CN110124105A - The biological 3D printing ink-manufacturing method of controllable gel-sol phase transition temperature - Google Patents

Abstract

The invention discloses a kind of biological 3D printing ink-manufacturing methods of controllable gel-sol phase transition temperature, the present invention by regulation gelatin/sodium alginate 3D printing aquogel system Sol-gel phase transition temperature, by control within the temperature range of most suitable cell is grown.Then the cartilage frame that high resolution is printed using cell 3D printing technique improves the printability and biocompatibility of hydrogel material.Inorganic nanoparticles are added in this basic aquogel system, the mechanical property of cartilage frame can be improved, to solve the technical problem that printing shaping temperature is mismatched with cell growth temperature and hydrogel material mechanical property is bad.

Description

Preparation method of 3D bioprinting ink with adjustable gel-sol phase transition temperature

technical field

The invention relates to the technical field of material preparation of artificial cartilage scaffolds prepared by biological 3D printing, in particular to a method for preparing cartilage scaffolds with sodium alginate/gelatin-based printing ink that can regulate the sol-gel transition temperature.

Background technique

There are more and more patients with cartilage defects caused by obesity, exercise, age or disease, and it has become a major killer of human health. At present, cartilage repair is still a major problem in clinical medicine. In recent years, the development of cell 3D printing technology has promoted the application of additive manufacturing technology in the field of tissue engineering. Cell 3D printing technology not only regulates the mechanical properties of the scaffold by controlling the microstructure, shape and composition of the tissue scaffold with high precision, but also has a certain impact on the number and distribution of cells.

Since polysaccharide matrices such as gelatin, sodium alginate, and chitosan are derived from natural tissues, they are the main components of the extracellular matrix and have good biocompatibility, so they are often used as bioprinting inks for 3D bioprinting. The sol-gel state transition of the gelatin/sodium alginate-based printing ink system is reversible. At a certain temperature, the printing ink will undergo a sol-gel state transition, allowing the scaffold to be printed. Therefore, gelatin/sodium alginate bioink has higher requirements on printing temperature. At the same time, cells are very sensitive to the temperature of the living environment, so the printing temperature is extremely high. However, the sol-gel transition temperature suitable for scaffold printing does not necessarily match the temperature range suitable for cell growth. If the two are not matched, the printability of the printing ink using mixed cells will be affected, and the activity of the cells encapsulated in the printing ink will also be adversely affected. The application of 3D printed cartilage scaffolds to repair damaged cartilage is limited. It is a challenge to develop bioprinting inks suitable for cell 3D printing for the preparation of cartilage scaffolds. Therefore, inventing a gelatin/sodium alginate-based printing ink with adjustable sol-gel phase transition temperature is a technical problem to be solved urgently in this field.

Contents of the invention

Insufficient in the prior art of the present invention, the purpose of the present invention is to prepare a gelatin/sodium alginate system with adjustable sol-gel phase transition temperature, and improve the printing accuracy of the mixed cell printing slurry. The invention can regulate the sol-gel phase transition temperature of the gelatin/sodium alginate 3D printing hydrogel system, and control it in the most suitable temperature range for cell growth. Then, a high-resolution cartilage scaffold was printed using cell 3D printing technology to improve the printability and biocompatibility of the hydrogel material. Adding inorganic nanoparticles to this basic hydrogel system can improve the mechanical properties of the cartilage scaffold, thereby solving the technical problems of the mismatch between the printing temperature and the cell growth temperature and the poor mechanical properties of the hydrogel material.

Technical scheme of the present invention is to realize like this:

The invention provides a method for regulating the sol-gel transition temperature of a temperature-sensitive hydrogel system based on gelatin/sodium alginate printing ink, and the preparation process is as follows:

Step (1) Dissolving or dispersing the gelatin powder in deionized water, heating in a water bath at a temperature of 65°C to 90°C, stirring at a speed of 10 to 30r/min, heating and stirring for 20 to 45min, and preparing a gelatin solution.

Step (2) Dissolve or disperse sodium alginate in the gelatin solution, heat in a water bath at a temperature of 65°C to 90°C, stir at a speed of 10 to 30r/min, heat and stir for 80 to 150min, and prepare a sodium alginate/gelatin solution Mix the hydrogel solution.

