Bionic magnesium alloy material for inducing synchronous bone-cartilage regeneration and preparation method thereof

Abstract

The application provides a kind of to induce bone-cartilage synchronous regeneration biomimetic magnesium alloy material and preparation method thereof, it is related to biomedical material technical field, the to induce bone-cartilage synchronous regeneration biomimetic magnesium alloy material includes porous magnesium alloy support and layered solidification in the pore of porous magnesium alloy support biomimetic hydrogel HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q;The HAMA-RGD@BMM-KGN is the biomimetic hydrogel formed by dispersing BMM loaded with KGN in HAMA-RGD solution;The HAMA-RGD@BMM-Q is the biomimetic hydrogel formed by dispersing BMM loaded with quercetin Q in HAMA-RGD solution.The to induce bone-cartilage synchronous regeneration biomimetic magnesium alloy material can provide stable three-dimensional mechanical support on macroscopic, reconstruct the spatial gradient structure of cartilage and subchondral bone on microscopic, realize "bone-cartilage" synchronous, zoned regeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a biomimetic magnesium alloy material that induces simultaneous bone-cartilage regeneration and its preparation method. Background Technology

[0002] The osteochondral unit is the functional core that maintains joint weight-bearing, cushioning, and stable movement. Its microstructure, from the articular surface downwards, consists of hyaline cartilage, calcified cartilage, and subchondral bone, exhibiting a clear compositional and mechanical gradient. Once a large-scale osteochondral defect occurs, penetrating the cartilage and subchondral bone, local stress distribution becomes unbalanced, and the cartilage matrix undergoes continuous degeneration, eventually progressing to severe pain, limited mobility, and even end-stage joint disease. Such large-scale osteochondral defects are common in trauma, sports injuries, and the progression of osteoarthritis; however, currently, there is a lack of ideal treatment methods that can simultaneously reconstruct the complete structure and function of both the cartilage and subchondral bone.

[0003] Current clinical repair strategies, such as microfracture techniques, autologous / allogeneic osteochondral transplantation, and autologous chondrocyte transplantation, can restore joint surface continuity to some extent in small, focal defects. However, they generally face problems such as insufficient donor sites, poor geometric and mechanical matching between grafts and recipient sites, poor integration of the cartilage-bone interface, and the fact that repaired tissue often resembles fibrocartilage. These shortcomings make it difficult for repaired tissue to withstand the complex weight-bearing environment of the joint over a long period, leading to collapse, re-injury, or disease recurrence. Therefore, how to provide stable three-dimensional mechanical support on a macroscopic level and reconstruct the spatial gradient structure of cartilage and subchondral bone on a microscopic level to achieve synchronous and regional regeneration of bone and cartilage is an urgent problem to be solved in the field of bone-cartilage repair technology. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention solves the following technical problem: providing a biomimetic magnesium alloy material and its preparation method for inducing synchronous bone-cartilage regeneration, which can provide stable three-dimensional mechanical support on a macroscopic level and reconstruct the spatial gradient structure of cartilage and subchondral bone on a microscopic level, thereby achieving synchronous and zoned regeneration of bone and cartilage.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a biomimetic magnesium alloy material for inducing synchronous bone-cartilage regeneration, characterized in that it comprises a porous magnesium alloy scaffold and biomimetic hydrogels HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q, which are layered and solidified in the pores of the porous magnesium alloy scaffold; wherein HAMA-RGD@BMM-KGN is a biomimetic hydrogel formed by dispersing BMM loaded with KGN in a HAMA-RGD solution; and HAMA-RGD@BMM-Q is a biomimetic hydrogel formed by dispersing BMM loaded with quercetin Q in a HAMA-RGD solution.

[0006] Preferably, the HAMA-RGD@BMM-KGN is cured on the upper 1 / 3 of the porous magnesium alloy scaffold, and the HAMA-RGD@BMM-Q is cured on the lower 2 / 3 of the porous magnesium alloy scaffold.

[0007] Preferably, the HAMA-RGD solution comprises 2% HAMA, 1% RGD, and 0.1% photoinitiator by mass-volume ratio.

[0008] Preferably, the porous magnesium alloy scaffold is a three-dimensional scaffold with a pore size of 500 μm.

[0009] Preferably, the porous magnesium alloy support is made of magnesium-zinc alloy, wherein, by mass fraction, the magnesium-zinc alloy comprises 95-99% magnesium and 1-5% zinc.

