RU2792573C1 - Method for stimulating osteoregeneration after surgical treatment of tuberculous osteitis in the experiment - Google Patents

Abstract

FIELD: medicine; experimental surgery.

SUBSTANCE: invention can be used to optimize the recovery of bone tissue after surgical treatment of destructive skeletal lesions of tuberculous etiology. After radical reconstructive surgery and healing of the surgical wound rabbits are administered pamidronic acid once at a dose of 1 mg/kg intravenously.

EFFECT: method provides inhibition of implant lysis, longer induced osteogenesis and neoangiogenesis in the area of bone resection due to administration of pamidronic acid.

1 cl, 8 dwg, 1 tbl

Description

The invention relates to medicine, namely to experimental surgery, and can be used to optimize the recovery of bone tissue after surgical treatment of destructive skeletal lesions of tuberculous etiology.

The basis of surgical treatment of osteoarticular tuberculosis is radical resection of destructive foci within healthy tissues [1-3]. Large post-resection cavities, especially those of the supporting (lower) limbs, require plastic filling, for which implants of the appropriate size are usually used [4, 5]. The goal of bone grafting is to restore the bone as completely as possible without the formation of a residual cavity filled with scar tissue [6].

Most often, bone grafts or non-biological resorbable materials are used for bone grafting, which, under conditions of postoperative adaptation, undergo natural biological processes, the first phase of which is osteolysis - resorption, which under the conditions of the initial infectious process can be quite pronounced or complete. At the same time, even a normally ongoing process of implant adaptation in large bone defects requires more time for recovery and, therefore, immobilization and limitation of the axial load on the affected segment, which worsens the conditions for revascularization and osteogenesis, slowing down the processes of osteoregeneration.

One of the methods for regulating bone formation after radical reconstructive surgery may be the inhibition of the activity of osteoclasts, which provide osteoresorption as one of the initial mechanisms of reparative osteoregeneration. This action is selectively possessed by bisphosphonates, drugs based on pyrophosphate, which not only have the effect of inhibiting the differentiation of osteoclasts from precursors, but also have anti-inflammatory activity [7,8]. Bisphosphonates are used in the treatment of both systemic genetic diseases - primary [9] and secondary osteoporosis [10], and in focal lesions of bone tissue - cysts, metastases of lytic tumors, non-bacterial osteomyelitis [11-14]. In the Russian Federation, pamidronic acid is the most commonly used bisphosphonate in children [15].

Pamidronic acid (pamidronate) - is a drug used from the first weeks of life in osteogenesis imperfecta, its efficacy and safety has been studied for more than 25 years in this disease, the use of pamidronate is included in the relevant clinical recommendations. Pamidronate is also used to treat non-bacterial osteomyelitis in children and to relieve pain from bone metastases in adults and children.

The objective of the invention is to develop a method for stimulating osteoregeneration after surgical treatment of tuberculous osteitis in the experiment.

The task is realized by the fact that rabbits on the background of anti-tuberculosis therapy are resected destructive foci within healthy tissues, the surgical defect is filled with plastic material, then, after the surgical wound has healed, a single injection of pamidronic acid solution is administered intravenously at the rate of 1 mg per kg of animal weight.

The proposed method for regulating osteoregeneration after surgical treatment of tuberculous osteitis in the experiment is new; previously, data on stimulating bone tissue recovery after surgical treatment of destructive skeletal lesions of tuberculous etiology have not been published.

EFFECT: pronounced osteolysis of grafts and longer induced osteogenesis. The method is illustrated by graphic material, where: Fig. 1. Dynamics of body weight in rabbits.

Fig. 2. Zone of implantation of the sample of group No. 4, 6 months of observation. A - reconstructed image, B - CT-section. The implant is visualized in the medial condyle of the right femur of a rabbit of heterogeneous density, with inclusions of CaSo4, growing through bone beams.

Fig. 3. The nature and extent of specific inflammation, Me, 3 months of follow-up.

Fig. 4. The nature and extent of specific inflammation, Me, 6 months of follow-up.

