WO2014058342A1

WO2014058342A1 - Calcium and magnesium biophosphate-based porous microspheres with controllable particle size for the regeneration of bone tissue

Info

Publication number: WO2014058342A1
Application number: PCT/RU2013/000071
Authority: WO
Inventors: Любовь Константиновна ПОЛЕЖАЕВА
Original assignee: Общество с ограниченной ответственностью "СТАЛВЕК"
Priority date: 2012-10-10
Filing date: 2013-02-01
Publication date: 2014-04-17
Also published as: RU2497548C1

Abstract

The invention relates to biophosphate ceramic materials for medicine, more specifically for traumatology, orthopedics, reconstructive surgery, cosmetology and dentistry, and to a drug delivery system. The porous microspheres are granules based on calcium phosphate, obtained from biological hydroxyapatite, and magnesium orthophosphate. The mixture from which the granules are formed by electrospinning also contains 1-3% sodium alginate solution in distilled water, and a curing agent, namely a saturated solution of calcium chloride. The granules are heat treated. The granules have a size within the range of from 10 μm to 1000 μm and exhibit biocompatibility, biodegradability and osteoinductive and osteoconductive properties. The granules have through pores with a size of 1-100 μm and have a total porosity of 40-75%.

Description

Porous microspheres based on calcium and magnesium biophosphates with adjustable particle size for bone tissue regeneration.

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

Calcium phosphates are the main constituent elements of bone and are widely used as materials for bone tissue regeneration and the manufacture of durable bone implants used in orthopedics, maxillofacial surgery, etc. Tricalcium phosphate - Ca ₃ (P ₄ ) are the most commonly used calcium phosphates in medicine. 2 (TCP) and hydroxyapatite - Ca ₅ (P0 ₄ ) ZON (HA) or biphasic materials based on the TKF-HA system. The 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 initial particles of the ceramic powder. One of the methods for increasing the mechanical characteristics and lowering the sintering temperature of ceramics is the use of ceramic ultrafine powders with a particle size of less than 1 μm.

(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) calcium phosphate materials based on TCF and HA from powders with an average crystal size of 0.5 μm. The obtained materials were characterized by a fine-crystalline structure with an average crystal size of 1-2 μm, porosity of less than 7%. The disadvantage of this material was the high firing temperature of 1220-1300 ° C. In addition, at temperatures above 1250 ° C a decrease in strength and partial decomposition of HA with the formation of toxic calcium oxide are observed.

Also known are calcium phosphate materials based on the tricalcium phosphate-hydroxyapatite system (S. Raynaud, E. Champion, D. Bernache-Assollant "Calcium phosphate appatites with variable Ca / P atomic ratio 2. Calcination and sistering", Biomaterials 23 (2002) 1073-1080 ) obtained by sintering nanocrystalline powders at a temperature of 1100-1150 ° C. In this work, we succeeded in lowering the sintering temperature by using nanocrystalline powders and using the hot pressing method. As a result, biphasic materials of the TKF-GA system were obtained, which were characterized by a finely dispersed structure with an average crystal of 200 nm. The disadvantage of this ceramic is the high firing temperature and the use of the hot pressing method, which leads to a significant increase in the cost of finished products.

Another calcium phosphate ceramic material is known from RU 2359708, June 27, 2009 for the manufacture of bone implants and / or the replacement of defects in various bone pathologies.

The material consists of tricalcium phosphate-hydroxyapatite ceramics with the addition of potassium carbonate and / or lithium carbonate or calcium carbonate in the following ratio of components in ceramics, mass%:

hydroxyapatite and / or tricalcium phosphate - 80-99.5

one of the above additives is 0.5-20.0

Known ceramic material has a particle size of not more than 10Onm and porosity (open) of less than 4% after firing up to 700 ° C.

