3D bio-printing osteochondral organ based on dynamic mechanical regulation and control as well as construction method and application of 3D bio-printing osteochondral organ

Abstract

The invention provides a 3D biological printing osteochondral organ based on dynamic mechanical regulation and control, and a construction method and application thereof. Compared with the prior art, the invention innovatively provides a biphasic scaffold optimization modification strategy: firstly, an osteochondral hydrogel scaffold with an accurate layered structure is prepared by adopting a 3D biological printing technology, then a cell mechanical transduction signal channel is activated by a dynamic pressure stimulation system, and cartilage / bone biphasic differentiation of stem cells is directionally regulated and controlled; and by constructing a bionic mechanical microenvironment, the obtained osteochondral organ not only keeps excellent biocompatibility, but also has gradient mechanical properties and a clear spatial layered structure (cartilage layer-bone layer), the directional cartilage formation / osteogenic differentiation efficiency of the osteochondral organ is remarkably improved, and the osteochondral organ has a good application prospect. The problems of spatial heterogeneity construction and accurate regulation and control of cell fate in osteochondral tissue engineering are effectively solved, and the method has important application value in the fields of osteochondral defect repair treatment and mechanical stimulation mechanism research.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and particularly relates to a 3D bioprinted osteochondral organoid based on dynamic mechanical regulation, a construction method thereof, and an application thereof. Background Art

[0002] Due to the avascular structure of articular cartilage tissue, articular cartilage injuries are often difficult to self-repair. Clinically, autologous chondrocyte transplantation, autologous or allogeneic cartilage transplantation are generally used to treat large-scale cartilage defects, but there are problems such as limited sources of materials. With the combined development of stem cell technology and osteochondral tissue engineering, stem cell-based in vitro osteochondral organoids have been successively developed successfully.

[0003] Currently, the studied articular osteochondral organoids only simply mix the scaffold with seed cells or chondrogenic induction factors, lacking a gradient arrangement structure similar to normal cartilage tissue. It is difficult to achieve good fusion between the scaffold and the surrounding tissues after implantation, thus affecting the flatness of the articular cartilage surface and the overall stress relationship, resulting in poor repair effects. Moreover, existing organoid culture technologies only consider biochemical and physical properties and do not fully consider the mechanical environment in which cells are located. In the in vitro osteochondral organoid culture technology, due to the lack of mechanical stimulation, it is difficult to induce mesenchymal stem cells (MSCs) to differentiate into cartilage and bone stratified structures spatially, directionally, and controllably. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a 3D bioprinted osteochondral organoid based on dynamic mechanical regulation, a construction method thereof, and an application thereof.

[0005] The present invention provides a construction method of a 3D bioprinted osteochondral organoid based on dynamic mechanical regulation, comprising the following steps:

[0006] S1) Providing cartilage layer bioink and bone layer bioink;

[0007] The cartilage layer bioink comprises 1% w / v - 5% w / v of a first hydrogel matrix, 5% w / v - 10% w / v of a second hydrogel matrix, 0.1% w / v - 0.5% w / v of a photoinitiator, 1×10 5 ~1×10 7 chondrocytes per mL and a culture medium;

[0008] The bone layer bioink comprises 1% w / v - 5% w / v of a first hydrogel matrix, 5% w / v - 10% w / v of a second hydrogel matrix, 0.1% w / v - 0.5% w / v of a photoinitiator, 1% w / v - 10% w / v of hydroxyapatite, 1×10 5 ~1×10 7Osteoblasts at a density of

[0009] The first hydrogel matrix is selected from hyaluronic acid-based hydrogel matrices and / or sodium alginate-based hydrogel matrices;

[0010] The second hydrogel matrix is selected from gelatin-based hydrogel matrices;

[0011] S2) Photocuring and crosslinking the bone layer bioink using a 3D printer to obtain a bone layer; then photocuring and crosslinking the cartilage layer bioink on the surface of the bone layer to form a cartilage layer, obtaining an osteochondral biphasic scaffold;

[0012] S3) Subjecting the osteochondral biphasic scaffold to dynamic pressure stimulation to obtain a 3D bioprinted osteochondral organoid.

[0013] Preferably, the first hydrogel matrix is selected from one or more of methacrylated hyaluronic acid, hyaluronic acid methacrylate, and methacrylated sodium alginate;

[0014] The second hydrogel matrix is selected from methacrylated gelatin;

[0015] The photoinitiator is selected from lithium phenyl-2,4,6-trimethylbenzoylphosphinate and / or 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone.

[0016] Preferably, the cartilage layer bioink comprises 2% w / v of the first hydrogel matrix, 7.5% w / v of the second hydrogel matrix, 0.2% w / v of the photoinitiator, 1×10 6 chondrocytes per mL and a culture medium;

[0017] The bone layer bioink comprises 2% w / v of the first hydrogel matrix, 7.5% w / v of the second hydrogel matrix, 0.2% w / v of the photoinitiator, 5% w / v of hydroxyapatite, 1×10 6 osteoblasts per mL and a culture medium.

