PL226650B1 - Method for preparing a synthetic hydroxyapatite containing silver ion - Google Patents

Description

The subject of the invention is a method of obtaining synthetic hydroxyapatite containing silver ions incorporated into its structure.

Synthetic hydroxyapatite has a crystal structure identical to the structure of the mineral component of bone tissue, tooth enamel and dentin, which makes it biocompatible. It is known from literature and practice that after implanting hydroxyapatite into a living organism, the components of the hydroxyapatite structure are quite easily exchanged with ions in body fluids.

In the current surgical practice, for the reconstruction of bone defects, synthetic hydroxyapatite, i.e. calcium phosphate, with a chemical and structural composition similar to the mineral component of bone tissue and teeth, is used on an increasing scale.

As the chemical composition and crystal structure of synthetic hydroxyapatite are similar to the mineral component of bone, it is effectively a substitute for bone tissue. It is used in the form of a powder or paste to fill bone defects caused by disease or an accident, in the form of ceramic fittings or in the form of coatings applied to metallic implants (e.g. hip or knee prostheses made of titanium alloys).

Synthetic hydroxyapatite implanted into a living organism is not resorbed by this organism, but instead, it fills the gap existing in the bone, or creates a bioactive layer between the metallic implant and the bone. The bioactivity of hydroxyapatite is based on the fact that new natural bone tissue grows on its surface as a result of the activity of osteoblasts.

Two most commonly used methods of hydroxyapatite synthesis are widely described in the literature

- dry and wet.

Dry methods consist in roasting calcium phosphate mixtures with a molar ratio of Ca / P = 1.67 in a water vapor atmosphere at temperatures above 1000 ° C.

Wet methods are based on precipitation of calcium phosphate with a Ca / P molar ratio close to 1.67 from an aqueous solution of phosphates with calcium compounds, and in some cases also roasting it at a temperature above 1000 ° C. These are reactions taking place in aqueous solutions or suspensions based on the neutralization reactions of acids and bases, e.g. H3PO4 and Ca (OH) 2 or calcium salts, e.g. CaCl2, Ca (NO3), (CH3COO) 2Ca with phosphates, e.g. Na2HPO4, NaH2PO4, (NH4) 2HPO4.

For example, the patents PL 103 759 and PL 190 486 describe in detail the methods of obtaining pure, monophasic, not doped with other compounds or ions, calcium phosphates by wet methods, commonly used in ceramics technology.

The patent specification PL 103 759 discloses a wet method for the production of an unadded powder of calcium phosphate - dicalcium orthophosphate with the chemical formula CaHPO ₄ XH ₂ O using orthophosphoric acid and calcium salts.

On the other hand, the patent description PL 190 486 describes the methods of wet production of calcium phosphate powders, which are a mixture of hydroxyapatite (Ca10 (PO4) 6 (OH) 2) and tricalcium phosphate (Ca3PO4) or pure tricalcium phosphate, while the synthesis of calcium phosphates is substrates in the form of calcium hydroxide suspension and orthophosphoric acid solution,

2a, final products may contain HPO4 ^2- ions in their structure, derived from the acid used in the synthesis.

A mechanochemical method of obtaining hydroxyapatite is also known, but it is not widely used.

The biological activity of silver has been known since ancient times. Diluted silver nitrate solution, due to its bactericidal properties, is used in the treatment of burns and all kinds of wounds.

Its antibacterial activity against bacteria such as Staphylococus aureus, Streptococus, Pseudomonas and Escherichia is described in the literature (Klasen H. J. Historical review of the use of silver in the treatment of burns. Early uses. Burns 26, (2000b), 117-130).

Diluted 0.01 wt.% silver nitrate solution is moderately antibacterial and can be used on mucous membranes, and at a concentration of 0.1% by weight. kills most of the microbial cells. Whereas a 5 wt.% Solution AgNO3 is used as a disinfectant for extensive burns.