[0010] Secondly, the present invention also provides a method for preparing a biomimetic magnesium alloy material that induces simultaneous bone-cartilage regeneration, the method comprising the following steps:

[0011] BMM loaded with KGN was dispersed in HAMA-RGD solution to form a biomimetic hydrogel HAMA-RGD@BMM-KGN;

[0012] BMM loaded with Q was dispersed in HAMA-RGD solution to form a biomimetic hydrogel HAMA-RGD@BMM-Q;

[0013] The HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q were layered and infused into the pores of a porous magnesium alloy scaffold, and then cured to obtain the biomimetic magnesium alloy material that induces synchronous bone-cartilage regeneration.

[0014] Preferably, the method for preparing BMM loaded with KGN includes: preparing a stock solution of KGN at a concentration of 10-20 mg / mL, mixing KGN and BMM at a mass ratio of 1:50, and incubating at 37°C for 1 hour to obtain BMM-KGN.

[0015] Preferably, the preparation method of the BMM loaded with Q includes: preparing a Q solution of 10-20 mg / mL, mixing the Q solution with BMM at a Q to BMM mass ratio of 1:50, and incubating at 37°C for 1 h to obtain BMM-Q.

[0016] Preferably, the specific steps for layering and injecting the HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q into the pores of a porous magnesium alloy scaffold and curing them to obtain the biomimetic magnesium alloy material that induces synchronous bone-cartilage regeneration include: injecting the HAMA-RGD@BMM-KGN into the upper 1 / 3 of the porous magnesium alloy scaffold and photocuring it, and then injecting the HAMA-RGD@BMM-Q into the lower 2 / 3 of the porous magnesium alloy scaffold and photocuring it, thereby obtaining the biomimetic magnesium alloy material that induces synchronous bone-cartilage regeneration.

[0017] Preferably, the photocuring conditions are: irradiation with light of wavelength 405nm for 30-60s.

[0018] Compared with existing technologies, the advantages of this invention are as follows: This invention constructs a biomimetic magnesium alloy material (MZ-Gel) targeting simultaneous bone-cartilage regeneration: Kartogenin (KGN) or quercetin (Q) is loaded onto stem cell membrane vesicles (BMM) to construct two types of biomimetic hydrogels targeting mesenchymal stem cells: HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q. The spatial layering of these hydrogels with a porous magnesium alloy scaffold creates a "cartilage-bone" functional gradient, primarily driving cartilage generation in the upper layer and enhancing osteogenic and bone remodeling in the lower layer. Simultaneously, the Mg²⁺ released during magnesium alloy degradation is utilized... + Synergistic regulation of the local microenvironment. Therefore, the biomimetic magnesium alloy material for inducing synchronous bone-cartilage regeneration of the present invention can provide stable three-dimensional mechanical support on a macroscopic level and reconstruct the spatial gradient structure of cartilage and subchondral bone on a microscopic level, thereby achieving synchronous and zoned regeneration of bone and cartilage. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the preparation process of the biomimetic magnesium alloy material for inducing synchronous bone-cartilage regeneration according to an embodiment of the present invention.

[0021] Figure 2 This is a particle size distribution diagram of NTA detection in BMM in Example 1 of the present invention;

[0022] Figure 3 This is a transmission electron microscope (TEM) image of the BMM in Embodiment 1 of the present invention;

[0023] Figure 4The BMM in Embodiment 2 of the present invention TEM image of KGN;

[0024] Figure 5 The BMM in Embodiment 2 of the present invention TEM image of Q;

[0025] Figure 6 This is a Micro-CT front view of the magnesium alloy stent with a nail-shaped structure in Embodiment 4 of the present invention;

[0026] Figure 7 This is a Micro-CT top view of the magnesium alloy stent with a nail-shaped structure in Embodiment 4 of the present invention;

[0027] Figure 8 This is a Micro-CT image of the porous magnesium alloy stent in Embodiment 4 of the present invention;

[0028] Figure 9 This is a SEM image of the porous magnesium alloy support in Embodiment 4 of the present invention;

[0029] Figure 10 This is a magnified SEM image of a porous magnesium alloy support in Embodiment 4 of the present invention.

[0030] Figure 11 This is a diagram showing the relative mRNA expression of COL2 in Example 5 of the present invention;

[0031] Figure 12 This is a diagram showing the relative mRNA expression of Runx2 in Example 5 of the present invention;

[0032] Figure 13 This is a diagram showing the relative mRNA expression of ALP in Example 5 of the present invention;

[0033] Figure 14 This is a diagram showing the relative mRNA expression of COL1 in Example 5 of the present invention;

[0034] Figures 11-14 The black dots represent the raw data of biological experiments. For example, if each bar chart contains 3 black dots, it means that each group performed 3 replicate experiments and obtained 3 raw data points.

