CN110604835A - A composite process method for preparing vascular stent - Google Patents

Abstract

The invention relates to a composite process method for preparing vascular stents. Two different processes are used to prepare a three-layer vascular stent structure. The inner layer materials are PLCL, PEG and endothelial growth factor. Electrospinning is used to form an axially ordered structure. Spinning; the middle layer is collagen, and the prepared collagen layer is more solid than collagen hydrogel by electrochemical method; the outermost layer is electrospun, and the material is PLCL, and the spinning is collected into a circumferential order. The blood vessel stent prepared by the invention well simulates the three-layer structure of the natural blood vessel, and has broad prospects in clinical application.

Description

A composite process method for preparing vascular stent

technical field

The invention relates to a composite process method for preparing a vascular stent, which is applied in the fields of biomanufacturing technology and tissue repair.

Background technique

Cardiovascular disease has become one of the main causes of death in the world. The main causes of cardiovascular disease are intimal hyperplasia and thrombosis, which lead to vascular occlusion. Once vascular occlusion occurs, it will lead to tissue ischemia far from the heart. make it lose its original organizational function. At present, large-diameter artificial blood vessels (>6mm) made of metal, nylon, polytetrafluoroethylene and other materials have achieved extensive success in the field of human large and medium-sized artery defect repair, but there is no suitable artificial blood vessel for small-diameter blood vessels (<6mm). Solution, now the main treatment method is blood vessel transplantation, but due to the limited source of autologous blood vessels and the high price of allogeneic decellularization technology, a large number of artificial blood vessels are clinically required as transplant substitutes.

With the extensive research on vascular stents in recent years, it is found that the best way to prepare vascular stents is to imitate the three-layer structure of natural blood vessels to achieve the purpose of vascular transplantation. The performance requirements of the bracket. At present, with the rapid development of materials science, tissue engineering, cell biology and clinical medicine, it is possible to construct tissue-engineered artificial blood vessels using composite technology, which has become the focus and hot spot of research at home and abroad.

An ideal tissue-engineered blood vessel should have the following conditions: 1) have or simulate the three-layer structure of the blood vessel wall in vivo (ie adventitia, media and intima); 2) have good biocompatibility, and are not prone to thrombosis and Immune rejection; 3) Biological properties, such as relaxation and contraction responses to drug stimulation; 4) Mechanical properties of blood vessels, that is, they can provide sufficient strength and have certain viscoelasticity.

Contents of the invention

In order to solve the deficiencies in the preparation of tissue engineering vascular stents at the present stage, the purpose of the present invention is to provide a composite process for preparing vascular stents, thereby solving the limitations brought by a single preparation process, and can better simulate natural blood vessels and The extracellular matrix makes the prepared stent more meet the requirements of various performances in clinical medicine.

In order to achieve the above-mentioned purpose, the technical scheme that the present invention adopts is as follows:

A composite process method for preparing vascular stents, comprising the following steps:

a. Dissolve PLCL (poly(L-lactic acid-ε-caprolactone)) and PEG (polyethylene glycol) in a mixed solution of calcium chloride and N,N-dimethylformamide at a volume ratio of 1:1 150mg/mL～350mg/mL solution is used as the oil phase, trimethylated chitosan loaded with endothelial growth factor is added as the water phase, the oil phase and the water phase are fully mixed, and stirred until a stable water emulsion is prepared solution;

b. Load the water-emulsion solution in a into the barrel for electrospinning, use a receiving rod to collect the electrospinning, and prepare a circumferentially arranged electrospinning layer with a thickness of 200-400 μm;

c. Cut the electrospinning layer collected in the circumferential direction with medical scissors, lay it flat in a petri dish filled with alcohol, and press it on the electrospinning layer with a sterilized square iron block. After 2~ After 3 days of cultivation, the flattened electrospun layer was rewound on another electrode rod with a diameter of 2 to 3 mm, and then the electrospun layer was sutured with PLA (polylactic acid) thread, and finally trimmed to obtain Desired axially aligned electrospinning;

