TPMS structure bionic bone scaffold with immune regulation and control function and preparation method of TPMS structure bionic bone scaffold

Abstract

The invention belongs to the technical field of bionic bone scaffolds, and particularly relates to a TPMS structure bionic bone scaffold with an immune regulation function and a preparation method of the TPMS structure bionic bone scaffold. Comprising the following steps: preparing a three-period extremely-small curved surface bionic scaffold and micro-tissues, loading micro-tissue spheres in a methacryloyl gelatin solution, then pouring the micro-tissue spheres into the three-period extremely-small curved surface bionic scaffold, and carrying out photo-crosslinking gel forming to obtain the three-period extremely-small curved surface structure bionic bone scaffold with the immune regulation and control function. The bionic bone scaffold disclosed by the invention can be used for accurately simulating components and structures of natural bones, has good immunoregulation and osteogenesis promoting capabilities, and is expected to cooperate with functionalized micro-tissues to construct an immune microenvironment and promote bone tissue regeneration.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bionic bone scaffolds, and particularly relates to a TPMS structure bionic bone scaffold with immune regulation function 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] The bone repair process is divided into hematoma inflammation and organization period, callus formation period and callus remodeling period. After bone tissue damage, the hematoma organization period is first entered, the microvascular network is broken to form a local hematoma, a large number of inflammatory cells are recruited to cause acute inflammatory reaction, and mesenchymal stem cells, vascular endothelial cells and fibroblasts are recruited to change the surrounding environment, and the hematoma is gradually organized and evolved into fibrous connective tissue. After entering the callus formation period, the related inflammatory factors stimulate the proliferation and differentiation of osteogenic related cells into bone cells, and the original callus is formed through intramembranous ossification and endochondral ossification. Then, in the callus remodeling period, the callus on the stress axis is continuously enhanced, and the callus on the non-stress axis is continuously removed, and finally the bone tissue with complete Haversian system is formed.

[0004] With the continuous development of biomaterials and additive manufacturing technology, bone tissue engineering is expected to replace natural bone grafts and become a feasible solution to solve the problem of bone defect treatment. The scaffold fills the defect site and provides mechanical support before bone defect treatment, and also provides structural support for cells to guide new tissue growth. However, previous scaffold research has focused on improving its ability to promote bone or blood vessels, and immune response as the starting stage of bone regeneration cannot be ignored in bone tissue repair. SUMMARY

[0005] The purpose of the embodiment of the present application is to provide a TPMS structure bionic bone scaffold with immune regulation function and a preparation method thereof. The bionic bone scaffold of the present application can accurately simulate the composition and structure of natural bone, has good immune regulation and bone formation ability, and is expected to construct an immune microenvironment by synergizing with functional microtissues to promote bone tissue regeneration.

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

[0007] In a first aspect, the present application provides a preparation method of a TPMS structure bionic bone scaffold with immune regulation function, comprising the following steps: preparing a three-period minimal surface bionic scaffold and microtissues, loading the microtissue spheres in a methacryl gelatin solution, then perfusing the three-period minimal surface bionic scaffold, and obtaining the three-period minimal surface structure bionic bone scaffold with immune regulation function by light crosslinking.

[0008] Further, the three-period minimal surface bionic scaffold is a bionic scaffold prepared based on biphasic calcium phosphate, and the physical properties of the three-period minimal surface bionic scaffold are adjusted by parameterized modeling to meet different bone defect repair requirements, the physical properties including porosity, pore size, mechanical strength, roughness and topological structure.

[0009] Further, the microtissues are prepared by bone marrow mesenchymal stem cells, and the microtissues are single-cell or multi-cell module units formed by cells through extracellular matrix, including cell sheets and cell spheres.

