A customizable biphasic hydrogel scaffold and methods of making and using the same
By fabricating customizable biphasic hydrogel scaffolds, the problems of difficult biomechanical property simulation, high cost, and long recovery period in the treatment of articular cartilage injury have been solved, achieving multifunctional therapeutic effects such as anti-oxidation, anti-inflammation, and promoting healing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGDONG ADAPTIVE BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-07-16
- Publication Date
- 2026-06-23
Smart Images
Figure CN120754328B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bone repair, specifically relating to a customizable biphasic hydrogel scaffold, its preparation method, and its application. Background Technology
[0002] Articular cartilage is connective tissue covering the epiphyseal plates of the elbow / knee joints. It possesses good elasticity, compressive strength, and lubrication, effectively reducing wear and tear between bones and withstanding high-frequency dynamic pressure. Articular cartilage damage is a common degenerative orthopedic condition, typically manifesting as joint pain and functional impairment. Because cartilage lacks blood vessels, lymphatic vessels, and nerves, its nutrition depends on synovial fluid and subchondral bone, resulting in a low regenerative capacity.
[0003] Currently, the treatment methods for articular cartilage damage are divided into three categories: conservative treatment, repair treatment, and regenerative treatment.
[0004] However, current treatment methods have many drawbacks: (1) Conservative treatment, such as arthroscopic lavage, can relieve pain in the short term, but cannot stop the progression of damage and has the risk of damaging the surrounding healthy cartilage. (2) Repair treatment includes methods such as bone marrow stimulation, which stimulates the formation of blood clots in the subchondral bone, promotes the migration of mesenchymal stem cells to the cartilage area, and generates fibrocartilage. However, fibrocartilage cannot replace the biomechanical properties of natural cartilage. Although autologous and allogeneic transplantation are effective, there are problems such as donor source, tissue integration, and immune response. (3) Regenerative treatment, such as autologous chondrocyte implantation (ACI) and matrix-assisted chondrocyte transplantation (MACI), can generate functionally stable cartilage by culturing chondrocytes in vitro and transplanting them to the defect site. However, the treatment cost is high, the recovery period is long, and the regeneration of the osteochondral interface and subchondral bone is still challenging.
[0005] Therefore, there is an urgent need to develop a multifunctional treatment method that mimics the biomechanical properties of natural bone, possesses antioxidant and anti-inflammatory properties, and promotes cartilage regeneration. This method could effectively address the complexities of articular cartilage defects, while also offering lower treatment costs, shorter recovery periods, and the ability to promote the regeneration of the osteochondral interface and subchondral bone, thus providing a more comprehensive and sustainable solution. Summary of the Invention
[0006] One objective of this invention is to address the above-mentioned technical problems by providing a customizable biphasic hydrogel scaffold that is suitable for customization, mimics the biomechanical properties of natural bone, and has antioxidant, anti-inflammatory, and healing-promoting effects.
[0007] Another object of the present invention is to provide a method for preparing the biphasic hydrogel scaffold.
[0008] Another object of the present invention is to provide the application of the biphasic hydrogel scaffold.
[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0010] In a first aspect, the present invention provides a customizable biphasic hydrogel scaffold comprising a hyaline cartilage layer and a subchondral bone layer, wherein the hyaline cartilage layer comprises methacrylamide type I recombinant collagen (Col1MA) and bone marrow mesenchymal stem cells (BMSCs), and the subchondral bone layer comprises methacrylamide type I recombinant collagen (Col1MA) and resveratrol-loaded bioactive glass (MBG@RES).
[0011] Preferably, in the hyaline cartilage layer, by weight-volume percentage, the content of methacrylamide type I recombinant collagen is 5-15%, and the content of bone marrow mesenchymal stem cells is 1-2 × 10⁻⁶. 5 per mL.
[0012] More preferably, in the hyaline cartilage layer, by weight-volume percentage, the content of methacrylamide type I recombinant collagen is 10%, and the content of bone marrow mesenchymal stem cells is 1 × 10⁻⁶. 5 per mL.
[0013] Preferably, in the subchondral bone layer, by mass-volume percentage, the amount of methacrylamide type I recombinant collagen is 7.5-15%, and the amount of resveratrol-loaded bioactive glass is 1-2%.
[0014] More preferably, in the subchondral bone layer, by mass-volume percentage, 10% is methacrylamide type I recombinant collagen and 2% is resveratrol-loaded bioactive glass.
