RU2317088C1 - Osteoinducing material (variants) - Google Patents

Abstract

FIELD: medicine, in particular agents for bone defect compensation.

SUBSTANCE: in the first embodiment claimed material includes hydroxyapatite with tricalcium phosphate and additionally it contains osteogenesis-inducing composition of non-collagen bone tissue proteins in specific component ratio per 100 g of material. In the second embodiment claimed material includes hydroxyapatite with tricalcium phosphate and/or collagen and additionally it contains osteogenesis-inducing composition of non-collagen bone tissue proteins. In the third embodiment claimed material includes hydroxyapatite with tricalcium phosphate and/or collagen and additionally it contains osteogenesis-inducing composition of non-collagen bone tissue proteins and double distilled water in specific component ratio per 100 g of material.

EFFECT: material with high osteoplastic ability.

9 cl, 3 ex, 6 dwg

Description

The invention relates to medicine, in particular to means for compensating for defects in bones (bone cysts, foci of osteonecrosis, osteomyelitic sequestration) by implanting in a defect material that stimulates bone tissue neoplasm.

At present, there are many known osteoplastic materials containing collagen and calcium phosphate substances individually or in combination: Hydroxyapol, Kolapol, Gapkol (ZAO NPO Polistom, RF); “KollapAn” (Firm “Intermedapatit”, RF); Allomatrix-implant (Connect Biopharm LLC, Russian Federation); Osteoplast (NPK Vitafarm-R LLC, RF); “Bio-Oss”, “Bio-Oss Collagen” (“Geistlich, Biomaterials” Switzerland); “Biobon”, “Endobon” (“Biomet Merck”, Germany); Osteoplant flex (Bioteck, Italy).

It is also known that the composition of non-collagenous proteins of bone tissue (NSC) obtained by extraction with a urea solution, liberation from water-soluble proteins by dialysis against water possesses osteoinductive properties (Patent of the Russian Federation No. 2050158, CL A61K 35/32. // (A61K 35/32 38:00, published on December 20, 1995).

The closest analogue to the proposed decision on the composition, physico-chemical and biological properties is a material for the compensation of bone defects, including collagen and / or calcium phosphate substances (RF patent No. 2034572, CL A61L 15/32, publ. 05/10/1995) .

The disadvantage of these drugs is the fact that collagen and calcium phosphate substances themselves do not have osteoinductive properties and acquire them after binding of peptide growth factors circulating in the blood [Tissue Engineering: Applications in Maxillofacial Surgery and Perriodontics / Eds. S.E. Linch, R. G. Genco, R. E. Marcs. - Chicago: Quintesstnce Publishing Co, Inc. - 1999. - 300 p.].

The technical result achieved by the claimed solution is to obtain a drug with high osteoplastic ability, due to the presence directly in it of protein substances isolated from bone tissue with osteoinductive properties.

The specified result is achieved by an osteoinductive material, including hydroxyapatite with tricalcium phosphate and / or collagen, the material additionally containing a composition of non-collagenous proteins of bone tissue, stimulating osteogenesis, in the following ratio of components per 100 g of material, g:

composition of non-collagenous proteins of bone tissue, stimulating osteogenesis 0.001-0.005 hydroxyapatite with tricalcium phosphate rest

And also due to the fact that the composition of hydroxyapatite with tricalcium phosphate additionally contains fluorohydroxyapatite and / or carbonatapatite and / or calcium fluoride.

And also due to the fact that the material is made in the form of granules or powder.

The indicated result is achieved by an osteoinductive material, including hydroxyapatites and / or collagen, the material additionally containing a composition of non-collagenous proteins of bone tissue that stimulates osteogenesis, in the following ratio of components per 100 g of material, g:

composition of non-collagenous proteins of bone tissue, stimulating osteogenesis 0.001-0.005 hydroxyapatite with tricalcium phosphate 20-90 collagen rest

And also due to the fact that the composition of hydroxyapatite with tricalcium phosphate additionally contains fluorohydroxyapatite, and / or carbonatapatite, and / or calcium fluoride.

And also due to the fact that the material is made in the form of a sponge or ceramic.

