A macroporous multi-scale tissue engineering scaffold based on electrospinning and a preparation method and application thereof

Abstract

The present application relates to the field of tissue engineering and biomedical materials, in particular to a macroporous multi-scale tissue engineering scaffold based on electrospinning and a preparation method and application thereof; first, a PU / PLCL blended electrospinning film is prepared, the electrospinning film is broken into a dispersion liquid and a gelatin solution, and the dispersion liquid and the gelatin solution are mixed at a certain ratio, and a three-dimensional tissue engineering scaffold is prepared by cross-linking the gelatin; the vascular tissue engineering scaffold has a macropore, good mechanical properties, a suitable swelling rate and a water contact angle, good biocompatibility, and to some extent, the structure of nanofibers is retained; and the present application can be applied to the field of artificial blood vessel materials and vascular transplantation biomedical materials.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering and biomedical materials, specifically to a large-pore, multi-scale tissue engineering scaffold based on electrospinning, its preparation method, and its application. Background Technology

[0002] Artificial vascular grafts are a common clinical treatment for vascular occlusion, but their development and application are greatly limited because small-diameter artificial blood vessels with an inner diameter of less than 6 mm are prone to restenosis (Pashneh-Tala S, MacNeil S, Claeyssens F. The tissue-engineered vascular graft-past, present, and future). Tissue Engineering Part B-Reviews , 2016, 22(1): 68-100). Electrospinning can be used to prepare ultrafine fiber membranes to simulate the extracellular matrix and promote cell adhesion (Awad NK, Niu H, AliU, Morsi YS, Lin T. Electrospun fibrous scaffolds for small-diameter bloodvessels: A review). Membranes , 2018, 8(1): 15). Among the materials used to prepare vascular tissue engineering scaffolds, synthetic aliphatic polyester materials poly(L-lactide-co-caprolactone) (PLCL) and polyurethane (PU) both have good biocompatibility and mechanical properties, and can be used in the field of vascular tissue engineering. Tissue engineering scaffolds traditionally prepared by electrospinning are dense two-dimensional membranes with limited thickness and low porosity. Poor connectivity between pores and the small pore size distributed on the surface of the nanofiber membrane greatly hinder cell infiltration within the scaffold, thus limiting the application of electrospun membranes in tissue engineering. Therefore, it is crucial to improve the preparation method to obtain 3D structured tissue engineering scaffolds with structures, pore sizes, and distributions suitable for cell growth (Han, S.; Nie, K.; Li, J.; Sun, Q.; Wang, X.; Li, X.; Li, Q., 3D Electrospun Nanofiber-Based Scaffolds: From Preparations and Properties to Tissue Regeneration Applications. Stem Cells Int 2021, 2021, 8790143.).

[0003] Hydrogels, as the most commonly used form of tissue engineering scaffold, possess inherent properties such as water content similar to soft tissue and variable mechanical properties. By selecting different components and cross-linking methods, and adjusting their concentrations and ratios, hydrogels that meet specific tissue repair needs in terms of structure and function can be constructed. However, hydrogels struggle to mimic the complex three-dimensional micro and nanofiber structures present in the extracellular matrix. In contrast, electrospun nanofibers can effectively simulate the structure of the extracellular matrix. Gelatin, a multi-stage hydrolysis product of type I collagen extracted from connective tissue, contains RGD sequences in its structure. It not only maintains cell membrane stability but also plays an important role in cell adhesion, and is widely used in tissue engineering and drug delivery systems. Introducing gelatin into the field of vascular tissue engineering has a positive promoting effect on the remodeling of damaged blood vessels. Summary of the Invention

[0004] This invention overcomes the shortcomings of existing technologies and provides a large-pore, multi-scale tissue engineering scaffold based on electrospinning. Using PU, PLCL, and gelatin as raw materials, this invention prepares PU / PLCL electrospun fiber membrane materials via emulsion electrospinning. The electrospun membrane is broken up using a mechanical stirrer and uniformly dispersed in isopropanol. Gelatin is dissolved in hexafluoroisopropanol and mixed with the electrospun membrane solution. Crosslinking is performed using glutaraldehyde, followed by freeze-drying to obtain a 3D vascular scaffold. This vascular scaffold possesses the advantages of large pore size and multi-scale structure, promoting endothelial cell adhesion and proliferation. This invention has excellent application prospects in the field of artificial blood vessel materials.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a large-pore multi-scale tissue engineering scaffold based on electrospinning, wherein the tissue engineering scaffold is composed of electrospun fibers and hydrogel, has a water content similar to that of soft tissue, and has a fibrous structure similar to that of extracellular matrix; the electrospun fibers are composed of fibers with a diameter of 0.5~5 μm, and the hydrogel has a pore size of 100~800 μm.