On the medical market, there are compresses under the trade name Silver Cel, Aquacell Ag, based on silver, which effectively release Ag ⁺ ions under the influence of exudate fluid from the wound.

PL 226 650 B1

The literature on the subject shows that silver when used in small amounts is not only non-toxic to the living organism, but also stimulates a number of living cells, such as lymphocytes, fibroblasts, and osteoblasts to increased activity (Bosetti M. et al Silver coated materials for external fixation devices; in vitro biocompatibility and genotoxicity, Biomaterials 23, (2002), 887-892).

The cited publication shows that the Ag element can play a beneficial role in bone surgery, firstly because it is biocompatible, and secondly because it has a beneficial effect on the activity of osteoblasts. Osteoblasts, in turn, are bone-forming cells, which, as their name suggests, are responsible for bone reconstruction.

Due to the above, the subject of research of numerous research centers is to obtain synthetic hydroxyapatite with silver content for biomedical applications. For example, in the publication of W. Chen, Y. Liu, H.S. Courtney, M. Betteng, C.M. Agrawal, JD Bumgardner, J. L Ong, "In vitro anti-bacterial and biological properties of magnetron co-sputtered silver-containing hydroxyapatite coating", Biomaterials, 27 (2006), 5512-5517) discloses the use of advanced coating deposition technology using magnetron sputtering to obtain a thin hydroxyapatite coating with metallic silver. In the presented method, the active targets (the so-called targets) were: hydroxyapatite (HAp) and silver (Ag), but the authors of the publication do not provide any specification regarding the origin and method of preparing the targets.

The technical issue still pending is the development of a simple and effective method of in situ wet modification of synthetic hydroxyapatite with silver ions (Ag ⁺ ) during its synthesis using the wet method, so that silver ions are introduced into the structure of the synthesized hydroxyapatite in the highest possible amount, but Thus, the parent structure of hydroxyapatite, which is responsible for its bioactive properties, was not disturbed, which is particularly important in biomedical applications of such hydroxyapatite material.

It was unexpectedly found that during the synthesis of hydroxyapatite by the wet method, it is possible to introduce a certain amount of silver in the ionic form into the structure of hydroxyapatite without disturbing the parent structure, which structure is responsible for its bioactive properties, the maximum amount of Ag ⁺ ions which it does not affect the structure of the produced hydroxyapatite at the level below 5.7 mol%. Ag. It was also found that above this level, the synthesis product ceases to be hydroxyapatite (it loses its parent structure) and becomes a mixture of hydroxyapatite and Ag3PO4.

According to the invention, a method for obtaining synthetic hydroxyapatite containing silver ions incorporated into its structure, in which the hydroxyapatite is precipitated from an aqueous solution containing phosphates with calcium compounds, and the precipitate is separated, washed with water and dried, characterized in that the phosphate-containing solution is di-sodium or di-potassium phosphate at a concentration of 0.04-0.2 M / I is slowly combined, preferably dropwise, at a temperature above 40 ° C, and preferably at boiling point, with the second solution, which contains the calcium salt, preferably calcium acetate at a concentration of 0.1-0.5 M / L and a highly water-soluble silver salt, in particular silver nitrate, chloride or acetate, in such an amount that in the second solution the Ag / Ca molar ratio is preferably from 0.01 / 1 to 0.05 / l, whereby both the solutions containing phosphate, calcium and silver ions are combined in such amounts that in the combined solutions the final Ca / P molar ratio is from 1 to 1.8, and the Ag / molar ratio is Ca no exceeded 0.1 / l.

During the slow combination of both solutions, calcium phosphate precipitates with the structure of hydroxyapatite and with a composition similar to the stoichiometric Ca10 (PO4) 6 (OH) 2 with an admixture of silver ions, characterized by a biocompatible structure.

It is understood that in the present process, both the first solution to the second and the second solution to the first can be gradually added, as long as they are brought together slowly enough to eliminate the risk of precipitation of phosphates other than hydroxyapatite.

