Highly bionic gradient artificial cartilage scaffold material, preparation method and application

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a highly-bionic gradient artificial cartilage scaffold material, a preparation method and application, the highly-bionic gradient artificial cartilage scaffold material comprises a hyaline cartilage layer, a calcified cartilage layer and a subchondral bone layer, the calcified cartilage layer is composed of mineralized I-type collagen fibers containing 30% of nano-hydroxyapatite, and the subchondral bone layer is composed of mineralized I-type collagen fibers containing 60% of nano-hydroxyapatite; the prepared scaffold highly simulates natural osteochondral tissues, has similar composition, nano structure and mechanical properties with the natural osteochondral tissues, and presents an obvious gradient; the hydrogel has excellent biocompatibility and biological activity, and cell proliferation and gradient differentiation of bone marrow mesenchymal stem cells can be remarkably promoted; in a rabbit osteochondral defect model, osteochondral tissue regeneration is remarkably promoted, and the compound can be applied to clinical treatment of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a highly biomimetic gradient artificial cartilage scaffold material, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a widespread degenerative joint disease affecting the health and quality of life of hundreds of millions of people worldwide. Osteochondrial defects are a key characteristic of severe osteoarthritis. Due to the limited self-regenerative capacity of osteochondral tissue, timely clinical intervention is necessary. Autologous tissue transplantation and allogeneic transplantation are common clinical methods for treating osteochondral defects, but they have significant drawbacks. The source of autologous grafts is extremely limited and donor complications exist, while allogeneic grafts carry risks such as immune rejection and disease transmission.

[0003] Biomimetic scaffold materials have attracted significant attention in osteochondral tissue engineering due to their ability to mimic the structure and function of natural tissues. Biphasic scaffold materials, by mimicking natural cartilage and subchondral bone, have been used to repair osteochondral defects. However, because of the significant differences in composition between the cartilage and bone layers, they cannot replicate the gradient structure of natural osteochondral tissue. Although multiphasic biomimetic scaffolds have also been developed for osteochondral tissue regeneration, their composition and structure still cannot replicate natural osteochondral tissue.

[0004] Natural osteocartilage tissue has a highly complex composition and structure, mainly consisting of a hyaline cartilage layer, a calcified cartilage layer, and a subchondral bone layer. The upper hyaline cartilage layer is primarily composed of type II collagen fibers and proteoglycans; the lower subchondral bone layer is mainly composed of type I collagen fibers and 60% in-situ mineralized hydroxyapatite; the calcified cartilage layer serves as the intermediate connecting region between the hyaline cartilage layer and the subchondral bone layer. From the hyaline cartilage layer to the subchondral bone layer, natural osteocartilage tissue exhibits a gradient composition and structure. Currently, developing gradient scaffold materials that can precisely mimic the composition, structure, and function of natural osteocartilage tissue remains an urgent and pressing problem to be solved.

[0005] The inventors unexpectedly prepared a highly biomimetic gradient artificial cartilage scaffold material using yak collagen, chondroitin sulfate, phosphate, and calcium hydroxide via a one-step freeze-drying technique. The scaffold's composition, nanostructure, and mechanical properties are highly similar to natural osteochondral tissue, exhibiting a clear gradient. This scaffold possesses excellent bioactivity, promoting the gradient differentiation of bone marrow mesenchymal stem cells into chondrocytes and osteoblasts. In a rabbit osteochondral defect model, this scaffold can promote osteochondral tissue regeneration, showing great potential for clinical treatment of osteoarthritis. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a highly biomimetic gradient artificial cartilage scaffold, comprising three layers: a hyaline cartilage layer, a calcified cartilage layer, and a subchondral bone layer. The hyaline cartilage layer is composed of collagen fibers and chondroitin sulfate, the calcified cartilage layer is composed of mineralized collagen fibers containing 30% nano-hydroxyapatite, and the subchondral bone layer is composed of mineralized collagen fibers containing 60% nano-hydroxyapatite.

[0007] Preferably, the collagen fiber is one or more of animal type I collagen fiber, type II collagen fiber, and type III collagen fiber.

[0008] Preferably, the collagen fibers of the transparent cartilage layer are yak type II collagen fibers.

[0009] Preferably, the nano-hydroxyapatite is hydroxyapatite formed by in-situ mineralization of phosphate and calcium hydroxide using collagen as a biological template.

[0010] Preferably, the mineralized collagen fiber is a complex of nano-hydroxyapatite and collagen fiber formed by simultaneous self-assembly and in-situ mineralization of collagen, phosphoric acid, and calcium hydroxide solution, wherein the collagen fiber is yak type I collagen fiber.

