Preparation method of a long-term alternative anti-fatigue meniscus

Abstract

The present invention discloses a preparation method of a long-term alternative anti-fatigue meniscus, belonging to the technical field of meniscus preparation, and comprising the following steps: S1 making a meniscus fiber framework; S2 making a molding die for pouring a PVA solution; S3 placing the meniscus fiber framework into the molding die and injecting the PVA solution to fill and wrap the meniscus fiber framework; S4 placing the molding die on top of a heat-conducting component, with the bottom of the heat-conducting component immersed in a freezing source, so that the water in the PVA solution freezes to form ice columns, and the PVA is concentrated between the ice columns; S5 placing the frozen meniscus in a freeze dryer for freeze-drying until all the ice columns are sublimated; S6 annealing the freeze-dried meniscus, and soaking the annealed meniscus in deionized water until it is saturated with water absorption; By setting a meniscus fiber framework inside the PVA solution and performing directional freezing, freeze-drying and annealing treatment on the PVA solution, the mechanical properties, service life and anti-fatigue performance of the prepared meniscus are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of meniscus preparation, and particularly to a method for preparing a long-term replacement anti-fatigue meniscus. Background Art

[0002] The meniscus is a pair of wedge-shaped semilunar fibrocartilages with a thick outer edge and a thin inner edge between the femoral condyle and the tibial plateau, playing key physiological and biomechanical functions in the knee joint. For example, it disperses pressure, absorbs shock, maintains the stability of the knee joint, maintains the proprioception of the knee joint, and lubricates the joint, etc. In addition, the meniscus can also disperse the load to the adjacent articular cartilage, thereby protecting the hyaline cartilage from wear. The defect of the meniscus means a reduction in the contact area between the femoral condyle and the meniscus, increasing the contact pressure of the joint surface, which leads to cartilage wear and gradually disappears within 10 years, and finally develops into osteoarthritis and irreparable knee joint injuries.

[0003] According to the statistical results of the Medscape website, nearly 2 million people need to be treated for meniscus injuries every year, and it mostly occurs in sports people and the elderly. To maintain the function of the knee joint and avoid pain, a series of treatment methods have been developed clinically according to the location and degree of meniscus injuries, such as: traditional Chinese medicine treatment, total (or partial) meniscectomy, meniscus repair, and meniscus transplantation and reconstruction, etc.

[0004] For patients with severe meniscus damage, only by replacing the entire meniscus can cartilage degeneration be avoided. Artificial menisci are mainly divided into degradable meniscus scaffolds and alternative meniscus scaffolds. Although commercial products of degradable meniscus scaffolds have been applied in clinical practice, the new tissue in the scaffold is only meniscus-like tissue, which does not have the physiological structure and function of the natural meniscus and cannot meet the requirements of the mechanical properties of the knee joint. Alternative meniscus scaffolds are a type of meniscus substitute that can exist in the body for a long time and have similar biomechanical and physiological properties to the natural meniscus. In recent decades, it has gradually become a research hotspot. The Fourth Medical Center of the General Hospital of the Chinese People's Liberation Army has disclosed a silk woven meniscus implant and its preparation method (CN113633827A), which uses silk as the weaving material and is then prepared by bone glue infusion molding. Yixing Xinli Weaving Co., Ltd. discloses a silk / gel multidirectional fiber sandwich meniscus and its preparation method (CN114214838A). In this invention, the tensile stiffness of the meniscus reaches more than 0.3N, the elastic modulus reaches more than 70MPa, and the porosity is more than 94%. The Institute of Chemistry of the Chinese Academy of Sciences discloses a photocuring 3D printing preparation method for a long-term replacement tissue engineering meniscus scaffold (CN112220968A). The meniscus scaffold provided by the invention has mechanical properties similar to those of the natural meniscus and has good biocompatibility and in vivo stability. The replacement meniscus scaffold should have the characteristics of structural stability, non-degradability, and fatigue resistance. However, the above inventions do not consider the fatigue resistance of the scaffold under long-term loading-unloading cycles of the knee joint. How to simultaneously give the artificial meniscus good mechanical properties, stability, fatigue resistance and the advantage of easy fixation in the knee joint is a problem that researchers at home and abroad have not solved. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a long-term replacement anti-fatigue meniscus in view of the defects and shortcomings in the prior art. By setting a meniscus fiber frame inside a PVA solution and subjecting the PVA solution to directional freezing, freeze drying and annealing treatment, the density of the PVA chain and the crystallinity of the PVA are improved, the mechanical properties of the hydrogel are improved, and then the mechanical properties of the meniscus are improved. Under the condition that the use state remains unchanged, the service life and fatigue resistance of the meniscus are improved.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides a method for preparing a long-term replacement anti-fatigue meniscus, comprising the following contents:

