Vasculation bone micro-tissue and preparation method thereof

Abstract

The invention belongs to the technical field of medicine, and discloses a vascularized bone micro-tissue and a preparation method thereof, the vascularized bone micro-tissue comprises umbilical cord mesenchymal stem cell spheres and bone marrow mesenchymal stem cells, and the bone marrow mesenchymal stem cells cover the umbilical cord mesenchymal stem cell spheres to form a core-shell structure. Compared with an anoxic environment, the inside of the micro-tissue sphere of the vascularization bone micro-tissue disclosed by the invention promotes formation of tubules, improves an internal inflammation micro-environment, increases extracellular matrix secretion, provides a good basis for bone mesenchymal stem cells (BMSCs) osteogenic differentiation, constructs a cell growth micro-environment, and accelerates bone regeneration in a stent.

Description

TECHNICAL FIELD

[0001] The present application relates to the medical technical field, and in particular to a vascularized osteogenic microtissue and a preparation method thereof. BACKGROUND

[0002] The description of the background of the present application belongs to the related art related to the present application, and is only used to illustrate and facilitate the understanding of the content of the present application, and should not be understood as the applicant's explicit recognition or presumption that the present application is the prior art on the date of the first filing of the application.

[0003] Microtissue is a single cell or multicellular module unit formed by cells through extracellular matrix, such as cell membrane sheet, cell spheroid, etc., which has been applied in the fields of stem cell therapy, tumor in vitro model construction, etc., and shows good application prospect. Compared with traditional two-dimensional adherent cultured cells, microtissue formed by 3D culture allows cells to adapt to their natural shape, increasing cell-to-cell contact and communication. Microtissue secretes a large amount of extracellular matrix, simulating the 3D environment in vivo, and has good biological functions, such as stable morphology and excellent metabolic function. Compared with two-dimensional cells, microtissue prepared by bone marrow mesenchymal stem cells (BMSCs) differentiates to osteogenic direction earlier.

[0004] Microtissue spheroid has a three-layer structure, the outer layer cells proliferate highly, the middle layer cells are relatively static, and the core area is composed of hypoxic and necrotic cells, and metabolic products accumulate. The cells in the central part are limited in contact with nutrients, and the exchange of oxygen and carbon dioxide is limited, and metabolic waste accumulates, resulting in the formation of an internal necrotic area in the central area of the cell spheroid. Therefore, it is urgent to effectively functionalize the microtissue, improve the core necrosis phenomenon, and improve the biological activity of the microtissue. SUMMARY

[0005] The purpose of the embodiment of the present application is to provide a vascularized osteogenic microtissue and a preparation method thereof. The relative hypoxic environment inside the microtissue spheroid of the vascularized osteogenic microtissue of the present application promotes tubule formation, improves the internal inflammatory microenvironment, increases the secretion of extracellular matrix, and provides a good foundation for the osteogenic differentiation of BMSCs, constructs a cell growth microenvironment, and accelerates the bone regeneration inside the scaffold.

[0006] The purpose of the embodiment of the present application is achieved by the following technical solutions:

[0007] A vascularized osteogenic microtissue, the vascularized osteogenic microtissue comprising a umbilical cord mesenchymal stem cell spheroid and bone marrow mesenchymal stem cells, the bone marrow mesenchymal stem cells covering the outside of the umbilical cord mesenchymal stem cell spheroid to form a core-shell structure.

[0008] Further, the particle size range of the umbilical cord mesenchymal stem cell spheroid is 100-300 μm.

[0009] A preparation method of a vascularized osteogenic microtissue, umbilical cord mesenchymal stem cells are prepared into umbilical cord mesenchymal stem cell spheroids, and bone marrow mesenchymal stem cells are covered on the umbilical cord mesenchymal stem cell spheroids to form a core-shell structure vascularized osteogenic microtissue.

[0010] Further, the umbilical cord mesenchymal stem cell spheroids are prepared by a low-adhesion U-shaped 96-well plate: 10000-30000 cell number of umbilical cord mesenchymal stem cell suspension is inoculated in a low-adhesion U-shaped 96-well plate, and the stem cell spheroids can be formed after overnight.