[0018] Preferably, the cartilage layer bioink is prepared according to the following method:

[0019] Mix 1% w / v - 5% w / v of the first hydrogel matrix, 5% w / v - 10% w / v of the second hydrogel matrix, 0.1% w / v - 0.5% w / v of the photoinitiator with a culture medium, filter and sterilize, and add 1×10 5 - 1×10 7 chondrocytes per mL to obtain the cartilage layer bioink;

[0020] The bone layer bioink is prepared according to the following method:

[0021] Mix 1% w / v - 5% w / v of the first hydrogel matrix, 5% w / v - 10% w / v of the second hydrogel matrix, 0.1% w / v - 0.5% w / v of a photoinitiator, 1% w / v - 10% w / v of hydroxyapatite with a culture medium, filter and sterilize, and add 1×10 5 ~1×10 7 osteoblasts per mL to obtain a bone layer bioink.

[0022] Preferably, in step S2), the temperature for photocuring crosslinking printing of the bone layer bioink is 20°C - 25°C; the extrusion speed of the bone layer bioink is 1.3 - 1.5 mm 3 / s; the printing speed of the bone layer bioink for photocuring crosslinking is 1 - 10 mm / s; the layer height of the bone layer bioink for photocuring crosslinking is 0.1 - 0.3 mm;

[0023] In step S2), the temperature for photocuring crosslinking printing of the cartilage layer bioink is 20°C - 25°C; the extrusion speed of the cartilage layer bioink is 1.3 - 1.5 mm 3 / s; the printing speed of the cartilage layer bioink for photocuring crosslinking is 1 - 10 mm / s; the layer height of the cartilage layer bioink for photocuring crosslinking is 0.1 - 0.3 mm.

[0024] Preferably, the thickness ratio of the bone layer to the cartilage layer in the osteochondral biphasic scaffold is (1 - 2):1.

[0025] Preferably, in step S3), during dynamic pressure stimulation, the pressure probe contacts the cartilage layer of the osteochondral biphasic scaffold; the downward pressing depth of the pressure probe is 5% - 15% of the thickness of the osteochondral biphasic scaffold.

[0026] Preferably, the frequency of the dynamic pressure stimulation is 0.5 - 2 Hz; the stimulation time for each dynamic pressure stimulation is 0.5 - 2 h, with a stop of 20 - 24 h; the time for dynamic pressure stimulation is 5 - 10 days.

[0027] Preferably, the frequency of the dynamic pressure stimulation is 1 Hz; the stimulation time for each dynamic pressure stimulation is 1 h, with a stop of 23 h; the time for dynamic pressure stimulation is 7 days.

[0028] The present invention also provides a 3D bioprinted osteochondral organoid based on dynamic mechanical regulation constructed by the above construction method.

[0029] The present invention also provides an application of the above 3D bioprinted osteochondral organoid based on dynamic mechanical regulation in the preparation of bone repair materials.

[0030] The present invention provides a method for constructing 3D bioprinted osteochondral organoids based on dynamic mechanical regulation, comprising the following steps: S1) providing a cartilage layer bioink and a bone layer bioink; the cartilage layer bioink includes 1% w / v to 5% w / v of a first hydrogel matrix, 5% w / v to 10% w / v of a second hydrogel matrix, 0.1% w / v to 0.5% w / v of a photoinitiator, 1×10 5 ~1×10 7 chondrocytes per mL and a culture medium; the bone layer bioink includes 1% w / v to 5% w / v of a hyaluronic acid-based photocurable crosslinking agent, 5% w / v to 10% w / v of a gelatin-based photocurable crosslinking agent, 0.1% w / v to 0.5% w / v of a photoinitiator, 1% w / v to 10% w / v of hydroxyapatite, 1×10 5 ~1×10 7 osteoblasts per mL and a culture medium; S2) subjecting the bone layer bioink to photocurable crosslinking printing using a 3D printer to obtain a bone layer; then subjecting the cartilage layer bioink to photocurable crosslinking printing on the surface of the bone layer to form a cartilage layer, thereby obtaining an osteochondral biphasic scaffold; S3) subjecting the osteochondral biphasic scaffold to dynamic pressure stimulation to obtain 3D bioprinted osteochondral organoids based on dynamic mechanical regulation. Aiming at the technical bottleneck in the spatial directed differentiation control of existing mesenchymal stem cells, the present invention innovatively proposes a strategy for optimizing and modifying the biphasic scaffold: first, a 3D bioprinting technology is used to prepare an osteochondral hydrogel scaffold with a precise layered structure, and then the cell mechanotransduction signaling pathway is activated through a dynamic pressure stimulation system to directionally regulate the chondrogenic / osteogenic biphasic differentiation of stem cells; at the same time, compared with the traditional static culture mode, this method constructs a biomimetic mechanical microenvironment, so that the obtained osteochondral organoids not only maintain excellent biocompatibility, but also have gradient mechanical properties and a clear spatial layered structure (cartilage layer - bone layer), and their directed chondrogenic / osteogenic differentiation efficiency is significantly improved. This breakthrough effectively solves the problems of spatial heterogeneity construction and precise regulation of cell fate in osteochondral tissue engineering, and has important application value in the fields of osteochondral defect repair treatment and mechanical stimulation mechanism research. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the construction process and characterization diagram of 3D bioprinted osteochondral organoids based on dynamic mechanical regulation provided by the present invention;

[0032] Figure 2 is the effect diagram after printing the biphasic scaffold;

