**3. In vivo studies**

The bone, in vivo, is exposed to mechanical stimulation by muscle contraction and body movements, and the mechanical load induces an increase in bone mass formation [38]. During body movement, the forces applied results in changes in hydrostatic pressure, fluid flow-induced shear stress, direct cell strain, and electric fields [38–40].

In order to identify the mechanical properties necessary for humans, it is important to study what kind of in vivo studies and which animal models have been considered in the literature (see **Table 1**). The choice of the most appropriate animal model is an important step in clinical translation, because it will help to better understand and propose innovative strategies for bone regeneration. Each animal model has pros and cons [41], and in each study, a specific set of parameters is used. That is why it is difficult to compare the different studies available [42, 43]. There are various models that were studied in vivo for full bone regeneration. Rabbit, rat, ovine (sheep or bovine), canine, and goat are the most used.

There are six studies on the literature where a rabbit model was considered. Nather and their co-workers [44] evaluated the effect of bone marrow mesenchymal stem cells (BMMSCs) on the biological healing of a 1.5 cm cortical bone allograft in the tibia of adult rabbits. In their study it was shown that BMMSCs can improve cortical allograft binding rate, reabsorption activity, bone formation, and osteocyte cell count. In 2013, Khojasteh et al. [45] developed a scaffold using particulate mineralized bone/fibrin glue/mesenchymal stem cells (MSCs). Through the alizarin staining method, they verified that there was a deposition of mineralized matrix. This was also demonstrated by RT-PCR analysis of osteocyte markers. At the end of 3 weeks, osteocalcin, osteopontin, and collagen I messenger RNA were produced. They concluded that this implant would be a promising combination for vertical bone augmentation around implants inserted simultaneously into the tibia of rabbits. Lee et al. [46] studied the effect of autologous BMMSCs seeded into gel foam on structural bone allograft healing in 1.5 cm femoral defect of white rabbits. They concluded that the use of MSCs influenced the bone formation, resorption, and angiogenesis. Jang et al. [47] extruded porous HAp scaffolds, which were set in a drill-cut femur rabbit bone. After 4 and 8 weeks of implantation, micro-CT scanning images showed material degradation and integration of the sample into the native bone. In this period, the morphological behavior was similar in bone tissuescaffold junction. Chowdhary et al. [48] had evaluated the early response of bone tissue to micro threads with an oxidized titanium implant (4 mm in diameter and 8 mm in length) between the macro threads. The study was tested in rabbit legs, tibia, and femur. The bone regeneration happened near the micro threads, and the bone growth in femur indicated that the cancellous bone seems to be more sensitive to micro thread stimulation. Recently, in 2018, Tovar and co-workers [49] used 3D printing, specifically robocasting/direct writing, to develop a scaffold with 100% beta-tricalcium phosphate (β-TCP) (350 μm pore diameter) in order to regenerate critical-sized rabbit radius defects in vivo. A 3 cm incision was made in a critical defect of 11 mm, approximately, in the radio, and the periosteum was resected to at least 1.5 cm proximal and distal to the defect. This scaffold proved to be good for bone tissue engineering (BTE) since at 8 weeks it showed bone formation with signs of resorption of the scaffold. The amount of bone formed was increased from week to week, regenerating the medullary space, and at 24 weeks the scaffold was significantly resorbed.

Rats are also another in vivo model referred in the literature. Saravanan et al. [50] introduced in an albino-Wistar rat with a critical-sized bone defect in the tibia a scaffold containing chitosan, gelatin, and graphene oxide by freeze drying.

**165**

formation.

**4. In vitro studies**

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

They concluded that this scaffold promoted increase in osteoblasts and increased the collagen content, accelerating the bridging of the rat tibial bone defect.

