阅读说明：本技术 复合外泌体Nidogen-1的组织工程支架及其制备方法 (Tissue engineering scaffold of composite exosome Nidougen-1 and preparation method thereof ) 是由 程朋真 刘斌 杨柳 曹天庆 宁芬茹 于 2020-07-30 设计创作，主要内容包括：本发明公开了一种复合外泌体Nidogen-1的组织工程支架及其制备方法,收集经过表达处理的骨髓间充质干细胞上清液,通过超速离心法分离外泌体Nidogen-1,调整其使用浓度为10-100μg/mL；将海藻酸钠、透明质酸、PEG-600溶于去离子水制成混合预聚物；添加外泌体Nidogen-1,从而制备获得复合外泌体Nidogen-1的组织工程支架。将该组织工程支架植入创伤部位或生物相容性组织工程材料中心,可促进缺损部位血管再生,改善材料中心代谢微环境,具有重要的临床应用价值。(The invention discloses a tissue engineering scaffold of a composite exosome Nidogen-1 and a preparation method thereof, wherein the tissue engineering scaffold is characterized in that bone marrow mesenchymal stem cell supernatant subjected to expression treatment is collected, the exosome Nidogen-1 is separated by an ultracentrifugation method, and the use concentration of the exosome Nidogen-1 is adjusted to be 10-100 mu g/mL; dissolving sodium alginate, hyaluronic acid and PEG-600 in deionized water to prepare a mixed prepolymer; adding an exosome Nidogen-1 to prepare the tissue engineering scaffold of the composite exosome Nidogen-1. The tissue engineering scaffold is implanted into a wound part or a biocompatible tissue engineering material center, can promote the regeneration of blood vessels of the defect part and improve the metabolism microenvironment of the material center, and has important clinical application value.)

1. A composite tissue engineering scaffold is characterized in that: the composite tissue engineering scaffold comprises a biocompatible matrix and an angiogenetic effect molecule loaded on the matrix, wherein the angiogenetic effect molecule is an exosome Nidoun-1; or the composite tissue engineering scaffold comprises a biocompatible matrix and vascular endothelial cells which are loaded on the matrix and are treated by the angioblasts.

2. The composite tissue engineering scaffold according to claim 1, wherein: the using concentration of the exosome Nidogen-1 is 10-100 mu g/mL calculated by the total protein contained, and the volume ratio of the exosome Nidogen-1 to the biocompatible matrix is 1: 5-10.

3. The composite tissue engineering scaffold according to claim 1, wherein: the biocompatible matrix is selected from a composite hydrogel or a matrigel.

4. A preparation method of a composite tissue engineering scaffold is characterized by comprising the following steps: the method comprises the following steps:

1) obtaining an angiogenetic effect molecule, wherein the angiogenetic effect molecule is an exosome Nidocen-1;

2) and loading the exosome Nidocen-1 into a biocompatible matrix to obtain the composite tissue engineering scaffold.

5. The preparation method of the composite tissue engineering scaffold according to claim 4, wherein the preparation method comprises the following steps: the step 1) specifically comprises the following steps: overexpression of Nidocen-1 in the bone marrow mesenchymal stem cells, and separation of vesicular bodies in a culture solution of the bone marrow mesenchymal stem cells by an ultracentrifugation method to obtain an exosome Nidocen-1.

6. The preparation method of the composite tissue engineering scaffold according to claim 4, wherein the preparation method comprises the following steps: the step 2) specifically comprises the following steps: mixing the exosome Nidogen-1 with liquid composite hydrogel or matrigel, wherein the using concentration of the exosome Nidogen-1 is 10-100 mu g/mL calculated by the total protein.

7. The preparation method of the composite tissue engineering scaffold according to claim 6, wherein the preparation method comprises the following steps: the liquid composite hydrogel is prepared by mixing 95-100mL of water, 0.5-2g of sodium alginate, 1-5g of hyaluronic acid and 1.5-6mL of PEG.

8. A bionic bone tissue scaffold is characterized in that: the composite tissue engineering scaffold comprises a mechanical load-bearing structure and a composite tissue engineering scaffold embedded in the mechanical load-bearing structure; the composite tissue engineering scaffold comprises a biocompatible matrix and an angiogenetic effect molecule loaded on the matrix, wherein the angiogenetic effect molecule is an exosome Nidoun-1; or the composite tissue engineering scaffold comprises a biocompatible matrix and vascular endothelial cells which are loaded on the matrix and are treated by the angioblasts.

9. The biomimetic bone tissue scaffold according to claim 8, wherein: the mechanical load-bearing structure is selected from hollow porous bioceramic made of tissue engineering bone materials.

10. Application of an exosome Nidocen-1 in preparation of a medicine or a tissue engineering scaffold for repairing soft tissue or bone tissue defects.

Technical Field

The invention belongs to the field of medical biomaterials, and particularly relates to a tissue engineering scaffold of a composite exosome Nidougen-1 and a preparation method thereof.

