A 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC synergistic differentiation and a preparation method and application thereof

Abstract

The application relates to a 3D-printed biphasic bone cartilage repair scaffold based on iPSC-BMSC synergistic differentiation and a preparation method and application thereof. The bone cartilage repair scaffold is a double-layer biomimetic structure, and specifically comprises a subchondral bone repair layer and an articular cartilage repair layer. The subchondral bone repair layer is an A-type composite hydrogel / polycaprolactone electrostatic spinning support framework loaded with bone marrow mesenchymal stem cells (BMSCs), and the A-type composite hydrogel comprises methacrylated hyaluronic acid, methacrylated sodium alginate and methacrylated gelatin. The articular cartilage repair layer is a B-type composite hydrogel loaded with induced pluripotent stem cells (iPSCs), and the B-type composite hydrogel comprises pig knee joint decellularized cartilage matrix, methacrylated hyaluronic acid, methacrylated chondroitin sulfate and methacrylated gelatin. Compared with the prior art, the bone cartilage repair scaffold can realize integrated regeneration of bone cartilage, and is expected to be used for regenerative repair treatment of bone cartilage injury.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering materials technology, and in particular to a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation, its preparation method, and its application. Background Technology

[0002] Osteochondrial defects (OCDs) are full-thickness injuries affecting both hyaline cartilage and subchondral bone in articular processes. They are a common pathological manifestation of osteoarthritis, acute and chronic sports injuries, and traumatic arthritis. Due to the avascular, nerve-free, and lymphatic-free physiological characteristics of articular cartilage, its physiological self-repair ability is almost completely lost. Functional reconstruction of osteocartilage defects has long been a major clinical challenge in orthopedics and sports medicine.

[0003] Current routine clinical treatments all have unavoidable limitations: conservative drug treatment can only relieve pain symptoms and cannot reverse cartilage damage or achieve structural regeneration in the defect area; bone marrow stimulation technology, with microfracture as its core, mainly produces fibrocartilage as the repair tissue, whose mechanical properties and wear resistance are far lower than those of natural hyaline cartilage, and its long-term degeneration rate is high; although autologous osteochondral transplantation can achieve hyaline cartilage-like repair, it is only suitable for small focal defects and is limited by the availability of donors and secondary damage to the donor site; total joint replacement is the final treatment option for patients with end-stage osteoarthritis, but it has complications such as limited prosthesis lifespan and postoperative wear and loosening, and is not suitable for young, highly active individuals.

[0004] In recent years, tissue engineering techniques based on biomaterials have provided new strategies for the regeneration and repair of osteochondral defects. However, traditional osteochondral repair scaffolds still face several technical bottlenecks: insufficient mechanical properties easily lead to scaffold collapse, poor bioactivity results in limited chondrogenic differentiation capacity, and poor hydrophilicity and biocompatibility affect host integration. Current mainstream bone marrow mesenchymal stem cell (BMSC) repair strategies face problems such as donor shortage, immune rejection, differentiation heterogeneity, and easy calcification and fibrosis of repaired tissues. Especially in cartilage-bone interface repair, BMSCs are prone to hypertrophy and ectopic calcification after chondrogenic differentiation, making it difficult to maintain a stable hyaline cartilage phenotype.

[0005] CN116763995A discloses an integrated osteochondral scaffold with a directional porous structure, employing a two-layer composite hydrogel scaffold including an upper composite hydrogel, a transition layer, and a lower composite hydrogel. Methacrylamide gelatin, chitosan, and silk fibroin are used to simulate cartilage tissue. The directional porous structure is prepared using directional cryo-curing and photocuring crosslinking techniques. The transition layer uses methacrylamide gelatin and dopamine chemical grafting to improve interfacial bonding strength. However, this approach still lacks accurate simulation of the natural osteochondral gradient structure, making it difficult to achieve gradient matching of mechanical properties between the subchondral bone layer and the articular cartilage layer; it lacks cartilage / bone-specific extracellular matrix signaling and growth factor loading capacity in its composition; and it also lacks research on the regulatory mechanisms for cartilage phenotypic stability.

[0006] Therefore, existing osteochondral repair scaffolds still have technical bottlenecks such as asynchronous osteochondral regeneration, easy hypertrophy and calcification of BMSCs after chondrogenic differentiation, mismatch between scaffold mechanical properties and natural tissues, sudden release of growth factors, and poor interfacial bonding. New osteochondral repair scaffolds that can precisely drive integrated osteochondral regeneration still need to be developed. Summary of the Invention

[0007] The purpose of this invention is to overcome at least one of the defects in the prior art and provide a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC synergistic differentiation, its preparation method and application. Through the synergistic combination of iPSC-BMSC and biomimetic gradient scaffold, integrated functional regeneration of osteochondral and long-term stable maintenance of cartilage phenotype can be achieved.

