CN111700710A

CN111700710A - Template for tissue engineering material and tissue engineering material

Info

Publication number: CN111700710A
Application number: CN202010381923.1A
Authority: CN
Inventors: 许鹏赟; 徐霁; 季旭东
Original assignee: Lingbo Biotechnology Hangzhou Co ltd
Current assignee: Lingbo Biotechnology Hangzhou Co ltd
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2020-09-25
Anticipated expiration: 2040-05-08
Also published as: CN111700710B

Abstract

The invention provides a template for tissue engineering materials, which comprises a support body, an anti-exposure layer, a reticular fiber framework layer and a protective layer, wherein the support body, the anti-exposure layer, the reticular fiber framework layer and the protective layer are sequentially arranged; and a hollow structure is arranged on the protective layer. Compared with the prior art, when the template provided by the invention is used for preparing the tissue engineering material, the anti-exposure layer is degraded to form a gap, and finally the acellular matrix material can be coated on the surface of the inner cavity of the reticular fibrous skeleton, so that the exposure of fibers is reduced, and the adhesion of blood coagulation components is reduced; the reticular fiber skeleton can provide good mechanical property, so that the tissue engineering material has good kink resistance, rebound resilience, explosion resistance, puncture closeness and sewability; meanwhile, the protective layer can protect the reticular fiber skeleton layer structure, the skeleton structure is prevented from being damaged when the prepared material is cultured, and the hollow structure on the protective layer can enable the prepared tissue engineering material to have an external ridge beam, enhance the suture strength, the blasting pressure and the anti-kink capacity of the blood vessel, and accelerate the integration with the surrounding tissue.

Description

Template for tissue engineering material and tissue engineering material

Technical Field

The invention belongs to the technical field of tissue engineering, and particularly relates to a template for a tissue engineering material and the tissue engineering material.

Background

Cardiovascular disease is the most fatal disease worldwide, and is often caused by reduced blood flow and nutrient deficiency due to narrowing or blockage of blood vessels, resulting in damage to tissues or organs, often manifested as coronary heart disease, cerebrovascular disease, peripheral arterial disease. According to the world health organization, the number of deaths worldwide from cardiovascular related diseases per year will increase to 2330 ten thousand by 2030. The blood vessel transplantation operation is still the conventional means for treating the diseases, and the collection of autologous blood vessels (such as great saphenous vein, bilateral internal thoracic artery, radial artery and the like) of a patient in the operation is still the gold standard of blood vessel transplantation at present. But only artificial vessels can be selected for replacement because autologous vessels have already been harvested or are not matched in length, caliber, or suffer from complex systemic vascular lesions.

At present, the artificial blood vessel is usually prepared by adopting artificial high molecular materials, such as polyethylene glycol terephthalate

Expanded polytetrafluoroethylene

The prepared blood vessel is directly implanted into a human body, and under the condition, the artificial high polymer material can be directly contacted with blood or tissue of the human body, and a certain rejection reaction can be generated, so that the conditions of thrombus, inflammation, intimal hyperplasia and the like are caused, and the transplantation failure is further caused.

The artificial blood vessel can be effectively constructed by applying an in vivo engineering method, and the basic principle is to utilize the spontaneous immune coating reaction of an organism to an implant. The conventional method is to implant the tubular object under the skin of the host, and the in vivo engineered blood vessel can be obtained after the host is wrapped by the tissue. The method has the advantages that the prepared blood vessel is composed of cells and tissues and has good biocompatibility, but because of lack of support of high polymer materials, the mechanical property of the blood vessel is poor, the tubular structure cannot be effectively maintained, the suturing difficulty is high, and aneurysm and suture end stenosis are easy to occur when the blood vessel is implanted into an arterial system. In the previous research, a fiber framework is manufactured on the surface of a silicone tube by using a high molecular material, then the fiber framework and the silicone tube are implanted into the subcutaneous part of an animal as a template for tissue engineering, and after tissue wrapping is formed, cell removal treatment is carried out, so that the polymer fiber framework-reinforced tissue engineering blood vessel is obtained, the mechanical property of the blood vessel material is remarkably improved, and the problems are effectively solved. In addition, the tissue engineering blood vessel prepared by the method has the acellular matrix, so that the biocompatibility of the blood vessel material is improved, compared with the artificial blood vessel constructed by a pure high polymer material, the rejection reaction is reduced, and the success rate of blood vessel transplantation is improved to a certain extent. However, in this process, the uniformity of the parallel spacing of the fibers in each layer of the fibers of the polymer skeleton is poor, which results in non-uniform pore size of the skeleton and thus poor vascular yield. In addition, the fiber structure of the framework is also easy to cause structural damage due to subcutaneous implantation operation or in vitro cell culture, and the yield of the blood vessel is reduced. On the basis of earlier research, the outer surface of the prepared polymer skeleton enhanced tissue engineering blood vessel is smooth, the blood vessel is easy to slide after being implanted into a body, the integration rate of the blood vessel and tissues around the implanted part needs to be improved, and part of skeleton fibers in the inner cavity of the polymer skeleton enhanced tissue engineering blood vessel prepared in the earlier stage are exposed, so that certain blood coagulation matrix adhesion can be caused.

Disclosure of Invention

In view of this, the technical problem to be solved by the present invention is to provide a template for tissue engineering material and a tissue engineering material; the tissue engineering material prepared by the template has stable mechanical parameters, good anti-kink capacity, rough outer surface, prevention of in-vivo sliding, easy integration with organism tissues, no fiber exposure in an inner cavity and difficult thrombosis.

The invention provides a template for tissue engineering materials, which comprises a support body, an anti-exposure layer, a reticular fiber framework layer and a protective layer, wherein the support body, the anti-exposure layer, the reticular fiber framework layer and the protective layer are sequentially arranged; and a hollow structure is arranged on the protective layer; the exposure prevention layer comprises a biodegradable material.

Preferably, the thickness of the protective layer is 300-3000 μm; the width of the hollow structure is 500-3000 mu m.

Preferably, the hollowed-out structures form thread-shaped, parallel straight lines or parallel curves along the direction of the protective layer; the distance between adjacent parallel hollow structures is 500-3000 mu m.

Preferably, the thickness of the anti-exposure layer is 50-500 μm.

Preferably, the exposure prevention layer comprises a synthetic polymer material and/or a natural polymer material; the synthetic high molecular material is selected from one or more of polyethylene glycol (PEO), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P (3HB-co-4HB)), poly (P-dioxanone (PDS) and poly (glycerol sebacate) (PGS); the natural polymer material is selected from one or more of gelatin, collagen, hyaluronic acid and fibrin glue.

Preferably, the thickness of the reticular fiber framework layer is 200-2000 μm; the mesh fiber skeleton layer is formed of fibers; the diameter of the fiber is 20-200 mu m; the crossing angle of the fibers forming the reticular fiber skeleton layer is 30-110 degrees.

Preferably, the template for the tissue engineering material is a template for a tissue engineering blood vessel.

The invention also provides a tissue engineering material which is prepared by taking the template for the tissue engineering material as a template.

Preferably, the composite material comprises an inner layer, a middle layer and an outer layer which are tightly attached; the inner layer is a acellular matrix layer; the middle layer consists of a reticular fiber framework and an acellular matrix filled in the reticular fiber framework; the outer layer is an acellular matrix with a convex structure; and the acellular matrix layer of the inner layer, the acellular matrix of the middle layer and the acellular matrix of the outer layer are integrally arranged.

The invention provides a template for tissue engineering materials, which is characterized by comprising a support body, an anti-exposure layer, a reticular fiber skeleton layer and a protective layer which are sequentially arranged; and a hollow structure is arranged on the protective layer; the exposure prevention layer comprises a biodegradable material. Compared with the prior art, when the template provided by the invention is used for preparing the tissue engineering material, the anti-exposure layer is degraded to form a gap, and finally the acellular matrix material can be coated on the surface of the inner cavity of the reticular fibrous skeleton to reduce exposure of fibers, so that thrombosis is reduced; the reticular fiber skeleton can provide good mechanical property, so that the tissue engineering material has good kink resistance, rebound resilience, explosion resistance, puncture closeness and sewability; meanwhile, the protective layer can protect the reticular fiber skeleton layer structure, the skeleton structure is prevented from being damaged when the materials are cultured and prepared, the tissue engineering material prepared by the protective layer can have an external ridge beam due to the arrangement of the hollow structure, the bursting pressure, the anti-torsion capacity and the suture strength of the blood vessel are enhanced, and the rough outer surface is not easy to move after being implanted in a body, so that the integration with surrounding tissues is accelerated.