[0035] Figures 11-14 The "T" symbol in the figure represents an error bar, used to describe the standard deviation (SD) of the three raw data.

[0036] Figure 15 These are toluidine blue staining images of each group in Example 5 of the present invention;

[0037] Figure 16 These are ALP staining images of each group in Example 5 of the present invention;

[0038] Figure 17 These are ARS staining images of each group in Example 5 of the present invention;

[0039] Figure 18 The images show toluidine blue staining after adding different concentrations of magnesium ions in Example 5 of this invention.

[0040] Figure 19 These are ALP staining images after adding different concentrations of magnesium ions in Example 5 of this invention;

[0041] Figure 20 These are ARS staining images of different concentrations of magnesium ions added in Example 5 of this invention;

[0042] Figure 21 This is a schematic diagram of the rabbit osteochondral defect model used in Embodiment 6 of the present invention;

[0043] Figure 22 This is a schematic diagram of implanting MZ-Gel in a rabbit osteochondral defect model in Embodiment 6 of the present invention;

[0044] Figure 23 These are CT three-dimensional reconstruction images of rabbit bones in each group after 8 weeks in Example 6 of this invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, 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.

[0046] This invention provides a biomimetic magnesium alloy material for inducing simultaneous bone-cartilage regeneration and its preparation method. A biomimetic magnesium alloy material (MZ-Gel) targeting simultaneous bone-cartilage regeneration is constructed: Kartogenin (KGN) or quercetin (Q) is loaded onto stem cell membrane vesicles (BMMs) to construct two types of biomimetic hydrogels targeting mesenchymal stem cells: HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q. The spatial layering of these hydrogels with a porous magnesium alloy scaffold creates a "cartilage-bone" functional gradient, primarily driving cartilage generation in the upper layer and enhancing osteogenic and bone remodeling in the lower layer. Simultaneously, the Mg²⁺ released during magnesium alloy degradation is utilized... +By synergistically regulating the local microenvironment, it can provide stable three-dimensional mechanical support on a macroscopic level and reconstruct the spatial gradient structure of cartilage and subchondral bone on a microscopic level, thereby achieving synchronous and zoned regeneration of bone and cartilage. This solves the technical problem in existing technologies that cannot provide stable three-dimensional mechanical support on a macroscopic level and reconstruct the spatial gradient structure of cartilage and subchondral bone on a microscopic level to achieve synchronous and zoned regeneration of bone and cartilage.

[0047] To achieve the above-mentioned technical effects, the overall concept of this invention is as follows:

[0048] On the one hand, this invention provides a biomimetic magnesium alloy material (MZ) that induces simultaneous bone-cartilage regeneration. The structure comprises a porous magnesium alloy scaffold and biomimetic hydrogels HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q, which are layered and solidified in the pores of the porous magnesium alloy scaffold. HAMA-RGD@BMM-KGN is a biomimetic hydrogel formed by dispersing BMM loaded with KGN in a HAMA-RGD solution. HAMA-RGD@BMM-Q is a biomimetic hydrogel formed by dispersing BMM loaded with Q in a HAMA-RGD solution.

[0049] This invention constructs a biomimetic magnesium alloy material (MZ-Gel) targeting simultaneous bone-cartilage regeneration: Kartogenin (KGN) or quercetin (Q) is loaded onto stem cell membrane vesicles (BMMs) to construct two types of biomimetic hydrogels targeting mesenchymal stem cells: HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q. These hydrogels, loaded with a porous magnesium alloy scaffold, form a "cartilage-bone" functional gradient, primarily driving cartilage generation in the upper layer and enhancing osteogenic and bone remodeling in the lower layer. Simultaneously, the uniform porous structure of the magnesium alloy scaffold provides both good mechanical support and controllable perfusion, and the degradation of the magnesium alloy generates Mg... 2+ It can promote osteoblast proliferation and differentiation, regulate bone remodeling-related signaling pathways, and facilitate new bone formation and mineralization. Therefore, the MZ-Gel of this invention can provide stable three-dimensional mechanical support on a macroscopic level and reconstruct the spatial gradient structure of cartilage and subchondral bone on a microscopic level, achieving synchronous and zoned regeneration of bone and cartilage.

