CN110585481A

CN110585481A - Preparation and application of nano magnesium hydroxide regional coating polylactic acid-caprolactone scaffold for repairing spinal injury

Info

Publication number: CN110585481A
Application number: CN201910803446.0A
Authority: CN
Inventors: 关燕清; 杨波; 班晴; 陈吾雅
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2019-08-28
Filing date: 2019-08-28
Publication date: 2019-12-20
Anticipated expiration: 2039-08-28
Also published as: CN110585481B

Abstract

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.

Description

Preparation and application of nano magnesium hydroxide regional coating polylactic acid-caprolactone scaffold for repairing 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.

EXAMPLE 1 Synthesis and characterization of Nano-sized magnesium hydroxide

First, experiment method

Preparing nano magnesium hydroxide by a chemical precipitation method, constructing a nano magnesium hydroxide material by the chemical precipitation method, coating the nano magnesium hydroxide material on one surface of a PCL-PLA support material, and using the coated surface as the inner surface of the support to be curled and arranged in the support to construct a tubular Mg-PCL-PLA support material. Freezing and storing, and carrying out subsequent experiments, wherein the flow chart is shown in figure 1.

The specific operation method comprises the following steps:

(1) with MgCl₂·6H₂O is prepared into 0.5mol/L solution;

(2) 20ml of 0.5mol/L MgCl₂Mixing the solution, 1-1.5 g of PEG2000 and 20ml of 85% ethanol to obtain a mixture;

(3) adding 26% ammonia water 15ml and 95% ethanol 10ml into the mixture, respectively, and magnetically stirring at 60 deg.C for 1.5 h;

(5) and (4) centrifugally washing the fully stirred liquid (repeating for 3 times), carrying out vacuum drying for 24 hours after centrifugally washing, and collecting the obtained powder for later use.

The transmission electron microscope is used for detecting the nano magnesium hydroxide, and the specific operation method comprises the following steps:

fixing the nano magnesium hydroxide sample on a sample table, placing the sample table on a transmission electron microscope sample chamber, vacuumizing the sample chamber, and observing.

Second, experimental results

The result of the transmission electron microscope experiment of the nano magnesium hydroxide particles is shown in fig. 2, the nano magnesium hydroxide particles prepared by the method have spherical structures, and under the transmission electron microscope, the nano magnesium hydroxide particles are dispersed and have uniform texture, and the average particle size is about 120 nm. The particles have low electron density, are translucent, and have a small number of irregular spherical shapes.

The result of the SEAD detection experiment of the nano magnesium hydroxide particles is shown in fig. 3, and the diffraction pattern of the electron beam after passing through the particle crystal is regular hexagon. The result shows that the crystal prepared by the method is a single crystal, and the crystal form is a regular hexahedron.

The results of the nano magnesium hydroxide particle size analysis experiments are shown in fig. 3, and the average particle size of the nano magnesium hydroxide particles is mainly concentrated between 100nm and 150nm, which is consistent with the TEM results.

Example 2 modification and characterization of PCL-PLA Material by Nano magnesium hydroxide

First, experiment method

(1) Modification of PCL-PLA material by nano magnesium hydroxide

Preparation of PCL-PLA base scaffold: the stent is cut into a square of 1cm × 1cm for use.

Regional coating of nano-magnesium hydroxide, taking a small amount of nano-magnesium hydroxide prepared in example 1; coating the viscous nano magnesium hydroxide precipitate on a square PCL-PLA bracket; preparing the square stent coated with the nano magnesium hydroxide and the blank stent not coated with the nano magnesium hydroxide into tubular structures by a physical method, so that the Mg-PCL-PLA tubular stent contains the nano magnesium hydroxide inside but does not contain magnesium material outside, and the coating weight of the nano magnesium hydroxide is about 0.7-0.8 g/cm²。

(1) Infrared spectroscopy

Drying each sample and KBr in a dryer, mixing 1-2 mg of the sample with 200mg of pure KBr, uniformly grinding, and grinding the mixture to a particle size of less than 2 μm so as to avoid the influence of scattered light. Placing the mixture in a mold, pressing the mixture into a transparent sheet on an oil press under the pressure of 5-10MPa, and placing the transparent sheet on the machine; and simultaneously, drying the blank bracket and the modified bracket, and performing on-machine determination.

