CN100431624C - Method for preparing injectable polyletic acid micro-carrier/chitosan hydrogel composite scaffold - Google Patents

Abstract

The invention discloses a preparation method of an injectable polylactic acid microcarrier/chitosan hydrogel composite support. Carbodiimide condensation method is adopted to graft methacrylic acid and lactic acid in chitosan molecular chain in sequence to obtain polymerizable water-soluble chitosan derivatives. The polylactic acid microcarrier is mixed with the chitosan hydrogel prepolymer, and at the same time, the thickener konjac glucomannan is added to increase the viscosity of the prepolymer solution to ensure the suspension of the polylactic acid microcarrier and facilitate the operation of the injection process. Ammonium persulfate and tetramethylethylenediamine oxidation-reduction initiation system are used to make the above compound gel in situ at body temperature to form a composite scaffold of cell microcarrier/chitosan hydrogel. The process of the invention is simple, and the prepared composite bracket not only solves the problem that the injectable microcarrier is not easy to form and wander in the body, but also can significantly improve the strength of the hydrogel, and has a synergistic effect. The composite scaffold is nontoxic, biodegradable and biocompatible, and is expected to be used as an injectable scaffold in tissue engineering.

Description

Preparation method of injectable polylactic acid microcarrier/chitosan hydrogel composite scaffold

technical field

The invention relates to a preparation method of an injectable polylactic acid microcarrier/chitosan hydrogel composite support, in particular to an injectable support used in tissue engineering.

technical background

Injectable scaffolds are composed of cells and a fluid, biocompatible material, and are directly injected into the defect site of the body through a syringe. The material can form a scaffold in situ with certain mechanical strength, certain shape and exchangeable with body fluids, in which cells grow and eventually form tissues. The material can also be directly injected into the body, and the cells in the surrounding tissue of the injection can be used to expand, grow and proliferate to form a tissue. Due to the characteristics of minimal invasiveness, in vivo culture environment and low cost, injectable stents have important clinical application value and good development prospects. Injectable scaffolds are mainly divided into injectable hydrogel scaffolds and injectable cell microcarriers. Injectable hydrogel scaffolds are mainly formed in vivo through sol-gel transition, which has good cell compatibility and easy molding, but has low strength and rapid degradation. Injectable cell microcarriers need to use liquid as a transport carrier to inject into the damaged part and build up in the body, which is easy to operate, but it is not easy to form, and there is a problem of wandering in the body. At present, both at home and abroad are basically focusing on the improvement of single-component performance, and there is no combination of the two to obtain optimal performance.

Contents of the invention

The purpose of the invention is to provide a method for preparing an injectable polylactic acid microcarrier/chitosan hydrogel composite scaffold for tissue engineering.

The preparation method of the injectable polylactic acid cell microcarrier/chitosan hydrogel composite support of the present invention comprises the following steps:

1) Dissolve chitosan (CS) in a solution containing methacrylic acid (MA) at room temperature, then add water-soluble carbodiimide (EDAC) to react at room temperature, water-soluble carbodiimide The molar ratio to methacrylic acid is 0.25 to 1.5. After the reaction is completed, dialyze in triple distilled water to remove unreacted substances and by-products, and freeze-dry to obtain chitosan (CM) grafted with methacrylic acid;

2) At room temperature, dissolve chitosan grafted with methacrylic acid in a solution containing lactic acid (LA), then add water-soluble carbodiimide, and react at room temperature, water-soluble carbodiimide and lactic acid The molar ratio is 0.3 to 1.8. After the reaction, dialyze in triple distilled water to remove unreacted substances and by-products, and freeze-dry to obtain chitosan derivatives (CML) grafted with methacrylic acid and lactic acid;

3) at room temperature, the chitosan derivatives grafted with methacrylic acid and lactic acid are dissolved in water or phosphate buffer saline (PBS), and prepared into a chitosan derivative solution with a concentration of 1 to 2.5%, and then Konjac glucomannan (KGM) powder is added, and the concentration of the konjac glucomannan in the chitosan derivative solution is 0.6-1%. After stirring evenly, add 2.5-15mM ammonium persulfate (APS) and tetramethylethylenediamine (TMEDA) into the initiation system, the molar ratio of ammonium persulfate and tetramethylethylenediamine is 1:1, and mix well;

4) Add the polylactic acid microcarriers to the mixed solution in step 3), wherein the weight-volume ratio of the polylactic acid microcarriers in the mixed solution is 2.5% to 10%, after mixing evenly, place it at 25°C to 45°C for reaction A polylactic acid microcarrier/chitosan hydrogel composite scaffold was formed.

