from Eq. 9.24 we have:

$$
\Delta \mathbf{P} = \Delta \mathbf{P}\_{\text{Tot}} - \Delta \mathbf{P}\_{\text{e}} \tag{27}
$$

For each of the straight lines and for the three points, the shear stress is calculated corrected *τcorr:*

The apparent shear stress is evaluated with:

$$\tau\_{\rm app} = \frac{\Delta \mathbf{P} \cdot \mathbf{D}}{4 \ast \mathbf{L}} \tag{28}$$

**Figure 15.** *Illustration of determining the value of the Bagley correction.*

**Figure 16.** *Bagley plot: Pressure drop vs. L/D for different shear rates [22].*

Corrected shear stress is as follows:

$$
\tau\_{\rm corr} = \left(\frac{-\Delta \mathbf{P}}{\mathbf{L} + \mathbf{e} \cdot \mathbf{D}}\right) \cdot \frac{\mathbf{D}}{4} \tag{29}
$$

#### **3.4 Weissenberg: rabinowitch correction**

This correction is applied to the shear rate, since a molten plastic does not behave like a Newtonian fluid that presents a parabolic velocity distribution [23], but rather in a pseudoplastic manner, presenting a non-parabolic velocity distribution [24], this is shown in the Eq. (30)

*Rheological Model of Materials for 3D Printing by Material Extrusion DOI: http://dx.doi.org/10.5772/intechopen.109630*

$$
\gamma\_{\rm corr} = \frac{\left(\clubsuit + \frac{1}{n}\right) \gamma\_{\rm app}}{4} \tag{30}
$$

where,

*γcorr* is the corrected shear rate in [s-1];

*n* is the slope of the relationship between shear rate and shear stress; *γapp* represents the apparent value of the shear rate on the tube wall;

$$\mathbf{n} = \frac{\mathbf{d} \mathbf{log} \mathbf{r}\_{\mathrm{app}}}{\mathbf{d} \dot{\mathbf{y}}\_{\mathrm{app}}} \tag{31}$$

*n* is 1 for a Newtonian fluid.

#### **3.5 Polymer rheological models for 3D printing using Cross-WLF parameters**

Cross-Williams-Landel-Ferry (WLF) model or Cross-WLF model [25] uses the results of the experimental data obtained in a capillary rheometer to describe the rheological behavior of polymeric materials, which allows the determination of their viscosity, to evaluate their processability under different conditions [26]. Within the manufacturing processes, the polymer is subjected to various shear conditions, which need to be evaluated and predicted [27]. Under conditions of low or practically zero shear, the viscosity of the material usually remains constant, presenting a Newtonian behavior. On the contrary, under high shear conditions, the viscosity decreases rapidly with the shear rate, showing a pseudoplastic behavior [28].

This model has been chosen because it can predict with a very good approximation the pseudoplastic behavior of the melt at high shear rates and the Newtonian behavior at low shear conditions [29], see **Figure 14**. This viscosity model is used in computeraided engineering (CAE) programs such as Autodesk Moldflow™ and Ansys PolyFlow™ [30] for simulation of the plastic injection process [31, 32], because it offers the best fit to the viscosity data [33]. This model also simplifies the calculations of the pseudoplastic region, favoring the interpretation of results by considering its slope linear on a logarithmic scale [34].

This viscosity model describes the dependence of viscosity as a function of temperature (K), the shear rate *Tm*ðγ(s�<sup>1</sup> ) and pressure p (Pa), which are the model dependent parameters.

The viscosity according to this model is expressed as:

$$\eta\left(\dot{\mathbf{y}},\mathbf{T}\_{\mathbf{m}},\mathbf{p}\right) = \frac{\eta\_0(\mathbf{T}\_{\mathbf{m}},\mathbf{p})}{\mathbf{1} + \left(\frac{\eta\_0(\mathbf{T}\_{\mathbf{m}},\mathbf{p})}{\mathbf{1}^\ast}\dot{\mathbf{y}}\right)^{(1-\mathbf{n})}}\tag{32}$$

where,

*η* is the viscosity of the molten material in [Pa.s];

*η*<sup>0</sup> is the zero shear viscosity or the 'Newtonian limit' where the viscosity approaches a constant at very low shear rates [Pa.s];

*τ* <sup>∗</sup> is the constant of the model that indicates the shear stress from which the pseudoplastic behavior of the material begins, determined by fitting the curve [Pa] with *<sup>K</sup>* <sup>¼</sup> *<sup>η</sup>*<sup>0</sup> *<sup>τ</sup>* <sup>∗</sup>*n* is the index of the power law in the high shear rate regime, determined by the curve fit that symbolizes the slope of the pseudoplastic behavior in the form of (*n* � 1), see **Figure 17**.

#### **Figure 17.**

*Approximation of the viscosity with the Cross-WLF model in Eqs. (32) and (33) [35].*

*γ*\_is the apparent shear rate in [s�<sup>1</sup> ].

This model is complemented by the Williams-Landel-Ferry (WLF) model. Bagley [20] which helps to determine the behavior of the material with respect to null shear phenomena and offers reliable results. His expression is:

$$\eta\_0(\mathbf{T}\_\mathbf{m}, \mathbf{p}) = \left\{ \begin{array}{c} \mathbf{1}\_\mathbf{D}, \mathbf{1}\_\mathbf{e} \left( \frac{\mathbf{1}\_\mathbf{1} \cdot (\mathbf{T}\_\mathbf{m} - \mathbf{T})}{\mathbf{1}\_\mathbf{1} + (\mathbf{T}\_\mathbf{m} - \mathbf{T})} \right) \\ \ast, \text{si } \mathbf{T}\_\mathbf{m} < \mathbf{T} \end{array} \right. \\ \text{si } \mathbf{T}\_\mathbf{m} < \mathbf{T} \end{array}$$

$$\mathbf{A}\_2 = \tilde{\mathbf{A}}\_2 + \mathbf{D}\_3 \bullet \mathbf{p}$$

$$\tilde{\mathbf{T}} = \mathbf{D}\_2 + \mathbf{D}\_3 \bullet \mathbf{p} \tag{33}$$

where:

*T*~ is the glass transition temperature of the material in [K], which depends on the pressure.

*D*<sup>1</sup> is the model constant that indicates the viscosity under zero shear conditions at the glass transition temperature of the material and atmospheric pressure, in [Pa.s].

*D*<sup>2</sup> is a model constant indicating the glass transition temperature of the material at atmospheric pressure, in [K].

*D*3, constant of the model that indicates the variation of the transition temperature of the material as a function of pressure, in [K/Pa].

*<sup>A</sup>*1, *<sup>A</sup>*~2, are model constants, in [�], [K], respectively.

*pe*s the pressure in [Pa].

To use this model, its seven parameters can be determined based on estimates given by various authors [36] or by analysis of the characteristics of the materials.

## **4. Conclusions**

It is important to know the viscoelastic and rheological properties of the materials for 3D printing by MEX, since this will allow to avoid performing an error trial

### *Rheological Model of Materials for 3D Printing by Material Extrusion DOI: http://dx.doi.org/10.5772/intechopen.109630*

process, which is long and expensive, being able to perform the process through the necessary parameters to simulate the process through computer-aided engineering.

The rheological parameters of a polymer or composite material for manufacturing by extrusion can be determined by using a capillary rheometer, due to its high cutting speeds and shear stresses.

Bagley and Rabinowitch corrections can be applied to the results obtained through the capillary rheometer to compensate the change in diameter in the capillary, and these results can be exported to simulation programs by computer to verify the technical feasibility of manufacturing with the composite material.

When the Cross WLF model is applied, we have a good approximation of the rheological properties of the material to any pressure and temperature condition, which allows us to extrapolate the results to adjust the properties in a 3D printing process by MEX.

## **Acknowledgements**

We thank the Central University of Ecuador for their support with time and funds to carry out this study.

## **Conflict of interest**

The author declares no conflict of interest.

