CN112933295A

CN112933295A - Chitosan composite slow-release stent for bone tissue engineering and preparation method thereof

Info

Publication number: CN112933295A
Application number: CN202110179886.0A
Authority: CN
Inventors: 蔡海波; 王进; 何武博; 谭文松
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2021-06-11

Abstract

The invention discloses a chitosan composite slow-release stent for bone tissue engineering and a preparation method thereof, wherein the chitosan composite slow-release stent consists of chitosan, sodium carboxymethylcellulose, montmorillonite and EGCG-encapsulated chitosan microspheres; wherein the mass ratio of the chitosan to the sodium carboxymethylcellulose to the montmorillonite to the EGCG-encapsulated chitosan microspheres is 5-50:1-10:1-10: 1-10. The chitosan composite slow-release scaffold prepared by the method has an obvious three-dimensional structure and good biodegradability and biocompatibility, and after qualitative and quantitative tests, the chitosan composite slow-release scaffold is proved to be capable of effectively promoting the proliferation and osteogenic differentiation of mesenchymal stem cells, and can be applied to the field of biomedical materials as bone tissue engineering materials.

Description

Chitosan composite slow-release stent for bone tissue engineering and preparation method thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a chitosan composite slow-release stent for bone tissue engineering and a preparation method thereof.

Background

Bone defects caused by tumors, wounds and the like are one of common injuries, and the treatment effect is difficult to achieve by conventional medicaments. The bionic scaffold, the osteogenesis inducing molecules and the osteoblasts are combined together to obtain a bone tissue engineering technology for regenerating bone tissues after main bone defects or other pathological changes, and the bone tissue engineering technology becomes a promising treatment means. Among them, mesenchymal stem cells have self-renewal, multi-lineage differentiation ability, low immunogenicity, immunoregulatory properties and homing ability, and hardly have carcinogenic risk and ethical problems, and are considered as ideal seed cells.

In terms of scaffold materials, although natural and synthetic scaffold materials are used to manufacture bone tissue engineering scaffolds, natural scaffold materials, such as gelatin, collagen, chitosan, silk fibroin, etc., are preferred due to their better biocompatibility and biodegradability. Among them, chitosan has good biocompatibility, biodegradability, antioxidant activity and other biochemical characteristics, and can be used for preparing various forms of tissue engineering materials, so that the chitosan has wide application in tissue engineering.

During the research and practice of the prior art, the inventor of the present invention found that when chitosan is used as a tissue engineering scaffold material alone, there is a defect of poor mechanical strength, and the material is easily broken when it is in a humid environment. Therefore, the chitosan is subjected to modification design, has high mechanical property, and becomes a scaffold material meeting the requirements of bone tissue engineering scaffolds, and becomes a key of bioengineering requirements.

The (2R,3R) -5, 7-dihydroxy-2- (3,4, 5-trihydroxyphenyl) chroman-3-yl 3,4, 5-trihydroxybenzoate (EGCG) is catechin monomer separated from tea leaves, and is the main component of green tea polyphenol. The catechin has the highest EGCG content, and is the strongest chemical defense and anticancer substance in green tea catechin. Research shows that EGCG has the effect of promoting the proliferation of mesenchymal stem cells, and can simultaneously improve the activity of Alkaline phosphatase (ALP) and stimulate the up-regulation of osteogenic genes so as to promote osteogenic differentiation. Therefore, EGCG can be widely applied as an osteogenesis inducer in bone tissue engineering. However, the direct addition of EGCG to a culture medium or a physiological environment for use easily causes the problems of low bioavailability and instability in neutral and alkaline environments of EGCG, reduces the effect thereof, and needs to construct a system with a slow release effect to solve the problems. On the other hand, the commonly used method for constructing the drug sustained-release system is to directly mix the drug into the stent material or the prepared stent, the long-time sustained-release effect of the method is not ideal, and the time requirement from the attaching proliferation of the mesenchymal stem cells to the completion of the differentiation cycle into osteogenesis cannot be well met, so the method for constructing the stent with the long-time drug sustained-release function becomes the key requirement for meeting the bone tissue engineering.