[0010] Further, the preparation of the microtissues by bone marrow mesenchymal stem cells specifically comprises the following steps: when the third generation of adherent growth BMSCs reaches 80%-90%, 0.25% trypsin is added for digestion to prepare a single-cell suspension, 10 4 , 3×10 4 , 5×10 4 The three cell number of BMSCs spheres are inoculated in a low-adhesion U-shaped 96-well plate, and the microtissues are formed after overnight culture.

[0011] Further, the loading of the microtissue spheres in the methacryl gelatin solution specifically comprises the following steps: gelatin is dissolved in PBS at 50°C and continuously stirred, and methacrylate is added; after 3h of light-free reaction, 2 times the volume of PBS is added for dilution, and the product is dialyzed with distilled water at 40°C for 5d; then the product is filtered with a 0.22μm filter and freeze-dried to form a white porous foam, which is stored at -20°C for further use;

[0012] After the preparation of the methacryl gelatin, the BMSCs spheres are uniformly mixed in the methacryl gelatin, and cured under ultraviolet light, then cultured to construct a bone marrow mesenchymal stem cell sphere delivery system.

[0013] A TPMS structure bionic bone scaffold with immune regulation function is prepared by the above preparation method.

[0014] Further, the three-period minimal surface structure bionic bone scaffold comprises a three-period minimal surface bionic scaffold and microtissues, and the three-period minimal surface bionic scaffold and the microtissues are connected by methacryl gelatin.

[0015] The three-period minimal surface biomimetic scaffold has a hollow tubular structure and is distributed in a stepped layer shape around the periphery, is connected between layers, and gradually decreases in porosity from the inside to the outside, and large pores, small pores and micropores coexist.

[0016] The embodiment of the present application has the following beneficial effects:

[0017] The TPMS biomimetic scaffold of the present application can accurately simulate the natural bone composition and structure, has good immunomodulatory and osteogenic ability, and is expected to construct an immune microenvironment in cooperation with functional microtissues to promote bone tissue regeneration.

[0018] The microtissues formed by 3D culture allow cells to adapt to their natural shape, increasing cell-cell contact and communication. Microtissues secrete a large amount of extracellular matrix, simulating the 3D environment in vivo, and have good biological functions, such as stable morphology and excellent metabolic function. The applicant successfully prepared microtissues by bone marrow mesenchymal stem cells (BMSCs), and compared with two-dimensional cells, the microtissues differentiated to the osteogenic direction earlier, not only that, the microtissues effectively promoted the polarization of macrophages to M2 type, becoming a reliable method for regulating the immune microenvironment of tissue engineering. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a structural schematic diagram of the three-period minimal surface biomimetic scaffold in the present application;

[0020] Figure 2 It is a general observation and diameter analysis of BMSCs spheroids in the present application, (A) 2D BMSCs and general observation of BMSCs spheroids under a microscope; (B) diameter of BMSCs spheroids with different cell numbers;

[0021] Figure 3 It is a scanning electron microscope and transmission electron microscope observation of BMSCs spheroids in the present application, (A) general observation of BMSCs under a scanning electron microscope; (B) microstructure of BMSCs under a scanning electron microscope; (C) transmission electron microscope graph of internal cells after sectioning of BMSCs spheroids; (D) transmission electron microscope graph of peripheral cells after sectioning of BMSCs spheroids;

[0022] Figure 4 It is a flow cytometry graph of 2D cultured cells and BMSCs spheroids with different cell numbers in the present application;

[0023] Figure 5 It is a growth change of 2D BMSCs and BMSCs spheroids loaded with GelMA in the present application;

[0024] Figure 6 It is an ALP staining of 2D cultured BMSCs and 3D BMSCs spheroids after osteogenic induction in the present application;

[0025] Figure 7 GelMA loaded 2D cultured BMSCs and 3D BMSCs spheroids after osteogenic induction in the present application were subjected to alizarin red S staining;

[0026] Figure 8 The use flowchart of the TPMS structure bionic bone scaffold with immune regulation function in the present application is shown. DETAILED DESCRIPTION

[0027] The present application will be further described below in conjunction with examples.