## **Appendix 1**

### Terms and symbols



## **Author details**

Jorge Mauricio Fuentes Fuentes Central University of del Ecuador, Quito, Ecuador

\*Address all correspondence to: jmfuentes@uce.edu.ec

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Rheological Model of Materials for 3D Printing by Material Extrusion DOI: http://dx.doi.org/10.5772/intechopen.109630*

## **References**

[1] Bachhar N, Gudadhe A, Kumar A, Andrade P, Kumaraswamy G. 3D printing of semicrystalline polypropylene: Towards eliminating warpage of printed objects. Bulletin of Materials Science. 2020;1-8. DOI: 10.1007/s12034-020-02097-4

[2] Hajikarimi P, Moghadas Nejad F. Mechanical models of viscoelasticity. In: Applications of Viscoelasticity. 2021. vol. 1. pp. 27-41. DOI: 10.1016/b978-0- 12-821210-3.00003-6

[3] Varna J, Pupure L. Characterization of Viscoelasticity, Viscoplasticity, and Damage in Composites. 2nd ed. London, UK: Elsevier; 2019. DOI: 10.1016/ B978-0-08-102601-4.00016-3

[4] Mainardi F, Spada G. Creep, relaxation and viscosity properties for basic fractional models in rheology. European Physical Journal: Special Topics. 2011;**193**(1):133-160. DOI: 10.1140/epjst/e2011-01387-1

[5] Gutierrez-Lemini D. Engineering viscoelasticity. Engineering Viscoelasticity. Vol. 1. 2014:1-353. DOI: 10.1007/978-1-4614-8139-3

[6] Fombuena V, Boronat L, Sánchez-Nácher L, García-Sanoguera D, Balart R. "Utilidad de los modelos de viscoelasticidad en el aprendizaje de la ingeniería de materiales poliméricos," Modelling in Science Education and Learning. Jan 2017;**10**(1):137. DOI: 10.4995/msel.2017.6315

[7] Hajikarimi P, Moghadas Nejad F. Mechanical models of viscoelasticity. In: Applications of Viscoelasticity. London, UK: Elsevier; 2021. pp. 27-61. DOI: 10. 1016/b978-0-12-821210-3.00003-6

[8] Mackay ME. "The importance of rheological behavior in the additive

manufacturing technique material extrusion." Journal of Rheology. Nov 2018;**62**(6):1549-1561. DOI: 10.1122/ 1.5037687

[9] Polychronopoulos ND, Vlachopoulos J. Polymer Processing and Rheology. In: Jafar Mazumder M, Sheardown H, Al-Ahmed A, editors. Functional Polymers. Polymers and Polymeric Composites: A Reference Series. Springer, Cham. 2019. DOI: 10.1007/978-3-319-95987-0\_4

[10] Irgens F. Linearly Viscoelastic Fluids. In: Rheology and Non-Newtonian Fluids. Springer, Cham. 2014. DOI: 10.1007/978-3-319-01053-3\_7

[11] CAMPUSplastics | datasheet POLYFORT® FIPP 30 T K1005. https:// www.campusplastics.com/campus/en/da tasheet/POLYFORT%C2%AE+FIPP+30 +T+K1005/LyondellBasell/103/21818245/ SI?pos=2 [Accessed: June 17, 2021]

[12] Prabhu R, Devaraju A. Recent review of tribology, rheology of biodegradable and FDM compatible polymers. Materials Today: Proceedings. 2020;**39**:781-788. DOI: 10.1016/j.matpr. 2020.09.509

[13] Picco S et al. Polymeric Additive Manufacturing: The Necessity and Utility of Rheology. London, UK, London, UK: IntechOpen; 2016. p. 13 no. tourism [Online]. Available: https://www.intechopen.com/ books/advanced-biometric-technologies/ liveness-detection-in-biometrics

[14] Carvalho C, Bom RP, Joinville C. de & Whirpool SA. Propriedades reológicas de abs e suas misturas oriundas de reciclagem primária. Anais Do 10o Congresso Brasileiro de Polímeros. Vol. 1. 2009:10

[15] Maysaa M, Rahamtalla E, Deen H. Viscosity Measurement by using Melt flow Index for Thermoplastic polymers. Sudan University of Science and Technology. 2014. Available from: http://repository.sustech.edu/handle/ 123456789/9099

[16] Morrison FA. Rheometry CM4650 Chapter 10: Rheometry. 2018. pp. 1–56

[17] Nikzad M, Masood SH, Sbarski I, Groth A. Rheological properties of a particulate-filled polymeric composite through fused deposition process. Materials Science Forum. 2010;**654–656**: 2471-2474. DOI: 10.4028/www. scientific.net/MSF.654-656.2471

[18] ASTM D 4402. Standard test method for measuring the viscosity of Mold powders above their melting point using a rotational viscometer. Control. 1999; **94**, no. Reapproved:5-7. DOI: 10.1520/ D4402

[19] Osswald T, Rudolph N. "Rheometry," in Polymer Rheology. Munich, Germany: Carl Hanser Verlag GmbH & Co. KG; 2014. pp. 187-220. DOI: 10.3139/9781569 905234.006

[20] Bagley EB. End corrections in the capillary flow of polyethylene. Journal of Applied Physics. 1957;**28**(5):624-627. DOI: 10.1063/1.1722814

[21] Dealy JM, Wang J. Methods Melt Rheology and its Applications in the Plastics Industry. Montreal, Canada: Springer; 2013. pp. 149-152 [Online]. Available: http://www.springer.com/ series/4604

[22] Montanes N et al. Modelización reológica mediante Cross-WLF de un nuevo material compuesto elaborado con bioPE y Thyme. Vol. 2. pp. 22–27, [Online]. Available: http://revista.aemac.org/

[23] Pranata dkk. Study of rheological. Thermal and Mechanical Behavior of Reprocessed Polyamide. 2013;**6**(53): 679-688

[24] M. Dees, M. Mangnus, N. Hermans, W. Thaens, A. S. Hanot, and P. van Puyvelde, "On the pressure correction of capillary melt rheology data." Rheologica Acta. Feb 2011;**50**(2):117-124. DOI: 10.1007/s00397-011-0529-2.

[25] Cross MM. Rheology of Non-Newtonian Fluids: A New Flow Equation for Pseudoplastic Systems. Journal of Colloid Science. 1965;**20**:417-437. DOI: 10.1016/0095-8522(65)90022-X

[26] Ferrándiz S, Arrieta MP, López J, Navarro R. Demostració práctica de la validesa dels models matematics en elements finits. Aplicaci'o al model de Cross. Modelling in Science Education and Learning. 2013;**6**(3):67-82

[27] Osswald T. Rudolf N. Polymer Rheology: Fundamentals and Applications. Munich, Germany: Hanser, 2015

[28] Irgens F. Rheology and nonnewtonian fluids. In: Rheology and Non-Newtonian Fluids. 2013;**9783319010**:1- 16. DOI: 10.1007/978-3-319-01053-3

[29] Reig MJ, Segui VJ, Zamanillo JD. Rheological behavior modeling of recycled ABS/PC blends applied to injection molding process. Journal of Polymer Engineering. 2005;**25**(5): 435-457. DOI: 10.1515/ POLYENG.2005.25.5.435

[30] Canonsburg TD. ANSYS POLYMAT user 's guide. Knowledge Creation Diffusion Utilization. 2012;**15317** (October):724-746

[31] Debbaut B. Rheology: from Process to Simulation. 2005;**13**:23-36

*Rheological Model of Materials for 3D Printing by Material Extrusion DOI: http://dx.doi.org/10.5772/intechopen.109630*

[32] Acedo J. Caracterización y simulación del comportamiento viscoelástico de materiales plásticos mediante el Método de Elementos Finitos. Valencia; Spain: Universidad Politécnica de Valencia; 2019

[33] Sanchez LC, Augusto C, Helena S, Bettini P, Costa LC. Rheological approach for an additive manufacturing printer based on material extrusion. The International Journal of Advanced Manufacturing Technology. Vol. 105. 2019. pp. 2403–2414. DOI:10.1007/ s00170-019-04376-.

[34] Piotr M. Numerical and Experimental Analysis of Filament-based Material Extrusion Additive Manufacturing Ph.D. thesis, Technical University of Denmark DTU, Mechanical Engineering Section of Manufacturing Engineering. 2020

[35] Ferri D, Perolo A, Nodari M. Cross-WLF parameters to predict rheological properties of polylactic acid. Annual Transactions of the Nordic Rheology Society. 2017;**25**(1):419-426

[36] Passaglia E, Martin GM. Variation of Glass Temperature With Pressure in Polypropylene. Journal of Research of the National Bureau of Standards. Section A, Physics and Chemistry. 1964 May-Jun;**68A**(3):273-276. DOI: 10.6028/ jres.068A.024. Epub 1964 Jun 1. PMID: 31834721; PMCID: PMC5327688

## 3D Printing for Tissue Regeneration

*Meghana Kasturi, Vidhi Mathur, Prachi Agarwal, Varadharajan Srinivasan and Kirthanashri S. Vasanthan*

## **Abstract**

Tissue engineering is an interdisciplinary field and 3D bioprinting has emerged to be the holy grail to fabricate artificial organs. This chapter gives an overview of the latest advances in 3D bioprinting technology in the commercial space and academic research sector. It explores the commercially available 3D bioprinters and commercially printed products that are currently available in the market. It provides a brief introduction to bioinks and the latest developments in 3D bioprinting various organs. The chapter also discusses the advancements in tissue regeneration from 3D printing to 4D printing.

**Keywords:** 3D bioprinting, bioink, tissue engineering, regenerative medicine, 4D bioprinting, scaffold

## **1. Introduction**

Tissue engineering is a branch of biomedical engineering that focuses on repairing and/or replacing diseased and damaged organs. This is done primarily via developing artificial organs using natural or synthetic materials. Organ shortage is a severe problem worldwide due to the non-availability of donors and tissue engineering strategies enable to produce a scaffold that mimics the organ of interest [1]. Threedimensional (3D) bioprinting has great potential in this field and was developed in the early 1990s, and has evolved ever since. It is an additive manufacturing (AM) technique that uses computer-aided design (CAD) models to deposit biomaterials on the substrate along with living cells, extracellular matrix (ECM) components, biochemical cues, and drugs. The three basic steps in the 3D bioprinting process are: (i) preprocessing - includes developing CAD models to develop in-vitro scaffolds or to develop organ blueprints from imaging modalities such as computer tomography (CT) and magnetic resonance imaging (ii) processing - produces a physical structure that mimics the organ/tissue of interest from the designed model (iii) postprocessing - improves the bioprinted organ model and scope for transplantation if required. Over recent years, there has been a huge demand and interest in 3D bioprinting due to its potential to produce high-throughput biomimetic organ scaffolds. Several technological advancements have come up in 3D bioprinting which are mentioned in **Figure 1.** The goal of 3D bioprinting is to provide alternative approaches to autologous and allogeneic implant treatments and avoid animal testing in drug studies and disease models. 3D bioprinting has several biological applications in the fields of

tissue engineering, materials science, pharmaceutical drug development and validation, cosmetics testing, personalized medicine, regenerative medicine, cell-based biosensors, and bionics.

