CN114767931B

CN114767931B - Tubular stent and preparation method and application thereof

Info

Publication number: CN114767931B
Application number: CN202210439129.7A
Authority: CN
Inventors: 牛玉清; 夏慧敏; 付铭; 赵彰; 邓富铭; 胡金华; 孙秀红; 孙玮棠
Original assignee: Guangzhou Women and Childrens Medical Center
Current assignee: Guangzhou Women and Childrens Medical Center
Priority date: 2022-04-25
Filing date: 2022-04-25
Publication date: 2023-01-03
Anticipated expiration: 2042-04-25
Also published as: CN114767931A

Abstract

The invention discloses a tubular stent and a preparation method and application thereof, wherein the tubular stent comprises a hollow gelatin inner layer and a poly-L-lactic acid layer wrapped on the outer surface of the gelatin inner layer; the poly-L-lactic acid layer comprises 2-7 poly-L-lactic acid sub-layers. The tubular scaffold provided by the invention has a bionic structure and excellent mechanical properties, the inner layer and the outer layer of the tubular scaffold are similar to the epithelial layer and the smooth muscle layer of a natural urethra, and the tubular scaffold shows J-shaped mechanical reaction characteristics and radial elasticity of the urethra of a young male life body; the gelatin inner layer of the tubular stent is prepared by adopting a solution blow-molding spinning method, and then the poly-L-lactic acid layer of the tubular stent is prepared by adopting a dip spinning method, so that the preparation method is simple and efficient, and has the advantage of batch production; the tubular stent can be widely applied to preparing materials for treating urethral injury diseases of young men.

Description

Tubular stent and preparation method and application thereof

Technical Field

The invention belongs to the field of materials, and particularly relates to a tubular stent, and a preparation method and application thereof.

Background

The urethra of a young boy is irreversibly damaged, so that the patient can not urinate, and even germ infection is caused in severe cases. The existing treatment method is to intervene through a reconstruction operation to recover the lost function of the urinary catheter. Similar to other long tubular tissues, urethral reconstruction in the current post-operative procedure may cause problems of stenosis, failure to completely treat urethral lesions, and impact on the life of young boys. In order to improve the life quality of patients with body blood vessel and urethra injury, the tubular stent manufactured by the tissue engineering technology is helpful to overcome the possible stenosis problem after urethra reconstruction. However, the existing tubular stent has the problem that the mechanical property is far different from the mechanical property of the urethra tissue of a human body, and the tubular stent cannot be applied to urethra treatment of young boys. Therefore, there is a need to develop a tubular stent with mechanical properties similar to those of human urethral tissues to solve the problem of urethral injury in young boys.

Disclosure of Invention

To overcome the problems of the prior art described above, it is an object of the present invention to provide a tubular stent; the second purpose of the invention is to provide a preparation method of the tubular stent; it is a further object of the present invention to provide the use of such a tubular stent.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a tubular stent, which comprises a hollow gelatin inner layer and a poly-L-lactic acid layer wrapped on the outer surface of the gelatin inner layer; the poly-L-lactic acid layer comprises 2-7 poly-L-lactic acid sub-layers.

Preferably, the thickness of the inner layer of the gelatin is 80-300 μm; further preferably, the thickness of the gelatin inner layer is 100-270 μm; still more preferably, the thickness of the inner layer of gelatin is 105 μm to 265. Mu.m. Wherein, the gelatin layer is less than 80 μm or more than 300 μm, which can not match the thickness of the natural urethra epithelium layer, resulting in larger difference between the mechanical properties of the tubular scaffold and the urethra tissue of young boys.

Preferably, the thickness of the poly-L-lactic acid layer is 180-360 μm; more preferably, the thickness of the poly-L-lactic acid layer is 200-350 μm; still more preferably, the thickness of the poly (L-lactic acid) layer is 210 to 330 μm. Wherein, the thickness of the poly-L-lactic acid layer is less than 180 μm or more than 360 μm, which can not match the thickness of the smooth muscle layer of the natural urethra, resulting in larger difference of the mechanical properties of the tubular stent and the urethra tissue of young boys.

Preferably, the gelatin layer is a layer structure.

Preferably, the length of the tubular stent is 15mm-90mm; further preferably, the tubular stent has a length of 20mm to 80mm.

Preferably, the wall thickness of the tubular stent is 260-660 μm; further preferably, the wall thickness of the tubular stent is 300 μm to 600 μm.

Preferably, the inner diameter of the tubular stent is 1300 μm to 4000 μm; further preferably, the inner diameter of the tubular stent is 1700 μm to 3700 μm.

Preferably, the outer diameter of the tubular stent is 1500 μm to 4700 μm; further preferably, the outer diameter of the tubular stent is 1930 μm to 4400 μm.

Preferably, the gelatin weight average molecular weight of the gelatin inner layer is 40kDa to 60kDa; further preferably, the gelatin weight average molecular weight of the gelatin inner layer is 45kDa to 55kDa.

