Cartilage-bone integrated organ and preparation method thereof

Abstract

The invention relates to a cartilage-bone integrated organ and a preparation method thereof. The cartilage-bone integrated organ comprises a bone-imitating layer and a cartilage-imitating layer, the bone-imitating layer is formed by printing bone-imitating matrix force responsive bio-ink constructed based on piezoelectric hydrogel, and the cartilage-imitating layer is formed by printing cartilage-imitating matrix force responsive bio-ink constructed based on piezoelectric hydrogel. According to the invention, a piezoelectric material is combined with bio-ink, and a double-ecological niche structure of a cartilage layer and a bone layer is accurately constructed by using a 3D biological printing technology. And the mechanical force is converted into a bio-electricity signal through the piezoelectric material, so that cell activity and tissue regeneration are promoted. Meanwhile, accurate structure control is achieved through the 3D biological printing technology, and a brand new strategy is provided for articular cartilage defect repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoids, and particularly relates to a cartilage-bone integrated organoid and a preparation method thereof. Background Art

[0002] Articular cartilage defects are common causes leading to osteoarthritis, dysfunction and even disability, seriously affecting the quality of life of patients. Existing treatment methods, such as platelet-rich plasma, microfracture, cartilage transplantation, high tibial osteotomy and bioactive materials, all show certain limitations. The treatment of platelet-rich plasma lacks standardization and has a short-lived effect; microfracture may cause intra-articular hemorrhage and limited functional recovery; cartilage transplantation faces donor limitations and graft anchoring problems; high tibial osteotomy can adjust biomechanics but has limited indications; bioactive materials need to be further optimized to improve clinical effects. The deficiencies of these treatment means highlight the urgency of developing new methods for articular cartilage repair in order to achieve more effective treatment effects and improve the quality of life of patients.

[0003] Although organoid technology shows potential in the field of articular cartilage repair, current research mainly focuses on the repair of single tissue types, that is, the independent construction of bone organoids and cartilage organoids. Bone organoids can mimic the complex structure and biological functions of bone tissue, helping to promote the healing of bone defects. However, although cartilage organoids show good effects in mimicking the cartilage microenvironment and promoting cartilage repair, there are challenges in post-transplant fixation. The independent construction and application of these two types of organoids have not fully met the needs of cartilage-bone integrated repair.

[0004] In the field of tissue engineering and regenerative medicine, a method of constructing a cartilage-bone integrated structure by encapsulating cells with chondrogenic and osteogenic differentiation potential in hydrogel materials has provided new ideas for the repair and regeneration of osteochondral composite defects (Chinese Patent CN115475281B). This method uses hydrogel materials to simulate the microenvironment of tissue growth and obtains mature tissue-engineered cartilage-bone composites through in vitro or in vivo culture, effectively solving the problems of low cell survival rate and poor tissue stability in traditional repair methods. However, the biomimicry and functionality of the cartilage-bone composites prepared by this method still need to be improved. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the present invention constructs cartilage-bone integrated organoids by using piezoelectric hydrogels and 3D bioprinting technology. By converting mechanical force into bioelectric signals, the cell activity and tissue regeneration ability are significantly enhanced, and the mechanical properties and functional performance of cartilage and bone tissues are effectively improved. At the same time, this technology realizes precise structural control of the cartilage layer and the bone layer, breaks through the limitations of traditional methods in simulating the natural cartilage-bone interface, significantly improves the biomimicry and functionality of the organoids, and provides an efficient and precise solution for the repair of articular cartilage defects.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] In the first aspect, the present invention provides a bioink for preparing cartilage-bone integrated organoids, including a bone matrix force-responsive bioink based on piezoelectric hydrogels and a cartilage matrix force-responsive bioink based on piezoelectric hydrogels;

[0008] The bone matrix force-responsive bioink based on piezoelectric hydrogels includes: GelMA hydrogel as a dispersion system, and piezoelectric material barium titanate BTO, nano-hydroxyapatite nHA, and bone marrow-derived stem cells dispersed in the GelMA hydrogel;

[0009] The cartilage matrix force-responsive bioink based on piezoelectric hydrogels includes: ChSMA hydrogel as a dispersion system, and piezoelectric material barium titanate BTO, transforming growth factor TGF-β, and bone marrow-derived stem cells dispersed in the ChSMA hydrogel.

[0010] In an embodiment of the present invention, the bone marrow-derived stem cells are bone marrow mesenchymal stem cells BMSCs derived from SD rats.

[0011] In an embodiment of the present invention, in the bone matrix force-responsive bioink based on piezoelectric hydrogels, the concentration of piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of nano-hydroxyapatite nHA is 50-70 mg / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, and the concentration of GelMA is 80-100 mg / mL.

[0012] In an embodiment of the present invention, the bone matrix force-responsive bioink based on piezoelectric hydrogels contains a photoinitiator, and the concentration of the photoinitiator is 2-2.5 mg / mL. The photoinitiator is selected as LAP.

