A growth factor controlled release carrier for tissue engineering meniscus scaffold and a preparation method thereof

Abstract

The application belongs to the technical field of meniscus injury repair materials, and particularly relates to a growth factor controllable release carrier for tissue engineering meniscus regeneration repair and a preparation method thereof. The application discloses a growth factor controllable release carrier for tissue engineering meniscus regeneration repair and a preparation method thereof, and proposes a shell-core structure coaxial electrostatic spinning carrier, specifically, different growth factors are loaded in the shell-core layer through coaxial electrostatic spinning, and the time sequence release of the growth factors is realized through the shell-core layer using different degradation rate carriers (the degradation rate of the core layer is greater than that of the shell layer), and the hollow fiber structure is realized with the lapse of time. The electrostatic spinning fibers are broken into short rod-shaped fibers, combined with biological ink, and through 3D printing, the precise heterogeneity structure and component bionics are realized. Through microscale degradation rate regulation and structure design, the technical bottlenecks of time sequence release and blood vessel guidance are broken through.

Description

Technical Field

[0001] This invention belongs to the field of meniscus injury repair materials technology, specifically relating to a growth factor controllable release carrier for tissue-engineered meniscus regeneration and repair and its preparation method. Background Technology

[0002] The meniscus, a fibrocartilaginous structure within the knee joint, plays a crucial role in cushioning shocks, stabilizing the joint, and distributing load. Due to its avascular nature (only 10%-30% of the peripheral area has blood supply), its self-healing ability is extremely weak after injury. Without timely intervention, it can easily lead to secondary diseases such as articular cartilage degeneration and osteoarthritis, severely impacting patients' motor function and quality of life. Clinical statistics show that meniscus injuries account for over 25% of sports injuries, and with the increasing aging population, the incidence of degenerative meniscus injuries is rising year by year, highlighting the urgent need for clinical treatment. Currently, treatments for meniscus injuries mainly fall into three categories: First, meniscus repair, which is only suitable for fresh injuries in areas with rich peripheral blood supply, showing extremely poor repair effects in areas with poor blood supply, and a postoperative healing rate of less than 60%; second, partial meniscectomy, which can quickly relieve pain, but leads to abnormal joint load distribution, with an incidence of osteoarthritis 3-5 times higher than in the general population 5-10 years post-surgery; and third, meniscus transplantation, whose clinical application is limited by donor shortages (mainly relying on allogeneic donation), immune rejection, and low post-transplant integration efficiency. These traditional therapies are all insufficient to achieve structural and functional regeneration of the meniscus, necessitating breakthroughs in new repair technologies. Currently, two commercially available meniscus support products are available: collagen meniscus implants (CMI) and Actift; however, related studies show that these supports suffer from problems such as volume shrinkage over time, predominantly scar tissue repair, and a tendency to induce joint effusion.

[0003] Existing tissue-engineered meniscus scaffold materials are mainly divided into natural polymers (such as collagen, chitosan, and hyaluronic acid) and synthetic polymers (such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid copolymer (PLGA), and polycaprolactone (PCL)). Natural polymers have excellent biocompatibility, but low mechanical strength and difficult-to-control degradation rates (e.g., collagen completely degrades in vivo within 2-4 weeks, unable to support long-term regeneration). Synthetic polymers can have their mechanical properties and degradability optimized through molecular design (e.g., PCL has a degradation cycle of 12-24 months), but poor bioactivity makes it difficult to induce directed cell differentiation and matrix deposition, requiring the loading of growth factors to enhance their function. Traditional carriers (such as microspheres and hydrogels) often use physical embedding methods to load growth factors. However, this can easily lead to a "burst release effect" (40%-60% of the total load is released within 24 hours post-procedure) due to material swelling or degradation. This results in excessively high early growth factor concentrations, triggering inflammatory responses, followed by insufficient concentrations to support matrix synthesis. While some carriers improve release profiles through chemical cross-linking, residual cross-linking agents reduce biocompatibility and hinder the phased release of multiple factors. Furthermore, existing carriers are mostly amorphous particles or block structures, which tend to aggregate after binding with scaffolds, failing to form a continuous "cell-factor-scaffold" interface. They also do not consider the vascularization requirements of meniscus regeneration. Blood supply to areas lacking blood vessels relies primarily on scaffold structures to guide vascular ingrowth, but traditional carriers lack specific structural designs to promote angiogenesis, leading to tissue degeneration due to insufficient nutrient supply.

