CN110152062A - A 3D bioprinted hydrogel scaffold for tissue regeneration and its preparation method - Google Patents

Abstract

The invention mainly relates to a printable 3D hydrogel support and a preparation method thereof, belonging to the field of tissue engineering. The 3D printable hydrogel scaffold provided by the present invention is composed of gellan gum with biocompatibility and shear thinning behavior characteristics and plastic soluble starch, and printed with smooth surface and complete hollow out by 3D printing technology. Composite scaffolds with prismatic structures for tissue injury repair and nerve regeneration. The scaffold has good stability and is printed from biodegradable and absorbable biomaterials. The scaffold highly mimics the three-dimensional growth space of cells in the body, which is conducive to the attachment of cells and allows the diffusion of nutrients and the discharge of waste, so that cells can fully grow and proliferate.

Description

A 3D bioprinted hydrogel scaffold for tissue regeneration and its preparation method

technical field

The invention relates to the field of tissue engineering, in particular to a 3D bioprinted hydrogel scaffold applied to tissue regeneration and a preparation method thereof, specifically belonging to the field of tissue engineering nerve regeneration.

Background technique

Peripheral nerves are the bridge connecting the nerve center and peripheral target structures. Its main function is to sense stimuli, transmit nerve impulses to the nerve center, and transmit the impulse of the nerve center to control muscle movement and gland secretion. Peripheral nerve defect refers to the disconnection of the peripheral nerve trunk caused by trauma or surgery. After restoring its "bioelasticity" in the functional position, there is still a distance between the broken ends. In daily production and life, peripheral nerve injuries caused by traffic accidents, industrial injuries, wars, earthquakes and clinical surgical accidents are common clinical symptoms. According to the statistics of the World Health Organization, there are about 360,000 new cases of peripheral nerve injury in the United States every year, and more than 300,000 new cases in Europe every year. There are about 1 million new cases of peripheral nerve injury in my country every year, and about 300,000 cases of peripheral nerve defect.

At present, traditional autologous nerves are still used as nerve bridges in clinical peripheral nerve defects. In recent years, great progress has been made in the use of tissue engineering nerve bridges to repair nerve defects, and many products have entered clinical or are about to enter clinical use.

However, the limited sources of traditional autologous nerve bridges, the mismatch of tissue size and structure, long-term denervation of the graft donor site, secondary injury and other reasons, so the application of autologous nerve transplantation has great limitations. Compared with autologous transplantation, the repairing effect of commonly used artificial nerve grafts is not very satisfactory, especially when repairing long-distance, thick or old nerve defects, its repairing effect is still far behind compared with autologous transplantation.

Therefore, the development of a new type of 3D bioprinted hydrogel scaffold that can be used for nerve regeneration, which can be compared to autologous nerve grafts, guide nerves to grow into the distal side, and shape new nerves, has broad clinical application prospects as artificial nerve grafts. .

Contents of the invention

The technical problem to be solved by the present invention is to provide a 3D bioprinted hydrogel scaffold for nerve regeneration and a preparation method thereof. After 3D printing, the printed bracket provided by the present invention has good gelation property.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

The invention provides a 3D bioprinted hydrogel scaffold applied to tissue regeneration and a preparation method thereof. The hydrogel scaffold is composed of gellan gum and plastic The characteristic soluble starch was mixed and prepared a composite scaffold with smooth surface and complete structure by means of 3D printing technology.

The invention provides a method for preparing a 3D bioprinted hydrogel scaffold applied to tissue regeneration, the specific steps are as follows:

Step 1: adding gellan gum powder into water to prepare a gellan gum solution;

Step 2: On the basis of step 1, take the soluble starch powder and prepare it into a mixed solution of gellan gum and starch;

Step 3: Pour the mixed solution in step 2 into a 50ml centrifuge tube, and place it to cool at room temperature;

Step 4: Put the cooled gellan gum and starch mixed solution in a 4°C refrigerator overnight to obtain raw materials for preparing 3D bioprinted hydrogel scaffolds.