[0028] 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.

[0029] Applicants have found through extensive research that bone regeneration is not a simple process involving bone formation and bone resorption, but a complex system connecting multiple systems such as bone, blood vessels and immune system. With the deepening of research on bone immunity in the field of bone regeneration, researchers have found that in the formation of local bone microenvironment, the change of immune microenvironment induced by the implantation of biomaterials in the body is one of the core factors determining bone formation and remodeling. A good immune response can regulate bone regeneration-related processes such as osteogenic differentiation, osteoclastic differentiation, fibrosis and vascularization by regulating various factors such as growth factors, chemokines and inflammatory factors, and plays a key role in bone regeneration. Therefore, using immune cells as the primary target cells in bone repair process, using biomaterials to drive immune cells to construct a local suitable bone immune microenvironment has become an important method for tissue engineering treatment of bone defects. Therefore, the present application constructs a bone tissue engineering scaffold-hydrogel microcarrier-osteogenic microtissue composite bone material.

[0030] Bone tissue engineering scaffolds can fill the defect site and provide mechanical support in the early stage of bone defect treatment, and at the same time provide structural support for cells to promote bone tissue regeneration. Inflammation changes induced by biomaterial implantation are considered to be an important reason for implant failure. Blindly reducing the host's immune response is not conducive to bone repair. By giving bone tissue engineering scaffolds immune regulation properties through various strategies, the host's bone immune microenvironment can be effectively intervened and precisely manipulated to promote bone tissue regeneration. The composition and structure design of the scaffold are very important for the exertion of its biological function.

[0031] The application relates to a preparation method of a TPMS structure bionic bone support with immune regulation function, which comprises the following steps: preparing a three-period minimal surface bionic support and a microtissue, loading the microtissue ball in a methacryl gelatin solution, then perfusing the three-period minimal surface bionic support, and obtaining the three-period minimal surface structure bionic bone support with immune regulation function through light crosslinking and gelation (as shown in Figure 1

[0032] In terms of components, calcium phosphate, as a main component of natural bone tissue, can effectively promote the polarization of macrophages to M2 type. Calcium phosphate can increase the calcium level in the extracellular environment, enhance the calcium-sensitive receptor-mediated macrophage driving effect, induce M2-like polarization, and regulate the adhesion, morphology, proliferation and expression of key cytokines of macrophages, and has good immunoregulatory and osteoinductive properties. At present, various calcium phosphate materials have been developed and used for bone regeneration, such as biphasic calcium phosphate (BCP), beta-tricalcium phosphate (TCP) and the like. The DLP printing technology is an effective method for preparing calcium phosphate support quickly and accurately. The applicant previously summarized the application of DLP printing technology in bone tissue engineering, and prepared various bionic supports based on the DLP printing technology, and successfully repaired bone defects. The bionic support prepared based on biphasic calcium phosphate has good 3D printing performance, biocompatibility and osteogenic ability. The preparation of the TPMS support in the project provides material and technical support.

[0033] In terms of structure, the TPMS bionic support accurately simulates the important structural characteristics of natural bone, adjusts the physical properties such as porosity, pore size, mechanical strength, roughness and topological structure, and realizes immune regulation in the bone repair process. Bone tissue is divided into two parts according to its structure. Cortical bone accounts for about 80%, the structure is dense, the porosity is 10%, and the bone plate is composed of multiple layers arranged closely; cancellous bone accounts for about 20%, the structure is loose, the porosity is 50-90%, the bone plates are supported by connecting structures, forming a porous reticular structure, and the average curvature is zero. The mesh center is filled with bone marrow, nerves and blood vessels. The trabeculae have the characteristics of large, small and micro pores, and such structural characteristics make the trabeculae have good elasticity and toughness when bearing pressure and tension. At the same time, it has good specific surface area, which is helpful for the adhesion and proliferation and differentiation of surface cells.