[0004] Electrospinning technology has become a preferred method for fabricating tissue engineering scaffolds due to its ability to prepare biomimetic nanofibers (50-500 nm in diameter, matching the scale of natural extracellular matrix fibers). However, traditional uniaxial electrospun scaffolds have two major drawbacks: first, growth factors are directly loaded inside the fibers, which are easily deactivated due to the high temperature and pressure during the spinning process (the activity retention rate is less than 50%); second, the fibers are solid structures, which cannot form channels for blood vessel ingrowth, and the release kinetics are singular, making it difficult to achieve time-sequential release.

[0005] Some existing meniscus repair scaffolds employ a macroscopic zoning design of 'medial white zone - lateral red zone', using synthetic polymers such as polycaprolactone and polylactic acid as framework materials. While this approach matches the spatial differences in the natural meniscus structure by loading different regions with transforming growth factor β3 and connective tissue growth factor, it suffers from several key limitations: First, this approach involves independent macroscopic regional distribution without microscopic encapsulation or nesting structures, making it impossible to regulate the release sequence of factors through differences in carrier degradation rates. Second, it lacks a 'differentiated degradation rate carrier' design, resulting in synchronous or undefined regulatory logic for material degradation in the medial and lateral regions. This only allows for synchronous or random release of factors, failing to meet the temporal requirements of 'early angiogenesis promotion and later cartilage regeneration promotion' in meniscus repair. Third, it lacks a specific structural design to guide vascular ingrowth, relying solely on regional blood supply for repair, resulting in lower regeneration efficiency and nutrient supply stability compared to controllable release carriers. Summary of the Invention

[0006] To address the aforementioned deficiencies, this invention discloses a controllable release carrier for meniscus regeneration and repair in tissue engineering, along with its preparation method. It proposes a shell-core coaxial electrospun carrier, specifically by loading different growth factors onto the shell and core layers through coaxial electrospinning. Furthermore, by using carriers with different degradation rates (the core layer's degradation rate is greater than the shell layer) in the shell and core layers, the sequential release of growth factors is achieved, resulting in a hollow fiber structure over time. The electrospun fibers are broken down into short rod-shaped fibers and combined with bio-ink, enabling precise heterogeneous structures and biomimetic composition through 3D printing. This application overcomes the technical bottlenecks of sequential release and vascular guidance through microscale degradation rate regulation and structural design.

[0007] This invention aims to provide a time-release carrier of growth factors for the regeneration and repair of tissue-engineered meniscus scaffolds. In the early stages, it rapidly releases pro-angiogenic growth factors to promote microvascular network formation and provide nutrients for subsequent tissue regeneration. During the subsequent repair cycle, it continuously releases growth factors that promote cartilage regeneration, fostering stem cell differentiation and cartilage matrix formation. The growth factor release carrier is precisely deposited in the tissue-engineered meniscus scaffold using 3D printing, biomimetically mimicking the natural meniscus fiber orientation, thereby regulating the regenerative structure of the meniscus.

[0008] To achieve the above objectives, the first technical solution of this application discloses a method for preparing a growth factor controllable release carrier for tissue-engineered meniscus regeneration and repair, comprising the following steps:

[0009] S1. Prepare solutions of the core material and the shell material respectively;

[0010] S2. The core material solution is connected to the inner coaxial tube and the shell material is connected to the outer coaxial tube to obtain the coaxial electrospinning method.

[0011] Both the core material and the shell material are biodegradable, with the core material degrading faster than the shell material. Furthermore, the core material is loaded with angiogenesis-promoting growth factors, while the shell material is loaded with cartilage regeneration-promoting growth factors.

[0012] The angiogenesis-promoting growth factors include one or more of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor-2 (FGF-2); the cartilage regeneration-promoting growth factors include one or more of transforming growth factor-β1 / 3 (TGF-β1 / 3), insulin-like growth factor-1 (IGF-1), and bone morphogenetic protein-7 (BMP-7).

[0013] Furthermore, the core material is any one or any combination of PLGA, PCL, PLA, TPU, gelatin, silk fibroin, hyaluronic acid and their modified products; the shell material is any one or any combination of PLGA, PCL, PLA, and TPU; wherein the degradation rate is controlled by adjusting the molecular weight and proportion of the added materials.

