Three-layer repair stent as well as preparation method and application thereof

Abstract

The invention discloses a three-layer repair scaffold and a preparation method and application thereof, and the three-layer repair scaffold comprises a first repair layer containing a first matrix and a first active component dispersed in the first matrix, a second repair layer containing a second matrix and a second active component dispersed in the second matrix, and a middle connecting layer, the first repairing layer is located between the first repairing layer and the second repairing layer and connected with the first matrix and the second matrix through intermolecular force; the first active component and the second active component are coated in the same or different sustained-release materials. The three-layer repair scaffold provided by the invention can better promote mesenchymal stem cells to differentiate towards cartilage and osteogenesis in different repair layers, so that the injury of cartilage and subchondral bone can be more effectively repaired.

Description

Technical Field

[0001] This invention relates to the field of bone and joint repair, and more particularly to a three-layer repair scaffold, its preparation method, and its uses. Background Technology

[0002] Knee osteoarthritis (KOA) is a common and frequently occurring disease in clinical practice. Currently, a diverse system of clinical treatments has been developed for KOA. On the one hand, anti-inflammatory and analgesic drugs are used to relieve pain and other symptoms; on the other hand, physical therapy is implemented to assist treatment through physical methods; additionally, there are local injections of hyaluronic acid and surgical procedures. The common goal of these treatments is to alleviate clinical symptoms and slow disease progression. When the condition worsens to a severe stage, joint replacement surgery is considered.

[0003] In recent years, with a deeper understanding of KOA, research on Chinese herbal monomers, and progress in cell engineering technology and biomaterials research, emerging regenerative and repair treatment strategies have emerged. Combining Chinese herbal monomers with cell engineering and biomaterials for research and innovation has become a new direction for KOA treatment.

[0004] Patent document 202310131565.2 discloses a full-thickness repair double-layer scaffold for bone and joint repair, wherein the cartilage and subchondral bone of the bone and joint are repaired by loading different active ingredients of traditional Chinese medicine, icariin and quercetin, into the upper and lower scaffolds.

[0005] Although the aforementioned double-layer stent technology has achieved relatively good repair results, the release of the active ingredients of traditional Chinese medicine is still relatively fast and the sustained-release effect is not good because the active ingredients of traditional Chinese medicine are only fixed in the stent through the adsorption of dopamine in the stent.

[0006] In addition, it is necessary to explore and develop new repair scaffold structure systems. Summary of the Invention

[0007] This invention provides a novel three-layer repair scaffold, its preparation method, and its applications.

[0008] A first aspect of the present invention provides a three-layer repair stent, comprising: The first repair layer comprises a first matrix and a first active ingredient dispersed in the first matrix. The second repair layer comprises a second matrix and a second active ingredient dispersed in the second matrix, and An intermediate connecting layer is located between the first repair layer and the second repair layer and is connected to the first matrix and the second matrix respectively through intermolecular forces; The first active ingredient and the second active ingredient are coated in the same or different sustained-release materials.

[0009] The three-layer repair scaffold provided by this invention encapsulates the active ingredients in a sustained-release material to form a sustained-release system of the active ingredients, which is then dispersed in a matrix. Compared with the prior art, which directly disperses the active ingredients in the matrix, the sustained-release effect of the active ingredients is significantly improved.

[0010] Furthermore, this invention innovatively constructs a three-layer scaffold structure, with an intermediate connecting layer between the first and second repair layers. By utilizing the intermolecular forces present between the intermediate connecting layer and both the first and second matrices, the connection strength between the layers is increased, effectively preventing layer detachment.

[0011] In some embodiments of the first aspect of the present invention, the first active ingredient is icariin and the second active ingredient is quercetin.

[0012] Icariin (ICA) is the main active ingredient of Epimedium and has a variety of complex pharmacological effects. Studies have found that Icariin has a certain regulatory effect on osteoblasts and osteoclasts in cartilage and bone tissues and can effectively repair cartilage lesions.

[0013] Quercetin can inhibit rankl-mediated osteoclastogenesis, osteoblast apoptosis, oxidative stress, and inflammatory responses, while promoting osteogenicity, angiogenesis, antioxidant expression, adipocyte apoptosis, and osteoclast apoptosis. Quercetin has a phytoestrogen-like effect in inhibiting bone resorption and competitively participates in the binding of the estrogen receptor (ER). Studies have shown that estrogen plays a role in stimulating the differentiation of primitive cells into osteoblasts in osteoblast cell lines, and has a significant effect on subchondral bone repair.

