RU2720838C1 - Method for simulating chronic osteomyelitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to orthopedics and experimental medicine for simulating chronic osteomyelitis of limb longitudinal bone for simulating chronic osteomyelitis in rabbit. A bone defect of rounded shape is formed in a proximal tibial bone metaphysis. Allograft bone pre-impregnated with microbial suspension of Staphylococcus aureus is introduced as infectious agent. Bone defect is formed by tetrahedral cone-shaped drill with diameter of 0.5 cm with limiter to depth of 0.5 cm. Allograft bone is impregnated by immersion of allogenic bone with weight of 1 gram for 15 minutes in solution of microbial suspension with concentration of microbial cells Staphylococcus aureus 6×10⁸ CFU/ml by a standard for optical standardization of bacteria 1 McFarland (McF), with a solution density of 1 g/ml. Staphylococcus aureus ATCC 29213 grown on a dense nutrient medium for 24 hours is used. Simulating chronic osteomyelitis is completed on 21st day.

EFFECT: method provides creating a model of a chronic osteomyelitic process by minimizing the risk of developing a purulent process in the surrounding soft tissues and the joint cavity; standardized size of the defect enables to obtain reproducible results when used in experiments aimed at studying the osteomyelitis treatment options.

5 cl, 7 dwg, 1 ex

Description

The invention relates to experimental medicine for modeling chronic osteomyelitis of the long bones of the extremities.

To date, the treatment of osteomyelitis remains very relevant. In the general structure of diseases of the musculoskeletal system, chronic osteomyelitis is 10-25%, and the frequency of unsatisfactory treatment results and relapses of this disease is up to 40%. Methods of surgical treatment are constantly being developed and improved, requiring the creation of new experimental models of osteomyelitis.

A known method of modeling osteomyelitis (Patent RU No. 2622369). For this, under aseptic conditions, a cavity is created in the metaepiphyseal area of the bone of a laboratory animal under anesthesia. Turunda moistened with a 1% ethoxysclerol solution and autocost fragments with subsequent infection of the cavity with staphylococcus aureus are introduced into this cavity. Infection is carried out on the 7th day by introducing into the resulting cavity a piece of the daily culture of Staphylococcus aureus in 2% agar-agar solution. In this case, the hole in the bone is filled with ercodont cement and the wound is covered with amoxiclav powder. On the 31st day after the introduction of the microbial culture, chronic osteomyelitis develops.

The disadvantage of this method is its complexity, due to the two-stage implementation, as well as an increase in the cost of the experiment due to the drugs used (ethoxysclerol, ercodont-cement).

A known method of modeling osteomyelitis Dotsenko I. A. (Patent RU No. 2578818). As a carrier, the authors used dacron tissue contaminated with a strain of a pathogenic microorganism by keeping a strip of dacron tissue in a suspension of the pathogen 1.5 McF (1 × 10 ⁸ CFU). After placing the strip in the bone defect, it was sealed with bone wax and the wound was sutured in layers. Strip removal was performed on the 7th day.

The disadvantage of this method is the need for additional surgical intervention to remove the bacterial carrier, and the use of surgical wax can aggravate the infectious process, leading to septic complications and death of the animal.

As a prototype, a method for modeling chronic purulent bone wounds (Patent RU 2499295) was selected, including the formation of a bone defect along the axis of the bone, placing in it a culture mixture of the museum strain of Staphylococcus Aureus No. 5 in the amount of 40-45 million CFU per 1 kg of body weight of the experimental animal and 0.1 ml of sterile silica sand. Three days after the operation, necrotic masses and pus are removed from the bone defect zone, three surgical sutures are removed in the middle third of the wound, forming a fistulous wound. Perform wound reinfection with the same culture at a dose of 20-25 million CFU per 1 kg of body weight. Subsequently, the wound is kept in fistulous form.

The disadvantage of the above method is the need for a two-stage intervention with additional infection of the wound, the inability to completely remove the carrier (silica sand) of Staphylococcus Aureus from the wound. The size of the defect formed is formed at the discretion of the researcher, so we can say “the size of the defect is not standardized”, which subsequently leads to distortion of the data obtained by the experimenter, the lack of reproducibility of the obtained results when using the model.

The objective of the invention is to create a model of a chronic osteomyelitis process that has a pathomorphological similarity with the osteomyelitis process in humans, allowing a long time to maintain this process, with a standardized size of the defect to obtain reproducible results when used in experiments aimed at studying treatment options.

The technical result of the proposed method is the creation of a model of a chronic osteomyelitis process having a pathomorphological similarity with the osteomyelitis process in a person who is devoid of the disadvantages of analogues and prototype.

The technical result is achieved by the fact that in the method of modeling chronic osteomyelitis, including the formation of a bone defect, the introduction of an infectious agent into it, the removal of part of the sutures from the middle third of the wound on the third day after surgery and the dilution of its edges to initiate fistulous passage, form a standardized round bone defect forms, allocost is pre-impregnated with a microbial suspension.

Preferably, a standardized bone defect is formed with a tetrahedral conical drill with a diameter of 0.5 cm with a limiter to a depth of 0.5 cm.

Preferably, the allocost is impregnated by immersing the allogeneic bone weighing 1 gram for 15 minutes in a microbial suspension solution with a concentration of microbial cells of Staphylococcus aureus 6 * 10 ⁸ CFU / ml according to the standard for optical standardization of bacteria 1 McFarland (McF), with a solution density of 1 g / ml.

Preferably, Staphylococcus aureus ATCC 29213 grown in solid nutrient medium for 24 hours is used.

Preferably, modeling of chronic osteomyelitis is completed on day 21.

