RU2279676C2 - Method for controlling reparative osteogenesis in animals - Google Patents

Abstract

FIELD: veterinary biochemistry, biochemistry of bony tissue.

SUBSTANCE: the present innovation deals with evaluating the activity of osteogenesis due to carrying out biochemical blood testing in the course of which it is necessary to detect the levels of total calcium, inorganic phosphate, magnesium and chlorine content (C) in blood serum during regeneration of diaphysial fractures of tubular bones without phenomenon of distraction to calculate the index of electrolytes (IE) according to the following formula: IE = C_Ca C_Mg C_Cl/C_PO4. At its value being above 150 (up to250 and more) one should conclude upon catabolic phase, and at its value being below 150 (up to 110 and less) - anabolic phase of osteogenesis should be stated upon.

EFFECT: higher efficiency of control.

1 ex, 2 tbl

Description

The invention relates to the field of veterinary biochemistry, in particular to the biochemistry of bone tissue. This method is necessary to increase the information content of biochemical blood tests in the treatment of injuries of the musculoskeletal system in animals. The method allows to determine the phase of the regenerative process in bone tissue, its duration, as well as activity.

To control the postoperative course in orthopedic patients, X-ray and biochemical studies are used. The traditional laboratory method for assessing the activity of reparative osteogenesis is the determination of alkaline phosphatase activity (EC 3.1.3.1.) [3]. Determining the activity of enzymes in veterinary laboratories is a laborious and expensive research method, which increases the cost of the services provided. In addition, this test can be considered truly informative in the partial measurement of the activity of the bone isoenzyme of alkaline phosphatase. For this purpose, electrophoretic separation of phosphomonoesterase isoenzymes 1 is used. With unconditional specificity and high accuracy, this method requires considerable time and special equipment, reagents, which greatly complicates the control of reparative osteogenesis.

The closest analogue of the invention is a laboratory monitoring system for postoperative course while eliminating axial deformities of the lower extremities by means of transosseous osteosynthesis in children [1]. Axial deformities of the lower extremities resulting from rickets, rickets-like diseases, Erlacher-Blount disease, etc., were eliminated by the method of transosseous compression-distraction osteosynthesis according to Ilizarov. The authors assessed the activity of reparative osteogenesis in the correction of axial deformities of the lower extremities using a neural network recognition program based on the work of F. Wassermen. The basic principle of this program is the assumption that a mathematical neural network models the biological from an information point of view (information model of a biological neural network). The network input is the vector of patient parameters (laboratory blood tests), the output is the conditional number of the diagnosis, and the network output dimension is equal to the number of diagnoses. The fact that the object belongs to the class is indicated by the equality to unity of the component of the output vector. Mathematical processing of laboratory research results includes an assessment of the uniformity of the samples (4 samples), the calculation of the average and its confidence interval, the significance of differences in the comparison groups by nonparametric methods. The neural network recognition program can be found on the Internet on a personal page at: http: mcl.kurgan.ru/igor.btm.

The disadvantage of this method in veterinary medicine is the large number of diagnoses of bone pathology incorporated into the program; a very large number of laboratory tests that must be performed in order to use the neural network recognition program. To perform laboratory analyzes, special equipment, expensive reagents are necessary, they are laborious and time-consuming.

The aim of the invention is the development of a method of controlling reparative osteogenesis in animals during the regenerative process after fractures of the tubular bones based on laboratory data. A method for controlling reparative osteogenesis in animals involves determining blood biochemical parameters, based on which the electrolyte index (IE) is calculated. The index increases the level of information content of laboratory research data (total calcium, inorganic phosphorus, magnesium, chlorides). The phase of the regenerative process is determined by the value of the electrolyte index: the catabolic phase of osteogenesis is accompanied by an increase in IE of more than 150 (up to 250 or more); the anabolic phase is characterized by a decrease in IE below 125 (to 110 or less), its duration, as well as osteogenesis activity.

