Cartilage repair scaffold and preparation method thereof

Abstract

The invention relates to the technical field of cartilage repair, and particularly discloses a cartilage repair stent and a preparation method thereof. The scaffold comprises a bone-facing surface part and a back bone surface part, wherein the bone-facing surface is a porous three-dimensional network structure formed by decellularizing bovine pericardium, porcine pericardium or horse pericardium, and I-type and III-type collagen is reserved; the back bone surface is a soft membrane layer made of cow leather, pigskin or horse skin and provides mechanical support. The preparation method comprises the following steps: selectively removing type II collagen through enzymolysis, enhancing the stability of the collagen through chemical treatment, and combining the two-phase structure through a freeze-drying process. After the stent is implanted, the regeneration of the autologous II-type collagen can be promoted in combination with a micro-fracture surgery, the mechanical property close to that of natural cartilage is formed, the problems of insufficient strength of fibrous cartilage, donor limitation, secondary injury and the like in traditional repair are effectively solved, and the cartilage repair effect and the life quality of a patient are remarkably improved.

Description

Technical Field

[0001] The present invention relates to cartilage repair technology, and more specifically, it relates to a cartilage repair scaffold and a preparation method thereof. Background Art

[0002] Affected by the increasing aging population and the increasing number of sports injuries, the number of patients with cartilage injuries has been growing year by year. Joint diseases (such as osteoarthritis) and sports traumas are the main inducements, especially highly prevalent among sports enthusiasts and the young and middle-aged groups. If cartilage injuries are not intervened in a timely manner, they are likely to trigger joint degenerative diseases, resulting in chronic pain and functional disorders, significantly reducing the quality of life of patients.

[0003] Currently, the treatment methods for cartilage injuries are mainly divided into two categories: conservative treatment and surgical treatment. Conservative treatment includes physical therapy, drug therapy, and injection therapy (such as hyaluronic acid, platelet-rich plasma, etc.). However, these methods usually only relieve symptoms and cannot achieve substantial repair of cartilage. Surgical treatment includes microfracture surgery, cartilage transplantation, etc. Although these methods have made certain progress in promoting cartilage repair, there are still many limitations. For example, microfracture surgery can relieve pain in the short term, but the repaired tissue is fibrocartilage with insufficient mechanical strength and is prone to recurrence. Autologous cartilage transplantation has problems such as limited repair effect, insufficient donor sources, and secondary injuries. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a novel cartilage scaffold and a preparation method thereof for use in the repair of cartilage injuries and to provide a better choice for the treatment of cartilage defects.

[0005] To achieve the above purpose, the present invention provides the following technical solutions: A cartilage repair scaffold includes a bone-facing part and a bone-backing part. The bone-facing part is formed by processing bovine pericardium, porcine pericardium, or equine pericardium into a three-dimensional network structure and has type I collagen and type III collagen. The bone-backing part is formed by processing bovine skin, porcine skin, or equine skin into a soft membrane layer structure. The bone-facing part and the bone-backing part are combined together by a freeze-drying process.

[0006] Further, the three-dimensional network structure has pores with a pore diameter of 100 - 200 microns.

[0007] Further, for preparing the bone-facing part, the steps of processing bovine pericardium, porcine pericardium, or equine pericardium are as follows: First, remove fat and connective tissues and rinse surface impurities with physiological saline; Immerse in an antibiotic solution or low-concentration hydrogen peroxide to kill microorganisms; Use a 0.1% - 1% sodium dodecyl sulfate solution for shaking treatment to dissolve cell membranes and remove cell debris; Treat with 0.5 - 1% glutaraldehyde to enhance the stability of collagen fibers.

[0008] Use type II collagen - specific enzyme to degrade type II collagen at pH 7 - pH 8 and 37 ± 5°C, retaining type I / III collagen.

[0009] Furthermore, the steps for preparing the dorsal bone surface part and treating cowhide, pigskin or horsehide are as follows: Remove fat and hair, and repeatedly rinse with saline to remove blood stains and impurities; Soak in a chloroform - methanol mixture for 24 h to remove lipid components; Use a 0.1% - 1% sodium dodecyl sulfate solution for shaking treatment to dissolve cell membranes and remove cell debris; Treat with 0.5 - 1% glutaraldehyde to enhance the stability of collagen fibers.

[0010] Treat with peracetic acid (0.1% - 0.5%) or β - propiolactone to destroy the virus envelope and nucleic acid.

[0011] Furthermore, the bone - facing part and the dorsal bone - facing part are combined together through a freeze - drying process to form a biphasic cartilage repair scaffold.

[0012] Furthermore, the volume ratio of the chloroform - methanol mixture is 2:1.