d. Fully dissolve the collagen in 200-300mL of distilled water at room temperature and stir for a long time, then use the electrode rod with a sutured inner layer in c as the negative electrode, and a concentric hollow cylindrical rod as the positive electrode. Attached to the electrospun layer, and then by adjusting the distance between the electrode rod (negative electrode) and the cylindrical rod (positive electrode), a compacted collagen layer of 0.4-0.5mm is obtained as the middle layer;

e. Take out the electrode rod prepared in d (wrapping the inner layer of electrospinning and the middle layer of collagen layer), remove the air bubbles in the collagen, and then soak the two layers of film and the receiving rod in alcohol for 2 hours and sterilize them by ultraviolet light , and then washed with PBS (phosphate buffer saline) solution for 3 to 5 times to replace the residual alcohol in the bracket;

f. Remove the electrode rod in e, and then use the receiving rod as the receiving platform, and then electrospin a layer of circumferentially arranged PLCL layer on the outer layer, and finally obtain a three-layer vascular stent.

In the step a, the mass ratio of PLCL to PEG is (5-10):1, and the volume ratio of the oil phase to the water phase is (10-20):1.

When making the inner layer of the vascular stent in the step b, the diameter of the receiving rod is 2 to 3 cm, and the length of the receiving range of the receiving rod to receive electrospinning on the shaft is 7 to 9 mm, and the material of the receiving rod is 316L stainless steel solid electrode rod.

When making the inner layer of the vascular stent in the steps b and c, the voltage between the receiving rod and the needle is 10-15kv, the injection speed is 0.8-1.2mL/h, and the distance between the tip of the needle and the receiving rod 10 ~ 15cm, electrospinning 1 ~ 3h.

When making the middle layer of the vascular stent in the step d and step e, the electrochemical voltage is 2.5-3.5V, the electrochemical time is 30-45min, and the cylindrical rod as the positive electrode is a 316L stainless steel hollow cylindrical electrode rod.

The hydrogen and oxygen bubbles generated electrochemically are placed under vacuum in step e.

When making the outer layer of the vascular stent in the step f, the electrospinning voltage is controlled to be 10-15kv, the injection speed is 3-5mL/h, the rotational speed of the electrode rod is controlled at 800-1000r/min, and the distance between the needle and the electrode rod is The distance is 10-15cm, and the electrospinning time is 1-3h.

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

1. The vascular stent prepared in the present invention adopts biocompatible and degradable biomaterials, so it has good biocompatibility.

2. Both the inner layer and the outer layer of the stent prepared by the present invention are made of PLCL material, which has sufficient mechanical strength and compliance, and the middle layer uses an electrochemical protein collagen layer, so it has sufficient strength.

3. The inner layer of the vascular stent prepared by the present invention adopts PGA (polyglycolic acid) and PLCL materials to contain endothelial growth factor, wherein the PGA material degrades faster, and can quickly release endothelial growth factor thereby accelerating endothelialization.

4. The inner layer of the vascular stent prepared by the present invention adopts an axial arrangement consistent with the direction of blood flow, which conforms to the habit of endothelial cell growth, adsorption and reproduction.

5. The middle layer of the vascular stent prepared by the present invention adopts an electrochemical collagen layer. Compared with the ordinary collagen layer, the collagen layer has higher strength and harder hardness, better simulates the natural vascular collagen layer, and is beneficial to smooth muscle cells. attached.

6. The outer layer of the blood vessel stent prepared in the present invention adopts the electrospun layer arranged in the circumferential direction, and the electrospun layer arranged in the circumferential direction is more conducive to the adsorption of smooth muscle cells.

Description of drawings

FIG. 1 is a schematic structural view of the electrospinning equipment used in the preparation of the inner layer structure of the vascular stent according to the present invention.

Fig. 2 is a schematic diagram of the present invention after the inner electrospinning layer is cut open and then pressed with an iron block and soaked in alcohol for disinfection.

Fig. 3 is a schematic diagram of the electrochemical structure used in the preparation of the middle layer structure of the vascular stent in the present invention.

Fig. 4 is a schematic diagram of the final three-layer structure of the vascular stent prepared in the present invention.

