**7. Optimized approach of a 3D scaffold**

*Design and Manufacturing*

ECM components.

**6. Biological components**

data, is the most appropriate.

The dogma of molecular biology is the basis for producing most bone cell and

Bone remodeling is divided into five stages: activation, resorption, reversal, formation, and, finally, mineralization (see **Figure 3**). It is a process in which the old bone is reabsorbed and there is new bone formation. The cells that are involved in bone remodeling are osteoblasts, osteoclasts, and osteocytes, which actively participate in osseointegration and repair. Osteoclasts activate bone resorption, while osteocytes regulate bone homeostasis and osteoblasts form bone [15, 110]. In addition to bone cells, there are other cell lines that can be used in bone regeneration, which are human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), stem cells, and fibroblasts. According to Kuhn [111] and other workers [112, 113], hESCS present a rapidly proliferating rate. However, their transplantation induces uncontrollable spontaneous differentiation and can the teratoma formation may occur. Another type of stem cell is iPSCs. They can differentiate into several cells. However, there are studies that show that these cells can also give rise to teratomas and, in studies that distinguish high-quality lines from the iPSCs, allowed the detection of large duplications of genes that could potentially affect the differentiation and pluripotency of these cells [28, 99]. For these reasons, these cells are not considered the best ones for bone regeneration. Compared to fibroblasts, stem cells have a greater ability to migrate, so these type of cells are the most suitable cells for bone regeneration. Kargozar [58] recently studied the osteogenic potential of different MSCs, such as those derived from human bone marrow, umbilical cord (UC-MSCs) and adipose (AD-MSCs). It concluded that BMMSCs, according to collected histological

The combination of scaffold, AM, and bioreactor culture shows great potential for creating automated production ecosystems that will enable the formation of commercially available products for BTE application. Efficient nutrient and oxygen transport are important for this type of applications. To this end, bioreactor systems have tried to overcome this difficulty. Rotating-wall vessels are limited to small scaffolds as they do not provide optimal mass transport to the center of the scaffold and are not efficient in osteogenic differentiation, due to shear stress values transmitted to cells.

**170**

**Figure 3.**

*Bone Remodeling Cycle.*

Scaffold architectures were designed in SolidWorks 2018 software. The design was bioinspired. This inspiration came from the natural organization of long bones, as represented in **Figure 4**. The diaphysis is composed of cortical bone (external region) which covers the trabecular bone (internal region). The trabecular bone has a larger surface area than the cortical bone and has a bone volume fraction ranging from 5% to a maximum of 60% [117]. It is known that the cortical zone corresponds to ~20% of the total diameter [118]. Bearing this in mind, it is expected that mimicking this type of organization, the mechanical behavior of the final scaffolds would be better and closer to the natural tissue.

The design considered has a height of 10 mm and diameter of 30 mm (see **Figure 5**). Thus, the cortical zone, the outer part of the scaffold, has a thickness of 6 mm and the trabecular zone, the inner part, has 18 mm. In the middle, there is a canal that corresponds to the medullar cavity. As happens in the native tissue, the region corresponding to the trabecular bone presents a higher porosity than the cortical one. So, the proposed scaffold has pores with different sizes between the different parts, bigger in the trabecular and smaller in the cortical

**Figure 4.** *Natural organization of long bones.*

#### *Design and Manufacturing*

(400 and 300 μm of pore diameters, respectively). According to Zhang et al. [119], these pores are within the required values, since exceeding the pore size of 400 μm, cells do not sense the 3D, resulting in poor ECM production. Moreover, they are organized in a radial way, with a significant difference between the cortical zone and the trabecular zone.

The projected scaffold presents a total porosity of 42%, whereas the cortical part has approximately 5% porosity. This porosity mimics the normal porosity in the native bone (see **Table 1**) as shown by Fernandez-Yague et al. [120] and Wang et al. [3]. The trabecular zone has a porosity of approximately 57%. This porosity is also in agreement with the authors previously mentioned. However, this value is closer to the lower limit. This porosity can be improved by the addition of horizontal channels, but its inclusion would decrease the mechanical behavior of the proposed scaffold.

