阅读说明：本技术 一种规则六面体碳酸钙材料及其制备方法和应用 (Regular hexahedral calcium carbonate material and preparation method and application thereof ) 是由 石硕 魏伊彤 于 2019-04-16 设计创作，主要内容包括：本发明提供了一种规则六面体碳酸钙材料及其制备方法和应用。在室温条件下通过化学沉淀法制备出长边为3.5±0.2μm,短边2.5±0.2μm的六面体碳酸钙材料。将该材料以一定浓度与骨髓间充质干细胞在正常培养液或成骨诱导液中共培养后,检测骨髓间充质干细胞的存活率；采用茜素红染色矿化实验、碱性磷酸酶活性测试评估规则六面体碳酸钙材料对骨髓间充质干细胞向成骨细胞分化的影响；利用实时荧光定量PCR检测三种成骨相关基因OCN、OPN、RUNX2的表达情况。结果表明,规则六面体碳酸钙材料具有良好的细胞相容性,并能够用于促进骨髓间充质干细胞向成骨细胞分化,明显提高骨髓间充质干细胞的成骨能力,此研究结果为骨组织工程提供数据支持,具有较大应用前景。(The invention provides a regular hexahedral calcium carbonate material and a preparation method and application thereof. The hexahedral calcium carbonate material with the long side of 3.5 +/-0.2 microns and the short side of 2.5 +/-0.2 microns is prepared by a chemical precipitation method at room temperature. Co-culturing the material and bone marrow mesenchymal stem cells in a normal culture solution or an osteogenesis inducing solution according to a certain concentration, and detecting the survival rate of the bone marrow mesenchymal stem cells; evaluating the influence of the regular hexahedron calcium carbonate material on the differentiation of the bone marrow mesenchymal stem cells into osteoblasts by adopting an alizarin red staining mineralization experiment and an alkaline phosphatase activity test; the expression conditions of three osteogenesis related genes, namely OCN, OPN and RUNX2, are detected by using real-time fluorescent quantitative PCR. The result shows that the regular hexahedral calcium carbonate material has good cell compatibility, can be used for promoting the differentiation of the bone marrow mesenchymal stem cells to osteoblasts and obviously improving the osteogenic capacity of the bone marrow mesenchymal stem cells, provides data support for bone tissue engineering, and has a wide application prospect.)

1. A preparation method of regular hexahedron calcium carbonate material is characterized by comprising the following steps: which comprises the following steps:

(1) mixing Na₂HPO₄Dissolving CTAB in water, stirring while dropping CaCl₂Adjusting the pH range of the solution to 9.5 +/-0.5 by using ammonia water in the dropwise adding process;

(2) stirring at room temperature, filtering, and adding anhydrous ethanol with the same volume into the filtrate;

(3) and (4) centrifuging, washing with water, and drying to obtain a white powder sample, namely the regular hexahedral calcium carbonate material.

2. A regular hexahedral calcium carbonate material is characterized in that: the regular hexahedral calcium carbonate material is prepared by the method of claim 1.

3. The regular hexahedral calcium carbonate material according to claim 2, characterized in that: the long side of the regular hexahedral calcium carbonate material is 3.5 +/-0.2 mu m, and the short side of the regular hexahedral calcium carbonate material is 2.5 +/-0.2 mu m.

4. Use of the regular hexahedral calcium carbonate material according to claim 2, in the culture of mesenchymal stem cells.

5. Use according to claim 4, characterized in that: the regular hexahedral calcium carbonate material is used as an additive of a culture solution or an osteogenesis inducing solution of the bone marrow mesenchymal stem cells to promote the differentiation of the bone marrow mesenchymal stem cells into osteoblasts.

6. Use according to claim 5, characterized in that: the culture solution is prepared from alpha-MEM, fetal calf serum and streptomycin mixed solution (100 +/-10): (10. + -.1): (1 plus or minus 0.1) in proportion; and/or the presence of a gas in the gas,

the osteogenesis inducing solution is prepared by adding 100 +/-10 nmol of dexamethasone, 50 +/-5 mu mol of ascorbic acid and 10 +/-1 mmol of beta-sodium glycerophosphate into the culture solution.

7. A preparation for promoting differentiation of mesenchymal stem cells into osteoblasts, comprising: the preparation contains regular hexahedral calcium carbonate according to claim 2.

8. The preparation for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 7, wherein: the regular hexahedral calcium carbonate has a concentration of 1-200. mu.g/ml in the formulation.

