Bio-printed tendon-fibrocartilage-bone composite repair material and its application

Abstract

The present invention provides a tendon-fibrocartilage-bone composite repair material based on bioprinting and its application, which relates to the technical field of tissue engineering. The composite repair material sequentially includes a bone layer, a fibrocartilage layer, and a tendon layer from bottom to top; the bone layer is obtained by bioprinting a first bioink composition containing bone marrow mesenchymal stem cells, decalcified cancellous bone, and a first bioink; the fibrocartilage layer is obtained by bioprinting a second bioink composition containing fibrocartilage cells, fibrocartilage extracellular matrix, and a second bioink; the tendon layer is obtained by bioprinting a third bioink composition containing tendon stem cells, tendon extracellular matrix, and a third bioink; the tendon stem cells and bone marrow mesenchymal stem cells are pretreated with pioglitazone. The composite repair material alleviates the insufficient energy regulation in the early stage of rotator cuff injury repair and can help reshape the gradient structure of the tendon-bone interface.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and particularly to a tendon-fibrocartilage-bone composite repair material based on bioprinting and its application. Background Art

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

[0003] Rotator cuff injury has a high incidence and serious harm, seriously affecting the quality of life and life health of patients. Rotator cuff injury is a common injury caused by persistent or high-intensity exercise. At present, clinical surgery is used to reconstruct the anatomical position of the injured tendon. However, due to the poor healing effect of the tendon-bone interface, the retear rate of the rotator cuff after repair can be as high as 20% - 94%. Especially for large and irreparable rotator cuff injuries, due to factors such as fatty infiltration and progressive atrophy of the tendon-bone interface tissue, the repair difficulty is extremely high. Therefore, the research and development of treatment products that can effectively repair the tendon-bone interface is a research hotspot in the field of sports medicine at home and abroad.

[0004] As a "bridge" structure connecting tendon and bone, the tendon-bone interface includes tendon, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone tissue from shallow to deep, which is a special area with different components, structural compositions, and mechanical properties. The four layers of tissue at the tendon-bone interface show a gradient change and interlace with each other. Its integrity can effectively reduce stress concentration and transfer the force from the tendon to the bone, thereby reducing the risk of tendon rupture and fracture.

[0005] Although traditional surgical and fixation methods in clinical practice can restore the continuity of the tendon-bone interface, the regenerated tissue is mainly scar tissue, resulting in weakened mechanical strength and increased retear risk after surgery. For ideal tendon-bone interface reconstruction, on the one hand, it is necessary to restore the spatial anisotropy of each tissue component, structure, and mechanics, including the good regeneration and gradual transition of tendon, fibrocartilage, and bone tissue; on the other hand, it is necessary to have a high repair timeliness and provide a good "regeneration microenvironment" for the tendon-bone interface in a timely manner. Therefore, starting the tendon-bone interface repair as early as possible and restoring the spatial gradient structure of the tendon-bone interface can achieve good functional reconstruction and reduce the occurrence of tendon retear after rotator cuff repair. Therefore, reconstructing the gradient structure of the tendon-bone interface with a gradual transition and providing a good "regeneration microenvironment" for the healing of the tendon-bone interface as early as possible has always been the key research direction for rotator cuff injury repair.

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

[0007] The purpose of the present invention is to provide a tendon-fibrocartilage-bone composite repair material based on bioprinting to alleviate the insufficient energy regulation in the early stage of rotator cuff injury repair and help reshape the gradient structure of the tendon-bone interface.

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

[0009] In a first aspect, a tendon-fibrocartilage-bone composite repair material based on bioprinting is provided. The composite repair material sequentially includes a bone layer, a fibrocartilage layer, and a tendon layer from bottom to top;

[0010] The bone layer is obtained by bioprinting a first bioink composition, and the first bioink composition includes bone marrow mesenchymal stem cells, decalcified cancellous bone, and a first bioink;

[0011] The fibrocartilage layer is obtained by bioprinting a second bioink composition, and the second bioink composition includes fibrocartilage cells, fibrocartilage extracellular matrix, and a second bioink;

[0012] The tendon layer is obtained by bioprinting a third bioink composition, and the third bioink composition includes tendon stem cells, tendon extracellular matrix, and a third bioink;

[0013] The tendon stem cells and the bone marrow mesenchymal stem cells are pretreated with pioglitazone.

[0014] In a second aspect, a set of bioink compositions is further provided. The set of bioink compositions includes the first bioink composition of the first aspect, the second bioink composition of the first aspect, and the third bioink composition of the first aspect, which are separately and independently packaged.

