**5. Material component**

*Design and Manufacturing*

counterparts [28, 64].

in order to find the best strategy.

nutrients [67].

hypotheses. However, cells cultured in vitro are not replicates of their in vivo

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

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

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

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

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 comple-

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

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.

compressive strength, compared to the other designs [68].

mented with the remodeling of the original bone morphology.

compared with scaffolds with prismatic geometries.

silk seeded with this type of cells [23].

**166**

For bone regeneration, it is necessary to consider that the scaffold must restore 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 the scaffold [36].

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 and additive manufacturing (AM).


#### **Table 2.**

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