Preparation Method and Application of Bone Defect Repair Material

Abstract

The present invention provides a preparation method and application of a bone defect repair material, relating to the field of biotechnology. The preparation method includes subjecting a cell-hydrogel composite to dynamic mechanical stimulation; the cell-hydrogel composite is a hydrogel embedded with bone marrow mesenchymal stem cells; the dynamic mechanical stimulation includes causing the cell-hydrogel composite to undergo periodic compressive deformation, the compressive deformation amount not exceeding 10% of its own volume, the compression frequency not exceeding 1 Hz, the time of each exposure to dynamic mechanical stimulation not exceeding 1 h, and the time interval between two adjacent exposures to dynamic mechanical stimulation being 18 to 30 h. The bone defect repair material prepared by this preparation method can effectively promote bone tissue regeneration.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a preparation method and application of a bone defect repair material. Background Art

[0002] The following statements only provide background information related to the present invention and do not necessarily constitute prior art.

[0003] The repair and regeneration of bone defects caused by various reasons have always been the key issues of clinical concern. At present, the main methods for bone defect repair are autologous bone or allogeneic bone transplantation. However, autologous bone transplantation has problems such as limited supply and possible bleeding, infection, etc.; at the same time, allogeneic bone transplantation also faces limitations such as donor shortage, high cost, and infection risk. Therefore, there is an urgent need to carry out research and development of alternative treatment strategies.

[0004] With the development of material preparation technology, various artificial synthetic bone repair materials have been widely studied, such as bioceramics, metal materials, polymer materials, and related derivative composite materials, etc. However, these artificial synthetic bone repair materials are difficult to achieve good bone defect repair effects due to factors such as insufficient biological activity, lack of local blood supply, and processing technology bottlenecks. Tissue engineering technology combines new biological materials / scaffolds with stem cells and growth factors to improve the biological activity of bone repair materials to improve the bone repair effect, but such a combination method is often a simple accumulation of materials, cells, and factors, and it is difficult to maintain the differentiation potential of stem cells in the materials and cannot form a perfect function.

[0005] Generally speaking, these materials are gradually replaced by bone organ repair materials due to various defects and limitations. Bone organs are three-dimensional self-renewing and self-organizing micro bone tissues with bionic spatial characteristics constructed based on bioactive materials. This kind of bone defect repair material can simulate the microenvironment under physiological or pathological conditions of the body, and then realize the efficient and timely regeneration of various tissues at the defect site, so as to achieve good bone defect repair effects.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide a preparation method of a bone defect repair material, and the bone defect repair material prepared by this preparation method can effectively promote bone tissue regeneration.

[0008] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0009] In a first aspect, a method for preparing a bone defect repair material is provided. The preparation method includes subjecting a cell-hydrogel composite to dynamic mechanical stimulation; the cell-hydrogel composite is a hydrogel embedded with bone marrow mesenchymal stem cells.

[0010] The dynamic mechanical stimulation includes causing the cell-hydrogel composite to undergo periodic compressive deformation, where the compressive deformation amount does not exceed 10% of its own volume, the compression frequency does not exceed 1 Hz, the time for each exposure to the dynamic mechanical stimulation does not exceed 1 h, and the time interval between adjacent exposures to the dynamic mechanical stimulation is 18 - 30 h.

[0011] In an optional embodiment, the dynamic mechanical stimulation includes causing the cell-hydrogel composite to undergo periodic compressive deformation, with a compressive deformation amount of 5% of its own volume, a compression frequency of 0.5 Hz, and a time of 0.5 h for each exposure to the dynamic mechanical stimulation.

[0012] In an optional embodiment, the preparation method includes subjecting the cell-hydrogel composite to the dynamic mechanical stimulation 3 - 14 times.

[0013] In an optional embodiment, the preparation method includes subjecting the cell-hydrogel composite to the dynamic mechanical stimulation 3 times.

[0014] In an optional embodiment, the density of bone marrow mesenchymal stem cells in the cell-hydrogel composite is 10 5 ~10 8 / cm 3 。

[0015] In an optional embodiment, the cell-hydrogel composite is cultured using a growth medium.

[0016] In an optional embodiment, the hydrogel includes a hydrogel containing proteins and / or polysaccharides.

[0017] In an optional embodiment, the preparation method includes: mixing bone marrow mesenchymal stem cells, an initiator, and a methacrylated gelatin solution, where the mass-volume fraction of methacrylated gelatin is 10%, and the density of bone marrow mesenchymal stem cells is 10 5 ~10 8 / cm 3 , and obtaining the cell-hydrogel composite through photocrosslinking;

[0018] Cultivate the cell-hydrogel complex in a growth medium. After 24 hours of cultivation, subject the cell-hydrogel complex to dynamic mechanical stimulation 3 to 14 times. The dynamic mechanical stimulation includes causing the cell-hydrogel complex to undergo periodic compressive deformation, with the compressive deformation amount being 5% of its own volume, the compression frequency being 0.5 Hz, the time for each exposure to the dynamic mechanical stimulation being 0.5 h, and the time interval between two consecutive exposures to the dynamic mechanical stimulation being 24 h.

[0019] In a second aspect, there is also provided a bone defect repair material prepared by the preparation method of the first aspect.

[0020] In a third aspect, there is also provided the application of the preparation method described in the first aspect, or the bone defect repair material of the second aspect, in the preparation of bone organoids.

[0021] In a fourth aspect, there is also provided a bone organoid, which contains the bone defect repair material of the first aspect.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention proposes to construct a bone defect repair material based on dynamic-static collaborative mechanical stimulation. First, a cell-hydrogel complex is constructed, and the hydrogel is used to simulate the extracellular matrix structure to provide a suitable static spatial mechanical stimulation for bone marrow mesenchymal stem cells. Further, a dynamic mechanical stimulation that generates periodic compressive deformation is applied to the cell-hydrogel complex.

[0024] The cells in the cell-hydrogel complex adhere to and fully stretch with the hydrogel during the cultivation stage. Their cytoskeleton structure can effectively sense the static spatial mechanical stimulation from the hydrogel. This mechanical stimulation activates the regulatory pathways related to cell differentiation, resulting in upregulation of the expression of early and late genes in the multi-directional differentiation of bone marrow mesenchymal stem cells. The static spatial mechanical stimulation promotes the multi-directional differentiation process of stem cells in the cell-hydrogel complex, and the experiment also confirms that the static spatial mechanical stimulation can also retain the differentiation ability of stem cells by inhibiting senescence.