[0014] This invention utilizes icariin and quercetin to load different repair layers, which is particularly beneficial for the repair of bone and joint injuries. The first repair layer containing icariin promotes the differentiation of mesenchymal stem cells into cartilage, thereby repairing the cartilage layer of the bone and joint. The second repair layer containing quercetin promotes the differentiation of mesenchymal stem cells into osteoblasts, thereby repairing the subchondral bone layer. Ultimately, it achieves a synchronous and integrated repair effect of cartilage and subchondral bone in the bone and joint.

[0015] In some embodiments, the sustained-release material used has both hydrophilic and hydrophobic groups. On the one hand, the hydrophobic groups of the sustained-release material are beneficial for encapsulating the active material therein. On the other hand, the hydrophilic groups are more conducive to the dispersion and stability of the formed sustained-release particles of the active material in an aqueous system.

[0016] In one specific embodiment, the sustained-release material used is shellac. Shellac is a known sustained-release material that naturally possesses both hydrophilic and hydrophobic groups, and can be used for encapsulating active materials without further modification, simplifying the preparation process of sustained-release particles and the three-layer repair scaffold.

[0017] In some embodiments, the intermolecular forces include hydrogen bonds, van der Waals forces, and / or electrostatic interactions.

[0018] As described above, the intermolecular interaction forces increase the bonding strength between layers, effectively preventing layer detachment.

[0019] In some embodiments, the first matrix and the second matrix are the same, both containing or composed of dopamine-modified sodium alginate.

[0020] This invention simplifies the fabrication process of a three-layer repair scaffold by using the same first and second matrices. Furthermore, the use of dopamine-modified sodium alginate, utilizing the polyhydroxy structure of dopamine to provide hydrogen bonds, increases the bonding strength.

[0021] In one specific embodiment, dopamine-modified sodium alginate is prepared by feeding sodium alginate and dopamine in a mass ratio of 1:3.

[0022] In some embodiments, the intermediate linker layer comprises, or is particularly composed of, catechol-modified chitosan. Catechol has a polyhydroxy structure similar to dopamine, which, in conjunction with the dopamine structure in the repair layer, can increase the bonding strength between layers.

[0023] Furthermore, chitosan is a polycation, while sodium alginate in the matrix is ​​a polyanion. Through the superposition and arrangement of anions / cations / anions, a more stable electrostatic attraction can be formed, further increasing the connectivity between the upper and lower layers and the stability of the three-layer repair scaffold.

[0024] A second aspect of the present invention provides a method for preparing a three-layer repair scaffold, characterized in that it comprises: The first active ingredient and the second active ingredient are respectively coated into the sustained-release material to form sustained-release particles of the first active ingredient and sustained-release particles of the second active ingredient. Sodium alginate was reacted with dopamine to prepare dopamine-modified sodium alginate as a matrix; The first active ingredient sustained-release particles and the second active ingredient sustained-release particles are respectively dispersed in the dopamine-modified sodium alginate to obtain the first repair layer material and the second repair layer material. Catechol and chitosan are reacted to obtain catechin-modified chitosan as an intermediate linking layer material; and The first repair layer material, the intermediate connecting layer material, and the second repair layer material are sequentially stacked to form a three-layered body, which is then cross-linked to obtain the three-layered repair scaffold.

[0025] In some embodiments, the first active ingredient is icariin, the second active ingredient is quercetin, and / or the sustained-release material is shellac.

[0026] In some embodiments, during the reaction of sodium alginate and dopamine, the mass ratio of sodium alginate to dopamine is 1:3.

[0027] The inventors unexpectedly discovered through experiments that the grafting amount of dopamine onto sodium alginate increases with the increase of dopamine content. More dopamine can provide more hydrogen bonds, thus providing a more stable scaffold structure. However, on the other hand, the inventors found that when the amount of dopamine is too large, for example, when the mass ratio of dopamine to sodium alginate is greater than 3:1, the resulting matrix material becomes brittle. When used to prepare the scaffold, it easily causes the scaffold to become brittle and prone to breakage. Moreover, such a matrix material has a very dark color and is not suitable for use as the matrix of the three-layer repair scaffold of this invention. Therefore, the optimal mass ratio of sodium alginate to dopamine is 1:3.