Since pore interconnectivity is considered by some authors a key point to cell migration and proliferation, another design is proposed, and (see **Figure 6**) it is inspired by a DNA strand. As the scaffold gains height, the base rotates, with a rotation angle of 36°. This was considered to guarantee that the end of the filaments was supported on all layers. Also, in this case, pores diverge gradually, so that the differences between cortical bone and trabecular bone can be noticed.

**Figure 5.** *Cylindrical scaffold.*

**173**

**Acknowledgements**

*Comprehensive Review on Full Bone Regeneration through 3D Printing Approaches*

In this scaffold, pores range from 50 to 1500 μm in each layer, which is in agreement with the Zadpoor [121] and Szpalski [39]. However, the minimum required pore size is 100 μm, to make it easier to transport oxygen and nutrients and discard waste products [39, 121, 122]. For proper cell propagation, according to Bael [123], pore size should not pass the 1000 μm. Compared to the previous design presented, this one presents a slightly lower porosity in total, of around 38%, in which the cortical and trabecular parts have 22% and 49% porosity, respectively. According to the authors Andrzejewska [34], Keaveny et al. [2], and Xiao et al. [124], the porosity of the trabecular zone is near the defined porosity values. However, the porosity of the zone corresponding to the cortical part is far above the maximum value of the defined values found in **Table 1**. In order to decrease this porosity, it is necessary to shrink the pore size used. Despite this limitation, this scaffold already has the advantage of fully interconnected pores, which will facilitate cell growth and the transport of oxygen and nutrients. The combination of all supports of TE, which were described above, could lead successfully to bone formation. As biomechanics and TE advanced, it is easy to foresee the development of a new model for bone formation in which the use of an

Bone defects are a constantly growing problem, affecting thousands of people around the world, which causes a loss in life quality, and most of the time, for an active population, it may take long periods of recovery. Until now, there are no synthetic substitutes that meet the mechanical and biological requirements for the longterm cure of critical-size bone defects. To overcome this health problem, the use of temporary biocompatible and biodegradable scaffolds becomes the best choice. Structures produced by AM have superior advantages compared to the conventional techniques, mainly due to better control over the desired architecture. Moreover, the choice of the AM technique to produce these scaffolds is essential to ensure control,

namely, in terms of biological, physicochemical, and mechanical properties.

Considering all types of materials available, associated with the desired bone regeneration and the use of synthetic polymers, as PCL or PLA, combined with collagen type I for the trabecular region and Hap for cortical region, seems to be the best strategy to follow. To obtain the designed structures with these biomaterials, the most suitable AM technique is the FFF. For the selection of the final scaffold within the two proposals, further studies need to be performed. However, a third option could be also considered, which would include the cortical region of the first proposed scaffold (ensuring the required mechanical resistance) and the trabecular zone of the second one (assuring a proper porosity and pore interconnectivity to

Among the most commonly used bioreactors for bone regeneration, perfusion bioreactors appear as the most suitable, because it improves osteogenic proliferation and differentiation due to improved mass transfer and adequate shear stress. When making a design proposal for bone regeneration, it is necessary to study the

This work is supported by the Fundação para a Ciência e Tecnologia (FCT)

and Centro2020 through the Project references UID/Multi/04044/2019,

*DOI: http://dx.doi.org/10.5772/intechopen.90864*

original scaffold leads to long bone fracture healing.

allow cell migration, nutrient, and oxygen exchange).

mechanical effects, such as stress and tension, and link them

**8. Conclusions**

**Figure 6.** *DNA chain-inspired cylindrical scaffold.*

#### *Comprehensive Review on Full Bone Regeneration through 3D Printing Approaches DOI: http://dx.doi.org/10.5772/intechopen.90864*

In this scaffold, pores range from 50 to 1500 μm in each layer, which is in agreement with the Zadpoor [121] and Szpalski [39]. However, the minimum required pore size is 100 μm, to make it easier to transport oxygen and nutrients and discard waste products [39, 121, 122]. For proper cell propagation, according to Bael [123], pore size should not pass the 1000 μm. Compared to the previous design presented, this one presents a slightly lower porosity in total, of around 38%, in which the cortical and trabecular parts have 22% and 49% porosity, respectively.

