**Abstract**

The study of immune responses of peripheral blood in bone regeneration for bone formation predicting complications is relevant. Studies were carried out on patients with the facial skeleton injury before and after stable osteosynthesis of the mandible in a fixing device for external fixation. Of the 136 patients, 17 people's bone tissue regeneration has been slow. Laboratory tests were carried out before and after the operation. The studies included the study of cellular immunity, humoral immunity, phagocytosis, acute phase proteins and cytokine status. The obtained data were processed using variation statistics methods and the modified theorem of T. Bayes. The study revealed that alternating stages of regeneration of bone tissue (inflammation, proliferation of osteoblasts, collagenogenesis and ossification) are accompanied by changes in the immunological status. A comparative study of the dynamics of immunological parameters at normal and slow osteogenesis had made it possible to establish criteria for delayed consolidation of bone tissue. Prognostic criteria before the operations include increasing the concentration of IgM and decreasing the concentration of C-reactive protein, in the early postoperative period-increasing the number of leukocytes, the concentration of tumor necrosis factor, IgM, as well as reducing the number of CD45<sup>+</sup> CD3<sup>+</sup> -cells, the complement activity and the amount of lactoferrin.

**Keywords:** monitoring of osteogenesis, prognostic criteria, nitro blue tetrazolium test, tumor necrosis factor, IgM, C-reactive protein

#### **1. Introduction**

Regulation of bone formation when damaged is regulated by a complex set of factors, including mechanical conditions for the formation of high-grade regeneration, vascular reactions, the influence of the neuroendocrine system, effects of metabolites and growth factors [1]. A reflection of the processes becomes dynamic of blood parameters, among which the most important are the changes in the immune status.

It is known that the immune system and bone tissue have broad functional and structural connections. The heterogeneity of cell population included in the immune system and the variety of regulators produced determine its ambiguous participation in the regulation of bone tissue regeneration.

Both macrophages and osteoclasts develop from the same progenitor cell of the monocytic line [2]. Both of these cells have the ability to destroy tissues, which leads to the activation of repair processes carried out by cells of mesenchymal origin, fibroblasts or osteoblasts. From stromal cells expressing the most important regulators of osteoclastogenesis and being a natural microenvironment for normal hematopoiesis, osteoblasts develop [3].

lymphocytes into the bone marrow [16]. These cells and bone tissue molecules, as it turned out, ultimately realize their effect on B lymphocytes through the transcription factor Pax5 (B-cell specific activator). In the absence of this factor, maturation of B lymphocytes does not occur. In this case, immature pre-B cells can differentiate into other cells of bone marrow origin, for example, into macrophages, osteoclasts and other cells [17]. The lack of mature lymphocytes in bone tissue is reliably associated with an increased volume of trabecular bone tissue [18]. It is interesting to note that under physiological conditions [19], as well as under the conditions of ontogenesis [20], the lack of mature B lymphocytes practically does not affect the cellular balance and metabolism in bone tissue. However, under pathological conditions or external stimuli, the absence of

*Immunological Monitoring of Osteogenesis Disorder DOI: http://dx.doi.org/10.5772/intechopen.92099*

mature B lymphocytes does not allow bone tissue to adequately respond to changing