The above-mentioned polylactic acid microcarriers are polylactic acid microspheres with collagen on the surface, which can be directly compounded, or can be compounded after pre-loading required cells (such as chondrocytes, osteoblasts or liver cells, etc.) on its surface.

The method of the invention has simple operation process and mild implementation conditions. The high-viscosity konjac glucomannan is used as a thickener to increase the viscosity of the chitosan solution grafted with methacrylic acid and lactic acid, so as to ensure the suspension of polylactic acid microcarriers in the solution, so as to facilitate the smooth progress of injection. The microcarriers are injected into the required parts with the gel prepolymer as the transport carrier, and gelled in situ to form a microcarrier/hydrogel composite scaffold with a certain shape. The hydrogel is conducive to the shaping of the microcarriers, and Wrapping it can prevent it from wandering in the body; at the same time, the microcarrier can effectively improve the strength of the hydrogel, and the combination of the two has a certain synergistic effect. The composite scaffold is nontoxic, biodegradable and biocompatible. The method of the invention provides a cell scaffold with a new structure and a preparation method thereof, which can be used in tissue engineering and plastic repair.

Description of drawings

Fig. 1 is the change curve of MA grafting amount under the mol ratio of different EDAC and MA;

Fig. 2 is the change curve of LA grafting amount under the mol ratio of different EDAC and LA;

Fig. 3 is at 37 ℃, the photo of polylactic acid microcarrier suspended in KGM/CML solution for 15 minutes; Wherein the concentration of CML is 1%, the mark in the figure is the concentration of KGM;

Figure 4 is the gel time of 1% CML with different KGM contents added at 37°C and 5mM APS/TMEDA;

Figure 5 is the equilibrium swelling ratio of 1% CML gels with different KGM contents prepared at 37°C and 5mM APS/TMEDA in water for 24h;

Figure 6 is the dynamic elastic modulus of 1% CML gels with different KGM contents prepared at 37°C and 5mM APS/TMEDA, and the KGM content is marked in the figure;

Fig. 7 is the dynamic modulus of elasticity of the 0.6%KGM/1%CML composite scaffold prepared under 37 DEG C and 5mM APS/TMEDA with different polylactic acid microcarrier content, marked as the weight volume content of microcarrier in the figure;

Figure 8 is the internal structure of the 0.6% KGM/1% CML composite scaffold with different polylactic acid microcarrier contents prepared at 37°C and 5mM APS/TMEDA after drying, and the weight volume content of the microcarrier is marked under the figure, (a) 2.5%, (b) 5%, (c) 10%;

Figure 9 shows the cell activity of chondrocytes in 5% microcarrier/0.6%KGM/1%CML composite scaffold, and the cell planting density is 600×10 ⁴ /ml;

Figure 10 is the observation of the growth behavior of chondrocytes in 5% microcarrier/0.6%KGM/1%CML composite scaffold by laser confocal microscope (CLSM), in the figure (a) is 1 day, (b) is 6 days, (c) 12 days;

Figure 11 is a scanning electron microscope (SEM) to observe the morphology of chondrocytes in 5% microcarrier/0.6%KGM/1%CML composite scaffold, in the figure (a), (b) are 1 day, (c), (d) is 6 days, (e), (f) are 12 days.

Figure 12 is the morphology of chondrocytes observed in 5% microcarrier/0.6%KGM/1%CML composite scaffold by SEM, in which (a) is a spherical cell, (b) is a cell mass, and (c) is a spread cell layer.