Background

The large-range tissue and organ defects caused by wounds and diseases have the characteristics of high incidence and high disability rate. The regenerative medicine and the tissue engineering technology break through continuously, and provide powerful support for using exogenous materials to transplant and repair the defective tissues. However, early vascularization remains a key factor limiting its repair. The tissue engineering scaffold product is generally constructed in vitro and applied in vivo, and researches prove that tissue fluid can only penetrate 300 mu m below the surface of a scaffold material, cells in the scaffold are difficult to obtain sufficient oxygen and nutrition supply, the proliferation, differentiation and secretion functions of the cells are influenced, and even central cells of the scaffold are induced to die, so that the reconstruction in the scaffold is blocked, and the bottleneck problem of restricting the tissue engineering graft to repair the defect of a host organ is caused. For this purpose, chinese patent CN111065731A accomplishes the repair by forming vascular organoids in vitro and then implanting them in vivo. However, there is a problem that the repair process is complicated as compared with the tissue engineering graft. And the patent uses exogenous stem cells for revascularization, and has the problems of immunological rejection, infection risk and tumorigenicity.

The exosomes derived from stem cells have functions similar to those of stem cells, such as promoting tissue repair and regeneration. Compared with pure cell therapy, exosome is easier to penetrate human body barriers to reach damaged parts, the risk of exogenous stem cell tumorigenesis is avoided, freeze thawing and storage are convenient, and the exosome can become a more ideal clinical bioremediation agent. However, due to the complex composition of the exosome, related tissue engineering composite materials perfectly fit with the exosome are rarely seen in the market, and the use efficiency and the application development process of the exosome as a new-generation biological product are restricted.

Nidogen Nidogen-1 is an extracellular matrix (ECM) protein mainly secreted by interstitial cells, is one of main components of a vascular basement membrane, and is reported in the literature that in the process of forming capillaries, Nidogen-1 participates in the process of forming tubular structures, can stabilize the structures of new blood vessels and promote the maturation of micro blood vessels. However, previous studies focused primarily on the direction in which Nidougen-1 maintains homeostasis as a structural protein, and Nidougen-1 is thought to be present in the extracellular matrix, not in exosomes.

In summary, the problems to be solved at present are: how to build blood circulation in the tissue engineering graft as early as possible to promote cell infiltration, proliferation and differentiation, thereby exerting the treatment advantages of the tissue engineering graft and improving the targeted repair effect.

Disclosure of Invention

In order to solve the problems of ischemia and hypoxia and slow metabolism in the tissue engineering graft, the invention provides a tissue engineering scaffold of a composite exosome Nidoun-1 and a preparation method thereof based on the first confirmation that structural protein Nidoun-1 exists in exosome and has the function of regulating protein.

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

a composite tissue engineering scaffold comprises a degradable biocompatible matrix and a angiogenetic effect molecule loaded in the matrix, wherein the angiogenetic effect molecule is an exosome Nidogen-1; or, the composite tissue engineering scaffold comprises a degradable biocompatible matrix and vascular endothelial cells loaded in the matrix and treated by the angioblasts.

Preferably, the exosome Nidogen-1 is isolated from a culture solution of bone marrow mesenchymal stem cells which are subjected to cell overexpression Nidogen-1 treatment.

Preferably, the use concentration of the exosome Nidogen-1 (based on the total protein contained in the exosome Nidogen-1) is 10-100 mu g/mL, and the volume ratio of the exosome Nidogen-1 to the biocompatible matrix is 1: 5-10.

Preferably, the biocompatible matrix is selected from a composite hydrogel or a matrigel.

Preferably, the composite hydrogel includes sodium alginate, hyaluronic acid, polyethylene glycol (e.g., PEG600), and water.

The preparation method of the composite tissue engineering scaffold comprises the following steps:

1) obtaining an angiogenetic effect molecule, wherein the angiogenetic effect molecule is an exosome Nidocen-1;

2) loading the exosome Nidocen-1 into a degradable biocompatible matrix to obtain the composite tissue engineering scaffold.

Preferably, the step 1) specifically comprises the following steps: overexpressing Nidocen-1 in the separated mesenchymal stem cells, and then separating the vesicular bodies in the culture solution of the mesenchymal stem cells by an ultracentrifugation method to obtain exosome Nidocen-1.

Preferably, the step 2) specifically comprises the following steps: the exosome Nidogen-1 with the use concentration of 10-100 mug/mL is mixed with liquid composite hydrogel or matrigel, and then post-treatment (stabilization of structure, coagulation, etc.) is carried out.

Preferably, the liquid composite hydrogel is prepared by mixing 95-100mL of water, 0.5-2g of sodium alginate, 1-5g of hyaluronic acid and 1.5-6mL of PEG.

Preferably, the liquid composite hydrogel is prepared by mixing 95-100mL of water, 1-2g of sodium alginate, 1-2g of hyaluronic acid and 3-6mL of PEG.

Preferably, the post-treatment specifically comprises the following steps: mixing the exosome Nidocen-1 with the liquid composite hydrogel, and then crosslinking by adopting calcium chloride.

