Bionic matrix hydrogel for constructing bone organs and preparation method thereof

Abstract

The invention relates to bionic matrix hydrogel for constructing bone organs and a preparation method of the bionic matrix hydrogel. The bionic matrix hydrogel is prepared from a bone acellular extracellular matrix, a salmon DNA (Deoxyribose Nucleic Acid) network and a calcium phosphate oligomer under the combination of photo-crosslinking and self-assembly physical crosslinking. The acellular extracellular matrix in the bionic matrix hydrogel can simulate organic components in natural bones, the calcium phosphate oligomer can simulate inorganic components in the natural bones, and sufficient phosphate ions can be provided for biomineralization due to introduction of a DNA network. The biomimetic matrix hydrogel can replace Matrigel to act in bone organoid culture, provide a living environment similar to bone tissue for three-dimensional culture cells, enhance substance exchange and signal transduction between cells, promote proliferation, adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells, and further accelerate the process of biomineralization. The invention provides an optional new material for preparing the functional bone organ.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials and organoids, and in particular to a biomimetic matrix hydrogel for constructing bone organoids and a preparation method thereof. Background Art

[0002] Bone tissue, as a dynamic load-bearing structure of the organism, regulates mineral homeostasis, blood production, and mechanically protects important internal organs. However, bone defects caused by tumors, trauma, infection, senile diseases, etc. can damage the integrity of the skeleton and further limit the normal activities of the body. Due to the limited inherent regenerative capacity of endogenous cells, the reconstruction of critical bone defects remains a major challenge in clinical orthopedics. Given that current clinical methods such as autologous bone and allogeneic bone transplantation are limited by donor scarcity and immune rejection, replicating the complex structure and function of specific bone tissue in vitro has become a long-term pursuit to achieve bone defect reconstruction. Bone organoids have received extensive attention in bone tissue repair due to their physiological properties similar to natural bone. Unlike soft tissue organoids, which are self-assembled by three-dimensional cells, bone organoids require a mineralized structure that responds to a special inorganic / organic hybrid extracellular matrix to mimic the load-bearing capacity of natural bone. More importantly, bone organoids must be able to respond to biochemical signals in a dynamic environment and support functions such as vascularization. These requirements increase the complexity of constructing functional bone organoids.

[0003] In order to create a biomimetic bone microenvironment to construct functional bone organoids, bone-mimicking matrix hydrogels should replicate the natural composition of bone tissue. Bone-derived decellularized extracellular matrix is ​​considered to be an ideal material that completely mimics the organic components of natural bone matrix. Many studies have shown that bone-derived decellularized extracellular matrix can promote the recruitment, proliferation and differentiation of bone marrow mesenchymal stem cells. In addition, inorganic bone-mimicking matrix materials are another important component of bone-mimicking hydrogel matrices. Although hydroxyapatite can be combined with organic matrices, its relatively high crystallinity and slow degradation rate are not conducive to the formation of bone organoids. Calcium phosphate oligomers have good degradation rate, osteogenesis and angiogenesis abilities, which can accelerate the mineralization and vascularization of bone marrow mesenchymal stem cells in bone-mimicking matrix, further promoting the formation of mineralized bone organoids. In addition. DNA contains abundant phosphate groups, which can enhance the binding to Ca released by calcium phosphate oligomers. 2+ interaction, thereby accelerating the construction of vascularized bone organoids. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a bionic matrix hydrogel for constructing bone organoids and a preparation method thereof. The bionic matrix hydrogel provided by the present invention can be used to construct bone organoids. The bionic matrix hydrogel can replace Matrigel in the culture of bone organoids, provide a living environment similar to bone tissue for three-dimensional cultured cells, enhance material exchange and signal conduction between cells, and promote the proliferation, adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells, thereby accelerating the process of biomineralization, thereby promoting the construction of bone organoids and bone defect repair.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] In a first aspect, the present invention provides a biomimetic matrix hydrogel for constructing bone organoids, wherein the biomimetic matrix hydrogel is a hydrogel formed by photo-crosslinking and self-assembly of a premix containing bone decellularized extracellular matrix, salmon DNA and calcium phosphate oligomers;

[0007] The premixed solution uses phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution as solvent, the concentration of bone decellularized extracellular matrix is ​​5-15wt%, the concentration of salmon DNA is 0.5-2%, and the concentration of calcium phosphate oligomer is 0.25-1wt%.

[0008] Preferably, the premixed solution uses phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution as a solvent, the concentration of bone decellularized extracellular matrix is ​​10wt%, the concentration of salmon DNA is 0.5%, and the concentration of calcium phosphate oligomer is 1wt%.

