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

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

The invention relates to medicine and the medical industry, and more specifically to obtaining biocompatible materials for osteoplasty, which can be used as osteoinductive funds to fill defects in bone and cartilage tissues in traumatology, orthopedics, dentistry, neurosurgery for congenital and acquired diseases.

Osteoplastic materials are widely used in wound-healing defects of locomotor apparatus, dentistry, maxillofacial surgery and traumatology. Among these materials are popular composites of collagen and a crystalline component, primarily hydroxyapatite and tricalcium phosphate. This is an important role for the properties of a mineral filler, which depend on its chemical composition, source, and method of synthesis. The effectiveness of the replacement implant material regenerating bone tissue is largely determined by its resorption. The hydroxyapatite animal enclosed in bone regenerate, long-term resorbed and is "trapped" in the newly formed bone tissue, in addition, the acute problem of infection with prion diseases, when using bone material of cattle.

Known SPO is about plastic bone cavity with a mixture of ceramics (tricalcium phosphate and hydroxyapatite (Weerakoon. Replacement of bone defects with ceramics on the basis of hydroxyapatite tumors and tumor-like diseases. Abstract of dissertation of candidate of medical Sciences. - M., 1998. - 21 S.).

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

Known methods of homoplastic bone cavities of bone splinters and shavings in cancellous bone (abstract of dissertation of candidate of medical Sciences. M., 1957. - 16 S.; Glasunov. Replacement of defects of tubular bones crushed bone. Abstract of dissertation of candidate of medical Sciences. L., 1963. - 40 S.).

The disadvantages of these methods are the possibility of nekrotizirovanne and rejection free, deprived of food bone fragments, the danger of allergic reactions and infection of the recipient (HIV, hepatitis b and C).

From the description to the patent of Russian Federation (RU) No. 2325170, 27.05.2008 known biocomposite material for osteoplasty, including bone matrix (40,0-95,0 wt.%), collagen (0.25 to 1.0 wt.%), pharmaceutically acceptable bestnet (0.1 to 10.0 wt.%) and water (the rest).

As a bone graft in this composition uses one of the following bone material or a mixture of these bone materials selected from the following groups: natural bone mineral, bone hydroxy shall Petit (hereinafter referred to Gap), synthetic HAP, pulverized demineralized bone of animals, pulverized demineralized bone man.

Biocomposite material provides osteoconductive and osteoinductive activity for the transplant and low antigenicity.

From the description to the patent of Russian Federation (RU) No. 2296575, 01.09.2004 known composition for the treatment of bone defects in injuries and inflammatory conditions, which contains a nutrient medium Needle MEM, 10%serum fruits cows, diploid cells of human fibroblasts and gel filler with the following content of components in 1 ml: suspension diploid cells human fibroblasts -1×10⁵-5×10⁷cell; culture medium Needle MEM is 0.01 to 0.9; 10%serum fruits cow - 0,01-0,2; gel filler - the rest is up to 1 ml as diploid cells human fibroblasts composition comprises a strain of diploid cells of human fibroblasts from lung tissue of embryo replacement therapy FAC 16-1 or the strain of diploid cells of human fibroblasts from skin and muscle tissue of the embryo replacement therapy FAC 16-2. As a gel filler can be used 5%polyethylene oxide. The use composition can accelerate the onset of remission and increase the length of remission chronic diseases.

From the description to PA is into Russian Federation (RU) No. 2301684, 21.11.2005 known for another transplant mixture containing (wt.%) freeze-dried allogenic bone - 65; allogenic hydroxyapatite - 10; platelet gel, autologous blood of the patient 20; metronidazole - 5.

This mix is mainly used in complex treatment of patients with parodontitis.

From the description of the published application of the Russian Federation (RU) No. 98119221, 10.09.1999 known solution inducer of osteogenesis for dentistry, including glycosaminoglycans, water, calcium salts and water-soluble derivatives of cellulose, in the following ratio, wt.%:

Calcium salt is 0.001 to 0.01;

Sulfated glycosaminoglycans - 0,1-1,0;

Water-soluble derivatives of cellulose - 0,05-2,5;

Water - the rest.