[0011] Preferably, the fabrication of the highly biomimetic gradient artificial cartilage scaffold mainly includes the following steps:

[0012] (1) Preparation of transparent cartilage layer: Dissolve yak type II collagen in acetic acid solution to prepare a collagen solution of 3-5 mg / mL, adjust the pH to 5.5-7.4, let stand at room temperature, centrifuge, remove the supernatant, add 0.1-1 wt% 1,4-butanediol diglycidyl ether for crosslinking at room temperature for 24-72 hours, centrifuge, wash, and mix collagen fibers and chondroitin sulfate at a mass ratio of 10:1 to obtain transparent cartilage layer gel;

[0013] (2) Preparation of calcified cartilage layer: Dissolve yak type I collagen in acetic acid solution to prepare a 10-15 mg / mL collagen solution, adjust the pH to 5.5-7.4, mix collagen and phosphoric acid solution at a mass ratio of 2.55:1 and stir evenly, then add it to calcium hydroxide suspension, control the calcium-to-phosphorus ratio to 5:3, stir and react at 25-37℃ for 5-24 hours, centrifuge, remove supernatant, crosslink the obtained precipitate with 0.1-1 wt% 1,4-butanediol diglycidyl ether at room temperature for 24-72 hours, centrifuge and wash to obtain calcified cartilage layer gel;

[0014] (3) Preparation of subchondral bone layer: Dissolve yak type I collagen in acetic acid solution to prepare a 10-15 mg / mL collagen solution, adjust the pH to 5.5-7.4, mix collagen and phosphoric acid solution at a mass ratio of 1:1.37 and stir evenly, then add it to calcium hydroxide suspension, control the calcium-to-phosphorus ratio to 5:3, stir and react at 25-37℃ for 5-24 hours, centrifuge, remove supernatant, crosslink the obtained precipitate with 0.1-1 wt% 1,4-butanediol diglycidyl ether at room temperature for 24-72 hours, centrifuge and wash to obtain subchondral bone layer gel;

[0015] (4) Place the gel of the subchondral bone layer at the bottom, the gel of the calcified cartilage layer in the middle layer, and the gel of the hyaline cartilage layer at the top. Pre-freeze the stacked three layers of gel at -35℃ for 10 hours and perform gradient freeze-drying. The procedure is as follows: -35℃ to -20℃, 5℃ / h; -20℃ to -15℃, 0.78℃ / h, and maintain at -15℃ for 1h; -15℃ to -5℃, 2℃ / h; -5℃ to 25℃, 7.5℃ / h; and maintain at 25℃ for 16hrs to obtain a highly biomimetic gradient artificial cartilage scaffold.

[0016] Preferably, the pH in step (1) is 5.5; the pH in step (2) is 6.5; and the pH in step (3) is 7.4.

[0017] A second objective of this invention is to provide the application of the highly biomimetic gradient artificial cartilage scaffold in promoting osteochondral tissue regeneration.

[0018] A third objective of this invention is to provide the application of the highly biomimetic gradient artificial cartilage scaffold in the treatment of osteoarthritis.

[0019] A fourth objective of this invention is to provide the application of the highly biomimetic gradient artificial cartilage scaffold in the fabrication of artificial cartilage.

[0020] A fifth objective of this invention is to provide the application of the highly biomimetic gradient artificial cartilage scaffold in the preparation of biomaterials and medical devices.

[0021] The beneficial effects of the present invention are: (1) The present invention provides a highly biomimetic gradient artificial cartilage scaffold, the scaffold comprising three layers: a hyaline cartilage layer, a calcified cartilage layer, and a subchondral bone layer; the hyaline cartilage layer is composed of type II collagen fibers and chondroitin sulfate, the calcified cartilage layer is composed of mineralized type I collagen fibers containing 30% nano-hydroxyapatite, and the subchondral bone layer is composed of mineralized type I collagen fibers containing 60% nano-hydroxyapatite; (2) The prepared scaffold can highly mimic the complex composition, microstructure, and mechanical properties of natural osteochondral tissue; (3) The scaffold exhibits a clear gradient from the hyaline cartilage layer to the subchondral bone layer; (4) The scaffold has excellent biocompatibility and can significantly promote cell proliferation; (5) The scaffold has excellent biological activity and can promote the gradient differentiation of bone marrow mesenchymal stem cells; (6) The scaffold has outstanding osteochondral tissue regeneration capacity and can be applied to the clinical treatment of osteoarthritis. Attached Figure Description

[0022] Figure 1 Morphological characterization of scaffold materials: Note: AC: hyaline cartilage; CC: calcified cartilage; SB: subchondral bone; BGC: biomimetic gradient artificial cartilage; Where: A is an anatomical photograph of the BGC scaffold material; B is a SEM image of the BGC scaffold material; C is a SEM image of the AC scaffold material; D is a SEM image of the CC scaffold material; E and F are SEM images of the SB scaffold material.

