A three-layer osteochondral repair composite hydrogel material with physical induction of chondrogenic-osteogenic function respectively and a preparation method thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-04-09
- Publication Date
- 2026-06-26
Smart Images
Figure CN122272907A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of preparation technology of osteochondral repair hydrogels, specifically relating to a three-layer osteochondral repair composite hydrogel material with physical induction of chondrogenesis and osteogenic function, and its preparation method. Background Technology
[0002] Articular cartilage damage and osteochondral defects are common orthopedic diseases, often caused by trauma, degenerative changes, metabolic diseases, or sports injuries. They frequently affect the entire functional unit, from the articular cartilage to the subchondral bone. Because articular cartilage tissue lacks blood vessels, lymphatic vessels, and nerves, and has low chondrocyte density and limited proliferative capacity, it is almost impossible for it to self-repair after injury. Without timely and effective intervention, local defects will progressively worsen, eventually developing into osteoarthritis, leading to loss of joint function or limb disability.
[0003] Natural osteochondral tissue possesses a complex layered gradient structure, consisting of a layer of hyaline cartilage, a layer of calcified cartilage, and a layer of subchondral bone, from the superficial to the deep layers. Each layer exhibits significant differences in extracellular matrix composition, mechanical properties, pore structure, and biological function. The hyaline cartilage layer is primarily responsible for bearing load and lubricating the joint, while the subchondral bone layer provides mechanical support and nutrient supply. The intermediate calcified cartilage layer acts as a functional interface unit, providing mechanical buffering and isolating the different cellular microenvironments of the adjacent layers. This unique layered structure suggests that an ideal osteochondral repair strategy should precisely mimic the natural layered gradient structure to achieve synchronous regeneration and functional integration of the cartilage and bone layers.
[0004] Current clinical treatments, such as microfracture techniques and autologous or allogeneic osteochondral transplantation, still have significant limitations. Fibrous cartilage formed through microfracture techniques lacks the biomechanical properties of hyaline cartilage; osteochondral transplantation faces challenges such as limited donor sites, donor site morbidity, graft-recipient bed thickness mismatch, and immune rejection. The rise of tissue engineering technology has provided new solutions for osteochondral regeneration, but how to simultaneously achieve functional construction of both cartilage and bone layers within the same scaffold system and ensure synergistic integration between the two layers remains a challenge in current research.
[0005] In recent years, researchers have begun to explore strategies for synergistically inducing the directed differentiation of stem cells through bioactive molecules and physical stimulation. In cartilage regeneration, KGN, a small molecule compound, can specifically induce mesenchymal stem cells to differentiate into chondrocytes by regulating the CBFβ-RUNX1 signaling pathway, promoting cartilage matrix synthesis. ZnO nanowires can generate local electrical signals under the subtle mechanical stimulation of physiological activities, promoting the expression of cartilage-related genes through electromechanical transduction. However, neither small molecule induction nor physical stimulation alone can achieve stable and efficient cartilage regeneration effects.
[0006] In bone regeneration, BMP-2, as the most classic osteogenic inducing factor, has been widely used in bone tissue engineering. However, its short in vivo half-life, high-dose administration, and tendency to induce ectopic osteogenic side effects are drawbacks. HA, as the main inorganic component of the bone matrix, possesses good osteoconductivity and biocompatibility. Fe3O4 nanoparticles, due to their magnetic response characteristics, can generate mechanical stimulation under an external magnetic field, triggering osteogenic signaling pathways such as MAPK and Wnt / β-catenin by activating mechanosensitive ion channels (such as Piezo1) on the cell membrane. Although each of these strategies has its advantages, the integrated scaffold design that combines the chemical induction of BMP-2, the osteoconductivity of HA, and the magnetomechanical regulation of Fe3O4 to achieve low-dose, high-safety, and high-efficiency osteogenic bone formation has not yet been fully developed.
[0007] Based on the aforementioned technical bottlenecks and existing problems, this invention designs a three-layered osteochondral repair composite hydrogel material: the upper layer is a HAMA hydrogel loaded with BMSCs / KGN / ZnO nanowires, which synergistically promotes cartilage differentiation through the chemical induction of KGN and the piezoelectric physical stimulation of ZnO nanowires; the lower layer is a GelMA hydrogel loaded with BMSCs / BMP-2 / HA / Fe3O4 nanoparticles, which combines the chemical induction of BMP-2, the osteoconduction of HA, and the magnetic response physical regulation of Fe3O4 to achieve multi-mechanism synergistic osteogenic effects; the middle layer is a high-concentration GelMA / HA composite dense layer, which serves as a functional interface unit to physically isolate the cellular microenvironment of the upper and lower layers. This invention, through the partitioned functional design of the three-layer structure and the synergistic integration between the layers, provides a new technical solution for achieving biomimetic and efficient repair of osteochondral tissue. Summary of the Invention
[0008] The purpose of this invention is to address the difficulty in repairing double-layered osteochondral in the prior art, and to provide a three-layered osteochondral repair composite hydrogel material and its preparation method that have physical induction of chondrogenesis and osteogenic functions respectively.
[0009] This invention is achieved using the following technical solution: A method for preparing a three-layer osteochondral repair composite hydrogel material with physical-induced chondrogenic and osteogenic functions includes the following steps: A middle-layer bio-ink was prepared, wherein the middle-layer bio-ink is a high-concentration GelMA (methacrylamide gelatin) solution composed of HA (hydroxyapatite); Prepare an upper layer bio-ink, wherein the upper layer bio-ink is a porous HAMA (methacrylamide hyaluronic acid) solution loaded with BMSCs (bone marrow mesenchymal stem cells), KGN (Kartogenin) and ZnO nanowires. A lower-layer bio-ink was prepared, wherein the lower-layer bio-ink was a low-concentration porous GelMA solution loaded with BMSCs, BMP-2, HA and Fe3O4 nanofillers; A multi-nozzle bio-3D printer was used to print the lower, middle, and upper layers sequentially. After each layer was printed, UV curing was performed immediately to set the shape of each layer. After the overall scaffold was printed, final curing was performed to ensure that the interfaces of the three-layer structure were fully cross-linked, resulting in the osteochondral repair composite hydrogel material including the upper cartilage region, the middle isolation region, and the lower bone region.
[0010] Furthermore, the preparation process of the intermediate layer bio-ink includes: Using HAMA with a substitution degree of 30-70% and HA with a particle size of 100-300nm as raw materials, under sterile and light-protected conditions, a medium-layer bio-ink containing 12-20wt% GelMA, 1-10wt% HA and 0.05-0.25wt% photoinitiator was prepared in a water bath at 50-70℃. After stirring evenly, the mixture was centrifuged to remove bubbles at a speed of 500-750rpm for 3-5min to obtain the medium-layer bio-ink.
[0011] Furthermore, the preparation process of the upper layer bio-ink includes: Using HAMA with a substitution degree of 30-70%, ZnO nanowires with a diameter of 50-150 nm and a length of 5-20 μm, and P3-P5 generation BMSCs as raw materials, KGN was first prepared into a 10 mM stock solution with DMSO under aseptic conditions and stored at 4°C in the dark. Under aseptic and light-protected conditions, a PBS solution containing 3-8 wt% HAMA, 0.25-1.5 wt% ZnO nanowires, and 0.05-0.25 wt% photoinitiator was prepared in a water bath at 50-70°C. After the solution cooled to 35-37°C, the KGN stock solution was added to a final concentration of 1-10 μM, and P3-P5 generation BMSCs were added to a final cell density of 5-10 × 10⁶ cells / mL. 6 After mixing the cells / mL thoroughly, centrifuge to remove bubbles at a speed of 500-750 rpm for 3-5 min to obtain the upper layer of bio-ink.