Detailed ways

Example 1

Weigh chitosan (CS) 800mg and place it in a 250ml Erlenmeyer flask, add 100ml three-distilled water and 420μl MA (0.48mmol), after the CS is completely dissolved, add 930mg EDAC (0.48mmol), then stir the reaction at room temperature 24h. In order to remove unreacted MA and other small molecular products, the reaction mixture was placed in a dialysis bag with a cut-off molecular weight of 10,000 Da, dialyzed in a large amount of triple-distilled water at room temperature for 3 days, and the triple-distilled water was changed 2 to 3 times a day. Finally, the liquid was frozen and freeze-dried to obtain MA-grafted chitosan (CM). The yield of CM is greater than 90%, and the grafting amount of MA is about 23%, as shown in Figure 1; it can be swollen in water. Dissolve the above 400mg CM in 50ml three-distilled water containing 210μl LA (0.2mmol), and add 460mgEDAC (0.24mmol) after completely dissolving. After the mixture was stirred at room temperature for 24 hours, the reaction mixture was placed in a dialysis bag with a cut-off molecular weight of 10,000 Da, dialyzed in a large amount of triple-distilled water at room temperature for 3 days, and the triple-distilled water was changed 2 to 3 times a day. Finally, the liquid was frozen and freeze-dried to obtain chitosan (CML) grafted with MA and LA. The yields of CML were all greater than 90%, and the grafting amount of LA was about 52%, as shown in Figure 2. Dissolve the chitosan derivative (CML) of the methacrylic acid of 100mg graft rate 23% and the lactic acid of graft rate 52% in water, be mixed with 10ml 1% CML solution, then add 60mg KGM powder, mix well . Add 50 μl of 1M ammonium persulfate (APS) and 50 μl of 1M and tetramethylethylenediamine (TMEDA) solution and mix well. Then add 500mg polylactic acid microcarrier, the weight volume ratio of microcarrier in the mixed solution is 5%, after mixing evenly, inject in the mold with 1ml syringe (no needle). Placed at 37°C for 8 minutes to obtain a microcarrier/hydrogel composite scaffold. The dynamic elastic modulus of the composite scaffold is 0.12-1.15 MPa at a frequency of 0.1-100 rad/s, as shown in Figure 7, and its internal structure after drying is shown in Figure 8b.

Example 2

Weigh 800mg of chitosan and place it in a 250ml Erlenmeyer flask, add 100ml triple distilled water and 420μl MA (0.48mmol), after the CS is completely dissolved, add 232.5mg EDAC (0.12mmol), then stir the reaction at room temperature for 24h . In order to remove unreacted MA and other small molecular products, the reaction mixture was placed in a dialysis bag with a cut-off molecular weight of 10,000 Da, dialyzed in a large amount of triple-distilled water at room temperature for 3 days, and the triple-distilled water was changed 2 to 3 times a day. Finally, the liquid was frozen and freeze-dried to obtain MA-grafted chitosan (CM). The CM yields were all greater than 90%, and the MA grafting amount was about 11.74%, as shown in Figure 1.

Example 3

The 400mg CM that the MA grafting amount that example 1 obtains is about 23% is dissolved in the 50ml three-distilled water that contains 210 μ l LA (0.2mmol), adds 115mg EDAC (0.06mmol) after dissolving completely. After the reaction mixture was stirred at room temperature for 24 hours, it was placed in a dialysis bag with a cut-off molecular weight of 10,000 Da, dialyzed in a large amount of triple-distilled water at room temperature for 3 days, and the triple-distilled water was changed 2 to 3 times a day. Finally, the liquid was frozen and freeze-dried to obtain MA and LA grafted chitosan (CML). The yield of CML was greater than 90%, and the LA grafting amount was about 43.2%, as shown in Figure 2.

Example 4

The 100mg CML that example 1 obtains is dissolved in water, is mixed with 10ml 1% CML solution, then adds 100mg KGM powder, mixes, and KGM content is 1%. Then add 250 mg of polylactic acid microcarriers and place them in a water bath at 37° C. After 15 minutes, it can be seen that the polylactic acid microcarriers are suspended in 1% KGM/1% CML solution, which facilitates the injection. See Figure 3.

Example 5

The 100mg CML that example 1 obtains is dissolved in water, is mixed with 10ml 1% CML solution, then adds 60mg KGM powder, mixes homogeneously. Add 50 μl of 1M ammonium persulfate (APS) and 50 μl of 1M tetramethylethylenediamine (TMEDA) solution, mix well, and place directly at 37°C. Its gel time is 5.4min, as shown in Figure 4. The equilibrium swelling ratio is 65, see Figure 5. The dynamic elastic modulus is 1.2KPa～24.4KPa when the frequency is 0.1～100rad/s, see Figure 6.

Example 6

The 100mg CML that example 1 obtains is dissolved in water, is mixed with 10ml 1% CML solution, then adds 60mg KGM powder, mixes homogeneously. Add 50 μl of 1M ammonium persulfate (APS) and 50 μl of 1M tetramethylethylenediamine (TMEDA) solution. Add 250mg of polylactic acid microcarrier, its microcarrier content is 2.5%, after mixing evenly, use 1ml syringe (no needle) to inject into the mold, put it at 37 ℃, after 8min, microcarrier/hydrogel composite scaffold can be obtained . The dynamic elastic modulus of the composite scaffold is 13KPa-258KPa when the frequency is 0.1-100rad/s, as shown in Figure 7, and its internal structure after drying is shown in Figure 8a.