[0028] The third aspect of this invention provides the application of a three-layer repair scaffold in repairing cartilage-subchondral bone defects of the knee joint.

[0029] In summary, the three-layer repair scaffold provided by this invention combines clinically effective active ingredients, such as icariin and quercetin, with the three-layer repair scaffold and slowly releases the drug through a sustained-release material, such as shellac, which can better promote the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts in different repair layers, thereby more effectively repairing damage to cartilage and subchondral bone. Attached Figure Description

[0030] Figure 1 The Alg-DA prepared in Example 1 of this invention is shown. 3.0 UV absorption spectrum and Alg-DA 3.0 with Alg-DA 1.0 Comparison of ungrafted Alg; Figure 2 The Alg-DA prepared in Example 1 of this invention is shown. 3.0 Infrared absorption spectrum and Alg-DA 3.0 Comparison with ungrafted Alg; Figure 3 Here are SEM images, where (a) shows a monolayer scaffold Alg-DA containing icariin shellac particles. 3.0-ICA, (b) shows a monolayer scaffold Alg-DA containing quercetin shellac particles. 3.0 - Que, (c) shows the upper layer (containing icariin) and middle layer of the three-layer repair scaffold prepared in Example 1, and (d) shows the middle layer and lower layer (containing quercetin) of the three-layer repair scaffold prepared in Example 1. Figure 4 Alg-DA 3.0 - ICA and Alg-DA 3.0 - Degradation curve of Que; Figure 5 Alg-DA was shown 3.0 - ICA and Alg-DA 3.0 - Que and the compression modulus of the three-layer repair scaffold; Figure 6 The cartilage defect repair status is shown at different time points; Figure 7 The image shows the bone defect repair status as examined by MicroCT at 3 months. Figure 8 HE staining at various time points is shown; Figure 9 The immunostaining combinations at various time points are shown. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0032] Example 1 Icariin (ICA) and quercetin (Q) ue ) Covering (1) Encapsulation of icariin 300 mg of shellac (Sh) was dissolved in 20 mL of ethanol, centrifuged at 6500-7000 rpm for 10 min, and insoluble impurities were removed using a 0.45 μm organic filter membrane to prepare the shellac stock solution. Subsequently, 20 mg of icariin was dissolved in the shellac stock solution and stirred until homogeneous. The resulting mixture was added dropwise to 100 mL of water (600 rpm) at a rate of 0.5 mL / min using a peristaltic pump. Solid insoluble matter was removed by suction filtration, followed by rotary evaporation to remove ethanol, and then lyophilized to obtain shellac particles coated with icariin.

[0033] (2) Coating of quercetin 300 mg of shellac (Sh) was dissolved in 20 mL of ethanol, centrifuged at 6500-7000 rpm for 10 min, and insoluble impurities were removed using a 0.45 μm organic filter membrane to prepare the shellac stock solution. Subsequently, 20 mg of quercetin was dissolved in the shellac stock solution and stirred thoroughly. The resulting mixture was added dropwise to 100 mL of water (600 rpm) at a rate of 0.5 mL / min using a peristaltic pump. Solid insoluble matter was removed by suction filtration, followed by rotary evaporation to remove ethanol, and then lyophilized to obtain shellac particles coated with quercetin.

[0034] Synthesis of dopamine-modified sodium alginate (Alg-DA) Weigh 1g of sodium alginate (Alg) and place it in a three-necked flask. Add 100 mL of PBS buffer (50 mM, pH=5.5) and stir to dissolve at room temperature. After complete dissolution, add 1.94 g of EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 2.32 g of NHS (N-hydroxysuccinimide), with n(COOH) / n(EDC) / n(NHS)=1 / 2 / 4. Activate at room temperature for 30 min, then add 3g of dopamine hydrochloride (DA·HCl). Repeat the vacuuming and nitrogen purging three times to remove oxygen from the reaction system and prevent DA oxidation. Then react at room temperature under a nitrogen atmosphere for 24 h. After stopping the reaction, dialyze in deionized water to remove the catalyst and unreacted monomers. After no UV absorption peak of DA is observed in the dialysate, freeze-dry to obtain a white cotton-like modified macromolecule, denoted as Alg-DA. 3.0 .

