CN101905040B - Method for preparing three-dimensional cell scaffold from elastic microsphere pore-forming agent - Google Patents

Abstract

A method for preparing a three-dimensional cell scaffold with an elastic microsphere porogen, characterized in that the steps are as follows: 1) preparing the elastic microsphere porogen, and preparing a scaffold material solution; 2) placing the elastic microsphere porogen in a mold , add the scaffold material solution to immerse the porogen, mix evenly to obtain the scaffold/elastic microsphere porogen compound, press filter the composite with a filter press plate with micropores, so that the elastic microsphere porogen is evenly distributed in the In the solution and in contact with each other, the complex was pre-frozen at -10°C or lower for 3 hours; 3) The complex was demolded and freeze-dried for 6-8 hours; 4) The complex was rehydrated in distilled water for 1 hour; 5) Remove the porogen. The scaffold preparation method of the invention can better realize the artificial controllability of the scaffold pore structure, and the preparation process is simple and easy.

Description

Method for preparing three-dimensional cell scaffold with elastic microsphere porogen

technical field

The invention belongs to the field of biomaterials, and in particular relates to a method for preparing a three-dimensional cell scaffold by combining an elastic microsphere porogen with a filter press forming technology.

Background technique

In tissue engineering, cells rely on scaffolds as growth templates. Cell scaffolds not only guide tissue regeneration, but also control the properties of tissues or organs. Therefore, three-dimensional porous scaffolds play a key role in tissue engineering and are one of the important fields of tissue engineering research. . A large number of studies have shown that the pore shape, size, and porosity of the cell scaffold directly affect the migration, differentiation, proliferation, and tissue formation of the planted cells. An ideal cell scaffold must have a certain range of pores and pore communication channels. It has been reported that the main factors affecting the growth of bone tissue into the pores of the material are the inter-pore connectivity and the diameter of the inter-pore channels. Pores with good connectivity and appropriate size of the communication channels are more conducive to the entry of blood vessels and osteoblasts (Chang BS, Lee CK, Hong KS, et al. Osteoconduction at porous hydroxyapatite with various spore configuration. Biomaterials, 2000, 21(12): 1291-8). Among the various traditional technologies for preparing cell scaffolds, the fiber bonding method can obtain scaffolds with good connectivity between pores, but the pore structure is difficult to control; although gas foaming and phase separation techniques can prepare large-volume porous scaffolds, the pores The complete connectivity cannot be guaranteed, and the size of the pores and the size of the channels between the pores cannot be controlled; the scaffolds prepared by the solvent casting/particle leaching method have poor connectivity and rough pore morphology.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art, and to provide a method for preparing three-dimensional cell scaffolds by combining elastic microsphere porogens and press-press molding techniques. The method can obtain spherical, pore size and pore size. The intercommunicating channel is an artificially controllable three-dimensional cell scaffold, and the preparation process is simple and easy.

The method for preparing a three-dimensional cell scaffold with an elastic microsphere porogen involved in the present invention is mainly based on the following principles: applying the filter press molding technology to the preparation of a three-dimensional cell scaffold, and combining it with the elastic microsphere porogen, which can be combined advantages of both. The invention uniformly mixes the support material solution and the elastic microsphere porogen to form a compound, and utilizes the fluidity of the support material solution and the deformability of the porogen to form the compound by pressure filtration. When a certain pressure is applied to the composite, the elastic microsphere porogens undergo elastic deformation and contact and squeeze each other, causing the volume of the composite system to decrease, and the excess polymer solution is filtered out. At this time, the shape of the microspheres and the degree of extrusion between them will be fixed. This method can control the pore size of the scaffold by controlling the size of the microspheres. After the elastic microspheres are pressed against each other, they will change from a point contact state to a surface contact state. After the porogen is removed, pores are formed at the position of the microspheres. , the mutual contact surface of the microspheres will be converted into the scaffold pore communication channel, and the size of the scaffold pore communication channel can be controlled by adjusting the contact area between the microspheres.