Neutrophils, while not playing a key role in the repair phase, at the same time have an effect on collagenosis and remodeling of the extracellular matrix of bone tissue. They produce factors that activate fibroblasts and metalloproteinases (collagenases, gelatinases and stromalysin), which can catabolize all the main products of extracellular matrix and play an important role in remodeling of bone matrix [23]. When studying the state of non-specific protection among the victims of severe mechanical polytrauma, it was found that regardless of the nature of the traumatic agent, the degree and severity of the damage, in almost all cases, certain changes in the immune system were observed. For example, in the first week after an injury, the polymorphonuclear neutrophil system was functionally inferior [6]. Currently, researchers are inclined to believe that the complex interactions between individual cells of the immune system during the entire post-traumatic period have not been sufficiently studied. However, it is widely known that bone damage, like any other traumatic effect, leads to an increase in the migratory ability of polymorphonuclear neutrophils [24]. At the same time, the mechanism, according to which a sharp decrease in this migratory ability is noted several times throughout the posttraumatic period, remains unclear [25]. A study of the role of polymorphonuclear leukocytes by other scientists showed that a decrease in the number of cells led to a significant decrease in the mechanical strength of the regeneration. This, according to the authors, was associated with the outflow of neutrophils into the focus of inflammation (hematoma revascularization), which was accompanied by the release of a large number of inflammatory mediators (serotonin, bradykinins and histamine). This process violated the revascularization of the forming bone callus

When bone tissue is damaged, reactive changes are noted not only in the cellular

but also in the humoral component of the immune system. It was found that activated lymphocytes responded with a significant reduction in proliferation when interacting with the main component of the extracellular matrix-collagen both in vitro and in vivo [27]. It is also interesting to note that bone tissue injury leads to the production of autoantibodies to oxidized low-density lipoproteins, and the concentration of these autoantibodies is quite strongly related to the intensity of post-traumatic osteogenesis [28]. In the framework of the study of immunoregulation of osteogenesis, a violation of the synthesis of immunoglobulins by lymphocytes with damage to bone tissue was established [29]. Some authors also obtained data on a decrease in the synthesis of immunoglobulins class G in severe mechanical

An important role in the regulation of bone tissue regeneration belongs to neutrophils. The impact of a damaging factor on the tissues leads to the development of acute inflammation, the main effectors of which are these cells. The out-

come of the process largely depends on the reactivity of neutrophils [22].

and led to a decrease in its mechanical strength [26].

conditions [21].

polytrauma [6].

**157**

Quantitative and qualitative changes in immunocompetent cells are due to lymphocyte-macrophage regulation of osteogenesis and are reflected regeneration of the bone-favorable or delayed.

An important role in the regulation of bone tissue regeneration belongs to immune cells-lymphocytes. In an animal experiment, it was shown that thymectomy leads to a significant delay in osteogenesis after a bone fracture [4]. Different lymphocyte populations influence the regulation of bone tissue regeneration ambiguously: when analyzing the cellular composition of the regeneration zone in difficultly fused fractures, a significant decrease in the number of T cells, the absence of mast cells and the predominance of fibroblasts and mononuclear cells was revealed [5]. There are reports that with severe mechanical polytrauma, deep disturbances of the cellular immunity are observed in the form of inhibition of the expression of CD2DR+ receptors and a decrease in the absolute number of cells with CD4<sup>+</sup> and CD8+ phenotypes [6]. It was also shown that the activity of NK cells is significantly reduced among the patients in the fracture zone, as well as in peripheral blood [7]. A number of authors have found that in animals with a deficiency of mature T lymphocytes, physiological metabolism in bone tissue is not disturbed [8]. However, under conditions when there was a need to increase bone metabolism (in trauma and after surgery), deficiency of T-lymphocyte function led to the formation of demineralized bone matrix and loss of density of existing bone tissue [9]. Clinical studies had also confirmed the role of T lymphocytes in the regulation of bone density. It was revealed that both CD4+ and CD8<sup>+</sup> cells can produce factors that affect osteoclastogenesis [10]. There are a number of studies that show the inhibitory role of T lymphocytes in relation to the formation of osteoclasts and resorptive changes in the bone [11]. It is indicated that activated T lymphocytes can reduce osteoclastogenesis by producing INF-γ. In vitro, the removal of CD8+ T lymphocytes from a cell culture containing both osteoclast precursors and bone marrow cells significantly increases the formation of mature osteoclasts [12]. Some in vivo data indicate the involvement of the prostaglandin mechanism in this process [13]. In various pathological conditions, the nature of the influence of T lymphocytes on osteoclastogenesis is determined by both a set of locally secreted cytokines and the degree of differentiation of T lymphocytes [14]. Bone resorption caused by some cytokines secreted by T lymphocytes is often accompanied by further increased bone tissue formation, which leads to an increase in bone metabolism [15]. The direct molecular mechanisms of influence, as well as the conditions that determine the stimulating or inhibitory role of T lymphocytes in the regulation of bone tissue regeneration, are under study.

