The Role of the Immune Cells in Fracture Healing

Long-bone fracture healing is likened to endochondral ossification seen during embryonic development, but a key difference is the presence of an inflammatory phase during the former. When a fracture occurs, there is a local disruption of the vascularization and soft tissue. In response to this vascular injury, a hematoma forms and will act as the future template for callus formation [11]. Immune cells, including platelets, neutrophils, and macrophages are then recruited to the site and activated [12, 13]. These cells invade the hematoma and secrete growth factors and cytokines, which help to recruit mesenchymal cells. The hematoma is reorganized and there is deposition of a fibrin thrombus [11, 12]. As capillaries invade the thrombus, granulation tissue replaces the fibrin clot. Neutrophils and macrophages remove dead cells and debris [14]. They also release factors that promote the recruitment of mesenchymal progenitor cells that originate from the periosteum, bone marrow, and systemic circulation [15, 16]. These cells, in turn, have an immunosuppressive character which helps to resolve the inflammation at the site and prepare it for the next stage of healing [17‐21].

During the inflammatory phase, mesenchymal progenitor cells are recruited to the site of injury and undergo chondrogenic differentiation. It is the decreased mechanical stability at the fracture site that promotes this chondrogenesis. The granulation tissue is replaced by a fibrocatilaginous callus which poses a semi-rigid quality to provide mechanical support [8, 11]. Initially, this cartilaginous callus is largely avascular; however, as healing proceeds, the callus is invaded by endothelial cells, promoting angiogenesis [22]. This induces terminal differentiation of chondrocytes, resulting in hypertrophy and the production of mineralized cartilaginous matrix [8, 23]. The fate of these chondrocytes is now debated as either undergoing apoptosis or undergoing trans-differentiation/dedifferentiation to osteogenic cells [24].

Upon calcification of the fracture callus, osteoprogenitor cells are recruited from the periosteum, bone marrow, vasculature, and surrounding tissue to initiate osteogenesis and the deposition of bone onto the calcified cartilage [8]. Simultaneously, osteoclasts are activated at the site and begin to resorb the cartilaginous callus. This results in the replacement of the cartilaginous callus with the bony callus, which is composed of woven bone and provides greater stability than the fibrocartilaginous callus [25]. Macrophages and T and B cells have been shown to play a role during mineralization (discussed below); however, their function has yet to be elucidated.

The remodeling phase marks the last stage of fracture repair. Woven bone within the callus is replaced with laminar bone, consisting of a highly organized matrix of collagen fibers; therefore restoring the original structure and function of the bone [11]. This process is driven by osteoclast-­-mediated bone resorption followed by osteoblast-­-mediated bone formation [8].

As described, fracture repair is a complex process requiring a well-organized response from multiple cell-types. During the inflammatory phase; clot formation, tissue granulation, and cell recruitment are necessary first steps which are dependent on the coordination of various immune cells. Throughout the different phases, hematopoietic cells appear to direct mesenchymal cell differentiation and activity. New information is rapidly being uncovered as osteoimmunity is a developing field. The following is a summary of immune cell types and their observed roles in fracture healing.

Hematopoietic cells arise from the mesoderm during embryonic development and locate to numerous sites in the human body. Along with the spleen, the bone marrow serves as a primary source for hematopoietic cells during adulthood: from within it all cells of the hematopoietic lineage can be differentiated. These cells play important roles in staving off infection and identifying foreign bodies; they are divided into two groups: cells of the lymphoid lineage and cells of the myeloid lineage. The majority of the cells located within the bone marrow cavity remain in a quiescent, multipotent state and are activated upon stimulus. Bone injury often results in bleeding; damage to the local vasculature serves as an activation step to recruit and activate these cells.

