阅读说明：本技术 一种磁响应的天然血管基质凝胶支架材料及其制备方法 (Magnetic-response natural vascular matrix gel scaffold material and preparation method thereof ) 是由 陈国宝 付强 黄湘 *** 夏斌 王富平 陈忠敏 于 2019-09-25 设计创作，主要内容包括：本发明公开了一种磁响应的天然血管基质凝胶支架材料及其制备方法,所述支架材料在冰浴条件下,将脱细胞化血管、壳聚糖、β-甘油磷酸钠和磁性纳米颗粒复合构建水凝胶体系,然后将其固化得到凝胶,再将所述凝胶冷冻干燥即得到磁响应的天然血管基质凝胶支架材料。本发明制备的凝胶支架材料具有良好的生物相容性,能保证细胞在支架上的粘附与生长,并且该支架可以在磁场作用下促进支架内细胞的代谢速率和细胞增殖,还可以诱导凝胶支架的血管生成。本发明还可以根据需要来调节四氧化三铁纳米颗粒和脱细胞血管基质的含量从而改变其磁响应性和血管生成能力,适用范围广,具有良好的应用价值。本发明原料来源广泛,成本低廉,易于保存,更易于临床推广。(The invention discloses a magnetic-response natural blood vessel matrix gel scaffold material and a preparation method thereof. The gel scaffold material prepared by the invention has good biocompatibility, can ensure the adhesion and growth of cells on the scaffold, can promote the metabolic rate and cell proliferation of the cells in the scaffold under the action of a magnetic field, and can also induce the angiogenesis of the gel scaffold. The invention can also adjust the content of ferroferric oxide nano particles and acellular vascular matrixes according to the requirement so as to change the magnetic responsiveness and the angiogenesis capacity of the ferroferric oxide nano particles and the acellular vascular matrixes, has wide application range and good application value. The invention has wide raw material source, low cost, easy preservation and easy clinical popularization.)

1. The magnetic-response natural vascular matrix gel scaffold material is characterized in that the scaffold material is prepared by compounding decellularized vascular matrix, chitosan, beta-sodium glycerophosphate and magnetic nanoparticles under an ice bath condition to construct a hydrogel system, solidifying the hydrogel system to obtain gel, and freeze-drying the gel to obtain the magnetic-response natural vascular matrix gel scaffold material.

2. The magnetically-responsive natural vascular matrix gel scaffold material of claim 1, wherein the mass ratio of the decellularized vascular matrix, chitosan, sodium beta-glycerophosphate and magnetic nanoparticles is 0.5 ~ 3:10 ~ 27:5 ~ 21:1 ~ 10.

3. The magnetically-responsive natural vascular matrix gel scaffold material of claim 1, wherein the magnetic nanoparticles have a particle size of less than or equal to 20 nm and are ferroferric oxide.

4. A method for preparing a magnetically-responsive natural vascular matrix gel scaffold material as claimed in any one of claims 1 ~ 3, comprising the steps of:

1) Dissolving pepsin in dilute hydrochloric acid to obtain a pepsin solution, then adding the acellular vascular matrix, and adjusting the pH value to be neutral to obtain an acellular vascular matrix solution;

2) slowly and dropwisely adding a beta-sodium glycerophosphate solution into a chitosan solution under an ice bath condition, uniformly mixing to obtain a mixed solution, then adding the acellular vascular matrix solution prepared in the step 1) into the mixed solution, and uniformly mixing to obtain a composite gel solution;

3) Adding the magnetic nanoparticles into the composite gel solution obtained in the step 2), performing ultrasonic dispersion until the mixture is uniform, adjusting the pH value, standing in a refrigerator at 4 ℃ to remove surface bubbles, then placing in a thermostat to gelatinize the system, and then performing freeze drying to obtain the natural vascular matrix gel scaffold material with magnetic response.