Regarding the canine model, two studies were published in the literature. In 1996, Bragdon and co-workers [52] showed that, in canine femurs, an oscillating motion of 20 μm does not affect osseointegration. However, 40 and 150 μm oscillating motion of implants does not support bone growth. Recently, Barba et al. [19] implanted in vivo, in a canine model calcium-deficient scaffold (0.3 mm height and 5 mm diameter) with Hap considering different pore architectures and compared with two ceramics, a biphasic calcium phosphate (BCP) and a β-TCP with rat mesenchymal stem cells (rMSCs). Pores ranged from 10 to 300 μm. With this, calcium-deficient scaffold and Hap triggered osteogenic differentiation of rMSCs. They concluded that calcium-deficient HAp foam scaffolds with a spherical concave

Animal studies are needed to understand bone regeneration. Variables such as the amount of bone formation and its kinetics, mechanical properties and safety obtained by the scaffold, including the presence of toxic degradation in different organs and in terms of inflammatory response need to be understood in detail [42]. However, bone fractures performed in animals do not represent the complexity of healing human fractures [23, 37]. The potential of each different type of cells both

Ko and co-workers studied the potential of human-induced pluripotent stem cells (hiPSCs) against the human bone marrow mesenchymal stem cells (hBMMSCs). Both cells were placed in rat bone defects, with a size of 2 cm, which is similar to the human value mentioned above. They concluded that both hIPSCs and hBMMSCs have osteogenic potential in vivo [53]. However, some authors showed the existence of risks of teratoma formation after transplantation in hIPSCs [28, 54–56]. In literature, the use of MSCs seems to significantly help bone regeneration in in vivo studies [31, 45, 57–59]. Some authors defend that the addition of growth factors to cell-scaffold constructs promotes bone regeneration [60]. Nevertheless, Kleinhans et al. [61] showed that a good culture capable of mimicking tissue

morphology, functionality, and biology, for example, using bioreactors, is sufficient

In vitro models are required to accurately record the physiology of healing at a site of bone fracture since bone takes weeks to differentiate in vitro [62] and wound healing can take weeks to months [23, 63]. In vitro studies are advantageous because they offer a controlled environment to experimental test molecular and cellular

There is a great deal of discussion today about the incorporation of growth factors. In this chapter, authors defend the nonnecessity of its incorporation, since upon the right environmental conditions, cells are actually able to secrete the optimal extracellular matrix (ECM) components. Therefore, a good mechanically stimulated culture combined with transcription factors influences cells to bone

to obtain a homogeneous cell distribution of soluble factors.

A model of an ovine with 35 mm tibial defect was then used by Smith et al. [51] to study full bone regeneration. They produced a scaffold using blending process of poly (L-lactic acid)-poly(ε-caprolactone) (PLLA: PCL 20:80) with and without marrow-derived skeletal stem cells. They divided the tests into three different groups: empty defect, scaffold alone, and scaffold with cells. Radiographic has shown poor bone formation upon 12 weeks. However, there was a greater tendency

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

for bone formation in the scaffold with cells.

macroporosity allow osteoinduction.

in vitro and in vivo plays here a key role.

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

They concluded that this scaffold promoted increase in osteoblasts and increased the collagen content, accelerating the bridging of the rat tibial bone defect.

A model of an ovine with 35 mm tibial defect was then used by Smith et al. [51] to study full bone regeneration. They produced a scaffold using blending process of poly (L-lactic acid)-poly(ε-caprolactone) (PLLA: PCL 20:80) with and without marrow-derived skeletal stem cells. They divided the tests into three different groups: empty defect, scaffold alone, and scaffold with cells. Radiographic has shown poor bone formation upon 12 weeks. However, there was a greater tendency for bone formation in the scaffold with cells.

Regarding the canine model, two studies were published in the literature. In 1996, Bragdon and co-workers [52] showed that, in canine femurs, an oscillating motion of 20 μm does not affect osseointegration. However, 40 and 150 μm oscillating motion of implants does not support bone growth. Recently, Barba et al. [19] implanted in vivo, in a canine model calcium-deficient scaffold (0.3 mm height and 5 mm diameter) with Hap considering different pore architectures and compared with two ceramics, a biphasic calcium phosphate (BCP) and a β-TCP with rat mesenchymal stem cells (rMSCs). Pores ranged from 10 to 300 μm. With this, calcium-deficient scaffold and Hap triggered osteogenic differentiation of rMSCs. They concluded that calcium-deficient HAp foam scaffolds with a spherical concave macroporosity allow osteoinduction.