[0033] Figure 3 is the scanning electron micrograph of the freeze-dried biphasic scaffold;

[0034] Figure 4 is the scanning electron micrograph of the freeze-dried cartilage scaffold and bone scaffold;

[0035] Figure 5 EDS analysis diagram of the biphasic scaffold;

[0036] Figure 6 Infrared spectrum diagram of the cartilage layer and bone layer;

[0037] Figure 7 Stress-strain curve diagram of the biphasic scaffold;

[0038] Figure 8 Physical diagram of the hydrogel compression device;

[0039] Figure 9 Confocal laser microscopy images of the biphasic scaffold with and without pressure stimulation;

[0040] Figure 10 Immunofluorescence images of the ability of the biphasic scaffold with and without pressure stimulation to regulate the osteogenic / chondrogenic ability of loaded stem cells;

[0041] Figure 11 RT-PCR images of the ability of the biphasic scaffold with and without pressure stimulation to regulate the osteogenic / chondrogenic ability of loaded stem cells;

[0042] Figure 12 Western Blot (WB) experimental result images of the biphasic scaffold with and without pressure stimulation;

[0043] Figure 13 Pairwise comparison diagram of the repair effect of osteochondral defect after implanting the organoids. Detailed implementation manner

[0044] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0045] The present invention provides a method for constructing 3D bioprinted osteochondral organoids based on dynamic mechanical regulation, including the following steps: S1) providing cartilage layer bioink and bone layer bioink; the cartilage layer bioink includes 1% w / v to 5% w / v of a first hydrogel matrix, 5% w / v to 10% w / v of a second hydrogel matrix, 0.1% w / v to 0.5% w / v of a photoinitiator, 1×10 5 ~1×10 7chondrocytes per mL and culture medium; the bone layer bioink comprises 1% w / v - 5% w / v hyaluronic acid-based photocurable crosslinking agent, 5% w / v - 10% w / v gelatin-based photocurable crosslinking agent, 0.1% w / v - 0.5% w / v photoinitiator, 1% w / v - 10% w / v hydroxyapatite, 1×10 5 ~1×10 7 osteoblasts per mL and culture medium; the first hydrogel matrix is selected from hyaluronic acid-based hydrogel matrix and / or sodium alginate-based hydrogel matrix; the second hydrogel matrix is selected from gelatin-based hydrogel matrix; S2) subject the bone layer bioink to photocurable crosslinking printing using a 3D printer to obtain a bone layer; then subject the cartilage layer bioink to photocurable crosslinking printing on the surface of the bone layer to form a cartilage layer, thereby obtaining a bone-cartilage biphasic scaffold; S3) subject the bone-cartilage biphasic scaffold to dynamic pressure stimulation to obtain a 3D bioprinted bone-cartilage organ based on dynamic mechanical regulation.

[0046] In the present invention, through 3D bioprinting, good stratification and good biocompatibility of the biphasic structure can be achieved. By pressure stimulation, the corresponding signal pathways are activated to induce and maintain the differentiation and phenotype of the bone-cartilage scaffold cells, thereby constructing a bone-cartilage organ with good stratification.

[0047] Among them, the present invention does not have any special restrictions on the sources of all raw materials, and they can be commercially available.

[0048] First, cartilage layer bioink and bone layer bioink are provided.

[0049] In a specific embodiment provided by the present invention, the cartilage layer bioink comprises 1% w / v - 5% w / v first hydrogel matrix, 5% w / v - 10% w / v second hydrogel matrix, 0.1% w / v - 0.5% w / v photoinitiator, 1×10 5 ~1×10 7 chondrocytes per mL and culture medium; where w / v refers to mg / mL; the first hydrogel matrix is preferably one or more of methacrylated hyaluronic acid, hyaluronic acid methacrylate, and methacrylated sodium alginate; the second hydrogel matrix is preferably methacrylated gelatin; the photoinitiator is preferably lithium phenyl-2,4,6-trimethylbenzoylphosphinate and / or 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959); the culture medium is preferably complete culture medium.

[0050] In a specific embodiment provided by the present invention, the cartilage layer bioink preferably comprises 1% w / v - 3% w / v first hydrogel matrix, 6% w / v - 8% w / v second hydrogel matrix, 0.1% w / v - 0.3% w / v photoinitiator, 0.5×105 ~0.5×10 6 chondrocytes per mL and culture medium; more preferably, it comprises 2% w / v of a first hydrogel matrix, 7.5% w / v of a second hydrogel matrix, 0.2% w / v of a photoinitiator, 1×10 6 chondrocytes per mL and culture medium.

[0051] In a specific embodiment provided by the present invention, the cartilage layer bioink comprises 1% w / v - 5% w / v of a first hydrogel matrix, 5% w / v - 10% w / v of a second hydrogel matrix, 0.1% w / v - 0.5% w / v of a photoinitiator, 1% w / v - 10% w / v of hydroxyapatite, 1×10 5 ~1×10 7 chondrocytes per mL and culture medium; where w / v refers to mg / mL; the first hydrogel matrix is preferably one or more of methacrylated hyaluronic acid, hyaluronic acid methacrylate, and methacrylated sodium alginate; the second hydrogel matrix is preferably methacrylated gelatin; the photoinitiator is preferably lithium phenyl-2,4,6-trimethylbenzoylphosphinate and / or 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959); the culture medium is preferably complete medium.

