CN107789667B - Porous composite scaffold for promoting cartilage regeneration and preparation method thereof - Google Patents
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Abstract
The invention discloses a porous composite scaffold for promoting cartilage regeneration, which is prepared from the following raw materials in parts by weight: 100-400 parts of glucan, 1-30 parts of nano microcrystalline cellulose and 3-21 parts of graphene oxide. The invention also discloses a preparation method of the porous composite scaffold. The porous composite scaffold prepared by the method comprises Dex, CNCs and GO, has excellent mechanical properties and good water absorption swelling property, the pore size is suitable for cell adhesion proliferation, meanwhile, Graphene Oxide (GO) can induce mesenchymal stem cells to differentiate into chondrocytes, glucan provides a good cell survival environment, and the porous composite scaffold can promote cartilage to regenerate more quickly and repair cartilage injury quickly by combining the three-dimensional porous scaffold and the graphene oxide surface activity.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a porous composite scaffold for promoting cartilage regeneration and a preparation method thereof.
Background
Degeneration and trauma are common reasons of articular cartilage injury, the orthopedic diseases are difficult to treat clinically, cartilage is rare in quantity, slow in proliferation and weak in bone self-repair capacity due to the fact that the cartilage is lack of blood supply, even tiny injury can aggravate the disease, at present, no good treatment method is available for cartilage, requirements for sources of transplanted replacement bone are high, and the defects of insufficient autologous bone transplant donors, allograft immunological rejection and the like can be overcome by applying bone tissue engineering technology to treat bone defects. Therefore, bone repair using bone tissue engineering is a hot spot for treating bone defects.
Dextran (Dextran, Dex), a natural polysaccharide, has good biocompatibility and good application prospects in biomedical materials, is often used as a drug carrier, exists in mucus secreted by certain microorganisms in the growth process, can be divided into alpha and beta dextrans according to the structure, and linear and triple helix structures can subdivide different functions of the Dextran, wherein the linear alpha Dextran has good solubility but poor mechanical properties, so the application of the Dextran is often limited in orthopedics.
Graphene Oxide (GO) Graphene is a new material with a single-layer sheet structure composed of carbon atoms. As one of the most important derivatives of graphene, the graphene has excellent properties of good water solubility, easy functionalization, low toxicity and the like due to the fact that the surface of the graphene is rich in oxygen-containing groups such as hydroxyl, carboxyl and the like, so that the graphene can be used as a drug carrier, and has good application prospects in the biomedical field (such as drug release) due to the unique structure and properties of the graphene; graphene oxide, however, tends to aggregate in saline solutions and serum, thereby limiting many applications in biology.
The existing method for treating cartilage injury generally injects hydrogel into cartilage injury to repair cartilage injury, however, since hydrogel is soft and solid, the method has poor effect of treating cartilage injury, low cure rate and slow curative effect.
Disclosure of Invention
Based on the above, the invention aims to overcome the defects of the prior art and provide the stent which has a faster curative effect and a higher cure rate for treating the cartilage injury.
In order to achieve the purpose, the invention adopts the technical scheme that: a porous composite scaffold for promoting cartilage regeneration is prepared from the following raw materials in parts by weight: 100-400 parts of glucan, 1-30 parts of nano microcrystalline cellulose and 3-21 parts of graphene oxide.
It should be noted that the nanocrystalline Cellulose (CNCs) is made of natural Cellulose, has good biocompatibility, low immunogenicity, low toxicity, and good mechanical properties, and is often used as a material surface enhancer, has a high surface area, is easy to agglomerate, and is weak to degrade in vivo, so it is suitable for trace use.
Preferably, the porous composite scaffold is of a three-dimensional porous structure, and the pore diameter of the porous composite scaffold is 80-230 microns.
Preferably, the nanocrystalline cellulose is in a rod-shaped structure, the length of the rod is 140-360 nm, and the width of the rod is 20-40 nm.
Preferably, the mass ratio of the glucan to the nanocrystalline cellulose to the graphene oxide is 20: 1: 1.
preferably, the mass percentage of the graphene oxide in the porous composite support is 3-7%. More preferably, the mass percentage content of the graphene oxide in the porous composite scaffold is 5%. Through a plurality of experiments, the inventor of the present application finds that when the mass ratio of the dextran to the nanocrystalline cellulose to the graphene oxide is 20: 1: 1, when the mass percentage of the graphene oxide in the porous composite scaffold is 5%, the porous composite scaffold has high cartilage regeneration promoting efficiency and low toxicity to cells.