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

Magnesium phosphate is known to be part of bone tissue. Magnesium acts as a regulator of cell growth; it is necessary at all stages of the synthesis of protein molecules. Magnesium is an indispensable element of the triad Ca, P, Mg, the exchange of which is closely interconnected. He takes part in phosphorus metabolism, energy metabolism, ACE synthesis, carbohydrate metabolism, regulates glycolysis, participates in the construction of bone tissue, provides the functional ability of nervous and muscle tissues.

Like calcium, it takes part in many metabolic processes. Enzymes, which include the magnesium ion in the active catalytic center, play an important role in providing the body with energy, transporting various substances through cell membranes, and synthesizing proteins and nucleic acids. Magnesium plays an important role in the processes of osteogenesis, can directly affect the function of bone cells.

From RU2292868, 02/10/2007, a material for filling bony maxillofacial and dental defects is known. The material is made on the basis of a reaction-hardening mixture of powders, it contains hydroxyapatite and tricalcium phosphate, a solution of magnesium phosphate and sodium phosphate in phosphoric acid is used as a mixing liquid at a certain quantitative content of them in the mixing liquid, and the amount of mixing liquid is equal to the amount of the reaction-hardening mixture is 0.25-0.65. Inexpensive starting components and high strength make it possible to widely use this material for correlation of fragments of the alveolar ridge, closure of cavities in bone tissues and treatment of various cracks of traumatic origin.

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

It can be selected as the closest analogue in terms of the number of common similar features.

The technical task of this invention is to obtain a material in the form of microspheres granules based on calcium and magnesium biophosphates with an adjustable particle size, having biocompatibility, biodegradation, osteoinductive and osteoconductive properties, which can be completely replaced by bone tissue.

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

According to the prior art it was found that the interaction of calcium and magnesium will be optimal when the ratio in the body is 1: 0.5-0.7, respectively. Otherwise, calcium will interfere with the absorption of magnesium. Therefore, we chose the ratio of magnesium orthophosphate powder and biological hydroxyapatite 0.5-1.0. These ratios are optimal. Below these values, the resulting mixture of magnesium orthophosphate, hydroxyapatite, when sodium alginate solution is added, becomes very liquid and the time of its outflow from the needle at 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 forced through a needle.

The heat treatment of the granules is carried out for 60 minutes at a temperature of 850 ° C. Using the electrospinning method to obtain granules allows the manufacture of 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 spray 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 the use of high temperatures, which makes it extremely attractive.

The essence of the method is as follows:

A high voltage of the order of 15-20 kV is applied to a drop of a viscous solution, in this case the liquid acquires a charge, the forces of electrostatic repulsion between the molecules begin to counteract the forces of surface tension, and the drop extends. In this case, a certain ratio of sodium alginate and biophosphates based on calcium and magnesium is selected, as well as voltage, so that electrospray of the liquid occurs. Depending on the applied voltage, it is possible to obtain granules of different sizes. At a voltage of less than 15 kV, granules of more than 1 μm are obtained, and an increase in voltage of more than 20 kV is impractical from the point of view of safety and economy. Drops are sprayed into a hardener bath containing a saturated solution of calcium chloride. The resulting granules are calcined for 60 minutes at a temperature of 850 ° C and sifted through sieves to obtain fractions with a certain granule size.

The electrospinning installation consists of a nozzle (this is a needle from a syringe) connected to a high voltage source, a pump and a grounded collector. Liquid is placed in the syringe: a solution of sodium alginate and biophosphates based on calcium and magnesium; then the liquid is squeezed out using a pump at a rate of 1 drop per second onto the tip of the needle.

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

The resulting hydroxyapatite is crushed to obtain powder particles with a size of not more than 40 microns. This method allows to obtain a fine powder of hydroxyapatite with a microelement composition as close as possible to the composition of human bone tissue. To increase the osteogenic properties of the material, magnesium orthophosphate is introduced into their composition.