From the description to the patent of Russian Federation (RU) No. 2335258, 27.05.2008-known implant for bone cavity plasty on the basis of the coarse collagen matrix of spongy bone decalcified, and on it are immobilization of antimicrobial drugs, cultures blast cells, stimulants biogenesis (for example, methyluracil, hydroxyapatite), vitamins (e.g. ascorbic acid). For cooking on a collagen matrix with immobilized therein drugs applied composition having adhesive properties (bioactive product containing polyvinyl alcohol, plasmarelaisbot the composition is).

The method of manufacturing the implant is quite complicated.

In recent years more and more are spread implants intended for fixation of hard tissue, in particular bone tissue with simultaneous stimulation of tissue regeneration.

From the description to the patent of Russian Federation (RU) No. 2153877, 10.08.2000 known agent that stimulates solid fabric in the form of a membrane, powder, gel, granules or solution, for which manufacturing using chitosan fixed on it a polysaccharide selected from heparin, heparan sulfate, chondroitin sulfates and doctranslate.

And from the description to the patent of Russian Federation (RU) No. 2301677, 27.06.2007 known biotransplant for the treatment of degenerative and traumatic diseases of the articular cartilage and intervertebral discs, characterized in that it contains a media that contains a basic substance, for example, chitosan, and/or alginate, and/or collagen and composite additive hyaluronic acid, chondroitin sulfate in the ratio of basic substances and composite additives 90-99%:1-10%, and the suspension of allogeneic or autologous cell cultures chondroblasts, fibroblasts and mesenchymal stem cells in their content in 1 ml of media 5-50 million cells; while the carrier further includes a matrix proteins: collagen types (I-XIX), integrin, fibronectin, laminin in the concentration is from 0.1 to 100 μg/ml; in addition, the carrier further includes a fibroblast growth factors, and/or transforming growth factor α and β, and/or a growth factor secreted by platelets, and/or a growth factor for hepatocytes in a concentration of from 1 to 300 ng/ml, and the medium further includes antiseptics selected from the group of ions of silver, copper, gold, and broad-spectrum antibiotics.

This transplant is quite complicated because of the use of stem cells, the allocation of which is associated with great difficulties.

Despite the diversity of osteoplastic materials of different content and properties today are among them, you cannot select "ideal" for use in dentistry and maxillofacial surgery.

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

In recent years, more attention is paid to substances introduced in osteoplastic composition with the aim to build bone tissue and its mineralization. But this is about the time it was not a comparative analysis of osteoplastic materials of different composition and origin, the most acceptable in their properties, for the directional reparative osteogenesis.

The technical objective of the claimed invention is to provide biocompatible, bioactive and degrading in the dynamics of the songs, modeled in accordance with the size and volume of the plastic zone and able to effectively stimulate the regeneration of bone tissue, to reduce the number of infectious complications and to shorten the time of treatment; and the expansion of the means with wound-healing effect in relation to the supporting tissues, bones of the craniofacial area and dentistry, and the restoration of the bone defect after surgery.

The technical problem is achieved by the creation of biocompatible composition having osteoinductive and osteoconductive properties to fill (treatment) partial and full defects in cartilage and bone tissue containing matrix comprising chitosan gel and an aqueous solution of the drug "Algalon"containing serum glycoprotein obtained from the blood serum of cattle possessing biological activity in midget doses

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

the matrix representing a chitosan gel to 90.0 to 99.9;

an aqueous solution of the listed drug, "Hell is the Elon" - 0,1-10,0 (per matrix).

The technical problem is also achieved by a method of obtaining a biocompatible composition having osteoinductive and osteoconductive properties to fill (treatment) partial and full defects in cartilage and bone tissue, which consists in the immobilization in chitosan gel drug "Algalon" in the form of an aqueous solution containing serum glycoprotein from the blood serum of cattle possessing biological activity in midget doses of 10^-9-10^-15mg/ml, and a salt of calcium.

Chitosan gel provides a bacteriostatic effect and contributes to the dosed release of the drug "Algalon" in the area of the defect. The gel has good ability to biodegradation.

