阅读说明：本技术 一种针对脊柱损伤修复的纳米氢氧化镁区域性涂布聚乳酸-己内酯支架的制备及应用 (Preparation and application of nano magnesium hydroxide regional coating polylactic acid-caprolactone scaffold for repairing spinal injury ) 是由 关燕清 杨波 班晴 陈吾雅 于 2019-08-28 设计创作，主要内容包括：本发明公开了一种针对脊柱损伤修复的纳米氢氧化镁区域性涂布聚乳酸-己内酯支架的制备及应用。本发明构建了一种Mg-PCL-PLA仿生骨组织支架,在PCL-PLA材料管内部加载有纳米氢氧化镁。本发明通过用纳米氢氧化镁修饰聚乳酸-聚己内酯的方法构建一种新型的仿生骨组织工程支架。该支架具有良好的力学性能,并可以对骨细胞、神经细胞的良好促生长作用,提供了一种新的,无毒副作用的细胞修复方法,为脊柱损伤的治疗能够做出贡献,可以推动骨组织工程的进步与发展。(The invention discloses preparation and application of a nano magnesium hydroxide regional coating polylactic acid-caprolactone stent aiming at spinal injury repair. The invention constructs a Mg-PCL-PLA bionic bone tissue scaffold, and nano magnesium hydroxide is loaded in the PCL-PLA material tube. The invention constructs a novel bionic bone tissue engineering scaffold by a method of modifying polylactic acid-polycaprolactone by using nano magnesium hydroxide. The scaffold has good mechanical property, can play a role in promoting growth of bone cells and nerve cells, provides a new cell repair method without toxic and side effects, can contribute to treatment of spinal injury, and can promote progress and development of bone tissue engineering.)

1. The Mg-PCL-PLA bionic bone tissue scaffold is characterized in that nano magnesium hydroxide is loaded in the PCL-PLA material tube.

2. The Mg-PCL-PLA bionic bone tissue scaffold as claimed in claim 1, wherein the loading amount of nano magnesium hydroxide is 0.604-0.942 g/cm²。

3. The Mg-PCL-PLA bionic bone tissue scaffold as claimed in claim 1, wherein the average particle size of the nano magnesium hydroxide is 100-150 nm.

4. A preparation method of a Mg-PCL-PLA bionic bone tissue scaffold is characterized in that nano magnesium hydroxide is coated in the interior of a PCL-PLA material tube.

5. The method according to claim 4, wherein the nano magnesium hydroxide is applied in an amount of 0.6 to 1.2g/cm²。

6. The preparation method according to claim 4, wherein the nano magnesium hydroxide is prepared by a chemical precipitation method.

7. The preparation method according to claim 6, wherein the preparation method of the nano magnesium hydroxide comprises the following steps:

S1.0.2～1.5mol/L MgCl₂mixing the solution, PEG2000 and 80-90% ethanol to obtain a mixed solution, wherein MgCl is contained₂The dosage ratio of the solution, PEG2000 and 85% ethanol is as follows: 15-50 ml: 1-1.5 g: 15-25 ml;

s2, mixing the mixed solution in the S1, 25-28% of ammonia water and 90-95% of ethanol, and stirring, wherein the volume ratio is 30-75 ml: 10-20 ml: 5-15 ml;

s3, centrifuging, collecting the precipitate, washing the precipitate and drying.

8. The Mg-PCL-PLA bionic bone tissue scaffold prepared by the preparation method of any one of claims 4 to 7.

9. Use of the Mg-PCL-PLA biomimetic bone tissue scaffold of any of claims 1 or 8 in the preparation of tissue substitutes, drug screens and/or case model studies to restore bone tissue damage, promote cell growth, promote cell viability, promote cell proliferation and/or promote cell differentiation.

10. The use of claim 9, wherein the bone tissue injury is a spinal injury.

Technical Field

The invention relates to the technical field of bionic bone tissue, in particular to preparation and application of a nano magnesium hydroxide regional coating polylactic acid-caprolactone scaffold aiming at spinal injury repair

Background

Bone tissue is one of the important components of human beings, and various bone tissue diseases cause serious physical and psychological trauma to a large number of patients: fracture is the most common bone tissue injury, and the traditional bone injury treatment method causes great inconvenience to people due to long treatment period, pain in the treatment process and the like; in addition, serious diseases such as bone cancer still threaten the physical and mental health of human beings. Especially, the spine injury causes great physical and psychological trauma to a great number of patients.

