阅读说明：本技术 CaCO3/MgO纳米复合物及其在骨修复中的应用 (CaCO3MgO nano composite and application thereof in bone repair ) 是由 阮静 范先群 杨大鹏 黄雅琢 于 2020-04-17 设计创作，主要内容包括：一种CaCO<Sub>3</Sub>/MgO纳米复合物,本质为MgO纳米颗粒掺杂的蛋壳颗粒,由蛋壳和乙酸镁经浸渍、煅烧制得。该材料具有天然、绿色和环保的特点,不仅可充分利用含有大量CaCO<Sub>3</Sub>的废弃蛋壳,还显著降低了镁离子的生物毒性,有利于发挥镁离子对骨再生的促进作用。以本发明的纳米复合物为原料制造的支架等医疗器械,可应用于骨修复。(CaCO (calcium carbonate) 3 MgO nanocomposite, essentially MgO nanoparticle-doped eggshell particles, prepared by leaching eggshells and magnesium acetateSoaking and calcining. The material has the characteristics of nature, environmental protection and capability of fully utilizing a large amount of CaCO 3 The waste eggshells also obviously reduce the biotoxicity of magnesium ions and are beneficial to playing the role of promoting the bone regeneration by the magnesium ions. Medical devices such as stents and the like manufactured by taking the nano-composite as a raw material can be applied to bone repair.)

1. A nano-composite characterized by being prepared by mixing magnesium ions and eggshells.

2. The nanocomposite as claimed in claim 1, wherein 0.06g of the magnesium ions is added per gram of the eggshell.

3. Nanocomposite according to claim 1, comprising MgO and CaCO₃MgO and CaCO₃Is a polycrystalline structure.

4. The nanocomposite as claimed in claim 1, which is prepared by calcining for 3 hours at a temperature of 600 ℃.

5. Nanocomposite according to claim 1, characterized in that C, O, Mg and Ca are evenly distributed over the surface of the material.

6. Nanocomposite according to claim 1, characterized in that 1.2g ground eggshell and 50ml of 0.05M magnesium acetate at a concentration are homogeneously mixed; then, washing with water for several times and drying; finally, calcining at 600 ℃ for 3 hours, wherein the calcining heating rate is mainly 2 ℃/min.

7. Use of a nanocomposite according to any one of claims 1 to 6 in the manufacture of a medical device.

8. A medical device, characterized in that it is produced from the nanocomposite according to any one of claims 1 to 6.

9. A scaffold, characterized in that it is produced from the nanocomposite according to any one of claims 1 to 6.

10. A bone meal produced from the nanocomposite according to any one of claims 1 to 6 as a raw material.

Technical Field

The invention relates to a material prepared from natural materials, in particular to CaCO prepared based on eggshells₃the/MgO nano composite has osteoinductive property and is suitable for being made into medical devices such as a bracket and the like.

Background

Bone defects caused by trauma, tumor resection and congenital diseases seriously affect the daily life of patients. The rapid development of tissue engineering brings vitality to the field of bone defect repair, and the scaffold material as an important component part of bone tissue repair becomes a hotspot of research in recent years. Because allogenic bone and autologous bone transplantation have insurmountable defects of immunological rejection, donor deficiency and the like, the scaffold material prepared by taking calcium phosphate/calcium carbonate and some biological macromolecules (such as chitosan, sodium alginate, collagen and the like) as main raw materials is produced at the same time, and is widely concerned due to good biocompatibility and mechanical property. However, the unmatched osteogenesis and degradation rate caused by the scaffold material without osteoinductive capacity still seriously restrict the bone defect repair effect, and bring great test for clinical treatment.

Magnesium has been reported to play a very important role in the osteogenesis process. In addition, magnesium stimulates osteoblast proliferation, differentiation and bone mineralization by regulating active calcium transport and activating phagocytic function. Magnesium deficiency may be associated with bone fragility and/or reduced bone growth. Therefore, the incorporation of Mg in appropriate amounts is beneficial for the regeneration of new bone, and many studies have confirmed the addition of Mg²⁺Can improve the osteogenic inductivity of the scaffold. However, Mg suddenly released from the scaffold²⁺Ions may cause problems of biological toxicity and the like, so that the method is left unused for a long time due to safety problems (Acta biometer.10 (2014) 2834-2842).

Disclosure of Invention

The invention aims to provide a nano-composite which is prepared by soaking and calcining eggshells and magnesium acetate and reduces the biological toxicity of magnesium ions.

Another aspect of the inventionAims at providing a nano composite which is prepared by soaking and calcining eggshell and magnesium acetate and contains CaCO₃And MgO, which has osteoinductive properties, promotes bone regeneration and tissue repair.

It is still another object of the present invention to provide a nanocomposite containing CaCO₃And MgO, so that the mechanical property of the prepared bracket is obviously improved.