Disclosure of Invention

The invention provides a chitosan composite slow-release bracket for bone tissue engineering and a preparation method thereof, which can solve the problems of poor stability and slow-release effect of the bone tissue engineering bracket in neutral and alkaline environments in the prior art.

The invention provides a chitosan composite slow-release stent for bone tissue engineering, which comprises the components of chitosan, sodium carboxymethyl cellulose, montmorillonite and EGCG-encapsulated chitosan microspheres; wherein the EGCG is (2R,3R) -5, 7-dihydroxy-2- (3,4, 5-trihydroxyphenyl) chroman-3-yl 3,4, 5-trihydroxybenzoate; the mass ratio of the chitosan to the sodium carboxymethylcellulose to the montmorillonite to the EGCG-encapsulated chitosan microspheres is 5-50:1-10:1-10: 1-10.

The invention also provides a preparation method of the chitosan composite slow-release bracket for bone tissue engineering, which comprises the following steps: s1) preparing EGCG-encapsulated chitosan microspheres; s2) adding the chitosan, the montmorillonite, the sodium carboxymethylcellulose and the EGCG-encapsulated chitosan microspheres into purified water according to the mass ratio of 5-50:1-10:1-10:1-10, mixing, and then adding glacial acetic acid to obtain a chitosan composite slow-release stent mixed solution; s3) freeze-drying the chitosan composite slow-release stent mixed solution to obtain a freeze-dried product of the chitosan composite slow-release stent; s4) placing the freeze-dried product of the chitosan composite sustained-release stent into a sodium hydroxide solution with the concentration of 30-50 mg/ml to soak for 2-5 hours, and then washing with a phosphate buffer solution to obtain the chitosan composite sustained-release stent.

Further, the step S1) includes the following steps: s11) adding EGCG into a chitosan solution with the mass concentration of 5-50 mg/mL to ensure that the mass concentration of the EGCG is 0.10-0.24 mg/mL, and uniformly mixing to obtain EGCG-chitosan mixed solution; s12) adding liquid paraffin and sorbitan oleate into a round-bottom flask, heating to 40-55 ℃, stirring and mixing, adding EGCG-chitosan mixed solution in the stirring process to ensure that the volume percentage of the liquid paraffin is 70-80%, the volume percentage of the sorbitan oleate is 1-5%, the volume percentage of the EGCG-chitosan mixed solution is 15-25%, and stirring for 1-2 hours to obtain reaction liquid; s13), adding a glutaraldehyde solution with the mass concentration of 25% into the reaction liquid in the step S12), wherein the volume ratio of the liquid paraffin, the sorbitan oleate, the EGCG-chitosan mixed liquid and the glutaraldehyde with the concentration of 25% is 70-80:1-5:15-25: 1-2; fully mixing and crosslinking for 30 minutes to obtain EGCG-chitosan microsphere mixed liquor; s14) placing the EGCG-chitosan microsphere mixed solution into a centrifuge, centrifuging for 3-10 minutes at the rotating speed of 1000-2000 rmp, and then removing supernatant to obtain precipitate; sequentially washing the precipitate with petroleum ether, ethanol and ultrapure water to obtain EGCG-chitosan microspheres; s15) freezing the EGCG-chitosan microspheres at-70 to-85 ℃ for 15 to 36 hours, taking out the EGCG-chitosan microspheres, putting the EGCG-chitosan microspheres into a freeze dryer, and freeze-drying the EGCG-chitosan microspheres at-40 to-50 ℃ for 45 to 50 hours to obtain the freeze-dried EGCG-encapsulated chitosan microspheres.

Further, in the chitosan composite slow-release stent mixed solution in the step S2), the components include 5mg/mL to 50mg/mL of chitosan by mass concentration; 1 mg/mL-10 mg/mL of the sodium carboxymethylcellulose; 1 mg/mL-10 mg/mL of the montmorillonite and 1 mg/mL-10 mg/mL of the EGCG-encapsulated chitosan microsphere.