Under the large background, the bone tissue engineering has great potential for treating bone-related injuries or diseases, and becomes a leading-edge method for repairing damaged organs, and has certain potential. In general, the study of bone tissue engineering contains three major elements: the research on scaffolds, cells and growth factors is one of the more active fields at present.

The existing biological materials applied to tissue engineering, whether natural or artificially synthesized, cannot meet the requirements of ideal extracellular matrix materials, and have certain defects, such as excessively high or excessively low in-vivo degradation rate, insufficient mechanical strength, poor biocompatibility, inflammation and the like. How to expand the sources of tissue engineering scaffold materials and economically and conveniently meet the requirements of patients becomes a subject to be solved.

Biodegradable high polymer is a research hotspot in the field of material science at present, polylactic acid (PLA) and Polycaprolactone (PCL) are both polyester-based degradable high polymer approved by the United states food and drug administration and can be used for clinic, and the PCL-PLA material has good biodegradation rate and biocompatibility and certain mechanical strength. The biodegradable polymer nano composite material is a novel composite material developed in recent years, is based on biodegradable high molecular polymers, is modified by using nano materials, and can be widely applied to research in multiple fields. The penetration of the nano material further improves various functions of the composite material.

Magnesium is involved in regulating various vital activities and plays an extremely important role in the body, for example: magnesium ions as an endogenous protective factor participate in important cell metabolism and function regulation in brain tissues, and have an obvious protective effect on nerve cells; magnesium can be involved in the prevention and treatment of certain neurological diseases in the body; the magnesium element can enhance the differentiation of the bMSCs to cartilage and has the function of supplementing chondrocytes to bone tissues with inflammation; the activity and differentiation of osteoblasts are promoted by magnesium ions (6-10 mM) with a certain concentration.

The magnesium resource is rich, the price is low, the magnesium is one of the lightest materials, and the density and the human compact bone density (1.75 g/cm)³) Quite equivalent, about 1.47g/cm³Its advantages are high specific rigidity and strength, and ideal machinability and toughness. The elastic modulus of magnesium metal and magnesium alloy is about 4SGPa, is close to that of hard bone tissue, and has proper mechanical property with the bone tissue. In addition, magnesium is one of the few metals that can be degraded in vivo.

Based on the matching of magnesium and human mechanical properties, magnesium alloy was used as an implant material in plastic repair and surgical operations in the last 40 th century. However, the corrosion rate of the magnesium alloy manufactured at that time in the human physiological environment is too high, and a large amount of hydrogen is generated, which has negative effects on cells such as osteogenesis and osteoclasts, and limits the application of the magnesium alloy as a bone implant material, so the magnesium alloy is replaced by the stainless steel which appears later. In the 90 s, with the continuous and intensive research on magnesium alloys, the corrosion resistance and the mechanical property of the alloys are greatly improved, and meanwhile, the corrosion resistance and the antibacterial property of the magnesium alloys are further improved through surface treatment, so that the magnesium alloys and magnesium-containing materials are expected to be widely applied to medical implant materials again.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides preparation and application of a nano magnesium hydroxide regionally coated polylactic acid-caprolactone stent for repairing spinal injuries.

Firstly, preparing nano magnesium hydroxide particles by using a chemical precipitation method, modifying a polylactic acid-caprolactone (PCL-PLA) material by using the nano magnesium hydroxide particle material to construct a novel Mg-PCL-PLA tubular bracket with the PCL-PLA outside and a nano magnesium hydroxide coating layer inside, and further finding the effect of promoting the growth and differentiation of bMSCs and PC12 by using the bracket.

The first purpose of the invention is to provide a Mg-PCL-PLA bionic bone tissue scaffold.

The second purpose of the invention is to provide a preparation method of the Mg-PCL-PLA bionic bone tissue scaffold.

The third purpose of the invention is to provide the Mg-PCL-PLA bionic bone tissue scaffold prepared by the preparation method.

The fourth purpose of the invention is to provide the application of the Mg-PCL-PLA bionic bone tissue bracket in preparing tissue substitutes for restoring bone tissue injury, promoting cell growth, promoting cell viability, promoting cell proliferation and/or promoting cell differentiation, drug screening and/or case model research.