[0013] In one embodiment of the present invention, in the bone matrix-mimicking force-responsive bioink based on piezoelectric hydrogel, the GelMA hydrogel as the dispersion system is obtained by co-incubating GelMA solid and photoinitiator LAP in a sterile complete α-MEM medium.

[0014] In one embodiment of the present invention, in the cartilage matrix-mimicking force-responsive bioink based on piezoelectric hydrogel, the concentration of piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of transforming growth factor TGF-β is 10-15 ng / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, and the concentration of ChSMA is 40-100 mg / mL.

[0015] In one embodiment of the present invention, the cartilage matrix-mimicking force-responsive bioink based on piezoelectric hydrogel contains a photoinitiator, and the concentration of the photoinitiator is 2-2.5 mg / mL. The photoinitiator is selected as LAP.

[0016] In one embodiment of the present invention, in the cartilage matrix-mimicking force-responsive bioink based on piezoelectric hydrogel, the ChSMA hydrogel as the dispersion system is obtained by co-incubating ChSMA solid and photoinitiator LAP in a sterile complete α-MEM medium.

[0017] In one embodiment of the present invention, the piezoelectric material barium titanate BTO is silanized barium titanate.

[0018] In one embodiment of the present invention, the preparation method of the silanized barium titanate is as follows:

[0019] Disperse 8-10 mg / mL barium titanate in absolute ethanol, ultrasonically treat for 20-30 min, then slowly add 0.1%-1% γ-methacryloxypropyltrimethoxysilane, and adjust the pH value to 4-5 with acetic acid; stir the mixture and react at 60-70 °C for 2-4 h, ultrasonically treat for 20-30 min every 30-40 min. After the reaction is completed, add ethanol to terminate the reaction, then collect the particles by centrifugation, wash the collected particles with ethanol to remove impurities, and dry to obtain silanized barium titanate.

[0020] In a second aspect, the present invention provides a cartilage-bone integrated organoid, which includes a bone-mimicking layer and a cartilage-mimicking layer. The bone-mimicking layer is printed with a bone matrix-mimicking force-responsive bioink based on piezoelectric hydrogel, and the cartilage-mimicking layer is printed with a cartilage matrix-mimicking force-responsive bioink based on piezoelectric hydrogel.

[0021] The cartilage-bone integrated organoid is an organoid with a dual ecological niche of an osteoid layer and a chondroid layer constructed from an osteoid matrix and a chondroid matrix force-responsive bioprinting ink.

[0022] In a third aspect, the present invention provides a method for preparing a cartilage-bone integrated organoid, comprising the following steps:

[0023] Using 3D bioprinting technology, the osteoid matrix force-responsive bioprinting ink based on piezoelectric hydrogel is printed and solidified into an osteoid layer.

[0024] Using 3D bioprinting technology, the chondroid matrix force-responsive bioprinting ink based on piezoelectric hydrogel is printed and solidified into a chondroid layer on the upper layer of the osteoid layer.

[0025] The printed structure is cultured in a complete α-MEM medium, and finally a cartilage-bone integrated organoid is obtained.

[0026] In an embodiment of the present invention, the method for preparing the osteoid matrix force-responsive bioprinting ink based on piezoelectric hydrogel is as follows:

[0027] Take GelMA solid and add it to a sterile complete α-MEM medium, and incubate at room temperature for 1-2 h; then add silanized BTO and nHA, and incubate at 37-50 °C and 300-500 rpm for 20-30 min; then, add BMSCs from SD rats and co-incubate at 37-40 °C for 20-30 min; finally, add the photoinitiator LAP and incubate at 37-50 °C and 300-500 rpm for 30 min-1 h to obtain the osteoid matrix force-responsive bioprinting ink based on piezoelectric hydrogel.

[0028] The concentration of the piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of nano-hydroxyapatite nHA is 50-70 mg / mL, the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, the concentration of GelMA is 80-100 mg / mL, and the concentration of the photoinitiator LAP is 2-2.5 mg / mL.

[0029] In an embodiment of the present invention, the method for preparing the chondroid matrix force-responsive bioprinting ink based on piezoelectric hydrogel is as follows:

[0030] Take solid ChSMA and add it to sterile complete α-MEM medium, and incubate at room temperature for 1-2 h; then add silanized BTO and TGF-β, and incubate at 37-50 °C and 300-500 rpm for 20-30 min; then, add BMSCs derived from SD rats and co-incubate at 37-40 °C for 20-30 min; finally, add the photoinitiator LAP and incubate at 37-50 °C and 300-500 rpm for 30 min-1 h to obtain a cartilage matrix force-responsive bioink based on a piezoelectric hydrogel.

[0031] The concentration of the piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of the transforming growth factor TGF-β is 10-15 ng / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, the concentration of ChSMA is 40-100 mg / mL, and the concentration of the photoinitiator LAP is 2-2.5 mg / mL.

