CE-SKP / CPH / P2G3 three-layer composite scaffold, preparation method and application

Abstract

The invention belongs to the technical field of biological composite materials, and particularly relates to a CE-SKP / CPH / P2G3 three-layer composite scaffold, a preparation method and application. The CE-SKP / CPH / P2G3 three-layer composite scaffold is characterized in that CPH hydrogel is placed under a CE-SKP composite scaffold system, a P2G3 nanofiber membrane is placed under the CPH hydrogel, a calcified cartilage layer of a physiological structure is simulated, relative independence of a cartilage repair microenvironment is kept, and mineralization of newborn cartilage cells is induced, so that the calcified cartilage layer of the physiological structure is formed; wherein the CE-SKP composite scaffold system is a seed cell-free composite scaffold system which is constructed by adsorbing self-assembled polypeptide nanofiber hydrogel Ac-(RADA) 4-CONH2 / Ac-(RADA) 4GGSKPPGTSS-CONH2 (RAD / SKP) to a natural cartilage extracellular matrix (ECM), and the composite scaffold system is a seed cell-free composite scaffold system which is constructed by adsorbing the self-assembled polypeptide nanofiber hydrogel Ac-(RADA) 4-CONH2 / Ac-(RADA) 4GGSKPPGTSS-CONH2 (RAD / SKP) to the natural cartilage extracellular matrix (ECM). The CE-SKP / CPH / P2G3 three-layer composite scaffold utilizes the three-layer composite structure to solve the technical problems that in the prior art, a cartilage layer and a calcified cartilage layer in a physiological structure cannot be well simulated, and independence of a repairing microenvironment cannot be kept, joint repairing of the cartilage layer and the calcified cartilage layer is achieved, and a better articular cartilage full-thickness defect repairing effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocomposite materials, and in particular relates to a CE-SKP / CPH / P2G3 three-layer composite stent, a preparation method and an application thereof. Background Art

[0002] Articular cartilage is an important component of joints. Due to the lack of blood vessels and nerves, damaged articular cartilage usually does not heal on its own. If isolated articular cartilage defects are not treated in time, osteoarthritis will be induced as the disease progresses. Musculoskeletal diseases, including OA, are the most disabling diseases in the world, and the related medical expenses are huge.

[0003] At present, the treatment of focal cartilage damage and early OA mainly focuses on relieving pain, improving joint movement function and preventing disease progression, while the treatment of late OA is mainly joint replacement, but its cost is very expensive. Early intervention of focal cartilage defects and OA is of great strategic significance, which can reduce medical costs while improving the quality of life of patients. In recent years, the strategy of three-dimensional bionic repair of cartilage damage combined with tissue engineering technology has received widespread attention. How to achieve single-stage surgery to complete the repair of articular cartilage damage, thereby avoiding secondary surgical processes such as cell acquisition, expansion culture and re-implantation, reducing the burden and risk of patients, and reducing medical costs, has become a new research goal.

[0004] The biocomposite material formed by articular cartilage, calcified cartilage and subchondral bone, called the osteochondral unit, has the unique ability to transmit loads during weight-bearing and joint movement. Changes in the composition or structure of any single component of this unit will lead to the destruction of joint integrity and loss of function. An important goal of tissue engineering strategies to repair articular cartilage is to restore the normal layered physiological structure of articular cartilage as much as possible, including articular cartilage as well as calcified cartilage. Therefore, designing a bionic cartilage scaffold containing a calcified cartilage layer for repairing cartilage defects is a technical problem that needs to be solved urgently in this field.

[0005] Articular cartilage lacks blood vessels and nerves, chondrocytes exist in a low oxygen tension environment, and compared with other cell types, chondrocytes have very low oxygen consumption. The surface of cartilage has only 10% of normal arterial oxygen tension, while the deep cartilage is less than 1%. In the late stage of OA, microcracks appear at the osteochondral junction and vascular components penetrate the calcified cartilage. The newly formed vascular channels that penetrate the tide line are accompanied by sensory nerves and sympathetic nerves, which may cause joint pain in the late stage of OA. Therefore, keeping the cartilage repair microenvironment independent may provide a more suitable microenvironment for cartilage growth and development, which helps to better regenerate cartilage.

