3D printing composite bio-ink for repairing cartilage defects and preparation method and application thereof

Abstract

The application provides a 3D printing composite biomaterial ink for repairing cartilage defects and a preparation method and application thereof, the composite biomaterial ink comprises the following components in mass fraction: a biopolymer material 25-30 wt.%, a decellularized matrix 10-30 wt.%, polyethylene oxide 10 wt.%, and the balance is ultrapure water; the composite biomaterial ink further comprises a crosslinking agent, the concentration of the crosslinking agent is 0.00125 g / ml, and the crosslinking agent is lithium phenyl-2,4,6-trimethylbenzoyl phosphinate. The cartilage tissue engineering scaffold prepared by using the 3D printing composite biomaterial ink for repairing cartilage defects provided by the application has good biological functions and can effectively promote the structure reconstruction and function recovery of the cartilage injury site.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, specifically to a 3D-printed composite bio-ink for repairing cartilage defects, its preparation method, and its application. Background Technology

[0002] Because articular cartilage lacks blood vessels, nutrients struggle to reach these areas, resulting in slow or even nonexistent self-repair processes. Therefore, finding a novel bio-ink for use in cartilage tissue engineering scaffolds, while possessing excellent biological functions, to better reconstruct and repair damaged cartilage sites, is of paramount importance.

[0003] Cartilage tissue engineering scaffolds have been widely used in cartilage repair. They can fill defective areas, provide mechanical support, and guide the growth of new tissue in the early treatment of bone defects. Commonly used cartilage tissue engineering materials mainly include the following: synthetic polymers, such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic-glycolic acid copolymer (PLGA), and polyethylene glycol diacrylate (PEGDA); and natural polymers, such as collagen, alginate, and chitosan (CTS).

[0004] 3D printing technology has become an ideal strategy for precisely constructing tissues or organs with complex spatial structures. Bio-ink materials used in 3D printing mainly include polyethylene glycol diacrylate (PEGDA), methacrylamide gelatin (GelMA), or other polymeric materials. While repair scaffolds printed with these bio-inks have good mechanical properties, their cell adhesion ability is insufficient, making it difficult for cells to proliferate, differentiate, and maintain normal physiological activities on the scaffold. Decellularized matrix (dECM), where cells have been removed, leaving only the extracellular matrix, is a natural scaffold material and therefore has good biocompatibility, making it widely used in tissue engineering. However, dECM has weaker mechanical properties, degrades more rapidly, is insufficient to maintain multi-layered scaffolds, and is not conducive to tissue regeneration. This necessitates the introduction of additional biomaterials to better utilize dECM in 3D printing. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a 3D-printed composite bio-ink for repairing cartilage defects, its preparation method, and its application.

[0006] The technical solution of this invention is as follows: In a first aspect, the present invention provides a 3D printing composite bio-ink for repairing cartilage defects, the composite bio-ink comprising the following components by mass fraction: 25-30 wt.% biopolymer material, 10-30 wt.% decellularized matrix, 10 wt.% polyethylene oxide, and the balance being ultrapure water; the composite bio-ink further comprises a crosslinking agent, the crosslinking agent concentration being 0.00125 g / ml, and the crosslinking agent being lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid.

[0007] More preferably, the decellularized matrix has a mass fraction of 30%.

[0008] More preferably, the biopolymer material is at least one of polyethylene glycol diacrylate, gelatin methacrylate, and polylactic acid.

[0009] More preferably, the biopolymer material is polyethylene glycol diacrylate.

[0010] More preferably, the polyethylene glycol diacrylate has a mass fraction of 30%.

[0011] In a second aspect, the present invention provides a method for preparing a 3D-printed composite bio-ink for repairing cartilage defects, the method comprising the following steps: S1. Disperse the biopolymer material, decellularized matrix, and polyethylene oxide in ultrapure water to obtain a mixture; S2. Add a crosslinking agent to the mixture obtained in step S1 and mix evenly to obtain a 3D printing composite bio-ink for repairing cartilage defects.

[0012] More preferably, in step S2, a magnetic stirrer is used to mix the rotor.

[0013] In a third aspect, the present invention provides the application of the above-described 3D printing composite bio-ink for repairing cartilage defects or the 3D printing composite bio-ink for repairing cartilage defects prepared by the above-described preparation method in the preparation of cartilage tissue engineering scaffolds.