[0025] Dynamic-static cooperative mechanical stimulation can upregulate the expression of genes related to bone regeneration and repair, induce osteogenic differentiation of cell-hydrogel composites, and secrete factors promoting lymphangiogenesis. The experimental results show that the static spatial mechanical stimulation from the hydrogel initiates the transcription of early expression genes related to bone regeneration, and the stem cells have entered the early stage of osteogenic differentiation. On this basis, applying appropriate dynamic mechanical stimulation, the stem cells will further differentiate into osteoblasts. Finally, the cell-hydrogel composites subjected to dynamic mechanical stimulation can promote bone regeneration. Transplanting the bone defect repair materials prepared by dynamic-static cooperative mechanical stimulation to the bone defect sites of the test animals, the newly formed bone tissue is significantly more than that of the bone defect repair materials obtained by only applying static spatial mechanical stimulation. Under the optimal dynamic mechanical stimulation conditions, the bone defect repair materials can recruit more lymphatic endothelial cells for lymphangiogenesis. Brief Description of the Drawings

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0027] Figure 1 Swelling of GelMA hydrogels with different mass-volume fractions in Example 1;

[0028] Figure 2 Degradation of GelMA hydrogels with different mass-volume fractions in Example 1;

[0029] Figure 3 Compressive modulus of GelMA hydrogels with different mass-volume fractions in Example 1 (* represents 0.01 < p < 0.05; ** represents 0.001 < p < 0.01; *** represents 0.0001 < p < 0.001)

[0030] Figure 4 Scanning electron microscope images of the microscopic morphology of GelMA hydrogels with different mass-volume fractions in Example 1, the scale bar is 200 μm;

[0031] Figure 5 Images of cell cytoskeleton (red fluorescence) / nucleus (blue fluorescence) staining of cells under static mechanical stimulation of GelMA hydrogels within 14 days in Example 2, the scale bar is 200 μm;

[0032] Figure 6Osteogenic, chondrogenic, and tenogenic gene expression profiles of two cell-hydrogel composites (3D group and Control group) in Example 2 at different time points of osteogenic induction (* represents 0.01 < p < 0.05, ** represents 0.001 < p < 0.01, *** represents 0.0001 < p < 0.001, **** represents p < 0.0001);

[0033] Figure 7 Cell senescence staining and quantitative analysis results of cells on a two-dimensional culture well plate (Plate group), cells on the surface of GelMA hydrogel (2D-coated group), and cells in GelMA hydrogel (3D group) in Example 2. The scale bar is 200 μm (ns represents no significant difference, *** represents 0.0001 < p < 0.001);

[0034] Figure 8 Cytoskeleton (red fluorescence) / nucleus (blue fluorescence) staining and live (green fluorescence) / dead (red fluorescence) of cells in 3D culture composites under different compression frequencies and deformation mechanical stimulation protocols in Example 3. The static culture group (Static) is the 3D culture composite that does not receive dynamic mechanical stimulation. The scale bar is 200 μm;

[0035] Figure 9 Cytoskeleton (red fluorescence) / nucleus (blue fluorescence) staining and live (green fluorescence) / dead (red fluorescence) of cells in 3D culture composites under different compression duration mechanical stimulation protocols in Example 3. The static culture group (Static) is the 3D culture composite that does not receive dynamic mechanical stimulation. The scale bar is 200 μm;

[0036] Figure 10 Osteogenic gene expression of cells in the cell-hydrogel composite under cyclic mechanical stimulation with different parameters in Example 3 (14-day cyclic mechanical stimulation) (ns represents no significant difference, * represents 0.01 < p < 0.05, ** represents 0.001 < p < 0.01, *** represents 0.0001 < p < 0.001, **** represents p < 0.0001);

[0037] Figure 11 Bubble matrix diagram of KEGG enrichment analysis of upregulated differential genes in the dynamic-static synergistic mechanical stimulation group (Mechanical) vs. the static mechanical stimulation culture group (Static) samples in Example 3;

[0038] Figure 12 Bubble matrix diagram of GO-BP enrichment analysis of upregulated differential genes in the dynamic-static synergistic mechanical stimulation group (Mechanical) vs. the static mechanical stimulation culture group (Static) samples in Example 3;

[0039] Figure 13 Transcriptome heatmap of the time trend of samples in the dynamic-static collaborative mechanical stimulation group (Mechanical) vs. the static mechanical stimulation culture group (Static);

[0040] Figure 14 To implement the performance of promoting bone-lymphatic vessel regeneration in vitro by dynamic mechanical stimulation in Example 4, the dynamic-static collaborative mechanical stimulation group (Mechanical) vs. the static mechanical stimulation culture group (Static), the scale bar is 200 μm, (ns represents no significant difference, * represents 0.01 < p < 0.05, ** represents 0.001 < p < 0.01, *** represents 0.0001 < p < 0.001, **** represents p < 0.0001);

[0041] Figure 15 H&E staining results of rat skulls in different implant groups in Example 4, the scale bar is 200 μm (Blank, no implant; GelMA, implanted pure hydrogel; Static, implanted with the complex of the static mechanical stimulation group; Mechanical, implanted with the bone organoid with self-promoting lymphatic vessel regeneration in the dynamic-static collaborative mechanical stimulation group);

[0042] Figure 16 Three-dimensional reconstruction images and quantitative analysis results of Micro-CT of rat skulls in different implant groups in Example 4 (Blank, no implant; GelMA, implanted pure hydrogel; Static, implanted with the complex of the static mechanical stimulation group; Mechanical, implanted with the bone organoid with self-promoting lymphatic vessel regeneration in the dynamic-static collaborative mechanical stimulation group; ns represents no significant difference, * represents 0.01 < p < 0.05, **** represents p < 0.0001);

[0043] Figure 17 IHC-LYVE1 staining and quantitative analysis results of rat skulls in different implant groups in Example 4, the scale bar is 200 μm (Blank, no implant; GelMA, implanted pure hydrogel; Static, implanted with the complex of the static mechanical stimulation group; Mechanical, implanted with the bone organoid with self-promoting lymphatic vessel regeneration in the dynamic-static collaborative mechanical stimulation group; ns represents no significant difference, * represents 0.01 < p < 0.05, ** represents 0.001 < p < 0.01, *** represents 0.0001 < p < 0.001, **** represents p < 0.0001). Detailed implementation manners

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0045] In a first aspect, a preparation method of a bone defect repair material is provided. The preparation method includes subjecting a cell-hydrogel complex to dynamic mechanical stimulation; the cell-hydrogel complex is a hydrogel embedded with bone marrow mesenchymal stem cells.