[0050] Among them, BMM refers to cell membrane vesicles derived from stem cells, which retain cell membrane proteins such as various integrins, CD44, etc.; Kartogenin (KGN) is a common chondrogenic agent that can induce bone marrow mesenchymal stem cells (BMSCs) to differentiate into cartilage; Quercetin (Q) has antioxidant, anti-inflammatory, and osteogenic effects; HAMA (Hyaluronic Acid Methacrylate); RGD is arginine. glycine Aspartic acid (Rrginine) Glycine Aspartic acid (RGD) peptide sequence.

[0051] This invention has found that hyaluronic acid (HA) has a high affinity for CD44, and the RGD peptide sequence can bind to a variety of integrins. Therefore, HAMA-RGD can form a biocompatible interface with integrins and CD44 retained by BMM, thereby enhancing tissue adhesion and retention.

[0052] The biomimetic hydrogel of this invention is a hyaluronic acid-based hydrogel that can mimic the high water content and network structure of the extracellular matrix of chondrocytes, providing a suitable microenvironment for cell adhesion, migration, and differentiation. By introducing adhesion-promoting short peptide sequences such as RGD, the biomimetic hydrogel can specifically bind to integrins on the surface of mesenchymal stem cells, enhancing cell adhesion and signal transduction. Furthermore, it is loaded with the chondrogenic molecule Kartogenin (KGN) and bioactive factors such as quercetin (Q), which has both anti-inflammatory and osteogenic effects. Targeted delivery and sustained release are achieved using biomimetic carriers such as cell membrane vesicles. This enables differentiated regulation of the cartilage and bone regions spatially and continuous, gentle signal stimulation temporally, thereby simultaneously driving stem cell chondrogenic and osteogenic differentiation.

[0053] Preferably, the HAMA-RGD@BMM-KGN is cured in the upper 1 / 3 of the porous magnesium alloy scaffold, and the HAMA-RGD@BMM-Q is cured in the lower 2 / 3 of the porous magnesium alloy scaffold. This invention utilizes BMM... KGN is positioned in the upper 1 / 3 of the magnesium alloy scaffold to promote cartilage differentiation, and BMM is placed in place. Q is positioned in the lower two-thirds of the magnesium alloy scaffold to promote osteogenic differentiation, thereby constructing a bone-fitting structure. Functional stratification of cartilage physiological structure enables synchronous regeneration of bone and cartilage.

[0054] Preferably, the HAMA-RGD solution comprises 2% HAMA, 1% RGD, and 0.1% photoinitiator by weight-volume ratio. This invention enables BMM (Browser-Modulated Metallic Mold) by mixing HAMA and RGD with a photoinitiator to form the HAMA-RGD solution. KGN, BMM Q is uniformly dispersed in HAMA-RGD solution to form a biomimetic hydrogel. When the biomimetic hydrogel is poured into the pores of the porous magnesium alloy scaffold, it can be solidified in the porous magnesium alloy scaffold under light conditions.

[0055] Preferably, the porous magnesium alloy scaffold is a three-dimensional scaffold with a pore size of 500 μm. Through research, this invention has found that the aforementioned pore size facilitates the uniform distribution of HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q within the porous magnesium alloy scaffold.

[0056] Preferably, the porous magnesium alloy support is made of magnesium-zinc alloy, wherein, by mass fraction, the magnesium-zinc alloy comprises 95-99% magnesium and 1-5% zinc.

[0057] Preferred, such as Figure 6-10 As shown, the porous magnesium alloy support has a nail-like structure, meaning its shape resembles that of a nail, to facilitate MZ Successful implantation of gel in bone defect surgery. The porous magnesium alloy scaffold includes a nail head at the top of the nail-shaped structure, a nail shank in the middle of the nail-shaped structure, and a nail tip at the bottom of the shaping structure. The nail head and the nail shank are both cylindrical structures, and the diameter of the nail head is larger than the diameter of the nail shank. The nail tip is a conical structure, and the diameter of the bottom of the cone is the same as the diameter of the nail shank.

[0058] The porous magnesium alloy scaffold features a porous structure on its nail head, into which biomimetic hydrogels HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q are infused. This porous structure is uniformly distributed across the upper and lower surfaces and the sides of the cylinder. Holes on the upper and lower surfaces penetrate the cylinder from the top and bottom, while holes on the sides penetrate the cylinder from the sides. This porous structure allows for the uniform release of active materials when the biomimetic hydrogels HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q are fixed within the porous magnesium alloy scaffold.