[0008] S1 Preparation of meniscus fiber frame: Prepare the meniscus fiber frame according to the patient's meniscus image;

[0009] S2 Preparation of the Molding Die: Prepare a molding die for pouring the PVA solution according to the meniscus image of the patient. The size of the cavity of the molding die is not less than the size of the meniscus fiber framework.

[0010] S3 Pouring the PVA Solution: Place the meniscus fiber framework in S1 into the molding die in S2, and inject the PVA solution so that the PVA solution fills and wraps the meniscus fiber framework.

[0011] S4 Directional Freezing Treatment: Place the molding die in S3 on top of a heat-conducting component, and immerse the bottom of the heat-conducting component in a freezing source so that the water in the PVA solution freezes to form ice columns, and the PVA is concentrated between the ice columns.

[0012] S5 Freeze-Drying Treatment: Place the meniscus after directional freezing in S4 into a freeze dryer for freeze-drying until all the ice columns have sublimated.

[0013] S6 Annealing Treatment: Perform annealing treatment on the meniscus after freeze-drying in S5 to improve the crystallinity of the PVA, and then soak the annealed meniscus in deionized water until it reaches a saturated state to obtain the desired fatigue-resistant meniscus.

[0014] Preferably, the temperature of freeze-drying in S5 is -54°C, the time of freeze-drying is 40 hours, the temperature of annealing treatment in S6 is 90°C to 110°C, and the time of annealing treatment is 90 minutes.

[0015] Preferably, the meniscus fiber framework in S1 is prepared by the method of manual fiber weaving.

[0016] Preferably, the method of manual fiber weaving includes the following:

[0017] a. Obtain the meniscus image of the patient in S1 by means of computed tomography or magnetic resonance imaging.

[0018] b. Use a computer to draw a three-dimensional model of the meniscus according to the meniscus image in a.

[0019] c. Horizontally and longitudinally segment the three-dimensional model in b to determine the interval between the horizontal layer and the vertical layer, and finally obtain the data of the weaving structure of each horizontal and vertical layer.

[0020] d. According to the data of the weaving structure of each horizontal and vertical layer obtained in c, use a biofiber material to perform layer-by-layer weaving to obtain the meniscus fiber framework.

[0021] Preferably, the meniscus fiber framework in S1 is prepared by the method of 3D printing.

[0022] Preferably, the 3D printing method includes the following:

[0023] a1. Obtain the meniscus image of the patient in S1 by means of computed tomography or magnetic resonance imaging;

[0024] b1. Use a computer to draw a three-dimensional model of the meniscus based on the meniscus image in a1;

[0025] c1. Design a 3D printing frame model for the three-dimensional model obtained in b1 through computer software;

[0026] d1. Slice the 3D printing frame model obtained in c1 into G-code format through computer software;

[0027] e1: Print into a meniscus fiber frame using a biofiber material impregnated with a bioresin through a 3D printer;

[0028] Preferably, during the weaving in e and the printing in e1, a section of fiber for fixing the meniscus to the bone is led out from each of the two corners of the meniscus fiber frame;

[0029] Preferably, customize the forming mold in S2 by means of CNC machining;

[0030] Preferably, the material of the forming mold is plastic;

[0031] Preferably, the heat-conducting component in S4 is a copper blank and the freezing source is liquid nitrogen.