Drawings

FIG. 1 is a schematic structural diagram of a template for tissue engineering materials according to the present invention;

FIG. 2 is a schematic cross-sectional view of a template support for tissue engineering material provided by the present invention;

FIG. 3 is a schematic longitudinal cross-sectional view of a template support layer for tissue engineering material provided by the present invention;

FIG. 4 is a schematic view showing a process of preparing a tissue engineering material using a template for a tissue engineering material according to the present invention;

FIG. 5 is a schematic structural view of the template for tissue engineering materials according to comparative examples 1 to 5 of the present invention;

FIG. 6 is a scanning electron microscope image of a network fiber skeleton in a template for a tissue engineering material prepared in examples 3 and 8 of the present invention;

FIG. 7 is a scanning electron microscope image of the inner surface of the tissue engineering blood vessel obtained in example 3 and comparative example 3 of the present invention;

FIG. 8 is a H & E staining picture of a cross section of a tissue-engineered blood vessel obtained in example 1 of the present invention;

FIG. 9 is a macroscopic view of the tissue-engineered blood vessels obtained in example 3 and comparative example 8 of the present invention;

FIG. 10 is a rebound resilience test chart of the tissue-engineered blood vessel obtained in example 3 and comparative example 8 of the present invention;

fig. 11 is an internal cavity diagram of the tissue engineered blood vessel obtained in example 9 and comparative example 19 of the present invention 10 days after the sheep neck arteriovenous fistula is performed.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, fig. 1 is a schematic structural diagram of a template for tissue engineering material provided by the present invention, wherein 1 is a support, 2 is an anti-exposure layer, 3 is a mesh-shaped fiber skeleton layer, and 4 is a protective layer; and 4a is a hollow structure arranged on the protective layer.

The template for the tissue engineering material provided by the invention determines the overall shape of the prepared tissue engineering material by using the support body; the mesh fiber framework layer is supported to enable the mesh fiber framework layer to keep the shape under subcutaneous or in-vitro culture environment; the support body can be selected according to the application of the tissue engineering material to be prepared, and can be of a planar structure or a tubular structure; when the support body is of a tubular structure, the template for the tissue engineering material is a template for a tissue engineering blood vessel; when the support body is of a plane structure, the support body is preferably made of hard inert medical materials, and more preferably made of one or more of a silica gel sheet, a rubber sheet, a stainless steel plate, a nylon plate, a polyurethane sheet, a polycaprolactone sheet or polystyrene; when the support body is a tubular structure, see fig. 2 and 3, fig. 2 is a schematic cross-sectional view of the support body; FIG. 3 is a schematic longitudinal sectional view of a support body, which may be a solid structure or a hollow structure, without limitation; when the support body is of a solid structure, the support body can be of an integral solid structure or a double-layer structure, because the support body is prepared from high-molecular polymer or silica gel or rubber or nylon, the compatibility of the template and cells is good during tissue culture, but when the pipe diameter is larger, the template is easy to deform under the action of pressure, and a hard inner core is required to be added for supporting to form the double-layer structure; the longitudinal section of the support body can be rectangular or trapezoidal, and can also be any special shape matched with the shape of the vessel wall, such as U-shaped, Y-shaped, arched branched and the like; when the longitudinal section of the support body is irregular, the support body can be obtained by the following method: and acquiring three-dimensional data of the blood vessel of the part needing blood vessel transplantation, constructing a corresponding blood vessel 3D model by using the acquired three-dimensional data of the blood vessel size through computer software, and printing the blood vessel 3D model to obtain the special-shaped support. When the support body is of a tubular structure, the outer diameter of the support body is preferably 2-12 mm, and more preferably 2-8 mm; when the support body is a hollow tubular structure, the support body is preferably made of one or more of polymer materials such as medical silica gel, rubber, nylon, stainless steel, alloy, Polyurethane (PU), Polycaprolactone (PCL), poly (lactide-caprolactone) (PLCL) copolymer and the like; when the support body is of a solid tubular structure, the support body is preferably made of one or more of medical silica gel, rubber, nylon, stainless steel, alloy, polyurethane, polycaprolactone, poly (lactide-caprolactone) copolymer (PLCL) and other high polymer materials; when the support body is of a double-layer structure, the outer layer can be made of one or more of high polymer materials such as medical silica gel, rubber, nylon, Polyurethane (PU), Polycaprolactone (PCL), poly (lactide-caprolactone) copolymer (PLCL) and the like, and the inner layer can be made of one or more of materials such as medical silica gel, rubber, nylon, stainless steel, alloy, Polyurethane (PU), Polycaprolactone (PCL), poly (lactide-caprolactone) copolymer (PLCL) and the like.

The support body is provided with an anti-exposure layer; the thickness of the anti-exposure layer is preferably 50-500 mu m; in some embodiments provided herein, the anti-exposure layer preferably has a thickness of 50 μm; in some embodiments provided herein, the anti-exposure layer preferably has a thickness of 200 μm; in some embodiments provided herein, the anti-exposure layer preferably has a thickness of 250 μm; in some embodiments provided herein, the anti-exposure layer preferably has a thickness of 100 μm; in other embodiments provided herein, the anti-exposure layer preferably has a thickness of 500 μm; the anti-exposure layer comprises a degradable material, so that the anti-exposure layer can be degraded in the process of preparing the tissue engineering material by utilizing the template for the tissue engineering material, the arrangement of the anti-exposure layer can prevent the reticular fiber framework layer from being combined with the support body too tightly, improve the migration and growth of cells to the surface of the support body, secrete extracellular matrix around the support body, and enable the finally prepared vascular material to be exposed without framework fibers; in the present invention, the exposure prevention layer preferably includes a synthetic polymer material and/or a natural polymer material; the synthetic polymer material is preferably one or more of polyethylene glycol (PEO), polyglycolic acid (PGA), poly-P-dioxanone (PDS), polylactic-co-glycolic acid (PLGA), poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P (3HB-co-4HB)) and Polysebacate (PGS); the natural polymer material is preferably one or more of fibrin glue, gelatin, collagen, hyaluronic acid and fibrin glue; the anti-exposure layer arranged on the support body can be prepared by the technologies of electrostatic spinning, pouring, spraying, melt spinning, wet spinning, freeze drying, 3D printing and the like.

The anti-exposure layer is provided with a reticular fiber skeleton layer; the unique structure of the reticular fiber framework layer can enable the finally prepared tissue engineering material to have the advantages of kink resistance, rebound resilience, pierceable closure and necessary burst strength and suture strength; the pores can meet the requirement of migration and growth of cells to the inside of the framework and fill the pores of the framework; the thickness of the reticular fiber framework layer is preferably 200-2000 mu m; in some embodiments provided herein, the thickness of the reticulated fiber skeleton layer is preferably 200 μm; in some embodiments provided herein, the thickness of the mesh fiber skeleton layer is preferably 400 μm; in some embodiments provided herein, the thickness of the mesh fiber skeleton layer is preferably 550 μm; in some embodiments provided herein, the thickness of the mesh fiber skeleton layer is preferably 700 μm; in other embodiments provided herein, the thickness of the reticulated fiber skeleton layer is preferably 2000 μm; the mesh fiber skeleton layer is preferably formed of fibers; the composite material can be prepared by electrostatic spinning, melt spinning, 3D printing, fiber weaving and other methods; in the electrostatic spinning, the melt spinning and the 3D printing, the parallelism of the fiber spacing between each layer of fibers of the reticular fiber framework can be improved by adding phase control, so that the uniformity of the framework aperture is increased, namely, the standard deviation of the framework average aperture is reduced.