[0059] Secondly, such as Figure 1 As shown, the present invention also provides a method for preparing a biomimetic magnesium alloy material that induces simultaneous bone-cartilage regeneration, the preparation method comprising the following steps:

[0060] BMM loaded with KGN was dispersed in HAMA-RGD solution to form a biomimetic hydrogel HAMA-RGD@BMM-KGN;

[0061] BMM loaded with Q was dispersed in HAMA-RGD solution to form a biomimetic hydrogel HAMA-RGD@BMM-Q;

[0062] The HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q were layered and infused into the pores of a porous magnesium alloy scaffold, and then cured to obtain the biomimetic magnesium alloy material that induces synchronous bone-cartilage regeneration.

[0063] Preferably, the method for preparing BMM loaded with KGN includes: preparing a stock solution of KGN at a concentration of 10-20 mg / mL, mixing KGN and BMM at a mass ratio of 1:50, and incubating at 37°C for 1 hour to obtain BMM-KGN.

[0064] Preferably, the preparation method of the BMM loaded with Q includes: preparing a Q solution of 10-20 mg / mL, mixing the Q solution with BMM at a Q to BMM mass ratio of 1:50, and incubating at 37°C for 1 h to obtain BMM-Q.

[0065] Preferably, ultrasound is used to assist in incubation, thereby increasing the incubation rate. The ultrasound conditions are: 20-40 kHz, 60-80 W, and ultrasound time of 10 min.

[0066] Preferably, the method for preparing BMM includes: taking bone marrow mesenchymal stem cells (BMSCs), using ultrasound to lyse and break the membrane to obtain cell membrane precipitate, sequentially extruding the cell membrane precipitate through a 200nm polycarbonate membrane, and repeating the above extrusion operation to obtain BMMs of uniform size.

[0067] Preferably, the specific steps for lysis using ultrasound include: placing the cell pellet in an ice bath, sonicating for 3 seconds, pausing for 3 seconds, and repeating this process 5 times. The ultrasound power is 100-120W, resulting in a bone marrow mesenchymal stem cell membrane. The obtained cell membrane is centrifuged at 3000×g for 5 minutes, and the supernatant is then centrifuged at 20000×g for 30 minutes. Finally, the supernatant is centrifuged at 100000×g for 40 minutes to obtain the cell membrane pellet.

[0068] Preferably, the specific steps for layering and injecting the HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q into the pores of a porous magnesium alloy scaffold and curing them to obtain the biomimetic magnesium alloy material that induces synchronous bone-cartilage regeneration include: injecting the HAMA-RGD@BMM-KGN into the upper 1 / 3 of the porous magnesium alloy scaffold and photocuring it, and then injecting the HAMA-RGD@BMM-Q into the lower 2 / 3 of the porous magnesium alloy scaffold and photocuring it, thereby obtaining the biomimetic magnesium alloy material that induces synchronous bone-cartilage regeneration.

[0069] Preferably, the method for preparing the porous magnesium alloy bracket of the present invention includes: processing a porous structure using magnesium alloy nails as a casting blank. In a specific embodiment, the processing equipment for the porous magnesium alloy bracket of the present invention is a Beijing Jingdiao five-axis machine tool, model JDGR200T, with a maximum machine speed of 24,000 rpm, using a 0.4mm diameter drill bit, a rotational speed of 7,000 rpm, a feed rate of 0.15 mm per revolution, and a feed speed of 15 mm per minute.

[0070] The following specific embodiments illustrate a biomimetic magnesium alloy material for inducing simultaneous bone-cartilage regeneration and its preparation method. All raw materials used in the embodiments of this invention are commercially available products.

[0071] Example 1: Preparation of BMM

[0072] (1) Bone marrow mesenchymal stem cells (BMSCs) were cultured to 80–90% confluence, washed with phosphate-buffered saline (PBS), and the cells were collected.

[0073] (2) Membrane disruption using ultrasonic lysis: The cell pellet was placed in an ice bath, ultrasonicated for 3 seconds, paused for 3 seconds, and the above steps were repeated 5 times. The ultrasonic power was 100-120W to obtain the bone marrow mesenchymal stem cell membrane. The obtained cell membrane was centrifuged at 3000×g for 5 minutes, and the supernatant was centrifuged at 20000×g for 30 minutes. The supernatant was then centrifuged at 100000×g for 40 minutes to obtain the BMSC cell membrane pellet.

[0074] (3) The cell membrane precipitate was resuspended in isotonic buffer and extruded sequentially through a 200 nm polycarbonate membrane for 50 cycles to obtain BMM.