[0032] The present invention has achieved the following technical effects compared with the prior art:

[0033] 1. In the meniscus preparation method of the present invention, first, PVA hydrogel is prepared by the method of directional freezing. As water freezes, PVA molecules are expelled from the ice columns, and they are concentrated in the microchannels between the ice columns. At this time, the molecular chain movement weakens, the contact time between the chains becomes longer, and the distance between the chains shortens, which is conducive to the formation of physical network cross-linking points between the hydroxyl groups on the molecular chains. Finally, a PVA hydrogel with high toughness and complete extrudability can be obtained; the freeze-drying step removes the ice columns in the PVA hydrogel; the annealing treatment improves the crystallinity of PVA. The PVA crystal regions serve as physical cross-linking points, enhancing the mechanical properties of the meniscus. The PVA crystal regions are formed during the directional freezing process, arranged along the growth direction of the ice columns, and increase and improve during the annealing process, finally forming an anisotropic hydrogel, improving the fatigue resistance of the hydrogel;

[0034] 2. By arranging a meniscus fiber frame inside the hydrogel, the present invention mimics the distribution of natural meniscus collagen fibers in the radial and circumferential directions, significantly enhancing the mechanical properties of the meniscus, especially the tensile properties;

[0035] 3. The alternative meniscus prepared by the method of the present invention has fibers led out on both sides, which is convenient for firmly fixing the meniscus on adjacent bones, and solves the problem that it is difficult to fix the conventional alternative meniscus.

[0036] 4. Based on the situation of each patient, the present invention obtains the meniscus image of the patient by means of computed tomography (CT) or magnetic resonance imaging (MRI), improves the adaptability of the manufactured meniscus to the patient, and can design different fiber densities and hydrogel strengths according to personal needs such as different weights and different exercise intensities during the manufacturing process, realizing the personalized customization of artificial meniscus. Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0038] Figure 1 It is a perspective structural schematic diagram of a hand-woven meniscus fiber framework;

[0039] Figure 2 It is a front view structural schematic diagram of a hand-woven meniscus fiber framework;

[0040] Figure 3 It is a structural schematic diagram of a hand-woven - PVA hydrogel meniscus;

[0041] Figure 4 It is a perspective structural schematic diagram of a 3D-printed meniscus fiber framework;

[0042] Figure 5 It is a front view structural schematic diagram of a 3D-printed meniscus fiber framework;

[0043] Figure 6 It is a structural schematic diagram of a 3D-printed - PVA hydrogel meniscus;

[0044] Figure 7 It is a schematic diagram of a directional freezing experiment and an internal structural schematic diagram of the frozen PVA solution;

[0045] Figure 8 It is an internal structural schematic diagram of the PVA hydrogel after freeze-drying;

[0046] Figure 9 It is an internal structural schematic diagram of the PVA hydrogel after annealing;

[0047] Figure 10SEM image of the PVA hydrogel prepared by the directional freezing method after freeze-drying (perpendicular to the ice column growth direction);

[0048] Figure 11 SEM image of the PVA hydrogel prepared by the directional freezing method after freeze-drying (parallel to the ice column growth direction);

[0049] Figure 12 Cyclic compression test results of the PVA hydrogel prepared by the directional freezing method at 0 - 50%;

[0050] Figure 13 Cyclic compression test results of the fiber - woven - PVA hydrogel prepared by the directional freezing method at 0 - 50%;

[0051] Figure 14 Cyclic compression test results of the 3D fiber - printed - PVA hydrogel prepared by the directional freezing method at 0 - 50%.