[0014] Furthermore, the coaxial electrospinning operating voltage range is 7-13.5kV, the current collector distance range is 8-14cm, the core layer spinning solution flow rate is 1 mL / h, the shell layer spinning solution flow rate is 3mL / h, the needle distance from the receiver is 10-15cm, and the receiving mold rotation speed is 1500-2000r / min.

[0015] Furthermore, the outer diameter of the carrier is 30-300 μm, the growth factor content is 20-50 ng / ml, the inner diameter is 10-100 μm, and the growth factor content is 10-30 ng / ml.

[0016] And, according to the above preparation method, a growth factor controllable release carrier for tissue engineering meniscus regeneration and repair.

[0017] The second technical solution of this application discloses the application of the controllable release carrier of growth factors for tissue engineering meniscus regeneration and repair in the preparation of 3D printed menisci.

[0018] Furthermore, the carrier is used to simulate the circumferential fiber structure distribution in a natural meniscus.

[0019] Furthermore, the application specifically involves: cutting the carrier into short nanofibers and mixing them with bio-ink, then printing them into the tissue-engineered meniscus scaffold of the target model according to the circumferential fiber distribution of the target model meniscus, thereby regulating the meniscus regeneration structure.

[0020] Furthermore, the amount of the nanofibers added to the bio-ink is 1 g / ml.

[0021] Furthermore, the printing temperature of the bio-ink is 20-25℃, the printing speed is 1-8mm / s, and the extrusion speed is 0.5-5mm. 3 / s.

[0022] Furthermore, the bio-ink is composed of 10-20% methacrylated gelatin (GelMA), a fiber content of 0.5-3 g / ml, 1%-5% hyaluronic acid (HA), and 0.05%-1% photoinitiator LAP.

[0023] Furthermore, the method for preparing the nanofibers is as follows: the carrier is cut into small fragments of 0.5 × 0.5 cm. These fragments are immersed in deionized water and treated in a high-speed homogenizer at 6000 rpm for 10 minutes to disperse the fragments into short fibers.

[0024] The aforementioned carrier is used to print the fiber distribution of the meniscus scaffold. Its core layer uses a rapidly degrading material and is loaded with angiogenic growth factors. In the early stages of repair, it rapidly degrades and releases growth factors to form a hollow fibrous structure, serving as a carrier to guide angiogenesis. The shell layer uses a slower-degrading material and is loaded with cartilage regeneration growth factors, which slowly degrade and release growth factors during the repair cycle.

[0025] Beneficial effects

[0026] This invention provides a time-release carrier of growth factors for the regeneration and repair of tissue-engineered meniscus scaffolds. In the early stages, it rapidly releases pro-angiogenic growth factors, promoting microvascular network formation and providing nutrients for subsequent tissue regeneration. During the subsequent repair cycle, it continuously releases growth factors that promote cartilage regeneration, fostering stem cell differentiation and cartilage matrix formation. The growth factor release carrier is precisely deposited in the tissue-engineered meniscus scaffold using 3D printing, biomimetically mimicking the natural meniscus fiber orientation, thereby regulating the regenerative structure of the meniscus. Compared with existing technologies, it has the following advantages:

[0027] 1. Based on the sequential role of angiogenesis and cartilage regeneration in meniscus regeneration and repair, i.e. angiogenesis is the prerequisite and foundation for cartilage-like repair, this invention designs a controllable release carrier that can release growth factors sequentially, so that the growth factors that promote angiogenesis are released faster than the growth factors that promote cartilage regeneration. Compared with the traditional synchronous release scheme, this further improves the effect of meniscus regeneration and repair.

