Silk fibroin 3D printing based biological scaffold and preparation method and application thereof

Abstract

The invention belongs to the field of tissue engineering and medical materials, and relates to a silk fibroin 3D printing based biological scaffold suitable for bronchial mucosa epithelial growth anda preparation method thereof. The biological scaffold is characterized in that silk fibroin / hydroxypropyl carboxymethyl cellulose is adopted as a scaffold material, a 3D printing and freeze drying combined technology is adopted, a bronchial epithelial cell line (BEAS-2B cells) is used as seed cells, the BEAS-2B cells grow on a porous 3D printed silk fibroin / hydroxypropyl carboxymethyl cellulose scaffold, and then the 3D printed silk fibroin / hydroxypropyl carboxymethyl cellulose scaffold is obtained. The silk fibroin 3D printing based scaffold can be used for tracheal defect repair and can provide meaningful reference for development of tissue engineering artificial trachea.

Description

technical field

[0001] The invention belongs to the field of tissue engineering and medical materials, and relates to a biological scaffold based on silk fibroin 3D printing and its preparation method and application, in particular to a biological scaffold adapted to the growth of bronchial mucosal epithelium based on silk fibroin 3D printing and its preparation method . Background technique

[0002] The trachea is a hollow cylindrical organ lined with a layer of pseudostratified ciliated columnar epithelium that is important for mucus clearance and protection against infection. At present, in clinical treatment, various types of materials such as silicone rubber prosthesis and polyethylene prosthesis are used to repair tracheal defects, but it is difficult to promote the regeneration of airway epithelium and easily lead to complications such as granulation tissue hyperplasia and infection. It is very important to develop scaffolds that can regenerate the epithelium. [00...

Examples

Embodiment 1

[0051] Embodiment 1 prepares silk fibroin solution and HPMC solution

[0052] Put silkworm cocoons in Na 2 CO 3 (0.5wt%) solution boiled for 45min, and then repeatedly washed with deionized water; put the silk in a 40°C oven to dry overnight; put the degummed silk into a LiBr (9.3mol / L) solution at 60°C, and heat for 1h ; Then filter the dissolved silk fibroin solution with gauze; pour the silk fibroin solution into a dialysis bag (molecular weight cut-off 14,000Da), dialyze with deionized water for 3 days, change the water 3-4 times a day, to remove LiBr in the solution Centrifuge the dialyzed silk fibroin solution (8000rpm, 8min), then filter the supernatant with gauze; calibrate the concentration of the silk fibroin to 4wt% by weighing method; concentrate with about 20-22% PEG (PEG20000) Silk fibroin solution, concentrate the silk fibroin solution to a concentration of about 33-34wt%; after concentrating for 16-18 hours, slowly collect the concentrated silk protein soluti...

Embodiment 2

[0053] The preparation of embodiment 2SF / HPMC gel

[0054] For 3D printing materials, suitable material viscosity plays an important role in 3D printing. On the one hand, it can enable the material to be extruded from the printer nozzle, and on the other hand, it can maintain the shape of the extruded filament; If the needle is blocked, the printing will be interrupted, and too low viscosity will make the material more difficult to form; this embodiment uses Na 2 CO 3 The silk fibroin solution is prepared by degumming, and the silk fibroin solution obtained by this method has a lower molecular weight and a lower viscosity; the silk fibroin can be molded at a concentration of 15-33 wt%, and the higher the concentration, the better the moldability;

[0055] The above-mentioned concentrated silk fibroin solution is diluted to 30wt% with deionized water (this solution can be diluted to the required concentration according to actual conditions), and then 10wt% HPMC is added in the...

