WO2017180019A1

WO2017180019A1 - Biologically active composite material for repairing bone defects and preparation method thereof

Info

Publication number: WO2017180019A1
Application number: PCT/RU2016/000735
Authority: WO
Inventors: Виталий Васильевич ГУЗЕЕВ; Андрей Александрович НЕСТЕРЕНКО
Original assignee: Виталий Васильевич ГУЗЕЕВ; Андрей Александрович НЕСТЕРЕНКО
Priority date: 2016-04-11
Filing date: 2016-10-27
Publication date: 2017-10-19
Also published as: RU2617050C1

Abstract

The invention relates to medicine and may be used for repairing and eliminating bone defects in traumatology, orthopedics, jaw, facial and dental surgery, and also in dentistry. The desired technical result consists in broadening the scope of application of a method, and in producing a composite material exhibiting enhanced biocompatibility with bone tissue, thus allowing for higher-quality repair of defects having complex shapes and for enhanced safety of use, and is achieved in a method for producing a material in the form of a cement fluid, containing water, magnesium phosphate, magnesium oxide, zinc oxide and sodium dihydrogen phosphate, and a reaction-hardened powder containing hydroxyapatite, tricalcium phosphate and brushite, with a corresponding ratio of components.

Description

BIOACTIVE COMPOSITE MATERIAL FOR REPLACEMENT OF BONE DEFECTS AND METHOD FOR ITS PRODUCTION

The invention relates to medicine and can be used in traumatology, orthopedics, maxillofacial, facial and dental surgery, as well as in dentistry and cosmetology to replace and eliminate bone defects.

The main requirement for bone-replacing materials is biological compatibility with bone tissues and the absence of negative reactions when implanting and being in them for a long time. Therefore, these materials should be close in structure and properties to the matrix of bone tissues in chemical composition, not to change the chemical composition in the area of contact with the body, to be reconstructed into bone material without changing the volume, to be able to replace bone defects of complex anatomical shape in a liquid or viscous state and during the operation, turn into a solid material with mechanical properties comparable to bone tissue.

In this regard, the development of new bone-replacing materials, the technology of their manufacture and their application in practice is an urgent task.

Nonmetallic bioactive materials most fully meet the specified requirements, since they combine biological compatibility and bioresorbability. Such materials include composites based on calcium, magnesium, potassium, sodium phosphates, biopolymers of various nature - proteins and polysaccharides.

In particular, cement materials are known (Loreley Morejo n-Alonso, Oscar Jacinto Bareiro Ferreira, Raurl Garcia Carrodeguas, Luis Alberto dos Santos Bioactive composite bone cement based on a-tricalcium phosphate / tricalcium silicate J Biomed Mater Res Part 2012: 100B: 94 -102), which is obtained by mixing the reaction-hardening powder (RTP) of α-TCP with a cement liquid containing sodium phosphates. The result is cement based on precipitated oxyhydroxyapatite (OGA).

The disadvantage of this material is its relatively low strength.

Composite material for bone tissue replacement is also known [RU 2122437, A 61 L 27/00, A61F2 / 28 27.1 1.1998], containing inorganic calcium phosphates and biopolymers, chondroitin sulfate (CS), and calcium hydroxylapatite (HA) is used as calcium phosphate in the form of granules with a diameter of 1.5 - 2.0 mm and powder of beta-tricalcium phosphate (TCF) with a particle diameter of not higher than 50 microns, chondroitin sulfate and collagen powders with a particle diameter not exceeding 50 microns and a 2% collagen solution are used as biopolymers in the amount of 1 ob.ch. solution for 2 vol.h. mixtures of powders and granules used, and GA, TKF, collagen and cholesterol are taken in the ratio of 12-20: 6-12: 30-40: 34-38 wt.h.

The disadvantage of this material is the relatively low mechanical properties of products from it.

Known biomaterials based on hydroxyapatite and biopolymer-collagen, for example, a composition for bioactive microporous material that simulates natural bone structures, [RU 2349289, CI, A61F2 / 28, A61L27 / 10, A61L27 / 12, A61L27 / 56, 03.20.2009] powdered medical glass, hydroxyapatite powder, carbonate pore former and zeolite grains with a grain size of less than 1 mm and a pore size of from 0.5 nm to 20 μm, granulated together with hydroxyapatite to a grain size of more than 50 μm to 1 mm, in the following ratio of components, wt.%:

Glass 19.9-80

Hydroxyapatite 10-50

Zeolite 10-40

Pore former 0.1-10

The disadvantage of this composition is the relatively low mechanical strength and relatively low porosity, which does not allow for sufficient penetration of intercellular fluids.