(2) White light interference

After the scaffold was prepared, its surface morphology was examined and analyzed by scanning white light interferometer (BMT EXPERT, German).

(3) Scanning electron microscope

And naturally air-drying the blank bracket and the modified bracket, adhering and fixing the air-dried bracket sample on a sample table, spraying gold, placing the sample in a sample chamber of a scanning electron microscope, vacuumizing the sample chamber, and observing the sample by the scanning electron microscope.

(4) Analysis of Nano magnesium hydroxide Loading

The loading amount of the nano-magnesium hydroxide in the stent is detected by using an acid-base titration method. Placing the Mg-PCL-PLA bracket to be detected and a proper amount of distilled water (immersing the bracket) into a beaker, heating at high temperature, and stirring to completely dissolve the bracket. The pH of the solution to be tested is adjusted to be approximately equal to 10 by using standard HCl and NAOH solution. And adding 1-2 drops of a standard chrome black T solution as an indicator (when preparing the chrome black T standard solution, fully mixing the chrome black T and sodium chloride in a ratio of 1:100, and fully grinding), wherein the solution to be detected is purple red. And then titrating the solution to be detected by using 0.02mol/L ethylenediaminetetraacetic acid disodium solution, stopping titrating when the solution is changed from purple red to blue and the color is not faded within a certain time, and recording the consumption of the ethylenediaminetetraacetic acid disodium solution. And calculating the loading amount of the nano magnesium hydroxide in the unit bracket according to the consumption amount of the disodium ethylene diamine tetraacetate.

(5) Mechanical testing

The tensile test and the compression test are respectively carried out on the bracket by adopting the methods of national quality inspection standards GB/T18258-2000 and GBT 531.1-2008.

(6) Thermogravimetric analysis of Mg-PCL-PLA scaffolds

The thermal stability of the Mg-PCL-PLA scaffold was examined and analyzed by a thermogravimetric analyzer (TGA, TG209F1, NETZSCSCH, Germany). The detection temperature range is 25-800 ℃.

Second, experimental results

(1) Infrared spectroscopy

In order to further understand the result of the stent modification, the infrared spectroscopy experiment is adopted to characterize the stent. The infrared spectrograms of the Mg-PCL-PLA novel bionic bracket and the blank PCL-PLA bracket are shown in figure 5, and the PCL-PLA bracket modified by nano magnesium hydroxide is 3679.19cm^-1、2856.18cm^-1The blank PCL-PLA scaffold showed two distinct absorption peaks. These two absorption peaks with Mg (OH)₂The characteristic peaks of (A) are very similar. Therefore, the nanometer magnesium hydroxide is considered to be successfully modified on a PCL-PLA bracket, and the Mg-PCL-PLA novel bionic bone tissue engineering bracket is successfully constructed.

(2) Thermogravimetric analysis

The result chart of the thermogravimetric analysis experiment of the Mg-PCL-PLA bionic scaffold and the blank PCL-PLA is shown in figure 6, and the thermogravimetric analysis results of the Mg-PCL-PLA novel bionic bone tissue engineering scaffold and the blank scaffold are not greatly different. Therefore, the stability of the Mg-PCL-PLA bionic bone tissue engineering scaffold is not changed in the modification process. The Mg-PCL-PLA bionic bone tissue engineering scaffold has good thermal stability at 25-38 ℃, the quality and the property of the novel scaffold can not change obviously due to the influence of the temperature in the organism, and the scaffold can be stably implanted into the organism.

(3) White light interference

The influence of nano magnesium hydroxide modification on the surface appearance of the PCL-PLA body bracket is represented by a white light interference experiment. As shown in the data of FIG. 7, the PCL-PLA stent has a complex surface appearance, a significant hole structure and a significant collapse (black). The stent body had a thickness of about 1 μm and the surface had very pronounced protrusions, which resulted in a large overall stent roughness with an average Ra value of about 90nm and an average Ry value of about 1.5. mu.m. Thereafter, the modification of the stent by the nano-magnesium hydroxide causes obvious change of the stent morphology, and we can understand that the modification is as follows: 1. the hollow hole of the bracket is filled; 2. raising the surface of the stent; and 3, the overall thickness of the bracket is increased. From the experimental results, the integral bracket becomes flat, and the surface color is not greatly different (white); the whole thickness is increased to more than 2.5 mu m; while Ra and Ry values were reduced to 10nm and 0.5 μm, respectively (see FIG. 8). Compared with a PCL-PLA blank bracket, the numerical values are statistically significant different, and the modification effect is obvious.