[0006] An important goal of repairing articular cartilage with tissue engineering strategies is to restore the normal physiological structure of articular cartilage as much as possible. Therefore, designing a bionic cartilage scaffold containing a calcified cartilage layer and ensuring an independent cartilage microenvironment for repairing cartilage defects is a technical problem that needs to be solved in this field. Summary of the invention

[0007] In order to solve the above problems existing in the prior art, the present invention provides a CE-SKP / CPH / P2G3 three-layer composite stent, a preparation method and an application thereof.

[0008] The technical solution adopted by the present invention to solve this problem is:

[0009] A CE-SKP / CPH / P2G3 three-layer composite scaffold, which is a CE-SKP composite scaffold system in which a CPH hydrogel is placed under the CE-SKP composite scaffold system, and a P2G3 nanofiber membrane is placed under the CPH hydrogel, so as to simulate a calcified cartilage layer of a physiological structure and maintain the relative independence of the cartilage repair microenvironment, and then induce the mineralization of new chondrocytes to form a calcified cartilage layer of a physiological structure;

[0010] Among them: CE-SKP composite scaffold system is composed of self-assembled polypeptide nanofiber hydrogel

[0011] Ac-(RADA) 4 -CONH 2 / Ac-(RADA) 4 GGSKPPGTSS-CONH 2 A seed-free cell-free composite scaffold system constructed by adsorbing (RAD / SKP) into the natural cartilage extracellular matrix (ECM).

[0012] In the above technical scheme, CPH hydrogel, which is chitosan (CS) / polyγ-glutamic acid (γ-PGA) / hydroxyapatite (HA) hydrogel, can promote the mineralization of mesenchymal stem cells (MSCs) both in vivo and in vitro.

[0013] In the above technical solution, the P2G3 nanofiber membrane, which is a polycaprolactone (PCL) / gelatin (GEL) nanofiber membrane, has the function of blocking cell migration and ensuring the relative independence of the microenvironment.

[0014] In the above technical solution, the CE-SKP composite scaffold system is used to simulate the cartilage layer of the physiological structure, wherein: the self-assembling polypeptide nanofiber hydrogel Ac-(RADA) 4 -CONH 2 / Ac-(RADA) 4 GGSKPPGTSS-CONH 2 (RAD / SKP) is used to simulate the structure of bone marrow homing peptides and recruit endogenous repair cells in situ. The natural chondrocyte extracellular matrix (ECM) is used to induce the chondrogenic differentiation of the recruited endogenous repair cells.

[0015] The second object of the present invention is to provide an application of a CE-SKP / CPH / P2G3 three-layer composite scaffold in the preparation of a repair material for cartilage defects. The CE-SKP / CPH / P2G3 three-layer composite scaffold is a bionic cartilage scaffold containing a calcified cartilage layer and ensuring the independence of the cartilage microenvironment for repairing cartilage defects.

[0016] The third object of the present invention is to provide an application of a CE-SKP / CPH / P2G3 three-layer composite scaffold in the preparation of a repair material for full-thickness defects of articular cartilage. The CE-SKP / CPH / P2G3 three-layer composite scaffold is applied to the damaged site of articular cartilage. By using its three-layer composite structure to simulate the physiological structures of articular cartilage and the calcified cartilage layer and maintaining a relatively independent repair microenvironment, it recruits repair cells in situ, induces chondrogenic differentiation and sequential hypertrophic calcification, so as to achieve the common repair of the cartilage layer and the calcified cartilage layer.

[0017] The fourth object of the present invention is to provide a preparation method of a CE-SKP / CPH bilayer composite scaffold, which is characterized by including the following steps:

[0018] S1. Prepare a functionalized RAD / SKP mixed polypeptide solution;

[0019] S2. Prepare a decellularized cartilage matrix (DCM) scaffold;

[0020] S3. Prepare a CE-SKP composite scaffold;

[0021] S4. Prepare a CPH hydrogel;

[0022] S5. Prepare P2G3 nanofibers;

[0023] S6. Arrange the CE-SKP composite scaffold, the CPH hydrogel, and the P2G3 nanofibers in sequence from top to bottom to synthesize a CE-SKP / CPH / P2G3 three-layer composite scaffold system.