[0011] Further, the umbilical cord mesenchymal stem cell spheroids are prepared by a low-adhesion U-shaped 96-well plate: 10000-30000 cell number of umbilical cord mesenchymal stem cell suspension is inoculated in a low-adhesion U-shaped 96-well plate, and the stem cell spheroids can be formed after overnight.

[0012] The embodiment of the present application has the following beneficial effects:

[0013] The relatively hypoxic environment in the microtissue spheroids of the vascularized osteogenic microtissue of the present application promotes the formation of small tubes, improves the internal inflammatory microenvironment, increases the secretion of extracellular matrix, and provides a good foundation for the osteogenic differentiation of BMSCs, constructs a cell growth microenvironment, and accelerates the bone regeneration in the scaffold. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a structural schematic diagram of the vascularized osteogenic microtissue in the present application;

[0015] Figure 2 It is a result diagram of microtissue cell live and dead staining;

[0016] Figure 3 It is the expression of osteogenic related mRNA after osteogenic induction of the microtissue;

[0017] Figure 4 It is a CT and three-dimensional reconstruction of the microtissue in the treatment of rat femoral condyle bone defects (A) CT image; (B) CT three-dimensional reconstruction; (b-e) CT data statistical analysis. DETAILED DESCRIPTION

[0018] The present application will be further described below in combination with examples.

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, in the following description, different "an embodiment" or "embodiments" do not necessarily refer to the same embodiment. Different embodiments can be replaced or combined, and other embodiments can be obtained by those skilled in the art without creative labor.

[0020] A vascularized osteogenic microtissue, comprising umbilical cord mesenchymal stem cell spheroids and bone marrow mesenchymal stem cells, wherein the bone marrow mesenchymal stem cells are coated on the outside of the umbilical cord mesenchymal stem cell spheroids to form a core-shell structure.

[0021] In some embodiments of the present application, the particle size of the umbilical cord mesenchymal stem cell spheroids is 100-300 μm.

[0022] A method for preparing a vascularized osteogenic microtissue, comprising: preparing umbilical cord mesenchymal stem cells into umbilical cord mesenchymal stem cell spheroids, and coating bone marrow mesenchymal stem cells on the umbilical cord mesenchymal stem cell spheroids to form a core-shell structure vascularized osteogenic microtissue.

[0023] In some embodiments of the present application, the umbilical cord mesenchymal stem cell spheroids are prepared by a low-adhesion U-shaped 96-well plate: 10000-30000 cell number of umbilical cord mesenchymal stem cell suspension is inoculated in a low-adhesion U-shaped 96-well plate, and after overnight, stem cell spheroids are formed.

[0024] In some embodiments of the present application, the umbilical cord mesenchymal stem cells on the umbilical cord mesenchymal stem cell spheroids are coated by a dynamic rotation method: the culture medium in the umbilical cord mesenchymal stem cell 96-well plate is removed, a bone marrow mesenchymal stem cell suspension with a cell number of 10000-30000 is prepared, and is added to the 96-well plate containing the umbilical cord stem cell spheroids, and is placed on a multi-amplitude shaker in a cell culture box, with a rotation speed of 60 rpm / min, for 24 hours, to prepare an osteogenic microtissue with umbilical cord mesenchymal stem cells as the inner layer and bone marrow mesenchymal stem cells as the outer layer.

[0025] Embodiment 1

[0026] A method for preparing a vascularized osteogenic microtissue, comprising the following steps: 10000-30000 cell number of umbilical cord mesenchymal stem cell suspension is inoculated in a low adhesion U-shaped 96-well plate, and after overnight, stem cell spheres can be formed. The culture medium in the 96-well plate prepared in the above step of umbilical cord mesenchymal stem cells is removed, a bone marrow mesenchymal stem cell suspension with a cell number of 10000-30000 is prepared, and is added to the 96-well plate containing the umbilical cord stem cell spheres, and is placed in a multi-amplitude shaker in a cell incubator, and the rotation speed is adjusted to 60 rpm / min, and is continuously prepared for 24 hours, so as to prepare an osteogenic microtissue with umbilical cord mesenchymal stem cells as the inner layer and bone marrow mesenchymal stem cells as the outer layer.

[0027] The vascularized osteogenic microtissue can be used for bone defect regeneration and is directly placed in a bone defect site.

