CN-122005941-A - Micro-environment-imitating 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 micro-environment-imitating composite scaffold and application thereof in anisotropic regeneration of meniscus cartilage. The composite scaffold with excellent bioactivity and mechanical stability is prepared by combining the extracellular matrix of meniscus source with the 3D printing polycaprolactone scaffold, and meanwhile, the precise spatial arrangement of fibrochondrocytes and fibroblasts is realized by utilizing the 'occupying sacrificial' strategy based on the thermosensitive poloxamer hydrogel, so that the hexagonal meniscus tissue engineering scaffold with a heterogeneous spatial continuous structure and no obvious interface is successfully constructed. Experiments prove that the scaffold effectively reproduces the regional heterogeneity of the natural meniscus, realizes the gradient distribution of the I/II type collagen and the sulfated glycosaminoglycan, achieves the bionic reconstruction of the structure and the function, and provides a very promising transformation path for the functional meniscus repair reconstruction.

Inventors

• SONG XINGQI
• XIE SHANHONG
• LEI DONG
• ZHOU GUANGDONG

Assignees

• 上海交通大学医学院附属第九人民医院

Dates

Publication Date

20260512

Application Date

20260409

Claims (10)

1. 1. The preparation method of the micro-environment-imitating composite scaffold is characterized by comprising the following steps of: Preparing Me-dECM aqueous solution, namely taking a pig meniscus tissue, freezing and grinding, sequentially treating with 0.1-0.5% trypsin/phosphate buffer, nuclease solution, 8-12mM Tris-HCl buffer (containing 8-12U/mL aprotinin) and 1-5% triton X-100/phosphate buffer, washing with phosphate buffer, freeze-drying, treating with 0.1-0.5% collagenase, dialyzing with deionized water, freeze-drying, adding phosphate buffer, and dissolving to obtain Me-dECM aqueous solution; Preparing Me-dECM/GelMA composite hydrogel, namely adding a photoinitiator into the Me-dECM aqueous solution and the solution of the methacryloylated gelatin in a mass ratio of 1:5-10 to obtain Me-dECM/GelMA composite hydrogel; Inoculating the inner layer and the outer layer, namely taking a meniscus-type structural bracket, filling poloxamer F-127 in the middle layer of the bracket at the temperature of 30-40 ℃, then injecting Me-dECM/GelMA composite hydrogel containing fibrochondrocytes into the inner layer, injecting Me-dECM/GelMA composite hydrogel containing fibroblastic cells and vascular endothelial growth factors into the outer layer, and carrying out ultraviolet crosslinking curing; And (3) inoculating an intermediate layer, namely transferring the scaffold to 2-4 ℃, removing poloxamer F-127, injecting Me-dECM/GelMA composite hydrogel containing fibrochondrocytes and fibroblasts into the intermediate layer of the scaffold, and carrying out ultraviolet crosslinking curing to obtain the micro-environment-imitating composite scaffold.
2. 2. The method of claim 1, wherein the nuclease solution in step (1) is 8-12 mM Tris-HCl buffer containing 30-60U/mL deoxyribonuclease and 1-5U/mL ribonuclease a, and the ph=6-8.
3. 3. The method for preparing the micro-environment-simulated composite scaffold according to claim 1, wherein the photoinitiator in the step (2) is phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate.
4. 4. The method for preparing the micro-environment-simulated composite scaffold according to claim 3, wherein the addition amount of the phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate is 0.1-0.5wt% of the Me-dECM/GelMA composite hydrogel.
5. 5. The method for preparing a composite scaffold according to claim 1, wherein in the step (3), the concentration of the fibrochondrocytes injected into the inner layer is 6×10 7 -10×10 7 cells/mL, the concentration of the fibroblasts injected into the outer layer is 6×10 7 -10×10 7 cells/mL, and the concentration of the vascular endothelial growth factor is 80-120ng/mL.
6. 6. The method of claim 1, wherein in step (4), the concentration of the intermediate layer injected fibrochondrocytes is 3×10 7 -5×10 7 cells/mL and the concentration of the fibroblasts is 3×10 7 -5×10 7 cells/mL.
7. 7. The preparation method of the micro-environment-imitating composite scaffold according to claim 1, wherein the scaffold is made of polycaprolactone, is a perpendicular, continuous and hexagonal meniscus-type structure bionic scaffold without obvious interfaces, has a regular micropore structure, and has parameters of 300-500 μm in diameter, 1-2cm in length and 1-2mm in height, 400-450 μm in thickness of an outer layer, 200-250 μm in thickness of an intermediate layer and 100-150 μm in thickness of an inner layer, 480-520 μm in wire diameter and 1150-1290 μm in aperture.
8. 8. A simulated microenvironment composite scaffold made by the method of any one of claims 1-7.
9. 9. Use of the simulated microenvironment composite scaffold of claim 1 in anisotropic regeneration of meniscal cartilage.
10. 10. The use of claim 9, wherein the use comprises a meniscal defect or a meniscal damage.