The closest in technical essence and the achieved result when using it is a composite material [RU

2484850, CI, A61L 24/02, A61L 27/02, A61L 27/12, A61F 2/28, 06/20/2013], made on the basis of calcium phosphate cement for filling bone defects in the form of a reaction-hardening powder containing ^and - tricalcium phosphate, and a cement fluid containing water, wherein the reaction-hardening powder further comprises particles of hydroxyapatite ranging in size from 50 to 220 microns, and the cement fluid further comprises phosphoric acid, magnesium phosphate, sodium phosphate and / or potassium in the following ratio of components in calcium phosphate cement:

in a reaction-hardening mixture of powders, wt.%:

Particles of hydroxyapatite with a size of 50-220 microns 5-50

Powder ^a- tricalcium phosphate 50-95

in the following ratio of components in cement fluid,

Magnesium Phosphate 30-60

Sodium and / or Potassium Phosphate 3.5-25 Phosphoric acid 0.5-3.0

Water the rest

and the amount of cement fluid (ml) to the amount of the reaction-hardening powder mixture (g) is in the range of 0.45-0.75.

The disadvantage of this material is the relatively low compatibility with bone tissue, due to the fact that the set of ingredients of this composition does not fully correspond to the micro- and macroelement composition of bone tissue and, therefore, has relatively low rates of biocompatibility, osteogenic and osteoinductive properties.

In addition, the composition has a relatively narrow scope, because after preliminary molding of the product to eliminate the bone tissue defect, its heat treatment is required, which changes the volume of the cement mass and the shape of the product, which complicates the replacement of a defect of complex shape and does not allow qualitatively replacing defects of complex shape.

In addition to these shortcomings, one can note its relatively low safety, since the known material is characterized by the presence of free phosphoric acid in the cement mortar, which can uncontrolled or locally create an increased acidity of the material and lead to necrosis of biological tissues.

The problem solved in the invention with respect to the material is to develop a material with higher compatibility with bone tissue, which provides the possibility of better replacement of defects of complex shape and has a higher safety of use.

The required technical result is to increase biocompatibility with bone tissue, increasing the possibility of more high-quality replacement of defects of complex shape and increased safety of use.

This technical result, on the whole, is ensured by creating a bioactive resorbable material to replace bone defects, which, after filling defects in them during curing, forms a substance that is a close analogue of the inorganic component of bone tissue, which has biocompatibility, bioactivity, high osteoinductive and osteoconductive properties, thanks to which it is processed by the body into natural bone tissue, effectively restoring existing bone defects and subsequently Epitaxial growth creates a bone matrix.

The problem is solved, and the required technical result regarding the material is achieved by the fact that, in the composite material made in the form of a cement liquid containing water and magnesium phosphate, as well as a reaction-hardening powder containing hydroxyapatite and tricalcium phosphate, according to the invention, a binder liquid is additionally introduced, containing a solution of native collagen type II and IV and lysine powder, while the reaction-hardening powder further comprises brushite, the cement fluid further comprises magnesium oxide, zinc oxide and sodium dihydrogen phosphate, and β-tricalcium phosphate is used as tricalcium phosphate in the reaction-hardening powder, in the following ratio,% May:

hardening powder:

hydroxyapatite - 50-60

β-tricalcium phosphate - 20 - 30

Brush - 10-20

binder fluid:

native collagen solution of type II and IV (40-50%) - 95-98

lysine powder - 2-5 cement fluid:

magnesium phosphate - 10-15

magnesium oxide - 0.5-3

zinc oxide - 0.5-3

sodium dihydrogen phosphate - 5 -15

water is the rest.

moreover, the amount of reaction-hardening powder (g) to the binder fluid (ml) and the amount of cement fluid (ml) is in the range 1: (0.2-0.3): (0.3-0.5)

Also known are methods for producing composite materials for the replacement of bone defects.

In particular, there is a known method for the formation of bone tissue based on a collagen matrix [RU 2053733, CI, A61L27 / 00, 02.20.1996], according to which the collagen matrix is placed in a liquid solution containing calcium, phosphate and hydroxide ions, and the process of the formation of hydroxylappatite on collagen matrix produced by electrophoresis. A feature of the method is that, the thickness of the hydroxylappatite deposited on the collagen matrix is formed in proportion to the current density.