[0008] The objective of this invention can be achieved through the following technical solutions: The first aspect of the present invention provides a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation, wherein the osteochondral repair scaffold is a bilayer biomimetic structure, specifically including a subchondral bone repair layer and an articular cartilage repair layer. The subchondral bone repair layer is an electrospun support framework of type A composite hydrogel / polycaprolactone (PCL) loaded with bone marrow mesenchymal stem cells (BMSCs). The type A composite hydrogel includes 0.5-5 wt% methacrylamide hyaluronic acid (HAMA), 0.5-5 wt% methacrylamide sodium alginate (AlgMA), and 2-12 wt% methacrylamide gelatin (GelMA). The articular cartilage repair layer is a type B composite hydrogel loaded with induced pluripotent stem cells (iPSCs). The type B composite hydrogel includes 0.2-4 wt% of porcine knee joint decellularized cartilage matrix (dECM), 0.5-5 wt% of methacryloyl hyaluronic acid (HAMA), 0.5-5 wt% of methacryloyl chondroitin sulfate (ChSMA), and 1-10 wt% of methacryloyl gelatin (GelMA). The subchondral bone repair layer and the articular cartilage repair layer are bonded together at the interface through GelMA / AlgMA photocrosslinking.

[0009] Furthermore, the components of the type A composite hydrogel and type B composite hydrogel can be adjusted as needed to meet the requirements of different bone defect repair scenarios for scaffold mechanical properties and degradation rates.

[0010] Furthermore, the subchondral bone repair layer is an A-type composite hydrogel / polycaprolactone electrospun support framework loaded with bone marrow mesenchymal stem cells (BMSCs). The A-type composite hydrogel includes 1-3 wt% methacrylamide hyaluronic acid, 1-3 wt% methacrylamide sodium alginate, and 4-8 wt% methacrylamide gelatin. The articular cartilage repair layer is a type B composite hydrogel loaded with induced pluripotent stem cells (iPSCs). The type B composite hydrogel includes 0.5-2 wt% of decellularized porcine knee joint cartilage matrix, 1-3 wt% of methacrylamide hyaluronic acid, 1-3 wt% of methacrylamide chondroitin sulfate, and 3-7 wt% of methacrylamide gelatin.

[0011] Furthermore, the pore size of the polycaprolactone electrospun support framework is 200~400 μm, preferably 300 μm; the fiber diameter is 100~300 μm, preferably 200 μm. The parameters of the above-mentioned polycaprolactone electrospun support framework can be adapted to the needs of stem cell adhesion, proliferation, and inward growth.

[0012] Furthermore, the type A composite hydrogel is loaded with BMP-2 and VEGF165.

[0013] Furthermore, the loading of BMP-2 (bone morphogenetic protein-2) is 80~120 ng / mL, preferably 100 ng / mL.

[0014] Furthermore, the loading of VEGF165 (vascular endothelial growth factor 165) is 20-30 ng / mL, preferably 25 ng / mL.

[0015] Furthermore, the type B composite hydrogel is loaded with BMP-2 and TGF-β3.

[0016] Furthermore, the loading of BMP-2 is 15~25 ng / mL, preferably 20 ng / mL.

[0017] Furthermore, the loading of TGF-β3 (transforming growth factor-β3) is 5~15 ng / mL, preferably 10 ng / mL.

[0018] Furthermore, the compressive modulus of the subchondral bone repair layer is 10~13MPa, which matches the mechanical properties of natural subchondral bone.

[0019] Furthermore, the compressive modulus of the articular cartilage repair layer is 0.5~1 MPa, which matches the mechanical properties of natural articular cartilage.

[0020] Furthermore, the decellularized cartilage matrix of the pig knee joint is prepared by the following method: fresh pig knee joint cartilage is taken and subjected to repeated freeze-thaw cycles, SDS treatment and enzyme digestion to completely remove cellular components.

[0021] Furthermore, the SDS treatment uses an SDS solution with a concentration of 0.4~0.6wt%, preferably a 0.5wt% SDS solution.

[0022] Furthermore, the enzyme digestion employs a DNase / RNase complex system.

[0023] A second aspect of this invention provides a method for preparing a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation, comprising the following steps: S1. Preparation of bio-inks: Prepare type A composite hydrogel bio-ink loaded with BMSCs and type B composite hydrogel bio-ink loaded with iPSCs respectively. S2. 3D bioprinting: Using a dual extrusion 3D bioprinting system, firstly, polycaprolactone is melt-extruded to prepare a polycaprolactone electrospun support skeleton, then type A composite hydrogel is printed to cover the skeleton to form a subchondral bone repair layer, and then type B composite hydrogel is printed on it to form an articular cartilage repair layer. S3. Interface cross-linking enhancement: A precursor solution containing methacrylamide gelatin and methacrylamide sodium alginate is dropped onto the interface between the subchondral bone repair layer and the articular cartilage repair layer. A complete osteocartilage repair scaffold is obtained through two-step ultraviolet light cross-linking.