(4) Scanning electron microscope

In order to further determine the change of the fine structure of the modified bracket material, a scanning electron microscope is adopted to characterize the bracket, and the result of the representation accords with the observation data of micron or millimeter level in a white light interference experiment, so that the nano magnesium hydroxide particles can fill the hollow hole of the PCL-PLA bracket, raise the surface of the bracket and increase the thickness of the bracket, and meanwhile, the nano material can enable the surface of the bracket to be smoother.

As shown in FIG. 9, the blank PCL-PLA scaffold has a single-layer thin-sheet structure, smooth surface of the sheet layer, a layer thickness of 200nm, very obvious holes and non-uniform pore size. After the nano magnesium hydroxide is modified, the thickness of the thin layer is obviously increased, and the number of cavities is obviously reduced. And the nano magnesium hydroxide forms granular protrusions on the originally smooth surface of the lamella, and the grain size of the nano magnesium hydroxide is consistent with the previous grain size detection data and is about 100 nm.

These results indicate that the magnesium hydroxide nanotube can indeed modify the PCL-PLA scaffold, and the modification process obviously causes the change of the morphology of the scaffold, makes up for the defects of the scaffold, and may provide a more ideal scaffold microenvironment for the culture and growth of subsequent cells.

(5) Analysis of Nano magnesium hydroxide Loading

In order to determine the total amount of the nano magnesium hydroxide loaded on the stent per unit volume, the measurement is performed by using an acid-base titration experiment method, the result of the acid-base titration experiment is shown in fig. 10, and the loading amount of the nano magnesium hydroxide on each square centimeter of the Mg-PCL-PLA stent is 0.759 g. The quantity of the nano magnesium hydroxide modified and loaded on the bracket reaches the standard of exciting the activity of osteoblasts and promoting the growth and differentiation of the osteoblasts.

(6) Mechanical testing

In order to test the mechanical properties of the stent, tensile and compression experiments were performed on the stent. The tensile test of the scaffold showed that the ability of Mg-PCL-PLA to withstand tensile forces was reduced compared to PCL-PLA (FIG. 11 a). The compression experiment of the bracket shows that in a certain compression strain range, the Mg-PCL-PLA load (compressive stress) is higher than that of PCL-PLA, the hardness of the Mg-PCL-PLA is improved to some extent (figure 11b), and the maximum value of the Mg-PCL-PLA load is 6.50911N. The Mg-PCL-PLA novel bionic bone tissue engineering scaffold has good stress bearing performance and can be stably applied to the repair and treatment of bone tissue damage of a bearing part.

Example 3 biological Effect of Mg-PCL-PLA scaffolds on PC12 and bMSCs

First, cell culture

PC12, bMSCs, was provided by the animal center of the college of medicine of Zhongshan university. The PCL-PLA scaffold prepared in example 2 and the Mg-PCL-PLA scaffold were placed in 24-well cell culture plates, respectively. In addition, a control group without a scaffold was set. After the cells were confluent-cultured in a culture flask to 80%, they were cultured at 1.75X 10⁴Density per well was inoculated onto 24-well plates and cultured for 2, 4, 6 days for subsequent experiments. Other cell culture conditions were: low-sugar DMEM medium containing 10% newborn calf serum and 5.0% CO at 37 DEG C₂。

II, DAPI staining of PC12

1. Experimental methods

After culturing the cells in the 24-well plate, both scaffolds and the blank control were washed 3 times for 5min each with a PBS solution shaker. Subsequently, we fixed the cells with 4% paraformaldehyde for 30 min. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 for 20 min; after PBS again washing, DAPI dye solution incubation for 5min, PBS washing, microscopic examination.

2. Results of the experiment

When the density of the PC12 cells is 90%, respectively inoculating the cells in the culture bottle to a Mg-PCL-PLA novel bionic bone tissue engineering scaffold and a blank PCL-PLA scaffold, simultaneously setting a control group without an inoculated scaffold, after culturing for 6 days, carrying out DAPI staining observation on the cells on the scaffolds by DAPI staining, wherein the results are shown in figure 12, the cells grow on the two scaffolds and the blank control, and compared with the blank control group and the PCL-PLA scaffold, the cells on the Mg-PCL-PLA scaffold grow more densely, the cell nucleus form is better, and the growth promotion effect of the Mg-PCL-PLA scaffold on the cells is more obvious.