5. The method for preparing the magnetic-response natural blood vessel matrix gel scaffold material according to claim 4, wherein the concentration of the pepsin solution is 0.5 ~ 2 mg/mL, and the concentration of the dilute hydrochloric acid is 0.01 ~ 0.05.05 mol/L.

6. The preparation method of the magnetic-response natural vascular matrix gel scaffold material according to claim 4, wherein the mass concentration of the acellular vascular matrix solution is 5 ~ 20 mg/mL.

7. The method for preparing the magnetic-response natural blood vessel matrix gel scaffold material according to claim 4, wherein the concentration of the chitosan solution is 2 ~ 4% (m/v), and the concentration of the sodium glycerophosphate solution is 30% ~ 60% (m/v).

8. the method for preparing the magnetically-responsive natural vascular matrix gel scaffold material according to claim 4, wherein the pH value in step 3) is 6.5 ~ 7.5.5.

9. The method for preparing the magnetic-response natural blood vessel matrix gel scaffold material according to claim 4, wherein the temperature of the constant temperature box is 35 ~ 40 ℃.

10. The method for preparing the magnetic-response natural blood vessel matrix gel scaffold material according to claim 4, wherein the freeze-drying temperature is-20 ~ -80 ℃ and the time is 12 ~ 48 h.

Technical Field

The invention relates to the technical field of vascular tissue engineering scaffold materials, in particular to a magnetic-response natural vascular matrix gel scaffold material and a preparation method thereof.

background

the tissue engineering technology is an emerging technology which appears in recent years and can reconstruct and promote the defective tissue, and the basic elements of the tissue engineering technology comprise seed cells, biological scaffolds, bioactive factors and the like. The biological scaffold provides a three-dimensional growth scaffold for the seed cells, so that the seed cells are proliferated and differentiated, and the biological scaffold plays a role of replacing extracellular matrixes of tissues or organs, so that proper spatial distribution and cell connection are formed among the cells, the cells become artificial simulated extracellular matrixes, and a microenvironment for cell growth is formed. The biological scaffold material for tissue engineering comprises: bone, cartilage, blood vessels, nerves, skin and artificial organs, such as liver, spleen, kidney, bladder and other tissue scaffold materials.

Currently, while encouraging results have been achieved with parenchymal tissue (e.g., engineered bladder), tissue engineering of dense tissue (e.g., heart) that relies on vascular supply remains a challenge. One of the major obstacles to designing thick complex tissues (e.g., muscles) is the need to vascularize the tissue in vitro. In vitro vascularization is important for maintaining cell viability during tissue growth, inducing structural tissues and promoting vascularization upon implantation.

The invention patent CN201810741671.1 discloses a preparation method of a double-layer artificial small-caliber blood vessel with a modified inner layer, which comprises the steps of dispersing carboxylated mesoporous silicon into NaOH to obtain MSN; dispersing the mixture into a mixed solution of MES and NaCl, stirring, and adding EDC, PEG and NHS to obtain MSN-PEG; dispersing the Heparin-Heparin solution to obtain MSN-PEG-Heparin; dispersing the mixture in HFIP to obtain a dispersion liquid; preparing inner and outer spinning solutions respectively, and spinning to obtain the intravascular stent. Although the nanofiber obtained by electrospinning provides a suitable surface morphology for the adhesion and growth of cells, which is beneficial to the adhesion and growth of cells on the scaffold, the scaffold prepared by electrospinning is not beneficial to the migration and proliferation of cells in the depth direction due to too small pore size, and the effective regulation and control of the distribution of cells on the fibrous scaffold are difficult to realize, which limits the application of the scaffold in the field of tissue regeneration medical treatment. It is noted that, as a scaffold for angiogenesis, in addition to the effect of supporting cell growth and proliferation, a scaffold having the ability to promote cell proliferation and metabolism and the ability to vascularize rapidly and efficiently is required.