Step (3) The sodium alginate/gelatin solution is mixed with the hydrogel solution, and the temperature is naturally cooled to 20° C. to 28° C. in a water bath; the sodium alginate/gelatin mixed hydrogel is obtained;

In step (4), the heating and cooling cycles of the sodium alginate/gelatin solution mixed hydrogel solution are performed multiple times; through different heating and cooling times; sodium alginate/gelatin mixed hydrogels with different phase transition temperatures are obtained, namely 3D bioprinting inks with different phase transition temperatures were obtained.

The heating method is as follows: the sodium alginate/gelatin solution mixed hydrogel solution is heated in a water bath at a temperature of 65°C to 90°C, stirring at a rotation speed of 10 to 30r/min, and heating and stirring for 50 to 120min;

The cooling method is as follows: the sodium alginate/gelatin solution mixed with the hydrogel solution is naturally cooled to 20°C to 28°C in a water bath.

As preferably, the temperature of the water bath in step (1) is 78°C;

As preferably, the rotating speed in step (1) is 20r/min;

As preferably, the total heating and stirring of step (1) and step (2) is 110-130min;

As preferably, all will stop stirring and cool down naturally in cooling process;

Preferably, the mass ratio of gelatin to sodium alginate is 4:1, and the concentrations of gelatin and sodium alginate in the mixed solution are 8%-8.8% and 2.0%-2.2%, respectively.

As a preference, during the cooling process of the sodium alginate/gelatin solution mixed hydrogel solution, first uniformly ultrasonically disperse the nanoparticles into the deionized medium at room temperature, when the sodium alginate/gelatin solution mixed hydrogel solution stops heating and stirring Afterwards, when the temperature was lowered to 50° C., the homogeneous suspension of nanoparticles was then added to the sodium alginate/gelatin solution mixed hydrogel solution and heated and stirred at 50° C. for 60 min.

The prepared gelatin/sodium alginate mixed printing ink containing inorganic nanoparticles is printed into a 3D cartilage scaffold at room temperature by a cell assembly machine, and ionically cross-linked by a calcium chloride solution to obtain a high biological activity and biological Compatible and precise composite hydrogel 3D porous cartilage scaffold.

The gelatin, sodium alginate, and inorganic nanoparticles mentioned above can be purchased commercially, and the deionized water is prepared through equipment.

Compared with the prior art, the present invention has the following advantages:

1) The present invention can regulate the sol-gel transition temperature and viscosity of the temperature-sensitive hydrogel material using gelatin/sodium alginate as the bioprinting slurry.

2) A variety of inorganic nanoparticles can be added to the hydrogel system based on gelatin/sodium alginate printing paste, which can enhance the mechanical properties of the hydrogel and have an effect on cell adhesion.

3) The process method of the present invention is simple, low in cost, convenient to be suitable for industrialized production, and can be widely used in the field of biomedicine.

4) Compared with the gelatin/sodium alginate hydrogel system prepared by ordinary methods, the present invention can balance 3D printing accuracy, scaffold activity and cell compatibility, and can prepare fine cartilage scaffolds with good mechanical properties for cartilage repair .

Description of drawings

Figure 1 is a trend diagram of storage modulus and loss modulus with temperature when the conversion temperature is 31.6°C;

Figure 2 is a graph showing the variation of storage modulus and loss modulus with temperature when the conversion temperature is 30.5°C;

Figure 3 is a trend diagram of storage modulus and loss modulus with temperature when the conversion temperature is 29.2°C;

Fig. 4 is the variation of the viscosity of the base printing slurry with temperature in different processing methods;

Fig. 5 is the phase change temperature diagram of the basic printing paste of different treatment methods;

Figure 6(a) and (b) are the structural diagrams printed at 21°C after performing three heating and cooling cycles;

Figure 7 is a structural diagram printed at 25°C after performing three heating and cooling cycles;

Figure 8 is a structural diagram printed at 25°C after performing two heating and cooling cycles;

Figure 9 is a diagram of the structure printed at 25 °C after performing one heating and cooling cycle.

Detailed ways

The technical solutions and technical effects of the present invention will be described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of the present invention.

In the following examples, gelatin, sodium alginate, nano-scale hydroxyapatite, and nano-cellulose are all commonly used materials in the field, and can be obtained commercially.

Several specific examples are provided below to help understand the present invention.