In the present invention, the reticular fiber skeleton layer is preferably prepared by melt spinning;

the fibers in the mesh-like fiber skeleton layer may be fibers well known to those skilled in the art, and are not particularly limited, and the fibers in the mesh-like fiber skeleton layer according to the present invention are preferably formed of one or more of polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), Polylactide (PLA), Polyhydroxyalkanoate (PHA), poly (p-dioxanone) (PDS), Polycaprolactone (PCL), poly (lactide-caprolactone) copolymer (PLCL), Polyurethane (PU), poly (glycerol sebacate) (PGS), and polyethylene glycol (PEO); the diameter of the fiber is preferably 20-200 μm, and more preferably 20-100 μm; in some embodiments provided herein, the fibers preferably have a diameter of 20 μm; in some embodiments provided herein, the fibers preferably have a diameter of 40 μm; in some embodiments provided herein, the fibers preferably have a diameter of 60 μm; in other embodiments provided herein, the fibers preferably have a diameter of 100 μm; the crossing angle of the fibers forming the net-like fiber skeleton layer is preferably 30 to 110 °; in some embodiments provided herein, the fibers forming the reticulated fiber skeleton layer preferably have an intersection angle of 30 °; in some embodiments provided herein, the fibers forming the reticulated fiber skeleton layer preferably have an intersection angle of 50 °; in some embodiments provided herein, the fibers forming the reticulated fiber skeleton layer preferably have an intersection angle of 60 °; in other embodiments provided herein, the fibers forming the reticulated fiber skeleton layer preferably have an intersection angle of 110 °; the standard deviation of the average pore diameter of the reticular fiber framework layer is preferably 5-30 mu m; in some embodiments provided herein, the standard deviation of the pore size average of the mesh-like fiber skeleton layer is preferably 5 μm; in some embodiments provided herein, the standard deviation of the pore size mean of the reticulated fiber skeleton layer is preferably 12 μm; in some embodiments provided herein, the standard deviation of the pore size average of the mesh-like fiber skeleton layer is preferably 21 μm; in other embodiments provided herein, the standard deviation of the pore size average of the mesh-like fiber skeleton layer is preferably 30 μm; the average pore diameter of the reticular fiber framework layer is preferably 75-500 mu m; in some embodiments provided herein, the mesh-like fiber skeleton layer preferably has an average pore size of 75 μm; in some embodiments provided herein, the mesh-like fiber skeleton layer preferably has an average pore size of 105 μm; in some embodiments provided herein, the mesh-like fiber skeleton layer preferably has an average pore size of 220 μm; in other embodiments provided herein, the mesh-like fiber skeleton layer preferably has an average pore size of 500 μm; the parallelism of the parallel intervals of the fibers of the reticular fiber skeleton determines the uniformity of the pore diameters of the same tissue engineering material at different positions, and further determines the uniformity degree of the anti-explosion performance of the material at different positions. By limiting the crossing angle, the fiber diameter and the aperture uniformity of fiber weaving, namely improving the spinning precision, the nonuniformity of the framework aperture caused by too large/too small fiber spacing is avoided, the rejection rate of the blood vessel material is further increased, and the aperture uniformity is improved, so that the qualification rate of the finished product of the blood vessel is greatly improved; meanwhile, the mechanical property and the cell migration of the blood vessel are controlled by optimizing and controlling the reticular fiber skeleton structure (wall thickness, fiber diameter, aperture, thickness and angle of crossed fibers), so as to reach the gold standard of the artificial blood vessel.

A protective layer is arranged on the reticular fiber framework layer; the protective layer can prevent the damage of the animal subcutaneous embedding operation or in-vitro cell inoculation and in-vitro culture process to the skeleton structure, and can control the thickness of the tissue engineering material to ensure the uniformity of the wall thickness of the prepared tissue engineering material; the thickness of the protective layer is preferably 300-3000 mu m; in some embodiments provided herein, the thickness of the protective layer is preferably 300 μm; in some embodiments provided herein, the protective layer preferably has a thickness of 500 μm; in some embodiments provided herein, the protective layer preferably has a thickness of 1000 μm; in other embodiments provided herein, the protective layer preferably has a thickness of 3000 μm; the thickness of the protective layer determines the height of the surface bulge of the tissue engineering material; when the supporting layer is of a tubular structure, the inner diameter of the protective layer is matched with the outer diameter of the tubular structure formed by the supporting layer, the anti-exposure layer and the reticular fiber skeleton layer; the protective layer is provided with a hollow structure; exposing the reticular fiber skeleton layer through the hollow structure; the width of the hollow structure is preferably 500-3000, and more preferably 500-2000 mu m; the shape of the hollow structure along the radial section of the protective layer can be rectangular, trapezoidal, square or any other shape; the shape of the hollow structure along the radial section of the protective layer determines the convex shape of the outer wall of the tissue engineering material; the hollow structure is preferably in a thread shape, parallel straight lines or parallel curves along the direction of the protective layer; when the supporting body is of a tubular structure, the hollow structure on the protective layer is in a thread shape; when the supporting body is planar, the hollow structure on the protective layer is in a parallel straight line or flat curve structure; the two opposite ends of the protective layer do not contain hollow structures, so that the protective layer keeps an integral structure; the distance between the adjacent parallel hollow structures, namely between two adjacent threads, and between two parallel straight lines or curves is preferably 500-3000 mu m; in some embodiments provided by the present invention, the distance between the adjacent parallel hollow structures is preferably 500 μm; in some embodiments provided by the present invention, the distance between the adjacent parallel hollow structures is preferably 1000 μm; in some embodiments provided by the present invention, the distance between the adjacent parallel hollow structures is preferably 1500 μm; in some embodiments provided by the present invention, the distance between the adjacent parallel hollow structures is preferably 3000 μm; the protective layer is provided with the hollowed-out structure, particularly the spiral hollowed-out structure, so that spiral protrusions can be formed outside the tissue engineering material on the premise of ensuring effective migration of cells, the anti-kink performance, the anti-explosion performance and the suture strength of the tissue engineering material can be enhanced, the tissue engineering material can be prevented from sliding after being implanted into a human body, and the tissue engineering material is easier to integrate with tissues around an implanted part.

When the template provided by the invention is used for preparing a tissue engineering material, the anti-exposure layer is degraded to form a gap, so that cells can migrate, grow and secrete extracellular matrix in the gap, and after cell removal treatment, the acellular matrix material is coated on the inner cavity surface of the reticular fibrous skeleton to reduce exposure of fibers, so that the adhesion of blood coagulation components is reduced; the reticular fiber skeleton can provide good mechanical property, so that the tissue engineering material has good kink resistance, rebound resilience, explosion resistance, puncture closeness and sewability; meanwhile, the protective layer can protect the reticular fiber framework layer structure, the framework structure is prevented from being damaged when the materials are cultured and prepared, the arrangement of the hollow structure on the protective layer can enable the prepared tissue engineering material to have an external ridge beam, the anti-kink performance, the anti-explosion performance and the suture strength are improved, the rough surface is more stable in vivo, and the integration with the surrounding tissues of the implanted part is accelerated.

The invention also provides a tissue engineering material, which is prepared by taking the template for the tissue engineering material as a template; the preparation method is preferably tissue engineering culture; the tissue engineering culture can be subcutaneous implantation culture or in vitro culture; the in vitro culture can be in vitro static culture or in vitro dynamic culture; in the invention, different culture methods can be selected according to the structure of the supporting layer, such as in-vitro dynamic culture, the supporting layer needs to be a hollow structure, preferably a hollow structure made of one or a mixture of several of elastic Polyurethane (PU), poly (lactide-caprolactone) (PLCL), silica gel and rubber, and the materials have elasticity and can respond to the pressure of liquid to beat; other culture methods are not so limited; when subcutaneous implantation culture is carried out, the hollow structure on the template protection layer for the tissue engineering material can meet the requirements of migration and growth of cells to the interior of the template, so that the cells are filled in gaps generated after the degradation of the exposure prevention layer, the pores of the reticular fibrous skeleton and the hollow parts on the protection layer; for in vitro cell culture, the hollow structure on the culture medium can play a role in promoting the exchange of oxygen, nutrient substances and cell metabolic waste.

In the present invention, it is preferably prepared specifically by the following method: implanting the template for the tissue engineering material under the animal skin, or placing the template for the tissue engineering material implanted with cells into a culture solution, and culturing in a bioreactor; enabling cells to migrate and grow to the reticular fiber skeleton layer until the hollow part of the protective layer is full of the cells, and enabling fiber pores of the reticular fiber skeleton and gaps formed by degradation of the anti-exposure layer to grow; taking out, and removing the protective layer and the supporting layer to obtain the tissue engineering material; the planted cells are preferably one or more of fibroblasts, smooth muscle cells and smooth muscle cells differentiated from stem cells; when in-vitro dynamic culture is adopted, the flow rate of the culture medium in the bioreactor is preferably adjusted to ensure that the pressure is 10-200 mmHg; the culture is preferably carried out at 37 ℃ and 20% O₂、5％CO₂Culturing is carried out under the conditions.

