CN102716516A - Polydatin modified collagen scaffold, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a collagen scaffold modified by polydatin and its preparation method and application. The polydatin is modified with adipic acid modified dextran to improve its solubility, and then the polydatin modified by dextran is mixed with collagen and passed through 1 -Crosslinking of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) followed by lyophilization yielded collagen-based scaffolds. Guided by the basic idea of tissue engineering, the present invention takes collagen with good biocompatibility as the main composition, cultures a three-dimensional collagen scaffold for the growth of chondrocytes in vitro, and improves the solubility of polydatin by modifying it. Tissue engineered scaffolds provide convenience.

Description

Collagen scaffold modified by polydatin and its preparation method and application

technical field

The invention relates to the field of tissue engineering scaffolds, in particular to a collagen scaffold and its preparation method and application.

Background technique

Joint disease and articular cartilage damage are one of the main health problems faced by human beings, which will directly lead to dysfunction of joints and partial or total loss of labor force. The most common joint disease is arthritis. According to the latest statistics, there are 20 million arthritis patients in the United States, and the patients are mainly elderly people over 65 years old. It is estimated that by 2030, there will be more than 70 million people over the age of 65 in the American society, accounting for 70% of the total population, and many of them will be at risk of developing arthritis. With the acceleration of aging, the number of patients with arthritis will increase year by year, and the treatment of cartilage diseases and articular cartilage damage has become one of the clinical problems to be solved urgently. So far, there is still no cure for arthritis, and the usual treatment is surgery. However, a recent survey involving 285 well-known surgeons from 46 countries around the world shows that 82% of doctors believe that the current cartilage treatment methods are short-term effective. The emergence and development of tissue engineering technology has brought new hope for the treatment of cartilage diseases. Three-dimensional scaffolds for the growth of chondrocytes can be cultivated in vitro using materials with good biocompatibility as the main component.

Studies have shown that nitric oxide (NO) is an important inflammatory mediator in the pathogenesis of arthritis, a newly discovered biological information transmitter, and one of the main hazards of cartilage damage. Steroids and polydatin have the effect of delaying and alleviating arthritis, but because polydatin is insoluble in water, its application and development in the field of tissue engineering scaffolds are limited.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, with the basic idea of tissue engineering, the basic theory as a guide, to provide a collagen scaffold modified by polydatin and its preparation method and application, mainly based on collagen with good biocompatibility The three-dimensional collagen scaffold for the growth of chondrocytes is cultured in vitro, and modified to improve the solubility of polydatin, so as to facilitate its modification of tissue engineering scaffolds.

The purpose of the present invention is achieved through the following technical solutions:

Collagen scaffold modified by polydatin, modify polydatin with adipic acid modified dextran to improve its solubility, then mix polydatin with dextran and collagen and pass through 1-(3-dimethylaminopropyl) -3-Ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) cross-linked to obtain collagen-based scaffolds after lyophilization.

First, step 1, N, N'-carbonyldiimidazole (CDI)-activated adipic acid and dextran are mixed and reacted, and then the product is reacted with N, N'-carbonyldiimidazole (CDI)-activated polydatin Reaction, obtains the polydatin of modification, and concrete reaction principle is as follows:

Pour dextran (DEX) into dimethyl sulfoxide, then seal the flask and place it in an oil bath at 80°C. After the DEX is completely dissolved, the reaction can be terminated.

N, N'-carbonyldiimidazole (CDI) is used to activate adipic acid, and dimethyl sulfoxide (DMSO) is used as a solvent, that is, adipic acid and CDI are respectively added to dimethyl sulfoxide (DMSO) to dissolve, Then mix and pour it into a flask, place it under normal temperature of 20-25 degrees Celsius and stir, because the reaction produces a large number of bubbles, so no sealing treatment is required. After the reaction no longer produces bubbles, pour the activated adipic acid into the above-mentioned container containing DEX, and mix and react at room temperature 20-25 degrees Celsius for at least 8 hours, preferably 8-12 hours, more preferably 10-12 hours. Finally, the product was put into a dialysis bag (molecular weight cut-off: 3500), and dialyzed for 3-4 days. The water in the dialyzed product was evaporated with a rotary evaporator, and freeze-dried for 2-3 days.

Choose dimethyl sulfoxide (DMSO) as the solvent, that is, polydatin and N, N'-carbonyldiimidazole (CDI) are respectively added to dimethyl sulfoxide (DMSO) to dissolve, and the two are mixed at 70-100 ° C, Preferably, the reaction is carried out in an oil bath at 80-100° C. for at least 3 hours, preferably 5-10 hours. After the reaction, use 10 times the volume of diethyl ether to extract the reaction system. It can be observed that the solution has two upper and lower layers.