[0075] (4) NTA (Nanoparticle Tracking Analysis) was used to detect the particle size distribution. The particle size distribution diagram of BMM is shown below. Figure 2 As shown, from Figure 2 It can be seen that the BMM obtained by the above method has a uniform particle size distribution; the morphology of the bilayer membrane vesicles was observed by transmission electron microscopy (TEM), and the TEM image of the BMM is shown below. Figure 3 As shown.

[0076] Example 2: BMM KGN and BMM The Construction of Q

[0077] (1) KGN and Q were prepared with DMSO to prepare KGN stock solution and Q stock solution at 12 mg / mL respectively.

[0078] (2) KGN mother liquor and BMM were mixed at a mass ratio of KGN:BMM of 1:50, incubated at 37℃ for 1 h, and then ultrasonically assisted (20-40 kHz, power 60-80 W) for 10 min to obtain BMM. KGN;

[0079] The mother liquor (Q) and BMM were mixed at a mass ratio of Q:BMM of 1:50 and incubated at 37°C for 1 hour. BMM was then obtained by ultrasonic treatment (20-40 kHz, 60-80 W) for 10 minutes. Q.

[0080] (3) TEM observation of BMM KGN and BMM The morphology of Q, BMM KGN's TEM image as follows Figure 4 As shown, BMM TEM image of Q as follows Figure 5 As shown.

[0081] Example 3: HAMA RGD@BMM-KGN and HAMA RGD@BMM Preparation of Q

[0082] (1) Prepare HAMA-RGD solution with HAMA 2% w / v, RGD 1% w / v, photoinitiator LAP 0.1% w / v, and the balance being double-distilled H2O.

[0083] (2) BMM KGN was added to the HAMA-RGD solution prepared in (1) above to obtain the biomimetic hydrogel HAMA. RGD@BMM KGN;

[0084] BMM Q is added to the HAMA-RGD solution prepared in (1) above to obtain the biomimetic hydrogel HAMA. RGD@BMM Q.

[0085] Example 4: Preparation of a uniformly porous magnesium alloy scaffold and layered injection

[0086] (1) The casting and processing of magnesium alloy brackets are carried out in the following steps:

[0087] Based on mass fraction, 98% magnesium and 2% zinc are cast into a magnesium-zinc alloy. This alloy is then stamped to form a nail-shaped structure. The Micro-CT image of the nail-shaped structure is shown below. Figure 6 and Figure 7 As shown ( Figure 6 This is the front view of the nail-shaped structure. Figure 7 (Top view of the nail-shaped structure). Holes were drilled into the nail heads of the nail-shaped structure. The drilling equipment used was a Beijing Jingdiao 5-axis machine tool, model JDGR200T, with a maximum speed of 24,000 rpm. A 0.4mm diameter drill bit was used, with a speed of 7,000 rpm, a feed rate of 0.15 mm per revolution, and a feed speed of 15 mm per minute, resulting in a porous 3D scaffold with a hole diameter of 500 μm. The Micro-CT image of the porous magnesium alloy scaffold is shown below. Figure 8 As shown, Figure 8 The left side of the image shows the front view of the porous magnesium alloy scaffold, and the right side shows the top view. The SEM image of the porous magnesium alloy scaffold is shown below. Figure 9 , Figure 10 As shown, Figure 9 This is a top view of a porous magnesium alloy support. Figure 10 This is a magnified view of a porous magnesium alloy scaffold.

[0088] (2) Under vacuum conditions, the biomimetic hydrogel was infused into the porous structure of the magnesium alloy scaffold. The specific steps are as follows:

[0089] First, the biomimetic hydrogel HAMA RGD@BMM KGN was infused to the top 1 / 3 and cured by irradiation with 405nm wavelength light for 60s; then the biomimetic hydrogel HAMA was applied. RGD@BMM Q is poured into the lower 2 / 3 and cured by irradiation with 405nm wavelength light for 60s to obtain MZ. Gel.

[0090] Example 5: In vitro functional verification

[0091] (1) BMSC was mixed with material HAMA respectively. RGD@BMM KGN, HAMA RGD@BMM Q. Jointly cultivate.