[0009] In one embodiment of the present invention, the decellularized extracellular matrix is ​​obtained by demineralizing and decellularizing natural porcine bones.

[0010] In one embodiment of the present invention, the methacrylated decellularized extracellular matrix is ​​obtained by modifying natural decellularized extracellular matrix with methacrylic anhydride.

[0011] In one embodiment of the present invention, the calcium phosphate oligomer is composed of CaCl 2 Obtained by the reaction of triethylamine and phosphoric acid.

[0012] In a second aspect, the present invention provides a method for preparing the biomimetic matrix hydrogel for constructing bone organoids, the specific steps comprising:

[0013] Step 1, Preparation of Methacrylylated Decellularized Extracellular Matrix:

[0014] Dissolution of decellularized extracellular matrix: using acetic acid and pepsin to dissolve the powdered decellularized extracellular matrix and remove insoluble matter;

[0015] Preparation of methacrylylated decellular extracellular matrix: after activating the decellular extracellular matrix with EDC and NHS, modifying the decellular extracellular matrix with methacrylic anhydride at pH=7.4 to obtain methacrylylated decellular extracellular matrix;

[0016] Step 2, preparation of calcium phosphate oligomers:

[0017] CaCl 2 ·2H 2 O is dissolved in ethanol, triethylamine is added under magnetic stirring, and next, an ethanol solution of phosphoric acid is prepared, and then the two solutions are mixed and reacted under magnetic stirring to obtain calcium phosphate oligomers, referred to as CPO.

[0018] Step 3, preparation of biomimetic matrix hydrogel:

[0019] Preparation of premix: Dissolve methacrylylated decellularized extracellular matrix and salmon DNA in phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution to obtain DNA-decellularized extracellular matrix premix, and add calcium phosphate oligomer to form a hydrogel premix;

[0020] Preparation of bionic matrix hydrogel: The hydrogel premix is ​​irradiated with ultraviolet light to form a bionic matrix hydrogel.

[0021] In one embodiment of the present invention, in step 1, 75% acetic acid and an appropriate amount of pepsin are used to dissolve the decellularized extracellular matrix.

[0022] In one embodiment of the present invention, in step 1, the decellularized extracellular matrix is ​​activated with EDC and NHS under magnetic stirring for 12 hours.

[0023] In one embodiment of the present invention, in step 1, methacrylic anhydride and bone decellularized extracellular matrix solution are reacted in a ratio of 1:3 for 24 hours in a dark environment. Subsequently, the solution is dialyzed in deionized water for one week using a dialysis membrane with a molecular weight cutoff of 500 Da. After freeze-drying, the methacrylated decellularized extracellular matrix is ​​obtained.

[0024] In one embodiment of the present invention, in step 2, 1.12 g CaCl 2 ·2H 2 O was dissolved in ethanol. Under magnetic stirring, 22.2 mL of triethylamine was added to the solution. Next, 0.4 mL of phosphoric acid was mixed into 8 mL of ethanol and the resulting solution was stirred at room temperature. The two solutions were then mixed and reacted for 12 hours under magnetic stirring to obtain CPO.

[0025] In one embodiment of the present invention, in step 2, the concentration of the calcium phosphate oligomer is 1 wt %.

[0026] In one embodiment of the present invention, in step 3, the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate in the premix is ​​0.25wt%, wherein phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is a photoinitiator, the concentration of bone decellularized extracellular matrix in the premix is ​​10wt%, the concentration of salmon DNA in the premix is ​​0.5%, and the concentration of calcium phosphate oligomer in the premix is ​​1wt%.

[0027] In one embodiment of the present invention, in step 3, the conditions of the ultraviolet light irradiation are: 405 nm wavelength, 8.0 mW cm -2 strength.

[0028] In one embodiment of the present invention, the method for preparing the biomimetic matrix hydrogel comprises the following specific steps:

[0029] Step 1, preparation of decellularized extracellular matrix-DNA biomimetic matrix hydrogel:

[0030] Preparation of decellularized extracellular matrix-DNA premix: 5-15wt% (preferably 10wt%) of methacrylylated decellularized extracellular matrix, 0.25wt% of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 0.25-1wt% (preferably 1wt%) of calcium phosphate oligomer, and 0.5-2% (preferably 0.5wt%) of salmon DNA are mixed evenly to obtain a premix, wherein the concentrations are all the final concentrations in the premix;

[0031] Preparation of biomimetic matrix hydrogel: The premixed solution was heated at 405 nm wavelength and 8.0 mW cm -2 The crosslinking was achieved by irradiating with ultraviolet light at 1000 nm for 10 seconds.