In order to achieve the purpose, the invention is realized by the following technical scheme:

firstly, preparing nano magnesium hydroxide by a chemical precipitation method; coating the polylactic acid-polycaprolactone material with the coating solution for modification; and preparing the tubular Mg-PCL-PLA bionic bone tissue engineering scaffold by a physical method. After the preparation of the stent is finished, the stent is subjected to physicochemical characterization by various means, including thermogravimetric analysis, infrared spectrum detection, particle size analysis, X-ray diffraction (XRD) and the like. The success of preparing the Mg-PCL-PLA bionic bone tissue engineering scaffold is proved.

Thereafter, biological effect experiments were performed, and first, in vitro experiments were performed, in which mesenchymal stem cells (bMSCs) were seeded on the scaffold and growth factors were grafted, and the effect of the scaffold in promoting proliferation and differentiation of stem cells was evaluated. Then, differentiated rat adrenal pheochromocytoma cells (PC12, PC12 cells have the characteristics of nerve cells after being induced to differentiate by nerve growth factors) are inoculated on the magnesium-containing region of the scaffold, and the effect of the scaffold on promoting the growth of the nerve cells is evaluated, so that the result shows that the bionic scaffold has good growth-promoting and differentiating effects on bMSCs cultured on the scaffold, and the PC12 cultured on the scaffold also shows growth benefits.

Therefore, the invention claims a Mg-PCL-PLA bionic bone tissue scaffold, and nano magnesium hydroxide is loaded in the PCL-PLA material tube.

Preferably, the loading amount of the nano magnesium hydroxide is 0.604-0.942 g/cm²。

More preferably, the loading of the nano magnesium hydroxide is 0.759g/cm²。

Preferably, the average particle size of the nano magnesium hydroxide is 100-150 nm.

More preferably, the nano magnesium hydroxide has an average particle size of 120 nm.

Further preferably, the Mg-PCL-PLA bionic bone tissue scaffold is prepared by loading nano magnesium hydroxide into the PCL-PLA material tube, wherein the loading amount is 0.759g/cm²The average grain diameter of the nano magnesium hydroxide is 120 nm.

A preparation method of a Mg-PCL-PLA bionic bone tissue scaffold comprises the step of coating nano magnesium hydroxide in the interior of a PCL-PLA material tube.

Preferably, the coating weight of the nano magnesium hydroxide is 0.6-1.2 g/cm²。

Preferably, the nano magnesium hydroxide is prepared by a chemical precipitation method.

More preferably, the preparation method of the nano magnesium hydroxide comprises the following steps:

S1.0.2～1.5mol/L MgCl₂mixing the solution, PEG2000 and 80-90% ethanol to obtain mixed solution, wherein MgCl is contained₂The dosage ratio of the solution, PEG2000 and 85% ethanol is as follows: 15-50 ml: 1-1.5 g: 15-25 ml;

s2, mixing the mixed solution in the S1, 25-28% of ammonia water and 90-95% of ethanol, and stirring, wherein the volume ratio is 30-75 ml: 10-20 ml: 5-15 ml;

s3, centrifuging, collecting the precipitate, washing the precipitate and drying.

Preferably, in step S1, MgCl₂The concentration was 0.5 mol/L.

Preferably, in step S1, the ethanol concentration in the mixed solution is made to be 85% ethanol.

Preferably, in step S1, MgCl₂The dosage ratio of the solution, PEG2000 and 85% ethanol is as follows: 20 ml: 1-1.5 g: 20 ml.

Preferably, in step S2, the ammonia water is 26% ammonia water.

Preferably, in step S2, the ethanol is 95% ethanol.

Preferably, in step S2, the volume ratio of the mixture in S1, ammonia water and ethanol is 40: 15: 10.

preferably, in step S2, the stirring is magnetic stirring.

Preferably, in step S2, the stirring is performed at 50-80 ℃ for 0.5-3 h.

More preferably, in step S2, the stirring condition is 60 ℃ for 1.5 h.

Preferably, in step S3, the washing the precipitate is washing the precipitate 3 times with water.

Preferably, in step S3, the drying is vacuum drying for not less than 24 hours.

Further, most preferably, the preparation method of the nano magnesium hydroxide comprises the following steps:

S1.0.5mol/L MgCl₂mixing the solution, PEG2000 and 85% ethanol to obtain mixed solution, wherein MgCl is added₂The dosage ratio of the solution, PEG2000 and 85% ethanol is as follows: 20 ml: 1-1.5 g: 20ml of the solution;

s2, mixing the mixed solution in the S1, 26% ammonia water and 95% ethanol, and stirring, wherein the volume ratio is 40: 15: 10;

s3, centrifuging, collecting the precipitate, washing the precipitate for 3 times, and drying in vacuum for 24 hours.

