Bone material and collagen composition for restoring injured articulations

FIELD: medicine.

SUBSTANCE: bone-and-mineral product contains porous bone mineral particles produced from natural bone and having crystalline structure practically corresponding to natural bone structure and practically containing no endogenous organic material. The particles have fibers of physiologically compatible type II resorbable collagen at least on their surface. Mass proportion of type II collagen fibers and porous bone mineral is at least equal to approximately 1:40.

EFFECT: enhanced effectiveness in recovering combined injuries of cartilage and bone tissue in articulations having defects.

8 cl, 6 dwg

 

Cross-reference to related application

This application claims the effect of the provisional application ser. No. 60/219009 filed July 19, 2000

Prerequisites to the creation of inventions

The technical field of the invention

The present invention relates to the field of restoration of the damaged or defective joints.

Description of the prior art

Articulated joints in mammals are covered with articular cartilage, which prevents direct contact of the opposite surfaces of the bone and provides a soft movement of the articulated bones relative to each other.

There are several ways to repair damage and defects in the articular cartilage. These include implantation grown in culture of chondrocytes in place of damaged cartilage and causing damage to the coating of collagen plaques.

In some cases, damage or defect articulated joint extends deeper articular cartilage in an underlying layer of bone. Thus, in this area there is a need to develop materials and methods recovery of articulated joints in which the damage or defect extends through the cartilage into bone.

Summary of the invention

The object of the present invention is a bone mineral product designed for the application of the Oia in the treatment of combined injuries or defects of cartilage and bone in the articulated joints, which includes particles of porous bone mineral derived from natural bone having a crystalline structure, practically corresponding to the structure of natural bone and practically does not contain any endogenous organic material, where the particles are at least on their surface fibers capable of resorption physiologically compatible, collagen type II, and the mass ratio of collagen type II and particles of porous bone mineral is at least about 1:40.

Brief description of drawings

1 shows, partly schematically, a vertical projection of the end of the healthy bones of the articulated joint.

2 shows, partly schematically, a vertical projection of the end of the bones of the articulated joint, in which there is damage (defect)extending through the cartilage into bone.

Figure 3 shows, partly schematically, a vertical projection, which presents bone-mineral/collagen matrix of the present invention, introduced in damage at the end of the articulated bones of a joint.

Figure 4 shows, partly schematically, a vertical projection, which presents a collagen or a synthetic membrane that covers damage to the joint which is filled with bone-mineral/collagen matrix on n the present invention.

Figure 5 schematically shows a side projection of the membrane intended for use according to one of embodiments of the present invention.

Figure 6 schematically shows a side projection of the other membranes intended for use according to the second variant of implementation of the present invention.

Detailed description of the invention

The present invention can be applied to correct tissue defects, including defects of cartilage and bone defects, first of all, articulated joints, such as knee joints and the joints of the vertebral column.

Bone mineral product according to the present invention includes particles of porous bone mineral and collagen type II is a substrate for germination as native chondrocytes and native osteocytes inside the matrix for the impact on the regeneration of cartilage and bone.

The matrix of collagen type II product of the present invention also strengthens brittle bone mineral.

The object of the present invention is a purified consisting of particles of bone mineral product designed for application in medicine, particles of this mineral practically do not contain any endogenous organic material and have at least on their surface capable of resorption physio is logicheskie compatible collagen type II.

Bones of killed animals are an inexpensive source product, which is available in large quantities. They contain 50-60% of very fine crystals of hydroxyapatite, United collagen tissue and containing a sufficient amount of protein and other material and related fat and muscle tissue. Considering they are created by biological crystalline structure they can also be considered as having high biocompatibility of the prosthesis intended to replace the bone. Given their large specific surface area, they can also be used, for example, as an adsorbent or substrates for drug slow release.

Natural bone mineral includes hydroxyapatite crystallites, in which the degree of crystallinity, shape and size of crystals (wrong resembling a ribbon crystal morphology, thickness 5-10 mm, a length of 10-50 mm) and chemical structure of the surface depend on the ratio of calcium and phosphate (37,5-38,0% calcium and 15.5-51.9% of phosphorus). In addition, natural bone mineral there are small amounts of non-crystalline inclusions and the other consisting of calcium phosphate crystalline phase, which includes the mineral brushite and nihilogic and actualitzat. Inorganic f is for bone contains pores, including ultrastructural void (10-100 mm) between the naturally occurring the crystallites and educated in the removal of the organic phase and the microscopic space (1-20 microns, which include osteocyte lacunae, canaliculi, vascular channels, the channels Volkman and channels gaverova systems (100-500 mm)). The specific surface area, which is a measure of porosity is from 50 to 100 m2/g in the determination using mercury porosimetry. The degree of crystallinity of bone mineral can be determined using the diffraction of x-rays, and the porosity and morphology, as well as the size of the crystals using electron microscopy. A small number Neapolitan crystals can be detected by using thermogravimetric analysis.

