Porous microspheres of calcium and magnesium biophosphate with adjusted particle size for bone tissue regeneration

FIELD: medicine.

SUBSTANCE: invention refers to porous microsphere granules with the adjusted particle size for bone tissue regeneration. The above microspheres have a size within the range of 1-1000 mcm, have through pores of the size of 1-100 mcm and total porosity 40-75%. The declared microsphere granules are prepared by granulation by electrospinning, and heat-treated. A mixture used to form the granules by electrospinning contains a mixture of magnesium orthophosphate and biological hydroxyapatite of bovine demineralised bones in ratio 0.5:1.0, as well as 1-3% sodium alginate in distilled water and a hardener representing saturated calcium chloride.

EFFECT: invention provides preparing the microsphere granules possessing biocompatibility, biodegradation, osteoinduction and osteoconduction properties and able to be substituted by the bone tissue.

2 ex

 

The invention relates to the field biophosphate ceramic materials for medicine, namely to traumatology, orthopedics, reconstructive surgery, cosmetology, dentistry and to the system of drug delivery.

Calcium phosphates are the main elements of the bone and are widely used as materials for bone regeneration and production of solid bone implants used in orthopedic, maxillofacial surgery, etc. To the most used in medicine to calcium phosphates include tricalcium phosphate - CA3(PO4)2(TCP) and hydroxyapatite - CA5(PO4)3HE (HA) or bi-phase materials based on the system TKF-HA. Biological and mechanical properties of calcium phosphate ceramics are determined by the phase composition and structure, which mainly depend on the firing temperature and the size of the original particles of the ceramic powder. One method to improve mechanical properties and reduce the sintering temperature of ceramics is the use of ceramic ultrafine powders with a particle size less than 1 micron.

(A. Tampieri, G. Celotti, F. Szontagh and Landi "Sistering and characterization of HA and TCP bioceramics with control of their strebght and phase purity". J. of Materials in Medicine 8 (1997 29-37) were obtained calcium phosphate materials based on TCP and HA powders with cf who ne the crystal size of 0.5 μm. The obtained materials were characterized by a crystalline structure with an average crystal size of 1-2 microns, a porosity of less than 7%. The disadvantage of this material was high firing temperature 1220-1300°C. in Addition, at temperatures above 1250°C, the decrease of the strength and partial decomposition of HA with the formation of toxic calcium oxide.

Also known calcium phosphate-based materials system tricalcium phosphate-hydroxyapatite (S. Raynaud, E. Champion, D. Bemache-Assollant "Calcium phosphate appatites with variable Ca/P atomic ratio is 2. Calcination and sistering", Biomaterials 23 (2002) 1073-1080)obtained by sintering of nanocrystalline powders at a temperature of 1100-1150°C. In this work to reduce the sintering temperature managed through the use of nanocrystalline powders and method of hot pressing. This has resulted in bi-phase materials system TKF-HA, which was characterized by a fine structure with an average crystal 200 nm. The disadvantage of this pottery is a high firing temperature and the application of the method of hot pressing, resulting in a considerable rise in prices of finished products.

From RU 2359708, 27.06.2009 other known calcium phosphate ceramic material intended for the manufacture of bone implants and/or replacement of defects in various bone pathologies.

Material sostoi is from tricalcium phosphate-hydroxiapatite ceramics with additives of potassium carbonate, and/or lithium carbonate, or calcium carbonate in the following ratio of components in ceramics, wt.%:

hydroxyapatite and/or tricalcium phosphate- 80-99,5
one of the above additivesof 0.5 to 20.0

Known ceramic material has a particle size of not more than 100 nm and the porosity (open) less than 4% after annealing up to 700°C.

The material does not have the necessary osteoinductive and osteoconductive properties.

It is known that the magnesium phosphate is included in the composition of bone tissue. Magnesium acts as a regulator of cell growth that is necessary at all stages of the synthesis of protein molecules. Magnesium is an essential element of the triad CA, P, Mg, currency are closely interrelated. He takes part in the metabolism of phosphorus, energy metabolism, synthesis of the enzyme, metabolism of carbohydrates, regulates glycolysis, involved in the construction of bone tissue, providing functional capacity of the nervous and muscular tissues.

Like calcium, it takes part in many metabolic processes. The enzymes in the composition of the active catalytic center which includes magnesium ion plays an important role in providing energy to the body, the transport of various substances through cell membranes, synthesis be the Cove and nucleic acids. Magnesium plays an important role in osteogenesis, can directly affect the function of bone cells.

