RU2622369C1 - Chronic traumatic osteomyelitis simulation method - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: to simulate traumatic osteomyelitis of long bones in aseptic conditions, a cavity is created in the metaepiphyseal area of the laboratory animal bone under anesthesia. A turunda moistened with 1% Aethoxysklerol solution and fragments of autologous bone are placed into this cavity, with subsequent cavity infection with the Staphylococcus aureus culture. Infection is performed on day 7 by introduction of a piece of an overnight Staphylococcus aureus culture in 2% solution of agar-agar into the resulting cavity. At that, the hole in the bone is sealed by ercodont cement, and the wound is poured by amoxiclav powder.

EFFECT: method provides simulation of a long, rapidly flowing osteomyelitic process, accompanied by fistulas, and the ability to maintain it for such a long time, which is necessary for the study and objective evaluation of the process using the means and methods of its treatment.

Description

The invention relates to medicine, namely to experimental surgery and traumatology, and can be used to model traumatic osteomyelitis of the tubular bones, bringing it closer to the clinical conditions for studying the pathogenesis, clinic and justification of the treatment of the disease.

In modern medicine, chronic osteomyelitis is understood as an “infectious inflammatory process involving structural elements of the bone, i.e. non-specific purulent-inflammatory and purulent-necrotic lesions of bone tissue, bone marrow, periosteum and surrounding soft tissues, with a protracted recurrent course "(Amiraslanov Yu.A. Modern principles of surgical treatment of chronic osteomyelitis (lecture) / Yu.A. Amiraslanov, AM Svetukhin , IV Borisov // Infection in surgery. - 2004. - T. 22. - P. 8-13; Gostishchev VK. Prediction of changes in the strength of long tubular bones in surgery of chronic osteomyelitis / V.K. Gostishchev // Surgery. - 2010. - No. 2. - S. 4-6). The term "osteomyelitis" belongs to Reynaud (1831). Despite the fact that in Greek “osteomyelitis” means inflammation of the bone marrow only, the term has firmly entered into surgical practice and is used to refer to the non-specific purulent-inflammatory process of all bone structures and surrounding soft tissues (Akzhigitov G.N. Osteomyelitis / T .N. Akzhigitov, VG Galeev, VG Sakhautdinov. - M., 1986. - 208 p .; Akzhigitov T.N. Hematogenous osteomyelitis / G.N. Akzhigitov, Ya.B. Yudin. - M ., 1998. - 287 p .; Nikitin GD Surgical treatment of osteomyelitis / GD Nikitin, A.V. Cancer, S.A. Linnik. - St. Petersburg, 2000. - 286 p.). The problem of treating patients with chronic osteomyelitis continues to be one of the most complex and relevant among the diseases of the musculoskeletal system (Alekseev D.G. Chronic osteomyelitis: features of complex treatment at the present stage / D.G. Alekseev, I.V. Ishutov, V.E. Batakov // Young scientists of the region’s health care: materials of scientific - practical conference - Saratov, 2005. - P. 237-238; Borisov IV Modern methods of surgical treatment of osteomyelitis of long bones in adults: abstract of diss. ... d -ra of medical sciences / I.V. Borisov. - M., 2007. - 48 p .; Vasiliev S .L. Revascularization operations in the treatment of patients with chronic osteomyelitis of the lower extremity: abstract of thesis ... Candidate of Medical Sciences: 01/14/17 / S.L. Vasiliev. - Novosibirsk, 2010. - 23 pp.; Gostischev V.K. Infections in Surgery, A Guide for Doctors / V.K. Gostischev, Moscow: GEOTAR-Media, 2007. 768 pp .; Sonis A.G. Results of the Use of Gravity Therapy in the Treatment of Patients with Lower Limb Osteomyelitis / A.G. Sonis // Bulletin of experimental and clinical surgery. - 2010. - T 3, No. 4. - S. 377-384). This is due to the peculiarities of localization of osteomyelitis cavities, the severity of the clinical course of the pathological process, and the high rate of relapse of the disease. Patients are often repeatedly subjected to unsuccessful surgical interventions (Goryunov S.V. Purulent surgery: atlas / S.V. Goryunov, D.V. Romashov, I.A. Butivshchenko. - M., 2004. - 219 p .; Usik S. F. Osteomyelitis: Clinic, Diagnostics, Treatment / S.F. Usik, M.M. Fedoseev, A.N. Bratiychuk.-Saratov, 2007.- 96 s .; Bhavan K.P. Hematogenous vertebral osteomyelitis / K.P. Bhavan, N. Kinnani // Mo Med. 2009. - Vol 106, No. 4. - P. 277-82).