[0052] In a specific embodiment provided by the present invention, the cartilage layer bioink is prepared by the following method: Mix 1% w / v - 5% w / v of a first hydrogel matrix, 5% w / v - 10% w / v of a second hydrogel matrix, 0.1% w / v - 0.5% w / v of a photoinitiator with the culture medium, filter and sterilize, and add 1×10 5 ~1×10 7 chondrocytes per mL to obtain the cartilage layer bioink; the filtration and sterilization preferably use a 0.45 μm bacterial filter.

[0053] In a specific embodiment provided by the present invention, the chondrocytes are preferably prepared by the following method: Take the bone marrow tissue of a neonatal rabbit, prepare a cell suspension with complete medium, and culture it. When the cell fusion is about 90%, add trypsin for digestion and passage to obtain bone marrow mesenchymal stem cells; inoculate the bone marrow mesenchymal stem cells into complete medium and culture until the cell solution is 80%, then change the medium to chondrogenic differentiation medium for culture to obtain chondrocytes.

[0054] In a specific embodiment provided by the present invention, the bone layer bioink preferably comprises 1% w / v - 3% w / v of a first hydrogel matrix, 6% w / v - 8% w / v of a second hydrogel matrix, 0.1% w / v - 0.3% w / v of a photoinitiator, 3% w / v - 8% w / v of hydroxyapatite, 0.5×10 5 ~0.5×10 6 osteoblasts per mL and a culture medium; more preferably, it comprises 2% w / v of the first hydrogel matrix, 7.5% w / v of the second hydrogel matrix, 0.2% w / v of the photoinitiator, 5% w / v of hydroxyapatite, 1×10 6 osteoblasts per mL and the culture medium.

[0055] In a specific embodiment provided by the present invention, the bone layer bioink is prepared according to the following method: Mix 1% w / v - 5% w / v of the first hydrogel matrix, 5% w / v - 10% w / v of the second hydrogel matrix, 0.1% w / v - 0.5% w / v of the photoinitiator, 1% w / v - 10% w / v of hydroxyapatite and the culture medium, then filter and sterilize, and add 1×10 5 ~1×10 7 osteoblasts per mL to obtain the bone layer bioink; the filtration and sterilization preferably use a 0.45 μm bacterial filter.

[0056] In a specific embodiment provided by the present invention, the osteoblasts are prepared according to the following method: Inoculate bone marrow mesenchymal stem cells into a complete culture medium and culture until the cell solution reaches 80%, then change the culture medium to an osteogenic differentiation medium for culture to obtain osteoblasts.

[0057] The bone layer bioink is subjected to photocuring crosslinking printing using a 3D printer to obtain a bone layer; the temperature of the photocuring crosslinking printing of the bone layer bioink is preferably 20°C - 25°C; the extrusion speed of the bone layer bioink is preferably 1.3 - 1.5 mm 3 / s, more preferably 1.4 mm 3 / s; the printing speed of the photocuring crosslinking of the bone layer bioink is preferably 1 - 10 mm / s, more preferably 3 - 8 mm / s, still more preferably 4 - 6 mm / s, and most preferably 5 mm / s; the layer height of the photocuring crosslinking printing of the bone layer bioink is preferably 0.1 - 0.3 mm, more preferably 0.2 mm; the photocuring intensity of the photocuring crosslinking printing of the bone layer bioink is preferably 2.97 - 9.48 mw / cm 2 , more preferably 3.42 - 5.78 mw / cm 2 .

[0058] Then, the chondral layer bioink is subjected to photocuring crosslinking printing on the surface of the osseous layer to form the chondral layer, obtaining an osteochondral biphasic scaffold; the temperature for photocuring crosslinking printing of the chondral layer bioink is preferably 20°C to 25°C; the extrusion speed of the chondral layer bioink is preferably 1.3 to 1.5 mm 3 / s, more preferably 1.4 mm 3 / s; the printing speed of the chondral layer bioink for photocuring crosslinking is preferably 1 to 10 mm / s, more preferably 3 to 8 mm / s, still more preferably 4 to 6 mm / s, and most preferably 5 mm / s; the layer height of the chondral layer bioink for photocuring crosslinking printing is preferably 0.1 to 0.3 mm, more preferably 0.2 mm; the photocuring intensity of the chondral layer bioink for photocuring crosslinking printing is preferably 2.97 to 9.48 mw / cm 2 2, more preferably 3.42 to 5.78 mw / cm 2 .

[0059] In a specific embodiment provided by the present invention, the thickness ratio of the osseous layer to the chondral layer in the osteochondral biphasic scaffold is preferably (1 to 2):1, more preferably 1.5:1.