Used the drug "Algalon" is known as a drug for treating, in particular, burns eyes, damage to the cornea (see, for example, the description of the published application of the Russian Federation (RU) No. 94008009, 20.06.1996; Ragendra and other Application Adelana in the treatment of penetrating wounds of the cornea in the experiment, "Bulletin of ophthalmology, 1997, t, No. 2, p.12-15, the description to the patent of Russian Federation (RU) No. 2220687, 10.01.2004).

Also, this drug "Algalon" is used to repair cartilage defects when injected into the site of injury (see, for example, Asseverate, Ontogeny 2000, t, No. 4, s-283).

Get prep the rat "Algalon" on the basis of serum glycoprotein, isolated from the blood of animals (description to the patent of Russian Federation (RU) No. 2136695, 10.09.1999; description to the patent of Russian Federation №2223781, 20.02.2004). In its composition (drug Aggelon") is a salt of calcium, particularly calcium sulfate.

So, the composition comprises chitosan gel (of 90.0 to 99.9%), which acts as a matrix for the settlement of its cells and molecules of the extracellular matrix during implantation and immobilized in its preparation "Algalon"containing serum glycoprotein. Upon receipt of a composition mixed chitosan gel with a solution "Algalon"containing serum glycoprotein and calcium salts. The composition provides improved ability to stimulate the proliferation of osteoblasts, reparative osteogenesis recovery of morphologically normal bone matrix.

Use a combination for implantation into the defect cavity bone tissue with further closing of the collagen membrane and the stitching of the periosteum and soft tissues. Gel base used for the preparation of peptides were dosed gradual release in the damaged area of bone tissue. After application of the gel to prevent rapid leaching area of the defect is closed by a collagen membrane and sutured gums.

The following examples illustrate the invention but do not restrict it.

Example 1 (preparation of compositions according to the invention)

Composition - osteoinduction and osteoconduction in the treatment of bone pathology in dental and maxillofacial surgery, was prepared in the following manner when the following ratio of initial components (wt.%):

1.1. on chitosan gel 99,5% (100 g) immobilizovana the drug for injection "Algalon" of 0.5% (0.5 g).

1.2. on chitosan gel 90,9% (10 g) immobilizovana the drug for injection "Algalon" 9,1% (1 g).

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

Example 2

Around the implant weak aseptic inflammatory reaction with the beginning of the formation of capsules of immature connective tissue. In adipose tissue around the implant is marked moderate edema, mild neutrophilic infiltration by leukocytes and macrophages. Dominated by macrophage elements. There is also the proliferation of fibroblasts, forming small bands, and the tumor vessels. Formed the foci of immature granulation tissue, which begins to form connective tissue capsule. There was a newly formed capillaries, reproducing by budding from preexisting.

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

The difference is obvious the ü weak reaction of the surrounding tissue on the implant: each fragment is surrounded by a relatively small number of cellular elements, mainly macrophages mixed with neutrophils. Around each fragment is observed weakly pronounced proliferation of fibroblasts.

So, 3 days after the implantation of the composition into the tissue are detected such signs of tissue reaction, as aseptic inflammation, macrophage reaction and starting macrophage resorption of the biomaterial fibroblastic reaction and the formation of new blood vessels around the implant with the beginning of the formation of a connective tissue capsule and growth of granulation tissue into the implant.

7 days after implantation

The implant is permeated with strands of granulation tissue. The strands that grow into the implant, the tissue is less Mature, it contains a large number of macrophages and a single multi-core giant cells surrounding the fragments of chitosan participating in their resorption. In granulation tissue growing into the implant, in addition to macrophages visible neovasculature caliber capillaries and fibers of proliferating fibroblasts. Fibroblasts are spindle-shaped and MNOGOOTRASLEVOE form. The maturity of the fabric lies in the predominance of fibroblasts over other cells, the presence of a large number of newly formed collagen fibers, increased acidic content of GAG. Exudative inflammatory reaction in the form of edema and neutrophil infiltration in ranolazine tissue and surrounding the implant tissue are weak. The space between the fragments of the implant is filled with a relatively Mature connective tissue. Thus, after 7 days after implantation signs of an inflammatory tissue reaction to implants reduced. Macrophage resorption and germination granulation tissue implant intensify.