[0046] According to the action mode, the mechanical stimulation acting on the bone can be divided into two categories: static mechanical stimulation and dynamic mechanical stimulation.

[0047] The spatial mechanical stimulation of the extracellular matrix on cells is a kind of static mechanical stimulation. This kind of stimulation will cause changes in the internal and external structures of cells, regulate gene expression, protein synthesis, and signal pathway activity, etc., thereby affecting cell functions and physiological processes and inducing cell fate decisions. The stimulation exerted by the hydrogel on the cells in the cell-hydrogel complex of the present invention is static mechanical stimulation. Dynamic mechanical stimulation refers to applying periodic or intermittent pressure, stretching, or vibration to an object, regulating cell signal transduction and gene expression, and participating in many important physiological processes such as bone development, tissue repair, and immune response. Mechanical stimulation stimulates the aggregation and differentiation of bone marrow mesenchymal stem cells in the initial stage of fracture healing, promotes the formation of callus tissue in the repair stage, and promotes tissue reconstruction in the remodeling stage. However, the existing bone organ repair materials often ignore the role of mechanical stimulation during the construction process.

[0048] The present invention proposes a bone defect repair material constructed based on the synergistic dynamic and static mechanical stimulation. First, a hydrogel with appropriate stiffness, morphology, and adhesion sites is constructed to simulate the extracellular matrix structure, providing appropriate spatial mechanical stimulation for bone marrow mesenchymal stem cells to construct a multi-directional cell-hydrogel complex. Further, dynamic mechanical stimulation is applied to the complex, and through an appropriate dynamic mechanical stimulation scheme, the cell-hydrogel complex is induced to differentiate osteogenically and secrete lymphangiogenesis-promoting factors to construct a new type of bone defect repair material, accelerating the regeneration and repair of bone defects by synergistically promoting the reconstruction of lymphatic vessels around the bone defect repair material.

[0049] The dynamic mechanical stimulation includes subjecting the cell-hydrogel composite to periodic compressive deformation, where the amount of compressive deformation does not exceed 10% of its own volume, and can be, for example, but not limited to, not exceeding 5%, 6%, 7%, 8%, 9%, and 10% of its own volume; the compression frequency does not exceed 1 Hz, and can be, for example, but not limited to, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, and 1 Hz; the time for each exposure to the dynamic mechanical stimulation does not exceed 1 h, and can be, for example, but not limited to, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, and 1 h.

[0050] In an alternative embodiment, the dynamic mechanical stimulation includes subjecting the cell-hydrogel composite to periodic compressive deformation, with the amount of compressive deformation being 5% of its own volume, the compression frequency being 0.5 Hz, and the time for each exposure to the dynamic mechanical stimulation being 0.5 h.

[0051] The time interval between two adjacent exposures to the dynamic mechanical stimulation is 18 - 30 h, and can be, for example, but not limited to, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, and 30 h. During the entire preparation process, the time intervals between two adjacent exposures to the dynamic mechanical stimulation can be the same or different. For example, the time interval between two adjacent exposures to the dynamic mechanical stimulation is 24 h each time, or the first time interval is 18 h, the second is 24 h, the third is 18 h, etc.

[0052] In an alternative embodiment, the time interval between two adjacent exposures to the dynamic mechanical stimulation is 24 h.

[0053] In an alternative embodiment, the time interval for each exposure to the dynamic mechanical stimulation is 24 h.

[0054] In an alternative embodiment, the preparation method includes subjecting the cell-hydrogel composite to the dynamic mechanical stimulation 3 - 14 times, and can be, for example, but not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times.

[0055] In an alternative embodiment, the preparation method includes subjecting the cell-hydrogel composite to the dynamic mechanical stimulation 3 times.

[0056] The structure of bone also includes the periosteum, bone substance, articular cartilage, bone marrow, blood vessels, nerves, etc. Existing bone defect repair research focuses on the regeneration of the above structures. However, the latest research shows that lymphatic vessels are an important component of bones. Lymphocytes secrete chemokine CXCL12 to communicate dynamically with hematopoietic stem cells and Myh11+CXCR4+ pericytes, promoting bone mass regeneration at the injury site and accelerating bone healing. Existing bone organoid construction methods mostly represent one function of bone, such as bone formation, bone resorption, or hematopoiesis, often neglecting the role of this important organizational structure of lymphatic vessels in the process of bone defect repair.

[0057] Experiments found that dynamic mechanical stimulation can upregulate the expression of related genes throughout the preparation process. However, since lymphatic vessel regeneration is an early regeneration event during the defect repair process, as the number of dynamic mechanical stimulations increases, the intensity of lymphangiogenic-related activities in the dynamic-static collaborative mechanical stimulation group gradually weakens. Experiments show that 3 times of dynamic mechanical stimulation is beneficial for the further osteogenic differentiation of stem cells and lymphangiogenic-related activities.

[0058] In an alternative embodiment, starting from culturing the cell-hydrogel composite with a growth medium for 18 - 36 h, for example, it can be but not limited to 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, and 30 h, the cell-hydrogel composite begins to receive the dynamic mechanical stimulation.

[0059] In an alternative embodiment, starting from culturing the cell-hydrogel composite with a growth medium for 24 h, the cell-hydrogel composite begins to receive the dynamic mechanical stimulation.

[0060] In an alternative embodiment, the density of mesenchymal stem cells in the cell-hydrogel composite is 10 5 ~10 8 / cm 3 。

[0061] In an alternative embodiment, the sources of mesenchymal stem cells in the cell-hydrogel composite include but are not limited to one or more of mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys, and humans.

[0062] In an alternative embodiment, the cell-hydrogel composite is cultured with a growth medium, and the growth medium can be a medium known in the art and optionally capable of culturing mesenchymal stem cells, such as αMEM medium or IMDM medium.

[0063] In an alternative embodiment, the hydrogel comprises a hydrogel containing proteins and / or polysaccharides. The proteins include, but are not limited to, one or more of gelatin, collagen, and fibroin. The polysaccharides include, but are not limited to, one or more of hyaluronic acid, sodium alginate, chitosan, chondroitin sulfate, and heparin.

[0064] In an alternative embodiment, the proteins and / or polysaccharides are methacrylated.

[0065] In an alternative embodiment, the hydrogel is methacrylated gelatin.

[0066] In an alternative embodiment, the mass-volume fraction of methacrylated gelatin in the hydrogel is 10%.