[0015] In a third aspect, a preparation method of the tendon-fibrocartilage-bone composite repair material based on bioprinting of the first aspect is further provided. The preparation method includes using a bioprinting device to print the first bioink composition, the second bioink composition, and the third bioink composition to form a composite repair material including the bone layer, the fibrocartilage layer, and the tendon layer.

[0016] In a fourth aspect, an application of the tendon-fibrocartilage-bone composite repair material of the first aspect, the bioink of the second aspect, or the preparation method of the third aspect in preparing a product for repairing rotator cuff injuries is further provided.

[0017] In a fifth aspect, a product for repairing rotator cuff injuries is further provided, including the tendon-fibrocartilage-bone composite repair material of the first aspect or the set of bioinks of the second aspect.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) In the composite repair material provided by the present invention, tendon stem cells and bone marrow mesenchymal stem cells are pretreated with pioglitazone, which enhances the production of mitochondria in these cells and improves the efficiency of mitochondrial delivery between cells. In vitro experiments have confirmed that engineered stem cells indirectly deliver mitochondria to regulate the energy metabolism level of related damaged cells (increase in ATP and MtDNA content) and enhance their biological behavior (increase in cell proliferation ability), thus achieving energy metabolism reprogramming of damaged tissues / cells.

[0020] (2) The decalcified cancellous bone, fibrochondrocyte extracellular matrix, and tendon extracellular matrix in the composite repair material provided by the present invention all have good biocompatibility, degradability, and biomechanics. They can provide a tissue-specific microenvironment for cell growth, provide chemical signals and biomechanical signals, induce and regulate cell adhesion, growth, proliferation, and differentiation, and promote tissue repair and regeneration without degradation and removal.

[0021] (3) Through multi-cell bioprinting technology, the composite repair material realizes the integrated construction of three bioink compositions according to three-layer regions (tendon region, fibrochondral region, and bone region) to improve the regeneration and repair effect of the tendon-bone interface tissue after rotator cuff injury.

[0022] (4) The composite repair material can not only better solve the disadvantages of low and non-persistent and efficient direct mitochondrial delivery efficiency, but also continuously and slowly release mitochondria in a long period. Moreover, it can reproduce the tendon-bone interface tissue structure with composition, structure, and cell bionics, so as to better repair rotator cuff injury. Brief Description of the Drawings

[0023] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is the research design diagram of Example 1;

[0025] Figure 2 It is the photo of the composite repair material prepared in Example 1;

[0026] Figure 3 It is the staining results of dead and live cells (A) and the semi-quantitative analysis results of cell survival rate (B) after the cells are treated with the leaching solution of the photo of the composite repair material prepared in Example 1;

[0027] Figure 4Staining results and adhesion morphology of dead and live cells in the bone layer of the composite repair material prepared in Example 1. A is the staining result of live cells, B is the staining result of dead cells, C is the adhesion and morphology result, Bar = 50 μm;

[0028] Figure 5 ATP levels (A) and mtDNA content (B) of fibrocartilage cells co-cultured with Pg-TSPCs and Pg-BMSCs, and proliferation ability (C);

[0029] Figure 6 6-week in vivo histological H&E staining and safranin O / fast green results of the implantation groups in each experimental group, Bar = 500 μm. Detailed implementation manners

[0030] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0031] In a first aspect, a tendon-fibrocartilage-bone composite repair material based on bioprinting is provided. The composite repair material sequentially includes a bone layer, a fibrocartilage layer, and a tendon layer from bottom to top.

[0032] The bone layer is obtained by bioprinting with a first bioink composition, and the first bioink composition includes bone marrow mesenchymal stem cells, decalcified cancellous bone, and a first bioink.

[0033] In an optional implementation manner, the content of bone marrow mesenchymal stem cells in the first bioink composition is 1 - 10×10 5 / mL, for example, it can be but is not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10×10 5 / mL; the content of decalcified cancellous bone is 1 - 5% wt, for example, it can be but is not limited to 1, 2, 3, 4, or 5% wt.

[0034] In an optional implementation manner, the content of bone marrow mesenchymal stem cells in the first bioink composition is 3×10 5 / mL, and the content of decalcified cancellous bone is 2% wt.

[0035] The bone marrow mesenchymal stem cells in the first bioink composition are pretreated with pioglitazone.

[0036] In an optional implementation manner, the pretreatment of bone marrow mesenchymal stem cells with pioglitazone includes culturing bone marrow mesenchymal stem cells with a medium containing pioglitazone;

[0037] In an alternative embodiment, the concentration of pioglitazone in the culture medium for pre-treating bone marrow mesenchymal stem cells is 5 to 20 μM, for example, but not limited to, 5, 10, 15 or 20 μM, and preferably 10 μM.