[0024] Further, in step S1, the loading amount of BMSCs in the type A composite hydrogel bio-ink is (0.5~1.5)×10⁻⁶. 6 cells / mL, preferably 1×10⁻⁶ 6 cells / mL.

[0025] Further, in step S1, the iPSC loading in the type B composite hydrogel bio-ink is (0.5~1.5)×10⁻⁶. 6 cells / mL, preferably 1×10⁻⁶ 6 cells / mL.

[0026] Furthermore, in step S2, the extrusion temperature of the melt-extruded polycaprolactone is 100~120 ℃, and the nozzle inner diameter is 300~500 μm.

[0027] Furthermore, in step S2, the printing pressure of the type A composite hydrogel is no greater than 3 bar, and the printing speed is 1~10 mm / s.

[0028] Furthermore, in step S2, the printing speed of the type B composite hydrogel is 15~25 mm / s, and the nozzle inner diameter is 380~430 μm.

[0029] Furthermore, in step S3, the concentrations of methacrylamide gelatin and methacrylamide sodium alginate in the precursor solution are each independently 3-7 wt%, preferably 5 wt%.

[0030] Furthermore, in step S3, the two-step ultraviolet crosslinking specifically involves: first using 3~7 mW / cm² light... 2 UV pre-crosslinking for 10-20 seconds, followed by UV light at 15-25 mW / cm². 2 Fully cross-linked under ultraviolet light for 25~35 s.

[0031] Furthermore, the two-step ultraviolet crosslinking specifically involves: first, using 5 mW / cm 2 UV pre-crosslinking for 15 s, followed by 20 mW / cm 2 Fully cross-linked under ultraviolet light for 30 seconds.

[0032] The third aspect of this invention provides the application of a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation in the preparation of osteochondral defect repair materials.

[0033] Furthermore, the osteocartilage defect is any one of the following: full-thickness osteocartilage defect of the knee joint, osteoarthritis-related osteocartilage injury, or traumatic osteocartilage defect.

[0034] Furthermore, the osteochondral repair scaffold stabilizes the cartilage phenotype and prevents hypertrophy and calcification by inhibiting the PI3K / Akt signaling pathway.

[0035] In terms of structural design, this invention uses 3D bioprinting technology to precisely construct a biomimetic osteochondral gradient structure and introduces PCL electrospun support skeleton to achieve gradient matching of mechanical properties between the subchondral bone layer and the articular cartilage layer.

[0036] In terms of composition, this invention introduces decellularized cartilage matrix (dECM) of porcine knee joint into the cartilage layer to provide cartilage-specific biological signals, loads BMP-2 and VEGF165 into the subchondral bone layer, and achieves bidirectional induction of osteochondrocytes through iPSC-BMSC synergistic differentiation.

[0037] In terms of application effects, this invention blocks the hypertrophy and calcification process of chondrocytes at the molecular level by inhibiting the PI3K / Akt signaling pathway, and achieves complete osteochondral regeneration in a rabbit knee joint osteochondral defect model in 16 weeks. The surface of the newly formed cartilage is smooth and flat, and it integrates seamlessly with the surrounding normal tissue.

[0038] Compared with the prior art, the present invention has the following technical advantages: (1) This invention uses 3D bioprinting to precisely construct a biomimetic osteocartilage gradient structure, utilizes the synergistic differentiation of iPSC and BMSC, and combines the time-dependent release of growth factors to inhibit the PI3K / Akt signaling pathway to stabilize the cartilage phenotype and avoid hypertrophy and calcification, thus successfully realizing the integrated functional regeneration of osteocartilage and the long-term stable maintenance of the cartilage phenotype.

[0039] (2) The biomimetic gradient structure design of the present invention can accurately replicate the mechanical gradient, microstructure and biochemical microenvironment of natural osteocartilage. The interface is firmly bonded, avoiding delamination and shedding. It can withstand the mechanical load of joint movement and provide stable structural support for tissue regeneration.

[0040] (3) This invention innovatively uses iPSCs for cartilage layer repair, which solves the core defect of traditional BMSCs that are prone to hypertrophy and calcification after chondrogenic differentiation, and can stably maintain the hyaline cartilage phenotype; BMSCs are used for subchondral bone layer repair, which efficiently realizes osteogenic differentiation and vascularization, and the two work together to achieve synchronous regeneration of osteochondrocytes and cartilage.

[0041] (4) The osteochondral repair scaffold of the present invention can achieve long-term stable sustained release of growth factors without obvious burst release effect. The release cycle completely covers the key window of stem cell differentiation and precisely regulates the directed differentiation process of osteogenic and chondrogenic processes.