As another aspect of the present invention, the present invention also provides a method for preparing a porous composite scaffold, comprising the steps of:
(1) preparing nano microcrystalline cellulose: weighing cellulose, adding the cellulose into 35-70 wt% of concentrated sulfuric acid, reacting in a constant-temperature water bath for 1-6 hours, then adding deionized water, cooling, dispersing, dialyzing by using the deionized water until the pH value is stable, and freeze-drying to obtain the nano microcrystalline cellulose;
(2) dissolving graphene oxide in deionized water and uniformly dispersing to obtain a graphene oxide solution;
(3) adding glucan into deionized water, heating in a water bath at 40-60 ℃ until the glucan is dissolved to obtain a glucan solution, adding the nano microcrystalline cellulose obtained in the step (1) into the glucan solution, mixing and stirring uniformly, adding a NaOH solution and a sodium trimetaphosphate solution, reacting for 5-16 minutes, adding the graphene oxide solution obtained in the step (2), stirring uniformly, and freeze-drying to obtain the porous composite scaffold. In addition, the inventor of the application finds that the uniform and uniform pore-forming of the scaffold can be ensured only when the value of glucan (mass)/V (deionized water) is between 7% and 15%. More preferably, the value of dextran (mass)/V (deionized water) is 7.1 to 14.3%.
Preferably, the cellulose in step (1) is plant fiber.
Preferably, the mass ratio of the glucan in the step (3) to the graphene oxide in the step (2) is 100: 5.
preferably, the mass ratio of the glucan to the nanocrystalline cellulose in the step (3) is 100: 5.
in conclusion, the beneficial effects of the invention are as follows:
(1) the porous scaffold comprises Dex, CNCs and GO, has excellent mechanical properties and good water absorption swelling property, the pore size is suitable for cell adhesion proliferation, meanwhile, Graphene Oxide (GO) can induce mesenchymal stem cells to be differentiated into chondrocytes, glucan provides a good cell survival environment, and the three-dimensional porous scaffold and graphene oxide surface activity are combined, so that cartilage can be promoted to regenerate more quickly, and cartilage injury can be repaired quickly;
(2) the porous support can be used as a drug carrier matrix, has simple preparation process, wide material source, high production efficiency and low cost, and can be applied to industrial mass production.
Drawings
FIG. 1 is a scanning electron microscope image of a porous composite scaffold according to the present invention;
FIG. 2 is an electron microscope photograph of a porous composite scaffold according to the present invention;
FIG. 3 is a transmission electron microscope image of nanocrystalline cellulose prepared in the present invention;
FIG. 4 is a graph showing the degradation of the porous composite scaffold of the present invention in PBS and dextranase solutions, respectively;
FIG. 5 is a graph of the compressive modulus of a porous composite scaffold of the present invention;
FIG. 6 is an absorbance value of cells after culturing in the porous composite scaffold of the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
One embodiment of the porous composite scaffold for promoting cartilage regeneration in the invention comprises the following raw materials in parts by weight: 1g of glucan, 10mg of nano microcrystalline cellulose and 30mg of graphene oxide. The mass percentage of the graphene oxide in the porous composite support is 1%.
The porous composite scaffold is of a three-dimensional porous structure, and the pore diameter of the porous composite scaffold is 80-230 microns as shown in figures 1 and 2. The nanocrystalline cellulose is in a rod-like structure, as shown in FIG. 3, the length of the rod is 140-360 nm, and the width of the rod is 20-40 nm.
The preparation method of the porous composite scaffold comprises the following steps:
step (a): weighing a certain amount of cellulose, adding a certain amount of 65% concentrated sulfuric acid, magnetically stirring in a constant-temperature water bath at 50 ℃, reacting for 6 hours, adding a large amount of deionized water to terminate the reaction, cooling, ultrasonically dispersing, dialyzing by using the deionized water until the pH value is stable, and freeze-drying at-80 ℃ to obtain the rodlike nano microcrystalline cellulose.