[0067] In an alternative embodiment, the preparation method includes: mixing bone marrow mesenchymal stem cells, an initiator, and a methacrylated gelatin solution, where the mass-volume fraction of methacrylated gelatin is 10%, and the density of bone marrow mesenchymal stem cells is 10 5 ~10 8 / cm 3 , and obtaining the cell-hydrogel composite by photocrosslinking. The cell-hydrogel composite is placed in a growth medium for culture. After 24 hours of culture, the cell-hydrogel composite is subjected to dynamic mechanical stimulation 3 to 14 times. The dynamic mechanical stimulation includes causing the cell-hydrogel composite to undergo periodic compressive deformation, with a compressive deformation amount of 5% of its own volume, a compression frequency of 0.5 Hz, and each time of receiving the dynamic mechanical stimulation for 0.5 hours, and the time interval between adjacent times of receiving the dynamic mechanical stimulation is 24 hours.

[0068] In a second aspect, there is also provided a bone defect repair material prepared by the preparation method of the first aspect.

[0069] In a third aspect, there is also provided the application of the preparation method of the first aspect, or the bone defect repair material of the second aspect, in the preparation of bone organoids.

[0070] In a fourth aspect, there is also provided a bone organoid, which contains the bone defect repair material of the first aspect.

[0071] In an alternative embodiment, the bone organoid further contains drugs known in the art and optionally used for injury repair, including but not limited to one or more of cytokines, small molecule compound drugs, and polynucleotides.

[0072] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0073] Example 1

[0074] Screen for a GelMA (methacrylated gelatin) hydrogel with an appropriate mass volume fraction for constructing a cell-hydrogel composite.

[0075] Measure and calculate the swelling rate and degradation rate of GelMA hydrogels with different mass volume fractions by the weighing method; use a universal testing machine to detect the compressive modulus of GelMA hydrogels with different mass volume fractions; observe the microscopic morphology of GelMA hydrogels with different mass volume fractions by scanning microscopy.

[0076] I. Experimental methods:

[0077] 1. Sample preparation:

[0078] Prepare GelMA hydrogel samples with mass volume fractions of 7.5% (w / v), 10.0% (w / v), and 12.5% (w / v) respectively. The sample diameter is 10 mm and the height is 2 mm.

[0079] (1) Prepare a 0.25% (w / v) initiator standard solution: Take 20 mL of PBS and add it to a brown bottle containing 0.05 g of LAP initiator; heat and dissolve it in a water bath at 40 - 50 °C for 15 minutes, shaking several times during this period;

[0080] (2) Prepare a GelMA solution: Take the required mass of GelMA and put it into a centrifuge tube; add the initiator standard solution to the above centrifuge tube and shake to fully wet the GelMA; heat and dissolve it in a water bath at 60 - 70 °C in the dark for 20 - 30 minutes, shaking several times during this period; immediately sterilize the GelMA solution with a 0.22 μm sterile needle filter, and pay attention to keeping it in the dark and warm during this process (to prevent low-temperature gelation);

[0081] (3) Inject the GelMA solution into a polytetrafluoroethylene mold with a diameter of 10 mm and a depth of 2 mm, and irradiate it with a 405 nm light source for 20 seconds to gelate it. Finally, prepare a cell-hydrogel composite with a diameter of 10 mm and a height of 2 mm.

[0082] 2. Swelling property:

[0083] Evaluate the swelling performance of GelMA hydrogel samples with different mass volume fractions by the weighing method. After vacuum freeze-drying the GelMA hydrogel samples with a diameter of 10 mm and a height of 2 mm for 24 hours, record the final dry mass (W f ) and then put them into a well plate. Add 2 mL of sterile PBS to the wells, soak the samples at 37 °C, and remove all the liquid at 0, 1, 2, 4, 6, 12, and 24 h and remove the excess liquid on the samples with absorbent paper. Then weigh the samples to obtain the swollen mass (W s ). The swelling rate is defined as: Swelling Ratio = (W s-W f ) / W f ×100%, weigh the mass with an analytical balance with an accuracy of 0.1 mg. Take 4 samples for each group for statistical analysis.

[0084] 3. Degradability:

[0085] Using the principle of accelerating the degradation of GelMA hydrogel by collagenase, the degradation performance of GelMA hydrogel samples with different mass-volume fractions was tested in vitro. Weigh the GelMA hydrogel sample with a diameter of 10 mm and a height of 2 mm, and record it as (W 0 ). Put the sample into a well plate, add 2 mL of sterile PBS solution with a collagenase concentration of 2 U / mL into the well, soak the sample at 37 °C, remove all the liquid at 0, 1, 2, 4, 6, 12 h, and remove the excess liquid of the sample with absorbent paper. Then weigh the sample and record the sample weight Wn (the sample weight at 0 h is W 0 , and the sample weight at the sequential time points is W n ). The degradation rate is defined as: Degradation Ratio = (W 0 -W n ) / W 0 ×100%, weigh the mass with an analytical balance with an accuracy of 0.1 mg. Take 4 samples for each group for statistical analysis.

[0086] 4. Compressive modulus:

[0087] Place the cylindrical photocured GelMA hydrogel sample with a diameter of 10 mm and a height of 2 mm on the platform of an electronic universal testing machine, and perform a compression test at a loading rate of 0.2 mm / min until the specimen ruptures. The temperature is kept at 25 °C during the test, and the compressive modulus is obtained by calculating the slope of the linear region of the 10% stress-strain curve of the sample. Take 4 samples for each group for statistical analysis.

[0088] 5. Scanning electron microscope:

[0089] After freeze-drying different mass-volume fraction GelMA hydrogel samples using a vacuum freeze dryer, stick the dried samples on a metal sample stage with a conductive adhesive or other adhesives, then place them in a vacuum evaporator to sputter a metal film on the surface of the samples, and then observe the microscopic surface morphology of the hydrogel with a scanning electron microscope and take images.

[0090] II. Experimental results:

[0091] Methacrylated collagen (GelMA) constructs a hydrogel by photocrosslinking to simulate the extracellular matrix. The physicochemical properties of GelMA hydrogel vary with the change of GelMA mass-volume fraction. As Figure 1As shown, the 12.5% (w / v) hydrogel is not conducive to the exchange of nutrients in the hydrogel due to its low swelling property, and the 7.5% (w / v) hydrogel is not suitable for the construction of cell-hydrogel complexes because of its relatively fast degradation rate and poor elastic deformation ability. The 10.0% (w / v) GelMA hydrogel is suitable for the subsequent construction of cell-hydrogel complexes due to its good swelling property ( Figure 1 ), degradation rate ( Figure 2 ), appropriate compressive modulus ( Figure 3 ), and suitable microporous structure ( Figure 4 ).