[0042] (5) The osteochondral repair scaffold of the present invention continuously inhibits the PI3K / Akt signaling pathway, thereby blocking the hypertrophy and calcification process of chondrocytes at the molecular level and ensuring that the repaired tissue maintains the functional characteristics of hyaline cartilage for a long time.

[0043] (6) The osteochondral repair scaffold of the present invention achieved complete integrated osteochondral regeneration after implantation in a rabbit knee joint osteochondral defect model for 16 weeks. The surface of the newly formed hyaline cartilage was smooth and flat, and it was seamlessly integrated with the surrounding normal tissue. The subchondral bone matured and mineralized, and the joint mechanical function and pain symptoms were significantly improved. It can be used for the repair and treatment of knee joint osteochondral defects and osteoarthritis-related cartilage damage, and solves the core bottleneck of the traditional repair strategy of asynchronous osteochondral regeneration and easy hypertrophy and calcification of chondrocytes. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the structure of the 3D-printed biphasic osteochondral repair scaffold of the present invention.

[0045] Figure 2 This is a microscopic morphological characterization diagram of the 3D-printed biphasic osteochondral repair scaffold of Embodiment 1 of the present invention.

[0046] Figure 3 The mechanical property curves are shown for the subchondral bone repair layer and the articular cartilage repair layer in the 3D-printed biphasic osteochondral repair scaffold of Embodiment 1 of the present invention.

[0047] Figure 4 This is the in vitro sustained-release curve of growth factors in the 3D-printed biphasic osteochondral repair scaffold of Embodiment 1 of the present invention.

[0048] Figure 5 The results of the biocompatibility test of the 3D-printed biphasic osteocartilage repair scaffold of Example 1 of the present invention are shown. A represents the CCK-8 cell viability test results, and B represents the live / dead cell staining fluorescence microscopy images (green: live cells; red: dead cells). The scale bar is 100 μm.

[0049] Figure 6 This is a diagram showing the test results of the osteogenic induction capacity of the subchondral bone repair layer in the 3D-printed biphasic osteochondral repair scaffold of Embodiment 1 of the present invention.

[0050] Figure 7 This is a diagram showing the test results of the osteogenic induction capacity of the articular cartilage layer in the 3D-printed biphasic osteochondral repair scaffold of Embodiment 1 of the present invention.

[0051] Figure 8 The results of the molecular mechanism study of the 3D-printed biphasic osteochondral repair scaffold in Example 1 of this invention are presented.

[0052] Figure 9 This is an in vivo observation diagram of the repair effect of the osteocartilage repair scaffold of the present invention.

[0053] Figure 10 Micro-CT three-dimensional reconstruction image of the in vivo repair effect of the osteocartilage repair scaffold of the present invention.

[0054] Figure 11 Histological staining image of the in vivo repair effect of the osteocartilage repair scaffold of the present invention. Detailed Implementation

[0055] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0056] Unless otherwise specified, the reagents, methods, instruments, and equipment used in this invention are conventional in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0057] This invention addresses the technical bottlenecks of existing osteocartilage repair scaffolds, such as asynchronous osteocartilage regeneration, easy hypertrophy and calcification of BMSCs after chondrogenic differentiation, mismatch between scaffold mechanical properties and natural tissues, growth factor burst release, and poor interfacial bonding. It provides a 3D-printed biphasic osteocartilage repair scaffold based on iPSC-BMSC co-differentiation, which achieves integrated functional regeneration of osteocartilage and long-term stable maintenance of cartilage phenotype.

[0058] like Figure 1 As shown, the 3D-printed biphasic osteochondral repair scaffold of the present invention is a double-layer gradient biomimetic structure that can accurately simulate the layered anatomical structure and biochemical microenvironment of natural osteochondral. Specifically, it includes a lower subchondral bone repair layer and an upper articular cartilage repair layer. The two layers are bonded together by GelMA / AlgMA photocrosslinking to form a strong interface.

[0059] In the subchondral bone repair layer, a composite system of type A composite hydrogel and PCL electrospun support framework is used to load bone mesenchymal stem cells (BMSCs), and can also carry BMP-2 and VEGF165. The PCL electrospun framework provides the main mechanical support, the type A composite hydrogel provides a suitable growth microenvironment for BMSCs, BMP-2 induces osteogenic differentiation of BMSCs, and VEGF165 promotes local angiogenesis, providing sufficient nutrient supply for new bone formation.

[0060] In the articular cartilage repair layer, a type B composite hydrogel is used to load iPSCs and can also carry low doses of BMP-2 and TGF-β3. The dECM in the type B composite hydrogel provides cartilage-specific biological signals, low-dose BMP-2 initiates chondrogenic differentiation of iPSCs, and TGF-β3 maintains the cartilage phenotype and inhibits hypertrophy. By continuously inhibiting the PI3K / Akt signaling pathway, the hypertrophy and calcification process of chondrocytes is blocked at the molecular level.