Fig. 5. A focus of nonspecific inflammation around the implant granules with beams with active osteoblasts, hematoxylin-eosin (B

- preparation of the pineal gland, D - CT image, E, E - preparation of the pineal gland, sw. 1000x).

Fig. Fig. 6. Patterns of regenerative osteogenesis, points (0-3), Me, 3 months of follow-up.

Fig. 7. Patterns of regenerative osteogenesis, points (0 - 3), 6 months of follow-up.

Fig. 8. An example of a scoring of CT signs of osteoregeneration and osteolysis. Preparation G: visualization of the implant in the epiphysis - 0 points, implant germination by bone beams - 0 points, structure of bone beams - 1 point, structure of the epiphyseal plate - 1 point, formation of a cortical endplate at the site of trepanation - 0 points; bone cavities - 2 points, microsequesters - 2 points.

Preparation 3: visualization of the implant in the epiphysis - 2 points, ingrowth of the implant by bone beams - 2 points, structure of the bone beams - 2 points, structure of the epiphyseal plate - 2 points, formation of the cortical endplate at the site of trepanation - 2 points; bone cavities - 0 points, microsequesters - 0 points.

Experimental studies were carried out in the federal state budgetary institution "St. Petersburg Research Institute of Phthisiopulmonology" of the Ministry of Health of the Russian Federation on 21 male rabbits of the "Soviet Chinchilla" breed. The creation of a model of tuberculous osteitis in the medial condyle of the right femur of rabbits was performed according to a previously developed and patented method [16] in modification. A three-week drug-susceptible reference strain of Mycobacterium tuberculosis H37Rv was used as an infectious agent. During the experiment, after modeling the bone focus of specific inflammation and confirming the development of the tuberculous process, incl. On the basis of positive skin tests with the recombinant tuberculosis allergen "Diaskintest", the animals were divided into groups depending on the surgical and subsequent drug treatment options:

group 1 (infection control, n=3) - rabbits with simulated tuberculous osteitis, who did not receive anti-tuberculosis therapy (PTT) and did not undergo surgical treatment;

groups 2-4 - rabbits with simulated tuberculous osteitis, who, after the development of a specific focus of tuberculous osteitis, were prescribed anti-tuberculosis therapy (PTT) in the amount of isoniazid 10 mg/kg, ethambutol and pyrazinamide - 20 mg/kg per os. Then the necrectomy of the destruction cavity was performed with the filling of the surgical defect with granules of plastic material Osteoset®2DBM Pellets. Further differentiation of groups was carried out taking into account the treatment option, including:

… group 2 (n=4 rabbits) - PTT continued until the end of the experiment;

… group 3 (n=7) - 10 days after the operation, pamidronic acid was administered once at a dose of 1 mg/kg intravenously slowly against the background of ongoing PTT;

... group 4 (n=7) - on the 10th day after the operation, pamidronic acid was administered once with simultaneous cancellation of further PTT.

The dose of the drug for pamidronate was 1 mg/kg, the drug was diluted with a 0.9% NaCL solution until a concentration of less than 90 mg/250 ml was reached, it was injected slowly intravenously at an infusion rate of not more than 1 mg/min. The maximum dose of the drug should not exceed 90 mg.

Observation of laboratory animals was carried out for a period of 3 and 6 months after surgical treatment.

After euthanasia, carried out in accordance with the requirements set forth in the European Commission Guidelines on euthanasia and specified in GOST 33215-2014, all animals underwent autopsy sampling of the right femur (operation area); the rabbits withdrawn from the experiment after 6 months were also sampled from the left (intact) femur. The extracted bone samples were fixed in 10% neutral buffered formalin solution (pH 7.4).

The assessment of the condition of model animals was carried out on the basis of the following criteria:

- consumption of water and food;

- periodic (at the beginning of the study and before the main stages - infection, operation, excretion) monitoring of the dynamics of the body weight of animals using electronic scales of the AdventurerTM model, weighing accuracy ± 0.1 g.

- physical activity (good/reduced);

- healing of the surgical wound (primary intention / secondary intention);

- complications of the surgical wound (abscesses, hematomas - yes / no); lameness (yes/no).