[0092] Example 2

[0093] Construction and characterization of cell-hydrogel complexes.

[0094] Based on the static spatial mechanical stimulation of the extracellular matrix mimic, a cell-hydrogel complex with multi-directional differentiation potential was constructed. The hydrogel was added to the cells that had been centrifuged and the supernatant removed to prepare a cell-hydrogel suspension, and the cell-hydrogel complex was prepared by photocrosslinking using a mold. The in vitro osteogenic performance of the cell-hydrogel complex was characterized by ALP (alkaline phosphatase) staining and AR (alizarin red) staining; the in vitro osteogenic, chondrogenic, and tenogenic performances of the cell-hydrogel complex were characterized by qRT-PCR technology.

[0095] I. Experimental methods:

[0096] 1. Cell culture:

[0097] Primary rat bone marrow mesenchymal stem cells (rBMSC) were purchased from the Stem Cell Bank of the Chinese Academy of Sciences. These cells were cultured in a growth medium of α-MEM containing 10% fetal bovine serum and a 1% mixture of penicillin and streptomycin, and the culture environment was a 37°C incubator containing 5% CO 2 2.

[0098] 2. The detailed steps for the preparation and culture of the cell-hydrogel complex are as follows:

[0099] (1) Prepare a 0.25% (w / v) initiator standard solution: Take 20 mL of PBS and add it to a brown bottle containing 0.05 g of LAP initiator; heat it in a water bath at 40 - 50°C for 15 minutes to dissolve, shaking several times during this period;

[0100] (2) Preparation of GelMA solution: Weigh the required amount of GelMA and place it in a centrifuge tube. Add the initiator standard solution to the above centrifuge tube and shake it to fully soak the GelMA. Heat it in a water bath at 60 - 70 °C in the dark for 20 - 30 minutes, shaking it several times during this period. Immediately sterilize the GelMA solution with a 0.22 μm sterile needle filter, and pay attention to keeping it in the dark and warm during this process (to prevent low-temperature gelation);

[0101] (3) After digesting, collecting, and centrifuging the bone marrow mesenchymal stem cells in the culture flask to discard all the supernatant, resuspend them with the GelMA solution preheated to 37 °C to prepare a GelMA suspension containing cells, with a cell density of 10 5 ~10 8 / cm 3 ;

[0102] (4) Inject the GelMA suspension containing cells into a polytetrafluoroethylene mold with a diameter of 10 mm and a depth of 2 mm, and irradiate it with a 405 nm light source for 20 seconds to gelate it, and finally prepare a cell-hydrogel composite with a diameter of 10 mm and a height of 2 mm;

[0103] (5) Take out the cell-hydrogel composite and place it in a culture well plate. Add culture medium to each well, incubate it in a 37 °C incubator for 5 minutes, remove the culture medium, wash the sample, and then add fresh culture medium and culture it for a long time, replacing the fresh culture medium every 2 - 3 days.

[0104] 3. The detailed steps for extracting cells from the cell-hydrogel composite are as follows:

[0105] (1) Prepare a GelMA lysate with a concentration of 0.3 mg / mL using complete cell culture medium;

[0106] (2) Transfer the cell-hydrogel composite to a low-adhesion culture well plate and use a sterile pipette tip to fully break it. The smaller the gel block, the faster the lysis speed;

[0107] (3) Add 1 - 2 mL of GelMA lysate with a concentration of 0.3 mg / mL to each well;

[0108] (4) Place it in a 37 °C incubator for aseptic lysis, and observe the lysis situation under a microscope every 15 minutes;

[0109] (5) After sufficient lysis, centrifuge it at 1000 rpm for 5 minutes, discard the supernatant, add 5 mL of complete culture medium, repeat the washing and centrifugation once, and the obtained cells can be further cultured or used for detection and analysis such as protein and nucleic acid extraction.

[0110] 4. Construction of a cell-hydrogel composite with multi-directional differentiation potential based on static spatial mechanical stimulation mimicking the extracellular matrix

[0111] Preparation methods of the Control group and the 3D group: Collect a sufficient number of bone marrow mesenchymal stem cells and divide them equally into two parts, A and B. The cells in part A are seeded in a two-dimensional culture well plate at an appropriate density, and the cells in part B are seeded in GelMA hydrogel at an appropriate density according to the method in the second part of this example for three-dimensional culture. Both parts of the cells are incubated in a growth medium in an incubator (37 °C, 5% CO 2 ) for 14 days, and the fresh medium is changed every 2 - 3 days. After 14 days, the cells A in the two-dimensional culture well plate are collected and seeded in GelMA hydrogel (10.0% (w / v)) according to the method in the second part of this example, and the resulting complex is the Control group. At this time, the cell-hydrogel complex after 14 days of three-dimensional culture is the 3D group. Then, both cell-hydrogel complexes made from the two parts of the cells are incubated in an osteogenic induction medium in an incubator (37 °C, 5% CO 2 ) for 14 days, and the fresh medium is changed every 2 - 3 days. The osteogenic induction medium is prepared by adding 10 mmol / L β-glycerophosphate, 10-7 mol / L dexamethasone, and 50 μg / L vitamin C to the growth medium.

[0112] Plate group: Collect a sufficient number of bone marrow mesenchymal stem cells and seed them in a two-dimensional culture well plate at an appropriate density. Incubate them in a growth medium in an incubator (37 °C, 5% CO 2 ) for 14 days, and the fresh medium is changed every 2 - 3 days.

[0113] 2D-coated group: Collect a sufficient number of bone marrow mesenchymal stem cells and seed them on the surface of GelMA hydrogel (10.0% (w / v)) (see Example 1 for the preparation method of GelMA hydrogel) at an appropriate density. Incubate them in a growth medium in an incubator (37 °C, 5% CO 2 ) for 14 days, and the fresh medium is changed every 2 - 3 days.

