阅读说明：本技术 可降解聚合物-碳酸钙复合骨修复材料及其制备方法 (Degradable polymer-calcium carbonate composite bone repair material and preparation method thereof ) 是由 王军 王一村 江辉 邱阳 柏茂盛 赵建宁 于 2021-09-29 设计创作，主要内容包括：公开了一种改性的球形碳酸钙复合材料,由以下方法制备：向聚丙烯酸溶液中加入CaCl-(2)溶液和Na-(2)CO-(3),得到球形碳酸钙；将十二水合磷酸氢二钠溶于水中,加入球形碳酸钙；悬浮液进行水热反应；加入九聚精氨酸,得到改性的球形碳酸钙复合材料。此外,还公开了一种具有良好的细胞粘附能力且孔隙率高的可降解聚合物-碳酸钙复合骨修复材料及其制备方法。(Discloses a modified spherical calcium carbonate composite material, which is prepared by the following method: adding CaCl into polyacrylic acid solution 2 Solution and Na 2 CO 3 Obtaining spherical calcium carbonate; dissolving disodium hydrogen phosphate dodecahydrate in water, and adding spherical calcium carbonate; carrying out hydrothermal reaction on the suspension; adding nonapolyarginine to obtain the modified spherical calcium carbonate composite material. In addition, the degradable polymer-calcium carbonate composite bone repair material with good cell adhesion capability and high porosity and the preparation method thereof are also disclosed.)

1. A modified spherical calcium carbonate composite material is prepared by the following method:

(1) adding 0.1-0.3M CaCl into polyacrylic acid solution₂Adjusting the pH value of the solution to 7-9; adding 0.1-0.3M Na₂CO₃Stirring and reacting for 2-6h, filtering, washing and drying to obtain spherical calcium carbonate;

(2) dissolving disodium hydrogen phosphate dodecahydrate in water, adjusting pH to 9-11, and adding spherical calcium carbonate to form a suspension;

(3) carrying out hydrothermal reaction on the suspension, and cooling to room temperature after the reaction is finished;

(4) adding nonapolyarginine into the suspension, and adjusting the pH to 9-11; stirring the suspension for reaction for 2-10 h; and carrying out suction filtration, washing and drying to obtain the modified spherical calcium carbonate composite material.

2. The composite material of claim 1, wherein the polyacrylic acid has a weight average molecular weight M_w5-15 kilodaltons; the concentration of the polyacrylic acid solution is 2-5 g/L.

3. The composite of claim 1, wherein the polyacrylic acid solution is reacted with CaCl₂The volume ratio of the solution is (1.5-3): 1.

4. the composite material according to claim 1, wherein the spherical calcium carbonate and disodium phosphate dodecahydrate have a theoretical Ca/P atomic number ratio of 1: (0.8-1.6).

5. The composite material of claim 1, wherein the hydrothermal reaction temperature is 140-180 ℃; the hydrothermal reaction time is 0.5-4 h.

6. The composite material of claim 1, wherein the weight ratio of nonapolyarginine to spherical calcium carbonate is (0.1-0.2): 1.

7. a degradable polymer-calcium carbonate composite bone repair material is prepared by the following method:

(1) mixing polylactic acid and a mixture of polylactic acid and polylactic acid in a volume ratio of 1:1, preparing a solution from chloroform/acetone, adding the modified spherical calcium carbonate composite material as described in any one of claims 1 to 6 and ammonium bicarbonate, uniformly mixing, standing at 0 to-10 ℃ for 4 to 8 days; then volatilizing the solvent at room temperature, and naturally drying and forming to obtain a material matrix;

(2) soaking the material matrix in water bath at 40-80 deg.c to eliminate ammonium bicarbonate and vacuum stoving to obtain the degradable polymer-calcium carbonate composite material.

8. The composite bone repair material according to claim 7 wherein the polylactic acid has a weight average molecular weight M_w40-60 kilodaltons; the concentration of the polylactic acid solution is 0.2-0.8 wt%.

9. The composite bone repair material according to claim 7, wherein the weight ratio of the modified spherical calcium carbonate composite, ammonium bicarbonate and polylactic acid is (1-3): (6-8): 1.

