**2. Material and methods**

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

*Clinical Implementation of Bone Regeneration and Maintenance*

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.

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.

**158**

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 surveyed participants in this test.

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, osteosynthesis was done by opening and draining the purulent chamber. Surgical treatment of purulent focus was conducted by conventional rules.

Permanent rigid fixation of bone fragments and permanent functional damage 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 wound healing, microbial composition of purulent discharge.

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

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 mandibular fractures make 34.5–39.7 days.

The main clinical indicators are as follows: the ratio in the group of patients by gender and by age, the amount of intraoperative blood loss, presence of concomitant pathology of the cardiovascular, respiratory, urinary systems and the gastrointestinal tract, and the presence of allergies and operations, which were previously carried out using steel structures in both groups significantly did not differ. Laboratory studies were performed preoperatively and after 3 and 10 days and 1 and 3 months after its execution. Blood was obtained in the morning fasting from subclavian vein using the phlebotomy method and from the cubital vein or veins on the back of the hand, to receive physiotherapy.

analyzers Vitek 2-compact and ATB-Expression (bioMérieux, France). Bacteriological examination most frequently detected *Staphylococcus aureus* (92–95% of cases), among others were *Pseudomonas aeruginosa*, *Burkholderia cepacia*, *Acinetobacter baumannii*, *Enterococcus faecalis* and *Proteus vulgaris* (the proportion of each of them

Statistical data processing was carried out using the "Microsoft Office Excel 2007" program and "Statistica for Windows v.6.1". It included an assessment of the data on the normality of the distribution in the test groups and was based on data on the mathematical expectation of 0 and the corresponding standard deviation equal to 1. Kolmogorov–Smirnov test, Lilliefors, Shapiro–Wilk and χ<sup>2</sup> were used to test the hypothesis that the sample has a Gaussian (normal) distribution. To compare the variance of the studied samples, F-Fisher criterion was used. Statistical hypothesis is considered confirmed with a significance level of p < 0.05. Modified theory of T. Bayes was used to identify the prediction criteria [56]. Calculation of diagnostic sensitivity, diagnostic specificity and diagnostic information content of immu-

nological tests was performed using the following Eqs. (1)–(3) [57].

Diagnostic sensitivity <sup>¼</sup> *TP*

Diagnostic specificity <sup>¼</sup> *TN*

Diagnostic informative laboratory test <sup>¼</sup> *TP*

**3. Results and discussion**

**consolidation**

CD3<sup>+</sup>


CD19<sup>+</sup>

CD45<sup>+</sup>

**161**

where TP is the true-positive results: the number of patients with complications who were correctly classified using this criterion; FP is the false-positive results: the number of patients without complications, which have been erroneously attributed to the number of patients with complications as a result of this test; TN is the truenegative results: the number of patients without the complications that were correctly classified with the help of this test; and FN is the false-negative results: the number of patients with complications who were misclassified using this test.

As a model for studying the dynamics of immunological parameters in normal and complicated osteogenesis, the study used laboratory parameters in patients with lesions of the facial skeleton. Injury of facial skeleton, especially with slow consolidation of bone tissue, leads to disturbances of body functions and generating esthetic defects. Therefore, the creation of forecasting system complications of osteogenesis in the recovery of fractures of the lower jaw becomes important. To address the issue of participation of immunological reactions in the restoration of bone tissue, first consider the dynamics of the main laboratory parameters during normal consolidation lesions of the mandible. Before operation values, immunolog-

ical parameters were compared to known literature data [58, 59].

**3.1 Features of immune responses of peripheral blood in normal bone**

Before surgery, along with a slight increase in the relative number of

(**Table 1**). There was also a decrease in the activity of oxygen-dependent (nitro blue


*TP* <sup>þ</sup> *FN* � <sup>100</sup> (1)

*TN* <sup>þ</sup> *FP* � <sup>100</sup> (2)

*TP* <sup>þ</sup> *FP* � 100 (3)

in the total number of cases was not more than 1%).

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

To assess the lymphocyte population composition, whole blood with K3EDTA anticoagulant (ethylenediaminetetraacetic acid) was used at a concentration of 1.6 mg/ml (tube S-Monovette® 2.7 ml, Sarstedt, Germany), for determining the phagocytic activity of the cells-the blood with the anticoagulant heparin Li (lithium heparin) at a concentration of 16 IU/ml (tube S-Monovette® 7.5 ml, Sarstedt, Germany). Serum was used for obtaining tubes S-Monovette® 7.5 ml (Sarstedt, Germany) with polystyrene beads as activators of coagulation.

