RU2676478C1 - Method of preparing filling mass for closure of bone defect - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, namely to traumatology and orthopedics, and can be used to close a defect in flat and tubular bones. Method of preparation of the filling mass to close the bone defect in a rat in the experiment involves grinding the coral of the Acropridae family to a granule size of not more than 145 mcm, which are filled with a gelatin capsule by two-thirds of the volume, then the capsule is filled with autologous blood to its full volume, with the wall of the capsule being pre-perforated with holes with a diameter of 0.1 mm in the amount of 4 x 1 mm, then the capsule is implanted under the skin of a rat in the region of the thoracic spine for 14 days, then a filling mass is removed and used to close the bone defect.EFFECT: invention provides the elimination of the need to perform biocompatibility control in connection with the use of autologous blood; reduction of time and simplification of the method of preparation of the filling mass for implantation, improving the quality of antibacterial protection; elimination of the need to use special expensive equipment and reagents for the preparation of a filling mass for implantation; and formation of callus the 14day after the operation.1 cl, 10 dwg

Description

The invention relates to medicine, namely to traumatology and orthopedics, and can be used to close the defect of flat and tubular bones.

Historically, the closure of a defect in a bone was given to its filling. According to T.Ya. Aryeva and G.D. Nikitin (1955), initially surgeons used substances that were completely foreign to the body: iodoform (Moseting-Moorhof, 1880); sponges (Hamilton, 1881); salicylic acid (Schmidt, 1882); copper amalgam (Maier, 1893); gypsum (Deesman, 1893); glass (Salzer, 1896); lead (Lesser, 1896); gelatin (Schepelman, 1918); petroleum jelly (Orr, 1919); glass wool and charcoal (Kummel, 1920); paraffin (Silvestrini, 1920); ointment A.V. Vishnevsky (A.M. Ryzhikh, 1943) [5]. In addition, the following were used: chicken yolk [18]; white clay, bismuth nitrate and sulphate, sawdust and peat, gutta-percha, sea sand, Poultry manure (bird droppings), ground coffee, a mixture of sugar and chloramine, wood glue, molten paraffin, bismuth nitrate and sulphate, and about 50 other types of fillers [5]. The results of using these materials as fillings for closing a bone defect did not meet the expectations of surgeons and patients. These fillings were foreign bodies in the bone cavity, and also had a toxic effect on a living organism. Therefore, all of the listed materials were rejected and caused suppuration of the wound. However, some substances have found their application in medicine at the present time.

Modern methods for closing a bone defect include: debridement of a bone defect with ultrasound, antibiotics, and chemicals, for example, a solution of disodium salt of ethylenediaminetetraacetic acid [3, 20, 26, 29, 37]; stimulation of bone regeneration; the introduction into the cavity of a complex polymer composition, the basis of which was polyacrylate (“bone cement”) with antibiotics [39]; the use of resorbable fillings with the ability to stimulate osteogenesis and exert anti-inflammatory and antimicrobial effects [11]. The composition of modern resorbable fillings can include both inorganic substances (hydroxyappatite) and polymeric organic compounds (polylactide, biositall, hydroxyapol) [17]. Antibiotics or molecules can be added to some resorbable fillings that contribute to a more pronounced osteogenic effect, for example, collagen [38].

One of the most popular modern biodegradable fillings is the combined drug “KollapAn” which includes hydroxyapatite, collagen and a complex of antimicrobial agents [8]. A feature of “KollapAn” is that on its surface a full-fledged bone tissue is formed without the formation of connective tissue layers between its granules [18].

Despite the variety of modern methods of filling residual bone cavities, the percentage of unsatisfactory results during treatment remains quite high: according to some authors, a positive effect is achieved only in 55-65% of cases [6].

Modern researchers agree that a more physiological way of replacing residual cavities, as compared to filling, is transplantation, that is, plastic surgery with biological tissues [1, 5, 7, 16, 19, 21, 23, 24, 40]; bone “granules”, the use of skin, muscle, bone, blood, blood clots and other substitute substances [22, 42].

To activate reparative osteogenesis use: calcium-phosphate material, for example, KollapAn - a biocomposite material based on synthetic hydroxyapatite, collagen and antibiotic (produced by the domestic company Intermedapatit); drug Ostim (Ostim) - synthetic hydroxyapatite ultra-high dispersion in the form of a paste ("Osartis", Germany); Chronos preparation (chronOs) - granules of β-tricalcium phosphate ceramics (Mathys Medical Ltd Switzerland); Cerosorb preparation - granules of β-tricalcium phosphate ceramics (Curasan, Germany) [8, 9, 15, 31, 32, 34, 35].