[0061] Current mainstream bone marrow mesenchymal stem cell (BMSC) repair strategies face challenges such as donor shortages, immune rejection, differentiation heterogeneity, and susceptibility to calcification and fibrosis in repaired tissues. Particularly in cartilage-bone interface repair, BMSCs are prone to hypertrophy and ectopic calcification after chondrogenic differentiation, making it difficult to maintain a stable hyaline cartilage phenotype. The osteochondral repair scaffold of this invention, based on the synergistic differentiation of iPSCs and BMSCs, fully leverages the advantages of iPSCs, such as long-term expansion, stable pluripotency, and homogeneous origin, effectively overcoming the limitations of BMSCs caused by aging, donor variability, and pathological conditions. The chondrocytes derived from iPSCs significantly reduce the risk of terminal differentiation, possess anti-mineralization properties, and can form an extracellular matrix in three-dimensional constructs that is highly similar to natural hyaline cartilage.

[0062] Example 1: This embodiment employs an integrated fabrication process combining 3D bioprinting and interfacial photocrosslinking to prepare a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC synergistic differentiation. The specific operation is as follows: 1. Preparation of decellularized cartilage matrix (dECM) for porcine knee joint: (1) Take fresh pig knee joints aged 6-12 months, and under sterile conditions, peel off the articular cartilage of the femoral condyle and tibial plateau, cut it into fragments of about 1 g, and rinse repeatedly with sterile PBS to remove residual blood.

[0063] (2) Perform three cycles of repeated freeze-thaw treatment: freeze at -80 ℃ for 12 h, thaw at room temperature, and completely lyse chondrocytes.

[0064] (3) Place the cartilage fragments in a 0.5% (w / v) SDS solution and stir at room temperature for 24 h to remove cell membrane and cytoplasmic components.

[0065] (4) Add DNase and RNase solutions to the cartilage fragment suspension respectively, so that the final concentration of DNase in the reaction system is 50 U / mL and the final concentration of RNase is 1 U / mL. Incubate at 37 °C for 6 h to degrade the residual nucleic acid.

[0066] (5) Rinse with sterile PBS for 72 h, and change the washing solution every 12 h to completely remove residual SDS and enzyme reagents.

[0067] (6) The treated cartilage fragments were dissolved in 0.1M acetic acid, stirred at 4 °C for 24 h, and centrifuged at 12000×g for 15 min. The supernatant was lyophilized to obtain dECM powder. The DNA residue in the prepared dECM was found to be 26.68 ng / mg, which is far below the safety threshold for clinical biomaterials, and it had no complete nucleus structure, only empty cartilage lacunae.

[0068] 2. Preparation of bio-ink: (1) Type A hydrogel bio-ink: Dissolve 2% (w / v) HAMA, 2% (w / v) AlgMA and 6% (w / v) GelMA in sterile PBS, add 0.25% (w / v) photoinitiator LAP, and stir in the dark until completely dissolved. Then add the isolated and cultured third-generation BMSCs and adjust the cell density to 1×10⁻⁶ cells / year. 6 cells / mL; finally, add BMP-2 (final concentration 100 ng / mL) and VEGF165 (final concentration 25 ng / mL), mix gently to obtain type A bio-ink.

[0069] (2) Type B hydrogel bio-ink: Dissolve 2% (w / v) HAMA, 2% (w / v) ChSMA, 5% (w / v) GelMA and 1% (w / v) dECM in sterile PBS, add 0.25% (w / v) photoinitiator LAP, and stir in the dark until completely dissolved. Then add qualified iPSCs and adjust the cell density to 1×10⁻⁶. 6 cells / mL. Finally, add BMP-2 (final concentration 20 ng / mL) and TGF-β3 (final concentration 10 ng / mL), mix gently, and obtain type B bio-ink.

[0070] The iPSCs (induced pluripotent stem cells) used in this embodiment were commercially available human iPSC cell lines (such as ATCCACS-1026 or equivalent), which were identified as having a normal karyotype (G-banding analysis), expressing the pluripotency markers OCT4, SOX2, and NANOG (immunofluorescence detection positive rate >95%), and possessing the ability to differentiate into the three germ layers (confirmed by in vitro embryomorphic body formation experiments). The iPSCs were cultured in Matrigel-coated culture dishes with mTeSR1 medium and passaged every 3-4 days. Before use, they were confirmed to be negative for mycoplasma. Those skilled in the art can also reprogram iPSCs themselves by referring to known methods (such as Takahashi K, et al. Cell, 2007;131(5):861-872), or use other validated iPSC cell lines.

