CN106729979A - A kind of preparation method of the de- cellular vascular support of organizational project - Google Patents

Abstract

A method for preparing tissue-engineered decellularized vascular stents. The micron-scale polymer stent is implanted into the subcutaneous or abdominal cavity of the host, and the vascular stent is wrapped by the host immune protection mechanism and then decellularized to obtain non-immunogenic Reinforced tissue engineered blood vessels. The advantages of the present invention are: the present invention solves the insufficient mechanical properties of decellularized tissue-engineered blood vessels, and the multi-layer cross-linked network polymer scaffold fully improves the mechanical properties of the original decellularized vascular scaffolds; Immunogenicity issues during allogeneic transplantation. The host animal constructs a stent vessel formed by migrating cells caused by the immune mechanism. After decellularization treatment, the immune response after transplantation is reduced, and the extracellular matrix is fully utilized to provide a good environment for cell regeneration; The method can also prepare different decellularized vascular scaffolds by designing scaffolds with different shapes and sizes, so as to be applied to different vascular grafting conditions.

Description

A kind of preparation method of tissue engineering decellularized vascular scaffold

technical field

The invention relates to a preparation method of tissue engineering decellularized vascular scaffold.

Background technique

Cardiovascular disease is the number one killer of human health, and the first choice for more than half of patients is vascular transplantation, but it is difficult for some patients to implement it due to the limitation of their own blood vessel sources. Allogeneic or xenograft blood vessels have been used as a substitute for autologous blood vessels to repair diseased or missing blood vessels, but the potential risk of disease transmission, acute immune rejection and limited donor sources limit their application in transplantation. The development of tissue engineering technology provides a new solution for the repair and reconstruction of human vascular defects. At present, a variety of synthetic and degradable materials have been developed for the construction of tissue engineering scaffolds, which have been applied clinically and achieved good results. Tissue engineered blood vessels are vascular substitutes with good biocompatibility and mechanical properties constructed by tissue engineering methods. The basic components are vascular scaffold materials, seed cells and signaling factors. The research on vascular scaffolds has always been Difficulties and key points in the research field of vascular tissue engineering. Among them, natural biological scaffold materials use autologous or allogeneic blood vessels to decellularize to obtain extracellular matrix. Due to the strong affinity between these extracellular matrices and cells, they can provide a similar in vivo tissue development environment for cell growth, reproduction, and differentiation, and are biocompatible. It has good resistance and compliance, and low immune rejection, so it has the potential to be applied to tissue engineered blood vessels.

However, when these allogeneic or xenogeneic decellularized scaffolds are applied to small-caliber (inner diameter less than 6mm) vascular grafts, there are problems such as insufficient mechanical properties in the environment of small-caliber and high-tension vessels, which may lead to severe aneurysms. Therefore, in order to realize the wide application of decellularized vascular scaffolds in small-caliber tissue engineered blood vessels, the problem of insufficient mechanical properties of the scaffolds must be solved. Technologies such as melt spinning and wet spinning can use some biocompatible polymer materials to prepare scaffolds with good mechanical properties. The fiber diameter and fiber orientation of this scaffold can be controlled, and it can It provides better mechanical support in the radial direction of blood vessels, so as to meet the mechanical performance requirements of blood flow stimulation in vivo, so it has been paid attention to in the research of tissue engineered blood vessels.

Contents of the invention

The purpose of the present invention is to address the above existing problems and combine decellularized tissue engineered blood vessels with mechanical scaffolds prepared by techniques such as melt spinning to invent a method for preparing tissue engineered decellularized blood vessel scaffolds that can effectively enhance mechanical properties The decellularized vascular stent prepared by the method has the characteristics of significantly enhanced mechanical properties, low immune response after transplantation in vivo, and vascular stents with different shapes can be designed to meet the actual clinical application.

Technical scheme of the present invention:

A preparation method of tissue engineering decellularized vascular scaffold, comprising the following steps:

1) Using a biodegradable polymer material as a raw material, a micron-scale or nano-scale ordered network vascular stent is prepared on a medical silicone tube by melt spinning or wet spinning technology, and the ordered network vascular stent is The fiber is spirally wound outside the silicone tube, the angle between the fibers is 45°, the thickness of the mesh vascular stent layer is 600 μm, and the diameter of the vascular stent is 2 mm;

2) Implant the above-mentioned fibrous scaffold with silicone tube into the subcutaneous part of the rabbit or rat experimental animal for 2 weeks to 3 months, the host initiates the immune response mechanism and causes fibroblasts to migrate to the scaffold, and finally forms a network composed of host cells and Tissue engineered vascular scaffold wrapped in extracellular matrix;

3) Take out the above-mentioned tissue-engineered vascular stent, remove the silicone tube, and perform the following treatment on the outer layer of the vascular material: sterilize with 75wt% alcohol for 20 minutes, transfer to the ultra-clean bench for operation, and use 1wt% dodecane Sodium sulfonate (SDS) was shaken on a shaker for 12 hours, and the SDS was completely washed away with double distilled water (DDH ₂ O), treated with 100 U of DNase and 40 U of RNase on a shaker for 24 hours, and the enzyme was decomposed with DDH ₂ O. Wash away completely to obtain the tissue engineered decellularized vascular scaffold.