10. a method for preparing the degradable polymer-calcium carbonate composite bone repair material according to any one of claims 7 to 9, which comprises the following steps:

(1) adding 0.1-0.3M CaCl into polyacrylic acid solution₂Adjusting the pH value of the solution to 7-9; adding 0.1-0.3M Na₂CO₃Stirring and reacting for 2-6h, filtering, washing and drying to obtain spherical calcium carbonate;

(2) dissolving disodium hydrogen phosphate dodecahydrate in water, adjusting pH to 9-11, and adding spherical calcium carbonate to form a suspension;

(3) carrying out hydrothermal reaction on the suspension, and cooling to room temperature after the reaction is finished;

(4) adding nonapolyarginine into the suspension, and adjusting the pH to 9-11; stirring the suspension for reaction for 2-10 h; and carrying out suction filtration, washing and drying to obtain the modified spherical calcium carbonate composite material.

(5) Mixing polylactic acid and a mixture of polylactic acid and polylactic acid in a volume ratio of 1:1, preparing a solution from chloroform/acetone, adding the modified spherical calcium carbonate composite material and ammonium bicarbonate, uniformly mixing, and standing for 4-8 days at a temperature of between 0 and 10 ℃ below zero; then volatilizing the solvent at room temperature, and naturally drying and forming to obtain a material matrix;

(6) soaking the material matrix in water bath at 40-80 deg.c to eliminate ammonium bicarbonate and vacuum stoving to obtain the degradable polymer-calcium carbonate composite material.

Technical Field

The invention belongs to the technical field of medical biomaterials, and particularly relates to a degradable polymer-calcium carbonate composite bone repair material and a preparation method thereof.

Background

Bones are important components of the human body and a skeleton supporting the weight of the human body. Bone is a material with a complex hierarchical structure, is composed of components with multiple scales and irregular arrangement and directions, has non-uniform chemical properties and structure, and has anisotropy. With respect to chemical composition, bone is composed primarily of bone minerals and collagen, in a ratio of about 2: 1. the former includes Hydroxyapatite (HA), carbonated hydroxyapatite, amorphous calcium phosphate, and the like.

When the skeleton is slightly damaged or has small-segment bone defect, the human bone tissue has certain self-regeneration capacity to deal with the bone defect and can maintain balance by osteoblasts, osteoclasts and other functional cells in a microenvironment. If serious injury or large bone defect occurs, the bone defect can not heal spontaneously only by means of the dynamic balance of the bone, and the bone defect can be healed only by repairing the bone defect with bone repair material.

The bone repair material comprises a metal material, a ceramic material, a natural polymer material and a synthetic polymer material. Metallic materials have excellent mechanical properties, are biologically inert, but have poor osteointegration and the potential for metal corrosion and ion release. The nano-scale calcium carbonate, hydroxyapatite, beta-tricalcium phosphate and other scaffold materials have similar chemical properties to bone mineral substances, have higher specific surface area, are easier to perform surface modification, can more effectively carry drugs or bioactive molecules, are more beneficial to cell adhesion, have the advantages of easier regulation and control of morphology and preparation conditions and the like, but are influenced by single application due to overlarge rigidity and brittleness, have longer degradation time and have uncontrollable degradation speed. The natural polymer material and the hydrogel scaffold thereof are biodegradable, have high biocompatibility and bioactivity, but are often limited by lower mechanical properties and excessively fast degradation speed. The synthetic polymer materials are various in variety, but the materials for bone repair at present are still mostly concentrated on biodegradable polylactic acid, polyglycolic acid, polylactide and other materials, the polylactic acid has good degradability, but the rapid degradation of the polylactic acid can cause the problems of too high concentration of local acidic substances of tissues, damage to bone repair microenvironment and the like. Thus, composite materials combining the advantages of various materials are receiving wide attention of researchers, but the variety of composite materials is more extensive, and the development of composite materials with good biocompatibility and biodegradability and excellent osteoinductive capacity is still the key.

Chinese patent application CN102343115A discloses a three-dimensional network-shaped chitosan-calcium carbonate composite nano-material and preparation and cell compatibility thereof. The preparation method takes chitosan as a carbon source, calcium chloride dihydrate, ammonium bicarbonate and secondary distilled water as raw materials, and obtains the chitosan-calcium carbonate composite nano material on the surface of stainless steel by a one-step co-electrodeposition method. The biocompatibility of the chitosan-calcium carbonate composite nano-net structure is researched through in vitro cell culture. The electrochemical deposition method is simple and controllable, the chitosan-calcium carbonate fiber net structure is firmly combined with the substrate, the mechanical property is strong, the biocompatibility is good, and the chitosan-calcium carbonate three-dimensional fiber net structure is used as a cell scaffold material, so that a good experimental result is obtained. Simple process, low cost and environmental protection, meets the standard of biological materials used as tissue engineering cell culture matrixes, and can be applied to bone repair.