Technical Field

The invention belongs to the technical field of biomedicine, relates to a regular hexahedral calcium carbonate material, and a preparation method and application thereof, and particularly relates to application of the regular hexahedral calcium carbonate material in promoting osteogenic differentiation of mesenchymal stem cells.

Background

Stem cells are immature cells with certain differentiation capacity, can realize rapid self-renewal and proliferation, and can be asymmetrically divided into precursor cells or somatic cells under certain external stimulation.

Bone marrow-derived Mesenchymal Stem Cells (MSCs) have a variety of differentiation potentials, are widely available, are easily obtained, have low foreign body rejection, and have recently received extensive attention from researchers. Research shows that the MSC has the capacity of differentiation across germ layers, can be differentiated into a series of tissues such as bones, cartilages, muscles, tendons, fat, nerves and the like, and still has good differentiation capacity even after continuous subculture and cryopreservation. Therefore, the MSC has good application prospect in the fields of cell therapy, tissue repair and the like. MSCs are commonly used as seed cells in bone tissue repair and bone tissue engineering, and can be clinically used for bone tissue repair and cell therapy. Based on these properties, MSCs play an important role in tissue repair and regeneration, and have great application potential in tissue engineering and regenerative medicine.

Calcium carbonate materials are common inorganic non-metallic materials in the biomedical field, and research on calcium carbonate materials mostly focuses on the aspects of the way in which calcium carbonate materials enter cells, distribution in cells, cytotoxicity, genotoxicity, slow release of drugs and the like. Although calcium carbonate materials have been studied in the above-mentioned respects, no studies have been found on the effects on the differentiation function of stem cells.

Disclosure of Invention

The invention aims to provide a regular hexahedral calcium carbonate material which has good biocompatibility and can induce and promote osteogenic differentiation of bone marrow mesenchymal stem cells, and a preparation method and application thereof. The calcium carbonate material is used as a biomedical material, and the research on the influence of the calcium carbonate material on the differentiation process of the bone marrow mesenchymal stem cells can provide data support and reference for the application of the biomedical fields such as tissue engineering and the like.

In order to achieve the above purpose, the solution of the invention is as follows:

a method for preparing regular hexahedral calcium carbonate material, which comprises the following steps:

(1) mixing Na₂HPO₄Dissolving CTAB in water, stirring while dropping CaCl₂Adjusting the pH range of the solution to 9.5 +/-0.5 by using 0.1M ammonia water in the dropwise adding process;

(2) stirring at room temperature, filtering, and adding anhydrous ethanol with the same volume into the filtrate;

(3) and (4) centrifuging, washing with water, and drying to obtain a white powder sample, namely the regular hexahedral calcium carbonate material.

Wherein the long side of the regular hexahedral calcium carbonate material is about 3.5 +/-0.2 μm, and the short side is about 2.5 +/-0.2 μm.

The regular hexahedral calcium carbonate material can be used for culturing mesenchymal stem cells. Specifically, the regular hexahedral calcium carbonate material is used as an additive to a culture solution or an osteogenesis inducing solution of the mesenchymal stem cells to promote differentiation of the mesenchymal stem cells into osteoblasts. The culture medium is prepared from alpha-MEM, fetal calf serum and streptomycin (100 +/-10): (10. + -.1): (1 plus or minus 0.1) in proportion. The osteogenesis inducing liquid is prepared by adding dexamethasone 100nmol, ascorbic acid 50 mu mol and beta-sodium glycerophosphate 10mmol into a culture solution.

Preferably, the preparation method of the regular hexahedral calcium carbonate material comprises the following steps:

(1) 1.25. + -. 0.25mM Na₂HPO₄0.45 +/-0.10 mM CTAB is dissolved in 240mL of distilled water to obtain a first mixed solution; na (Na)₂HPO₄The concentration in the first mixed solution was 5.21. + -. 1mmol/L, and the concentration of CTAB (a Chinese name: cetyltrimethylammonium Bromide; an English name: Hexadecyl trimethyammonium Bromide) in the first mixed solution was 1.88. + -. 0.4 mmol/L;

(2) while stirring the first mixed solution, slowly adding 60mL of CaCl₂The solution is dripped dropwise (CaCl)₂CaCl in solution₂The concentration is 4.99 +/-0.1 mM), and a second mixed solution is obtained after the dropwise addition is finished; adjusting the pH range of the second mixed solution to 9.5 +/-0.5 by using 0.1M ammonia water in the dropwise adding process;

(3) stirring the second mixed solution at room temperature for 24-48h, filtering with a 0.22 μm filter membrane, and filtering to remove impurities to obtain filtrate; adding absolute ethyl alcohol with the same volume as the filtrate into the filtrate to obtain a third mixed solution; and (3) centrifuging the third mixed solution for 10min at 14000rpm by using a centrifuge, washing the third mixed solution for multiple times by using distilled water, and drying the third mixed solution to obtain a white powder sample (the micron-sized regular hexahedral calcium carbonate material).