[0114] II. Experimental results:

[0115] Rat bone marrow mesenchymal stem cells (rBMSCs) were implanted in 10.0% (w / v) GelMA hydrogel to construct cell-hydrogel composites. After the cells spread and adhered in the hydrogel, their cytoskeletons would rearrange under the influence of the hydrogel's spatial structure. The cytoskeleton is a fibrous network composed of the nuclear skeleton, cytoplasmic skeleton, cell membrane skeleton, cross-linking factors, and extracellular matrix, providing the framework for the basic cell morphology and connecting all mechanosensitive components. The assembly of the cytoskeleton requires a certain space. If the cell spreading space is restricted, even if the microenvironment can provide sufficient balance force, the cytoskeleton cannot be assembled, and the intracellular traction force is inhibited. Therefore, a fully extended and structurally intact cytoskeleton is the basis for cells to efficiently sense spatial mechanical stimuli. Cells have contact inhibition during growth, which refers to the avoidance behavior and growth arrest phenomenon shown when cells come into contact with each other. In three-dimensional space, when the cytoskeletons between cells extend to contact each other, it means that the cytoskeletons of individual cells are fully extended. The cytoskeleton / nucleus staining results are as Figure 5 shown. As the growth culture time increased, the cytoskeleton area of individual cells gradually increased. When cultured until day 14, the cytoskeletons between cells were in full contact, indicating that the cytoskeletons of individual cells were fully extended at this time and could efficiently sense the static spatial mechanical stimuli from the microporous structure of the hydrogel.

[0116] Cells were seeded into GelMA hydrogel and cultured for 14 days to construct a 3D group that could fully sense the static spatial mechanical stimuli of the hydrogel; the composite obtained without culturing the cells for 14 days before seeding them into GelMA hydrogel was the Control group. Since the skeletons of the cells in the Control group could not be fully extended and adhered, they could not well sense the static spatial mechanical stimuli brought by the microporous structure of the hydrogel. The above two Control group and 3D group cell-hydrogel composites were cultured in osteogenic induction medium for 14 days. At days 3, 7, and 14 of osteogenic induction, the RNA of the cells in the cell-hydrogel was extracted for gene expression analysis. Here, osteogenesis-related genes (OSTERIX, OCN), chondrogenesis-related genes (ACAN, COL2), and tendonogenesis-related genes (TNMD, SCX) were selected for research.

[0117] The experimental results are as Figure 6As shown, for the genes OSTERIX, ACAN, and TNMD that are early expressed in osteogenesis, chondrogenesis, and tenogenesis, the cells in the cell-hydrogel composites in the 3D group showed higher expression levels on the third day than those in the cell-hydrogel composites in the Control group. As the osteogenic induction time increased, the expression levels of the genes early expressed in osteogenesis, chondrogenesis, and tenogenesis in the cells of the cell-hydrogel composites in the 3D group all showed a downward trend, while the expression levels of the genes early expressed in osteogenesis, chondrogenesis, and tenogenesis in the cells of the cell-hydrogel composites in the Control group all showed an upward trend. For the genes OCN, COL2, and SCX that are late expressed in osteogenesis, chondrogenesis, and tenogenesis, throughout the 14 days of osteogenic induction, the cells in the cell-hydrogel composites in the 3D group all showed higher expression levels than those in the cell-hydrogel composites in the Control group. As the osteogenic induction time increased, the expression levels of the genes late expressed in each group gradually increased. This is because the cells in the cell-hydrogel composites in the 3D group adhered to the hydrogel and fully extended during the growth medium culture stage, and their cytoskeleton structures effectively sensed the static spatial mechanical stimuli from the hydrogel. This mechanical stimulus activated the regulatory pathways related to cell differentiation, resulting in the gradual upregulation of the genes early expressed in cell differentiation in all directions, that is, the static spatial mechanical stimulus promoted the multi-directional differentiation process of stem cells in the cell-hydrogel composites.

[0118] Stem cell senescence is an important indicator affecting the multi-directional differentiation ability of stem cells. During the process of cell senescence, the activity of β-Gal increases significantly. Under the action of β-Gal, X-Gal is hydrolyzed to produce a blue product. To prove that static spatial mechanical stimuli retain the differentiation ability of stem cells by inhibiting senescence, in this example, mesenchymal stem cells were respectively seeded on two-dimensional culture well plates (Plate group), on the surface of GelMA hydrogels (2D-coated group), and in GelMA hydrogels (3D group), and cell senescence staining was performed after culturing in growth medium for 14 days. The experimental results are as Figure 7 shown. There was no significant difference in the cell senescence rate between the Plate group and the 2D-coated group, while the cell senescence rate in the 3D group was significantly lower than that in the Plate group and the 2D-coated group, indicating that spatial mechanical stimuli rather than the components of the hydrogel itself can maintain the multi-directional differentiation potential of stem cells by inhibiting stem cell senescence.

[0119] Example 3 Construction of Bone Organoids with Self-Promoting Lymphangiogenesis Function Based on Dynamic-Static Synergistic Mechanical Stimuli

[0120] Based on the osteogenic differentiation of cell-hydrogel composites induced by dynamic-static collaborative mechanical stimulation and the secretion of lymphangiogenesis-promoting factors, bone organoids with self-promoting lymphangiogenesis function were constructed. Cell viability / death staining, cytoskeleton / nucleus staining, qRT-PCR technology, and RNA-seq were used to evaluate the dynamic compression mechanical protocol composed of compression deformation, compression frequency, daily compression duration, and compression days. ALP (alkaline phosphatase) staining, AR (alizarin red) staining, and qRT-PCR technology were used to characterize the in vitro osteogenic performance of cells in the dynamic-static collaborative mechanical stimulation group and the static spatial mechanical stimulation group; the culture supernatants of the two groups were collected as conditioned media to verify the promotion of lymphangiogenesis in vitro by dynamic-static collaborative mechanical stimulation; methods such as Micro-CT and IHC staining were used to verify the promotion of bone-lymphangiogenesis in vivo in the samples of the dynamic-static collaborative mechanical stimulation group.

[0121] I. Experimental methods:

[0122] 1. Different dynamic compression mechanical stimulations

[0123] The key parameters (compression frequency Frequency, compression deformation Deformation, daily compression duration Time, and compression days) were studied, and cell viability / death staining experiments, cytoskeleton / nucleus staining experiments, qRT-PCR experiments, and RNA-seq were used to observe the effects of dynamic-static collaborative mechanical stimulation on cell survival, spreading adhesion, osteogenic differentiation, and lymphangiogenesis.

[0124] The cell-hydrogel composites in this example were prepared according to the following method:

[0125] Bone marrow mesenchymal stem cells were collected and seeded in GelMA hydrogel for three-dimensional culture at a density of 10 5 ~10 8 / cm 3 according to the method in the second part of this example. They were incubated in a growth culture medium in an incubator (37 °C, 5% CO 2 2), and the medium was replaced with fresh medium every 2 - 3 days. During the culture period, dynamic stimulation was carried out according to the protocols in Tables 1 - 4 below. The compression days indicate the number of consecutive days of dynamic mechanical stimulation starting from 24 h after culture and at the same time every day. The dynamic mechanical stimulation device was a BioDynamic 5200 three-dimensional tissue culture and testing system.