The biodegradable polymer material is polycaprolactone (PCL), poly L-lactide-co-caprolactone (PLCL), polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA) , polyhydroxyalkanoate (PHA) and polyurethane (PU) or a mixture of two or more in any proportion.

An application of the prepared tissue engineering decellularized blood vessel scaffold is used for blood vessel transplantation in experimental animals.

The advantages of the present invention are:

1. The multi-layer cross-linked network polymer scaffold has fully improved the mechanical properties of the original decellularized vascular scaffold, and successfully solved the problems of aneurysm after vascular transplantation; 2. This method constructs a immune mechanism in the host animal The scaffold blood vessels formed by the migrating cells are then decellularized, which not only retains the mesh scaffold to improve the mechanical properties of blood vessels, but also reduces the immune response after transplantation, and makes full use of the extracellular matrix to provide a good environment for cell regeneration;3 The method can also prepare different decellularized vascular scaffolds by designing scaffolds of different shapes and sizes, so as to be applied to different vascular grafting conditions.

Description of drawings:

Figure 1 is a schematic diagram of the three-dimensional structure of decellularized blood vessels.

Fig. 2 is an enlarged schematic diagram of the cross-sectional structure of the decellularized vascular scaffold.

In the figure: 1, immune wrapping material; 2, polymer fiber; 3, medical silicone tube.

detailed description

Example 1:

A preparation method of tissue engineering decellularized vascular scaffold, adopting melt spinning technology to prepare with PCL as raw material, comprising the following steps:

1) Add 20 grams of PCL with a molecular weight of 8000 into the barrel of the melt spinning machine, raise the temperature of the barrel to 210°C and keep it for 1h, adjust the distance between the needle of the barrel and the medical silicone tube with an outer diameter of 2mm to 1cm, set The propulsive flow rate of the fixed barrel is 0.5mL/h, the speed of the receiving silicone tube is 300rpm, the horizontal moving speed is 10mm/s, the moving distance is 10cm, and the spinning time is 12min. Mesh vascular stent;

2) Cut the above-mentioned PCL ordered mesh vascular stent with silicone tubes into 2cm sections, sterilize with ethylene oxide, anesthetize the rats, shave the sides of the back to grow a 6cm long, 2cm wide rectangular surface, and use Cut a 1 cm long incision with scissors, use flat scissors to separate the cortex from the muscle tissue into a cavity with a length of 4 cm, a width of 1 cm, and a depth of 0.3 cm, and bury the PCL fiber scaffold in the cavity. One month after the implantation, the rats were anesthetized, and the hair removal incision was made on the other side of the implanted scaffold material. The material was stripped from the surrounding tissue and then taken out. After removing the excess connective tissue, a tissue wrapped by host cells and extracellular matrix was obtained. Tissue engineered vascular stents;

3) Disinfect the above-obtained stent with 75wt% alcohol for 20min, transfer it to a clean bench, treat it with 1wt% sodium dodecylsulfonate (SDS) for 12h, and distill it with double distilled water (DDH ₂ O) SDS was completely washed away, treated with 100U of DNase and 40U of RNase for 24 hours, and the enzymes were completely washed away with DDH ₂ O to obtain decellularized vascular scaffolds, which were put into phosphate-buffered saline for later use. The phosphate-buffered saline Composition: NaCl 137mmol/L, KCl 2.7mmol/L, Na ₂ HPO ₄ 10mmol/L, KH ₂ PO ₄ 2mmol/L; pH of the solution is 7.2-7.4.

The prepared tissue engineered acellular vascular stent is used for vascular transplantation in experimental animals. The method is as follows: the obtained acellular vascular stent is subjected to rat abdominal aortic vascular transplantation experiments, and the tissue phase is evaluated by hematoxylin and eosin staining and immunofluorescence staining. Capacitance and vascular regeneration, the shape and patency of blood vessels in rats were detected by small animal Doppler ultrasound imager, and the mechanical properties of transplanted blood vessels were evaluated. Histological staining analysis shows that there is no obvious immune rejection reaction between the decellularized vascular scaffold and the body, and the histocompatibility is good; Doppler ultrasound imaging shows that the shape of the blood vessels remains normal and the blood flow is smooth; the mechanical test results show that the decellularized tissue The mechanical properties such as burst pressure and tensile strength of engineered vascular stents can meet physiological needs.