[0034] ​Triply Periodic Minimal Surfaces (TPMS) is a minimal surface that repeats periodically in three dimensions. Compared with traditional scaffolds (based on struts / lattices), TPMS biomimetic scaffolds have similar topological structure to natural bone trabecula, interconnected pores and good permeability, which helps the flow of nutrients and the discharge of metabolic waste, and builds a good immune microenvironment. At the same time, the zero-curvature property of TPMS reduces stress concentration during bone regeneration and improves the negative effects of sharp edges of traditional scaffolds on cell adhesion. In addition, TPMS scaffolds have good specific surface area, which helps cell adhesion, proliferation and differentiation. Studies have shown that TPMS scaffolds exhibit good immune regulation and osteogenic ability during bone repair, and are expected to promote the repair and regeneration of defective bone tissue. The various unit cell structures of TPMS can be adjusted by parameterized modeling to achieve specific porosity, specific surface area and mechanical properties, etc. to meet different bone defect repair needs. Referring to the lamellar bone, the applicant previously constructed a TPMS model through the Gryoid unit cell, which has a hollow tubular structure and is distributed in a stepped layer around the scaffold. The layers are connected to each other, and the porosity gradually decreases from the inside to the outside, and there are large, small and micro pores. Therefore, the TPMS biomimetic scaffold can accurately simulate the composition and structure of natural bone, has good immune regulation and osteogenic ability, and is expected to construct an immune microenvironment with functional microtissues and promote bone tissue regeneration.

[0035] The TPMS biomimetic scaffold of the application can accurately simulate the composition and structure of natural bone, has good immune regulation and osteogenic ability, and is expected to construct an immune microenvironment with functional microtissues and promote bone tissue regeneration.

[0036] The microtissues formed by 3D culture allow cells to adapt to their natural shape, increasing cell-to-cell contact and communication. Microtissues secrete a large amount of extracellular matrix, simulating the 3D environment in vivo, and have good biological functions such as stable morphology and excellent metabolic function. The applicant successfully prepared microtissues by bone marrow mesenchymal stem cells (BMSCs), which differentiated earlier to the osteogenic direction compared with two-dimensional cells. In addition, microtissues effectively promote the polarization of macrophages to M2 type, which is a reliable method for tissue engineering to regulate the immune microenvironment.

[0037] In some embodiments of the application, the triply periodic minimal surface biomimetic scaffold is a biomimetic scaffold prepared based on biphasic calcium phosphate. Parameterized modeling is used to adjust the physical properties of the triply periodic minimal surface biomimetic scaffold to meet different bone defect repair needs, including porosity, pore size, mechanical strength, roughness and topological structure.

[0038] In some embodiments of the present application, the microtissue is a single cell or multi-cell module unit formed by cells aggregating through extracellular matrix, such as cell 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 the traditional two-dimensional adherent culture cells, the microtissue formed by 3D culture allows cells to adapt to their natural shape, increases cell-to-cell contact and communication. The microtissue secretes a large amount of extracellular matrix, simulates the 3D environment in vivo, has good biological functions, such as stable morphology and excellent metabolic function. The applicant successfully prepared microtissue by bone marrow mesenchymal stem cells (BMSCs), and compared with two-dimensional cells, the microtissue differentiated to osteogenic direction earlier, not only that, the microtissue effectively promoted the polarization of macrophages to M2 type, becoming a reliable method for regulating immune microenvironment of tissue engineering.

[0039] In some embodiments of the present application, the preparation of microtissue by bone marrow mesenchymal stem cells specifically comprises the following steps: BMSCs microtissue preparation: when the third generation of adherent growth BMSCs reaches 80%-90%, 0.25% trypsin is added for digestion to prepare a single cell suspension, 10 4 , 3x10 4 , 5x10 4 cells of BMSCs spheroid cells are inoculated in low-adhesion U-shaped 96-well plates, and microtissues can be formed after overnight culture.