[0126] 1.1 Implementation plan of mechanical stimulation with different compression frequencies and deformations:

[0127] Table 1

[0128]

[0129]

[0130] 1.2 Implementation scheme of mechanical stimulation schemes with different compression durations:

[0131] Table 2

[0132] Group Compression deformation amount Compression frequency (Hz) Daily compression duration (h) Compression days (days) 1 5％ 0.1 0.5 14 2 5％ 0.5 0.5 14 3 10％ 0.1 0.5 14 4 10％ 0.5 0.5 14 5 5％ 0.1 1 14 6 5％ 0.5 1 14 7 10％ 0.1 1 14 8 10％ 0.5 1 14

[0133] 1.3 Example of mechanical stimulation scheme for osteogenic performance

[0134] Table 3

[0135] Group Compression deformation amount Compression frequency (Hz) Daily compression duration (h) Compression days (days) 1 5％ 0.1 0.5 14 2 5％ 0.5 0.5 14 3 10％ 0.1 0.5 14 4 10％ 0.5 0.5 14

[0136] 1.4 Example of mechanical stimulation scheme with different compression days:

[0137] Table 4

[0138] Group Compression deformation amount Compression frequency (Hz) Daily compression duration (h) Compression days (days) 1 5％ 0.5 0.5 3 2 5％ 0.5 0.5 7 3 5％ 0.5 0.5 14

[0139] II. Experimental results:

[0140] The 3D culture complex under continuous static spatial mechanical stimulation was subjected to dynamic compression mechanical stimulation using a three-dimensional tissue culture and testing system. An osteo-organoid with self-promoted lymphatic vessel regeneration was constructed through dynamic-static collaborative mechanical stimulation. First, the 3D culture complex was subjected to dynamic compression mechanical stimulation for 3 days. It was found that a 20% compression deformation, a compression frequency of 1 Hz, and a compression of 0.5 h per day for 3 consecutive days had a significant inhibitory effect on cell survival ( Figure 8 ).

[0141] Furthermore, the 3D culture complex was subjected to dynamic mechanical stimulation for 14 days. It was found that a compression duration of 1 h per day had a significant inhibitory effect on the extension of the cytoskeleton, as shown in Figure 9 .

[0142] The 3D culture complex was subjected to dynamic mechanical stimulation for 14 days. It was found that a stimulation scheme of 5% compression deformation, 0.5 Hz compression frequency, 0.5 h compression per day, and continuous compression for 14 days (5%-0.5 Hz-0.5 h-14 days) was beneficial to the expression of osteogenesis-related genes COL-I, BSP, TGF-β, ALP, OCN, and BMP-2 ( Figure 10 ), that is, 5%-0.5 Hz-0.5 h might induce the complex to differentiate in the osteogenic direction.

[0143] Under the stimulation protocol of 5% - 0.5 Hz - 0.5 h, the 3D culture complexes were subjected to dynamic mechanical stimulation for different days. At 3 days, 7 days, and 14 days, rBMSCs in the dynamic-static synergistic mechanical stimulation group (Mechanical, 3D culture complexes receiving dynamic mechanical stimulation) and the static mechanical stimulation culture group (Static, 3D culture complexes not receiving mechanical stimulation) were extracted for RNA-seq. KEGG enrichment analysis and GO-BP enrichment analysis were performed on the up-regulated differentially expressed genes at the above three time points. Then, according to the enrichment results of the differentially expressed genes at each time point, a KEGG enrichment analysis bubble matrix diagram ( Figure 11 ) and a GO enrichment analysis bubble matrix diagram ( Figure 12 ) were made respectively. In the KEGG enrichment analysis, the up-regulated differentially expressed genes in the dynamic-static synergistic mechanical stimulation group VS the static mechanical stimulation culture group samples at each time point were mainly enriched in pathways such as protein digestion and absorption, extracellular matrix receptor interaction, etc. In the GO-BP enrichment analysis, the up-regulated differentially expressed genes in the dynamic-static synergistic mechanical stimulation group VS the static mechanical stimulation culture group samples at each time point were mainly enriched in aspects such as bone regeneration and repair. More importantly, in the third day, the up-regulated differentially expressed genes in the dynamic-static synergistic mechanical stimulation group VS the static mechanical stimulation culture group samples were also enriched in the key activity of lymphatic vessel development.

[0144] The up-regulated differences at each time point in the above two parts of bone regeneration and repair and lymphatic vessel regeneration and repair were summarized and analyzed, and a time trend transcriptome heat map ( Figure 13 ) was made. The results showed that at each time point in the dynamic-static synergistic mechanical stimulation group, the osteogenesis-related activities of the cells in the dynamic-static synergistic mechanical stimulation group were more active than those in the static mechanical stimulation culture group samples, and the difference increased with the prolongation of the dynamic-static synergistic mechanical stimulation time. This was because the dynamic-static synergistic mechanical stimulation promoted osteogenic differentiation, and as the synergistic mechanical stimulation time became longer, the expression difference of osteogenesis-related genes between the dynamic-static synergistic mechanical stimulation group and the static mechanical stimulation culture group gradually became larger.

[0145] For the related activities of lymphangiogenesis, dynamic mechanical stimulation could up-regulate the expression of related genes throughout the experimental stage. However, since lymphatic vessel regeneration is an early regeneration event during the defect repair process, the intensity of the related activities of lymphangiogenesis in the dynamic-static synergistic mechanical stimulation group gradually weakened with the prolongation of the dynamic mechanical stimulation time.

[0146] In summary, applying a 3-day dynamic mechanical stimulation to the 3D-cultured complex with the stimulation protocol of 5% - 0.5 Hz - 0.5 h is beneficial for the further osteogenic differentiation of stem cells and the related activities of lymphatic vessel generation. Therefore, the 5% - 0.5 Hz - 0.5 h - 3-day dynamic compression mechanical stimulation protocol is used to construct bone organoids with the function of promoting lymphatic vessel regeneration by itself.