[0032] The third aspect of the present invention provides a method for constructing bone organoids based on a biomimetic matrix hydrogel, the specific steps comprising:

[0033] Step 1, loading of bone marrow mesenchymal stem cells: bone marrow mesenchymal stem cells were loaded at a rate of (1-10)×10 6 The density of cells / mL was added to the premixed solution for forming the biomimetic matrix hydrogel, and the premixed solution containing bone marrow mesenchymal stem cells was cross-linked under light to obtain the biomimetic matrix hydrogel loaded with bone marrow mesenchymal stem cells, which was pre-cultured in vitro using osteogenic culture medium for 3 days.

[0034] Step 2, in vitro dynamic culture of mineralized bone organoids: The pre-cultured biomimetic matrix hydrogel loaded with bone marrow mesenchymal stem cells is cultured using a dynamic culture system, and mineralized bone organoids are obtained after long-term induction in osteogenic culture medium for 30 days.

[0035] Step 3, in vivo heterotopic ossification to construct vascularized bone organoids: biomimetic matrix hydrogel loaded with bone marrow mesenchymal stem cells pre-cultured in nude mice subcutaneous heterotopic ossification culture was used, and vascularized bone organoids were obtained after long-term culture for 4 weeks.

[0036] In one embodiment of the present invention, a method for preparing a premix for forming a biomimetic matrix hydrogel is as follows: methacrylylated decellularized extracellular matrix and salmon DNA are dissolved using phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution to obtain a DNA-decellularized extracellular matrix premix, and calcium phosphate oligomers are added thereto to form a hydrogel premix, wherein the premix uses phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution as a solvent, the concentration of bone decellularized extracellular matrix is ​​5 to 15 wt%, the concentration of salmon DNA is 0.5 to 2%, and the concentration of calcium phosphate oligomers is 0.25 to 1 wt%.

[0037] A fourth aspect of the present invention provides use of the bionic matrix hydrogel or the vascularized mineralized bone organoid in the preparation of a medicament or medical device for repairing bone defects.

[0038] The decellularized extracellular matrix in the biomimetic matrix hydrogel of the present invention can simulate the organic components in natural bones, the calcium phosphate oligomers can simulate the inorganic components in natural bones, and the introduction of the DNA network can provide sufficient phosphate ions for biomineralization. The biomimetic matrix hydrogel can replace Matrigel in the culture of bone organoids, provide a living environment similar to bone tissue for three-dimensional cultured cells, enhance the material exchange and signal conduction between cells, and promote the proliferation, adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells, thereby accelerating the process of biomineralization, thereby promoting the construction of bone organoids and bone defect repair. The present invention provides a new material for preparing biomineralized bone organoids.

[0039] Compared with the prior art, the present invention has the following effects:

[0040] First, the decellularized extracellular matrix of the present invention can significantly simulate the organic components in the natural structure of bones;

[0041] Secondly, the calcium phosphate oligomers of the present invention can significantly simulate the inorganic components in the natural structure of bones;

[0042] Thirdly, the introduction of DNA in the present invention can provide recombinant phosphate ions for biomineralization and bone repair. The exposed phosphate groups in the hydrogel are located on the DNA molecules and can react with the Ca released by the calcium phosphate oligomers. 2 +interaction, thereby accelerating the formation of bone organoids;

[0043] Fourthly, the biomimetic matrix hydrogel described in the present invention can provide a living environment similar to bone tissue for three-dimensional cultured cells, enhance material exchange and signal transduction between cells, and promote the proliferation, adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells, thereby accelerating the process of biomineralization, thereby promoting bone defect repair.

[0044] Fifth, the present invention utilizes biomimetic matrix hydrogel to sequentially construct vascularized mineralized bone organoids, which can achieve rapid repair of the skull through the construction of bone organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 . Flowchart for the preparation of biomimetic matrix hydrogels and the construction of vascularized bone organoids.

[0046] Figure 2 .Preparation verification diagram of biomimetic matrix hydrogel.

[0047] Figure 3 .Characterization results of different biomimetic matrix hydrogels.

[0048] Figure 4 .Life, death and distribution of bone marrow mesenchymal stem cells in biomimetic matrix hydrogel.

[0049] Figure 5 .The skeleton of bone marrow mesenchymal stem cells in biomimetic matrix hydrogel.

[0050] Figure 6 .Image of the effect of biomimetic matrix hydrogel on osteogenic differentiation of bone marrow mesenchymal stem cells.