[0024] In the above technical solution, in step S1, the preparation of the functionalized RAD / SKP mixed polypeptide solution includes the following steps:

[0025] S101. Functionalize the self-assembling peptide RADA16-I by directly extending the C-terminus with the functional motif SKP (SKPPGTSS) that mimics the bioactivity of bone marrow homing polypeptides to form the designed functionalized Ac-(RADA) 4 -CONH 2 / Ac-(RADA) 4 GGSKPPGTSS-CONH 2 (RAD / SKP) polypeptide;

[0026] S102. Dissolve 10 mg of RAD / SKP polypeptide powder in 1 ml of distilled water and mix well to obtain a homogeneous polypeptide solution of 1% (w / v, pH 3 - 4). After sterilization with a filter, store the polypeptide solution at 4 °C until the next use;

[0027] S103. Mix the functionalized RAD / SKP polypeptide with a 1% RADA16-I solution at a volume ratio of 1:1 to obtain a functionalized RAD / SKP mixed polypeptide solution.

[0028] In the above technical solution, in step S2, a decellularized cartilage matrix scaffold is prepared by the freeze-drying method. Cartilage sections are taken from fresh pig joints and transported to the laboratory on ice with a sterile PBS solution at pH 7.6. The cells are removed by pulverization under sterile conditions. The specific method includes the following steps:

[0029] S201. Wash and pulverize the cartilage blocks in PBS containing 3.5% (w / v) PMSF and 0.1% (w / v) EDTA to inhibit protease activity;

[0030] S202. Suspend the cartilage fragment suspension and rotate it at 2000 rpm for 5 minutes, then remove the precipitate. Centrifuge the new suspension at 7000 rpm again for 5 minutes;

[0031] S203. Incubate the cartilage microfilaments in a hypotonic Tris-HCl containing 1% TritonX-100 at 4 °C with gentle stirring for 12 h. After washing in PBS without protease inhibitors, stir in 10 mM Tris-HCl (pH 7.5) containing 50 U / ml deoxyribonuclease and 1 U / ml ribonuclease at 37 °C for 12 h;

[0032] S204. Rinse the decellularized cartilage matrix microfilaments with sterile PBS and make a 3% (w / v) suspension;

[0033] S205. Add the suspension to a cylindrical mold, place it at -80 °C for 1 h, and then freeze-dry it under vacuum for 48 h;

[0034] S206. Crosslink the scaffold under 258 nm ultraviolet light for 6 h, and sterilize the scaffold with a diameter of 3 mm and a height of 1.5 mm by irradiating it with 60Co at 5 mrad.

[0035] In the above technical solution, in step S3, the DCM scaffold is combined with the RAD / SKP mixed polypeptide solution to prepare the biofunctional bone marrow-specific homing scaffold CE-SKP composite scaffold. The specific method includes the following steps:

[0036] S301. Add the RAD / SKP mixed polypeptide solution to a cell culture dish to immerse the porous DCM scaffold;

[0037] S302. Add a culture medium, trigger the polypeptide gel by self-assembly inside the DCM scaffold, and incubate at 37 °C for 1 h to obtain a biofunctional 3D scaffold system with a gel solid two-phase;

[0038] S303. Before in vivo implantation and in vitro experiments, change the medium again to balance the gel to physiological pH and incubate at 37 °C overnight.

[0039] In the above technical solution, in step S4, the preparation of the CPH hydrogel includes the following steps: Mix a 5% γ-PGA and CS (1:1, w / w) solution with 1% HA, dissolve it and then add 1% (v / v) acetic acid to form a CS / γ-PGA / HA (CPH) hydrogel. After freeze-drying, soak it in deionized water to remove residual acidic substances.

[0040] In the above technical solution, in step S5, the preparation of the P2G3 nanofibers includes the following steps: Dissolve 10 wt% PCL and GEL in a hexafluoroisopropanol solution (P / G = 2:3, w / w; P2G3), then add 0.2% (v / v) acetic acid to the mixed solution to obtain a uniform and stable mixed solution. Prepare a P2G3 nanofiber membrane by electrospinning technology.

[0041] The advantages and positive effects of the present invention are as follows: The CE-SKP / CPH / P2G3 three-layer composite scaffold in the present invention utilizes its three-layer composite structure to solve the problems in the prior art that it is impossible to well simulate the cartilage layer and the calcified cartilage layer in the physiological structure and it is impossible to maintain the independence of the repair microenvironment, realizes the common repair of the cartilage layer and the calcified cartilage layer, and achieves a better repair effect for full-thickness articular cartilage defects.