Hydrogels exhibit good biocompatibility when in contact with blood, body fluids, and human tissues because of their high water content (typically greater than 80% of the total mass) and structural similarity to biological tissues, e.g., some soft tissues in the human body (e.g., tendons, ligaments, meniscal cartilage, etc.) are also composed of gel materials. The current strategies for promoting neovascularization mainly include: 1) adding an angiogenesis factor or a drug that activates an angiogenesis signaling pathway to the implant; 2) the implant binds endothelial cells/mesenchymal stem cells, stimulating the generation of new blood vessels in vivo by prevascularization of cells in vitro in the implant; 3) injecting a plasmid encoding an angiogenic factor to transduce the plasmid into the angiogenic factor; 4) modulation of ROS/NO activates angiogenic pathways. At present, a plurality of researchers develop and develop hydrogel vascular regeneration stents, for example, the invention patent CN201810986445.X discloses a tissue engineering stent with a vascular structure and a preparation method thereof, and the preparation method comprises the following steps: s1, constructing a three-dimensional network model, and printing a sugar scaffold with a three-dimensional network structure through 3D; s2, immersing the sugar scaffold printed in the step S1 into an ethanol mixed solution of calcium salt, and volatilizing the ethanol to obtain the sugar scaffold with the calcium salt loaded on the surface; s3, filling the sugar support obtained in the step S2 with modified photocuring macromolecules, and performing photocuring to form a cured hydrogel support; and S4, placing the cured hydrogel scaffold in water, PBS or cell culture medium to remove the sugar scaffold, thereby obtaining the tissue engineering scaffold with the bionic vascular network. The invention patent CN201811326186.4 discloses a vascularization full-layer tissue engineering skin assembled by hydrogel, a nano-fiber scaffold and skin cells layer by layer and a preparation method thereof, the artificial tissue engineering skin comprises an epidermal layer and a dermal layer, wherein the epidermal layer is formed by alternately laminating the nano-fiber scaffold positioned above the dermal layer and a seed cell; the dermis layer consists of a lower layer nanofiber scaffold, an upper layer hydrogel scaffold and three kinds of seed cells, wherein the seed cells are distributed on the surface of the nanofiber scaffold and the inside and the surface of hydrogel. The patent uses the in vitro cell prevascularization mode to stimulate the sprouting of new blood vessels, but the method is controversial because the survival rate of cells in vivo cannot be determined, the manufacturing process of the scaffold is complex and the time consumption is long, and the growth factor which plays a role in promoting angiogenesis is expensive and easy to inactivate. The transduction of angiogenic factors by plasmids is limited in its application due to its low efficiency and possible accompanying inflammatory responses. The means of activating angiogenic pathways using ROS/NO are currently still in the laboratory. It can thus be seen that the prior art has not completely overcome the problem of promoting neovascularization in damaged tissue.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a magnetic-response natural vascular matrix gel scaffold material, which solves the problems of high cost, low efficiency and possible inflammatory reaction in promoting angiogenesis of the conventional scaffold material, and also can not completely overcome the problem of promoting the rapid and effective generation of new blood vessels in damaged tissues.

The invention also provides a preparation method of the magnetic-response natural vascular matrix gel scaffold material, and solves the problems of complex operation method, high cost and difficult industrial production of the existing preparation method.

In order to solve the technical problems, the invention adopts the following technical scheme: a natural blood vessel matrix gel scaffold material with magnetic response is characterized in that a hydrogel system is constructed by the aid of decellularized blood vessel matrix, chitosan, beta-sodium glycerophosphate and magnetic nanoparticles in an ice bath condition, then the hydrogel system is solidified to obtain gel, and the gel is frozen and dried to obtain the natural blood vessel matrix gel scaffold material with magnetic response.

Further, the mass ratio of the decellularized vascular matrix, the chitosan, the sodium beta-glycerophosphate and the magnetic nanoparticles is 0.5 ~ 3:10 ~ 27:5 ~ 21:1 ~ 10.

Further, the particle size of the magnetic nano-particles is less than or equal to 20 nm, and the magnetic nano-particles are ferroferric oxide.