B lymphocytes are also involved in the regulation of bone tissue regeneration. In the bone marrow, the earliest precursors of B lymphocytes are in close contact with the endosteal surface of the bone, and the most mature cells of this series are located in the center of the bone marrow. Such a spatial organization of B lymphopoiesis in the bone marrow suggests that the cells of the osteoblastic germ located in the endosteum, as well as stromal cells located in the connective tissue stroma of the bone marrow, produce factors that influence the early precursors of B lymphocytes. It has been suggested that molecules like vascular cell adhesion molecules (VCAMs), expressed on the surface of stromal cells, mediate the binding of lymphocyte precursors, predominantly B lymphocytes, since the latter form an essential part of the entire lymphoid component of the bone marrow. It was also revealed that this molecule plays an important role in the phenomenon of homing of

#### *Immunological Monitoring of Osteogenesis Disorder DOI: http://dx.doi.org/10.5772/intechopen.92099*

to the activation of repair processes carried out by cells of mesenchymal origin, fibroblasts or osteoblasts. From stromal cells expressing the most important regulators of osteoclastogenesis and being a natural microenvironment for normal hema-

*Clinical Implementation of Bone Regeneration and Maintenance*

Quantitative and qualitative changes in immunocompetent cells are due to lymphocyte-macrophage regulation of osteogenesis and are reflected regeneration

An important role in the regulation of bone tissue regeneration belongs to immune cells-lymphocytes. In an animal experiment, it was shown that thymectomy leads to a significant delay in osteogenesis after a bone fracture [4]. Different lymphocyte populations influence the regulation of bone tissue regeneration ambiguously: when analyzing the cellular composition of the regeneration zone in difficultly fused fractures, a significant decrease in the number of T cells, the absence of mast cells and the predominance of fibroblasts and mononuclear cells was revealed [5]. There are reports that with severe mechanical polytrauma, deep disturbances of the cellular immunity are observed in the form of inhibition of the expression of CD2DR+ receptors and a decrease in the absolute number of cells with CD4<sup>+</sup> and CD8+ phenotypes [6]. It was also shown that the activity of NK cells is significantly reduced among the patients in the fracture zone, as well as in peripheral blood [7]. A number of authors have found that in animals with a deficiency of mature T lymphocytes, physiological metabolism in bone tissue is not disturbed [8]. However, under conditions when there was a need to increase bone metabolism (in trauma and after surgery), deficiency of T-lymphocyte function led to the formation of demineralized bone matrix and loss of density of existing bone tissue [9]. Clinical studies had also confirmed the role of T lymphocytes in the regulation of bone density. It was revealed that both CD4+ and CD8<sup>+</sup> cells can produce factors that affect osteoclastogenesis [10]. There are a number of studies that show the inhibitory role of T lymphocytes in relation to the formation of osteoclasts and resorptive changes in the bone [11]. It is indicated that activated T lymphocytes can reduce osteoclastogenesis by producing INF-γ. In vitro, the removal of CD8+ T lymphocytes from a cell culture containing both osteoclast precursors and bone marrow cells significantly increases the formation of mature osteoclasts [12]. Some in vivo data indicate the involvement of the prostaglandin mechanism in this process [13]. In various pathological conditions, the nature of the influence of T lymphocytes on osteoclastogenesis is determined by both a set of locally secreted cytokines and the degree of differentiation of T lymphocytes [14]. Bone resorption caused by some cytokines secreted by T lymphocytes is often accompanied by further increased bone tissue formation, which leads to an increase in bone metabolism [15]. The direct molecular mechanisms of influence, as well as the conditions that determine the stimulating or inhibitory role of T lymphocytes in

the regulation of bone tissue regeneration, are under study.