14 days after implantation

The implant is fully couched connective tissue, in which is imbedded fragments of chitosan gel. Connective tissue that fills the space between the fragments of the implant has a more Mature character than in the previous period (it has more fibroblasts and collagen fibers), but in this tissue still present in macrophages. Inflammation (neutrophil) infiltration is reduced in comparison with the previous period. In addition to collagen fibers, the formation of adipose tissue. Thus, after 14 days the dynamics of morphological patterns evidenced by a decrease in inflammation, maturation of connective tissue and increased resorption of materials.

30 days after implantation

On the site of the implant is fully restored fatty tissue, in which are visible only small areas of fibrous tissue. In the center of this tissue remains small fragments of collagen. In the surrounding tissue inflammatory infiltration is virtually nonexistent. Thus, n is the period there is a significant restoration of the structure of the subcutaneous tissue. This term inflammatory infiltration decreases, and some animals disappear completely, the original structure of the subcutaneous tissue is restored. Fibrosarcoma collagen tissue, which fills the space between collagen fragments in the previous period, at the present time, in the reverse development of collagen tissue, turns into loose fiber.

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

2 months after implantation

On the site of the implant structure of fatty tissue is mostly restored. Thus, at this time the structure of the subcutaneous tissue is restored. In adipose tissue, there are only a small fibrous thickening, inflammatory reaction is absent. This suggests that the two-month term structure of cellulose recovered.

Morphological study using histological and histochemical methods allowed us to study the structural features of the composition in the dynamics of its destruction, and tissue reaction when her subcutaneous implantation.

It is shown that the cellular elements (neutrophils, macrophages, fibral the texts and other) penetrate into the implant, forming granulation tissue, and then the more Mature fibrous connective tissue, growing and filling the space between the structural elements. As resorption of the implant, it is more and more replaced by connective tissue, and from the second week after implantation of the tissue gradually undergoes involution (reverse development), and in the implantation site recovers the structure of the subcutaneous tissue. The composition causes a relatively weak and gradually decreasing the inflammatory response in the surrounding implant tissue.

The decrease in the volume of the implant occurs due to the cell (mostly macrophage) resorption. After 2 months on the site of the implant, there are no traces of the operation.

Example 3

In the experiment studied the effect of composition on recovery of bone at its introduction in artificially induced bone defect. Traditional biomedical research method for assessing the impact of osteoplastic materials on healing of bone defects is the creation of artificial defect in the distal epiphysis of the femur of the rat, followed by the study of the dynamics of bone tissue healing. In the experiment studied for regenerative processes in bone defects under the influence of these materials, the nature of the interaction of composition and regeneration in bone wound, the effects of the influence of composition on the regenerative process in bone wound.

In experiments took outbred albino rats (males) weighing 180-210 g In the region of the distal epiphysis of the femur under geksenalovy anesthesia reproduced using boron standard boundary defects in diameter on the surface of 2.0 mm and a depth of about 2 mm Bone defects in the control did not fill in the experience of the injected composition. To prevent the fall of materials from the inlet was closed sterile thin collagen membrane. The operation was conducted under aseptic conditions. The timing of breeding animals from experiments - 15, 30, 60, 90 days. In each period was 4-5 rats from the group. Tissue blocks decalzinirute in EDTA and subjected to standard histological processing to fill in the paraffin coating of paraffin sections with hematoxylin and eosin.

In experiments on rats were observed for bone reparative processes when making the osteoinductive and osteoconductive composition in standard bone defects, reproduced in the proximal epiphysis of the tibia bone.

Within 15 days after any defect in the composition of bone defect in large areas was filled cellular fibrous connective tissue. Quite often it was possible to see the micro-cavities lined seals of the basic substance containing a band of fibroblastic elements and thin the x reticular, as well as collagen fibers. There was a formation of osteogenic cells and their subsequent differentiation.

Towards the depth of the defect was detected signs build new bone substance. The newly formed bone structures were represented by deposits Stevenage substances, and places of trabecular widely looping systems of fibrous bone beams.