However, the composition and structure of natural bone mineral cannot be accurately reproduced in the products obtained in vitro, or by using the previously obtained naturally occurring of hydroxiapatite. It was proposed two methods for the purification of natural bone mineral, such as calcining (burning) and extraction solvent.

The temperature required in the process of calcination for annealing the organic components of the bone, is quite high. This drive is t to extensive recrystallization of the mineral component with the formation of numerous more coarse-grained crystals. Obtained as a result of this process, the material has a relatively small specific surface area. Consequently, such material cannot be easily reconstructed with getting a new bone at the implant and the implant can save unclear from the perspective of reverse engineering properties, although in some cases they can be used.

The extraction of proteins removed from degreased bones with an acceptable solvent. The resulting bone mineral is then washed to remove solvent.

In both cases, when natural bone remove organic impurities in order to obtain only the bone mineral, the strength of the material is greatly reduced and individual pieces purified bone mineral are very fragile. This complicates the handling of the material and can lead to undesirable effects during implantation.

In a jointly owned patent US 5573771 (incorporated in the present description by reference) describes a medical product in the form of bone mineral in which the bone mineral reinforced by a matrix of collagen type I (collagen I) or a mixture of collagen type I and collagen type III, collagen I and collagen III).

Animal collagen is present in several forms, and various tissues containing the different proportions of collagen of the appropriate type. The collagen sponge material, which is used in medicine and cosmetics, as a rule produced from skin and tendons, and it consists mainly of collagen I and/or collagen III. Bone collagen consists mainly of collagen I and collagen II.

The collagen material II of the present invention may include in addition to almost pure collagen II different proportions of collagen I, collagen III and their mixtures with collagen II. For example, the material of collagen II may be mixed with about 0.1 to 10 wt.% (preferably from about 0.1 to 5 wt.%) collagen III and/or from about 1-50 wt.% collagen I.

The material of collagen II according to the present invention can be impregnated each of the individual types of particles to improve the properties during the processing of a product during production and use. In this case, the mass ratio of the material of collagen II and purified bone mineral is preferably greater than 1:40, preferably is in the range from more than 1:8 to less than 4:1, preferably less than 1:2. Preferably, the share of collagen II accounts for about 1-30 wt.% in terms of bone mineral product according to the present invention, preferably about 5-15%. Material II collagen penetrates into the porous structure of the bone mineral and effectively replaces a part of the natural protein material previously available is the future in natural bone, and although this adds strength, but also leads to immunological reactions of the tissue at the implantation of bone mineral.

The material of collagen II can be used to obtain a matrix consisting of particles of bone mineral, which can be obtained from the joints of a particular form. In this case, perhaps the use of collagen II in combination with the gel-forming macromolecular substance such as gelatin. The mass ratio of the fibrous material and bone mineral may be in the range from 1:40 to 3:20, for example about 1:10. On gel phase preferably have from 2 to 20 wt.% in terms of bone mineral, such as about 5%. When the gel phase using gelatin, then he may have a small amount of cross-linking, for example, formed by the use of 0.28 parts of formaldehyde.

Bone mineral is preferably obtained from trabecular bone and tie with the fibers of collagen II in order to enhance the physical strength of the matrix. According to preferred variants of the implementation bone-mineral/collageneous product of the present invention is used as a matrix for the regeneration of cartilage defects in the articulated joints, which also has an additional bone loss.

Bone-mineral product at present is in the invention can be used for the regeneration of cartilage in the knee joints, the joints of the foot, spine, etc., and as used for the correction of implant or prosthesis for replacement of bone, for example, in orthopedic surgery, including the reversion of the hip, replacement of lost bone, such as in trauma, surgery for oral and maxillofacial surgery or filling defect periodontal and holes after removal of the tooth, including capacity bumps. Thus, impregnated consisting of particles of the material according to the invention can be used for filling cavities in the bone, and his increased strength (reduced fragility) is important for processing and filling procedure.