[0060] The osteochondral biphasic scaffold is subjected to dynamic pressure stimulation to obtain a 3D bioprinted osteochondral organoid based on dynamic mechanical regulation; when performing the dynamic pressure stimulation, the pressure probe is in contact with the chondral layer of the osteochondral biphasic scaffold; in the present invention, the pressure probe is preferably used after being sequentially sterilized by ultraviolet light, soaked in alcohol, and washed with PBS; the downward pressing depth of the pressure probe is preferably 5% to 15% of the thickness of the osteochondral biphasic scaffold, more preferably 8% to 12%; the frequency of the dynamic pressure stimulation is preferably 0.5 to 2 Hz, more preferably 0.5 to 1.5 Hz, still more preferably 1 Hz; the dynamic pressure stimulation is specifically a repeated pressure stimulation and stop step, and the duration of each stimulation is preferably 0.5 to 2 h, more preferably 0.5 to 1.5 h, still more preferably 1 h; stop for 20 to 24 h, preferably stop for 21 to 24 h, still more preferably 22 to 23 h, and most preferably 23 h; the duration of the dynamic pressure stimulation is preferably 5 to 10 days, more preferably 6 to 8 days, still more preferably 7 days. Mechanical stimulation can promote the differentiation and development of chondrocytes and osteocytes and maintain their cell phenotypes.

[0061] In a specific embodiment provided by the present invention, the 3D bioprinted osteochondral organoid based on dynamic mechanical regulation is constructed and characterized according to the Figure 1 process shown.

[0062] The present invention innovatively proposes a strategy for optimizing and modifying a biphasic scaffold: First, a 3D bioprinting technique is used to prepare an osteochondral hydrogel scaffold with an accurate hierarchical structure. Subsequently, a dynamic pressure stimulation system is used to activate the cell mechanotransduction signaling pathway, and the chondrogenic / osteogenic biphasic differentiation of stem cells is directionally regulated. At the same time, compared with the traditional static culture mode, this method constructs a biomimetic mechanical microenvironment, enabling the obtained osteochondral organoids to not only maintain excellent biocompatibility but also possess gradient mechanical properties and a clear spatial hierarchical structure (chondral layer - bone layer). The efficiency of their directional chondrogenic / osteogenic differentiation is significantly improved. This breakthrough effectively solves the problems of spatial heterogeneity construction and precise regulation of cell fate in osteochondral tissue engineering and has important application value in the fields of osteochondral defect repair treatment and mechanical stimulation mechanism research.

[0063] The present invention also provides a 3D bioprinted osteochondral organoid based on dynamic mechanical regulation constructed by the above construction method.

[0064] The present invention also provides an application of the above 3D bioprinted osteochondral organoid based on dynamic mechanical regulation in the preparation of bone repair materials.

[0065] To further illustrate the present invention, the following examples are used to describe in detail a 3D bioprinted osteochondral organoid based on dynamic mechanical regulation, its construction method, and its application provided by the present invention.

[0066] The reagents used in the following examples are all commercially available; Gelma (Shuhe (Wenzhou) Biotechnology Co., Ltd.), lap (Yuanye (Shanghai) Biotechnology Co., Ltd.), osteogenic medium (Procell (Wuhan) Life Science Co., Ltd.), chondrogenic medium (Procell (Wuhan) Life Science Co., Ltd.) were used in the examples. Live / Dead kit: Beyotime (Shanghai) Biotechnology Co., Ltd.;

[0067] Complete medium: 89% F12 basal medium; 10% fetal bovine serum; 1% penicillin - streptomycin;

[0068] MeHA was synthesized according to the following steps: First, 500 mg of hyaluronic acid was weighed and dissolved in 50 mL of deionized water. Under ice bath conditions, an acylation reaction was carried out with 1.5 mL of methacrylic anhydride, and the pH value of the reaction system was maintained between 8 - 9 by dropping NaOH solution. After a 24 - hour ice bath reaction, gradient purification was carried out using a dialysis bag with a molecular weight cut - off of 3500 (changing water every 1 - 2 hours initially and gradually extending to 8 hours / time), and finally, a white flocculent Me - HA product was obtained by freeze - drying. This functional modification endows the material with photocuring characteristics, and the hardness of the hydrogel matrix can be regulated by adjusting the HA molecular weight, and precise cross - linking of the three - dimensional network structure can be achieved under specific wavelength light irradiation with a photoinitiator.

[0069] Example 1

[0070] 1.1 Preparation of Bone-Chondral Bioink Loaded with Bone Marrow Mesenchymal Stem Cells (BMSCs)

[0071] 1.1.1 Cell Culture and Predifferentiation Treatment

[0072] Newborn rabbits within 1 week of birth were selected and sacrificed by cervical dislocation. They were completely immersed in a beaker containing 75% ethanol for 20 minutes and then transferred to a laminar flow hood. The bilateral femurs and tibias were separated and transferred to a petri dish containing PBS. The attached muscles and tissues were carefully removed. Subsequently, both ends were cut intermittently to expose the medullary cavity. A 1 mL syringe was used to aspirate the complete medium, and the medullary cavity was repeatedly rinsed and the bone marrow tissue was blown into a cell suspension, which was placed in an incubator at 5% CO 2 , 37 °C for culture. After 3 days, half of the medium was changed. When the cell confluence reached about 90%, the medium was discarded, and the cells were rinsed twice with PBS. 2 mL of trypsin solution was added, shaken well, and placed in the incubator for digestion for 1 minute. When the cells shrank and the cell gaps increased, the trypsin was discarded, and 2 mL of complete medium was added to terminate the digestion and passage was carried out.

