**3. Molecular stimulators of fracture repair**

Four sources of molecular stimulators and regulators of bone healing could be distinguished:


mature bone. Thus, this process is, in fact, a regeneration, not healing, as its goal is to restore

Clinically, healing manifests with remission of pain corresponding with gradually increasing stiffness enabling transduction of mechanical loads. Radiographically-with formation and remodeling of the callus in-between its gap. Monitoring of this process indicates the advance

From the histological point of view, restitution of the bone continuity proceeds due to accumulation and activation of mesenchymal stem cells (MSCs) that, transforming into osteoblasts and maturating into osteocytes, synthesize and release proteins forming the extracellular

In the vast majority of cases, MSCs that settle hematoma differentiate into chondrocytes. But revascularization, due to the ingrowth of blood vessels from the vasculature of the adjacent tissues, improves local oxygenation enabling the transformation of newly inflowing progenitors into osteoblasts that, maturating into osteocytes, initiate ossification forming bone cuff around the fracture gap. Starting from its periphery, it moves toward the center replacing the soft callus with woven bone [1, 2]. Later on, it's remodeling rebuilds the microarchitecture to the mature bonecancellous or compact, depending on the type of the bone that was primarily at the fracture gap. The described above process, the endochondral ossification, proceeds in ca. 97–98% of all fractures, whereas remaining 2–3% heal due to the direct osteonal growth in the process called primary bone healing basing on the intramembranous ossification [3]. The latter one is possible, when the volume of the fracture's gap is minimal, thus in non-displaced or impacted fractures only. In those cases, the short distance between bone fragments enables osteonal

remodeling toward the fracture gap restoring its vascularization and mineralizing it.

MSCs residue several tissues, including bone marrow, endosteum, and periosteum. They are abundantly represented in adipose tissue surrounding the extremity with subcutaneous fat and form a subpopulation of its leukocytes in peripheral blood as well [4]. Thus, extravasated into posttraumatic hematoma and recruited from adjacent tissues they form a population of

Their accumulation proceeds due to chemotactic stimulation. Stromal-derived factor-1 (SDF-1; also known as CXC-motif chemokine 12: CXCL12) is one of the most potent attractants of MSCs. Widely distributed in bone marrow, it splits in-between neighboring tissues, when bone continuity is broken, recruiting progenitors that accumulate at the sites of its highest concentration, the fracture gap. Inflowing cells multiply under the mitogenic stimulation of

MSCs are precursors of various cells of mesenchymal origin, including chondrocytes, fibroblasts, adipocytes, neurons, and myocytes. The direction of their differentiation depends upon molecular regulation and local physicochemical conditions. When stimulated improperly or

not only the bone's continuity but also its structure.

**2. Cellular aspects of fracture healing**

precursors for reparative processes.

platelet-derived growth factor (PDGF) [5].

of the reparative processes.

matrix (ECM).

4 Trauma Surgery

Platelets are the abundant source of molecular substances of blood origin. Released from granules into a posttraumatic hematoma, those substances activate, together with mediators released from nerve endings, and cellular events proceeding in the fracture gap.

Platelets participate in various reparative processes, being involved in the restoration of traumatized mucous and epithelia, healing various soft tissues (i.e. muscle) and the bone, and restoration of the vascularity in the process of angiogenesis. An influence of other hematoma products, including fibrin clot and activated clotting factors, hemoglobin, complement cascade and subcellular structures such as subcellular fragments of blood cells increases, giving an insight into a complex role of several hematoma compounds in the healing [7].

Traumatized tissues provide molecular stimuli that are released in response to injury. Damage - associated molecular pattern molecules (DAMPs) are the most potent activators of the sterile, traumatic inflammation ("first hit"), whereas the later one ("second hit") mostly dependents on molecules provided by the inflowing immune cells. Those molecules activate immune system directly through toll-like receptors (TLRs) [8]. So far, several DAMPs have been distinguished, including heat-shock proteins (HSPs), high-mobility group box 1 (HMGB-1), monosodium urate, heparan sulfate, adenosine triphosphate (ATP), polysaccharides, proteoglycan, phospholipids, and deoxyribonucleic acid (DNA). Similar capabilities possess hyaluronian fragments released from disintegrated ECM [9, 10].

Nerve endings provide neuromediators that participate in fracture repair, including calcitonin gene - related peptide and neuropeptide-Y [11]. Released in response to mechanical (injury) and physicochemical (hypoxia, acidosis) stimuli, they participate in the molecular regulation of cellular events during the reparative phase and callus mineralization [12]. However, they were also found to control remodeling [13].

The later abundant source of molecular stimulators are leukocytes originating from the bloodforming hematoma and inflowing from the peripheral circulation. Granulocytes are the first

probably prevent from auto aggression against infiltrating progenitors, thus enabling them to proliferate and differentiate into bone forming cells. Depletion of Breg cells, analogically to splenectomy, results in the delay of the fracture healing [16, 17]. Moreover, T cells were

Fracture Repair: Its Pathomechanism and Disturbances http://dx.doi.org/10.5772/intechopen.76252 7

Immune cells are an ample source of several molecular substances, including cytokines (i.e. IL-6 and IL-8) and growth factors (PDGF, fibroblast growth factor; FGF, TGF-β, and bone morphogenetic proteins—BMPs) [19]. Together with molecules released from nerve endings and bone marrow, they regulate cellular events stimulating proliferation and differentiation of MSCs.