Step 5: Put the cooled mixed raw material into the extrusion-based 3D printer barrel, and print according to the imported model to obtain a composite bracket.

Step 6: Sterilize the printed scaffold.

Step 7: Modify the sterilized scaffold to achieve the purpose of modifying the scaffold function, and then inoculate cells.

In one embodiment of the present invention, the water is ultrapure water.

In one embodiment of the present invention, in the step 1, gellan gum (GG) is added into ultrapure water, stirred and dissolved in a water bath, and an aqueous solution of gellan gum is prepared.

In one embodiment of the present invention, the mass ratio of ultrapure water and gellan gum in the step 1 is 70-100:16.

In one embodiment of the present invention, the step 2 is to add a certain proportion of soluble starch powder to the gellan gum aqueous solution obtained in step 1 to prepare a mixed solution of gellan gum and soluble starch.

In one embodiment of the present invention, the mass ratio of ultrapure water, gellan gum and soluble starch in the step 2 is 70-100:16:4-16.

In one embodiment of the present invention, in the step 2, the temperature of the water bath for preparing the mixed solution is 90-100°C.

In one embodiment of the present invention, in the step 2, the preparation of the mixed solution requires stirring for 30-40 minutes.

In one embodiment of the present invention, in the step 2, a stirring speed of 1000 rpm is required for preparing the mixed solution.

In one embodiment of the present invention, in step 3, the prepared mixed solution needs to be cooled at room temperature for about 2 hours, and then put into a 50ml centrifuge tube to continue cooling for 1 hour.

In one embodiment of the present invention, in step 5, the imported 3D printing models are cuboids or cubes with different side lengths, the printing speed of the 3D printer is 15-20mm/s, the nozzle temperature is 240°C, and the platform temperature is 17°C , the diameter of the printing needle is 0.74mm, the thickness of the printing layer is 1mm, the filling distance is 2mm, and the air pressure is 0.11~0.3MPa.

In one embodiment of the present invention, in the step 6, the printed scaffold is frozen in a -20°C refrigerator for 48 hours, then placed in a vacuum freeze dryer at -50°C for 36 hours, and finally sterilized by ultraviolet light for 2 hours.

In one embodiment of the present invention, the modification method adopted in step 7 is to graft the sterilized scaffolds with different concentrations (0.5-10 μg/ml) of YIGSR polypeptides, and the grafting time is 1 h.

Compared with the prior art, a hydrogel scaffold for 3D bioprinting of the present invention and its preparation method have the following beneficial effects:

1. The hydrogel scaffold of the present invention can simulate the three-dimensional growth environment of cells, facilitate the attachment of cells, and promote the growth and proliferation of cells;

2. In the present invention, the composite scaffold of biocompatible, non-toxic and shear-thinning gellan gum and plastic soluble gellan gum can meet the requirements of scaffold transplantation and can be applied to nerve regeneration.

Description of drawings

1 is a schematic diagram of the preparation of a 3D bioprinted hydrogel scaffold for tissue regeneration in the present invention;

2 is a schematic diagram of the 3D bioprinted hydrogel scaffold for tissue regeneration obtained in Example 1 of the present invention;

Fig. 3 is a schematic diagram of the 3D bioprinted hydrogel scaffold for tissue regeneration obtained in Example 2 of the present invention.

Detailed ways

The present invention will be described in further detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Example 1:

The composite scaffold 3D printing material is made from the following raw materials in parts by mass:

16 parts gellan gum

4 parts soluble starch

80 parts of water

In the example of the present invention, the water is ultrapure water, which can avoid the damage of ions to the bracket.

In the example of the present invention, gellan gum is food grade, has good heat resistance and acid resistance, and has high stability to enzymes. It dissolves into a transparent solution when heated, and forms a transparent and solid gel after cooling. Suitable for melt extrusion 3D printing.