Step (b): 60mg of graphene oxide is weighed and dissolved in 2ml of deionized water, and the graphene oxide is uniformly dispersed in an ultrasonic instrument at 100 Hz.
And (c) dissolving 1g of glucan in 14ml of deionized water, placing the mixture in a water bath at 50 ℃ for heating and magnetically stirring for dissolving, weighing 10mg of nano microcrystalline cellulose, adding the nano microcrystalline cellulose into the glucan solution, mixing and stirring uniformly, adding 1ml of 10mol/L NaOH solution, adding 1ml of 0.3g/ml sodium trimetaphosphate solution, adding 2ml of 15mg/ml graphene oxide solution after reacting for 10 minutes, quickly placing the mixture at minus 80 ℃ for freezing after fully stirring uniformly for 10 minutes, and then freeze-drying the mixture at minus 80 ℃ to obtain the Dex/CNCs/GO porous composite scaffold with wt 1% of graphene oxide.
Example 2
One embodiment of the porous composite scaffold for promoting cartilage regeneration in the invention comprises the following raw materials in parts by weight: 2g of glucan, 75mg of nano microcrystalline cellulose and 90mg of graphene oxide. The mass percentage of the graphene oxide in the porous composite support is 3%.
The porous composite scaffold is of a three-dimensional porous structure, as shown in figures 1 and 2, and the pore diameter of the porous composite scaffold is 80-230 microns. The nanocrystalline cellulose is in a rod-like structure, as shown in FIG. 3, the length of the rod is 140-360 nm, and the width of the rod is 20-40 nm.
The preparation method of the porous composite scaffold comprises the following steps:
step (a): weighing a certain amount of cellulose, adding a certain amount of 65% concentrated sulfuric acid, magnetically stirring in a constant-temperature water bath at 50 ℃, reacting for 6 hours, adding a large amount of deionized water to terminate the reaction, cooling, ultrasonically dispersing, dialyzing by using the deionized water until the pH value is stable, and freeze-drying at-80 ℃ to obtain the rodlike nano microcrystalline cellulose.
Step (b): weighing 90mg of graphene oxide, dissolving in 2ml of deionized water, and performing ultrasonic dispersion uniformly in an ultrasonic instrument at 100 Hz.
And (c) dissolving 2g of glucan in 28ml of deionized water, placing the mixture in a water bath at 50 ℃ for heating and magnetically stirring for dissolving, weighing 75mg of nano microcrystalline cellulose, adding the nano microcrystalline cellulose into the glucan solution, mixing and stirring uniformly, adding 1ml of 10mol/L NaOH solution, adding 1ml of 0.3g/ml sodium trimetaphosphate solution, adding 2ml of 45mg/ml graphene oxide solution after reacting for 10 minutes, quickly placing the mixture at-80 ℃ for freezing after fully stirring uniformly for 10 minutes, and freeze-drying the mixture at-80 ℃ to obtain the Dex/CNCs/GO porous composite scaffold with 3 wt% of graphene oxide.
Example 3
One embodiment of the porous composite scaffold for promoting cartilage regeneration in the invention comprises the following raw materials in parts by weight: 3g of glucan, 150mg of nano microcrystalline cellulose and 150mg of graphene oxide. The mass percentage of the graphene oxide in the porous composite support is 5%.
The porous composite scaffold is of a three-dimensional porous structure, and the pore diameter of the porous composite scaffold is 80-230 microns as shown in figures 1 and 2. The nanocrystalline cellulose is in a rod-shaped structure, as shown in fig. 3, the length of the rod is 140-360 nm, and the width of the rod is 20-40 nm.
The preparation method of the porous composite scaffold comprises the following steps:
step (a): weighing a certain amount of cellulose, adding a certain amount of 65% concentrated sulfuric acid, magnetically stirring in a constant-temperature water bath at 50 ℃, reacting for 6 hours, adding a large amount of deionized water to terminate the reaction, cooling, ultrasonically dispersing, dialyzing by using the deionized water until the pH value is stable, and freeze-drying at-80 ℃ to obtain the rodlike nano microcrystalline cellulose.
Step (b): weighing 150mg of graphene oxide, dissolving in 2ml of deionized water, and performing ultrasonic dispersion uniformly in an ultrasonic instrument at 100 Hz.
