Bionic heterogeneous tissue engineering meniscus stent and preparation method thereof

Abstract

The invention discloses a bionic heterogeneous tissue engineering meniscus stent and a preparation method thereof, and discloses application of the bionic heterogeneous tissue engineering meniscus stent in the field of meniscus repair. Based on a multi-nozzle 3D printing technology, a biodegradable high polymer material and bio-ink loaded with growth factors / stem cells are innovatively adopted, and the highly bionic heterogeneous tissue engineering meniscus is constructed. Specifically, degradable macromolecular polycaprolactone (PCL) is used as a raw material, and annular and radial fibers are printed to form a stent frame by accurately regulating and controlling a printing path and simulating a natural meniscus collagenous fiber distribution rule; on the basis, biological ink loaded with differentiated growth factors and stem cells is printed in a staggered manner according to different physiological characteristics of a red region and a white region of the meniscus, so that partitioned precise inoculation of seed cells and controllable partitioned release of the growth factors are realized, and a cell microenvironment with spatial specificity is provided for meniscus regeneration; the tissue repair efficiency and the bionic integrating degree are obviously improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of bionic heterogeneity tissue engineering meniscus scaffold and its preparation method, belong to meniscus injury repair material technical field. BACKGROUND

[0002] Meniscus injury caused by sports injury, accidental trauma, degenerative arthritis and obesity and aging is an important reason for joint functional disorder. Although the current clinical use of meniscus suture, partial or complete meniscectomy and allograft transplantation can relieve symptoms to some extent, but there are problems such as incomplete functional recovery, limited donor and tissue rejection. Meniscus is a wedge-shaped fibrocartilage tissue located between femoral condyle and tibial plateau, which tolerates and coordinates the mechanical state of vertical compression, sliding, rotation, twisting between femoral condyle and tibial plateau of knee joint, plays a role in stress transmission, joint stabilization, lubrication and protection of articular cartilage, and ensures the completion of knee function. The special function of meniscus is determined by its composition and structural heterogeneity: from synovial margin to free edge, from rich blood vessels to avascular, fibrocartilage to hyaline cartilage, type I collagen to type II collagen, circular arrangement to radial arrangement. This complex heterogeneity composition and structure make the treatment and regeneration of meniscus injury extremely difficult. With the in-depth understanding of the structure and function of joint, some different types of biomaterial scaffolds have been developed for meniscus regeneration, such as collagen-based meniscus implant (CMI, Germany) and polyurethane meniscus scaffold (Actifit, UK), which have been commercialized in the United States and Europe respectively and have been clinically applied. However, the scaffold prepared by traditional technology cannot simulate the complex heterogeneous microstructure of natural meniscus, lacks the anisotropic mechanical properties of natural meniscus, and has the problems of insufficient recruitment and colonization of endogenous cells, which leads to slow meniscus remodeling and uncertain long-term repair effect, and cannot be used for whole meniscectomy repair and reconstruction.

[0003] Around structure bionics, mechanical bionics and induced regeneration, the tissue engineering scaffold is transformed from single phase to multi phase, from linear gradient to variable gradient, from simple filling replacement to active induction of tissue regeneration, which is expected to change the predicament of meniscus regeneration. Biological 3D printing realizes the integration of multi-cell and multi-material through precise digital manufacturing of biological ink, and constructs an implant that not only has suitable anisotropic biomechanical properties, but also can adapt to the composition and structural heterogeneity of knee meniscus. As an FDA-approved thermoplastic polymer, polycaprolactone (PCL) has excellent mechanical properties and suitable degradation characteristics, making it an ideal choice for meniscus scaffold. However, single PCL material, although providing good mechanical support, has problems such as lack of cell adhesion sites and severe wear of cartilage surface. But due to the lack of material bionics, the function and long-term effect of the new meniscus still need to be investigated.