Weigh the adipic acid-dextran product, dissolve it in DMSO, mix it with the above-mentioned ready-to-use solution, and react at room temperature at 20-25 degrees Celsius for at least 12 hours, preferably 20-24 hours. After the above reaction is finished, dialyze (molecular weight cut-off is 3500) for 3-4 days, and then rotary evaporate. The product can be dried in a freeze dryer to obtain the final product.

Next, step 2, the modified polydatin and collagen were passed through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS ) after cross-linking and freeze-drying to obtain a collagen-based scaffold, the specific reaction principle is as follows (Liu Y, Griffith M, Matsky M.A, et al.Properties of porcine and recombinant human collagen matrices for optically clear tissue engineering applications. Biomaromolecules, 2006, 7 (6): 1819-1828.):

Specifically, the cross-linking reaction can be carried out at room temperature of 20-25 degrees Celsius, the mass ratio of collagen to dextran-modified polydatin is (1-3): 1, and EDC and NHS can be added according to the ratio of collagen cross-linking or Slight excess to ensure cross-linking effect.

Characterized by the Nova NanoSEM high-resolution scanning electron microscope of the US FEI company, as shown in Figure 1, it can be seen that the morphology of the scaffold is basically maintained and has a porous structure; the NMR characterization is carried out by the 500MHz high-resolution superconducting NMR spectrometer Varian INOVA in the United States , as shown in Figure 2, choose dimethyl sulfoxide as the solvent, a is dextran, b is polydatin, c is polydatin grafted dextran, and c comprehensively reflects the structural characteristics of polydatin and polydatin , the characteristic peak of chemical shift 9.5 is the characteristic peak of phenolic hydroxyl group on polydatin, indicating that dextran and polydatin achieve the purpose of reaction. In addition, the product after dialysis can be dissolved in pure water, and it can be tested that it can change the color of the ferric chloride aqueous solution, indicating that there are phenolic hydroxyl groups on polydatin in the intercepted macromolecules, which further proves that dextran and polydatin have reacted Purpose.

The infrared spectrum of the bracket was measured by the NICOLET Nexu6700 infrared spectrometer in the United States. As shown in Figure 3, 2800-3000cm ^-1 is the vibration absorption of saturated hydrocarbons in the polymer main chain, and 1649cm ^-1 is the amide I band, which belongs to the stretching vibration of the CO double bond. 1546cm ^-1 is the amide II band, which belongs to the rocking vibration of NH. At 3300cm ^-1 on the figure, it is the absorption peak of polydatin phenolic hydroxyl group. It shows that the modified polydatin molecular chain is contained in the scaffold, so that the target product is produced.

Nitric oxide (NO) is an important inflammatory mediator in the pathogenesis of arthritis, a newly discovered biological information transmitter, and one of the main hazards of cartilage damage. Polydatin can inhibit the activity of nitric oxide synthase (NOS), thereby reducing the damage of articular cartilage. Taking advantage of Polydatin's ability to slow the arthritis process, it is added to the collagen scaffold to aid in cartilage repair. In the technical scheme of the present invention, adipic acid-modified polydatin is used to modify polydatin to improve its solubility, and then the collagen support is immersed in the solution of modified polydatin, and the modified polydatin is used to fill the collagen support. Collagen with good biocompatibility is the main component, and the three-dimensional collagen scaffold for the growth of chondrocytes is cultivated in vitro to facilitate its modification of the tissue engineering scaffold.

Description of drawings

Figure 1 SEM photos of the morphology of the scaffold, where a is magnified 50 times; b is magnified 100 times (NovaNanoSEM high-resolution scanning electron microscope from FEI Company, USA).

The NMR spectrogram of Fig. 2 polydatin grafted dextran, wherein (a) dextran is made the nuclear magnetic pattern when dimethyl sulfoxide is solvent; (b) polydatin is when dimethyl sulfoxide is made solvent NMR image; (c) NMR image of polydatin-grafted dextran in dimethyl sulfoxide (500MHz high-resolution superconducting NMR spectrometer Varian INOVA, USA).

The infrared spectrogram of Fig. 3 bracket (US NICOLET Nexu6700 infrared spectrometer).