The used laboratory research methods are simple to implement, their implementation does not require special equipment and specific reagents. Mathematical processing of laboratory data increases their information content, which significantly increases the availability of test results to a wide range of practicing veterinarians.

This goal is achieved by calculating the index of electrolytes (IE) according to the level of data of electrolytes in blood serum according to the formula:

where C _Ca is the concentration of total calcium in serum, mmol / l;

With _Mg - the concentration of magnesium in blood serum, mmol / l;

With _Cl - the concentration of chloride in the blood serum, mmol / l;

With _PO4 - the concentration of inorganic phosphorus in the blood serum, mmol / L.

The numerator of the electrolyte index (IE) is a product of the concentrations of elements, which are one of the main products of bone tissue demineralization. The denominator reflects the content of inorganic phosphorus in the internal environment of the body, the level of which is directly related to the formation of amorphous phosphate in bone tissue, which subsequently transforms into hydroxyapatite (the main component of the bone mineral phase). The quantitative value of the electrolyte index reflects the ratio in the body of bone resorption and bone mineralization processes.

In healthy purebred dogs, in our studies, IE ranged from 125 to 150, depending on body weight and age.

The process of reparative osteogenesis of bone tissue consists of two phases: catabolic and anabolic. The phases have clear boundaries that can be determined based on the level of serum electrolytes and the IE calculated on their basis.

In our studies, the catabolic phase of healing of experimental fractures, depending on the method of osteosynthesis and the nature of the fracture, was accompanied by an increase in IE to 250 or more. The increase in the electrolyte index is due not only to an increase in the concentration of total calcium, magnesium, and chloride in the blood serum, but also to a drop in the level of inorganic phosphate. This is due to the processes of dissolution and lysis of bone tissue at the ends of fragments of the damaged bone and adjacent areas. In this case, the mineral components are washed out of the bone tissue into the extracellular fluid. At the same time, renal clearance is reduced, which also contributes to an increase in the concentration of these elements in the blood.

The anabolic phase of regeneration is characterized by a drop in the level of IE to 110 and below. The decrease in the electrolyte index is due to a decrease in the levels of total calcium, magnesium, chlorides and an increase in the concentration of inorganic phosphate caused by the precipitation of calcium orthophosphate, dissolved in tissue fluid, in the newly formed bone regenerate. We found that the phase transition of tricalcium orthophosphate to bone regenerate is characterized by a high level of inorganic phosphate in the blood serum and a low concentration of calcium.

The catabolic phase in osteogenesis precedes the anabolic one. The more intense it is, the faster the conditions are prepared for the transition of osteogenesis to the next phase, which shortens the healing time of fractures. The sequence of phase changes in the regulation of the regenerative process in bone tissue clearly reflects the dynamics of changes in IE.

The biological meaning of this index comes down to the fact that in the catabolic phase of osteogenesis, its value is higher than the average physiological value as a result of accumulation of bone tissue demineralization products in the blood, while in the anabolic phase, on the contrary, it is lower.

As a result of a comparative analysis of the proposed solution with the prototype, we can conclude that the claimed method for determining the nature of the course of osteogenesis allows us to evaluate its condition as accurately as when using the neural network recognition program. But this does not require a large number of laboratory studies that require special equipment and specific reagents, have statistics on a large number of diagnoses of bone pathology. The applied laboratory research methods are easily feasible in any veterinary biochemical laboratory; common reagents are used for their implementation. In veterinary biochemistry of bone tissue, methods for controlling reductive osteogenesis based on the calculation of the electrolyte index are not known. Therefore, the proposed method meets the criterion of "novelty."

Features that distinguish the proposed technical solution from the prototype:

1. A method for monitoring reparative osteogenesis is used only for animals with a diagnosis of "Fracture of tubular bones."