[0042] In addition, as the creative auxiliary evidence of the present invention, it is also reflected in the following important aspects:

[0043] 1. The aging population and the increasing participation in sports are the main driving forces in the market for cartilage defect repair. With the aging of the population, the incidence of cartilage damage diseases such as osteoarthritis is rising continuously, which has led to an increased demand in the cartilage repair market. At the same time, the increasing global sports participation has also increased the incidence of cartilage injuries, further driving the development of the market. The cartilage tissue engineering technology used in this project makes cartilage repair and regeneration more efficient and precise, not only improving the success rate of surgeries but also reducing the recovery time of patients, thereby enhancing the quality of life of patients. The technical solution of this project is expected to gain wide market acceptance after transformation, achieving an increase in market commercial value. At the same time, the improvement of patients' quality of life will also bring long-term benefits to society.

[0044] 2. The technical solution of the present invention solves the technical problems in the prior art that it is impossible to well simulate the cartilage layer and the calcified cartilage layer in the physiological structure and that it is impossible to maintain the independence of the repair microenvironment, realizes the co-repair of the cartilage layer and the calcified cartilage layer, achieves a better repair effect for full-thickness articular cartilage defects, and solves the technical problems that people have been eager to solve but have never succeeded in. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The technical solution of the present invention will be further described in detail below in conjunction with the drawings and embodiments. However, it should be understood that these drawings are only designed for explanatory purposes and thus do not limit the scope of the present invention. In addition, unless otherwise specified, these drawings are only intended to conceptually illustrate the structural configurations described herein and are not necessarily drawn to scale.

[0046] Figure 1 It is a characterization introduction diagram of the CE-SKP / CPH / P2G3 three-layer composite scaffold; wherein: Figure 1 A is the general appearance diagram of each layer of the scaffold; Figure 1 B is the live-dead staining image of each layer of the scaffold; Figure 1 C is the statistical chart of the proportion of live and dead cells in each layer of the scaffold;

[0047] Figure 2 It is a characterization introduction diagram of the CE-SKP / CPH / P2G3 three-layer composite scaffold; wherein: Figure 2 A is the crystal violet staining picture of cells in the transwell migration experiment; Figure 2 B is the statistical chart of the number of cell migrations; Figure 2 C is the statistical chart of glycosaminoglycan content; Figure 2 D is the statistical chart of the PCR results of COLⅡ, SOX9 and ACAN; Figure 2 E is the immunoblotting diagram of COLⅡ and SOX9; Figure 2 F is the alizarin red staining picture of chondrocytes; Figure 2 G is the statistical chart of alkaline phosphatase activity;Figure 2 H is a statistical chart of the PCR results of OCN, COLⅠ, COLⅩ, and RUNX2; Figure 2 I is a western blot of COLⅠ, COLⅩ, and RUNX2; Figure 2 J is a crystal violet staining picture of cells in the transwell migration assay; Figure 2 K is a statistical chart of the number of cell migrations;

[0048] Figure 3 are the results pictures after the implantation of the CE-SKP / CPH / P2G3 three-layer composite scaffold in vivo; where: Figure 3 A is a gross observation picture; Figure 3 B is an International Cartilage Repair Society (ICRS) cartilage repair score picture; Figure 3 C is a micro-CT picture of the cartilage surface;

[0049] Figure 4 are the section staining pictures of the cartilage surface; where: Figure 4 A is a hematoxylin-eosin (HE) staining picture; Figure 4 B is a safranin-fast green staining picture. Detailed implementation manners

[0050] First of all, it should be noted that the following will specifically illustrate the specific structure, characteristics, advantages, etc. of the present invention by way of examples. However, all descriptions are only for the purpose of illustration and should not be construed as forming any limitation to the present invention. In addition, any single technical feature described or implied in the various embodiments mentioned in this article, or any single technical feature shown or implied in the various drawings, can still be arbitrarily combined or deleted between these technical features (or their equivalents), so as to obtain more other embodiments of the present invention that may not be directly mentioned in this article. In addition, for the sake of simplifying the drawings, the same or similar technical features may only be marked at one place in the same drawing.

