Fracture healing, which is a very complex process, can generally be examined as three overlapping stages. These are the inflammatory, the repair (soft and hard callus), and the remodeling stages. Bone tissue is extremely strong as a structure but fractures occur by excessive forces. When there is a fracture, not only bone tissue is injured, but also the periosteum, muscle tissues, and endosteal and periosteal vascular structures are damaged. The cortex, periosteum, bone marrow, and surrounding soft tissue in the field of injury all contribute to the healing process. A fracture, which is a mechanical event, triggers a reduction in local oxygen and nutrients at the site and secretion of various factors to the area, which will lead to biological events [4, 5].

The inflammatory stage starts with following trauma and the hematoma which is necessary to healing process formed with the cells containing bone marrow of peripheral and intramedullary origin. The major actors in the inflammatory process are cytokines, platelets, bone morphogenetic protein (BMP), and mesenchymal stem cells (MSCs). The inflammatory phase continues for approximately 1 week and progresses with cellular accumulation and macrophages and thrombocytes coming quickly to the injury site. Many growth factors are expressed from the cells. Primarily, these may include platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), interleukin-1 and interleukin-6, and cytokines such as prostaglandin E₂ [3–5].

The initial pro-inflammatory response includes tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), IL-6, IL-11, and IL-18 secretion. These pro-inflammatory cytokines are not only expressed from the macrophages but are also secreted from the MSCs in the periosteum. The secretions peak in the first 24 h after any fracture, and while the levels reduce during the period of cartilage formation, an increase is seen again during the bone remodeling phase [8]. These factors contribute to the accumulation of inflammatory cells and angiogenesis. In addition, cytokines play a role in endochondral bone formation and in the regulation of remodeling.

TNF-α is expressed from the macrophages and the other inflammatory cells, and it is believed that it plays a role in the formation of secondary inflammatory signals and the accumulation of chemotactic factors. IL-1 and IL-6 are the most important interleukins in fracture healing. In addition to the primary cartilaginous callus production of these two interleukin molecules and the angiogenesis-stimulating effects in the injury area, IL-6 also stimulates osteoblast and osteoclast differentiation [3]. All these factors play an active role in starting of the repair process with activation of the cells in the bone marrow, periosteum, and fracture hematoma [3, 5, 9–15].

In the early stage of bony formation, periosteal pre-osteoblasts and local osteoblasts differentiate to new bone with the expression of osteocalcin gene. As mesenchymal cells and fibroblasts proliferate with early new bone formation, they swap with fracture hematoma. The stem cells, which play an effective role in fracture healing, are believed to originate from the bone marrow, periosteum, local muscle, soft tissues, and vascular tissues [4–6, 11]. After maturation of fracture hematoma, a newly formed blood vessel network advances to the healing fracture callus. This vessel network forms a source for the growth factors and progenitor cells which differentiate the mesenchymal cells. The collagenous matrix develops in fracture hematoma and it contains major collagen types. These collagens are Type I and Type II collagens and are responsible for the continuity of the non-cartilaginous and cartilaginous extracellular matrix (ECM), respectively [5, 12]. In the soft callus period, which can last up to 3 weeks, the mesenchymal cells are differentiated from the chondrocytes which will create cartilaginous callus formation. Mature and hypertrophic chondrocytes release Type X collagen and proteases which fragment ECM. At the same time, hypertrophic chondrocytes express transcription factor called as runx2 which regulates ECM proteins such as osteocalcin and osteopontin which are necessary for ossification [4, 5, 12, 16–19].

The differentiation of the hypertrophic cartilage to the bone is noticeable in the advanced stages of fracture healing. This final stage is known as the hard callus period, and soft callus tissue gradually changes to hard callus tissue which will be solid. In this period, the chondrocyte differentiation, apoptosis, ECM fragmentation, revascularization, and bone formation occur [5]. Consequently, cartilage tissue in the medium becomes calcified. At this point, new blood vessels enter the medium, and transformation of the cartilage to bone tissue continues. Finally, matured new bone cannot be distinguished from the original bone, and this period is called as remodeling stage of fracture healing [3–5, 12, 13, 20].

For an ideal fracture healing, there are four main biological requirements [5, 9, 14]. Firstly, it is necessary to have a series of the progenitor or MSCs in the right place at the right time. These cells also originate from trabecular bone, surrounding soft tissue including synovia and synovial fluid, the hematopoietic system, bone marrow, and particularly the deep layers of the periosteum, and they play an important role in fracture healing process [5]. Previous studies have supported that osteoblasts and osteocytes, which have a role in the formation of new bone, originate from periosteum, bone marrow, and endosteum, while chondrocytes within the fracture callus primarily originate from the periosteum [5, 15, 16]. Traumatic damage or subtraction of the periosteum leads to the loss of the local source of the multipotent MSCs. Absence of these cells consequences a delay in fracture healing. For bone regeneration, it is necessary to have an accumulation of MSCs that they proliferate and that they differentiate to osteogenic cells. Recent publications have emphasized that accumulation of the circulating systemic MSCs, particularly at the inflammatory response stage, plays an extremely important role in repair process [17–19]. In other words, as a response to the signaling associated with tissue damage, MSCs are sent to the fracture site and participate in the repair process. Primary source of these MSCs originating in the bone marrow is the periosteum cambium layer in fracture healing. Buckwalter et al. demonstrated that, if periosteum is removed, the fracture callus capacity is reduced [20].