2. The level of electrolytes is determined in blood serum, and not in plasma [2].

3. In the blood serum, the content of total calcium, inorganic phosphorus, magnesium is determined, and not their ionic forms in mEq / l [2].

4. For the treatment of fractures of limbs of sick animals do not use the phenomenon of distraction.

5. For the implementation of the control method does not need to perform a large number of studies: analysis of the cellular composition of peripheral blood; acid and alkaline phosphatase activity with determination of partial values of their bone isoenzymes; lactate dehydrogenase with electrophoretic fractionation and subsequent calculation of the fractions of the H- and M-subunits of the enzyme; concentration of lactic and pyruvic acid.

6. Assessment of the nature of the course of osteogenesis is carried out on the basis of the calculation of the index of electrolytes according to the formula:

where C _Ca is the concentration of total calcium in serum, mmol / l;

With _Mg - the concentration of magnesium in blood serum, mmol / l;

With _Cl - the concentration of chloride in the blood serum, mmol / l;

With _PO4 - the concentration of inorganic phosphorus in the blood serum, mmol / L.

Analysis of the known technical solutions in the field of veterinary biochemistry of bone tissue allows us to conclude that there are no signs in them that are similar to the distinguishing features in the claimed method. This allows us to conclude that it meets the criterion of "significant differences".

Execution example.

When testing the method in outbred dogs, an experimental transverse tibia osteotomy was modeled. The experimental groups were formed from animals aged 2–3 years, body weight 16–20 kg, and tibia length according to an X-ray of 10–12 cm. Two types of osteotomy were used as a model:

1. without displacement of the ends of the fragments, n = 10 (1 group);

2. with the displacement of the ends of the fragments along the length, width, accompanied by significant damage to the skeletogenic tissues and vessels feeding the bone, n = 10 (group 2).

The nature of the displacement of bone fragments was evaluated by x-ray method. Osteosynthesis was performed using an external fixation apparatus, the Ilizarov apparatus.

In the blood serum of experimental animals on the 1st, 3-4th, 10-12th, 40-45th and 60th days of osteogenesis, the level of electrolytes was determined: calcium, phosphorus, magnesium and chlorides. As a control, we used data from the determination of the same indicators in experimental groups of animals before osteotomy. Based on laboratory analysis data, an electrolyte index was calculated.

The results of determining the level of electrolytes in blood serum and calculated on the basis of their index of electrolytes (IE) are presented in tables 1 and 2.

Table 1 The content of electrolytes in blood serum, IE in the 1st experimental group of dogs (n = 10) during osteogenesis, M ± m Research days
after operation IE ELECTROLYTES, MOL / L Ca ²⁺ PO ₄ ^3- Mg ²⁺ Cl ^- The control 150.6 ± 4.8 2.7 ± 0.34 1.5 ± 0.24 0.9 ± 0.14 93 ± 5,4 1st day 236.9 ± 4.6 * 3.23 ± 0.27 * 1.7 ± 0.18 1.17 ± 0.26 * 106.6 ± 4.6 * 3-4th day 233.8 ± 8, * 2.4 ± 0.16 * 1.34 ± 0.35 1.21 ± 0.17 * 107.9 ± 4.1 * 10-12th day 100.0 ± 5.7 * 2.53 ± 0.42 2.03 ± 0.19 * 0.79 ± 0.1 90.2 ± 2.6 40-45th day 108.9 ± 3.8 * 2.59 ± 0.39 1.8 ± 0.13 * 0.83 ± 0.12 91.2 ± 4.1 60th day 144.8 ± 6.2 2.66 ± 0.29 1.47 ± 0.2 0.87 ± 0.16 92.0 ± 0.47 Note: the * indicates indicators that differ from control values with a confidence level of at least P <0.05