The invention also provides a preparation method of the magnetic response natural blood vessel matrix gel scaffold material, which comprises the following steps:

1) Dissolving pepsin in dilute hydrochloric acid to obtain a pepsin solution, then adding the acellular vascular matrix, and adjusting the pH value to be neutral to obtain an acellular vascular matrix solution;

2) Slowly and dropwisely adding a beta-sodium glycerophosphate solution into a chitosan solution under an ice bath condition, uniformly mixing to obtain a mixed solution, then adding the acellular vascular matrix solution prepared in the step 1) into the mixed solution, and uniformly mixing to obtain a composite gel solution;

3) adding the magnetic nanoparticles into the composite gel solution obtained in the step 2), performing ultrasonic dispersion until the mixture is uniform, adjusting the pH value, standing in a refrigerator at 4 ℃ to remove surface bubbles, then placing in a thermostat to gelatinize the system, and then performing freeze drying to obtain the natural vascular matrix gel scaffold material with magnetic response.

Further, the concentration of the pepsin solution is 0.5 ~ 2 mg/mL, and the concentration of the dilute hydrochloric acid is 0.01 ~ 0.05.05 mol/L.

Further, the mass concentration of the acellular vascular matrix solution is 5 ~ 20 mg/mL.

Further, the concentration of the chitosan solution was 2 ~ 4% (m/v).

Further, the concentration of the sodium glycerophosphate solution was 30% ~ 60% (m/v).

further, the pH value in the step 3) is 6.5 ~ 7.5.5.

Further, the oven temperature was 35 ~ 40 ℃.

Further, the freeze-drying temperature is-20 ~ -80 ℃, and the time is 12 ~ 48 h.

Compared with the prior art, the invention has the following beneficial effects:

1. The magnetic response natural vascular matrix gel scaffold prepared by the invention takes the decellularized vascular matrix, chitosan, beta-sodium glycerophosphate and magnetic nanoparticles as raw materials, and a hydrogel system of the chitosan and the beta-sodium glycerophosphate carries the decellularized blood vessels and the magnetic nanoparticles to carry out gelation in situ to form a surface porous structure, so that the fixing force of the decellularized blood vessels and the magnetic nanoparticles on the hydrogel is increased, and the porous structure is more favorable for the adhesion of cells on the scaffold and the growth of the cells in the depth direction. The ferroferric oxide nano particles with different concentrations are compounded to enable the stent to have magnetic response capability, and under the action of a magnetic field, cells can sense stimulation applied by an external magnetic field to promote the proliferation and metabolic capability of vascular endothelial cells on the stent; the added acellular vascular matrix is used for removing cells in blood vessels by a physical, chemical or enzymatic method, eliminating immunogenicity, retaining natural extracellular matrix components of the blood vessels, and the natural extracellular matrix components contain bioactive components which have the capacity of promoting the differentiation of the cells to a vascular lineage, endowing a scaffold with the capacity of inducing the regeneration of blood vessels, and simultaneously avoiding the use of growth factors. Therefore, the scaffold disclosed by the invention has synergistic compatibility among the decellularized blood vessel, the chitosan, the beta-sodium glycerophosphate and the magnetic nanoparticles, not only can promote the proliferation and metabolic capacity of cells in the blood vessel, but also is beneficial to migration of the cells and differentiation to a vascular common system in the processes of blood vessel regeneration and wound healing, and the speed of blood vessel regeneration and repair is improved. The invention provides a new idea for promoting the generation of new blood vessels in damaged tissues and has good application prospect.

2. The magnetic response natural vascular matrix gel scaffold prepared by the invention has good biocompatibility and controllable degradation speed, can be discharged out of the body through the kidney, cannot be accumulated in the body, has no cellular immunocompetence and high safety, can finish the conversion from sol to gel in a short time, has better formability, overcomes the defect of insufficient mechanical properties of natural polymer hydrogel, has bioactivity, generates new extracellular matrix and is full of cells after being implanted into the subcutaneous tissue of an SD rat for 28 days, and simultaneously generates a large number of blood vessels with different apertures in the interior. The invention can also adjust the content of ferroferric oxide nano particles and acellular vascular matrixes according to the requirement, thereby changing the magnetic responsiveness and the angiogenesis capacity of the stent and having wide application prospect in the field of biomedicine.