[0004] This application is submitted in response to the above-mentioned issues. Summary of the Invention

[0005] The purpose of this invention is to solve the problems that traditional meniscus structures and components cannot regenerate and repair the meniscus, and are difficult to produce in a customized manner. The invention provides a biomimetic heterogeneous tissue-engineered meniscus scaffold and its preparation method.

[0006] The primary objective of this invention is to provide a method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold. The method involves using biodegradable polymer materials and hydrogel bio-inks containing bone marrow mesenchymal stem cells (BMSCs) and growth factors to print the biomimetic heterogeneous tissue-engineered meniscus scaffold. Based on the collagen fiber distribution of the natural meniscus, a meniscus scaffold framework is constructed by printing circumferential and radial fibers using biodegradable polymers. Hydrogel bio-inks loaded with different growth factors and bone marrow mesenchymal stem cells are then printed alternately in the red and white areas of the meniscus scaffold framework.

[0007] Using the above technical solution, biodegradable polycaprolactone (PCL) is used as the raw material. By precisely controlling the printing path, the distribution pattern of collagen fibers in the natural meniscus is simulated, and circumferential and radial fibers are printed to form a scaffold framework. On this basis, bio-inks loaded with differentiated growth factors and stem cells are printed alternately to target the different physiological characteristics of the red and white areas of the meniscus. This enables precise regional seeding of seed cells and controllable regional release of growth factors, providing a spatially specific cellular microenvironment for meniscus regeneration, significantly improving tissue repair efficiency and biomimetic compatibility.

[0008] Preferably, the growth factors include red zone growth factors and white zone growth factors. The red zone growth factors are one or more of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), and connective tissue growth factor (CTGF). The white zone growth factors are one or more of transforming growth factor-β1 / 3 (TGF-β-1 / 3), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), and bone morphogenetic protein-7 (BMP-7).

[0009] Preferably, the amount of growth factor added to the red zone is 10-30 ng / mg, the amount of growth factor added to the white zone is 5-15 ng / mg, and the amount of bone marrow mesenchymal stem cells added is 1×10⁻⁶. 6 -5×10 6 .

[0010] Preferably, the growth factor is loaded onto a microsphere carrier, and then the microsphere carrier loaded with the growth factor is added to the hydrogel bio-ink.

[0011] Preferably, the load is specifically: Step 1: Prepare 20ml of 4% PVA (polyvinyl alcohol) solution and 400ml of 0.4% PVA solution. Place 20 ml of 4% PVA solution and 400 ml of 0.4% PVA solution in a 90°C water bath and heat. Stir at 300 rpm until completely dissolved. Continue stirring and cool to room temperature for later use. Step 2: Preparation of oil phase substances Mix 200 mg PLGA (polylactic acid-glycolic acid copolymer) with 6 ml DCM (dichloromethane) in a beaker, cover the mouth of the beaker with plastic wrap (one layer inside and one layer outside) to slow down the volatilization of dichloromethane, and dissolve it by stirring at 750 rpm under ice bath conditions until it is completely dissolved to obtain an oil phase substance; Step 3: Preparation of aqueous phase substances Mix 1.67 μg / ml growth factor with 2 ml UP water and 75 mg BSA (bovine serum albumin), and stir at 750 rpm under ice bath conditions until completely dissolved; Step 4: Preliminary mixing Under ice bath conditions, the aqueous phase substance is added to the oil phase substance with stirring at 500 rpm, and the substance is slowly and uniformly dripped below the liquid surface. Step 5: Ultrasonic Treatment The emulsion formed in step four is sonicated for 10 seconds, then stopped for 20 seconds to prevent the microspheres from overheating and rupturing due to prolonged sonication. This process is repeated until 90 seconds are obtained to produce the colostrum. Step 6: Pour the colostrum into 20ml of 4% PVA solution and stir at 1000rpm for 0.5h; Step 7: Pour the emulsion obtained in Step 6 into 400ml of 0.4% PVA solution and stir at 1000rpm for 4 hours to allow DCM to evaporate; Step 8: Centrifugation, freezing, and lyophilization Centrifuge the substance obtained in step seven at 10,000 rpm for 5 minutes, repeat the centrifugation 4 times to remove residual PVA, then freeze it at -20°C, and finally freeze-dry it externally for 2-3 days before storing it in a -20°C freezer.