[0038] In an alternative embodiment, the culture time for culturing bone marrow mesenchymal stem cells using the culture medium containing pioglitazone is 100 to 140 h, for example, but not limited to, 100, 110, 120, 130 or 140 h, and preferably 120 h.

[0039] The fibrous cartilage layer is obtained by bioprinting with a second bioink composition, and the second bioink composition comprises fibrous cartilage cells, fibrous cartilage extracellular matrix and a second bioink.

[0040] In an alternative embodiment, the content of fibrous cartilage cells in the second bioink composition is 1 to 10×10 5 / mL, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10×10 5 / mL; the content of fibrous cartilage extracellular matrix is 1 to 5% wt, for example, but not limited to, 1, 2, 3, 4 or 5% wt.

[0041] In an alternative embodiment, the content of fibrous cartilage cells in the second bioink composition is 3×10 5 / mL, and the content of fibrous cartilage extracellular matrix is 2% wt.

[0042] The tendon layer is obtained by bioprinting with a third bioink composition, and the third bioink composition comprises tendon stem cells, tendon extracellular matrix and a third bioink.

[0043] In an alternative embodiment, the content of tendon stem cells in the third bioink composition is 1 to 10×10 5 / mL, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10×10 5 / mL; the content of tendon extracellular matrix is 1 to 5% wt, for example, but not limited to, 1, 2, 3, 4 or 5% wt.

[0044] In an alternative embodiment, the content of tendon stem cells in the third bioink composition is 3×10 5 / mL, and the content of tendon extracellular matrix is 2% wt.

[0045] The tendon stem cells in the third bioink composition are pre-treated with pioglitazone.

[0046] In an alternative embodiment, the pre-treatment of tendon stem cells with pioglitazone includes culturing tendon stem cells in a medium containing pioglitazone;

[0047] In an alternative embodiment, the concentration of pioglitazone in the medium used for pre-treating tendon stem cells is 5 - 20 μM, for example, it can be but is not limited to 5, 10, 15 or 20 μM, and preferably 10 μM.

[0048] In an alternative embodiment, the culturing time of tendon stem cells in a medium containing pioglitazone is 100 - 140 h, for example, it can be but is not limited to 100, 110, 120, 130 or 140 h, and preferably 120 h.

[0049] In an alternative embodiment, the first bioink, the second bioink, and the third bioink each independently include a hydrogel. The hydrogel compositions constituting the first bioink, the second bioink, and the third bioink can be the same or different. The hydrogel can include hydrogels known in the art that can optionally be used as bioinks, including but not limited to one or more of alginate, gelatin, chitosan, silk fibroin, fibrinogen, hyaluronic acid, cellulose, polyethylene glycol, polyglycolic acid, polylactic acid, and polylactic acid - polyglycolic acid.

[0050] In an alternative embodiment, the first bioink includes a photocurable hydrogel, preferably a GelMa (methacrylated gelatin) hydrogel. In an alternative embodiment, the first bioink is a GelMa hydrogel with a concentration of 2 - 10% wt (for example, it can be but is not limited to 2, 3, 4, 5, 6, 7, 8, 9 or 10% wt), and preferably a GelMa hydrogel with a concentration of 5% wt. Optionally, the first bioink also contains a photoinitiator.

[0051] In an alternative embodiment, the second bioink includes a photocurable hydrogel, preferably a GelMa hydrogel. In an alternative embodiment, the second bioink is a GelMa hydrogel with a concentration of 2 - 10% wt (for example, it can be but is not limited to 2, 3, 4, 5, 6, 7, 8, 9 or 10% wt), and preferably a GelMa hydrogel with a concentration of 5% wt. Optionally, the second bioink also contains a photoinitiator.

[0052] In an alternative embodiment, the third bioink includes a photocurable hydrogel, preferably a GelMa hydrogel. In an alternative embodiment, the third bioink is a GelMa hydrogel with a concentration of 2 - 10% wt (for example, it can be but is not limited to 2, 3, 4, 5, 6, 7, 8, 9 or 10% wt), and preferably a GelMa hydrogel with a concentration of 5% wt. Optionally, the third bioink also contains a photoinitiator.

[0053] In an alternative embodiment, the photoinitiator includes LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate).

[0054] In an alternative embodiment, a photocuring bioprinting device is used for layer-by-layer 3D printing, and the printing parameters include: light intensity of 5-10 mw / cm 2 , exposure time of 10-15 s, number of base layers of 5-10, and exposure time of the base layer of 8-12 s.

[0055] In an alternative embodiment, the printing parameters include: light intensity of 8 mw / cm 2 , exposure time of 12 s, number of base layers of 6, and exposure time of the base layer of 10 s.