[0006] The vascular tissue engineering scaffold of this invention has a large pore size, good mechanical properties, suitable swelling ratio, and water contact angle. The PU / PLCL fiber membrane of this invention is characterized by a tensile strength of over 10 MPa, a thickness of 10-70 μm, and is composed of 1-2 μm fibers.

[0007] This invention also provides a method for preparing a large-pore, multi-scale tissue engineering scaffold based on electrospinning, comprising the following steps:

[0008] (1) Poly(L-lactide-co-caprolactone) and polyurethane were dissolved in a mixture of chloroform and N,N-dimethylformamide at a certain mass ratio and stirred to obtain a stable electrospinning solution.

[0009] (2) Electrospin the electrospinning solution obtained in step (1) to obtain an electrospinned fiber membrane;

[0010] (3) Remove the aluminum foil from the electrospun fiber membrane obtained in step (2), cut it into the same pieces with scissors, add the pieces to a beaker containing isopropanol, and obtain a piece dispersion.

[0011] (4) The fragment dispersion obtained in step (3) is broken up using a high-speed disperser at a speed of 10,000 r / min until the fiber membrane is completely broken up and uniformly dispersed in isopropanol to obtain 1% w / v Electrospun membrane dispersion;

[0012] (5) Dissolve gelatin in hexafluoroisopropanol to obtain a gelatin solution;

[0013] (6) The electrospun membrane dispersion obtained in step (4) and the gelatin solution obtained in step (5) are mixed in a beaker in a certain proportion to obtain a mass formed by the combination of gelatin and electrospun membrane fragments.

[0014] (7) Wipe off the remaining solvent on the clumps obtained in step (6), put them into ultrapure water and stir magnetically for 10~20 min to obtain electrospun membrane dispersion / gelatin mixed solution.

[0015] (8) Add glutaraldehyde to the mixed solution obtained in step (7) to perform cross-linking for 5 h;

[0016] (9) The solution obtained in step (8) is placed in a -20℃ freezer for pre-freezing, and then placed in a freeze dryer for freeze drying for 12 hours to obtain a three-dimensional vascular tissue engineering scaffold.

[0017] Furthermore, in step (1), the mass ratio of poly(L-lactide-co-caprolactone) to polyurethane is 1:1~9, the volume ratio of chloroform to N,N-dimethylformamide is 4~8:1, and the concentration of the electrospinning solution is 100~200 mg / mL.

[0018] Furthermore, the poly(L-lactide-co-caprolactone) mentioned in step (1) has a number-average molecular weight of 200 kDa and a molar ratio (lactide:caprolactone) of 70:30; the density of the polyurethane is 1.18 g / mL at 25℃.

[0019] Furthermore, the electrospinning conditions in step (2) are as follows: the stainless steel needle is 18~20 G, the injection rate is 0.5~1.5 mL / h, the electrospinning voltage is 10~15 kV, the receiving distance is 15~20 cm, the electrospinning time is 8~12 h, and the thickness of the electrospinned fiber membrane is 10~70 μm.

[0020] Furthermore, in step (3), the mass of the fragment is 0.2~1 g, and the volume of isopropanol is 10~20 mL.

[0021] Furthermore, in step (5), the mass of gelatin is 0.5~1.2 g, and the volume of hexafluoroisopropanol is 10~20 mL, yielding 12% w / v A gelatin solution; the gelatin has a solubility of 50 mg / mL.

[0022] Furthermore, in step (6), the electrospun membrane dispersion obtained in step (4) and the gelatin solution obtained in step (5) are mixed in a volume ratio of 1:0.1 to 1:2, and the volume ratio of ultrapure water in step (7) to the volume of fragments in step (3) is 5 mL:0.2 to 1 g.

[0023] Furthermore, in step (8), the volume ratio of glutaraldehyde to ultrapure water in step (7) is 0.2~0.5:5.