[0051] Figure 7 .Image of the effect of biomimetic matrix hydrogel on the angiogenic ability of human umbilical vein endothelial cells.

[0052] Figure 8 .Macroscopic image of the in vitro dynamic culture system of biomimetic matrix hydrogel.

[0053] Fig. 9 .Macroscopic images of bone marrow mesenchymal stem cells after dynamic three-dimensional culture in vitro on biomimetic matrix hydrogel.

[0054] Fig.10 . Macroscopic images of bone marrow mesenchymal stem cells after three-dimensional culture in vivo in biomimetic matrix hydrogel.

[0055] Fig.11 .Image of the degradation ability of biomimetic matrix hydrogel in mice.

[0056] Fig.12 .3D modeling image of mouse skull defect treated with biomimetic matrix hydrogel. DETAILED DESCRIPTION

[0057] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0058] The contents of the present invention have not been completed. The experimental reagents, materials or instruments used in the examples were all purchased from commercial products unless otherwise specified.

[0059] Example 1 Preparation of biomimetic matrix hydrogel

[0060] Step 1, Preparation of Methacrylylated Decellularized Extracellular Matrix:

[0061] Dissolution of decellularized extracellular matrix: 1 g of powdered decellularized extracellular matrix was dissolved using 75% acetic acid and 10 mg of pepsin to remove insoluble matter;

[0062] Preparation of methacrylylated decellularized extracellular matrix: After activating the decellularized extracellular matrix with 287.5 mg EDC and 172.5 mg NHS at pH = 7.4 and magnetic stirring for 12 h, methacrylic anhydride and bone decellularized extracellular matrix solution were reacted in a ratio of 1:3 for 24 h in a dark environment. Subsequently, the solution was dialyzed in deionized water at 37 ° C for one week using a dialysis membrane with a molecular weight cutoff of 500 Da. The methacrylylated decellularized extracellular matrix was obtained after freeze drying.

[0063] Step 2, preparation of calcium phosphate oligomers:

[0064] 1.12 g CaCl 2 ·2H 2 O was dissolved in 160 mL of ethanol. Under magnetic stirring, 22.2 mL of triethylamine was added to the solution. Next, 0.4 mL of phosphoric acid was mixed into 8 mL of ethanol and the resulting solution was stirred at room temperature. The two solutions were then mixed and reacted for 12 hours under magnetic stirring to obtain calcium phosphate oligomer CPO.

[0065] Step 3, preparation of biomimetic matrix hydrogel:

[0066] Preparation of premix: The methacrylylated decellularized extracellular matrix prepared in step 1 and salmon DNA are dissolved in a 0.25% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution in a ratio of 10 wt% and 0.5% (i.e., the methacrylylated decellularized extracellular matrix in the premix is ​​10 wt% and the salmon DNA in the premix is ​​0.5%) to obtain a DNA-decellularized extracellular matrix premix, and 1% of calcium phosphate oligomer is added thereto (i.e., the final mass concentration of calcium phosphate oligomer in the hydrogel premix) to form a hydrogel premix;

[0067] Preparation of biomimetic matrix hydrogel: The hydrogel premix was heated at 405 nm wavelength and 8.0 mW cm -2 A biomimetic matrix hydrogel is formed under high-intensity ultraviolet light irradiation.

[0068] The biomimetic matrix hydrogel process prepared in this example is referenced Figure 1 As shown, the successful preparation of biomimetic matrix hydrogels is Figure 2 Verified by means of 1 H NMR analysis confirmed the successful grafting of methacrylylated decellularized extracellular matrix. Compared with bone-derived decellularized extracellular matrix, methacrylylated decellularized extracellular matrix showed new characteristic peaks at about 5.3 and 5.5 ppm, indicating that methacrylate groups were successfully grafted ( Figure 2 A). Next, the salmon DNA in the biomimetic matrix hydrogel was stained with SYBR Green I dye, demonstrating the uniform distribution of DNA ( Figure 2 B). Furthermore, ATR-FTIR analysis revealed that the addition of calcium phosphate oligomers resulted in a new peak at 1444 cm-1 compared to the methacrylylated decellularized extracellular matrix, which confirmed the successful introduction of calcium phosphate oligomers ( Figure 2 C). In addition, it was found in the macroscopic image that after UV irradiation, the biomimetic matrix hydrogel can change from a liquid state to a gel state ( Figure 2 D).

[0069] Example 2

[0070] This example mainly examines the properties of the biomimetic matrix hydrogel.

[0071] Referring to the description of Example 1, when preparing different biomimetic matrix hydrogels, the difference mainly lies in whether DNA and calcium phosphate oligomers are added during the preparation of the premix.