Further, in the step S2), the volume percentage of the glacial acetic acid is 0.5% to 1.5%.

Further, the step S3) includes adding the obtained chitosan composite slow-release scaffold mixed solution into a template, freezing the template at-15 to-25 ℃ for 15 to 36 hours, then placing the template in a freeze dryer, and freeze-drying at-45 to-55 ℃ for 45 to 50 hours to obtain the chitosan composite slow-release scaffold freeze-dried product.

The invention has the beneficial effects that: the chitosan composite slow-release scaffold for bone tissue engineering provided by the invention has the advantages that EGCG is encapsulated in chitosan, so that the chitosan composite slow-release scaffold which has a good slow-release effect and has the effect of promoting differentiation of human mesenchymal stem cells to osteoblasts is formed. The preparation method of the chitosan composite slow-release bracket for bone tissue engineering has the advantages of easily obtained materials, simple and convenient operation, low preparation cost and good repeatability. The chitosan composite slow-release stent prepared by the preparation method has an obvious three-dimensional structure and good microscopic appearance. The composite sustained-release stent has good biodegradability and biocompatibility, and after the tests of biodegradation rate, EGCG release rate, cell growth curve, qualitative and quantitative tests and the like, the composite sustained-release stent can effectively promote the proliferation and osteogenic differentiation of mesenchymal stem cells, and can be applied to the field of biomedical materials as bone tissue engineering materials.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a flow chart of the preparation of chitosan composite sustained-release scaffolds provided in examples 1 and 2 of the present invention;

fig. 2 is a flowchart of the preparation step S1) of the chitosan composite sustained-release stent provided in examples 1 and 2 of the present invention;

FIG. 3 is a scanning electron microscope image of the chitosan composite sustained-release stent provided in example 1 of the present invention;

FIG. 4 is an enlarged view of the position of the box in FIG. 3 in accordance with the present invention;

FIG. 5 is a scanning electron microscope image of the chitosan composite sustained-release stent provided in example 2 of the present invention;

FIG. 6 is an enlarged view of the position of the box in FIG. 5 in accordance with the present invention;

FIG. 7 is a scanning electron microscope image of a composite sustained-release stent prepared by a conventional comparative example;

FIG. 8 is a graph comparing biodegradation rates of composite sustained-release stents of examples of the present invention and comparative examples;

FIG. 9 is a graph comparing the EGCG release rates of composite sustained-release stents of examples of the present invention and comparative examples;

FIG. 10 is a graph comparing cell viability of composite sustained-release scaffolds of examples of the present invention and comparative examples;

FIG. 11 is a graph comparing cell growth curves of composite sustained-release scaffolds of examples of the present invention and comparative examples;

FIG. 12 is a graph comparing cell distribution of composite sustained-release scaffolds of examples and comparative examples of the present invention;

FIG. 13 is a graph comparing cell distribution of alkaline phosphatase and calcium deposition of composite sustained-release scaffolds according to examples and comparative examples of the present invention;

FIG. 14 is a graph comparing the determination of alkaline phosphatase activity of the composite sustained-release stents of examples of the present invention and comparative examples;

fig. 15 is a graph comparing the determination of calcium content of the composite sustained-release stents of examples of the present invention and comparative examples.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention provides a chitosan composite slow-release bracket for slow release of bone tissue engineering and a preparation method thereof. The following are detailed below. It should be noted that the following description of the embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present invention, the term "including" means "including but not limited to". The terms first, second, third and the like are used merely as labels, and do not impose numerical requirements or an established order. Various embodiments of the invention may exist in a range of versions; it is to be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention; accordingly, the described range descriptions should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, it is contemplated that the description of a range from 1 to 6 has specifically disclosed sub-ranges such as, for example, from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within a range such as, for example, 1, 2, 3,4,5, and 6, as applicable regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any number (fractional or integer) recited within the indicated range.