[0012] Preferably, the microsphere carrier is one or more of polylactic acid-glycolic acid copolymer (PLGA), chitosan, and sodium alginate microspheres.

[0013] Preferably, the matrix material of the hydrogel bio-ink is selected from one or more of human recombinant collagen and methacrylated gelatin (GelMA), and the hydrogel bio-ink contains 1%-5% nanocellulose (CNC) and 0.05%-0.1% photoinitiator (LAP).

[0014] Preferably, the biodegradable polymer material is polycaprolactone (PCL) with a molecular weight of 20,000 to 80,000.

[0015] Preferably, the specific steps are as follows: S1. Based on the shape and size of the natural meniscus, establish the radial and circumferential fiber structure of the PCL frame structure; S2. In the middle of the 3D printed PCL framework fibers layer by layer, establish printing fibers loaded with bio-ink of growth factors / bone marrow mesenchymal stem cells, wherein the outer 1 / 3 is filled with bio-ink printing fibers loaded with red zone growth factors / bone marrow mesenchymal stem cells, and the inner 2 / 3 is filled with bio-ink printing fibers loaded with white zone growth factors / bone marrow mesenchymal stem cells. S3. Combine the above models to generate a multi-nozzle printing G-code file, in which the PCL frame is set to be printed by the T0 nozzle, the printing temperature is 65-95℃, the printing speed is 5-15 mm / s, and the extrusion speed is 0.5-5 mm. 3 The bio-ink for red zone growth factors / bone marrow mesenchymal stem cells was printed using a T1 printhead at a printing temperature of 20-25 ℃, a printing speed of 1-8 mm / s, and an extrusion speed of 0.5-5 mm / s. 3 / s; Bio-ink for white zone growth factors / bone marrow mesenchymal stem cells was printed using a T2 printhead at a printing temperature of 20-25℃, a printing speed of 1-8 mm / s, and an extrusion speed of 0.5-5 mm / s. 3 / s; S4. Add PCL material to printhead T0, add red area hydrogel bio-ink to printhead T1, add white area hydrogel bio-ink to printhead T2, import multi-printhead printing path control file G-code file, and print under sterile conditions to obtain biomimetic heterogeneous tissue-engineered meniscus scaffold.

[0016] Preferably, in step S1, the circumferential fiber diameter spacing is 400-800 μm, the included angle between radial fibers is 5°-15°, the height of each layer is designed to be 100-500 μm, and the number of layers is adjusted according to the thickness of the meniscus.

[0017] Using the above technical solution, the collagen fiber orientation of the natural meniscus is simulated, and the printed PCL frame is composed of annular and radial fibers, with the fiber diameter adjustable from 50 to 500 μm. The porosity of the frame structure is adjustable in zones from 30% to 60% between the free edge and the synovial edge, and the pore size is adjustable in zones from 50% to 600 μm between the free edge and the synovial edge.

[0018] This invention, based on a multi-level biomimetic strategy, optimizes the preparation method of ink for bio-3D printing of meniscus scaffolds while considering both printing performance and cell compatibility requirements during the preparation process. Utilizing a newly developed multi-nozzle 3D printing device, it simultaneously prints PCL framework material and methacryloyl gelatin / natural meniscus extracellular matrix bio-ink loaded with bone marrow mesenchymal stem cells / growth factors, achieving more precise biomimetic heterogeneous structure and composition. Ultimately, it reshapes the heterogeneous and complex tissue structure and composition of the meniscus to ensure it possesses the required biological functions and mechanical properties.

[0019] A second objective of the present invention is to provide a biomimetic heterogeneous tissue-engineered meniscus scaffold prepared by the above method.