## **2. Commercial 3D bioprinters**

A 3D bioprinter is an automated device that enables the development of functional tissue and organ models. The 3D bioprinting technology is generally classified into three types – (i) droplet-based bioprinters (ii) extrusion-based bioprinter (iii) light-based bioprinter (**Figure 1**). Extrusion-based bioprinters are widely used and are based on the principle of depositing the material layer by layer. Laser-based 3D bioprinters deposit the bioink drop by drop, the principle is like an inkjet 3D bioprinter. Some companies and universities have developed 3D bioprinting technologies that cannot be easily classified into widely known technologies. For example, Cyfuse Biomedics has developed a technique where cells are 3D printed on a needle array. A scaffold is not required in this method instead only a cluster of cells (not mixed with other biomaterials) are skewered onto vertical needles to fabricate 3D tissue structures. Companies like rainbow biosciences have developed a bioprinter called BiOassay where biocompatible magnetic nanoparticles are used to print the 3D structures and use the working principle of magnetic levitation [2]. There are many emerging bioprinting technologies being developed by researchers across the world to make the process more efficient and cost-effective. Currently, the 3D-printed organs can be used for research only; however, in future, they can be transplanted into human patients.

The wide range of applications has driven many companies/universities to develop bioprinting technology. The following are the types of business models utilized by these companies that exploit bioprinting technology – (i) Manufacturing bioprinters (ii) Providing bioprinting services (iii) providing cell therapies that utilize bioprinting technology. Commercially available 3D bioprinters have increased in the market over the past decade and have rapidly advanced the tissue engineering field. The 3D Bioprinting Market is expected to reach USD 3261.31 Million by 2027, from USD 796.9 Million in 2020 growing at a compound annual growth rate of 22.3% during

2021–2027 [3]. The growth of this market is due to a limited number of organ donors, and an increase in the aging population with chronic diseases. The rise in R&D investment in this sector, advancement in commercially available products, and increment in the incidence of chronic diseases are other vital factors that are likely to boost market growth during the coming years [4]. **Table 1** provides a list of commercially available bioprinters in the market.

## **3. Formulation of bioinks**

In bioprinting, cells are placed at user-defined coordinates, along with biomaterials that are either (i) mixed with cells before printing, or (ii) printed simultaneously with one print head while the cells are deposited via the other print head (**Figure 2A** and **B**). Materials used in bioprinting that contain cells in the mixture are termed as bioink. The biomaterial is usually a polymer (natural or synthetic) that has biocompatible components and provides favorable rheological properties for the desired organ of interest. Hydrogels are the most used bioinks. However, hydrogel precursors are widely in use these days as they can be cross-linked into hydrogels post-biofabrication. Another method is to crosslink the precursor solution to obtain a viscous ink, followed by crosslinking the scaffold post-printing [5].

An ideal bioink should have the desired physicochemical properties to print mechanically stable scaffolds which mimic the organ of interest (**Figure 2C**). These properties are determined by the mechanical strength of the scaffold, viscosity of the ink, chemical structure of the polymer, and biological characteristics of the desired tissue. These properties should lead to: (i) mechanically stable scaffolds, that have the mechanical strength similar to the native tissue (ii) adjustable rheological properties (gelation, viscosity) to help in ease of bioprinting the constructs while retaining the desired structural shape (**Figure 3**); (iii) biocompatibility, biodegradability if required; (iv) not be cytotoxic to be suitable for in vivo studies and possible transplantation in future; and (v) large scale reproducibility of the ink [6]. Optimizing the bioink formulation is a vital step toward successful bioprinting and this is represented in **Figure 4** in the form of a flowchart. **Table 2** provides a few examples of different polymers that have been used in the formulation of bioinks.

## **4. 3D bioprinting for hard tissues**

### **4.1 Bone**

Bone is a complex tissue that has mechanical, metabolic, and hemopoietic functions. It provides structure and function to the surrounding tissues. Currently, bone defect repairs are treated by grafts: Autologous grafts, allografts, and synthetic grafts. Alternative methods like cadaver allografts and xenografts are available but they have poor biological properties like lower biocompatibility and risk of infection. Osteoconductive properties are seen in synthetic grafts but are degraded by osteoclasts and hence are suitable for small defect repairs only. Bone tissue engineering offers solutions to treat bone defects and one effective way is via 3D bioprinting. However, a major concern is to provide a solution that overcomes the challenges faced in conventional treatments by improving osteoinduction and osteoconduction. Studies have shown that 3D bioprinted bone constructs avoid the possibility of immune rejection which was



**Table 1.**

*List of commercially available 3D bioprinters.*

## *3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*

#### **Figure 2.**

*(A) Bioprinted scaffolds with cells in ink (B) cells seeded on scaffolds after bioprinting (C) properties to consider for the formulation of an ideal bioink.*

#### **Figure 3.**

*Ideal strength and degradation profile of a bioprinted scaffold.*

observed earlier in the use of grafts which may have otherwise led to inflammation, fibrosis, scarring, and transplant failure. The advantage of 3D bioprinting over the current grafting technique is that the cells are spatially distributed within the construct, thus optimizing tissue regeneration. 3D-bioprinted bone tissues have a huge impact

*3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*

*Flowchart depicts the process of bioink development.*

on clinical practice as it makes reconstruction of bone defects with complex shapes efficient and less time-consuming, by translating defect data from image modalities like CT to CAD designs which make it possible for patient-specific bioprinting. The ideal


#### **Table 2.**

*Polymers used in bioinks.*

scaffold should mimic the bone structure and composition, have a good resorption rate, allow for vascularization, and have a higher bone healing/formation ability compared to ceramics and metals. Fabrication of bone constructs with various geometries, porosity, and sizes, which are specific to each patient's features is possible via 3D bioprinting. It also helps to fabricate osteoinductive scaffolds [7].

Bioink is necessary to bioprint bone and it should have good mechanical strength without losing cell viability and bioactivity. Bioinks can be classified in three categories- (i) first generation – materials that are bioinert and biocompatible. Chances of rejection are minimized in this case. The scaffold remains in vivo to provide mechanical support and does not degrade, e.g., metals (stainless steel and titanium) and polymers (ii) second generation – materials that are biocompatible and bioactive simultaneously. They allow mineralization and biodegradation over time so that the cells can replace the scaffold. (iii) third generation – bioresponsive materials. They contain growth factors and stimulatory molecules that trigger osteoblast differentiation including bone morphogenetic proteins (BMP) and fibroblast growth factors (FGF). A composite bioink is most beneficial for use since it combines the best of all three generation of bioinks i.e., a balance between mechanical and functional properties is obtained to meet the needs of the desired tissue [8].

Bone regeneration requires osteoinductive cues which include growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF), bone

#### *3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*

morphogenetic proteins (BMPs), parathyroid hormone, and platelet-derived growth factor (PDGF). Growth factors are delivered in 3D-printed bone scaffolds during or after the printing process. The biochemical factors can be delivered on already printed scaffolds by adding them on the top surface after printing or within the micropores during printing. These osteogenic factors help in enhanced cell proliferation, differentiation, and angiogenesis. Bioinks were formulated using BMP-2 loaded PLGA nanoparticles, alginate, and mesenchymal stem cell (MSC). Composite bioink showed enhanced printability and yielded stable constructs post-printing. Sustained in vitro release for up to two weeks was observed in BMP2-loaded PLGA nanoparticles and was also noted to induce osteogenesis of the MSCs [9]. Bone healing is linked to the relationship between blood vessels and bone cells. It is known that VEGF is released during fracture healing. VEGF inhibition has been shown to interfere with fracture repairs and bone defects. However, it was not sufficient to heal large defects. Furthermore, it was observed that VEGF did not drive progenitor cells toward the chondrogenic or osteogenic lineage. Hence, combination therapies with BMPs are being developed to advance the regeneration of large bone defects. VEGF and BMP-2 were delivered to enhance the regeneration of large bone defects. The release of these growth factors was studied by 3D bioprinting alginate-based bioinks with nanoparticles. The bone formation was enhanced in vivo by slowing the release of BMP-2. Enhanced vascularization was found in vivo when VEGF was introduced in the study. Accelerated large bone defect healing was observed in this case using 3D-printed implants containing VEGF and BMP-2 [10].