Example 7

The 100mg CML that example 1 obtains is dissolved in water, is mixed with 10ml 1% CML solution, then adds 60mg KGM powder, mixes homogeneously. Add 50 μl of 1M ammonium persulfate (APS) and 50 μl of 1M tetramethylethylenediamine (TMEDA) solution. Add 1g of polylactic acid microcarrier, the weight volume content of which is 10%. After mixing evenly, inject it into the mold with a 1ml syringe (no needle), place it at 37°C, and get the microcarrier/hydrogel after 8min Composite bracket. The dynamic elastic modulus of the composite scaffold is 0.87-2.15 MPa at a frequency of 0.1-100 rad/s, as shown in Figure 7, and its internal structure after drying is shown in Figure 8c.

Example 8

The CML and KGM obtained in Example 1 are sterilized by ultraviolet light irradiation; polylactic acid microcarriers are sterilized by soaking in 75% alcohol; Sterilize by membrane filtration. Chondrocytes were pre-planted on the surface of 250mg polylactic acid microcarriers, and the cells were overgrown after 7 days for use. Dissolve 50mg of CML in 5ml of PBS first to make a 1% CML/PBS solution, then add 30mg of KGM powder and mix well. A PBS solution of initiator APS/TMEDA was added to a final concentration of 5 mM. First add 0.5ml of high-concentration cell suspension into the gel prepolymer, mix well, and the cell planting density is 600×10 ⁴ /ml. Cell-loaded microcarriers were then added at a content of 5%. The mixture was injected into the mold with a 1ml syringe and placed at 37°C for 8 minutes to form a composite scaffold. The composite scaffold is transferred to a 24-well culture plate, cultured in a culture medium containing 20% calf serum, and the medium is changed every 2-3 days. The activity of chondrocytes first increased and then remained unchanged, as shown in Figure 9. Some cells died during the gelation and mixing process, and the cells migrated, adhered, spread and proliferated to the surface of the microcarrier, and there were cell clusters, as shown in Figure 10 and Figure 11. The cells in the gel are round and there are cell clusters, and the cells on the surface of the microcarriers are a spread cell layer, as shown in Figure 12.

Claims (3)

1. the preparation method of injectable polylactic acid microcarrier/aquagel compound rest may further comprise the steps:

1) at normal temperatures, chitosan is dissolved in the solution that contains methacrylic acid, add water-soluble carbodiimide then, under room temperature, react, the mol ratio of water-soluble carbodiimide and methacrylic acid is 0.25～1.5, after reaction finishes, place the tri-distilled water dialysis to remove unreacting substance and by-product, lyophilizing obtains the chitosan of grafting methacrylic acid;

2) at normal temperatures, the chitosan of grafting methacrylic acid is dissolved in the solution that contains lactic acid, add water-soluble carbodiimide then, under room temperature, react, the mol ratio of water-soluble carbodiimide and lactic acid is 0.3～1.8, after reaction finishes, place the tri-distilled water dialysis to remove unreacting substance and by-product, lyophilizing obtains the chitosan derivatives of grafting methacrylic acid and lactic acid;

3) at room temperature, the chitosan derivatives of grafting methacrylic acid and lactic acid is dissolved in water or the phosphate buffer, be mixed with concentration and be 1～2.5% chitosan derivative solution, add the Rhizoma amorphophalli glucomannan powder then, the concentration of Rhizoma amorphophalli glucomannan in chitosan derivative solution is 0.6～1%, after stirring, adds 2.5～15mM initiator system Ammonium persulfate. and tetramethylethylenediamine, the mol ratio of Ammonium persulfate. and tetramethylethylenediamine is 1: 1, mix homogeneously;

4) the polylactic acid microcarrier is joined in the mixed liquor of step 3), wherein the w/v of polylactic acid microcarrier in mixed liquor is 2.5%～10%, behind the mix homogeneously, reacts formation polylactic acid microcarrier/aquagel compound rest down in 25 ℃～45 ℃.

2. the preparation method of injectable polylactic acid microcarrier according to claim 1/aquagel compound rest is characterized in that the polylactic acid microcarrier is the polylactic acid microsphere that the surface has collagen.

3. the preparation method of injectable polylactic acid microcarrier according to claim 2/aquagel compound rest is characterized in that at polylactic acid microcarrier area load cell.

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