[0023] Figure 2 TEM characterization of scaffold materials: Note: A is the TEM image of the AC layer; B is the TEM image of the CC layer; C is the TEM image of the SB layer.

[0024] Figure 3 Characterization of stent material composition: Note: A is the FT-IR image of the stent material; B is the XRD pattern of the stent material; C is the TGA image of the stent material.

[0025] Figure 4 Mechanical properties of scaffold materials

[0026] Note: A represents the compressive strength of the support material; B represents the compressive modulus of the support material.

[0027] Figure 5 Live / dead cell staining images of bone marrow mesenchymal stem cells cultured on scaffold material.

[0028] Figure 6Expression of cartilage and osteogenic-related genes in bone marrow mesenchymal stem cells cultured on scaffold materials. Note: A: Relative expression level of Acan gene on scaffold material; B: Relative expression level of Col2a1 gene on scaffold material; C: Relative expression level of Sox9 gene on scaffold material; D: Relative expression level of ALP gene on scaffold material; E: Relative expression level of Col1a1 gene on scaffold material; F: Relative expression level of Runx2 gene on scaffold material.

[0029] Figure 7 Osteochondrial tissue regeneration at 6 and 12 weeks after scaffold material implantation into the defect. Note: A is the visual image; B is the Micro-CT image; C and D are the BMD, BV / TV, and Tb.N values, respectively.

[0030] Figure 8 Histological staining images of rabbit cartilage defects repaired with scaffold material

[0031] Note: A is an H&E staining image; B is a Masson staining image. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] It should be noted that, unless otherwise specified, all reagents used in the following examples can be purchased commercially.

[0034] In the following embodiments, it should be noted that AC is short for hyaline cartilage; CC is short for calcified cartilage; SB is short for subchondral bone; and BGC is short for biomimetic gradient artificial cartilage. In some embodiments and accompanying drawings, the abbreviations are used directly instead of the full names, and they have the same meaning.

[0035] Example 1: Preparation of hyaline cartilage (AC) scaffold

[0036] Yak type II collagen was dissolved in acetic acid solution to form a homogeneous solution of 5 mg / mL. The pH was adjusted to 5.5, and the solution was allowed to stand at room temperature. The precipitate obtained by centrifugation was cross-linked with 1 wt% 1,4-butanediol diglycidyl ether (BDDE) at room temperature for 24 hours. After centrifugation and washing, collagen fibers and chondroitin sulfate were mixed at a mass ratio of 10:1. After pre-freezing at -35°C for 10 hours, the solution was freeze-dried according to the following program: -35°C to -20°C, 5°C / h; -20°C to -15°C, 0.78°C / h, and maintained at -15°C for 1 hour; -15°C to -5°C, 2°C / h; -5°C to 25°C, 7.5°C / h; and maintained at 25°C for 16 hours to obtain hyaline cartilage (AC) scaffold.

[0037] Example 2: Preparation of calcified cartilage (CC) scaffold

[0038] Yak type I collagen was dissolved in acetic acid solution to form a homogeneous solution of 15 mg / mL. The pH was adjusted to 6.5. Collagen and phosphoric acid solution were mixed at a mass ratio of 2.55:1 and stirred until homogeneous. Then, it was added to calcium hydroxide suspension, and the calcium-to-phosphorus ratio was controlled at 5:3. After stirring and reacting at 25°C for 24 hours, the precipitate obtained by centrifugation was cross-linked with 1 wt% BDDE at room temperature for 24 hours. After centrifugation and washing, it was pre-frozen at -35°C for 10 hours. Then, it was freeze-dried according to the following program: -35°C to -20°C, 5°C / h; -20°C to -15°C, 0.78°C / h, and maintained at -15°C for 1h; -15°C to -5°C, 2°C / h; -5°C to 25°C, 7.5°C / h; and maintained at 25°C for 16 hours to obtain calcified cartilage (CC) scaffold.

[0039] Example 3: Preparation of subchondral bone (SB) scaffold

[0040] Yak type I collagen was dissolved in acetic acid solution to form a homogeneous solution of 15 mg / mL. The pH was adjusted to 7.4. Collagen and phosphoric acid solution were mixed at a mass ratio of 1:1.37 and stirred until homogeneous. Then, it was added to calcium hydroxide suspension, and the calcium-to-phosphorus ratio was controlled at 5:3. After stirring and reacting at 25°C for 24 hours, the precipitate obtained by centrifugation was cross-linked with 1 wt% BDDE at room temperature for 24 hours. After centrifugation and washing, it was pre-frozen at -35°C for 10 hours. Then, it was freeze-dried according to the following program: -35°C to -20°C, 5°C / h; -20°C to -15°C, 0.78°C / h, and maintained at -15°C for 1h; -15°C to -5°C, 2°C / h; -5°C to 25°C, 7.5°C / h; and maintained at 25°C for 16 hours to obtain subchondral bone (SB) scaffold.

