LV14592B - Calcium phosphate bone cement and method for its preparation - Google Patents

Abstract

Proposed calcium phosphate bone cement and method for its preparation contributes to the field of medicine and can be used for surgical reconstruction of bone defects.Composition of calcium phosphate bone cement paste consists of a solid and a liquid phase. The solid phase consists of synthesized alpha-tricalcium phosphate (alpha-TCP) and beta-TCP, while the liquid phase consists of aqueous solutions of sodium or potassium dihydrogen phosphates. Molar ratio of calcium (Ca) and phosphorus (P) remains constant in solid phase, while the concentration of sodium (Na+) and potassium (K+) ions are changing in liquid phase.

Description

Description of the Invention

The present invention relates to the medical field and is useful in the reconstruction of bone damage in non-load-bearing areas, such as bone tissue engineering or drug delivery systems.

Recent literature [1-4] provides a general overview of calcium phosphate (CaP) cements and their composite materials, describes their diversity, their properties and their applications in the biomedical field. The information obtained confirms that over the last 20 years there has been active and versatile research into the development of new formulations of CaP bone cement and the development of technologies aimed at improving the quality and applicability of these materials, ensuring their biocompatibility and bioactivity, bioresorbability, bone substitute, surgical requirements for the cementitious mass of cement and suitable curing time [3].

Calcium phosphate (CaP) cements are known to be prepared from calcium-containing salts, including calcium phosphates, as solid phase but aqueous solutions of phosphate salts or organic acids as the liquid phase [5]. In the solid phase, the ratio of calcium (Ca) to phosphorus (P) has been reported to vary from 1.3: 1 to 1.8: 1.

A disadvantage of these materials is that the wide range of Ca: P ratios given can include virtually all CaPs that have various effects on the end products of the cementitious curing process and their properties. Depending on the chemical composition of the liquid phase, the curing time of the cement mass varies from 2.5 to 14 minutes. The disadvantage of these cements is that they are characterized by relatively low compressive strength - 10.7 ± 0.8 - 6.6 ± 0.7 mPa after curing time of 2 - 4 h.

Also known is a US patent patenting a composition for replacing hard tissue (filling defects in bone tissue) [6]. Calcium phosphates (CaP) of variable composition Ca _{n + 2} (PO4) ₂ O _n .i, where n is used as a solid phase composite material. = 1.9 - 4.9. Aqueous solutions of various organic acids (containing TCA cycles) or polymers and organic acids (citric, tartaric, malic, etc.) (25-60% by weight)

%). The curing time of the plastic mass depends on the ratio of solid to liquid phase in the cement mass C / H = 1-2.2 g / lg and the pH of the liquid phase, which varies from 6.92 to 7.10. The disadvantage of this material is the relatively short cure time (1-7 min) and, when the organic acid solutions or their polymers are added to the plastic mass of the cement, their decomposition products can cause inflammation in living tissues before complete acid neutralization. In addition, depending on the molar ratio (4.8) of the solid phase raw materials (CaCO ₃ and γ - Ca ₂ P ₂ C> 7), the cement solid phase Can + ₂ (PO 4) ₂ O _n .i synthesis temperature is relatively high at 1650 ° C. .

Topical research on bone cements based on α-tricalcium (α-TCP). Since α-TCP is thermodynamically less stable than β-TCP, α-TCP is chemically highly reactive and recrystallizes from non-stoichiometric hydroxylapatite (HAp) at a solid phase Ca / P ratio of 1.5-1.67 as a result of the cement paste hardening reaction. [1,2], Non-stoichiometric HAp is biologically active and, with appropriate bioresorption, natural bone tissue completely replaces the implant material.

Known composite materials for fast-setting calcium phosphate cements using various calcium phosphates as solid phase, including α-TCP as solid phase and aqueous solutions of inorganic, organic acids or their salts as liquid phase [7] size, liquid phase chemical composition and concentration, and is in the range of 2 to 90 minutes. Depending on the above parameters, different crystalline phases of CaP, characterized by different properties and structure, crystallize in the cement curing process depending on the above mentioned parameters. A disadvantage of the patented implant materials is that with the use of different liquid phase compositions and different CaP solid Szes without specifying solid to liquid phase ratios, it is not possible to predict a certain cement hardening time, which is important in practical surgery.