[0071] 3. Fabrication of 3D-printed biphasic osteochondral repair scaffolds: (1) Preparation of PCL electrospun support skeleton: PCL (molecular weight 90 k) was dissolved in methanol to prepare a 10% (w / v) solution. PCL electrospun membrane with fiber diameter of about 200 μm and pore size of about 300 μm was prepared by electrospinning technology and cut into the required size as support skeleton.

[0072] (2) Dual-extrusion 3D printing: A 3D bioprinter equipped with high-temperature and low-temperature nozzles was used. First, PCL particles were added to the high-temperature nozzle and melted and extruded at 100-120 °C to print a mesh-like support skeleton. Then, type A bio-ink was loaded into the low-temperature nozzle and printed at a pressure of <3 bar and a speed of 1-10 mm / s to uniformly coat the PCL skeleton and form a subchondral bone layer with a thickness of 2 mm. Finally, type B bio-ink was loaded into the other low-temperature nozzle and printed at a speed of 20 mm / s on the subchondral bone layer to obtain a 2 mm thick articular cartilage layer, resulting in a 6 mm diameter and 4 mm height double-layer scaffold prototype.

[0073] (3) Interfacial crosslinking enhancement: A mixed precursor solution containing AlgMA and GelMA (the final concentrations of AlgMA and GelMA in the precursor solution are each 5% (w / v), and they are mixed in equal volumes) is dropped onto the interface of the bilayer scaffold to ensure thorough wetting and removal of air bubbles. First, place it at 5 mW / cm². 2 Pre-crosslinking was performed by irradiation under a UV lamp for 15 s to fix the interface morphology; then it was placed under a 20 mW / cm² UV lamp. 2 Irradiation under ultraviolet light for 30 seconds enables full cross-linking, resulting in a strong bond between the two layers.

[0074] (4) Sterilization and storage: The prepared scaffold is placed in a clean bench and sterilized by irradiation with ultraviolet light for 30 min. Then it is rinsed three times with sterile PBS to remove uncrosslinked monomers and impurities. It is stored at 4 ℃ for later use.

[0075] like Figure 2 As shown, the final osteochondral repair scaffold fabricated in this embodiment has an overall diameter of approximately 6 mm and a height of approximately 4 mm, which is compatible with the standardized modeling parameters of the rabbit knee joint osteochondral defect model. Scanning electron microscopy (SEM) observation revealed that the subchondral bone layer (Type A) exhibits a porous network structure in which PCL electrospun fibers and hydrogel interpenetrate, with fiber diameters ranging from 100 to 300 μm and pore sizes from 200 to 400 μm, which is conducive to the adhesion, migration, and nutrient exchange of BMSCs. The articular cartilage layer (Type B) exhibits a uniform and dense hydrogel microstructure, with iPSCs evenly distributed within it, exhibiting plump cell morphology and good integration with the surrounding hydrogel matrix. The interface transition between the two layers is continuous, with no obvious delamination, indicating that the interface cross-linking enhancement process effectively achieved the integrated bonding of the subchondral bone layer and the articular cartilage layer.

[0076] Based on the successful preparation of the above-mentioned 3D printed biphasic osteocartilage repair scaffold, the present invention further conducts the following tests and characterizations on the prepared osteocartilage repair scaffold.

[0077] 1. Mechanical property testing: The bracket was subjected to compression testing using a universal testing machine.

[0078] like Figure 3 As shown, the results indicate that the compressive modulus of the subchondral bone layer is 11.35 MPa, and the compressive modulus of the articular cartilage layer is 0.85 MPa, which are highly consistent with the mechanical properties of natural subchondral bone and articular cartilage.

[0079] 2. Sustained-release performance test: The in vitro release curve of the growth factor was detected by ELISA.

[0080] like Figure 4 As shown, the results indicate that BMP-2, VEGF165, and TGF-β3 can be continuously released for more than 21 days without significant burst release effects, and the release concentration can meet the needs of stem cell directed differentiation.

[0081] 3. Biocompatibility evaluation: The cytotoxicity of the scaffold was detected by the CCK-8 assay and live / dead cell staining method.

[0082] like Figure 5 As shown, the results indicate that within the concentration range of 0.01-0.1 g / mL, the survival rate of both BMSCs and iPSCs is >95%, the cell morphology is intact, and there is no obvious cell death, indicating that the scaffold has excellent biocompatibility.

[0083] 4. Evaluation of the scaffold's ability to induce differentiation in vitro: (1) Osteogenic induction capacity of the subchondral bone layer: BMSCs were co-cultured with type A scaffold extract, and osteogenic-related indicators were detected by scratch assay, ALP staining, Alizarin Red S staining, and RT-qPCR.

[0084] like Figure 6 As shown, the type A scaffold extract can significantly promote the migration of BMSCs, enhance ALP activity and calcium nodule formation ability, and upregulate the expression of osteogenic-related genes such as Runx2, Osterix and Col1a1, indicating that it has excellent osteogenic induction ability.