More preferably, after removing the protective layer and the support body, a decellularization treatment step is preferably performed to obtain the tissue engineering material, namely the tissue engineering material comprises an inner layer, a middle layer and an outer layer which are tightly attached; the inner layer is a acellular matrix layer; the middle layer consists of a reticular fiber framework and an acellular matrix filled in the reticular fiber framework; the outer layer is an acellular matrix with a convex structure; and the acellular matrix layer of the inner layer, the acellular matrix of the middle layer and the acellular matrix of the outer layer are integrally arranged.

The decellularization treatment is preferably performed by an SDS method or a liquid nitrogen freeze-thaw method.

Preferably, the SDS method comprises the steps of: soaking the sample in 1% SDS solution, shaking on a shaker at room temperature for 12h, washing residual SDS on the tissue wrap with sterile physiological saline, and placing in sterile mixed solution of DNase and RNase (enzyme liquid system is 40ml, and buffer solution thereof is 0.2mol/L MgCl)₂，0.2mol/LCaCl₂And 0.1mol/L Tris-HCl with pH 6.4 and ultrapure water, wherein the concentration of DNase is 50U/ml and the concentration of RNase is 1U/ml), shaking the mixture on a shaking table at room temperature for 24 hours, then washing the residual DNase and RNase on the tissue wrappage by using sterile physiological saline, and finally placing the obtained product in sterile PBS for storage at 4 ℃.

Preferably, the liquid nitrogen freeze-thaw method comprises the following steps: the samples were snap frozen in liquid nitrogen for 20s, thawed at room temperature for 60s, repeated 5 times, then rinsed 4-5 times with sterile physiological saline, and the cell debris was rinsed clean. The material was then placed in a sterile mix of DNase and RNase (40 ml enzyme solution, buffer from 0.2mol/L MgCl)₂，0.2mol/L CaCl₂And 0.1mol/L Tris-HCl with pH 6.4 and ultrapure water, wherein the concentration of DNase is 50U/ml and the concentration of RNase is 1U/ml), shaking the mixture on a shaking table at room temperature for 24 hours, then washing the residual DNase and RNase on the tissue wrappage by using sterile physiological saline, and finally placing the prepared product in sterile PBS for storage at 4 ℃.

Referring to fig. 4, fig. 4 is a schematic view of a process of preparing a tissue engineering blood vessel by tissue culture using the template for tissue engineering material provided by the present invention, wherein 1 is a support, 2 is an anti-exposure layer, 3 is a reticular fiber skeleton layer, and 4 is a protective layer; 4a is a hollow structure arranged on the protective layer, 5 is cells and extracellular matrix for filling the template, 5a is the cells and extracellular matrix for filling the hollow part of the template protective layer, 5b is the cells and extracellular matrix for filling the gaps generated by the degradation of the bare-proof layer of the template, 5c is the cells and extracellular matrix for filling the fiber pores of the reticular fiber skeleton layer of the template, 6 is the decellularized matrix of the prepared blood vessel, 6a is the convex decellularized matrix of the outer layer of the prepared blood vessel, 6b is the inner layer of the decellularized matrix of the prepared blood vessel, and 6c is the decellularized matrix of the middle layer of the prepared blood vessel for filling the fiber pores of the reticular fiber skeleton layer. In the culture process, the anti-exposure layer (2) is degraded, a gap is formed between the support body (1) and the reticular fiber skeleton layer (3), then the grooves (4a) of the protective layer, the pores of the reticular fiber skeleton (3) and the gap formed by degradation of the anti-exposure layer (2) are filled with cells and extracellular matrix (5), then the protective layer (4) and the support body (1) are removed, the part without a convex structure is cut off after cell removal treatment, and the tissue engineering blood vessel with an inner acellular matrix layer (6b), a middle layer fiber skeleton filled with an acellular matrix (6c) and an outer layer with a convex acellular matrix (6a) is obtained, and the acellular matrix layer (6b) of the inner layer, the acellular matrix (6c) of the middle layer and the convex acellular matrix (6a) of the outer layer are integrally arranged.

According to the invention, the tissue engineering material obtained after the cell removal treatment is preferably loaded with bioactive substances, so that the smoothness of the biological engineering material can be improved; the bioactive material is preferably loaded on the tissue engineering material by covalent or physical adsorption; the kind of the bioactive substance is preferably one or 2 mixed kinds of heparin and hirudin.

In the invention, the prepared tissue engineering material is preferably a tissue engineering blood vessel or a tissue engineering patch; when the tissue engineering material is a tissue engineering patch, the tissue engineering material can be used for a blood vessel patch, a hernia patch, a heart patch, a bladder patch, a urethra patch or an intestine patch.

The reticular fiber skeleton in the tissue engineering material obtained by the invention mainly plays a role in improving good mechanical properties, so that the tissue engineering material has good kink resistance, rebound resilience, bursting resistance, puncture closeness and suture property; the acellular matrix compounded on the reticular fibrous skeleton provides good biocompatibility on the whole, can promote the regeneration of vascular tissues and the integration with tissues of an implanted part, has divided functions according to the position of the acellular matrix, and the extracellular matrix filled in the gaps of the skeleton also plays a role in effectively blocking the leakage of blood and biological macromolecules; moreover, the tissue engineering material prepared by the invention can also be loaded with anticoagulant substances, so that the smoothness is improved, and acute coagulation is not easy to occur.

The tissue engineering material provided by the invention not only meets the specific mechanical requirements required by an implanted part, but also has good biocompatibility, is beneficial to cell adhesion and growth, and can be used for clinical blood vessel replacement, blood vessel bypass establishment or arteriovenous fistulation.

In order to further explain the present invention, the following describes a template for tissue engineering material and a tissue engineering material in detail with reference to the examples.

The reagents used in the following examples are all commercially available.

Example 1

The template for the tissue engineering material provided by the embodiment is a tissue engineering blood vessel template, and comprises a support body, namely an inner core, an anti-exposure layer, a reticular fiber skeleton layer and a protective layer, namely an outer sleeve from inside to outside; the protective layer is provided with a hollow structure; the protective layer is arranged outside the reticular fiber framework layer, and the inner diameter of the protective layer is matched with the outer diameter of the reticular fiber framework layer.

The template preparation process comprises the following steps:

1. selecting a silicone tube as a support body, wherein the outer diameter is 2mm, and the inner diameter is 1 mm; the outer diameter of the support body determines the inner diameter of the prepared tissue engineering blood vessel.

2. The method comprises the following steps of (1) performing PDS: gelatin is 1:2 (mass ratio) as a raw material, and a biodegradable layer is prepared on the surface of the silicone tube by adopting a spin coating method. And inserting a stainless steel rod with a proper caliber into the silicone tube to serve as a receiving rod, and connecting the receiving rod with a rotating motor. Hexafluoroisopropanol as solvent, PDS: gelatin is 1:2 (mass ratio) as a solute, a coating solution with the concentration of 0.3G/ml is prepared, a 14G needle is adopted, the coating solution is extruded and coated on the outer wall of a silica gel tube which rotates at the rotating speed of 150r/min and the horizontal moving speed of 2mm/s by an injector according to the injection speed of 15ml/h, the distance from the needle to the outer wall of the silica gel tube is 5mm, and the needle vertically faces downwards to the axis of the silica gel tube. After the coating is finished, the silicone tube coated with the PDS/gelatin mixture is placed in a fume hood, after the solvent is volatilized, the silicone tube is placed in a vacuum pump to remove the residual solvent, and the PDS/gelatin anti-exposure layer with the thickness of 50 microns can be obtained on the surface of the silicone tube.