Preferably, the number average molecular weight of the poly-L-lactic acid layer is 350kDa to 500kDa; further preferably, the number average molecular weight of the poly-L-lactic acid layer is 400kDa to 440kDa.

Preferably, the tubular stent has an ultimate tensile strength of 1300kPa to 2700kPa; further preferably, the tubular stent has an ultimate tensile strength of 1500kPa to 2500kPa.

Preferably, the elongation at break of the tubular stent is 420% -800%; further preferably, the tubular stent has an elongation at break of 450% to 650%.

Preferably, the elastic modulus of the tubular stent is 800kPa to 7000kPa; further preferably, the tubular stent has an elastic modulus of 2000kPa to 6000kPa.

In a second aspect, the present invention provides a method for preparing a tubular stent according to the first aspect of the present invention, comprising the steps of:

preparing the gelatin inner layer of the tubular stent by using a solution blow spinning method, and then preparing the poly-L-lactic acid layer of the outer layer of the tubular stent by using a dip spinning method to obtain the tubular stent.

Preferably, the mass concentration of the gelatin solution adopted by the solution blow spinning method is 5-12%; more preferably, the mass concentration of the gelatin solution adopted by the solution blow spinning method is 7-9%.

Preferably, the mass concentration of the poly-L-lactic acid solution adopted by the dip spinning method is 5-12%; more preferably, the mass concentration of the poly-L-lactic acid solution adopted by the dip spinning method is 8-10%.

Preferably, the solvent of the gelatin solution comprises at least one of halogenated alcohol solvents; further preferably, the solvent of the gelatin solution comprises at least one of hexafluoroisopropanol and trifluoroethanol; still further preferably, the solvent of the gelatin solution is hexafluoroisopropanol and trifluoroethanol in a volume ratio (0.2-2): 1; still more preferably, the solvent of the gelatin solution is hexafluoroisopropanol.

Preferably, the solvent of the poly-L-lactic acid solution comprises at least one of an amine solvent, a halogenated alkane solvent, an alcohol solvent and a furan solvent; further preferably, the solvent of the poly-L-lactic acid solution comprises at least one of dimethylformamide, dichloromethane, methanol and tetrahydrofuran; still further preferably, the solvent of the poly-L-lactic acid solution comprises a mixed solvent composed of dimethylformamide and dichloromethane; more preferably, the solvent of the poly-L-lactic acid solution is dimethylformamide and dichloromethane in a volume ratio of (40-60): 50 of the solvent mixture.

Preferably, the dissolution temperature of the gelatin solution or the poly-L-lactic acid solution is 15-30 ℃.

Preferably, the dissolution time of the gelatin solution or the poly-L-lactic acid solution is 8-16 h.

Preferably, the spinning temperature of the solution blow spinning method or the solution dipping spinning method is 15-30 ℃; further preferably, the solution blow-spinning method or the dip-spinning method has a spinning temperature of 20 ℃ to 25 ℃.

Preferably, the relative humidity of the solution blow spinning method or the solution dipping spinning method is 30-60%; further preferably, the solution blow spinning method or the solution dip spinning method has a spinning relative humidity of 40% to 50%.

Preferably, the dip spinning method has a spinning frequency of 2 to 7.

Preferably, the feeding rate of the solution blow spinning method is 90-150 mu L/min; further preferably, the solution blow spinning process has a feed rate of 100 μ L/min to 140 μ L/min.

Preferably, the high voltage of the solution blow spinning method is 10kV-15kV; further preferably, the high voltage of the solution blow spinning process is 11kV to 13kV.

Preferably, the low pressure of the solution blow spinning method is-2 kV- (-1) kV; further preferably, the low pressure of the solution blow spinning process is-1.8 kV- (-1.4) kV.

Preferably, the solution blow spinning process further comprises the use of a precipitation bar.

Preferably, the distance between the sedimentation rod and the touch nozzle is 20cm-30cm.

Preferably, the solution is blow spun and the rotational speed of the precipitation rod during collection spinning is 300rpm to 500rpm.

Preferably, the high voltage of the dipping spinning method is 12kV-16kV; further preferably, the high voltage of the dip spinning method is 13kV to 15kV.

Preferably, the low pressure of the dipping spinning method is-1.8 kV- (-0.8) kV; further preferably, the low pressure of the dip spinning method is-1.4 kV- (-1.0) kV.

Preferably, after the dip spinning, the rotational speed of the precipitation rod during collection spinning is 700rpm to 1100rpm.

Preferably, the solution blow spinning and dip spinning deposition time is 8h to 50h.

In a third aspect, the invention provides the use of a tubular stent according to the first aspect of the invention in the manufacture of a material for the treatment of a disease associated with urethral damage in a young male.

Preferably, the urethral injury disease in young men is irreversible volume urethral injury disease in young men.