The disadvantage of this method is the relatively narrow scope, since the material obtained using this method has a relatively low biological activity and relatively low mechanical strength.

Closest to the technical nature of the proposed is a method for producing composite materials for the replacement of bone defects [RU 2484850, CI, A61L 24/02, A61L 27/02, A61L 27/12, A61F 2/28, 06/20/2013], according to which the reaction -hardening powder containing 0.5 g of ceramic particles of hydroxyapatite with a size of 56-82 μm and a powder of 0.5 g of α-tricalcium phosphate, mixed with 0.25 ml of cement liquid (50% May, magnesium phosphate and 5% May, potassium phosphate, 5% May, sodium phosphate, phosphoric acid 1% May, the rest is water) for 1-2 minutes with a metal spatula on the glass until sour cream-like state, after which the mixture placed in a cylindrical mold with a diameter of 8 mm for 15-20 minutes, and the molded sample is transferred to a thermostat at a temperature of 37 ° C in a solution of SBF (Simulated Body Fluid), corresponding to human blood plasma for 24 hours.

The disadvantage of this method is the relatively narrow scope, since the material obtained using this method has a relatively low biological activity and relatively low safety.

The problem solved by the invention in relation to the method is to expand the scope of its application with the aim of providing the possibility of obtaining a material having higher compatibility with bone tissue, which enables better replacement of defects of complex shape and has a higher safety of use.

The required technical result is to expand the scope in order to ensure the possibility of obtaining a material having increased biocompatibility with bone tissue, increased ability to better replace defects of complex shape and increased safety of use.

The problem is solved, and the required technical result is achieved by the fact that, according to the proposed method, a plastic mass is formed, which is mixed by rubbing in a mortar a reaction-hardening powder containing hydroxyapatite, β-tricalcium phosphate and brushite, with a binder liquid containing a solution of native collagen II and type IV and lysine powder, and immediately before the use of the composite material, a cement liquid is prepared, for which magnesium phosphate, magnesium oxide, zinc oxide and sodium dihydrogen phosphate are mixed in double-distilled water, and, when using composite material, the formed plastic mass is mixed with cement liquid and solidified for 24 hours.

Bioactive composite material for the replacement of bone defects consists of three components:

1. Reactive hardening powder

2. Binder fluid

3. Cement fluid

The ratio between components in parts by weight is 1: (0.2-0.3): (0.3-0.5).

As the reaction-hardening powder, calcium phosphate material is used in the form of a biological calcium phosphate compound (CFS) obtained from bones of cattle and pigs by the method of their demineralization in hydrochloric acid with a concentration of 0.5-3 M, followed by precipitation of 1-6 M NaOH solution to pH = 7-8.5, by filtering the precipitate of calcium phosphates, washing it repeatedly by pulping by distillation in distilled water. The resulting precipitate was subjected to stepwise heat treatment: drying at 80-120 ° C and calcining at 750-830 ° C. The obtained biological CFS has a polydisperse composition with a particle size of from 0.1-400 microns, which are agglomerates of grains with a size of 15 to 50 nm. Sterilization of CFS occurs when it is calcined directly during production at 750-780 ° C. The binder material additionally contains an amino acid - lysine, which provides transport of bioresorbable calcium to the site of restoration of the bone defect and promotes epitaxial bone growth.

The composition of the calcium phosphate material is controlled, for example, by X-ray phase and X-ray fluorescence analysis. Calcium phosphate the material contains phases: hydroxyapatite (amorphous and crystalline phases) (50-60%), β-tricalcium phosphate (20-30) and brushite Ca (НР0 ₄ ) (Н ₂ 0) with a ratio of Ca / P = 1.50-1, 67.

The hardening powder and cement liquid are sterilized before use by heating to 750-830 ° C and 500 ° C, respectively, and before use, the binder liquid is sealed in polyethylene and sterilized by ionizing gamma radiation from a cobalt-60 isotope (Co ⁶⁰ ) with a dose of (18000 -23000) ± 500 gray.

The biological calcium phosphate compound (CFS) obtained by the above method contains the main trace elements accompanying natural bone, the value of which can be estimated only by in vivo testing of the material, during which it showed high resorbability and remarkable osteoinductive and osteoconductive properties.