Example 1: Swell 4g of gelatin powder in a beaker filled with 20ml of deionized water for 30min, put the second beaker into a water bath magnetic stirrer with a water temperature of 78°C and heat and stir at a speed of 20r/min for 30min to obtain a concentration of 20.0% (W/V) gelatin aqueous solution, add 1g sodium alginate powder, 25ml deionized water in the beaker that fills gelatin aqueous solution, the heating temperature of magnetic stirrer keeps constant, and the rotating speed is adjusted to 10r/min, heating and stirring 80min, finally Obtain the sodium alginate of concentration about 2.2% (W/V) and the gelatin composite solution of concentration about 8.8% (W/V); Gradually lower to 20°C, take out and refrigerate for later use. 3D cartilage hydrogel scaffolds were printed at room temperature using a cell-controlled assembly machine. The printed scaffold was cross-linked in 4% CaCl ₂ solution, the scaffold was taken out of the CaCl ₂ solution, and the scaffold was rinsed with deionized water for 3 times to obtain a hydrogel scaffold with better mechanical properties.

Embodiment 2: 4g gelatin powder is filled in the beaker of 20ml deionized water and swells 30min, and the beaker is put into the water bath magnetic stirrer that water temperature is 78 ℃ and is heated and stirred at the speed of 20r/min for 30min to make concentration be 20.0% ( W/V) gelatin aqueous solution, add 1g sodium alginate powder, 25ml deionized water in the beaker that is filled with gelatin aqueous solution, the heating temperature of magnetic stirrer keeps constant, and the rotating speed is adjusted to 14r/min, heats and stirs 90min, finally obtains A compound solution of sodium alginate with a concentration of about 2.2% (W/V) and gelatin with a concentration of about 8.8% (W/V); adjust the heating temperature of the magnetic stirrer to 28°C, and set the speed to 0r/min, and compound The temperature of the solution gradually decreased to 28°C along with the water temperature; again, the heating temperature was set to 78°C, and the rotation speed was set to 14r/min. After heating and stirring for 60 minutes, the temperature was lowered again, and the solution was taken out and refrigerated for later use. Take it out and heat it to room temperature, and take out a part of the material for rheological performance test. The change of its loss modulus and storage modulus with temperature is shown in Figure 2. 3D cartilage hydrogel scaffolds were printed at room temperature using a cell-controlled assembly machine. The printed scaffold was cross-linked in 4% CaCl ₂ solution for 10 min, the scaffold was taken out from the CaCl ₂ solution, and the scaffold was rinsed with deionized water for 3 times to obtain a hydrogel scaffold with good mechanical properties.

Example 3: Swell 4g of gelatin powder in a beaker filled with 20ml of deionized water for 30min, put the second beaker into a water bath magnetic stirrer with a water temperature of 78°C and heat and stir at a speed of 20r/min for 30min to obtain a concentration of 20.0% (W/V) gelatin aqueous solution, add 1g sodium alginate powder, 25ml deionized water in the beaker that is filled with gelatin aqueous solution, the heating temperature of magnetic stirrer keeps constant, and the rotating speed is adjusted to 14r/min, heating and stirring for 90min, finally Obtain a sodium alginate with a concentration of about 2.2% (W/V) and a gelatin composite solution with a concentration of about 8.8% (W/V); the heating temperature of the magnetic stirrer is adjusted to 28°C, and the rotating speed is set to 0r/min, The temperature of the composite solution gradually decreased to 28°C along with the water temperature; the heating temperature was set to 78°C again, the rotation speed was set to 14r/min, and the temperature was lowered again after heating and stirring for 60 minutes; Take it out and heat it to room temperature, and take out a part of the material for rheological performance test. The change of loss modulus and storage modulus with temperature is shown in Figure 3. 3D cartilage hydrogel scaffolds were printed at room temperature using a cell-controlled assembly machine. The printed scaffold was cross-linked in 4% CaCl ₂ solution for 10 min, the scaffold was taken out from the CaCl ₂ solution, and the scaffold was rinsed with deionized water for 3 times to obtain a hydrogel scaffold with good mechanical properties.