3. Polycaprolactone (PCL) is used as a raw material, a reticular fiber framework layer is formed on the surface of the anti-exposure layer by utilizing a melt spinning technology, the fiber diameter of the reticular fiber framework layer is 20 mu m, the crossing angle between the fibers is 30 degrees, and the thickness of the reticular fiber framework layer is 200 mu m. The specific method of melt spinning comprises the following steps: connecting the receiving rod with a rotating motor; placing the PCL in a closed stainless steel injector wrapped by a hot melting device, heating at 210 ℃ for 1h, and then spinning; the stainless steel syringe is matched with a 20G stainless steel needle, the distance between the syringe needle and the receiving rod is 10mm, the flow rate of the PCL melt is 0.1ml/h, the rotating speed of the receiving rod is set to be 300r/min, and the translation speed is set to be 7 mm/s. The spin acceptance thickness was 200 μm.

4. The support body in the embodiment is a silicone tube, the wall thickness of the support body is 0.5mm, the inner diameter of the support body is 1mm, and the support body is strong in self-supporting performance, so that the stainless steel rod in the silicone tube is pulled out after the framework main body is prepared, and only the silicone tube is reserved as the support body.

5. PCL is used as a raw material, a protective layer is prepared by a 3D printing (Allevi3, Allevi, America) technology, two ends of the protective layer are of hollow-out-free structures, and the middle of the protective layer is of a threaded hollow-out structure.

6. The protective layer is sleeved outside the prepared fiber framework, the framework is completely positioned at the hollowed-out part, the rubber rod is made into a plug and is blocked at two ends of the protective layer, and the fiber framework is prevented from moving inside the protective layer in the process of subcutaneous implantation or in vitro cell culture.

Specific parameters of the template obtained in example 1 are shown in Table 1.

In this embodiment, in addition to the above template preparation process, the method for preparing a tissue engineering blood vessel includes the following specific steps:

1. the prepared tissue engineering blood vessel template is integrally implanted under the sheep skin and is taken out after 60 days.

2. And after the blood vessel template is taken out, removing the protective layer and the support body, cutting off the parts without the raised threads at the two tail ends of the blood vessel, and then performing cell removal treatment.

The cell removing treatment step adopts an SDS method: soaking the sample in 1% SDS solution, shaking on a shaker at room temperature for 12h, washing residual SDS on the tissue wrap with sterile physiological saline, and placing in sterile mixed solution of DNase and RNase (enzyme liquid system is 40ml, and buffer solution thereof is 0.2mol/L MgCl)₂，0.2mol/L CaCl₂And 0.1mol/L Tris-HCl with pH 6.4 and ultrapure water, wherein the concentration of DNase is 50U/ml and the concentration of RNase is 1U/ml), shaking the mixture on a shaking table at room temperature for 24 hours, then washing the residual DNase and RNase on the tissue wrappings with sterile physiological saline, and finally placing the obtained product in sterile PBS.

3. And (3) after cell removal treatment, cross-linking heparin is carried out, and the corresponding tissue engineering blood vessel can be obtained. Preparing sterile MES (2-morpholine ethanesulfonic acid) buffer solution with the pH value of 5.6 and the concentration of 0.05M by using deionized water, and then preparing crosslinking reaction liquid by using MES, wherein the concentration of each component in the reaction liquid is as follows: EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) 2.0 mg/ml; NHS (N-hydroxysuccinimide 1.2 mg/ml; heparin sodium 2.0 mg/ml. then, the crosslinking reaction is carried out, firstly, the acellular product is soaked in MES buffer solution with the pH value of 5.6 and the concentration of 0.05M for 20 minutes, then, the reaction solution is placed at 37 ℃ for reaction for 10 minutes to activate carboxyl, after 10 minutes, the acellular product is taken out of the MES buffer solution and is directly placed in the reaction solution, the reaction solution is gently shaken at 37 ℃ (20-40 rpm/min) for 4 hours, and then, 0.1M sterile Na is used for₂HPO₄(containing 1% PS) continuously washing the material for 2 hours, and then continuously washing the material for 24 hours at 4 ℃ by using sterile 4M NaCl at 60-80 rpm/min; then continuously washing the obtained product for 24 hours at 4 ℃ by using sterile water (containing 1% PS) at 60-80 rpm/min. Finally, the material was placed in sterile PBS solutionAnd preserving at middle temperature and 4 ℃ for later use.

Example 2:

the template preparation process comprises the following steps:

1. a polyurethane tube with an outer diameter of 4mm and an inner diameter of 2mm is used as a support body, and the outer diameter of the support body determines the inner diameter of the prepared tissue engineering blood vessel.

2. PLGA and PEO are used as raw materials, and a high-voltage electrostatic spinning technology is adopted to prepare an anti-exposure layer on the surface of a polyurethane tube, and the specific method comprises the following steps: a stainless steel rod with an appropriate diameter was inserted into the polyurethane tube as a receiving rod, and chloroform: methanol-5: 1 (volume ratio) as solvent, PLGA: PEO is 1:1 (mass ratio) and is used as a solute, an electrospinning solution with the concentration of 0.28G/mL is prepared, a 20G needle is adopted, the flow rate is set to be 8mL/h, a direct current voltage of 16kV is applied to the electrospinning needle, a grounded stainless steel plate is adopted as a conductive receiving plate, a receiving rod is arranged between the conductive receiving plate and the needle, the distance between the receiving rod and the conductive receiving plate is 1cm, and the distance between the conductive plate and the needle is 8 cm. The fibers were collected by a polyurethane tube during the process of gathering to the conductive plate, and a PLGA/PEO composite anti-exposure layer having a thickness of 200 μm was collected.

3. The method comprises the steps of taking poly (lactide-caprolactone) copolymer (PLCL) as a raw material, and forming a first reticular fiber framework layer on the surface of an exposure prevention layer by utilizing a melt spinning technology, wherein the diameter of fibers of the reticular fiber framework layer is 40 mu m, the intersection angle between the fibers is 50 degrees, and the thickness of the reticular fiber framework layer is 400 mu m. The specific method of melt spinning comprises the following steps: sleeving the polyurethane pipe provided with the anti-exposure layer obtained in the step 2 on a stainless steel rod to serve as a receiving rod, and connecting the receiving rod with a rotating motor; placing PLCL in a closed stainless steel injector wrapped by a hot melting device, heating at 220 ℃ for 1h, and then spinning; the stainless steel syringe is matched with a 20G stainless steel needle, the distance between the syringe needle and the receiving rod is 13mm, the flow rate of the melt is 0.25ml/h, the rotating speed of the receiving rod is set to be 200r/min, and the translation speed is set to be 18 mm/s. The spin acceptance thickness was 400 μm.

4. The stainless steel rod in the polyurethane tube is drawn out and a silica gel rod with proper size is inserted to play a supporting role.

Specific parameters of the template obtained in example 2 are shown in Table 1.

In this embodiment, in addition to the above template preparation process, a preparation method of a tissue engineering blood vessel is also included, and the specific process is the same as embodiment 1, which is not described herein again.

Example 3:

1. the silicone tube with the outer diameter of 5mm and the inner diameter of 3mm is used as a support body, and the outer diameter of the support body determines the inner diameter of the prepared tissue engineering blood vessel.

2. P (3HB-co-4HB) and PEO are used as raw materials, a high-voltage electrostatic spinning technology is adopted, and an anti-exposure layer is prepared on the surface of a silicone tube, and the specific method comprises the following steps: inserting a stainless steel bar with a proper caliber into the silicone tube to serve as a receiving bar, and adding chloroform: methanol 5:1 (volume ratio) as solvent, P (3HB-co-4 HB): PEO is 1:0.5 (mass ratio) as a solute, an electrospinning solution with a concentration of 0.3G/mL is prepared, a 19G needle is adopted, a flow rate is set to be 8mL/h, a direct current voltage of 14kV is applied to the needle, a grounded stainless steel plate is adopted as a conductive receiving plate, a silicone tube is arranged between the conductive receiving plate and the needle, the distance between the silicone tube and the conductive receiving plate is 1cm, the distance between the conductive plate and the needle is 11cm, a receiving rod rotates at a rotating speed of 100r/min, fibers sprayed by electrospinning are received in front of the conductive receiving plate, and a P (3HB-co-4HB)/PEO anti-exposure layer with the thickness of 250 mu m is collected.