... evaluating the effectiveness of the tuberculous osteitis model (Diaskintest, GTPR-diagnosis (positive/negative result)).

The most frequently used markers of osteogenesis processes were evaluated in blood serum dynamics: concentrations of total alkaline phosphatase, albumin, osteocalcin, β-Crosslaps, RANKL, sclerostin. Blood sampling was carried out from the marginal ear vein at the beginning of the study, 6 weeks after infection, and in the postoperative period - after 10 days, 3 and 6 months.

Quantitative and qualitative assessment in the autopsy material of the implantation zone, the contralateral condyle of the right femur, as well as the medial condyle of the left femur (radiological studies were performed using a Skyscan 1172 microtomograph (Bruker) with a pixel size of 13 and 27 µm) was carried out according to the following parameters:

- the optical density of the bone tissue was assessed in Hounsfield units in the RadiAnt DICOM Viewer medical imaging data viewer on an area of 0.001 cm ² ;

In accordance with the conditional qualitative-quantitative scale developed by the authors, five signs characterizing osteoregeneration and two signs of osteolysis were evaluated. The following were considered signs of osteoregeneration: preservation of visualization and ingrowth of the implant by bone beams, restoration of the structure of the cortical end and epiphyseal plates, structure of bone beams; osteolysis - the presence of bone cavities and microsequesters. Depending on the severity of the trait, points are assigned from 0 to 2, where 0 - the trait is absent, 1 - moderately pronounced, 2 - pronounced:

1. Visualization of the implant in the epiphysis: not visualized - 0 points, visualized small fragments of the implant up to 0.5 cm ³ - 1 point, visualized fragments of the implant more than 0.5 cm ³ - 2 points;

2. Germination of the implant by bone beams: absent - 0 points, visualized partially - 1 point, visualized throughout - 2 points;

3. Structure of bone beams: bone beams are structureless on the area of more than 50% of the epiphysis - 0 points, the structure of bone beams is preserved on 50% - 90% of the area of the epiphysis - 1 point, the structure of bone beams is preserved on more than 90% of the area of the epiphysis;

4. Structure of the epiphyseal plate: the epiphyseal plate is not visualized or is visualized on an area of less than 50% - 0 points, the epiphyseal plate is visualized on an area of 50% -90% - 1 point, the epiphyseal plate is visualized on an area of more than 90% - 2 points;

5. Formation of the cortical endplate at the site of trepanation: absent - 0 points, signs of formation with defects - 1 point, fully formed - 2 points;

6. Bone cavities: occupy more than 20% of the area of the epiphysis - 2 points, occupy 10 - 20% of the area of the epiphysis - 1 point, absent or occupy up to 10% - 0 points;

7. Microsequesters: multiple - 2 points; single (up to 5 on a 1 mm section) - 1 point, absent - 0 points.

If the sum of points of osteoregeneration signs is from 7 to 10, pronounced regeneration of bone tissue is stated, with the sum of points of signs of osteolysis from 2 to 4 - active lysis of bone tissue.

Standard histological examination of micropreparations and morphological analysis of inflammatory and regenerative processes in tissues were performed using preparations prepared according to the standard method and stained with hematoxylin and eosin and by the Ziehl-Nelsen method, as well as using images of preparations digitized on a Pannoramic scanner using Orbit Image Analysis and Panoramic Viewer.

Descriptive method evaluated:

- prevalence, degree/severity/stage of specific infiltrate (in points from 0 to 5);

- the presence of AFB (in points from 0 to 3);

- formation of foci of decay, organization phenomena, fibrosis, the presence of perifocal inflammation (yes, no);

- thickness of the fibrous capsule (in points from 0 to 3);

- dystrophic and regenerative changes in bone tissue (yes, no);

- structural formation of bone beams in the area of formed callus (yes, no).