The invention has the beneficial effects that:

the tubular scaffold provided by the invention has a bionic structure and excellent mechanical properties, the inner layer and the outer layer of the tubular scaffold are similar to the epithelial layer and the smooth muscle layer of a natural urethra, and the tubular scaffold shows J-shaped mechanical reaction characteristics and radial elasticity of the urethra of a young male life body; the gelatin inner layer of the tubular stent is prepared by adopting a solution blow-molding spinning method, and then the poly-L-lactic acid layer of the tubular stent is prepared by adopting a dip spinning method, so that the preparation method is simple and efficient, and has the advantage of batch production; the tubular stent can be widely applied to preparing materials for treating urethral injury diseases of young men.

Drawings

FIG. 1 is a scanning electron microscope image of the tubular stent prepared in example 3.

FIG. 2 is a graph of the stress strain curves of the tubular scaffolds prepared in examples 2, 4 and 7 and male baby rabbit urethral tissue.

FIG. 3 is a graph of longitudinal stress-strain curves of the outer layer of the poly (L-lactic acid) fiber layer of the tubular scaffold prepared in example 7 and the smooth muscle layer of the male young rabbit urethra.

FIG. 4 is a circumferential stress-strain graph of the outer poly (L-lactic acid) fiber layer of the tubular stent prepared in example 7 and the urethral smooth muscle layer of male young rabbit.

FIG. 5 is a graph of longitudinal stress-strain curves of the gelatin fiber layer of the inner layer of the tubular stent prepared in example 7 and the epithelial layer of male young rabbit urethra.

FIG. 6 is a circumferential stress-strain graph of the gelatin fiber layer of the inner layer of the tubular stent prepared in example 7 and the epithelial layer of male young rabbit urethra.

Fig. 7 is a cell viability assay of tubular scaffolds prepared in example 7 incubated with urothelial cells for 7 days.

Detailed Description

The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically detailed, are all those that can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available through commercial purchase.

All natural urethra used in the examples of the present application is urethra of young male rabbits of New Zealand of 8 weeks old.

Example 1

The specific preparation method of the tubular stent of this example is as follows:

the preparation method of the spinning solution comprises the following steps: gelatine has a relative molecular mass of about 50000, and is dissolved in hexafluoroisopropanol at room temperature for about 12 hours to reach a final Gelatine concentration of 7% (w/v). PLLA had a number average molecular weight (Mn) of 420000 and was dissolved in a mixture of dimethylformamide and dichloromethane (50/50V/V) at room temperature for about 12 hours to reach a final PLLA concentration of 8% (w/V).

Preparing a gelatin layer of the tubular stent by a solution blow spinning method: the dissolved gelatine solution was drawn up with a 10 ml spinning syringe, the syringe was mounted in a precision syringe pump, the feed rate was set at 120 μ L/min, both the compressed air and gelatine solution lines were connected to the spray apparatus, and the nozzle system could be placed at different positions and angles relative to the circumferential axis of a 2.7 mm precipitation rod (collecting a 2.7 mm inner diameter tubular stent). The high voltage was set to 12kV and the low voltage to-1.6 kV. The distance between the nozzle and the precipitation bar was kept constant at 25cm to ensure sufficient solvent evaporation before fiber deposition. The rotational speed of the precipitation rod for collecting gelatine spun yarn was 390rpm.

Preparing a poly-L-lactic acid layer of the tubular stent by using a dip spinning method: the deposition time for the Gelatine solution blowing and dip spinning process was 48 hours, then the dissolved PLLA solution was sucked up with a 5 ml spinning syringe, the syringe was fixed in a precision injection pump, the feed rate was set at 150 μ L/min, the high pressure was set at 14kV, the low pressure was set at-1.2 kV, the distance between the nozzle and the precipitation bar was kept constant at 25cm, and the rotation speed for the precipitation bar during the PLLA spinning (at this time, a layer of Gelatine spinning had been blown onto the precipitation bar) was 900rpm. The time for PLLA solution blow and dip spinning deposition was 10 hours. After completion of the PLLA solution blow and dip spinning deposition, the desired outer sublayer was obtained after 2 PLLA dip spinning depositions. The spinning conditions were 18-25 ℃ and 45% (relative humidity). After finishing, the spun yarn was peeled off gently from the precipitation bar to obtain a tubular stent, which was then trimmed to the desired length using surgical scissors and a scale according to the actual length required, and the resulting tubular stent was placed in a vacuum oven for 24 hours before further use to remove any residual organic solvent before further use, to obtain a tubular stent of the present example.

Example 2

the preparation method of the spinning solution comprises the following steps: gelatine has a relative molecular mass of about 50000 and is dissolved in hexafluoroisopropanol at room temperature for about 12 hours to reach a final Gelatine concentration of 8% (w/v). PLLA, number average molecular weight (Mn) 420000, was dissolved in a mixture of dimethylformamide and dichloromethane 50/50 (V/V) at room temperature for about 12 hours to reach a final PLLA concentration of 9% (w/V).