[0014] More preferably, the cartilage tissue engineering scaffold is in the shape of a cylindrical mesh cage.

[0015] More preferably, the cartilage tissue engineering scaffold is in the shape of a multi-layered cylindrical mesh cage.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The 3D printing composite bio-ink provided by this invention for repairing cartilage defects exhibits excellent cell compatibility. When mouse chondrocytes are seeded into a cartilage tissue engineering scaffold prepared using the bio-ink provided by this invention, the seeded cells show good proliferation characteristics and high activity, with a more significant proliferation effect compared to the control group.

[0017] 2. The 3D-printed composite bio-ink for repairing cartilage defects provided by this invention has the potential to promote cartilage injury repair. When bone marrow stem cells were seeded into a cartilage tissue-engineered scaffold prepared using the bio-ink provided by this invention, experimental results showed a significant increase in the expression of cartilage-related biomarkers (such as Collagen II and GAGs), indicating that the 3D-printed composite bio-ink for repairing cartilage defects provided by this invention can promote the healing of cartilage injury sites, providing an effective tissue engineering solution for the reconstruction and repair of cartilage injury sites. Attached Figure Description

[0018] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The diagram shows the composition of the 3D-printed composite bio-ink for repairing cartilage defects provided in the first embodiment of the present invention (PEGDA refers to ethylene glycol diacrylate; dECM refers to decellularized matrix). Figure 2 A schematic diagram of a multi-layer cylindrical mesh-shaped cartilage tissue engineering scaffold prepared using 3D printing equipment, provided in the first embodiment of the present invention; Figure 3 A schematic diagram showing the results of cell viability detection using the CCK-8 assay provided in the experimental examples of this invention; Figure 4 This diagram illustrates the detection results of qRT-PCR for cartilage-related products in the experimental examples of this invention (Collagen II refers to type II collagen; GAGs refers to glycosaminoglycans). Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific embodiments.

[0020] The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

[0021] First Embodiment This embodiment provides a 3D printing composite bio-ink for repairing cartilage defects and its preparation method.

[0022] The 3D printing composite bio-ink for repairing cartilage defects provided in this embodiment comprises the following components by mass fraction: 30 wt.% biopolymer material, 30 wt.% decellularized matrix, 10 wt.% polyethylene oxide (PEO), and the balance being ultrapure water. The crosslinking agent is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid with a concentration of 0.00125 g / ml.

[0023] The biopolymer material is at least one of polyethylene glycol diacrylate, gelatin methacrylate, and polylactic acid.

[0024] In this embodiment, the selected biopolymer material is polyethylene glycol diacrylate (PEGDA).

[0025] like Figure 1 As shown, in this embodiment, the 3D-printed composite bio-ink used to repair cartilage defects consists of PEGDA, dECM, PEO, a crosslinking agent, and ultrapure water. PEGDA can improve the mechanical properties and anti-degradation properties of the bio-ink; dECM gives the bio-ink good biocompatibility and cell compatibility; PEO can increase the viscosity of the bio-ink; the mass fractions of PEGDA, dECM, and PEO are 30%, 30%, and 10%, respectively, with the remainder being ultrapure water; the crosslinking agent is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, with a concentration of 0.00125 g / ml.

[0026] The preparation method of the 3D printing composite bio-ink for repairing cartilage defects in this embodiment includes the following steps: A mixture of polyethylene glycol diacrylate, decellularized matrix, and polyethylene oxide in a mass fraction of 30%:30%:10%, with the remainder being ultrapure water, was prepared. Then, lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid was added as a crosslinking agent at a concentration of 0.00125 g / ml. The mixture was then stirred using a magnetic stirrer to obtain a 3D printing composite bio-ink for repairing cartilage defects.

[0027] Second Embodiment This embodiment provides a 3D printing composite bio-ink for repairing cartilage defects. The difference from the first embodiment is that this embodiment includes the following composition by mass fraction: 30 wt.% polyethylene glycol diacrylate, 10 wt.% decellularized matrix, 10 wt.% polyethylene oxide, with the balance being ultrapure water, and the concentration of the crosslinking agent lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid is 0.00125 g / ml.