[0085] (2) Articular cartilage layer chondrogenic induction capacity: BMSCs and iPSCs were co-cultured with B-type scaffold extracts, and cartilage-forming and osteogenic-related markers were detected by toluidine blue staining, alizarin red S staining, RT-qPCR, and Western blot.

[0086] like Figure 7 As shown, the B-type scaffold extract can efficiently induce chondrogenic differentiation of iPSCs, upregulate the expression of chondrogenic genes such as SOX9 and COL2A1, and no obvious calcium nodule formation is observed. The expression of osteogenic genes Runx2, Osterix and Col1a1 is not significantly increased. In contrast, BMSCs can undergo chondrogenic differentiation under the same conditions, but at the same time, they exhibit obvious osteogenic transdifferentiation and hypertrophic phenotypes.

[0087] (3) Molecular mechanism research: Time-series transcriptome sequencing was performed on iPSCs. For example... Figure 8 As shown, the results indicate that the PI3K / Akt signaling pathway is continuously suppressed in the mid-to-late stages of chondrogenic differentiation, and the expression of downstream target genes SGK2 and CREB3L3, which mediate cartilage hypertrophy, is significantly downregulated, revealing the molecular mechanism by which iPSCs can stably maintain the cartilage phenotype.

[0088] 5. Evaluation of in vivo repair effect of stent: Animal model establishment and grouping: 12-week-old male New Zealand white rabbits, weighing approximately 2 kg, were selected. A full-thickness osteochondral defect model with a diameter of 3 mm and a depth of 3 mm was prepared in the trochlear groove of the right knee joint. The experimental rabbits were randomly divided into 4 groups: blank control group (Def-BC), scaffold-only group (Def-MC), BMSC single-load group (Def-B-EX), and iPSC+BMSC dual-load group (Def-BI-EX), with 6 rabbits in each group.

[0089] Stent implantation and postoperative management: The corresponding stent was implanted into the defect area, and the joint capsule and skin were sutured layer by layer. Antibiotics and analgesics were administered for two consecutive days postoperatively. The experimental rabbits were sacrificed at 8 and 16 weeks postoperatively, and samples were collected for testing.

[0090] Repair effect evaluation: General observation: such as Figure 9 As shown, at 16 weeks post-surgery, the defect area in the Def-BI-EX group was completely healed, with a smooth and even surface of newly formed cartilage. The color and texture were not significantly different from the surrounding normal cartilage, and the boundaries were indistinguishable. In contrast, the other three groups all showed varying degrees of residual defects, with uneven surfaces of newly formed tissue and poor integration with surrounding tissues.

[0091] Micro-CT detection: such as Figure 10 As shown, the subchondral bone in the Def-BI-EX group was completely regenerated, and the bone volume / total volume (BV / TV) ratio was significantly higher than that in other groups, while the bone density was basically consistent with that of the surrounding normal bone tissue.

[0092] Histological staining: such as Figure 11 As shown, H&E, Safranin O-Fixed Green and Toluidine Blue staining results showed that the defect area in the Def-BI-EX group was covered by mature hyaline cartilage tissue, the chondrocytes were neatly arranged, and the cartilage layer was closely connected with the subchondral bone layer, which met the histological criteria for integrated osteochondral repair.

[0093] Immunofluorescence staining: The expression levels of SOX9 and Col2a1 in the Def-BI-EX group were significantly higher than those in other groups, indicating that the newly formed cartilage has a typical hyaline cartilage phenotype.

[0094] Functional evaluation: The mechanical pain threshold and thermal pain threshold of rabbits in the Def-BI-EX group were significantly higher than those in other groups, indicating that the weight-bearing function of the knee joint was significantly improved and the pain symptoms were effectively relieved.

[0095] Safety evaluation: 16 weeks post-operation, there were no significant abnormalities in blood routine tests, liver and kidney function, and myocardial enzyme levels in the experimental rabbits of each group, and no obvious pathological damage was observed in the visceral tissues after HE staining, indicating that the scaffold has good in vivo biocompatibility.

[0096] In summary, this invention precisely constructs a biomimetic osteochondral gradient structure using 3D bioprinting. Utilizing the synergistic differentiation of iPSCs and BMSCs, combined with the time-dependent release of growth factors, it inhibits the PI3K / Akt signaling pathway to stabilize cartilage phenotype and prevent hypertrophy and calcification, achieving integrated osteochondral regeneration. This scaffold exhibits mechanical properties matching natural tissue, excellent biocompatibility, and stable growth factor release. Complete repair was achieved in 16 weeks in a rabbit knee osteochondral defect model. It can be used for the repair and treatment of knee osteochondral defects and osteoarthritis-related cartilage damage, overcoming the core bottlenecks of traditional repair strategies, such as asynchronous osteochondral regeneration and easy hypertrophy and calcification of chondrocytes.