A method for preparing a three-dimensional cell scaffold with an elastic microsphere porogen involved in the present invention, the steps are as follows:

1) preparing an elastic microsphere porogen, and preparing a scaffold material solution;

2) Put the elastic microsphere porogen in the mold, add the scaffold material solution to immerse the porogen, mix evenly to obtain the scaffold/elastic microsphere porogen composite, and use a filter press plate with micropores to prepare the composite Press filtration, so that the elastic microsphere porogen is evenly distributed in the solution and can be in contact with each other, and the composite is pre-frozen at -10°C or lower for 3 hours;

3) Demoulding the compound and freeze-drying for 6-8 hours;

4) Place the complex in distilled water and rehydrate for 1 hour;

5) Remove the porogen.

Above-mentioned 1) the preparation of elastic microsphere porogen has following two kinds of methods in the step:

The first method adopts polyvinyl alcohol preparation, and its steps are as follows:

(a) preparing a polyvinyl alcohol solution with a mass fraction of 7% to 12%;

(b) High-voltage electrostatic method to prepare polyvinyl alcohol elastic microspheres, the high voltage range is 10-15KV, and the distance between electrodes is 2-3cm; it is 2:1 chloroform and acetone mixed with a volume ratio lower than -25°C The solution is used as a solidifying liquid to solidify the microspheres; during the preparation process, a large amount of steam is passed through the outflow port of the polyvinyl alcohol solution to moisturize;

(c) repeatedly freezing the polyvinyl alcohol elastic microspheres in the solidified liquid 4 to 6 times, the freezing temperature is -25°C or lower, the freezing time is 10 hours each time, and the thawing time is 1 hour;

(d) Soak the polyvinyl alcohol elastic microspheres in saturated sodium chloride or saturated potassium chloride solution at normal temperature for 20-60 minutes; wash the elastic microspheres with distilled water for 2-3 times, and dry under normal pressure to remove the surface moisture;

(e) Sieving to obtain elastic microsphere porogens with a diameter of 100-1000 microns.

The second method adopts sodium alginate preparation, and its steps are as follows:

(a) preparing a sodium alginate solution with a mass fraction of 3% to 5%;

(b) Prepare calcium alginate elastic microspheres by high-voltage electrostatic method, the high-voltage range is 10-13KV, and the distance between electrodes is 2-3cm; Sodium microspheres are solidified into calcium alginate microspheres; during the preparation process, a large amount of steam is passed through the outflow port of the sodium alginate solution to moisturize;

(c) Soak the calcium alginate elastic microspheres in the solidification solution at room temperature for 30-90 minutes; wash the elastic microspheres with distilled water for 2-3 times, and dry under normal pressure to remove the surface moisture;

(d) Sieving to obtain calcium alginate elastic microsphere porogens with a diameter of 100-1000 microns.

The main advantages of the present invention are: (1) the method can obtain three-dimensional cell scaffolds whose pores are spherical, the size of the pores and the communication channels between the pores are artificially controllable; (2) the elastic microsphere porogen and the filter press forming technology are combined , which has the advantages of both. The pore size of the scaffold is controlled by the size of the elastic microsphere porogen, and the size of the connecting channel between the pores of the scaffold is controlled by the contact area between the microspheres after press filtration; (3) the preparation process is simple and easy.

Description of drawings

Figure 1 is a schematic diagram of the three-dimensional cell scaffold prepared by combining the elastic microsphere porogen with the filter press molding technology.

Fig. 2 is a topographic microscopic picture of a polyvinyl alcohol elastic microsphere porogen with a diameter of 100-1000 microns.

Figure 3 is a picture of the appearance of the three-dimensional cell scaffold.

Figure 4 is a microscope picture of the pore structure of the three-dimensional cell scaffold, with a magnification of 4 times.

Fig. 5 is a scanning electron microscope picture of a three-dimensional cell scaffold.

Detailed ways

Below in conjunction with accompanying drawing and embodiment the present invention is further described as follows:

Example 1

1) Prepare the polyvinyl alcohol elastic microsphere porogen, prepare a dioxane solution of polylactic acid with a mass fraction of 3% as the scaffold material solution; the preparation steps of the polyvinyl alcohol elastic microsphere porogen are as follows:

(a) preparation mass fraction is the polyvinyl alcohol solution of 7%;

(b) Prepare polyvinyl alcohol elastic microspheres by high-voltage electrostatic method, the voltage is 10KV, and the distance between electrodes is 3cm. During the preparation process, a large amount of steam is passed through the outflow port of polyvinyl alcohol solution to moisturize, and the volume ratio at -25 ° C is 2:1. The mixed solution of chloroform and acetone is used as the solidifying solution to solidify the microspheres;

(c) Freeze the microspheres 6 times repeatedly, the freezing temperature is -25°C, the freezing time is 10 hours each time, and the thawing time is 1 hour;

(d) Soak the microspheres in a saturated saline solution for 20 minutes, rinse with distilled water for 2 to 3 times, and dry under normal pressure;

(e) Sieving to obtain polyvinyl alcohol elastic microsphere porogens with a diameter of 100-1000 microns.