[0015] Preferably, the methacrylamide type I recombinant collagen is prepared by the following steps:
[0016] Dissolve 2g of type I collagen in 100mL of deionized water, adjust the pH to 7, add 2mL of methacrylic anhydride, react at room temperature for 24h, dialyze the product in deionized water, centrifuge at 10000rpm for 15min to remove impurities, freeze-dry the supernatant to obtain the methacrylamide type I recombinant collagen (Col1MA).
[0017] Preferably, the resveratrol-loaded bioactive glass is prepared by the following steps:
[0018] 1 g of resveratrol was dissolved in 50 mL of deionized water to prepare a 2% resveratrol solution. 1 g of bioactive glass was added to the resveratrol solution and ultrasonically dispersed for 15 min. Then, the mixture was stirred at 40 °C. After the reaction was completed, the glass was washed with deionized water to remove unbound resveratrol and freeze-dried to obtain the final product, resveratrol bioactive glass (MBG@RES).
[0019] More preferably, the bioactive glass is prepared by the following steps:
[0020] Dissolve 0.7 g of cetyltrimethylammonium bromide (CTAB) in 33 mL of deionized water, add 10 mL of ethyl acetate, stir for 30 minutes, then add 7 mL of 5 M ammonia solution and continue stirring for 15 minutes. Slowly add 3.6 mL of tetraethyl orthosilicate and 0.36 mL of triethyl phosphate to the above solution. Dissolve 2.28 g of calcium nitrate tetrahydrate in 5 mL of deionized water and slowly add it to the above solution. Stir at room temperature for 4 hours. Collect the reaction solution, centrifuge at 5000 rpm for 5 minutes to obtain a white precipitate. Wash the precipitate three times with anhydrous ethanol and deionized water, respectively, and dry at 60 °C for 24 hours. Then calcine the dried white powder at 600 °C with a heating rate of 1 °C / min for 5 hours to obtain the bioactive glass.
[0021] Preferably, the bone marrow mesenchymal stem cells are prepared through the following steps:
[0022] Rat limb bones were collected, and the muscle was removed in PBS buffer containing 1% penicillin-streptomycin solution. Small incisions were made at both ends of the bones in α-MEM basal medium to flush out the bone marrow. The bone marrow-medium mixture was collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and red blood cell lysis buffer was added for 10 min. The reaction was terminated by adding PBS, and the cells were centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in α-MEM complete medium and cultured at 37℃ and 5% CO2 for 48 h. The medium was changed, and the cells were passaged when they reached 80% confluence. After 2-3 passages, the cells were washed 3 times with PBS to obtain the bone marrow mesenchymal stem cells.
[0023] Secondly, the present invention also provides a method for preparing the biphasic hydrogel scaffold, which includes the following steps:
[0024] S1. Preparation of the hyaline cartilage layer:
[0025] A photoinitiation solution was prepared, and methacrylamide type I recombinant collagen was dissolved in the above photoinitiation solution to obtain a methacrylamide type I recombinant collagen solution, which was used as the bio-ink for the first layer of the hydrogel scaffold. The cultured bone marrow mesenchymal stem cells were digested and resuspended with 0.25% trypsin and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the bone marrow mesenchymal stem cells were resuspended with the bio-ink, mixed evenly, and 3D printed.
[0026] S2. Prepare the subchondral bone layer:
[0027] Prepare a photoinitiating solution, dissolve methacrylamide type I recombinant collagen in the above photoinitiating solution to obtain a methacrylamide type I recombinant collagen solution; add the bioactive glass loaded with resveratrol, vortex for 30s to mix evenly, and use the bio-ink as the second layer of the hydrogel scaffold. After the first layer is printed, continue to print the second layer.
[0028] Preferably, the curing conditions for the first layer of 3D printing are a light intensity of 17 mW / cm². 2 Exposure time: 20s; substrate exposure time: 24s; curing conditions for printing the second layer: light intensity 18mW / cm². 2 The exposure time is 22 seconds, and the exposure time for the grassroots level is 27 seconds.
[0029] Thirdly, the present invention also provides the use of the biphasic hydrogel scaffold in the preparation of drugs that promote wound healing or bone repair or have antioxidant or anti-inflammatory effects.
[0030] Compared with the prior art, the technical solution of the present invention has the following advantages:
[0031] (1) Performance of simulating the microenvironment of natural bone: The internal and external structures of the scaffold can be designed to be gradient through 3D printing technology or other manufacturing methods to simulate the hardness gradient of natural bone, thereby providing corresponding mechanical support for the repair of bone tissue in different parts and at different degrees.