table 2 The content of electrolytes in blood serum, IE in the 2nd experimental group of dogs (n = 10) during osteogenesis, M ± m Research days after surgery IE ELECTROLYTES, MOL / L Ca ²⁺ PO ₄ ^3- Mg ²⁺ Cl ^- 1st day 230.6 ± 4.3 * 2.97 ± 0.17 * 1.73 ± 0.24 1.21 ± 0.12 * 111.0 ± 2.9 * 3-4th day 236.0 ± 5.0 * 2.43 ± 0.14 * 1.35 ± 0.19 1.28 ± 0.24 * 102.7 ± 1.7 * 10-12th day 119.9 ± 3.7 * 2.59 ± 0.14 * 1.74 ± 0.17 * 0.87 ± 0.18 92.6 ± 2.4 * 40-45th day 121.1 ± 3.7 * 2.51 ± 0.15 1.62 ± 0.21 0.84 ± 0.11 93.5 ± 3.4 60th day 140.6 ± 5.3 2.56 ± 0.25 1.65 ± 0.13 0.96 ± 0.16 94.4 ± 3.3 Note: the * indicates indicators that differ from control values with a confidence level of at least P <0.05

The data of our studies showed that in the catabolic phase of regeneration, the IE level is higher than the average physiological value (150). It rises to 240, which is associated with the release of minerals into the blood as a result of the destruction of the bone matrix. Its value is the higher, the stronger the process of disorganization in bone tissue. This is determined by the severity of the injury and damage to the osteogenic tissues and intraosseous vessels. In the anabolic phase, on the contrary, the IE level is less than the average physiological value (decreases to 108) as a result of the active accumulation of these ions in the newly formed bone tissue (Tables 1, 2).

Thus, the phase of the regenerative process and its duration, i.e. IE informatively reflects the nature of the course of regenerative osteogenesis, depending on the stage and severity of the injury. The application of simple rules for processing laboratory data based on the use of mathematical modeling and biology in the field of regulation of mineral metabolism and skeletal homeostasis, the theory of reparative processes, makes such an assessment available to a medical practitioner.

Using the proposed method for controlling reparative osteogenesis in animals provides the following advantages compared to the existing method:

1. The proposed methodology for processing laboratory data allows you to objectively and accurately assess the phase of osteogenesis during the treatment of fractures of tubular bones and, if necessary, adjust treatment tactics.

2. Performing laboratory tests does not require expensive equipment and reagents.

3. To determine the phase of osteogenesis, you do not need to perform a large number of laboratory tests.

4. It is not necessary to have a large number of statistical data from laboratory studies of diseases with a general diagnosis of bone disease.

If necessary, this method of controlling reparative osteogenesis in animals can be used to evaluate the effectiveness of treatment in the development of new methods of treating injuries of the musculoskeletal system in animals and to predict the course of regeneration of the site of injury.

Information sources

1. Desyatnichenko K.S., Sklyar L.V., Gaydyshev I.P., Kuznetsova L.S., Talashchova I.A., Alekberov D.A. Laboratory monitoring of the postoperative course in eliminating axial deformities of the lower extremities by means of transosseous osteosynthesis // Mat. scientific and practical. conf., St. Petersburg, 2000. - S. 129-132.

2. Desyatnichenko KS, Gracheva L.I., Kuznetsova L.S. Functional biochemical studies in the clinic of orthopedics and traumatology // Method, recommended., Kurgan, 1990. - 27 p.

3. Murray R., Grenner D., Meyes P., Rodwell V. Human biochemistry, M .: Mir, 1993. - P.64-65.

Claims (1)

A method for assessing the activity of ostegenesis, which consists in conducting biochemical blood tests, characterized in that the blood electrolyte index is used to evaluate the activity, which is calculated by the level of total calcium, inorganic phosphate, magnesium, chlorine in the blood serum during the regeneration of diaphyseal fractures of tubular bones without occurrence distraction according to the following formula:

with an index value of more than 150 (up to 250 and above), the catabolic phase is established, below 150 (up to 110 and less) - the anabolic phase of osteogenesis.