2) Put the polyvinyl alcohol elastic microsphere porogen prepared in step 1) into the mold, add the scaffold material solution to immerse the porogen, and mix evenly to obtain the scaffold/polyvinyl alcohol elastic microsphere porogen composite, Use a filter plate with micropores to press filter the compound until it reaches 90% of the initial volume of the compound, and quickly transfer the filtered compound to a medical low-temperature box at -10°C for 3 hours;

3) Demoulding the compound, freeze-drying for 8 hours;

4) Place the complex in distilled water and rehydrate for 1 hour;

5) Put the compound into distilled water and heat it with microwave repeatedly for 5-10 times, each time for 2-3 minutes; dry under normal pressure to remove excess water, so as to obtain a formed three-dimensional scaffold.

Example 2

1) Prepare the polyvinyl alcohol elastic microsphere porogen, prepare the dioxane/chloroform solution of polylactic acid with a mass fraction of 3.5% as the scaffold material solution, and the volume ratio of dioxane and chloroform is 5: 2; The preparation steps of polyvinyl alcohol elastic microsphere porogen are as follows:

(a) preparation mass fraction is the polyvinyl alcohol solution of 10%;

(b) Prepare elastic microspheres by high voltage electrostatic method, the voltage is 12KV, and the distance between electrodes is 2.5cm. A mixed solution of methyl chloride and acetone is used as a solidifying solution to solidify the microspheres;

(c) Repeatedly freezing the microspheres 5 times, the freezing temperature is -25°C, the freezing time is 10 hours each time, and the thawing time is 1 hour;

(d) Soak the microspheres in a saturated saline solution for 40 minutes, rinse with distilled water for 2 to 3 times, and dry under normal pressure;

(e) Sieving to obtain elastic microsphere porogens with a diameter of 100-1000 microns.

2) Put the polyvinyl alcohol elastic microsphere porogen prepared in step 1) into the mold, add the scaffold material solution to immerse the porogen, and mix evenly to obtain the scaffold/polyvinyl alcohol elastic microsphere porogen composite, Use a filter plate with micropores to press filter the compound to 80% of the initial volume of the compound, and quickly transfer the filtered compound to a medical low-temperature box at -25°C for 3 hours;

3) Demoulding the compound and freeze-drying for 7 hours;

4) Place the complex in distilled water and rehydrate for 1 hour;

5) Put the compound into distilled water and heat it with microwave repeatedly for 5-10 times, each time for 2-3 minutes; dry under normal pressure to remove excess water, so as to obtain a formed three-dimensional scaffold.

Example 3

1) Prepare the polyvinyl alcohol elastic microsphere porogen, prepare the dioxane/chloroform solution of polylactic acid with a mass fraction of 4% as the scaffold material solution, and the volume ratio of dioxane and chloroform is 5: 2; The preparation steps of polyvinyl alcohol elastic microsphere porogen are as follows:

(a) preparation mass fraction is the polyvinyl alcohol solution of 12%;

(b) Elastic microspheres are prepared by high-voltage electrostatic method, the voltage is 15KV, and the distance between electrodes is 2cm. A mixed solution of methane and acetone is used as a solidification solution to solidify the microspheres;

(c) Freeze the microspheres 4 times repeatedly, the freezing temperature is -25°C, the freezing time is 10 hours each time, and the thawing time is 1 hour;

(d) Soak the microspheres in a saturated saline solution for 60 minutes, rinse with distilled water for 2 to 3 times, and dry under normal pressure;

(e) Sieving to obtain elastic microsphere porogens with a diameter of 100-1000 microns.