In Figures 1 to 4:

1—square iron block, 2—cut the inner membrane of the stent, 3—alcohol,

4—petri dish, 5—collagen solution, 6—receiving rod,

7—PLCL and PGA mixed layer, 8—collagen layer, 9—outer layer of stent,

10—the middle layer of the blood vessel stent, 11—the inner layer of the blood vessel stent.

Detailed ways

Below in conjunction with accompanying drawing, above-mentioned technical scheme is described further, and preferred embodiment of the present invention is described in detail as follows:

Embodiment one:

A composite process method for preparing vascular stents, comprising the following steps:

a. Dissolve PLCL and PEG in a mixed solution of calcium chloride and N,N-dimethylformamide with a volume ratio of 1:1 according to the mass ratio of 5:1, and prepare a 150 mg/mL solution as the oil phase, and add The endothelial growth factor trimethylated chitosan is used as the water phase, the oil phase and the water phase are fully mixed at a ratio of 10:1, and stirred until a stable water-emulsion solution is prepared.

b. Load the water-emulsion solution in a into the barrel for electrospinning, as shown in Figure 1, use a 316L stainless steel solid electrode rod with a diameter of 2.5cm as a receiving rod to collect the electrospinning, and control the receiving rod to receive The length of the receiving range of electrospinning on the shaft is 7mm, the voltage between the receiving rod and the needle is 10kv, the injection speed is 0.8mL/h, the distance between the tip of the needle and the receiving rod is 10cm, electrospinning for 1h, and the preparation A circumferentially aligned electrospun layer with a thickness of 150 μm was obtained.

c. Cut the electrospun layer collected in the circumferential direction with medical scissors, lay it flat on the petri dish 4 filled with alcohol 3, and press it on the electrospun layer 2 with a sterilized square iron block 1 , after 3 days of cultivation, the flattened electrospun layer was rewound on the electrode rod with a diameter of 2mm, and then the electrospun layer was sutured with PLA thread, and finally trimmed to obtain the desired axial arrangement. Electrospinning, as shown in Figure 2.

d. Collagen is fully dissolved in distilled water (200mL) at room temperature and stirred for a long time, then the electrode rod 6 with a sutured inner layer in c is used as a negative electrode, and a concentric hollow cylindrical rod 9 is used as a positive electrode, and 2.5 A voltage of V is applied between the two, and the collagen is attached to the electrospun layer by electrosorption. 20min to obtain a compacted collagen layer 8 as the middle layer, as shown in FIG. 3 .

e. Take out the two-layer membrane and electrode rod prepared in d and place them in a vacuum environment to remove the air bubbles in the collagen, then soak the two-layer membrane and electrode rod in alcohol for 2 hours and sterilize them with ultraviolet light, and then use The PBS solution was washed 5 times to replace the residual alcohol in the stent.

f. Remove the two layers of film and the receiving rod in e, and then use the electrode rod as the receiving platform, and then electrospin a layer of circumferentially arranged PLCL layer 9 on the outer layer. The specific electrospinning conditions are: the voltage is 15kv, and the injection The speed was 3mL/h, the rotational speed of the electrode rod was controlled at 800r/min, the distance between the needle and the electrode rod was 10cm, and the electrospinning time was 1h. Finally, a three-layer vascular stent was obtained (PLCL layer 9, collagen layer 10 and PLCL/ PEG layer), as shown in Figure 4.

Embodiment two:

This embodiment is basically the same as Embodiment 1, a composite process method for preparing a vascular stent, comprising the following steps:

a. Dissolve PLCL and PEG in a mixed solution of calcium chloride and N,N-dimethylformamide with a volume ratio of 1:1 according to the mass ratio of 7:1, and prepare a 250mg/mL solution as the oil phase, and add Endothelial growth factor trimethylated chitosan is used as the water phase, the oil phase and the water phase are fully mixed at a ratio of 15:1, and stirred until a stable water-emulsion solution is prepared.

b. Load the water-emulsion solution in a into the barrel for electrospinning, use a 316L stainless steel solid electrode rod with a diameter of 2.5cm as the receiving rod to collect the electrospinning, and control the receiving rod to receive the electrospinning on the shaft The length of the receiving range is 8mm, the voltage between the receiving rod and the needle is 10kv, the injection speed is 1mL/h, the distance between the tip of the needle and the receiving rod is 12cm, and the electrospinning is 1.5h. The spinning layer is 170um.