Wherein the length of the long side of the calcium carbonate crystal in the micron-sized regular hexahedron calcium carbonate material is about 3.5 +/-0.2 mu m, and the length of the short side is about 2.5 +/-0.2 mu m. The calcium carbonate crystal is hexahedron, and the length of the long side is different from that of the short side. Regular means the size rule of the microparticles.

The micron-sized regular hexahedral calcium carbonate material can be used for culturing rat bone marrow mesenchymal stem cells, namely, the micron-sized regular hexahedral calcium carbonate material is used as a culture solution of the rat bone marrow mesenchymal stem cells or an additive of osteogenesis inducing solution.

The culture method of the micron-sized regular hexahedral calcium carbonate material for promoting osteogenic differentiation of the mesenchymal stem cells comprises the following steps: after rat bone marrow mesenchymal stem cells adhere to the wall, a sterilized regular hexahedron calcium carbonate material is added into a culture solution or osteogenic induction liquid, and then the regular hexahedron calcium carbonate material and the rat bone marrow mesenchymal stem cells are co-cultured for 7-14 days, wherein the culture environment is a culture solution microenvironment or osteogenic induction liquid microenvironment. The distribution concentration of the regular hexahedral calcium carbonate material in the culture solution or osteogenesis inducing solution may be 1-200. mu.g/ml, or 50-150. mu.g/ml, or 100. mu.g/ml.

Wherein the length of the side of the micron-sized regular hexahedral calcium carbonate is about 3.5 + -0.2 μm at the long side and about 2.5 + -0.2 μm at the short side

Wherein, rat bone marrow mesenchymal stem cells grow to 70-80% after being attached to the wall, and then are cultured with the regular hexahedron calcium carbonate material together, and are induced to become osteoblasts. Under the condition of culture solution or osteogenesis inducing solution, regular hexahedron calcium carbonate material is used for promoting the differentiation of bone marrow mesenchymal stem cells to osteoblasts.

Wherein the sterilization mode of the materials is ultraviolet sterilization.

Wherein, in the method for promoting the bone marrow mesenchymal stem cells to directionally differentiate into the osteoblasts, the components of the culture solution are alpha-MEM, fetal calf serum and streptomycin mixed solution, and the culture solution is the mixture solution of the alpha-MEM, the fetal calf serum and the streptomycinThe volume ratio is (100 +/-10): (10. + -.1): (1. + -. 0.1). alpha-MEM is available from Gibco under the product name MEM alpha, nucleotides, GlutaMAX^TM(ii) a Fetal Bovine Serum was purchased from ScienCell corporation under the product name of Fetal Bovine Serum; the Penicillin mixed Solution is purchased from ScienCell, and has a product name of Penicilin/Streptomycin Solution (wherein the concentration of the sodium salt of Penicillin is 1x 10)⁷units/L; the streptomycin concentration is 10 g/L; NaCl concentration 8.5 g/L).

The osteogenesis inducing solution is prepared by adding dexamethasone 100 + -10 nmol, ascorbic acid 50 + -5 μmol and beta-sodium glycerophosphate 10 + -1 mmol into culture solution.

A preparation for promoting the differentiation of bone marrow mesenchymal stem cells into osteoblasts contains the micron-sized regular hexahedral calcium carbonate. The concentration of the micron-sized regular hexahedral calcium carbonate in the preparation is 1-200 mug/ml.

Due to the adoption of the scheme, the invention has the beneficial effects that:

the invention provides an application of a regular hexahedral calcium carbonate material in mesenchymal stem cells. The regular hexahedron calcium carbonate materials with different concentrations have no obvious influence on the survival rate of the mesenchymal stem cells. Regular hexahedron calcium carbonate material with certain concentration is added into the culture solution and the osteogenesis inducing solution to promote the mineralization of rat bone marrow mesenchymal stem cells, enhance the activity of alkaline phosphatase (ALP) and up-regulate the expression of osteogenesis related genes OCN, OPN and RUNX 2. The invention provides data support and reference for the application in biomedical fields such as tissue engineering and the like.