[0032] (2) Customizable scaffold materials: Cartilage tissue lacks blood vessels and other channels, has a high extracellular matrix density, and has gradient heterogeneity, making it very suitable for precise customization of scaffolds using 3D printing technology to repair (bone) cartilage defects.
[0033] (3) Design and preparation of composite materials: By combining methacrylamide type I recombinant collagen (Col1MA) with bioactive glass (MBG), a composite material with structural stability and biocompatibility was formed. Methacrylamide type I collagen enhanced the stability and integration of the interface between the bone layer and the cartilage layer. Through modification of the composite material, the effective loading and controlled release of RES (resveratrol) were achieved, thereby enhancing its antioxidant and anti-inflammatory functions.
[0034] (4) Improved biocompatibility: Natural materials Col1MA and MBG are used to improve biocompatibility. Compared with traditional synthetic materials, it can significantly reduce the occurrence of local inflammation or allergic reactions. In addition, by adjusting the content of Col1MA and MBG, the scaffold can simulate the biomechanical properties of natural bone, improve the repair effect, and accelerate bone tissue regeneration.
[0035] (5) The addition of resveratrol enhances antioxidant capacity, effectively scavenging free radicals, slowing down cell damage, and protecting cells from oxidative stress.
[0036] (6) Enhanced anti-inflammatory and healing-promoting effects: The design of this material not only focuses on biomechanical properties but also integrates anti-inflammatory and healing-promoting components. RES not only helps control the inflammatory response but also promotes osteogenic differentiation of BMSCs, thereby promoting bone tissue regeneration and repair to meet clinical needs.
[0037] (7) Multifunctional design: It can be customized, has antioxidant, anti-inflammatory and healing properties. The material design integrates multiple functions such as stability, antioxidant, anti-inflammatory and bone tissue regeneration to meet the complex needs of cartilage repair. Attached Figure Description
[0038] Figure 1 An image of the hydrogel scaffold is shown.
[0039] Figure 2 The maximum compressive strength of the hydrogel is shown.
[0040] Figure 3 The drug loading rate of RES is shown.
[0041] Figure 4 The cumulative release rate of RES is shown.
[0042] Figure 5 The results of the cell compatibility evaluation are shown.
[0043] Figure 6 The results of MDA content determination are shown.
[0044] Figure 7 The results of the anti-inflammatory experiment are shown. Detailed Implementation
[0045] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0046] Unless otherwise specified, the reagents and instruments used in the embodiments of the present invention are all conventional reagents and instruments existing in the art.
[0047] 1. Preparation of methacrylamide type I recombinant collagen (Col1MA):
[0048] Weigh 2g of type I collagen and dissolve it in 100mL of deionized water. Adjust the pH to 7, add 2mL of methacrylic anhydride, and react at room temperature for 24h. The product, methacrylamide type I recombinant collagen (Col1MA), is dialyzed in deionized water using a 3500Da dialysis bag. Centrifuge at 10000rpm for 15 minutes to remove impurities. Freeze-dry the supernatant and store at -20℃ until use.
[0049] 2. Preparation of bioactive glass (MBG):
[0050] 0.7 g of hexadecyltrimethylammonium bromide (CTAB) was dissolved in 33 mL of deionized water. 10 mL of ethyl acetate was added, and the mixture was stirred for 30 minutes. Then, 7 mL of ammonia (5M) was added, and stirring continued for 15 minutes. 3.6 mL of tetraethyl orthosilicate (TEOS) and 0.36 mL of triethyl phosphate were slowly added dropwise to the above solution. 2.28 g of calcium nitrate tetrahydrate was dissolved in 5 mL of deionized water and slowly added dropwise to the above solution. The mixture was stirred vigorously at room temperature for 4 hours. The reaction mixture was collected and centrifuged at 5000 rpm for 5 minutes to obtain a white precipitate. The precipitate was washed three times each with anhydrous ethanol and deionized water, and dried at 60 °C for 24 hours. The dried white powder was then calcined at 600 °C at a heating rate of 1 °C / min for 5 hours to obtain MBG.
[0051] 3. Preparation of resveratrol-loaded bioactive glass (MBG@RES):
[0052] 1 g of resveratrol (RES) was dissolved in 50 mL of deionized water or a suitable solvent to prepare a 2% RES solution. 1 g of bioactive glass was added to the RES solution and ultrasonically dispersed for 15 min, followed by stirring at 40 °C to promote the interaction between RES and the bioactive glass surface. After the reaction was complete, the mixture was washed with deionized water to remove unbound RES. The modified bioactive glass (MBG@RES) was freeze-dried for 48 h to obtain the final product.