The indicated result is achieved by an osteoinductive material, including hydroxyapatite with tricalcium phosphate and / or collagen, the material additionally containing a composition of non-collagenous proteins of bone tissue, stimulating osteogenesis, and bidistilled water in the following ratio of components per 100 g of material, g:

composition of non-collagenous proteins of bone tissue, stimulating osteogenesis 0.001-0.005 hydroxyapatite with tricalcium phosphate 10-80 collagen 1-2 bidistilled water rest

And also due to the fact that the composition of hydroxyapatite with tricalcium phosphate additionally contains fluorohydroxyapatite, and / or carbonatapatite, and / or calcium fluoride.

And also due to the fact that the material is made in the form of a gel.

The invention is illustrated by drawings, where figure 1 shows the regenerate in a hole defect in the femur of a rat after 75 days of implantation of the material INDOST granules (hematoxylin and eosin, vol.10, ok.10); figure 2 - regenerate in the hole defect of the femur of the rat after 75 days of implantation of the material HYDROXIAPOL HAP-99 (Picrofuxin, vol. 10, ok.10), figure 3 - regenerate in the diaphyseal defect of the tibia of the rat after 60 days after implantation of calcium phosphate-collagen sponge (control), (eosin and hematoxylin, vol. 20, approx. 10), Fig. 4 - regenerate in the diaphyseal defect of the tibia of the rat 60 days after implantation of the INDOST-sponge (experiment), (icrosirius , vol.20, ok.10), figure 5 - bone regenerate in the area of the defect of the lower jaw of the rat after 90 days lantation of the gel HYDROKIAPOL-collagen (eosin and hematoxylin, vol. 40, approx. 10), Fig. 6 - bone regenerate in the zone of the defect of the lower jaw of the rat after 90 days of implantation INDOST-gel (eosin and hematoxylin, vol. 40, approx. 10).

The preparation and use of the claimed material is illustrated by non-exhaustive examples.

Execution Example 1

Prepare an osteoinductive material (INDOST-granules) composition, g:

composition of non-collagenic proteins, inducing osteogenesis 0.001-0.005 HYDROXYAPOL GAP-99 rest

HYDROXYAPOL HAP-99 (manufactured by NPO POLISTOM) is a composition of hydroxyapatite with tricalcium phosphate.

Biological testing of osteoinductive materials is carried out on rats, which, subject to the rules of aseptic and anesthesia with a low-speed drill with a dental bur, create a hole defect with a diameter of 2.5 mm in the area of the distal pineal gland of the femur. The defect is filled with INDOST-granule material moistened with 0.15 M NaCl solution (experiment) or HYDROXIAPOL GAP-99 granules without NSC composition (control). Animals are removed from the experiment at a time of 14, 30 and 75 days after surgery. The operated limb fragment is prepared and the sections are dewaxed in xylene and stained with hematoxylin and eosin, picrosirius, according to Van Gieson and Schiff's reagent. The significance of differences in the activity of the regenerative process in different groups was evaluated nonparametrically using the SP.SS 8-02 software package.

Based on the morphological and functional analysis of the totality of preparations obtained from 30 experimental animals, a conclusion is made about the osteoinductive activity of a material such as the bone formation process excited by it, and the effect on the biodegradation rate of implant particles.

In all cases of implantation of experimental and control materials into a hole defect in the bone, a typical histological picture develops: The defect was filled due to fibrous (experience) or fibro-chondroid (control) proliferate, along the periphery of which young bone beams gradually formed, the number of which on the 15th day after surgery, the morphometric analysis was significantly higher in the experiment.

Basophilic-colored particles of the implant, as well as cystic cavities formed as a result of its biodegradation due to osteoclastic resorption of the particles of the implanted material, were visualized inside both the fibrous and fibrous-chondroid regenerates. In the dynamics of the experiment, a young bone continued to form on the basis of the primary regenerate and by the end of the observation period (75 days), the bone wound in the experiment was performed by morphologically mature spongy bone tissue, the intertrabular spaces contained red bone marrow (Fig. 1). In the control, the regenerate is represented by less mature bone tissue, which was in the phase of reparative remodeling - young bone beams in it, differentiating by enchondral osteogenesis, are distinguished by the variability of tinctorial properties - evidence of adaptive remodeling of the structure of bone regenerate (Fig. 2).