c. Cut the electrospun layer collected in the circumferential direction with medical scissors, lay it flat on the petri dish 4 filled with alcohol 3, and press it on the electrospun layer 2 with a sterilized square iron block 1 , after 2 days of cultivation, then the flattened electrospun layer was rewound on the electrode rod with a diameter of 2.5mm, then the electrospun layer was sutured with PLA thread, and finally trimmed to obtain the desired axial arrangement electrospinning.

d. Dissolve the collagen in distilled water (250mL) at room temperature and stir it for a long time, then use the electrode rod in c as the negative electrode, find a concentric solid cylindrical rod 9 as the positive electrode, and apply a voltage of 3V between the two Between the two, the collagen is attached to the electrospun layer by electroadsorption, and the duration of electrification is controlled at 40 minutes, and then by regulating the distance between the electrode rod 6 (negative pole) and the concentric solid cylindrical rod 9 (positive pole), electrochemistry 40min, thereby Obtain a compacted collagen layer as an intermediate layer.

e. Take out the two-layer membrane and electrode rod prepared in d and place them in a vacuum environment to remove the air bubbles in the collagen, then soak the two-layer membrane and electrode rod in alcohol for 2 hours and sterilize them with ultraviolet light, and then use The PBS solution was washed 4 times to replace the residual alcohol in the stent.

f. Remove the two layers of film and the electrode rod in e, and then use the electrode rod as the receiving platform, and then electrospin a layer of circumferentially arranged PLCL layer on the outer layer. The specific electrospinning conditions are: voltage 15kv, injection speed The rotational speed of the receiving rod was controlled at 900r/min, the distance between the needle and the receiving rod was 12cm, and the electrospinning time was 2h. Finally, a three-layer vascular stent was obtained.

Embodiment three:

This embodiment is basically the same as Embodiment 1, a composite process method for preparing a vascular stent, comprising the following steps:

a. Dissolve PLCL and PEG in a mixed solution of calcium chloride and N,N-dimethylformamide with a volume ratio of 1:1 according to the mass ratio of 10:1, and prepare a 350 mg/mL solution as the oil phase. The endothelial growth factor trimethylated chitosan is used as the water phase, the oil phase and the water phase are fully mixed at a ratio of 20:1, and stirred until a stable water-emulsion solution is prepared.

b. Load the water-emulsion solution in a into the barrel for electrospinning, use a 3cm diameter 316L stainless steel solid electrode rod as a receiving rod to collect the electrospinning, and control the receiving rod to receive the electrospinning on the shaft The length of the receiving range is 9mm, the voltage between the receiving rod and the needle is 15kv, the injection speed is 1.2mL/h, the distance between the tip of the needle and the receiving rod is 15cm, the electrospinning is 3h, and finally a circumferentially arranged electrospun Layer 200um.

c. Cut the electrospun layer collected in the circumferential direction with medical scissors, lay it flat on the petri dish 4 filled with alcohol 3, and press it on the electrospun layer 2 with a sterilized square iron block 1 , after 3 days of cultivation, then rewind the flattened electrospun layer on the electrode rod 6 with a diameter of 3mm, then suture the electrospun layer with PLA thread, and finally trim it to obtain the desired axial arrangement electrospinning.

d. Dissolve the collagen in distilled water (300mL) at room temperature, fully dissolve and stir for a long time, then use the sutured inner layer electrode rod 6 in c as the negative electrode, and then find a concentric solid cylindrical rod 9 as the positive electrode. A voltage of 3.5V is applied between the two, and the collagen is attached to the electrospun layer by electrosorption, and the electrification time is controlled at 45 minutes. The distance is 45min, so as to obtain a compacted collagen layer as the middle layer.

e. Take out the two-layer membrane and electrode rod prepared in d and place them in a vacuum environment to remove the air bubbles in the collagen, then soak the two-layer membrane and electrode rod in alcohol for 2 hours and sterilize them with ultraviolet light, and then use The PBS solution was washed 5 times to replace the residual alcohol in the stent.

f. Remove the two layers of film and the electrode rod in e, and then use the electrode rod as the receiving platform, and then electrospin a layer of circumferentially arranged PLCL layer on the outer layer. The specific electrospinning conditions are: voltage 15kv, injection speed The rotation speed of the electrode rod was controlled at 1000r/min, the distance between the needle and the electrode rod was 15cm, and the electrospinning time was 3h. Finally, a three-layer vascular stent was obtained.