[0053] 4. Extraction and culture of bone marrow mesenchymal stem cells (BMSCs)
[0054] Three- to four-week-old SD rats were euthanized by dislocation and then immersed in 75% alcohol for 5 minutes for disinfection. The rats were then transferred to a laminar flow hood, and the bones of the limbs were removed using autoclaving instruments. Muscles were removed from the bones in PBS buffer (containing 1% penicillin-streptomycin solution). Small incisions were made at both ends of the bones in α-MEM basal medium, and the bone marrow was flushed out using a 1 mL syringe. The bone marrow-medium mixture was collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and erythrocyte lysis buffer was added for 10 minutes. The reaction was terminated by adding PBS, and the mixture was centrifuged at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells in α-MEM complete medium, and culture in a 37°C CO2 incubator (containing 5% CO2) for 48 h. Change the medium after each culture. Once the cells reach 80% confluence, passage them for 2-3 generations. After passage, wash the cells three times with PBS, digest them with trypsin, and once the cells become rounded, add α-MEM complete medium. Collect the cell suspension and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in bio-ink.
[0055] Example 1:
[0056] First, prepare the photoinitiation solution: Add 1 mg of LAP (lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, photoinitiator) and 0.6 mg of lemon yellow (light blocker) to 1 mL of deionized water, and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Digest and resuspend the cultured BMSCs cells with 0.25% trypsin, and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0057] Next, 0.075g of Col1MA was weighed and dissolved in the above solution, and then 0.01g of MBG@RES was added to prepare the second layer of bio-ink.
[0058] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0059] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0060] Example 2:
[0061] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0062] Next, 0.075g of Col1MA was weighed and dissolved in the above solution, and then 0.015g of MBG@RES was added to prepare the second layer of bio-ink.
[0063] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0064] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0065] Example 3:
[0066] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0067] Next, 0.075g of Col1MA was weighed and dissolved in the above solution, and then 0.02g of MBG@RES was added to prepare the second layer of bio-ink.
[0068] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0069] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0070] Example 4:
[0071] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0072] Next, 0.1g of Col1MA was weighed and dissolved in the above solution, and then 0.01g of MBG@RES was added to prepare the second layer of bio-ink.
[0073] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0074] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0075] Example 5:
[0076] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0077] Next, 0.1g of Col1MA was weighed and dissolved in the above solution, and then 0.015g of MBG@RES was added to prepare the second layer of bio-ink.
[0078] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²).2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0079] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0080] Example 6:
[0081] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0082] Next, 0.1g of Col1MA was weighed and dissolved in the above solution, and then 0.02g of MBG@RES was added to prepare the second layer of bio-ink.
[0083] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0084] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0085] Example 7:
[0086] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0087] Next, 0.15g of Col1MA was weighed and dissolved in the above solution, and then 0.01g of MBG@RES was added to prepare the second layer of bio-ink.
[0088] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0089] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0090] Example 8:
[0091] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0092] Next, 0.15g of Col1MA was weighed and dissolved in the above solution, and then 0.015g of MBG@RES was added to prepare the second layer of bio-ink.
[0093] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²).2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0094] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0095] Example 9:
[0096] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0097] Next, 0.15g of Col1MA was weighed and dissolved in the above solution, and then 0.02g of MBG@RES was added to prepare the second layer of bio-ink.
[0098] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0099] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0100] Comparative Example 1:
[0101] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0102] Next, 0.1g of Col1MA was weighed and dissolved in the above solution to prepare the second layer of bio-ink.
[0103] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0104] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0105] Comparative Example 2:
[0106] First, prepare the photoinitiator solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water, and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in the solution to prepare the first layer of bio-ink.
[0107] Next, 0.1g of Col1MA was weighed and dissolved in the above solution, and then 0.02g of MBG@RES was added to prepare the second layer of bio-ink.
[0108] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0109] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0110] Comparative Example 3:
[0111] First, prepare the photoinitiation solution: Add 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) to 1 mL of deionized water and vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in this solution as the bio-ink for the upper layer of the hydrogel scaffold. Resuspend the cultured BMSCs cells after digestion with 0.25% trypsin and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in the upper bio-ink at a density of 1 × 10⁻⁶ cells / mL. 5 The cell suspension at a density of 1 cell / mL is rapidly mixed with the upper layer of bio-ink to prepare the first layer of bio-ink.
[0112] Next, 0.1g of Col1MA was weighed and dissolved in the above solution, and then 0.02g of RES was added to prepare the second layer of bio-ink.