Description

Micro-environment-imitating composite scaffold and application thereof in anisotropic regeneration of meniscus cartilage Technical Field The invention belongs to the technical field of medical materials for meniscus regeneration, and relates to a microenvironment-imitating composite scaffold and application thereof in anisotropic regeneration of meniscus cartilage. Background Meniscus injury is one of the common sports injuries. As a fibrocartilage tissue between the femoral condyle of the knee joint and the tibial plateau, the meniscus plays an irreplaceable role in maintaining the mechanical stability of the joint, transmitting load, absorbing impact, lubricating the joint and the like. The tissue mainly comprises cells and extracellular matrix (ECM), and comprises various components such as collagen, elastin, glycosaminoglycan and the like. The meniscus is extremely poor in its ability to self-repair due to its own maldistribution of blood supply (i.e. "outer red-inner white" areas). Once damaged, the stress distribution of the joint is abnormal, the lubrication function is reduced and the stability is lost, so that the serious consequences such as cartilage degeneration, osteoarthritis and the like are caused. Currently, the clinical treatments for meniscal lesions mainly include suturing, partial or total excision and allograft. However, these methods have significant limitations in that suturing is only suitable for red-zone injuries with abundant blood supplies, resection can change the normal mechanical environment of the joint, accelerate joint degeneration, and allograft can face problems of serious shortage of donors, transmission of potential diseases, immune rejection, difficulty in size matching and the like. Therefore, developing an active meniscus alternative that can achieve both structural and functional repair has become a critical clinical challenge in the fields of sports medicine and orthopedics. Tissue engineering and regenerative medicine provide new ideas for functional regeneration of meniscus. Over the last two decades, researchers have tried to inoculate bone marrow mesenchymal stem cells (BMSCs) or chondrocytes onto scaffolds constructed of materials such as Polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA) or decellularized extracellular matrix (dECM), and to achieve regeneration of meniscus tissue in animal models such as rabbits, sheep, etc. However, these strategies have focused on building homogenized tissue. The natural meniscus has a typical spatial heterogeneous structure that an outer vascular region (red region) is rich in type I collagen, blood vessels and nerves and mainly plays roles of repairing and partial bearing, and an inner avascular region (white region) is mainly composed of type II collagen and glycosaminoglycans (GAGs) and has the core functions of bearing pressure, dispersing load and buffering impact. The prior homogenization engineering scaffold cannot reproduce the complex spatial gradient (comprising cell composition, ECM component and mechanical property), so that the interface integration of the new tissue and the host tissue is poor, the mechanical property is insufficient, and early degeneration is easy to occur. In order to simulate the heterogeneity of menisci, 3D printing and localized controlled release of growth factors have been introduced into this field in recent years. For example, there are studies on the use of a dual jet printing system to release Connective Tissue Growth Factor (CTGF) and transforming growth factor-beta 3 (TGF-beta 3) in different regions of the scaffold, respectively, to induce region-specific collagen expression, and studies on the use of a localized acellular matrix (Me-dECM) bio-ink to construct a dual-layer scaffold, achieving a type I/II collagen ratio similar to that of natural tissue. These methods have progressed in creating biochemical gradients, but often neglect biological continuity between the natural meniscus regions and synergy of the cellular microenvironment, resulting in poor interfacial integration within the regenerated tissue and incomplete functional recovery. In summary, the prior art still faces the core challenges of reconstructing a bionic microenvironment capable of simulating the continuous anisotropy of a natural meniscus on multiple scales of a macroscopic structure, a microscopic morphology, a molecular signal and the like, and realizing the spatial precise arrangement and fate control of different cell types in the microenvironment so as to drive the regeneration of functional meniscus tissues with physiological heterogeneity and mechanical gradient. Solving these challenges is of great importance in achieving thorough repair of meniscus injury. Disclosure of Invention In order to solve the problems in the prior art, the first object of the invention is to provide a micro-environment-imitating composite scaffold, which combines a decellularized extracellular matrix (Me-dECM) f