Run Example 2

Prepare the material INDOST-sponge composition, g:

The composition of the proteins inducing osteogenesis 0.001-0.005 HYDROXYAPOL GAP-99 20-90 collagen I lyophilized rest

Biological testing of osteoinductive materials is carried out on rats, which, subject to the rules of asepsis and anesthesia with a circular saw, create a diaphyseal defect 2 mm high in the middle part of the lower leg bones. Retrograde intramedullary osteosynthesis is performed with a 0.5 mm diameter spoke, onto which a fragment of an INDOST sponge is strung, which compensates for a defect (experiment) or material of a similar composition, but not containing a NSC composition (control). Animals are removed from the experiment at 7, 14, and 60 days after surgery. An operated fragment of the limb is prepared and histological preparations are prepared, as is customary. Based on the morphological and functional analysis of the totality of preparations obtained from 30 experimental animals, a conclusion is drawn about the osteoinductive activity of a material such as the bone formation process excited by it.

According to a comparative morphological study of the compensation of a tubular bone defect under the conditions of the described model, the typical dynamics of reparative osteogenesis at the site of implantation of a material with osteoinductive properties is reproduced. However, its morphofunctional features had characteristic features in the “control” and “experience” group.

In the control, by the end of the first week after surgery, the presence of a mixed thrombus is noted in the intervention zone, the lumen of the bone marrow canal is filled with loose connective tissue. Among the fibers of the connective tissue there is a significant number of thin-walled vessels with a large clearance. In cell populations macrophage and lymphoid cells appear. In the diastasis between the fragments, hematoma organization, proliferation of granulation tissue, cell infiltration by lymphocytes and plasma cells of the bone marrow were noted. The ends of the fragments are subject to pronounced reactive transformations - young isoforms of collagen predominate in their composition, tinctorial properties are heterogeneous, there are a large number of resorption lacunae.

In the experiment, by the end of 7 days, a network of weakly mineralized bone beams appears in the loose connective tissue filling the interfragmental space. Among the fibroblastic elements, macrophages and lymphoid cells were found.

By the end of the 2nd week, in the control group, the diastasis between the fragments was filled with coarse-fibrous connective tissue, in which foci of chondroid proliferate were marked, giving a pronounced SIR reaction, and their edges undergo active rearrangement. During this period, at the ends of the fragments, a network of immature bone beams, a dense coarse-fibrous connective tissue with significant areas of cartilage, was noted. A bulk periosteal callus with a fibro-chondroid structure was also found.

In the experimental group, immature, decomposed bone beams that begin to form an intermediate callus were revealed during this period. In the regeneration zone between the formed bone beams, osteoblasts and also osteoclasts are revealed. An increase in the number of dilated vessels was noted, which indicates adaptive remodeling of bone microarchitectonics. Diastasis is blocked by a pronounced periosteal callus with a developed network of bone trabeculae with foci of a chondroid, while the regenerate in the inter-fragmental fissure consists of more mature bone structures.

On the 60th day after the creation of the bone defect in the control group, it was found that on the basis of the cartilage and coarse fibrous connective tissue filling the bone defect, processes of intensive osteogenesis were revealed - the appearance of a network of rather mature bone beams located at the ends of the fragments along the periosteal and endostal surfaces of the cortical plate . Diastasis is completely performed by bone beams of varying degrees of morphological maturity, among which there are sections of fibrous and cartilage tissue. The consolidation of fragments occurs on the basis of volumetric bone-fibro-cartilaginous preventive callus (Fig.3).

At the same time after the intervention in the experiment, the fragments are firmly fixed by mature bone tissue, the periosteal callus is practically absent. The regenerate contains collagen heterogeneous in the degree of morphological maturity and orientational ordering, which indicates the completion of the processes of reparative osteogenesis (figure 4).

Run Example 3

Prepare the material INDOST-gel composition, g:

The composition of the proteins inducing osteogenesis 0.001-0.005 HYDROXYAPOL GAP-99 15-20 collagen I 4-5 bidistilled water rest

24 Wistar rats weighing 180-220 g are divided into 2 groups: control and experimental. Animals of both groups in the lower jaw branch area under hexenal anesthesia created a through defect with carbide bur No. 3 at low speeds. A gel from HYDROXIAPOL HAP-99 g and collagen I without NSC was introduced into the jaw defect in rats of the 1st group (control). In the 2nd group (experiment), an INDOST gel was introduced into a bone defect. During the operation, the gel from the syringe was inserted through a thick needle into the defect of the jaw branch and, to prevent leakage, were closed on both sides with collagen membranes, which are used to make Parodonkol osteoplastic material (NPO POLISTOM). Animals were withdrawn from the experiment in terms of 15, 30, 60 and 90 days by the introduction of an excess dose of hexenal. The jaw was isolated, fixed in 10% neutral formalin, and after conventional histological treatment, it was embedded in paraffin. Sections 7-8 μm thick were stained with hematoxylin and eosin and analyzed using light microscopy.