Osteoinductive materials are preferred for studies in bone regeneration as they enhance regenerative properties. A study has shown that the NICE bioink (7.5% methacrylated gelatin, 1% kappa carrageenan, 2% nano silicates) showed both high print performance and enzymatic degradability. This bioink provided cell friendly microenvironment for bone bioprinting [11]. Jakus et al. developed a bioink composed of 90% HAP and 10% PCL or PLGA. It was found that the bioprinted scaffolds promoted osteogenic differentiation without additional biochemical factors. Higher biocompatibility, tissue integration, vascularization, and mineralization were observed in animal studies with no immune rejection [12].

Cell adhesion, viability, and metabolism are influenced by a scaffold's internal architecture like pore size and shape which in turn affect the bone regeneration capacity. A major challenge in developing a scaffold is to obtain a balance between the mechanical strength of the scaffold and mimicking the native strength of the tissue. A comparative study was done in the internal architecture of the scaffold- (i) continuous pattern (ii) zigzag-Spiral pattern, in treating bone defects treatments. It was found that the printed scaffolds showed characteristics like that of a native bone – permeability, porosity, and mechanical properties which are owed to the microarchitecture of the scaffold design. Human mesenchymal stem cells seeded scaffolds determined the effects of geometrical microstructure on cell attachment and morphology. The cells in the scaffold with a zigzag pattern infilled pores quickly in comparison with the other pattern [13]. **Tables 3** and **4** outline the latest developments in 3D bioprinting of bone in-vivo and in-vitro studies respectively.

## **4.2 Teeth**

Teeth are the hardest part of the human body and have limited capacity for repair and regeneration. The gold standard treatment for permanent tooth loss by disease, defect, or injury has been dental implants. Titanium alloys are widely used to


**Table 3.**

*3D bioprinting for bone: in vivo studies.*

#### *Advances in 3D Printing*

**220**

### *3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*


**Table 4.** *3D bioprinting for bone: in-vitro studies.*

manufacture dental implants. The tooth has multiple internal organs and its connection to surrounding tissues (periodontal ligament and alveolar bone) is important for its function. Hence, the entire tooth unit i.e., tooth root and the adjacent connecting tissues should be considered for tooth regeneration. The 3D printing technique is beneficial for tooth regeneration and building patient-specific supporting structures for teeth (e.g., dentures, dental implants, aligners etc). Dentistry applications of scaffolds are in the limelight of research with the aim of enhancing the regeneration of dental tissues. Bioprinting dental and periodontal tissues is a primary focus of research in dental regeneration [34].

In the native tooth, two mineralized tissues are present – dentin and enamel. Dentin provides strength and toughness while enamel is hard and resistant to both fracture and wear. Titanium alloys are commonly used implants, and they exceed the required strength and stiffness that is normally found in native teeth. This leads to alveolar bone resorption post-implantation. A study used collagen, agarose, and fibrin-based bioink to bioprint dental pulp. The study was successful in vascular tube formation at the root. Human dental pulp cells and Human primary umbilical vein endothelial cells were used in the bioink. Injecting the prepared bioink by the handheld bioprinter in-vitro showed vascularization and proved to be effective in comparison to filling up the canals with inert materials and sacrificing the tooth [35]. Yi-Ting Lin et al., developed calcium silicate-reinforced gelatin methacrylate bioink and bioprinted dental pulp stem cells along the scaffolds for dental regeneration. The release of silicon ions from the scaffolds contributed to enhanced regenerative properties by upregulating the expression of various odontogenic-related biomarkers. It was also found that these increased calcium mineralization. The developed bioink enhanced the mechanical properties of the scaffold and contributed to increased regenerative properties [36]. Jonghyeuk Han et al., developed a new Demineralized Dentin Matrix particle (DDMp) bio-ink. This bioink is composed of human tooth-derived DDMp, fibrinogen–gelatin mixture, and dental cells. It was found that the DDMp bio-ink improved odontogenic differentiation [37]. **Tables 5** and **6** outline the latest developments in 3D bioprinting of teeth in-vivo and in-vitro studies, respectively.

## **5. 3D bioprinting for liver**

Liver disorders like acute liver failure, chronic liver disease, liver fibrosis, viral hepatitis, and carcinoma have led to high mortality which requires liver transplantation [58]. 3D printing has been used as an alternative strategy to generate organs in vitro as being a shortage of organ donors [59]. 3D printed patient-specific liver models are being used and are showing great potential in disease treatment while the constructs having scaffolds and cells (bioprinted) are being used for the fabrication of liver tissue-like constructs and whole artificial livers [60].

It is very important to select the appropriate kind of cells and scaffold when considering 3D printing of liver tissues [61]. The viability of the hepatocytes reduces in vitro and there is a loss of hepatic phenotype [62]. Many studies have focused on liver regeneration using patient-specific functional cells and pluripotent stem cells. Valve-based inkjet bioprinting has been used by Faulker-jones et al. to print human induced pluripotent stem cells and human embryonic stem cells. The cells were able to differentiate into hepatocyte-like cells post-printing, and there were positive results for nuclear factor 4-alpha and albumin secretion. The cells were compatible to fabricate mini livers as drug testing models [63]. Primary


*3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*

**Table 5.**

 *3D bioprinting for teeth: in vivo studies.*


**Table 6.** *3D bioprinting for teeth: in-vitro studies.* rat hepatocytes, HUVECs, and human lung fibroblasts were bioprinted by Lee et al. using multiple nozzle-based extrusion printing. Collagen-based bioink was mixed with the cells and a 3D construct was fabricated by infusing the bioink into PCL framework. There was enhanced survival and functionality of the HCs in the printed liver construct due to the 3D environment-induced interaction among cells. This study showed potential in the liver tissue regeneration field for the capillarylike networked 3D constructs [64].

Robbins et al., used iPSC-derived HLCs, and endothelial and hepatic stellate cells to fabricate highly reproducible 3D liver constructs. These constructs had high viability, multi-layered architecture, tissue-like cell density. There was improved reproducibility, durability, and biological complexity in a study conducted by Nguyen et al. the liver constructs were able to show more biological functions including storing lipids and glycogens and retaining their viability, and compartmentalized structure. Kim et al., used an alginate scaffold and primary mouse hepatocytes to fabricate liver constructs [65]. The cells were viable for 14 days and there was an increase in albumin, HNF-α. Zhong et al., fabricated 3D-printed hydrogel and they were implanted in mice in different groups acting as a control, hydrogel, hydrogel with cells, and hydrogel with hepatocyte growth factor. The viability of the cells was not affected by the hydrogel. The group implanted with cells showed significant improvement in levels of albumin, bilirubin, and the group with HGF, had the longest survival time [66].

## **6. 3D bioprinting for tubular organs**

Tubular organs like Esophagus, blood vessels, urethra, etc. are very prone to infection and can be treated via surgery, stent insertion, and organ transplant that is dependent on suitable donors and autologous organs [67]. Tissue engineering has emerged as an alternative approach for developing grafts and scaffolds.

## **6.1 Blood vessels**

Vascular systems are the most common tissue-engineered structures in the body. Development and discoveries have happened in the past years toward the fabrication of vascular networks in all organ systems. An arterial scaffold consisting of three layers of polydioxanone, fibrin, and gelatin was fabricated by Thomas et al. [68]. The Polydioxanone (PDS) layer provided mechanical integrity and the protein layers had a similar functional extracellular matrix as blood vessels. Nguyen et al., fabricated a tubular scaffold made up of PCL/PU using electrospinning for artificial blood vessels, which demonstrated improved cell attachment and proliferation [69].

## **6.2 Trachea**

Tracheal disorders are rare but still life-threatening like tracheal stenosis and narrowing, and such disorders require immediate treatment. 3D printing of trachea constructs is gaining popularity in the field of regenerative medicine [70]. 3D bioprint tracheal constructs were fabricated by Taniguhi et al. using chondrocytes and mesenchymal cells [71]. Spheroids were fabricated and matured in a bioreactor; then as tracheal grafts transplanted in rats. Silicone stents were used as a framework to provide support and prevent collapsing of the stent. Vascular and epithelium networks

were observed over the grafts thus successfully making a way in tracheal engineering. Goa et al., fabricated a porous PCL construct that would mimic the native trachea of rabbits [72]. The graft was cytocompatible as it was observed when seeded with chondrocytes. There was successful formation of cartilage tissue in the subcutaneous spaces of the mice. Later, it was transplanted into the rabbit, and the survival time was observed as 10 weeks. The fabricated scaffolds can be used for tracheal replacement therapies and for repairing whole-segment tracheal defects.