Embodiment 3

[0056] Example 3 Preparation of 3D printed SF / HPMC scaffold

[0057] Import the pre-designed model file whose output format is STL into the 3D printer control software; put the barrel containing 30wt% SF / HPMC thixotropic gel into the nozzle of the printer, and set the printing parameters: needle diameter 0.11-0.51 mm, printing air pressure 0.1-0.5MPa, printing speed 15-60mm s -1 ;When the gel is ejected from the nozzle, it forms a rod-like filament, and the nozzle of the printer moves along the XY axis on the printing platform. After the first layer is printed, the nozzle of the printer moves up along the Y axis to print the second layer. This is repeated layer by layer, and finally the designed model is printed; then the printed 30wt% SF / HPMC gel scaffold is frozen in a -20°C refrigerator for 24 hours, and then freeze-dried in a freeze dryer for 2 days, and finally the freeze-dried The 30wt% SF / HPMC scaffolds were soaked in 75% ethanol for 1h to induce the SF / HPMC scaffolds ...

technical field

[0001] The invention belongs to the field of tissue engineering and medical materials, and relates to a biological scaffold based on silk fibroin 3D printing and its preparation method and application, in particular to a biological scaffold adapted to the growth of bronchial mucosal epithelium based on silk fibroin 3D printing and its preparation method . Background technique

[0002] The trachea is a hollow cylindrical organ lined with a layer of pseudostratified ciliated columnar epithelium that is important for mucus clearance and protection against infection. At present, in clinical treatment, various types of materials such as silicone rubber prosthesis and polyethylene prosthesis are used to repair tracheal defects, but it is difficult to promote the regeneration of airway epithelium and easily lead to complications such as granulation tissue hyperplasia and infection. It is very important to develop scaffolds that can regenerate the epithelium. [00...

Examples

Embodiment 1

[0051] Embodiment 1 prepares silk fibroin solution and HPMC solution

[0052] Put silkworm cocoons in Na 2 CO 3 (0.5wt%) solution boiled for 45min, and then repeatedly washed with deionized water; put the silk in a 40°C oven to dry overnight; put the degummed silk into a LiBr (9.3mol / L) solution at 60°C, and heat for 1h ; Then filter the dissolved silk fibroin solution with gauze; pour the silk fibroin solution into a dialysis bag (molecular weight cut-off 14,000Da), dialyze with deionized water for 3 days, change the water 3-4 times a day, to remove LiBr in the solution Centrifuge the dialyzed silk fibroin solution (8000rpm, 8min), then filter the supernatant with gauze; calibrate the concentration of the silk fibroin to 4wt% by weighing method; concentrate with about 20-22% PEG (PEG20000) Silk fibroin solution, concentrate the silk fibroin solution to a concentration of about 33-34wt%; after concentrating for 16-18 hours, slowly collect the concentrated silk protein soluti...

Embodiment 2

[0053] The preparation of embodiment 2SF / HPMC gel

[0054] For 3D printing materials, suitable material viscosity plays an important role in 3D printing. On the one hand, it can enable the material to be extruded from the printer nozzle, and on the other hand, it can maintain the shape of the extruded filament; If the needle is blocked, the printing will be interrupted, and too low viscosity will make the material more difficult to form; this embodiment uses Na 2 CO 3 The silk fibroin solution is prepared by degumming, and the silk fibroin solution obtained by this method has a lower molecular weight and a lower viscosity; the silk fibroin can be molded at a concentration of 15-33 wt%, and the higher the concentration, the better the moldability;

[0055] The above-mentioned concentrated silk fibroin solution is diluted to 30wt% with deionized water (this solution can be diluted to the required concentration according to actual conditions), and then 10wt% HPMC is added in the...

Embodiment 3

[0056] Example 3 Preparation of 3D printed SF / HPMC scaffold

[0057] Import the pre-designed model file whose output format is STL into the 3D printer control software; put the barrel containing 30wt% SF / HPMC thixotropic gel into the nozzle of the printer, and set the printing parameters: needle diameter 0.11-0.51 mm, printing air pressure 0.1-0.5MPa, printing speed 15-60mm s -1 ;When the gel is ejected from the nozzle, it forms a rod-like filament, and the nozzle of the printer moves along the XY axis on the printing platform. After the first layer is printed, the nozzle of the printer moves up along the Y axis to print the second layer. This is repeated layer by layer, and finally the designed model is printed; then the printed 30wt% SF / HPMC gel scaffold is frozen in a -20°C refrigerator for 24 hours, and then freeze-dried in a freeze dryer for 2 days, and finally the freeze-dried The 30wt% SF / HPMC scaffolds were soaked in 75% ethanol for 1h to induce the SF / HPMC scaffolds ...

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