And (c) dissolving 3g of glucan in 28ml of deionized water, placing the mixture in a water bath at 50 ℃ for heating and magnetically stirring for dissolving, weighing 150mg of nano microcrystalline cellulose, adding the nano microcrystalline cellulose into the glucan solution, mixing and stirring uniformly, adding 1ml of 10mol/L NaOH solution, adding 1ml of 0.3g/ml sodium trimetaphosphate solution, adding 2ml of 75mg/ml graphene oxide solution after reacting for 10 minutes, quickly placing the mixture at-80 ℃ for freezing after fully stirring uniformly for 10 minutes, and then freeze-drying the mixture at-80 ℃ to obtain the Dex/CNCs/GO porous composite scaffold with 5 wt% of graphene oxide.
Example 4
One embodiment of the porous composite scaffold for promoting cartilage regeneration in the invention comprises the following raw materials in parts by weight: 4g of glucan, 300mg of nano microcrystalline cellulose and 210mg of graphene oxide. The mass percentage of the graphene oxide in the porous composite support is 7%.
The porous composite scaffold is of a three-dimensional porous structure, and the pore diameter of the porous composite scaffold is 80-230 microns as shown in figures 1 and 2. The nanocrystalline cellulose is in a rod-like structure, as shown in FIG. 3, the length of the rod is 140-360 nm, and the width of the rod is 20-40 nm.
The preparation method of the porous composite scaffold comprises the following steps:
step (a): weighing a certain amount of cellulose, adding a certain amount of 65% concentrated sulfuric acid, magnetically stirring in a constant-temperature water bath at 50 ℃, reacting for 6 hours, adding a large amount of deionized water to terminate the reaction, cooling, ultrasonically dispersing, dialyzing by using the deionized water until the pH value is stable, and freeze-drying at-80 ℃ to obtain the rodlike nano microcrystalline cellulose.
Step (b): 210mg of graphene oxide is weighed and dissolved in 2ml of deionized water, and the graphene oxide is uniformly dispersed by ultrasonic under 100Hz in an ultrasonic instrument.
And (c) dissolving 4g of glucan in 28ml of deionized water, placing the mixture in a water bath at 50 ℃ for heating and magnetically stirring for dissolving, weighing 150mg of nano microcrystalline cellulose, adding the nano microcrystalline cellulose into the glucan solution, mixing and stirring uniformly, adding 1ml of 10mol/L NaOH solution, adding 1ml of 0.3g/ml sodium trimetaphosphate solution, adding 2ml of 105mg/ml graphene oxide solution after reacting for 10 minutes, quickly placing the mixture at minus 80 ℃ for freezing after fully stirring uniformly for 10 minutes, and then freeze-drying the mixture at minus 80 ℃ to obtain the Dex/CNCs/GO porous composite scaffold with wt 7% of graphene oxide.
Example 5 testing of the Performance of porous composite scaffolds in the present invention
(1) Degradation testing of porous composite scaffolds
Test objects: the Dex/CNCs/GO composite scaffolds with wt 1%, 3%, 5% and 7% GO contents are prepared by the above embodiment, and because GO occupies a small relative mass, the scaffold with 5% GO content is selected to be in a PBS buffer solution and a human body simulation environment containing dextranase for degradation performance test;
the test method comprises the following steps: weighing a certain mass of the composite scaffold, respectively placing the composite scaffold in PSB (pH 7.4) buffer solution and trypsin-containing buffer solution (sample: PBS: pancreatin 10mg:2ml:100 mu L), dynamically culturing for 1, 3, 5, 7, 10, 14 and 21 days at 37 ℃, replacing fresh solution every other half a day, and preparing 3 parallel samples in each group.
And (3) testing results:
as shown in fig. 4, the experiment shows that the scaffold of the present invention degrades slowly in the buffer solution and degrades faster in the enzyme solution (glucanase); meanwhile, the compressive property of the support material is tested, and the experimental result is shown in fig. 5, so that the mechanical property of the support is slightly enhanced along with the increase of the GO content.