## **6.3 Excretory organs**

Many organs in the excretory systems are hollow and tubular in morphology including Bowman's capsule, tubules in renal nephron urethra, etc. 3D printing has been utilized and applied in the fabrication of tissues and organs in this organ system. Zhang et al., fabricated cell-laden urethra using PCL and poly (lactide-co-εcaprolactone) (PLCL) polymers having spiral scaffold design that could mimic the native properties of the urethra of rabbits [73]. Urothelial cells and smooth muscle cells of the bladder were added to the hydrogel comprising gelatin, Dulbecco's Modified Eagle Medium (DMEM) and hyaluronic acid, and the cell-laden hydrogel was fabricated. The urethra was 3D printed by adding PCL/PLCL polymers blend in one syringe and cell-laden hydrogels in another. The polymers provided with the structural framework and the cell-laden hydrogels contributed to mimic the microenvironment. It was observed that the scaffold had the mechanical properties equivalent to native rabbit urethra and the hydrogel was able to maintain a suitable microenvironment and the results set up a strong foundation for future studies on 3d bioprinting of urethra. Pi et al., used a coaxial extrusion-based printing technique to fabricate complex tubular hollow fibers which were made up of blend bioink consisting of PEG, and GelMA/alginate hydrogel [74]. The main objective of this study was to avoid the pre/post-processing step as the coaxial nozzle allows the printing of multiple layers in one step. The team was successfully able to print cannular urothelial tissue constructs using human urothelial cells and human bladder smooth muscle cells. This kind of fabrication is a fundamental step toward creating human cannular tissues.

## **6.4 Gastrointestinal tract**

The esophagus is the tubular tube connecting throat to stomach. Many congenital and acquired disorders of GI tract have only esophageal replacement as the treatment option. 3D printed scaffolds are being considered to repair damaged esophagus. Esophageal reconstruction has been done using resorbable materials, acellular matrices, decellularized patches, and implants of synthetic polymers [70]. Pisani et al., fabricated a biodegradable patch using PLA-PCL polymer. Two different techniques- electrospinning and temperature-induced precipitation were used to develop the cellularized patch. The protocol was repeatable, reproducible, and simple [75]. Haghdel et al., fabricated a flexible esophageal stent to treat esophageal strictures using PLA, polyurethane, and Polyvinyl alcohol (PVA) [76]. The stent was assessed in vitro and in vivo canine esophagus. The stent was implanted in a 16-year-old boy who had esophageal stricture, and it was observed for 2 months. No major inflammatory effects and cytotoxicity were observed, and the mechanical tests revealed that the nature and behavior did not change significantly. This biocompatible polymeric stent can be used as an individualized treatment for treating esophageal structures.

## **7. Commercial 3D bioprinted products**

Manufacturing companies have been using 3D printing for years, mostly to create product prototypes. Models and molds are produced by several manufacturers using huge and quick 3D printers referred to as "rapid prototyping machines" [77]. There are many.stl files that may be used for business. Many of these printed goods are comparable to those that are made traditionally [78]. There are now businesses that employ 3D printing for industrial medical purposes [79]. These include Organovo, Helisys, and Ultimateker, a business that creates living human tissue through 3D printing. The use of 3D printing in medicine, however, is still relatively new. The market value of 3D printing is \$700 million out of which only 1.6% of it is devoted to medical uses. If we look at the numbers, it is anticipated that the market value of 3D printing will expand to be a sector of \$8.9 billion in the next 10 years out of which 21% of it is estimated to go toward its usage in medical applications [78].

The democratization of product design and production is another advantageous aspect of 3D printing [80].

A significant shift has been made in the manner hearing aids are made, currently 99% of hearing aids that fit in the human ear are fabricated via 3D printers. Every individual has a unique ear canal shape, and 3D printers make it possible to build custom-shaped devices quickly and affordably [81]. Another productive commercial use of 3D printing is the production of 50,000 Invisalign braces per day. Each user's set of these transparent, removable, 3D-printed orthodontic braces is unique and is created to order. This item serves as an excellent illustration of how 3D printing can be utilized effectively and commercially to create unique, personalized, complex items [80].

In 2010, Organovo made its first noteworthy business using just primary human cells to successfully bioprint entirely functional blood arteries. The year 2014 saw the introduction of Organovo's ExVive™ 3D bioprinted human liver tissue models. There were histological and functional resemblances to the natural liver, and albumin, ATP, and CYP3A4 activity are consistently expressed for up to 28 days. Drugs like Valproic acid and Monocrotaline have their therapeutic effects demonstrated using tissue models [79].

Organovo released ExVive™ Human Kidney Tissue in 2016, a complete threedimensional bioprinted human tissue made of primary renal fibroblasts and endothelial cells at the tubule-interstitial interface, which is rich in collagen IV, and polarized primary renal proximal tubule epithelial cells (RPTECs) in the apical layer [77]. ExVive™ Human Kidney Tissue displays in vivo-like renal transporter expression, barrier function, and the production of the crucial enzyme gamma glutamyl transferase (GGT). When subjected to the chemical Cisplatin, this bioprinted kidney tissue produces kidney damage indicators and shows transporter-dependent (OCT2) drug uptake [82]. The world's first animal thyroid gland was successfully 3D printed by 3D Bioprinting Solutions (3dbio) in March 2015 and then implanted into the mouse when it was alive. In addition, to create artificial tissues in the International Space Station using a magnetic 3D bioprinter, 3dbio has been collaborating with Russia's national space agency, United Rocket and Space Corporation (URSC). The company hopes to fabricate synthetic thyroid and kidney tissue using this technology [83].

The most recent RX1TM bioprinting from Aspect Biosystems makes use of their exclusive Lab-on-a-Printer™ microfluidic technology. Contains a coaxial flowfocusing system that guarantees the direct extrusion of biological fibers in a range

of diameters. The device was utilized to show how to fabricate the 3DBioRingTM artificial airway. Primary human airway smooth muscle cells make up contractile smooth muscle tissue that lines the airway. When histamine is present, the airway tissue responds with proper and repeatable contractions, and when pharmacological stimuli are present, it dilates (B2-agonist) [84].

A new biotech company called BIOLIFE4D was established in 2015. The business hopes to 3D bioprint patient-specific, perfectly operational hearts for secure and reasonably priced organ transplantation. They are a strong group of biomedical researchers and businesspeople that are now supporting their research through equity crowdfunding. The goal of the BIOLIFE4D technique is to 3D bioprint a human heart using adult induced pluripotent stem cells (iPSCs), following a complete MRI (Magnetic Resonance Imaging) scan to determine the precise dimensions needed for its production [85].

Poietis makes use of INSERM and the University of Bordeaux technology. The business focuses on D laser-assisted bioprinting technology and collaborates with BASF and L'Oréal to develop bioprinted skin models and hair follicles, respectively [86]. Their NGB 17.03 bioprinting machine, which has an eight-axis motion, can print 3D models down to the level of a single cell. Early in 2018, Poietis introduced the first bioprinted human full-skin model made with their NGB bioprinter, called Poieskin® [87].

In collaboration with scientists at Sichuan University's West China Hospital, Revotek has had success implanting 3D-printed arteries within simian test subjects. In 30 rhesus monkeys, a replacement of a 2-centimeter portion of the abdominal artery was done with a 3D-printed blood conduit, and the stem cell bioink was created using the monkeys' own autologous adipose mesenchymal stem cells (ADSCs) [88]. Using a print head with two nozzles, the printer can presently manufacture 10-centimeter blood arteries in about two minutes [89].

TeVido biodevices make use of patented Clemson University technology. TeVido's initial product is a bioprinted nippular-areola implant for breast reconstructive surgery. In two to three years, clinical trials are expected to begin. The second product from TeVido is intended for Vitiligo sufferers who desire to print skin tissues to lessen the contrast in colors [90].

## **8. Advancements in 4D bioprinting**

The fourth dimension (4D) which is 4D printing incorporates time, and it is an improved production method based on 3D printing. With this technique, external stimulation can cause the printed constructions to alter form over time. 4D bioprinting refers to the recent expansion of 4D printing to include the printing of complex constructions from biocompatible materials or even live cells. If one of the following criteria is met, 4D printing can be referred to as 4D bioprinting. 1) Biomedical engineering may make use of printed products, such as biomedical gadgets. 2) The printed materials are transplantable into the human body and are biocompatible. 3) The printed materials are loaded with living cells. When using 4D bioprinting, the bio constructs size, form, and/or functionality might vary over time [91].

The benefits that 4D printing has over 3D printing might prove to be the necessary proof of concept and accelerate wider adoption. More precisely, 4D printing enables the implementation of micro or nano actuators by providing sensation, knowledge of the movement, and programmability embedded into the material

#### *3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*

without any requirement for an external source or system like the batteries, wires, engines etc. Additional advantages of these systems include decreased installation time, expense, human effort, mistakes, storage, and the number of components in a prototype or system.

There have been reports of 4D printing applications in several industries, including medicinal devices, security, the creation of precisely patterned surfaces for optics, electrical devices, constructions with multidirectional capabilities, and soft actuators. Recent years have seen a huge increase in the popularity of soft robotics, which attempts to emulate biology by building flexible and rigid controlled objects, notably for the medical industry. Researchers have recently been more interested in the usage of Shape memory alloys and electroactive polymers which are the materials that change their shape and size according to the temperature and electric field respectively [92], pressurized fluid or gas-operable elastomers, chemical stimuli, and light-sensitive materials with a focus on soft robotics and the biomedical area. As a result, many new opportunities and chances are anticipated to materialize soon as the development of 4D printing technology would open several new possibilities [93].