Chinese patent application CN106421894A discloses a bone tissue engineering scaffold material, which is a composite fiber membrane prepared from stearic acid modified hydroxyapatite powder and poly-L-lactic acid, wherein the mass percentage concentration of the stearic acid modified hydroxyapatite powder in the composite fiber membrane is 1-15%. The preparation method comprises three steps of carrying out surface modification on hydroxyapatite powder by using stearic acid, blending the hydroxyapatite powder and a poly-L-lactic acid solution and carrying out electrostatic spinning on the blended solution. The composite fiber membrane of the bone tissue engineering scaffold has a porous communication structure, the hydroxyapatite powder and the polylactic acid matrix are firmly combined, the hydroxyapatite powder is not easy to fall off in the degradation process, the degradation product has little influence on the pH value of a human body, and the like, and the porous structure is superior to the existing material in the aspects of facilitating the transmission of nutrients and metabolites and the like.

Chinese patent application CN111184916A discloses a method for preparing a hydroxyapatite/levorotatory polylactic acid composite bone scaffold, which comprises the steps of carrying out surface modification on levorotatory polylactic acid powder by utilizing polydopamine, soaking the modified powder in simulated body fluid to generate hydroxyapatite on the surface of the levorotatory polylactic acid powder in situ, filtering, cleaning, drying and grinding the obtained powder to obtain composite powder, and preparing the hydroxyapatite/levorotatory polylactic acid composite bone scaffold by selective laser sintering of the obtained composite powder. In the composite bone scaffold prepared by the method, hydroxyapatite is uniformly distributed in the levorotatory polylactic acid matrix, and strong interface bonding is realized between the hydroxyapatite and the levorotatory polylactic acid.

Chinese patent application CN104524630A discloses a composite bone repair material and a preparation method thereof. The bone repair material is formed by compounding degradable lactic acid-basic amino acid copolymer and calcium silicate, wherein the calcium silicate accounts for 10-50% of the total mass of the bone repair material, the lactic acid-basic amino acid copolymer is formed by polymerizing L-lactic acid and at least one alpha-basic amino acid, and the basic amino acid accounts for 5-30% of the total molar weight of the copolymer. Mixing L-lactic acid and basic amino acid, dehydrating, performing prepolymerization reaction in the presence of a catalyst at a lower temperature, raising the temperature, completing polymerization reaction to obtain a lactic acid-basic amino acid copolymer, and performing composite reaction with calcium silicate to obtain a target product. The bone repair material can be prepared into products for bone repair in different forms required clinically through extrusion molding or injection molding. The material is degradable in vivo, can provide calcium and silicon ions for bone tissues, has higher bioactivity, has obvious advantages in the aspects of promoting collagen synthesis, cell proliferation and differentiation and the like, and degradation products have no obvious influence on the surrounding environment.

As a bone repair material, it is required to satisfy technical characteristics in various aspects, not only to have suitable mechanical strength and plasticity, good biocompatibility, good biodegradability; it is also desirable to have a porous structure (certain pore size and porosity) and good cell adhesion capability. The porous structure is not only beneficial to nutrient transportation and discharge of metabolic waste, but also provides space for cell migration, blood vessel growth and bone growth; and the good cell adhesion capability can maintain the normal growth, proliferation and differentiation of cells. The contradiction that fish and bear paw can not be obtained exists between the different technical characteristics.

On the basis of the prior art, the invention provides a degradable polymer-calcium carbonate composite bone repair material with good cell adhesion capability and high porosity and a preparation method thereof.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a degradable polymer-calcium carbonate composite bone repair material having good cell adhesion ability and high porosity, and a preparation method thereof.

In order to achieve the purpose, on one hand, the invention adopts the following technical scheme: a modified spherical calcium carbonate composite material is prepared by the following method:

(1) adding 0.1-0.3M CaCl into polyacrylic acid solution₂Adjusting the pH value of the solution to 7-9; adding 0.1-0.3M Na₂CO₃Stirring and reacting for 2-6h, filtering, washing and drying to obtain spherical calcium carbonate;

(2) dissolving disodium hydrogen phosphate dodecahydrate in water, adjusting pH to 9-11, and adding spherical calcium carbonate to form a suspension;

(3) carrying out hydrothermal reaction on the suspension, and cooling to room temperature after the reaction is finished;

(4) adding nonapolyarginine into the suspension, and adjusting the pH to 9-11; stirring the suspension for reaction for 2-10 h; and carrying out suction filtration, washing and drying to obtain the modified spherical calcium carbonate composite material.

The composite material of the present invention, wherein polyacrylic acid has a weight average molecular weight M_w5-15 kilodaltons; the concentration of the polyacrylic acid solution is 2-5 g/L.