[0073] Osteogenic Differentiation: The third-generation rabbit BMSCs were seeded in a 6-well plate at a cell density of 1×10 6 cells / mL. When the cell confluence reached 80%, the complete medium was replaced with osteogenic differentiation medium, and the osteogenic differentiation medium was changed every 3 days for a total of 1 week of culture.

[0074] Chondrogenic Differentiation: The third-generation BMSCs were seeded in a 6-well plate at a cell density of 1×10 6 cells / mL. When the cell confluence reached 80%, the complete medium was replaced with chondrogenic differentiation medium, and the chondrogenic differentiation medium was changed every 3 days for a total of 1 week of culture.

[0075] 1.1.2 Preparation of Stem Cell-Loaded Bioink

[0076] Bioink for the cartilage layer: 2 w / v% methacrylated hyaluronic acid (MeHA), 7.5 w / v% methacrylated gelatin (GelMA), and 0.2 w / v% photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP). Taking the preparation of 1 mL of bioink as an example: 20 mg of Me-HA, 75 mg of GelMA, and 2 mg of LAP were added to 1 mL of complete medium, and magnetic stirring was carried out until completely dissolved. After complete dissolution, it was filtered using a 0.45 μm bacterial filter. The BMSCs after chondrogenic predifferentiation treatment were mixed with the bioink at a density of 1×10 6 cells / mL.

[0077] Bone layer bio-ink: 2w / v% methacryloyl hyaluronic acid (MeHA), 7.5w / v% methacryloyl gelatin (GelMA), 5w / v% nanohydroxyapatite (nHA) and 0.2w / v% photoinitiator (phenyl-2,4,6-trimethylbenzoylphosphonate lithium, LAP). Take 1 mL of bio-ink preparation as an example: add 20 mg of Me-HA, 75 mg of GelMA, 50 mg of nHA and 2 mg of LAP to 1 mL of complete culture medium and stir magnetically until completely dissolved. After fully dissolved, filter with a 0.45 μm bacterial filter. After osteogenic pre-differentiation treatment, 1×10 6 The density of 1000 g / mL was mixed with the bio-ink.

[0078] 1.2 Dual-phase osteochondral scaffold printing

[0079] The overall osteochondral scaffold is designed to have a square area of ​​10 mm × 10 mm, with an overall thickness of 5 mm; the thickness of the cartilage layer is 2 mm; and the thickness of the bone layer is 3 mm. The SunP BioMaker 2i bio-3D printer from China was used for photocuring cross-linking printing. After printing the bone layer, the cartilage layer was printed on its surface to obtain an osteochondral biphasic scaffold.

[0080] The temperature of the cartilage layer printing syringe was 20°C; the extrusion speed was 1.4 mm 3 / s; printing speed 5 mm / s; printing needle inner diameter 0.25 mm; printing layer height 0.2 mm; light curing intensity 3.42 mw / cm 2 ; The number of printing layers is 10.

[0081] The temperature of the bone layer printing syringe was 20°C; the extrusion speed was 1.4 mm 3 / s; printing speed 5 mm / s; printing needle inner diameter 0.25 mm; printing layer height 0.2 mm; light curing intensity 3.42 mw / cm 2 ; The number of printing layers is 15.

[0082] The effect of the dual-phase bracket after printing is as follows Figure 2 As shown, the left picture is the side effect picture, the middle picture is the top effect picture, and the right picture is the bottom effect picture.

[0083] Cartilage hydrogel and bone hydrogel were printed separately using the same printing method as above as controls.

[0084] 1.3 SEM and EDS analysis of biphasic scaffolds

[0085] The morphology of the printed biphasic scaffold was analyzed using SEM. According to the sample preparation procedure outlined in the previous section, the samples were frozen and freeze-dried. The freeze-dried samples were cut from the side to observe the microtopography of the upper and lower cartilage layers and bone layers, and their scanning electron micrographs were obtained as shown in Figure 3 and Figure 4 shown, where Figure 3 the left figure in Figure 4 is the freeze-dried biphasic scaffold diagram, the middle is the SEM 21 magnification diagram of the biphasic scaffold, and the right figure is the SEM 100 magnification diagram of the biphasic scaffold; Figure 5 the left figure in

[0086] is the cartilage scaffold, and the right figure is the bone scaffold. To improve the conductivity of the material, the samples were coated with Pt before being placed in the SEM chamber for imaging. The images were captured using a scanning electron microscope at different magnifications with a voltage of 5 kV. The device was equipped with an EDS analyzer for elemental analysis, and its EDS analysis diagram was obtained as shown in

[0087] The freeze-dried samples of the cartilage hydrogel and bone hydrogel were placed in an agate mortar, ground evenly and pressed into tablets, and then scanned in the range of 400 cm-1 - 4000 cm-1 using a Fourier transform infrared spectrometer to obtain the infrared absorption spectra of the test samples, as shown in Figure 6 shown, Figure 6 where the left figure is the infrared spectrum of the cartilage layer and the right figure is the infrared spectrum of the bone layer.