[0113] Then, the prepared first layer of bio-ink is poured into the groove of the 3D printer, and the printing model and its size are selected for printing (light intensity: 17mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 24s), after molding, a second layer is printed on this hydrogel layer (light intensity: 18mW / cm). 2 Exposure time: 22s, number of base layers: 1, base layer exposure time: 27s).
[0114] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.
[0115] Test Results
[0116] 1. Appearance of the hydrogel scaffold
[0117] Figure 1 An image of the hydrogel scaffold of Example 1 immersed in PBS is shown. The hydrogel scaffolds of Examples 2-9 have a similar appearance.
[0118] 2. Swelling rate test:
[0119] Record the initial weight of the printed hydrogel, denoted as W0. Place the prepared hydrogel sample in a 24-well plate, and add PBS buffer using a pipette to completely submerge the hydrogel sample. After 8 hours, remove the sample, carefully wipe off excess moisture from the outside of the hydrogel with weighing paper, place it on a plastic petri dish, weigh it, and record the weight of the hydrogel, denoted as W. t .
[0120] The swelling ratio is calculated using the following formula:
[0121]
[0122] Table 1. Swelling Rate
[0123] Grouping swelling rate Example 1 97.3±1.7％ Example 2 84.2±1.3％ Example 3 72.9±0.9％ Example 4 132.1±2.2％ Example 5 125.3±2.3％ Example 6 116.4±1.5％ Example 7 192.5±1.6％ Example 8 181.1±0.8％ Example 9 164.6±1.6％ Comparative Example 1 187.3±1.5％ Comparative Example 2 110.9±0.8％ Comparative Example 3 180.5±0.9％
[0124] The results indicate that an excessively high swelling ratio may cause the material to expand in volume after absorbing water in vivo, compressing surrounding tissues or organs and affecting post-implantation stability and biocompatibility. Furthermore, an excessively high swelling ratio may lead to material softening, reducing its mechanical strength and affecting its supporting function in bone repair. Table 2 shows that the swelling ratio gradually increases with increasing Col1MA content; however, the swelling ratio of the hydrogel significantly decreases after the addition of MBG@RES. This is because MBG releases CaO in the hydrogel. 2+ MBG can interact with negatively charged groups (such as carboxyl or phosphate groups) in the hydrogel matrix to form ionic crosslinks. Ionic crosslinking increases the crosslinking density between polymer chains, making the hydrogel more compact when absorbing water, thus limiting the swelling of the hydrogel. However, excessive MBG may also partially block the pores in the hydrogel, thereby reducing the swelling rate.
[0125] 3. Compressive strength
[0126] Test method: Place the sample on the sample stage, take the printed hydrogel support, and adjust the height of the sample stage so that both the upper and lower surfaces are in contact with the fixture. Compress at a constant rate of 0.05 mm / min. The Young's modulus is calculated using the following formula:
[0127]
[0128] In the formula, F is the compressive force on the support, A is the cross-sectional area, L0 is the initial height of the support before compression, and ΔL is the change in the height of the support.
[0129] Figure 2 The maximum compressive strength of each specimen is shown.
[0130] Results Explanation:
[0131] Ideally, the compressive strength of a hydrogel should be between 1 MPa and 2 MPa. This range provides biomechanical properties similar to cartilage, capable of withstanding dynamic joint loads while maintaining a degree of flexibility, mimicking the elasticity and durability of cartilage. The maximum compressive strength of the hydrogel scaffold is as follows: Figure 2As shown, the mechanical strength of the scaffold gradually increases with the increase of Col1MA content; in addition, MBG@RES also plays an important role in improving the mechanical strength of the scaffold, and this effect can be clearly seen in all embodiments and Comparative Example 2. This may be because the addition of MBG@RES can make the three-dimensional network structure inside the sponge more compact and stable, which is beneficial for the hydrogel scaffold to resist large deformations.
[0132] 4. Resveratrol loading
[0133] Test method: Dissolve 1 mg RES in 1 mL of anhydrous ethanol to prepare a 1 mg / mL solution. Then, serially dilute with pure water to 500, 250, 125, 62.5, and 36.25 μg / mL. Measure the absorbance at 305 nm using a UV spectrophotometer and plot a standard curve. Centrifuge the supernatant obtained after loading RES and measure the absorbance at 305 nm using a UV spectrophotometer. Calculate the drug content in the supernatant.
[0134] Calculation formula: Drug loading (%) = (Total drug mass / Total MBG mass) × 100%
[0135] Figure 3 The drug loading rate of RES is shown.