As shown in fig. 1, the present invention provides a method for preparing a chitosan composite sustained-release scaffold for bone tissue engineering, comprising the following steps: s1) preparing EGCG-encapsulated chitosan microspheres; as shown in fig. 2, the step S1) includes the following steps: s11) adding EGCG into a chitosan solution with the mass concentration of 5-50 mg/mL to ensure that the mass concentration of the EGCG is 0.10-0.24 mg/mL, and uniformly mixing to obtain EGCG-chitosan mixed solution; s12) adding liquid paraffin and sorbitan oleate into a round-bottom flask, heating to 40-55 ℃, stirring and mixing, adding EGCG-chitosan mixed solution in the stirring process to ensure that the volume percentage of the liquid paraffin is 70-80%, the volume percentage of the sorbitan oleate is 1-5%, the volume percentage of the EGCG-chitosan mixed solution is 15-25%, and stirring for 1-2 hours to obtain reaction liquid; s13), adding a glutaraldehyde solution with the mass concentration of 25% into the reaction liquid in the step S12), wherein the volume ratio of the liquid paraffin, the sorbitan oleate, the EGCG-chitosan mixed liquid and the glutaraldehyde with the concentration of 25% is 70-80:1-5:15-25: 1-2; fully mixing and crosslinking for 30 minutes to obtain EGCG-chitosan microsphere mixed liquor; s14) placing the EGCG-chitosan microsphere mixed solution into a centrifuge, centrifuging for 3-10 minutes at the rotating speed of 1000-2000 rmp, and then removing supernatant to obtain precipitate; sequentially washing the precipitate with petroleum ether, ethanol and ultrapure water to obtain EGCG-chitosan microspheres; s15) freezing the EGCG-chitosan microspheres for 15 to 36 hours at-70 to-85 ℃; and then taking out and putting into a freeze dryer, and freeze-drying for 45-50 hours at the temperature of-40-50 ℃ to obtain the freeze-dried EGCG-encapsulated chitosan microspheres.

S2) adding the chitosan, the montmorillonite, the sodium carboxymethylcellulose and the EGCG-encapsulated chitosan microspheres into purified water according to the mass ratio of 5-50:1-10:1-10:1-10, mixing, and then adding glacial acetic acid, wherein the volume percentage of the glacial acetic acid is 0.5-1.5%, so as to obtain the chitosan composite sustained-release stent mixed solution. In the chitosan composite slow-release stent mixed solution in the step S2), the components comprise 5 mg/mL-50 mg/mL of chitosan according to mass concentration; 1 mg/mL-10 mg/mL of the sodium carboxymethylcellulose; 1 mg/mL-10 mg/mL of the montmorillonite and 1 mg/mL-10 mg/mL of the EGCG-encapsulated chitosan microsphere.

S3) freeze-drying the chitosan composite slow-release stent mixed solution to obtain a freeze-dried product of the chitosan composite slow-release stent. The step S3) comprises the steps of adding the obtained chitosan composite slow-release bracket mixed solution into a template, and freezing the template for 15 to 36 hours at a temperature of between 15 ℃ below zero and 25 ℃ below zero; and then placing the template in a freeze dryer, and freeze-drying for 45-50 hours at the temperature of-45-55 ℃ to obtain the chitosan composite slow-release stent freeze-dried product.

S4) placing the freeze-dried product of the chitosan composite sustained-release stent into a sodium hydroxide solution with the concentration of 30-50 mg/ml to soak for 2-5 hours, and then washing with a phosphate buffer solution to obtain the chitosan composite sustained-release stent.

The chitosan composite sustained-release scaffold for bone tissue engineering of the present invention will be further described with reference to the following specific examples.

Example 1

As shown in fig. 1, an embodiment of the present invention provides a method for preparing a chitosan composite sustained-release scaffold for bone tissue engineering, including steps S1) -S4).