Zhang et al. modified cellulose with stearoyl moieties to create a material that responds to moisture. They created a film out of this material that, when exposed to an environment with a moisture gradient, would bend because of the non-uniform absorption of moisture [94]. To expand its biological uses or improve the control of printing accuracy, certain novel techniques are emerging. In certain ways, recent advances in 4D bioprinting have resolved issues that were once seen as obstacles, such as the development of microscale vascular models and medication delivery systems for the stomach and muscular actuators. Now that 4D bioprinting is more understood, it has drawn a lot of attention to the research of tissue regeneration and biomedical devices [95]. The fact that 4D bioprinting can better suit the physiological aspects of the body is now widely acknowledged by experts. Instead of being in a static environment like 3D printing, the 4D bioprinted devices may integrate dynamic modification. It has been demonstrated that 4D bioprinting has enormous potential to change tissue engineering, medication delivery, and other sectors. It offers up a new path for bio fabrication. We have a thorough grasp of the biomedical area thanks to the innovative features of 4D bioprinting, not only in terms of tissue and organ regeneration but also in terms of illness therapy. It totally advances the idea of biomedicine while innovating traditional industrial techniques. The tissues in the human body are exceedingly malleable, non-static, and have special roles that are ideal for dynamic alterations. Conventional 3D-printed objects may have certain forms, topologies, or cells, but they are unable to demonstrate dynamic processes. Given this, 4D bioprinting effectively satisfies the need for biomedicine. To the greatest degree possible, 4D bioprinting aims to emulate biological functions in vivo. The bodily reaction cues that trigger the shift should be secure and simple to manage [96].

## **9. Conclusion**

In this chapter, we have reviewed the basics of 3D printing, the various types of bioprinters available like droplet-based, extrusion-based, light-based bioprinters. There are many commercially available bioprinters discussed, developed by various companies fulfilling the requisites of bioprinting. Bioinks are the core part of bioprinting, and it is important to formulate them properly to get the constructs that can be stable, biodegradable, biocompatible, and able to mimic the native

microenvironment of the tissue. Numerous studies have been mentioned where 3D printing has been used to fabricate bone grafts, dental implants, liver disease models, liver tissue constructs, vascular structures, tracheal constructs, cell-laden urethra, and esophageal stent. Some of the mentionable commercially available 3D printed products available include ExVive™ 3D bioprinted human liver tissue model, ExVive™ human kidney tissue, animal thyroid gland by 3dbio, etc. The future of 3D printing is 4D printing which utilizes time as a fourth dimension. The smart materials used for 4D printing change the shape and size under the influence of an external stimulus. 4D printing will open several new possibilities in the field of biomedicine.

## **10. Future scope**

3D printing is the latest technology creating a buzz in all fields including artificial intelligence, advanced simulations, biomedicine, and engineering. The scope embraces objects like human organs, aircraft components, and much more. The technique is being widely accepted due to the several advantages its offers including patient-specific design, high complexity, cost-effective fabrication, and high productivity. The possible uses of 3D printing are endless now, from decreasing the cost of health care to the construction of houses. The cost of production of the prosthetic limb has been reduced to 75% by using 3D printing by the company Mercuris. Rice university has developed a 3D bioprinter that can print narrow blood vessels and which led to developing lung model. 3D printing is being explored by various researchers but now many are working around 4D printing as it is the upcoming technique that is beginning to establish. There are still many challenges and hurdles that must be addressed including the lack of multi-material printers, lack of low-cost printers, and smart materials. The area is still new and unexplored.

## **Acknowledgements**

The authors wish to acknowledge the Department of Science and Technology-Science Engineering Research Board, Early Career Research Award (ECR/2018/000709) and Indian Council for Medical Research (ICMR Adhoc -5/3/8/53/2020 – ITR) for funding the research.

## **Conflict of interest**

The authors declare no conflict of interest.

*3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*

## **Author details**

Meghana Kasturi1 , Vidhi Mathur1 , Prachi Agarwal1 , Varadharajan Srinivasan<sup>2</sup> and Kirthanashri S. Vasanthan1 \*

1 Manipal Centre for Biotherapeutics Research, Manipal Academy of Higher Education, Manipal, Karnataka, India

2 Department of Civil Engineering, JSS Academy of Higher Education, Noida, Uttar Pradesh, India

\*Address all correspondence to: kirthanashri.sv@manipal.edu; kirthanasv@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Mhanna R, Hasan A. Introduction to tissue engineering. In: Tissue Engineering for Artificial Organs: Regenerative Medicine, Smart Diagnostics and Personalized Medicine. Vol. 1. Germany. 8 May 2017. pp. 1-34

[2] Magnetic Levitation Aids 3D Bioprinting [Internet]. Engineering.com. Available from: https://www.engineering. com/story/magnetic-levitation-aids-3d-bioprinting. [Accessed: October 10, 2022]

[3] \$3.26 Billion 3D Bioprinting Market, 2027 by Technology (Inkjet, Magnetic Levitation, Laser-assisted), Component (3D Bioprinters, Biomaterials, Scaffolds), Material, Application-ResearchAndMarkets. com [Internet]. 2021. Available from: https://www.businesswire. com/news/home/20210422005702/ en/3.26-Billion-3D-Bioprinting-Market-2027-by-Technology-Inkjet-Magnetic-Levitation-Laser-assisted-Component-3D-Bioprinters-Biomaterials-Scaffolds-Material-Application--- ResearchAndMarkets.com. [Accessed: October 10, 2022]

[4] 3D Bioprinting Market Size & Share Report, 2022-2030 [Internet]. Available from: https://www.grandviewresearch. com/industry-analysis/3d-bioprintingmarket. [Accessed: October 10, 2022]

[5] Groll J, Burdick JA, Cho DW, Derby B, Gelinsky M, Heilshorn SC, et al. A definition of bioinks and their distinction from biomaterial inks. Biofabrication. 2018;**11**(1):013001

[6] Gungor-Ozkerim PS, Inci I, Zhang YS, Khademhosseini A, Dokmeci MR. Bioinks for 3D bioprinting: An overview. Biomaterials Science. 2018;**6**(5):915-946

[7] Genova T, Roato I, Carossa M, Motta C, Cavagnetto D, Mussano F. Advances on bone substitutes through 3D bioprinting. International Journal of Molecular Sciences. 2020;**21**(19):7012

[8] Salah M, Tayebi L, Moharamzadeh K, Naini FB. Three-dimensional bioprinting and bone tissue engineering: Technical innovations and potential applications in maxillofacial reconstructive surgery. Maxillofacial Plastic and Reconstructive Surgery. 2020;**42**(1):18

[9] Choe G, Lee M, Oh S, Seok JM, Kim J, Im S, et al. Three-dimensional bioprinting of mesenchymal stem cells using an osteoinductive bioink containing alginate and BMP-2-loaded PLGA nanoparticles for bone tissue engineering. Biomaterial Advanced. 2022;**136**:212789

[10] Freeman FE, Pitacco P, van Dommelen LHA, Nulty J, Browe DC, Shin JY, et al. 3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration. Science Advances. 2020;**6**(33):eabb5093

[11] Chimene D, Miller L, Cross LM, Jaiswal MK, Singh I, Gaharwar AK. Nanoengineered Osteoinductive bioink for 3D bioprinting bone tissue. ACS Applied Materials & Interfaces. 2020;**12**(14):15976-15988

[12] Jakus AE, Rutz AL, Jordan SW, Kannan A, Mitchell SM, Yun C, et al. Hyperelastic "bone": A highly versatile, growth factor–free, osteoregenerative, scalable, and surgically friendly biomaterial. Science Translational Medicine. 2016;**8**(358):358ra127-358ra127

## *3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*

[13] Fallah A, Altunbek M, Bartolo P, Cooper G, Weightman A, Blunn G, et al. 3D printed scaffold design for bone defects with improved mechanical and biological properties. Journal of the Mechanical Behavior of Biomedical Materials. 2022;**134**:105418

[14] Nulty J, Freeman FE, Browe DC, Burdis R, Ahern DP, Pitacco P, et al. 3D bioprinting of prevascularised implants for the repair of critically-sized bone defects. Acta Biomaterialia. 2021;**126**:154-169

[15] Shokouhimehr M, Theus AS, Kamalakar A, Ning L, Cao C, Tomov ML, et al. 3D bioprinted bacteriostatic Hyperelastic bone scaffold for damagespecific bone regeneration. Polymers. 2021;**13**(7):1099

[16] Calcium silicate nanowirescontaining multicellular bioinks for 3D bioprinting of neural-bone constructs - ScienceDirect [Internet]. Available from: https://www. sciencedirect.com/science/article/abs/ pii/S1748013222002122?via%3Dihub [Accessed: November 13, 2022]

[17] Zhang X, Liu Y, Zuo Q, Wang Q, Li Z, Yan K, et al. 3D bioprinting of biomimetic Bilayered scaffold consisting of Decellularized extracellular matrix and silk fibroin for Osteochondral repair. International Journal of Bioprinting. 2021;**7**(4):401

[18] Korn P, Ahlfeld T, Lahmeyer F, Kilian D, Sembdner P, Stelzer R, et al. 3D printing of bone grafts for cleft alveolar Osteoplasty–In vivo evaluation in a preclinical model. Frontiers in Bioengineering and Biotechnology. 2020;**8**:217