The method is as follows. The position of the animal on the back. Perform combined anesthesia. Hairline is removed along the inner surface of the proximal third of the leg, and the surgical field is treated three times with a 0.5% alcohol solution of chlorhexidine. A skin incision of 2.0 cm is performed, followed by sharp and blunt accesses through all layers of soft tissues to the proximal metaepiphysis of the tibia. Perform wound hemostasis. Then form a standardized bone defect. In particular, the defect is formed by a tetrahedral conical drill with a diameter of 0.5 cm with a limiter to a depth of 0.5 cm. Allocity prepared by immersing allogeneic bone weighing 1 gram for 15 minutes in a solution of microbial suspension with a concentration of microbial is immersed in the defect zone. cells 6 × 10 ⁸ CFU / ml standard for optical standardization of bacteria 1 McFarland (McF), with a solution density of 1 g / ml Produce layered wound closure. On the 3rd day after surgery, part of the sutures are removed from the middle third of the wound and its edges are diluted to initiate the fistulous course. Observe the animal for 21 days.

Example 1

Extract from the protocol of experiment No. 16. Rabbit of breed "Chinchilla", male, weight 2.5 kg. Under combined anesthesia with preparations “Zoletil 50” at a dose of 15 mg / kg i / m, “Xila” at a dose of 0.1 mg / kg i / m and local anesthesia with 0.25% novocaine solution - 4 ml in the position of the animal on the back The hair was removed from the inner surface of the proximal third of the lower leg, the surgical field was treated three times with a 0.5% alcohol solution of chlorhexidine and a 2.0 cm skin incision was made, followed by sharp and blunt accesses through all layers of soft tissues to the proximal tibia meta-epiphysis. Wound hemostasis was performed. Then a standardized bone defect was formed with a tetrahedral cone-shaped drill 0.5 cm in diameter with a limiter to a depth of 0.5 cm. 1 gram of a previously prepared infected microbial suspension of allostia was immersed in the defect zone. Made a layered wound closure. Computed tomography was performed for the purpose of postoperative control (Fig. 1).

To prepare the impregnated allosty, a 1 gram fragment of allogeneic bone was immersed for 15 minutes in a solution of microbial suspension with a concentration of microbial cells of 6 × 10 ⁸ CFU / ml according to the standard for optical standardization of bacteria 1 McFarland (McF) with a solution density of 1 g / ml. Conducted control weighing (Laboratory balance VK 300.1). The mass of absorbed fluid per 1 gram of bone and the concentration of microbial cells in it were determined. 1 gram of bone absorbed 0.01 gram of a solution of microbial suspension with a concentration of microbial cells of 1.0 × 10 ⁸ CFU / ML confirmed by the data of bacteriological culture according to the method of serial dilutions. On the 3rd day after surgery, part of the sutures were removed from the middle third of the wound and its edges were diluted to initiate the fistulous course.

In the animal after surgery, there was a decrease in appetite, physical activity, exhaustion, fever, impaired function of the operated segment. On the 21st day, in the area of the postoperative wound there was an unhealed fistulous course, with a profuse purulent discharge of a curdled consistency (Fig. 2). According to computed tomography performed on day 21 after the operation, a cavity with uneven sclerotic edges filled with bone sequesters was visualized in the proximal metaepiphysis of the tibia. The cortical layer is unevenly thickened. Edema of adjacent soft tissues surrounding the focus, with the presence of fistulous passage (Figure 3). According to laboratory studies, a general analysis of blood showed a tendency to a decrease in hemoglobin, red blood cells and an increase in white blood cells. According to a bacteriological study, the growth of a monoculture of Staphylococcus aureus was revealed.

On day 21, the animal was withdrawn from the experiment. In histological preparations (Hematoxylin-eosin staining, × 200), a bone defect with areas of necrosis and marked leukocyte infiltration (Figure 4), fragments of melting bone-cartilaginous tissue (Figure 5), proliferation of connective tissue surrounding the focus of chronic suppurative inflammation (Fig .6), with areas of necrosis and marked infiltration by segmented neutrophils, the formation of the fistulous course (Fig.7), which confirms the formation of chronic osteomyelitis.

Thus, the proposed method for modeling chronic osteomyelitis avoids the generalization of the osteomyelitis process, ensures the survival of animals throughout the entire duration of the experiment, including through accurate dosing of the infecting agent, to complete the simulation for 21 days, is simple in technical design, the simulated process corresponds to pathomorphological changes characteristic of person. The localization of the defect minimizes the risk of the development of a purulent process in the surrounding soft tissues and joint cavity. The standardized size of the defect allows reproducible results when used in experiments aimed at exploring treatment options for osteomyelitis.

Claims (5)

1. A method for modeling chronic osteomyelitis in a rabbit, including the formation of a bone defect in the tibia metaphysis, the introduction of Staphylococcus aureus microbial cells into it, the removal of part of the sutures from the middle third of the wound on the third day after surgery, and diluting its edges to initiate fistulous passage, characterized in that a rounded bone defect is formed in the proximal tibia metaphysis, allocost is pre-impregnated with a microbial suspension as an infecting agent. 2. The method according to p. 1, characterized in that the bone defect is formed by a tetrahedral conical drill with a diameter of 0.5 cm with a limiter to a depth of 0.5 cm 3. The method according to PP. 1, 2, characterized in that the allogeneity is impregnated by immersing allogeneic bone weighing 1 gram for 15 minutes in a solution of microbial suspension with a concentration of microbial cells of Staphylococcus aureus 6 × 108 CFU / ml according to the standard for optical standardization of bacteria 1 McFarland (McF), with a density solution of 1 g / ml. 4. The method according to PP. 1-3, characterized in that they use Staphylococcus aureus ATCC 29213, grown in solid nutrient medium for 24 hours. 5. The method according to PP. 1-4, characterized in that the modeling of chronic osteomyelitis is completed on 21 days.

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