[0052] Among them, 1, meniscus fiber framework; 2, molding die; 3, hydrogel; 4, heat - conducting component; 5, freezing source; 6, ice column; 7, PVA chain; 8, PVA crystal region; 9, hydrogen bond; 10, amorphous PVA chain. Detailed implementation manners

[0053] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0054] To make the above - mentioned objects, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners.

[0055] As Figures 1 to 14 shown, the present invention provides a preparation method for a long - term alternative anti - fatigue meniscus, including the following:[[]]

[0056] S1 Prepare the meniscus fiber framework 1: Make the meniscus fiber framework 1 according to the meniscus image of the patient;

[0057] Prepare the meniscus fiber framework 1 by means of manual weaving or 3D printing.

[0058] S2 Prepare the molding die 2: Make the molding die 2 for pouring the PVA solution according to the meniscus image of the patient. The cavity size of the molding die 2 is not less than the size of the meniscus fiber framework 1;

[0059] In order to enable the meniscus fiber framework 1 prepared in S1 to be placed into the molding die 2, the cavity of the molding die 2 has the same shape as the meniscus fiber framework 1, and the cavity of the molding die 2 is not smaller than the size of the meniscus fiber framework 1.

[0060] S3 Pour the PVA solution: Place the meniscus fiber framework 1 in S1 into the molding die 2 in S2, and inject the PVA solution so that the PVA solution fills and wraps the meniscus fiber framework 1;

[0061] S4 Directional freezing treatment: Place the molding die 2 in S3 on top of the heat-conducting component 4, and immerse the bottom of the heat-conducting component 4 in the freezing source 5 so that the water in the PVA solution freezes to form ice columns 6, and the PVA chains 7 are concentrated between the ice columns 6;

[0062] As a preferred embodiment of the present invention, the heat-conducting component 4 is a copper blank, and the freezing source 5 is liquid nitrogen. Immerse half of the copper blank into the liquid nitrogen. At this time, the low temperature of the liquid nitrogen conducts upward along the copper blank, and a temperature gradient from bottom to top is formed in the molding die 2, so that the water in the lowest layer is frozen first, and then gradually upward, forming several parallel upward ice columns 6, and the PVA chains 7 are concentrated between the ice columns 6; The advantages of this temperature transfer method are: Since the ice grows perpendicular to the upper surface of the copper blank, several mutually parallel ice columns 6 are generated along the height direction of the molding die 2. Under the restriction of the ice columns 6, the PVA chains 7 are arranged in an oriented manner; Secondly, the oriented PVA chains 7 are between the ice columns 6, which increases the concentration of the PVA chains 7 in the region where the PVA chains 7 are located. Finally, a PVA hydrogel with high toughness and complete extrudability can be obtained.

[0063] S5 Freeze-drying treatment: Place the frozen meniscus in S4 in a freeze-dryer for freeze-drying until all the ice columns 6 are sublimated;

[0064] As a preferred embodiment of the present invention, the meniscus sample is placed in a freeze-dryer at -54 °C for 40 hours for freeze-drying. After freezing, both ice columns 6 and PVA chains 7 exist in the meniscus sample. In order to anneal the meniscus sample, it is necessary to first freeze-dry the frozen meniscus sample so that the ice columns 6 can be sublimated. Freeze-drying can maintain the shape of the meniscus sample and prevent it from shrinking and collapsing.

[0065] S6 Annealing treatment: Anneal the freeze-dried meniscus in S5 to increase the crystallinity of PVA, and then soak the annealed meniscus in deionized water until it reaches a saturated state to obtain the required anti-fatigue meniscus. The anti-fatigue meniscus has a meniscus fiber framework 1 inside and is wrapped with a hydrogel 3 outside.