In the example of the present invention, the starch is a soluble starch, insoluble in cold water, dissolved in boiling water, and forms a gel after cooling. The ratio of soluble starch to gellan gum is 4:1. At this ratio, the printing material can be smoothly extruded to form a filamentous substance, and piled up according to the printing path to form a composite scaffold with a smooth surface and a complete structure.

Specific steps are as follows:

Step 1: adding 16g of gellan gum powder to ultrapure water to prepare a gellan gum solution;

Step 2: Based on the basis of step 1, weigh 4g of soluble starch powder and prepare it into a mixed solution of gellan gum and starch. The temperature is 90°C, the stirring speed is 1000rpm/min, and the stirring time is 40min. Cold, can be stirred for 1h;

Step 3: Pour the mixed solution in step 2 into a 50ml centrifuge tube, and place it to cool at room temperature;

Step 4: Put the cooled gellan gum and starch mixed solution in a 4°C refrigerator overnight to obtain raw materials for preparing 3D bioprinted hydrogel scaffolds;

Step 5: Put the cooled mixed raw material into the extrusion-based 3D printer barrel, and print according to the imported model. The imported 3D printing model is a cuboid of 1.5cm×1.5cm×0.3cm. The printing speed of the 3D printer is 15mm/s, nozzle temperature 240°C, platform temperature 17°C, print needle diameter 0.74mm, print layer thickness 1mm, filling distance 2mm, air pressure 0.11MPa to obtain a composite stent;

Step 6: Put the printed scaffold at -20°C for finalization, and then sterilize;

Step 7: Modify the sterilized scaffold to achieve the purpose of modifying the scaffold function, and then inoculate cells.

Example 2:

The composite scaffold 3D printing material is made from the following raw materials in parts by mass:

16 parts gellan gum

Soluble starch 5.33 parts

8.67 parts of water

In the example of the present invention, the starch is a soluble starch, insoluble in cold water, dissolved in boiling water, and forms a gel after cooling. The ratio of soluble starch to gellan gum is 3:1. At this ratio, the printing material can be smoothly extruded to form a filamentous substance, and piled up according to the printing path to form a composite scaffold with a smooth surface and a complete structure.

In the example of the present invention, the provided 3D printing material has a reasonable component distribution ratio, a fast gelation speed during the printing process, rapid prototyping, and a complete structure of the printed scaffold.

The preparation method and follow-up treatment of 3D printing material of the present invention comprise:

Step 1: adding 16g of gellan gum powder to ultrapure water to prepare a gellan gum solution;

Step 2: Based on the basis of step 1, weigh 5.33g of soluble starch powder and prepare it into a mixed solution of gellan gum and starch. The temperature is 90°C, the stirring speed is 1000rpm/min, and the stirring time is 40min. If the weather If it is colder, it can be stirred for 1 hour;

Step 3: Pour the mixed solution in step 2 into a 50ml centrifuge tube, and place it to cool at room temperature;

Step 4: Put the cooled gellan gum and starch mixed solution in a 4°C refrigerator overnight to obtain raw materials for preparing 3D bioprinted hydrogel scaffolds.

Step 5: Put the cooled mixed raw material into the extrusion-based 3D printer barrel, and print according to the imported model. The imported 3D printing model is a cuboid of 1.5cm×1.5cm×0.3cm. The printing speed of the 3D printer is 15mm/s, nozzle temperature 240°C, platform temperature 17°C, print needle diameter 0.74mm, print layer thickness 1mm, filling distance 2mm, air pressure 0.11MPa to obtain a composite stent.

Step 6: Place the printed scaffold at -20°C for finalization and then sterilize.

Step 7: Modify the sterilized scaffold to achieve the purpose of modifying the scaffold function, and then inoculate cells.

Example 3:

The composite scaffold 3D printing material is made from the following raw materials in parts by mass:

16 parts gellan gum

8 parts soluble starch

76 parts of water

In the example of the present invention, the starch is a soluble starch, insoluble in cold water, dissolved in boiling water, and forms a gel after cooling. The ratio of soluble starch to gellan gum is 2:1. At this ratio, the printing material can be smoothly extruded to form a filamentous substance, and piled up according to the printing path to form a composite scaffold with a smooth surface and a complete structure.