According to one variant of the method according to the invention, the bone-mineral product/material II collagen is injected into the injury site of the bone and then bone-mineral/collagen product and the injury of the bone close collagen membrane for recovery of the tissue. Suitable collagen membranes known in the field and can be a single layer of collagen membrane, described in WO 96/25961 (application for US patent No. 08/894517), two-layer membrane made of collagen I/III, described in US patent No. 5837278, having a compact smooth non-porous outer barrier surface and the opposite fibrous, surface, or layered membrane, which includes the outdoor guncotton the th layer matrix of collagen II and at least one barrier layer made of collagen I, collagen III or a mixture thereof, as described in the application for US patent No. 09/545465 filed April 7, 2000, which corresponds to PCT/GB98/02976, etc. of the Above documents are incorporated into this description by reference.

According to one preferred embodiments, the membrane has an outer smooth barrier surface, which inhibits cell adhesion and acts as a barrier to the passage through it of the cells, and the opposite side of the membrane is fibrous and soft surface, through which cells can grow. One such product is Bio-Gide®supplied by the company Ed. Geistlich Söhne AG für Chemishe Industrie, Switzerland. Figure 5 shows the product Bio-Gide®having a smooth outer barrier surface 20 and a soft fibrous surface 22. When applied according to the present invention a soft fibrous surface placed on the injury site, and smooth outer barrier surface feature on the outside. Product Bio-Gide®described in US patent No. 5837278 (above), and it includes approximately 95-99% of collagen I and about 1-5% collagen III.

According to another preferred variant implementation of the present invention used a multilayer membrane described in the above patent application U.S. No. 09/545465. Such multi-layer membrane can be prepared by applying a suspension of collagen II on the soft fibrous surface of the above-described product Bio-Gide ®. Such a product is shown in Fig.6 and comprises an outer barrier surface 20 and the matrix layer, consisting mainly of collagen II having an open subcatalog texture 24, which is applied in the form of suspension on the soft fibrous surface 22 and dried. When the product is shown in Fig.6, is used according to the present invention, the outdoor subcategory consisting of collagen II layer 24 is applied directly to the injury site, and smooth outer barrier surface 20 have outside.

As noted above, the invention primarily relates to the regeneration of the defects of the articulated joint, which differ by the presence of damage as cartilage and underlying bone. Bone-mineral/collagen product according to the invention can be used to fill the damaged area of bone, and full damage to the bone can then be covered with a collagen membrane.

To enhance regeneration in bone-mineral/collagen matrix according to the invention prior to implantation can be added extracellular grown in culture the chondrocytes, and enriched by chondrocytes of the matrix can be implanted during surgery in an outdoor body or arrendatario. Alternatively, or in addition to the previous implanted matrix may shall be covered with a collagen membrane, consisting of collagen I, II and/or III, or covered with a synthetic membrane. Alternatively, or in addition to the previous one in such a collagen membrane or synthetic membrane can be added grown in culture the chondrocytes, and the membrane may be applied to fill the damaged bone, the implant during surgical intervention in an outdoor body or arrendatario.

Purified bone material may, for example, to represent the product described in the international patent application WO 86/07265 (PCT/GB86/00310). Such products can be obtained by the almost complete degreasing of certain bones, such as femurs bulls, and treated with ammonia or an organic amine to split the remaining protein followed by intense washing with water. This material retains the ability to resorption in the application that facilitates the process of correction.

Purified bone mineral can also be obtained by the calcination of a certain cancellous or cortical bone layer, for example, at 900°within 24 hours of Such calcinated bone mineral finds application when required constant pressively implants, for example, adding bumps. In any case, after removal of the organic material bone mill is becomes extremely fragile, and its strength can be significantly improved by the process according to the invention.

If the bone is intended for use as a carrier of drugs, as described in international patent application, it is advisable to bone mineral bore one or more of the absorbed drug or other physiologically active substances. According to one of embodiments of the product according to the invention includes at least one absorbed pharmaceutically or biologically active substance or mesenchymal stem cells, capable of differentiation into cells designed for the regeneration of cartilage or bone.

Physiological active substances that can be adsorbed on the bone mineral, preferably are at least partially water-soluble material and include antibacterial substances, such as antibiotics, such as penicillins, cephalosporins, aminoglycosides, etc., sulfonamides and, above all, the condensation products of formaldehyde and forenamed or N-substituted forenamed. The latter compounds may be represented by the formula

where R1denotes hydrogen or C1-C4alkyl group, and R2denotes hydrogen or a group of four who uly

where R1has the above values.

The compound of formula (I)in which R1and R2denote hydrogen, represents taurultam, and the compound in which R1denotes hydrogen and R2has the structure of formula (II)represents taurolidine. These compounds act as agents of transfer methylol, and they are effective not only against gram-negative and gram-positive bacteria, but also against inactivation of endotoxins and exotoxins produced by bacteria.