[0066] As a preferred embodiment of the present invention, the annealing temperature is 90°C to 110°C, and the annealing time is 90 minutes. During the annealing process, the PVA chains 7 are in a relaxed state, and PVA crystal regions 8 can be formed or perfected between the relaxed PVA chains 7 as physical cross-linking points. As the number of cross-linking points increases, the mechanical properties of the hydrogel 3 are continuously improved; before the annealing process, all the ice columns 6 in the meniscus sample have sublimated. Figures 7 to 9 Respectively show the distribution states of hydrogen bonds 9, amorphous PVA chains 10, and PVA crystal regions 8 in the hydrogel after directional freezing, freeze-drying, and annealing.

[0067] As a preferred embodiment of the present invention, the method for hand-weaving a meniscus with fibers includes the following:

[0068] a. Obtain the meniscus image of the patient in S1 by means of computed tomography or magnetic resonance imaging;

[0069] b. Use a computer to draw a three-dimensional model of the meniscus based on the meniscus image in a;

[0070] c. Horizontally and longitudinally segment the three-dimensional model in b to determine the interval between the horizontal and vertical layers, and finally obtain the data of the weaving structure of each horizontal and vertical layer;

[0071] d. According to the data of the weaving structure of each horizontal and vertical layer obtained in d, use a biofiber material to weave layer by layer to obtain the meniscus fiber framework 1.

[0072] The method for fabricating a meniscus by 3D printing includes the following:

[0073] a1. Obtain the meniscus image of the patient in S1 by means of computed tomography or magnetic resonance imaging;

[0074] b1. Use a computer to draw a three-dimensional model of the meniscus based on the meniscus image in a1;

[0075] c1. Design a 3D printing frame model for the three-dimensional model obtained in b1 through computer software;

[0076] d1. Slice the 3D printing frame model obtained in c1 into G-code format through computer software;

[0077] e1: Use a biofiber material impregnated with a bioresin to print into the meniscus fiber framework 1 through a 3D printer.

[0078] Among them, the biofiber material can adopt the existing technology, and the present invention does not make improvements thereto. The purpose of impregnating the bio-resin outside the biofiber material during 3D printing is to improve the adhesiveness of the biofiber material, so that the meniscus fiber framework 1 formed by 3D printing becomes an integral structure formed by the mutual adhesion of each biofiber. Secondly, whether the meniscus is made by fiber hand-weaving or 3D printing, a section of fiber for fixing the meniscus to the bone needs to be led out from each of the two corners of the meniscus fiber framework 1.

[0079] As a preferred embodiment of the present invention, when preparing the molding die 2, first, the meniscus image of the patient is obtained in the manner of a / a1; secondly, the computer is used to design the molding die 2 by using the meniscus image of the patient to obtain the three-dimensional data of the molding die 2; then, the molding die 2 is made by CNC machining according to this three-dimensional data. The material of the molding die 2 is selected as a plastic sheet with a suitable thickness, preferably a transparent acrylic sheet, which is convenient to directly observe the change of the substance in the mold cavity through the side wall of the mold.

[0080] The fatigue test results are as Figures 12 to 14 shown, Figure 12 are the cyclic compression test results of the hydrogel 3 at 0-50%. After 10,000 compression cycles, the stress drops from 9.8 MPa to 8.7 MPa, with a loss exceeding 11.2%; Figure 13 are the cyclic compression test results of the fiber braided-hydrogel 3 at 0-50%. After 10,000 compression cycles, the stress drops from 11.4 MPa to 10.7 MPa, with a loss of 6.1%; Figure 14 are the cyclic compression test results of the 3D fiber printed-hydrogel 3 at 0-50%. After 10,000 compression cycles, the stress drops from 13.3 MPa to 12.4 MPa, with a loss of 6.8%. After 10,000 cyclic compressions, the performance of the sample can still be maintained well, indicating that this artificial meniscus has excellent anti-fatigue performance. In addition, the addition of fibers not only improves the mechanical properties of the hydrogel. Among them, the compression stress (at 50%) of the fiber braided-hydrogel 3 increases from 9.8 MPa to 11.4 MPa, and the compression stress (at 50%) of the 3D fiber printed-hydrogel 3 increases from 9.8 MPa to 13.3 MPa, increasing by 16.3% and 35.7% respectively; moreover, during transplantation, the fibers led out from the edge of the meniscus can conveniently fix the meniscus to the surrounding bones, solving the problem that it is difficult to fix the meniscus in the past.