[0041] Example 4: Fabrication of a highly biomimetic gradient artificial cartilage (BGC) scaffold

[0042] (1) Yak type II collagen was dissolved in acetic acid solution to form a homogeneous solution of 3 mg / mL. The pH was adjusted to 5.5, and the solution was allowed to stand at room temperature. The precipitate obtained by centrifugation was cross-linked with 0.1 wt% BDDE at room temperature for 72 hours. After centrifugation and washing, collagen fibers and chondroitin sulfate were mixed at a mass ratio of 10:1 to obtain a gel of the transparent cartilage layer.

[0043] (2) Yak type I collagen was dissolved in acetic acid solution to form a homogeneous solution of 10 mg / mL. The pH was adjusted to 6.5. Collagen and phosphoric acid solution were mixed at a mass ratio of 2.55:1 and stirred evenly. Then it was added to calcium hydroxide suspension and the calcium-to-phosphorus ratio was controlled at 5:3. After stirring and reacting at 37°C for 5 hours, the precipitate obtained by centrifugation was cross-linked with 0.1 wt% BDDE at room temperature for 72 hours. After centrifugation and washing, the gel of calcified cartilage layer was obtained.

[0044] (3) Yak type I collagen was dissolved in acetic acid solution to form a homogeneous solution of 10 mg / mL. The pH was adjusted to 7.4. Collagen and phosphoric acid solution were mixed at a mass ratio of 1:1.37 and stirred evenly. Then it was added to calcium hydroxide suspension and the calcium-to-phosphorus ratio was controlled at 5:3. After stirring and reacting at 37°C for 5 hours, the precipitate obtained by centrifugation was cross-linked with 0.1 wt% BDDE at room temperature for 72 hours. After centrifugation and washing, the subchondral bone layer gel was obtained.

[0045] (4) Place the gel of the subchondral bone layer at the bottom, the gel of the calcified cartilage layer in the middle layer, and the gel of the hyaline cartilage layer at the top. Then, pre-freeze the stacked three layers of gel material at -35℃ for 10 hours. Finally, perform gradient freeze-drying. The procedure is as follows: -35℃ to -20℃, 5℃ / h; -20℃ to -15℃, 0.78℃ / h, and maintain at -15℃ for 1h; -15℃ to -5℃, 2℃ / h; -5℃ to 25℃, 7.5℃ / h; and maintain at 25℃ for 16hrs. A highly biomimetic gradient artificial cartilage (BGC) scaffold can be obtained.

[0046] The morphology of the prepared BGC scaffold is as follows Figure 1 As shown in Figure A, the results indicate that the BGC scaffold exhibits a gradient white sponge-like structure and possesses stable interfacial bonding.

[0047] Example 5: Preparation of Type I Collagen (Col I) Scaffold

[0048] Yak type I collagen was dissolved in acetic acid solution to form a homogeneous solution of 5 mg / mL. The pH was adjusted to 5.5, and the solution was allowed to stand at room temperature. The precipitate obtained by centrifugation was cross-linked with 0.1 wt% BDDE at room temperature for 72 hours. After centrifugation and washing, the solution was pre-frozen at -35°C for 10 hours and then freeze-dried according to the following program: -35°C to -20°C, 5°C / h; -20°C to -15°C, 0.78°C / h, and maintained at -15°C for 1 hour; -15°C to -5°C, 2°C / h; -5°C to 25°C, 7.5°C / h; and maintained at 25°C for 16 hours to obtain type I collagen (Col I) scaffold.

[0049] Example 6: Preparation of a type I collagen and hydroxyapatite (Col I-HAp) composite scaffold

[0050] Yak type I collagen was dissolved in acetic acid solution to form a homogeneous solution of 3 mg / mL. The pH was adjusted to 7.4, and then 2 volumes of PBS solution were added. After incubation at 37°C for 4 hours, the precipitate obtained by centrifugation was cross-linked with 0.1 wt% BDDE at room temperature for 72 hours. After centrifugation and washing, collagen and hydroxyapatite nanoparticles were mixed homogeneously at a mass ratio of 4:6 and pre-frozen at -35°C for 10 hours. Then, the mixture was freeze-dried according to the following program: -35°C to -20°C, 5°C / h; -20°C to -15°C, 0.78°C / h, and maintained at -15°C for 1 hour; -15°C to -5°C, 2°C / h; -5°C to 25°C, 7.5°C / h; and maintained at 25°C for 16 hours to obtain the type I collagen and hydroxyapatite (Col I-HAp) composite scaffold.

[0051] Example 7: Morphological characterization of a highly biomimetic gradient artificial cartilage (BGC) scaffold

[0052] The BGC scaffold prepared in Example 4 was fixed on the sample stage, and after sputtering gold for 25 seconds, the morphology of the sample was detected by scanning electron microscopy (SEM) at a voltage of 5.0 kV.