[0051] 1. Design of the CE-SKP / CPH / P2G3 three-layer composite scaffold

[0052] The CE-SKP / CPH / P2G3 three-layer composite scaffold utilizes its three-layer composite structure to solve the problem in the prior art that it is impossible to well simulate the cartilage layer and the calcified cartilage layer in the physiological structure, and it is impossible to maintain the independence of the repair microenvironment, realizing the co-repair of the cartilage layer and the calcified cartilage layer, and achieving a better repair effect for full-thickness defects of articular cartilage.

[0053] Wherein:

[0054] Self-assembling polypeptide nanofiber hydrogel Ac-(RADA) 4 -CONH 2 / Ac-(RADA)4 GGSKPPGTSS-CONH 2 (RAD / SKP) is adsorbed onto the seed cell-free composite scaffold system CE-SKP constructed from the extracellular matrix (ECM) of natural chondrocytes to simulate the cartilage layer of the physiological structure. RAD / SKP mimics the structure of the bone marrow homing peptide, which can recruit endogenous repair cells in situ. The ECM can induce the chondrogenic differentiation of the recruited endogenous repair cells, thereby achieving the repair of full-thickness cartilage defects in the trochlear groove of the rabbit femur.

[0055] Chitosan (CS) / poly-γ-glutamic acid (γ-PGA) / hydroxyapatite (HA) hydrogel (CPH hydrogel) can promote the mineralization of mesenchymal stem cells (MSCs) both in vitro and in vivo. Place CPH under CE-SKP to simulate the calcified cartilage layer of the physiological structure. CPH induces the mineralization of newly formed chondrocytes to form the calcified cartilage layer of the physiological structure.

[0056] Polycaprolactone (PCL) / gelatin (GEL) nanofiber membrane (P2G3 nanofiber membrane) has the function of blocking cell migration, which can ensure the relative independence of the microenvironment. Place it under CPH to maintain the independence of the cartilage repair microenvironment.

[0057] Combine the three-layer materials to form the CE-SKP / CPH / P2G3 three-layer composite scaffold, which can recruit repair cells in situ, induce chondrogenic differentiation and sequential hypertrophic calcification, and maintain a relatively independent repair microenvironment, achieving a better repair effect for full-thickness defects of articular cartilage.

[0058] 2. Preparation of the CE-SKP / CPH / P2G3 three-layer composite scaffold

[0059] First, prepare the polypeptide solution. Use the functional motif SKP (SKPPGTSS) that mimics the biological activity of the bone marrow homing polypeptide to directly extend the C-terminus of the self-assembling peptide RADA16-I for functionalization to form the designed functional RAD / SKP polypeptide. Dissolve 10 mg of RAD / SKP polypeptide powder in 1 ml of distilled water and mix well to obtain a homogeneous polypeptide solution of 1% (w / v, pH 3 - 4). After sterilization with a filter, store the polypeptide solution at 4 °C until the next use. Mix the functionalized RAD / SKP polypeptide with a 1% RADA16-I solution with a volume ratio of 1:1 to obtain the functionalized RAD / SKP mixed polypeptide solution.

[0060] Next, prepare the acellular cartilage matrix scaffold. The acellular cartilage matrix (DCM) scaffold was prepared by freeze-drying method. Cartilage slices were taken from fresh pig joints and transported to the laboratory in ice with sterile PBS solution at pH 7.6. The acellularization was carried out by pulverization under sterile conditions as follows: The cartilage blocks were washed and pulverized in PBS containing 3.5% (w / v) PMSF and 0.1% (w / v) EDTA to inhibit protease activity. The cartilage fragment suspension was rotated at 2000 rpm for 5 minutes, then the precipitate was removed, and the new suspension was centrifuged again at 7000 rpm for 5 minutes. The cartilage microfilaments were incubated in hypotonic Tris-HCl containing 1% TritonX-100 at 4 °C with gentle stirring for 12 h. After washing in PBS without protease inhibitor, they were stirred at 37 °C in 10 mM Tris-HCl (pH 7.5) containing 50 U / ml deoxyribonuclease and 1 U / ml ribonuclease for 12 h. The acellular cartilage matrix microfilaments were rinsed with sterile PBS and made into a 3% (w / v) suspension. The suspension was added to a cylindrical mold, placed at -80 °C for 1 h, and then freeze-dried under vacuum for 48 h. Then the scaffold was cross-linked under 258 nm ultraviolet light for 6 h, and the scaffolds with a diameter of 3 mm and a height of 1.5 mm were sterilized by irradiation with 60Co at 5 mrad.

