**2. Regeneration procedure**

Replace the entirety of this text with the main body of your chapter. The body is where the author explains experiments, presents and interprets data of one's research. Authors are free to decide how the main body will be structured. However, you are required to have at least one heading. Please ensure that either British or American English is used consistently in your chapter. Since a complete understanding of the bone healing process will help in finding the best bone replacement, this section describes this procedure. Bone healing procedure is a complex process that involves different cellular and molecular events. The bone healing process can be divided into four main steps: (1) hematoma formation, (2) fibrocartilaginous callus formation, (3) bony callus formation, and (4) bone remodeling [9]. This process requires a systematic order and the failure of any of the steps can lead to the failure of the treatment process. The capability of bone regeneration depends on several factors such as the age of the patient, the condition of the patient, the type of bone, the size of the damaged area, the severity of the damage, blood sources, etc. [10]. The bone healing process involves two main pathways: intramembranous (IM) and endochondral (EC) ossification [11].

In the embryo, the endochondral ossification procedure lead to cartilaginous formation as a template and progressive mineralization in long bones [12]. Therefore, as a result, this process is also effective in-directly in bone repair. After the injury

#### *Recent Advances, Challenges and Future Opportunities for the Use of 3D Bioprinting in Large… DOI: http://dx.doi.org/10.5772/intechopen.111495*

during the endochondral ossification process, inflammatory reactions occur between the hematoma at the end of the fracture and its surrounding environment, which results in the creation of temporary granulation tissue, fibrous tissue, and cartilage, and finally, osteoblasts replace the cartilage tissue leads to bone tissue regeneration [13]. Whereas, in intramembranous ossification, the MSCs directly converted to bone cells without cartilage formation [13, 14].

There are various factors such as cell types, growth factors, cytokines, and chemokines that involves in the bone healing process and can facilitate bone healing. Hematoma is the first step of the healing process. It can act as a temporal natural scaffold and some specific cell types such as macrophages and T cells actives at this level and secretes cytokines and begins the regeneration procedure [15]. The acute inflammatory phase usually accelerates in the first 24 hours and finishes 1 week after injury [16]. Chen et al. [17] mentioned Beta-Catenin as an effective signaling in the healing process of bone injuries. This factor acts differently in various levels of fractures. In the first steps, it is necessary for pluripotent mesenchymal cell differentiation to osteoblasts and chondrocytes and after differentiation, it regulates osteoblasts' behaviors.

The secretion of several factors involves in this level includes TNF-Alpha, bone morphogenetic proteins (BMP), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, IL-11, and IL-18. The secretion of these factors starts angiogenesis [16]. As the healing process continues, chondrocytes, osteoblasts, and endothelial cells are activated and a cartilaginous callus (soft callus) is created to cover the created gap [18]. The regeneration of large bone defects is challenging in surgery and needs extra attention. Infection followed by long bone implant surgery in femoral, tibial, and humeral fractures is another important issue that should be considered. The treatment is performed according to the infection stage. Stage I infections can be treated by antibiotic administration with/without debridement. In Stage II debridement, IM reaming, antibiotic nails, antibiotics can be useful and the stage III infection is usually treated with debridement, exchange nailing, and systemic antibiotic administration [19]. In fact, large bone defects cannot be treated completely without intervention and need extra medical care [20].