[0088] 1.4 Fourier transform infrared spectroscopy analysis of the biphasic scaffold

[0089] The mechanical properties of the biphasic scaffold were evaluated using a microcomputer-controlled electronic universal testing machine with a capacity of 20 N. Before starting the procedure, the diameter and height of the samples were measured to determine the cross-sectional area. Compression evaluation was carried out at a speed of 1 mm min−1 and stopped when the strain-stress diagram indicated the fracture point, and the strain-stress curve of the biphasic scaffold was obtained as shown in Figure 7 shown.

[0090] 1.6 Design of the pressure stimulation device

[0091] The hydrogel compression device consists of a base, a lifting device, a pressure sensor, a compression probe, and a control device. The compression probe is made of anodized aluminum alloy, which can prevent degradation due to the reaction between the probe and ions in the culture medium during compression, ensuring the stability of the compression probe. The pressure sensor can report the values generated by compression through an external computer. The device base is designed according to the length and width of the 6-well plate to fix the 6-well plate. The control device can set the compression depth, the lifting speed of the probe, and the cycle time to meet the experimental requirements. See Figure 8 and Figure 8It is a physical diagram of the hydrogel compression device.

[0092] 1.7 Constructing osteochondral organoids with pressure stimulation

[0093] Before each compression of the biphasic scaffold, first sterilize the pressure stimulation device. Place the whole device in the ultra-clean bench and sterilize it with ultraviolet light overnight. The next day, add 75% alcohol to the 6-well plate, immerse the compression probe in the alcohol for 30 minutes, then rinse the probe 3 times with PBS, and use it after sucking off the residual PBS on the probe.

[0094] Place the prepared biphasic scaffold in a 6-well plate, zero the pressure sensor device, control the probe to move down. After the probe contacts the hydrogel (when the mechanical sensor just shows a number), zero the pressure sensor again. Set the overall downward pressure depth to 8% - 12% of the thickness of the osteochondral scaffold. The frequency is 1 Hz, and the duration is 1 hour. Withdraw the compression device and stop for 23 hours, then culture it in the cell incubator. The number of days of dynamic compression stimulation is 7 days (stimulate for 1 hour every day).

[0095] Culture the prepared biphasic scaffold for 7 days under the same conditions as a control.

[0096] After 7 days of culture, perform live / dead staining on the biphasic scaffold with pressure stimulation and the biphasic scaffold without pressure stimulation. Use a live / dead kit (Beyotime Biotechnology Co., Ltd. (Shanghai)). Immerse the scaffold in the live / dead staining solution at room temperature for 30 minutes, then gently wash it 3 times with sterile PBS and suck off the PBS solution. Take pictures and observe under a laser confocal microscope to obtain the laser confocal micrograph as Figure 9 shown, Figure 9 In the left picture in the following is the biphasic scaffold with pressure stimulation, and the right picture is the biphasic scaffold without pressure stimulation.

[0097] Perform immunofluorescence detection on BMSCs in the scaffolds of the non-dynamic compression group and the dynamic compression stimulation group for type II collagen Col-II (chondrogenesis-related protein) and osteocalcin OCN (osteogenesis-related protein), and obtain the immunofluorescence micrograph of the osteochondral biphasic scaffold regulating the osteogenic / chondrogenic ability of loaded stem cells before and after mechanical stimulation as Figure 10 shown. It was found that the expression of chondrogenic and osteogenic proteins increased after dynamic compression stimulation.

[0098] Extract the RNA of BMSCs in the scaffolds of the non-dynamic compression group and the dynamic compression stimulation group, and perform chondrogenesis- and osteogenesis-related gene detection to obtain the RT-PCR micrograph of the osteochondral biphasic scaffold regulating the osteogenic / chondrogenic ability of loaded stem cells before and after mechanical stimulation as Figure 11 shown. It was found that the expression of the chondrogenic gene Aggrecan and the osteogenic gene Runx2 increased significantly after dynamic compression stimulation.

[0099] To explore whether pressure stimulation can activate the FAK-ROCK mechanotransduction pathway in stem cells, thereby achieving directional regulation of the chondrogenic / osteogenic biphasic differentiation of stem cells, BMSCs proteins in the scaffolds of the non-dynamic compression group and the dynamic compression stimulation group were extracted respectively, and Western Blot (WB) experiments were carried out. As Figure 12 shown, pressure stimulation successfully activated the mechanosensitive pathway FAK-ROCK.

[0100] Three- to four-month-old skeletally mature New Zealand white rabbits were used. After anesthesia, the joint capsule was incised along the medial incision of the knee joint, the femoral condyle was exposed, and a bone cartilage defect was modeled on the femoral condyle with a trephine. The diameter of the model was 5 mm and the depth was 5 mm. Subsequently, the organoid scaffolds after dynamic pressure stimulation and the organoid scaffolds without stress stimulation were implanted respectively. At 8 weeks after surgery, the New Zealand white rabbits were euthanized, and their femoral condyle specimens were collected. Micro-CT scans were performed on the collected specimens to evaluate the repair effect of the osteochondral defect, and the repair effect of the osteochondral defect after implanting the organoids was obtained as shown in Figure 13 shown.