[0035] Preparation of catechin-modified chitosan Chitosan (0.5 g) was dissolved in a dilute hydrochloric acid solution (50 mL, pH = 5.5). Catechol (0.591 g) and EDC (1.244 g) were dissolved in an aqueous ethanol solution (ethanol to water volume ratio 1:1, 60 mL). The aqueous ethanol solution was then added to the chitosan-hydrochloric acid solution. The reaction solution was stirred vigorously at room temperature for 12 hours. 1 M hydrochloric acid solution was added to maintain the pH of the reaction solution at 5.5. After the reaction was completed, the reaction solution was purified by dialyzing in dilute hydrochloric acid solution at pH = 5.5 and in deionized water for 48 h and 12 h, respectively, and then lyophilized.

[0036] Preparation of a three-layer repair scaffold (1) Preparation of the first repair layer material solution containing icariin 600mg of Alg-DA 3.0 Dissolve in 50ml of ultrapure water. After complete dissolution, add 0.4mg of icariin shellac particles and disperse evenly to obtain the first repair layer material solution.

[0037] (2) Preparation of a second repair layer material solution containing quercetin Dissolve 600 mg of Alg-DA3.0 in 50 ml of ultrapure water. After complete dissolution, add 0.5 mg of quercetin shellac particles and disperse evenly to obtain the second repair layer material solution.

[0038] (3) Preparation of intermediate connecting layer material liquid Dissolve 600 mg of catechin-modified chitosan in 20 ml of ultrapure water, then mix well using a vortex mixer, sonicate for 10 minutes to enrich the bubbles on the liquid surface, and then degas under vacuum for 10 minutes for later use.

[0039] (4) Using a needleless syringe, first draw up the first repair layer material solution containing icariin prepared in step (1), add it to a cylindrical mold to a height of 2 mm, and freeze it rapidly with liquid nitrogen. Then draw up the intermediate connecting layer material solution prepared in step (3), cover it on the frozen first layer to a height of 1 mm, and freeze it rapidly with liquid nitrogen. Finally, draw up the second repair layer material solution containing quercetin prepared in step (2), cover it on the frozen intermediate layer to a height of 5 mm, and freeze it rapidly with liquid nitrogen. Finally, place the three-layer repair scaffold in a freeze dryer for freeze drying. The dried three-layer repair scaffold is cross-linked with CaCl2 (select a concentration of 1 wt%; if the concentration is too high, the shrinkage will be severe). After cross-linking, it is rinsed with deionized water. The cross-linked scaffold is then freeze-dried again for 48 h to obtain the three-layer repair scaffold loaded with icariin and quercetin.

[0040] The resulting three-layer repair stent has the following layer heights: the first repair layer is approximately 1 mm, the intermediate connecting layer is less than 1 mm, and the second repair layer is approximately 4 mm. In this paper, the three-layer repair stent is also referred to as a drug-eluting stent.

[0041] Example 2 The three-layer repair scaffold of Example 2 was prepared according to the method of Example 1. The difference between Example 2 and Example 1 is that the amount of quercetin shellac particles added during the preparation of the second repair layer material solution is 0.4 mg.

[0042] Example 3 The three-layer repair scaffold of Example 2 was prepared according to the method of Example 1. The difference between Example 2 and Example 1 is that the amount of quercetin shellac particles added during the preparation of the second repair layer material solution is 0.3 mg.

[0043] Example 4 Preparation of monolayer scaffolds containing icariin shellac particles According to the description in Example 1, a first repair layer material solution containing icariin was prepared.

[0044] 0.2 mL of the first repair layer material solution was injected into a cylindrical mold using a syringe and rapidly frozen with liquid nitrogen. The mold was then dried using a freeze dryer. After 48 h, the scaffold material was cross-linked with CaCl2 solution. The cross-linked scaffold was then freeze-dried again for 48 h to obtain a single-layer scaffold containing icariin shellac particles, denoted as Alg-DA. 3.0 - ICA.

[0045] Example 5 Preparation of monolayer scaffolds containing quercetin shellac particles According to the description in Example 1, a first repair layer material solution containing quercetin was prepared.