Improving the quality of treatment is associated with the introduction of new treatment methods and techniques into practice. An important stage in their development is an experimental study, which is based on modeling of the pathological process in animals.

A known method of modeling osteomyelitis S.M. Derizhanova (Vengerovsky I.S. Osteomyelitis in children / I.S. Vengerovsky. - Moscow, 1964. - 271 p.). The model is based on preliminary sensitization of the animal with an allogeneic protein, followed by the induction of aseptic inflammation of the bone by striking with a wooden stick on the periosteum. The author caused sensitization of rabbits with horse serum. Then, introducing a crucial dose of serum into the bone marrow cavity, he received aseptic allergic osteomyelitis at the site of injury. Based on these experiments, S.M. Derizhanov believed that bacterial emboli do not play any role in the pathogenesis of osteomyelitis. The disease develops only on the basis of sensitization of the body and the occurrence of aseptic inflammation in the bone, which comes from a variety of reasons. The disadvantages of this model are low productivity, complications in the form of a septic process in various organs caused by a large dose of introduced microorganisms (several billion microbial bodies). In addition, sensitization by an allogeneic protein also caused changes in the immune system that were not associated with the pathological process.

A known method of modeling Stetsula V.I. and Shtin V.P. (Vengerovsky I.S. Osteomyelitis in children / I.S. Vengerovsky. - Moscow, 1964. - 271 p.). The experiments were conducted on outbred dogs. The authors filled the lumen of the main nourishing artery of the humerus of dogs with a 50% suspension of metallic silver in a 2% gelatin solution. Subsequently, obstruction of the intraosseous vessels was obtained. This led to widespread necrosis of the bone followed by sclerosis. The disadvantage of this model is that obstruction of the entire vascular system that feeds the bone is mandatory. This does not allow the use of this model for experimental studies on the treatment of osteomyelitis.

A known method of modeling chronic osteomyelitis (RF patent No. 2049434), as follows. The dog's tibia was chosen as the object of the experiment. An activator was carried out through the middle of the diaphysis of the tibia using the technique of transosseous osteosynthesis and served with the Kirschner surgical needle, which later became the positive electrode (anode). The cathode in the proposed experiment can be a second spoke drawn through the same segment, an transosseous fixation device, and an electrode placed on the skin. The current source is a 1.5 V battery, current strength 50 mA. The needle was inserted through the through hole at an angle of 60 degrees to the axis of the canal, and the infectious material was introduced at the intersection of the spoke and the axis of the medullary canal through an additional hole in the cortical bone layer. The disadvantages of this method is that it is bulky and differs in the complexity of the technical design.

Closest to the proposed is a method of modeling Ponomareva I.P. et al. (Ponomarev I.P. Development of an experimental model of chronic osteomyelitis / I.P. Ponomarev, D.V. Maksimenko, E.V. Aliyeva // Proceedings of the SSMA. - Smolensk, 2004. - P. 58) that performed the study on 30 sexually mature rabbits of the Chinchilla breed weighing 3.0-3.5 kg and 36 non-linear white rats weighing 200-250 g. Modeling of chronic osteomyelitis included two stages: creating a morphological substrate of the pathological process - the bone cavity with the area of perifocal sclerosis and sequestration and her infection. As a sclerosing agent, a 20% alcohol solution of silver nitrate is proposed. Under anesthesia under aseptic conditions, a cavity was created in the metaepiphyseal zone of one of the bones of the forearm, into which turunda moistened with a solution of silver nitrate and fragments of autobone were introduced. Aseptic inflammation, a zone of perifocal sclerosis was formed on the 15-30th day. A culture of Staphylococcus aureus in saline was introduced into the resulting cavity. Within 2-3 weeks, most animals developed chronic osteomyelitis with a characteristic clinical and radiological picture. The disadvantage of this model is the duration of the experiment, as well as the fact that upon contact with surrounding soft tissues of a 20% alcohol solution of silver nitrate, their expressed sclerosis occurs, which contributes to the further development of infection in soft tissues.

The technical result of the proposed method is the modeling of a long, rapidly proceeding osteomyelitis process, accompanied by fistulas, and the possibility of maintaining it for such a long time, which is necessary for studying and objective evaluation of the process, in order to obtain means and methods of its treatment.

The specified technical result is achieved due to the fact that the authors first proposed the use of ethoxysclerol for the formation of a zone of perifocal sclerosis with the subsequent introduction of Staphylococcus aureus, immured in a special solution.

The method is as follows.