Magnesium orthophosphate powder is prepared by mixing magnesium oxide and 70% phosphoric acid in a ratio of 1: 1.18. The resulting magnesium orthophosphate was ground in a ball mill and sieved through sieves with a mesh size of not more than 40 μm. Magnesium orthophosphate prepared in this way gives the granules plastic properties.

It was experimentally established that obtaining particles with a size of more than 40 microns does not allow them to be injected through an insulin syringe under the skin or into small bone defects. In addition, particles with sizes not exceeding 40 μm, as is known from the prior art, have a high specific surface area of about 80 m / g, which is one of the key factors in their bioactivity.

The following is a specific example of the preparation of porous microspheres granules of calcium and magnesium biophosphates with an adjustable particle size for bone tissue regeneration according to the invention. This example illustrates the invention, but does not limit it.

Example 1

The obtained fractions of powders of magnesium orthophosphate and biological hydroxyapatite are mixed in a 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 a mixture of magnesium orthophosphate and biological hydroxyapatite, granules are formed using the electrospinning method. A saturated solution of calcium chloride is used as a hardener. The ratio of the components is as follows: a mixture of powders of magnesium orthophosphate and biological hydroxyapatite 0.5: sodium alginate solution 2: calcium chloride solution 5.

Example 2

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

So, the used method of electrospinning, namely, the method of electrospraying a liquid at a voltage of about 15-20 kV, followed by their capture in a bath with hardener calcium chloride and heat treatment of granules at a temperature of 830 ° C.

The grinding of the obtained powders is carried out in a ball mill for 30 minutes to obtain particles no larger than 40 microns. This particle size of the powder is optimal for the subsequent process of obtaining microspheres.

The heat treatment of the granules is carried out for 60 minutes. The microspheres contain through pores 1-100 microns in size, with a total porosity of 40-75%.

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

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

Formed pore space in the microspheres provides tissue growth into the pore space.

Based on numerous studies on the use of ceramic materials in medicine, it has been established that biophosphates belong to the class of bioactive materials with surface-active properties and high biocompatibility. The most convenient form for use in medicine are microspheres. So, for example, in cosmetology, when the material is introduced (since the injection is carried out with an insulin syringe, the microspheres should have a size of no more than 30-35 μm), fibroblasts are stimulated into the dermis of the biophosphate microspheres and the process of neocollagenesis is started. In the case of the introduction of microspheres under the periosteum, particles of biophosphates stimulate osteoblasts and the process of osteosynthesis starts.

The production of granules from synthesized hydroxyapatite is currently quite well mastered on a large scale. Obtaining granules from calcium and magnesium biophosphates of natural origin suggests increasing biocompatibility and osteogenic properties.

To study the properties of biocompatibility, biodegradation, as well as the ability to osteoinduction and osteoconduction of the material, studies were conducted on 20 female rats of the Wistan breed at the age of 3 months. In order to identify the systemic reactions of the body to the material, a set of laboratory studies was carried out, including the determination of the necessary biological parameters, X-ray examinations of the RCT were performed, and preparations were also made and studied. histological preparations of tissues surrounding the material. During the study, it was convincingly proved that the material has well-defined biocompatible, biodegradable properties. In addition, the material showed high osteoinductive and osteoconductive ability, which is confirmed by X-ray, histological and biochemical research methods.

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

Claims

Claim

1. Porous microspheres granules with adjustable particle size for bone tissue regeneration and characterized in that they have a size in the range of 1-1000 μm, have through pores with a size of 1-100 μm and a total porosity of 40-75%, granulated by electrospinning and heat-treated and at the same time obtained from a mixture containing magnesium orthophosphate powder and biological hydroxyapatite from demineralized bones of cattle at a ratio of 0.5: 1.0, as well as a 1-3% solution of sodium alginate in distilled water to obtain plastic mass, with a ratio of powder, liquid phase and hardener, a saturated solution of calcium chloride, as 0.5-1.5: 2-5: 5-20, respectively.