The Mg-PCL-PLA bionic bone tissue scaffold prepared by any one of the preparation methods.

The Mg-PCL-PLA bionic bone tissue scaffold is applied to preparation of tissue substitutes for restoring bone tissue injury, promoting cell growth, promoting cell viability, promoting cell proliferation and/or promoting cell differentiation, drug screening and/or case model research.

Preferably, the bone tissue injury is a spinal injury.

Preferably, the cells include but are not limited to murine adrenal pheochromocytoma cells PC12, bone marrow mesenchymal stem cells bMSCs and other various cells and cell lines.

Preferably, the cell differentiation is into bMSCs to osteoblast differentiation.

Compared with the prior art, the invention has the following beneficial effects:

the invention constructs a novel bionic bone tissue engineering scaffold by a method of modifying polylactic acid-polycaprolactone by using nano magnesium hydroxide. The scaffold has good mechanical property, can promote growth of bone cells and nerve cells, provides a new cell repair method without toxic and side effects, provides a new treatment method without toxic and side effects for spinal injuries, and can promote progress and development of bone tissue engineering.

Drawings

FIG. 1 is a schematic diagram of Mg-PCL-PLA scaffold preparation and subsequent study.

FIG. 2 is a Transmission Electron Microscopy (TEM) experiment of magnesium oxide nanotubes.

FIG. 3 is a selected area electron diffraction pattern of magnesium oxide nanoparticles.

FIG. 4 is a particle size analysis of magnesium hydroxide nanoparticles.

FIG. 5 is an infrared spectrum of the scaffold.

FIG. 6 is a thermogravimetric analysis of the scaffold.

FIG. 7 is a 3-dimensional image of a scaffold constructed by a white light interference experiment.

Fig. 8 is a stent surface roughness calculation.

FIG. 9 is a scanning electron microscope examination of the scaffolds.

FIG. 10 is the loading of nano-magnesium per unit volume of scaffold.

Fig. 11 shows the tension and compression test of the stent.

FIG. 12 is a graph showing the results of DAPI staining 6 days after the PC12 inoculation of the scaffolds.

FIG. 13 shows the results of cell counts of PC12 and bMSCs on the scaffolds after 6 days of culture.

FIG. 14 shows the growth rate of PC12 after 6 days of culture.

FIG. 15 shows the expression of BMP-2, β -actin protein detected by immunoblotting.

FIG. 16 shows the expression level of BMP-2, β -actin protein on the scaffold.

FIG. 17 shows the establishment of a spinal injury model in SD rats.

FIG. 18 shows the X-ray detection of the tail of SD rat.

FIG. 19 shows the routine detection of SD rat blood.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

1. Cell line

Rat bone marrow mesenchymal stem cells (bMSCs) and rat adrenal pheochromocytoma cells (PC12) were provided by the animal center of medical college of Zhongshan university and subcultured in this laboratory.

2. Primary reagent

MgCl₂·6H₂O; PEG 2000; absolute ethanol (analytically pure); ultrapure water; chrome black T; disodium ethylene diamine tetraacetate; NaCl; NaOH; concentrated hydrochloric acid; nitrogen (N)₂) (ii) a PBS solution; 75% alcohol; ammonia water; polylactic acid polycaprolactone scaffold material (PCL-PLA) was purchased from Jinan Dai handle Bio-technology Ltd; type I collagen fiber (Col I), pancreatin and low-sugar DMEM culture medium are all products of GIBCOBRL company; newborn calf serum is purchased from Hangzhou ilex chinensis bioengineering materials, Inc.; the 24-well polystyrene tissue culture substrate is a product of Corning corporation, usa.

3. Instrument for measuring the position of a moving object

Scanning electron microscope for field emission from LEO of Germany LEO 1530VP, Nikon microscope, optical microscope of Olympus of JapanMicroscopy, Sigma32184 high speed refrigerated centrifuge, Thermo CO₂An incubator, 78-1 magnetic stirrers of medical instrument factories of Jintan city, Jiangsu province, HV-85 autoclave, a sterile operating platform, a constant temperature water bath kettle of Guangzhou Keqiao experiment technology equipment Limited company and the like.

4. Statistical analysis

In the experiment, a ss19.0 statistical software is adopted for variance analysis, analysis functions are LSD and Duncan, and P <0.05 shows that the difference is obvious.