Cement composition based on a- and / TTCP with a Ca / P molar ratio of 1,4 1,498 [8] has also been developed. Solutions of sodium salts (succinates, lactates, acetylates, chlorides, etc.) or water-soluble polymers have been used as liquid phase in cement . The ratio of solid to liquid phase in the cement composition is 1.0 - 2.5. 1.0, but in the solid phase, α-TCP is 97-50: 3-50 (w / w) in relation to // - TCP. The curing time of the plastic mass of the cement is regulated by the method of preparation of α-TCP (from Ca ₂ P ₂ C> 7, CaCO ₃ , H ₃ PO ₄ in high temperature synthesis 1200 ° C) and the amount of β-TCP in the cement composition. The curing time of the plastic mass of these cements varies over a wide range from 9 min to 80 min.

These cement compositions lack be noted that the a- and / (- TCP extraction process is released CO _2, which increases the loss to the environment. The technological process is time consuming because TCP extraction process control a- and / (- TCP acquired mass ratio. A disadvantage is that the Ca / P ratio in the solid phase is <1.5, which negatively affects the bioresorption of the cementitious composition as an implant material.

Also known as calcium phosphate-based hydraulic cement for use in reconstructive surgery [9]. Mechanically activated (grinding) solid phase crystalline powders (α-TCP, // - TCP, oxyapatite Caio (P0)) were used as the solid phase ₄ ) 60, tetracalcium phosphate - Ca ₄ (PO ₄ ) ₂ O, etc.) to amorphous calcium phosphate. Sodium hydrogen phosphate (0.15 mol / l), sodium hyaluronate solutions, polyvinyl alcohol and other organic solutions were used as liquid phase. The molar ratio Ca / P in the plastic mass of the cement varies from 1.5 to 2.0, while the ratio of solid to liquid phase C / S = 1-2.5.1.0 (volume fraction). The curing time of the plastic mass of cement depends on the chemical composition of the solid and liquid phases, their interrelationship and the solid phase preparation process (specific surface, temperature). Depending on the factors mentioned above, the curing time of the plastic mass of the cement is between 5.6 min and 11.3 min (at 37 ° C).

The disadvantage of this material is that bioresorption does not occur in the volume of the material, but only on its surface. In order to facilitate the growth of bone tissue in volume, specially made granules of CaSO4HbO must be added to the plastic mass of the cement, which dissolves over time to form pores that facilitate the growth of new bone tissue in the implant material.

The technological process for obtaining a solid phase is time consuming and energy consuming, as well as making its economic parameters (cost price) more expensive when used as a raw material, such as α-TCP, with a commercial price of lg between 12.35 EUR and 48.70 EUR. Aldrich) [10],

The new calcium phosphate cement composition and its preparation method have been chosen as the prototype [11]. The solid phase of the developed calcium phosphate cement composition contains 2 chemically synthesized components, one of which is α-TCP (Ca ₃ (PO ₄ ) 2, 95% by weight) + stoichiometric hydroxylapatite - HAp (Caio (PO ₄ ) e (OH) ₂ - 5% by weight Calcium hydrogen phosphate dihydrate - DCPD (CaHP0.1-2H> 0) Ca / P molar ratio in the solid phase of 1.35-1.40 is used as the second solid phase component.

The following raw materials were used for the synthesis of one component of the solid phase: calcium nitrate Ca (NO ₃ ) ₂ solution, ammonium hydrogen phosphate (NHAHPCL solution, NH 4 OH ammonia solution). The maximum temperature of synthesis of the raw material mixtures is 1200 ° C. prepared by mixing solutions of two compounds - Na2HPO4 • 2H ₂ O and KH2PO4 Ca (NO ₃₎ 2. the reaction end product CAHP ₄ -2H ₂ O is the second plastic cement weight solids component. the two solids component of 95% α-TCP and 5 % HAp (component a) and CaHPO4-2H ₂ O (component B) is mixed with the liquid phase - 3%

Na ₂ HPO 4-2H ₂ O, solid phase to liquid phase C / H = 2.5-2.22 g / ml. CaP cement solid phase particle size <40 pm (100% by volume of powder). Cement paste hardening time 10-12 min, cement implantable up to 10 min.

The shortcomings of the prototype are:

- the low Ca / P molar ratio (1.35-1.40) in the solid phase of the cement's plastic mass carries the risk of too rapid resorption of the implant material, which does not ensure bone regeneration but may cause soft connective tissue to grow at the site of the defect;

- the concentration of the liquid phase limits the ability to adjust the pH of the liquid phase, which in turn does not allow the cement to be set with a controlled curing time;

the ratio of solid to liquid phase in the plastic mass of cement (C / H - 2.5 - 2.22 g / ml) reduces pore formation in the cement hardening process, which limits its applicability as a backbone for bone engineering or drug delivery systems;

the process of solid phase synthesis and liquid phase preparation is time and energy consuming and environmentally unfriendly.