By the method according to the invention, Ag ⁺ ions can be introduced into the synthetic hydroxyapatite in an amount up to 5.7 mol%, which on the one hand allows to maintain the current structure of hydroxyapatite, i.e. in other words to maintain its current biocompatibility, and on the other hand gives the hydroxyapatite supplemented bactericidal properties.

After introducing into the living organism, Ag ⁺ ions dispersed in the structure of hydroxyapatite may be exchanged with other ions, e.g. Na ⁺ , Ca ²⁺ , over time (gradually) released into the body fluid, thus acting as a bactericidal agent in natural tissue, surrounding an implanted hydroxyapatite biomaterial obtained by the present method. Moreover, since hydroxyapatite is insoluble, the process of releasing silver ions from the hydroxy4 structure

Apatite to the environment is based on the slow diffusion of Ag ⁺ ions, the use of hydroxyapatite containing Ag ⁺ ions as a biomaterial does not cause toxic effects for the living organism due to the low concentration of released silver ions.

The subject matter of the invention is illustrated below in some embodiments.

P r z k ł a d 1

A first solution was prepared by dissolving 3.56 g in 750 ml of distilled water

Na 2 HPO 4 2 H 2 O (0.02 Mol P).

A second solution was then prepared by dissolving 3.52 g in 250 ml of distilled water

CaH ₆ C ₄ O ₄ 4 H ₂ O (0.02 mol Ca), and then an additional 35 mg of AgNO3 (0.0002 mol Ag) was dissolved in the same solution.

The first solution was heated to reflux. The second solution was added dropwise to the boiling solution. A precipitate formed during the dropwise addition of the second solution. The precipitated solid was filtered off, washed several times with water and dried at 110 ° C. Approx. 2 g of dry solids were obtained. The sediment was identified by X-ray method using a Philips X 'Pert diffractometer equipped with a graphite monochromator PW 1752/00, Cu Ka 1.54, Ni filter (40 kV, 30 mA), as calcium phosphate with the structure of hydroxyapatite: Ca10 (PO4) 3 (OH ) 2.

Chemical analysis showed that the precipitate contained 0.8 mol%. Ag. The determination was made by atomic absorption spectrometry using the AAnalyst 300 Perkin Elmer apparatus. The determinations were carried out using an air-acetylene flame and a flame atomizer was used. The molar ratio in the combined solutions was as follows:

Ca: P = 1: 1;

Ag: Ca = 0.01: 1 which is 1 mol%. Ag versus Ca.

P r z k ł a d 2

A first solution was prepared by dissolving 17.8 g of Na 2 HPO 4 2 H 2 O (0.1 Mol P) in 1500 ml of distilled water.

A second solution was then prepared by dissolving 17.6 g in 500 ml of distilled water

CaH ₆ C ₄ O ₄ 4 H ₂ O (0.1 mol Ca), then an additional 340 mg of AgNO3 (0.002 mol of Ag) was dissolved in the same solution.

The first solution was heated to reflux. The second solution was added dropwise to the boiling solution. A precipitate formed during the dropwise addition of the second solution. The precipitated solid was filtered off, washed several times with water and dried at 110 ° C. 10.3 g of dry solids were obtained. The precipitate was identified (as in example 1) by X-ray method as calcium phosphate with the structure of hydroxyapatite: Ca10 (PO4) 3 (OH) 2.

Chemical analysis by atomic absorption spectrometry showed that the precipitate contained 1.5 mol%. Ag. The determination was carried out as in example 1.

The molar ratio in the combined solutions was as follows:

Ca: P = 1: 1;

Ag: Ca = 0.02: 1 representing 2 mol%. Ag versus Ca.

P r z k ł a d 3

A first solution was prepared by dissolving 7.12 g in 1660 ml of distilled water

Na 2 HPO 4 2 H 2 O (0.04 Mol P).