3. Polycaprolactone (PCL) is used as a raw material, a first reticular fiber framework layer is formed on the surface of the exposure-proof layer by utilizing a melt spinning technology, the fiber diameter of the reticular fiber framework layer is 60 mu m, the crossing angle between the fibers is 50 degrees, and the thickness of the reticular fiber framework layer is 550 mu m. The specific method of melt spinning comprises the following steps: sleeving the silicone tube provided with the P (3HB-co-4HB)/PEO anti-exposure layer obtained in the step 2 on a stainless steel rod with the diameter of 3.8mm to serve as a receiving rod, and connecting the receiving rod with a rotating motor; placing the PCL in a closed stainless steel injector wrapped by a hot melting device, heating at 210 ℃ for 1h, and then spinning; the stainless steel injector is matched with a 17G stainless steel needle, the distance between the injector needle and the receiving rod is 15mm, the flow rate of the PCL melt is 0.7ml/h, the rotation speed of the receiving rod is set to be 180r/min, and the translation speed is set to be 21 mm/s. The spin acceptance thickness was 550 μm.

4. The stainless steel rod in the silicone tube is drawn out and the silicone rod with proper size is inserted to play a supporting role.

Specific parameters of the template obtained in example 3 are shown in Table 1.

Example 4:

the template preparation process comprises the following steps:

1. the nylon tube with the outer diameter of 6mm and the inner diameter of 4mm is used as a support body, and the outer diameter of the support body determines the inner diameter of the prepared tissue engineering blood vessel.

2. The method is characterized in that PDS and gelatin are used as raw materials, a spraying technology is adopted, and an anti-exposure layer is prepared on the surface of a nylon tube, and the specific method comprises the following steps: a stainless steel rod of an appropriate diameter was inserted into the nylon tube as a receptor, and the receptor was immersed in a 2mg/mL dopamine solution prepared in 10mm tris buffer (ph8.5) for 24 hours to form an adhesive layer on the surface of the nylon tube. And (3) mixing the PDS: gelatin 1:1 (mass ratio) was dissolved in hexafluoroisopropanol to prepare a 0.3g/ml spray solution. The solution was sprayed vertically onto a rotating nylon tube at a flow rate of 0.2mL/s using an air pump spray gun, and a 100 μm thick anti-exposure layer of composite PDS and gelatin was collected on the surface of the nylon tube.

3. PCL is used as a raw material, a melt spinning technology is adopted, a reticular fiber framework layer is formed on the surface of an exposure prevention layer, the fiber diameter of the reticular fiber framework layer is 100 mu m, the intersection angle between fibers is 60 degrees, and the thickness of the reticular fiber framework layer is 700 mu m. The specific method of melt spinning comprises the following steps: sleeving the nylon pipe provided with the anti-exposure layer obtained in the step 2 on a stainless steel rod to serve as a receiving rod, and connecting the receiving rod with a rotating motor; placing the PCL in a closed stainless steel injector wrapped by a hot melting device, heating at 210 ℃ for 1h, and then spinning; the stainless steel syringe is matched with a 15G stainless steel needle, the distance between the syringe needle and the receiving rod is 17mm, the flow rate of the PCL melt is 2.4ml/h, the rotating speed of the receiving rod is set to be 150r/min, and the translation speed is set to be 27 mm/s. The spin acceptance thickness was 700 μm.

4. The stainless steel rod in the nylon is drawn out and the silica gel rod with proper size is inserted to play a supporting role. .

Specific parameters of the template obtained in example 4 are shown in Table 1.

Example 5:

the template preparation process comprises the following steps:

1. a stainless steel bar with the outer diameter of 8mm is used as a support body, and the outer diameter of the support body determines the inner diameter of the prepared tissue engineering blood vessel.

2. The method is characterized in that hyaluronic acid is used as a raw material, a freeze drying technology is adopted, and an anti-exposure layer is prepared on the surface of stainless steel, and the specific method comprises the following steps: preparing 25mg/ml hyaluronic acid solution by using distilled water as a solvent, fixing a mould outside a stainless steel round bar to enable the distance between the surface of the stainless steel and the mould to be 500 mu m, pouring the hyaluronic acid solution between the stainless steel round bar and the mould, freezing the solution at the temperature of minus 80 ℃ for 24 hours, and then carrying out vacuum freeze drying for 72 hours. After the mold is removed, a hyaluronic acid anti-exposure layer with the thickness of 500 mu m can be obtained on the surface of the stainless steel round bar.

3. PLA is used as a raw material, a first reticular fiber framework layer is formed on the surface of the anti-exposure layer by utilizing a melt spinning technology, the fiber diameter of the reticular fiber framework layer is 100 mu m, the crossing angle between the fibers is 110 degrees, and the thickness of the reticular fiber framework layer is 2000 mu m. The specific method of melt spinning comprises the following steps: taking the stainless steel cylinder with the diameter of 8mm and the anti-exposure layer obtained in the step 2 as a receiving rod, and connecting the receiving rod with a rotating motor; placing PLA in a closed stainless steel injector wrapped by a hot melting device, heating at 240 ℃ for 1h, and then spinning; the stainless steel syringe is matched with a 15G stainless steel needle, the distance between the syringe needle and the receiving rod is 19mm, the flow rate of the PLA melt is 10ml/h, the rotating speed of the receiving rod is set to be 120r/min, and the translation speed is set to be 72 mm/s. The spin acceptance thickness was 2000. mu.m.

4. PCL is used as a raw material, a protective layer is prepared by a 3D printing (Allevi3, Allevi, America) technology, two ends of the protective layer are of hollow-out-free structures, and the middle of the protective layer is of a threaded hollow-out structure.

5. The protective layer is sleeved outside the prepared fiber framework, the framework is completely positioned at the hollowed-out part, the rubber rod is made into a plug and is blocked at two ends of the protective layer, and the fiber framework is prevented from moving inside the protective layer in the process of subcutaneous implantation or in vitro cell culture.

Specific parameters of the template obtained in example 5 are shown in Table 1.

The specific parameters of the template for tissue engineering blood vessels described in examples 1 to 5 are shown in the following table 1:

TABLE 1 parameter Table of tissue engineering blood vessel template in examples 1 to 5

Examples 6 to 10

Examples 6 to 10 correspond to examples 1 to 5 in sequence, and each example is different only in that phase control is started when a reticular fiber framework is prepared, that is, at a folding point of each layer of spinning process, the phase control device is used for repositioning, and the angular displacement of a single-pass starting position is accurately controlled by controlling a step angle and the number of pulses, so that fibers are distributed on a support body at equal intervals, the uniformity of the pore size of the fiber framework is further improved, and the standard deviation of the average pore size of the obtained reticular fiber framework is reduced, and the specific difference is shown in a parameter selection table in table 2.

TABLE 2 parameters of templates for tissue engineering vessels in examples 6 to 10

Comparative examples 1 to 5

Comparative examples 1 to 5 correspond to examples 1 to 5 in sequence, and are different only in that no anti-exposure layer is provided, referring to fig. 5, fig. 5 is a schematic structural diagram of the template for tissue engineering blood vessels prepared in comparative examples 1 to 5, wherein 1 is a support, 2 is a reticular fiber skeleton layer, 4 is a protective layer, and 4a is a hollow structure provided on the protective layer. The specific differences are shown in the following table 3 parameter selection table.

TABLE 3 parameter conditions of templates for tissue engineering vessels in comparative examples 1 to 5

Comparative examples 6 to 10

Comparative examples 6 to 10 correspond to examples 1 to 5 in sequence, and the differences are only that no protective layer is arranged, and specific differences are shown in a comparative example parameter selection table in table 4 below.

TABLE 4 parameters of templates for tissue engineering vessels in comparative examples 6 to 10

Comparative examples 11 to 15

Comparative examples 11 to 15 correspond to examples 1 to 5 in sequence, and the differences are only that no protective layer is provided and no anti-exposure layer is provided, and the specific differences are shown in a comparative example parameter selection table in table 5 below.

TABLE 5 parameters of templates for tissue engineering vessels in comparative examples 11 to 15

Comparative examples 16 to 20

Comparative examples 16 to 20 correspond to examples 6 to 10 in sequence, and the differences are only that no anti-exposure layer is arranged, and specific differences are shown in a comparative example parameter selection table in table 6 below.

TABLE 6 parameter conditions for templates for tissue engineering vessels in comparative examples 16-20

Comparative examples 21 to 25

Comparative examples 21 to 25 correspond to examples 6 to 10 in sequence, and the difference is only that no protective layer is provided, and the specific difference is shown in a comparative example parameter selection table in table 7 below.