[0032] In one embodiment of the present invention, during printing, the specific parameters are a light intensity of 15-20 mW cm -2 and an exposure time of 16-20 s. The number of base layers is set to 2, each base layer has an exposure time of 8-10 s, and the penetration depth of the material is controlled at 50-70 μm. During the entire printing process, the temperature of the platform and the resin tank for placing the bioink is maintained at 37-40 °C.

[0033] In one embodiment of the present invention, after printing, the cartilage-bone integrated organoids are cultured in complete α-MEM medium at 37 °C.

[0034] Compared with the prior art, the beneficial effects of the present invention are reflected in the following aspects:

[0035] The innovation of the present invention lies in the successful construction of cartilage-bone integrated organoids, and particularly emphasizes the key role of electrical signal stimulation in tissue regeneration. By designing a bone-mimicking and cartilage-mimicking matrix force-responsive bioink based on a piezoelectric hydrogel, the present invention not only realizes the dual niche structure of the cartilage layer and the bone layer, but also converts mechanical force into bioelectrical signals through the piezoelectric material, significantly promoting cell activity and tissue regeneration. The combination of this mechanical stimulation and electrophysiological signals provides a microenvironment closer to the physiological state for the regeneration of cartilage and bone tissues, thereby achieving the functional restoration of cartilage-bone.

[0036] In addition, the present invention utilizes 3D bioprinting technology to achieve precise stratification of cartilage and bone layers, breaking through the limitations of traditional methods in terms of size and integration, and significantly enhancing the potential and stability of organoids in defect repair. The present invention not only has significant advantages in simulating the functions of natural cartilage-bone, but also provides higher biomimicry and functionality through the combination of piezoelectric effect and 3D bioprinting technology, providing a more comprehensive and effective solution for the repair of articular cartilage defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Voltage generated by the piezoelectric hydrogel prepared in Example 1 at a pressure level of 0.1 N;

[0038] Figure 2 Voltage generated by the piezoelectric hydrogel prepared in Example 1 at a pressure level of 0.5 N;

[0039] Figure 3 Voltage generated by the piezoelectric hydrogel prepared in Example 1 at a pressure level of 1 N;

[0040] Figure 4 Butterfly curve of the piezoelectric hydrogel prepared in Example 1;

[0041] Figure 5 Hysteresis curve of the piezoelectric hydrogel prepared in Example 1;

[0042] Figure 6 Expression levels of cartilage differentiation-related gene SOX9 under different material conditions after in vitro culture of the cartilage-bone integrated organoids prepared in Example 1;

[0043] Figure 7 Expression levels of cartilage differentiation-related gene Col2A1 under different material conditions after in vitro culture of the cartilage-bone integrated organoids prepared in Example 1;

[0044] Figure 8 Bone density of the cartilage-bone integrated organoids prepared in Example 1 after 4 weeks and 8 weeks of in vitro culture;

[0045] Figure 9 Bone volume / total volume ratio of the cartilage-bone integrated organoids prepared in Example 1 after 4 weeks and 8 weeks of in vitro culture;

[0046] Figure 10 Trabecular bone thickness of the cartilage-bone integrated organoids prepared in Example 1 after 4 weeks and 8 weeks of in vitro culture;

[0047] Figure 11 Trabecular bone number of the cartilage-bone integrated organoids prepared in Example 1 after 4 weeks and 8 weeks of in vitro culture. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention will be further described by means of embodiments. The following embodiments are only used to illustrate the present invention and do not limit the present invention.

[0049] In a first aspect, the present invention provides a bioink for preparing cartilage-bone integrated organoids, including an osteoid matrix force-responsive bioink based on a piezoelectric hydrogel and a chondroid matrix force-responsive bioink based on a piezoelectric hydrogel;

[0050] The osteoid matrix force-responsive bioink based on a piezoelectric hydrogel includes: a GelMA hydrogel as a dispersion system, and barium titanate BTO (a piezoelectric material), nano-hydroxyapatite nHA, and bone marrow-derived stem cells dispersed in the GelMA hydrogel;

[0051] The chondroid matrix force-responsive bioink based on a piezoelectric hydrogel includes: a ChSMA hydrogel as a dispersion system, and barium titanate BTO (a piezoelectric material), transforming growth factor TGF-β, and bone marrow-derived stem cells dispersed in the ChSMA hydrogel.

[0052] In an embodiment of the present invention, the bone marrow-derived stem cells are bone marrow mesenchymal stem cells BMSCs derived from SD rats.

[0053] In an embodiment of the present invention, in the osteoid matrix force-responsive bioink based on a piezoelectric hydrogel, the concentration of the piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of nano-hydroxyapatite nHA is 50-70 mg / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, and the concentration of GelMA is 80-100 mg / mL.

[0054] In an embodiment of the present invention, the osteoid matrix force-responsive bioink based on a piezoelectric hydrogel contains a photoinitiator, and the concentration of the photoinitiator is 2-2.5 mg / mL. The photoinitiator is selected as LAP.