[0040] The cell morphology is observed by inverted microscope, the primary culture of BMSCs adheres to the wall after 6 hours, and almost completely adheres to the wall after 24 hours, but a large number of suspended cells can be seen, the cell morphology is stable after multiple liquid changes and subculture to the third generation, and is long spindle-shaped. The third generation of BMSCs is inoculated in low-adhesion U-shaped well plate, and the cells aggregate into spheroids after about 12 hours, and no senescent or dead cells are observed in both groups Figure 2 A).

[0041] The diameters of the cell spheroids formed by different cell numbers inoculated in low-adhesion U-shaped well plates are different, we select 10 4 , 3x10 4 , 5x10 4 cells to aggregate into spheroids and measure the spheroid diameters. The results are shown in Figure 2 B, the diameters of the cell spheroids of 10 4 , 3x10 4 , 5x10 4 cells are 161.19±11.45 μm, 267.33±25.15 μm and 357.67±33.62 μm, respectively.

[0042] The surface of the cell spheroid is observed microscopically by scanning electron microscope, a large number of cells are seen to form spheroids by hugging together Figure 3A), cells were connected with each other and secreted a large amount of extracellular matrix, and a small amount of round-shaped dead cells were observed Figure 3 B) To observe the change of cell morphology, we performed tissue sectioning on the spheroids and observed the cell morphology by transmission electron microscopy, Figure 3 C is the cell morphology inside the spheroid, and 4-2D is the cell morphology at the periphery of the spheroid. It was found that the cells inside the spheroid were closely connected, and the shape was not a classic long spindle shape. The cells at the periphery of the spheroid were still a classic long spindle shape.

[0043] The cell viability of 2D adherent cells and BMSCs spheroids with three diameters was detected by flow cytometry, as shown in Figure 4 The survival rate of 2D adherent cells was 95.66%, while the survival rates of BMSCs spheroid cells with three diameters were 90.55%, 82.09% and 46.70%, respectively. With the increase of cell diameter, more and more cells died. There was no statistically significant difference (P>0.05) between the survival rate of 2D adherent cells and the survival rate of BMSCs spheroid cells (the number of cells was 10 4 ).

[0044] In some embodiments of the present application, the microtissue spheroids are loaded in a methacryl gelatin solution, which specifically comprises the following steps: preparation of GelMA

[0045] Dissolve gelatin in PBS at 50°C and continuously stir. Add methacrylate (MA). After 3h of reaction in the dark, dilute with 2 times the volume of PBS, and dialyze with distilled water at 40°C for 5 days. Then filter the product with a 0.22μm filter and freeze-dry it to form a white porous foam, which is stored at -20°C for further use.

[0046] After preparing GelMA, equal amounts of 2D adherent cells and BMSCs spheroids (100000 / ml) were mixed in GelMA (5% concentration) and cured under ultraviolet light, and then cultured to construct a BMSCs spheroid delivery system. As shown in Figure 5 Most of the 2D adherent cells cultured in GelMA remained in a round state on the first day, and had not yet extended tentacles. A small amount of tentacles extended on the third day, and the shape changed to a spindle shape on the fifth day, and the cells were connected with each other. The cells around the cell spheroids in GelMA extended tentacles on the first day, and migrated to the inside of the glue. On the third day, the number of cells around the spheroids increased, and on the fifth day, a large number of cells were observed, and the size of the spheroids did not change significantly. The results suggest that compared with adherent cells, cell spheroids can adapt to the 3D microenvironment inside GelMA earlier and make changes in the early stage.