[0053] The results of scanning electron microscopy are as follows Figure 1 Figures B and F show SEM images of the BGC scaffold material, with BB being the SEM image of the AC scaffold material, C the SEM image of the CC scaffold material, and E and F the SEM images of the SB scaffold material. The results show that each layer of the BGC scaffold has clear boundaries and stable interfaces; the AC, CC, and SB layers of the BGC scaffold all exhibit a highly interconnected porous structure with gradually decreasing pore size; in the SB layer of the BGC scaffold, a large number of minerals are uniformly distributed along the collagen fibers. These results indicate that the structure of the BGC scaffold is similar to that of natural osteochondral tissue.

[0054] Example 8: Nanostructure characterization of a highly biomimetic gradient artificial cartilage (BGC) scaffold

[0055] The AC, CC and SB gels prepared in (1), (2) and (3) of Example 4 were dispersed in an aqueous solution, then dropped onto a copper grid and air-dried. Their nanostructures were characterized by transmission electron microscopy (TEM) at 200 kV.

[0056] Results of transmission electron microscopy as follows Figure 2 The images shown are TEM images of layers AC, CC, and SB, respectively. The results indicate that layer AC contains only small-sized type II collagen fibers; layer CC shows a small amount of in-situ mineralized hydroxyapatite crystals distributed along type I collagen fibers; and layer SB displays larger-sized type I collagen fibers with abundant in-situ hydroxyapatite crystals on their surface. These results suggest that the size gradient of collagen fibers in the BGC scaffold increases from layer AC to layer SB, and the content of in-situ mineralized hydroxyapatite gradually increases.

[0057] Example 9: Compositional Characterization of a Highly Biomimetic Gradient Artificial Cartilage (BGC) Scaffold

[0058] (1) The AC, CC and SB layers in the BGC scaffold prepared in Example 4 were mixed and ground with potassium bromide particles at a mass ratio of 1:100. Then the powder was pressed into a transparent sheet and Fourier transform infrared (FT-IR) was measured.

[0059] The results are as follows Figure 3 As shown in Figure A, the AC bracket is at 1660cm. -1 The C=O peak is characteristic of collagen; the CC scaffold is at 1032 cm⁻¹. -1 603cm -1 and 562cm -1 The characteristic peaks that appeared belonged to phosphate groups; the SB scaffold at 1032 cm⁻¹ -1 603cm -1 and 562cm -1 The characteristic peaks that appeared were significantly enhanced. The results indicate that the BGC scaffold is mainly composed of collagen and hydroxyapatite, and exhibits a gradient distribution.

[0060] (2) The AC, CC and SB layers of the BGC scaffold prepared in Example 4 were placed on a powder X-ray diffractometer (XRD) and their crystal structure types were determined at a scanning rate of 0.02° / s.

[0061] The results are as follows Figure 3As shown in Figure B, the SB scaffold exhibits distinct characteristic peaks at 26.4°, 32.3°, 39.8°, 46.9°, and 49.8°, corresponding to the (002), (211), (310), (222), and (213) diffraction planes of hydroxyapatite, respectively. The CC scaffold shows relatively weaker characteristic peaks at 26.4°, 32.3°, 39.8°, 46.9°, and 49.8°, while the AC scaffold does not show any characteristic peaks of hydroxyapatite. The results indicate that the hydroxyapatite content in the BGC scaffold gradually increases from AC to SB.

[0062] (3) The AC, CC and SB layers of the BGC scaffold prepared in Example 4 were placed in the crucible of a thermogravimetric analyzer (TGA) and the mass change between 50°C and 800°C was measured at a scanning rate of 5°C / min.

[0063] The results are as follows Figure 3 As shown in Figure C, the SB scaffold has approximately 60% remaining mineral content; the CC scaffold has approximately 30% remaining mineral content; while the AC scaffold has virtually no remaining mineral content. The results indicate that the inorganic content and distribution in the BGC scaffold are similar to those of natural osteochondral tissue, exhibiting a clear gradient.

[0064] Example 10: Mechanical Properties of a Highly Biomimetic Gradient Artificial Cartilage (BGC) Scaffold

[0065] AC, CC, SB, and BGC stents with a diameter of 15 mm and a thickness of 12 mm were prepared according to the steps in Examples 1-4, and finally a compression test was performed at a speed of 1 mm / min.

[0066] The results are as follows Figure 4 As shown in the figure, 4A represents the compressive strength of the stent material; 4B represents the compressive modulus of the stent material. The results show that the compressive strengths of the AC, CC, SB, and BGC stents are 0.16 MPa, 0.30 MPa, 0.65 MPa, and 0.41 MPa, respectively, and their compressive moduli are 0.20 MPa, 0.47 MPa, 0.95 MPa, and 0.59 MPa, respectively. The results indicate that the BGC stent possesses excellent mechanical strength, and its mechanical properties gradually increase from AC to SB.