Figure 4 The growth of bone cells on the 14th day after implanting the scaffold, wherein a and c are blank collagen membranes, a is magnified 10 times, and c is magnified 20 times; b and d are collagen membranes added with polydatin, b is 10 times, and d is 20 times.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with specific embodiments. The atelopeptide pig skin type I collagen and dextran 40000 (DEXT40) were used for the experiment.

First, N, N'-carbonyldiimidazole (CDI)-activated adipic acid and dextran are mixed and reacted, and then the product is reacted with N, N'-carbonyldiimidazole (CDI)-activated polydatin to obtain an improved Sexual polydatin:

1. Take two 25ml flasks, 2 stoppers and 2 magnets, wash and dry them, and put them in an oven.

2. Weigh about 3g of adipic acid and add dimethyl sulfoxide (DMSO) to dissolve. Weigh about 1 g of N,N'-carbonyldiimidazole (CDI), and dissolve it in DMSO. After the above two substances are dissolved respectively, mix them and pour them into a flask, put them into a magnet, and stir them at room temperature. Since the reaction produces a large number of air bubbles, no sealing is required

3. Weigh 1g of dextran (DEX) in a flask, pour 6-15ml DMSO into it, and seal the flask

4. Observe that there is no bubble overflow in the reaction of adipic acid, then pour it into the flask containing DEX. After mixing the above solutions, react at room temperature for about 10 hours, put the product into a 3500 dialysis bag, and dialyze for 3-4 days.

5. Use a rotary evaporator to evaporate the water in the dialyzed product, put the distilled product into a small dish and wait for freeze-drying. After freeze-drying for 2-3 days, the product was taken out and the yield was calculated.

6. Weigh 0.4g polydatin (PD) and dissolve it with DMSO. Weigh 0.162g of CDI, dissolve it with DMSO, mix the two into a 50ml dry flask, and put it in an oil bath at 80°C for 3 hours.

7. Extract the above product with 10 times the volume of the reactant ether, and observe that the solution is divided into upper and lower layers. Pour the lower layer of yellow transparent oil from the lower end of the separatory funnel and put it into a 50ml flask to obtain activated PD.

8. Weigh the adipate-dextran product, dissolve it in DMSO, mix it with activated PD and continue to react for 12 hours.

9. After the above reaction is completed, after 3-4 days of dialysis, rotary steaming. The product can be dried in a freeze dryer to obtain the final product. Dissolve the product with pure water, and check that it can change the color of ferric chloride, indicating that the purpose of the reaction is achieved.

The modified polydatin and collagen were frozen by cross-linking with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) After drying, obtain a collagen-based scaffold: (1) Weigh 3-6mg NHS, 10mgEDC into a 1ml centrifuge tube, dissolve with 50ml ultrapure water; (2) modify the quality of polydatin according to collagen and dextran The ratio is (1-3): 1 Weigh dextran-modified polydatin (0.06g) and collagen (0.06g, 0.12g, 0.18g) respectively, dissolve them in ultrapure water, and mix the solution in (1) Pour in and mix well, and carry out cross-linking reaction at room temperature; you can use a plexiglass sheet (10cm*10cm*0.5mm) or a mold (500μl thick, 12mm in diameter) for reaction; then place the scaffold at room temperature under 100% humidity For 16 hours, move to 37°C and continue to mature for 5 hours. Open the mold and take out the stent sample, soak it in 10mM phosphate buffered saline (PBS pH=7.4), replace the PBS every hour, wash it for 7 days, put it into centrifuge tubes with holes respectively, put them into Freeze-dry in a freeze dryer, and the final product collagen scaffold can be obtained after two or three days.

Preparation of pure collagen by lyophilization after cross-linking of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), respectively Scaffold, the collagen scaffold modified by polydatin prepared above. Each of the two groups made five slices with a thickness of 500 μm and soaked them in PBS. After 3-4 days, treat the above-mentioned scaffold with a puncher with a diameter of 12 μm, then move it to 75 wt% alcohol aqueous solution for disinfection, soak it for 1-2 days, and put it into a cell culture dish.

After a known number of chondrocytes were centrifuged, the cells were dispersed with a fibrinogen solution to prepare a 500,000/mL fibrinogen solution (20 mg/mL). The cellular fibrinogen solution is introduced into the porous scaffold under the action of negative pressure. Then, a calcium chloride solution (5 U/mL) of thrombin was introduced under negative pressure. Use sterile gauze to absorb the liquid on the surface of the scaffold, and put it into a 24-well culture plate; put the culture plate into a constant temperature incubator at 37 degrees Celsius, and fully incubate for 30 minutes. After the fibrinogen is completely gelled, add 2 mL of DMEM to each well for culture. base.