[0046] 0.2 mL of the first repair layer material solution was injected into a cylindrical mold using a syringe and rapidly frozen with liquid nitrogen. The mold was then dried using a freeze dryer. After 48 h, the scaffold material was cross-linked with CaCl2 solution. The cross-linked scaffold was then freeze-dried again for 48 h to obtain a single-layer scaffold containing quercetin shellac particles, denoted as Alg-DA. 3.0 -Que.

[0047] Comparative Example 1 Preparation of a three-layer scaffold without active ingredients Prepare 3 wt% Alg-DA 3.0 An aqueous solution of Alg-DA and an aqueous solution of 3 wt% catechin-modified chitosan. First, using a syringe, 0.2 mL of Alg-DA was added... 3.0 The solution was injected into a cylindrical mold and rapidly frozen with liquid nitrogen to form the first layer. Then, a catechol-modified chitosan solution was added to the surface of the frozen first layer and rapidly frozen with liquid nitrogen. Finally, 0.2 mL of Alg-DA was added. 3.0 The solution was added on top of the second layer of frozen material and rapidly frozen using liquid nitrogen. After the third layer of solution was frozen, the scaffold material was placed in a freeze dryer for drying. The dried scaffold material was cross-linked with a 5 wt% CaCl2 solution, and the cross-linked scaffold was freeze-dried again for 48 h to obtain a three-layer scaffold without active ingredients. In this article, it is also referred to as a drug-free scaffold.

[0048] Characterization and Analysis For Alg-DA 3.0 Characterization was performed using ultraviolet and infrared absorption spectroscopy, such as... Figures 1-2 As shown.

[0049] Figure 1 It is the ultraviolet spectrum, from Figure 1 As can be seen, compared with unmodified Alg, Alg-DA 1.0 (Prepared by reacting Alg and DA·HCl in a mass ratio of 1:1) and Alg-DA3.0 A distinct UV absorption peak was observed at 280 nm, primarily due to the presence of catechol groups in DA, which generate UV absorption at 280 nm. This result preliminarily demonstrates the successful incorporation of DA into the Alg macromolecular chain. Furthermore, with increasing DA dosage, the intensity of the absorption peak of Alg-DA at 280 nm gradually increased, indicating that the degree of DA substitution in Alg-DA gradually increased with increasing DA dosage. Further, as mentioned earlier, when the dosage of dopamine is too high, for example, when the mass ratio of dopamine to sodium alginate is greater than 3:1, the resulting matrix material becomes brittle and dark in color, making it unsuitable for use as the matrix of the three-layer repair scaffold of this invention.

[0050] Figure 2 The image shows an infrared absorption spectrum. As can be seen, after grafting dopamine, characteristic infrared absorption peaks of -C=O and -NH- appear, which is consistent with... Figure 1 The results were consistent, proving that DA had been incorporated into the Alg macromolecular chain.

[0051] Scanning electron microscopy (SEM) analysis was performed on the three-layer repair scaffold prepared in Example 1 and the drug-loaded single-layer scaffolds prepared in Examples 4 and 5. The results are as follows: Figure 3 As shown. Figure 3 (a) Figure 3 In (b), the pore size of the single-layer scaffold ranges from 50 to 200 μm, a pore size favorable for cell growth. From the upper layer (first repair layer) and the middle layer (…) of the three-layer scaffold… Figure 3 (c) and the middle layer and the lower layer (second repair layer) Figure 3 The SEM image in (d) shows obvious structural differences between different layers.

[0052] The degradation performance of the scaffold material was studied using simulated human body fluid (SBF) to mimic a physiological environment. The results are as follows: Figure 4 As shown in the figure, the in vitro degradation curves of the monolayer scaffold materials prepared in Examples 4 and 5 after 56 days are as follows, with a degradation rate of approximately 71%. The undegraded portion is due to the easy oxidative cross-linking of the catechol groups of Alg-DA, which gives the scaffold material a relatively dense internal structure, effectively slowing down the flow of degradation solution inside the scaffold material.