Close to the proposed method is a technical solution, the essence of which is the use of mechanically ground natural coral (NK) to a particle size of 300-600 microns. Coral sterilization is performed by γ irradiation at a dose of 25 KGy. The assessment of acute cytotoxicity and matrix properties of the surface of NK is checked on a culture of immortalized normal human fibroblasts (PS). For this, one day before the start of the in vitro experiment, NK particles were placed in 96 well culture plates and filled with the following complete growth medium (ORS) of the following composition: DMEM (PanEco Russia), 10% fetal calf serum (HyClone, USA) ), glutamine (292 mg / l), gentamicin (50 mg / l) (PanEco Russia). Then, the medium with NK samples (experiment) is decanted and a PS suspension is added in a volume of 200 μl of ORS (seeding density of 70 thousand cells per well). The control is a culture of PS on polystyrene (culture plastic). The cultivation is carried out at 37 ° C and an atmosphere of moist air containing 5% CO ₂ . Change ORS in tablets is carried out twice a week [33].

NK cytotoxicity is determined after 24 hours. The matrix properties of corals are evaluated in the dynamics of culturing cells on them for 3, 7, and 14 days. The viability of the PS at the stages of the experiments was assessed using the MTT test [41], for which a pool of viable cells (VFAs) was calculated at any given time according to the optical density of the formazan solution, in comparison with the control.

To study the biocompatibility of NK samples in in vivo experiments, a subcutaneous transplantation model is used. To do this, female rats of the Wistar strain weighing 180-200 g — under anesthesia, a skin incision is made in the region of the thoracic spine and a 120 mg NC sample is implanted in the formed “pocket”. The wound is closed with knotted sutures. Animals are removed from the experiment after 3, 6, 9 and 12 weeks. A visual and histological assessment (staining with hematoxylin and eosin) of a tissue fragment with NK is performed.

In vivo studies of osteo-substituting potencies of NK are carried out on a defect after edge resection of the tibia. In rats, at the border of the upper and middle third of the leg bone, a “window defect” (length 6–8 mm, width 1.5–2 mm, depth 2.5–3 mm) is formed with the help of boron to the lower cortical layer. The defect area is filled with sterile ND particles. The surgical wound is sutured in layers. To conduct morphological studies of the zone of animal defect derived from the experiment after 3, 6, 9 and 12 weeks. (2 animals per term), produce histological studies [33].

On micropreparations from the peripheral part of this zone, almost complete replacement of the NK substance with compact bone tissue with the formation of organotypic structures (osteons) was revealed. In separate fields of view between the osteons there were remains of coral matter, areas of accumulation of osteoblasts, active neovascularization. In the center of some osteons, cavities with adipose tissue and chains of osteoblasts along the periphery of the cavity were determined, according to the authors, with the rudiments of the bone marrow matrix. The authors believed that the experimental data on the marginal resection of the tibia of the rat when the bone defect was closed with an implant based on natural coral indicate pronounced osteoreparative potentials of the coral skeleton from the Acropridae family [33].

The disadvantages of the known technical solutions [33] include: the long-term and difficult preparation of coral to close the defect in the bone, which requires special expensive equipment and reagents to prepare the filling mass for implantation; the use of a culture of immortalized normal human fibroblasts, which necessitated the control of NK for biocompatibility; the danger of an allergic reaction to the culture of immortalized normal human fibroblasts; increased risk of infectious complications associated with numerous manipulations in preparing coral for implantation. Identified voids on histological preparations, according to the applicants of the alleged invention, could be associated with a large amount of coral grana. What could be a significant disadvantage of the prototype.

The objective of the proposed technical solution was: to eliminate the disadvantages of the prototype by simplifying the method of preparing the filling mass to close the bone defect; elimination of the need to perform a biocompatibility control of the applied filling mass; reduction of time for its preparation for implantation; improving the quality of antibacterial protection; the exclusion of the use of special expensive equipment and reagents for preparing the filling mass for implantation; determination of the optimal size of coral granules for a quality osteoreparative process.

In FIG. 1-10 shows the argumentation of the main positions of the technical solution:

in FIG. 1 is a diagram of a perforated wall gelatin capsule;

in FIG. 2 - view of the wound after implantation of a gelatin capsule with crushed coral of the Acropridae family, soaked in autoblood;

in FIG. 3 - 14 days of incubation of the filling mass in the tissues of the animal: type and size of the extracted implant;

in FIG. 4 - 7 days of incubation: a histological preparation of the implant based on crushed coral with a granule size of 200-320 microns and larger;

in FIG. 5 - 7th day of incubation: a histological preparation of the implant based on crushed coral with a granule size of less than 145 microns;

in FIG. 6-14 days of incubation: a histological preparation of the implant based on crushed coral with a granule size of 200-320 microns and larger;

in FIG. 7-14 days of incubation: a histological preparation of the implant based on crushed coral with a granule size of less than 145 microns;

in FIG. 8 - 21 days of incubation: a histological preparation of the implant based on crushed coral with a granule size of 200-320 microns and larger;

in FIG. 9 - 21 days of incubation: a histological preparation of the implant based on crushed coral with a granule size of less than 145 microns;

in FIG. 10-14 days after closing the bone defect with filling mass: histological preparation of the bone in the projection of the defect.

