阅读说明：本技术 一种三维血管网络水凝胶的构建方法 (Construction method of three-dimensional vascular network hydrogel ) 是由 邱小忠 李创坤 王乐禹 于 2019-10-11 设计创作，主要内容包括：本发明实施例提供的三维血管网络水凝胶的构建方法,先将血管填充剂灌注到离体器官的血管中,待血管填充剂硬化后,剥离所述离体器官组织,得到所述离体器官的三维血管模型,再将三维血管模型放入模具中,向所述模具中倒入水凝胶预聚物低温聚合后,得到水凝胶模型,抽出所述水凝胶模型中的三维血管模型,即得所述三维血管网络,该方法工艺简单,无需复杂的加工过程,对设备要求低,构建的三维血管模型结构和生物体微血管网络一样,具有完全仿生的特点,且构建的三维血管模型具有较高的拉伸强度,弹性好,易于抽出,不会残留在凝胶内,生物相容性好,解决了现有技术中构建三维血管网络所存在的问题。(The construction method of the three-dimensional vascular network hydrogel provided by the embodiment of the invention comprises the steps of firstly infusing the vascular filler into the blood vessel of the isolated organ, after the vascular filler is hardened, the isolated organ tissue is peeled off to obtain a three-dimensional vascular model of the isolated organ, then the three-dimensional vascular model is put into a mould, pouring hydrogel prepolymer into the mold for low-temperature polymerization to obtain a hydrogel model, extracting the three-dimensional blood vessel model from the hydrogel model, the three-dimensional blood vessel network is obtained, the method has simple process, does not need complex processing process, has low requirement on equipment, and the constructed three-dimensional blood vessel model has the same structure as the organism micro-blood vessel network and has the characteristic of complete bionics, the constructed three-dimensional vascular model has higher tensile strength, good elasticity, good biocompatibility and high possibility of being drawn out, can not remain in gel and solves the problems existing in the construction of a three-dimensional vascular network in the prior art.)

1. A method for constructing three-dimensional vascular network hydrogel is characterized by comprising the following steps:

s1: perfusing a vascular filler into a blood vessel of an isolated organ;

s2: after the vascular filler perfused in the step S1 is hardened, stripping the tissue of the isolated organ to obtain a three-dimensional vascular model of the isolated organ;

s3: putting the three-dimensional blood vessel model into a mold, pouring hydrogel prepolymer into the mold, and polymerizing at low temperature to obtain a hydrogel model;

s4: and extracting the three-dimensional blood vessel model from the hydrogel model to obtain the three-dimensional blood vessel network hydrogel.

2. The construction method according to claim 1, wherein the vascular filler comprises the following components in percentage by volume:

dibutyl phthalate: 1 to 2 percent of the total amount of the catalyst,

color paste: 5 to 10 percent of the total weight of the composition,

the balance being natural latex.

3. The method for constructing a hydrogel, according to claim 1, wherein the hydrogel prepolymer comprises the following components in percentage by mass:

hydroxyethyl methacrylate: 4 to 6 percent of the total weight of the steel,

a crosslinking agent: 8 to 10 percent of the total weight of the steel,

initiator: 0.5 to 0.7 percent,

initiation accelerator (b): 0.3 to 0.5 percent.

4. The method of construction according to claim 3, wherein the cross-linking agent comprises: at least one of N, N-methylenebisacrylamide, 2-hydroxyalkylamide, N-methylolacrylamide, diacetone acrylamide, formaldehyde, and glutaraldehyde.

5. The build method of claim 3, wherein the initiator comprises: at least one of ammonium persulfate, potassium persulfate, sodium persulfate, benzoyl peroxide, tert-butyl peroxybenzoate, and diisopropyl peroxydicarbonate.

6. The build method of claim 3, wherein the initiation promoter comprises tetramethylethylenediamine.

7. The method of claim 1, wherein the low temperature polymerization is carried out at a temperature of-18 to-25 ℃.

8. The construction method according to claim 1, wherein the time of the low-temperature polymerization is not less than 24 hours.

9. The building method according to claim 1, characterized in that the building method comprises: in step S3, the hydrogel model is washed.

Technical Field

The invention belongs to the technical field of tissue biological manufacturing, and particularly relates to a construction method of three-dimensional vascular network hydrogel.