[0147] Example 4

[0148] In this example, the 3D-cultured cell-hydrogel composite was subjected to dynamic mechanical stimulation under the optimal parameter conditions of Example 3 (i.e., the sample was compressed for 3 days under the conditions of a compressive strain of 5%, a compression frequency of 0.5 Hz, and a daily compression duration of 0.5 h), and the following effect verification was carried out on the product:

[0149] I. The 3D-cultured cell-hydrogel composite was subjected to dynamic mechanical stimulation under the optimal parameter conditions of Example 3 (i.e., the sample was compressed for 3 days under the conditions of a compressive strain of 5%, a compression frequency of 0.5 Hz, and a daily compression duration of 0.5 h). Figure 14 (A) The results of ALP staining and AR staining showed that dynamic mechanical stimulation was beneficial to the osteogenic differentiation of stem cells. The qRT-PCR technique was used to characterize the expression of osteogenesis-related genes in the cells of the cell-hydrogel composite in the dynamic-static synergistic mechanical stimulation group and the static mechanical stimulation culture group. The results were as Figure 14 (B) shown. Dynamic mechanical stimulation promoted the expression of osteogenesis-related intermediate (ALP) and late (OCN) genes, indicating that the static spatial mechanical stimulation from the hydrogel initiated the transcription of early expression genes related to bone regeneration, and the stem cells had entered the early stage of osteogenic differentiation. On this basis, appropriate dynamic mechanical stimulation was applied, and the stem cells would further differentiate into osteoblasts. Finally, the cell-hydrogel composite subjected to dynamic mechanical stimulation could promote bone regeneration. The culture supernatants of the cell-hydrogel composites in the dynamic-static synergistic mechanical stimulation group and the static mechanical stimulation culture group were used as conditioned media for the lymphangiogenesis experiment. The results were as Figure 14 (C) and 14(D) shown. Dynamic mechanical stimulation was beneficial to the expression and secretion of lymphangiogenesis-promoting factors and induced the generation of lymphatic vessels by lymphatic endothelial cells.

[0150] II. The bone organoids with self-promoting lymphatic vessel regeneration function constructed by the above dynamic-static synergistic stimulation and the cell-hydrogel composite samples cultured by static mechanical stimulation were respectively implanted into the skull defect sites of rats, and the bone repair and lymphatic vessel regeneration effects of the two groups of samples at the in-vivo defect sites were observed. The results of H&E staining ( Figure 15 ) showed that in the early stage of implantation (2 weeks), there was no obvious difference in the newly formed bone tissue at the skull defect sites between the dynamic-static synergistic mechanical stimulation group (Mechanical) and the static mechanical stimulation culture group (Static). Until the late stage of implantation (6 weeks), the newly formed bone tissue at the bone defect sites in the dynamic-static synergistic mechanical stimulation group was significantly more than that in the static mechanical stimulation culture group. This result was in the Micro-CT detection and quantitative analysis results ( Figure 16) It was also confirmed in []. The results of Micro-CT examination showed that the blue part was the newly formed bone tissue (A). The quantitative analysis results of the newly formed bone in the bone defect area (B) indicated that the newly formed bone tissue in the bone defect area of the dynamic-static synergistic mechanical stimulation group was significantly more than that of the static culture group.

[0151] III. The lymphatic endothelial cell surface marker LYVE1 was selected as the target protein for IHC staining. The results showed ( Figure 17 ) that in the early stage of implantation (2 weeks), more lymphatic endothelial cells could be recruited around the samples of the dynamic-static synergistic mechanical stimulation group for lymphatic vessel regeneration. Until the late stage of implantation (6 weeks), there were more circular positive staining areas in the newly formed bone at the bone defect site of the dynamic-static synergistic mechanical stimulation group. This indicated that the implants in the dynamic mechanical stimulation group effectively accelerated lymphatic vessel regeneration. During the entire bone defect repair process, similar to angiogenesis, the lymphatic vessels at this site also occurred in the early stage. The vascular system that reconstructed the complete structure at the bone defect site and could conduct effective material transport and exchange played an important role in bone defect repair. In contrast, the ability of the implants in the static mechanical stimulation culture group to recruit lymphatic endothelial cells was relatively lagged, and lymphatic endothelial cells were not recruited until the late stage of implantation, delaying lymphatic vessel regeneration and also affecting bone defect repair.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a bone defect repair material, characterized in that: The method comprises subjecting a cell-hydrogel complex to dynamic mechanical stimulation; the cell-hydrogel complex is a hydrogel in which bone marrow mesenchymal stem cells are embedded; the hydrogel is methacrylated gelatin; The dynamic mechanical stimulation includes causing the cell-hydrogel complex to undergo periodic compression deformation, with the compression deformation amount being 5% of its own volume, the compression frequency being 0.5 Hz, the dynamic mechanical stimulation time being 0.5 h each time, and the time interval between two adjacent dynamic mechanical stimulations being 24 h; the cell-hydrogel complex is subjected to the dynamic mechanical stimulation 3 times.

2. The preparation method according to claim 1, characterized in that: The density of bone marrow mesenchymal stem cells in the cell-hydrogel complex is 10 5 ~10 8 / cm 3 .

3. The preparation method according to claim 1, characterized in that: The cell-hydrogel complex was cultured using growth medium.

4. The preparation method according to claim 3, characterized in that: After the cell-hydrogel complex is cultured in the growth medium for 18 to 36 hours, the cell-hydrogel complex begins to receive the dynamic mechanical stimulation.

5. The preparation method according to claim 4, characterized in that: After the cell-hydrogel complex was cultured with the growth medium for 24 hours, the cell-hydrogel complex began to receive the dynamic mechanical stimulation.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The mass volume fraction of methacryloyl gelatin in the hydrogel is 10%.

7. The preparation method according to claim 6, characterized in that: include: Bone marrow mesenchymal stem cells, initiators and methacrylated gelatin solution were mixed, the mass volume fraction of methacrylated gelatin was 10%, and the density of bone marrow mesenchymal stem cells was 10. 5 ~10 8 / cm 3 , obtaining the cell-hydrogel complex through photocrosslinking; The cell-hydrogel complex is cultured in a growth medium. After 24 hours of culture, the cell-hydrogel complex is subjected to dynamic mechanical stimulation three times. The dynamic mechanical stimulation includes subjecting the cell-hydrogel complex to periodic compression deformation, with the compression deformation amount being 5% of its own volume and the compression frequency being 0.5 Hz. Each dynamic mechanical stimulation lasts for 0.5 hours, and the time interval between two adjacent dynamic mechanical stimulations is 24 hours.

8. The bone defect repair material obtained according to the preparation method according to any one of claims 1 to 7.

9. Use of the preparation method according to any one of claims 1 to 7, or the bone defect repair material according to claim 8 in the preparation of bone organoids.

10. A bone organoid, characterized in that Contains the bone defect repair material according to claim 8.

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