The object of the present invention is to provide a composition of calcium phosphate (CaP) bone cement and an economical technological method for its preparation, which provides the use of these cements for regeneration of bone tissue in non-load bearing areas with predictable and adjustable curing time. would be equivalent to natural bone with good biocompatibility and suitable bioresorption that affects the growth of natural bone cell cells in the implant material, as well as being used as a backbone for bone tissue engineering or drug delivery systems.

The object of the invention is achieved by developing a composition of cement mass of cement with a constant solid (C) and liquid (S) phase ratio C / S = 1.75 g / ml, constant calcium (Ca) and phosphorus (P) molar ratio in solid phase Ca / P = 1.5 but with variable concentration of sodium (Na ⁺ ) or potassium (K ⁺ ) ions in the liquid phase.

The plastic mass compositions of the developed CaP cements are shown in Table 1.

Table 1

Plastic mass composition of CaP cement

Composition of the liquid phase Composition conscious jam Solid (C) to liquid (N) phase ratio, C / N = 1.75 g / ml Na ₂ HPO ₄ 2H ₂ O or K ₂ HPO ₄ 2H ₂ O, g / l NaH ₂ PO ₄ 2H ₂ O or KH ₂ PO ₄ 2H ₂ O, g / l Phosphate ion concentration, mol / l Concentration of sodium or potassium ions, mol / l Liquids phases pH 1 1 g of α-TCP per 0.57 ml 24.1 56.9 0.5 0.64 6.0 2 1 g of α-TCP per 0.57 ml 41.8 41.4 0.5 0.73 6.5 3 1 g of α-TCP per 0.57 ml 65.3 20.8 0.5 0.87 7.0 4 1 g of α-TCP per 0.57 ml 79.6 8.2 0.5 0.95 7.5 5 1 g of α-TCP per 0.57 ml 85.5 3.0 0.5 0.98 8.0

At such technological parameters, the curing time of the cement can be predicted and regulated depending on the specifics of the practical application of the cement (bone pathology, injuries, etc. bone damage). Experimentally it was found that increasing the concentration of Na ⁺ (or K ⁺ ) ions in the liquid phase from 0.64 mol / l to 0.98 mol / l changes the pH of the cementitious plastic curing process from 6.0 to 8.0, which in turn influences cement curing time. If the initial pH of the medium is acidic, ie pH <7.0, the final product of the cement hardening is not precipitated by hydroxylapatite (HAp) with Ca deficiency, but by octa-calcium phosphate (Cag (HPO ₄ ) ₂ (PO ₄ ) ₄ -5H ₂ O), which could delay nucleation of apatite crystals with subsequent non-stoichiometric HAp crystallization. Consequently, the curing time of the plastic mass of the cement is prolonged.

If the pH of the medium is basic (pH> 7.0), the solubility of the solid phase «-TCP decreases, which causes a decrease in the calcium and phosphate ions in the solution and a slower recrystallization, which in turn prolongs the curing time of the plastic mass.

The technological process for the preparation of CaP bone cement plastic mass consists of the following steps:

- solid phase synthesis;

- mechanical activation (grinding) of the solid phase surface;

solid phase drying;

- preparation of the liquid phase;

- sterilization of the solid and liquid phases (separately);

- preparing the plastic mass of the cement by mixing the solid phase (C) with the liquid phase (S), adding medication as needed.

Α-TCP (Ca3 (PO4) 2) Synthesized as Base Material for Solid Phase (C) Synthesis - Calcium Carbonate (CaCO ₃ ) (Sigma-Aldrich, Cat.No. 31208) and Calcium Hydrogen Phosphate Dihydrate (CaHPCL FLO) (Sigma-Aldrich, CatNo. 307653) in a molar ratio of 1: 2. The synthesis cycle lasts 8 h. The maximum synthesis temperature is 1400 ° C for 1 h. After roasting the powdered ingredients, the mixture is cooled rapidly to ambient temperature by pouring on a steel plate. The synthesized product consists of 90% <α-TCP <99% and / 7-TCP <10% (product composition calculated by X-ray diffractometry by semi-quantitative method). After solid phase synthesis, the powder is comminuted by milling for 1 h in a planetary ball mill "Pulverisette 5" in isopropanol medium. Experimentally it has been found that the optimum solid phase granulometric composition, which promotes a suitable and appropriate curing time (3-14 min), is from 10 to 3 pm - 50% - 70% of the particle volume and particles smaller than 3 pm - 50% - 30% by volume of the particles. When synthesized α-, β-TCP powders with a particle size of less than 3 µm and more than 50% by weight are used for the preparation of the solid phase, the curing time is reduced to less than 3 minutes, which is considered insufficient for the mass could be implanted qualitatively. Conversely, if the occupied volume of such size particles (<3 pm) in the plastic mass of cement is <30%, the cure time increases above 40 minutes at the phosphate ion concentration in the liquid phase of 0.4-0.6 mol / l.