Preparing a gelatin layer of the tubular bracket by a solution blow-molding spinning method: the dissolved gelatine solution was drawn up with a 10 ml spinning syringe, the syringe was mounted in a precision syringe pump, the feed rate was set at 120 μ L/min, both the compressed air and gelatine solution lines were connected to the spray apparatus, and the nozzle system could be placed at different positions and angles relative to the circumferential axis of a 2.7 mm precipitation rod (collecting a 2.7 mm inner diameter tubular stent). The high voltage was set to 12kV and the low voltage to-1.6 kV. The distance between the nozzle and the precipitation bar was kept constant at 25cm to ensure sufficient solvent evaporation prior to fiber deposition. The rotational speed of the precipitation rod for collecting gelatine spun yarn was 390rpm.

Preparing a poly-L-lactic acid layer of the tubular stent by using an immersion spinning method: the deposition time for the Gelatine solution blowing and dip spinning process was 48 hours, then the dissolved PLLA solution was sucked up with a 5 ml spinning syringe, the syringe was fixed in a precision syringe pump, the feed rate was set at 150 μ L/min, the high pressure was set at 14kV, the low pressure was set at-1.2 kV, the distance between the nozzle and the precipitation bar was kept constant at 25cm, and the rotational speed of the precipitation bar during continued precipitation of PLLA spinning (at which a layer of Gelatine spinning had been blown onto the precipitation bar) was 900rpm. The time for PLLA solution blow and dip spinning deposition was 10 hours. After completion of the PLLA solution blow and dip spinning deposition, the desired outer sublayer was obtained after 2 PLLA dip spinning depositions. The spinning conditions were 18-25 ℃ and 45% (relative humidity). After the end, the spun yarn was gently peeled off from the setting rod to obtain a tubular stent, which was then trimmed to the desired length of the tubular stent according to the actual length required using surgical scissors and a scale, and the resulting tubular stent was placed in a vacuum oven for 24 hours before further use to remove any residual organic solvent before further use, to obtain a tubular stent of this example.

Example 3

the preparation steps of the spinning solution are as follows: gelatine has a relative molecular mass of about 50000, and is dissolved in hexafluoroisopropanol at room temperature for about 12 hours to reach a final Gelatine concentration of 9% (w/v). PLLA, number average molecular weight (Mn) 420000, was dissolved in a mixture of dimethylformamide and dichloromethane 50/50 (V/V) at room temperature for about 12 hours to reach a final PLLA concentration of 10% (w/V).

Preparing a gelatin layer of the tubular bracket by a solution blow-molding spinning method: the dissolved gelatine solution was drawn up with a 10 ml spinning syringe, the syringe was mounted in a precision syringe pump, the feed rate was set at 120 μ L/min, both the compressed air and gelatine solution lines were connected to the spray apparatus, and the nozzle system could be placed at different positions and angles relative to the circumferential axis of a 2.7 mm precipitation rod (collecting a 2.7 mm inner diameter tubular stent). The high voltage was set to 12kV and the low voltage to-1.6 kV. The distance between the nozzle and the precipitation bar was kept constant at 25cm to ensure sufficient solvent evaporation prior to fiber deposition. The rotational speed of the precipitation bar at which the gelatine spun was collected was 390rpm.

Preparing a poly-L-lactic acid layer of the tubular stent by using a dip spinning method: the deposition time for the Gelatine solution blowing and dip spinning process was 48 hours, then the dissolved PLLA solution was sucked up with a 5 ml spinning syringe, the syringe was fixed in a precision injection pump, the feed rate was set at 150 μ L/min, the high pressure was set at 14kV, the low pressure was set at-1.2 kV, the distance between the nozzle and the precipitation bar was kept constant at 25cm, and the rotation speed for the precipitation bar during the PLLA spinning (at this time, a layer of Gelatine spinning had been blown onto the precipitation bar) was 900rpm. The time for the PLLA solution blow and dip spinning deposition was 10 hours. After completion of the PLLA solution blow and dip spinning deposition, the desired outer sublayer was obtained after 4 PLLA dip spinning depositions. The spinning conditions were 18-25 ℃ and 45% (relative humidity). After finishing, the spun yarn was peeled off gently from the precipitation bar to obtain a tubular stent, which was then trimmed to the desired length using surgical scissors and a scale according to the actual length required, and the resulting tubular stent was placed in a vacuum oven for 24 hours before further use to remove any residual organic solvent before further use, to obtain a tubular stent of the present example.

Example 4

Preparing a gelatin layer of the tubular bracket by a solution blow-molding spinning method: the dissolved gelatine solution was drawn up with a 10 ml spinning syringe, the syringe was mounted in a precision syringe pump, the feed rate was set at 120 μ L/min, both the compressed air and gelatine solution lines were connected to the spray apparatus, and the nozzle system could be placed at different positions and angles relative to the circumferential axis of a 2.8 mm precipitation rod (collecting a 2.8 mm inner diameter tubular stent). The high voltage was set to 12kV and the low voltage to-1.6 kV. The distance between the nozzle and the precipitation bar was kept constant at 25cm to ensure sufficient solvent evaporation before fiber deposition. The rotational speed of the precipitation rod for collecting gelatine spun yarn was 390rpm.