[0067] Example 11 Cell proliferation of a highly biomimetic gradient artificial cartilage (BGC) scaffold

[0068] Adherent rat bone marrow mesenchymal stem cells (BMSCs) were digested with trypsin to form a density of 1×10⁻⁶. 6A suspension of cells / mL was prepared. These suspensions were then seeded onto the surfaces of AC, CC, and SB scaffold materials and cultured in α-MEM complete medium for 1, 7, and 14 days, respectively. Live and dead cells were stained with calcein-acetylacetyl methyl ester (Calcein-AM) and propidium iodide (PI) at 37°C for 1 hour. Finally, cell fluorescence images were acquired using a laser confocal microscope.

[0069] The results are as follows Figure 5 The images show staining patterns of live and dead BMSCs cultured on AC, CC, and SB scaffolds for 1, 7, and 14 days, respectively. The results indicate that the number of live cells on the AC, CC, and SB scaffolds increased with increasing culture time. These results demonstrate that the BGC scaffold exhibits excellent biocompatibility and significantly promotes cell proliferation.

[0070] Example 12 Cell Differentiation of a Highly Biomimetic Gradient Artificial Cartilage (BGC) Scaffold

[0071] (1) The density is 1×10 6 Cell suspensions of [number] cells / mL were seeded onto Col I, AC, CC, and SB scaffold materials and cultured in α-MEM complete medium for 14 days. The control group used the same cell number and culture time but without scaffold material. Total RNA was extracted using an RNA isolation kit, cDNA was synthesized using the PrimeScript RT reagent Kit and gDNA Eraser, gene expression levels were analyzed using TB Green Premix Ex Taq II, and the expression of cartilage-specific genes was detected using a Real-Time PCR System. Finally, 2 [units of measurement missing] were used. -ΔΔCT The relative expression level of the target gene was calculated using a method with β-actin as the internal reference gene, and the expression level was normalized based on the gene expression of the blank group on day 14.

[0072] The results are as follows Figure 6 The figures show the relative expression levels of chondrocyte differentiation genes Acan, Col2a1, and Sox9 in Col I, AC, CC, and SB scaffolds, respectively. The results indicated that after 14 days of co-culture with the scaffolds, Acan, Col2a1, and Sox9 genes showed the highest expression in the AC scaffold, while the SB scaffold showed the lowest expression; furthermore, the gene expression in the AC scaffold was significantly higher than that in the Col I scaffold. These results suggest that the BGC scaffold significantly promotes graded chondrocyte differentiation of bone marrow mesenchymal stem cells, and the highly biomimetic AC scaffold (containing type II collagen fibers and chondroitin sulfate) exhibits superior chondrocyte differentiation capacity compared to the Col I scaffold (containing type I collagen fibers).

[0073] (2) The density is 1×106 Cell suspensions of [number] cells / mL were seeded onto AC, CC, SB, and Col-HAp scaffold materials and cultured in α-MEM complete medium for 14 days. The control group used the same cell number and culture time but without scaffold material. Total RNA was extracted using an RNA isolation kit, cDNA was synthesized using the PrimeScript RT reagent Kit and gDNAEraser, gene expression levels were analyzed using TB Green Premix Ex Taq II, and the expression of cartilage-specific genes was detected using a Real-Time PCR System. Finally, 2 [units of measurement missing] were used. -ΔΔCT The relative expression level of the target gene was calculated using a method with β-actin as the internal reference gene, and the expression level was normalized based on the gene expression of the blank group on day 14.

[0074] The results are as follows Figure 6 As shown in Figure DF, the relative expression levels of ALP, Col1a1, and Runx 2 genes in AC, CC, SB, and Col-HAp scaffolds are, respectively. The results showed that after 14 days of co-culture with the scaffolds, ALP, Col1a1, and Runx 2 genes had the highest expression in the SB scaffold, while the osteogenic gene expression was lowest in the AC scaffold; and the gene expression in the SB scaffold was significantly higher than that in the Col I-HAp scaffold. These results indicate that the BGC scaffold significantly promotes graded osteogenic differentiation of bone marrow mesenchymal stem cells, and the highly biomimetic SB scaffold (a mixture of type I collagen fibers and in-situ mineralized hydroxyapatite) exhibits superior osteogenic differentiation capacity compared to the Col I-HAp scaffold (a mixture of type I collagen and nano-hydroxyapatite particles).