Example 4: Swell 4g of gelatin powder in a beaker filled with 20ml of deionized water for 30min, put the second beaker into a water bath magnetic stirrer with a water temperature of 78°C and heat and stir at a speed of 20r/min for 20min to obtain a concentration of 20.0% (W/V) gelatin aqueous solution, add 1g sodium alginate powder, 25ml deionized water in the beaker that is filled with gelatin aqueous solution, the heating temperature of magnetic stirrer keeps constant, and the rotating speed is adjusted to 14r/min, heating and stirring for 90min, finally Obtain a sodium alginate with a concentration of about 2.2% (W/V) and a gelatin composite solution with a concentration of about 8.8% (W/V); the heating temperature of the magnetic stirrer is adjusted to 28°C, and the rotating speed is set to 20r/min, The temperature of the composite solution gradually decreased to 28°C along with the water temperature; again, the heating temperature was set to 78°C, and the rotation speed was set to 14r/min. After heating and stirring for 60 minutes, the temperature was lowered again, and it was taken out and refrigerated for later use. Take out the gelatin/sodium alginate basic composite hydrogel printing paste, heat it in a water bath to 50°C, add 0.55g of nano-sized hydroxyapatite to the basic hydrogel system and heat and stir for 1 hour. Cool at room temperature to room temperature, and then store the gelatin/sodium alginate hydrogel containing nano-hydroxyapatite in cold storage for future use. 3D cartilage scaffolds were printed at room temperature using a cell-controlled assembler. The printed scaffold was placed in 4% CaCl ₂ solution for cross-linking for 10 min, the scaffold was taken out of the CaCl ₂ solution, and the scaffold was rinsed with deionized water for 3 times to obtain a hydrogel scaffold with better mechanical properties.

Example 5: Swell 4g of gelatin powder in a beaker filled with 23ml of deionized water for 30min, put the second beaker into a water bath magnetic stirrer with a water temperature of 65°C and heat and stir at a speed of 10r/min for 30min to obtain a concentration of 20.0% (W/V) gelatin aqueous solution, add 1g sodium alginate powder, 25ml deionized water in the beaker that fills gelatin aqueous solution, the heating temperature of magnetic stirrer keeps constant, and the rotating speed is adjusted to 10r/min, heating and stirring 80min, finally Obtain a sodium alginate with a concentration of about 2.2% (W/V) and a gelatin composite solution with a concentration of about 8.8% (W/V); the heating temperature of the magnetic stirrer is adjusted to 20°C, and the rotating speed is set to 10r/min, The temperature of the composite solution gradually decreased to 20°C along with the water temperature; again, the heating temperature was set to 65°C, and the rotation speed was set to 10r/min. After heating and stirring for 50 minutes, the temperature was lowered again, and it was taken out and refrigerated for later use. 3D cartilage scaffolds were printed at room temperature using a cell-controlled assembler. Put the printed scaffold in 4% CaCl ₂ solution for cross-linking, take the scaffold out of the CaCl ₂ solution, rinse the scaffold 3 times with deionized water, and obtain the gelatin/sodium alginate hydrogel scaffold.

Example 6: Swell 4g of gelatin powder in a beaker filled with 25ml of deionized water for 30min, put the second beaker into a water bath magnetic stirrer with a water temperature of 90°C and heat and stir at a speed of 30r/min for 45min to obtain a concentration of 20.0% (W/V) gelatin aqueous solution, add 1g sodium alginate powder, 25ml deionized water in the beaker that is filled with gelatin aqueous solution, the heating temperature of magnetic stirrer keeps constant, and the rotating speed is adjusted to 30r/min, heating and stirring 150min, finally Obtain a sodium alginate with a concentration of about 2.2% (W/V) and a gelatin composite solution with a concentration of about 8.8% (W/V); the heating temperature of the magnetic stirrer is adjusted to 28°C, and the rotating speed is set to 30r/min, The temperature of the composite solution gradually decreased to 28°C along with the water temperature; again, the heating temperature was set to 90°C, and the rotation speed was set to 30r/min. After heating and stirring for 120min, the temperature was lowered again, and the solution was taken out and refrigerated for later use. 3D cartilage scaffolds were printed at room temperature using a cell-controlled assembler. Put the printed scaffold in 4% CaCl ₂ solution for cross-linking, take the scaffold out of the CaCl ₂ solution, rinse the scaffold 3 times with deionized water, and obtain the gelatin/sodium alginate hydrogel scaffold.

It can be seen from Figure 4 that increasing the heating and cooling times of the gelatin-sodium-alginate mixed hydrogel solution leads to a significant decrease in the viscosity of the gelatin-sodium-alginate mixed hydrogel.

It can be seen from Figure 5 that increasing the heating and cooling times of the gelatin-sodium-alginate mixed hydrogel solution leads to a significant decrease in the sol-gel transition temperature.

In Fig. 5 and 6, A: process once B: process twice C: process three times.