A second solution was then prepared by dissolving 11.8 g in 340 ml of distilled water

CaH ₆ C ₄ O ₄ 4 H ₂ O (0.067 mol Ca), then an additional 340 mg of AgNO3 (0.002 mol of Ag) was dissolved in the same solution.

The first solution was heated to reflux. The second solution was added dropwise to the boiling solution. A precipitate formed during the dropwise addition of the second solution. The precipitated solid was filtered off, washed several times with water and dried at 110 ° C. 6.1 g of dry solids were obtained. The precipitate was identified (as in example 1) by X-ray method as calcium phosphate with the structure of hydroxyapatite: Ca10 (PO4) 3 (OH) 2.

Chemical analysis showed that the precipitate contained 2.3 mol%. Ag. The determination was carried out as in example 1.

The molar ratio in the combined solutions was as follows:

Ca: P = 1.675: 1;

Ag: Ca = 0.03: 1 representing 3 mol%. Ag versus Ca.

PL 226 650 B1

P r z k ł a d 4

A first solution was prepared by dissolving 17.4 g of K2HPO4 (0.1 Mol P) in 1500 ml of distilled water.

A second solution was then prepared by dissolving 17.6 g in 500 ml of distilled water

CaH ₆ C ₄ O ₄ 4 H ₂ O (0.1 mol Ca), then an additional 340 mg of AgNO3 (0.002 mol of Ag) was dissolved in the same solution.

The first solution was heated to reflux. The second solution was added dropwise to the boiling solution. A precipitate formed during the dropwise addition of the second solution. The precipitated solid was filtered off, washed several times with water and dried at 110 ° C. 10.3 g of dry solids were obtained. The precipitate was identified by X-ray method (as in example 1) as calcium phosphate with the structure of hydroxyapatite: Ca10 (PO4) 3 (OH) 2.

Chemical analysis showed that the precipitate contained 1.9 mol%. Ag. The determination was carried out as in example 1.

The molar ratio in the combined solutions was as follows:

Ca: P = 1: 1;

Ag: Ca = 0.02: 1 representing 2 mol%. Ag versus Ca.

P r z k ł a d 5

A first solution was prepared by dissolving 8.7 g of K2HPO4 (0.05 Mol P) in 750 ml of distilled water.

A second solution was then prepared by dissolving 8.8 g in 250 ml of distilled water

CaH ₆ C ₄ O ₄ 4 H ₂ O (0.05 mol Ca), then an additional 425 mg of AgNO3 (0.0025 mol of Ag) was dissolved in the same solution.

The first solution was heated to reflux. The second solution was added dropwise to the boiling solution. A precipitate formed during the dropwise addition of the second solution. The precipitated solid was filtered off, washed several times with water and dried at 110 ° C. 5.3 g of dry solids were obtained. The precipitate was identified (as in example 1) by X-ray method as calcium phosphate with the structure of hydroxyapatite: Ca10 (PO4) 3 (OH) 2.

Chemical analysis showed that the precipitate contained 3.5 mol%. Ag. The determination was carried out as in example 1.

The molar ratio in the combined solutions was as follows:

Ca: P = 1: 1;

Ag: Ca = 0.05: 1 representing 5 mol%. Ag versus Ca.

P r z k ł a d 6

A first solution was prepared by dissolving 8.7 g of K2HPO4 (0.05 Mol P) in 750 ml of distilled water.

A second solution was then prepared by dissolving 8.8 g in 250 ml of distilled water

CaH ₆ C ₄ O ₄ 4 H ₂ O (0.05 Mol Ca) and then an additional 170 mg of AgNO3 (0.001 Mol Ag) was dissolved in the same solution.

The second solution was heated to reflux. The first solution was added dropwise to the boiling solution. A precipitate formed during the dropwise addition of the first solution.