2) Put the polyvinyl alcohol elastic microsphere porogen prepared in step 1) into the mold, add the scaffold material solution to immerse the porogen, and mix evenly to obtain the scaffold/polyvinyl alcohol elastic microsphere porogen composite, Press filter the compound with a filter plate with micropores until it reaches 70% of the initial volume of the compound, and quickly transfer the filtered compound to a medical low-temperature box at -25°C for 3 hours;

3) Demoulding the compound and freeze-drying for 6 hours;

4) Place the complex in distilled water and rehydrate for 1 hour;

5) Put the compound into distilled water and heat it with microwave repeatedly for 5-10 times, each time for 2-3 minutes; dry under normal pressure to remove excess water, so as to obtain a formed three-dimensional scaffold.

Example 4

1) Prepare the calcium alginate elastic microsphere porogen, prepare a polylactic acid dioxane solution with a mass fraction of 3% as the scaffold material solution; the preparation steps of the calcium alginate elastic microsphere porogen are as follows:

(a) preparation mass fraction is the sodium alginate solution of 3%;

(b) Prepare calcium alginate elastic microspheres by high-voltage electrostatic method, the voltage is 10KV, and the distance between electrodes is 3cm; use calcium chloride solution with a mass fraction of 20% at room temperature as the solidification solution to solidify sodium alginate microspheres into seaweed Calcium acid microspheres; during the preparation process, a large amount of steam is passed through the outflow port of the sodium alginate solution to moisturize;

(c) Soak the calcium alginate elastic microspheres in the solidification solution at room temperature for 30 minutes; wash the elastic microspheres with distilled water for 2 to 3 times, and dry under normal pressure to remove the surface moisture;

(d) Sieving to obtain calcium alginate elastic microsphere porogens with a diameter of 100-1000 microns.

2) Put the calcium alginate elastic microsphere porogen prepared in step 1) into the mold, add the scaffold material solution to immerse the porogen, and mix evenly to obtain the scaffold/calcium alginate elastic microsphere porogen composite, Use a filter plate with micropores to press filter the compound until it reaches 90% of the initial volume of the compound, and quickly transfer the filtered compound to a medical low-temperature box at -10°C for 3 hours;

3) Demoulding the compound, freeze-drying for 8 hours;

4) Place the complex in distilled water and rehydrate for 1 hour;

5) Soak the composite in EDTA solution with pH=7 for 2 hours, then put the scaffold/porogen composite in distilled water, and heat it with microwave repeatedly for 3-5 times, each time for 2-3 minutes.

Example 5

1) Prepare the calcium alginate elastic microsphere porogen, prepare a polylactic acid dioxane/chloroform solution with a mass fraction of 3.5% as a scaffold material solution, and the volume ratio of dioxane and chloroform is 5: 2; The preparation steps of calcium alginate elastic microsphere porogen are as follows:

(a) preparation mass fraction is the sodium alginate solution of 4%;

(b) Prepare calcium alginate elastic microspheres by high-voltage electrostatic method, the voltage is 12KV, and the distance between electrodes is 2.5cm; the calcium chloride solution with a mass fraction of 20% at normal temperature is used as the solidification solution to solidify the sodium alginate microspheres into Calcium alginate microspheres; during the preparation process, a large amount of steam is passed through the outflow port of the sodium alginate solution to moisturize;

(c) Soak the calcium alginate elastic microspheres in the curing solution at room temperature for 60 minutes; wash the elastic microspheres with distilled water for 2 to 3 times, and dry under normal pressure to remove the surface moisture;

(d) Sieving to obtain calcium alginate elastic microsphere porogens with a diameter of 100-1000 microns.

2) Put the calcium alginate elastic microsphere porogen prepared in step 1) into the mold, add the scaffold material solution to immerse the porogen, and mix evenly to obtain the scaffold/calcium alginate elastic microsphere porogen composite, Use a filter plate with micropores to press filter the compound to 80% of the initial volume of the compound, and quickly transfer the filtered compound to a medical low-temperature box at -25°C for 3 hours;

3) Demoulding the compound and freeze-drying for 7 hours;

4) Place the complex in distilled water and rehydrate for 1 hour;

5) Soak the composite in EDTA solution with pH=7 for 2.5 hours, then put the scaffold/porogen composite in distilled water, and heat it with microwave repeatedly for 3-5 times, each time for 2-3 minutes.