Animal studies are needed to understand bone regeneration. Variables such as the amount of bone formation and its kinetics, mechanical properties and safety obtained by the scaffold, including the presence of toxic degradation in different organs and in terms of inflammatory response need to be understood in detail [42]. However, bone fractures performed in animals do not represent the complexity of healing human fractures [23, 37]. The potential of each different type of cells both in vitro and in vivo plays here a key role.

Ko and co-workers studied the potential of human-induced pluripotent stem cells (hiPSCs) against the human bone marrow mesenchymal stem cells (hBMMSCs). Both cells were placed in rat bone defects, with a size of 2 cm, which is similar to the human value mentioned above. They concluded that both hIPSCs and hBMMSCs have osteogenic potential in vivo [53]. However, some authors showed the existence of risks of teratoma formation after transplantation in hIPSCs [28, 54–56]. In literature, the use of MSCs seems to significantly help bone regeneration in in vivo studies [31, 45, 57–59]. Some authors defend that the addition of growth factors to cell-scaffold constructs promotes bone regeneration [60]. Nevertheless, Kleinhans et al. [61] showed that a good culture capable of mimicking tissue morphology, functionality, and biology, for example, using bioreactors, is sufficient to obtain a homogeneous cell distribution of soluble factors.

There is a great deal of discussion today about the incorporation of growth factors. In this chapter, authors defend the nonnecessity of its incorporation, since upon the right environmental conditions, cells are actually able to secrete the optimal extracellular matrix (ECM) components. Therefore, a good mechanically stimulated culture combined with transcription factors influences cells to bone formation.

### **4. In vitro studies**

In vitro models are required to accurately record the physiology of healing at a site of bone fracture since bone takes weeks to differentiate in vitro [62] and wound healing can take weeks to months [23, 63]. In vitro studies are advantageous because they offer a controlled environment to experimental test molecular and cellular

*Design and Manufacturing*

**3. In vivo studies**

fields [38–40].

The bone, in vivo, is exposed to mechanical stimulation by muscle contraction and body movements, and the mechanical load induces an increase in bone mass formation [38]. During body movement, the forces applied results in changes in hydrostatic pressure, fluid flow-induced shear stress, direct cell strain, and electric

In order to identify the mechanical properties necessary for humans, it is important to study what kind of in vivo studies and which animal models have been considered in the literature (see **Table 1**). The choice of the most appropriate animal model is an important step in clinical translation, because it will help to better understand and propose innovative strategies for bone regeneration. Each animal model has pros and cons [41], and in each study, a specific set of parameters is used. That is why it is difficult to compare the different studies available [42, 43]. There are various models that were studied in vivo for full bone regeneration. Rabbit, rat,