[0055] In an embodiment of the present invention, in the osteoid matrix force-responsive bioink based on a piezoelectric hydrogel, the GelMA hydrogel as a dispersion system is obtained by incubating GelMA solid and the photoinitiator LAP in a sterile complete α-MEM medium.

[0056] In an embodiment of the present invention, in the chondroid matrix force-responsive bioink based on a piezoelectric hydrogel, the concentration of the piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of transforming growth factor TGF-β is 10-15 ng / mL, and the concentration of bone marrow-derived stem cells is 106 ~10 7 cells / mL, the concentration of ChSMA is 40 - 100 mg / mL.

[0057] In one embodiment of the present invention, the cartilage matrix - like force - responsive bioink based on piezoelectric hydrogel contains a photoinitiator, and the concentration of the photoinitiator is 2 - 2.5 mg / mL. The photoinitiator is selected as LAP.

[0058] In one embodiment of the present invention, in the cartilage matrix - like force - responsive bioink based on piezoelectric hydrogel, the ChSMA hydrogel as a dispersion system is obtained by incubating ChSMA solid and the photoinitiator LAP in a sterile complete α - MEM medium.

[0059] In one embodiment of the present invention, the piezoelectric material barium titanate BTO is silanized barium titanate.

[0060] In one embodiment of the present invention, the preparation method of the silanized barium titanate is as follows:

[0061] Disperse 8 - 10 mg / mL barium titanate in absolute ethanol, ultrasonically treat for 20 - 30 min, then slowly add 0.1% - 1% γ - methacryloxypropyltrimethoxysilane, and adjust the pH value to 4 - 5 using acetic acid; stir the mixture and react at 60 - 70 °C for 2 - 4 h, ultrasonically for 20 - 30 min every 30 - 40 min. After the reaction is completed, add ethanol to terminate the reaction, then collect the particles by centrifugation, wash the collected particles with ethanol to remove impurities, and dry to obtain silanized barium titanate.

[0062] In a second aspect, the present invention provides a cartilage - bone integrated organoid, which includes an osteoid - like layer and a cartilage - like layer. The osteoid - like layer is printed from an osteoid matrix - like force - responsive bioink based on piezoelectric hydrogel, and the cartilage - like layer is printed from a cartilage matrix - like force - responsive bioink based on piezoelectric hydrogel.

[0063] The cartilage - bone integrated organoid is an organoid with a dual - niche of an osteoid - like layer and a cartilage - like layer constructed from an osteoid matrix and a cartilage matrix - like force - responsive bioink.

[0064] In a third aspect, the present invention provides a preparation method of a cartilage - bone integrated organoid, comprising the following steps:

[0065] Use 3D bioprinting technology to print and solidify an osteoid matrix - like force - responsive bioink based on piezoelectric hydrogel into an osteoid - like layer,

[0066] Using 3D bioprinting technology, a cartilage-mimicking matrix force-responsive bioink based on piezoelectric hydrogel is printed and cured into a cartilage-mimicking layer on the upper layer of the bone-mimicking layer;

[0067] The printed structure is cultured in a complete α-MEM medium, and finally a cartilage-bone integrated organoid is obtained.

[0068] In one embodiment of the present invention, the preparation method of the bone-mimicking matrix force-responsive bioink based on piezoelectric hydrogel is as follows:

[0069] Take GelMA solid and add it to a sterile complete α-MEM medium, and incubate at room temperature for 1-2 h; then add silanized BTO and nHA, and incubate at 37-50 °C and 300-500 rpm for 20-30 min; then, add BMSCs from SD rats and co-incubate at 37-40 °C for 20-30 min; finally, add the photoinitiator LAP and incubate at 37-50 °C and 300-500 rpm for 30 min-1 h to obtain a bone-mimicking matrix force-responsive bioink based on piezoelectric hydrogel;

[0070] The concentration of the piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of nano-hydroxyapatite nHA is 50-70 mg / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, the concentration of GelMA is 80-100 mg / mL, and the concentration of the photoinitiator LAP is 2-2.5 mg / mL.

[0071] In one embodiment of the present invention, the preparation method of the cartilage-mimicking matrix force-responsive bioink based on piezoelectric hydrogel is as follows:

[0072] Take ChSMA solid and add it to a sterile complete α-MEM medium, and incubate at room temperature for 1-2 h; then add silanized BTO and TGF-β, and incubate at 37-50 °C and 300-500 rpm for 20-30 min; then, add BMSCs from SD rats and co-incubate at 37-40 °C for 20-30 min; finally, add the photoinitiator LAP and incubate at 37-50 °C and 300-500 rpm for 30 min-1 h to obtain a cartilage-mimicking matrix force-responsive bioink based on piezoelectric hydrogel,

[0073] The concentration of the piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of transforming growth factor TGF-β is 10-15 ng / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7cells / mL, the concentration of ChSMA is 40 - 100 mg / mL, and the concentration of the photoinitiator LAP is 2 - 2.5 mg / mL.