Background

With the development of tissue engineering, research related to tissue engineering, such as the manufacture of skin, ear, cartilage, and the like, has been significantly developed in recent years. However, there are still more problems with constructing large volumes of tissue, where vascularization is one of the major difficulties currently faced.

In the constructed engineered tissue, cells must be close enough (100-200 μm) to the vascular network to obtain oxygen and nutrient supply, thereby preventing the formation of necrotic cores. However, when the engineered tissue is implanted into a host, the speed of budding of host capillaries into the engineered scaffold is slow, and therefore the most important issue in the tissue fabrication process is the formation of a three-dimensional vascular network. CN109172039A discloses a method for preparing a vascular network-like channel by a composite process, the method combines an electrostatic spinning technology and a mold-like composite process to prepare the vascular network-like channel with a composite structure, the required materials are easy to obtain, the prepared vascular network-like channel with the composite structure is similar to an organism micro-vascular structure and has the bionic characteristic, an electrospinning layer in the composite vascular network-like channel is beneficial to the adhesion growth and differentiation proliferation of cells, and the strength and toughness of the structure are enhanced by a composite formed tissue structure. Although the method provided by the invention can solve the problem of vascularization of a massive tissue structure at present, the preparation process is complex and difficult to control.

In addition, in the prior art, hydrogels with micro-grooves or micro-channels can also be manufactured by photolithography. However, the microchannels formed by these methods are usually limited to two-dimensional planes, and rely on multiple layer-by-layer assembly steps, and the preparation process is complex, which easily results in poor alignment of the interfaces in the engineering tissues. The microchannel can also be manufactured by utilizing a biological printing technology, for example, a designed three-dimensional vascular network model is printed by using Pluronic F127, sodium alginate, agarose and the like, then the template is immersed in the hydrogel prepolymer, and the sacrificial template is removed after the hydrogel is formed, so that the stent with the three-dimensional vascular network is formed.

Disclosure of Invention

In order to solve the problems existing in the prior art of constructing a three-dimensional vascular network, embodiments of the present invention provide a method for constructing a three-dimensional vascular network.

In order to achieve the purpose, the embodiment of the invention adopts the following technical scheme:

a method for constructing three-dimensional vascular network hydrogel comprises the following steps:

s1: perfusing a vascular filler into a blood vessel of an isolated organ;

s2: after the vascular filler perfused in the step S1 is hardened, stripping the isolated organ tissue to obtain a three-dimensional vascular model of the isolated organ;

s3: putting the three-dimensional blood vessel model into a mold, pouring hydrogel prepolymer into the mold, and polymerizing at low temperature to obtain a hydrogel model;

s4: and extracting the three-dimensional blood vessel model from the hydrogel model to obtain the three-dimensional blood vessel network hydrogel.

The method has simple process, does not need complex processing process, has low requirement on equipment, has the same structure of the constructed three-dimensional blood vessel model as an organism micro-blood vessel network, has the characteristic of complete bionics, has the breaking elongation of 246.62 percent and the maximum tensile strength of 20.1MPa, shows that the three-dimensional blood vessel model has higher tensile strength, good elasticity, easy extraction, no residue in gel and good biocompatibility, is suitable for popularization and application in blood vessel tissue engineering, and is beneficial to solving the problem of vascularization network in the human tissue organ reconstruction problem in clinical medicine.

Preferably, the vascular filler comprises the following components in percentage by volume:

dibutyl phthalate: 1 to 2 percent of the total amount of the catalyst,

color paste: 5 to 10 percent of the total weight of the composition,

the balance being natural latex.

Preferably, the hydrogel prepolymer comprises the following components in percentage by mass:

hydroxyethyl methacrylate: 4 to 6 percent of the total weight of the steel,

a crosslinking agent: 8 to 10 percent of the total weight of the steel,

initiator: 0.5 to 0.7 percent,

initiation accelerator (b): 0.3 to 0.5 percent.

Further preferably, the crosslinking agent comprises: at least one of N, N-methylenebisacrylamide, 2-hydroxyalkylamide, N-methylolacrylamide, diacetone acrylamide, formaldehyde, and glutaraldehyde.