[0013] By adopting the above - mentioned technical solutions, the beneficial effects of the present invention are as follows: Biphasic synergistic repair: The porous structure (pore size 100 - 200 μm) of the bone - facing part of the scaffold provides a three - dimensional growth space for type II collagen regeneration, and the soft membrane layer on the dorsal bone surface enhances mechanical support, avoiding the damage to the repair layer caused by postoperative activities, and realizing dual repair of structure and function.

[0014] Autologous regeneration induction: Activate local blood supply through micro - fracture surgery, and guide the autologous type II collagen of the patient to regenerate in the pores of the scaffold, forming transparent cartilage similar to natural cartilage, and avoiding the mechanical defects and recurrence risks of fibrous cartilage.

[0015] High biocompatibility: Adopt natural pericardium and animal skin materials, and after decellularization, virus inactivation and other treatments, retain the natural biological activity of collagen, and reduce the immune rejection reaction.

[0016] Controllable preparation process: Selectively remove type II collagen through specific enzymatic hydrolysis (pH 7 - 8, 37 ± 5°C), and combine glutaraldehyde cross - linking to enhance stability, ensuring the structural integrity of the scaffold after long - term implantation.

[0017] Wide clinical application: Can replace traditional autologous transplantation, solve the problem of insufficient donors, and is applicable to various cartilage defect repair scenarios such as osteoarthritis and sports injuries. Brief Description of the Drawings

[0018] Figure 1 This is a schematic structural view of the cartilage repair scaffold according to an embodiment of the present invention. Detailed Description of the Embodiments

[0019] 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. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0021] The cartilage repair scaffold includes a bone-facing part 1 and a bone-backing part 2. The bone-facing part 1 is formed by processing bovine pericardium, porcine pericardium or equine pericardium into a three-dimensional network structure, retaining type I collagen and type III collagen, and removing type II collagen; the bone-backing part 2 is formed by processing cowhide, pigskin or horsehide into a soft film layer structure; the bone-facing part and the bone-backing part are combined together through a freeze-drying process.

[0022] Embodiment 1:

[0023] Preparation of the bone-facing surface Take fresh bovine pericardium, manually remove fat and connective tissue, and rinse with physiological saline until there is no impurity. Immerse it in a 0.3% hydrogen peroxide solution containing antibiotics for 6 hours for sterilization treatment. Place it in a 0.5% sodium dodecyl sulfate solution and shake for 12 hours to remove cell debris. Crosslink it with a 0.8% glutaraldehyde solution for 4 hours to enhance the stability of collagen fibers. Under the conditions of pH 7.5 and 37 °C, add a type II collagen-specific enzyme and react for 24 hours to degrade type II collagen and retain type I / III collagen, forming a porous network structure with a pore size of 150 μm.

[0024] Preparation of the bone-backing surface Take cowhide, manually remove the hair and subcutaneous fat, and rinse with physiological saline until there is no blood or dirt. Soak it in a chloroform-methanol mixture (volume ratio 2:1) for 24 hours to completely degrease. Treat it with 0.5% sodium dodecyl sulfate by shaking for 8 hours to remove residual cell components. Crosslink with 0.7% glutaraldehyde for 6 hours, and then soak it in 0.3% peracetic acid for 2 hours to inactivate the virus.

[0025] Scaffold assembly Overlay the bone-facing surface and the back-bone surface, place them in a freeze dryer, pre-freeze at -40°C for 4 hours, and vacuum dry for 24 hours to form a biphasic composite scaffold.

[0026] Example 2:

[0027] Preparation of the bone-facing surface Take fresh pig pericardium, manually remove the fat and connective tissue, and rinse with physiological saline until there are no impurities. Soak it in 0.2% hydrogen peroxide solution containing antibiotics for 8 hours for sterilization treatment. Place it in 0.6% sodium dodecyl sulfate solution and shake for 10 hours to remove cell debris. Crosslink with 0.9% glutaraldehyde solution for 3 hours to enhance the stability of collagen fibers. Under the conditions of pH 7.2 and 36°C, add type II collagen-specific enzyme and react for 20 hours to degrade type II collagen and retain type I / III collagen to form a porous network structure with a pore size of 120μm.

[0028] Preparation of the back-bone surface Take pigskin, manually remove the hair and subcutaneous fat, and rinse with physiological saline until there is no blood or dirt. Soak it in a chloroform-methanol mixture (volume ratio 2:1) for 20 hours to completely degrease. Treat it with 0.6% sodium dodecyl sulfate by shaking for 6 hours to remove residual cell components. Crosslink with 0.8% glutaraldehyde for 5 hours, and then soak it in 0.2% peracetic acid for 3 hours to inactivate the virus.