S1) preparing EGCG-encapsulated chitosan microspheres; as shown in fig. 2, steps S11) -S15 are included in the step S1): s11) adding EGCG into a chitosan solution with the mass concentration of 20mg/mL to ensure that the mass concentration of the EGCG is 0.12mg/mL, and uniformly mixing to obtain an EGCG-chitosan mixed solution; s12) adding liquid paraffin and sorbitan oleate into a round-bottom flask, heating to 50 ℃, stirring and mixing, adding an EGCG-chitosan mixed solution in the stirring process to enable the volume percentage of the liquid paraffin to be 75%, the volume percentage of the sorbitan oleate to be 3%, the volume percentage of the EGCG-chitosan mixed solution to be 20%, and stirring for 1.5 hours to obtain a reaction solution; s13), adding a glutaraldehyde solution with the mass concentration of 25% into the reaction liquid in the step S12), wherein the volume ratio of the liquid paraffin, the sorbitan oleate, the EGCG-chitosan mixed liquid and the glutaraldehyde with the concentration of 25% is 75:4:20: 1; fully mixing and crosslinking for 30 minutes to obtain EGCG-chitosan microsphere mixed liquor; s14) placing the EGCG-chitosan microsphere mixed solution into a centrifuge, centrifuging for 5 minutes at 1500rmp, and then removing supernatant to obtain precipitate; sequentially washing the precipitate with petroleum ether, ethanol and ultrapure water to obtain EGCG-chitosan microspheres; s15) freezing the EGCG-chitosan microspheres for 24 hours at-80 ℃; and then taking out and putting into a freeze dryer, and freeze-drying for 48 hours at the temperature of minus 50 ℃ to obtain the freeze-dried EGCG-encapsulated chitosan microspheres.

S2) adding the chitosan, the montmorillonite, the sodium carboxymethylcellulose and the EGCG-encapsulated chitosan microspheres into purified water according to the mass ratio of 20:3:4:4, mixing, and then adding glacial acetic acid, wherein the volume percentage of the glacial acetic acid is 1.0%, so as to obtain the chitosan composite slow-release stent mixed solution. In the chitosan composite slow-release stent mixed solution in the step S2), the mass concentration of the chitosan is 20 mg/mL; the mass concentration of the sodium carboxymethylcellulose is 3.0 mg/mL; the mass concentration of the montmorillonite is 4.0 mg/mL; the mass fraction of the EGCG-encapsulated chitosan microspheres is 4.0 mg/mL.

S3) freeze-drying the chitosan composite slow-release stent mixed solution to obtain a freeze-dried product of the chitosan composite slow-release stent. The step S3) comprises the steps of adding the obtained chitosan composite slow-release stent mixed solution into a template, and freezing the template for 24 hours at the temperature of-20 ℃; and then placing the template in a freeze dryer, and freeze-drying for 48 hours at the temperature of minus 50 ℃ to obtain the chitosan composite slow-release stent freeze-dried product.

S4) soaking the chitosan composite sustained-release stent freeze-dried product in a sodium hydroxide solution with the concentration of 40mg/ml for 4 hours, and then washing for 3 times by using a phosphate buffer solution to obtain the chitosan composite sustained-release stent.

Example 2

The present invention also provides another preparation method of a chitosan composite sustained-release scaffold for bone tissue engineering, which is different from example 1 in that, in step S11), the step of adding 0.12mg/mL EGCG is removed.

Comparative example

The invention provides a preparation method of a commonly used chitosan composite slow-release bracket for bone tissue engineering, which comprises the following steps:

0.12mg/mL EGCG, 4.0mg/mL sodium carboxymethylcellulose, 4.0mg/mL montmorillonite and 20mg/mL chitosan are mixed with ultrapure water and stirred for 1 hour, and 1% glacial acetic acid is added to dissolve the chitosan, so that chitosan composite slow-release stent mixed solution is obtained.

And (3) taking a 24-hole plate, adding 250 mu L of chitosan composite slow-release scaffold mixed solution into each hole, freezing for 24 hours at the temperature of minus 20 ℃, putting into a freeze drier, and freeze-drying for 48 hours at the temperature of minus 50 ℃ to obtain a freeze-dried product of the chitosan composite slow-release scaffold.

Soaking the chitosan composite sustained-release stent freeze-dried product in 40mg/ml sodium hydroxide for 4 hours, washing for 3 times by using a phosphate buffer solution, freezing for 24 hours at the temperature of minus 20 ℃, then putting the product into a freeze dryer, and freeze-drying for 48 hours at the temperature of minus 50 ℃ to obtain the chitosan composite sustained-release stent.