3. The magnetic response natural vascular matrix gel scaffold prepared by the invention has the advantages of simple preparation process, wide raw material source, low cost and easy storage. The gel scaffold has irreversible gelation and better mechanical property, simultaneously can change the rigidity of the scaffold by changing the proportion of raw materials, and because cells of different tissues can generate different responses to the rigidity of the environment, the adhesion proliferation and the metabolism of the cells are directly influenced, so that the application range of the scaffold can be expanded by the method.

Drawings

FIG. 1 is a H-E staining pattern of fresh and decellularized blood vessels, A being fresh; b is a decellularized blood vessel;

FIG. 2 is a FTIR plot of a magnetically responsive native vascular matrix gel scaffold of the present invention and its components;

FIG. 3 is a graph of the magnetic response of the magnetically responsive native vascular matrix gel scaffold of the present invention;

FIG. 4 is a H-E staining pattern of the magnetic response natural vascular matrix gel stent of the present invention implanted into SD rat subcutaneously for 28 days. A is chitosan/beta-sodium glycerophosphate-ferroferric oxide nanoparticle gel scaffold; b is chitosan/beta-sodium glycerophosphate-ferroferric oxide nano-particles-acellular vascular matrix gel scaffold.

Detailed Description

The present invention will be described in further detail with reference to examples. The reagents used in the examples are not specifically described and are commercially available.

The acellular blood vessels for preparing the natural vascular matrix can be prepared from blood vessels of different parts of different animals as materials as long as the requirements of the blood vessels to be repaired are met. For the sake of brevity, the present invention is described primarily in terms of decellularized blood vessels in porcine aorta, and other animals and portions of blood vessels may employ the same principles. The preparation method specifically comprises the following steps:

S1: cutting fresh pig aorta obtained in a slaughterhouse, soaking in deionized water overnight, placing in a gas bath constant temperature oscillator for oscillation bath for 12h, cleaning the fresh pig aorta, replacing the deionized water for 2 times during cleaning, and carrying out oscillation bath on the cleaned pig aorta in a Triton X-100 solution with the concentration of 3% for 12 h; and then carrying out degreasing treatment on the porcine aorta for 24h by using methanol, wherein the methanol needs to be replaced for three times during the treatment, deionized water-free oscillation immersion bath is not needed for 30min after degreasing, and redundant methanol is cleaned.

S2: the defatted porcine aorta was incubated with DNase I for 2h at 37 ℃ in step S1 and kept agitated, washed with PBS solution and the washing process kept slightly agitated. Then washing with deionized water, and performing shaking bath on pig aorta for 30 min; then soaking the pig aorta into absolute ethyl alcohol for shaking bath for 4h, and replacing the alcohol once in the midway; immersing porcine aorta with a volume of deionized water, and shaking the immersed acellular aorta in a gas bath constant temperature shaker; placing the processed acellular pig aorta in a vacuum drying oven at 37 ℃ for drying; grinding the completely dried acellular porcine aorta into powder by using a mortar to obtain the acellular blood vessel, and packaging the acellular blood vessel in a refrigerator at 4 ℃ for later use.

Fresh blood vessels and the resulting decellularized blood vessels were subjected to H-E staining, in which hematoxylin staining solution stained the nuclei blue and eosin staining solution stained the cytoplasm pink, as shown in FIG. 1.

As can be seen from the figure, the fresh blood vessel originally contains a large number of cell nuclei, which indicates that the inside contains a large number of cells, and after the decellularization treatment, the cells are basically removed and the extracellular matrix is completely reserved, which indicates that the decellularization method can effectively achieve the purpose of obtaining the complete extracellular matrix.