**156**

B lymphocytes are also involved in the regulation of bone tissue regeneration. In the bone marrow, the earliest precursors of B lymphocytes are in close contact with the endosteal surface of the bone, and the most mature cells of this series are located in the center of the bone marrow. Such a spatial organization of B lymphopoiesis in the bone marrow suggests that the cells of the osteoblastic germ located in the endosteum, as well as stromal cells located in the connective tissue stroma of the bone marrow, produce factors that influence the early precursors of B lymphocytes.

It has been suggested that molecules like vascular cell adhesion molecules

that this molecule plays an important role in the phenomenon of homing of

(VCAMs), expressed on the surface of stromal cells, mediate the binding of lymphocyte precursors, predominantly B lymphocytes, since the latter form an essential part of the entire lymphoid component of the bone marrow. It was also revealed

topoiesis, osteoblasts develop [3].

of the bone-favorable or delayed.

lymphocytes into the bone marrow [16]. These cells and bone tissue molecules, as it turned out, ultimately realize their effect on B lymphocytes through the transcription factor Pax5 (B-cell specific activator). In the absence of this factor, maturation of B lymphocytes does not occur. In this case, immature pre-B cells can differentiate into other cells of bone marrow origin, for example, into macrophages, osteoclasts and other cells [17]. The lack of mature lymphocytes in bone tissue is reliably associated with an increased volume of trabecular bone tissue [18]. It is interesting to note that under physiological conditions [19], as well as under the conditions of ontogenesis [20], the lack of mature B lymphocytes practically does not affect the cellular balance and metabolism in bone tissue. However, under pathological conditions or external stimuli, the absence of mature B lymphocytes does not allow bone tissue to adequately respond to changing conditions [21].

An important role in the regulation of bone tissue regeneration belongs to neutrophils. The impact of a damaging factor on the tissues leads to the development of acute inflammation, the main effectors of which are these cells. The outcome of the process largely depends on the reactivity of neutrophils [22]. Neutrophils, while not playing a key role in the repair phase, at the same time have an effect on collagenosis and remodeling of the extracellular matrix of bone tissue. They produce factors that activate fibroblasts and metalloproteinases (collagenases, gelatinases and stromalysin), which can catabolize all the main products of extracellular matrix and play an important role in remodeling of bone matrix [23]. When studying the state of non-specific protection among the victims of severe mechanical polytrauma, it was found that regardless of the nature of the traumatic agent, the degree and severity of the damage, in almost all cases, certain changes in the immune system were observed. For example, in the first week after an injury, the polymorphonuclear neutrophil system was functionally inferior [6]. Currently, researchers are inclined to believe that the complex interactions between individual cells of the immune system during the entire post-traumatic period have not been sufficiently studied. However, it is widely known that bone damage, like any other traumatic effect, leads to an increase in the migratory ability of polymorphonuclear neutrophils [24]. At the same time, the mechanism, according to which a sharp decrease in this migratory ability is noted several times throughout the posttraumatic period, remains unclear [25]. A study of the role of polymorphonuclear leukocytes by other scientists showed that a decrease in the number of cells led to a significant decrease in the mechanical strength of the regeneration. This, according to the authors, was associated with the outflow of neutrophils into the focus of inflammation (hematoma revascularization), which was accompanied by the release of a large number of inflammatory mediators (serotonin, bradykinins and histamine). This process violated the revascularization of the forming bone callus and led to a decrease in its mechanical strength [26].