[0075] Example 13: Tissue Regeneration of a Highly Biomimetic Gradient Artificial Cartilage (BGC) Scaffold

[0076] (1) Male New Zealand white rabbits (2.5–3 kg) were housed in a standard laboratory. Anesthesia was administered via intravenous injection of 2% (w / v) sodium pentobarbital (30 mg / kg). After shaving and disinfection of the surgical site, the skin and muscle tissue covering the right knee of each rabbit were incised with a scalpel to expose the trochlea. A cylindrical osteochondral defect with a diameter of 4 mm and a height of 3 mm was created in the trochlea of ​​the right knee of each rabbit using an electric drill. BGC scaffold material was implanted in the experimental group, while no treatment was given in the control group. The muscles and skin of the surgical site in both the experimental and control groups were sutured sequentially. Twelve weeks after implantation, the rabbits were euthanized with an overdose of sodium pentobarbital, and samples were collected.

[0077] The results are as follows Figure 7As shown in Figure A, at 6 weeks post-operation, the Blank group showed a large vacancy, while the BGC group showed significant osteochondral defect repair. At 12 weeks post-operation, the Blank group regenerated a small amount of osteochondral tissue, but significant defects were still visible. The regenerated cartilage in the BGC group had a smooth surface and fused well with the surrounding tissue. The results indicate that the BGC group has outstanding osteochondral regeneration capacity.

[0078] (2) After the collected specimens were fixed in 4% formaldehyde for 48 hours, the femoral structure was scanned using a Micro-CT imaging device. The scanning voltage was 40kV, the current was 250μA, the exposure time was 240ms, and the tomographic thickness was 0.1mm.

[0079] The results are as follows Figure 7 Figure BE shows the results, where B represents the 3D and 2D images from Micro-CT; C represents the bone mineral density (BMD) measurement; D represents the bone volume / total volume (BV / TV) measurement; and E represents the trabecular bone number (Tb.N) measurement. The results showed that at 12 weeks post-surgery, the Blank group still had significant defects, while the defects in the BGC group were completely filled by newly formed osteochondral tissue. The BMD values ​​for the Blank and BGC groups were 0.22 g / cc and 0.51 g / cc, respectively; the BV / TV values ​​for the Blank and BGC groups were 10.04% and 26.38%, respectively; and the Tb.N values ​​for the Blank and BGC groups were 0.56 I / mm and 1.22 I / mm, respectively. These results indicate that the BGC scaffold significantly promotes osteochondral tissue regeneration.

[0080] Example 14: Histological staining of osteochondral regeneration induced by a highly biomimetic gradient artificial cartilage (BGC) scaffold.

[0081] (1) The fixed samples were treated in 10% EDTA solution for 14 days to completely decalcify. Then the samples were dehydrated in alcohol in a gradient, embedded in paraffin, and sectioned to form 5 μm thin sections. The tissue sections were stained with hematoxylin and eosin (H&E).

[0082] The results are as follows Figure 8 As shown in Figure A, at 6 weeks post-surgery, the Blank group exhibited significant tissue defects accompanied by severe inflammation, while the BGC group regenerated a small amount of osteochondral tissue. At 12 weeks post-surgery, the Blank group regenerated a small amount of osteochondral tissue, while the defects in the BGC group were completely filled by the newly generated osteochondral tissue, resembling natural osteochondral tissue and containing a large number of mature chondrocytes. These results indicate that the BGC scaffold possesses excellent osteochondral defect repair capabilities.

[0083] (2) The fixed samples were treated in 10% EDTA solution for 14 days to completely decalcify. Then the samples were dehydrated in alcohol in a gradient, embedded in paraffin, and sectioned to form 5 μm thin sections. Masson staining was performed on the tissue sections.

[0084] The results are as follows Figure 8 As shown in Figure B, at 6 weeks post-surgery, the Blank group exhibited minimal collagen fiber formation, while the BGC group showed relatively abundant collagen fiber formation. At 12 weeks post-surgery, the Blank group showed increased collagen fiber regeneration, while the BGC group showed abundant collagen fiber formation. These results indicate that the BGC scaffold significantly promotes collagen regeneration in osteochondral tissue.

[0085] In summary, this invention provides a highly biomimetic gradient artificial cartilage scaffold, comprising three layers: a hyaline cartilage layer, a calcified cartilage layer, and a subchondral bone layer. The hyaline cartilage layer is composed of type II collagen fibers and chondroitin sulfate; the calcified cartilage layer is composed of mineralized type I collagen fibers containing 30% nano-hydroxyapatite; and the subchondral bone layer is composed of mineralized type I collagen fibers containing 60% nano-hydroxyapatite. The prepared highly biomimetic gradient artificial cartilage scaffold is white and spongy, with stable interfacial bonding and a biomimetic nanoporous structure. The scaffold mainly consists of collagen fibers and in-situ mineralized hydroxyapatite, the content, distribution, and structure of which are similar to natural bone and cartilage tissue, exhibiting a gradient trend. It possesses excellent mechanical properties, with its strength gradually increasing from AC to SB. The scaffold exhibits significant biocompatibility, significantly promoting cell proliferation; and demonstrates outstanding biological activity, promoting the gradient differentiation of bone marrow mesenchymal stem cells. The scaffold described has outstanding tissue regeneration capacity and can significantly promote the repair of osteocartilage defects, and can be applied to the clinical treatment of osteoarthritis.