Preparing a poly-L-lactic acid layer of the tubular stent by using a dip spinning method: the deposition time for the Gelatine solution blowing and dip spinning process was 48 hours, then the dissolved PLLA solution was sucked up with a 5 ml spinning syringe, the syringe was fixed in a precision syringe pump, the feed rate was set at 150 μ L/min, the high pressure was set at 14kV, the low pressure was set at-1.2 kV, the distance between the nozzle and the precipitation bar was kept constant at 25cm, and the rotational speed of the precipitation bar during continued precipitation of PLLA spinning (at which a layer of Gelatine spinning had been blown onto the precipitation bar) was 900rpm. The time for the PLLA solution blow and dip spinning deposition was 10 hours. After completion of the PLLA solution blow and dip spinning deposition, the desired outer sublayer was obtained after 6 PLLA dip spinning depositions. The spinning conditions were 18-25 ℃ and 45% (relative humidity). After the end, the spun yarn was gently peeled off from the setting rod to obtain a tubular stent, which was then trimmed to the desired length of the tubular stent according to the actual length required using surgical scissors and a scale, and the resulting tubular stent was placed in a vacuum oven for 24 hours before further use to remove any residual organic solvent before further use, to obtain a tubular stent of this example.

Example 5

Preparing a gelatin layer of the tubular stent by a solution blow spinning method: the dissolved gelatine solution was aspirated by a 10 ml spinning syringe, the syringe was mounted in a precision syringe pump, the feed rate was set at 120 μ L/min, both the compressed air and gelatine solution lines were connected to a spray device, and the nozzle system could be placed at different positions and angles relative to the circumferential axis of a 2.9 mm precipitation rod (collecting a 2.9 mm inner diameter tubular stent). The high voltage was set to 12kV and the low voltage to-1.6 kV. The distance between the nozzle and the precipitation bar was kept constant at 25cm to ensure sufficient solvent evaporation before fiber deposition. The rotational speed of the precipitation bar at which the gelatine spun was collected was 390rpm.

Preparing a poly-L-lactic acid layer of the tubular stent by using a dip spinning method: the deposition time for the Gelatine solution blowing and dip spinning process was 48 hours, then the dissolved PLLA solution was sucked up with a 5 ml spinning syringe, the syringe was fixed in a precision syringe pump, the feed rate was set at 150 μ L/min, the high pressure was set at 14kV, the low pressure was set at-1.2 kV, the distance between the nozzle and the precipitation bar was kept constant at 25cm, and the rotational speed of the precipitation bar during continued precipitation of PLLA spinning (at which a layer of Gelatine spinning had been blown onto the precipitation bar) was 900rpm. The time for the PLLA solution blow and dip spinning deposition was 10 hours. After the PLLA solution blow and dip spinning deposition was completed, the desired outer sub-layer was obtained after 3 PLLA dip spinning depositions. The spinning conditions were 18-25 ℃ and 45% (relative humidity). After the end, the spun yarn was gently peeled off from the setting rod to obtain a tubular stent, which was then trimmed to the desired length of the tubular stent according to the actual length required using surgical scissors and a scale, and the resulting tubular stent was placed in a vacuum oven for 24 hours before further use to remove any residual organic solvent before further use, to obtain a tubular stent of this example.

Example 6

Preparing a gelatin layer of the tubular bracket by a solution blow-molding spinning method: the dissolved gelatine solution was aspirated by a 10 ml spinning syringe, the syringe was mounted in a precision syringe pump, the feed rate was set at 120 μ L/min, both the compressed air and gelatine solution lines were connected to a spray device, and the nozzle system could be placed at different positions and angles relative to the circumferential axis of a 3.0 mm precipitation rod (collecting a 3.0 mm inner diameter tubular stent). The high voltage was set to 12kV and the low voltage to-1.6 kV. The distance between the nozzle and the precipitation bar was kept constant at 25cm to ensure sufficient solvent evaporation prior to fiber deposition. The rotational speed of the precipitation rod for collecting gelatine spun yarn was 390rpm.

Preparing a poly-L-lactic acid layer of the tubular stent by using a dip spinning method: the deposition time for the Gelatine solution blowing and dip spinning process was 48 hours, then the dissolved PLLA solution was sucked up with a 5 ml spinning syringe, the syringe was fixed in a precision injection pump, the feed rate was set at 150 μ L/min, the high pressure was set at 14kV, the low pressure was set at-1.2 kV, the distance between the nozzle and the precipitation bar was kept constant at 25cm, and the rotation speed for the precipitation bar during the PLLA spinning (at this time, a layer of Gelatine spinning had been blown onto the precipitation bar) was 900rpm. The time for the PLLA solution blow and dip spinning deposition was 10 hours. After the PLLA solution blow and dip spinning deposition was completed, the desired outer sub-layer was obtained after 6 PLLA dip spinning depositions. The spinning conditions were 18-25 ℃ and 45% (relative humidity). After the end, the spun yarn was gently peeled off from the setting rod to obtain a tubular stent, which was then trimmed to the desired length of the tubular stent according to the actual length required using surgical scissors and a scale, and the resulting tubular stent was placed in a vacuum oven for 24 hours before further use to remove any residual organic solvent before further use, to obtain a tubular stent of this example.