It is yet another object of the present invention to provide a medical device employing a composition containing CaCO₃And MgO nano-composite as raw material, has the function of promoting bone regeneration, and is suitable for bone repair scaffolds and the like.

A nanometer composite is prepared from eggshell and magnesium acetate by soaking and calcining.

The other nano composite is prepared by soaking and calcining eggshell and magnesium acetate, and 0.06g of magnesium ions is added into each gram of eggshell.

The other nanometer composite is prepared from eggshell and magnesium acetate by soaking and calcining, and contains MgO and CaCO₃And 0.06g of magnesium ions is added into each gram of eggshell.

Another nanometer compound is prepared by soaking eggshell in magnesium acetate, and calcining (such as heating at 600 deg.C for 3 hr at 2 deg.C/min) to obtain nanometer compound containing MgO and CaCO₃And 0.06g of magnesium ions is added into each gram of eggshell.

Another nanocomposite is prepared by the following preparation method:

first, 1.2g of ground eggshell and 0.05M magnesium acetate (50ml) are mixed homogeneously (for example: stirring for 3 hours at room temperature). Then, the mixture was washed several times with water and dried. Finally, calcining at 600 deg.C (heating rate of 2 deg.C/min) for 3 hr.

The nanocomposite of the present invention contains MgO and CaCO₃The magnesium oxide is in a polycrystalline structure, elements such as C, O, Mg, Ca and the like are uniformly distributed on the surface of the material, the magnesium oxide is positioned on the surface of calcium carbonate, and the characteristic peak of the X-ray diffraction 2 theta of the calcium carbonate is mainly 29.5 degrees and is also positioned at 23.0 degrees, 31.4 degrees, 35.8 degrees, 39.4 degrees, 43.16 degrees, 47.2 degrees, 47.5 degrees, 57.3 degrees, 60.8 degrees, 64.6 degrees and the like. The characteristic peaks of magnesium oxide X-ray diffraction 2 theta are 57.5 degrees and 63.3 degrees.

The nanocomposite of the present invention has an activity of promoting bone regeneration, such as: and the organic/inorganic composite material is formed with any polymer of PLGA, PLA, PEG, cyclodextrin and chitosan, and then prepared into various scaffolds or bone powder for bone repair.

Medical instruments using the CaCO of the present invention₃the/MgO nano composite is prepared by taking the raw material.

A stent made of CaCO₃The preparation method comprises the steps of dissolving a/MgO nano composite and carboxymethyl chitosan (CMC) in water, crosslinking the mixed solution by using carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogel, and freeze-drying. The support has a porous structure, and the pore diameter is 50-80 μm.

A bone meal prepared from CaCO₃the/MgO nano material is compounded with collagen (or growth factor) and used for preparing the artificial bone powder.

The technical scheme of the invention has the following beneficial effects:

the material containing calcium ions and magnesium ions provided by the invention is prepared by adding magnesium ions into eggshells, has the characteristics of nature, greenness and environmental protection, and not only can make full use of the material containing a large amount of CaCO₃The waste eggshells also obviously reduce the biotoxicity of magnesium ions and are beneficial to playing the role of promoting the bone regeneration by the magnesium ions.

The mechanical strength of the stent material prepared from the material containing calcium ions and magnesium ions provided by the invention is obviously improved.

The bone meal material prepared from the material containing calcium ions and magnesium ions provided by the invention has good bone regeneration capacity.

CaCO provided by the invention₃the/MgO nano compound is prepared by dipping and calcining eggshells and magnesium acetate, has the characteristics of natural green environmental protection and effectively improves Mg²⁺Biological activity in osteoinduction, making Mg²⁺The inorganic substance has relative chemical stability, biological activity, biocompatibility and other characteristics and is applied to bone repair.

Drawings

FIG. 1 shows a schematic view of the present invention CaCO₃XRD spectrum result diagram of the/MgO nano composite material;

FIG. 2 is CaCO according to the present invention₃Characterization graph of/MgO nanocomposite; wherein, the image A is SEM image under low magnification, the images B and C are SEM images under high magnification, and the image D is CaCO in the material₃Quantitative EDS spectrum of MgO;

FIG. 3 is CaCO according to the present invention₃A structural representation diagram of the/MgO nano composite material; wherein, fig. A, B is TEM, fig. C is HRTEM, and fig. B is inset SAED image;

FIG. 4 shows CaCO according to the present invention₃A preparation process sample diagram of the/MgO/CMC stent;

FIG. 5 shows CaCO produced by the present invention₃Morphological and physical characterization plots of/MgO/CMC scaffolds; wherein Panel A is CaCO co-cultured with free cells₃SEM image of/MgO/CMC stent, graph B is stress-strain curve of stent, graph C is Young's modulus of stent, graph D is compressive strength; (. p < 0.001)

FIG. 6 is CaCO₃Biocompatibility of a stent made of/MgO nano composite material; wherein, the graph A shows that the cells are in CaCO₃Proliferation on/MgO/CMC for 72 hours; panel B shows cells in CaCO₃Live/dead staining and scanning electron microscopy images after incubation on MgO and CMC scaffolds for 3 d; (. p < 0.05,. p < 0.01,. p < 0.001)

FIG. 7 is CaCO₃Osteoinductive effect of the scaffold made of/MgO nanocomposite; wherein panel A is ALP staining after 14 days of cell culture on scaffolds; panel B is ARS staining after 21 days of cell culture on scaffolds.