The composite material of the invention is prepared by mixing polyacrylic acid solution and CaCl₂The volume ratio of the solution is (1.5-3): 1.

the composite material according to the invention, wherein the theoretical Ca/P atomic number ratio of spherical calcium carbonate to disodium hydrogen phosphate dodecahydrate is 1: (0.8-1.6).

The composite material provided by the invention has the hydrothermal reaction temperature of 140-180 ℃; the hydrothermal reaction time is 0.5-4 h.

The composite material provided by the invention is characterized in that the weight ratio of nonapolyarginine to spherical calcium carbonate is (0.1-0.2): 1.

further, the invention provides a degradable polymer-calcium carbonate composite bone repair material with good cell adhesion capability and high porosity, which is prepared by the following method:

(1) mixing polylactic acid and a mixture of polylactic acid and polylactic acid in a volume ratio of 1:1, preparing a solution from chloroform/acetone, adding the modified spherical calcium carbonate composite material and ammonium bicarbonate, uniformly mixing, and standing for 4-8 days at 0-10 ℃; then volatilizing the solvent at room temperature, and naturally drying and forming to obtain a material matrix;

(2) soaking the material matrix in water bath at 40-80 deg.c to eliminate ammonium bicarbonate and vacuum stoving to obtain the degradable polymer-calcium carbonate composite material.

The composite bone repair material according to the present invention, wherein the polylactic acid has a weight average molecular weight M_w40-60 kilodaltons; the concentration of the polylactic acid solution is 0.2-0.8 wt%.

The composite bone repair material provided by the invention is characterized in that the weight ratio of the modified spherical calcium carbonate composite material to the ammonium bicarbonate to the polylactic acid is (1-3): (6-8): 1.

in still another aspect, the present invention also provides a method for preparing the degradable polymer-calcium carbonate composite bone repair material according to the present invention, comprising:

(1) adding 0.1-0.3M CaCl into polyacrylic acid solution₂Adjusting the pH value of the solution to 7-9; adding 0.1-0.3M Na₂CO₃Stirring and reacting for 2-6h, filtering, washing and drying to obtain spherical calcium carbonate;

(2) dissolving disodium hydrogen phosphate dodecahydrate in water, adjusting pH to 9-11, and adding spherical calcium carbonate to form a suspension;

(3) carrying out hydrothermal reaction on the suspension, and cooling to room temperature after the reaction is finished;

(4) adding nonapolyarginine into the suspension, and adjusting the pH to 9-11; stirring the suspension for reaction for 2-10 h; and carrying out suction filtration, washing and drying to obtain the modified spherical calcium carbonate composite material.

(5) Mixing polylactic acid and a mixture of polylactic acid and polylactic acid in a volume ratio of 1:1, preparing a solution from chloroform/acetone, adding the modified spherical calcium carbonate composite material and ammonium bicarbonate, uniformly mixing, and standing for 4-8 days at a temperature of between 0 and 10 ℃ below zero; then volatilizing the solvent at room temperature, and naturally drying and forming to obtain a material matrix;

(6) soaking the material matrix in water bath at 40-80 deg.c to eliminate ammonium bicarbonate and vacuum stoving to obtain the degradable polymer-calcium carbonate composite material.

Compared with the prior art, the degradable polymer-calcium carbonate composite material has higher porosity and good cell adhesion capacity.

Detailed Description

The following examples are merely illustrative of embodiments of the present invention and do not limit the scope of the invention.

Example 1

Weighing 3g of weight average molecular weight M_wPolyacrylic acid PAA of 10 kilodaltons was formulated as a 3g/L PAA solution. To 1L of PAA solution was added 500mL of 0.2M CaCl₂The solution was stirred to mix well and the pH was adjusted to 8. Then 500mL of 0.2M Na was added rapidly₂CO₃. The reaction was stirred at 300rpm for 4 h. After the reaction is finished, performing suction filtration, washing the filter cake for 3 times by using absolute ethyl alcohol and deionized water respectively, and then drying in vacuum to obtain spherical calcium carbonate; and (5) standby.

13.0g of disodium hydrogen phosphate dodecahydrate was weighed out and dissolved in 1L of deionized water, and the pH was adjusted to 10. Then 3g of spherical calcium carbonate was added and mixed well with stirring to form a suspension. Transferring the suspension into a hydrothermal reaction kettle, and carrying out hydrothermal reaction at 160 ℃ for 2 h. After the reaction is finished, the hydrothermal reaction kettle is cooled to room temperature along with the furnace. Transferring the suspension to an open container, adding 0.45g nonapolyarginine, and adjusting the pH to 10; the suspension was stirred at 800rpm for 6 h. And after the reaction is finished, performing suction filtration, washing the filter cake for 3 times by using absolute ethyl alcohol and deionized water respectively, and then drying in vacuum to obtain the modified spherical calcium carbonate composite material. SEM and XRD tests show that the spherical calcium carbonate has calcite type calcium carbonate as the inner layer and hydroxyapatite as the outer layer.