[0074] In one embodiment of the present invention, during printing, the specific parameters are a light intensity of 15 - 20 mW / cm -2 and an exposure time of 16 - 20 s. The number of base layers is set to 2, each base layer has an exposure time of 8 - 10 s, and the penetration depth of the material is controlled within 50 - 70 μm. During the entire printing process, the temperature of the platform and the resin tank for placing the bioink is maintained at 37 - 40°C.

[0075] In one embodiment of the present invention, after printing, the printed structure is placed in a complete α-MEM medium and cultured at 37°C.

[0076] The innovation of the present invention is mainly reflected in the successful construction of a cartilage-bone integrated organoid, and at the same time, it particularly emphasizes the important role of mechanical stimulation in tissue regeneration. Through the piezoelectric hydrogel and 3D bioprinting technology, a dual-niche structure of the cartilage layer and the bone layer is achieved. The application of the piezoelectric material barium titanate BTO enables mechanical force to be converted into bioelectric signals, significantly promoting cell activity and tissue regeneration, and providing a microenvironment closer to the physiological state for the regeneration of cartilage and bone tissues. In addition, the precision of the 3D bioprinting technology realizes the precise stratification of the cartilage and bone layers, breaking through the limitations of traditional methods in terms of size and integration, and significantly enhancing the potential and stability of the organoid in defect repair.

[0077] The present invention has significant advantages in simulating the functions of natural cartilage-bone. By combining the piezoelectric effect and 3D bioprinting technology, it provides higher biomimicry and functionality, and provides a more comprehensive and effective solution for the repair of articular cartilage defects.

[0078] The following exemplarily illustrates the preparation method of the cartilage-bone integrated organoid provided by the present invention.

[0079] A preparation method of a cartilage-bone integrated organoid, the preparation method mainly includes the following steps:

[0080] (1) Silanization of barium titanate:

[0081] Disperse 1 g of BTO in 100 mL of absolute ethanol and sonicate for 30 min. Subsequently, slowly add 1 mL of γ-methacryloxypropyltrimethoxysilane and adjust the pH value to 4 - 5 with acetic acid. Stir the mixture and react at 60 - 70 °C for 2 - 4 h, with ultrasonic treatment for 30 min every 30 min to prevent particle aggregation. After the reaction, add 100 mL of ethanol to terminate the process, and collect the particles by centrifugation at 8000 - 10000 rpm. Wash the collected particles 3 - 5 times with ethanol to remove impurities, and then dry them in vacuum at 50 - 60 °C. Store the final product in a sealed container to obtain the silanized barium titanate.

[0082] (2) Preparation of bone matrix-like force-responsive bioink based on piezoelectric hydrogel. Mix the silanized barium titanate BTO, nano-hydroxyapatite nHA, bone marrow-derived stem cells and GelMA hydrogel by co-incubation at room temperature to obtain the bone matrix-like force-responsive bioink based on piezoelectric hydrogel.

[0083] Specifically, take 0.1 - 0.5 g of GelMA and add it to 1 - 5 mL of sterile complete α-MEM medium, incubate at room temperature for 1 - 2 h; then add the silanized BTO and nHA, incubate at 37 - 50 °C and 300 - 500 rpm for 20 - 30 min; then, add bone marrow-derived stem cells and co-incubate at 37 - 40 °C for 20 - 30 min; finally, add the photoinitiator LAP and incubate at 37 - 50 °C and 300 - 500 rpm for 30 min - 1 h to obtain the bone matrix-like force-responsive bioink based on piezoelectric hydrogel.

[0084] (3) Preparation of cartilage matrix-like force-responsive bioink based on piezoelectric hydrogel. Mix the silanized barium titanate BTO, transforming growth factor TGF-β, bone marrow-derived stem cells and ChSMA hydrogel by co-incubation at room temperature to obtain the cartilage matrix-like force-responsive bioink based on piezoelectric hydrogel.

[0085] Specifically, take 0.1 - 0.5 g of ChSMA and add it to 1 - 5 mL of sterile complete α-MEM medium, incubate at room temperature for 1 - 2 h; then add the silanized BTO and TGF-β, incubate at 37 - 50 °C and 300 - 500 rpm for 20 - 30 min; then, add bone marrow-derived stem cells and co-incubate at 37 - 40 °C for 20 - 30 min; finally, add the photoinitiator LAP and incubate at 37 - 50 °C and 300 - 500 rpm for 30 min - 1 h to obtain the cartilage matrix-like force-responsive bioink based on piezoelectric hydrogel.

[0086] Specifically, the concentration of the piezoelectric material BTO is 90 - 100 mg / mL, the concentration of nano-hydroxyapatite nHA is 50 - 70 mg / mL, the concentration of transforming growth factor TGF-β is 10 - 15 ng / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL.

