RU2383564C1 - Composition - osteoinducer and osteoconductor in treatment of bone pathology in dentistry and oral surgery and method of obtaining composition - osteoinducer and osteoconductor in treatment of bone pathology in dentistry and oral surgery - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: composition includes chitosan gel, which has bactericidal properties, representing matrix for including in it of water solution for "Adgelon" injection, which contains serum glycoprotein from blood of livestock, possessing biological activity in supersmall doses 10-9-10-15 mg/ml, and calcium salt - preparation "Adgelon". In obtaining composition medicine "Adgelon" is immobilised on chitosan gel. ^ EFFECT: composition provides increased ability to stimulate proliferation of osteoblasts, reparative processes of osteoformation with recovery by means of morphologically normal bone matrix. ^ 2 cl, 3 ex

Description

The invention relates to medicine and the medical industry, and more specifically to the production of biocompatible materials for osteoplasty, which can be used as osteoinducing agents, for filling defects in bone and cartilage tissues in traumatology, orthopedics, dentistry, neurosurgery for congenital and acquired diseases.

Osteoplastic materials are widely used in the wound healing of defects of the musculoskeletal system, in dentistry, maxillofacial surgery and traumatology. Among these materials, composites from collagen and a crystalline component, primarily hydroxyappatite and tricalcium phosphate, are very popular. An important role is played by the properties of the mineral filler, which depend on its chemical composition, source of production, and synthesis method. The replacement efficiency of the implant material with regenerating bone tissue is largely determined by its resorption. Hydroxyappatite of animal origin, enclosed in bone regenerate, is not resorbed for a long time and is "walled up" in the newly formed bone tissue, and there is also an acute problem of infection with prion diseases when using bone material from cattle.

There is a known method for bone grafting with a mixture of ceramics (tricalcium phosphate) and hydroxyappatite (V.I. Makunin. Replacing bone defects with ceramics based on hydroxyappatite for tumors and tumor-like diseases. Abstract of the dissertation of the candidate of medical sciences. - M., 1998. - 21 p.).

The disadvantages of the method are the complexity of manufacturing ceramics and slow (within 3-4 years) resorption of the drug.

Known methods for homoplasty of bone cavities with bone fragments and chips of spongy substance of tubular bones (Abstract of the dissertation of the candidate of medical sciences. M., 1957. - 16 p .; GL Lusanov. Replacing defects of tubular bones with crushed bone tissue. Abstract of the dissertation of the candidate of medical sciences. L., 1963. - 40 p.).

The disadvantages of these methods are the possibility of necrotic and rejection of free, malnourished bone fragments, the risk of allergic reactions and infection of the recipient (HIV, hepatitis B and C).

From the description of the patent of the Russian Federation (RU) No. 2325170, 05.27.2008 a biocomposite material for osteoplasty is known, including bone matrix (40.0-95.0 wt.%), Collagen (0.25-1.0 wt.%), pharmaceutically acceptable bisphonate (0.1-10.0 wt.%) and water (rest).

As a bone graft in this composition, one of the following bone materials is used or a mixture of these bone materials selected from the following group: natural bone mineral, bone hydroxyappatite (hereinafter HAP), synthetic HAP, crushed demineralized animal bone, crushed demineralized human bone.

The biocomposite material provides osteoconductive and osteoinductive activity during transplantation and low antigenicity.

From the description of the patent of the Russian Federation (RU) No. 2296575, September 1, 2004, there is known a composition for treating bone defects in inflammatory etiology injuries, which contains the nutrient medium MEM needle, 10% serum from cow fetuses, diploid cells of human fibroblasts and gel filler, with the following content of components in 1 ml: suspension of diploid cells of human fibroblasts -1 × 10 ⁵ -5 × 10 ⁷ cells; nutrient medium Needle MEM - 0.01-0.9; 10% blood serum of cow fruits - 0.01-0.2; gel filler - the rest is up to 1 ml. As diploid cells of human fibroblasts, the composition comprises a strain of diploid cells of human fibroblasts from lung tissue of the embryo for replacement therapy of PEC 16-1 or a strain of diploid cells of human fibroblasts of human skin and muscle tissue of the embryo for replacement therapy of PEC 16-2. As a gel filler, 5% polyethylene oxide can be used. Using the composition allows you to accelerate the onset of remission and increase the duration of remission of a chronic disease.

From the description of the patent of the Russian Federation (RU) No. 2301684, November 21, 2005, another transplant mixture is known containing (in wt.%) Lyophilized allogeneic bone tissue - 65; allogeneic hydroxyappatite - 10; platelet gel of the patient’s autologous blood - 20; metronidazole - 5.