From the DAPI staining results, it was found that the PCL-PLA scaffold and the Mg-PCL-PLA scaffold indeed have the effect of promoting the growth of PC12 cells, and among them, the Mg-PCL-PLA has the best effect of promoting the growth of cells.

The cells seeded on the scaffold were counted and the results are shown in FIG. 13, whereAfter being cultured on the Mg-PCL-PLA bracket for 6 days, the number of the PC12 cells per unit bracket area reaches 6.35 multiplied by 10⁴And (4) respectively. Meanwhile, the growth of the bMSCs inoculated on the bracket is also favorable, and the cell number of the bMSCs per unit area reaches 6.95 multiplied by 10 after the bMSCs are cultured on the bracket for 6 days⁴And (4) respectively.

Third, MTT method for detecting cell proliferation

1. Experimental methods

Cells were seeded on the scaffolds and cultured for 48h, with 20. mu.l MTT added 4h in advance. The cells were cultured for 4h, the waste solution was aspirated, the scaffolds were washed twice with PBS, 100. mu.l DMSO was added, the treatment was carried out for 10min, and the absorbance was measured using a microplate reader.

2. Results of the experiment

In order to detect the growth status of the PC12 cells on each scaffold, after culturing the cells for 2 days, the survival rate of the cells was analyzed by MTT assay, and the results are shown in FIG. 14, wherein the increase times of the cells on the PCL-PLA scaffold and the Mg-PCL-PLA scaffold are both greater than that of the blank control group. The cell growth on the Mg-PCL-PLA bracket is nearly 3 times, and the cell cultured on the Mg-PCL-PLA bracket shows higher cell activity and proliferation capability.

Fourthly, immunoblotting (Western Blot) to detect the differentiation of bMSCs to osteoblasts

1. Experimental methods

The bMSCs treated for different periods of time on each scaffold were harvested, added with equal amounts of extraction buffer (125mM Tris. Cl, pH6.8, 2% SDS, 4M urea, 20% glycerol, 5% beta-mercaptoethanol), and placed on crushed ice for cell disruption by ultrasound. After crushing, centrifuging at 12000r/min for 10min at 4 ℃, and taking supernatant, namely protein suspension for low-temperature storage at minus 20 ℃.

Drawing a Coomassie brilliant blue standard curve, then carrying out protein quantification on the protein extraction suspension, determining the protein loading amount by taking a lowest concentration tube as a reference, adding equal volume of 2 xSDS-PAGE gel electrophoresis loading buffer (100mM Tris & Cl, pH6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol and 2% beta-mercaptoethanol), mixing, carrying out denaturation at 100 ℃ for 10min, uniformly blowing, and then taking a sample with a proper volume for carrying out SDS-PAGE discontinuous electrophoretic separation. Performing constant-pressure 80V electrophoresis for 30min by using 3% concentrated gel, performing constant-current 120V electrophoresis for about 70min by using 10% separation gel, taking one gel for membrane transfer, dyeing the other gel for 1h by using Coomassie brilliant blue R-250, observing under visible light after decoloration, and taking pictures.

After different proteins are separated by 10% SDS-PAGE gel electrophoresis, transferring the proteins to a Nitrocellulose (NC) membrane by a constant current of 300mA for 1 h; staining the transferred gel with Coomassie brilliant blue R-250, and observing the film transfer effect; blocking solution (3% bovine serum albumin) was soaked overnight at 4 ℃ and then reacted with primary antibody (1: 500) at room temperature for 2h, and the membrane was washed 3 times with TBS (20mmol/L Tris. Cl, 150mmol/L NaCl, pH8.0) for 10min each. Reacted with alkaline phosphatase-labeled goat anti-rabbit IgG enzyme-labeled antibody (anti-IgG AP conjugate,1:10000) at room temperature for 2h, followed by washing the membrane with TBS 4 times for 5min each, and finally developing with NBT/BCIP until bands appear, i.e., stopping the staining with 200. mu.L, 0.5mol/L EDTA (pH8.0), and 50ml PBS. If no color band is present on the film, the film is negative, and if a color band is present, the film is positive.