Healthy white laboratory rats were chosen to simulate chronic osteomyelitis due to their susceptibility to modeling of chronic osteomyelitis and ease of handling. When conducting experimental studies, the rules of laboratory practice of the Russian Federation were strictly observed (order of the Ministry of Health of the Russian Federation No. 267 of 06/19/2003), the principles set out in order of the USSR Ministry of Health No. 755 of August 12, 1977 "On measures to further improve the forms of work using laboratory animals ”and in the Convention for the Protection of Vertebrate Animals Used for Experimental and Other Purposes (Strasbourg, France, 1986).

Modeling of chronic osteomyelitis included the creation of a bone cavity with a zone of perifocal sclerosis with subsequent infection.

Under anesthesia (Zolitil-100 at a dose of 8 mc / kg / according to the manufacturer’s instructions /) under aseptic conditions, a laboratory animal performs a linear incision of the skin, subcutaneous fatty tissue, fascia, and muscle on a section of the outer surface of the lower third of the thigh shaved from wool 1.5 cm long, the metaepiphyseal area of the femur is exposed, where a cavity with a diameter of 3.5 mm is created using a hand-held micromotor surgical instrument with a protective device. After that, turunda moistened with 1% ethoxysclerol solution is introduced into the cavity (the active substance of ethoxysclerol is lauromacrogol 400, which causes endothelial sclerosis), and fragments of autobone. One suggestive suture with a silk thread of size 1.0 is applied to the skin.

On the 7th day, the postoperative scar is dissected, muscles are spread out in a blunt way, soft tissues are removed using single-tooth hooks and a piece of the daily culture of Staphylococcus aureus in 2% agar-agar containing about 150-200 thousand microbodies is introduced into the cavity using a Kassirsky needle. In order to prevent the development of purulent infection of soft tissues, the hole in the bone is filled with ercodont cement, the wound is covered with 500 mg amoxiclav powder.

Each time, the same strain of Staphilococcus aureus is used to model chronic osteomyelitis.

Preparation of a microbial culture is as follows: from a daily culture of Staphilococcus aureus grown on blood agar, a suspension is prepared in sterile physiological saline (pH 7.2) with a concentration of microbial cells of 10 ⁸ CFU / ML according to the optical turbidity standard; 1 ml of 2% molten and cooled to 50 ° agar-agar was mixed in a test tube with 1 ml of a suspension of a daily culture of Staphilococcus aureus (4 billion microbodies).

On the 31st day after the introduction of the microbial culture, chronic osteomyelitis develops. In 100% of cases, fistulas are formed with a purulent exudate compartment.

The development of chronic osteomyelitis was confirmed by clinical, laboratory, radiological and morphological data.

Evaluation of clinical data showed that the animals had exhaustion, loss of appetite, depression of the general condition, weak motor activity, a pronounced fragility of the coat was observed. Pallor and dryness of the mucous membranes were noted. Fistulas functioned around which a zone of intense hyperemia was determined, an increase in local temperature. From fistulous passages, a moderate amount of purulent exudate of creamy consistency was distinguished.

Among laboratory parameters, hemoglobin, erythrocytes, leukocytes, leukocyte formula, erythrocyte sedimentation rate (ESR), free radical oxidation and antioxidant protection were evaluated.

In laboratory animals, a decrease in the level of hemoglobin and red blood cells was noted. The hemoglobin level was 104.57 ± 2.99 g / l. Red blood cell counts corresponded to quantitative changes in the number of hemoglobin and amounted to 4.25 ± 0.03 × 10 ¹² / L. Marked anemia was toxic in nature and was associated with a chronic process.

Against the background of a pronounced purulent process, the leukocyte count corresponded to 13.00 ± 0.24 × 10 ⁹ / L. The course of the pathological process was accompanied by changes in the leukocyte formula. There was an increase in the number of stab, segmented neutrophils, as well as a moderate shift of the leukocyte formula to the left, monocytosis and lymphopenia. In animals, the content of stab and segmented neutrophils was 6.00 ± 1.29% and 64.14 ± 1.35%, respectively. These changes were accompanied by moderate monocytosis (2.57 ± 0.98%), indicating the presence of a purulent necrotic process in the tissues, and lymphopenia (27.29 ± 2.06%), indicating immunodeficiency in severe and long-lasting infectious and inflammatory process.

An increase in ESR was noted, this indicator in intact rats more than threefold and amounted to 6.57 ± 1.27 mm / hour.

Among the indicators of free radical oxidation processes that underlie the metabolism of all cells and determine the adaptive ability of the body to the action of damaging factors, lipid and protein peroxidation (modification) was evaluated.

As an indicator of lipid peroxidation activity, malondialdehyde was determined, which is the final product of free radical lipid oxidation and formed during the oxidative destruction of lipid hydroperoxides.