[0061] Finally, prepare the CE-SKP scaffold. The DCM scaffold was further combined with the RAD / SKP mixture to prepare the biofunctional bone marrow-specific homing scaffold CE-SKP. The polypeptide solution was added to the cell culture dish to immerse the porous DCM scaffold. Next, the culture medium was added to trigger the polypeptide gel by self-assembly within the DCM scaffold and incubated at 37 °C for 1 h to obtain a biofunctional 3D scaffold system with a gel-solid two-phase. Before in vivo implantation and in vitro experiments, the medium was changed again to balance the gel to physiological pH and incubated at 37 °C overnight. The prepared CE-SKP scaffold is as Figure 1 shown in A.

[0062] Prepare the CPH hydrogel. A 5% solution of γ-PGA and CS (1:1, w / w) was mixed with 1% HA, and after dissolution, 1% (v / v) acetic acid was added to form the CS / γ-PGA / HA (CPH) hydrogel. After freeze-drying, it was soaked in deionized water to remove residual acidic substances. The synthesized CPH is as Figure 1 shown in A.

[0063] Prepare the P2G3 nanofibers. 10 wt% PCL and GEL were dissolved in hexafluoroisopropanol solution (P / G = 2:3, w / w; P2G3), and then 0.2% (v / v) acetic acid was added to the mixed solution to obtain a uniform and stable mixed solution. The P2G3 nanofiber membrane was prepared by electrospinning technology. The prepared P2G3 is as Figure 1 shown in A.

[0064] Arrange CE-SKP, CPH, and P2G3 in sequence from top to bottom to synthesize a three-layer composite scaffold of CE-SKP / CPH / P2G3.

[0065] 3. Application of the three-layer composite scaffold of CE-SKP / CPH / P2G3

[0066] The three-layer composite scaffold of CE-SKP / CPH / P2G3 has the ability to recruit stem cells and induce the chondrogenic differentiation of stem cells, can sequentially induce the formation of a calcified cartilage layer, and can simultaneously maintain the independence of the cartilage repair microenvironment to achieve better cartilage defect repair effects.

[0067] First, as Figure 1 shown in B, we verified the biocompatibility of the three-layer scaffold. Live / dead imaging and semi-quantitative analysis showed that, as shown in the results Figure 1 C, synovial mesenchymal stem cells co-cultured with P2G3, CPH, and CE-SKP had good viability at both 24 h and 72 h. Next, we explored the function of the CE-SKP scaffold in recruiting mesenchymal stem cells (MSCs) and inducing their differentiation into chondrocytes. According to the results Figure 2 A and Figure 2 B, the CE-SKP scaffold recruited more cells compared to the blank control group, demonstrating its significant recruitment effect on stem cells. Next, we co-cultured the CE-SKP scaffold with SMSCs and detected them on the 7th and 14th days respectively. As Figure 2 shown in C, the CE-SKP scaffold co-culture group had a higher content of glycosaminoglycan (GAG) at both time points. As Figure 2 shown in D, compared to the blank control group, the CE-SKP scaffold had significantly increased expression of chondrogenic differentiation-related genes COLⅡ, SOX9, and ACAN. As Figure 2 shown in E, the Western bolt experiment indicated that CE-SKP induced the expression of higher levels of SOX9 and COLⅡ proteins compared to the control group.

[0068] Secondly, we explored the function of CPH in inducing the hypertrophic differentiation and calcification of chondrocytes. As Figure 2 shown in F, when CPH was co-cultured with chondrocytes, alizarin red staining on the 14th day indicated that more mineralized nodules were formed in the CPH co-culture. At the same time, as shown in the results Figure 2 G, we detected the ALP activity that plays an important role in the initial stage of mineralization on the 7th day of co-culture. Compared to the control group, CPH had higher ALP activity. PCR was performed on the 7th and 14th days. As Figure 2 shown in H, the chondrocytes co-cultured with CPH had higher expression of chondrocyte hypertrophy-related genes OCN, COLⅠ, COLⅩ, and RUNX2. The results showed asFigure 2 I. Western blot assays indicated that CPH induced higher expression levels of COLⅩ, COLⅠ, and RUNX2 proteins compared to the control group.