[0072] Without adding DNA and calcium phosphate oligomers, the methacrylylated decellularized extracellular matrix prepared in step 1 of Example 1 was directly dissolved in 0.25% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution as the decellularized extracellular matrix premix, and the obtained biomimetic matrix hydrogel was recorded as mECM.

[0073] DNA was added, and the methacrylylated decellularized extracellular matrix prepared in step 1 of Example 1 and salmon DNA were mixed in a ratio of 10 wt % and 0.5 wt % (i.e., the methacrylylated decellularized extracellular matrix in the premix was 10 wt %, and the salmon DNA in the premix was 0.5 wt %), and dissolved in 0.25 % phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution to obtain a DNA-decellularized extracellular matrix premix. The obtained biomimetic matrix hydrogel was recorded as mECMD.

[0074] DNA and calcium phosphate oligomers were added. The methacrylylated decellularized extracellular matrix prepared in step 1 of Example 1 and salmon DNA were dissolved in a 0.25% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution at a ratio of 10 wt% and 0.5%, respectively, to obtain a DNA-decellularized extracellular matrix premix, and 1% calcium phosphate oligomers were added thereto to form a hydrogel premix. The resulting biomimetic matrix hydrogel was recorded as BMH.

[0075] The above three different premixes have the same conditions for preparing biomimetic matrix hydrogels: the hydrogel premix is ​​heated at a wavelength of 405 nm and 8.0 mW cm -2 A biomimetic matrix hydrogel is formed under high-intensity ultraviolet light irradiation.

[0076] Figure 3 Characterization of hydrogels with different biomimetic matrices.

[0077] It can be seen that after adding salmon DNA and calcium phosphate oligomers to the hydrogel, all the gels formed have porous structures, which are conducive to the transportation of cell nutrients and the discharge of metabolic waste, as well as cell adhesion ( Figure 3 A). In addition, the energy dispersive spectrometer can also observe that the elements in the biomimetic matrix hydrogel are evenly distributed ( Figure 3 B). Secondly, the hydrogel was evenly dropped between two glass slides and then irradiated with ultraviolet light to make it gel. After gelation, the hydrogel was placed in an oven until completely dry. Subsequently, the sample was placed on a contact angle goniometer and 3 μL of pure water was dropped onto the surface of the hydrogel. After the water droplet came into contact with the hydrogel surface, a camera was used to capture the contact angle between the water droplet and the hydrogel surface. The experiment showed that the addition of DNA and calcium phosphate oligomers did not significantly change the hydrophilicity of the hydrogel ( Figure 3 C). However, after the hydrogel was made into a cylinder, the hardness of the hydrogel was tested using a nanoindenter. The experimental results showed that the hardness of the biomimetic matrix hydrogel increased significantly with the introduction of salmon DNA and calcium phosphate oligomers ( Figure 3D). After the hydrogel was gelled, the rheological properties were tested by a small amplitude frequency sweep test at 37 °C to evaluate the storage modulus (G′), loss modulus (G″), and viscoelasticity in the frequency range of 0.01 to 100 rad / s. In the frequency range of 0.01 to 100 rad / s, the elastic shear storage modulus (G′) of the biomimetic matrix hydrogel always exceeded the viscous shear loss modulus (G″), which proved the relatively stable structure of the biomimetic matrix hydrogel. In addition, the addition of salmon DNA and CPO led to an increase in G′, thereby enhancing the elasticity of the biomimetic matrix hydrogel ( Figure 3 E). This observation was further confirmed by analyzing the viscoelastic curves obtained from rheological experiments ( Figure 3 F). In addition, the swelling properties of the hydrogels were determined gravimetrically, recording the initial dry weight of the hydrogel (M0) and the weight after swelling in PBS at room temperature (M1), making sure to remove excess surface water with absorbent paper before weighing. The swelling ratio was determined using the following formula: Swelling ratio = (M1 / M0) × 100%. In the swelling experiments, all samples showed similar behavior, reaching swelling equilibrium in approximately 24 h with a swelling ratio of approximately 900%. However, the addition of salmon DNA and calcium phosphate oligomers resulted in a slower swelling rate for the biomimetic matrix hydrogel ( Figure 3 G). In addition, the degradation capacity of the hydrogel was determined by the weight method. In order to evaluate the degradation of the hydrogel, the initial mass of the hydrogel degradation was (M0), and then the hydrogel was soaked in PBS at 37°C and placed on a shaker. At different time intervals, the remaining hydrogel was freeze-dried and weighed (M1). The weight degradation capacity of the hydrogel was calculated as follows: Weight degradation rate = [(M0-M1) / M0] × 100%. All hydrogels had similar degradation behavior and were degraded in about 28 days ( Figure 3 H).