Example 6

1) Prepare the calcium alginate elastic microsphere porogen, prepare a polylactic acid dioxane/chloroform solution with a mass fraction of 4% as a scaffold material solution, and the volume ratio of dioxane and chloroform is 5: 2; The preparation steps of calcium alginate elastic microsphere porogen are as follows:

(a) preparation mass fraction is the sodium alginate solution of 5%;

(b) Prepare calcium alginate elastic microspheres by high-voltage electrostatic method, the voltage is 13KV, and the distance between electrodes is 2cm; use calcium chloride solution with a mass fraction of 20% at room temperature as the solidification solution to solidify sodium alginate microspheres into seaweed Calcium acid microspheres; during the preparation process, a large amount of steam is passed through the outflow port of the sodium alginate solution to moisturize;

(c) Soak the calcium alginate elastic microspheres in the solidification solution at room temperature for 90 minutes; wash the elastic microspheres with distilled water for 2 to 3 times, and dry under normal pressure to remove the surface moisture;

(d) Sieving to obtain calcium alginate elastic microsphere porogens with a diameter of 100-1000 microns.

2) Put the calcium alginate elastic microsphere porogen prepared in step 1) into the mold, add the scaffold material solution to immerse the porogen, and mix evenly to obtain the scaffold/calcium alginate elastic microsphere porogen composite, Press filter the compound with a filter plate with micropores until it reaches 70% of the initial volume of the compound, and quickly transfer the filtered compound to a medical low-temperature box at -25°C for 3 hours;

3) Demoulding the compound and freeze-drying for 6 hours;

4) Place the complex in distilled water and rehydrate for 1 hour;

5) Soak the composite in EDTA solution with pH=7 for 3.5 hours, then put the scaffold/porogen composite in distilled water, and heat it with microwave for 3-5 times, 2-3 minutes each time.

Fig. 2 is a topographic microscopic picture of a polyvinyl alcohol elastic microsphere porogen with a diameter of 100-1000 microns. Through the analysis of the experimental results of the preparation of the elastic microsphere porogen, the main factors affecting the particle size of the elastic microsphere porogen are: the voltage and the distance between the electrodes. The larger the microspheres, the smaller the average particle size of the prepared microspheres; the main factors affecting the elasticity and mechanical properties of the porogen are: solution concentration, salt solution soaking time, the greater the solution concentration, the longer the salt solution soaking time, and the mechanical strength of the microspheres. and elastic properties are better. In addition, repeated freezing will enhance the mechanical strength and elastic properties of polyvinyl alcohol elastic microspheres. When the number of freezing times reaches 5-6 times, the mechanical strength and elastic properties of the microspheres can basically meet the requirements of the experiment; The soaking time of the ball in saturated salt solution should not be too long, otherwise the elastic microsphere will lose too much water, which will reduce the elasticity and fail to meet the requirements of the experiment.

Figure 3 is a picture of the appearance of the three-dimensional cell scaffold, showing the uniformity of the appearance of the scaffold.

Fig. 4 is a microscope picture of the pore structure of the three-dimensional cell scaffold, showing the pore structure of the scaffold and the connectivity between the pores.

Fig. 5 is a scanning electron microscope picture of a three-dimensional cell scaffold. It can be clearly seen from the picture that there are spherical pores formed due to the removal of the elastic microsphere porogen (that is, the macropores on the cross-section of the scaffold in Figure 5, referred to as macropores for short), and there are many connections between the macropores. Hole (that is, the small hole on the wall of the large hole in Figure 5, referred to as the communicating hole). The existence of interconnected pores makes each macropore interconnected, indicating that the contact surface of the elastic microsphere porogens during the press filtration process is better transformed into a communication channel between the pores of the scaffold after the press filtration, ensuring Porosity of three-dimensional cellular scaffolds.