[0069] Next, to verify the function of the P2G3 nanofiber membrane, we coated a layer of P2G3 nanofiber membrane on the inner side of a transwell chamber and seeded BMSCs. The results are shown in Figure 2 J and Figure 2 K. Compared to the control group, almost no cells migrated to the opposite side in the P2G3 group.

[0070] As shown in Figure 3 A and Figure 3 B, gross observation showed that the CE-SKP / CPH / P2G3 three-layer composite scaffold filling had a significant therapeutic effect compared to the control group, with a higher repair depth, better integration with the surrounding cartilage, and a better gross appearance. Over time, the cartilage repair effect became better and corresponded to the ICRS score. The CE-SKP / CPH / P2G3 three-layer composite scaffold had a better repair effect, fewer cartilage cracks, better integration with the surrounding cartilage, and a smoother and more intact cartilage surface. As shown in Figure 3 C, Micro-CT reconstruction images more intuitively reflected the three-dimensional structure of the cartilage surface, further corroborating the gross observation images.

[0071] As shown in Figure 4 A, HE staining showed that there was no obvious severe inflammatory infiltration in both groups. The CE-SKP / CPH / P2G3 three-layer composite scaffold filling had a better tissue defect filling effect compared to the blank group, with a flatter cartilage surface. Over time, the cartilage surface filled with the CE-SKP / CPH / P2G3 three-layer composite scaffold became smoother, restored the cartilage layer structure well, and showed no obvious degeneration at 24 weeks. As shown in Figure 4 B, safranin-fast green staining showed that the CE-SKP / CPH / P2G3 three-layer composite scaffold filling induced more cartilage tissue (red) compared to the control group, and the regenerated calcified cartilage layer could be observed.

[0072] The above embodiments have described the present invention in detail, but the content described is only the preferred embodiments of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of the present invention application should still fall within the scope covered by the patent of the present invention.

Claims

1. A CE-SKP / CPH / P2G3 three-layer composite stent, characterized in that: The method is to place CPH hydrogel under the CE-SKP composite scaffold system and place P2G3 nanofiber membrane under the CPH hydrogel to simulate the calcified cartilage layer of physiological structure and maintain the relative independence of the cartilage repair microenvironment, and then induce the mineralization of new chondrocytes to form a calcified cartilage layer of physiological structure. Among them: The CE-SKP composite scaffold system is a seed cell-free composite scaffold system constructed by adsorbing the self-assembled polypeptide nanofiber hydrogel Ac-(RADA)4-CONH2 / Ac-(RADA)4GGSKPPGTSS-CONH2 onto the natural cartilage extracellular matrix.

2. The CE-SKP / CPH / P2G3 three-layer composite stent according to claim 1, characterized in that: CPH hydrogel, which is a chitosan / poly-gamma-glutamic acid / hydroxyapatite hydrogel, can promote the mineralization of mesenchymal stem cells both in vivo and in vitro; P2G3 nanofiber membrane, which is a polycaprolactone / gelatin nanofiber membrane, has the function of blocking cell migration and ensuring the relative independence of the microenvironment; The CE-SKP composite scaffold system is used to simulate the cartilage layer of the physiological structure, wherein: the self-assembling polypeptide nanofiber hydrogel Ac-(RADA)4-CONH2 / Ac-(RADA)4GGSKPPGTSS-CONH2 is used to simulate the structure of the bone marrow homing peptide to recruit endogenous repair cells in situ, and the natural cartilage extracellular matrix is ​​used to induce the recruited endogenous repair cells to differentiate into cartilage.

3. Use of the CE-SKP / CPH / P2G3 three-layer composite scaffold as described in claim 1 or 2 in the preparation of repair materials for cartilage defects.

4. Use of the CE-SKP / CPH / P2G3 three-layer composite scaffold as described in claim 1 or 2 in preparing a repair material for full-thickness defects of articular cartilage.