[0028] Third Embodiment This embodiment provides a 3D printing composite bio-ink for repairing cartilage defects. The difference from the first embodiment is that this embodiment includes the following composition by mass fraction: 30 wt.% polyethylene glycol diacrylate, 20 wt.% decellularized matrix, 10 wt.% polyethylene oxide, with the balance being ultrapure water, and the concentration of the crosslinking agent lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid is 0.00125 g / ml.

[0029] Fourth embodiment This embodiment provides a 3D printing composite bio-ink for repairing cartilage defects. The difference from the first embodiment is that this embodiment includes the following composition by mass fraction: 25 wt.% polyethylene glycol diacrylate, 10 wt.% decellularized matrix, 10 wt.% polyethylene oxide, and the balance being ultrapure water. The concentration of the crosslinking agent, lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, is 0.00125 g / ml.

[0030] Fifth Embodiment This embodiment provides a 3D printing composite bio-ink for repairing cartilage defects. The difference from the first embodiment is that this embodiment includes the following composition by mass fraction: 25 wt.% polyethylene glycol diacrylate, 20 wt.% decellularized matrix, 10 wt.% polyethylene oxide, with the balance being ultrapure water, and the concentration of the crosslinking agent lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid is 0.00125 g / ml.

[0031] Sixth Embodiment This embodiment provides a 3D printing composite bio-ink for repairing cartilage defects. The difference from the first embodiment is that this embodiment includes the following composition by mass fraction: 25 wt.% polyethylene glycol diacrylate, 30 wt.% decellularized matrix, 10 wt.% polyethylene oxide, with the balance being ultrapure water, and the concentration of the crosslinking agent lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid is 0.00125 g / ml.

[0032] Application examples This application example demonstrates the use of 3D-printed composite bio-inks for repairing cartilage defects in the fabrication of cartilage tissue engineering scaffolds.

[0033] The 3D printing composite bio-ink for repairing cartilage defects provided in the first embodiment was printed using 3D printing equipment according to the 3D printing model, such as... Figure 2 As shown, the 3D-printed composite bio-ink for repairing cartilage defects provided in the first embodiment is fabricated into a multi-layered cylindrical mesh-shaped cartilage tissue engineering scaffold using a 3D printing model. In this application example, the 3D printing model is constructed as follows: a 3D printing model is created using 3D printing software. The 3D printing model is a multi-layered cylindrical cage shape, and the path gaps of each layer are adjusted as needed.

[0034] In this application example, the operation process of the 3D printing equipment is as follows: the process parameters of the 3D bioprinting equipment are adjusted, the barrel air pressure is 200 kPa, the extrusion speed is 0.02 mm / s, the metal platform moving speed is 40 mm / s, the printing needle inner diameter is 160 μm, the layer height is 96 μm, the printing temperature is 25℃, the printed structure is controlled by the printing path, the printing path is 0 / 90°, and the solid infill rate is 18%.

[0035] In this application example, the specific process of 3D printing is as follows: 3D printing composite bio-ink for repairing cartilage defects is placed in the barrel of the 3D printing equipment, the relevant data of the 3D printing model is input into the 3D printing equipment, the equipment is started to perform 3D printing, and a cartilage tissue engineering scaffold is obtained.

[0036] Experimental Example The biological characteristics of the tissue-engineered scaffolds provided in the corresponding use cases were evaluated through the following experiments.

[0037] The 3D printing composite bio-ink used to prepare tissue engineering scaffolds was set as the experimental group and the control group.

[0038] Experimental group: A mixture of polyethylene glycol diacrylate, decellularized matrix, and polyethylene oxide was prepared in a mass fraction of 30%:30%:10%. A crosslinking agent, lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (0.00125 g / ml), was then added, with the remainder being ultrapure water. The mixture was then stirred using a magnetic stirrer to obtain a 3D printing composite bio-ink for repairing cartilage defects. A tissue engineering scaffold was prepared using the printing method provided in the application example.

[0039] Control group: A mixture of polyethylene glycol diacrylate and polyethylene oxide at a mass fraction of 30%:10% was prepared, and then a crosslinking agent of lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LiPF2) at a concentration of 0.00125 g / ml was added, with the remainder being ultrapure water. After mixing, the mixture was stirred with a magnetic stirrer to obtain the control group 3D printing composite bio-ink, which was then used to prepare a tissue engineering scaffold according to the printing method provided in the application example.