[0136] Results Explanation:
[0137] The drug loading of MBG was calculated to be 20.50 ± 0.23% by measuring the absorbance of the supernatant after loading RES.
[0138] 5. Resveratrol release
[0139] Test method: The printed hydrogel was placed in 1 mL of sterile PBS solution and incubated in a constant temperature shaking incubator at 37℃ and 100 rpm. Samples were taken at different time points (1, 3, 6, 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 h), with 1 mL of culture medium taken and replaced with an equal volume of fresh PBS. The samples were centrifuged at 4000 rpm for 5 min. The absorbance of resveratrol in PBS solution was measured using ultraviolet spectrophotometry at an excitation wavelength of 305 nm.
[0140] Calculation formula: Cumulative release rate (%) = (Cumulative drug release amount / Drug dosage) × 100%
[0141] Figure 4 The cumulative release rate of RES is shown.
[0142] Results show that RES loaded in aminated mesoporous bioactive glass (MBG@RES) and dispersed in Col1MA hydrogel, drug release in Example 6 exhibited two distinct phases: rapid release within the first 5 days, with approximately 31.03% of the drug released; subsequently, the release rate gradually slowed, reaching a cumulative release of 57.61% by day 14, and nearly 92.54% by day 46. Hydrogels from other examples also showed similar drug release patterns.
[0143] The initial rapid release may be due to the loose adsorption of drug molecules on or near the surface of the carrier, facilitating diffusion into the external environment. Furthermore, the rapid swelling of the hydrogel material upon contact with liquid also accelerates initial drug release. Over time, the drug is slowly released from deep within the carrier, gradually slowing the release rate. This may be because the mesoporous structure of the carrier restricts drug diffusion, while the network structure of the hydrogel also delays drug release. This release behavior is well-suited for long-term drug delivery applications. The early rapid release provides sufficient drug concentration for rapid therapeutic effect, while the later slow release maintains the drug's long-term effect. In Comparative Example 3, RES exhibited a burst release phenomenon, reaching 95.61% at 14 days. This indicates that the MBG@RES / Col1MA hydrogel composite material has excellent sustained-release properties, making it particularly suitable for therapeutic scenarios requiring long-term stable drug supply.
[0144] 6. In vitro degradation test
[0145] Test method: The printed hydrogel scaffold was freeze-dried, weighed, and recorded as W0. Then, the freeze-dried hydrogel sample was placed in a 24-well plate, and a fixed volume of PBS buffer was added using a pipette to completely submerge the hydrogel sample. The plate was then placed in a 37°C shaker. The degradation buffer was replaced every 2 days. On day 14, the sample was removed, excess degradation buffer was discarded, the sample was freeze-dried, weighed, and the weight of the hydrogel was recorded as W. t The degradation rate is calculated using the following formula:
[0146]
[0147] Table 2. Degradation rate
[0148] Grouping Degradation rate Example 1 64.1±4.6％ Example 2 61.5±5.8％ Example 3 54.4±2.6％ Example 4 47.4±1.8％ Example 5 41.3±5.3％ Example 6 30.2±3.5％ Example 7 42.6±4.7％ Example 8 28.2±3.8％ Comparative Example 1 75.2±1.9％ Comparative Example 2 69.3±6.1％ Comparative Example 3 71.1±3.3％
[0149] Results show that the ideal hydrogel degradation rate should be 20%-40% at 14 days, gradually degrading to near complete degradation within 4-6 weeks. This design allows the hydrogel scaffold to effectively support initial cartilage repair, subsequently providing sufficient space and environment for newly formed cartilage tissue to occupy the scaffold's position, ultimately completing the repair process. The addition of Col1MA significantly resisted degradation; this resistance increased with increasing dosage. Simultaneously, the MBG@RES content also significantly affected the degradation rate, possibly because a higher MBG@RES content can form a denser and more stable three-dimensional network structure, increasing the hydrogel's cross-linking degree and stability. Higher cross-linking density is generally associated with a slower degradation rate, as a tighter molecular network restricts water molecule penetration, thus delaying material degradation.
[0150] 7. Cell compatibility evaluation
[0151] Test method: Cultured BMSCs cells were digested and resuspended with 0.25% trypsin, and then cultured at a density of 1×10⁻⁶ cells / cells. 5 Cell suspension at 1 / mL was rapidly mixed with sterilized hydrogel, printed in a 3D printer, and placed in 24-well plates for further culture in α-MEM complete medium. Each group had at least 3 wells. Cell viability was quantitatively analyzed using CCK8. After 24 hours of culture, the plates were removed, and 300 μL of CCK8 working solution was added to each well. The 3D-printed scaffold was thoroughly broken up using a pipette tip. The plates were incubated at 37°C in a CO2 incubator (containing 5% CO2) for 1–2 hours. The absorbance (OD) was measured at 450 nm using a microplate reader, and cell viability was calculated using the formula:
[0152]
[0153] Figure 5 Cell compatibility evaluation is shown.