There are six studies on the literature where a rabbit model was considered. Nather and their co-workers [44] evaluated the effect of bone marrow mesenchymal stem cells (BMMSCs) on the biological healing of a 1.5 cm cortical bone allograft in the tibia of adult rabbits. In their study it was shown that BMMSCs can improve cortical allograft binding rate, reabsorption activity, bone formation, and osteocyte cell count. In 2013, Khojasteh et al. [45] developed a scaffold using particulate mineralized bone/fibrin glue/mesenchymal stem cells (MSCs). Through the alizarin staining method, they verified that there was a deposition of mineralized matrix. This was also demonstrated by RT-PCR analysis of osteocyte markers. At the end of 3 weeks, osteocalcin, osteopontin, and collagen I messenger RNA were produced. They concluded that this implant would be a promising combination for vertical bone augmentation around implants inserted simultaneously into the tibia of rabbits. Lee et al. [46] studied the effect of autologous BMMSCs seeded into gel foam on structural bone allograft healing in 1.5 cm femoral defect of white rabbits. They concluded that the use of MSCs influenced the bone formation, resorption, and angiogenesis. Jang et al. [47] extruded porous HAp scaffolds, which were set in a drill-cut femur rabbit bone. After 4 and 8 weeks of implantation, micro-CT scanning images showed material degradation and integration of the sample into the native bone. In this period, the morphological behavior was similar in bone tissuescaffold junction. Chowdhary et al. [48] had evaluated the early response of bone tissue to micro threads with an oxidized titanium implant (4 mm in diameter and 8 mm in length) between the macro threads. The study was tested in rabbit legs, tibia, and femur. The bone regeneration happened near the micro threads, and the bone growth in femur indicated that the cancellous bone seems to be more sensitive to micro thread stimulation. Recently, in 2018, Tovar and co-workers [49] used 3D printing, specifically robocasting/direct writing, to develop a scaffold with 100% beta-tricalcium phosphate (β-TCP) (350 μm pore diameter) in order to regenerate critical-sized rabbit radius defects in vivo. A 3 cm incision was made in a critical defect of 11 mm, approximately, in the radio, and the periosteum was resected to at least 1.5 cm proximal and distal to the defect. This scaffold proved to be good for bone tissue engineering (BTE) since at 8 weeks it showed bone formation with signs of resorption of the scaffold. The amount of bone formed was increased from week to week, regenerating the medullary space, and at 24 weeks the scaffold was

Rats are also another in vivo model referred in the literature. Saravanan et al. [50] introduced in an albino-Wistar rat with a critical-sized bone defect in the tibia a scaffold containing chitosan, gelatin, and graphene oxide by freeze drying.

ovine (sheep or bovine), canine, and goat are the most used.

**164**

significantly resorbed.

hypotheses. However, cells cultured in vitro are not replicates of their in vivo counterparts [28, 64].

When the bone is subjected to a mechanical force, electrical potentials are generated, which play an important role in bone remodeling. To mimic this natural process, bioreactors were created and are nowadays widely used. These 3D systems allow the control of various parameters, such as temperature, pH, oxygen concentration, growth factors, and mechanical stimuli, among others, and modulate cell growth more easily. These bioreactors can simulate the human bone environment and allow the study of the role of various factors in scaffolds or preculture scaffolds in vivo. In addition, to provide adequate nutrition and removing residues from all cells in the scaffold, fluid flow can be manipulated to physically stimulate bone growth [39]. Bone is constantly exposed to mechanical stimulation due to muscle contractions and body movements that result in changes in hydrostatic pressure, direct cell strain, fluid flow-induced shear stress, and electric fields. In addition, bone cells are more sensitive to mechanical stimulation. Therefore, providing physical stimulation in bioreactors becomes a key component of BTE strategies [65].

The following studies demonstrate the importance of performing in vitro testing in order to find the best strategy.

Jang et al. [66] developed a HAp scaffold to mimic native bone through a multipass extraction process with the addition of osteoblast-like cells, with pores of 150 ± 20 μm in diameter and with a pore structure of 50 ± 10 μm which is thin enough for rapid bone resorption. With in vivo tests and in vitro tests, they confirmed that the scaffold used is appropriate for graft without inflammatory reactions and bone formation after 8 weeks of implantation. The scaffold's porosity is a critical parameter enabling medium exchange and nutrient diffusion, which is a key role in cell proliferation. So, the optimization of the scaffold's porosity is important to help cell growth, formation of vascularization, and the diffusion of nutrients [67].

Roohani-Esfahani and their co-workers developed a glass–ceramic scaffold, with dimension size 6x6x6mm, by direct ink writing mimicking cortical bone with 600 μm custom-made nozzle. In the work, they concluded that a scaffold with hexagonal pore shapes (450 μm, 550 μm, 900 μm, and 1200 μm) present the highest compressive strength, compared to the other designs [68].