5. A method for preparing a CE-SKP / CPH double-layer composite stent, characterized in that: The following steps are involved: S1. preparing a functionalized RAD / SKP mixed peptide solution; S2, preparing acellular cartilage matrix scaffold, i.e., DCM scaffold; S3, preparing CE-SKP composite scaffold; S4, preparing CPH hydrogel; S5, preparing P2G3 nanofibers; S6. Arrange the CE-SKP composite scaffold, CPH hydrogel, and P2G3 nanofibers in sequence from top to bottom to synthesize the CE-SKP / CPH / P2G3 three-layer composite scaffold system.

6. The method for preparing a CE-SKP / CPH double-layer composite stent according to claim 5, characterized in that: In step S1, preparing a functionalized RAD / SKP mixed polypeptide solution comprises the following steps: S101, using the functional motif SKP that mimics the biological activity of bone marrow homing peptide to directly extend the C-terminus of the self-assembling peptide RADA16-I to form a designed functionalized RAD / SKP peptide; S102, dissolve 10 mg of polypeptide powder in 1 ml of distilled water and mix well to obtain a 1% uniform polypeptide solution with a pH of 3-4. After filter sterilization, store the polypeptide solution at 4°C until use in the next step; S103. Mix the functionalized RAD / SKP polypeptide and 1% RADA16-I solution in a volume ratio of 1:1 to obtain a functionalized RAD / SKP mixed polypeptide solution.

7. The method for preparing a CE-SKP / CPH double-layer composite stent according to claim 5, characterized in that: In step S2, DCM scaffolds were prepared by freeze drying. Cartilage slices were taken from fresh pig joints, transported to the laboratory on ice with sterile PBS solution with a pH of 7.6, and crushed and decellularized under sterile conditions. The specific method includes the following steps: S201, cartilage blocks were washed and pulverized in PBS containing 3.5% PMSF and 0.1% EDTA to inhibit protease activity; S202, the cartilage fragment suspension was spun at 2000 rpm for 5 minutes, then the precipitate was removed, and the new suspension was centrifuged again at 7000 rpm for 5 minutes; S203, cartilage microfilaments were incubated in hypotonic TriseHCl containing 1% TritonX-100 with gentle stirring at 4°C for 12 h, washed in PBS without protease inhibitors, and stirred in 10 mM TriseHCl containing 50 U / ml DNase and 1 U / ml RNase at 37°C for 12 h; S204, decellularized cartilage matrix microfilaments were rinsed with sterile PBS and prepared into a 3% suspension; S205, adding the suspension into a cylindrical mold, placing it at -80°C for 1 hour, and then vacuum freeze-drying it for 48 hours; S206. Cross-link the stent under 258 nm ultraviolet light for 6 h, and sterilize the stent with a diameter of 3 mm and a height of 1.5 mm using 5 mrad of 60Co irradiation.

8. The method for preparing a CE-SKP / CPH double-layer composite stent according to claim 5, characterized in that: In step S3, the DCM scaffold is combined with the RAD / SKP mixed polypeptide solution to prepare a biological functional bone marrow specific homing scaffold CE-SKP composite scaffold, and the specific method includes the following steps: S301, adding the RAD / SKP mixed polypeptide solution into the cell culture dish to immerse the porous DCM scaffold; S302, adding culture medium to trigger the polypeptide gelation by self-assembly in the DCM scaffold, and incubating at 37°C for 1 hour to obtain a biofunctional 3D scaffold system with two phases of gel and solid; S303. Before in vivo implantation and in vitro experiments, the medium was replaced again to allow the gel to equilibrate to physiological pH and incubated at 37°C overnight.

9. The method for preparing a CE-SKP / CPH double-layer composite stent according to claim 5, characterized in that: In step S4, the preparation of CPH hydrogel includes the following steps: 5% γ-PGA and CS solution are mixed with 1% HA in a ratio of 1:1, 1% acetic acid is added after dissolution to form CPH hydrogel, and after freeze-drying, it is soaked in deionized water to remove residual acidic substances.

10. The method for preparing a CE-SKP / CPH double-layer composite stent according to claim 5, characterized in that: In step S5, the preparation of P2G3 nanofibers includes the following steps: dissolving 10 wt % of PCL and GEL in a hexafluoroisopropanol solution at a ratio of 2:3, then adding 0.2 % of acetic acid to the mixed solution to obtain a uniform and stable mixed solution, and preparing a P2G3 nanofiber membrane by electrospinning technology.

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