[0087] (4) Preparation of cartilage-bone integrated organoids. Using a 3D DLP stereolithography printer, the bone matrix-mimicking and cartilage matrix-mimicking force-responsive bioinks based on piezoelectric hydrogels are successively subjected to stereolithography printing to construct a dual-niche integrating a bone layer and a cartilage layer; finally, it is placed in a cell incubator for culture to obtain the cartilage-bone integrated organoids.

[0088] Specifically, the bone matrix-mimicking force-responsive bioink and the cartilage matrix-mimicking force-responsive bioink are respectively placed in a resin tank, and then executed according to the set printing parameters; then the printed structure is placed in a complete α-MEM culture medium at 37°C for culture to obtain the cartilage-bone integrated organoids.

[0089] In some embodiments, the specific parameters of 3D stereolithography printing are a light intensity of 15 - 20 mW cm -2 and an exposure time of 16 - 20 s. The number of base layers is set to 2, each base layer has an exposure time of 8 - 10 s, and the penetration depth of the material is controlled at 50 - 70 μm. During the entire printing process, the temperature of the platform and the resin tank for placing the bioink is maintained at 37 - 40°C.

[0090] The cartilage matrix-mimicking and bone matrix-mimicking force-responsive bioinks constructed based on piezoelectric hydrogels provided by the present invention respectively mimic cartilage and bone matrices. The cartilage matrix uses ChSMA hydrogel, which has good mechanical properties, supports chondroid differentiation, and acts together with barium titanate and transforming growth factor TGF-β to promote cartilage differentiation. The bone matrix uses GelMA hydrogel combined with nano-hydroxyapatite nHA. GelMA hydrogel can promote osteogenic differentiation, while nHA not only provides the structural stability of the bone matrix, but also promotes the mineralization process and enhances the mechanical properties of bone tissue.

[0091] In the present invention, barium titanate BTO is used as a piezoelectric material and incorporated into the hydrogel. Utilizing its characteristic of generating bioelectric signals when stressed, it further promotes the maturation of organoids and cell activities. The introduction of the piezoelectric material provides bioelectric stimulation to cartilage and bone tissues, enhances the bioactivity of organoids and improves the integration with surrounding tissues.

[0092] The present invention draws on the concept of microfracture surgery and uses 3D bioprinting technology to develop cartilage-bone integrated organoids. By using piezoelectric bioinks (ChSMA / BTO / TGF-β for cartilage, GelMA / BTO / nHA for bone), the directional differentiation of bone marrow-derived stem cells (BMSCs) is promoted. ChSMA combined with TGF-β supports cartilage differentiation, GelMA combined with nHA promotes osteogenic differentiation, while BTO converts mechanical force into bioelectric signals under mechanical stimulation, enhancing cell activity and promoting organoid maturation. This dual niche model mimics the structure of the natural cartilage-bone interface, enhances the repair potential of organoids, and promotes the regenerative application of cartilage and bone tissues.

[0093] The present invention uses 3D bioprinting technology to combine cartilage and bone matrices to construct a dual niche model. This model uses a single type of stem cell and forms cartilage and bone layers through layer-by-layer printing technology to mimic the structure and function of the natural cartilage-bone interface. This method has achieved breakthroughs in the size and integration of organoids, improving their potential and stability in defect repair and promoting the repair effect of cartilage-bone integrated organoids.

[0094] Further examples are listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description in this article, rather than being limited to the specific values in the following examples.

[0095] Unless otherwise specified, various reagents and raw materials used in the present invention are commercially available products or products that can be prepared by well-known methods.

[0096] Example 1

[0097] The preparation method of cartilage-bone integrated organoids is as follows:

[0098] (1) Preparation of silanized barium titanate. Disperse 1 g of barium titanate (BTO) in 100 mL of absolute ethanol and sonicate for 30 min. Then, slowly add 1 mL of γ-methacryloxypropyltrimethoxysilane and adjust the pH value to 5 using acetic acid. Stir the mixture and react at 60 °C for 2 h, sonicating for 30 min every 30 min to prevent particle aggregation. After the reaction is completed, add 100 mL of ethanol to terminate the reaction, and then collect the particles by centrifugation (10,000 rpm). The collected particles are washed 5 times with ethanol to remove impurities and dried in vacuo at 50 °C. The final product is stored in a sealed container to obtain silanized barium titanate.

[0099] (2) Preparation of bone matrix-mimicking force-responsive bioink based on piezoelectric hydrogel. Add 0.5 g of GelMA solid to 5 mL of sterile complete α-MEM medium and incubate at room temperature for 1 h; then add 0.45 g of silanized BTO and 0.25 g of nHA, and incubate at 45 °C and 500 rpm for 30 min; then, add 5×10 6 BMSCs derived from SD rats and co-incubate at 37 °C for 20 min; finally, add 12.5 mg of photoinitiator LAP and incubate at 37 °C and 450 rpm for 30 min to obtain bone matrix-mimicking force-responsive bioink based on piezoelectric hydrogel (the concentration of BTO is 90 mg / mL, the concentration of nHA is 50 mg / mL, and the concentration of BMSCs is 10 6 cells / mL).