Abbot and co-workers, in 2016, developed a silk scaffold with osteoblasts to evaluate in vitro culture that stimulated bone differentiation and regeneration. In the end, they concluded that it was evident the mineralization in the scaffold with silk seeded with this type of cells [23].

Tovar and co-workers [49] had developed a cylindrical scaffold with 10.5 mm length, 4.5 mm outside diameter and 2.25 mm inside diameter, 330 μm struts, and around 400 μm pore spacing. They used a 330-μm-diameter extrusion nozzle with a velocity of 8 mm/s. The existence of macrometric and micrometric porosity in the scaffold helped in its degradation, which allowed the biomechanical load to the healing bone. This may explain the rapid development of bone properties in the regenerated tissue that is highly indicative of complete healing when it is complemented with the remodeling of the original bone morphology.

Recently, Barba et al. [19, 69] concluded that the geometric parameters of the scaffold, like curvature, influence bone tissue regeneration. They demonstrated that spongy scaffolds with concave pores attracted a large amount of ectopic bone compared with scaffolds with prismatic geometries.

Through the existing studies, both in vivo and in vitro, one can get an idea of both material and biological components essentials to a proper bone regeneration. In the first phase, it is necessary to understand which scaffold design is the most appropriate and which biomaterials are to combine it with the AM technique.

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**Table 2.**

**Ideal scaffolds**

(a natural with a synthetic one)

*Characteristics of the different materials used to produce a scaffold.*

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

For bone regeneration, it is necessary to consider that the scaffold must restore

Having into consideration the referred above and combining this information with the natural organization of bone (trabecular and cortical), the best strategy for BTE should pass by the use of collagen type I in the trabecular bone region and Hap in the cortical zone [80, 81]. Despite their advantages and the fact that they are already present in the bone native structure, their proper manipulation is only possible through their combination with synthetic polymers. The most suitable are poly(ε-caprolactone) (PCL) or polylactic acid (PLA) because they are both approved by the Food and Drug Administration. PCL is a stable, biocompatible, biodegradable polymer (from 12 to 48 months) and easy to handle to achieve the desired mechanical properties. Due to its low melting point (60°C) [82, 83], it can be easily combined with the collagen. PLA is a biocompatible polymer, more hydrophilic than PCL, and its handling is similar to the use of PCL. Hydrophilicity accelerates polymer degradation as it accelerates polymer and scaffold moisture [84]. However, it has a high melting point, which could be combined with Hap. With these materials, scaffolds can be produced with two methods: conventional

**Scaffolds References**

appropriate mechanical behavior. At a microscale it presents the architecture, 3D composition, and active molecular reactive groups. In a macroscale, they have porosity, stiffness, and elasticity

minimal movement between the implant and the host tissue and provide physiological loading functionality to the implant site

Ceramics Have been used because of their ability to sustain compressive loads [6, 74, 75]

can positively support cell adhesion and function

degradation, bioactivity, mechanical kinetics, tissue nonspecificity, and their intrinsic structural similarity to the extracellular matrix of native tissues. They also promote biological recognition, which

[71, 72]

[6]

[15, 73]

[76–79]

Natural polymers Biomaterials are widely used because of their biocompatibility,

Synthetic polymers Easy to manipulate the properties of the material to achieve the

Metals Are used in long bones to better attach to the bones where there is

Should exhibit the adequate mechanical properties, pore size, and biological activity, serve as cell support, and guarantee new bone formation and thus the use of more than one material

the normal biomechanical role of the tissue. **Table 2** shows different types of existing materials and their characteristics and some guidelines about how to obtain an ideal scaffold. However, there are other important features that need to be taken into account related to the different biological and physical signals involved in order to simulate the mechanism of remodeling in a natural environment, but more importantly, the scaffold must have the exact mechanical properties to withstand the loads the original bone held [6, 70]. The biomaterials used in the scaffolds must have a suitable rate of degradation in order to support bone regeneration. This rate of degradation depends on the corrosion resistance of the material used, which is affected by the chemical and physical characteristics of

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

**5. Material component**

the scaffold [36].

and additive manufacturing (AM).

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