[0056] The demineralized cancellous bone (DCB) and decellularized extracellular matrix (dECM) in the tendon-fibrocartilage-bone composite repair material based on bioprinting provided in the first aspect are excellent biomaterials for tissue regeneration. The fibrochondrocyte extracellular matrix (FCECM) of decellularized cartilage and the tendon extracellular matrix (TECM) of decellularized tendon have good biocompatibility and can promote the proliferation and chondrogenic / tendinous differentiation of stem cells (MSCs). DCB is one of the bio-derived scaffold materials, which mainly uses allogeneic or xenogeneic organs / tissues to obtain a decellularized matrix material through decellularization and antigen removal treatment. This material has extracellular matrix components and contains type I collagen, which is a good scaffold for cell adhesion and growth and is a good biogenic material for bone repair.

[0057] To mimic natural tissues, the chemical and mechanical properties of 3D tissue constructs should mimic the characteristics of natural tissues. Gradients of cell types, biomolecules, and other structural and compositional components also need to be developed in some types of bioprinted tissue constructs. To meet these requirements, precise control of the shape, flow, and composition of fibers carrying cells is needed during bioprinting. Therefore, methods are needed that can more precisely control the organization of materials, cells, and biomolecules in the resulting 3D structure to accurately mimic the composition of natural tissues. Bioprinting helps to adjust the structural and compositional properties of tissue constructs during printing. 3D bioprinting systems can be used to control cell and molecular deposition, flow, mixing, and gradient formation in the final 3D structure to complete the reconstruction of transitional complex tendon-bone interface tissues.

[0058] Delivery of normal-function mitochondria into damaged tissue cells to replenish dysfunctional mitochondria and achieve energy metabolism remodeling is called mitochondrial replacement therapy (MRT). Using cells as carriers to deliver mitochondria can avoid the risks of additional mitochondrial isolation, transfer, and loss of activity. Suitable carrier cells should meet the requirements of targeted migration to damaged tissues, spontaneous mitochondrial delivery, strong mitochondrial biogenesis ability, and low energy demand of their own. Mesenchymal stem cells (MSCs) have a low bioenergy demand in the glycolytic state, which is the basis for their delivery of their own mitochondria to damaged tissue cells. Human placenta-derived mesenchymal stem cells (hMSCs) treated with pioglitazone (pg) can activate continuous mitochondrial production in hMSCs by activating the PGC-1a-NRF1-TFAM pathway and can promote mitochondrial transfer between cells to a certain extent. Therefore, introducing pg-treated mesenchymal stem cells (MSCs) into damaged tissues can promote tissue healing, especially at the tendon-bone interface after rotator cuff injury.

[0059] 3D bioprinting can achieve precise position control of cells and extracellular matrix components and further create complex and functional multicellular tissues or organs in a 3D environment, which is helpful for the application of scaffold-based or scaffold-free tissue and organ structures, microorganisms, and single-chip organ model systems. 3D bioprinting uses inkjet, extrusion, and laser-based methods to create tissues with complex geometries and scalability. As an emerging technology, 3D bioprinting can construct complex hard and soft tissue structures by gradually depositing seed cells, metal ions, growth factors, and other biomaterials, thereby simulating the structural, cellular, and compositional characteristics of the tendon-bone interface.

[0060] 3D bioprinting also has the potential to repeatedly develop 3D structured tissues by controlling and optimizing the microenvironment, where the intrinsic cell morphology and structure can be reconstructed. The higher-order assembly of multicellular bioprinted structures can be developed through organized spatial patterns and tissue-specific gene expression. In addition, 3D bioprinting provides the potential to fabricate multicellular complex tissue structures by sequentially depositing different cell types, different active molecules, and biomaterials located in specific regions.

[0061] The tendon-fibrocartilage-bone composite repair material based on bioprinting provided by the first aspect designs a composite repair material constructed from three bioink components through multicellular bioprinting technology. Based on the good biocompatibility and differentiation-promoting effect of the extracellular matrix material, it can print a tissue-derived bioink composition containing bone marrow mesenchymal stem cells (pg pretreatment), fibrocartilage cells, and tendon stem cells (pg pretreatment) from bottom to top by regulating and mixing on the basis of bone, fibrocartilage, and tendon extracellular matrix bioinks. In a preferred embodiment, the composite repair material can take into account the gradual seed cells, extracellular components, and structural characteristics, effectively reconstruct the tendon area, fibrocartilage area, and bone area, and deliver mitochondria from pg-MSCs to the cells at the damaged site to reprogram the energy metabolism at the damaged site. It promotes the good reconstruction of the tendon-bone interface after rotator cuff injury from both spatial and temporal perspectives.