[0092] (2) Evaluation of chondrogenic differentiation marker COL2 and toluidine blue staining, as well as mRNA expression and alkaline phosphatase (ALP) and alizarin red S (ARS) staining of osteogenic differentiation markers COL1, Runx2, and OCN. Bone marrow mesenchymal stem cells (BMSCs) were co-cultured with HAMA-RGD, HAMA-RGD-BMM, HAMA-RGD@BMM-KGN, and HAMA-RGD@BMM-Q, respectively. After 7 days of culture, mRNA was collected for qPCR detection of related RNA expression and ALP staining. After 14 days of culture, toluidine blue and ARS staining were performed.

[0093] Figure 11 The relative expression of COL2 mRNA, Figure 12 The relative expression of Runx2 mRNA, Figure 13 The relative expression of ALP mRNA. Figure 14 The relative expression of COL1 mRNA; Figure 15 Stain each group with toluidine blue. Figure 16ALP was stained in each group. Figure 17 ARS staining was performed on each group.

[0094] exist Figures 11-17 In the middle, from left to right (or 1 to 5 in the figure), they are the blank group, HAMA-RGD group, HAMA-RGD-BMM group, HAMA-RGD@BMM-KGN group, and HAMA-RGD@BMM-Q group, respectively.

[0095] Figure 11 In the HAMA-RGD@BMM-KGN group, the relative expression of COL2 mRNA was the highest. Figure 12 In the HAMA-RGD@BMM-Q group, the relative expression of Runx2 mRNA was the highest. Figure 13 In the HAMA-RGD@BMM-Q group, the relative expression of ALP mRNA was the highest. Figure 14 Among them, the HAMA-RGD@BMM-Q group had the highest relative expression of COL1 mRNA.

[0096] Toluidine blue staining is a staining method for assessing chondrogenic differentiation. The proteoglycans secreted by chondrocytes bind to toluidine blue, resulting in a blue-purple color. The appearance and intensity of this blue-purple color directly reflect the accumulation of chondrogenic extracellular matrix (proteoglycans), a key marker of chondrogenic differentiation of BMSCs. Figure 15 Among them, the HAMA-RGD@BMM-KGN group showed the bluest color, indicating that HAMA-RGD@BMM-KGN has a strong effect on promoting chondrogenic differentiation. ALP is an early osteogenic differentiation indicator. The cytoplasm of ALP-positive cells (i.e., BMSCs that are differentiating into osteoblasts) will be stained blue or bluish-purple. The darker the color, the stronger the ALP activity of a single cell and the better the osteogenic differentiation effect. Figure 16 In the image, the HAMA-RGD@BMM-Q group showed the bluest color, indicating that HAMA-RGD@BMM-Q had a superior effect on promoting osteogenic differentiation. ARS staining represents calcified nodules, an indicator of late-stage osteogenic differentiation; the redder the color in the image, the more calcified nodules there are, and the better the osteogenic differentiation effect. Figure 17 Among them, the HAMA-RGD@BMM-Q group was the reddest, indicating that HAMA-RGD@BMM-Q has a better effect on promoting bone differentiation.

[0097] Therefore, from Figures 11-17 It can be seen that HAMA-RGD@BMM-KGN can promote chondrogenic differentiation of BMSCs, while HAMA-RGD@BMM-Q can promote osteogenic differentiation.

[0098] (3) Adding different concentrations of Mg 2+We simulated magnesium alloy degradation products to investigate their promoting threshold and synergistic range for cartilage / osteogenic induction.

[0099] like Figure 18-20 As shown, Mg 2+ The higher the concentration, the more obvious the staining; magnesium alloy degradation products Mg 2+ It can promote osteogenic and chondrogenic differentiation. When the magnesium ion concentration is 25 mM, magnesium ions already have a strong effect on promoting osteogenic and chondrogenic differentiation. Further increasing the magnesium ion concentration beyond this level will not yield better results.

[0100] Example 6: In vivo evaluation

[0101] (1) MZ implanted in a rabbit osteochondral defect model (4 mm in diameter) Gel, taken at 8 weeks;

[0102] like Figure 21 The image shows a schematic diagram of a rabbit osteochondral defect model. Figure 22 A schematic diagram of MZ-Gel implantation into a rabbit bone cartilage defect.

[0103] (2) micro CT scan to assess bone regeneration.

[0104] Figure 23 CT 3D reconstructed image of rabbit bone. Figure 23 In the diagram, A represents the modeling group, where rabbit bones are not treated in any way except for drilling holes to create a rabbit bone-cartilage defect model; B represents the MZ group, where a magnesium alloy scaffold is implanted into the rabbit bone-cartilage defect; and C represents the MZ-Gel group, where the MZ obtained in Example 4 of this invention is implanted into the rabbit bone-cartilage defect. Gel.