[0028] 2. Based on the important role of vascularization in cartilage regeneration and repair, this invention designs a coaxial electrospun fiber, in which the core structure degrades faster than the shell structure, thereby forming a hollow fiber structure over time. This actively guides vascular regeneration, solves the problem of unstable blood supply and nutrient supply in the dependent area, and achieves directional arrangement of fibers through 3D printing, ensuring tissue regeneration of the meniscus. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The surface morphology of the carrier prepared by electrospinning;

[0031] Figure 2 A schematic diagram illustrating the coaxial effect of the carrier prepared by coaxial electrospinning;

[0032] Figure 3 Schematic diagram of carriers prepared by coaxial electrospinning loaded with different growth factors;

[0033] Figure 4 A schematic diagram of a method for preparing a carrier by coaxial electrospinning;

[0034] Figure 5 Photographs and SEM images of carriers prepared by 3D computer extrusion coaxial electrospinning;

[0035] Figure 6 A schematic diagram of the preparation of a carrier for coaxial electrospinning. Detailed Implementation

[0036] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0037] Example 1: Preparation of a growth factor controllable release carrier for tissue-engineered meniscus regeneration and repair

[0038] like Figure 4 As shown, it includes the following steps:

[0039] (1) Preparation of spinning solution for coaxial electrospinning:

[0040] LA:GA=50:50 PLGA was dissolved in dichloromethane at 100 mg / ml, and LA:GA=75:25 PLGA was dissolved in hexafluoroisopropanol at 120 mg / ml. The high GA ratio solution served as the core layer solution for coaxial electrospinning and contained 10 ng / ml CTGF, while the low GA ratio solution served as the shell layer solution for coaxial electrospinning and contained 20 ng / ml TGF-β3. The solutions were then shaken overnight.

[0041] (2) 3D printing of electrospinning fixation device:

[0042] The diameter and length of the coaxial electrospinning nozzle were measured to obtain relevant data. A 3D model of the electrospinning fixture was created using SolidWorks software, and the printing path was generated using Simplify3D software. The electrospinning fixture was then fabricated using a 3D printer.

[0043] (3) Production of coaxial electrospun fibers:

[0044] Assemble the electrospinning fixator. Using a 2.5ml disposable sterile syringe, draw a certain amount of high-GA ratio solution and connect it to the core layer channel of the coaxial electrospinning needle. Using a 2.5ml disposable sterile syringe, draw a certain amount of low-GA ratio solution and connect it to the shell layer channel of the coaxial electrospinning needle. The coaxial electrospinning parameters are: operating voltage range of 10kV, core layer spinning solution flow rate of 1 mL / h, shell layer spinning solution flow rate of 3 mL / h, needle distance from receiver of 12cm, receiving mold rotation speed of 2000 r / min. The prepared carrier is shown below. Figure 1 , Figure 2 As shown, its growth factor loading is as follows: Figure 3 As shown

[0045] (4) Production of coaxial electrospun short fibers:

[0046] The prepared fibers were cut into small fragments of 0.5 × 0.5 cm. These fragments were immersed in deionized water and processed in a high-speed homogenizer at 6000 rpm for 10 minutes to disperse the fragments into short fibers. The fibers were then freeze-dried for 24 hours to obtain dried short fibers.

[0047] (5) Production of 3D printing bio-ink:

[0048] Dissolve methacrylated gelatin (GelMA) in PBS at a concentration of 10-15%, and add 0.5%-5% decellularized meniscus matrix (dECM) and 0.05%-1% photoinitiator LAP. Add short fibers to the ink at a concentration of 1 g / ml. Use a homogenizer at 500 rpm for 3 minutes to evenly distribute the fibers in the ink.

[0049] (5) 3D printing of scaffolds

[0050] A suitable model was established for the structure of natural meniscus circumferential fibers using SolidWorks, and the printing path was controlled by converting the 3D slices into G-Code files using Simplify3D. The printing temperature of the bio-ink was 20-25℃, the printing speed was 1-8mm / s, and the extrusion speed was 0.5-5mm3 / s.

[0051] The fiber morphology prepared in this embodiment is shown in Figures 1 and 2 (schematic diagrams of coaxial electrospun core-shell structure, scale bar 0.5 μm). Coaxial electrospinning technology successfully prepared nanofibers with a core-shell structure. Compared to traditional uniaxial electrospinning, this embodiment achieved the sequential release of growth factors through the difference in degradation rates between the shell and core layers. Simultaneously, by designing different degradation rates for the core and shell, a transformation from a core-shell structure to a hollow fiber structure was achieved after the core layer was completely degraded, forming hollow channels. This hollow structure can serve as a 'directional guiding channel' for blood vessel ingrowth, promoting the formation of microvascular networks within the scaffold and providing nutritional support for subsequent cartilage regeneration. Furthermore, the aforementioned shell-core nanofibers were cut into 0.5 × 0.5 cm short fibers and dispersed in bio-ink (ink containing 12% GelMA, 3% HA, and 0.5% photoinitiator LAP). Extrusion 3D printing technology was used (printing temperature 22-24℃, printing speed 3-5 mm / s, extrusion speed 1-3 mm³ / s, conforming to claim 10). (With limitations), the fibers are oriented according to the natural meniscus's arrangement pattern of 'dominant circumferential fibers and auxiliary radial fibers,' thus improving the biomimetic fit between the printed tissue-engineered meniscus scaffold and the natural meniscus to over 85%.