The most effective MSCs stimulators are the granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage-colony-stimulating factor (GM-CSF), cytokines (IL-1, IL-3, IL-7, IL-8, and IL-12), stem cell factor (SCF), Flt3 (fms like tyrosine kinase 3) ligand, macrophage inflammatory protein-1 (MIP-1) and the chemokines GROβ (growth-regulated oncogene β; also known as CXC-2) and SDF-1 [20, 21]. Each of them evokes the unique effect promoting migration, division, activating synthesis, and release of molecules forming an appropriate environment or fulfilling the specialized biological function. They enable migration, multiplication, and differentiation of progenitors into desired cell lineage. The most potent stimulators of MSCs differentiation into, according to the local physicochemical environment, chondrocytes, or osteoblasts are TGF-β, several BMPs (2, 4, 6, 7, 13, and 14), IGF-1 (insulin-like growth factor), and FGF [22, 23]. The final outcome in the form of fracture healing results from the convergent actions of numerous factors

Mineralization of the ECM restores mechanical properties of the gap bringing back its ability to carry body weight. Briefly, it consists in the deposition of calcium and phosphate precipitates, hydroxyapatite, around the mesh of ECM proteins, namely collagens [24]. The process takes place in matrix vesicles; subcellular structures of approx. 20–200 nm in diameter that contains a number of compounds, including annexins (annexin V), alkaline phosphatase, calbindin-D9k, pyrophosphatases, carbonic anhydrase, AMP-ases, bone sialoprotein-1 (BSP-1),

Matrix vesicles concentrate inorganic substrates for mineralization due to annexin-formed calcium channels and Na/Pi phosphate transporters (NPT3/Pit1; natrium-phosphate transporter 3/POU domain class 1 transcription factor 1). High concentration of those ions results in their spontaneous precipitation to amorphous octa-Ca/Pi crystals that later on, when released from the vesicles, are converted by osteonectin, osteocalcin, and bone sialoprotein-1 (BSP-1) into hydroxyapatite. Hydroxyapatite crystals are deposited into the ECM at the outer and inner surface of the collagen fibrils [26]. In consequence, the collagen forming ECM being responsible for bone elasticity also serves as a scaffold for inorganic substances [27]. Their remodeling by matrix metalloproteinases sets the direction of trabecular bone remodeling, creating it is three-dimensional structure according to the direction of mechanical loads, and thus optimizing its microarchitecture for the most effective resistance [28, 29]. Finally, remodeling restores the structure of the primary callus to the mature bone identical to that primarily present at the fracture gap.

influencing the target cells in an appropriate time sequence and place (**Figure 1**).

shown to promote maturation of the osteoblasts [18].

**4. Extracellular matrix mineralization**

osteonectin, osteocalcin, and several growth factors [25].

**Figure 1.** Schematic representation of recruitment, proliferation, multiplication, and differentiation of MSCs into osteoblasts or chondroblasts.

cellular population that actively populates the fracture gap. Those cells infiltrate the wound as early as at the sixth hour after injury providing its innate immune protection against pathogens, but also participating in reparative processes [14]. Being followed by lymphocytes and monocytes/macrophages they form an inflammatory phase of the healing cascade.

The significance of the inflammatory cells for the reparative processes is inappreciable, as they eradicate pathogens, remove wound debris and, partially, foreign bodies, but also supply the fracture gap with molecules regulating forthcoming cellular events. They also provide immune regulation of the healing, as the response of the lymph node draining the fracture gap was shown to reflect its cellular and molecular processes [35]. This mechanism seems to depend on regulatory B and T lymphocytes (Bregs and Tregs), as they were shown to participate in fracture healing. Bregs were presented to suppress the inflammatory phase secreting antiinflammatory cytokines IL-10 (interleukin-10) and TGF-β (transforming growth factor-β), and enhancing maturation of Tregs [15]. At the early phase of the reparative processes, they probably prevent from auto aggression against infiltrating progenitors, thus enabling them to proliferate and differentiate into bone forming cells. Depletion of Breg cells, analogically to splenectomy, results in the delay of the fracture healing [16, 17]. Moreover, T cells were shown to promote maturation of the osteoblasts [18].

Immune cells are an ample source of several molecular substances, including cytokines (i.e. IL-6 and IL-8) and growth factors (PDGF, fibroblast growth factor; FGF, TGF-β, and bone morphogenetic proteins—BMPs) [19]. Together with molecules released from nerve endings and bone marrow, they regulate cellular events stimulating proliferation and differentiation of MSCs.

The most effective MSCs stimulators are the granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage-colony-stimulating factor (GM-CSF), cytokines (IL-1, IL-3, IL-7, IL-8, and IL-12), stem cell factor (SCF), Flt3 (fms like tyrosine kinase 3) ligand, macrophage inflammatory protein-1 (MIP-1) and the chemokines GROβ (growth-regulated oncogene β; also known as CXC-2) and SDF-1 [20, 21]. Each of them evokes the unique effect promoting migration, division, activating synthesis, and release of molecules forming an appropriate environment or fulfilling the specialized biological function. They enable migration, multiplication, and differentiation of progenitors into desired cell lineage. The most potent stimulators of MSCs differentiation into, according to the local physicochemical environment, chondrocytes, or osteoblasts are TGF-β, several BMPs (2, 4, 6, 7, 13, and 14), IGF-1 (insulin-like growth factor), and FGF [22, 23]. The final outcome in the form of fracture healing results from the convergent actions of numerous factors influencing the target cells in an appropriate time sequence and place (**Figure 1**).