A bionic bone tissue scaffold comprises a mechanical load-bearing structure and the composite tissue engineering scaffold embedded in the mechanical load-bearing structure.

Preferably, the mechanical load-bearing structure is selected from hollow porous bioceramics made of tissue engineered bone material (e.g., calcium phosphate, hydroxyapatite, collagen).

The exosome Nidocen-1 is applied to preparation of a medicine or a tissue engineering scaffold for repairing defects of soft tissues such as skin.

Preferably, the drug or tissue engineering scaffold is implanted into the wound site by injection or coating.

The exosome Nidocen-1 is applied to preparation of a medicine or a tissue engineering scaffold for bone tissue defect repair.

Preferably, the bone tissue defect is selected from the group consisting of critical bone defects, and the length of the defect is 1.5 times or more the circumference of the defect.

The invention has the beneficial effects that:

the composite tissue engineering scaffold provided by the invention takes exosome Nidocen-1 as an angiogenetic effect molecule, can be independently used for treating soft tissue wounds such as skin and the like, can also form a bionic bone tissue scaffold, and accelerates bone defect repair by improving metabolic microenvironment. Compared with the traditional cell tissue engineering product, the exosome Nidogen-1 adopted by the invention belongs to the exosome enriched with the Nidogen-1, avoids the infection, the tumorigenic risk and the immune response brought by exogenous cells, and can stably play the roles of treatment and regulation. The composite tissue engineering scaffold can remarkably promote the effective response of biological materials inside and outside a body and accelerate the regeneration process of blood vessels, thereby constructing a high-activity tissue engineering bionic alternative material.

According to the active effect of an exosome component Nidogen-1 in the processes of promoting endogenous and exogenous cell migration and vascularization, the exosome rich in Nidogen-1, namely the exosome Nidogen-1 is loaded into a biocompatible matrix, and the formed composite tissue engineering scaffold can be implanted into soft tissue of a wound part or a tissue engineering bone material center, promotes angiogenesis in defective tissue repair, improves the material center metabolism microenvironment, so that the targeted repair efficiency is improved, the development trend of accurate drug delivery is met, and the composite tissue engineering scaffold has important clinical application value.

Furthermore, the tissue engineering bone material used in the invention has good biocompatibility, safety and no toxicity.

Furthermore, the bone marrow mesenchymal stem cell-derived exosome Nidogen-1 is generated by overexpression treatment, a high-abundance effect molecule component Nidogen-1 can be obtained, and the abundance of the Nidogen-1 in the exosome Nidogen-1 is more than 1.5 times that of the bone marrow mesenchymal stem cell-derived conventional exosome which is not subjected to overexpression treatment.

Drawings

FIG. 1 is a flow chart of the preparation of the composite tissue engineering scaffold in example 1 of the present invention.

FIG. 2 is a transmission electron microscope identification image (a) of the exosome enriched with Nidougen-1 in example 1 of the present invention, the Nidougen-1 western blot abundance in exosome (b), the detection result of the exosome surface marker CD9 (c), and the NTA particle size analysis (d) of the exosome enriched with Nidougen-1.

FIG. 3 shows the results of in vitro migration test of non-induced vascular endothelial cells (a), the results of in vitro migration test of vascular endothelial cells induced by conventional exosomes (b), the results of migration test of vascular endothelial cells induced by exosome Nidogen-1 (c) and the results of migration test of vascular endothelial cells induced by blocking exosomes (d).

FIG. 4 is the results of in vitro vascularization of non-induced vascular endothelial cells (a), in vitro vascularization of vascular endothelial cells induced by conventional exosomes (b), in vitro vascularization of vascular endothelial cells induced by exosomes Nidogen-1 (c), in vitro vascularization of vascular endothelial cells induced by blocking exosomes (d), and statistics of the number of nodes of tubular structure for each set of in vitro vascularization experiments (e); wherein: p <0.05, P <0.01, ns indicates no significant difference.

FIG. 5 is the appearance of the hydrogel prepared in example 1 of the present invention, wherein: (a) uncrosslinked liquid composite hydrogel, (b) crosslinked and cured hydrogel scaffold.

FIG. 6 shows the results of in vivo angiogenesis of vascular endothelial cells without induction (nude mice subcutaneous injection gel model) (a), the results of in vivo angiogenesis of vascular endothelial cells induced by conventional exosomes (b), and the results of in vivo angiogenesis of vascular endothelial cells induced by exosome-1 (c).

Fig. 7 shows the side appearance (a) of the ceramic material, the side appearance (b) of the tissue engineering bone composite scaffold and the cross-sectional appearance (c) of the tissue engineering bone composite scaffold used in the preparation of the tissue engineering bone composite scaffold in example 1 of the present invention.

FIG. 8 shows HE stained sections (a) of tissue engineering bone composite scaffold material center HE stained sections (a) without exosome injection, HE stained sections (b) of tissue engineering bone composite scaffold material center hemangioblast injected with conventional exosome, and HE stained sections (c) of tissue engineering bone composite scaffold material center hemangioblast injected with exosome Nidougen-1; wherein: the arrows within the black box indicate vascularized regions.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.