[0100] (3) Preparation of cartilage matrix-mimicking force-responsive bioink based on piezoelectric hydrogel. Add 0.5 g of ChSMA solid to 5 mL of sterile complete α-MEM medium and incubate at room temperature for 1 h; then add 0.45 g of silanized BTO and 50 ng of TGF-β, and incubate at 45 °C and 500 rpm for 30 min; then, add 5×10 6 BMSCs derived from SD rats and co-incubate at 37 °C for 20 min; finally, add 12.5 mg of photoinitiator LAP and incubate at 37 °C and 450 rpm for 30 min to obtain cartilage matrix-mimicking force-responsive bioink based on piezoelectric hydrogel (the concentration of BTO is 90 mg / mL, the concentration of TGF-β is 10 ng / mL, and the concentration of BMSCs is 10 6 cells / mL).

[0101] (4) Construction of bone-cartilage integrated organoids. Place the bone matrix-mimicking force-responsive bioink and cartilage matrix-mimicking force-responsive bioink into resin tanks respectively, and then print according to the set 3D photocuring printing parameters. The specific printing parameters are a light intensity of 15 mW·cm-2 and an exposure time of 16 s. The number of base layers is set to 2, the exposure time for each layer is 10 s, and the material penetration depth is controlled at 50 μm. The temperature of the platform and resin tank is maintained at 37 °C during the whole printing process. Then place the printed structure in complete α-MEM medium at 37 °C for culture, and finally obtain cartilage-bone integrated organoids.

[0102] Figure 1 、 2 、3 are the voltages generated by the piezoelectric hydrogel prepared in Example 1 at different pressure levels (0.1, 0.5, and 1 N); among them, Figure 1 、 2, the ordinate of 3 is voltage. As can be seen from the figure, as the pressure increases from 0.1 N to 1 N, the piezoelectric hydrogel always generates significantly higher voltages, highlighting its superior piezoelectric properties.

[0103] Figure 4 and Figure 5 are the butterfly curve and hysteresis curve of the piezoelectric hydrogel prepared in Example 1; among them, Figure 4 the ordinate of is amplitude, Figure 5 the ordinate of is phase. As can be seen from the figure, the piezoelectric (BTO composite) hydrogel exhibits a significant piezoelectric effect, including reversible mechanical deformation and polarization hysteresis phenomena.

[0104] Figure 6 and Figure 7 are the effect diagrams of the expression levels of cartilage differentiation-related genes SOX9 and Col2A1 under different material conditions after 4 weeks of in vitro culture of the cartilage-bone integrated organoids prepared in Example 1; Figure 6 the ordinate of is the expression level of SOX9, Figure 7 the ordinate of is the expression level of Col2A1. As can be seen from the figure, the TGF-β / BTO combination significantly enhances the expression levels of cartilage differentiation-related genes.

[0105] Figure 8 , 9 , 10, 11 are the effect diagrams of bone density, bone volume / total volume ratio, trabecular thickness and trabecular number of the cartilage-bone integrated organoids prepared in Example 1 after 4 weeks and 8 weeks of in vitro culture. As can be seen from the figure, although the monolayer bone organoids show higher indicators in terms of bone density, bone volume / total volume ratio, trabecular thickness and trabecular number from week 4 to week 8, the bilayer cartilage-bone integrated organoids also show gradual improvement in mineralization performance.

[0106] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. Obviously, 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 labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A bio-ink for preparing cartilage-bone integrated organoids, characterized in that: Including bone matrix force-responsive bio-ink based on piezoelectric hydrogel and cartilage matrix force-responsive bio-ink based on piezoelectric hydrogel; The bone matrix force-responsive bio-ink constructed based on piezoelectric hydrogel includes: GelMA hydrogel as a dispersion system and piezoelectric materials barium titanate BTO, nanohydroxyapatite nHA and bone marrow-derived stem cells dispersed in the GelMA hydrogel; The cartilage matrix force-responsive biological ink constructed based on piezoelectric hydrogel includes: a ChSMA hydrogel as a dispersed system and piezoelectric materials barium titanate BTO, transforming growth factor TGF-β and bone marrow-derived stem cells dispersed in the ChSMA hydrogel.

2. The biological ink for preparing cartilage-bone integrated organoids according to claim 1, characterized in that: In the bone matrix force-responsive bio-ink constructed based on piezoelectric hydrogel, the concentration of piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of nanohydroxyapatite nHA is 50-70 mg / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, and the concentration of GelMA was 80-100 mg / mL; The bone matrix force-responsive biological ink constructed based on piezoelectric hydrogel contains a photoinitiator, and the concentration of the photoinitiator is 2-2.5 mg / mL.