The temperature of the 3D printing environment in Figure 6 is 21°C. Due to the relatively high phase transition temperature of bio-inks that have been processed less frequently, the accumulation of broken filaments occurs when printing at a lower temperature, as shown in the two brackets on the left in Figure 6; The stent that has been heated three times has a relatively low transformation temperature, and the printing accuracy is relatively high under the condition of 21 °C, and no wire breakage occurs.

The gelatin/sodium alginate hydrogel solution was processed by different processing methods to obtain 3D printing bioinks with different phase transition temperatures. Printed at room temperature of 25°C, it can be seen from Figure 7 that the filaments extruded from the bio-3D printing ink processed three times are not easy to solidify and the scaffolds are easy to collapse. This is because the bio-ink obtained three times has a low sol-gel Gel conversion temperature; as can be seen from Figure 9, bio-3D printing inks that are processed once are prone to broken filaments, and the molding effect is not good, which is due to the higher conversion temperature of bio-3D printing inks that have been processed less times; from Figure 8 As can be seen in the figure, the 3D printing bio-ink with the best molding effect is processed twice. Therefore, at a specific temperature, the phase transition temperature of the hydrogel material can be adjusted to make the molding effect at this temperature the best.

Claims (7)

1. the biological 3D printing ink-manufacturing method of controllable gel-sol phase transition temperature, which is characterized in that this method is specifically wrapped Include following steps:

Gelatin powder is dissolved or dispersed in deionized water by step (1), and the temperature of heating water bath is 65 DEG C~90 DEG C, and revolving speed is 10~30r/min stirring, 20~45min of heating stirring are made into gelatin solution；

Sodium alginate is dissolved or dispersed in gelatin solution by step (2), and the temperature of heating water bath is 65 DEG C~90 DEG C, and revolving speed is 10~30r/min stirring, 80~150min of heating stirring are made into sodium alginate/glutin solution mixing hydrogel solution；

Sodium alginate/glutin solution mixing hydrogel solution in water-bath is stopped stirring and Temperature fall is to 20 by step (3) DEG C~28 DEG C；Obtain sodium alginate/glutin mixing hydrogel；

Step (4) again executes the heating of sodium alginate/glutin solution mixing hydrogel solution and cooling cycle multiple；By difference Heating and cooling number；Obtain having the sodium alginate/glutin mixing hydrogel of different phase transition temperatures to get to different phases The biological 3D printing ink of temperature；

Wherein heating means are as follows: by 65 DEG C~90 DEG C of temperature of sodium alginate/glutin solution mixing hydrogel solution heating water bath, Revolving speed is 10~30r/min stirring, 50~120min of heating stirring；

Cooling means are as follows: sodium alginate/glutin solution mixing hydrogel solution is stopped into stirring and Temperature fall in water-bath To 20 DEG C~28 DEG C.

2. the biological 3D printing ink-manufacturing method of controllable gel-sol phase transition temperature according to claim 1, special Sign is: the water bath heating temperature in step (1) is 78 DEG C.

3. the biological 3D printing ink-manufacturing method of controllable gel-sol phase transition temperature according to claim 1, special Sign is: the revolving speed in step (1) is 20r/min.

4. the biological 3D printing ink-manufacturing method of controllable gel-sol phase transition temperature according to claim 1, special Sign is: step (1) and step (2) heating stirring summation are 110-130min.

5. the biological 3D printing ink-manufacturing method of controllable gel-sol phase transition temperature according to claim 1, special Sign is: being intended to stop stirring and Temperature fall in cooling procedure.

6. the extendible ink-manufacturing method of 3D printing of controllable gel-sol phase transition temperature according to claim 1, It is characterized in that: in sodium alginate/glutin solution mixing hydrogel solution cooling procedure, first at room temperature by nano particle Uniform ultrasonic disperse after sodium alginate/glutin solution mixing hydrogel solution stops heating stirring, works as temperature into deionization When being reduced to 50 DEG C, then nano particle unit for uniform suspension is added in sodium alginate/glutin solution mixing hydrogel solution 50 DEG C of heating stirring 60min.

7. the biological 3D printing ink-manufacturing method of controllable gel-sol phase transition temperature according to claim 1, special Sign is: the mass ratio of the gelatin and sodium alginate is 4:1, and gelatin and sodium alginate remix the mass concentration in solution Respectively 8%~8.8% and 2.0%~2.2%.