The osteoprogenitor cells also produce some molecules such as BMPs, stromal cell-derived factor 1 (SDF-1), and hypoxia-inducible factor-1 α (HIF-α) [3]. These molecules also play an important role in bone repair. While BMP-2 is crucial in bone repair, other BMPs such as BMP-7 are more important in the accumulation of the progenitor cells. In an experimental rat model, the levels of BMP-2, BMP-4, and BMP-7 showed an increase in the early stages of intramembranous ossification [21].

The second requirement for bone repair is the structure of ECM that will create the scaffold to which the progenitor cells can attach. ECM is a dynamic structure and shows continuous changes in order to provide differentiation by gathering together progenitor cells in the bone regeneration process. The main structure of ECM is formed by collagens in cartilage and bone tissues [5]. The main collagen components are Types II, IX, X, and XI for the cartilage matrix and Types I and VI for of the bone matrix. During the bone healing process, ECM keeps on remodeling constantly. This cascade is primarily organized due to the resorption of mineralized cartilage started by macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin, and TNF-α. The most important role of TNF-α is to trigger chondrocyte apoptosis. The absence of TNF-α results in a delayed resorption of mineralized cartilage and prevention of the formation of new bone. Some studies showed that while the level of RANKL is very low in intact bone, it is very high in levels after any fracture [8, 12]. In time, the cartilage tissue is replaced with bone tissue, and this substitution occurs in endochondral ossification; this is one of the most spectacular examples of tissue remodeling [5]. The fracture can gain a more stable structure from soft callus formed from cartilaginous callus in areas of relatively weak mechanical stability. This central hard callus and the bridging which is formed allow weight-bearing on the fracture and ultimately represent a semirigid structure.

The third requirement is some molecular structures which play an effective role in the process of cartilage formation and ossification at different times. Runx2, BMP, TGF-β, and IGF are well-known molecular structures which play a role in osteoblast differentiation [5, 12]. Of these factors, there is a greater understanding of the importance of factor runx2, which plays a direct role in osteoblast differentiation.

The final requirement is to have sufficient blood supply in the region. For successful fracture healing, it is necessary to have a good blood supply and revascularization. Endochondral fracture healing does not remain only within the angiogenesis pathways but also includes cartilaginous degradation to remove the ECM and cells and chondrocyte apoptosis which is necessary for the development of blood vessels at the fracture site [5]. Nowadays, the effect of vascularization of the injured area on repair is a well-known issue. Because of this, it has been attempted to make the surgical methods used for fracture fixation and implant designs appropriate to protect of vascularization in the last few decades [22, 23]. In the endochondral ossification process, the replacement of the hypertrophic cartilage structure with bone tissue requires good vascularization of the environment. The vascular ingrowth within the developing callus tissue is regulated by fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and angiopoietins 1 and 2 [3]. The two molecular pathways regulating the vascularization process are the angiopoietin-dependent pathway and the VEGF-dependent pathway [10]. As angiopoietins 1 and 2 are primary vascular morphogenetic proteins, they are expressed in the early stages of the healing cycle. In contrast, VEGF is expressed in the advanced stages of endochondral bone formation. VEGF plays a key role in the regulation of vascular regeneration and is thought to be the most effective factor [3, 24]. VEGF is expressed from both osteoblasts and hypertrophic chondrocytes. Thus, the opportunity is provided for transformation of the avascular cartilaginous matrix to vascularized bone tissue and blood vessel invasion. At the same time, VEGF is an essential mediator in the neo-angiogenesis and revascularization of the fracture site. In addition to VEGF, some other molecules such as TGF, BMP, FGF, and PDGF also play a regulatory role in the vascularization process [4, 5, 24].

There are two different ways of healing as direct (primary) and indirect (secondary) bone healing [3–5]. Direct healing is achieved by cortical healing cycle after complete anatomic reduction of the ends of fracture. The continuity of the fractured cortexes helps to intersect conic extensions. The bone structure formed without remodeling allows anatomic lamellar bone and rapid Haversian regeneration. If there is any form of movement in the fracture line, indirect fracture healing occurs.

For many years, scientists have hoped to accelerate fracture healing, and various studies have been conducted related to this. Generally, the methods used to enhance process of the fracture healing can be classified under the two subheadings of biological and physical methods. Biological stimulation is provided with osteoinductive, osteoconductive, or osteogenic factors [1–6, 11, 13, 14, 20, 25, 28]. While the osteoinductive effect has the meaning of stimulating bone formation, the osteoconductive effect is stated for the factors forming an appropriate skeletal frame for fracture healing. The osteogenic effect is stated to mean the factors stimulated for osteogenesis directly from the cells [4, 12, 29]. In addition, the electrical effect and the application of ultrasound can be included in the physical modalities used on the fracture site [2–5, 20, 28].