Morphometric method in 10 fields of view at x1000 magnification was used to evaluate:

- the number and distribution of cells that characterize osteoregeneration and remodulation (osteoblasts, osteoclasts, osteocytes);

- maturity of bone tissue in the lesion (activity of osteoblasts and osteocytes, maturity of granulation tissue,

fibrosis, mesenchyme, newly formed bar structures, sequesters) - in points from 0 to 3. Morphometric analysis in the Orbit Image Analysis program and Pannoramic Viewer evaluated:

- parameters of bone tissue areas (% of bone area);

- parameters of areas of specific inflammation divided into zones of necrosis, infiltrate and fibrosis (% of bone area and % of total inflammation area);

- the thickness of the bone beams (µm) in the epiphyseal part of the distal parts of the femur.

The obtained data were subjected to statistical processing by methods of variation statistics using Microsoft Excel and STATISTICA 10 programs with the determination of the mean value (M), confidence interval (p), differences were considered significant at p<0.07. To compare the samples, nonparametric Kruskal-Wallis and Mann-Whitney tests were used for independent samples. Correlation analysis was performed using the Pearson correlation coefficient.

The effectiveness of the method is illustrated by the following results:

no lethal outcomes, undesirable reactions to the introduction of PTT and BF were registered. Healing of the surgical wound occurred by primary intention. All animals, except for one from the control group (generalized tuberculosis infection), were in a satisfactory condition by the time of hatching;

body weight gain was observed in all rabbits ranging from 4.8 to 15.3% over the entire observation period (Fig. 1). At the same time, in groups 3 and 4, who received pamidronic acid, the maximum weight gain was noted, which in group 3 + 15.3% for the entire observation period and + 10.6% from the moment of surgical treatment to the end of the experiment (p = 0.04 ); in group 4 - +7.7% and +10.6%, for the same periods.

The distinguishing radiation features of groups 3 and 4 are the preservation of the image of the implant in 85.7-100% of cases even in the presence of bone cavities, as well as its ingrowth with bone beams (Fig. 2) (for comparison, in group 2, the implant at a follow-up period of 3 months is determined only in 50% of cases, for a period of 6 months - is not visible in any preparation);

an increase in the sum of scores of signs characteristic of osteoregeneration is observed in dynamics among all treated groups (2-4). At a period of 3 months, their minimum values are determined in group 2 (2.5 points), the maximum - in group 3. At the same time, at a period of 6 months in group 4, there are maximum values of osteoregeneration patterns (7.5 points) and minimum - osteolysis (0 .5 points) (Table 1);

manifestations of specific inflammation are maximally noted in groups 2-4 at the 3rd month of observation and occupy from 10% to 50% of the area of the epiphysis, incl. AFB were found in most lesions (Fig. 3). Against the background of PTT, BP and their combination, by 6 months, a significant positive trend was noted in the form of leveling of specific inflammation in 50-75% of cases (Fig. 4).

A characteristic histological feature of groups 3 and 4 is:

detection of stromal strands represented by fibroblast-fibrocyte-like cells, collagen structures that radiate from the zones of residual inflammation (around the implant granules); an increased number of full-blooded vessels in these areas. On the bone beams, the number of active osteoblasts is increased (Fig. 5);

the presence of signs of active remodeling of bone tissue in the area of inflammation, which has more granulation tissue, active mesenchyme and full-blooded vessels for a period of 3 months (Fig. 6). For a period of 6 months, the largest amount of granulation tissue and full-blooded vessels was noted in group 4 (Fig. 7);

when assessing the ratios of the density of the implant, the number of osteoclasts in the area of inflammation and the average per drug, a strong negative correlation (Pearson's coefficient - 1) is determined at a period of 3 months between groups 2, 3 and 4, and at a period of 6 months - between groups 2 and 4.

Thus, a comprehensive analysis of the results of a morphometric study allows us to conclude that the preparations of groups 3 and 4 (animals treated with pamidronic acid) have both less pronounced graft osteolysis and more prolonged induced osteogenesis.

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Claims (1)

A method for stimulating osteoregeneration after surgical treatment of tuberculous osteitis in an experiment, characterized in that rabbits undergo resection of destructive foci within healthy tissues against the background of anti-tuberculous chemotherapy, fill the surgical defect with plastic material, then, after healing of the surgical wound, a single intravenous injection of a solution of pamidronic acid at a rate of 1 mg per kg of animal weight.

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