[0154] The results show that the hydrogel scaffolds did not produce any toxicity to the cells, and the cell viability was all above 90%, even exceeding 100% (Examples 6-8). This indicates that the prepared hydrogel scaffolds have no obvious toxicity to the cells and can promote cell proliferation to a certain extent.
[0155] 8. Determination of MDA (malondialdehyde) content:
[0156] Test method:
[0157] BMSCs cells were loaded at 2×10 4Cells were seeded at a density of 1 / 24 wells into four groups: Control, Example 6, Comparative Example 1, and Comparative Example 3. After cell adhesion, appropriate materials were added to each group for co-culturing. The culture medium was changed after 12 hours. Except for the Control group, the other four groups were treated with 100 μM H2O2 for 4 hours to induce oxidative stress. Cells were collected into centrifuge tubes, and the supernatant was discarded after centrifugation. 1 mL of extraction buffer was added per 4 million cells, and bacteria or cells were sonicated (20% power, 3 seconds sonication, 10-second interval, repeated 30 times). The cells were then centrifuged at 8000g, 4°C for 10 minutes, and the supernatant was collected and placed on ice. Subsequently, the cells were tested using an MDA kit (Maclean's).
[0158] Calculation formula: MDA content (nmol / 10) 4 cells)=[ΔA×V 反总 / (ε×d)×10 9 ] / (400×V 样 / V 样总 )=0.0645×ΔA
[0159] In the above formula, V 反总 Total volume of the reaction system, 8 × 10⁻⁶ -4 L; ε: Malondialdehyde molar extinction coefficient, 155 × 10⁻⁶ 3 L / mol / cm; d: optical path of the cuvette, 1cm; V 样 Add the sample volume, 0.2 mL; V 样总 : Volume of extract solution added; 400: Total number of cells, 4 million.
[0160] Results indicate: From Figure 6 It can be seen that the MDA levels in Example 6 and the Control group were comparable, while Comparative Example 1 and Comparative Example 3 both had higher MDA levels. This indicates that the hydrogel scaffold in Example 6 can effectively reduce the level of lipid peroxide MDA. This may be because the silicon, calcium, and phosphorus ions in the bioactive glass, when interacting with body fluids or cells, can promote the effective neutralization of free radicals in the body by antioxidant enzymes (such as superoxide dismutase and catalase) and resveratrol, thus slowing down cellular oxidative damage. The two work synergistically to inhibit oxidative stress and provide a stable microenvironment for bone tissue. The hydrogel scaffolds in other examples also showed similar effects in reducing lipid peroxide MDA levels as in Example 6.
[0161] 8. Anti-inflammatory evaluation:
[0162] Test Method: After anesthetizing rats, the surface cartilage layer was removed using a scalpel or micro-drill, extending down to the subchondral bone. A defect of 2-3 mm in diameter was typically created. Some bleeding was usually observed at this stage; after hemostasis with cotton swabs, a hydrogel scaffold was placed, and the wound was closed with 7-0 absorbable sutures. Ten days post-surgery, bone tissue samples were collected, washed in pre-chilled PBS (0.02 mol / L, pH 7.0-7.2) to remove blood, and weighed. A 1.0 g tissue block was transferred to a glass homogenizer, and 5 mL of pre-chilled PBS was added for thorough homogenization on ice. The resulting homogenate was subjected to two freeze-thaw cycles. The homogenate was then centrifuged at 5000 × g for 5 minutes, and the supernatant was collected for ELISA detection.
[0163] Figure 7 The evaluation of the anti-inflammatory effect is shown.
[0164] Results indicate: From Figure 7 It can be seen that, compared with the saline group and Comparative Example 1, the hydrogel scaffold loaded with Example 6 can effectively reduce the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β, providing a stable microenvironment for bone tissue. The hydrogel scaffolds of other examples also showed similar effects as Example 6 in reducing the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β.