Example 2:

A preparation method of tissue engineering decellularized vascular scaffold, adopting wet spinning technology to prepare PLCL as raw material, comprising the following steps:

1) Dissolve 1 gram of PLCL into 10 mL of hexafluoroisopropanol, stir at room temperature until completely dissolved, connect a medical silicone tube with a diameter of 2 mm to the rotating motor, draw the PLCL solution into the syringe, and put the needle of the syringe into the ethanol coagulation bath The middle distance is 1cm from the silicone tube. Set the solution flow rate to 2ml/h, receive the silica gel tube at a speed of 500rpm, and spin the tube for 40 minutes to obtain an orderly network vascular stent with spirally wound fibers outside the silicone tube. After the preparation is completed, the vascular stent is vacuum-dried for later use;

2) The above-obtained ordered mesh vascular stent with an inner diameter of 2mm was cut and grown into a vascular stent of 3cm, sterilized with 75wt% medical alcohol and replaced with sterile saline for later use. After the rabbit was anesthetized, both sides of the back were shaved Use scissors to cut a 1cm-long incision on a rectangular surface with a length of 6cm and a width of 4cm. Use flat scissors to separate the cortex from the muscle tissue to form a cavity with a length of 4cm, a width of 1cm, and a depth of 0.3cm. After the subcutaneous implantation time reaches 3 weeks, the rabbit is anesthetized, and the hair removal incision is made on the other side of the implanted stent, and the stent is stripped from the surrounding tissue and taken out;

3) Disinfect the obtained stent with 75wt% alcohol for 20min, transfer it to a clean bench, treat it with 1wt% sodium dodecylsulfonate (SDS) for 12h, and distill the SDS with double distilled water (DDH ₂ O) Wash it off completely, treat it with 100U of DNase and 40U of RNase for 24 hours, wash off the enzyme completely with double distilled water (DDH ₂ O), and obtain the decellularized vascular scaffold, put it in phosphate buffered saline solution for later use (the composition is the same as that in the implementation example 1).

The prepared tissue engineered acellular vascular stent is used for vascular transplantation in experimental animals. The method is as follows: the obtained acellular vascular stent is subjected to rabbit carotid artery vascular transplantation experiments, and its histocompatibility is evaluated by hematoxylin and eosin staining and immunofluorescence staining. The morphology and patency of blood vessels in rats were detected by small animal Doppler ultrasound imager, and the mechanical properties of transplanted blood vessels were evaluated. Histological staining analysis showed that there was no obvious immune rejection reaction between the acellular vascular scaffold and the body, and the tissue compatibility was good; Doppler ultrasound imaging showed that the shape of the blood vessels remained normal and the blood flow was smooth; the mechanical test results showed that the acellular tissue engineering The burst pressure, tensile strength and other mechanical properties of the vascularized stent can meet the physiological needs.

Claims (3)

1. a preparation method of tissue engineering decellularized vascular scaffold, it is characterized in that comprising the following steps: 1) Using a biodegradable polymer material as a raw material, a micron-scale or nano-scale ordered network vascular stent is prepared on a medical silicone tube by melt spinning or wet spinning technology, and the ordered network vascular stent is The fiber is spirally wound outside the silicone tube, the angle between the fibers is 45°, the thickness of the mesh vascular stent layer is 600 μm, and the diameter of the vascular stent is 2 mm; 2) Implant the above-mentioned fibrous scaffold with silicone tube into the subcutaneous part of the rabbit or rat experimental animal for 2 weeks to 3 months, the host initiates the immune response mechanism and causes fibroblasts to migrate to the scaffold, and finally forms a network composed of host cells and Tissue engineered vascular scaffold wrapped in extracellular matrix; 3) Take out the above-mentioned tissue-engineered vascular stent, remove the silicone tube, and perform the following treatment on the outer layer of the vascular material: sterilize with 75wt% alcohol for 20 minutes, transfer to the ultra-clean bench for operation, and use 1wt% dodecane Sodium sulfonate (SDS) was shaken on a shaker for 12 hours, and the SDS was completely washed away with double distilled water (DDH ₂ O), treated with 100 U of DNase and 40 U of RNase on a shaker for 24 hours, and the enzyme was decomposed with DDH ₂ O. Wash away completely to obtain the tissue engineered decellularized vascular scaffold. 2. according to the preparation method of the described tissue engineering decellularized vascular scaffold of claim 1, it is characterized in that: described biodegradable polymer material is polycaprolactone (PCL), poly L-lactide-co-caprolactone Polyester (PLCL), polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), polyhydroxyalkanoate (PHA) and polyurethane (PU), or a mixture of two or more in any proportion. 3. An application of the tissue engineering decellularized vascular scaffold prepared in claim 1, characterized in that it is used for vascular transplantation in experimental animals.