To evaluate the immune status, a standard set of laboratory tests has been used complete with modern diagnostic methods [47]. The number of leukocytes was determined using hematology analyzer Cell-Dyn 1700 (Abbot, USA), reagents and equipment from the firm Abbott. Differentiation of population composition of leukocytes was carried out in the Romanovsky-Giemsa stained smears. Stab neutrophil and segmented neutrophil cell ratio was calculated [48]. Lymphocyte subpopulations were determined by flow cytometry on the device Coulter®Epics®XL (Beckman Coulter, USA), using a monoclonal antibody produced by Beckman Coulter. Lysis of erythrocytes was performed by using the sample preparation station Coulter®Q-Prep (Beckman Coulter, USA) and reagents Immunoprep, manufactured by Beckman Coulter. Quality control was performed using gauge particles Flow Check. In vitro activation of T lymphocytes with phytohemagglutinin was performed and evaluated in the reaction of inhibition of leukocyte migration [49]. The metabolic activity of neutrophils was evaluated in the reduction reaction of nitro blue tetrazolium peroxide radicals under the action of cells and evaluated by light microscopy [50]. Spontaneous and latex-stimulated activity was determined, their ratio was calculated (stimulation index). The ability of the neutrophils to kill was determined using cytochemical cell study data. Myeloperoxidase activity was determined by Grantham-Knoll [51], and the result was expressed by the average cytochemical coefficient of Kaplow [52]. The level of the lysosomal cationic cytoplasmic proteins was determined by reaction with bromophenol blue, and the results are also expressed as the average cytochemical factor [53]. The content of serum immunoglobulin classes A, M and G was determined by enzyme immunoassay. Lysozyme activity was evaluated by gel-diffusion test for the ability to lyse culture *M. lysodeikticus* [54], and the functional state of the complement system was adjusted to 50% by assessment of hemolysis of sheep erythrocytes [49]. Contents lactoferrin and cytokines (IL-1α, IL-8, TNF-α, IL-10 and IL-1ra) were determined using the method of two-site "sandwich" ELISA-variant using test systems from firms "Protein contour" (St. Petersburg, Russia), "Cytokine" (St. Petersburg, Russia), "Vector-Best" (Novosibirsk, Russia) and BioSourse International (USA) on immunoassay equipment Stat Fax (Awareness Technology Inc., USA). The concentration of C-reactive protein was determined by turbidimetric and ceruloplasmin - at Ravin [55].

The selection of antibiotic therapy in osteomyelitis was performed based on bacteriological examination of wound and aspirate purulent contents of the cavity. The evaluation included a microscopic examination of a Gram stain, inoculation of culture media production by bioMérieux (France). Identification of microorganisms and determination of their sensitivity to antibiotics were carried out by the

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

The main clinical indicators are as follows: the ratio in the group of patients by gender and by age, the amount of intraoperative blood loss, presence of concomitant pathology of the cardiovascular, respiratory, urinary systems and the gastrointestinal tract, and the presence of allergies and operations, which were previously carried out using steel structures in both groups significantly did not differ. Laboratory studies were performed preoperatively and after 3 and 10 days and 1 and 3 months after its execution. Blood was obtained in the morning fasting from subclavian vein using the phlebotomy method and from the cubital vein or veins on the

To assess the lymphocyte population composition, whole blood with K3EDTA anticoagulant (ethylenediaminetetraacetic acid) was used at a concentration of 1.6 mg/ml (tube S-Monovette® 2.7 ml, Sarstedt, Germany), for determining the phagocytic activity of the cells-the blood with the anticoagulant heparin Li (lithium heparin) at a concentration of 16 IU/ml (tube S-Monovette® 7.5 ml, Sarstedt, Germany). Serum was used for obtaining tubes S-Monovette® 7.5 ml (Sarstedt,