[0024] In addition, the present invention also provides the application of the large-pore multi-scale tissue engineering scaffold obtained by the above preparation method in vascular transplantation.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention, a large-pore, multi-scale tissue engineering scaffold, overcomes the inherent limitations of hydrogels in simulating the microstructure of the extracellular matrix (ECM) at the nanoscale and the difficulty of providing a three-dimensional cell culture environment with electrospun fibers. It can simulate the ECM of vascular tissue and construct structures suitable for tissue regeneration. Poly(L-lactide-co-caprolactone), polyurethane, and gelatin are selected as materials. The toughness of poly(L-lactide-co-caprolactone) and the strength of polyurethane are utilized to improve the mechanical properties of the electrospun fibers, making them more similar to the mechanical properties of natural blood vessels. Simultaneously, the addition of gelatin improves the hydrophilicity of the tissue engineering scaffold, making it more conducive to cell adhesion. Through freeze-drying, the pore size of the scaffold is significantly increased, facilitating cell migration into the interior and deeper layers of the scaffold and promoting the formation of multilayer cells, thereby benefiting vascular repair. This invention has applications in the fields of artificial blood vessel materials and biomedical materials for vascular transplantation. Attached Figure Description

[0027] Figure 1 The appearance morphology of the large-pore multi-scale artificial blood vessel tissue engineering scaffold prepared in Example 1.

[0028] Figure 2 Stress-strain curves of the stents prepared in Examples 1-3.

[0029] Figure 3 The bar chart shows the elastic modulus of the scaffolds prepared in Examples 1-3.

[0030] Figure 4 The porosity histograms are for the scaffolds prepared in Examples 1-3. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. Example 1

[0032] Poly(L-lactide-co-caprolactone) and polyurethane were dissolved in a 1:1 mass ratio of chloroform and N,N-dimethylformamide (DMF) in a 1:1 volume ratio to prepare a solution with a concentration of 130 mg / mL. The solution was stirred to obtain a stable electrospinning emulsion.

[0033] The obtained solution was electrospun under the following conditions: 20 G stainless steel needles (ID=0.6 mm, OD=0.9 mm), injection rate of 1.2 mL / h, electrospinning voltage of 14 kV, receiving distance of 20 cm, electrospinning time of 8 h, and electrospun fiber membrane thickness of 70 μm, composed of fibers with a diameter of 1175±236 nm.

[0034] Remove the aluminum foil from the electrospun membrane and cut it into nearly identical pieces. Add the pieces to a beaker containing 0.2 g of isopropanol in 20 mL of isopropanol to obtain a dispersion. Use a high-speed disperser at 10,000 rpm until the fiber membrane is completely broken up and uniformly dispersed in the isopropanol, yielding a 1% dispersion. w / v An electrospun membrane dispersion was prepared. Gelatin was dissolved in hexafluoroisopropanol (1.2 g gelatin, 10 mL hexafluoroisopropanol), yielding a 12% gelatin-to-isopropanol solution. w / v Gelatin solution.

[0035] Electrospun membrane dispersion and gelatin solution were mixed in a beaker at a volume ratio of 1:0.3 to obtain clumps of gelatin and electrospun membrane fragments. Residual solvent was wiped off the clumps, and the mixture was placed in ultrapure water (5 mL volume) and magnetically stirred for 10 min to obtain an electrospun membrane dispersion / gelatin mixed solution. Glutaraldehyde (0.2 mL volume, 50% mass ratio) was added to the mixed solution for crosslinking over 5 h. The solution was pre-frozen at -20°C and then freeze-dried for 12 h to obtain a large-pore, multi-scale artificial vascular tissue engineering scaffold.

[0036] The SEM image of this large-pore multi-scale artificial blood vessel tissue engineering scaffold is shown in the figure. Example 2

[0037] Poly(L-lactide-co-caprolactone) and polyurethane were dissolved in a 1:1 mass ratio of chloroform and N,N-dimethylformamide (DMF) in a 1:1 volume ratio to prepare a solution with a concentration of 130 mg / mL. The solution was stirred to obtain a stable electrospinning emulsion.

[0038] The obtained solution was electrospun under the following conditions: 20 G stainless steel needles (ID=0.6 mm, OD=0.9 mm), injection rate of 1.2 mL / h, electrospinning voltage of 14 kV, receiving distance of 20 cm, electrospinning time of 8 h, and electrospun fiber membrane thickness of 70 μm, composed of fibers with a diameter of 1175±236 nm.