Claims (7)

1. A compound process method for preparing vascular stent, is characterized in that, comprises the following steps: a. Dissolve PLCL and PEG together in a mixed solution of calcium chloride and N,N-dimethylformamide with a volume ratio of 1:1, make a 150mg/mL~350mg/mL solution as the oil phase, add endothelial The growth factor trimethylated chitosan is used as the water phase, the oil phase and the water phase are fully mixed and stirred until a stable water-emulsion solution is prepared; b. Load the water-emulsion solution in a into the barrel for electrospinning, use a receiving rod to collect the electrospinning, and prepare a circumferentially arranged electrospinning layer with a thickness of 200-400 μm; c. Cut the electrospun layer collected in the circumferential direction with medical scissors, lay it flat in a petri dish filled with alcohol, and press it on the electrospun layer with a sterilized square iron block. After 2~ After 3 days of culture, rewind the flattened electrospun layer on another electrode rod with a diameter of 2~3mm, then suture the electrospun layer with PLA thread, and finally trim it to get the required shaft Aligned electrospinning; d. Fully dissolve the collagen in 200~300mL of distilled water at room temperature and stir for a long time, then use the sutured inner electrode rod in c as the negative electrode, and a concentric hollow cylindrical rod as the positive electrode, and the collagen is attached by electrosorption On the electrospun layer, then by adjusting the distance between the electrode rod and the cylindrical rod, a compacted collagen layer of 0.4~0.5mm is obtained as the middle layer; e. Take out the electrode rod wrapped with the inner layer of electrospinning and the middle layer of collagen layer in d, remove the air bubbles in the collagen, then soak the electrode rod in alcohol for 2 hours and sterilize it according to the ultraviolet lamp, and then wash it with PBS solution 3~5 times, replace the residual alcohol in the bracket; f. Remove the electrode rod in e, and then use the electrode rod as a receiving platform, and then electrospin a layer of circumferentially arranged PLCL layer on the outer layer, and finally obtain a three-layer vascular stent. 2. The composite process method for preparing vascular stents according to claim 1, characterized in that: in the step a, the mass ratio of PLCL and PEG is (5-10): 1, and the volume of the oil phase and the water phase used is The ratio is (10~20):1. 3. according to claim 1, prepare the composite process method of vascular stent, it is characterized in that: when making vascular stent inner layer in described step b, the diameter of described receiving rod is 2 ~ 3cm, and the control receiving rod receives electrospun The length of the receiving range of the wire on the shaft is 7-9mm, and the material of the receiving rod is a 316L stainless steel solid electrode rod. 4. according to claim 1, the compound process method for preparing vascular stent is characterized in that: when making vascular stent inner layer in described step b and step c, the voltage between the described receiving rod and the needle is 10～15kv , the injection speed is 0.8~1.2mL/h, the distance between the tip of the needle and the receiving rod is 10~15cm, and the electrospinning is 1~3h. 5. The composite process method for preparing vascular stent according to claim 1, characterized in that: when making the middle layer of vascular stent in said step d and step e, its electrochemical voltage is 2.5 ~ 3.5V, and the electrochemical duration is 30 ~ 45min, the cylindrical rod used as the positive electrode is a 316L stainless steel hollow cylindrical electrode rod. 6. The composite process method for preparing vascular stent according to claim 1, characterized in that: in the step e, the hydrogen and oxygen bubbles generated by electrochemistry are processed under vacuum. 7. according to claim 1, prepare the compound process method of vascular stent, it is characterized in that: when making vascular stent outer layer in the described step f, the voltage of control electrospinning is 10～15kv, and injection speed is 3～5mL/ h, the rotational speed of the receiving rod is controlled at 800~1000r/min, the distance between the needle and the receiving rod is 10~15cm, and the electrospinning time is 1~3h.