(2) Cytological evaluation of porous composite scaffolds
Test objects: the porous composite scaffolds of examples 1-4;
the test method comprises the following steps: bone marrow mesenchymal stem cell BMSCs are respectively planted on the porous composite scaffold material in the embodiments 1-4, and the toxicity of the material and the induced differentiation condition of the scaffold material on the bone marrow mesenchymal stem cells can be judged according to the cell proliferation condition;
and (3) testing results:
as shown in fig. 6, the level of absorbance reflects the proliferation amount status of the cells, and the higher the absorbance, the higher the proliferation amount of the cells; the experimental result shows that the porous composite scaffold of the invention has good biocompatibility, does not seriously hinder cell proliferation, but 7% GO shows larger toxicity, and the porous composite scaffold of the embodiment 3 is an optimal target scaffold by combining the analysis, and has high induced differentiation efficiency and smaller toxicity on the mesenchymal stem cells.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. The porous composite scaffold for promoting cartilage regeneration is characterized in that raw materials for preparing the porous composite scaffold comprise the following components in parts by weight: 100-400 parts of glucan, 1-30 parts of nano microcrystalline cellulose and 3-21 parts of graphene oxide; the mass percentage of the graphene oxide in the porous composite support is 3-7%.
2. The porous composite scaffold according to claim 1, wherein the porous composite scaffold is a three-dimensional porous structure, and the pore size of the pores is 80-230 μm.
3. The porous composite scaffold according to claim 1, wherein the nanocrystalline cellulose is in a rod-like structure, the length of the rod is 140 to 360nm, and the width of the rod is 20 to 40 nm.
4. The porous composite scaffold according to claim 1, wherein the mass ratio of dextran, nanocrystalline cellulose and graphene oxide is 20: 1: 1.
5. the porous composite scaffold according to claim 1, wherein the mass percentage of graphene oxide in the porous composite scaffold is 5%.
6. The method for preparing a porous composite scaffold according to any one of claims 1 to 5, comprising the steps of:
(1) preparing nano microcrystalline cellulose: weighing cellulose, adding the cellulose into 35-70 wt% of concentrated sulfuric acid, reacting in a constant-temperature water bath for 1-6 hours, then adding deionized water, cooling, dispersing, dialyzing by using the deionized water until the pH value is stable, and freeze-drying to obtain the nano microcrystalline cellulose;
(2) dissolving graphene oxide in deionized water and uniformly dispersing to obtain a graphene oxide solution;
(3) adding glucan into deionized water, heating in a water bath at 40-60 ℃ until the glucan is dissolved to obtain a glucan solution, adding the nano microcrystalline cellulose obtained in the step (1) into the glucan solution, mixing and stirring uniformly, adding a NaOH solution and a sodium trimetaphosphate solution, reacting for 5-16 minutes, adding the graphene oxide solution obtained in the step (2), stirring uniformly, and freeze-drying to obtain the porous composite scaffold.
7. The method according to claim 6, wherein the cellulose in the step (1) is a plant fiber.
8. The preparation method according to claim 6, wherein the mass ratio of the glucan in the step (3) to the graphene oxide in the step (2) is 100: 5.
9. the preparation method according to claim 6, wherein the mass ratio of the glucan to the nanocrystalline cellulose in the step (3) is 100: 5.
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CN105521525A (en) * | 2015-12-16 | 2016-04-27 | 广州创赛生物医用材料有限公司 | Porous composite scaffold for bone tissue engineering and preparation method therefor |
CN106146899A (en) * | 2016-06-21 | 2016-11-23 | 华南理工大学 | A kind of stannic oxide/graphene nano microcrystalline cellulose composite material thin film and preparation method thereof |
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CN101820929A (en) * | 2007-10-11 | 2010-09-01 | 国家健康与医学研究院 | Preparation is used for the method for the porous support of organizational project |
CN104857569A (en) * | 2015-05-06 | 2015-08-26 | 浙江大学 | Preparation method of fibroin and graphene oxide composite bracket material |
CN105521525A (en) * | 2015-12-16 | 2016-04-27 | 广州创赛生物医用材料有限公司 | Porous composite scaffold for bone tissue engineering and preparation method therefor |
CN106146899A (en) * | 2016-06-21 | 2016-11-23 | 华南理工大学 | A kind of stannic oxide/graphene nano microcrystalline cellulose composite material thin film and preparation method thereof |
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