To determine the optimal size of coral pellets, experiments were performed on outbred male rats weighing 300-340 g, which were divided into two groups. In animals of the first group, crushed coral with a granule size of 200-320 μm and larger was tested. In animals of the second group, ground coral was tested with a granule size of not more than 145 μm. Portions of crushed coral were placed in gelatin capsules with a perforated wall (Fig. 1) having 4 holes per 1 mm ² with a diameter of up to 0.1 mm. Capsules in compliance with the rules of asepsis and antiseptics filled two-thirds of their volume with coral chips. Then, blood was taken from the tail vein of the animals, which was filled with coral chips until the gelatin capsule was completely filled. Each animal under general anesthesia under the skin in the area of the thoracic spine implanted capsule with crushed coral treated with autoblood. The skin wound was closed with knotted sutures (1) tightly (Fig. 2). The skin suture was treated with a liquid antiseptic.

The implants were removed from four rats from each group on the 7th, 14th and 21st day of incubation. In the extracted implant, the gelatin capsule was absorbed during the incubation process and was replaced by a dense tissue formation with dimensions larger than the gelatin capsule (Fig. 3). The implant was fixed in 10% acid formalin, embedded in paraffin, and serial sections 12-15 μm thick were made. Sections were stained with hematoxylin and eosin. Histological specimens were described under a Leica DM2500 digital imaging microscope.

On the 7th day of incubation in the implants of animals of the first (Fig. 4) and second (Fig. 5) groups, the development of stroma from loose fibrous connective tissue (2) was determined from the periphery to the center of the preparation, which surrounded the voids (3) remaining from the granules decalcified coral. Void sizes from crushed coral with a granule size of less than 145 microns were significantly smaller than from granules with a size of 200-320 microns and larger. In addition, in animals of the second group (Fig. 5), the implant had a capsule consisting of compact (4 _A ) and loose (4B) connective tissue 88.86-136.03 μm thick, which was penetrated by numerous blood vessels (5), whose diameter ranged from 21.98-34.92 microns. Single stromal blood vessels (6), which were oriented closer to the connective tissue capsule (Fig. 5), passed through the stroma from loose fibrous connective tissue (2).

After a 14-day incubation on the histological preparations of the implants of animals of the first group (Fig. 6), an abundance of fibroblasts (7) and lymphocytes (8) was revealed. On the histological specimens of animal implants of the second group (Fig. 7), an accumulation of fibroblasts (7) was found around the periphery, which surrounded large foci of formation of cartilaginous (9) and young bone tissue (10).

On the 21st day of incubation, the implants of the animals of the first group (Fig. 8) were well vascularized, an abundance of fibroblasts (7) and lymphocytes (8) were revealed. On histological specimens of animal implants of the second group (Fig. 9), the formation of compact bone tissue (11) with haversian canals (12) was determined.

An analysis of the obtained experimental data gave us the opportunity to fulfill the task by preparing a filling mass using crushed coral of the Acropridae family with a granule size of not more than 145 μm, with which a gelatin capsule was filled into two-thirds of the volume, and then autoblood was introduced until it was completely filled, and the capsule wall was preliminarily perforated holes with a diameter of 0.1 mm in an amount of 4 x 1 mm ^2, and the gelatin capsule was used instead of collagen, and to complete the cooking process plombirovo hydrochloric mass of a gelatin capsule with the contents, considering bioethics [43], aseptic and antiseptic regulations implanted under the skin of the thoracic spine of rat 14-five days.

In order to assess the effectiveness of the use of filling mass to close the bone defect prepared according to the developed method, the authors of the proposed method, an experiment was performed. Under general anesthesia with observance of aseptic and antiseptic using a spherical dental cutter, a defect measuring 8 × 2 × 2.5 mm (40 mm ³ ) was formed on the lateral side of the rat in the middle third of the femur. The formed bone defect was filled with the prepared filling mass and covered with adjacent muscle tissue. After hemostasis, the wound was closed tightly in layers. The skin suture was treated with a liquid antiseptic.

On the 14th day after the operation, the animals were taken out of the experiment. Histological examination (staining with hematoxylin-eosin and according to Mallory) revealed the formation of bone callus, along the inner surface of which penetrated into the bone tissue of the femur of the young bone (10) and fibrous (13) tissues. The bone marrow matrix was represented by compact bone tissue (11) having osteons with blood vessels passing through the haversian canals (12) (Fig. 10).

The positive effect when applying the method was: to eliminate the need to perform a biocompatibility control in connection with the use of autologous blood; to reduce time and simplify the method of preparing the filling mass for implantation, which increased the quality of antibacterial protection; the fact that special expensive equipment and reagents were not used to prepare the filling mass; in the formation of bone marrow already on the 14th day after the operation.

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Claims (1)

A method of preparing a filling mass for closing a bone defect in a rat in an experiment, comprising grinding the coral of the Acropridae family to a granule size of not more than 145 μm, with which a gelatin capsule is filled into two-thirds of the volume, then the capsule is filled with autoblood to its full volume, the capsule wall being pre-punched with holes with a diameter of 0.1 mm in an amount of 4 x 1 mm ^2, more capsule is implanted under the skin of rats in the thoracic spine in 14-day five, then recovered and used Filling weight d I'm closing the bone defect.

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