[0029] Scaffold assembly Overlay the bone-facing surface and the back-bone surface, place them in a freeze dryer, pre-freeze at -45°C for 3 hours, and vacuum dry for 20 hours to form a biphasic composite scaffold.

[0030] Example 3:

[0031] Preparation of the bone-facing surface Take fresh horse pericardium, manually remove the fat and connective tissue, and rinse with physiological saline until there are no impurities. Soak it in 0.4% hydrogen peroxide solution containing antibiotics for 5 hours for sterilization treatment. Place it in 0.8% sodium dodecyl sulfate solution and shake for 14 hours to remove cell debris. Crosslink with 1.0% glutaraldehyde solution for 5 hours to enhance the stability of collagen fibers. Under the conditions of pH 7.8 and 38°C, add type II collagen-specific enzyme and react for 28 hours to degrade type II collagen and retain type I / III collagen to form a porous network structure with a pore size of 180μm.

[0032] Preparation of the dorsal bone surface Take horse skin, manually remove the hair and subcutaneous fat, and rinse with physiological saline until there is no blood and dirt. Immerse it in a chloroform-methanol mixture (volume ratio 2:1) for 28 hours to completely degrease. Treat it with 0.8% sodium dodecyl sulfate by shaking for 10 hours to remove residual cell components. Crosslink with 1.0% glutaraldehyde for 7 hours, and then soak it in 0.4% β-propiolactone solution for 1.5 hours to inactivate the virus.

[0033] Assembly of the scaffold Overlay the bone-facing side and the dorsal bone surface, place it in a freeze dryer, pre-freeze at -35°C for 5 hours, and vacuum dry for 28 hours to form a biphasic composite scaffold.

[0034] Clinical application Perform microfracture surgery on the cartilage damage area of the patient to form micropores with a diameter of 2 mm. Trim the scaffold to match the defect size, suture and fix it after implantation. Since the pericardium removes type II collagen, the pericardium will present a porous three-dimensional network structure. Due to the microfracture surgery, the body's self-regulatory ability will concentrate blood supply to the damaged area, form its own type II collagen, and the generated type II collagen can attach to the three-dimensional network structure, combine with type I collagen and type II collagen, thereby achieving cartilage regeneration.

[0035] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any ordinary changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention should be included in the protection scope of the present invention.

Claims

1. A cartilage repair scaffold, characterized in that Comprising The bone-facing part, which is made of bovine pericardium, porcine pericardium or equine pericardium after treatment to form a three-dimensional network structure and has type I collagen and type III collagen; The back bone-facing part, which is made of bovine skin, porcine skin or equine skin after treatment to form a soft membrane layer structure; The bone-facing part and the back bone-facing part are combined together.

2. The cartilage repair scaffold according to claim 1, wherein, The three-dimensional network structure has pores with a pore diameter of 100-200 microns.

3. The preparation method of a cartilage repair scaffold according to claim 1, characterized in that, Comprising the preparation of the bone-facing part, and the steps of treating bovine pericardium, porcine pericardium or equine pericardium are: First, remove fat and connective tissue, and rinse the surface impurities with physiological saline; Soak in an antibiotic solution or low-concentration hydrogen peroxide to kill microorganisms; Use a 0.1% - 1% sodium dodecyl sulfate solution for shaking treatment to dissolve cell membranes and remove cell debris; Use 0.5 - 1% glutaraldehyde for treatment to enhance the stability of collagen fibers; Use type II collagen-specific enzyme to degrade type II collagen at pH 7 - pH 8 and 37 ± 5°C, and retain type I / III collagen.

4. A method for preparing a cartilage repair scaffold according to claim 1 or 3, characterized in that, Comprising the preparation of the back bone-facing part, and the steps of treating bovine skin, porcine skin or equine skin are: Remove fat and hair, and repeatedly rinse with physiological saline to remove blood stains and impurities; Soak in a chloroform-methanol mixture for 24 h to remove lipid components; Use a 0.1% - 1% sodium dodecyl sulfate solution for shaking treatment to dissolve cell membranes and remove cell debris; Use 0.5 - 1% glutaraldehyde for treatment to enhance the stability of collagen fibers; Treat with peracetic acid (0.1% - 0.5%) or β-propiolactone to destroy the virus envelope and nucleic acid.

5. A method for preparing a cartilage repair scaffold according to claim 4, characterized in that, The bone-facing part and the back bone-facing part are combined together through a freeze-drying process to form a biphasic cartilage repair scaffold.

6. The preparation method of a cartilage repair scaffold according to claim 4, characterized in that The volume ratio of the chloroform-methanol mixture is 2:1.

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