Platelets are non-nucleated cells of the myeloid lineage. Their primary function lies in blood clotting; however, they have been shown to have a role in fracture healing [26]. Soon after injury, circulating platelets arrive at the affected site and are activated by the thrombin released in response to injured vasculature. Activated platelets take part in creating the fibrin thrombus. This acts as a scaffold for cellular engraftment as platelets secrete inflammatory cytokines (IL-1, IL-6, TNF-α) and growth factors (PDGF, TGF-beta) to recruit other immune cells (neutrophils and monocytes) and mesenchymal progenitor cells respectively [27‐29].

Neutrophils are phagocytic cells of the myeloid lineage. As described, neutrophils are recruited by IL-1, TNF-α secreted by platelets [30, 31]. The role of neutrophils in fracture healing is still being elucidated and involves many aspects of tissue repair. During early stages of the inflammatory phase, neutrophils have been shown to contribute to the fibrin thrombus by depositing a fibronectin matrix [32]. During the later stages of the inflammatory phase, neutrophils take part in removing cellular and tissue debris and are implicated in removal of the thrombus [33‐35]. However, their most significant role seems to involve the secretion of cytokines (IL-1, IL-6, IL-10, TNF-α, MCP-1, CXCL-1α, MIP-1) to attract monocytes, which will differentiate to macrophages [36‐39].

Macrophages are phagocytic cells of the myeloid lineage. They are differentiated from monocytes. These cells play an integral part in bone homeostasis as well as bone fracture repair. During homeostasis, macrophages likely act as niche cells to osteoblasts and to osteoclasts, taking part in the crosstalk and communication to maintain the balance in bone remodeling. Indeed, our work shows that ablation of macrophages retards early skeletal growth and development resulting in decreased trabecular number and decreased bone mineral density and later leads to osteoporosis [40•].

The importance of macrophages in fracture healing is still being investigated. The depletion of macrophages during bone fracture healing delays bone union [5, 40•, 41]. Fracture calluses from mice in which macrophages had been ablated developed smaller, under-mineralized fracture calluses with increased amounts of fibrotic tissue. Furthermore, depletion of macrophages decreased the number of mesenchymal progenitor cells and inhibited the ability of these cells to differentiate to osteoblasts [40•, 42••].

In fracture healing, monocytes are recruited to the site of injury by MIP-1 (also known as CXCL2) primarily, and by IL-1 and TNF-α. They subsequently differentiate to macrophages that have a sliding scale of functional attributes dependent on their “polarization”, which is induced by extracellular signals and is thought to be reversible in vivo [43]. At one end, macrophages undergo programming to become classically activated M1 macrophages when exposed to inflammatory cytokines (IL-1, TNF-α). These are inflammatory macrophages that further secrete IL-1, IL-6, TNF-α, MCP-1, and MIP-1 to maintain recruitment of monocytes. They perform phagocytosis to remove necrotic cells as well as the fibrin thrombus [45]. At the other end of the sliding scale are the alternatively activated M2 macrophages, which are functional after exposure to IL-4. These cells initiate an anti-inflammatory response in the later stages of inflammation as they secrete tissue repair signals (IL-10, TGF-beta, BMP-2, and VEGF), recruit mesenchymal progenitor cells, induce osteochondral differentiation, and prompt angiogenesis [46‐50].

The importance of macrophages in tissue homeostasis has be confirmed in other tissues as well [44‐47]. Interestingly, tissue-resident macrophages have been found to be of benefit to tissue health while macrophages derived from circulating monocyte have been found to be less efficient [48]. Recently, the existence of tissue resident macrophages in bone (termed Osteomacs) has been proposed [42••, 49]. The comparative importance of tissue-resident versus monocyte-driven macrophages in bone biology has yet to be elucidated.

Osteoclasts are multinucleated cells of the myeloid lineage; they differentiate directly from monocytes, although they can also arise from macrophages [50].

Although osteoclasts are not traditionally thought of as immune cells, they are able to act as innate immune cells within bone as inflammatory signals lead to differentiation and activation of osteoclasts [51].