In the example of the present invention, the provided 3D printing material has a reasonable component distribution ratio, a fast gelation speed during the printing process, rapid prototyping, and a complete structure of the printed scaffold.

The preparation method and follow-up treatment of 3D printing material of the present invention comprise:

Step 1: adding 16g of gellan gum powder to ultrapure water to prepare a gellan gum solution;

Step 2: Based on the basis of step 1, weigh 8g of soluble starch powder and prepare it into a mixed solution of gellan gum and starch. The temperature is 90°C, the stirring speed is 1000rpm/min, and the stirring time is 40min. Cold, can be stirred for 1h;

Step 3: Pour the mixed solution in step 2 into a 50ml centrifuge tube, and place it to cool at room temperature;

Step 4: Put the cooled gellan gum and starch mixed solution in a 4°C refrigerator overnight to obtain raw materials for preparing 3D bioprinted hydrogel scaffolds.

Step 5: Put the cooled mixed raw material into the extrusion-based 3D printer barrel, and print according to the imported model. The imported 3D printing model is a cuboid of 2cm×2cm×0.5cm, and the printing speed of the 3D printer is 20mm/ s, the nozzle temperature is 240°C, the platform temperature is 17°C, the diameter of the printing needle is 1mm, the thickness of the printing layer is 1mm, the filling distance is 2mm, and the air pressure is 0.2MPa to obtain a composite scaffold.

Step 6: Place the printed scaffold at -20°C for finalization and then sterilize.

Step 7: Modify the sterilized scaffold to achieve the purpose of modifying the scaffold function, and then inoculate cells.

Example 4:

The composite scaffold 3D printing material is made from the following raw materials in parts by mass:

16 parts gellan gum

16 parts of soluble starch

70 parts of water

In the example of the present invention, the starch is a soluble starch, insoluble in cold water, dissolved in boiling water, and forms a gel after cooling. The ratio of soluble starch to gellan gum is 1:1. At this ratio, the printing material can be smoothly extruded to form a filamentous substance, and piled up according to the printing path to form a composite scaffold with a smooth surface and a complete structure.

In the example of the present invention, the provided 3D printing material has a reasonable component distribution ratio, a fast gelation speed during the printing process, rapid prototyping, and a complete structure of the printed scaffold.

The preparation method and follow-up treatment of 3D printing material of the present invention comprise:

Step 1: adding 16g of gellan gum powder to ultrapure water to prepare a gellan gum solution;

Step 2: Based on the basis of step 1, weigh 16g of soluble starch powder, and prepare it into a mixed solution of gellan gum and starch, wherein the temperature is 90°C, the stirring speed is 1000rpm/min, and the stirring time is 40min. Cold, can be stirred for 1h;

Step 3: Pour the mixed solution in step 2 into a 50ml centrifuge tube, and place it to cool at room temperature;

Step 4: Put the cooled gellan gum and starch mixed solution in a 4°C refrigerator overnight to obtain raw materials for preparing 3D bioprinted hydrogel scaffolds.

Step 5: Put the cooled mixed raw material into the extrusion-based 3D printer barrel, and print according to the imported model. The imported 3D printing model is a cuboid of 2cm×2cm×0.5cm, and the printing speed of the 3D printer is 20mm/ s, the nozzle temperature is 240°C, the platform temperature is 17°C, the diameter of the printing needle is 1mm, the thickness of the printing layer is 1mm, the filling distance is 2mm, and the air pressure is 0.3MPa to obtain a composite scaffold.

Step 6: Place the printed scaffold at -20°C for finalization and then sterilize.

Step 7: Modify the sterilized scaffold to achieve the purpose of modifying the scaffold function, and then inoculate cells.