Claims
1. A customizable biphasic hydrogel scaffold, characterized in that, The biphasic hydrogel scaffold comprises a hyaline cartilage layer and a subchondral bone layer. The hyaline cartilage layer comprises methacrylamide type I recombinant collagen and bone marrow mesenchymal stem cells. The subchondral bone layer comprises methacrylamide type I recombinant collagen and bioactive glass loaded with resveratrol. In the hyaline cartilage layer, by weight-volume percentage, 5-15% is methacrylamide type I recombinant collagen and 1-2 × 10⁻⁶ is bone marrow mesenchymal stem cells. 5 cells / mL; In the subchondral bone layer, by mass-volume percentage, 7.5-15% is methacrylamide type I recombinant collagen and 1-2% is resveratrol-loaded bioactive glass; The methacrylamide type I recombinant collagen was prepared by the following steps: 2 g of type I collagen was dissolved in 100 mL of deionized water, the pH was adjusted to 7, 2 mL of methacrylic anhydride was added dropwise, and the reaction was carried out at room temperature for 24 h. The product was dialyzed in deionized water, centrifuged at 10000 rpm for 15 minutes to remove impurities, and the supernatant was freeze-dried to obtain the methacrylamide type I recombinant collagen. The resveratrol-loaded bioactive glass is prepared by the following steps: 1 g of resveratrol was dissolved in 50 mL of deionized water to prepare a 2% resveratrol solution; 1 g of bioactive glass was added to the resveratrol solution and ultrasonically dispersed for 15 min, then stirred and mixed at 40 °C. After the reaction was completed, the glass was washed with deionized water to remove unbound resveratrol and freeze-dried to obtain the final product. The bioactive glass is prepared by the following steps: Dissolve 0.7 g of hexadecyltrimethylammonium bromide in 33 mL of deionized water, add 10 mL of ethyl acetate, stir for 30 minutes, then add 7 mL of 5 M ammonia solution and continue stirring for 15 minutes. Slowly add 3.6 mL of tetraethyl orthosilicate and 0.36 mL of triethyl phosphate to the above solution. Dissolve 2.28 g of calcium nitrate tetrahydrate in 5 mL of deionized water and slowly add it to the above solution. Stir at room temperature for 4 hours. Collect the reaction solution, centrifuge at 5000 rpm for 5 minutes to obtain a white precipitate. Wash the precipitate three times with anhydrous ethanol and deionized water, respectively, and dry at 60 °C for 24 hours. Then calcine the dried white powder at 600 °C at a heating rate of 1 °C / min for 5 hours to obtain the bioactive glass. The biphase hydrogel scaffold was prepared through the following steps: S1. Preparation of the hyaline cartilage layer: A photoinitiation solution was prepared, and methacrylamide type I recombinant collagen was dissolved in the above photoinitiation solution to obtain a methacrylamide type I recombinant collagen solution, which was used as the bio-ink for the first layer of the hydrogel scaffold. The cultured bone marrow mesenchymal stem cells were digested and resuspended with 0.25% trypsin and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the bone marrow mesenchymal stem cells were resuspended with the bio-ink, mixed evenly, and 3D printed. S2. Prepare the subchondral bone layer: Prepare a photoinitiating solution by dissolving methacrylamide type I recombinant collagen in the above photoinitiating solution to obtain a methacrylamide type I recombinant collagen solution; add the bioactive glass loaded with resveratrol, vortex for 30 s to mix evenly, and use the bio-ink as the second layer of the hydrogel scaffold. After the first layer is printed, continue printing the second layer.
2. The biphase hydrogel scaffold according to claim 1, characterized in that, The bone marrow mesenchymal stem cells were prepared through the following steps: Rat limb bones were collected, and the muscle was removed in PBS buffer containing 1% penicillin-streptomycin solution. Small incisions were made at both ends of the bones in α-MEM basal medium to flush out the bone marrow. The bone marrow-medium mixture was collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and red blood cell lysis buffer was added for 10 min. The reaction was terminated by adding PBS, and the cells were centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in α-MEM complete medium and cultured at 37℃ and 5% CO2 for 48 h. The medium was changed, and the cells were passaged when they reached 80% confluence. After 2-3 passages, the cells were washed 3 times with PBS when they reached 80% confluence to obtain the bone marrow mesenchymal stem cells.
3. The biphase hydrogel scaffold according to claim 1, characterized in that, The curing conditions for the first layer of 3D printing were a light intensity of 17 mW / cm². 2 Exposure time was 20 s, and substrate exposure time was 24 s; the curing conditions for printing the second layer were a light intensity of 18 mW / cm². 2 The exposure time is 22 seconds, and the exposure time for the grassroots level is 27 seconds.
4. The use of the biphasic hydrogel scaffold as described in any one of claims 1 to 3 in the preparation of medicaments that promote wound healing or bone repair or have antioxidant or anti-inflammatory effects.