[0078] Example 3

[0079] In order to explore the effect of biomimetic matrix hydrogel on the behavior of bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells were cultured at a rate of 5×10 6 The cells were cultured in hydrogel at a density of 100 / mL and stained with live-dead reagent in the dark for 30 min on days 1, 3, and 7.

[0080] Bone marrow mesenchymal stem cells were cultured at a rate of 5×10 6 The method of culturing the cells in hydrogel at a density of cells / mL is as follows:

[0081] Bone marrow mesenchymal stem cells were cultured at a rate of 5×10 6 The density of 1000 μg / mL was added to different hydrogel premixes, and then the different hydrogel premixes were heated at 405 nm wavelength and 8.0 mW cm -2Different biomimetic matrix hydrogels containing bone marrow mesenchymal stem cells were formed under ultraviolet light of different intensities.

[0082] Wherein, without adding DNA and calcium phosphate oligomers, the methacrylylated decellularized extracellular matrix prepared in step 1 of Example 1 was directly dissolved in 0.25% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution as the decellularized extracellular matrix premix, and the obtained biomimetic matrix hydrogel was recorded as mECM.

[0083] DNA was added, and the methacrylylated decellularized extracellular matrix prepared in step 1 of Example 1 and salmon DNA were mixed in a ratio of 10 wt % and 0.5 wt % (meaning that the methacrylylated decellularized extracellular matrix in the premix was 10 wt % and the salmon DNA in the premix was 0.5 wt %), and dissolved in 0.25 % phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution to obtain a DNA-decellularized extracellular matrix premix. The obtained biomimetic matrix hydrogel was recorded as mECMD.

[0084] DNA and calcium phosphate oligomers were added. The methacrylylated decellularized extracellular matrix prepared in step 1 of Example 1 and salmon DNA were mixed in a ratio of 10 wt % and 0.5 wt % (meaning that the methacrylylated decellularized extracellular matrix was 10 wt % in the premix and the salmon DNA was 0.5 wt % in the premix). The DNA-decellularized extracellular matrix premix was obtained by dissolving it in 0.25 % phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution. 1 % calcium phosphate oligomer was added thereto to form a hydrogel premix. The obtained biomimetic matrix hydrogel was recorded as BMH.

[0085] The different biomimetic matrix hydrogels obtained above were pre-cultured in vitro using osteogenic culture medium.

[0086] The experimental results are as follows Figure 4 As shown in the figure, it can be seen that the biomimetic matrix hydrogel formed by adding DNA and calcium phosphate oligomers to the hydrogel has good activity in the biomimetic matrix hydrogel within 7 days of culture. Subsequently, after 14 days of culture, the nucleus and skeleton of the bone marrow mesenchymal stem cells were stained, and the experimental results are shown in Figure 1. Figure 5 As shown in the figure, it can be seen that a skeleton network of bone marrow mesenchymal stem cells is formed in the bionic matrix hydrogel, which is beneficial to the signal transduction of cells in the bionic matrix hydrogel.

[0087] Example 4

[0088] In order to explore the effect of biomimetic matrix hydrogel on the osteogenic ability of bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells and biomimetic matrix hydrogel were co-cultured using transwell chambers, in which the upper chamber was 100 μm biomimetic matrix hydrogel and the lower chamber was 5×104 Bone marrow mesenchymal stem cells with a density of 100 / mL were co-cultured with osteogenic induction matrix and upper hydrogel, fixed with 4% paraformaldehyde at 7 and 14 days, and stained with alkaline phosphatase and alizarin red staining reagent for 30 minutes to obtain staining images. The experimental results are shown in Figure 6 As shown in the figure, the experimental results show that biomimetic matrix hydrogel can significantly accelerate the osteogenic differentiation ability of bone marrow mesenchymal stem cells. In order to explore the effect of biomimetic matrix hydrogel on the angiogenesis ability of human umbilical vein endothelial cells, human umbilical vein endothelial cells were co-cultured with biomimetic matrix hydrogel. The experimental results are shown in the figure. Figure 7 As shown, the biomimetic matrix hydrogel can accelerate angiogenesis and produce a vascular network in about 8 hours.

