## 4 How Bone Healing Occurs after Bone Injury  
Bone healing is the process that restores the anatomy and function of bone after injury (fracture or osteotomy); it can be divided into primary and secondary healing based on differences in the mobility between the fracture fragments [41; 42]. Primary healing happens when bone injured surfaces are juxtaposed and fixed through surgery and bone remodeling through the original fracture line leads to bone healing. Secondary healing takes place in all other circumstances and is usually divided into four stages that partially overlap each other \begin{table}
\begin{tabular}{l l l l} \hline \hline \multicolumn{1}{c}{\multirow{2}{*}{**Cytology and Histology**}} & \multicolumn{2}{c}{**Molecular Biology and**} & \multicolumn{1}{c}{**Physics**} \\ \multicolumn{1}{c}{} & \multicolumn{1}{c}{**Biochemistry**} & & \\ \hline Histologic parameters & PCR & Temperature rise \\ \(\bullet\) & cell composition & BMP, Wnt, osteogenesis, & \(\bullet\) & thermocouples \\ \(\bullet\) & blood clot and vessels & inflammatory cytokines, & \(\bullet\) & infrared thermography \\ \(\bullet\) & connective tissue, bone neformation, & apoptosis, growth-factors; & \(\bullet\) & thermosensors \\  & microcracks, lacunae, bridges & Hsp70 & \(\bullet\) & fiber optic thermometer \\ \hline \multirow{3}{*}{Histomorphometric} & ELISA & \multirow{3}{*}{Micro-CT} \\  & & test on the perisulcular fluid to & \\ \cline{1-1}  & and Micromorphometric Analysis & search for RANKL and OPG & \\ \cline{1-1}  & & levels & \\ \hline Immunohistochemistry & & & \\ \(\bullet\) & VEGF, CAS3, OPG, RANKL, OC, NADH & & CBCT \\  & and NADPH2, diaphorase activity & & \\ \hline Scanning electron microscopy (SEM) & & Laser 3D scanning \\ \hline Transmission electron microscopy (TEM) & & Laser profilometry \\ \hline Intravital microscopy & & & \\ \hline \hline \end{tabular} BMP: Bone Morphogenetic Protein.  
\end{table} 
Table 1: Main investigations for heat-related bone tissue necrosis. The first stage is called the \"inflammatory stage\": after injury, bone blood vessels release blood within the fracture site and hematoma develops between bone fragments. This lasts a few days and is characterized by pain and swelling. The lack of blood supply to adjacent bone leads to bone necrosis that is characterized histologically by empty osteocyte lacunae. After blood clot formation, cytokines (such as PDGF, TGF-81, VEGF, PGE1 and E2) released from platelets and mast cells stimulate neoangiogenesis and the formation of a granulation tissue that replaces the blood clot. Mononuclear phagocytes derived from new vessels assist the removal of necrotic bone and blood clots and aid the construction of the soft callus that will follow. Macrophages are also believed to play a fundamental role in fracture repair since they secrete several growth factors, such as fibroblast growth factor (FGF) that initiates fibroplasia both in soft tissue as well as in bone repair [43]. The second stage is called the \"soft callus stage\": it is characterized by a fibrous tissue whose cells are derived from endosteum, periosteum, bone marrow and adjacent soft tissues. From this stage on, a recapitulation of bone histogenesis occurs, leading to the third stage, called the \"hard callus stage\", whose progression strictly depends on the presence of blood supply. If a good blood supply is present, new osteoblasts differentiate and start to lay down the bone matrix during \"intramembranous ossification\". During intramembranous ossification, both in physiological organogenesis and in pathologic conditions, it has been demonstrated for the first time [8; 44; 45; 46; 47] that two different processes of bone formation exist, occurring in sequence, named static osteogenesis (SO) and dynamic osteogenesis (DO).