[19] Wang C, Lai J, Li K, Zhu S, Lu B, Liu J, et al. Cryogenic 3D printing of dual-delivery scaffolds for improved

bone regeneration with enhanced vascularization. Bioactive Material. 2021;**6**(1):137-145

[20] 3DBioprinted Integrated Osteochondral Scaffold-Mediated Repair of Articular Cartilage Defects in the Rabbit Knee, SpringerLink. Available from: https://link.springer.com/ article/10.1007/s40846-019-00481-y

[21] Long Zhisheng XL. Effect of artificial bone with multi-scale hydroxyapatite/ chitosan microtubule structure on rabbit bone defect repair and angiogenesis. Chinese Journal of Tissue Engineering Research. 2022;**26**(34):5436

[22] Zhao L, Luo Y, Wang Y, Zhao F, Chen X, Cai D. Three-dimensional printed BGS treat a large bone defect in a rabbit model. Doklady. Biochemistry and Biophysics. 2021;**497**(1):123-129

[23] Sun X, Ma Z, Zhao X, Jin W, Zhang C, Ma J, et al. Three-dimensional bioprinting of multicell-laden scaffolds containing bone morphogenic protein-4 for promoting M2 macrophage polarization and accelerating bone defect repair in diabetes mellitus. Bioactive Material. 2021;**6**(3):757-769

[24] Chiesa I, Maria CD, Lapomarda A, Fortunato GM, Montemurro F, Gesù RD, et al. Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting. Biofabrication. 2020;**12**(2):025013

[25] Zhang J, Eyisoylu H, Qin XH, Rubert M, Müller R. 3D bioprinting of graphene oxide-incorporated cellladen bone mimicking scaffolds for promoting scaffold fidelity, osteogenic differentiation and mineralization. Acta Biomaterialia. 2021;**121**:637-652

[26] Daly AC, Cunniffe GM, Sathy BN, Jeon O, Alsberg E, Kelly DJ. 3D bioprinting of developmentally inspired templates for whole bone organ engineering. Advanced Healthcare Materials. 2016;**5**(18):2353-2362

[27] Wei M, Quinnell SP, Bendtsen ST. Development of a novel alginatepolyvinyl alcohol-hydroxyapatite hydrogel for 3D bioprinting bone tissue engineered scaffolds. Journal of Biomedical Materials Research. 2017;**105**:1457-1468

[28] Murphy C, Kolan K, Li W, Semon J, Day D, Leu M. 3D bioprinting of stem cells and polymer/bioactive glass composite scaffolds for bone tissue engineering. International Journal of Bioprinting. 2017;**3**(1):005

[29] Ojansivu M, Rashad A, Ahlinder A, Massera J, Mishra A, Syverud K, et al. Wood-based nanocellulose and bioactive glass modified gelatin–alginate bioinks for 3D bioprinting of bone cells. Biofabrication. 2019;**11**(3):035010

[30] Kondiah PJ. A 3D bioprinted pseudo-bone drug delivery scaffold for bone tissue engineering. Pharmaceutics. 2020;**12**:166

[31] Anada T, Pan CC, Stahl AM, Mori S, Fukuda J, Suzuki O, et al. Vascularized bone-mimetic hydrogel constructs by 3D bioprinting to promote Osteogenesis and angiogenesis. International Journal of Molecular Sciences. 2019;**20**(5):1096

[32] Park J, Lee SJ, Jung TG, Lee JH, Kim WD, Lee JY, et al. Surface modification of a three-dimensional polycaprolactone scaffold by polydopamine, biomineralization, and BMP-2 immobilization for potential bone tissue applications. Colloids and Surfaces. B, Biointerfaces. 2021;**199**:111528

[33] Rajput M, Mondal P, Yadav P, Chatterjee K. Light-based 3D bioprinting of bone tissue scaffolds with tunable mechanical properties and architecture from photocurable silk fibroin. International Journal of Biological Macromolecules. 2022;**202**:644-656

[34] Morrison DG, Tomlinson RE. Leveraging advancements in tissue engineering for bioprinting dental tissues. Bioprinting. 2021;**23**:e00153

[35] Duarte Campos DF, Zhang S, Kreimendahl F, Köpf M, Fischer H, Vogt M, et al. Hand-held bioprinting for de novo vascular formation applicable to dental pulp regeneration. Connective Tissue Research. 2020;**61**(2):205-215

[36] Lin YT, Hsu TT, Liu YW, Kao CT, Huang TH. Bidirectional differentiation of human-derived stem cells induced by biomimetic calcium silicate-reinforced gelatin methacrylate bioink for odontogenic regeneration. Biomedicine. 2021;**9**(8):929

[37] Han J, Jeong W, Kim MK, Nam SH, Park EK, Kang HW. Demineralized dentin matrix particle-based bio-ink for patient-specific shaped 3D dental tissue regeneration. Polymers. 2021;**13**(8):1294

[38] Kim K, Lee CH, Kim BK, Mao JJ. Anatomically shaped tooth and periodontal regeneration by cell homing. Journal of Dental Research. 2010;**89**(8):842-847

[39] Gong H, Zhao Y, Chen Q, Wang Y, Zhao H, Zhong J, et al. 3D bio-printing of photocrosslinked anatomically tooth-shaped scaffolds for alveolar ridge preservation after tooth extraction. Journal of Materials Chemistry B. 2022;**10**(41):8502-8513

[40] Tang H, Bi F, Chen G, Zhang S, Huang Y, Chen J, et al. 3D-bioprinted recombination structure of Hertwig's epithelial root sheath cells and

## *3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*

dental papilla cells for alveolar bone regeneration. International Journal of Bioprinting. 2022;**8**(3):512

[41] Feng S, Liu J, Ramalingam M. 3D printing of stem cell responsive Ionically-Crosslinked polyethylene glycol Diacrylate/alginate composite hydrogels loaded with basic fibroblast growth factor for dental pulp tissue engineering: A preclinical evaluation in animal model. Journal of Biomaterial and Tissue Engineering. 2019;**9**(12):1635-1643

[42] Chen RS, Hsu SH, Chang HH, Chen MH. Challenge tooth regeneration in adult dogs with dental pulp stem cells on 3D-printed hydroxyapatite/Polylactic acid scaffolds. Cell. 2021;**10**(12):3277

[43] Yi K, Li Q, Lian X, Wang Y, Tang Z. Utilizing 3D bioprinted platelet-rich fibrin-based materials to promote the regeneration of oral soft tissue. Regenerative Biomaterial. 2022;**9**:rbac021

[44] Park CH, Rios HF, Jin Q, Bland ME, Flanagan CL, Hollister SJ, et al. Biomimetic hybrid scaffolds for engineering human tooth-ligament interfaces. Biomaterials. 2010;**31**(23):5945-5952

[45] Park CH et al. Image-based, fiber guiding scaffolds: A platform for regenerating tissue interfaces. Tissue Engineering Part C: Methods. 2013;**20**:533-542

[46] Lee CH et al. Three-dimensional printed multiphase scaffolds for regeneration of periodontium complex. Tissue Engineering Part A. 2014;**20**:1342-1351

[47] Park CH, Rios HF, Jin Q, Sugai JV, Padial-Molina M, Taut AD, et al. Tissue engineering bone-ligament complexes using fiber-guiding scaffolds. Biomaterials. 2012;**33**(1):137-145

[48] Cresswell Boyes AJ et al. Composite 3D printing of biomimetic human teeth. Scientific Reports. 2022;**12**:7830

[49] Han J, Kim DS, Jang H, Kim H-R, Kang H-W. Bioprinting of threedimensional dentin–pulp complex with local differentiation of human dental pulp stem cells. Journal of Tissue Engineering. 2019:3-7

[50] Mousavi Nejad Z, Zamanian A, Saeidifar M, Vanaei HR, Salar AM. 3D bioprinting of Polycaprolactone-based scaffolds for pulp-dentin regeneration: Investigation of physicochemical and biological behavior. Polymers. 2021;**13**(24):4442

[51] Park JH, Gillispie GJ, Copus JS, Zhang W, Atala A, Yoo JJ, et al. The effect of BMP-mimetic peptide tethering bioinks on the differentiation of dental pulp stem cells (DPSCs) in 3D bioprinted dental constructs. Biofabrication. 2020;**12**(3):035029

[52] Vurat MT, Şeker Ş, Lalegül-Ülker Ö, Parmaksiz M, Elçin AE, Elçin YM. Development of a multicellular 3D-bioprinted microtissue model of human periodontal ligament-alveolar bone biointerface: Towards a preclinical model of periodontal diseases and personalized periodontal tissue engineering. Genes and Diseases. 2022;**9**(4):1008-1023

[53] Athirasala A, Tahayeri A, Thrivikraman G, França CM, Monteiro N, Tran V, et al. A dentinderived hydrogel bioink for 3D bioprinting of cell laden scaffolds for regenerative dentistry. Biofabrication. 2018;**10**(2):024101

[54] Dutta SD, Bin J, Ganguly K, Patel DK, Lim KT. Electromagnetic field-assisted cell-laden 3D printed poloxamer-407 hydrogel for enhanced osteogenesis. RSC Advances. 2021;**11**(33):20342-20354