[0089] Example 5

[0090] Bone marrow mesenchymal stem cells were cultured at a rate of 5×10 6 The bone organoids were constructed by implanting bone mesenchymal stem cells at a density of 100 / mL into a biomimetic matrix hydrogel (bone marrow mesenchymal stem cells were first added to the premix and then exposed to light to form the hydrogel), and then pre-cultured with osteogenic induction medium for 3 days. First, a dynamic culture system was used to induce osteogenics for 1 month to construct bone organoids. Figure 8 As shown in the figure, the biomimetic matrix hydrogel loaded with bone marrow mesenchymal stem cells and the osteogenic culture medium are placed in the two connected channels respectively, and the culture medium is circulated in the culture system by the power provided by the gas to simulate the natural bone microenvironment. Fig. 9 As shown, the experimental results show that the bone organoids constructed with biomimetic matrix hydrogels have extremely strong biomineralization capabilities, and the dynamic culture system can also significantly accelerate the process of bone organoid biomineralization. Secondly, the pre-cultured biomimetic matrix hydrogels loaded with bone marrow mesenchymal stem cells were cultured using the subcutaneous heterotopic ossification of nude mice to construct in vivo vascularized bone organoids. The experimental operation is as follows: After the nude mice are anesthetized, the pre-cultured biomimetic matrix hydrogels loaded with bone marrow mesenchymal stem cells are placed subcutaneously. After suturing the wounds, the nude mice are killed 2 weeks and 4 weeks after implantation, and the vascularized bone organoids are taken out. The experimental results are shown in the figure. Fig.10 As shown, the experimental results indicate that the biomimetic matrix hydrogel can accelerate the biomineralization of bone organoids and can significantly accelerate the increase of blood vessel adhesion in bone organoids in vivo.

[0091] Example 6

[0092] In order to evaluate the in vivo degradation ability of the biomimetic matrix hydrogel, 500 μL of biomimetic matrix hydrogel (without bone marrow mesenchymal stem cells) was implanted in C57BL / 6 mice. The experimental results are shown in Figure 2. Fig.11As shown. The biomimetic matrix hydrogel was first soaked in Cy5.5 NHS solution for 1 hour to label the hydrogel, and then rinsed with PBS to remove excess dye. The labeled hydrogel was then implanted into the back of C57BL / 6 mice. Then, the in vivo imaging system was used to monitor the retention of the hydrogel in vivo, with the excitation and emission wavelengths set to 675nm and 740nm, respectively. In the in vivo degradation experiment, after adding salmon DNA and calcium phosphate oligomers, the biomimetic matrix hydrogel was completely degraded after 6 weeks of implantation, and the group with only salmon DNA added was basically completely degraded after 5 weeks. This degradation trend is consistent with the trend observed in the in vivo degradation study.

[0093] Example 7

[0094] (1) Construction of mouse skull defect model: C57 male mice aged 5-6 weeks were taken and, after general anesthesia, a skull defect with a diameter of 3 mm was constructed in the skull using a punch.

[0095] (2) Application of biomimetic matrix hydrogel to repair skull defects: Bone marrow mesenchymal stem cells were injected at a rate of 5×10 6 The mice were implanted into the biomimetic matrix hydrogel at a density of 100 μg / mL and pre-cultured with osteogenic induction medium for 3 days before being implanted into the defect site. Each defect site was implanted with 5 μL of biomimetic matrix hydrogel. The mice were raised normally after surgery and were killed after 6 and 12 weeks to analyze the repair effect.

[0096] This example mainly investigates the repair effect of biomimetic matrix hydrogel on skull defects. Fig.12 The skull CT scans of each group of animal experiments at 6 and 12 weeks. Among them, the biomimetic matrix hydrogel with added DNA and calcium phosphate oligomers has the strongest repair effect, which proves the repair effect of biomimetic matrix hydrogel on skull defect repair.

[0097] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A biomimetic matrix hydrogel for constructing bone organoids, characterized in that: The biomimetic matrix hydrogel is a hydrogel formed by photo-crosslinking and self-assembly of a premix containing bone decellularized extracellular matrix, salmon DNA and calcium phosphate oligomers; The premixed solution uses phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution as solvent, the concentration of bone decellularized extracellular matrix is ​​5-15wt%, the concentration of salmon DNA is 0.5-2%, and the concentration of calcium phosphate oligomer is 0.25-1wt%.

2. The biomimetic matrix hydrogel for constructing bone organoids according to claim 1, characterized in that: The premixed solution uses phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution as a solvent, the concentration of bone decellularized extracellular matrix is ​​10wt%, the concentration of salmon DNA is 0.5%, and the concentration of calcium phosphate oligomer is 1wt%.