Further preferably, the initiator comprises: at least one of ammonium persulfate, potassium persulfate, sodium persulfate, benzoyl peroxide, tert-butyl peroxybenzoate, and diisopropyl peroxydicarbonate.

Further preferably, the initiation promoter comprises tetramethylethylenediamine.

Preferably, the temperature of the low-temperature polymerization is-18 to-25 ℃.

Preferably, the time of the low-temperature polymerization is more than or equal to 24 hours.

Preferably, the construction method comprises: in step S3, the hydrogel model is washed.

When cleaning, the hydrogel model is soaked in deionized water for 48h, and water is replaced every 12h to remove monomers and other impurities which do not participate in polymerization.

The embodiment of the invention has the beneficial effects

1. The method for constructing the three-dimensional vascular network hydrogel provided by the embodiment of the invention comprises the steps of firstly infusing a vascular filler into blood vessels of an isolated organ, stripping tissues of the isolated organ after the vascular filler is hardened to obtain a three-dimensional vascular model of the isolated organ, then placing the three-dimensional vascular model into a mould, pouring hydrogel prepolymer into the mould for low-temperature polymerization to obtain a hydrogel model, and extracting the three-dimensional vascular model from the hydrogel model to obtain the three-dimensional vascular network hydrogel, wherein the method has the advantages of simple process, no need of complex processing process, low requirement on equipment, same structure of the constructed three-dimensional vascular model as a biological micro vascular network, complete bionic characteristics, higher tensile strength, good elasticity, easy extraction, no residue in the gel and good biological compatibility, the problem of constructing a three-dimensional vascular network in the prior art is solved;

2. the method for constructing the three-dimensional vascular network hydrogel provided by the embodiment of the invention has the advantages of simple process, no need of complex processing process, low requirement on equipment, and completely bionic characteristics of the constructed three-dimensional vascular model with the same structure as the biological micro-vascular network, wherein the elongation at break of the constructed three-dimensional vascular model is 246.62%, and the maximum tensile strength is 20.1MPa, which indicates that the three-dimensional vascular model has higher tensile strength, good elasticity, easy extraction and no residue in the gel;

3. the three-dimensional vascular network obtained by the construction method of the embodiment of the invention has good biocompatibility, is suitable for being popularized and applied to vascular tissue engineering, and is beneficial to solving the problem of vascularization network in the human tissue organ reconstruction problem in clinical medicine.

Drawings

Fig. 1 is a stress-strain graph of the three-dimensional vascular model of example 2.

FIG. 2 is a blood vessel model extraction test of the three-dimensional blood vessel network hydrogel in example 2.

FIG. 3 is an infrared spectrum of the three-dimensional vascular network hydrogel of example 2.

FIG. 4 is a graph showing the results of the biocompatibility test for the three-dimensional vascular network hydrogel in example 2 after 1 day.

FIG. 5 is a graph showing the results of the three-dimensional vascular network hydrogel biocompatibility test in example 2 after 3 days.

FIG. 6 is a graph showing the results of the biocompatibility test for the three-dimensional vascular network hydrogel in example 2 after 7 days.

FIG. 7 is a schematic diagram of the three-dimensional vascular network hydrogel biocompatibility testing of the living cell rate in example 2.

Detailed Description

The embodiment of the invention provides a method for constructing three-dimensional vascular network hydrogel, which comprises the steps of firstly infusing a vascular filler into blood vessels of an isolated organ, stripping tissues of the isolated organ after the vascular filler is hardened to obtain a three-dimensional vascular model of the isolated organ, then placing the three-dimensional vascular model into a mold, pouring hydrogel prepolymer into the mold for low-temperature polymerization to obtain a hydrogel model, and extracting the three-dimensional vascular model from the hydrogel model to obtain the three-dimensional vascular network hydrogel. The method has simple process, does not need complex processing process, has low requirement on equipment, and the constructed three-dimensional blood vessel model has higher tensile strength, good elasticity, good biocompatibility, easy extraction, no residue in gel, and is suitable for popularization and application in blood vessel tissue engineering, and is favorable for solving the vascularization network problem in human tissue organ reconstruction problem in clinical medicine.

In order to better understand the above technical solutions, the above technical solutions will be described in detail with reference to specific embodiments.