To impart viscous flowing properties to the composite material, a binder liquid was used — a solution of native collagen of type II and IV, which was isolated from animal bones in the process of obtaining biological CFS. An amino acid, lysine, was used as an additive to the binder fluid, which ensures the transport of bioresorbable calcium to the site of restoration of the bone defect and promotes epitaxial bone growth. Lysine is involved in two very important processes: tissue repair and collagen formation. Lysine is widely used in the period of postoperative recovery, as well as the treatment of injuries of a different nature. It improves the absorption of calcium from the blood and its transport to bone tissue. The prepared mixture of a solution of collagen and lysine is sealed in a plastic film and sterilized with ionizing gamma radiation from the cobalt-60 isotope (Co ⁶⁰ ) with a dose of 18000-23000 ± 0.5 Gray. For the cement fluid, chemical reagents were used: magnesium phosphate, magnesium oxide, zinc oxide, sodium dihydrogen phosphate, which were qualified as “pure for analysis” and “especially pure”. The ratio between magnesium and zinc oxides and sodium dihydrogen phosphate is chosen so that, during the formation of the cement fluid, the oxides are neutralized with sodium dihydrogen phosphate to form mixed magnesium, sodium, and zinc phosphates, which provide good reaction hardening properties of the material. Sterilization of powders of magnesium and zinc oxides, magnesium phosphate and sodium dihydrogen phosphate is carried out by calcining in an oven at 500 ° C for one hour.

A method of obtaining a bioactive composite material for replacing bone defects consists in the sequence of operations: starting components: CFS powder and a binder solution of native collagen with lysine are mixed by grinding in an agate mortar to a homogeneous mass, then a cement solution was added to the mixture with continuous stirring. After thorough mixing, a plastic mass similar to plasticine is formed, which is easily molded. The necessary products are formed manually or in molds from the resulting mass - plates, sticks, balls (for example, for chemical, biological and mechanical tests). The curing time of the obtained material depends on the ratio of the components and varies from 5 to 30 minutes. In addition, with manual molding, the material does not cure for a longer time while it is in the hands of a surgeon or researcher.

The test results showed that, in contrast to the known cements, the proposed material has full biocompatibility with bone tissue, biodegradability, creates the conditions for reducing the need for bone grafting and overall duration surgical intervention, eliminate the need for additional fixation of bone fragments (debris).

Example.

A powder of calcium phosphate compounds (CFS) containing 1 g of particles with a size of 0.1-400 μm is mixed with 0.2 g of a mixture of collagen and lysine and 0.3 g of cement fluid. Mixing is carried out in the indicated sequence by grinding in an agate mortar, then the plastic mass is subjected to manual molding. Of the half of the resulting plastic mass, a ball is formed and placed in a cylindrical shape with a diameter of 8 mm for mechanical testing. Curing of the mixture occurs at room temperature almost instantly. Preliminary curing of the material occurs in 10-15 minutes, and complete occurs after 24 hours. By this method, samples were prepared for mechanical tests and for preclinical testing. Cement liquid is prepared immediately before the test by mixing the powders of phosphates and oxides that are part of bidistilled water.

Similarly prepared samples having compositions within the claimed limits of the CFS. If a binder component (collagen solution with lysine) is added to the mixture in an amount less than 0.2, the plasticity of the material decreases, and exceeding this amount more than 0.3 leads to an increase in the curing time of the composite. The addition of a cement fluid in an amount of less than 0.3 to the reaction hardening mixture complicates the uniformity of mixing and, accordingly, the uniform distribution of CFS in the plastic mass, which crumbles, and the addition of more than 0.5 can lead to a decrease in the biological activity of the material due to a quantitative decrease in composition of bioactive CFS. The addition of lysine in an amount of 2-5 is justified by the following: when using lysine in an amount of less than 2% in May. has little effect on the effect of his participation in restoration of a bone defect having a bone structure, and when the content is above 5%, an increase in the setting time and hardening of the material is observed.

An elemental analysis of the inventive bioactive composite material with a composition of 1: 0.25: 0.4 is presented in table 1, and showed the presence of a basic and microelement composition characteristic of bone tissue.

Heavy metals in its composition are absent.

Table 1 - Results of elemental analysis of bone cement

The evaluation of the parameters of the porous structure and specific surface area is given in table 2. The inventive bioactive composite material with a composition of 1: 0.25: 0.4 is a medium-porous material with a high specific surface area and an average pore size of 3 nm.

Table 2 - Evaluation of the parameters of the porous structure and specific surface of the bioactive composite material with a composition of 1: 0.25: 0.4

Mechanical tests of a bioactive composite material with a composition of 1: 0.25: 0.4 indicate its rather high strength properties, close to the strength of the cancellous bone.