This mixture is mainly used in the complex treatment of patients with periodontitis.

From the description of the published application of the Russian Federation (RU) No. 98119221, 09/10/1999, a solution-inducer of osteogenesis for dentistry is known, including glycosaminoglycans, water, calcium salts and water-soluble cellulose derivatives, in the following ratio, wt.%:

Calcium Salts - 0.001-0.01;

Sulfated glycosaminoglycans - 0.1-1.0;

Water-soluble cellulose derivatives - 0.05-2.5;

Water is the rest.

From the description of the patent of the Russian Federation (RU) No. 2335258, May 27, 2008, an implant for bone plastic surgery based on a coarse collagen matrix from decalcified spongy bone is known, and antibacterial preparations, blast cell cultures, biogenesis stimulators (e.g. methyluracil, hydroxyappatite) are immobilized on it ), vitamins (e.g. ascorbic acid). For preparation, a composition possessing adhesive properties is applied to a collagen matrix with preparations immobilized on it (bioactive preparation containing polyvinyl alcohol, plasmogen gel composition).

A method of manufacturing this implant is quite complicated.

Recently, implants designed to fix hard tissues, in particular bone tissue with simultaneous stimulation of tissue regeneration, are becoming more common.

From the description of the patent of the Russian Federation (RU) No. 2153877, 08/10/2000, an agent is known that stimulates hard tissue in the form of a membrane, powder, gel, granules or solution, for the manufacture of which chitosan with a polysaccharide fixed on it, selected from heparin, heparan sulfate, chondroitin sulfates, is used and dextransulfate.

And from the description of the patent of the Russian Federation (RU) No. 2301677, 06/27/2007 known biotransplant for the treatment of degenerative and traumatic diseases of the articular cartilage and intervertebral discs, characterized in that it contains a carrier that includes a base substance, for example, chitosan, and / or alginate, and / or collagen and composite additives - hyaluronic acid, chondroetin sulfate with a ratio of the basic substance and composite additive of 90-99%: 1-10%, as well as a suspension of allogeneic or autogenic cell culture of chondroblasts, fibroblasts and mesenchymal st Tin cells when their contents in 1 ml medium 5-50 million cells; the carrier further includes matrix proteins: collagen of various types (I-XIX), integrin, fibronectin, laminin in a concentration of from 0.1 to 100 μg / ml; in addition, the carrier further includes fibroblast growth factors, and / or transforming growth factor α and β, and / or platelet-derived growth factor and / or hepatocyte growth factor at a concentration of from 1 to 300 ng / ml, and the carrier further includes antiseptics selected from the group of silver, copper, gold ions and broad-spectrum antibiotics.

This transplant is quite complicated due to the use of stem cells in it, the selection of which is associated with great difficulties.

Despite the variety of osteoplastic materials of various contents and properties, today it is impossible to distinguish among them the “ideal” for use in dentistry and maxillofacial surgery.

Differences in the effects of osteoplastic materials are due to different properties of minerals, as well as collagen of various types. However, most materials do not have predictable and sufficiently pronounced osteoplastic properties, especially in patients with poor reparative osteogenesis, due to hereditary or acquired qualities and as a result of exposure to pathogenic factors.

In recent years, more attention has been paid to substances introduced into osteoplastic compositions in order to enhance the construction of bone tissue and its mineralization. But until now, a comparative analysis of osteoplastic materials of various composition and origin, the most acceptable in their properties, for directed reparative osteogenesis has not been carried out.

The technical task of the claimed invention is the creation of biocompatible, bioactive and dynamic degrading compositions, modeled in accordance with the size and volume of the plastic zone and able to effectively stimulate bone regeneration, reduce the number of infectious complications and reduce treatment time; as well as expanding the arsenal of funds with a wound healing effect in relation to supporting tissues, bones of the craniofacial area and dentistry, and restoration of a bone defect after surgery.

The technical problem is achieved by creating a biocompatible composition with osteoinductive and osteoconductive properties to replenish (treat) partial and complete defects of cartilage and bone tissue, containing a matrix comprising chitosan gel and an aqueous solution of the drug "Adgelon" containing serum glycoprotein obtained from serum of cattle ultra low doses of livestock

10 ^-9 -10 ^-15 mg / ml, and calcium salt in the following ratios of the starting components in wt.%:

a matrix representing a chitosan gel - 90.0-99.9;

an aqueous solution of the specified drug "Adgelon" - 0.1-10.0 (based on the matrix).

