**4. Extracellular matrix mineralization**

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

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

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

monocytes/macrophages they form an inflammatory phase of the healing cascade.

osteoblasts or chondroblasts.

6 Trauma Surgery

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

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.