The precipitated solid was filtered off, washed several times with water and dried at 110 ° C. 5.2 g of dry solids were obtained. The precipitate was identified (as in example 1) by X-ray method as calcium phosphate with the structure of hydroxyapatite: Ca10 (PO4) 3 (OH) 2.

Chemical analysis showed that the precipitate contained 1.9 mol%. Ag. The determination was carried out as in example 1.

The molar ratio in the combined solutions was as follows:

Ca: P = 1: 1;

Ag: Ca = 0.02: 1 representing 2 mol%. Ag versus Ca.

P r z k ł a d 7

A first solution was prepared by dissolving 3.56 g of Na2HPO4 (0.02 Mol P) in 250 ml of distilled water.

A second solution was then prepared by dissolving 3.52 g of CaH ₆ C ₄ O ₄ ^ H ₂ O (0.02 Mol Ca) in 750 ml of distilled water, and then an additional 238 mg of AgNO3 (0.0014 Mol of Ag) was dissolved in the same solution. ).

The second solution was heated to reflux. The first solution was added dropwise to the boiling solution. A precipitate formed during the dropwise addition of the first solution.

PL 226 650 B1

The precipitated solid was filtered off, washed several times with water and dried at 110 ° C. 2.1 g of dry solids were obtained. The precipitate was identified (as in example 1) by X-ray method as calcium phosphate with the structure of hydroxyapatite: Ca10 (PO4) 3 (OH) 2.

Chemical analysis showed that the precipitate contained 3.9 mol%. Ag. The determination was carried out as in example 1.

The molar ratio in the combined solutions was as follows:

Ca: P = 1: 1;

Ag: Ca = 0.7: 1 which is 7 mol%. Ag versus Ca.

P r z k ł a d 8 (comparative)

A first solution was prepared by dissolving 3.56 g in 750 ml of distilled water

Na 2 HPO 4 2 H 2 O (0.02 Mol P).

A second solution was then prepared by dissolving 3.52 g in 250 ml of distilled water

CaH ₆ C ₄ O? H ₂ O (0.02 mol Ca), then an additional 340 mg of AgNO3 (0.002 mol of Ag) was dissolved in the same solution.

The first solution was heated to reflux. The second solution was added dropwise to the boiling solution. A precipitate formed during the dropwise addition of the second solution. The precipitated solid was filtered off, washed several times with water and dried at 110 ° C. 1.9 g of dry solids were obtained. The precipitate was identified (as in example 1) by X-ray method as a mixture of: calcium phosphate with the structure of hydroxyapatite Ca10 (PO4) 3 (OH) 2 and silver phosphate Ag3PO4

Chemical analysis showed that the precipitate contained 5.7% Ag mol. The determination was carried out as in example 1.

The molar ratio in the combined solutions was as follows:

Ca: P = 1: 1.

Ag: Ca = 0.1: 1 representing 10 mol%. Ag versus Ca.

Claims (2)

Method for the preparation of synthetic hydroxyapatite containing silver ions, incorporated into its structure, in which hydroxyapatite is precipitated from an aqueous solution containing phosphates with calcium compounds, and the precipitate is separated, washed with water and dried, characterized in that the solution containing di-sodium phosphate or phosphate is di-potassium, with a concentration of 0.04-0.2 M / l is slowly combined, preferably dropwise, at a temperature above 40 ° C, and preferably at boiling point, with the second solution, which contains a calcium salt, preferably calcium acetate, at a concentration of 0.1-0 , 5 M / L and a highly water-soluble silver salt in such an amount that in the second solution the Ag / Ca molar ratio is preferably from 0.01 / 1 to 0.05 / 1, both solutions being combined in such amounts that in the combined solutions, the final Ca / P molar ratio ranged from 1 to 1.8, and the Ag / Ca molar ratio did not exceed 0.1 / 1. 2. The method according to p. The process of claim 1 wherein the silver ion source is silver nitrate, chloride or acetate.