[0012] Furthermore, the preparation process of the lower layer bio-ink includes: Using GelMA with a substitution degree of 35-65%, HA nanoparticles with a particle size of 50-200 nm, Fe3O4 nanoparticles with a diameter of 10-30 nm, and P3-P5 generation BMSCs as raw materials, BMP-2 was prepared into a stock solution of 100 μg / mL in PBS containing 0.1% bovine serum albumin under aseptic conditions. Under aseptic and light-protected conditions, a PBS solution containing 3-8 wt% GelMA, 0.01-0.1 wt% Fe3O4 nanoparticles, 1-5 wt% HA nanoparticles, and 0.05-0.25 wt% photoinitiator was prepared in a water bath at 50-70℃. After the solution cooled to 35-37℃, the BMP-2 stock solution was added to a final concentration of 50-200 ng / mL, and P3-P5 generation BMSCs were added to a final cell density of 5-10 × 10⁶ cells / mL. 6 After mixing the cells / mL thoroughly, centrifuge to remove bubbles at a speed of 500-750 rpm for 3-5 min to obtain the lower layer of bio-ink.
[0013] Furthermore, the photoinitiator is one or more of LAP (lithium phenyl-2,4,6-trimethylbenzoyl phosphate), Irgacure 2959, and Eosin Y.
[0014] Furthermore, the multi-nozzle bio-3D printing and curing process includes: The lower, middle, and upper layers of bio-ink are preheated to 30-35℃ and loaded into the independent temperature-controlled nozzles of a multi-nozzle bio-3D printer. The nozzle temperatures for the lower and middle layers of bio-ink are controlled at 35-37℃, and the nozzle temperature for the upper layer of bio-ink is controlled at 25-30℃. The printing platform temperature is set to 8-12℃. Each nozzle is automatically calibrated non-contactly to ensure that the X / Y / Z axis positioning error is ≤0.05 mm. The lower, middle, and upper layers are printed sequentially, each consisting of several thin layers stacked together. After each layer is printed, it is cured using 405 nm UV light. After all the structures are printed, the entire scaffold is finally cured to ensure full cross-linking between the layers, resulting in the osteochondral repair composite hydrogel material.
[0015] Furthermore, the printing and curing parameters for each layer include: When printing the lower layer structure, a nozzle with a diameter of 200-300 μm is used, the extrusion pressure is 80-180 kPa, and the printing speed is 5-12 mm / s. The thickness of the thin layer is set to 70-85% of the nozzle diameter, and it is printed layer by layer according to a preset porous mesh structure path, with the mesh pore size being 200-400 μm. During curing, the curing strength of the first thin layer in contact with the printing platform is 8-10 mW / cm². 2The curing time is 20-30 seconds, and the curing strength of the remaining thin layers is 4-8 mW / cm. 2 The curing time is 10-20 seconds, and the total thickness of the lower layer structure is controlled at 1-2 mm. When printing the middle layer structure, a nozzle with a diameter of 250-400 μm is used, the extrusion pressure is 200-350 kPa, and the printing speed is 2-6 mm / s. The thickness of the thin layer is set to 60-75% of the nozzle diameter. During curing, the curing strength of each thin layer is 5-8 mW / cm². 2 The curing time is 20-30 seconds, the total thickness of the middle layer structure is controlled at 0.5-1.2 mm, and the middle layer structure is a dense, non-porous structure. When printing the upper layer structure, a nozzle with a diameter of 200-300 μm is used, the extrusion pressure is 40-120 kPa, and the printing speed is 8-18 mm / s. The thickness of the thin layer is set to 80-100% of the nozzle diameter. During curing, the curing strength of each thin layer is 3-5 mW / cm². 2 The curing time is 10-15 seconds to minimize photo-oxidative damage to the upper BMSCs, while ensuring that the HAMA network is fully cross-linked to form a stable scaffold, and the total thickness of the upper structure is controlled at 1-2 mm. After the overall support frame is printed, it undergoes final curing, with a curing strength of 4-6 mW / cm². 2 The curing time is 30-60 seconds to ensure full cross-linking of the three-layer structure interface.
[0016] A three-layer osteochondral repair composite hydrogel material with physical induced chondrogenesis and osteogenic functions is prepared by the above method. The osteochondral repair composite hydrogel material consists of three layers: an upper layer with piezoelectric and chemical chondrogenic functions, a middle layer with functional units of calcified cartilage layer that mimic natural osteochondral, and a lower layer with magnetic and chemical chondrogenic functions.
[0017] Furthermore, the osteochondral repair composite hydrogel material can induce osteogenic formation under a dynamic cyclic magnetic field; the dynamic cyclic magnetic field is a sinusoidal cyclic magnetic field with a frequency of 1-3 Hz and an intensity of 200-400 mT, and a magnetic field gradient ≤10mT / mm; starting from the 3rd day after magnetic field treatment, the magnetic field is applied 1-2 times a day, each time lasting 15-45 minutes, for 2-3 consecutive weeks.
[0018] Compared with the prior art, the present invention has the following advantages: 1) This invention uses GelMA, HAMA, HA nanoparticles, ZnO nanowires, Fe3O4 nanoparticles, BMSCs, KGN, and BMP-2 as core raw materials, and combines them with a gradient concentration formulation strategy, temperature-responsive multi-nozzle bio-3D printing, interlayer gradient UV curing, time-sequential cell loading process, and magnetically responsive remote physical control to prepare a three-layer osteochondral repair composite hydrogel material. The selection of raw materials and the combination of processes mentioned above are original to this invention. For the first time, this invention integrates an upper layer of chemical-piezoelectric synergistic cartilage-promoting, a lower layer of chemical-osteoconduction-magnetic responsive synergistic bone-promoting, and a middle layer of temperature-sensitive dense interface isolation into a single scaffold system. Furthermore, by controlling the printing temperature gradient, it achieves spatiotemporal decoupling of the in-situ physical pre-gelation and chemical cross-linking of the GelMA material, providing an innovative process solution for solving the problem of high-precision printing of cell-containing bio-inks.
[0019] 2) The three-layer structure designed in this invention deeply simulates the layered gradient characteristics of natural osteochondral tissue. Each layer presents a differentiated design in terms of material composition, concentration ratio, pore structure, mechanical properties and functional positioning, and achieves overall synergy through interlayer interface chemical cross-linking: a. The upper layer promotes the multi-mechanism synergistic design of the cartilage layer: The upper layer uses 3-8wt% low concentration HAMA as matrix material, loaded with BMSCs, KGN small molecules and ZnO nanowire piezoelectric materials. The biomimetic principle and functional mechanism of its structural design are as follows: ① Adaptability of topology structure to cell migration: 3-8wt% low polymer concentration combined with layer height setting of 80-100% nozzle diameter (160-300 μm) and grid-like printing path to construct a through-pore macroporous network with a pore size of 200-400 μm. This pore size range has been optimized and screened—pores smaller than 200 μm easily hinder cell migration, while pores larger than 400 μm have insufficient surface area, affecting cell adhesion—perfectly matching the migration size requirements of BMSCs (cell diameter approximately 20-30 μm, requiring a pore size ≥ 5 times the cell diameter for unimpeded migration), while simultaneously reserving sufficient space for the deposition of newly formed cartilage matrix. ② This invention directly incorporates KGN in a free form into the HAMA network, utilizing the swelling properties of HAMA hydrogel to achieve sustained release of KGN (release cycle approximately 2-3 weeks), perfectly matching the signal demand window during the early stages of cartilage differentiation (0-14 days). ③ The piezoelectric-electrochemical coupling mechanism of ZnO nanowires: The piezoelectric effect of ZnO nanowires (diameter 50-150 nm, length 5-20 μm) originates from the non-centrosymmetry of their wurtzite crystal structure. Under the minute mechanical stimulation generated by joint physiological activities (frequency 0.1-3 Hz, strain 1-10%), ZnO nanowires undergo bending deformation, leading to the separation of positive and negative charge centers and generating transient piezoelectric potential. More importantly, the one-dimensional high aspect ratio structure of ZnO nanowires allows them to form a penetrating piezoelectric network even at low doping concentrations (0.25-1.5 wt%), achieving efficient conversion of mechanical signals into electrical signals, thereby first and foremost efficiently promoting cartilage through physical means. ④ Synergistic effect of upper-level mechanisms: The chemical signal of KGN lays the foundation for differentiation direction, while the piezoelectric physical signal of ZnO enhances differentiation efficiency, synergistically activating cartilage matrix genes. b. Quadruple synergistic design of the lower layer to promote bone layer: The lower layer uses 3-8wt% low concentration GelMA as matrix material, loaded with BMSCs, BMP-2 growth factor, HA nanoparticles and Fe3O4 magnetic nanoparticles. The biomimetic principle and functional mechanism of its structural design are as follows: ① Cascade enhancement effect of dual nanoparticles: HA nanoparticles and Fe3O4 nanoparticles form a dual nanocomposite system in the lower matrix.The main functions of HA nanoparticles include: a) Bone conduction scaffold: The surface of HA is rich in calcium ion binding sites, which mediate the adhesion and spread of BMSCs; b) Local calcium and phosphorus pool: HA slowly releases calcium and phosphate ions in a weakly acidic microenvironment (pH≈6.5-6.8 during the inflammatory phase), locally increasing the calcium and phosphorus concentration and promoting the formation of hydroxyapatite mineralization nuclei; c) Affinity anchoring of BMP-2: HA has a natural affinity for BMP-2 (through the electrostatic interaction between the heparin binding domain of BMP-2 and calcium ions on the surface of HA), which can achieve the enrichment and sustained release of BMP-2 and prolong its biological half-life. Fe3O4 nanoparticles endow the underlying magnetic response function, and their superparamagnetism enables them to generate directional movement and rotation under the action of an external magnetic field. Magnetic drive generates localized mechanical force through the rotational vibration of Fe3O4 nanoparticles, which is transmitted to the cytoskeleton via cell-matrix adhesion sites. This activates surface physical responses such as integrin aggregation, cytoskeleton remodeling, and ion channel opening. This synergistic effect, combined with the chemical induction of BMP-2 and the osteoconductive action of HA, drives the osteogenic differentiation process. This purely physical, remote control method enables non-invasive, controllable, and programmable intervention in the osteogenic process. ②HA's affinity anchoring of BMP-2 slows its release and prolongs its duration of action. Therefore, the lower layer of this invention can achieve osteogenic effects comparable to traditional high-dose methods at BMP-2 concentrations of only 50-200 ng / mL, significantly reducing the risk and cost of ectopic osteogenic formation. ③ Triple Synergistic Spatiotemporal Integration: The lower layer achieves synergistic integration of four mechanisms: chemical induction by BMP-2, osteoconduction by HA, magnetic-mechanical conversion by Fe3O4, and mechanotransduction of calcium signals. HA provides adhesion sites and calcium-phosphorus sources (days 0-7), BMP-2 initiates the osteogenic differentiation program (days 0-14), and Fe3O4 magnetically drives continuous mechanical stimulation (days 2-28). These three mechanisms are highly synergistic in time and space, jointly driving efficient osteoogenesis. c. Precise Structural Design of the Middle Physical Isolation Layer: The middle layer uses 12-20wt% high-concentration GelMA combined with 1-10wt% HA nanoparticles to construct a low-porosity, dense structure. The scientific and functional mechanisms of its structural design include: ① Critical Effect of Dual Concentration Windows: Increasing the GelMA concentration from 3-8wt% (upper and lower layers) to 12-20wt% (middle layer) produces a critical transition—the polymer chain entanglement density exceeds the permeability threshold, forming a continuous, dense network. This concentration-driven structural phase transition is key to achieving physical isolation. ② Pore-filling effect of HA nanoparticles: 1-10 wt% HA nanoparticles are uniformly dispersed in the GelMA network, further filling the network gaps and blocking cell migration channels. At the same time, the hydrogen bonding interaction between HA particles and GelMA chains enhances network stability, increasing the compressive modulus of the middle layer to 3-5 times that of the upper and lower layers, mimicking the mechanical buffering function of natural calcified cartilage layers.③ Chemical fusion mechanism of interlayer interface: This invention employs an interlayer UV curing process. After the middle layer is printed onto the surface of the lower layer, UV irradiation is performed immediately, causing the incompletely cross-linked residual double bonds in the lower layer to covalently cross-link with the methacryloyl groups in the middle layer, forming an interpenetrating network. This chemically fused interface significantly improves the interlayer bonding strength compared to physically superimposed interfaces, preventing interlayer delamination after implantation. ④ Dual functional balance of isolation and communication: Although the middle layer is dense and isolates cell migration, it still allows limited diffusion of small molecule signaling substances (such as BMP-2 and KGN), enabling signal communication between the upper and lower layers. BMP-2 secreted by the lower layer can diffuse in trace amounts to the middle layer interface, forming a signal gradient and guiding the directional alignment of cells at the interface; cartilage matrix molecules secreted by the upper layer can also diffuse to the middle layer, forming a natural transition zone. This selective permeability mimics the functional characteristics of natural calcified cartilage layers.
[0020] 3) This invention develops a temperature-responsive multi-nozzle collaborative printing process for the three-layer material based on its differentiated rheological properties and biological requirements. Its innovation is reflected in: (1) Precise control mechanism of nozzle-platform temperature gradient: For the temperature-sensitive physical gel properties of GelMA, this invention adopts a 15-25℃ temperature difference design between the nozzle (35-37℃) and the platform (8-12℃) to achieve the following synergistic effects: ① High temperature of the nozzle maintains liquid state: Ensures that the GelMA ink is kept in a low viscosity fluid state in the nozzle, avoids needle blockage, and ensures printing continuity; ② Low temperature of the platform induces pre-gel: After the extruded GelMA filament comes into contact with the low temperature platform, it quickly undergoes physical cross-linking (forming physical hydrogel), and completes the transformation from liquid to solid state within a few seconds, which helps to preserve the structure and avoid collapse; ③ Spatiotemporal decoupling of physical pre-gel and chemical cross-link: Physical pre-gel provides temporary structural stability, and UV photochemical cross-link provides permanent network fixation. The two are separated by temperature control, which ensures both printing accuracy and cell activity. This temperature-driven physical-chemical dual crosslinking timing regulation is the core process innovation of this invention to achieve high-precision printing of cell-containing GelMA. (2) Precise matching of layer pressure and intensity: This invention designs a viscosity-matching printing parameter system to address the differences in viscosity of the three layers of ink: ① Pressure gradient: From the upper layer to the lower layer and then to the middle layer, the pressure increases step by step to compensate for the extrusion resistance caused by the increase in viscosity; ② Speed gradient: From the upper layer (8-18 mm / s) to the lower layer (5-12 mm / s) and then to the middle layer (2-6 mm / s), the speed decreases step by step to ensure that the extrusion amount of each layer is precisely controllable; ③ Layer height gradient: From the upper layer (80-100% of the nozzle diameter) to the lower layer (70-85%) and then to the middle layer (60-75%), the layer height decreases step by step to compensate for the insufficient spreading of high-viscosity materials. This multi-parameter synergistic optimization enables high-precision printing of all three layers of materials under the same nozzle diameter. (3) Gradient UV curing process: This invention designs an interlayer gradient UV curing process for three layers with different cell loads and thickness requirements: ① High-intensity curing of the first layer (8-10 mW / cm²) 2 ① 20-30 seconds: Ensure the lower layer adheres firmly to the low-temperature platform to prevent the support from slipping during printing; ② Medium-strength curing of the intermediate layer (4-8 mW / cm²) 2 10-20 seconds): Balancing cross-linking efficiency and cell activity; ③ Low-intensity curing of the upper layer (3-5mW / cm) 2 10-15 seconds): Maximizes protection of the upper BMSCs from photo-oxidative damage; ④ Final curing (4-6 mW / cm²) 2 (30-60 seconds): Overall reinforcement and interface integration.
[0021] 4) This invention is the first to combine the Fe3O4 magnetic response system with a three-layer osteochondral scaffold to achieve remote, non-invasive, and programmable osteogenic regulation after implantation: (1) Biological adaptation of dynamic magnetic field parameters: This invention selects a dynamic cyclic magnetic field (frequency 1-3 Hz, intensity 200-400 mT, sine wave) instead of a static magnetic field. The mechanism is based on the following: ① Frequency matching cell response window: 1-3 Hz is consistent with the physiological gait frequency (1-2 Hz), which can simulate the periodic mechanical load borne by the subchondral bone during normal walking; ② Intermittent stimulation to avoid adaptive desensitization: The dynamic magnetic field generates intermittent mechanical stimulation (1-2 times a day, 15-45 min each time), avoiding cell desensitization and signal attenuation due to continuous stimulation; ③ Sine wave to simulate physiological load curve: The sine wave is similar in shape to the stress-strain curve during joint movement, and is more easily perceived by cells as a physiological signal. (2) Matching the timing of magnetic drive with the healing process: The present invention is designed to start magnetic drive from the 3rd day after implantation. The biological basis includes: ① Avoidance of the inflammation period: The 0-3 days after implantation are the acute inflammation period, and the cells are in a state of stress. Applying physical stimulation too early may aggravate the inflammatory response; ② Cell adhesion stabilization period: By the 3rd day, BMSCs have completed the initial adhesion, spreading and integrin expression, and have the ability to sense and respond to mechanical stimulation; ③ Differentiation initiation window: The 3rd to 21st day is the critical window period for the directed differentiation of stem cells. Magnetic drive stimulation can maximize the differentiation induction efficiency.
[0022] 5) This invention achieves multi-dimensional and multi-scale functional synergy through the differentiated design and integrated construction of a three-layer structure: (1) Synergy of spatial partitioning and interface integration: The three layers perform their respective functions in space (upper cartilage zone, middle isolation zone, and lower bone zone), but form a whole through interface chemical fusion, avoiding the interlayer delamination problem of traditional multilayer scaffolds. The chemical interpenetrating network at the interface also plays a signal gradient conduction function - the BMP-2 of the lower layer can diffuse in small amounts to the bottom of the middle layer to form an osteogenic signal gradient, guiding the cells at the interface to differentiate in the direction of osteoogenesis; the cartilage matrix molecules of the upper layer diffuse to the top of the middle layer to form a cartilage signal gradient. This signal gradient interface makes the three layers form a natural transition zone, avoiding stress concentration and functional failure caused by steep interfaces. (2) Temporal synergy of chemical-physical multi-mechanisms: The multiple mechanisms within each layer of this invention exhibit programmed synergy over time: ① Early stage (0-7 days): Chemical signals of KGN and BMP-2 dominate, laying the foundation for differentiation direction; ② Middle stage (7-21 days): The piezoelectric signal of ZnO (upper layer) and the magnetic driving signal of Fe3O4 (lower layer) are enhanced, and physical stimulation strengthens differentiation efficiency; ③ Late stage (after 21 days): Ions released by HA and ZnO / Fe3O4 (Zn 2+ Ca 2+ Fe 3+(3) Interlayer signal communication and functional interaction: There is bidirectional signal communication between the three layers: ① Mechanical signal from top to bottom: The joint pressure borne by the upper layer is transmitted to the lower layer through the middle layer, simulating the force transmission path of the natural joint; ② Biochemical signal from bottom to top: VEGF secreted by osteoblasts in the lower layer can diffuse to the upper layer, promote blood vessel ingrowth, and provide nutrition for cartilage regeneration; ③ Signal relay function of the middle layer: The middle layer not only isolates cells, but also regulates the diffusion rate of signal molecules through its dense network, forming a natural signal gradient. This interlayer signal dialogue makes the three layers not isolated functional units, but an organically integrated functional whole.
[0023] 6) This invention addresses the clinical challenge of osteochondral defects with the following breakthrough advantages: ① It solves the problem of interface confusion in traditional scaffolds: Traditional integrated osteochondral scaffolds lack effective interface isolation design, often leading to cell migration and signal interference between the cartilage and bone regions after implantation, ultimately resulting in a blurred interface of regenerated tissue (the appearance of fibrocartilage mixed areas). This invention, through the design of a dense middle isolation layer, fundamentally blocks cell migration across layers while allowing limited diffusion of signaling molecules, making the interface between regenerated cartilage and bone clear and structurally orderly, mimicking the tidal line structure of natural osteochondral tissue. ② It achieves efficient induction of low-dose growth factors: Through synergistic chemical-physical multi-mechanism, this invention can achieve efficient osteoproliferation at BMP-2 concentrations of only 50-200 ng / mL, significantly reducing the risk of ectopic osteoogenesis, inflammatory reactions, and tumors that may be induced by high-dose BMP-2. Similarly, KGN's small molecule alternative growth factor strategy avoids the high cost and instability problems of growth factors. ③ Remote non-invasive control for personalized treatment: This invention utilizes the Fe3O4 magnetic response system to achieve remote, non-invasive, programmable physical control after implantation. Doctors can dynamically adjust magnetic field parameters (intensity, frequency, and duration of action) based on the patient's postoperative recovery (such as bone healing progress shown in imaging) to enhance bone formation as needed. This post-implantation adjustability is a core advantage unmatched by traditional scaffolds. ④ Integrated 3D printing for precise customization: Employing multi-nozzle bio-3D printing, this invention can precisely customize the three-layer thickness, diameter, and pore distribution of the scaffold according to the patient's individual defect dimensions (obtained through CT / MRI 3D reconstruction), achieving personalized and precise repair. The temperature gradient design and interlayer photocuring of the printing process ensure the structural fidelity of complex-shaped scaffolds. ⑤ Complete material degradation and tissue regeneration are synchronized: GelMA, HAMA, HA, ZnO, and Fe3O4 are all degradable or metabolizable materials: GelMA / HAMA degrades through ester bond hydrolysis (degradation cycle 2-4 weeks), HA degrades through cell-mediated phagocytosis (degradation cycle 4-12 weeks), and ZnO and Fe3O4 release ions through dissolution and are then excreted through metabolism (degradation cycle 8-16 weeks). The degradation rate matches the tissue regeneration rate—as the scaffold gradually degrades, new cartilage and bone tissue gradually grow in, ultimately achieving complete self-tissue replacement and avoiding foreign body residue. Attached Figure Description
[0024] Figure 1 This is a schematic diagram of the preparation process of the three-layer osteochondral repair composite hydrogel material with physical induction of chondrogenesis and osteogenic functions according to the present invention.
[0025] Figure 2 The results of CCK-8 culture of BMSCs in the upper and lower hydrogels of Examples 1 and 3 of this invention are shown.
[0026] Figure 3The results show the fluorescence of BMSCs in the hydrogel in Examples 1 and 3 of this invention.
[0027] Figure 4 Immunofluorescence results of BMSCs after chondrogenic differentiation in the upper gel (a corresponds to Example 1, b corresponds to Example 1).
[0028] Figure 5 The results show the PCR results of osteogenic / chondrogenic differentiation of BMSCs in the upper and lower gel layers. Detailed Implementation
[0029] The present invention will now be described in further clarity and detail with reference to the accompanying drawings and specific examples.
[0030] Comparative Example 1: 1) Preparation of the upper layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% HAMA (approximately 50% substitution degree) and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, add BMSCs (P3 generation) suspension until the final cell density is 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0031] 2) Preparation of the lower layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% GelMA (approximately 50% substitution degree) and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, add BMSCs (P3 generation) suspension until the final cell density is 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0032] 3) Multi-nozzle bio-3D printing to construct a double-layer scaffold: After preheating the two types of bio-inks to 32°C, they are loaded into the independent temperature-controlled nozzles of the bio-printer: the lower ink is placed in nozzle 1, and the upper ink is placed in nozzle 2. The temperatures of nozzle 1 and nozzle 2 are set to 37°C and 28°C respectively, and the printing platform temperature is set to 10°C. Automatic calibration of the dual nozzles is performed to ensure positioning accuracy.
[0033] a. Lower Layer Printing: Start printhead 1, select a 250μm nozzle, set the extrusion pressure to 120kPa, printing speed to 8mm / s, and layer height to 200μm. Print the next layer layer by layer according to the preset porous mesh structure path (total thickness approximately 1.5mm). After each layer is printed, immediately turn on the 405nm UV light source for interlayer curing: first layer light intensity 8mW / cm². 2The duration is 25 seconds; the illumination intensity of subsequent layers is adjusted to 6 mW / cm². 2 The time is 15 seconds.
[0034] b. Upper Layer Printing: After the lower layer has cured, switch to printhead 2. Select a 250μm nozzle, set the extrusion pressure to 60kPa, printing speed to 12mm / s, and layer height to 220μm. Print the upper layer layer by layer (total thickness approximately 1.5mm) according to the preset porous mesh structure path. For each layer printed, use 405nm UV light for curing, reducing the light intensity to 4mW / cm². 2 The time is 12 seconds to reduce damage to cells.
[0035] d. Post-processing: After both layers are printed and the interlayer curing is completed, the entire composite material undergoes a final curing process using 405nm UV light at an intensity of 5mW / cm². 2 The time is 45 seconds, which makes the interface bonding stronger, thus obtaining the desired double-layer osteochondral repair composite hydrogel material.
[0036] 4) Mechanical property testing: The compressive modulus of the composite hydrogel was tested using a universal testing machine. The results showed that its compressive modulus was approximately 260±19 kPa, indicating good mechanical support properties. Biocompatibility testing: (e.g., ...) Figure 2 As shown, BMSCs were encapsulated in the upper and lower hydrogel layers and co-cultured for 7 days. The cell viability rates were 95.3% (upper layer) and 94.1% (lower layer), respectively, indicating that the material has good cell compatibility. In situ three-dimensional immunofluorescence confocal imaging of the upper layer BMSCs showed relatively low Col2 fluorescence signal (e.g., ...). Figure 4 After separating and lysing the upper and lower gel layers, BMSCs were subjected to chondrogenic / osteogenic RT-qPCR detection, and the results are as follows: Figure 5As shown. Animal experiment: A rabbit knee joint osteochondral defect model was constructed, and the prepared double-layer osteochondral repair composite hydrogel material was implanted into the defect area. Starting from the 3rd day after implantation, a dynamic circulating magnetic field (2Hz frequency, 300mT intensity sine wave, direction perpendicular to the defect plane) was applied to the defect area using a magnetic drive device, once daily for 30 minutes each time, for 3 consecutive weeks. Twelve weeks postoperatively, samples were taken for Micro-CT, MRI (magnetic resonance imaging), and histological staining (Safranin O, Fast Green, and type II collagen immunohistochemistry). Micro-CT results showed that the subchondral bone defect area was filled with only a small amount of new bone tissue, the trabecular structure was sparse and disordered, and there was a significant difference compared with normal bone tissue, indicating unsatisfactory osteogenic repair. MRI results showed that the signal intensity of the cartilage layer defect area was significantly different from that of the surrounding normal cartilage, the new tissue filling was incomplete, and no typical cartilage signal characteristics were observed. Histological staining (Safranin O-Fixed Green staining) revealed that the regenerated tissue was mainly disorganized fibrous tissue with very weak Safranin O staining and no hyaline cartilage-like extracellular matrix deposition. Immunohistochemical staining for type II collagen was negative, indicating that neither the upper nor lower layers effectively induced the formation of functional cartilage or bone tissue. These results suggest that the bilayer composite hydrogel material, lacking functional nanofillers, active factors, and a biomimetic intermediate calcified layer structure, exhibited unsatisfactory repair effects in both layers, failing to achieve integrated osteochondral regeneration and repair.
[0037] Example 1: like Figure 1 As shown, the preparation process of a three-layer osteochondral repair composite hydrogel material with physical-induced chondrogenic and osteogenic functions is as follows: 1) Preparation of intermediate-layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 15wt% GelMA (approximately 50% substitution), 5wt% HA (approximately 200nm particle size), and 0.15wt% photoinitiator LAP. After preparation, centrifuge at 600rpm for 4min to remove bubbles, then store at 4°C for later use.
[0038] 2) Preparation of the upper layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% HAMA (approximately 50% substitution), 0.8 wt% ZnO nanowires (approximately 100 nm in diameter and 10 μm in length), and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, add KGN stock solution to a final concentration of 5 μM, and then add BMSCs (P3 generation) suspension to a final cell density of 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0039] 3) Preparation of the lower layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% GelMA (approximately 50% substitution), 3 wt% HA nanoparticles (approximately 100 nm in diameter), 0.05 wt% Fe3O4 nanoparticles (approximately 20 nm in diameter), and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, quickly add BMP-2 stock solution to a final concentration of 100 ng / mL, and then add BMSCs (P3 generation) suspension to a final cell density of 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0040] 4) Multi-nozzle bio-3D printing to construct a three-layer scaffold: After preheating the three types of bio-inks to 32°C, they are loaded into the independent temperature-controlled nozzles of the bio-printer: the lower layer ink is placed in nozzle 1, the middle layer ink in nozzle 2, and the upper layer ink in nozzle 3. The temperatures of nozzles 1 and 2 are set to 37°C, the temperature of nozzle 3 to 28°C, and the printing platform temperature is set to 10°C. Automatic calibration of the three nozzles is performed to ensure positioning accuracy.
[0041] a. Lower Layer Printing: Start printhead 1, select a 250μm nozzle, set the extrusion pressure to 120kPa, printing speed to 8mm / s, and layer height to 200μm. Print the next layer layer by layer according to the preset porous mesh structure path (total thickness approximately 1.5mm). After each layer is printed, immediately turn on the 405nm UV light source for interlayer curing: first layer light intensity 8mW / cm². 2 The duration is 25 seconds; the illumination intensity of subsequent layers is adjusted to 6 mW / cm². 2 The time is 15 seconds.
[0042] b. Middle Layer Printing: After the lower layer has cured, switch to printhead 2. Select a 300μm nozzle, set the extrusion pressure to 250kPa, printing speed to 4mm / s, and layer height to 200μm. Print a non-porous middle layer (total thickness approximately 0.8mm). Each layer is cured using 405nm UV light at a light intensity of 6mW / cm². 2 The time is 25 seconds.
[0043] c. Upper Layer Printing: After the middle layer has cured, switch to printhead 3. Select a 250μm nozzle, set the extrusion pressure to 60kPa, printing speed to 12mm / s, and layer height to 220μm. Print the upper layer layer by layer (total thickness approximately 1.5mm) according to the preset porous mesh structure path. For each layer printed, use 405nm UV light for curing, reducing the light intensity to 4mW / cm². 2 The time is 12 seconds to reduce damage to cells.
[0044] d. Post-processing: After all three layers are printed and interlayer curing is completed, the entire composite material undergoes a final curing process using 405nm UV light with an intensity of 5mW / cm². 2 The time is 45 seconds, which makes the three-layer interface more firmly bonded, thus obtaining the desired three-layer osteochondral repair composite hydrogel material with physical induction of chondrogenesis and osteogenic function.
[0045] 5) Mechanical property testing: The compressive modulus of the composite hydrogel was tested using a universal testing machine. The results showed that its compressive modulus was approximately 290±25 kPa, indicating good mechanical support properties. Biocompatibility testing: BMSCs were encapsulated in the upper and lower layers of the hydrogel and co-cultured for 7 days. The cell viability rates were 92.4% (upper layer) and 90.1% (lower layer), respectively, indicating that the material has good cell compatibility (e.g., ...). Figure 2 , Figure 3 As shown). In situ three-dimensional immunofluorescence confocal imaging of the upper BMSCs revealed a significant enhancement of the Col2 fluorescence signal (e.g., ...). Figure 4 After separating and lysing the upper and lower gel layers, BMSCs were subjected to chondrogenic / osteogenic RT-qPCR detection. The results showed that the expression of chondrogenic genes was significantly upregulated in the upper layer cells and significantly upregulated in the lower layer cells (compared to Comparative Example 1, e.g.) Figure 5 Animal experiments: A rabbit knee joint osteochondral defect model was constructed, and the three-layer composite hydrogel material prepared above was implanted into the defect area. Starting from the 3rd day after implantation, a dynamic circulating magnetic field (2Hz frequency, 300mT sine wave, direction perpendicular to the defect plane) was applied to the defect area using a magnetic drive device, once daily for 30 minutes each time, for 3 consecutive weeks. Twelve weeks post-operation, samples were harvested for Micro-CT, MRI (magnetic resonance imaging), and histological staining (Safranin O, Fast Green, and type II collagen immunohistochemistry). Micro-CT results showed that the subchondral bone plate was intact and continuous, the trabecular bone structure was dense and regularly arranged, and the bone volume fraction was significantly increased. MRI results showed that the signal intensity of the newly formed cartilage tissue was similar to that of the surrounding normal cartilage, the cartilage layer thickness was uniform, and the surface was smooth and flat. Histological staining (Safranin O-Fixed Green staining) revealed that the regenerated tissue exhibited a typical hyaline cartilage-like appearance, with normal chondrocyte morphology and orderly arrangement, and abundant proteoglycans in the extracellular matrix. Immunohistochemical staining for type II collagen showed strong positivity, indicating that the main component of the newly formed cartilage matrix was functional type II collagen. These results demonstrate that the three-layer composite hydrogel material successfully achieved integrated regeneration and repair of osteochondrocytes and cartilage.
[0046] Example 2: 1) Preparation of intermediate-layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 15wt% GelMA (approximately 50% substitution), 5wt% HA (approximately 200nm particle size), and 0.15wt% photoinitiator LAP. After preparation, centrifuge at 600rpm for 4min to remove bubbles, then store at 4°C for later use.
[0047] 2) Preparation of the upper layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% HAMA (approximately 50% substitution), 0.8 wt% ZnO nanowires (approximately 100 nm in diameter and 10 μm in length), and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, add KGN stock solution to a final concentration of 5 μM, and then add BMSCs (P3 generation) suspension to a final cell density of 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0048] 3) Preparation of the lower layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% GelMA (approximately 50% substitution), 3 wt% HA nanoparticles (approximately 100 nm in diameter), 0.05 wt% Fe3O4 nanoparticles (approximately 20 nm in diameter), and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, quickly add BMP-2 stock solution to a final concentration of 100 ng / mL, and then add BMSCs (P3 generation) suspension to a final cell density of 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0049] 4) Multi-nozzle bio-3D printing to construct a three-layer scaffold: After preheating the three types of bio-inks to 32°C, they are loaded into the independent temperature-controlled nozzles of the bio-printer: the lower layer ink is placed in nozzle 1, the middle layer ink in nozzle 2, and the upper layer ink in nozzle 3. The temperatures of nozzles 1 and 2 are set to 37°C, the temperature of nozzle 3 to 28°C, and the printing platform temperature is set to 10°C. Automatic calibration of the three nozzles is performed to ensure positioning accuracy.
[0050] a. Lower Layer Printing: Start printhead 1, select a 250μm nozzle, set the extrusion pressure to 120kPa, printing speed to 8mm / s, and layer height to 200μm. Print the next layer layer by layer according to the preset porous mesh structure path (total thickness approximately 1.5mm). After each layer is printed, immediately turn on the 405nm UV light source for interlayer curing: first layer light intensity 8mW / cm². 2The duration is 25 seconds; the illumination intensity of subsequent layers is adjusted to 6 mW / cm². 2 The time is 15 seconds.
[0051] b. Middle Layer Printing: After the lower layer has cured, switch to printhead 2. Select a 300μm nozzle, set the extrusion pressure to 250kPa, printing speed to 4mm / s, and layer height to 200μm. Print a non-porous middle layer (total thickness approximately 0.8mm). Each layer is cured using 405nm UV light at a light intensity of 6mW / cm². 2 The time is 25 seconds.
[0052] c. Upper Layer Printing: After the middle layer has cured, switch to printhead 3. Select a 250μm nozzle, set the extrusion pressure to 60kPa, printing speed to 12mm / s, and layer height to 220μm. Print the upper layer layer by layer (total thickness approximately 1.5mm) according to the preset porous mesh structure path. For each layer printed, use 405nm UV light for curing, reducing the light intensity to 4mW / cm². 2 The time is 12 seconds to reduce damage to cells.
[0053] d. Post-processing: After all three layers are printed and interlayer curing is completed, the entire composite material undergoes a final curing process using 405nm UV light with an intensity of 5mW / cm². 2 The time is 45 seconds, which makes the three-layer interface more firmly bonded, thus obtaining the desired three-layer osteochondral repair composite hydrogel material with physical induction of chondrogenesis and osteogenic function.
[0054] 5) Compared to Example 1, this example reduces the stimulation of the dynamic circulating magnetic field. Animal experiment: A rabbit knee joint osteochondral defect model was constructed, and the three-layer composite hydrogel material prepared above was implanted into the defect area. Twelve weeks post-operation, samples were taken for Micro-CT, MRI (magnetic resonance imaging), and histological staining (Safranin O-Fixed Green, type II collagen immunohistochemistry). Micro-CT results showed that the subchondral bone defect area was filled with new bone tissue, but the trabecular structure was relatively sparse and irregularly arranged, and the bone volume fraction was significantly lower than that in Example 1. Although the osteogenic effect was not as good as the magnetically driven group, it still showed a certain bone conduction and repair capacity. MRI results showed that the defect area was filled with new tissue, and the signal intensity was slightly lower than that of the surrounding normal cartilage. Histological staining (Safranin O-Fixed Green staining) showed that the regenerated tissue was mainly fibrocartilage-like tissue, with a small amount of hyaline cartilage-like matrix deposition; type II collagen immunohistochemical staining was positive, confirming the effectiveness of the material in chemically inducing chondrogenesis. The above results indicate that, in the absence of physical magnetic stimulation, the three-layer composite hydrogel material can still achieve partial repair of osteochondral defects through its chemical induction function, but the quality and maturity of the newly formed bone tissue are significantly lower than those of Example 1 with applied magnetic field.
[0055] Example 3: 1) Preparation of intermediate-layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 15wt% GelMA (approximately 50% substitution), 5wt% HA (approximately 200nm particle size), and 0.15wt% photoinitiator LAP. After preparation, centrifuge at 600rpm for 4min to remove bubbles, then store at 4°C for later use.
[0056] 2) Preparation of the upper layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% HAMA (approximately 50% substitution), 0.8 wt% ZnO nanowires (approximately 100 nm in diameter and 10 μm in length), and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, add a suspension of BMSCs (P3 generation) until the final cell density reaches 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0057] 3) Preparation of the lower layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% GelMA (approximately 50% substitution), 3 wt% HA nanoparticles (approximately 100 nm in diameter), 0.05 wt% Fe3O4 nanoparticles (approximately 20 nm in diameter), and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, add BMSCs (P3 generation) suspension until the final cell density is 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0058] 4) Multi-nozzle bio-3D printing to construct a three-layer scaffold: After preheating the three types of bio-inks to 32°C, they are loaded into the independent temperature-controlled nozzles of the bio-printer: the lower layer ink is placed in nozzle 1, the middle layer ink in nozzle 2, and the upper layer ink in nozzle 3. The temperatures of nozzles 1 and 2 are set to 37°C, the temperature of nozzle 3 to 28°C, and the printing platform temperature is set to 10°C. Automatic calibration of the three nozzles is performed to ensure positioning accuracy.
[0059] a. Lower Layer Printing: Start printhead 1, select a 250μm nozzle, set the extrusion pressure to 120kPa, printing speed to 8mm / s, and layer height to 200μm. Print the next layer layer by layer according to the preset porous mesh structure path (total thickness approximately 1.5mm). After each layer is printed, immediately turn on the 405nm UV light source for interlayer curing: first layer light intensity 8mW / cm². 2 The duration is 25 seconds; the illumination intensity of subsequent layers is adjusted to 6 mW / cm².2 The time is 15 seconds.
[0060] b. Middle Layer Printing: After the lower layer has cured, switch to printhead 2. Select a 300μm nozzle, set the extrusion pressure to 250kPa, printing speed to 4mm / s, and layer height to 200μm. Print a non-porous middle layer (total thickness approximately 0.8mm). Each layer is cured using 405nm UV light at a light intensity of 6mW / cm². 2 The time is 25 seconds.
[0061] c. Upper Layer Printing: After the middle layer has cured, switch to printhead 3. Select a 250μm nozzle, set the extrusion pressure to 60kPa, printing speed to 12mm / s, and layer height to 220μm. Print the upper layer layer by layer (total thickness approximately 1.5mm) according to the preset porous mesh structure path. For each layer printed, use 405nm UV light for curing, reducing the light intensity to 4mW / cm². 2 The time is 12 seconds to reduce damage to cells.
[0062] d. Post-processing: After all three layers are printed and interlayer curing is completed, the entire composite material undergoes a final curing process using 405nm UV light with an intensity of 5mW / cm². 2 The time is 45 seconds, which makes the three-layer interface more firmly bonded, thus obtaining the desired three-layer osteochondral repair composite hydrogel material with physical induction of chondrogenesis and osteogenic function.
[0063] 5) Compared to Example 1, this example reduces the introduction of KGN and BMP-2 in both the upper and lower layers. The mechanical properties are similar to those of Example 1, exhibiting good mechanical support. Biocompatibility testing: BMSCs were encapsulated in the above-mentioned upper and lower hydrogels and co-cultured for 7 days. The cell viability rates were 88.4% (upper layer) and 86.8% (lower layer), respectively, indicating that the material has good cell compatibility (e.g., ...). Figure 2 , Figure 3(As shown). Animal experiment: A rabbit knee joint osteochondral defect model was constructed, and the three-layer composite hydrogel material prepared above was implanted into the defect area. Starting from the 3rd day after implantation, a dynamic circulating magnetic field (2Hz frequency, 300mT intensity sine wave, direction perpendicular to the defect plane) was applied to the defect area using a magnetic drive device, once daily for 30 minutes each time, for 3 consecutive weeks. Twelve weeks postoperatively, samples were taken for Micro-CT, MRI (magnetic resonance imaging), and histological staining (Safranin O, Fast Green, and type II collagen immunohistochemistry). Micro-CT results showed that the subchondral bone plate was intact and continuous, the trabecular bone structure was dense and regularly arranged, and the bone volume fraction was significantly increased. MRI results showed that the signal intensity of the newly formed cartilage tissue was similar to that of the surrounding normal cartilage, the cartilage layer thickness was uniform, and the surface was smooth and flat. Histological staining (Safranin O-Fixed Green staining) revealed that the regenerated tissue exhibited a typical hyaline cartilage-like appearance, with normal chondrocyte morphology and orderly arrangement, and abundant proteoglycans in the extracellular matrix. Immunohistochemical staining for type II collagen showed strong positivity, indicating that the main component of the newly formed cartilage matrix was functional type II collagen. These results demonstrate that the three-layer composite hydrogel material successfully achieved integrated regeneration and repair of osteochondrocytes. Micro-CT results showed that new bone tissue filled the subchondral bone region, with relatively dense and regularly arranged trabecular bone structure. The bone volume fraction was significantly higher than the blank control group, but still less than in Example 1, indicating that physical magnetic stimulation compensated for the BMP-2 deficiency to some extent, inducing osteogenic differentiation, but the osteogenic effect was slightly inferior to that of Example 1 with the synergistic effect of chemical factors. MRI results showed that new tissue filled the cartilage layer defect area, with signal intensity close to that of the surrounding normal cartilage, but the cartilage layer thickness was slightly thinner and the surface smoothness was slightly worse. Histological staining (Safranin O-Fix Green staining) revealed that the regenerated tissue was mainly hyaline cartilage, with relatively normal chondrocyte morphology and decent arrangement, and abundant proteoglycan deposition in the extracellular matrix. Immunohistochemical staining for type II collagen was positive, confirming the effectiveness of the synergistic induction of chondrogenesis by physical magnetic stimulation and the piezoelectric effect of ZnO, but the staining intensity and extent were weaker than in Example 1. These results indicate that, in the absence of KGN and BMP-2 chemical inducing factors, this composite hydrogel material can still effectively repair osteochondral defects by relying on its three-layer biomimetic structure, piezoelectric effect, and magnetically driven physical signals. However, the repair quality (including the density of the newly formed bone tissue, the thickness and maturity of the cartilage layer) is somewhat inferior to that of Example 1, which utilizes a synergistic chemical-physical approach.
[0064] Example 4: 1) Preparation of the upper layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% HAMA (approximately 50% substitution), 0.8 wt% ZnO nanowires (approximately 100 nm in diameter and 10 μm in length), and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, add KGN stock solution to a final concentration of 5 μM, and then add BMSCs (P3 generation) suspension to a final cell density of 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0065] 2) Preparation of the lower layer bio-ink: Under sterile, light-protected conditions and in a 60°C water bath, prepare a PBS solution containing 5 wt% GelMA (approximately 50% substitution), 3 wt% HA nanoparticles (approximately 100 nm in diameter), 0.05 wt% Fe3O4 nanoparticles (approximately 20 nm in diameter), and 0.15 wt% photoinitiator LAP. After the solution cools to 37°C, quickly add BMP-2 stock solution to a final concentration of 100 ng / mL, and then add BMSCs (P3 generation) suspension to a final cell density of 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.
[0066] 3) Multi-nozzle bio-3D printing to construct a double-layer scaffold: After preheating the two types of bio-inks to 32°C, they are loaded into the independent temperature-controlled nozzles of the bio-printer: the lower ink is placed in nozzle 1, and the upper ink is placed in nozzle 2. The temperature of nozzle 1 is set to 37°C, the temperature of nozzle 2 is set to 28°C, and the printing platform temperature is set to 10°C. Automatic calibration of the two nozzles is performed to ensure positioning accuracy.
[0067] a. Lower Layer Printing: Start printhead 1, select a 250μm nozzle, set the extrusion pressure to 120kPa, printing speed to 8mm / s, and layer height to 200μm. Print the next layer layer by layer according to the preset porous mesh structure path (total thickness approximately 1.5mm). After each layer is printed, immediately turn on the 405nm UV light source for interlayer curing: first layer light intensity 8mW / cm². 2 The duration is 25 seconds; the illumination intensity of subsequent layers is adjusted to 6 mW / cm². 2 The time is 15 seconds.
[0068] b. Upper Layer Printing: After the lower layer has cured, switch to printhead 2. Select a 250μm nozzle, set the extrusion pressure to 60kPa, printing speed to 12mm / s, and layer height to 220μm. Print the upper layer layer by layer (total thickness approximately 1.5mm) according to the preset porous mesh structure path. For each layer printed, use 405nm UV light for curing, reducing the light intensity to 4mW / cm². 2 The time is 12 seconds to reduce damage to cells.
[0069] d. Post-processing: After both layers are printed and the interlayer curing is completed, the entire composite material undergoes a final curing process using 405nm UV light at an intensity of 5mW / cm². 2 The time is 45 seconds, which makes the interface bonding stronger, thus obtaining the desired double-layer osteochondral repair composite hydrogel material with physical induction of chondrogenesis and osteogenic function.
[0070] 4) Compared to Example 1, this example reduces the design of the middle layer. Mechanical property testing: The compressive modulus of the composite hydrogel was tested using a universal testing machine. The results showed that its compressive modulus was approximately 270.3 ± 25 kPa, indicating good mechanical support properties. Animal experiments: A rabbit knee joint osteochondral defect model was constructed, and the prepared bilayer composite hydrogel material was implanted into the defect area. Starting from the 3rd day after implantation, a dynamic circulating magnetic field (2Hz frequency, 300mT intensity sine wave, direction perpendicular to the defect plane) was applied to the defect area using a magnetic drive device, once daily for 30 minutes each time, for 3 consecutive weeks. Twelve weeks post-operation, samples were taken for Micro-CT, MRI (magnetic resonance imaging), and histological staining (Safranin O, Fast Green, and type II collagen immunohistochemistry). Micro-CT results showed that the subchondral bone plate was intact and continuous, the trabecular bone structure was dense and regularly arranged, and the bone volume fraction was significantly increased. MRI results showed that the signal intensity of the newly formed cartilage tissue was similar to that of the surrounding normal cartilage, the cartilage layer thickness was uniform, and the surface was smooth and flat. Histological staining (Safranin O-Fixed Green staining) revealed that the regenerated tissue exhibited a typical hyaline cartilage-like appearance, with normal chondrocyte morphology and orderly arrangement, and abundant proteoglycans in the extracellular matrix. Immunohistochemical staining for type II collagen showed strong positivity, indicating that the main component of the newly formed cartilage matrix was functional type II collagen. These results demonstrate that the bilayer composite hydrogel material successfully achieved integrated regeneration and repair of osteochondrocytes and cartilage. Micro-CT results showed that the subchondral bone region was filled with new bone tissue, and the trabecular structure was relatively dense. However, the new bone tissue abnormally extended into the upper layer, causing bone tissue to invade the space that should have been occupied by cartilage tissue. MRI results showed that although the cartilage layer defect area was filled with new tissue, the cartilage signal and bone tissue signal were mixed, and the interface between the cartilage layer and bone tissue was blurred, making it difficult to clearly distinguish the tide line position. Histological staining (Safranin O-Fixed Green staining) revealed a disordered intermingling of osteogenic and chondrogenic regions in the regenerated tissue. Extensive bone tissue intrusion was observed in the upper layer, while occasional island-like cartilage-like tissue was seen in the lower layer. The lack of a clear calcified layer at the interface between the upper and lower layers resulted in the disappearance or significant shift of the tide line structure. Type II collagen immunohistochemical staining showed positive areas mainly concentrated in the upper layer, but the distribution was uneven, with a mixture of cartilage and bone matrix at the interface with bone tissue. These results indicate that in the absence of a biomimetic intermediate calcified layer, the lack of a physical barrier and functional transition between the upper and lower layers leads to interference between osteogenic and chondrogenic differentiation signals, abnormal bone tissue intrusion into the upper layer, unclear cartilage layer interface, and disordered tide line structure, failing to reconstruct the gradient structure and clear interface of natural osteochondral tissue.
Claims
1. A method for preparing a three-layered osteochondral repair composite hydrogel material, each possessing physical induction of chondrogenesis and osteogenic function, characterized in that, Includes the following steps: A middle-layer bio-ink was prepared, wherein the middle-layer bio-ink was a high-concentration GelMA solution composed of HA; An upper-layer bio-ink was prepared, wherein the upper-layer bio-ink was a porous HAMA solution loaded with BMSCs, KGN and ZnO nanowires; A lower-layer bio-ink was prepared, wherein the lower-layer bio-ink was a low-concentration porous GelMA solution loaded with BMSCs, BMP-2, HA and Fe3O4 nanofillers; A multi-nozzle bio-3D printer was used to print the lower, middle, and upper layers sequentially. After each layer was printed, UV curing was performed immediately to set the shape of each layer. After the overall scaffold was printed, final curing was performed to ensure that the interfaces of the three-layer structure were fully cross-linked, resulting in the osteochondral repair composite hydrogel material including the upper cartilage region, the middle isolation region, and the lower bone region.
2. The preparation method according to claim 1, characterized in that, The preparation process of the middle layer bio-ink includes: Using HAMA with a substitution degree of 30-70% and HA with a particle size of 100-300nm as raw materials, under sterile and light-protected conditions, a medium-layer bio-ink containing 12-20wt% GelMA, 1-10wt% HA and 0.05-0.25wt% photoinitiator was prepared in a water bath at 50-70℃. After stirring evenly, the mixture was centrifuged to remove bubbles, thus obtaining the medium-layer bio-ink.
3. The preparation method according to claim 1, characterized in that, The preparation process of the upper layer bio-ink includes: Using HAMA with a substitution degree of 30-70%, ZnO nanowires with a diameter of 50-150 nm and a length of 5-20 μm, and P3-P5 generation BMSCs as raw materials, KGN was first prepared into a 10 mM stock solution with DMSO under aseptic conditions and stored at 4°C in the dark. Under aseptic and light-protected conditions, a PBS solution containing 3-8 wt% HAMA, 0.25-1.5 wt% ZnO nanowires, and 0.05-0.25 wt% photoinitiator was prepared in a water bath at 50-70°C. After the solution cooled to 35-37°C, the KGN stock solution was added to a final concentration of 1-10 μM, and P3-P5 generation BMSCs were added to a final cell density of 5-10 × 10⁶ cells / mL. 6 The cells / mL were mixed evenly and then centrifuged to remove bubbles, thus obtaining the upper layer of bio-ink.
4. The preparation method according to claim 1, characterized in that, The preparation process of the lower layer bio-ink includes: Using GelMA with a substitution degree of 35-65%, HA nanoparticles with a particle size of 50-200 nm, Fe3O4 nanoparticles with a diameter of 10-30 nm, and P3-P5 generation BMSCs as raw materials, BMP-2 was prepared into a stock solution of 100 μg / mL in PBS containing 0.1% bovine serum albumin under aseptic conditions. Under aseptic and light-protected conditions, a PBS solution containing 3-8 wt% GelMA, 0.01-0.1 wt% Fe3O4 nanoparticles, 1-5 wt% HA nanoparticles, and 0.05-0.25 wt% photoinitiator was prepared in a water bath at 50-70℃. After the solution cooled to 35-37℃, the BMP-2 stock solution was added to a final concentration of 50-200 ng / mL, and P3-P5 generation BMSCs were added to a final cell density of 5-10 × 10⁶ cells / mL. 6 The cells / mL were mixed evenly and then centrifuged to remove bubbles, yielding the lower layer of bio-ink.
5. The preparation method according to any one of claims 2-4, characterized in that, The photoinitiator is one or more of LAP, Irgacure 2959, and Eosin Y.
6. The preparation method according to claim 1, characterized in that, The multi-nozzle bio-3D printing and curing process includes: The lower, middle, and upper layers of bio-ink are preheated to 30-35℃ and loaded into the independent temperature-controlled nozzles of a multi-nozzle bio-3D printer. The nozzle temperatures for the lower and middle layers of bio-ink are controlled at 35-37℃, and the nozzle temperature for the upper layer of bio-ink is controlled at 25-30℃. The printing platform temperature is set to 8-12℃. Each nozzle is automatically calibrated non-contactly to ensure that the X / Y / Z axis positioning error is ≤0.05 mm. The lower, middle, and upper layers are printed sequentially, each consisting of several thin layers stacked together. After each layer is printed, it is cured using 405 nm UV light. After all the structures are printed, the entire scaffold is finally cured to ensure full cross-linking between the layers, resulting in the osteochondral repair composite hydrogel material.
7. The preparation method according to claim 6, characterized in that, The printing and curing parameters for each layer include: When printing the lower layer structure, a nozzle with a diameter of 200-300 μm is used, the extrusion pressure is 80-180 kPa, and the printing speed is 5-12 mm / s. The thickness of the thin layer is set to 70-85% of the nozzle diameter, and it is printed layer by layer according to a preset porous mesh structure path. The curing strength of the first thin layer in contact with the printing platform is 8-10 mW / cm. 2 The curing time is 20-30 seconds, and the curing strength of the remaining thin layers is 4-8 mW / cm. 2 The curing time is 10-20 seconds, and the total thickness of the lower layer structure is controlled at 1-2 mm. When printing the middle layer structure, a nozzle with a diameter of 250-400 μm is used, the extrusion pressure is 200-350 kPa, and the printing speed is 2-6 mm / s. The thickness of the thin layer is set to 60-75% of the nozzle diameter; the cured strength of each thin layer is 5-8 mW / cm². 2 The curing time is 20-30 seconds, the total thickness of the middle layer structure is controlled at 0.5-1.2 mm, and the middle layer structure is a dense, non-porous structure. When printing the upper layer structure, a nozzle with a diameter of 200-300 μm is used, the extrusion pressure is 40-120 kPa, and the printing speed is 8-18 mm / s. The thickness of the thin layer is set to 80-100% of the nozzle diameter; the cured strength of each thin layer is 3-5 mW / cm². 2 The curing time is 10-15 seconds, and the total thickness of the upper structure is controlled at 1-2 mm. After the overall support frame is printed, it undergoes final curing, with a curing strength of 4-6 mW / cm². 2 The curing time is 30-60 seconds to ensure full cross-linking of the three-layer structure interface.
8. A three-layered osteochondral repair composite hydrogel material, each possessing physical induction of chondrogenesis and osteogenic function, characterized in that, The osteochondrogenic composite hydrogel material, prepared by any one of claims 1-7, comprises a three-layer structure, including: an upper layer structure with piezoelectric and chemically facilitating cartilage, a middle layer structure with functional units mimicking the calcified cartilage layer of natural osteochondrogenic cartilage, and a lower layer structure with magnetic and chemically facilitating bone function.
9. The osteochondral repair composite hydrogel material according to claim 8, characterized in that, The osteochondrogenic composite hydrogel material can induce osteogenic formation under a dynamic cyclic magnetic field. The dynamic cyclic magnetic field is a sinusoidal cyclic magnetic field with a frequency of 1-3 Hz and an intensity of 200-400 mT, and a magnetic field gradient of ≤10 mT / mm. Starting from the third day after magnetic field treatment, the magnetic field is applied 1-2 times a day, each time for 15-45 minutes, for 2-3 consecutive weeks.