The technical problem is also achieved by a method for producing a biocompatible composition having osteoinductive and osteoconductive properties for replenishing (treating) partial and complete defects of cartilage and bone tissue, which consists in immobilizing the drug Adgelon in the form of an aqueous solution containing serum glycoprotein from serum from the chitosan gel blood of cattle, which has biological activity in ultra-low doses of 10 ^-9 -10 ^-15 mg / ml, and calcium salt.

Chitosan gel provides a bacteriostatic effect and contributes to the dosed output of the drug "Adgelon" in the area of the defect. The gel also has good biodegradability.

The used drug "Adgelon" is known as a drug for the treatment of, in particular, eye burns, damage to the cornea of the eye (see, for example, the description of the published application of the Russian Federation (RU) No. 94008009, 06/20/1996; R.A. Gundorova and others. Adgelona in the treatment of penetrating wounds of the cornea in the experiment, "Bulletin of Ophthalmology", 1997, t.113, No. 2, p.12-15, description of the patent of the Russian Federation (RU) No. 2220687, 01/10/2004).

Also, this drug “Adgelon” is used to restore cartilage defects when injected into the lesion area (see, for example, A.S. Neverkovich, Ontogenesis 2000, v.31, No. 4, p. 282-283).

Get the drug "Adgelon" on the basis of serum glycoprotein isolated from animal blood (description of the patent of the Russian Federation (RU) No. 2136695, 09/10/1999; description of the patent of the Russian Federation No. 2223781, 02/20/2004). Its composition (the drug "Adgelon") includes a calcium salt, in particular calcium sulfate.

So, the composition includes chitosan gel (90.0-99.9%), which acts as a matrix for populating it with cells and extracellular matrix molecules during implantation and the Adgelon preparation containing serum glycoprotein immobilized in it. Upon receipt of the composition, the chitosan gel is mixed with the Adgelon solution containing serum glycoprotein and calcium salts. The composition provides increased ability to stimulate the proliferation of osteoblasts, reparative processes of bone formation with the restoration of morphologically normal bone matrix.

A combination is used for implantation into the defective cavity of the bone tissue with further closure of the collagen membrane and suturing of the periosteum and soft tissues. The gel base is used so that the peptide preparation has a dosed gradual yield in the area of bone damage. After applying the gel to prevent its rapid leaching, the defect area is closed with a collagen membrane and the gum is sutured.

The following examples illustrate the invention, but do not limit it.

Example 1. (preparation of a composition according to the invention)

The composition of the osteoinductor and osteoconductor in the treatment of bone pathology in dentistry and maxillofacial surgery was prepared as follows with the following ratio of starting components (wt.%):

1.1. on a chitosan gel of 99.5% (100 g), the preparation for injection “Adgelon” 0.5% (0.5 g) was immobilized.

1.2. on the chitosan gel, 90.9% (10 g) immobilized the drug for injection "Adgelon" 9.1% (1 g).

The use of this composition for healing bone defects is characterized by the following examples.

Example 2

Around the implant, a mild aseptic inflammatory reaction with the onset of capsule formation from immature connective tissue. In the fatty tissue around the implant, moderate edema, weak infiltration by neutrophilic leukocytes and macrophages are noted. Macrophage elements predominate. The proliferation of fibroblasts forming small cords and vascular neoplasm is also noted. Foci of immature granulation tissue form, from which the connective tissue capsule begins to form. Separate newly formed capillaries appear, multiplying by budding from pre-existing ones.

All this indicates a mild aseptic inflammation around the implant as the initial tissue reaction to it.

The difference is a very weak reaction of the surrounding tissue to the implant: each fragment is surrounded by a relatively small number of cellular elements, mainly macrophages with an admixture of neutrophils. Around each fragment, mild fibroblast proliferation is observed.

Thus, on the 3rd day after implantation of the composition in the tissue, such signs of a tissue reaction as aseptic inflammation, macrophage reaction and beginning macrophage resorption of the biomaterial, fibroblastic reaction and vascular neoplasm around the implant with the onset of the formation of connective tissue capsule and germination of granulation tissue into the implant are revealed.

7 days after implantation

The implant is pierced by strands of granulation tissue. In the strands growing into the interior of the implant, the tissue is less mature, it contains a large number of macrophages and single multinucleated giant cells surrounding fragments of chitosan involved in their resorption. In the granulation tissue sprouting into the implant, in addition to macrophages, newly formed vessels of the caliber of capillaries and strands of proliferating fibroblasts are visible. Fibroblasts have a fusiform and multi-process shape. Maturity of the tissue lies in the predominance of fibroblasts over other cells, the presence of a large number of newly formed collagen fibers, and an increased content of acidic GAGs. Exudative inflammatory reaction in the form of edema and neutrophilic infiltration in granulation tissue and in the tissue surrounding the implant is weak. The space between the implant fragments is filled with relatively mature connective tissue. Thus, 7 days after implantation, signs of an inflammatory tissue reaction to implants are reduced. Macrophage resorption and germination by granulation tissue of the implant are enhanced.