[0020] The beneficial effects of this invention are: (1) The present invention constructs circumferential and radial fibers based on the distribution of collagen fibers in the natural meniscus, and prints a biodegradable polymer PCL framework accordingly, realizing the gradient change of porosity, pore size and mechanical properties of the meniscus support from the free edge to the synovial edge.

[0021] (2) The present invention designs bio-hydrogel inks loaded with growth factors / bone marrow mesenchymal stem cells for the white area of ​​the meniscus. Through 3D printing path design, the growth factors and bone marrow mesenchymal stem cells are seeded and released in a partitioned manner, ensuring the tissue partition regeneration and functional partition remodeling of the meniscus.

[0022] (3) This invention achieves the interlaced printing of hydrogel bio-ink loaded with growth factors / bone marrow mesenchymal stem cells and biodegradable polymer materials through a newly designed multi-material printing path and filament diameter control model, forming a double braided network, which effectively avoids the problem of scaffold delamination caused by the non-fusion of interfaces of different materials in traditional heterogeneous scaffolds. Attached Figure Description

[0023] Figure 1 This is a design drawing of the circumferential and radial fibers of the PCL frame of the present invention.

[0024] Figure 2 This invention provides 3D-printed PCL frames with different pore sizes and porosities.

[0025] Figure 3 This is a schematic diagram of the meniscus repair scaffold of the present invention, which includes red and white bio-ink zones and a PCL frame.

[0026] Figure 4 This invention relates to a biomimetic heterogeneous tissue-engineered meniscus (large pore size) 3D printed using a multi-nozzle process.

[0027] Figure 5 This invention relates to a biomimetic heterogeneous tissue-engineered meniscus (small aperture) 3D printed by multiple nozzles.

[0028] Figure 6 This invention illustrates the distribution relationship between PCL and hydrogel bio-ink in a biomimetic heterogeneous tissue-engineered meniscus produced by multi-nozzle 3D printing.

[0029] Figure 7 This is a comparison image of the printed PCL frame and the natural meniscus in this invention.

[0030] Figure 8 This is a microscopic image showing the gradual increase in the aperture of the support structure from the white area to the red area in this invention.

[0031] Figure 9 This relates to the relationship between the spacing of the support wires and the tensile strength of the present invention.

[0032] Figure 10 This invention relates to the relationship between the spacing of the support wires and the compressive strength.

[0033] Figure 11 This is a photograph of the actual object during the 3D printing process of this invention. Detailed Implementation

[0034] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0035] Example 1 The system employs a PCL framework structure with a circumferential fiber spacing of 600 μm and a radial fiber angle of 12°. Each layer is 300 μm high, with a total of 25 layers, matching the average thickness of an adult meniscus (approximately 7.5 mm). The red zone bio-ink contains 10 ng / mg connective tissue growth factor CTGF and... BMSCs, with white blood cells containing 10 ng / mg TGF-β3 and BMSCs, with a hydrogel substrate of 15 wt% human recombinant collagen, 5% CNC and 0.08% LAP. During printing, the T0 printhead constructs the PCL framework at 80°C and 10 mm / s, while the T1 / T2 printheads simultaneously fill the red and white areas with bio-ink at 22°C and 5 mm / s. This design achieves a continuous gradient change in porosity from the outer to the inner layer, with an initial compressive modulus of 40 MPa, suitable for repairing meniscus defects without significant inflammation, such as... Figures 1-8 As shown.

[0036] Example 2 The difference from Example 1 is that the circumferential fiber spacing was reduced to 400 μm (a 33% decrease), the radial angle remained constant at 12°, the height of each layer was 300 μm, and the total number of layers was 25, resulting in a significant increase in the circumferential tensile strength of the stent. The CTGF concentration in the red zone was increased to 30 ng / mg (the upper limit), and the BMSCs density increased to [missing value]. (Upper limit), TGF-β3 in the white zone increased to 15 ng / mg (upper limit), with other parameters the same as in Example 1. Although the porosity was reduced, the mechanical strength of the scaffold was improved, allowing it to withstand greater impact, while simultaneously increasing the content of stem cells and growth factors to ensure sufficient regenerative performance. (e.g.) Figure 5 and Figure 11 (As shown).