[0062] In the second aspect, a set of bioink compositions is also provided. The set of bioink compositions includes the first bioink composition, the second bioink composition, and the third bioink composition in the first aspect, each independently packaged. The set of bioink compositions can be used to construct the tendon-fibrocartilage-bone composite repair material based on bioprinting in the first aspect.

[0063] In an optional embodiment, the content of bone marrow mesenchymal stem cells in the first bioink composition is 1 - 10×10 5 / mL, and the content of decalcified cancellous bone is 1 - 5% wt; the content of fibrocartilage cells in the second bioink composition is 1 - 10×10 5 / mL, and the content of fibrocartilage extracellular matrix is 1 - 5% wt; and the content of tendon stem cells in the third bioink composition is 1 - 10×10 5 / mL, and the content of tendon extracellular matrix is 1 - 5% wt.

[0064] In an optional embodiment, the content of bone marrow mesenchymal stem cells in the first bioink composition is 3×10 5 / mL, and the content of decalcified cancellous bone is 2% wt; the content of fibrocartilage cells in the second bioink composition is 3×10 5 / mL, and the content of fibrocartilage extracellular matrix is 2% wt; and the content of tendon stem cells in the third bioink composition is 3×10 5 / mL, and the content of tendon extracellular matrix is 2% wt.

[0065] In an alternative embodiment, the mesenchymal stem cells in the first bioink composition are mesenchymal stem cells cultured in a medium containing 5-20 μM pioglitazone for 100-140 h, preferably mesenchymal stem cells cultured in a medium containing 10 μM pioglitazone for 120 h.

[0066] In an alternative embodiment, the tendon stem cells in the third bioink composition are tendon stem cells cultured in a medium containing 5-20 μM pioglitazone for 100-140 h, preferably tendon stem cells cultured in a medium containing 10 μM pioglitazone for 120 h.

[0067] In an alternative embodiment, the first bioink, the second bioink, and the third bioink comprise a photocurable hydrogel, preferably GelMa.

[0068] In an alternative embodiment, the first bioink composition, the second bioink composition, and the third bioink composition further contain a photoinitiator, and the photoinitiator preferably comprises LAP.

[0069] In a third aspect, a method for preparing the tendon-fibrocartilage-bone composite repair material based on bioprinting according to the first aspect is further provided. The preparation method includes using a bioprinting device to print the first bioink composition, the second bioink composition, and the third bioink composition to form a composite repair material including the bone layer, the fibrocartilage layer, and the tendon layer.

[0070] In an alternative embodiment, a photocurable bioprinting device is used for layer-by-layer 3D printing, and the printing parameters include: light intensity 5-10 mw / cm 2 , exposure time 10-15 s, number of base layers 5-10, and base layer exposure time 8-12 s.

[0071] In an alternative embodiment, the printing parameters include: light intensity 8 mw / cm 2 , exposure time 12 s, number of base layers 6, and base layer exposure time 10 s.

[0072] In a fourth aspect, an application of the tendon-fibrocartilage-bone composite repair material according to the first aspect, the set of bioinks according to the second aspect, or the preparation method according to the third aspect in the preparation of a product for repairing rotator cuff injuries is further provided.

[0073] In a fifth aspect, a product for repairing rotator cuff injuries is further provided. The product for repairing rotator cuff injuries includes the tendon-fibrocartilage-bone composite repair material according to the first aspect or the set of bioinks according to the second aspect.

[0074] In an alternative embodiment, the rotator cuff injury repair product includes the tendon-fibrocartilage-bone composite repair material of the first aspect, and may also optionally include a pharmaceutical ingredient for relieving or treating rotator cuff injuries that is selected arbitrarily in the art.

[0075] In an alternative embodiment, the rotator cuff injury repair product includes the set of bioinks of the second aspect. When in use, the set of bioinks is printed to obtain a corresponding rotator cuff injury composite repair material according to the shape of the graft required at the affected area.

[0076] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any way.

[0077] Example 1

[0078] This example provides a tendon-fibrocartilage-bone composite repair material based on bioprinting, and the research design diagram is as Figure 1 shown.

[0079] 1. Preparation of engineered MSCs:

[0080] (1) Tendon stem cells (TSPCs) were inoculated and cultured overnight, and then co-cultured with a 10 μM Pg (pioglitazone) solution for 5 days to obtain Pg-TSPCs (engineered tendon stem cells).

[0081] (2) Bone marrow mesenchymal stem cells (BMSCs) were inoculated and cultured overnight, and then co-cultured with a 10 μM Pg (pioglitazone) solution for 5 days to obtain Pg-BMSCs (engineered bone marrow mesenchymal stem cells).