From RU 2292868, 10.02.2007 known material for filling bone maxillofacial and dental defects. The material is made on the basis of the reaction-hardening mixture of powders, contains hydroxyapatite and tricalcium phosphate, as shuts the fluid used in the solution of magnesium phosphate and phosphate in phosphoric acid at a certain quantitative content of them shuts the liquid, the quantity shuts fluid to the number of reaction-hardening mixture is 0,25-0,65. Budget source components and high strength allow a wide use of this material for the correlation of the fragments of the alveolar ridge, closing cavities in the bone tissues and treatment of various traumatic fractures of Genesis.

Known material has limited use and does not provide all the necessary properties, namely the necessary biocompatibility, biodegradation, necessary osteoinductive and osteoconductive properties.

It can be selected as the closest analogue to the total number of similar features.

The technical purpose of this invention to provide a material in the form of microspheres granules based biophosphate the calcium and magnesium with adjustable particle size, possessing biocompatibility by biodegradation, osteoinductive and osteoconductive properties, is able to completely replaced by bone tissue.

The goal of the project and achieved technical result is achieved by a porous granules microspheres with controlled particle size for the regeneration of bone tissue and is characterized by the fact that have a size in the range of 1-1000 μm, the through pores of a size of 1-100 microns and a total porosity of 40-75%, granular method of electrospinning, heat treated and obtained from a mixture containing a powder of magnesium orthophosphate and biological hydroxyapatite powder from demineralized bone of cattle at a ratio of 0.5-1.0, and 1-3%solution in distilled water of sodium alginate to obtain a plastic mass, the ratio of powder, liquid phase and hardener, a saturated solution of calcium chloride, 0.5 to 1.5:2-5:5-20, respectively.

According to the prior art it was found that interaction of calcium and magnesium will be optimal when the ratio in the body 1:0,5-0,7 respectively. Otherwise, the calcium will interfere with the absorption of magnesium. Therefore, we chose the ratio of powder orthophosphate magnesium and biological hydroxyapatite 0.5 to 1.0. These ratios are optimal. Below these values the resulting mixture of orthophosphate mage is Oia, hydroxyapatite adding a solution of sodium alginate is very liquid and the time to expiration of the needle when the applied voltage is so fast that the granules do not have time to form. Above these values the resulting mixture is so viscous that it cannot be pushed through the needle.

The heat treatment of the pellets within 60 minutes at a temperature of 850°C. Using the method of electrospinning to obtain pellets allows to produce very small particles of biophosphates, changing the applied voltage.

Electrospinning at the moment can be called the most advanced and fastest way to obtain granules. Electrospinning is a dispersion of liquid and the formation of granules from a viscous medium under the influence of an electric field. The process is quite simple and does not require high temperatures, which makes it extremely attractive. The essence of the method consists in the following:

To drop viscous solution is applied a high voltage of 15-20 kV, in this case, the liquid acquires a charge, the electrostatic force of repulsion between the molecules begin to counteract the forces of surface tension and the drop is drawn. In this case, selected a certain ratio of sodium alginate and biophosphates on the basis of calcium and magnesium, and also the voltage going to the ILO elektrorazpredelenie fluid. Depending on the applied voltage, it is possible to obtain granules of different sizes. When voltage is less than 15 kV are obtained granules than 1 μm, and the increase in voltage of more than 20 kV impractical from the standpoint of safety and economy. The droplets are sprayed into the tub with a hardener containing a saturated solution of calcium chloride. The obtained granules prokalyvayutsya for 60 minutes at a temperature of 850°C and sieved through sieves to obtain fractions with a specific granule size.

Installation for electrospinning consists of nozzles (syringe needle)connected to the high voltage source, a pump and a grounded collector. The syringe is placed in a liquid: a solution of sodium alginate and biophosphates on the basis of calcium and magnesium; the liquid is then pumped is extruded at a rate of 1 drop per second at the tip of the needle.

The powder used in the invention biological hydroxyapatite obtained as follows. First get biological hydroxyapatite from the bones of cattle by demineralization of bone tissue in 1 M hydrochloric acid solution and subsequent precipitation of hydroxyapatite precipitator, for example 5 M sodium hydroxide solution, filtration, heat treatment at a temperature of 830°C.

The obtained hydroxyapatite is crushed to obtain powder particles of the size of the ROM is not more than 40 μm. This method allows you to obtain a fine powder of hydroxyapatite and trace element composition, as close as possible to the composition of human bone tissue. To enhance osteogenic properties of the material in their composition is administered magnesium orthophosphate.

Powder orthophosphate magnesium is prepared by mixing magnesium oxide and 70% orthophosphoric acid in the ratio of 1:1,18. The resulting orthophosphate magnesium were ground in a ball mill and sifted through a sieve with a mesh size not more than 40 μm. Prepared in this way the orthophosphate magnesium gives the granules plastic properties.