[0039] Remove the aluminum foil from the electrospun membrane and cut it into nearly identical pieces. Add the pieces to a beaker containing 0.2 g of isopropanol in 20 mL of isopropanol to obtain a dispersion. Use a high-speed disperser at 10,000 rpm until the fiber membrane is completely broken up and uniformly dispersed in the isopropanol, yielding a 1% dispersion. w / v An electrospun membrane dispersion was prepared. Gelatin was dissolved in hexafluoroisopropanol (1.2 g gelatin, 10 mL hexafluoroisopropanol), yielding a 12% gelatin-to-isopropanol solution. w / v Gelatin solution.

[0040] Electrospun membrane dispersion and gelatin solution were mixed in a beaker at a volume ratio of 1:0.5 to obtain clumps of gelatin and electrospun membrane fragments. Residual solvent was wiped off the clumps, and the mixture was placed in ultrapure water (5 mL volume) and magnetically stirred for 10 min to obtain an electrospun membrane dispersion / gelatin mixed solution. Glutaraldehyde (0.2 mL volume, 50% mass ratio) was added to the mixed solution for crosslinking over 5 h. The solution was pre-frozen at -20°C and then freeze-dried for 12 h to obtain a large-pore, multi-scale artificial vascular tissue engineering scaffold. Example 3

[0041] Poly(L-lactide-co-caprolactone) and polyurethane were dissolved in a 1:1 mass ratio of chloroform and N,N-dimethylformamide (DMF) in a 1:1 volume ratio to prepare a solution with a concentration of 130 mg / mL. The solution was stirred to obtain a stable electrospinning emulsion.

[0042] The obtained solution was electrospun under the following conditions: 20 G stainless steel needles (ID=0.6 mm, OD=0.9 mm), injection rate of 1.2 mL / h, electrospinning voltage of 14 kV, receiving distance of 20 cm, electrospinning time of 8 h, and electrospun fiber membrane thickness of 70 μm, composed of fibers with a diameter of 1175±236 nm.

[0043] Remove the aluminum foil from the electrospun membrane and cut it into nearly identical pieces. Add the pieces to a beaker containing 0.2 g of isopropanol in 20 mL of isopropanol to obtain a dispersion. Use a high-speed disperser at 10,000 rpm until the fiber membrane is completely broken up and uniformly dispersed in the isopropanol, yielding a 1% dispersion. w / v An electrospun membrane dispersion was prepared. Gelatin was dissolved in hexafluoroisopropanol (1.2 g gelatin, 10 mL hexafluoroisopropanol), yielding a 12% gelatin-to-isopropanol solution. w / v Gelatin solution.

[0044] Electrospun membrane dispersion and gelatin solution were mixed in a beaker at a volume ratio of 1:1 to obtain clumps of gelatin and electrospun membrane fragments. Residual solvent was wiped off the clumps, and the mixture was placed in ultrapure water (5 mL volume) and magnetically stirred for 10 min to obtain an electrospun membrane dispersion / gelatin mixed solution. Glutaraldehyde (0.2 mL volume, 50% mass ratio) was added to the mixed solution for crosslinking over 5 h. The solution was pre-frozen at -20°C and then freeze-dried for 12 h to obtain a large-pore, multi-scale artificial vascular tissue engineering scaffold.

[0045] The large-pore, multi-scale artificial blood vessel tissue engineering scaffolds prepared in Examples 1-3 were subjected to performance testing, such as... Figure 2-4 As shown, Figure 2 The figures show the stress-strain curves of the scaffolds with electrospun membrane dispersion: gelatin solution ratios of 1:0.3, 1:0.5, and 1:1. As can be observed from the figures, the scaffold with electrospun membrane dispersion: gelatin solution ratio of 1:0.3 shows a rapid increase in compressive strength after 20% ≤ strain ≤ 25%, while the scaffolds with the other two ratios show a slow increase in compressive strength in this range, only showing some improvement after strain ≥ 30%.

[0046] Figure 3 The graph shows the elastic modulus of the scaffolds. The scaffold with an electrospun membrane dispersion: gelatin solution ratio of 1:0.3 has a much higher elastic modulus than the other two ratios, which is 5.18 MPa.