Other acceptable physiologically active substances include proteins and polypeptides, which can promote bone regeneration, primarily collagenase proteins originating from bone matrix and bone cells. They include mitogenic factors, such as factor, skeletal growth and morphogenetic factors and angiogenesis factors, and transforming growth factor bone. Especially valuable are the growth factors of the natural bone matrix, such as ossein or more preferably osteopontin.

According to one of embodiments pharmaceutically active substance is chosen from the group comprising bone morphogenetic proteins (BMP)such as BMP-2-8, or other molecules skeletal matrix,as well as signal peptides, such as the transforming factor-β growth of TGF-β, TGF-β1, vascular endothelial growth factor (VEGF), insulinogenic growth factor (IGF), a protein related parathyroid hormone (PTHrP) and platelet-derived growth factor (PGGF).

The product according to the invention can also be used as a carrier for stem cells, specific lines of differentiation, such as cells of the articular cartilage or bone. Such stem cells can be cultured in vitro to increase their number and be in places destined for recovery in matrices media with growth factors or without them.

It should be obvious that according to other variant or in addition to the above physiologically active substance can be included in the macromolecular substance, for example, impregnated gelatin. This is especially important for proteins, such as the aforementioned growth factors bone.

Bone mineral, usually has the form of particles, the average diameter of which ranges from 0.1 to 10 mm Particles for incorporation into collagen fiber II is preferably obtained from trabecular bone, and they usually must have a size of 0.1-5 mm, preferably 0.5 to 2 mm May be appropriate dense packing of particles of bone mineral to apply a mixture of particles of two or a large size is s, for example, from 0.25 to 1 mm 1 to 2 mm, or in a wider range, for example from 0.25 to 2 mm.

Purified bone mineral can be obtained, for example, using the method described in the above international patent application. For example, the fat can be removed using one or more conventional fat solvents, such as ethers, for example dimethyl ether; ketones, such as acetone; or hydrocarbons or halogenated hydrocarbons, such as heptane or methylcyclohexane or tolyl.

It may be appropriate before further processing to remove the extracting agent, such as toluene, by the intermediate extraction miscible with water solvent, such as ethanol. The collagen material can be dissolved using proteolytic agents such as bases, such as potassium hydroxide in glycerol, or organic bases, such as amines, such as Ethylenediamine, or amides, such as formamide, preferably at elevated temperatures. Such agents are preferably miscible with water substances to facilitate their removal by washing with water. Especially good results were obtained using extraction bone Ethylenediamine at a temperature of reflux distilled.

The extraction of appropriate conduct at each stage, p and necessary replacement of the solvent until while no longer extracted additional material, for example, within 1 or 2 weeks. It may be appropriate to implement the additional grinding after the initial removal of protein, because the bone is easier to break at this stage than before extraction. After treatment with base excess solvent is carefully removed, for example, by evaporation and/or, if necessary, by washing with water.

The material is usually subjected to a stage of drying. It may be appropriate at this stage to sterilize the material, for example, hot processing, which may lead to additional treatment. The absorption and/or adsorption physiologically active substance is preferably carried out by immersing the treated bone mineral in an aqueous solution of the substance, preferably under sterile conditions. The concentration of the active substance preferably is relatively high for easy absorption and/or adsorption and depends also on the solubility of the active product.

The invention will be described below in more detail with reference to the drawings.

Figure 1 shows the articular end of the bone 10 with cartilage 12, which is healthy and has no damage.

Figure 2 shows articular bone 10 with cartilage 12, in which there is damage 14 extending from the cartilage 12 in the guard 10.

Figure 3 shows bone-mineral/cartilage matrix 16 according to the invention, which can be entered or not entered chondrocytes, filling the 14 damage in the cartilage 12 and bones 10.

Figure 4 shows a collagen or synthetic plaque 18, which can be entered or not entered chondrocytes, covering damage 14 filled with new bone and mineral/collagen matrix 16 according to the invention for the regeneration of injuries such as cartilage 12 and bones 10. Plaque 18 can be attached by any suitable means 19, such as surgical sutures, surgical pins or glue.

The invention is explained using the following examples, which are given only for illustrative purposes.

Example 1

Femur bull boiled in hot water to clean, crushed to a particle size of 5-10 mm and was extracted with toluene at the temperature of reflux distilled within 24 hours to conventional Soxhlet extractions. The additional material was extracted with ethanol to remove toluene and then was extracted at elevated temperature with azeotropic mixture of Ethylenediamine and water (85:15) for 8 days with several changes of solvent until until no longer was extracted no additional organic material. Then the product was dried in air at 100°C.