[0082] (3) Fibrocartilage cells: Rat knee joint meniscus tissue was taken, and the medial 2 / 3 region of the meniscus tissue was excised in a sterile culture dish. After washing 3 times with PBS, the meniscus was cut into 1 mm 3 fragments with a sterile ophthalmic scissors, and then 0.2% type II collagenase was added, and it was placed in an incubator at 37°C and 5% CO 2 for digestion for 60 min with the help of a magnetic stirrer until the tissue fragments disappeared; the digestion was terminated with DMEM culture medium containing 10% FBS, and it was cultured until the 2nd - 3rd passage for standby.

[0083] 2. Preparation of decalcified cancellous bone (DCB):

[0084] Fresh porcine scapulae were used, the periosteum, cartilage and soft tissues were removed, and the cancellous bone was taken and prepared into a round cake shape. It was repeatedly washed by stirring with running water to remove bone marrow, blood stains and surface grease. After washing with tap water, it was stored at -80°C for 3 days, and then the following steps were carried out in sequence:

[0085] (1) Dehydrate with absolute ethanol for 2 h and air dry.

[0086] (2) Defat with chloroform / methanol (1:1, v / v) for 4 h and air dry.

[0087] (3) Decalcify with 0.6 mol / L hydrochloric acid for 6 h (hydrochloric acid / spongy bone: 20 ml / g), and air dry.

[0088] (4) Dehydrate with absolute ethanol for 2 h and air dry.

[0089] (5) Defat with chloroform / methanol (1:1, v / v) for 4 h and air dry.

[0090] (6) Immerse in 10% PBS (pH = 7.4) at 37 °C for 3 d, change the solution twice in the middle, and grind to powder after air drying for standby.

[0091] 3. Preparation of fibrochondrocyte extracellular matrix (FCECM) and tendon extracellular matrix (TECM):

[0092] (1) Take fresh porcine meniscus, and rinse it 3 times with phosphate buffer solution (PBS, pH 7.4).

[0093] (2) Immerse the fibrochondrocyte in electrolyzed oxidizing water (EOW) for primary sterilization three times, 10 minutes each time.

[0094] (3) Under sterile conditions, cut the cartilage slices into small pieces about 1 mm with ophthalmic scissors and rinse them 3 times with sterile distilled water.

[0095] (4) Immerse in 3% hydrogen peroxide for re-sterilization for 30 min, and rinse with sterile distilled water 3 times, 30 minutes each time.

[0096] (5) Put it into a crusher to break it up, add 2 times sterile triple-distilled water, and repeatedly crush the cartilage homogenate at low temperature.

[0097] (6) Add 10 times triple-distilled water for hypotonic mixing. Freeze the hypotonic cell homogenate at -20 °C and then thaw it at room temperature for 4 freeze-thaw cycles.

[0098] (7) Centrifuge at a gradient. Centrifuge the crushed homogenate at 2000 rpm for 30 minutes and take the supernatant.

[0099] (8) Then centrifuge at 3000 rpm for 30 minutes and take the supernatant.

[0100] (9) Centrifuge at 4000 rpm for 30 minutes and take the supernatant. Repeat 5 times to fully wash away cell debris and residual substances until the pH reaches 7.0.

[0101] (10) Take the upper homogenate and centrifuge it at 10,000 rpm for 30 minutes at 4°C. Discard the supernatant and collect the precipitate as cell-free nanoscale ECM slurry. After freeze-drying, grind it into powder for later use.

[0102] The preparation method of tendon extracellular matrix is the same as that of the above-mentioned fibrocartilage extracellular matrix. Treat the tendon tissue using the above steps to obtain tendon extracellular matrix.

[0103] 4. Preparation of bioink composition:

[0104] (1) Bone layer bioink (the first bioink composition): GelMa hydrogel (5% GelMa, 0.25 g / L LAP) containing engineered bone marrow mesenchymal stem cells (3×10 5 / mL) and DCB (2% wt).

[0105] (2) Fibrocartilage layer bioink (the second bioink composition): GelMa hydrogel (5% GelMa, 0.25 g / L LAP) containing fibrocartilage cells (3×10 5 / mL) and FCECM (2% wt).

[0106] (3) Tendon layer bioink (the third bioink composition): GelMa hydrogel (5% GelMa, 0.25 g / L LAP) containing engineered tendon stem cells (3×10 5 / mL) and TECM (2% wt).