[0052] It should be noted that the coaxial electrospinning carrier described in this application is a "functionalized fiber unit of a 3D printed scaffold," such as... Figure 5 , Figure 6 As shown, it only undertakes the functions of "temporal release of growth factors" and fiber orientation through printing and extrusion; the "mechanical support" function is undertaken by the subsequent 3D printed bio-ink matrix and framework.

[0053] Example 2

[0054] The preparation method in this embodiment is the same as in Example 1. Compared with Example 1, the core layer solution is prepared by dissolving gelatin in hexafluoroisopropanol at a concentration of 80 mg / ml. Compared with Example 1, the core-shell structure in this embodiment is clearer, which can further improve the time variability of its sequential release of growth factors.

[0055] Example 3

[0056] The preparation method in this embodiment is the same as that in Example 1. Compared with Example 1, 5% hyaluronic acid is added to the bio-ink to increase the viscosity of the ink, so that the fibers can be dispersed more evenly in the ink and avoid the fibers settling to the bottom.

[0057] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. The application of a controllable release carrier of growth factors for tissue-engineered meniscus regeneration and repair in the preparation of tissue-engineered meniscus scaffolds, characterized in that, The method for preparing the controllable release carrier of growth factors for tissue-engineered meniscus regeneration and repair includes the following steps: S1. Prepare solutions of the core material and the shell material respectively; S2. The core material solution is connected to the inner coaxial tube and the shell material is connected to the outer coaxial tube to obtain the coaxial electrospinning method. Both the core material and the shell material are biodegradable, with the core material degrading faster than the shell material. Furthermore, the core material is loaded with angiogenesis-promoting growth factors, while the shell material is loaded with cartilage-regenerating growth factors. The angiogenesis-promoting growth factor is any one or more of vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor-2; the cartilage regeneration-promoting growth factor is any one or more of transforming growth factor-β1 / 3, insulin-like growth factor-1, and bone morphogenetic protein-7. The content of pro-angiogenic growth factor in the core material solution is 10-30 ng / ml, and the content of cartilage regeneration growth factor in the shell material solution is 20-50 ng / ml. The outer diameter of the carrier is 30-300 μm, and the inner diameter is 10-100 μm; The core material preferentially degrades to form hollow fiber channels, which are used to guide blood vessel ingrowth. The core material is any one or any combination of PLGA, PCL, PLA, TPU, gelatin, silk fibroin, hyaluronic acid and their modified products; the shell material is any one or any combination of PLGA, PCL, PLA, and TPU; wherein the degradation rate is controlled by adjusting the molecular weight and proportion of the added materials.

2. The application according to claim 1, characterized in that, The coaxial electrospinning working voltage range is 7-13.5kV, the current collector distance range is 8-14 cm, the core layer spinning solution flow rate is 1 mL / h, the shell layer spinning solution flow rate is 3 mL / h, the needle distance from the receiver is 10-15 cm, and the receiving mold rotation speed is 1500-2000 r / min.

3. The application according to claim 1, characterized in that, The carrier is used to simulate the circumferential fiber structure distribution in a natural meniscus.

4. The application according to claim 1, characterized in that, The specific application involves cutting the carrier into short nanofibers, mixing them with bio-ink, and then printing them into the tissue-engineered meniscus scaffold of the target model according to the circumferential fiber distribution of the target model meniscus, thereby regulating the meniscus regeneration structure.

5. The application according to claim 4, characterized in that, The amount of the nanofibers added to the bio-ink is 1 g / ml.

6. The application according to claim 4, characterized in that, The bio-ink is printed at a temperature of 20-25℃, a printing speed of 1-8 mm / s, and an extrusion speed of 0.5-5 mm / s. 3 / s.

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