14 days after implantation

The implant is completely germinated by connective tissue into which fragments of the chitosan gel are walled. The connective tissue filling the space between the implant fragments is more mature than in the previous period (there are more fibroblasts and collagen fibers in it), however, macrophages are still present in this tissue. Inflammatory (neutrophilic) infiltration is reduced compared with the previous period. In addition to collagen fibers, fatty tissue forms. Thus, after 14 days, the dynamics of the morphological picture indicates a decrease in inflammation, maturation of connective tissue and increased resorption of materials.

30 days after implantation

In place of the implant, fatty tissue is completely restored, in which only small areas of fibrous tissue are visible. Small fragments of collagen remain at the center of this tissue. In the surrounding tissue, inflammatory infiltration is practically absent. Thus, a significant restoration of the structure of subcutaneous tissue occurs during this period. At this time, inflammatory infiltration decreases, and in some animals it disappears completely, the initial structure of subcutaneous tissue is restored. Fibrosed collagen tissue, which filled the space between collagen fragments in the previous period, is currently being converted into loose fiber as a result of the reverse development of collagen tissue.

Thus, 30 days after implantation, in some groups, the implants are resorbed, the connective tissue undergoes partially reverse development (involution), and subcutaneous tissue is restored at the site of implantation.

2 months after implantation

At the site of the implant, the structure of fatty tissue is mainly restored. Thus, for this period, the structure of the subcutaneous tissue is restored. In the fatty tissue, only small fibrous thickenings are found, there is no inflammatory reaction. This indicates that by two months the fiber structure has recovered.

A morphological study using histological and histochemical methods allowed us to study: structural features of the composition in the dynamics of its destruction, as well as the tissue reaction during its subcutaneous implantation.

It was shown that cellular elements (neutrophils, macrophages, fibroblasts, etc.) penetrate the implant, forming granulation tissue, and then more mature fibrous connective tissue, which grows and fills the space between the structural elements. As the implant is resorbed, it is increasingly replaced by connective tissue, and from the second week after implantation, this tissue is gradually subjected to involution (reverse development), and the structure of subcutaneous tissue is restored at the site of implantation. The composition causes a relatively weak and gradually decreasing inflammatory response in the tissue surrounding the implant.

A decrease in the volume of the implant occurs due to cellular (mainly macrophage) resorption. After 2 months, there is no sign of surgery at the site of the implant.

Example 3

In the experiment, the effect of the composition on the restoration of bone tissue was studied when it was introduced into an artificially caused bone defect. A traditional method in biomedical research for assessing the effect of osteoplastic materials on the healing of bone defects is the creation of an artificial defect in the distal pineal gland of the rat thigh followed by a study of the dynamics of bone healing. In the experiment, the course of regenerative processes in bone defects was studied under the influence of these materials, the nature of the interaction of the composition and the regenerate in the bone wound, and the effect of the composition on the regenerative process in the bone wound.

Outbred white rats (males) weighing 180-210 g were taken into experiments. In the area of distal epiphyses of the femur, standard edge defects with a diameter of 2.0 mm and a depth of about 2 mm were reproduced using boron under hexenal anesthesia. Bone defects in the control were not filled with anything; the composition was introduced in the experiment. To prevent materials from falling out of the inlet, it was closed with a sterile thin collagen membrane. The operation was performed under aseptic conditions. The time for removing animals from the experiments is 15, 30, 60, 90 days. In each term, there were 4-5 rats from the group. Tissue blocks were decalcified in an EDTA solution and subjected to standard histological treatment with paraffin embedding and staining of the paraffin sections with hematoxylin and eosin.

In experiments on rats, the course of bone-repair processes was observed when the osteoinductive and osteoconductive composition was introduced into standard bone defects reproduced in the proximal tibia epiphyses.

Fifteen days after the composition was introduced into the defect, the bone defect in large areas was filled with cell-fibrous connective tissue. Quite often, it was possible to see microcavities in it, bordered by seals of the main substance, containing a belt of fibroblastic elements and thin reticular as well as collagen fibers. The formation of osteogenic cells occurred with their subsequent differentiation.

In the direction into the depth of the defect, signs of building a new bone substance were found. The newly formed bone structures were represented by deposits of osteoid substance, and in some places trabecular widely looped systems from fibrous bone glands.