[0053] The compressive properties of the three-layer repair scaffold prepared in Example 1 and the drug-loaded single-layer scaffold prepared in Examples 4 and 5 were tested. The compressive modulus of the scaffold material at 30% deformation was as follows: Figure 5 As shown in the figure. It can be seen from the figure that Alg-DA 3.0 -Que and Alg-DA 3.0The compressive strengths of -ICA are 0.53 MPa and 0.54 MPa, respectively. The catechol groups in Alg-DA can undergo oxidative cross-linking, which can significantly improve the degree of cross-linking of the scaffold material and endow it with better mechanical properties.

[0054] Experimental Example: Animal Joint Repair Experiment Experimental animal grouping Forty male New Zealand white rabbits (8 months old, weighing 2.5-3.0 kg) were used. They were randomly divided into an experimental group (drug-loaded scaffold group, using the three-layer repair scaffold prepared in Example 1), a control group (non-drug-loaded scaffold group, using the three-layer repair scaffold without icariin and quercetin prepared in Comparative Example 1), and a blank control group. Time points: 1 month (n=10), 2 months (n=10), 3 months (n=10), and 6 months (n=10) after the experiment. Osteochondrial defect models were created on both knee joints of the hind legs of the New Zealand white rabbits.

[0055] Animal modeling Weigh the New Zealand White Rabbits and mark their ears with pen numbers for recording.

[0056] Anesthesia: New Zealand white rabbits were anesthetized with 1% sodium pentobarbital at a concentration of 3 ml / kg.

[0057] After successful anesthesia, the rabbits were placed in a supine position with their limbs secured to the operating table with ropes. The hair on both knees of the hind legs was shaved, and the surgical area was disinfected with povidone-iodine. In both groups, a straight incision was made on the medial side of the knee joint, cutting through the skin and fascia, and then dissecting to the distal femur. The joint capsule was opened to expose the femoral condyle, causing the patella to dislocate laterally. Using specialized surgical instruments, a full-thickness osteochondral defect (5mm in diameter, 6mm deep) was created in the trochlear grooves of both femurs of the rabbits. After thoroughly cleaning the surgical site with saline and confirming no active bleeding, each group placed a drug-loaded scaffold prepared in Example 1 and an unloaded scaffold prepared in Comparative Example 1 at the modeling site, as per the preoperative design. The osteochondral defect in the untreated knee served as a blank control. The incisions were sutured layer by layer. All New Zealand white rabbits were returned to their postoperative cages and allowed free movement. Postoperatively, 400,000 units of penicillin were injected intraperitoneally for three consecutive days.

[0058] Specimen processing Material sourcing and preservation Samples were collected at four time points: 1 month, 2 months, 3 months, and 6 months post-surgery. The original incisions of both knee joints were made to fully expose the distal femoral articular surface. The distal femur was cut off vertically from the femoral shaft using bone forceps above the femoral condyle. Surrounding soft tissue was removed, and the specimens were placed in test tubes and preserved with paraformaldehyde.

[0059] HE staining ①Tissue blocks were collected, fixed, routinely embedded in paraffin, and sectioned at 4µm.

[0060] ②The sections were routinely dewaxed with xylene and washed with various grades of ethanol to water: xylene (I) 5 min, xylene (II) 5 min, 100% ethanol 2 min, 95% ethanol 1 min, 80% ethanol 1 min, 75% ethanol 1 min, and distilled water 2 min.

[0061] ③ Stain with hematoxylin for 5 minutes, then rinse with tap water.

[0062] ④ Differentiate with hydrochloric acid and ethanol for 30 seconds, then insert and withdraw several times.

[0063] ⑤ Soak in tap water for 15 minutes or in warm water at about 50℃ for 5 minutes.

[0064] ⑥ Place in eosin solution for 2 minutes.

[0065] ⑦ Routine dehydration, clearing, and sealing.

[0066] Immunohistochemical staining ①Dewaxing paraffin sections to water ② Incubate with 3% H2O2 at room temperature for 5-10 minutes to eliminate the activity of endogenous peroxidase. ③ Rinse with distilled water, then soak in PBS for 5 minutes twice. ④ Block with 5%-10% normal goat serum (diluted with PBS), incubate at room temperature for 10 min, discard the serum, do not wash. Add primary antibody working solution and incubate at 37°C for 1-2 hours or at 4°C overnight. ⑤ Rinse with PBS, 5 min * 3 times ⑥ Add an appropriate amount of biotin-labeled secondary antibody working solution and incubate at 37°C for 10-30 minutes. ⑦ Rinse with PBS, 5 min * 3 times ⑧ Add an appropriate amount of horseradish enzyme or alkaline phosphatase-labeled streptavidin working solution and incubate at 37°C for 10-30 min. Wash with PBS three times, 5 min each time. ⑨ Develop color with colorimetric reagent for 3-15 minutes (DAB or NBT / BCIP) ⑩ Rinse thoroughly with tap water, re-dye, dehydrate, clarify, and seal.