Observe the appearance and microstructure of the scaffolds prepared in Examples 1-6, and measure the pore size and pore communication channel size with a ruler. The results are shown in Table 1:

Table 1

Degree of filtration Pore size of scaffold (μm) Channel size between scaffold pores (μm) 70% 639 326±26.6(8) 80% 539 283±52.0(8) 90% 593 257±36.2(8)

From the above experimental results, it can be known that: (1) The size of the elastic microsphere porogen affects the pore size of the scaffold. The larger the particle size of the porogen, the larger the pore size of the prepared scaffold; the greater the pressure filtration degree of the porogen/scaffold composite, the greater the elastic deformation of the porogen, and the larger the pore size of the prepared scaffold. The pore size of the scaffold can be controlled by controlling the size of the elastic microspheres. (2) The effect of the degree of filtration on the size of the connecting channels between the pores of the scaffold. It can be seen from the above table that the greater the pressure filtration degree of the porogen/scaffold complex, the larger the contact area of the microspheres, and the larger the size of the inter-pore communication channel formed after the removal of the porogen. By adjusting the degree of filtration, the size of the communication channels between the pores of the scaffold can be controlled.

Claims (3)

1. A method for preparing a three-dimensional cell scaffold with an elastic microsphere porogen, characterized in that the steps are as follows 1) preparing an elastic microsphere porogen, and preparing a scaffold material solution; 2) Put the elastic microsphere porogen in the mold, add the scaffold material solution to immerse the porogen, mix evenly to obtain the scaffold/elastic microsphere porogen composite, and use a filter press plate with micropores to prepare the composite Press filtration, so that the elastic microsphere porogen is evenly distributed in the solution and can be in contact with each other, and the composite is pre-frozen at -10°C or lower for 3 hours; 3) Demoulding the compound and freeze-drying for 6-8 hours; 4) Place the complex in distilled water and rehydrate for 1 hour; 5) Remove the porogen. 2. a kind of method for preparing three-dimensional cell scaffold with elastic microsphere porogen according to claim 1, is characterized in that: in the 1st) step, elastic microsphere porogen adopts polyvinyl alcohol to prepare, and its steps are as follows: (a) preparing a polyvinyl alcohol solution with a mass fraction of 7% to 12%; (b) High-voltage electrostatic method to prepare polyvinyl alcohol elastic microspheres, the high voltage range is 10-15KV, and the distance between electrodes is 2-3cm; it is 2:1 chloroform and acetone mixed with a volume ratio lower than -25°C The solution is used as a solidifying liquid to solidify the microspheres; during the preparation process, a large amount of steam is passed through the outflow port of the polyvinyl alcohol solution to moisturize; (c) repeatedly freezing the polyvinyl alcohol elastic microspheres in the solidified liquid 4 to 6 times, the freezing temperature is -25°C or lower, the freezing time is 10 hours each time, and the thawing time is 1 hour; (d) Soak the polyvinyl alcohol elastic microspheres in saturated sodium chloride or saturated potassium chloride solution at normal temperature for 20 to 60 minutes; rinse the elastic microspheres with distilled water for 2 to 3 times, and dry under normal pressure to remove the surface moisture; (e) sieving to obtain an elastic microsphere porogen with a diameter of 100 to 1000 microns; Step 5) The porogen removal method is to put the scaffold/polyvinyl alcohol elastic microsphere porogen complex into distilled water, and repeat the microwave heating for 5-10 times, each time for 2-3 minutes. 3. a kind of method for preparing three-dimensional cell scaffold with elastic microsphere porogen according to claim 1, is characterized in that: in the 1st) step, elastic microsphere porogen adopts sodium alginate to prepare, and its steps are as follows: (a) preparing a sodium alginate solution with a mass fraction of 3% to 5%; (b) Prepare calcium alginate elastic microspheres by high voltage electrostatic method, the voltage range is 10-13KV, and the distance between electrodes is 2-3cm; The microspheres are solidified into calcium alginate microspheres; during the preparation process, a large amount of steam is passed through the outflow port of the sodium alginate solution to moisturize; (c) Soak the calcium alginate elastic microspheres in the solidification solution at room temperature for 30-90 minutes; wash the elastic microspheres with distilled water for 2-3 times, and dry under normal pressure to remove the surface moisture; (d) sieving to obtain a calcium alginate elastic microsphere porogen with a diameter of 100 to 1000 microns; Step 5) The porogen removal method is to put the scaffold/calcium alginate elastic microsphere porogen complex into the EDTA solution with pH=7 and soak for 2-3.5h, and then pore-forming the scaffold/calcium alginate elastic microsphere The agent complex was placed in distilled water, and microwave heating was repeated for 3 to 5 times, each time for 2 to 3 minutes. the

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