The chitosan composite sustained-release scaffold for bone tissue engineering of the present invention was measured and explained in connection with examples 1 and 2 and comparative examples.

First, observing the appearance

The chitosan composite sustained-release scaffolds of example 1, example 2 and comparative example were placed in a scanning electron microscope to observe the microscopic morphology, and the characterization results are shown in fig. 3 to fig. 7.

Second, Performance measurement

(1) And (3) measuring the biodegradation rate:

after weighing, incubating in phosphate buffer solution containing lysozyme with the activity of 10000U/L, continuously oscillating at the temperature of 37 ℃ and the rotating speed of 100rpm, and replacing the phosphate buffer solution every two days. The chitosan composite sustained-release stent after shaking for 7 days, 14 days and 21 days is taken, respectively frozen and dried, and then weighed, and the biodegradation rate is calculated, and the result is shown in fig. 8.

(2) And (3) slow release curve determination:

the chitosan composite slow-release scaffold of example 1 and the chitosan composite slow-release scaffold of the comparative example are placed in a 24-well plate, 1mL of phosphate buffer solution is added into each well, a blank solute with the same volume is added after 200 mu L of sampling is carried out every day, sampling is continuously carried out for 24 days, and the EGCG content in the samples is respectively measured by using an ultraviolet spectrophotometer, wherein the result is shown in figure 9.

(3) And (3) measuring the biocompatibility:

the chitosan composite sustained-release scaffolds of example 1, example 2 and comparative example were sterilized and placed in a 24-well plate with a cell density of 2 x 10⁴cells/cm²Intermittent charging ofThe results of inoculating the plasma stem cells in a 24-well plate, adding 1mL of α MEM medium (containing 10% fetal bovine serum) to each well, culturing for 48 hours, and then measuring the biocompatibility of the chitosan composite sustained-release scaffold by the MTT method are shown in fig. 10.

(4) Cell proliferation capacity assay:

the chitosan composite sustained-release scaffolds of example 1, example 2 and comparative example were sterilized and placed in a 24-well plate, and the cell density was set to 0.5 x 10⁴cells/cm²The mesenchymal stem cells (2) were seeded in a 24-well plate, 1mL of α MEM medium (containing 10% fetal bovine serum) was added to each well, and samples were taken at 1 day, 3 days, 5 days, and 7 days of culture time, and the cell growth was measured by the CCK-8 method, and the results are shown in FIG. 11. The samples cultured for 1 day, 3 days, and 5 days were stained with calcein-AM (live cells/green) and PI (dead cells/red), and the growth of the cells was examined, and the results are shown in fig. 12.

(5) Alkaline phosphatase and calcium deposition assay:

the chitosan composite sustained-release scaffolds of example 1, example 2 and comparative example were sterilized and placed in a 24-well plate with a cell density of 1 x 10⁴cells/cm²The mesenchymal stem cells are inoculated in a 24-well plate, 1mL of alpha MEM culture medium (containing 10% fetal bovine serum) is added into each well, samples are respectively taken when the culture time is 7 days and 14 days, and qualitative test is carried out after the samples are dyed by an NBT/BCIP kit and alizarin red dye solution, and the result is shown in figure 13; quantitative test is carried out on the mesenchymal stem cells in the sample by utilizing the ALP enzyme activity kit and the calcium determination kit, and the results are shown in fig. 14 and fig. 15.

Third, test results

As shown in fig. 3 to 7, after characterization by scanning electron microscopy, it can be observed that the chitosan composite sustained-release scaffolds in examples 1 and 2 have obvious spherical structures, while the chitosan composite sustained-release scaffolds in comparative examples have no obvious spherical structure, which proves that the chitosan microspheres have been formed in the chitosan composite sustained-release scaffolds in examples 1 and 2.