[0028] Figure 1 As a structural schematic diagram, the applicant prepares a functionalized osteogenic microtissue with a "core-shell structure", the inner layer is umbilical cord mesenchymal stem cells (HUMSCs), and the outer layer is bone marrow mesenchymal stem cells (BMSCs). The relative hypoxic environment in the sphere promotes HUMSCs to form small tubes, improves the internal hypoxic environment, improves the biological activity of cells Figure 1 , Figure 2 ), strengthens the immune regulation ability, and provides a good foundation for BMSCs osteogenic differentiation.

[0029] Figure 2 As a microtissue cell live and dead staining diagram, after the BMSCs microtissue and the BMSCs / HUMSCs microtissue are cultured for 3 days, live and dead staining of the cell spheres is performed, and the results show that compared with the BMSCs microtissue, the BMSCs / HUMSCs microtissue sphere center has significantly fewer dead cells.

[0030] Figure 3 As a microtissue osteogenic induction osteogenic related mRNA expression situation, after 2D culture of BMSCs, BMSCs microtissue, and BMSCs / HUMSCs microtissue are induced for 5 days and 10 days, mRNA is extracted to detect the expression of osteogenic related genes Col, OPN, Runx2, and OSX, and the results show that the osteogenic related mRNA expression is: BMSCs / HUMSCs microtissue > BMSCs microtissue > 2D culture BMSCs, which indicates that the vascularized osteogenic microtissue effectively promotes the biological activity of cells to be improved.

[0031] Figure 4CT and three-dimensional reconstruction of microtissue treatment of rat femoral condyle bone defect. After anesthesia, the rat femoral condyle bone defect model was established with a diameter of 3 cm and a depth of 4 cm; BMSCs microtissue and BMSCs / HUMSCs microtissue were implanted into the defect site for treatment, and the material was taken out for CT analysis 8 weeks after the operation. CT and three-dimensional reconstruction of microtissue treatment of rat femoral condyle bone defect. (A) CT image; (B) CT three-dimensional reconstruction; (b-e) CT data statistical analysis.

[0032] It should be explained that the above examples can be freely combined according to needs. The above introduction is only the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A vascularized osteogenic microtissue, characterized in that, The vascularized osteogenic microtissue comprises umbilical cord mesenchymal stem cell spheroids and bone marrow mesenchymal stem cells, and the bone marrow mesenchymal stem cells are covered outside the umbilical cord mesenchymal stem cell spheroids to form a core-shell structure.

2. The vascularized osteogenic microtissue of claim 1, wherein, The particle size of the umbilical cord mesenchymal stem cell spheroids ranges from 100 to 300 microns.

3. A method for preparing a vascularized osteogenic microtissue, characterized by, The umbilical cord mesenchymal stem cells are prepared into umbilical cord mesenchymal stem cell spheroids, and the bone marrow mesenchymal stem cells are covered on the umbilical cord mesenchymal stem cell spheroids to form a core-shell structure vascularized osteogenic microtissue.

4. The method for preparing a vascularized osteogenic microtissue according to claim 3, wherein, The umbilical cord mesenchymal stem cell spheroids are prepared by a low-adhesion U-shaped 96-well plate: 10,000-30,000 cell number of umbilical cord mesenchymal stem cell suspension is inoculated in a low-adhesion U-shaped 96-well plate, and the stem cell spheroids are formed after overnight.

5. The method of claim 3, wherein the step of culturing the vascularized osteogenic microtissues is performed in a bioreactor. 5 The umbilical cord mesenchymal stem cells are covered on the umbilical cord mesenchymal stem cell spheroids by a dynamic rotation method: the culture medium in the 96-well plate for preparing umbilical cord mesenchymal stem cells is removed, a bone marrow mesenchymal stem cell suspension with a cell number of 10,000-30,000 is prepared, and the 96-well plate containing the umbilical cord stem cell spheroids is placed in a multi-amplitude shaker in a cell culture box, the rotation speed is adjusted to 60 rpm / min, and the preparation is continued for 24 hours, so that the umbilical cord mesenchymal stem cell spheroids are prepared into an osteogenic microtissue with umbilical cord mesenchymal stem cells as the inner layer and bone marrow mesenchymal stem cells as the outer layer.

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