It was established experimentally that the receipt of the particles larger than 40 microns is not possible to enter them through an insulin syringe under the skin or in small bone defects. In addition, particles with a size not more than 40 μm, as is known from the prior art, have high specific surface area of about 80 m2/g, which is one of the key factors of their bioactivity.

Below is a specific example of production of porous microspheres granules of biophosphates calcium and magnesium with adjustable size particles for bone tissue regeneration according to the invention. This example illustrates the invention but does not limit it.

Example 1.

The fractions obtained powders of magnesium orthophosphate and biological hydroxyapatite mixed in zootoxin and 0.5 to 1.0 and mixed in a ball mill for 30 minutes. Then prepare a plastic mass containing 1-3% solution of sodium alginate dissolved in distilled water and the mixture orthophosphate magnesium and biological hydroxyapatite granules are formed by using the method of electrospinning. As the hardener used a saturated solution of calcium chloride. The ratio of components as follows: a mixture of powders of magnesium orthophosphate and biological hydroxyapatite 0,5: a solution of sodium alginate 2; solution of calcium chloride 5.

Example 2.

The fractions obtained powders of magnesium orthophosphate and biological hydroxyapatite mixed in the ratio of 0.5-1.0 and mixed in a ball mill for 30 minutes. Then prepare a plastic mass containing 1-3% solution of sodium alginate dissolved in distilled water and the mixture of powders of magnesium orthophosphate and biological hydroxyapatite granules are formed by using the method of electrospinning. As the hardener used a saturated solution of calcium chloride. The ratio of components as follows: a mixture of powders of biophosphates calcium and magnesium to 1.5: solution of sodium alginate 5: a solution of calcium chloride 20.

So, the method used for electrospinning, namely, the method of electrocapillary fluid at a voltage of 15-20 kV, followed by their capture in the tub with hardener calcium chloride and heat the processing of the granules at a temperature of 830°C.

Grinding the obtained powders is carried out in a ball mill for 30 minutes to obtain a particle size of not more than 40 μm. Such particle size of the powder is optimal for the subsequent process of obtaining microspheres.

The heat treatment of the pellets within 60 minutes. The microspheres contain through-pore size of 1-100 μm, the total porosity of 40-75%.

Used heat treatment temperature sufficient to completely remove the organic component and complete crystallization of the amorphous forms of calcium phosphate compounds and, in addition, necessary and sufficient for the formation of the pore space in the microspheres.

The selected ratio of the components (between calcium phosphate and magnesium phosphate and other components) is necessary for optimal leakage of the fluid through the needle, and get biophosphate microspheres containing the through pores of a certain size.

Formed pore space in the microspheres provides the germination of tissues deep into the pore space.

Based on numerous research on the medical use of ceramic materials is established that biophosphate belong to the class of bioactive materials with surface-active properties and high biocompatibility. The most convenient form for use in medicine are microspheres. For example, the cosmetology with the introduction of the material (because the introduction is insulin syringe, the microspheres should be no more than 30-35 μm) in the dermis of the microspheres of biophosphates stimulate fibroblasts and starts the process of neocollagenesis. In the case of the introduction of microspheres under the periosteum, particles biophosphates stimulate osteoblasts and start the process of osteosynthesis.

The production of granules synthesized hydroxyapatite now well enough developed on a large scale. Obtaining granules of biophosphates calcium and magnesium naturally expects to increase biocompatibility and osteogenic properties.

To study the properties of biocompatibility, biodegradation, as well as the ability to osteoinductive and osteoconductive material studies have been conducted on 20 rats female breed "Wistar" at the age of 3 months. In order to identify systemic reactions of the body of the material was carried out laboratory studies, including the necessary biological indicators were carried out x-ray studies of CT and also prepared and studied histological preparations surrounding the tissue material. Research has conclusively proven that the material has a well-defined biocompatible, biodegradable properties. Furthermore, the material showed high osteoinductive and osteoconductive ability, confirm that the Deno x-ray, histological and biochemical methods.

In addition, there was a higher concentration of calcium in serum, in comparison with the control group of animals that is the most important predictor of successful osteogenesis.

Porous granules-microspheres with controlled particle size for the regeneration of bone tissue, and is characterized by the fact that have a size in the range of 1-1000 μm, have continuous pores with a size of 1-100 microns and a total porosity of 40-75%, granular method of electrospinning and comb, and obtained from a mixture containing a powder of magnesium orthophosphate and biological hydroxyapatite from demineralized bone of cattle at a ratio of 0.5:1.0, and also 1-3%solution of sodium alginate in distilled water to obtain a plastic mass, the ratio of powder, liquid phase and hardener, a saturated solution of calcium chloride as of 0.5-1.5:2-5:5-20, respectively.



 

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2 cl, 1 ex

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EFFECT: invention increases strength of the composite material 8-9-fold.

3 ex, 1 tbl

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