2. Results of the experiment

In order to evaluate the effect of the scaffold on promoting the differentiation of the bMSCs into osteoblasts, a Western blotting experiment is carried out, the expression condition of related proteins is shown in figure 15, and the amount of beta-actin protein of BMP-2 expressed by cells on the Mg-PCL-PLA scaffold is obviously more than that of a blank control experiment group and a PCL-PLA scaffold experiment group. This result shows that the bMSCs have begun to differentiate into osteoblasts on the Mg-PCL-PLA scaffold and have a faster differentiation rate.

The related protein expressed by the cells on the scaffold is quantitatively analyzed, the analysis result is shown in figure 16, the BMP-2 protein expression level on the Mg-PCL-PLA scaffold is obviously improved, and the expression amount is about 2 times of that of a control group. Meanwhile, beta-actin protein also shows high expression on the scaffold.

Example 4 Mg-PCL-PLA scaffold repairing Effect on spinal injury in SD rats

First, experiment method

1. SD rat culture and establishment of rat spinal injury model

Four week old SD rats were provided by southern university of medical laboratory animal center. Raising to eight weeks of age under standard SPF-grade raising conditions, and then performing surgery molding.

SD rats were preoperatively fasted for 12h, weighed, and anesthetized with 2% sodium pentobarbital solution 2 ml/kg. Surgery was performed on anesthetized SD rats at a standard sterile operating table. After the tail of the rat was sterilized with 75% alcohol, the rat tail vertebrae were exposed with a surgical instrument and a wound having a size of about 1cm × 2mm × 0.5cm was made, followed by sterilization with 75% alcohol and suturing, and an injury model was established.

2. Stent implantation

Two scaffold materials: PCL-PLA and Mg-PCL-PLA were prepared into a tube shape as described in 2.2.3, and implanted into the injured part of the caudal vertebra of rats, and a blank control group (subjected to surgery without implanting a stent) was set. After 28 days of post-operative culture, subsequent experiments were performed.

3. SD rat X-ray detection and blood analysis

After 28 days of post-operative culture, the rat tails were examined by X-ray (uDR 580I, United Imaging, China). Blood samples were collected 12h after SD rats surgery and sent to Wuhan Severe organisms for routine blood testing.

Second, experimental results

As shown in fig. 17, after the injury model was established, the activity of the tail activity of SD rats was reduced compared to that before the model was created.

After implantation of the stent, the tail of the SD rat 28D was subjected to X-ray examination, and the results are shown in fig. 18. The results show that compared with the tail vertebrae of SD rats in the control group and the PCL-PLA group, the tail vertebrae of SD rats have swollen bone tissues and certain double images around the bones, which are caused by tail inflammation and immune reaction caused by operation or injury, and the results of subsequent blood tests are consistent with the characteristics. In addition, the control group (without stent implantation) showed some deformity of the tail vertebrae of the rats, some bending of the damaged tail vertebrae compared to the normal bone, and the tail suture of the rats of the control group had slower healing of the damage and a gap in the X-ray results. After the caudal vertebra of the Mg-PCL-PLA group is recovered by 28D after operation, the caudal vertebra has no difference with the normal caudal vertebra, and does not initiate inflammation or acute immunoreaction at the caudal vertebra, and the non-toxic side effect of the Mg-PCL-PLA is reflected. In conclusion, the Mg-PCL-PLA stent has better healing promoting effect on the injury of the tail spine of the SD rat than the PCL-PLA stent and the stent-free group.

After SD rats are operated for 12h, blood of three groups of rats is collected and subjected to blood routine detection, and the result shows that (shown in figure 19) after the SD rats and the PCL-PLA rats are operated for 12h, the contents of white blood cells, lymphocytes and neutrophils in the blood are higher than those in the Mg-PCL-PLA group. This indicates that the inflammation and subsequent immunoreaction of the damaged caudal vertebra of the first two groups of rats appear within 12h after operation, which are not favorable for the rapid repair of the damaged caudal vertebra. And the level of each cell in the blood of the Mg-PCL-PLA group is stable, which shows that the group of rat caudal vertebra injuries do not cause the phenomena of inflammation and the like, and proves that the Mg-PCL-PLA bionic scaffold has the characteristic of no toxic or side effect.

In conclusion, the Mg-PCL-PLA novel bionic bone tissue engineering scaffold shows a good effect of repairing bone tissue damage, and provides a new treatment method without toxic or side effect for spinal injury.

Claims

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;

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.