In some areas there were signs resorptive changes, in particular macrophages were found. The prevailing element of the regenerate bone defect, everywhere typically remained cell-fibrous, sometimes well fibrillazione, connective tissue, comprising particles of implantable material.

In the deeper parts of the defect in the composition of the fibrillar components of the regenerate was dominated by collagen fibers.

Cellular elements were represented mainly by fibroblasts.

At the entrance to the defect cellularity of the regenerate was slightly increased due to impurities in the fibroblasts of such cells, like macrophages and occasionally eosinophils. Pay attention to how happened ingrowth of cellular fibrous tissue in composition and naturally occurring formation at the injection composition Stevenage substances and fibrous bone trabeculae. Ingrowth of cell-fiber connection is additional tissue between the implant and the development of intensive giant cell reaction characterized within a specified time observations of the condition of the tissue complex in the area of the defect as active proliferative-reactive the process of restoring the osteogenic tissue.

30 days after graft in the defect noted the intensive formation of bone tissue, osteoinductive process became apparent. As a rule, at the entrance of the bone defect in cellular fibrous tissue to regenerate was the formation of bone structures, and microbacteria space in them were occupied by cellular fibrous tissue. Visible giant cells and osteopenia balocchi. It is interesting to note that largely ran up to the formation of cartilaginous tissue, often between fragments korepodobnoj substances actively growing cellular fibrous connective tissue. Education osteogenic beams is an important element of the processes occurring in the bone defect. In the newly formed bone tissue was dominated by shirokolisty mesh from young bone trabeculae, which naprashivaetsa on maternal bone. The newly formed bone beams near the parent bone was totalsales, the matrix has acquired a fine lamellar in nature. Bone substance had somewhere osteopenia, and in most of the fields of view - coarse-fibered structure.

60 days of the newly formed bone substance undergoes secondary reconstruction, separate parcels undergo resorption, with the participation of giant osteoclasts.

After 60 and 90 days of observation in the regenerate, began to dominate the bone structure, though in these terms in the input area in the defect mentioned site, preserved features of connective tissue substrate. Bone regenerate subjected to gradual maturation, in the depth of the defect he was unstable welded to the parent bone. Its substance was acquired lamellar in nature, outside the matrix of bone beams remained fibrous.

Only 90 day observations, bone regenerate found the tendency to form separate osteonic structures. The structure and tinctorial properties of newly formed bone was not changed significantly corresponded to the characteristics of bone to regenerate, developing in terms of 60-90 days. There was a formation of extensive fields of young bone tissue, represented by a mesh of fibrous bone trabeculae forming the narrow, then wide arcade, naprashivayuschiesya on the edge of the parent bone. Bone substance was characterized by a high degree is to tell differentiation and had a lamellar structure.

Conclusion

As shown by the results of the study, the composition is caused in the area of graft formation in bone defects of cellular fibrous tissue. Usually around fragments implantable material was observed focal lymphomacrophagal infiltrates, often you could see a giant multinucleated cells. At the same time on the 15th day of the experiment there were signs of varying degrees of extent bone regeneration activity, although connective tissue component prevails over the bone. In terms of 30 to 90 days was the replacement of bone defects of the newly formed bone tissue. They showed themselves the ability to osteoinductive, which is especially clearly manifested in the later stages, when abudals intensive formation of bone. It should be specified at a very early signs of reparative osteogenesis, which indicates a fairly high osteoporosi implant. Thus, the intensity of reparative osteogenesis was quite high. The recovery was due to nevoobrazimaja bone elements, easy replacement chitosan based composition.

1. Composition - osteoinduction and osteoconduction, in the treatment of bone pathology in dental and maxillofacial surgery, containing the matrix, pre the excitation chitosan gel and immobilized thereon an aqueous solution for injection "Algalon", containing serum glycoprotein obtained from the blood serum of cattle possessing biological activity in midget doses of 10^-9-10^-15mg/ml, and a salt of calcium in the following ratio of initial components, wt.%:

2. The method of obtaining the composition of osteoinductive and osteoconductive, in the treatment of bone pathology in dental and maxillofacial surgery according to claim 1, including immobilization of an aqueous solution of the drug "Algalon" on chitosan gel.