[0101] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing 3D bioprinted osteochondral organoids based on dynamic mechanical regulation, characterized in that: The following steps are involved: S1) providing cartilage layer bio-ink and bone layer bio-ink; The cartilage layer bio-ink comprises 1% w / v to 5% w / v of a first hydrogel matrix, 5% w / v to 10% w / v of a second hydrogel matrix, 0.1% w / v to 0.5% w / v of a photoinitiator, 1×10 5 ~1×10 7 / mL chondrocytes and culture medium; The bone layer bio-ink comprises 1% w / v to 5% w / v of a first hydrogel matrix, 5% w / v to 10% w / v of a second hydrogel matrix, 0.1% w / v to 0.5% w / v of a photoinitiator, 1% w / v to 10% w / v of hydroxyapatite, 1×10 5 ~1×10 7 Osteoblasts / mL and culture medium; The first hydrogel matrix is ​​selected from a hyaluronic acid hydrogel matrix and / or a sodium alginate hydrogel matrix; The second hydrogel matrix is ​​selected from a gelatin-based hydrogel matrix; S2) using a 3D printer to perform photocuring and cross-linking printing on the bone layer bio-ink to obtain a bone layer; then performing photocuring and cross-linking printing on the surface of the bone layer on the cartilage layer bio-ink to form a cartilage layer to obtain a bone-cartilage biphasic scaffold; S3) subjecting the osteochondral biphasic scaffold to dynamic pressure stimulation to obtain 3D bioprinted osteochondral organoids.

2. The construction method according to claim 1, characterized in that: The first hydrogel matrix is ​​selected from one or more of methacrylated hyaluronic acid, hyaluronic acid methacrylate and methacrylated sodium alginate; The second hydrogel matrix is ​​selected from methacrylated gelatin; The photoinitiator is selected from lithium phenyl-2,4,6-trimethylbenzoylphosphonate and / or 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone.

3. The construction method according to claim 1, characterized in that: The cartilage layer bio-ink comprises 2% w / v of a first hydrogel matrix, 7.5% w / v of a second hydrogel matrix, 0.2% w / v of a photoinitiator, 1×10 6 / mL chondrocytes and culture medium; The bone layer bio-ink comprises 2% w / v of a first hydrogel matrix, 7.5% w / v of a second hydrogel matrix, 0.2% w / v of a photoinitiator, 5% w / v of hydroxyapatite, 1×10 6 cells / mL osteoblasts and culture medium.

4. The construction method according to claim 1, characterized in that: The cartilage layer bio-ink is prepared according to the following method: 1% w / v~5% w / v of the first hydrogel matrix, 5% w / v~10% w / v of the second hydrogel matrix, and 0.1% w / v~0.5% w / v of the photoinitiator were mixed with the culture medium, filtered and sterilized, and 1×10 5 ~1×10 7 / mL chondrocytes, and cartilage layer bio-ink was obtained; The bone layer bio-ink is prepared according to the following method: 1% w / v~5% w / v of the first hydrogel matrix, 5% w / v~10% w / v of the second hydrogel matrix, 0.1% w / v~0.5% w / v of the photoinitiator, and 1% w / v~10% w / v of hydroxyapatite were mixed with the culture medium, filtered and sterilized, and 1×10 5 ~1×10 7 / mL osteoblasts to obtain bone layer bio-ink.

5. The construction method according to claim 1, characterized in that: In the step S2), the temperature of the bone layer bio-ink photocuring cross-linking printing is 20°C to 25°C; the extrusion speed of the bone layer bio-ink is 1.3 to 1.5 mm 3 / s; the speed of photocuring cross-linking printing of bone layer bio-ink is 1~10 mm / s; the layer height of photocuring cross-linking printing of bone layer bio-ink is 0.1~0.3 mm; The temperature of the photocuring cross-linking printing of the cartilage layer bio-ink in step S2) is 20°C to 25°C; the extrusion speed of the cartilage layer bio-ink is 1.3 to 1.5 mm 3 / s; the speed of photocuring and cross-linking printing of cartilage layer bio-ink is 1~10 mm / s; the layer height of photocuring and cross-linking printing of cartilage layer bio-ink is 0.1~0.3 mm.

6. The construction method according to claim 1, characterized in that: The thickness ratio of the bone layer to the cartilage layer in the osteocartilage biphasic scaffold is (1-2):

1.

7. The construction method according to claim 1, characterized in that: During the dynamic pressure stimulation in step S3), the pressure probe is in contact with the cartilage layer of the osteochondrium biphasic scaffold; the pressure probe is pressed down to a depth of 5% to 15% of the thickness of the osteochondrium biphasic scaffold; The frequency of the dynamic pressure stimulation is 0.5-2 Hz; the duration of each dynamic pressure stimulation is 0.5-2 h, and the rest time is 20-24 h; the duration of the dynamic pressure stimulation is 5-10 days.

8. The construction method according to claim 1, characterized in that: The frequency of the dynamic pressure stimulation is 1 Hz; each dynamic pressure stimulation lasts for 1 hour and stops for 23 hours; the duration of the dynamic pressure stimulation is 7 days.

9. A 3D bioprinted osteochondral organoid based on dynamic mechanical regulation constructed by the construction method described in any one of claims 1 to 8.

10. Use of the 3D bioprinted osteochondroid organ based on dynamic mechanical regulation constructed by the construction method of any one of claims 1 to 8 or the 3D bioprinted osteochondroid organ based on dynamic mechanical regulation as described in claim 9 in the preparation of bone repair materials.

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