According to a comparative morphological study of the compensation of a defect in the flat bones of the facial skull under the conditions of the described model, the typical dynamics of reparative osteogenesis at the site of implantation of a material with osteoinductive properties is reproduced. However, its morphological and functional signs show that in the “control” group, restructuring of the newly formed bone, which is taking place towards the end of the observation period, continues. By 90 days there is an increase in the number of anastomoses of the barrels of the newly formed bone tissue and the formation of a common bone regenerate filling the entire zone of the defect (Fig. 5).

At the same time in the experiment, only narrow gaps between the bone beams accounted for the share of fibrous connective tissue, the newly formed bone beams were fused to the lamellar bone tissue preserved here. The newly formed beams themselves, both in color and structure, did not differ from preserved bone tissue. The border between them became almost indistinguishable. Adipose tissue was found between the bone beams. In some beams there were individual osteoclasts (Fig.6).

Thus, the proposed materials have significantly more pronounced properties to stimulate bone formation than the means from which it differs in the content of protein compositions capable of inducing osteogenesis. This ability is due to the combination of a number of biological factors:

1. To stimulate the involvement of undifferentiated pluripotent cells at the site of implantation of osteoplastic material.

2. To increase the number of these cells at the site of implantation through their proliferation.

3. Upon reaching a certain density of these cells, promote their differentiation into osteogenic and stimulate the expression of bone extracellular matrix.

The mass of the protein that binds to collagen 1 and / or calcium phosphates is determined by the degree of affinity (affinity) of the latter for these proteins. The content of these proteins in the modified material is sufficient to obtain an osteoinductive effect, but does not reach the values at which their antigenic properties are manifested, an immune response is possible. Therefore, to obtain compositions of NSC and plasma proteins, allo- and xenomaterials can be used.

The forms in which these materials can be prepared are convenient for filling as defects in the form of cavities (bone cysts, osteomyelitic sequestra) - granules, powders, and for implantation in diastasis between bone fragments (for example, post-traumatic or post-resection diaphyseal defects) - ceramics sponge with fixation by immersion or extraosseous devices. The use of the proposed tool in medicine does not require the development of new operational techniques in comparison with the currently used materials.

Claims (9)

1. Osteoinductive material, including hydroxyapatite with tricalcium phosphate, characterized in that it contains a composition of non-collagenous proteins of bone tissue, stimulating osteogenesis, in the following ratio of components, per 100 g of material, g: composition of non-collagenous proteins of bone tissue, stimulating osteogenesis 0.001-0.005 hydroxyapatite with tricalcium phosphate rest 2. Osteoinductive material according to claim 1, characterized in that the composition of hydroxyapatite with tricalcium phosphate further comprises fluorohydroxyapatite and / or carbonatapatite, and / or calcium fluoride. 3. Osteoinductive material according to claim 1, characterized in that it is made in the form of granules or powder. 4. Osteoinductive material, including hydroxyapatites and collagen, characterized in that it contains a composition of non-collagenous proteins of bone tissue, stimulating osteogenesis, in the following ratio of components, per 100 g of material, g: composition of non-collagenous proteins of bone tissue, stimulating osteogenesis 0.001-0.005 hydroxyapatite with tricalcium phosphate 20-90 collagen rest 5. Osteoinductive material according to claim 4, characterized in that the composition of hydroxyapatite with tricalcium phosphate further comprises fluorohydroxyapatite and / or carbonatapatite and / or calcium fluoride. 6. Osteoinductive material according to claim 4, characterized in that it is made in the form of a sponge or ceramic. 7. Osteoinductive material, including hydroxyapatite with tricalcium phosphate and collagen, characterized in that it contains a composition of non-collagenous proteins of bone tissue, stimulating osteogenesis, and bidistilled water, in the following ratio of components, per 100 g of material, g: composition of non-collagenous proteins of bone tissue, stimulating osteogenesis 0.001-0.005 hydroxyapatite with tricalcium phosphate 10.0-80.0 collagen 1.0-2.0 bidistilled water rest 8. Osteoinductive material according to claim 7, characterized in that the composition of hydroxyapatite with tricalcium phosphate additionally contains fluorohydroxyapatite and / or carbonatapatite, and / or calcium fluoride. 9. Osteoinductive material according to claim 7, characterized in that it is made in the form of a gel.

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