3. The biomimetic matrix hydrogel for constructing bone organoids according to claim 1, characterized in that: The decellularized extracellular matrix is ​​obtained by demineralizing and decellularizing natural pig bones; The decellularized extracellular matrix is ​​obtained by modifying natural decellularized extracellular matrix with methacrylic anhydride.

4. The biomimetic matrix hydrogel for constructing bone organoids according to claim 1, characterized in that: The calcium phosphate oligomer is obtained by reacting CaCl2, triethylamine and phosphoric acid.

5. A method for preparing a biomimetic matrix hydrogel for constructing bone organoids according to any one of claims 1 to 4, characterized in that: The specific steps include: Step 1, Preparation of Methacrylylated Decellularized Extracellular Matrix: Dissolution of decellularized extracellular matrix: using acetic acid and pepsin to dissolve the powdered decellularized extracellular matrix and remove insoluble matter; Preparation of methacrylylated decellular extracellular matrix: after activating the decellular extracellular matrix with EDC and NHS, modifying the decellular extracellular matrix with methacrylic anhydride at pH=7.4 to obtain methacrylylated decellular extracellular matrix; Step 2, preparation of calcium phosphate oligomers: CaCl2·2H2O was dissolved in ethanol, triethylamine was added under magnetic stirring, and then an ethanol solution of phosphoric acid was prepared, and then the two solutions were mixed and reacted under magnetic stirring to obtain calcium phosphate oligomers, referred to as CPO; Step 3, preparation of biomimetic matrix hydrogel: Preparation of premix: Dissolve methacrylylated decellularized extracellular matrix and salmon DNA in phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution to obtain DNA-decellularized extracellular matrix premix, and add calcium phosphate oligomer to form a hydrogel premix; Preparation of bionic matrix hydrogel: The hydrogel premix is ​​irradiated with ultraviolet light to form a bionic matrix hydrogel.

6. The method for preparing a biomimetic matrix hydrogel for constructing bone organoids according to claim 5, characterized in that: The preparation method of the biomimetic matrix hydrogel specifically comprises the following steps: Step 1, preparation of decellularized extracellular matrix-DNA biomimetic matrix hydrogel: Preparation of decellularized extracellular matrix-DNA premix: 10 wt% of methacrylylated decellularized extracellular matrix, 0.25 wt% of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 1% of calcium phosphate oligomer, and 0.5% of salmon DNA were mixed evenly to obtain a premix, where the concentrations are the final concentrations in the premix; Preparation of biomimetic matrix hydrogel: The premixed solution was heated at 405 nm wavelength and 8.0 mW cm -2 The ultraviolet light was irradiated again for 10 seconds to achieve crosslinking.

7. A method for constructing bone organoids based on biomimetic matrix hydrogel, characterized in that: The specific steps include: Step 1, loading of bone marrow mesenchymal stem cells: bone marrow mesenchymal stem cells were loaded at a rate of (1-10)×10 6 The premixed solution containing bone marrow mesenchymal stem cells is cross-linked under light to obtain a biomimetic matrix hydrogel loaded with bone marrow mesenchymal stem cells, and then pre-cultured in vitro using an osteogenic medium; Step 2, in vitro dynamic culture of mineralized bone organoids: using a dynamic culture system to culture the pre-cultured biomimetic matrix hydrogel loaded with bone marrow mesenchymal stem cells, and obtaining mineralized bone organoids after long-term induction in osteogenic culture medium; or, Step 3, in vivo heterotopic ossification to construct vascularized bone organoids: biomimetic matrix hydrogel loaded with bone marrow mesenchymal stem cells pre-cultured in nude mice subcutaneous heterotopic ossification culture, and vascularized bone organoids were obtained after long-term culture.

8. The method for constructing bone organoids based on biomimetic matrix hydrogel according to claim 7, characterized in that: The preparation method of the premix for forming a biomimetic matrix hydrogel is as follows: methacrylylated decellularized extracellular matrix and salmon DNA are dissolved in phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution to obtain a DNA-decellularized extracellular matrix premix, and calcium phosphate oligomers are added thereto to form a hydrogel premix, wherein the premix uses phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution as a solvent, the concentration of bone decellularized extracellular matrix is ​​5-15wt%, the concentration of salmon DNA is 0.5-2%, and the concentration of calcium phosphate oligomers is 0.25-1wt%.

9. A bone organoid constructed based on a biomimetic matrix hydrogel prepared according to the method of claim 7 or 8.

10. Use of the biomimetic matrix hydrogel according to claims 1 to 4 or the bone organoid according to claim 9 in the preparation of a medicament or medical device for repairing bone defects.

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