TABLE 7 parameter conditions of templates for tissue engineering vessels in comparative examples 21 to 25

Comparative examples 26 to 30

Comparative examples 25 to 30 correspond to examples 6 to 10 in sequence, and the differences are only that no protective layer is provided and no anti-exposure layer is provided, and the specific differences are shown in a comparative example parameter selection table in table 8 below.

TABLE 8 parameter conditions for templates for tissue engineering vessels in comparative examples 26 to 30

And (3) acellular matrix filling detection:

the blood vessels prepared in examples 1 to 10 and comparative examples 1 to 30 were tested for acellular matrix filling, and the specific method was as follows:

1. detection of inner cavity acellular matrix coverage: after the prepared tissue engineering blood vessel is frozen and dried, the covering of the acellular matrix in the lumen of the blood vessel and the exposure of fibers are observed by using a scanning electron microscope.

2. And (3) detecting the acellular matrix filling condition of the fiber skeleton: the obtained tissue engineering blood vessel was dehydrated and embedded in paraffin, then paraffin sections were cut, the sections were stained with H & E, and the filling of the pores of the fibrous skeleton with acellular matrix was observed by a microscope (Leica DM 4B).

3. Detecting the filling condition of the acellular matrix at the hollow part of the protective layer: and (5) observing with naked eyes.

The obtained scanning electron microscope pictures are shown in FIGS. 6 to 7; the resulting H & E stained image is shown in FIG. 8; a macroscopic image of its macroscopic camera is obtained as shown in fig. 9.

As can be seen from the Scanning Electron Microscope (SEM) image in fig. 6, the mesh-like fiber skeleton of example 3 obtained without phase control in the preparation process of the fiber skeleton has poor fiber pitch parallelism and non-uniform pore diameter, while the mesh-like fiber skeleton of example 8 obtained after phase control is used has good fiber pitch parallelism and uniform pore diameter.

As can be seen from the SEM picture of fig. 7, the inner cavity of example 3 provided with the anti-exposure layer is completely covered by the acellular matrix, and no fiber is exposed in the inner cavity, while the inner cavity of comparative example 3 not provided with the anti-exposure layer is only partially covered by the acellular matrix, and the fiber is exposed in the inner cavity.

As can be seen from fig. 8H & E stained pictures, the lumen of the tissue engineered blood vessel prepared in example 1 has a complete acellular matrix inner layer, and the pores in the fibrous skeleton have been completely filled with the acellular matrix. (the white dotted line is the boundary between the exposure prevention layer and the mesh-like fiber skeleton layer, and the upper left is the exposure prevention layer)

As can be seen from fig. 9, the protective layer provided in example 3 has a spiral hollow structure, so that the exterior of the finally prepared tissue engineering blood vessel has a raised spiral acellular matrix structure, while the protective layer is not provided in comparative example 8, so that the exterior of the finally prepared tissue engineering blood vessel is smooth and has no raised spiral acellular matrix structure.

The results of the above acellular matrix filling tests are shown in table 9 below:

TABLE 9 acellular matrix filling test results

From the above detection data, it can be seen that:

1. phase control is set, pore size uniformity of the fiber skeleton is improved, cell migration and extracellular matrix filling are not affected, and finally obtained fiber skeletons of all blood vessels are filled with acellular matrixes.

2. The exposed layer is arranged to degrade during the subcutaneous implantation. Along with the degradation, the generated cavity can be replaced by cell tissues, and the inner cavity of the blood vessel obtained after the decellularization treatment can be covered by the decellularized matrix, so that the final tissue engineering blood vessel inner cavity can not be exposed to fibers.

3. And the outer sleeve is arranged, and finally, a raised acellular matrix structure corresponding to the shape of the hollow structure can be generated on the outer wall of the tissue engineering blood vessel.

And (3) physical property detection:

the tissue engineering blood vessels prepared in the examples 1 to 10 and the comparative examples 1 to 30 are subjected to the following physical property detection, and specific detection items and methods are as follows:

1. and (3) detecting a finished product of the blood vessel prepared by using the template: the blood vessels are slightly kneaded from one end to the other end by a thumb and a forefinger, if the parts of the blood vessels are easy to collapse or distort (the situation is usually caused by nonuniform pore diameters among fibers of a reticular fiber framework layer caused by a spinning process), or the macroscopic fibers are in a nonuniform structure (the situation is mostly caused by the damage of the structure of the reticular fiber framework layer caused by a subcutaneous implantation process, the blood vessel material becomes white and is slightly transparent after the decellularization treatment, and the approximate structure of the reticular fiber framework layer can be seen through the decellularization matrix), the unqualified blood vessels are obtained, and the yield of the blood vessels prepared by utilizing the template is calculated. The following tests were all performed using qualified blood vessels.

2. And (3) kink radius detection: and placing the sample on the radius gauge, gradually reducing the radius of the radius gauge until the blood vessel sample is slightly narrowed or kinked, and recording the radius of the radius gauge at the moment, namely the kinking radius of the sample.

3. And (3) detecting the burst strength: carefully placing a balloon with a proper size into the obtained blood vessel sample, connecting the balloon with a three-way interface through a catheter, respectively connecting the other two interfaces of the three-way interface with a pressure recording device and a pressurizing device, pressurizing the balloon through the pressurizing device to expand the blood vessel until the blood vessel is ruptured, recording the ruptured pressure value of the blood vessel sample, and converting the pressure into mmHg to be used as a measurement result unit.

4. And (3) detecting the stitching strength: a section of blood vessel sample is cut along the axial direction, a 6-0 suture line penetrates through the blood vessel wall 2mm below the edge of one end of the blood vessel and is sewn into a semi-ring, the tail end of the suture line is fixed on a clamp of a tensile machine, the other end of the blood vessel sample is fixed on the other clamp of the tensile machine, and the suture line is stretched at the speed of 50 mm/min. The amount of tension pulling the suture out of the vessel wall is recorded. The stitching strength is calculated in units of N.

5. And (3) resilience detection: and (3) pinching the blood vessel sample by using a forceps for 2 seconds, immediately loosening the forceps, and observing whether the blood vessel can restore to the original appearance by using naked eyes.

The obtained resilience detection image is shown in fig. 10, and whether the tissue engineering blood vessel prepared in example 3 and comparative example 8 has an external bulge structure or not, the fibrous skeleton structure in the blood vessel can enable the prepared blood vessel to have good resilience.

The physical property test results are shown in Table 10;

TABLE 10 results of physical Properties measurements

From the above detection data, it can be seen that:

1. the phase control can improve the uniformity of the pore diameter of the fibrous skeleton, further improve the yield of tissue engineering materials, and improve the average bursting strength of the prepared qualified blood vessels, and the average bursting strength of all the detected qualified blood vessels is more than 1600mmHg, thereby meeting the requirements of being used as artificial blood vessels.

2. The protective layer is arranged to enable the outer wall of the tissue engineering blood vessel to form a thread convex structure acellular matrix, so that the kinking radius of the tissue engineering blood vessel is reduced, and the kink resistance of the tissue engineering blood vessel is improved.

3. The protective layer is arranged to form a thread bulge structure acellular matrix on the outer wall of the tissue engineering blood vessel, so that the suture strength of the prepared blood vessel can be improved

4. Suture strength tests show that the average value of the suture strength of all types of blood vessels is more than 2N, and the suture strength more than 1.7N is reported in the literature to meet the requirement of transplantation, so that all blood vessels have good suture strength.