According to the authors Andrzejewska [34], Keaveny et al. [2], and Xiao et al. [124], the porosity of the trabecular zone is near the defined porosity values. However, the porosity of the zone corresponding to the cortical part is far above the maximum value of the defined values found in **Table 1**. In order to decrease this porosity, it is necessary to shrink the pore size used. Despite this limitation, this scaffold already has the advantage of fully interconnected pores, which will facilitate cell growth and the transport of oxygen and nutrients.

The combination of all supports of TE, which were described above, could lead successfully to bone formation. As biomechanics and TE advanced, it is easy to foresee the development of a new model for bone formation in which the use of an original scaffold leads to long bone fracture healing.

## **8. Conclusions**

*Design and Manufacturing*

and the trabecular zone.

proposed scaffold.

(400 and 300 μm of pore diameters, respectively). According to Zhang et al. [119], these pores are within the required values, since exceeding the pore size of 400 μm, cells do not sense the 3D, resulting in poor ECM production. Moreover, they are organized in a radial way, with a significant difference between the cortical zone

The projected scaffold presents a total porosity of 42%, whereas the cortical part has approximately 5% porosity. This porosity mimics the normal porosity in the native bone (see **Table 1**) as shown by Fernandez-Yague et al. [120] and Wang et al. [3]. The trabecular zone has a porosity of approximately 57%. This porosity is also in agreement with the authors previously mentioned. However, this value is closer to the lower limit. This porosity can be improved by the addition of horizontal channels, but its inclusion would decrease the mechanical behavior of the

Since pore interconnectivity is considered by some authors a key point to cell migration and proliferation, another design is proposed, and (see **Figure 6**) it is inspired by a DNA strand. As the scaffold gains height, the base rotates, with a rotation angle of 36°. This was considered to guarantee that the end of the filaments was supported on all layers. Also, in this case, pores diverge gradually, so that the

differences between cortical bone and trabecular bone can be noticed.

**172**

**Figure 6.**

**Figure 5.** *Cylindrical scaffold.*

*DNA chain-inspired cylindrical scaffold.*

Bone defects are a constantly growing problem, affecting thousands of people around the world, which causes a loss in life quality, and most of the time, for an active population, it may take long periods of recovery. Until now, there are no synthetic substitutes that meet the mechanical and biological requirements for the longterm cure of critical-size bone defects. To overcome this health problem, the use of temporary biocompatible and biodegradable scaffolds becomes the best choice. Structures produced by AM have superior advantages compared to the conventional techniques, mainly due to better control over the desired architecture. Moreover, the choice of the AM technique to produce these scaffolds is essential to ensure control, namely, in terms of biological, physicochemical, and mechanical properties.

Considering all types of materials available, associated with the desired bone regeneration and the use of synthetic polymers, as PCL or PLA, combined with collagen type I for the trabecular region and Hap for cortical region, seems to be the best strategy to follow. To obtain the designed structures with these biomaterials, the most suitable AM technique is the FFF. For the selection of the final scaffold within the two proposals, further studies need to be performed. However, a third option could be also considered, which would include the cortical region of the first proposed scaffold (ensuring the required mechanical resistance) and the trabecular zone of the second one (assuring a proper porosity and pore interconnectivity to allow cell migration, nutrient, and oxygen exchange).

Among the most commonly used bioreactors for bone regeneration, perfusion bioreactors appear as the most suitable, because it improves osteogenic proliferation and differentiation due to improved mass transfer and adequate shear stress. When making a design proposal for bone regeneration, it is necessary to study the mechanical effects, such as stress and tension, and link them

## **Acknowledgements**

This work is supported by the Fundação para a Ciência e Tecnologia (FCT) and Centro2020 through the Project references UID/Multi/04044/2019,

PAMI—ROTEIRO/0328/2013 (NO. 022158), and MATIS (CENTRO-01-0145- FEDER-000014-3362). It is also funded by the projects insitu.Biomas (POCI-01-0247-FEDER-017771), Bone2Move (PTDC/CVT-CVT/31146/2017), and Stimuli2BioScaffolds (PTDC/EME-SIS/32554/2017).