The weight average molecular weight M_wPreparing a 1 wt% PLLA chloroform solution from 50 kilodalton polylactic acid PLLA; according to the volume ratio of 1:1, adding acetone; the mixture was mixed by sonication. To the above solutions were added 2 times and 7 times the weight of PLLA based modified spherical calcium carbonate composite and ammonium bicarbonate, respectively, mixed well by sonication, and placed in a refrigerator at-5 ℃ for 6 days. And taking out the solution, volatilizing the solvent at room temperature, and naturally drying and forming to obtain the material matrix. Soaking the material matrix in water bath at 60 deg.c to eliminate ammonium bicarbonate and vacuum stoving to obtain the degradable polymer-calcium carbonate composite material.

Comparative example 1

Weighing 3g of weight average molecular weight M_wPolyacrylic acid PAA of 10 kilodaltons was formulated as a 3g/L PAA solution. To 1L of PAA solution was added 500mL of 0.2M CaCl₂The solution was stirred to mix well and the pH was adjusted to 8. Then 500mL of 0.2M Na was added rapidly₂CO₃. The reaction was stirred at 300rpm for 4 h. After the reaction is finished, performing suction filtration, washing the filter cake for 3 times by using absolute ethyl alcohol and deionized water respectively, and then drying in vacuum to obtain spherical calcium carbonate; and (5) standby.

13.0g of disodium hydrogen phosphate dodecahydrate was weighed out and dissolved in 1L of deionized water, and the pH was adjusted to 10. Then 3g of spherical calcium carbonate was added and mixed well with stirring to form a suspension. Transferring the suspension into a hydrothermal reaction kettle, and carrying out hydrothermal reaction at 160 ℃ for 2 h. After the reaction is finished, the hydrothermal reaction kettle is cooled to room temperature along with the furnace. And (3) performing suction filtration, washing the filter cake for 3 times by using absolute ethyl alcohol and deionized water respectively, and then performing vacuum drying to obtain the modified spherical calcium carbonate composite material. SEM and XRD tests show that the spherical calcium carbonate has calcite type calcium carbonate as the inner layer and hydroxyapatite as the outer layer.

The weight average molecular weight M_wPreparing a 1 wt% PLLA chloroform solution from 50 kilodalton polylactic acid PLLA; according to the volume ratio of 1:1, adding acetone; the mixture was mixed by sonication. To the above solution were added 2 times and 7 times, respectively, the modified spherical calcium carbonate composite and ammonium hydrogen carbonate (particle size 200- & ltmu.m. & gt, 300 μm) based on the weight of PLLA, mixed uniformly by sonication, and placed in a refrigerator at-5 ℃ for 6 days. Taking out the solution, and volatilizing the solvent at room temperatureAnd naturally drying and forming to obtain the material matrix. Soaking the material matrix in water bath at 60 deg.c to eliminate ammonium bicarbonate and vacuum stoving to obtain the degradable polymer-calcium carbonate composite material.

Evaluation of Performance

The porosity was measured at 30 ℃ using a pycnometer method using absolute ethanol as a solvent.

The cell adhesion test was evaluated using the following method: rat bone marrow stromal cell MSC cells were seeded at a cell concentration of 1.0X 10 onto 6-well plates packed with glass slides of the degradable polymer-calcium carbonate composite of example 1 and comparative example 1⁵Cells/well. Then 3mL of medium (DMEM/F12 ═ 1:1) was added to each well and mixed well with the cells. At 37 deg.C, 5% CO₂And incubation in an incubator at 90% relative humidity for 3 days, washing 3 times with PBS, followed by fixation with 3% glutaraldehyde in PBS solution for 8min, washing, and staining with crystal violet (0.1mL, 9%). The area of adhesion was analyzed using an inverted microscope and image analysis software.

The results show that the porosities of example 1 and comparative example 1 are 72.6% and 68.3%, respectively; the adhesion areas of example 1 and comparative example 1 were 47.8% and 39.2%, respectively.

It should be understood that the detailed description of the invention is merely illustrative of the spirit and principles of the invention and is not intended to limit the scope of the invention. Furthermore, it should be understood that various changes, substitutions, deletions, modifications or adjustments may be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents are also within the scope of the invention as defined in the appended claims.