5. The structure of the fiber framework is good, so that all types of detection blood vessels have resilience.

And (3) carrying out implantation detection in the vascular animal body:

according to the experience of previous animal experiments, the calibers of the blood vessels prepared in examples 1 and 6 and comparative examples 1, 6, 11, 16, 21 and 26 are 2mm, and rat abdominal aorta transplantation should be performed; the vessel calibers prepared in examples 2, 7 and comparative examples 2, 7, 12, 17, 22, 27 were 4mm, and beagle carotid artery transplantation should be performed; the vessel calibers prepared in examples 3, 8 and comparative examples 3, 8, 13, 18, 23, 28 were 5mm, and a carotid artery graft of sheep (about 30kg) should be performed; the vessel calibers prepared in examples 4 and 9 and comparative examples 4, 9, 14, 19, 24 and 29 were 6 mm; a cervical arteriovenous fistula of sheep (about 50kg) should be made; the calibers of the vessels prepared in examples 5 and 10 and comparative examples 5, 10, 15, 20, 25 and 30 were 8mm, and beagle abdominal aorta transplantation should be performed. However, following the "3R" principle (reduction, optimization, substitution) of animal experiments, the use of animals was minimized with the precursors to demonstrate beneficial effects. Therefore, the invention only selects a canine carotid artery transplantation and a sheep neck arteriovenous fistulization model to detect related caliber blood vessels for the following reasons: 1. the canine carotid artery transplantation is a large animal blood vessel transplantation experiment, is more representative than a small animal (rat abdominal aorta transplantation), and the size of the used blood vessel is closer to the clinical use condition, so that the obtained result is more significant. 2. The dog carotid artery transplantation uses a blood vessel with the caliber of 4mm, which is a typical small-caliber artificial blood vessel, for the small-caliber artificial blood vessel, the problems of thrombus, intimal hyperplasia and the like are easy to appear as the caliber is smaller, so that the transplantation failure is caused, and therefore, the blood vessel with the caliber of 4mm can better reflect the performance of the blood vessel than the blood vessel with the caliber of 5mm and the blood vessel with the caliber of 8 mm. 3. Sheep neck arteriovenous fistulization is different from above-mentioned 3 other animal blood vessel transplantation models, and the fistulization is to link to each other artificial blood vessel and artery and vein for hemodialysis uses, and the puncture closure nature of artificial blood vessel is implanted to the detectable, and other 3 animal blood vessel transplantation models all carry out the connection of artery and artery with artificial blood vessel, can not be used to detect vascular puncture closure nature, consequently need remain sheep neck arteriovenous fistulization model.

The detection after the blood vessel is transplanted to dog carotid artery and sheep neck arteriovenous fistulization mainly comprises: thrombus incidence, patency rate, puncture closure performance and with the surrounding tissue integration speed, specifically as follows:

1. animal selection: the calibers of the blood vessels prepared in examples 2 and 7 and comparative examples 2, 7, 12, 17, 22 and 27 were 4mm, and beagle carotid artery grafts were performed with the length of the implanted blood vessel being 4 cm; the calibers of the blood vessels prepared in examples 4 and 9 and comparative examples 4, 9, 14, 19, 24 and 29 were 6mm, and arteriovenous fistulation of the neck of sheep (about 50kg) was performed, and the length of the implanted blood vessel was 7 to 10 cm. For dogs, each animal was subjected to bilateral carotid vascular grafts, with 4 replicates per group vessel per time point; for sheep, one-sided cervical arteriovenous fistulation was performed per animal to prevent heart failure in the animals, and 4 samples were repeated per time point for each group of vessels.

2. Speed of integration with peripheral tissues test: at the time point of 10 days after operation, the anesthetized animals are subjected to material taking, and the integration condition of the implanted blood vessel and the surrounding tissues in the material taking process is analyzed. The implanted blood vessel is easy to separate from the surrounding tissues, namely is not integrated; the implanted blood vessel is not easy to separate from the surrounding tissues, and no inflammation occurs, and the signs of swelling are the integration completion.

3. And (3) detecting the thrombus condition: and detecting whether thrombus is formed in the inner cavity of the tissue engineering blood vessel taken out at the time point of 10 days after the operation by a body type microscope. The incidence of mild thrombosis (non-occluded) and the incidence of thrombosis in the occluded blood stream were calculated based on the severity of the thrombus, respectively.

4. And (3) detecting the patency: animals were anesthetized 1 month after implantation and blood vessel patency was assessed using color doppler ultrasound (M9, mai rui, china).

5. Puncture closure detection: after anaesthetizing sheep, puncturing the tissue engineering blood vessel for performing arteriovenous fistula at the neck of the sheep by 16G dialysis, drawing out the puncture needle after blood flows out, immediately pressing the puncture needle by using sterile absorbent cotton for hemostasis, simultaneously starting a timer, confirming that no blood flows out from the puncture hole under the condition of no need of pressing after pressing is finished, stopping timing, and recording time, wherein the time is puncture hemostasis time to reflect the puncture hemostasis performance of the blood vessel, and the time is taken as a result unit.

A lumen pattern of the tissue engineered blood vessels prepared in example 9 and comparative example 19 was obtained 10 days after the sheep neck arteriovenous fistula was performed, as shown in fig. 11.

As can be seen from the stereomicroscope picture in fig. 11, a part of the lumen of the blood vessel prepared in example 9 after 10 days of the mobile venous fistula has slight thrombosis, but most of the lumen of the blood vessel has no thrombosis and has no serious thrombosis blocking the blood flow, while a part of the lumen of the blood vessel prepared in comparative example 19 after 10 days of the mobile venous fistula has serious thrombosis blocking the blood flow, which indicates that the inner layer of the blood vessel is covered by a decellularized layer, thereby reducing the exposure of lumen fibers and helping to reduce the thrombosis.

The results of the in vivo implantation test of the obtained vascular animals are shown in table 11:

TABLE 11 results of the vascular graft assay

From the above detection data, it can be seen that:

1. the acellular matrix inner layer generated by the anti-exposure layer is arranged, so that the exposure of the inner cavity fiber skeleton is reduced, and the occurrence of thrombus is inhibited.

2. The uniformity of the aperture of the skeleton fiber is improved through phase control, so that the time for pressing hemostasis after puncture can be reduced, and the vascular puncture closure is improved.

3. The protective layer is arranged to enable the outer wall of the tissue engineering blood vessel to form a thread bulge structure acellular matrix, and the integration speed of the blood vessel and surrounding tissues is improved.

4. The results show that the thread-shaped acellular matrix protruding from the outer layer of the tissue engineering blood vessel is helpful for reducing the time for pressing for hemostasis after puncture and improving the closure of the blood vessel puncture.

Claims

1. The template for the tissue engineering material is characterized by comprising a support body, an anti-exposure layer, a reticular fiber framework layer and a protective layer which are sequentially arranged; and a hollow structure is arranged on the protective layer; the exposure prevention layer comprises a biodegradable material.

2. The template for a tissue engineering material according to claim 1, wherein the thickness of the protective layer is 300 to 3000 μm; the width of the hollow structure is 500-3000 mu m.

3. The template for tissue engineering material according to claim 1, wherein the hollowed-out structure is in a thread shape, parallel straight lines or parallel curves along the direction of the protective layer; the distance between adjacent parallel hollow structures is 500-3000 mu m.

4. The template for a tissue engineering material according to claim 1, wherein the thickness of the anti-exposure layer is 50 to 500 μm.

5. The template for tissue engineering material according to claim 4, wherein the exposure prevention layer comprises a synthetic polymer material and/or a natural polymer material; the synthetic high molecular material is selected from one or more of polyethylene glycol, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly (3-hydroxybutyrate-co-4-hydroxybutyrate), poly (p-dioxane-hexanone) and polysebacic acid glyceride; the natural polymer material is selected from one or more of gelatin, collagen, hyaluronic acid and fibrin glue.

6. The template for a tissue engineering material according to claim 1, wherein the thickness of the reticular fiber skeleton layer is 200-2000 μm; the mesh fiber skeleton layer is formed of fibers; the diameter of the fiber is 20-200 mu m; the crossing angle of the fibers forming the reticular fiber skeleton layer is 30-110 degrees.

7. The template for tissue engineering material according to claim 1, wherein the standard deviation of the average value of the pore diameters of the reticular fiber skeleton layer is controlled to be 5-30 μm.

8. The template for tissue engineering material according to claim 1, wherein the template for tissue engineering material is a template for tissue engineering blood vessels.

9. A tissue engineering material prepared by using the template for tissue engineering material according to any one of claims 1 to 8 as a template.

10. The tissue engineering material of claim 9, comprising an inner layer, a middle layer and an outer layer which are closely attached; the inner layer is a acellular matrix layer; the middle layer consists of a reticular fiber framework and an acellular matrix filled in the reticular fiber framework; the outer layer is an acellular matrix with a convex structure; and the acellular matrix layer of the inner layer, the acellular matrix of the middle layer and the acellular matrix of the outer layer are integrally arranged.