[0067] Observation indicators ① General observation Observe postoperative diet, gait, range of motion of joints, incision condition, and general condition.

[0068] ② Gross observation of joint repair The color and texture of the joint defect and surrounding tissues.

[0069] ③ Observation under a light microscope Histological changes in the defect area and adjacent areas stained with HE; Tissue samples were obtained at 3 months for CT scans; osteochondral defects were assessed at 1, 2, 3, and 6 months by HE staining and COL I, COL II, SOX9, and OCN immunohistochemical staining.

[0070] Research results are as follows Figure 6-9 As shown: Six months after the model was established in New Zealand white rabbits, the depressions in the blank control group became shallower and the local area became rougher. The repaired tissue in the unloaded scaffold group was smooth and translucent, and visible connections with the surrounding normal cartilage were observed, indicating that the defect was not completely repaired by the repaired tissue. In the drug-loaded three-layer repair scaffold group, no model depressions were observed, and the local repaired tissue was consistent and similar to the adjacent normal cartilage. Micro-CT 3D reconstruction showed that the drug-loaded scaffold group had the best bone repair effect, while the unloaded scaffold group had a better bone repair effect than the blank control group. Conclusion: Through experiments, we found that the three-layer repair scaffold loaded with icariin and quercetin has good biocompatibility and can effectively repair damage to articular cartilage and subchondral bone, showing promising application prospects.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application 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 this application.

Claims

1. A three-layer repair stent, characterized in that, include: The first repair layer comprises a first matrix and a first active ingredient dispersed in the first matrix. The second repair layer comprises a second matrix and a second active ingredient dispersed in the second matrix, and An intermediate connecting layer is located between the first repair layer and the second repair layer and is connected to the first matrix and the second matrix respectively through intermolecular forces; The first active ingredient and the second active ingredient are coated in the same or different sustained-release materials.

2. The three-layer repair stent according to claim 1, characterized in that, The first active ingredient is icariin, and the second active ingredient is quercetin.

3. The three-layer repair stent according to claim 1 or 2, characterized in that, The slow-release material has hydrophilic and hydrophobic groups, and is preferably shellac.

4. The three-layer repair stent according to claim 1 or 2, characterized in that, The intermolecular forces include hydrogen bonds, van der Waals forces, and / or electrostatic interactions.

5. The three-layer repair stent according to claim 4, characterized in that, The first matrix and the second matrix are the same, both containing dopamine-modified sodium alginate.

6. The three-layer repair stent according to claim 5, characterized in that, The intermediate connecting layer contains catechin-modified chitosan.

7. A method for preparing a three-layer repair scaffold, characterized in that, include: The first active ingredient and the second active ingredient are respectively coated into the sustained-release material to form sustained-release particles of the first active ingredient and sustained-release particles of the second active ingredient. Sodium alginate was reacted with dopamine to prepare dopamine-modified sodium alginate as a matrix; The first active ingredient sustained-release particles and the second active ingredient sustained-release particles are respectively dispersed in the dopamine-modified sodium alginate to obtain the first repair layer material and the second repair layer material. Catechol and chitosan were reacted to obtain catechin-modified chitosan, which served as an intermediate linking layer material. and The first repair layer material, the intermediate connecting layer material, and the second repair layer material are sequentially stacked to form a three-layered structure, which is then cross-linked to obtain the three-layered repair scaffold.

8. The method according to claim 7, characterized in that, The first active ingredient is icariin, the second active ingredient is quercetin, and / or the sustained-release material is shellac.

9. The method according to claim 7 or 8, characterized in that, In the reaction process of sodium alginate and dopamine, the mass ratio of sodium alginate to dopamine is 1:

3.

10. The application of the three-layer repair scaffold according to any one of claims 1-6 in repairing cartilage-subchondral bone defects of the knee joint.

Images