As shown in fig. 8, the measurement results show that after 21 days of culture, the biodegradation rates of the chitosan composite sustained-release scaffolds in examples 1 and 2 can reach more than 50%, which is significantly higher than that of the comparative example, and show that the chitosan composite sustained-release scaffold provided in example 1 of the present invention has good biodegradability.

As can be seen from fig. 9, compared with the comparative example, the EGCG release rate of the chitosan composite sustained-release stent prepared in example 1 slowly and regularly increases with the increase of the culture days, the culture days when the EGCG release rate reaches the maximum value is 22 days, and the EGCG release rate is obviously prolonged compared with the 14 days of the comparative example, which indicates that the chitosan composite sustained-release stent prepared in example 1 has better EGCG sustained-release capability and longer action time.

As shown in fig. 10, the result of the biocompatibility test indicates that the chitosan composite sustained-release scaffolds in examples 1 and 2 and the comparative example have good biocompatibility after being cultured, wherein the chitosan composite sustained-release scaffold in example 1 has the highest cell viability.

As can be seen from fig. 11, the chitosan composite sustained-release scaffold prepared in example 1 has a more significant ability to promote proliferation of mesenchymal stem cells than the comparative example and example 2. As can be seen from fig. 12, after culturing for 7 days and 14 days, the number of mesenchymal stem cells on the chitosan composite sustained-release scaffold prepared in example 1 is the largest, the growth state is the best, the distribution is more uniform, and the density is the largest, which proves that the chitosan composite sustained-release scaffold prepared in example 1 has the best cell proliferation promoting capability.

As can be seen from fig. 13, 14 and 15, the chitosan composite sustained-release scaffolds prepared in example 1 and the comparative example have significantly better effects on promoting osteogenic differentiation of mesenchymal stem cells than those of example 2. The ALP activity and the calcium content of the chitosan composite sustained-release stent prepared in example 1 are higher than those of the chitosan composite sustained-release stent prepared in comparative example and example 2, which shows that the chitosan composite sustained-release stent prepared in example 1 has the strongest promotion effect on the osteogenic differentiation of mesenchymal stem cells.

In conclusion, the chitosan composite sustained-release stent prepared in the embodiment 1 provided by the invention has an obvious spherical structure, and the surface of the sphere is smooth, the particle size distribution is uniform, and the shape is good. The determination of biodegradation rate, slow release curve, biocompatibility, cell proliferation capacity and alkaline phosphatase and calcium deposition proves that the chitosan composite slow release stent has excellent capacity of promoting mesenchymal stem cell proliferation, can effectively delay the release of EGCG, improves the action period of the chitosan composite slow release stent, and can be applied to bone tissue engineering as a biomedical material.

Of course, the present invention is not limited to the above embodiments, and the mass ratio or volume percentage of each component may be adjusted to form other possible schemes. For example, when preparing the EGCG-encapsulated chitosan microspheres, the volume ratio of the liquid paraffin, the sorbitan oleate, the chitosan-EGCG mixed solution and the 25% glutaraldehyde may be 70:4:10: 1; or 75:5:20: 2; or 75:3:15: 2; or 70:2:20: 2; the chitosan composite slow-release stent comprises the components of chitosan, sodium carboxymethylcellulose, montmorillonite and EGCG-encapsulated chitosan microspheres in a mass ratio of 30:10:10: 1; or 40:5:10: 10; or 5:10:10: 5; or 50:10:5: 5; the above embodiments are only used to illustrate the present invention and not to limit the present invention.

The chitosan composite sustained-release scaffold for bone tissue engineering and the preparation method thereof provided by the embodiment of the invention are described in detail above, and the specific embodiment is applied in the text to explain the principle and the implementation mode of the invention, and the description of the embodiment is only used for helping to understand the method and the core idea of the invention; meanwhile, for those skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Claims

1. A chitosan composite slow release stent for bone tissue engineering is characterized in that the components of the chitosan composite slow release stent comprise chitosan, sodium carboxymethyl cellulose, montmorillonite and EGCG-encapsulated chitosan microspheres; wherein the EGCG is (2R,3R) -5, 7-dihydroxy-2- (3,4, 5-trihydroxyphenyl) chroman-3-yl 3,4, 5-trihydroxybenzoate; the mass ratio of the chitosan to the sodium carboxymethylcellulose to the montmorillonite to the EGCG-encapsulated chitosan microspheres is 5-50:1-10:1-10: 1-10.