[0107] 5. Preparation of gradient gradient bioprinted hydrogel:

[0108] Using the photocuring 3D bioprinting technology, printing parameters: Adopt DLP (Digital Light Processing) technology to perform layer-by-layer 3D printing on the scaffold through a photocuring printer. Respectively add the bone layer bioink, fibrocartilage layer bioink, and tendon layer bioink prepared in the previous step into the 3D printer material cup. Set the parameters: light intensity 8 mw / cm 2 ; exposure time 12 s; number of base layers 6, exposure time of base layers 10 s. Print successfully at room temperature respectively to achieve the simultaneous hybrid printing of the three bioinks in the tendon-fibrocartilage-bone integrated scaffold and the tendon layer and bone layer containing mitochondrial engineered MSCs, and finally achieve the multicellular bioprinting of the tendon area, fibrocartilage area, and bone area. The photo of the composite repair material obtained in this example is as Figure 2 shown.

[0109] 6. Effect verification:

[0110] (1) The extract of the composite hydrogel repair material prepared using the above steps was used to culture cells for 1, 3, and 7 days, and the biocompatibility of the repair material was observed by dead / live cell staining. Specifically, the chondrocytes cultured in a 6-well plate were immersed in 2 mL of PBS working solution containing 4 mM ethidium homodimer-1 and 2 mM calcein-AM for 30 min. The excitation wavelengths of 556 nm and 488 nm were selected using a fluorescence microscope to detect the imaging of ethidium homodimer-1 (red represents dead cells) and calcein-AM (green represents live cells). The staining results and the semi-quantitative analysis results of cell viability are as Figure 3 shown. It can be seen that there is no significant difference between the extract of the composite repair material and the control group, indicating that it has no cytotoxicity and does not affect the survival rate of cells. The composite repair material is safe.

[0111] (2) Further, chondrocytes were cultured on the composite repair material prepared in the above steps, and dead / live cell staining and cytoskeleton staining were performed. Specifically, after culturing for 1 day, 4',6-diamidino-2-phenylindole (DAPI) and phalloidin (Cytoskeleton, USA) were used for staining to further evaluate the morphology of chondrocytes on the repair material. The dead / live staining results and the cytoskeleton staining results are as Figure 4 shown. It can be seen from Figure 4 that the printing process does not damage the survival of cells in the bioink composition, and the cell viability in the prepared composite repair material is good.

[0112] (3) Pg-TSPCs and Pg-BMSCs were respectively cultured in the upper layer using a Transwell chamber, and the chondrocytes stimulated with IL-1β were placed in the lower layer of the chamber for co-culture with the engineered stem cells. After co-culturing for 7 days, the ATP level and mtDNA content were detected. The results are as Figure 5 shown in A and B, indicating that the ATP level and mtDNA content in the Pg-TSPCs and Pg-BMSCs groups were significantly increased compared with the damaged control group. This finding is related to the number of transferred mitochondria, indicating that the mitochondrial energy metabolism of damaged chondrocytes is upregulated. Further, the engineered stem cells and chondrocytes were co-cultured for 1, 3, 5, and 7 days, and the proliferation ability after culture was detected using a CCK-8 reagent. The results showed that the cell proliferation ability in the Pg-TSPCs and Pg-BMSCs groups was significantly increased compared with the control group.

[0113] (4) Implant the composite repair material prepared in the above steps into the rat rotator cuff injury model. The animal operation is as follows: Anesthetize the rats, and prepare the skin around the left shoulder joint and perform partial disinfection. Expose the deltoid muscle surgically, separate and incise the deltoid muscle to expose the supraspinatus muscle. Separate the supraspinatus muscle and cut the tendon at the junction of the supraspinatus tendon and the humerus. Resuture the supraspinatus muscle to the greater tubercle. In the composite material implantation group, directly transplant the material into the gap at the tendon-bone interface. Close each layer of tissue of the wound layer by layer to complete the rotator cuff injury repair model.

[0114] The experimental groups are divided as follows:

[0115] Number of groups Group Quantity 1 Sham operation group 4 2 Simple gel group (control) 4 3 Engineered stem cell gel group 4