[0097] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation, characterized in that, The osteochondral repair scaffold is a double-layer biomimetic structure, specifically including a subchondral bone repair layer and an articular cartilage repair layer; The subchondral bone repair layer is an A-type composite hydrogel / polycaprolactone electrospun support framework loaded with bone marrow mesenchymal stem cells (BMSCs). The A-type composite hydrogel includes 0.5-5 wt% methacrylamide hyaluronic acid, 0.5-5 wt% methacrylamide sodium alginate, and 2-12 wt% methacrylamide gelatin. The articular cartilage repair layer is a type B composite hydrogel loaded with induced pluripotent stem cells (iPSCs). The type B composite hydrogel includes 0.2-4 wt% of decellularized cartilage matrix of porcine knee joint, 0.5-5 wt% of methacrylamide hyaluronic acid, 0.5-5 wt% of methacrylamide chondroitin sulfate, and 1-10 wt% of methacrylamide gelatin. The subchondral bone repair layer and the articular cartilage repair layer are bonded together at the interface through photocrosslinking of methacrylamide gelatin / methacrylamide sodium alginate.

2. The 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation according to claim 1, characterized in that, The pore size of the polycaprolactone electrospun support skeleton is 200~400 μm, and the fiber diameter is 100~300 μm.

3. The 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation according to claim 1, characterized in that, The type A composite hydrogel is loaded with BMP-2 and VEGF165, with BMP-2 loading at 80~120 ng / mL and VEGF165 loading at 20~30 ng / mL. The type B composite hydrogel is loaded with BMP-2 and TGF-β3, with BMP-2 loading at 15~25 ng / mL and TGF-β3 loading at 5~15 ng / mL.

4. The 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation according to claim 1, characterized in that, The compressive modulus of the subchondral bone repair layer is 10~13 MPa, and the compressive modulus of the articular cartilage repair layer is 0.5~1 MPa.

5. The 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation according to claim 1, characterized in that, The decellularized cartilage matrix of the pig knee joint was prepared by the following method: fresh pig knee joint cartilage was taken and subjected to repeated freeze-thaw cycles, SDS treatment and enzyme digestion to completely remove cellular components; The SDS treatment uses an SDS solution with a concentration of 0.4~0.6wt%; The enzyme digestion uses a DNase / RNase complex system.

6. A method for preparing a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Preparation of bio-inks: Prepare type A composite hydrogel bio-ink loaded with BMSCs and type B composite hydrogel bio-ink loaded with iPSCs respectively. S2. 3D bioprinting: Using a dual extrusion 3D bioprinting system, firstly, polycaprolactone is melt-extruded to prepare a polycaprolactone electrospun support skeleton, then type A composite hydrogel is printed to cover the skeleton to form a subchondral bone repair layer, and then type B composite hydrogel is printed on it to form an articular cartilage repair layer. S3. Interface cross-linking enhancement: A precursor solution containing methacrylamide gelatin and methacrylamide sodium alginate is dropped onto the interface between the subchondral bone repair layer and the articular cartilage repair layer. A complete osteocartilage repair scaffold is obtained through two-step ultraviolet light cross-linking.

7. The method for preparing a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation according to claim 6, characterized in that, In step S1, the loading amount of BMSCs in the type A composite hydrogel bioink is (0.5~1.5)×10⁻⁶. 6 cells / mL; The iPSC loading in the type B composite hydrogel bio-ink is (0.5~1.5)×10⁻⁶. 6 cells / mL.

8. The method for preparing a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation according to claim 6, characterized in that, In step S2, the extrusion temperature of the melt-extruded polycaprolactone is 100~120 ℃, and the nozzle inner diameter is 300~500 μm; The printing pressure of the type A composite hydrogel is no more than 3 bar, and the printing speed is 1~10 mm / s; The printing speed of the type B composite hydrogel is 15~25 mm / s, and the nozzle inner diameter is 380~430 μm.

9. The method for preparing a 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation according to claim 6, characterized in that, In step S3, the concentrations of methacrylamide gelatin and methacrylamide sodium alginate in the precursor solution are each independently 3-7 wt%. The two-step ultraviolet crosslinking process specifically involves: first, using 3~7 mW / cm² light... 2 UV pre-crosslinking for 10-20 seconds, followed by UV light at 15-25 mW / cm². 2 Fully cross-linked under ultraviolet light for 25~35 s.

10. The application of the 3D-printed biphasic osteochondral repair scaffold based on iPSC-BMSC co-differentiation as described in any one of claims 1-5 in the preparation of osteochondral defect repair materials, characterized in that, The osteocartilage defect is any one of the following: full-thickness osteocartilage defect of the knee joint, osteoarthritis-related osteocartilage injury, or traumatic osteocartilage defect.

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