Claims

1. A highly biomimetic gradient artificial cartilage scaffold material, characterized in that, The scaffold comprises three layers: a hyaline cartilage layer, a calcified cartilage layer, and a subchondral bone layer. The hyaline cartilage layer is composed of collagen fibers and chondroitin sulfate. The calcified cartilage layer is composed of mineralized collagen fibers containing 30% nano-hydroxyapatite. The subchondral bone layer is composed of mineralized collagen fibers containing 60% nano-hydroxyapatite.

2. The highly biomimetic gradient artificial cartilage scaffold as described in claim 1, characterized in that, The collagen fibers mentioned are one or more of animal type I collagen fibers, type II collagen fibers, and type III collagen fibers.

3. The highly biomimetic gradient artificial cartilage scaffold as described in claim 1, characterized in that, The collagen fibers in the transparent cartilage layer are yak type II collagen fibers.

4. The highly biomimetic gradient artificial cartilage scaffold as described in claim 1, characterized in that, The nano-hydroxyapatite is formed by in-situ mineralization of phosphate and calcium hydroxide using collagen as a biological template. The mineralized collagen fiber is a complex of nano-hydroxyapatite and collagen fiber formed by simultaneous self-assembly and in-situ mineralization of collagen, phosphate and calcium hydroxide solution. The collagen fiber is yak type I collagen fiber.

5. A method for preparing the highly biomimetic gradient artificial cartilage scaffold according to any one of claims 1-4, characterized in that, The method includes the following steps: (1) Preparation of a transparent cartilage layer: Dissolve yak type II collagen in acetic acid solution to prepare a collagen solution of 3-5 mg / mL, adjust the pH to 5.5-7.4, let stand at room temperature, centrifuge, remove the supernatant, add 0.1-1 wt% 1,4-butanediol diglycidyl ether to crosslink at room temperature for 24-72 hours, centrifuge, wash, and mix collagen fibers and chondroitin sulfate at a mass ratio of 10:1 to obtain a transparent cartilage layer gel; (2) Preparation of calcified cartilage layer: Dissolve yak type I collagen in acetic acid solution to prepare a 10-15 mg / mL collagen solution, adjust the pH to 5.5-7.4, mix collagen and phosphoric acid solution at a mass ratio of 2.55:1 and stir evenly, then add it to calcium hydroxide suspension, control the calcium-to-phosphorus ratio to 5:3, stir and react at 25-37℃ for 5-24 hours, centrifuge, remove supernatant, crosslink the obtained precipitate with 0.1-1 wt% 1,4-butanediol diglycidyl ether at room temperature for 24-72 hours, centrifuge and wash to obtain calcified cartilage layer gel; (3) Preparation of subchondral bone layer: Dissolve yak type I collagen in acetic acid solution to form a homogeneous solution of 10-15 mg / mL, adjust the pH to 5.5-7.4, mix collagen and phosphoric acid solution at a mass ratio of 1:1.37 and stir evenly, then add it to calcium hydroxide suspension, control the calcium-to-phosphorus ratio to 5:3, stir and react at 25-37℃ for 5-24 hours, centrifuge, remove supernatant, crosslink the obtained precipitate with 0.1-1 wt% 1,4-butanediol diglycidyl ether at room temperature for 24-72 hours, centrifuge and wash to obtain subchondral bone layer gel; (4) Place the gel of the subchondral bone layer at the bottom, the gel of the calcified cartilage layer in the middle layer, and the gel of the hyaline cartilage layer at the top. Pre-freeze the stacked three layers of gel at -35℃ for 10 hours, and then perform gradient freeze drying. The procedure is as follows: -35℃ to -20℃, 5℃ / h; -20℃ to -15℃, 0.78℃ / h, and maintain at -15℃ for 1h; -15℃ to -5℃, 2℃ / h; -5℃ to 25℃, 7.5℃ / h; maintain at 25℃ for 16hrs. A highly biomimetic gradient artificial cartilage scaffold can be obtained.

6. The application of the highly biomimetic gradient artificial cartilage scaffold as described in any one of claims 1-4 in promoting osteochondral tissue regeneration.

7. The application of the highly biomimetic gradient artificial cartilage scaffold as described in any one of claims 1-4 in the treatment of osteoarthritis.

8. The application of the highly biomimetic gradient artificial cartilage scaffold as described in any one of claims 1-4 in the preparation of artificial cartilage.

9. The application of the highly biomimetic gradient artificial cartilage scaffold as described in any one of claims 1-4 in the preparation of biomaterials and medical devices.

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