It has also been found that such multi-modal particle partitioning in the cementitious plastic mass solidifies pores in the cement volume, which facilitates bioresorption of the implant material and, when medicated in the plastic mass of the cement, open porosity also contributes to their slow release into the body. After solid phase grinding, the solid phase suspension is dried at 70 ° C. The drying cycle is carried out with thinning and lasts for 5 hours.

Liquid phase injection as a source of raw material sodium (or potassium) phosphate dihydrate Na2HPO4-2H2O (Enola, Cat.Nr. N-2092) and sodium (or potassium) phosphate monobasic dihydrate NaH ₂ PO4-2H ₂ O (Enola, Cat.Nr. N -2112) solutions of water (deionised, purified by Crystal E apparatus, R> 0.055) with variable concentrations of sodium ions (NaG or potassium ions (K ⁺ ) between 0.64 and 0.98 mol / L (Table 1) but Changing the concentration of Na ⁺ or K ⁺ ions causes a change in the pH of the liquid phase as the concentration of these ions increases, the liquid phase of the cement paste becomes more basic and its pH rises to 8.0.

After preparation of the solid and liquid phases, they are sterilized by surgical techniques (elevated temperature (~ 300 ° C), ionizing radiation, autoclaving, etc.). The solid and liquid phases of the plastic mass of cement are sterilized separately. During the sterilization process it should be taken into account that the solid phase of the cement plastic mass reacts with water, so that during sterilization of the solid phase, it must be ensured that it is not in contact with moisture. After sterilization of the solid and liquid phases, the plastic mass of the cement is prepared by mixing the solid phase with the liquid phase relative to the solid phase: liquid phase - C / H - 1.75 g / ml, which promotes porosity and a suitable rate of bone regeneration. If necessary, the necessary medication (peptides, antibiotics, morphogenetic proteins, etc.) can be added to the mixing process.

The molar ratio of Ca / P in the plastic mass of cement is constant - 1.5. Mixing time of cement plastic mass 0.5 - 2 min. It depends on the amount of paste to be prepared. Cement is formed by the physico-chemical reactions of the solid and liquid solidification processes of the paste.

The properties of the experimentally prepared CaP bone cements are shown in Table 2.

Table 2

Properties of developed CaP bone cement compositions

Composition conscious jam Plastic mass cohesive time, min, at 21 ° C Initial setting time of the cement, min, at 21 ° C Cement cure time ¹ min, at 21 ° c Compressive strength after 48 h maturation, MPa Porosity after 48 h of maturity,% 1 7th 14th 25 (7) 8.6 ± 1.0 54 2 3 5 17 (7) 9.7 ± 1.0 54 3 1 3 7 (7) 9.8 ± 1.2 55 4 3 6th 17 (7) 10.3 ± 0.9 54 5 7th 14th 35 (10) 11.2 ± 0.9 52

^x - Brackets - Stirring at 21 ° C and placing at 37 ° C after 2 min of mixing.

From the experimental results it can be concluded that by changing the concentration of Na ⁺ (or K ⁺ ) ions in the liquid phase from 0.64 to 0.98 mol / l, the pH of the liquid phase changes from 6.0 to 8.0 and these criteria (parameters) depend on the curing time of the cement. The initial setting time of the cement is 3 minutes at pH 7.0. If the curing time of liquid phase 7.0 <pH <7.0 increases to 14 minutes. Predictability and controllability of cement curing time ensures practical use of these cements for individual reconstructive surgery needs and needs, depending on the nature of the bone defect. It has been found that at the temperature of the biological organism (human), the final cure time decreases, which is positive because it allows the wound to be closed faster. CaP bone cements developed after 48 h maturity have a compressive strength of 8.6 ± 1.0 to 11.2 ± 0.9 MPa. As the basicity of the curing medium increases, the compressive strength values increase. Based on the results of compressive strength values, CaP bone cement developed is used in reconstructive surgery to eliminate trabecular bone defects in non-load-bearing areas. Open microporousness is between 52% and 55%. The lowest is for cements with higher compressive strength. Open porosity with interconnected pores contributes to the bioresorption of the implant and positively affects the gradual release of topically applied drugs over time.