The degree of oxidative modification of proteins (OMB) was evaluated by the content of carbonyl groups in the reaction with 2,4-dinitrophenylhydrazine, which are formed mainly as a result of direct oxidation of certain amino acid residues by free radicals, as well as in the interaction with lipid peroxidation products.

In the course of the studies, it was found that in laboratory animals the MDA level was 42.52 ± 5.19 nmol / L. This is 2.9 times higher than this indicator in intact animals and testified to the intensification of LPO processes. As a result of the activation of lipid peroxidation and the accumulation of free radicals, oxidative modification of blood plasma lipoproteins occurs, a violation of the structural and functional integrity of cell membranes, and the release of lysosomal enzymes.

When studying the oxidative modification of proteins, which plays a key role in the molecular mechanisms of oxidative stress and is the trigger for the oxidative degradation of other molecules, an increase in the level of 2,4-dinitrophenylhydrazones (DNPH) to 80.36 ± 3.22 nm / mg of protein was noted.

Strict regulation of SRO reactions is achieved through the functioning of a coordinated system of enzymatic and non-enzymatic mechanisms for controlling the content of reactive oxygen species, free radicals, and molecular products of SRO.

Superoxide dismutase is central to the body's enzymatic antioxidant defense system. One of the indicators of the non-enzymatic link are sulfhydryl groups.

In the course of the studies, an increase in the level of SH-groups was found. However, this activation of the nonenzymatic AOS link was insufficient to inhibit free radical processes. The level of SH groups was 107.45 ± 3.12 mg. A statistically significant decrease in the level of AOS enzymatic link was observed. The content of SOD was 0.40 ± 0.03 srvc An imbalance of physiological oxidative-antioxidant balance occurred.

Radiologically in laboratory animals, the soft tissues of the thigh of the affected limb are increased in volume. The bone is thickened due to periosteal layers. Foci of destruction with uneven sclerotic walls were found. Against the background of the osteoporosis zone with a sclerotic rim, areas of increased density 0.2-0.4 cm in diameter are visualized - sequestration in the sequestral box. The medullary canal is sclerosed.

A morphological assessment reveals areas of loose, unformed connective tissue between sequesters. The cellular component is represented mainly by inflammatory cells. Spreading in the inter-trabecular spaces, the inflammatory infiltrate causes pH changes. Acidosis, local edema and leukocyte activity increase bone necrotization. Against the background of the spread of inflammation through the Haversian and nutritional channels, thrombosis of the vessels passing through them develops. Vascular obstruction further enhances necrotization of large areas of bone. There are sequesters containing a significant number of trabeculae with numerous osteoblasts. Sequestration in the environment of a cavity with purulent necrotic contents is noted. From this cavity are fistulous passages, reaching the periosteum. Violation of the integrity of the periosteum causes the formation of abscesses in the surrounding soft tissues.

The detection of hyaluronic acid showed that the bone beams in the focal zone contain a moderate number of round or oval-shaped cells with short processes, with a cytoplasm saturated with hyaluronic acid (HA). The intercellular substance of the beams contains many SIC-positive fibers. In the preparations, the growth of fragments of the old bone is determined, which are practically devoid of HA and neutral mucopolysaccharides (NPS), there are fragments whose cellular elements have a strongly vacuolated cytoplasm and lack a clearly defined nucleus. The periosteum of fragments of the old bone contains a significant amount of small GK-positive cells and fibers. In the area of the bone defect, an extensive, friable mass of GK-positive and SHIK-positive fibers, interspersed by small cells with a narrow rim of the GK-positive cytoplasm, is determined.

Thus, a new model of chronic osteomyelitis was obtained that meets modern requirements and is characterized by morphological changes in the bone that correspond to those in humans, as well as good productivity, the absence of generalization of infection and mortality, and low cost of the experiment. The experiment made it possible to observe and study the course of the disease, made it possible to control the initial state of the objects of experience.

The theoretical justification of the developed method was carried out on the basis of the Research Institute of EBM GBOU VPO VSMU them. N.N. Burdenko of the Ministry of Health of Russia.

Claims (1)

A method for modeling chronic traumatic osteomyelitis, including the creation under aseptic conditions of a cavity in the metaepiphyseal area of the bone of a laboratory animal under anesthesia and the introduction of turunda moistened with a solution of sclerosing agent and fragments of autobone into this cavity, followed by infection of the cavity with Staphylococcus aureus culture, characterized in that as a sclerosing agent use a 1% solution of ethoxysclerol and infection is carried out on the 7th day by introducing into the obtained a piece of a daily culture of Staphylococcus aureus in 2% agar-agar solution, while the hole in the bone is filled with ercodont cement and the wound is covered with amoxiclav powder.