In some areas, signs of resorptive changes were observed, in particular macrophages were detected. The predominant regenerate element in the bone defect, as a rule, everywhere remained cell-fibrous, sometimes well fibrillated, connective tissue, which included particles of planted material.

In the deeper parts of the defect, collagen fibers predominated in the composition of the fibrillar component of the regenerate.

Cell elements were represented mainly by fibroblasts.

At the entrance to the defect, the cellularity of the regenerate was somewhat increased due to impurities to fibroblasts of cells such as macrophages and, occasionally, eosinophils. Attention was drawn to how the growth of cell-fibrous tissue into the composition and the naturally occurring formation of osteoid substance and fibrous bone trabeculae at the injection site of the composition occurred. The growth of cell-fibrous connective tissue between the structures of the implant and the development of an intense giant-cell reaction characterized the state of the tissue complex in the defect area as an active proliferative-reactive process of osteogenic tissue reconstruction during the indicated periods of observations.

30 days after replanting, an intensive neoplasm of bone tissue was noted in the defect, the osteoinductive process became pronounced. As a rule, at the entrance to the bone defect in the cell-fibrous tissue of the regenerate there was a neoplasm of the bone structures, and the intertrabular spaces in them were occupied by cell-fibrous tissue. Giant cells and osteoid beams are visible. It is interesting to note that the formation of cartilage tissue reached a significant degree, and often between the fragments of a bone-like substance, cell-fibrous connective tissue actively grows. The formation of osteoid rolls is an important element of the processes that occur in a bone defect. In the newly formed bone tissue, a wide-looped network of young bone trabeculae prevailed, which was layered on the mother's bone. The newly formed bone beams near the maternal bone were thickened, their matrix acquired a thin-fiber plate-like character. The bone substance in some places had an osteoid, and in most fields of view, a coarse-fibrous structure.

On the 60th day, the newly formed bone substance undergoes secondary rearrangement, its individual sections undergo resorption with the participation of giant osteoclasts.

In some areas, the process of maturation deep in the bone marrow led to the appearance of osteon systems. In general, the reparative process in the bone defect was distinguished by the intensity and general tendency to active secondary rearrangement and maturation of the bone substance.

After 60 and 90 days of observation, bone structures began to predominate in the regenerate, although even at this time in the area of entry into the defect, territories were preserved that retained the features of a connective tissue substrate. Bone regenerate underwent gradual maturation; in the depth of the defect, it turned out to be fused with the mother's bone. Here, his substance acquired a lamellar character; outwardly, the matrix of bone beams remained fibrous.

Only on the 90th day of observations did the bone substance of the regenerate show a tendency to form individual osteon structures. At the same time, the structure and tinctorial properties of the newly formed bone substance did not undergo significant changes and corresponded to the characteristics inherent in bone regenerate, which develops for a period of 60-90 days. The formation of extensive fields of young bone tissue, represented by a network of fibrous bone trabeculae, forming either narrow or wide arcades that form on the edge of the mother’s bone, took place. Bone substance was characterized by a high degree of differentiation and had a lamellar structure.

Conclusion

As the results of the study showed, the composition caused the formation of cell-fibrous tissue in bone defects in the transplantation area. As a rule, focal lymphomacrophagic infiltrates were observed around fragments of planted material; giant multinuclear cells were often seen. At the same time, already on the 15th day of the experiment, signs of varying severity of bone formation activity were observed, although the connective tissue component prevails over the bone. In the period from 30 to 90 days, bone defects were replaced with newly formed bone tissue. At the same time, the ability to osteoinduction manifested itself, which was especially pronounced in the late stages, when intensive formation of bone substance was observed. Of particular note is the rather early appearance of signs of reparative osteogenesis, which indicates a rather high osteopotency of the implant. Thus, the intensity of reparative osteogenesis was quite high. Recovery was due to newly formed bone elements that easily replaced the chitosan base of the composition.

Claims (2)

1. The composition is an osteoinductor and osteoconductor, in the treatment of bone pathology in dentistry and maxillofacial surgery, containing a matrix that is a chitosan gel and an aqueous solution for injection Adgelon immobilized on it, containing serum glycoprotein obtained from cattle blood serum having biological activity in ultra-low doses of 10 ^-9 -10 ^-15 mg / ml, and calcium salt in the following ratio of starting components, wt.%:
chitosan gel matrix 90.0-99.9 the drug above "Adgelon" in the form of an aqueous solution (based on the matrix) 0.1-10.0 2. A method of obtaining a composition of an osteoinductor and an osteoconductor, in the treatment of bone pathology in dentistry and maxillofacial surgery according to claim 1, including the immobilization of an aqueous solution of the drug "Adgelon" on a chitosan gel.