Osteoclasts are specialized cells as they are the only cells that resorb bone matrix [52]. Their primary role is that of a ‘bone phagocyte’. They are responsible for debridement, resorption of the cartilaginous callus, resorption of the bony callus, resorbing the tunnels required for vasculature and nerves, and together with osteoblasts balance bone remodeling [25]. Upon activation, osteoclasts adhere to the bone surface, form a ruffled border, and create a tight seal with the mineralized surface termed a resorption pit [52]. This sealed compartment is then acidified by pumping in hydrogen ions to dissolve the hydroxyapatite crystal and lysosomal enzymes are secreted into the resorption pit to digest the proteinaceous material.

Recently, studies have further elucidated the immune cell signaling that regulates osteoclast activation. During fracture healing, monocytes are recruited to the site and differentiate to osteoclasts. Osteoclasts, which reside on mineralized bone surfaces, are primarily activated by receptor activator of nuclear factor kappa-B ligand (RANKL) binding to the osteoclast cell surface receptor RANK. Osteoblasts appear to be the primary source of RANKL during homeostasis and fracture healing; however, NK cells and activated T cells are also able to produce RANKL during fracture healing. Conversely, osteoprotegerin (OPG) is a decoy receptor that binds to RANK and inhibits RANKL binding, thereby preventing osteoclast activation. OPG is secreted by osteoblasts during homeostasis and fracture healing and by B cells during fracture healing.

This inflammatory signaling combines with a resorption-based signaling to create the communication mechanism that regulates bone remodeling. As osteoclasts resorb the bone matrix, proteins such as bone sialoprotein and osteopontin, which are intercalated within the mineralized matrix and bound to collagen and hydroxyapatite, are freed and able to signal to local osteoblasts as the RGD motif of these proteins binds to the α_vβ₃ cell surface receptor [53, 54].

T lymphocytes and B lymphocytes (also known as T cells and B cells) are hematopoietic cells of the lymphoid lineage. They constitute the two cell types of adaptive immunity and while their lineage can be further classified and subdivided, for the purpose of this review they will simply be classified as T cells and B cells. Depletion of T cells or of B cells leads to diminished bone health and decreased fracture healing [55, 56]. Mice lacking T cells and B cells have been shown to have stiffer bones that are more susceptible to fracture [57].

T cells and B cells seem to have cell-signaling roles near the end of the inflammatory phase and again during the mineralization phase [58]. During later stages of the inflammatory phase, T cells produce RANKL to recruit, differentiate, and activate osteoclasts; likely in an effort to remove the fibrin thrombus in preparation of the cartilaginous callus. During this time, B cells are involved in suppression of the pro-inflammatory signals IFN-γ, TNF-α, and IL-2 [59]. Concurrently, B cells produce OPG, thereby regulating osteoclastic differentiation and activity [60‐62].

Recent findings have placed more attention on the role of the cytokine IL-17 secreted by T cells. IL-17 has been shown to be an immunomodulator, able to induce anti-inflammatory functions from mesenchymal stromal cells [63] and has been shown to induce osteogenic differentiation and activity, aiding in osteoblast maturation [56]. Furthermore, IL-17 plays a role during the remodeling phase of the fracture callus as it increases expression and secretion of RANKL, leading to enhanced proliferation and activation of osteoclasts [64].

Natural killer cells (NK cells) are hematopoietic cells of the lymphoid lineage. The immunological function of NK cells is to recognize foreign or virally infected cells and induce apoptosis or cell lysis through cytotoxic granules [65]. Little is known about the function of natural killer cells in fracture healing. It is possible that NK cells play a role in removal of damaged cells located at the site of injury; however, conditions at the fracture site have been shown to inhibit NK cell-based cell lysis [66]. It is more likely that NK cells play a signaling role in debridement of the injured tissue recruiting inflammatory cells and osteoclasts as they are known to produce IFN-γ and RANKL [67]. NK cells may also play a role in tissue deposition through recruitment of mesenchymal progenitor cells at a later stage of fracture repair as they secrete CXCL7 [68].