[0037] Example 3 Unlike the standard gradient of Example 1, this example increases the circumferential fiber spacing to 800 μm (a 33% increase), the radial angle to 12°, and the height of each layer to 300 μm. The red zone CTGF concentration is increased to 20 ng / mg, and the cross-linking degree is improved by increasing the LAP content to 0.1%, significantly extending the growth factor sustained-release period. The PCL frame printing speed is reduced to 5 mm / s (lower limit) to ensure the stability of the large-pore structure. The increased pore size and lower elastic modulus of this scaffold, compared to Example 1, reduce wear on the patient's articular cartilage in the early stages of implantation, making it suitable for patients with arthritis or joint damage. Furthermore, the high growth factor content and long release period after implantation make this scaffold more suitable for chronic degenerative injuries with insufficient blood supply.

[0038] Example 4 While maintaining the fiber parameters of Example 1 (600 μm spacing, 12° angle), the hydrogel substrate was replaced with 20 wt% methacrylated gelatin (Example 1 uses human collagen), and the CNC addition was increased to 5% (upper limit), resulting in a 3-fold increase in ink yield stress. The densities of red and white BMSCs were adjusted to... (Lower limit) To avoid the inhibition of cell activity by high CNC content, the resulting scaffold has better elasticity and stronger protective effect on cartilage.

[0039] Experimental Example 1: Effect of different wire spacings on the tensile and compressive strength of the product Adjust the wire spacing to different values ​​of 0.6mm, 0.8mm, 1.0mm, 1.4mm and 1.6mm, and measure the tensile strength and compressive strength of the corresponding products.

[0040] refer to Figure 9 and Figure 10It can be seen that the tensile strength and compressive strength are different for different wire spacings. As the wire spacing increases, both the tensile strength and compressive strength of the product decrease.

[0041] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0042] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold, characterized in that, A biomimetic heterogeneous tissue-engineered meniscus scaffold was fabricated using biodegradable polymer materials and hydrogel bio-inks containing bone marrow mesenchymal stem cells and growth factors. Based on the collagen fiber distribution of the natural meniscus, the meniscus scaffold framework was constructed by printing circumferential and radial fibers with biodegradable polymers. Hydrogel bio-inks loaded with different growth factors and bone marrow mesenchymal stem cells were then printed alternately in the red and white areas of the meniscus scaffold framework.

2. The method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold as described in claim 1, characterized in that, The growth factors include red zone growth factors and white zone growth factors. The red zone growth factors are one or more of vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor-2, and connective tissue growth factor. The white zone growth factors are one or more of transforming growth factor-β1 / 3, basic fibroblast growth factor, insulin-like growth factor-1, and bone morphogenetic protein-7.

3. The method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold as described in claim 2, characterized in that, The amount of growth factor added to the red zone is 10-30 ng / mg, the amount of growth factor added to the white zone is 5-15 ng / mg, and the amount of bone marrow mesenchymal stem cells added is 1×10⁻⁶. 6 -5×10 6 .