To evaluate the immune status, a standard set of laboratory tests has been used complete with modern diagnostic methods [47]. The number of leukocytes was determined using hematology analyzer Cell-Dyn 1700 (Abbot, USA), reagents and equipment from the firm Abbott. Differentiation of population composition of leukocytes was carried out in the Romanovsky-Giemsa stained smears. Stab neutrophil and segmented neutrophil cell ratio was calculated [48]. Lymphocyte subpopulations were determined by flow cytometry on the device Coulter®Epics®XL (Beckman Coulter, USA), using a monoclonal antibody produced by Beckman Coulter. Lysis of erythrocytes was performed by using the sample preparation station Coulter®Q-Prep (Beckman Coulter, USA) and reagents Immunoprep, manufactured by Beckman Coulter. Quality control was performed using gauge particles Flow Check. In vitro activation of T lymphocytes with phytohemagglutinin was performed and evaluated in the reaction of inhibition of leukocyte migration [49]. The metabolic activity of neutrophils was evaluated in the reduction reaction of nitro blue tetrazolium peroxide radicals under the action of cells and evaluated by light microscopy [50]. Spontaneous and latex-stimulated activity was determined, their ratio was calculated (stimulation index). The ability of the neutrophils to kill was determined using cytochemical cell study data. Myeloperoxidase activity was determined by Grantham-Knoll [51], and the result was expressed by the average cytochemical coefficient of Kaplow [52]. The level of the lysosomal cationic cytoplasmic proteins was determined by reaction with bromophenol blue, and the results are also expressed as the average cytochemical factor [53]. The content of serum immunoglobulin classes A, M and G was determined by enzyme immunoassay. Lysozyme activity was evaluated by gel-diffusion test for the ability to lyse culture *M. lysodeikticus* [54], and the functional state of the complement system was adjusted to 50% by assessment of hemolysis of sheep erythrocytes [49]. Contents lactoferrin and cytokines (IL-1α, IL-8, TNF-α, IL-10 and IL-1ra) were determined using the method of two-site "sandwich" ELISA-variant using test systems from firms "Protein contour" (St. Petersburg, Russia), "Cytokine" (St. Petersburg, Russia), "Vector-Best" (Novosibirsk, Russia) and BioSourse International (USA) on immunoassay equipment Stat Fax (Awareness Technology Inc., USA). The concentration of C-reactive protein was determined by turbidimetric and ceruloplas-

The selection of antibiotic therapy in osteomyelitis was performed based on bacteriological examination of wound and aspirate purulent contents of the cavity. The evaluation included a microscopic examination of a Gram stain, inoculation of culture media production by bioMérieux (France). Identification of microorganisms and determination of their sensitivity to antibiotics were carried out by the

back of the hand, to receive physiotherapy.

*Clinical Implementation of Bone Regeneration and Maintenance*

min - at Ravin [55].

**160**

Germany) with polystyrene beads as activators of coagulation.

analyzers Vitek 2-compact and ATB-Expression (bioMérieux, France). Bacteriological examination most frequently detected *Staphylococcus aureus* (92–95% of cases), among others were *Pseudomonas aeruginosa*, *Burkholderia cepacia*, *Acinetobacter baumannii*, *Enterococcus faecalis* and *Proteus vulgaris* (the proportion of each of them in the total number of cases was not more than 1%).

Statistical data processing was carried out using the "Microsoft Office Excel 2007" program and "Statistica for Windows v.6.1". It included an assessment of the data on the normality of the distribution in the test groups and was based on data on the mathematical expectation of 0 and the corresponding standard deviation equal to 1. Kolmogorov–Smirnov test, Lilliefors, Shapiro–Wilk and χ<sup>2</sup> were used to test the hypothesis that the sample has a Gaussian (normal) distribution. To compare the variance of the studied samples, F-Fisher criterion was used. Statistical hypothesis is considered confirmed with a significance level of p < 0.05. Modified theory of T. Bayes was used to identify the prediction criteria [56]. Calculation of diagnostic sensitivity, diagnostic specificity and diagnostic information content of immunological tests was performed using the following Eqs. (1)–(3) [57].

$$\text{Diagnostic sensitivity} = \left(\frac{\text{TP}}{\text{TP} + \text{FN}}\right) \times 100\tag{1}$$

$$\text{Diagnostic specificity} = \left(\frac{TN}{TN + FP}\right) \times 100\tag{2}$$

$$\text{Diagnostic information laboratory test} = \left(\frac{TP}{TP + FP}\right) \times 100\tag{3}$$

where TP is the true-positive results: the number of patients with complications who were correctly classified using this criterion; FP is the false-positive results: the number of patients without complications, which have been erroneously attributed to the number of patients with complications as a result of this test; TN is the truenegative results: the number of patients without the complications that were correctly classified with the help of this test; and FN is the false-negative results: the number of patients with complications who were misclassified using this test.