3. The biological ink for preparing cartilage-bone integrated organoids according to claim 1, characterized in that: In the cartilage-like matrix force-responsive bio-ink constructed based on piezoelectric hydrogel, the concentration of piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of transforming growth factor TGF-β is 10-15 ng / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, the concentration of ChSMA is 40-100 mg / mL; The cartilage-mimicking matrix force-responsive biological ink constructed based on piezoelectric hydrogel contains a photoinitiator, and the concentration of the photoinitiator is 2-2.5 mg / mL.

4. The biological ink for preparing cartilage-bone integrated organoids according to claim 1, characterized in that: The piezoelectric material barium titanate BTO is silanized barium titanate; The preparation method of the silanized barium titanate is: 8-10 mg / mL barium titanate is dispersed in anhydrous ethanol, and ultrasonically treated for 20-30 minutes. Then, 0.1%-1% γ-methacryloxypropyltrimethoxysilane is slowly added, and the pH value is adjusted to 4-5 with acetic acid; the mixture is stirred and reacted at 60-70°C for 2-4 hours, and ultrasonically treated for 20-30 minutes every 30-40 minutes. After the reaction is completed, ethanol is added to terminate the reaction, and then particles are collected by centrifugation. The collected particles are washed with ethanol to remove impurities and dried to obtain silanized barium titanate.

5. A cartilage-bone integrated organoid, characterized in that: The cartilage-bone integrated organoid comprises a bone-mimicking layer and a cartilage-mimicking layer, wherein the bone-mimicking layer is printed by a bone-mimicking matrix force-responsive biological ink constructed based on piezoelectric hydrogel, and the cartilage-mimicking layer is printed by a cartilage-mimicking matrix force-responsive biological ink constructed based on piezoelectric hydrogel; The bone matrix force-responsive biological ink constructed based on piezoelectric hydrogel and the cartilage matrix force-responsive biological ink constructed based on piezoelectric hydrogel are the biological inks defined in any one of claims 1-4.

6. A method for preparing cartilage-bone integrated organoids according to claim 5, characterized in that: The following steps are involved: The 3D bioprinting technology is used to print and solidify the bone-like matrix force-responsive bio-ink based on piezoelectric hydrogel into a bone-like layer. Using 3D bioprinting technology, a cartilage-like matrix force-responsive bio-ink constructed based on piezoelectric hydrogel is printed and solidified on the upper layer of the bone-like layer to form a cartilage-like layer; The printed structure was cultured in complete α-MEM medium to eventually obtain cartilage-bone integrated organoids.

7. The method for preparing cartilage-bone integrated organoids according to claim 6, characterized in that: The preparation method of bone matrix force-responsive bio-ink based on piezoelectric hydrogel is as follows: Take GelMA solid and add it to sterile complete α-MEM culture medium, incubate at room temperature for 1-2 hours; then add silanized BTO and nHA, incubate at 37-50°C, 300-500rpm for 20-30 minutes; then add BMSCs from SD rats, and incubate at 37-40°C for 20-30 minutes; finally, add photoinitiator LAP, incubate at 37-50°C, 300-500rpm for 30 minutes to 1 hour, and obtain bone matrix force-responsive bio-ink constructed based on piezoelectric hydrogel; The concentration of the piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of nanohydroxyapatite nHA is 50-70 mg / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, the concentration of GelMA is 80-100 mg / mL, and the concentration of photoinitiator LAP is 2-2.5 mg / mL.

8. The method for preparing cartilage-bone integrated organoids according to claim 6, characterized in that: The preparation method of cartilage-like matrix force-responsive bio-ink based on piezoelectric hydrogel is as follows: Take the ChSMA solid and add it to the sterile complete α-MEM culture medium, incubate it at room temperature for 1-2 hours; then add the silanized BTO and TGF-β, incubate it at 37-50°C and 300-500rpm for 20-30 minutes; then add BMSCs from SD rats and incubate it at 37-40°C for 20-30 minutes; finally, add the photoinitiator LAP, incubate it at 37-50°C and 300-500rpm for 30 minutes to 1 hour, and obtain the cartilage matrix force-responsive bio-ink constructed based on piezoelectric hydrogel. The concentration of the piezoelectric material barium titanate BTO is 90-100 mg / mL, the concentration of transforming growth factor TGF-β is 10-15 ng / mL, and the concentration of bone marrow-derived stem cells is 10 6 ~10 7 cells / mL, the concentration of ChSMA is 40-100 mg / mL, and the concentration of photoinitiator LAP is 2-2.5 mg / mL.

9. The method for preparing cartilage-bone integrated organoids according to claim 6, characterized in that: When printing, the specific parameters are 15-20mW cm -2 The light intensity and exposure time were set to 16-20 s, the number of base layers was set to 2, each base layer had an exposure time of 8-10 s, the penetration depth of the material was controlled at 50-70 μm, and the temperature of the platform and the resin tank for placing the bio-ink was maintained at 37-40 °C during the entire printing process.

10. The method for preparing cartilage-bone integrated organoids according to claim 6, characterized in that: After printing, the cartilage-bone integrated organoids were cultured in complete α-MEM medium at 37°C.

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