[55] Zhang C, Chen Z, Liu J, Wu M, Yang J, Zhu Y, et al. 3D-printed pretapped-hole scaffolds facilitate one-step surgery of predictable alveolar bone augmentation and simultaneous dental implantation. Composites. Part B, Engineering. 2022;**229**:109461

[56] Zhang S et al. Three-dimensional cell printed lock-key structure for Oral soft and hard tissue regeneration. Tissue Engineering Part A. 2022;**28**:13-26

[57] Thattaruparambil Raveendran N, Vaquette C, Meinert C, Samuel Ipe D, Ivanovski S. Optimization of 3D bioprinting of periodontal ligament cells. Dental Materials. 2019;**35**(12):1683-1694

[58] Picon RV, Bertol FS, Tovo CV, de Mattos ÂZ. Chronic liver failureconsortium acute-on-chronic liver failure and acute decompensation scores predict mortality in Brazilian cirrhotic patients. World Journal of Gastroenterology. 2017;**23**(28):5237

[59] Tanaka K. Resection versus transplantation for hepatocellular carcinoma exceeding Milan criteria within increasing donor shortage. Hepatobiliary Surgery and Nutrition. 2017;**6**(4):280

[60] Mitsouras D, Liacouras P, Imanzadeh A, Giannopoulos AA, Cai T, Kumamaru KK, et al. Medical 3D printing for the radiologist. Radiographics. 2015;**35**(7):1965

[61] Lewis PL, Green RM, Shah RN. 3D-printed gelatin scaffolds of differing pore geometry modulate hepatocyte function and gene expression. Acta Biomaterialia. 2018;**69**:63-70

[62] Wang JZ, Xiong NY, Zhao LZ, Hu JT, Kong DC, Yuan JY. Review fantastic

medical implications of 3D-printing in liver surgeries, liver regeneration, liver transplantation and drug hepatotoxicity testing: A review. International journal of surgery. 2018;**56**:1-6

[63] Faulkner-Jones A, Fyfe C, Cornelissen DJ, Gardner J, King J, Courtney A, et al. Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D. Biofabrication. 2015;**7**(4):044102

[64] Cho JW, Choi YJ, Yong WJ, Pati F, Shim JH, Kang KS, et al. Development of a 3D cell printed construct considering angiogenesis for liver tissue engineering. Biofabrication. 2016;**8**(1):015007

[65] Kim Y, Kang K, Jeong J, Paik SS, Kim JS, Park SA, et al. Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure. Annals of surgical treatment and research. 2017;**92**(2):67-72

[66] Zhong C, Xie HY, Zhou L, Xu X, Zheng SS. Human hepatocytes loaded in 3D bioprinting generate mini-liver. Hepatobiliary & Pancreatic Diseases International. 2016;**15**(5):512-518

[67] Góra A, Pliszka D, Mukherjee S, Ramakrishna S. Tubular tissues and organs of human body—Challenges in regenerative medicine. Journal of Nanoscience and Nanotechnology. 2016;**16**(1):19-39

[68] Thomas V, Zhang X, Vohra YK. A biomimetic tubular scaffold with spatially designed nanofibers of protein/ PDS® bio-blends. Biotechnology and Bioengineering. 2009;**104**(5):1025-1033

[69] Nguyen TH, Padalhin AR, Seo HS, Lee BT. A hybrid electrospun PU/PCL scaffold satisfied the requirements of blood vessel prosthesis in terms

## *3D Printing for Tissue Regeneration DOI: http://dx.doi.org/10.5772/intechopen.109141*

of mechanical properties, pore size, and biocompatibility. Journal of Biomaterials Science, Polymer Edition. 2013;**24**(14):1692-1706

[70] Farhat W, Chatelain F, Marret A, Faivre L, Arakelian L, Cattan P, et al. Trends in 3D bioprinting for esophageal tissue repair and reconstruction. Biomaterials. 2021;**267**:120465

[71] Taniguchi D, Matsumoto K, Tsuchiya T, Machino R, Takeoka Y, Elgalad A, et al. Scaffold-free trachea regeneration by tissue engineering with bio-3D printing. Interactive cardiovascular and thoracic surgery. 2018;**26**(5):745-752

[72] Gao M, Zhang H, Dong W, Bai J, Gao B, Xia D, et al. Tissue-engineered trachea from a 3D-printed scaffold enhances whole-segment tracheal repair. Scientific Reports. 2017;**7**(1):1-2

[73] Zhang K, Fu Q, Yoo J, Chen X, Chandra P, Mo X, et al. 3D bioprinting of urethra with PCL/PLCL blend and dual autologous cells in fibrin hydrogel: An in vitro evaluation of biomimetic mechanical property and cell growth environment. Acta Biomaterialia. 2017;**50**:154-164

[74] Pi Q, Maharjan S, Yan X, Liu X, Singh B, van Genderen AM, et al. Digitally tunable microfluidic bioprinting of multilayered cannular tissues. Advanced Materials. 2018;**30**(43):1706913

[75] Pisani S, Dorati R, Conti B, Modena T, Bruni G, Genta I. Design of copolymer PLA-PCL electrospun matrix for biomedical applications. Reactive and Functional Polymers. 2018;**124**:77-89

[76] Haghdel M, Alizadeh AA, Ghasemi Y, Hosseinpour H, Foroutan H, Shahriarirad S, et al. Utilization of 3D-printed polymer stents for benign esophageal strictures in patients with caustic ingestion. Journal of 3D Printing in Medicine. Mar 2021;**5**(1):11-21

[77] Hoy MB. 3D printing: Making things at the library. Medical reference services quarterly. 2013;**32**(1):93-99

[78] Schubert C, Van Langeveld MC, Donoso LA. Innovations in 3D printing: A 3D overview from optics to organs. British Journal of Ophthalmology. 2014;**98**(2):159-161

[79] Klein GT, Lu Y, Wang MY. 3D printing and neurosurgery--ready for prime time? World Neurosurgery. 2013;**80**(3-4):233-235

[80] Mertz L. Dream it, design it, print it in 3-D: What can 3-D printing do for you? IEEE Pulse. 2013;**4**(6):15-21

[81] Banks J. Adding value in additive manufacturing: Researchers in the United Kingdom and Europe look to 3D printing for customization. IEEE Pulse. 2013;**4**(6):22-26

[82] King SM, Higgins JW, Nino CR, Smith TR, Paffenroth EH, Fairbairn CE, et al. 3D proximal tubule tissues recapitulate key aspects of renal physiology to enable nephrotoxicity testing. Frontiers in Physiology. 2017;**8**:123

[83] Choudhury D, Anand S, Naing MW. The arrival of commercial bioprinters– towards 3D bioprinting revolution! International Journal of Bioprinting. 2018;**4**(2):139

[84] Ravanbakhsh H, Karamzadeh V, Bao G, Mongeau L, Juncker D, Zhang YS. Emerging technologies in multi-material bioprinting. Advanced Materials. 2021;**33**(49):2104730

[85] Biolife4D. About BIOLIFE4D. 2018. Available from: https://biolife4d.com/ about/

[86] L'Oréal and Poietis sign an exclusive research partnership to develop bioprinting of hair. 2016. Available from: https://www.poietis.com/en/postrelease.php?id=6&view=0.

[87] Poieskin. 2018. Available from: http:// poietis.com/fr/poieskin/welcome.php.

[88] Wang S, Hunt K. Chinese Company Implants 3-D Printed Blood Vessels into Monkeys. Hong Kong: CNN; 2017

[89] Davies S. Chinese medical researchers create natural blood vessels using 3D bioprinter. 2016. Available from: https://www. tctmagazine.com/api/content/55948f80 c07b-11e6b59b-0aea2a882f79/

[90] TeVido Vitiligo. 2018. Available from: http://tevidobiodevices.com/vitiligo/

[91] Zolfagharian A, Kouzani AZ, Khoo SY, Moghadam AA, Gibson I, Kaynak A. Evolution of 3D printed soft actuators. Sensors and Actuators A: Physical. 2016;**250**:258-272

[92] Jani JM, Leary M, Subic A, Gibson MA. A review of shape memory alloy research, applications and opportunities. Materials & Design. 2014;**56**:1078-1113

[93] Ahmed K, Shiblee MN, Khosla A, Nagahara L, Thundat T, Furukawa H. Recent progresses in 4D printing of gel materials. Journal of The Electrochemical Society. 2020;**167**(3):037563

[94] Zhang K, Geissler A, Standhardt M, Mehlhase S, Gallei M, Chen L, et al. Moisture-responsive films of cellulose stearoyl esters showing reversible shape transitions. Scientific Reports. 2015;**5**(1):1-3

[95] Melocchi A, Uboldi M, Inverardi N, Briatico-Vangosa F, Baldi F, Pandini S, et al. Expandable drug delivery system for gastric retention based on shape memory polymers: Development via 4D printing and extrusion. International Journal of Pharmaceutics. 2019;**571**:118700

[96] Mathur V, Agarwal P, Srinivasan V, Panwar A, Vasanthan KS. Facet of 4D printing in biomedicine. Journal of Materials Research. 15 Nov 2022:1-7

## **Chapter 10**