Table 3 - Res results of mechanical tests

To assess the systemic effect of the implantable bioactive composite material with a composition of 1: 0.25: 0.4 on the body, preclinical tests were performed on rats of the Wistar breed (30 pcs. Weighing 218-226 g) obtained from the experimental biomodeling department of the Scientific Research Institute of Hygiene and on rabbits 6 months (10 pcs. Weighing 2.5-3 kg).

Tests of a bioactive composite material based on biological CFS showed that in rats after 1 month, and in rabbits 1.5 months after the operation, it was found that vessels of different diameters and nerve fibers were detected in the newly formed tissue surrounding and growing into the matrix. Throughout the entire thickness of the implant as a result of its biodegradation, the material is replaced by newly formed bone trabeculae. Blood vessels are full-blooded, which indicates the connection of blood vessels with the bloodstream. In world practice, there are reports of restoration of the structure of bones at the site of a bone defect with germination of blood vessels, but there is practically no information on the restoration of nerve fibers in the inert matrix. When using the proposed material, bone is restored with blood vessels and nerve fibers.

Thus, thanks to the improvement of the known method, the scope of its application is expanding, which allows to achieve the required technical result with respect to the composite material obtained, which has increased biocompatibility with bone tissue, which provides better replacement of defects of complex shape and increased safety of use.

Claims

Claim

1. Bioactive composite material for the replacement of bone defects, made in the form of a cement fluid containing water and magnesium phosphate, and a reaction-hardening powder containing hydroxyapatite and tricalcium phosphate, characterized in that an additional binder fluid containing a solution of native collagen II and IV is added type and lysine powder, while the reaction-hardening powder additionally contains brushite, the cement fluid additionally contains magnesium oxide, zinc oxide and sodium dihydrogen phosphate, and as tricalcium β-tricalcium phosphate is used in the reaction-hardening powder in the following ratio of components,% May:

hardening powder:

hydroxyapatite - 50-60

β-tricalcium phosphate - 20 - 30

Brush - 10-20

binder fluid:

native collagen solution of type II and IV (40-50%) - 95-98

lysine powder - 2-5

cement fluid:

magnesium phosphate - 10-15

magnesium oxide - 0.5-3

zinc oxide - 0.5-3

sodium dihydrogen phosphate - 5 -15

water is the rest. moreover, the amount of reaction-hardening powder (g) to the binder fluid (ml) and the amount of cement fluid (ml) is in the range 1: (0.2-0.3): (0.3-0.5)

2. The material according to claim 1, characterized in that, as a reaction-hardening powder containing hydroxyapatite, β-tricalcium phosphate and brushite, use calcium phosphate material in the form of a biological calcium phosphate compound (CFS), obtained from bones of cattle and pigs, by the method of their demineralization in hydrochloric acid with a concentration of 0.5-3 M, followed by precipitation of 1-6 M NaOH solution to pH = 7-8.5, filtration of calcium phosphate precipitate, its repeated washing by pulping in distilled water, after the resulting precipitate is subjected to a heat treatment step comprising drying at 80-120 ° C and calcination at 750-830 ° C.

3. The material according to p. 1, characterized in that the reaction-hardening powder has a polydisperse composition with a particle size of 0.1 ^ 00 microns, which are agglomerates of grains ranging in size from 15 to 50 nm.

4. The material according to claim 1, characterized in that a solution of native collagen of type II and IV is used in the binder fluid, which is isolated from animal bones in the process of obtaining a biological calcium phosphate compound.

5. A method of producing a bioactive composite material for replacing bone defects according to claim 1, which consists in forming a plastic mass, for which the reaction-hardening powder containing hydroxyapatite, β-tricalcium phosphate and brushite is mixed by grinding with a binder, containing a solution of native collagen type II and IV and lysine powder, and a cement liquid is prepared immediately before the composite material is used, for which it is mixed in bidistilled water magnesium phosphate, magnesium oxide, zinc oxide and sodium dihydrogen phosphate, moreover, when using a composite material, the formed plastic mass is mixed with cement liquid and solidified for 24 hours.

6. The method according to claim 5, characterized in that the reaction-hardening powder and cement liquid before use are sterilized by heating to 750-830 ° C and 500 ° C, respectively.

7. The method according to claim 5, characterized in that before use, the binder liquid is sealed in polyethylene and sterilized by ionizing gamma radiation from the cobalt-60 isotope (With ⁶⁰ ) radiation dose (18000 -23000) ± 500 Gray.