[0116] The in vivo histological staining results are as Figure 6 shown. H&E and safranin O / fast green staining were used to evaluate the histology of the regenerated tissue. In the sham operation group, the collagen fibers in the fibrocartilage area were arranged neatly. At 6 weeks after surgery, H&E staining showed that the repaired tendon in the control group had not healed, with a large number of inflammatory cell infiltrations, almost absent fibrocartilage cells, angiogenesis, and discontinuous collagen fibers, and its morphology was consistent with the scar tissue state: loose and irregular arrangement. In the experimental group, there were fewer inflammatory cell infiltrations in the repair area, an increase in fibrocartilage-like cells, and neat arrangement of collagen fibers. At 12 weeks, the repair process was completed, and almost no inflammatory cells were seen in each group. In the control group, some fibrocartilage-like cells and regenerated fibrocartilage tissue were visible, and the collagen fibers were arranged relatively orderly. In the experimental group, the fibrocartilage tissue was wider, the tendon-bone interface was obvious, and the anatomical structure was closer to that of the sham operation group. Safranin O / fast green staining was used to evaluate the polysaccharide deposition at the tendon-bone interface; at 6 weeks, only a small amount of scattered polysaccharides were seen in the control group, while good polysaccharide deposition was seen in the experimental group at 6 weeks. At 12 weeks, uneven staining was still visible in the control group, while a large amount of polysaccharides were seen in the experimental group. It shows that the composite repair material prepared in this example can effectively promote the healing of the tendon-bone interface after rotator cuff injury.

[0117] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: They can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tendon-fibrocartilage-bone composite repair material based on bioprinting, characterized in that: From bottom to top, it includes the bone layer, fibrocartilage layer and tendon layer; The bone layer is obtained by bioprinting a first bio-ink composition, wherein the first bio-ink composition is composed of 1×10 5 ~10×10 5 / mL, 1-5%wt of decalcified cancellous bone and the first bio-ink; The fibrocartilage layer is obtained by bioprinting a second bio-ink composition, wherein the second bio-ink composition is composed of 1×10 5 ~10×10 5 / mL, 1-5%wt of fibrocartilage extracellular matrix, and the second bio-ink; The tendon layer is obtained by bioprinting a third bio-ink composition, wherein the third bio-ink composition is composed of 1×10 tendon stem cells. 5 ~10×10 5 / mL, tendon extracellular matrix 1~5%wt and the third bio-ink; The first bio-ink, the second bio-ink and the third bio-ink are composed of GelMa hydrogel and a photoinitiator; The tendon stem cells and the bone marrow mesenchymal stem cells are pretreated with pioglitazone; Pretreatment of tendon stem cells with pioglitazone included culturing tendon stem cells with medium containing 5-20 μM pioglitazone for 100-140 h; Bone marrow mesenchymal stem cells were pretreated with pioglitazone, including culturing the bone marrow mesenchymal stem cells in a medium containing 5-20 μM pioglitazone for 100-140 h.

2. The composite repair material according to claim 1, characterized in that: The content of bone marrow mesenchymal stem cells in the first bio-ink composition is 3×10 5 / mL, and the content of decalcified cancellous bone was 2%wt.

3. The composite repair material according to claim 1, characterized in that: The content of fibrochondrocytes in the second bio-ink composition is 3×10 5 / mL, and the content of fibrocartilage extracellular matrix was 2%wt.

4. The composite repair material according to claim 1, characterized in that: The content of tendon stem cells in the third bio-ink composition is 3×10 5 / mL, and the content of tendon extracellular matrix was 2%wt.

5. The composite repair material according to claim 1, characterized in that: In the culture medium containing pioglitazone, the concentration of pioglitazone is 10 μM.

6. The composite repair material according to claim 1, characterized in that: The culture time was 120 h.

7. The composite repair material according to claim 1, characterized in that: The concentration of GelMa hydrogel in the first bio-ink, the second bio-ink and the third bio-ink is independently 2~10%wt.

8. The composite repair material according to claim 1, characterized in that: The concentration of GelMa hydrogel in the first bio-ink, the second bio-ink and the third bio-ink is independently 5%wt.

9. The composite repair material according to any one of claims 1 to 8, characterized in that: Use a light-curing bioprinting device for layer-by-layer 3D printing. The printing parameters include: light intensity 5~10 mw / cm 2 , exposure time 10~15 s, number of base layers 5~10 and base layer exposure time 8~12 s.

10. The composite repair material according to claim 9, characterized in that: Printing parameters include: light intensity 8mw / cm 2 , exposure time 12s, number of base layers 6 and base layer exposure time 10s.

11. The method for preparing the tendon-fibrocartilage-bone composite repair material based on bioprinting according to any one of claims 1 to 10, characterized in that: The method comprises printing the first bio-ink composition, the second bio-ink composition and the third bio-ink composition using a bio-printing device to form a composite repair material comprising the bone layer, the fibrocartilage layer and the tendon layer.

12. Use of the tendon-fibrocartilage-bone composite repair material according to any one of claims 1 to 10, or the tendon-fibrocartilage-bone composite repair material prepared by the preparation method according to claim 11 in preparing products for repairing rotator cuff injuries.

13. A product for repairing rotator cuff injury, characterized in that: The tendon-fibrocartilage-bone composite repair material comprises the tendon-fibrocartilage-bone composite repair material according to any one of claims 1 to 10.

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