[0081] It should be noted that for those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention, and any reference signs in the claims should not be regarded as limiting the claims involved.

Claims

1. A preparation method of a long-term alternative anti-fatigue meniscus, characterized in that: It includes the following steps: S1 Prepare the meniscus fiber framework: Make the meniscus fiber framework according to the meniscus image of the patient; wherein, at each of the two corners of the meniscus fiber framework, a section of fiber for fixing the meniscus to the bone is led out; and in the horizontal direction, the meniscus fiber framework is formed by the intersection of mutually staggered fibers. S2 Prepare the molding die: Make a molding die for pouring the PVA solution according to the meniscus image of the patient, and the cavity size of the molding die is not less than the size of the meniscus fiber framework. S3 Pour the PVA solution: Place the meniscus fiber framework in S1 into the molding die in S2, and inject the PVA solution so that the PVA solution fills and wraps the meniscus fiber framework. S4 Directional freezing treatment: Place the molding die in S3 on the top of the heat-conducting component, and the bottom of the heat-conducting component is immersed in the freezing source, so that the water in the PVA solution freezes to form ice columns, the PVA is concentrated between the ice columns, and a number of mutually parallel ice columns are generated along the height direction of the molding die. S5 Freeze-drying treatment: Place the meniscus after directional freezing in S4 in a freeze-dryer for freeze-drying until all the ice columns sublimate. S6 Annealing treatment: Perform annealing treatment on the meniscus after freeze-drying in S5 to improve the crystallinity of the PVA, and then soak the meniscus after annealing treatment in deionized water until it reaches the saturation state to obtain the required anti-fatigue meniscus.

2. The preparation method according to claim 1, wherein: The temperature of freeze-drying in S5 is -54°C, and the time of freeze-drying is 40 hours. The temperature of annealing treatment in S6 is 90°C to 110°C, and the time of annealing treatment is 90 minutes.

3. The preparation method according to claim 1, characterized in that: The meniscus fiber framework in S1 is prepared by the method of manual fiber weaving.

4. The preparation method according to claim 3, characterized in that: The method of manual fiber weaving includes the following steps: a. Obtain the meniscus image of the patient in S1 by means of computed tomography or magnetic resonance imaging. b. Use a computer to draw a three-dimensional model of the meniscus according to the meniscus image in a. c. Horizontally and longitudinally segment the three-dimensional model in b to determine the interval between the horizontal layer and the longitudinal layer, and finally obtain the data of the weaving structure of each horizontal and longitudinal layer. d. According to the data of the weaving structure of each horizontal and longitudinal layer obtained in c, use biofiber materials to weave layer by layer to obtain the meniscus fiber framework.

5. The preparation method according to claim 1, characterized in that: The meniscus fiber framework in S1 is prepared by the method of 3D printing.

6. The preparation method according to claim 5, characterized in that: The method of 3D printing includes the following steps: a1. Obtain the meniscus image of the patient in S1 by means of computed tomography or magnetic resonance imaging. b1. Use a computer to draw a three-dimensional model of the meniscus according to the meniscus image in a1. c1. Design a 3D printing framework model for the three-dimensional model obtained in b1 through computer software. d1. Slice the 3D printing framework model obtained in c1 into the G-code format through computer software. e1: Use a biofiber material impregnated with a bioresin to print into a meniscus fiber framework through a 3D printer.

7. The preparation method according to claim 1, characterized in that: The molding die in S2 is customized by means of CNC machining.

8. The preparation method according to claim 7, characterized in that: The material of the molding die is plastic.

9. The preparation method according to claim 1, wherein: In S4, the heat-conducting component is a copper blank and the refrigerating source is liquid nitrogen.

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