[0047] ALP and alizarin red S (osteogenesis)

[0048] BMSCs spheroids and 2D adherent cells were loaded in GelMA, and the results of ALP and alizarin red staining after 7 days and 14 days of osteogenic induction are shown in Figure 6 、 Figure 7 With the increase of osteogenic induction time, the ALP and alizarin red S staining intensity at the two time points increased; among them, the ALP staining intensity of BMSCs spheroids was higher than that of 2D cells at 7 days and 14 days, and the amount of calcium nodules was higher than that of 2D cells at 14 days, and the difference was statistically significant (P<0.05), which proved that in GelMA, BMSCs spheroids could better perform osteogenic differentiation in the early stage. Figure 8 The use flowchart of the TPMS structure bionic bone scaffold with immune regulation function is shown.

[0049] A TPMS structure bionic bone scaffold with immune regulation function, wherein the TPMS structure bionic bone scaffold with immune regulation function is prepared by the preparation method.

[0050] In some embodiments of the present application, the TPMS structure bionic bone scaffold includes a TPMS structure bionic scaffold and microtissues, and the TPMS structure bionic scaffold and microtissues are connected by methacrylated gelatin.

[0051] The TPMS structure bionic scaffold has a hollow tubular structure and is distributed in a stepped layer shape around the periphery, the layers are connected to each other, the porosity gradually decreases from the inside to the outside, and large pores, small pores and micro-pores coexist (as shown in Figure 1 ).

[0052] It should be noted that the above embodiments can be freely combined as needed. The above introduction is only for 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. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a TPMS structure bionic bone scaffold with immunoregulatory function, characterized in that, The method comprises the following steps: preparing a tri-periodic minimal surface biomimetic scaffold and microtissues, loading the microtissue spheres in a methacrylated gelatin solution, and then perfusing the solution into the tri-periodic minimal surface biomimetic scaffold, and obtaining the tri-periodic minimal surface structure biomimetic bone scaffold with immune regulation function by light crosslinking.

2. The method for preparing the TPMS biomimetic bone scaffold with immune regulation function according to claim 1, characterized in that, The tri-periodic minimal surface biomimetic scaffold is a biomimetic scaffold prepared based on biphasic calcium phosphate, and the physical properties of the tri-periodic minimal surface biomimetic scaffold are adjusted by parameterized modeling to meet different bone defect repair requirements, and the physical properties include porosity, pore size, mechanical strength, roughness and topological structure.

3. The method for preparing the TPMS biomimetic bone scaffold with immune regulation function according to claim 1, characterized in that, The microtissues are prepared by bone marrow mesenchymal stem cells, and the microtissues are single-cell or multi-cell module units formed by cells through extracellular matrix, including cell sheets and cell spheres.

4. The method for preparing the TPMS biomimetic bone scaffold with immune regulation function according to claim 3, characterized in that, The preparation of microtissues by bone marrow mesenchymal stem cells comprises the following steps: when the third generation of BMSCs adherent growth reaches 80%-90%, 0.25% trypsin is added for digestion to prepare a single cell suspension, 10 4 , 3 x 10 4 , 5 x 10 4 BMSCs of three cell numbers are inoculated in a low-adhesion U-shaped 96-well plate, and microtissues are formed after overnight culture.

5. The method for preparing the TPMS biomimetic bone scaffold with immune regulation function according to claim 1, characterized in that, The microtissue spheres are loaded in a methacrylated gelatin solution, and the specific steps include the following: dissolving gelatin in PBS at 50 DEG C with continuous stirring, and adding methacrylate; after 3 hours of light-free reaction, diluting with 2 times the volume of PBS, and dialyzing with distilled water at 40 DEG C for 5 days; then filtering the product with a 0.22 mu m filter and freeze-drying to form white porous foam, and storing at -20 DEG C for further use; After preparing the methacrylated gelatin, the BMSCs spheres are mixed in the methacrylated gelatin, and after curing under ultraviolet light, culture is carried out to construct a bone marrow mesenchymal stem cell sphere delivery system.

6. A TPMS structure bionic bone scaffold with immunoregulatory function, characterized in that, The tri-periodic minimal surface structure biomimetic bone scaffold with immune regulation function is prepared by the preparation method in any one of claims 1-5.

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