The dried product was additionally crushed to obtain particles sosrednim size of 0.2-2 mm and sterilized in the autoclave. Pieces of spongy bone of the thigh bulls, usually with a diameter of 10 mm, was purified using a similar process without the final granulation.

Example 2

Frozen cartilage from a freshly slaughtered pigs were soaked in cold water, thoroughly washed and mechanically cleaned from the remnants of meat, bones and solid pieces. The material is then washed for 30 min in running water.

Then the material was ground three times in the homogenizer. Visible to the naked eye the particle size at the end of the grinding stage was approximately 8 mm

Pieces of cartilage were obezvozhivani by four washing with acetone, each time for 8 hours Then the cartilage was degreased by four extraction with n-hexane. Each treatment lasted for at least 8 hours a Ratio of hexane and cartilage was 1:10.

After degreasing the cartilage was allowed to swell in water for drinking. The ratio of water and the material was 10:1. The processing time was 24 hours

Then the material was treated with NaOH (5 wt%), the ratio of cartilage and fluid was 1:4, and the treatment continued for 32 hours during the processing of the pieces of cartilage are well mixed. Then the cartilage was washed from alkali. Thus, initial pH value, equal to 14, fell to 9-11. Dissolved impurities are washed and separated from the cartilage. Obtained after treatment with alkali liquid collected on the I restore glycosaminoglycans and collagen.

Then the collagen material was treated with concentrated HCl (about 3 wt.%) first, when the pH value is below 1.0. The processing time was 4-6 hours

Then the material was washed with cold water up until the pH value was not increased to 3-3 .5. All impurities were removed and the resulting product was desterrado salt of collagen mass, suitable for the production of sponge or other collagenous material. For this purpose cartilaginous mass according to the invention may be subjected to degreasing, freezing and drying by freezing.

Example 3

The extract obtained after alkaline treatment as described in example 2, contained glycosaminoglycan, alkali denatured proteins and salts. The first extract was neutralized with HCl, the pH after neutralization was 6. Then the extract was treated with an auxiliary means for filtering, namely diatomaceous earth, which is used to remove denaturirovannykh proteins. In the extract was injected with 0.5 wt.% diatomaceous earth and was removed by filtration together with the denatured protein.

Then the supernatant was subjected to ultrafiltration using a membrane impermeable to particles with a molecular weight above about 10,000 Da. Thus, removing the salt, leaving cleared of glycosaminoglycan.

Example 4

2.0 g of the material of collagen II, polucen the th in example 3, crushed in a mixer with 500 g of distilled water. This dispersion was centrifuged and the supernatant water was removed. To the resulting suspension of collagen fibers was added to 17.5 g of granulated cortical bone of the bull, purified using the process described in example 1, followed by thorough mixing and removing water under vacuum (70 mm RT. column). Granulated bone had a size of from 0.5 to 1.0 mm After removal of water was added 5 ml of 9 wt.% aqueous solution of gelatin (cross linked with 0,6% 35%aqueous formaldehyde) and the mixture was again dried under vacuum.

The spongy mass is cut into pieces and dried under vacuum at 60°C. Pieces of sponge Packed in plastic containers and sterilized by irradiation with gamma rays.

1. Consisting of particles of bone and mineral product to recover the combined damage of cartilage and bone defects, where the product comprises particles of porous bone mineral derived from natural bone having a crystalline structure, practically corresponding to the structure of natural bone, and practically does not contain any endogenous organic material, where the particles are at least on their surface fibers capable of resorption physiologically compatible, collagen type II, and the mass ratio of collagen fibers type and particles of porous bone mineral is at least about 1:40.

2. The product according to claim 1, where the particles have an average diameter of from about 0.1 to about 5 mm

3. The product according to claim 1, additionally containing at least one absorbed pharmaceutically or biologically active substance or mesenchymal stem cells, capable of differentiation into cells designed for the regeneration of cartilage or bone.

4. The product according to claim 3, in which the pharmaceutically active substance is chosen from the group comprising taurolidine, taurultam and their mixture.

5. The product according to claim 1, further comprising gelatin in the gel phase, and collagen type II are in the gel phase and the gel phase is about 2-20 wt.% in terms of bone mineral.

6. The product according to claim 3, in which the physiologically active substance is chosen from the group comprising bone morphogenetic proteins (BMP), other molecules skeletal matrix and signal peptides.

7. The product according to claim 6, in which the physiologically active substance is chosen from the group comprising BMP-2-8, TGF-β, TGF-β1, VEGF, IGF, PTHrP and PGGF.

8. The product according to claim 3, additionally comprising stem cells of the articular cartilage or stem cells of the bone.



 

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