Example 7

the preparation method of the spinning solution comprises the following steps: gelatine has a relative molecular mass of about 50000, and is dissolved in hexafluoroisopropanol at room temperature for about 12 hours to reach a final Gelatine concentration of 7% (w/v). PLLA, number average molecular weight (Mn) 420000, was dissolved in a mixture of dimethylformamide and dichloromethane 50/50 (V/V) at room temperature for about 12 hours to reach a final PLLA concentration of 8% (w/V).

Preparing a poly-L-lactic acid layer of the tubular stent by using a dip spinning method: the deposition time for the Gelatine solution blowing and dip spinning process was 48 hours, then the dissolved PLLA solution was sucked up with a 5 ml spinning syringe, the syringe was fixed in a precision injection pump, the feed rate was set at 150 μ L/min, the high pressure was set at 14kV, the low pressure was set at-1.2 kV, the distance between the nozzle and the precipitation bar was kept constant at 25cm, and the rotation speed for the precipitation bar during the PLLA spinning (at this time, a layer of Gelatine spinning had been blown onto the precipitation bar) was 900rpm. The time for PLLA solution blow and dip spinning deposition was 10 hours. After the PLLA solution blow and dip spinning deposition was completed, the desired outer sub-layer was obtained after 4 PLLA dip spinning depositions. The spinning conditions were 18-25 ℃ and 45% (relative humidity). After the end, the spun yarn was gently peeled off from the setting rod to obtain a tubular stent, which was then trimmed to the desired length of the tubular stent according to the actual length required using surgical scissors and a scale, and the resulting tubular stent was placed in a vacuum oven for 24 hours before further use to remove any residual organic solvent before further use, to obtain a tubular stent of this example.

Comparative example 1

The tubular stent of the present example is the tubular stent disclosed in patent application CN 109847101A.

Performance testing

1. Scanning electron microscope testing of tubular stents

The morphology of the cross section of the tubular stent prepared in example 7 (length 90mm, inner diameter 2.7 mm) was observed with a scanning electron microscope (SEM, TM-1000, hitachi) at an accelerating voltage of 8kV to 10 kV. Prior to imaging, the samples were sputter plated with gold for 50 seconds to increase conductivity. FIG. 1 is a scanning electron microscope image of the tubular stent prepared in example 3. The tubular stent of example 3 was found to have a wall thickness of 550 μm, an inner gelatin thickness of 210 μm and an outer layer thickness of 340 μm from the SEM image using ImageJ software.

2. Tensile testing of tubular stents

The inner and outer layers of the tubular stent containing the poly-L-lactic acid (PLLA)/gelatin (gelatine) fiber sub-layer were tested under uniaxial tension using a texture analyzer equipped with a 5N weighing cell. Rectangular samples of the structure were tested to determine their longitudinal and circumferential stress-strain profiles. Three samples were cut and tested in both the longitudinal and circumferential directions of each layer. The thickness and width of the sample were measured using a micrometer with an accuracy of 10 μm. For the pre-treated samples, the structure was subjected to stretching at a constant speed of 10 mm/min.

FIG. 2 is a graph of stress strain curves of the tubular scaffolds prepared in examples 2, 4 and 7 and male young rabbit urethral tissue. Wherein, the tubular stents prepared in the embodiments 2, 4 and 7 have the length of 30mm, the thickness of 0.47-0.65 mm and the width of 8.5-8.8 mm; the male young rabbits are 6 weeks old, and the urethra of the male young rabbits is taken as a control group, wherein the length of the male young rabbits is 30mm, the thickness of the male young rabbits is 0.47mm, and the width of the male young rabbits is 8.5-8.8 mm.

FIG. 3 is a graph of longitudinal stress-strain curves of the outer layer of the poly (L-lactic acid) fiber layer of the tubular scaffold prepared in example 7 and the smooth muscle layer of the male young rabbit urethra. Using surgical scissors to trim the tubular fibrous scaffold along the precipitation rod to obtain a tubular scaffold with a length of 3cm, then using surgical operation to cut along the long axis direction to obtain a fibrous membrane with a length of 3cm, a thickness of 0.6cm and a width of 8.8mm, and then using surgical sharp forceps to slightly pull apart the gelatin fiber layers of the concentric fibrous sheets on the gelatin fiber layers, namely obtaining two fiber films of the gelatin fiber layer and the poly-L-lactic acid fiber layer. FIG. 4 is a circumferential stress-strain graph of the outer poly (L-lactic acid) fiber layer of the tubular stent prepared in example 7 and the urethral smooth muscle layer of male young rabbit. Wherein the length of the inner spinning wall of the inner gelatin fiber layer is 30mm, the thickness is 0.35mm, and the width is 8.5mm; the length of the inner spinning wall of the outer poly-L-lactic acid fiber layer is 30mm, the thickness is 0.36mm, and the width is 8.5mm; the length of the epithelial layer of the young rabbit is 30mm, the thickness is 0.36mm, and the width is 8.5mm.