[0047] Figure 4 The graph shows the porosity of the scaffolds at three different ratios. It can be observed that the porosity of the scaffolds with the electrospun membrane dispersion: gelatin solution ratios of 1:0.3, 1:0.5, and 1:1 are 80%, 81%, and 68%, respectively. The porosity of the scaffold with the electrospun membrane dispersion: gelatin solution ratio of 1:1 is significantly lower than that of the other two groups.

Claims

1. A method for fabricating a large-pore, multi-scale tissue engineering scaffold based on electrospinning, characterized in that, Includes the following steps: (1) Poly(L-lactide-co-caprolactone) and polyurethane are dissolved in a mixed solution of chloroform and N,N-dimethylformamide at a mass ratio of 1:1 to 9, wherein the volume ratio of chloroform to N,N-dimethylformamide is 4 to 8:

1. The mixture is stirred to obtain a stable electrospinning solution with a concentration of 100 to 200 mg / mL. The poly(L-lactide-co-caprolactone) has a number-average molecular weight of 200 kDa and a molar ratio (lactide:caprolactone) of 70:

30. The density of polyurethane at 25°C is 1.18 g / mL. (2) Electrospin the electrospinning solution obtained in step (1) to obtain an electrospinned fiber membrane; (3) Remove the aluminum foil from the electrospun fiber membrane obtained in step (2), cut it into the same pieces with scissors, add the pieces to a beaker containing isopropanol, and obtain a piece dispersion. (4) The fragment dispersion obtained in step (3) is broken up using a high-speed disperser at a speed of 10,000 r / min until the fiber membrane is completely broken up and uniformly dispersed in isopropanol to obtain 1% w / v Electrospun membrane dispersion; (5) Dissolve gelatin in hexafluoroisopropanol to obtain 12% w / v gelatin solution; (6) The electrospun membrane dispersion obtained in step (4) and the gelatin solution obtained in step (5) are mixed in a beaker at a volume ratio of 1:0.1 to 1:2 to obtain a mass of gelatin and electrospun membrane fragments. (7) Wipe off the remaining solvent on the clumps obtained in step (6), put them into ultrapure water and stir magnetically for 10~20 min to obtain electrospun membrane dispersion / gelatin mixed solution. (8) Add glutaraldehyde to the mixed solution obtained in step (7) to perform cross-linking for 5 h; (9) The solution obtained in step (8) is placed in a -20℃ freezer for pre-freezing, and then placed in a freeze dryer for freeze drying for 12 hours to obtain a three-dimensional vascular tissue engineering scaffold.

2. The method for preparing a large-pore, multi-scale tissue engineering scaffold based on electrospinning according to claim 1, characterized in that, The electrospinning conditions in step (2) are as follows: the stainless steel needle is 18~20 G, the injection rate is 0.5~1.5 mL / h, the electrospinning voltage is 10~15 kV, the receiving distance is 15~20 cm, the electrospinning time is 8~12 h, and the thickness of the electrospinned fiber membrane is 10~70 μm.

3. The method for preparing a large-pore, multi-scale tissue engineering scaffold based on electrospinning according to claim 1, characterized in that, In step (3), the mass of the fragment is 0.2~1 g and the volume of isopropanol is 10~20 mL.

4. The method for preparing a large-pore, multi-scale tissue engineering scaffold based on electrospinning according to claim 2, characterized in that, In step (5), the mass of gelatin is 0.5~1.2 g, the volume of hexafluoroisopropanol is 10~20 mL, and the solubility of the gelatin is 50 mg / mL.

5. The method for preparing a large-pore, multi-scale tissue engineering scaffold based on electrospinning according to claim 1, characterized in that, The volume ratio of ultrapure water in step (7) to the volume ratio of fragments in step (3) is 5 mL: 0.2~1 g.

6. The method for preparing a large-pore, multi-scale tissue engineering scaffold based on electrospinning according to claim 1, characterized in that, In step (8), the volume ratio of glutaraldehyde to ultrapure water in step (7) is 0.2~0.5:

5.

7. A large-pore multi-scale tissue engineering scaffold obtained by the preparation method according to any one of claims 1-6.

8. The application of a large-pore multi-scale tissue engineering scaffold obtained by the preparation method according to any one of claims 1-6 in the preparation of biomedical materials for vascular transplantation.

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