2. A preparation method of a chitosan composite slow-release bracket for bone tissue engineering is characterized by comprising the following steps:

s1) preparing EGCG-encapsulated chitosan microspheres;

s2) adding the chitosan, the montmorillonite, the sodium carboxymethylcellulose and the EGCG-encapsulated chitosan microspheres into purified water according to the mass ratio of 5-50:1-10:1-10:1-10, mixing, and then adding glacial acetic acid to obtain a chitosan composite slow-release stent mixed solution;

s3) freeze-drying the chitosan composite slow-release stent mixed solution to obtain a freeze-dried product of the chitosan composite slow-release stent;

3. The method for preparing the chitosan composite sustained-release scaffold for bone tissue engineering according to claim 2, wherein the step S1) comprises the steps of:

s11) adding EGCG into a chitosan solution with the mass concentration of 5-50 mg/mL to ensure that the mass concentration of the EGCG is 0.10-0.24 mg/mL, and uniformly mixing to obtain EGCG-chitosan mixed solution;

s12) adding liquid paraffin and sorbitan oleate into a round-bottom flask, heating to 40-55 ℃, stirring and mixing, adding EGCG-chitosan mixed solution in the stirring process to ensure that the volume percentage of the liquid paraffin is 70-80%, the volume percentage of the sorbitan oleate is 1-5%, the volume percentage of the EGCG-chitosan mixed solution is 15-25%, and stirring for 1-2 hours to obtain reaction liquid;

s13), adding a glutaraldehyde solution with the mass concentration of 25% into the reaction liquid in the step S12), wherein the volume ratio of the liquid paraffin, the sorbitan oleate, the EGCG-chitosan mixed liquid and the glutaraldehyde with the concentration of 25% is 70-80:1-5:15-25: 1-2; fully mixing and crosslinking for 30 minutes to obtain EGCG-chitosan microsphere mixed liquor;

s14) placing the EGCG-chitosan microsphere mixed solution into a centrifuge, centrifuging for 3-10 minutes at the rotating speed of 1000-2000 rmp, and then removing supernatant to obtain precipitate; sequentially washing the precipitate with petroleum ether, ethanol and ultrapure water to obtain EGCG-chitosan microspheres;

s15) freezing the EGCG-chitosan microspheres at-70 to-85 ℃ for 15 to 36 hours, taking out the EGCG-chitosan microspheres, putting the EGCG-chitosan microspheres into a freeze dryer, and freeze-drying the EGCG-chitosan microspheres at-40 to-50 ℃ for 45 to 50 hours to obtain the freeze-dried EGCG-encapsulated chitosan microspheres.

4. The method for preparing the chitosan composite sustained-release scaffold for bone tissue engineering according to claim 2, wherein in the chitosan composite sustained-release scaffold mixed solution of the step S2), the components by mass concentration comprise:

5 mg/mL-50 mg/mL of chitosan;

1 mg/mL-10 mg/mL of sodium carboxymethylcellulose;

1 mg/mL-10 mg/mL of montmorillonite; and

the EGCG-coated chitosan microsphere is 1 mg/mL-10 mg/mL.

5. The method for preparing a chitosan composite sustained-release scaffold for bone tissue engineering according to claim 2, wherein in the step S2), the volume percentage of glacial acetic acid is 0.5-1.5%.

6. The method for preparing the chitosan composite sustained-release scaffold for bone tissue engineering according to claim 2, wherein the step S3) comprises adding the obtained chitosan composite sustained-release scaffold mixed solution into a template, freezing the template at-15 to-25 ℃ for 15 to 36 hours, and then placing the template in a freeze-drying machine, and freeze-drying at-45 to-55 ℃ for 45 to 50 hours to obtain the chitosan composite sustained-release scaffold freeze-dried product.