FIG. 5 is a graph of longitudinal stress-strain curves of the gelatin fiber layer of the inner layer of the tubular stent prepared in example 7 and the epithelial layer of male young rabbit urethra. FIG. 6 is a circumferential stress-strain graph of the gelatin fiber layer of the inner layer of the tubular stent prepared in example 7 and the urothelial layer of male young rabbit. Wherein, the length of the inner spinning wall of the inner gelatin fiber layer is 8.5mm, the thickness is 0.27mm, and the width is 0.5mm. The young rabbit smooth muscle layer has a length of 8.5mm, a thickness of 0.27mm and a width of 0.5mm.

The longitudinal tensile strength of the gelatin layer of the inner layer of the tubular stent prepared in example 7 of the application is about 1000kPa, the elongation at break is about 580 percent, and the elastic modulus is about 2800kPa, which is very close to the longitudinal mechanical performance parameter of the urethral epithelium layer (see figure 5); the tubular stent prepared in example 7 has a tensile strength in the circumferential direction of about 500kPa, an elongation at break of about 450%, and an elastic modulus of about 1400kPa, which is very close to the circumferential mechanical performance parameters of the urethral epithelium layer (see FIG. 6); the outer layer of the tubular stent prepared in example 7 (containing 3 PLLA wire sublayers) had a longitudinal tensile strength of about 2500kPa, an elongation at break of about 580% and an elastic modulus of about 6900kPa, very close to the longitudinal tensile parameter of the urethral smooth muscle layer (see fig. 3); the outer layer of the tubular stent prepared in example 7 had a tensile strength of about 1500kPa in the circumferential direction, an elongation at break of about 700% and an elastic modulus of about 4600kPa, which is very close to the circumferential mechanical performance parameters of the urothelial layer (see fig. 4). The sub-layer and the whole mechanical property of the urethral stent are close to those of the natural urethra.

Table 1 shows the results of mechanical property tests of the tubular scaffolds and male young rabbit urethral tissues prepared in the examples and comparative example 1. As can be seen from Table 1, the tubular stent prepared by the method can more accurately simulate the mechanical reactivity of the natural urethra, while the mechanical property parameters of the tubular stent prepared by the method in the comparative example 1 are too high in tensile strength and elastic modulus, and much lower in elongation at break than the natural urethra, so that the urethral stent prepared by the comparative example 1 is very high in rigidity and poor in flexibility and is not suitable for the use requirement of diseased urethra reconstruction of young organisms.

TABLE 1 mechanical Performance test results of the tubular scaffolds and male young rabbit urethral tissues prepared in examples and comparative example 1

The tensile strength of the natural urethra (including an epithelial layer and a smooth muscle layer) of the young New Zealand rabbit used in the test is 2000-2200 kPa, the elongation at break is 480-580%, and the elastic modulus is 5000-6000 kPa; the tubular stent (the inner layer comprises 1 gelatine silk sub-layer, and the outer layer comprises 3 PLLA silk sub-layers) manufactured in example 7 of the application has the mechanical properties that the tensile strength is 1800-2200 kPa, the elongation at break is 480-550%, and the elastic modulus is 5000-7000 kPa, which shows that the tubular stent manufactured in example 7 is similar to the mechanical properties of the natural urethra of the young New Zealand rabbit.

3. Cell compatibility testing of tubular scaffolds

In order to evaluate the cellular compatibility of the prepared tubular scaffolds, primary urothelial cells were isolated by first performing a bladder biopsy on young male New Zealand rabbits. The CCK-8 assay was then used to quantify cellular metabolic activity in the tubular scaffold. The method comprises the steps of suturing two ends of a tubular support with surgical suture lines, inoculating urothelial cells on the inner wall of the tubular support through the unstitched end after the tubular support with the length of 5cm and primary urothelial cells are incubated together, and then suturing the other end of the tubular support and strengthening the tubular support with gel glue. After 24 hours, after the cells had adhered to the wall, both ends of the tubular scaffold were cut with sterile surgical scissors and cut into 0.5cm long tubular scaffolds, which were transferred to a 96-well plate for further culture, and the original culture medium in the 96-well plate was aspirated, replaced with 100. Mu.L of fresh DMEM containing 20. Mu.L of CCK-8 reagent, and incubated at 37 ℃ for 1 hour, after which the tubular scaffolds were removed from the 96-well plate, and their absorbance of the culture medium was measured at 450nm in a SynergyTM H1 multimode plate reader. Positive controls are cells in petri dishes, blank wells are negative controls, and all samples are tested in triplicate. Calculating the formula: cell viability = (cell scaffold group-blank wells)/(positive control wells-blank wells) × 100%.

Fig. 7 is a cell viability assay of tubular scaffolds prepared in example 7 incubated with urothelial cells for 7 days. As can be seen from fig. 7, the tubular scaffold prepared in example 7 has excellent biocompatibility, and after being incubated with urothelial cells for 7 days, the urothelial cells still have high bioactivity.