The porosity depends on the ratio of solid to liquid phase and the granulometric composition of the solid phase. As these parameters are constant for experimental compositions, the differences in porosity value are insignificant.

The structure of the cement, after dissolution and recrystallization of the solid phase of the raw materials, with subsequent cement maturation for 48 h, is formed by non-stoichiometric hydroxylapatite (HAp) crystals in the form of plates. Experimental investigation of the morphology of the non-stoichiometric structure of HAp has shown that HAp plates are thinner and have a larger surface area when the acidic liquid phase (pH 6) is used, but they become thicker and smaller in size when the basic liquid phase (pH 8) is used. At the liquid phase, the pH = 6 plates have a surface area of 0.5 µm x 0.5 µm and a thickness of 0.02 µm. At the liquid phase pH = 8, the surface area of the plates decreases to 0.1 pm x 0.2 pm but the thickness increases to 0.05 pm.

Cement structure morphology studies confirm that the degree of crystallinity is higher when the liquid phase of the cement is more basic, and that the surface area of the crystalline structure of the implant is larger and accelerates bioresorption.

In vitro tests of the developed CaP bone cement showed that the number of live cells (human osteoblasts) in the implant material after 72h compared to the sample in the control plot was 112%. The most effective process was found for CaP bone cements for the preparation of liquid phase pH = 7.0, ie with Na ⁺ (or K ⁺ ) ion concentration of 0.87 mol / l. In vitro studies using a rabbit model with a 4mm bone defect confirm that the rate of bioresorption of the developed CaP cements is adequate for bone regeneration.

In vivo studies have shown that the developed CaP bone cements improve the body's quality of life in a relatively short period of time because the implant material is completely absorbed after 3 months and is identical to natural bone.

LITERATURE USED

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Claims (7)

1. Calcium phosphate (CaP) bone cement is made of a plastic mass consisting of a solid (C) and liquid (S) phase which results in the formation of bone cement as a result of the curing reactions, characterized in that the solid phase of the cement mass synthesized a-tricalcium phosphate (α-TCP) arβ-TCP impurities, and the liquid phase - sodium or potassium hydrogen phosphate dihydrate Na ₂ HPO4><2H ₂ O (or Κ ₂ ^ΗΡ04 χ 2Η20) and sodium or potassium dihydrogen phosphate dihydrate NaH2PC> 4 ^x 2H2O (or KH ₂ PO 4 ^X 2H ₂ O) aqueous solution. CaP bone cement according to claim 1, characterized in that the molar ratio of calcium to phosphorus in the solid phase of the cement mass is 1.5 and the ratio of solid phase (C) to liquid phase (C) is C / S = 1, 75 g / ml. The process for the synthesis of a plastic mass solid phase of CaP bone cement according to claim 1 or 2, characterized in that it is obtained by heating a mixture of calcium carbonate (CaCO ₃ ) and calcium hydrogen phosphate dihydrate (Ca ₂ HPO 4 ^x 2 H ₂ O). to a maximum temperature of 1400 ° C with a cycle time of ~ 8h followed by rapid cooling of the powder mixture to room temperature, milling in a planetary ball mill lh in isopropanol and drying the resulting slurry at 70 ° C for 5h. The process for the synthesis of a solid phase plastic mass of CaP bone cement according to claim 3, wherein a product containing 90% <α-TCP <99% and βTCP <10% is obtained. The plastic phase solid state synthesis product of CaP bone cement according to claim 4, characterized in that it contains particles of different sizes (multimodal particle distribution): 10-3 pm = 50-70% and <3 pm = 50-30% of the total volume of particles. The process for preparing the liquid phase of CaP bone cement plastic mass according to claim 1 or 2, characterized in that it is prepared using aqueous solutions of sodium or potassium hydrogen phosphate or dihydrogen phosphate 8-9%, with a liquid phase pH of 6.0 to 8. , 0, which can be achieved by changing the concentration of sodium or potassium ions at constant concentration of phosphate ions - 0.5 mol / l. The plastic mass of CaP bone cement according to claim 1, characterized in that it may additionally contain medicaments.