The invention relates to the field of tissue engineering, and provides a method for preparing a composite scaffold of gellan gum and soluble starch that can be applied to tissue regeneration 3D printing. The printing material is to mix gellan gum and starch in different proportions to print a composite scaffold with a smooth surface and a complete structure. Specifically, combining the biocompatibility and shear-thinning behavior characteristics of gellan gum and the plasticity characteristics of starch, the scaffold required for composite tissue engineering regeneration is prepared. Simulate the three-dimensional growth space of cells in vivo, so that cells can fully grow and proliferate.

Claims (10)

1. a kind of 3 biometric print hydrogel scaffolds applied to regeneration, it is characterised in that: described applied to regeneration 3D biometric print hydrogel scaffold is combined by gellan gum and soluble starch.

2. a kind of preparation method of 3D biometric print hydrogel scaffold applied to regeneration according to claim 1, It is characterized in that comprising the steps of:

Step 1: gellan gum powder being added in ultrapure water, prepares gellan gum solution；

Step 2: based on the basis of step 1, weigh soluble starch powder, be added into gellan gum solution be configured to tie it is cold Glue and starch mixed solution；

Step 3: the mixed solution of step 2 pouring into 50ml centrifuge tube, places cooling at room temperature；

Step 4: gellan gum and starch mixed solution after will be cooled be put into 4 DEG C of refrigerator overnights, obtain being used to prepare 3D biology Print the raw material of hydrogel scaffold；

Step 5: cooling mixed raw material being put into the 3D printer barrel based on extrusion, is carried out according to the model having been introduced into Printing is to get compound rest；

Step 6: printed bracket is carried out shaping into sterilization treatment；

Step 7: processing being modified to the bracket to have sterilized, achievees the purpose that be modified cradling function, then can be inoculated with thin Born of the same parents.

3. a kind of preparation method of 3D biometric print hydrogel scaffold applied to regeneration according to claim 2, It is characterized by: the mass ratio of ultrapure water and gellan gum is 70-100:16 in the step 1.

4. a kind of preparation method of 3D biometric print hydrogel scaffold applied to regeneration according to claim 2, It is characterized by: the mass ratio of ultrapure water, gellan gum and soluble starch is 70 ~ 100:16:4 ~ 16 in the step 2；System The bath temperature of standby gellan gum and starch mixed solution is 90 ~ 100 DEG C；Wherein, gellan gum and starch mixed solution needs are prepared Stir 30 ~ 40min.

5. a kind of preparation method of 3D biometric print hydrogel scaffold applied to regeneration according to claim 2, It is characterized by: prepare gellan gum in the step 2 and starch mixed solution to need mixing speed be 1000rpm.

6. a kind of preparation method of 3D biometric print hydrogel scaffold applied to regeneration according to claim 2, It is characterized by: after the gellan gum and starch mixed solution that prepare in the step 3 need to cool down 2h at room temperature, then It is put into 50ml centrifuge tube and continues cooling 1h.

7. a kind of preparation method of 3D biometric print hydrogel scaffold applied to regeneration according to claim 2, It is characterized by: the 3D printing model imported in the step 5 is the cuboid or square of different side lengths, used mould Type Software for producing is Solidworks.

8. a kind of preparation method of 3D biometric print hydrogel scaffold applied to regeneration according to claim 2, It is characterized by: in the step 5 3D printer setting: print speed be 15 ~ 20mm/s, 240 DEG C of nozzle temperature, platform temperature 17 DEG C of degree, printing needle diameter are 0.74mm, print thickness 1mm, the filling distance 2mm, and air pressure is 0.11 ~ 0.3MPa.

9. a kind of preparation method of 3D biometric print hydrogel scaffold applied to regeneration according to claim 2, It is characterized by: printed bracket is put into -20 DEG C of refrigerator freezing 48h in the step 6, then put it into -50 DEG C true 36h in vacuum freecing-dry machine, last ultraviolet sterilization 2h.

10. a kind of preparation method of 3D biometric print hydrogel scaffold applied to regeneration according to claim 2, It is characterized by: modification processing method employed in the step 7 is the bracket physical absorption various concentration that will have been sterilized The YIGSR polypeptide of (0.5-10 μ g/ml), grafting time 1h.