In a word, the regular hexahedral calcium carbonate material has good cell compatibility, promotes the differentiation of the bone marrow mesenchymal stem cells to osteoblasts, and obviously improves the osteogenic capacity of the bone marrow mesenchymal stem cells.

Drawings

FIG. 1 is a scanning electron micrograph of a regular hexahedral calcium carbonate according to the present invention;

FIG. 2 is an X-ray powder diffraction spectrum of the regular hexahedral calcium carbonate material of the present invention and a corresponding PDF standard card;

FIG. 3 is a Fourier infrared spectrum of a regular hexahedral calcium carbonate material of the present invention;

FIG. 4 is a graph showing the survival rate of mesenchymal stem cells measured by MTT method at 24h and 72h after adding regular hexahedral calcium carbonate of different concentrations;

FIG. 5 is a photograph showing alizarin red staining after adding regular hexahedral calcium carbonate with different concentrations and co-culturing with rat bone marrow mesenchymal stem cells for 7 days and 14 days under the condition of culture solution;

FIG. 6 is a photograph showing alizarin red staining after adding regular hexahedral calcium carbonate with different concentrations and rat bone marrow mesenchymal stem cells for co-culture for 7 days and 14 days under the condition of osteogenic induction liquid;

FIG. 7 is a photograph showing the quantitative staining of alizarin red after adding regular hexahedron calcium carbonate with different concentrations to co-culture with rat bone marrow mesenchymal stem cells for 14 days under the condition of culture solution;

FIG. 8 is a quantitative photograph of alizarin red staining after adding regular hexahedral calcium carbonate with different concentrations to co-culture with rat bone marrow mesenchymal stem cells for 14 days under the condition of osteogenic induction liquid;

FIG. 9 is a graph showing alkaline phosphatase activity data after adding 50. mu.g/ml regular hexahedral calcium carbonate to co-culture rat bone marrow mesenchymal stem cells for 7 days under a culture medium condition;

FIG. 10 is a graph showing alkaline phosphatase activity data after adding 50. mu.g/ml regular hexahedral calcium carbonate to co-culture rat bone marrow mesenchymal stem cells for 7 days under the condition of an osteogenic induction solution;

FIG. 11 is a graph showing the expression data of the osteogenesis-related gene OCN after co-culturing with rat bone marrow mesenchymal stem cells for 7 days and 10 days by adding 50. mu.g/ml regular hexahedral calcium carbonate under the condition of an osteogenesis inducing solution;

FIG. 12 is a graph showing the expression data of osteogenesis-related gene OPN after co-culturing with rat bone marrow mesenchymal stem cells for 7 days and 10 days by adding 50. mu.g/ml regular hexahedral calcium carbonate under the condition of an osteogenesis inducing solution;

FIG. 13 is a graph showing the expression data of the osteogenesis-related gene RUNX2 after co-culturing with rat bone marrow mesenchymal stem cells for 7 days and 10 days in the presence of an osteogenesis inducing solution in which 50. mu.g/ml regular hexahedral calcium carbonate was added.

Detailed Description

The invention provides a regular hexahedral calcium carbonate material and a preparation method and application thereof. The micron-sized regular hexahedral calcium carbonate material is prepared by a chemical precipitation method at room temperature. Co-culturing the material and bone marrow mesenchymal stem cells in a normal culture solution or an osteogenesis inducing solution according to a certain concentration, and detecting the survival rate of the bone marrow mesenchymal stem cells; evaluating the influence of the regular hexahedron calcium carbonate material on the differentiation of the bone marrow mesenchymal stem cells into osteoblasts by adopting an alizarin red staining mineralization experiment and an alkaline phosphatase activity test; the expression conditions of three osteogenesis related genes, namely OCN, OPN and RUNX2, are detected by using real-time fluorescent quantitative PCR. The result shows that the regular hexahedron calcium carbonate material has good cell compatibility, promotes the differentiation of the bone marrow mesenchymal stem cells to osteoblasts, obviously improves the osteogenic capacity of the bone marrow mesenchymal stem cells, provides data support for bone tissue engineering, and has a great application prospect.

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples.