Microenvironment-imitated composite scaffold and application thereof in anisotropic regeneration of meniscus cartilage

Abstract

The invention belongs to the field of tissue regeneration materials, and relates to a microenvironment-imitating composite scaffold and application thereof in anisotropic regeneration of meniscus cartilage. According to the scaffold, a meniscus-derived acellular extracellular matrix and a 3D printing polycaprolactone scaffold are combined, and the composite scaffold with excellent biological activity and mechanical stability is prepared; meanwhile, by utilizing a space occupying sacrifice strategy based on the temperature-sensitive poloxamer hydrogel, precise spatial arrangement of fibrochondrocytes and fibroblasts is realized, and the hexagonal meniscus tissue engineering scaffold with a heterogeneity spatial continuous structure and without an obvious interface is successfully constructed. Experiments prove that the stent effectively reproduces regional heterogeneity of natural meniscus, realizes gradient distribution of I / II type collagen and sulfated glycosaminoglycan, achieves bionic reconstruction of structure and function, and provides a promising conversion path for repair and reconstruction of functional meniscus.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology for meniscus regeneration, and relates to a microenvironment-mimicking composite scaffold and its application in anisotropic regeneration of meniscus cartilage. Background Technology

[0002] Meniscus injury is one of the most common sports injuries. As the fibrocartilaginous tissue between the femoral condyle and tibial plateau in the knee joint, the meniscus plays an irreplaceable role in maintaining joint mechanical stability, transmitting loads, absorbing shock, and lubricating the joint. Its tissue is mainly composed of cells and extracellular matrix (ECM), containing various components such as collagen, elastin, and glycosaminoglycans. Due to the uneven blood supply distribution of the meniscus (i.e., the "red on the outside, white on the inside" zone), its self-repair ability is extremely poor. Once injured, it easily leads to abnormal intra-articular stress distribution, decreased lubrication function, and loss of stability, subsequently causing serious consequences such as cartilage degeneration and osteoarthritis.

[0003] Currently, clinical treatments for meniscus injuries mainly include suturing, partial or total resection, and allogeneic transplantation. However, these methods all have significant limitations: suturing is only suitable for injuries in the highly vascularized red zone; resection alters the normal biomechanical environment of the joint, accelerating joint degeneration; and allogeneic transplantation faces problems such as severe donor shortages, potential disease transmission, immune rejection, and difficulties in size matching. Therefore, developing a viable meniscus replacement that can achieve both structural and functional repair has become a key clinical challenge that urgently needs to be addressed in the fields of sports medicine and orthopedics.

[0004] Tissue engineering and regenerative medicine have provided new insights into the functional regeneration of the meniscus. Over the past two decades, researchers have attempted to seed bone marrow mesenchymal stem cells (BMSCs) or chondrocytes onto scaffolds constructed from materials such as polycaprolactone (PCL), polylactic-glycolic acid copolymer (PLGA), or decellularized extracellular matrix (dECM), achieving meniscus tissue regeneration in animal models such as rabbits and sheep. However, these strategies primarily focus on constructing homogenized tissue. The natural meniscus exhibits a typical spatial heterogeneity: the lateral vascular zone (red zone) is rich in type I collagen, blood vessels, and nerves, primarily functioning in repair and partial load-bearing; the medial avascular zone (white zone) is dominated by type II collagen and glycosaminoglycans (GAGs), whose core functions are bearing pressure, dispersing loads, and buffering shocks. Existing homogenized engineered scaffolds cannot replicate this complex spatial gradient (including cellular composition, ECM components, and mechanical properties), resulting in poor interfacial integration between the newly formed tissue and the host tissue, insufficient mechanical properties, and a predisposition to early degeneration.

[0005] To mimic the heterogeneity of the meniscus, technologies such as 3D printing and regionalized controlled release of growth factors have been introduced into this field in recent years. For example, some studies have used a dual-nozzle printing system to release connective tissue growth factor (CTGF) and transforming growth factor-β3 (TGF-β3) in different regions of the scaffold to induce region-specific collagen expression; other studies have used regionalized decellularized matrix (Me-dECM) bio-ink to construct bilayer scaffolds, achieving a type I / II collagen ratio similar to that of natural tissue. These methods have made progress in creating biochemical gradients, but they often neglect the biological continuity between different regions of the natural meniscus and the synergistic effects of the cellular microenvironment, leading to poor interfacial integration within the regenerated tissue and incomplete functional recovery.

[0006] In summary, current technologies still face the following core challenges: how to reconstruct a biomimetic microenvironment capable of mimicking the "continuous anisotropy" of the natural meniscus at multiple scales, including macroscopic structure, microscopic morphology, and molecular signals; and how to achieve precise spatial arrangement and fate regulation of different cell types within this microenvironment, thereby driving the regeneration of functional meniscus tissue with physiological heterogeneity and mechanical gradients. Solving these challenges is crucial for achieving complete repair of meniscus injuries. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, the primary objective of this invention is to provide a microenvironment-mimicking composite scaffold. This scaffold combines decellularized extracellular matrix (Me-dECM) derived from the meniscus with a 3D-printed polycaprolactone (PCL) scaffold, creating a composite scaffold possessing both excellent bioactivity and mechanical stability. Simultaneously, utilizing a "site-sacrifice (POS)" strategy based on thermosensitive poloxamer F-127 hydrogel, precise spatial arrangement of fibrochondrocytes (FCs) and fibroblasts (FBs) is achieved, successfully constructing a hexagonal meniscus tissue-engineered scaffold with a heterogeneous spatially continuous structure and no obvious interfaces. In vitro and in vivo experiments confirm that this scaffold can effectively replicate the regional heterogeneity of the natural meniscus, achieving a gradient distribution of type I / II collagen (COL I / II) and sulfated glycosaminoglycans (GAGs), thus achieving biomimetic reconstruction of structure and function.

[0008] The second objective of this invention is to provide the application of the above-mentioned microenvironment-mimicking composite scaffold in the anisotropic regeneration of meniscal cartilage, which realizes meniscal regeneration with excellent mechanical properties and gradient heterogeneity, and provides a very promising transformation path for functional meniscal repair and reconstruction.

[0009] The present invention discloses a method for preparing a microenvironment-mimicking composite scaffold, comprising the following steps: Preparation of Me-dECM (meniscus-derived decellularized matrix) aqueous solution: Porcine meniscus tissue was taken, and after cryogenic grinding, it was treated sequentially with 0.1%-0.5% trypsin / phosphate buffer (PBS), nuclease solution, 8-12 mM Tris-HCl buffer (containing 8-12 U / mL aprotinin), and 1%-5% Triton X-100 / phosphate buffer. After washing with phosphate buffer, it was freeze-dried and treated with 0.1%-0.5% collagenase. After dialyzing with deionized water, it was freeze-dried again and dissolved in phosphate buffer to obtain Me-dECM aqueous solution. Preparation of Me-dECM / GelMA composite hydrogel: A photoinitiator was added to a solution of the aforementioned Me-dECM aqueous solution and methacrylamide gelatin (GelMA) in a mass ratio of 1:5-10 to obtain Me-dECM / GelMA composite hydrogel. Inner and outer layer inoculation: Take a meniscus-shaped scaffold, and at 30-40℃, first fill the middle layer of the scaffold with poloxamer F-127, then inject Me-dECM / GelMA composite hydrogel containing fibrochondrocytes into the inner layer and Me-dECM / GelMA composite hydrogel containing fibroblasts and vascular endothelial growth factor into the outer layer, and then cure it by ultraviolet cross-linking. Intermediate layer seeding: The scaffold is placed at 2-4℃, poloxamer F-127 is removed, and Me-dECM / GelMA composite hydrogel containing fibrochondrocytes and fibroblasts is injected into the intermediate layer of the scaffold. After UV cross-linking and curing, a microenvironment-mimicking composite scaffold is obtained.

[0010] In a specific embodiment of the present invention, it is further preferred that the nuclease solution in step (1) is an 8-12 mM Tris-HCl buffer containing 30-60 U / mL deoxyribonuclease and 1-5 U / mL ribonuclease A, and the pH is 6-8.

[0011] In a specific embodiment of the present invention, it is further preferred that the photoinitiator in step (2) is lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP).

[0012] In a specific embodiment of the present invention, it is further preferred that the amount of lithium phenyl-2,4,6-trimethylbenzoylphosphinate added is 0.1-0.5 wt% of the Me-dECM / GelMA composite hydrogel.

[0013] In a specific embodiment of the present invention, it is further preferred that, in step (3), the concentration of fibrochondrocytes injected into the inner layer is 6 × 10⁻⁶. 7 -10×10 7 cells / mL.

[0014] In a specific embodiment of the present invention, it is further preferred that, in step (3), the concentration of fibroblasts injected into the outer layer is 6 × 10⁻⁶. 7 -10×10 7 The concentration of vascular endothelial growth factor was 80-120 ng / mL.

[0015] In a specific embodiment of the present invention, it is further preferred that, in step (4), the concentration of fibrochondrocytes injected into the intermediate layer is 3 × 10⁻⁶. 7 -5×10 7 cells / mL, fibroblast concentration was 3×10 7 -5×10 7 cells / mL.

[0016] In a specific embodiment of the present invention, it is further preferred that the material of the scaffold is polycaprolactone, and the scaffold is a biomimetic scaffold with a vertical, continuous, and interface-free hexagonal meniscus structure, having a regular microporous structure; the parameters of the scaffold are: diameter 300-500μm, length 1-2cm, height 1-2mm, outer layer thickness 400-450μm, middle layer thickness 200-250μm and inner layer thickness 100-150μm, filament diameter 480-520μm, and pore size 1150-1290μm.

[0017] The present invention also discloses a microenvironment-inspired composite scaffold prepared by the above-described method.

[0018] This invention also discloses the application of the above-mentioned microenvironment-mimicking composite scaffold in anisotropic regeneration of meniscal cartilage, including meniscal defects or meniscal damage.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The composite scaffold of the present invention adopts a continuous hexagonal structure without obvious interfaces, and for the first time realizes the biomimetic construction of the "red outside and white inside" transition area of ​​the meniscus, avoiding the interface separation problem of traditional partitioned scaffolds. The COLⅠ / Ⅱ gradient distribution is consistent with the height of the natural meniscus.

[0020] 2. By combining the POS strategy with the thermosensitive F-127, precise spatial arrangement of FCs and FBs is achieved, resulting in high cell survival rate and clear partition boundaries, thus solving the problems of operational complexity and accuracy in multi-cell patterning.

[0021] 3. The scaffold combines the mechanical stability of PCL with the bioactivity of Me-dECM. Its mechanical properties match the anisotropic characteristics of the natural meniscus, its degradation rate is controllable, it does not require a second surgery to remove it, and it has excellent biocompatibility.

[0022] 4. This invention can effectively promote vascularization and functional matrix deposition in meniscus defect areas, and is especially suitable for refractory white area defects, providing a novel biomimetic strategy and transformation scheme for meniscus tissue engineering. Attached Figure Description

[0023] Figure 1 This is a fabrication roadmap and region-specificity diagram of a microenvironment-mimicking composite scaffold as described in Example 1. (A) Fabrication roadmap of the microenvironment-mimicking composite scaffold; (B) Flowchart of the preparation of a region-specific meniscus-derived decellularized extracellular matrix (Me-dECM) aqueous solution via decellularization and enzymatic hydrolysis steps; (C) HE staining and DAPI staining images of fibrocartilage tissue before and after decellularization, scale bar: 500 μm; (DF) Quantitative analysis of DNA, GAG, and collagen (COL) content in fibrocartilage tissue before and after decellularization (*p<0.05); (G) Analysis of the DAPI fluorescence intensity ratio in fibrocartilage tissue before and after decellularization (*p<0.05); (H) Gel formation process of the Me-dECM group under 365 nm UV irradiation for 30 s.

[0024] Figure 2 The cell compatibility and in vivo regeneration capacity of the Me-dECM / GelMA composite hydrogel in Example 3 were evaluated. (A) Live / dead staining images of FCs and FBs in the GelMA and Me-dECM composite hydrogels on days 1, 3, 5, and 7, scale bar: 500 μm; (B) Fluorescence intensity ratio analysis of live FCs; (C) Fluorescence intensity ratio analysis of live FBs; (D) Cross-sectional photographs of GelMA and Me-dECM hydrogels after implantation in nude mice; (E) Overall morphology of the regenerated fibrocartilage constructs in nude mice at 4 / 8 weeks, scale bar: 500 μm; (FG) HE, toluidine blue (TB), and safranin O (SO) staining images of fibrocartilage tissue in the GelMA and Me-dECM groups at 4 / 8 weeks, scale bar: 2 mm, 100 μm.

[0025] Figure 3 This is an in vivo experimental evaluation of the Me-dECM / GelMA composite hydrogel for repairing rabbit meniscus defects in Example 4. (A) A schematic diagram of the surgical procedure for injecting injectable Me-dECM into a rabbit medial meniscus defect model; (B) A gross photograph of the rabbit knee joint at 8 weeks post-surgery; (C) HE, SO, and TB stained images of regenerated fibrocartilage tissue in the GelMA group and the Me-dECM group at 8 weeks. Scale bar: 100 μm.

[0026] Figure 4Example 5 shows the 3D printing fabrication and mechanical property characterization of the microenvironment-mimicking composite scaffold, including: (A) a schematic diagram of the 3D printing process of the meniscus scaffold; (B) top and side views of the meniscus scaffold, scale bar: 1 cm; (C) a photograph of the composite state of GelMA and Me-dECM hydrogel on the scaffold, scale bar: 1 cm; (D) scanning electron microscope (SEM) images of the PCL frame at different magnifications, scale bars: 1 mm, 500 μm, and 200 μm; (EF) images of GelMA / PCL and Me-dECM at different magnifications. Optical microscope images of the / PCL composite scaffold, scale bars: 1mm, 500μm, 200μm; (G) Quantitative analysis of the aperture and wire diameter of the Me-dECM / PCL scaffold; (H) Stress-strain curves of the composite scaffold under uniaxial compression test; (I) Histograms of the elastic modulus of the composite scaffold at 5% and 30% compressive strain; (J) Cyclic compression test curves of the composite scaffold at the maximum strain of 40%; (K) Curves showing the change in elastic recovery rate of the composite scaffold with the number of cycles in the first ten cycles (elastic recovery rate = rebound stress / compressive stress).

[0027] Figure 5 Example 5 shows the partitioned scaffold construction and cell patterning seeding based on the POS strategy, wherein (A) is a schematic diagram of two-dimensional patterning construction in vitro using the POS strategy; (B) is a schematic diagram of the inner and outer double-layer structure of the partitioned scaffold and structural detail photographs at different magnifications; (C) is a schematic diagram of F-127-assisted partitioned cell seeding, scale bar: 1cm; (D) is a confocal micrograph of spatially patterned cells on the PCL scaffold (green: FCs; red: FBs), scale bar: 1cm.

[0028] Figure 6 For the in vivo regeneration effect evaluation of the space-engineered Me-dECM / PCL implants in Example 5, (A) photographs of space-engineered Me-dECM / PCL implants loaded with FCs (inner) and VEGF loaded with FBs (outer); (B) gross images of BIM and dp-BAM after 8 weeks, scale bar: 1cm; (CD) histological staining images of BIM and dp-BAM constructs with HE, Masson, SO, TB and COLI / II staining, showing regional matrix heterogeneity, scale bar: 1cm, 100μm.

[0029] Figure 7Example 5 shows the evaluation of tp-BAM meniscus regeneration based on a 3D assembly strategy. (A) Schematic diagram of tp-BAM meniscus regeneration using a 3D assembly strategy; (B) Appearance of the tp-BAM meniscus before implantation and overall morphology photographs of nude mice after 8 weeks of subcutaneous culture; (C) Histological images of natural meniscus stained with HE and SO; (D) Histological images of tp-BAM stained with HE, Masson, SO, TB, COLI, and COLII; (E) Immunofluorescence observation images of specific differentiation of the COLI (red) and COLII (green) regions of the meniscus. Scale bar: 1 cm, 100 μm. Detailed Implementation

[0030] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0031] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0032] Unless otherwise specified, all reagents used in this invention are commercially available.

[0033] The present invention provides a microenvironment-mimicking composite scaffold, the design principle of which is as follows: Figure 1 As shown in Figure A, an aqueous solution of meniscus-derived acellular extracellular matrix (Me-dECM) was first obtained from porcine meniscus tissue. This solution was then mixed with methacrylamide gelatin (GelMA) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP) to prepare a Me-dECM / GelMA composite hydrogel. This invention combines meniscus-derived acellular extracellular matrix (Me-dECM) with a 3D-printed polycaprolactone (PCL) scaffold to prepare a composite scaffold with both excellent bioactivity and mechanical stability. Simultaneously, utilizing a "seat sacrifice (POS)" strategy based on thermosensitive poloxamer F-127 hydrogel, precise spatial arrangement of fibrochondrocytes (FCs) and fibroblasts (FBs) was achieved, successfully constructing a hexagonal meniscus tissue engineering scaffold with a heterogeneous spatially continuous structure and no obvious interface. In vitro and in vivo experiments have confirmed that the scaffold can effectively replicate the regional heterogeneity of the natural meniscus, achieve a gradient distribution of type I / II collagen (COLⅠ / Ⅱ) and sulfated glycosaminoglycans (GAGs), and achieve biomimetic reconstruction of structure and function.

[0034] Example 1: Preparation of Me-dECM / GelMA composite hydrogel Preparation of meniscus-derived decellularized extracellular matrix (Me-dECM) aqueous solution: To construct a bioactive tissue-specific matrix microenvironment, porcine meniscus tissue was first decellularized, such as... Figure 1 As shown in B. Specifically, fresh porcine meniscus tissue was pulverized into powder using a cryo-grinder and treated with 0.5% trypsin / phosphate buffer (PBS, mass / volume ratio) for 24 h. Subsequently, it was treated sequentially with nuclease solution (containing 50 U / mL deoxyribonuclease and 1 U / mL ribonuclease A, dissolved in 10 mM Tris-HCl buffer, pH=7.5) for 4 h, 10 mM Tris-HCl buffer (containing 10 U / mL aprotinin) for 20 h, and 1% Triton X-100 / PBS solution (volume ratio) for 24 h. The tissue was washed with PBS 6 times, 8 h each time, and then lyophilized before being treated with 0.15% collagenase for 24 h. The resulting solution was dialyzed against deionized water for 3 days, then lyophilized again and dissolved in phosphate buffer to obtain Me-dECM aqueous solution.

[0035] Preparation of Me-dECM / GelMA composite hydrogel: 0.11g of Me-dECM aqueous solution and 1g of methacrylamide gelatin (GelMA) were mixed and then 0.002g of phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) was added to obtain Me-dECM / GelMA composite hydrogel.

[0036] HE staining results showed that natural cartilage tissue contained normal cellular structures with nuclei, while the Me-dECM in Example 1 was a decellularized sample, containing only residual vacuolar structures; simultaneously, DAPI staining showed that the original... Blue fluorescent cell nuclei were present in the cartilage tissue, but the Me-dECM / GelMA composite hydrogel in Example 1 did not show this signal. Figure 1 As shown in C. Figure 1 As shown in D and 1G, quantitative DNA analysis and semi-quantitative DAPI fluorescence analysis of the meniscus tissue further confirmed the effectiveness of decellularization. The DNA content of the Me-dECM / GelMA composite hydrogel in this embodiment was significantly reduced compared to the natural meniscus, and collagen and GAGs components were preserved during the decellularization process. Figure 1 As shown in EF. Figure 1 As shown in H, the Me-dECM / GelMA composite hydrogel at 365 nm and 20 mW / cm 2 It can be rapidly converted into a gel state under ultraviolet light irradiation.

[0037] Example 2: Preparation of fibrochondrocytes (FCs) and fibroblasts (FBs) Rabbits were anesthetized intravenously and thoroughly disinfected with povidone-iodine. Under aseptic conditions, meniscus tissue was obtained from the knee joint and dermal tissue was excised from the abdomen. The collected tissues were placed in sterile centrifuge tubes containing high-glucose DMEM medium and transported to the laboratory at 4°C for the separation of FCs and FBs.

[0038] Under aseptic conditions, the surrounding connective tissue was dissected with surgical scissors to expose the fibrocartilage and dermis; the cartilage and dermis were then cut into 1-2 mm pieces. 3 Small pieces of the cells were placed in 50 mL centrifuge tubes containing 0.25% chloramphenicol solution and soaked for 30 min, followed by rinsing three times with PBS. 30-40 mL of 0.15% collagenase solution was added to the cartilage tissue, and the cells were digested at 37°C and 100 rpm for 8-12 h. The dermal tissue was treated similarly, with 30-40 mL of 0.15% collagenase solution added, and digested at 37°C and 100 rpm for 1-4 h. After digestion, the undigested tissue was filtered through a 100 μm cell sieve, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded to obtain primary rabbit FCs and FBs (P0 generation). The cells were then cultured at 1×10⁻⁶ cells per cell line. 5 Cells were resuspended at a concentration of 10 cells / mL in high-glucose DMEM medium containing 10% fetal bovine serum and 1% triple antibodies. 10 mL of cell suspension was seeded into 10 cm culture dishes and cultured in an incubator at 37°C, 5% CO2, and saturated humidity. The medium was changed every 3 days. When the cell confluence reached 90% after 3-4 days, the cells could be passaged.

[0039] Example 3: Biocompatibility and subcutaneous cartilage regeneration capacity of Me-dECM / GelMA composite hydrogel To verify the biocompatibility of the Me-dECM / GelMA composite hydrogel, in vitro live / dead staining experiments and nude mouse subcutaneous cartilage regeneration experiments were conducted in this embodiment. FCs were isolated from the meniscus tissue of Example 2, and FBs were isolated from the abdominal dermis tissue, and amplified to the second generation according to the established method.

[0040] In this embodiment, the GelMA group and the Me-dECM group were selected for cell viability testing. The GelMA group was a composite hydrogel of methacrylamide gelatin (GelMA) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (containing 0.2% LAP photoinitiator); the Me-dECM group was the Me-dECM / GelMA composite hydrogel of Example 2.

[0041] Both the GelMA and Me-dECM composite hydrogels were sterilized using a 0.22 μm filter. FCs and FBs were then separated at a ratio of 1.0 × 10⁻⁶. 7Cells were uniformly mixed at a density of cells / mL in the composite hydrogel and cultured at 37°C. After 1, 4, and 7 days of culture, the cell viability of the cell-loaded hydrogel construct was assessed using a live / dead cell viability assay kit (Invitrogen) according to the manufacturer's instructions. Cell viability was observed and calculated using a fluorescence microscope.

[0042] In vitro cell experiments showed that both FCs and FBs proliferated well in the GelMA and Me-dECM groups, and both hydrogels exhibited high cell viability within a 7-day culture period, confirming their excellent cell compatibility. Figure 2 As shown in Figure A. Semi-quantitative analysis indicated that the Me-dECM group had a more significant promoting effect on the proliferation of fibrochondrocytes (FCs), such as... Figure 2 As shown in B, there was no statistically significant difference in the effect on fibroblast (FB) growth, such as... Figure 2 As shown in C. Compared to the GelMA group, the Me-dECM group retained complex signals such as natural specific collagen (e.g., type II collagen), GAGs, and various growth factors (e.g., TGF-β, BMPs) derived from chondrocytes.

[0043] To further evaluate the in vivo cartilage regeneration capacity of the Me-dECM group, FCs were administered at a rate of 8 × 10⁻⁶. 7 Inoculations were performed at a density of [number] cells / mL in the GelMA and Me-dECM groups to create 1 cm diameter disc-shaped structures, which were then implanted into nude mice. Samples were harvested at 4 and 8 weeks, respectively. Figure 2 As shown in D. A general observation reveals that the hydrogel maintains its intact morphology and exhibits no significant absorption, as... Figure 2 As shown in Figure E. Histological analysis revealed that, compared to the GelMA group, the Me-dECM group exhibited richer extracellular matrix deposition in the regenerated tissue, with strong positive staining for SO and TB, suggesting a higher content of GAGs. Figure 2 As shown in FG. The above results confirm that the natural ECM components (including type II collagen, specific GAGs and growth factors) retained in the Me-dECM group can effectively enhance the ability of FCs to secrete cartilage matrix and accelerate cartilage formation.

[0044] Example 4: Meniscus Heterogeneity Regeneration Assessment Experiment Using Me-dECM / GelMA Composite Hydrogel Me-dECM / GelMA composite hydrogel was used for in-situ repair of meniscus cartilage. To evaluate the repair effect of Me-dECM / GelMA composite hydrogel on meniscus cartilage, healthy adult New Zealand white rabbits were used for in vivo experiments in this embodiment. After the rabbits were fixed in a supine position, an anterolateral incision was made in the knee joint, exposing the lateral meniscus layer by layer. A full-thickness cylindrical defect with a diameter of 2 mm was prepared in the body using ophthalmic scissors and a corneal trephine. In the Me-dECM group, 10% (w / v) Me-dECM / GelMA composite hydrogel aqueous solution was injected in situ into the defect. In the GelMA group, a prepolymer of a composite hydrogel containing 10% (w / v) methacrylamide gelatin (GelMA) and phenyl-2,4,6-trimethylbenzoyl lithium phosphine sulfate (LAP) (containing 0.2% LAP photoinitiator) was injected. Both were treated with 365 nm ultraviolet light (20 mW / cm²). 2 Irradiation for 30 seconds for curing; post-operative intramuscular injection of penicillin (400,000 U / animal) for 3 days to prevent infection; individual cage housing with free movement. Figure 3 As shown in Figure A. Eight weeks post-surgery, the defect area in the Me-dECM group was completely filled with smooth, milky-white new tissue, with a flat surface and no cracks. The repair effect was significantly better than that in the GelMA group, as shown in Figure A. Figure 3 As shown in Figure B, HE, SO, and TB histological staining revealed a dense distribution of FCs in the regenerated tissue of the Me-dECM group, with strong positive staining for SO and TB, consistent with a typical chondrogenic phenotype characterized by abundant GAG aggregation. Figure 3 As shown in Figure C. In summary, the Me-dECM / GelMA composite hydrogel can effectively promote cartilage repair in localized small meniscus defects in rabbits.

[0045] Example 5: Fabrication of a Microenvironment-Simulated Composite Scaffold 5.1 Fabrication of 3D Printed Scaffold Using the continuous extrusion technology of the Regenovo 3D bioprinter, polycaprolactone (PCL) material was fabricated into a vertical, continuous, and interface-free hexagonal meniscus-shaped biomimetic scaffold with a regular microporous structure. The scaffold's parameters are: diameter 500 μm, length 2 cm, height 2 mm, outer layer thickness 400 μm, middle layer thickness 250 μm, and inner layer thickness 100 μm; filament diameter 500 μm; and pore size 1200 μm. Figure 4 As shown in B, the photocrosslinked GelMA composite hydrogel and Me-dECM composite hydrogel can precisely fill the macroscopic pores of the PCL scaffold, serving as a bioactive filling phase, such as... Figure 4As shown in Figure C. Compared to the GelMA group, the three-dimensional network structure formed after freeze-drying of the Me-dECM group is more loose and porous. This filling strategy optimizes the microstructure of the scaffold while preserving its permeability, thus creating a local microenvironment that is more conducive to cell adhesion and proliferation.

[0046] This embodiment compares three types of scaffolds: a pure PCL scaffold, a GelMA / PCL composite scaffold, and a Me-dECM / PCL bioactive scaffold. SEM images show that the PCL filaments are vertically stacked with interconnected gaps, forming numerous micropores on the scaffold surface and interior, exhibiting high porosity and a rough surface. Figure 4 As shown in Figure D, optical microscopy images reveal that the composite hydrogels of the GelMA and Me-dECM groups are uniformly distributed within the PCL scaffold. After lyophilization, the GelMA / PCL and Me-dECM / PCL scaffolds effectively maintain the original multilayered framework structure of the PCL, exhibiting a clear hierarchical organization. Figure 4 As shown in E and 4F. Measurements showed that the micropore size of the scaffold was 1220.31 ± 69.11 μm, and the diameter of the column formed by adjacent filaments was 307.81 ± 17.99 μm. Furthermore, there was no significant change in pore size before and after freeze-drying, indicating that the material structure was stable. Figure 4 As shown in G.

[0047] To further evaluate its mechanical properties, uniaxial compression and cyclic compression tests were conducted in this embodiment, such as... Figure 4 As shown in H, 4J. The results show that this composite strategy effectively combines the bioactivity of the Me-dECM composite hydrogel with the mechanical support advantages of PCL; stress-strain curve analysis shows that the Me-dECM composite hydrogel of the PCL scaffold provides the necessary mechanical support. In this embodiment, the elastic modulus of the material under small strain (5%, simulating initial contact) and large strain (30%, simulating physiological load) was calculated and compared. It was found that the compressive modulus of the Me-dECM / PCL under both strain conditions is comparable to that of the natural meniscus. Figure 4 As shown in Figure I, in the cyclic compression test, the Me-dECM group / PCL showed a better elastic recovery rate than the natural meniscus in the first 10 cycles, indicating that it possesses higher structural stability, excellent fatigue resistance, and can adapt to repeated cyclic loads during joint movement. Figure 4 As shown in K.

[0048] In summary, the Me-dECM / PCL scaffold possesses suitable porous structure, mechanical stability, and elastic characteristics, making it an ideal biomimetic scaffold for the meniscus.

[0049] 5.2 Multicellular Patterned Spatial Biomimetic Construction. To construct a biomimetic meniscus multicellular partitioned structure, this embodiment utilizes a POS strategy based on the thermosensitive hydrogel F-127 to achieve spatially patterned arrangement of multiple cells. For example... Figure 5 As shown in Figure A, this strategy consists of three steps: First, 30% concentration F-127 is filled into a specific area of ​​a 3D-printed PCL scaffold in gel form at 37°C to form a physical barrier; second, hydrogels loaded with FCs are seeded in unoccupied areas; finally, the F-127 hydrogel is liquefied and removed at 4°C, and hydrogels loaded with FBs are seeded in the vacated areas. This process achieves precise spatial arrangement of FCs and FBs on a macroscopic scale, confirming that the POS strategy can effectively guide the partitioned distribution of cell-loaded hydrogels. To verify the accuracy of the multicellular partitioned structure, this embodiment uses hydrogels of different colors to construct inner and outer partitioned structures, such as... Figure 5 As shown in Figure B, the boundaries between the different colored hydrogels are clearly defined. Subsequent construction of multi-cell partitions loaded with cell hydrogels confirmed the feasibility of this cell patterning distribution method. Figure 5 As shown in C. This embodiment further validates the cell patterning distribution effect using fluorescently labeled cells. The results show that DiO-labeled FCs (green) and DiI-labeled FBs (red) form well-defined and stable partitioned structures within the scaffold, as shown in Figure C. Figure 5 As shown in D.

[0050] 5.3 Construction of anisotropic menisci.

[0051] The BIM group consisted of a single FC hydrogel; the dp-BAM group was composed of an outer FBs / VEGF region and an inner FCs region; the tp-BAM group was continuously composed of an outer FBs / VEGF region, a middle FCs / FBs mixed region, and an inner FCs region. All hydrogels were loaded into prefabricated PCL porous scaffolds for mechanical support. Cells were spaced at a density of 8 × 10⁶ cells / cm². 7 Cells per mL were resuspended in Me-dECM / GelMA composite hydrogel to prepare "Colloid 1 (FCs hydrogel)"; the cell density was set at 8 × 10⁻⁶ cells / mL. 7 Cells / mL of FBs and 100 ng / mL of vascular endothelial growth factor (VEGF) were resuspended in Me-dECM / GelMA composite hydrogel to prepare "Colloid 2 (FBs / VEGF hydrogel)"; cells were taken at a density of 4 × 10⁻⁶ cells / mL. 7Cell-forming cells (FCs) and free radicals (FBs) per mL were resuspended in Me-dECM / GelMA composite hydrogel to prepare "Colloid 3 (FCs and FBs mixed cell hydrogel)". Using thermosensitive poloxamer F-127 as a sacrificial material, its gelation at 37°C and melting at 4°C were utilized to achieve biomimetic construction of multi-region implants. During the construction of the BIM group, 200 μL of colloid 1 was directly injected into the Me-dECM group / PCL scaffold and cured. During the construction of the dp-BAM group, F-127 gel was first injected into the outer region of the Me-dECM group / PCL scaffold, and then colloid 1 was inoculated and cured in the inner region. After cooling and removing F-127, colloid 2 was inoculated in the outer region. During the construction of the tp-BAM group, F-127 was first used to occupy the middle region of the Me-dECM group / PCL scaffold. Then, colloid 1 and colloid 2 were inoculated and cured in the inner and outer regions, respectively. Finally, the middle F-127 template was removed and colloid 3 was inoculated to form a sandwich-structured multi-zone biomimetic implant.

[0052] To verify the potential of bioprinted scaffolds in promoting in vivo cartilage regeneration, cartilage formation and regeneration were assessed after 8 weeks. Tissue samples were collected for histological staining to analyze the degree of meniscus regeneration. The tissue and immunohistochemical analysis process was as follows: First, the biomimetic meniscus was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm thick sections. The sections were stained with HE, SO, TB, COL I, and COL II according to the manufacturer's instructions to assess the regenerated tissue structure. In addition, after antigen retrieval and serum blocking, the sections were incubated overnight at 4°C with COL I (1:300) and COL II (1:50) primary antibodies, followed by incubation with secondary antibodies for 1 h. The staining results were observed using a fluorescence microscope. Finally, a semi-quantitative histological scoring system was used to further evaluate the meniscus regeneration effect.

[0053] 5.3.1 In vivo heterogeneous regeneration of dual-partition BAM (dp-BAM).

[0054] To simulate the morphology and tissue heterogeneity of the natural meniscus, two implants were constructed: a homogeneous meniscus scaffold (BIM) and a two-layered meniscus scaffold (dp-BAM). These were subcutaneously implanted into nude mice for in vivo evaluation over 8 weeks. Figure 6 As shown in Figure A.

[0055] Gross observation showed that both implants maintained their initial morphology and structural integrity, such as Figure 6 As shown in B; histological evaluation of dp-BAM showed that its cell phenotype and extracellular matrix deposition had more significant region specificity, such as Figure 6As shown in CD, the medial region is dominated by a cartilage matrix composed of type II collagen and proteoglycans, while the lateral region is characterized by a fibrous matrix primarily composed of type I collagen. Immunohistochemical results show that type I collagen gradually decreases from the lateral to the medial region, while type II collagen increases accordingly. This distribution is consistent with the natural meniscus, which is characterized by type I collagen dominating the lateral region and type II collagen dominating the medial region. The histological results of the dp-BAM regenerated tissue exhibit a region-specific cell phenotype, consistent with the anisotropic tissue characteristics of the natural meniscus ECM; however, BIM did not show similar differences in cell structure. Notably, the medial region of dp-BAM is rich in globular chondrocytes, while the lateral region is dominated by spindle-shaped fibrous cells, similar to the tissue structure distribution of the natural meniscus.

[0056] The results indicate that this partitioning strategy achieves spatially controllable cell distribution, promotes the formation of heterogeneous extracellular matrix, and effectively mimics the histological structure and molecular composition of the natural meniscus. In summary, the POS strategy can efficiently achieve multi-cell patterning and in vivo heterogeneous regeneration.

[0057] 5.3.2 In vivo heterogeneous regeneration of tripartite meniscus (tp-BAM). In reality, the natural meniscus is a continuous tissue without clear boundaries, and its tissue sample is obtained from... Figure 7 As shown in Figure A. To more accurately simulate the continuous morphology and tissue anisotropy of the natural meniscus, a TP-BAM implant was constructed and subcutaneously implanted into nude mice for in vivo evaluation over 8 weeks, as shown in Figure A. Figure 7 As shown in B. Histological sections of a natural meniscus show a gradient distribution of components in its inner, middle, and outer regions, such as... Figure 7 As shown in C; histological analysis of the tp-BAM regenerated meniscus shows that it can reproduce the region-specific structure of natural tissue, such as... Figure 7 As shown in D, the cell phenotype and ECM distribution exhibit anisotropic characteristics highly similar to those of the natural meniscus: the medial region shows a large number of spherical chondrocytes embedded in a cartilage matrix rich in type II collagen and proteoglycans; the lateral region is dominated by spindle-shaped fibroblasts, accompanied by a regularly arranged type I collagen fiber matrix; more importantly, the tp-BAM partition structure contains an intermediate transitional region containing a transitional cell population of mixed chondrocytes and fibroblasts, which is more consistent with the physiological structure of the natural meniscus, while neither BIM nor dp-BAM can present such a continuous cell structure.

[0058] In summary, the tp-BAM implant successfully replicates the region-specific structure of the natural meniscus and the anisotropic distribution of the extracellular matrix (type I / II collagen, aggregates, and glycans), effectively solving the core challenges of simulating the continuity and tissue heterogeneity of the natural meniscus, and providing a promising biomimetic strategy for clinical meniscus repair.

[0059] The microenvironment-mimicking composite scaffold of the present invention has the following characteristics: I. Composition and Structural Design of Microenvironment-Simulated Composite Scaffold The composite scaffold of this invention adopts a two-layer structure of "mechanical support phase - bioactive filling phase": the mechanical support phase is a 3D printed PCL frame, which provides mechanical strength and pore structure matching the natural meniscus by precisely controlling the printing parameters (filament diameter, pore size, layer thickness), ensuring cell adhesion and nutrient transport; the bioactive filling phase is a composite hydrogel of Me-dECM and GelMA. Me-dECM retains meniscus tissue-specific collagen (COLⅠ / Ⅱ), sulfated glycosaminoglycans (GAGs), and growth factors (such as TGF-β and BMPs), which can induce FCs / FBs to differentiate in a targeted manner, while GelMA enhances the cross-linking stability and cell compatibility of the hydrogel.

[0060] The scaffold is a continuous, hexagonal structure without obvious interfaces: the outer layer matches the red zone of the natural meniscus, mainly composed of fibroblasts (FBs) and Coryl Group I (COLI), with VEGF added to promote vascularization; the inner layer matches the white zone, mainly composed of chondrocytes (FCs), Coryl Group II (COLII), and glycoproteins (GAGs), enhancing its compressive strength; the middle layer is a transition zone, a mixture of FCs and FBs, achieving a gradient transition between cellular and matrix components, avoiding interface separation, and replicating the structural continuity of the natural meniscus. By regulating the secretion of COLII and GAGs by FCs and the secretion of COLI by FBs, a gradient decrease in COLI from the outer to the inner layer and a gradient increase in COLII from the outer to the inner layer are achieved, replicating the heterogeneity of the natural meniscus. This promotes chondrocyte maturation, vascular infiltration, and matrix deposition, accelerating the cartilage regeneration process.

[0061] II. Position Sacrifice (POS) Strategy Based on the thermosensitive F-127, a "Position Sacrifice (POS)" strategy is used to solve the problem of precise multi-cell partitioning: F-127 is in a gel state at high temperatures, which can act as a physical barrier to occupy specific areas of the scaffold, enabling cell seeding in the target area; after liquefaction and removal at low temperatures, another type of cell can be seeded in the vacated area. Through the steps of "positioning-seeding-removal-reseeding", precise arrangement of FCs and FBs in a continuous structure is achieved, with a cell survival rate of ≥90%, clear partition boundaries, and no cell damage.

[0062] III. Stent Performance and Mechanism of Action (1) Physicochemical properties: The porosity of the composite scaffold meets the needs of cell infiltration and nutrient exchange; the mechanical properties are anisotropic, the tensile modulus of the outer layer matches the tensile resistance requirements of the red zone of the meniscus, and the compressive modulus of the inner layer matches the compressive resistance requirements of the white zone. After 10 cycles of compression, the elastic fatigue resistance is excellent; in the simulated joint fluid environment, the degradation rate of the scaffold can be controlled to provide growth space for cells.

[0063] (2) Biological properties: Me-dECM can increase the secretion of COLII and GAGs in FCs. The multi-cell patterned design makes COLI decrease from the outside to the inside and COLII increase from the inside, thus replicating the molecular heterogeneity of the natural meniscus. The VEGF in the outer layer can promote the migration of vascular endothelial cells and solve the problem of nutrient supply in the inner white area.

[0064] (3) Mechanism of action: The scaffold induces directed cell differentiation through tissue-specific signals of Me-dECM, and combined with multicellular spatial arrangement, it constructs a biomimetic microenvironment with "cell-matrix-mechanics" synergy; by activating the PI3K-Akt signaling pathway, it regulates actin scaffold remodeling, promotes FC maturation and collagen fiber arrangement of FBs, and accelerates anisotropic regeneration of the meniscus.

[0065] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing a microenvironment-mimicking composite scaffold, characterized in that, Includes the following steps: Preparation of Me-dECM aqueous solution: Porcine meniscus tissue was taken, and after freeze-milling, it was treated sequentially with 0.1%-0.5% trypsin / phosphate buffer, nuclease solution, 8-12mM Tris-HCl buffer (containing 8-12 U / mL aprotinin) and 1%-5% Triton X-100 / phosphate buffer. After washing with phosphate buffer, it was freeze-dried and treated with 0.1%-0.5% collagenase. After dialyzing with deionized water, it was freeze-dried again and dissolved in phosphate buffer to obtain Me-dECM aqueous solution. Preparation of Me-dECM / GelMA composite hydrogel: A photoinitiator was added to a solution of the aforementioned Me-dECM aqueous solution and methacrylamide gelatin in a mass ratio of 1:5-10 to obtain Me-dECM / GelMA composite hydrogel. Inner and outer layer inoculation: Take a meniscus-shaped scaffold, and at 30-40℃, first fill the middle layer of the scaffold with poloxamer F-127, then inject Me-dECM / GelMA composite hydrogel containing fibrochondrocytes into the inner layer and Me-dECM / GelMA composite hydrogel containing fibroblasts and vascular endothelial growth factor into the outer layer, and then cure it by ultraviolet cross-linking. Intermediate layer seeding: The scaffold is placed at 2-4℃, poloxamer F-127 is removed, and Me-dECM / GelMA composite hydrogel containing fibrochondrocytes and fibroblasts is injected into the intermediate layer of the scaffold. After UV cross-linking and curing, a microenvironment-mimicking composite scaffold is obtained.

2. The method for preparing the microenvironment-mimicking composite scaffold according to claim 1, characterized in that, The nuclease solution described in step (1) is an 8-12 mM Tris-HCl buffer containing 30-60 U / mL deoxyribonuclease and 1-5 U / mL ribonuclease A, with a pH of 6-8.

3. The method for preparing the microenvironment-mimicking composite scaffold according to claim 1, characterized in that, The photoinitiator mentioned in step (2) is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

4. The method for preparing the microenvironment-mimicking composite scaffold according to claim 3, characterized in that, The amount of phenyl-2,4,6-trimethylbenzoyl lithium phosphinate added is 0.1-0.5 wt% of the Me-dECM / GelMA composite hydrogel.

5. The method for preparing the microenvironment-mimicking composite scaffold according to claim 1, characterized in that, In step (3), the concentration of fibrochondrocytes injected into the medial layer is 6 × 10⁻⁶. 7 -10×10 7 cells / mL; the concentration of fibroblasts injected into the outer layer was 6 × 10⁻⁶ cells / mL. 7 -10×10 7 The concentration of vascular endothelial growth factor was 80-120 ng / mL.

6. The method for preparing the microenvironment-mimicking composite scaffold according to claim 1, characterized in that, In step (4), the concentration of fibrochondrocytes injected into the intermediate layer is 3 × 10⁻⁶. 7 -5×10 7 cells / mL, fibroblast concentration was 3×10 7 -5×10 7 cells / mL.

7. The method for preparing the microenvironment-mimicking composite scaffold according to claim 1, characterized in that, The scaffold is made of polycaprolactone and is a biomimetic scaffold with a vertical, continuous, and interface-free hexagonal meniscus structure, featuring a regular microporous structure. The parameters of the scaffold are: diameter 300-500μm, length 1-2cm, height 1-2mm, outer layer thickness 400-450μm, middle layer thickness 200-250μm, and inner layer thickness 100-150μm, filament diameter 480-520μm, and pore size 1150-1290μm.

8. A microenvironment-inspired composite scaffold prepared by any one of the preparation methods of claims 1-7.

9. The application of the microenvironment-mimicking composite scaffold as described in claim 1 in anisotropic regeneration of meniscal cartilage.

10. The application according to claim 9, characterized in that, The applications described include meniscus defects or damage.

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