FIG. 8 is a tissue staining map of rat skull; wherein panel a is HE staining in response to vascularization; panel B shows staining of Masson in response to type I collagen deposition.

Detailed Description

The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

The test methods used in the following examples of the invention are specifically illustrated below:

1)CaCO₃preparation of/MgO nanocomposite

First 1.2g of ground eggshell and 0.05M magnesium acetate (50ml) were dissolved in a beaker and stirred at room temperature for 3h to achieve homogeneous mixing. The homogeneously mixed solution was washed several times with ultrapure water and dried. Finally, the product was calcined in a muffle furnace at 600 ℃ for 3 hours. The heating rate was 2 ℃/min.

2)CaCO₃Preparation of/MgO/CMC scaffolds

Dissolving 2% (W/V) carboxymethyl chitosan (CMC) in 5% (W/V) CaCO₃To the MgO solution, 0.1m EDC and 0.025m NHS were then added to the solution. The mixture was incubated at room temperature for 30 minutes to allow complete cross-linking. After the mixed solution is modified into hydrogel, the porous CaCO is obtained by freeze drying and ultrasonic cleaning₃a/MgO/CMC stent.

3) Cell experiment method

The obtained human adipose tissue was cut into small pieces, digested with 0.1% collagenase I (Sigma Aldrich, st. louis, MO, USA), and the digested tissue was incubated at 37 ℃ while gently shaking overnight. Finally, the precipitated cells were resuspended in α -MEM (Invitrogen, Carlsbad, CA, USA), added with 10% FBS (Gibco, USA) and 100 units/ml penicillin streptomycin (Invitrogen), and cultured at 37 ℃ under an atmosphere of 5% CO 2. The culture medium was changed every 3 days, and experiments were performed using 3 rd to 5 th generation cells.

At 1 × 10 in 100. mu.l of medium per well⁴Cell density cells were seeded in scaffolds and proliferation of cells was detected using the CCK-8 cell counting kit (Dojindo, Japan) after 0, 24, 48 and 72 hours of culture. Detecting cell viability by live/dead method (Invitrogen), culturing in scaffold for 3 days, observing cell viability and mineralization degree of scaffold by scanning electron microscope, culturing cells and scaffold for 3 days, fixing scaffold with 2.5% glutaraldehyde, dehydrating in gradient ethanol series, and coating with JFC-1200 fine coatingThe machine was sputtered with gold at 30 mA. The sample was observed under a Scanning Electron Microscope (SEM) at 5kv in a high vacuum mode.

4) Detection of osteogenesis-related genes

At cell confluence around 60-70%, the medium was changed and recorded as the onset of osteogenic differentiation. After osteoblast culture for 7 days, relative expression of Bone Sialoprotein (BSP), Osteocalcin (OCN), Osteopontin (OPN) and bone silk (osterix) osteogenesis differentiation related gene mRNA is detected by a real-time qPCR method. All experiments were performed in triplicate and all data were normalized to GAPDH expression. The primer sequences used are shown in Table 1.

TABLE 1

5) Animal model

Cells and a scaffold are co-cultured for 3 days before operation, male SD rats of 8 weeks are selected, intraperitoneal injection of pentobarbital (Nembutal 3.5mg/100mg) is performed for anesthesia, and a sagittal incision of 1.0cm at the center of the scalp is used for exposing the skull. Two defects of critical size were created on both sides of the scalp using trephines (novag, Goldach, Switzerland) of 5mm diameter, and the scaffold/cell complex was then implanted into the defects. Finally, the incision is closed and cleaned. 15 rats were randomly divided into 3 groups: CaCO₃MgO/CMC, blank control. Animals were sacrificed 8 weeks post-surgery and the cranium was harvested and fixed with 4% PFA. After decalcification, paraffin embedding was carried out, and a section was prepared.

6) Dyeing method

Tissue sections were stained with hematoxylin/eosin and Masson trichrome stains, respectively, according to the manufacturing instructions. Tissue sections were visualized by optical microscopy (Olympus BX51)

7) Statistical analysis

All data presented in this study are presented as mean ± standard deviation unless otherwise specifically indicated. Each experiment was repeated at least three times. Statistical analysis was performed on the data using one-way anova. P < 0.05 is statistical significance.