The anatomical structure of the natural urethra consists of an epithelial layer, a smooth muscle layer of an inner longitudinal ring and an outer longitudinal ring and a urethra cavernous body, the tubular scaffold structure prepared in the embodiment of the application is inspired by the configuration of ECM structures in the natural urethral epithelial layer and the smooth muscle layer, a gelatin layer of an inner layer is opposite to a standard epithelial layer, and 2-7 layers of an outer layer are opposite to the anatomical structural characteristics (thickness) of the longitudinal ring and the outer ring of the standard smooth muscle; the structure and mechanical properties of the tubular stent prepared by the method of this example perfectly match the epithelial layer and smooth muscle layer of the natural urethra, and therefore the tubular stent comprises an inner gelatin layer and an outer poly-L-lactic acid layer, and the outer poly-L-lactic acid layer comprises 2-7 sublayers, so that the prepared stent can simulate the structural configuration of the natural urethra and show the J-shaped mechanical reaction and radial elasticity of the natural urethra.

The tubular scaffold provided by the invention has a bionic structure and excellent mechanical properties, the thicknesses of the inner layer and the outer layer of the tubular scaffold are respectively 80-300 mu m and 180-360 mu m, and are similar to the thicknesses of the epithelial layer and the smooth muscle layer of the natural urethra of male infants (aged 0.2-7 years) (85-300 mu m and 180-360 mu m). Both the inner and outer layers of the tubular stent exhibit J-shaped mechanical response characteristics and radial elasticity similar to those of the urothelial and smooth muscle layers of young male rabbits (fig. 2-6); this animal model is superior to the dog model because the penis of a rabbit has no penile bone and is therefore anatomically and functionally closer to the human penis. The gelatin inner layer of the tubular stent is prepared by adopting a solution blow-molding spinning method, and then the levorotatory poly-L-lactic acid layer of the tubular stent is prepared by adopting a dip spinning method, so that the preparation method is simple and efficient, and has the advantage of batch production; the tubular stent can be widely applied to the preparation of materials for treating urethral injury diseases of young men.

The manufacturing method disclosed in this application is a new automated production method that combines dip spinning to deposit a concentric fiber layer tubular scaffold, and a solution blow spinning device suitable for intercalation of aligned reinforcing nanofibers, this additional manufacturing method allowing the assembly of concentric tubular scaffolds with specific fiber orientation and number of fibers. The tubular stent prepared by the preparation method shows J-shaped mechanical reaction and radial elasticity of the urethra of a young male life body.

The above examples are preferred embodiments of the present invention, but the present invention is not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and they are intended to be included in the scope of the present invention.

Claims

1. A preparation method of a tubular urethral stent is characterized by comprising the following steps:

preparing a spinning solution: relative molecular mass of gelatin 50000, dissolved in hexafluoroisopropanol at room temperature for 12 h to reach a final gelatin concentration of 7%; poly (L-lactic acid) having a number average molecular weight of 420000, dissolved in dimethylformamide and dichloromethane at a volume ratio of 50:50 for 12 hours to reach the final poly-L-lactic acid concentration of 8%;

preparing a gelatin layer of the tubular stent: sucking the dissolved gelatin solution by a 10 mL spinning injector, fixing the injector in a precise injection pump, setting the feeding rate to be 120 mu L/min, connecting compressed air and a gelatin solution pipeline into a spraying device, and placing a nozzle system at different positions and angles relative to the circumferential axis of a 2.7 mm precipitation rod; the high voltage is set to 12kV, and the low voltage is set to-1.6 kV; the distance between the nozzle and the precipitation bar was kept constant at 25cm to ensure sufficient solvent evaporation before fiber deposition, and the rotation speed of the precipitation bar at which the gelatin filaments were collected was 390 rpm;

preparation of the poly-L-lactic acid layer of the tubular scaffold: the deposition time of the gelatin solution blowing and dip spinning method is 48 h, then a 5 mL spinning injector is used for sucking the dissolved poly-L-lactic acid solution, the injector is fixed in a precise injection pump, the feeding rate is set to be 150 muL/min, the high pressure is set to be 14kV, the low pressure is set to be-1.2 kV, the distance between a nozzle and a precipitation rod is kept constant at 25cm, the rotation speed of the precipitation rod is 900rpm when the poly-L-lactic acid is continuously precipitated, and the deposition time of the poly-L-lactic acid solution blowing and dip spinning method is 10 h;

after the poly-L-lactic acid solution is deposited by a blow molding and dip spinning method, obtaining a required outer sub-layer after 4 times of poly-L-lactic acid solution dip spinning method deposition, wherein the spinning environmental conditions are 18 to 25 ℃ and 45% relative humidity; after the end, the spinning is slightly peeled off from the precipitation rod to obtain a tubular stent, then the tubular stent is trimmed to the required length of the tubular stent according to the actual required length by using surgical scissors and a scale, and the obtained tubular stent is placed in a vacuum drying oven for 24 hours before further use so as to remove residual organic solvent before further use, thereby obtaining the urethral tubular stent.

2. Use of a urethral tubular stent prepared by the method of claim 1 for the preparation of a material for the treatment of a urethral injury disorder in a young male.