When bone tissue is damaged, reactive changes are noted not only in the cellular but also in the humoral component of the immune system. It was found that activated lymphocytes responded with a significant reduction in proliferation when interacting with the main component of the extracellular matrix-collagen both in vitro and in vivo [27]. It is also interesting to note that bone tissue injury leads to the production of autoantibodies to oxidized low-density lipoproteins, and the concentration of these autoantibodies is quite strongly related to the intensity of post-traumatic osteogenesis [28]. In the framework of the study of immunoregulation of osteogenesis, a violation of the synthesis of immunoglobulins by lymphocytes with damage to bone tissue was established [29]. Some authors also obtained data on a decrease in the synthesis of immunoglobulins class G in severe mechanical polytrauma [6].

Lactoferrin is an important regulator of osteocyte activity that increases bone formation in vivo [30]. Usually, lactoferrin is secreted under the influence of stimuli caused by inflammation, as it is contained in neutrophil secretory granules [31]. Lactoferrin affects the synthesis of chemokines, plays an important immunomodulatory function to reduce the high concentration of osteolytic cytokines such as TNF-α and IL-1α [32] and stabilizes binding complexes [33]; therefore, its direct effects on the activity and development of osteocytes are apparently supplemented by these intermediary effects [30]. Reliable data have now been obtained, and it shows that lactoferrin stimulates osteoblastic growth and acts as a powerful factor in the survival of osteoblasts, preventing cell apoptosis. In addition, lactoferrin enhances the function of differentiated osteoblasts [34]. The effect of lactoferrin on the development of osteoclasts was evaluated in mouse's bone marrow cultures; as a result, it was found to exceed the response to such strong growth factors as insulin, amylin, IL-18, adrenomedullin, C-terminal telopeptides and calcitonin [35]. After local injection of lactoferrin, active bone growth was established, as well as the fact that it is a powerful factor in osteoblast growth, which can reduce bone resorption [30]. Lactoferrin acts on preosteoclasts and a large number of mature cells of this origin; however, it has no effect on bone resorption by isolated mature osteoclasts [30]. There is also a report on the effects of lactoferrin leading to bone resorption, which demonstrates that lactoferrin reduces bone function: a mixed rabbit bone cell culture is resorbed in a manner independent of RANK (receptor activator of NF-kB)/RANKL (receptor activator of NF-kB ligand)/OPG (osteoprotegerin) system [36]. Obviously, the identification of the mechanisms by which lactoferrin acts on bone cells is important due to the revealed possibilities of the demonstrated effects, and therefore this direction is being actively studied. The putative lactoferrin receptor is known to have been identified [37]. There is evidence that lactoferrin acts through a receptor-bound protein related to the low-density lipoprotein family (LRP) [38]. Identification of the LRP1 receptor as a functional lactoferrin receptor in osteoblasts explains the interaction mechanism and makes it possible to regulate the physiological or pharmacological effects on the bone, as well as the introduction of vitamins and other necessary substances into the bone tissue [39]. The same applies to the revealed LRP5 and LRP6 receptors, structurally associated with LRP1, which are necessary regulators of osteoblast functions [40]. Thus, lactoferrin, on the one hand, provides bone growth and, on the other hand, can perform the therapeutic function of a local agent to restore bone integrity after damage.

**2. Material and methods**

*Immunological Monitoring of Osteogenesis Disorder DOI: http://dx.doi.org/10.5772/intechopen.92099*

surveyed participants in this test.

The study included 136 patients with lesions of the facial skeleton before and after stable osteosynthesis of the mandible in a fixing device for external fixation. Limitation of the injury was on average 12.0 3.0 days. Indications for the use of external fixation devices in damaged bone were complicated by primary and secondary shifts: mandibular fractures, in 92 persons (67.6%); disjoint fractures, in 20 persons (14.7%); fused fractures, in 21 persons (15.4%); and gunshot defects, in 3 people (2.2%). A study was authorized by the Ethics Committee, guided by the order of operation, standard operating procedures and international instruments, which are based on "Declaration of Helsinki of the World Medical Association" and its subsequent editions, UN documents and of the Council of Europe documents relating to the rights of the patient, Consolidated Guideline for Good Clinical Practice and the National Russian Federation Standard "Good Clinical Practice" GOST R 52379-2005 from 01.04.2006. Informed consent to participate in a research project in accordance with the "Statement of Ethical Control" had been obtained from all

The diagnosis is based on clinical and instrumental learning techniques using X-ray data, orthopantomography and CT. The volume of surgical intervention determines the nature and localization of lesions. External or intraoral access was used. The second type of access was a priority, since its implementation in the postoperative period was excluded orostoma development. The inner edge of the subsystems was fixed at 1 cm from the skin surface to avoid damaging the soft tissue. An important moment during the use of external fixation device was to restore a fractured bone axis creating contact bone fragments across the fracture surface and compression at the junction of the bone fragments. Some patients have

been diagnosed with osteomyelitis. When the osteomyelitis was detected,

treatment of purulent focus was conducted by conventional rules.

wound healing, microbial composition of purulent discharge.

term results of treatment patients were followed up to 4 years.

lar fractures make 34.5–39.7 days.

**159**

osteosynthesis was done by opening and draining the purulent chamber. Surgical

to the load of the lower jaw were created to organize the normal course of the recovery process and a successful fight against purulent infection. These conditions of bone tissue restoration were achieved by the periodic-every 5–7 days-tension of weakened rods and displacement of rings of external retainer relative to each other.

When dressing wounds and fistulas, great importance had been attached to providing constant drainage of purulent separable. Medical dressings are replaced in a timely and high-quality manner, taking into account the nature and stage of

An apparatus for external fixation after treatment was taken off when the following clinical signs of coalescence bone appeared: disappearance of soft tissue swelling, lack of mobility at the junction of the bone fragments during the clinical trial on the motility and on the basis of radiographic criteria: identifying fuzziness of contour ends of the fragments and improving optical densities in the gap region of bone damage. Terms of use of metal structures averaged 15.0 3.0 days. Long-

Of the total patients (136 people) based on retrospective analysis of data, a group with slow regeneration of bone tissue was isolated, because of osteomyelitis presence. It amounted to 17 people or 12.5% of all patients. Consolidation of bone tissue in this group was observed after an average 43.0 1.0 postoperative day. The control group consisted of patients whose postoperative period was uneventful. Their consolidation of bone tissue occurs on average through 29.0 2.0 days after the imposition of an external fixation device. It occurred somewhat earlier than in the treatment with the other ways. Usually, the terms of consolidation of mandibu-

Permanent rigid fixation of bone fragments and permanent functional damage

Integrins for extracellular matrix proteins also take part in the regulation of bone tissue regeneration. These receptors are a substrate for adhesion, migration and differentiation of fibroblasts and osteogenic cells [41]. Receptors provide a link between the cytoskeleton and the extracellular matrix, transmitting information about stretching and compression of bone tissue through, the cell membrane [42] and activate certain signaling pathways, affecting gene expression [43]. It was found that during the recirculation process, when the cells of the immune system migrate through tissues and interact with one of the main components of the extracellular matrix (collagen), and integrins act as receptors, the activation signal is combined with an antigen-recognizing receptor and is able to change the direction of action of immunocompetent cells [44]. It is known that the metabolism of bone tissue during damage is provided by numerous cytokines-IL-1 (interleukin), IL-3, IL-4, IL-6, IL-11, TNF-α (tumor necrosis factor), TNF-β, colony stimulating factors, leukemia inhibitory factor, INF-γ (interferon), TGF-β (transforming growth factor) [45, 46]. There is no doubt that the study of blood immune responses in patients with damage to the bone will assess their relationship with the passage of bone formation and find the results obtained practical application. Thus, current is the study of blood immunological reactions during the regeneration of bone tissue to predict complications osteogenesis.