4. The method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold as described in claim 1, characterized in that, The growth factor is loaded onto a microsphere carrier, and then the microsphere carrier loaded with the growth factor is added to the hydrogel bio-ink. The load is specifically: Step 1: Prepare 20ml of 4% PVA solution and 400ml of 0.4% PVA solution. Place 20 ml of 4% PVA solution and 400 ml of 0.4% PVA solution in a 90°C water bath and heat. Stir at 300 rpm until completely dissolved. Continue stirring and cool to room temperature for later use. Step 2: Preparation of oil phase substances Mix 200 mg PLGA and 6 ml DCM in a beaker, cover the mouth of the beaker with plastic wrap, and dissolve by stirring at 750 rpm under ice bath conditions until completely dissolved to obtain the oil phase substance; Step 3: Preparation of aqueous phase substances Mix 1.67 μg / ml growth factor with 2ml UP water and 75mg BSA, and stir at 750rpm under ice bath conditions until completely dissolved; Step 4: Preliminary mixing Under ice bath conditions, the aqueous phase substance is added to the oil phase substance with stirring at 500 rpm, and the substance is slowly and uniformly dripped below the liquid surface. Step 5: Ultrasonic Treatment The emulsion formed in step four is sonicated for 10 seconds, stopped for 20 seconds, and the process is repeated until 90 seconds are obtained to obtain colostrum. Step 6: Pour the colostrum into 20ml of 4% PVA solution and stir at 1000rpm for 0.5h; Step 7: Pour the emulsion obtained in Step 6 into 400ml of 0.4% PVA solution and stir at 1000rpm for 4 hours; Step 8: Centrifugation, freezing, and lyophilization Centrifuge the substance obtained in step seven at 10,000 rpm for 5 minutes, repeat the centrifugation 4 times, then freeze it at -20°C, and finally freeze-dry it externally for 2-3 days before storing it in a -20°C freezer.

5. The method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold as described in claim 4, characterized in that, The microsphere carrier is one or more of polylactic acid-glycolic acid copolymer (PLGA), chitosan, and sodium alginate microspheres.

6. The method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold as described in claim 5, characterized in that, The matrix material of the hydrogel bio-ink is selected from one or more of human recombinant collagen and methacrylated gelatin, and the hydrogel bio-ink contains 10%-25% matrix material, 1%-5% nanocellulose and 0.05%-0.1% photoinitiator.

7. The method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold as described in claim 6, characterized in that, The biodegradable polymer material is polycaprolactone with a molecular weight of 20,000-80,000.

8. The method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold as described in claim 7, characterized in that, The specific steps are as follows: S1. Based on the shape and size of the natural meniscus, establish the radial and circumferential fiber structure of the PCL frame structure; S2. In the middle of the 3D printed PCL framework fibers layer by layer, establish printing fibers loaded with bio-ink of growth factors / bone marrow mesenchymal stem cells, wherein the outer 1 / 3 is filled with bio-ink printing fibers loaded with red zone growth factors / bone marrow mesenchymal stem cells, and the inner 2 / 3 is filled with bio-ink printing fibers loaded with white zone growth factors / bone marrow mesenchymal stem cells. S3. Combine the above models to generate a multi-nozzle printing G-code file, in which the PCL frame is set to be printed by the T0 nozzle, the printing temperature is 65-95℃, the printing speed is 5-15 mm / s, and the extrusion speed is 0.5-5 mm. 3 The bio-ink for red zone growth factors / bone marrow mesenchymal stem cells was printed using a T1 printhead at a printing temperature of 20-25 ℃, a printing speed of 1-8 mm / s, and an extrusion speed of 0.5-5 mm / s. 3 / s; Bio-ink for white zone growth factors / bone marrow mesenchymal stem cells was printed using a T2 printhead at a printing temperature of 20-25℃, a printing speed of 1-8 mm / s, and an extrusion speed of 0.5-5 mm / s. 3 / s; S4. Add PCL material to printhead T0, add red area hydrogel bio-ink to printhead T1, add white area hydrogel bio-ink to printhead T2, import multi-printhead printing path control file G-code file, and print under sterile conditions to obtain biomimetic heterogeneous tissue-engineered meniscus scaffold.

9. The method for preparing a biomimetic heterogeneous tissue-engineered meniscus scaffold as described in claim 8, characterized in that, In step S1, the circumferential fiber spacing is 400-800 μm, the radial fiber angle is 5°-15°, and the height of each layer is designed to be 100-500 μm. The number of layers is adjusted according to the thickness of the meniscus. The PCL frame aperture size shows a gradient increase from 50-1500 μm from the white area to the red area, exhibiting a partitioned structural feature.

10. A biomimetic heterogeneous tissue-engineered meniscus scaffold prepared by the method as described in any one of claims 1-9.

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