[0105] from Figure 23 It can be seen that implanting MZ into cartilage defects in rabbits... Eight weeks after gel treatment, the cartilage and bone of the rabbit bones differentiated and regenerated synchronously, and the defects were basically repaired.

[0106] It should be noted that, in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0107] This invention is not limited to the embodiments described above. Those skilled in the art can make various improvements and refinements without departing from the principles of this invention, and these improvements and refinements are also considered within the scope of protection of this invention. Contents not described in detail in this specification are prior art known to those skilled in the art.

Claims

1. A biomimetic magnesium alloy material for inducing simultaneous bone-cartilage regeneration, characterized in that, The invention includes a porous magnesium alloy scaffold and biomimetic hydrogels HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q, which are layered and solidified in the pores of the porous magnesium alloy scaffold. HAMA-RGD@BMM-KGN is a biomimetic hydrogel formed by dispersing stem cell membrane vesicles (BMM) loaded with KGN in a HAMA-RGD solution. HAMA-RGD@BMM-Q is a biomimetic hydrogel formed by dispersing BMM loaded with quercetin Q in a HAMA-RGD solution. Among them, HAMA is hyaluronic acid methacrylate, RGD is an arginine-glycine-aspartic acid peptide sequence, and KGN is chondroitin.

2. The biomimetic magnesium alloy material for inducing synchronous bone-cartilage regeneration as described in claim 1, characterized in that, The HAMA-RGD@BMM-KGN is cured on the upper 1 / 3 of the porous magnesium alloy scaffold, and the HAMA-RGD@BMM-Q is cured on the lower 2 / 3 of the porous magnesium alloy scaffold.

3. The biomimetic magnesium alloy material for inducing simultaneous bone-cartilage regeneration as described in claim 1, characterized in that, The HAMA-RGD solution comprises 2% HAMA, 1% RGD, and 0.1% photoinitiator by mass-volume ratio.

4. The biomimetic magnesium alloy material for inducing simultaneous bone-cartilage regeneration as described in claim 1, characterized in that, The porous magnesium alloy scaffold is a three-dimensional scaffold with a pore size of 500 μm.

5. The biomimetic magnesium alloy material for inducing simultaneous bone-cartilage regeneration as described in claim 1, characterized in that, The porous magnesium alloy support is made of magnesium-zinc alloy, wherein, by mass fraction, the magnesium-zinc alloy comprises 95-99% magnesium and 1-5% zinc.

6. The method for preparing the biomimetic magnesium alloy material for inducing simultaneous bone-cartilage regeneration as described in any one of claims 1 to 5, characterized in that, The preparation method includes the following steps: BMM loaded with KGN was dispersed in HAMA-RGD solution to form a biomimetic hydrogel HAMA-RGD@BMM-KGN; BMM loaded with Q was dispersed in HAMA-RGD solution to form a biomimetic hydrogel HAMA-RGD@BMM-Q; The HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q were layered and infused into the pores of a porous magnesium alloy scaffold, and then cured to obtain the biomimetic magnesium alloy material that induces synchronous bone-cartilage regeneration.

7. The preparation method according to claim 6, characterized in that, The method for preparing BMM loaded with KGN includes: preparing a stock solution of KGN at a concentration of 10-20 mg / mL, mixing KGN and BMM at a mass ratio of 1:50, and incubating at 37°C for 1 hour to obtain BMM-KGN.

8. The preparation method according to claim 6, characterized in that, The method for preparing BMM loaded with Q includes: preparing a Q solution of 10-20 mg / mL, mixing the Q solution with BMM at a Q to BMM mass ratio of 1:50, and incubating at 37°C for 1 h to obtain BMM-Q.

9. The preparation method according to claim 6, characterized in that, The specific steps for obtaining the biomimetic magnesium alloy material that induces synchronous bone-cartilage regeneration by layering and injecting the HAMA-RGD@BMM-KGN and HAMA-RGD@BMM-Q into the pores of a porous magnesium alloy scaffold and curing them include: injecting the HAMA-RGD@BMM-KGN into the upper 1 / 3 of the porous magnesium alloy scaffold and photocuring it, and then injecting the HAMA-RGD@BMM-Q into the lower 2 / 3 of the porous magnesium alloy scaffold and photocuring it, thereby obtaining the biomimetic magnesium alloy material that induces synchronous bone-cartilage regeneration.

10. The preparation method according to claim 9, characterized in that, The conditions for photocuring are: irradiation with light of wavelength 405nm for 30-60s.

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