Use a fluorescence microscope (Olympus BX51 fluorescent inverted microscope) to observe the growth of chondrocytes. From the fluorescence microscope images of chondrocytes after 14 days (Figure 4), it can be seen that the growth of chondrocytes in the experimental group added polydatin was significantly better than that of blank collagen Group. The number of chondrocytes in the blank collagen group on the left side of the figure continues to increase, but they are elongated and fibrous, and have lost the ability to continue to differentiate. Such cells cannot repair the damaged cartilage. The photo on the right side of the figure shows that after adding polydatin, the number of chondrocytes continued to increase, and the cell density was high, but the cells were still round or polygonal, indicating that the chondrocytes in this group had strong vitality and good growth conditions. At the same time, it has also been proved that polydatin does have the effect of stimulating the growth of chondrocytes.

"Day" mentioned in this application refers to 24 hours.

The present invention has been described as an example above, and it should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacements that can be made by those skilled in the art without creative labor all fall within the scope of this invention. protection scope of the invention.

Claims (10)

1. the collagen scaffold modified by polydatin, it is characterized in that, modify polydatin with adipic acid modified dextran to improve its solubility, then mix polydatin and collagen with dextran and pass through 1-(3- The collagen-based scaffold is obtained after cross-linking with dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and freeze-drying. 2. The preparation method of the collagen scaffold modified by polydatin is characterized in that, it is carried out according to the following steps: First, step 1, N, N'-carbonyldiimidazole-activated adipic acid and dextran are mixed and reacted, and then the product is reacted with N, N'-carbonyldiimidazole-activated polydatin to obtain modified polydatin; Next, namely step 2, freeze modified polydatin and collagen after cross-linking with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide Get a stent after drying. 3. the preparation method of the collagen scaffold of polydatin modification according to claim 2, is characterized in that, in described step 1, selects N, N '-carbonyldiimidazole to activate adipic acid, selects dimethyl methylene Sulfone is used as a solvent, that is, adipic acid and N, N'-carbonyldiimidazole are respectively added to dimethyl sulfoxide to dissolve, then mixed and poured into a flask, and stirred at room temperature at 20-25 degrees Celsius. 4. the preparation method of the collagen scaffold of polydatin modification according to claim 2 is characterized in that, in described step 1, the adipic acid after activation is poured in the above-mentioned container that dextran is housed, and normal temperature 20 Mix and react at -25 degrees Celsius for at least 8 hours; finally put the product into a dialysis bag for dialysis for 3-4 days, use a rotary evaporator to evaporate the water in the dialyzed product, and freeze-dry for 2-3 days. 5. The preparation method of polydatin-modified collagen scaffold according to claim 4, characterized in that, in the step 1, the mixing reaction at room temperature is 20-25 degrees Celsius, preferably 8-12 hours, more preferably 10-12 hours. 6. the preparation method of the collagen scaffold modified by polydatin according to claim 2, is characterized in that, in described step 1, polydatin and N, N'-carbonyldiimidazole are respectively added to dissolve in dimethyl sulfoxide After the two are mixed, react in an oil bath at 70-100°C for at least 3 hours; after the reaction is completed, use 10 times the volume of ether in the reaction system to extract, and pour the yellow transparent oil in the lower layer into the container from the lower end of the separatory funnel to wait use. 7. the preparation method of the collagen scaffold modified by polydatin according to claim 6, is characterized in that, in described step 1, polydatin and N, N'-carbonyldiimidazole are respectively added to dissolve in dimethyl sulfoxide , After mixing the two, react in an oil bath at 80-100°C for 5-10 hours. 8. the preparation method of the collagen scaffold of polydatin modification according to claim 2, is characterized in that, in described step 2, takes adipic acid-dextran product, dissolves with N , after the polydatin activated by N'-carbonyldiimidazole is mixed, react at room temperature for at least 12 hours at 20-25 degrees Celsius, preferably 20-24 hours; Steam; place the product in a freeze dryer to dry to the final product. 9. the preparation method of polydatin-filled collagen scaffold according to claim 2, is characterized in that, in described step 2, select room temperature 20-25 degree Celsius to carry out cross-linking reaction, collagen and dextran modify the quality of polydatin The ratio is (1-3): 1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide can be added according to the ratio of collagen crosslinking Or a slight excess to ensure the cross-linking effect. 10. The application of the polydatin-modified collagen scaffold as claimed in claim 1 as a cartilage tissue scaffold.

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