[0040] 1) Cell proliferation detection Culture of seed cells: Using DMEM / F12 as a reference, a DMEM / F12 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum was prepared, and mouse chondrocytes were cultured in a cell culture incubator at 37°C and 5% CO2.

[0041] Seed cell cultivation: 100 µL of a 1×10⁻⁶ solution was dropped onto the cartilage tissue engineering scaffold. 5 Mouse chondrocytes were seeded onto two different tissue engineering scaffolds using a droplet of medium at a concentration of / mL. The seeded scaffolds were then cultured in a cell culture incubator for 1 hour to promote initial attachment of the mouse chondrocytes. Culture medium was then slowly added until the entire scaffold was covered. The experimental and control groups were incubated at 37°C with 5% CO2 for 1, 3, 7, and 14 days. Cell proliferation and viability in both groups were assessed using the CCK-8 assay.

[0042] The results are as follows Figure 3 As shown, cell proliferation and cell viability in the experimental group were significantly greater than those in the control group, indicating that the cells on the cartilage tissue engineering scaffold provided by this invention exhibit good proliferation behavior and high cell viability. This demonstrates that the 3D printing composite bio-ink provided by this invention for repairing cartilage defects has excellent cell compatibility.

[0043] 2) qRT-PCR detection of cartilage-related products (Collagen II and GAGs) Collagen II is a specific type of collagen that is one of the main components of the cartilage matrix. It is abundant in cartilage and is essential for maintaining its structure and function. It provides cartilage with resistance to pressure and elasticity, making it a key protein for cartilage health.

[0044] GAGs are an abbreviation for glycosaminoglycans, a class of linear polysaccharides that bind to proteins in cartilage to form proteoglycans. GAGs include hyaluronic acid (HA), chondroitin sulfate (CS), and keratin sulfate (KS). These molecules play a role in lubrication, shock absorption, and moisture retention in cartilage, and are crucial for its elasticity and wear resistance.

[0045] Collagen II and GAGs together constitute the extracellular matrix of cartilage, and they interact to maintain the integrity and function of cartilage. In cartilage damage, degenerative diseases (such as osteoarthritis), or other pathological conditions, the content and structure of Collagen II and GAGs may change, affecting cartilage health. Therefore, they are also important targets for research and treatment of cartilage diseases.

[0046] After culturing the experimental and control groups for 7 days, qRT-PCR was performed to detect the expression of Collagen II and GAGs in the experimental and control groups.

[0047] The results are as follows Figure 4 As shown, the expression levels of cartilage-related products (Collagen II and GAGs) in the experimental group were significantly higher than those in the control group, indicating that the 3D-printed composite bio-ink provided by this invention for repairing cartilage defects has the potential to promote cartilage damage repair.

[0048] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A 3D-printed composite bio-ink for repairing cartilage defects, characterized in that, The composite bio-ink comprises the following components by mass fraction: The composite bio-ink comprises 25-30 wt.% biopolymer material, 10-30 wt.% decellularized matrix, and 10 wt.% polyethylene oxide. It also includes a crosslinking agent with a concentration of 0.00125 g / ml, the crosslinking agent being lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, with the remainder being ultrapure water.

2. The 3D printing composite bio-ink for repairing cartilage defects according to claim 1, characterized in that, The decellularized matrix has a mass fraction of 30%.

3. The 3D printing composite bio-ink for repairing cartilage defects according to claim 1, characterized in that, The biopolymer material is at least one of polyethylene glycol diacrylate, gelatin methacrylate, and polylactic acid.

4. The 3D printing composite bio-ink for repairing cartilage defects according to claim 3, characterized in that, The biopolymer material is polyethylene glycol diacrylate.

5. The 3D printing composite bio-ink for repairing cartilage defects according to claim 3, characterized in that, The mass fraction of the polyethylene glycol diacrylate is 30%.

6. A method for preparing a 3D printing composite bio-ink for repairing cartilage defects as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Disperse the biopolymer material, decellularized matrix, and polyethylene oxide in ultrapure water to obtain a mixture; S2. Add a crosslinking agent to the mixture obtained in step S1 and mix evenly to obtain a 3D printing composite bio-ink for repairing cartilage defects.

7. The method for preparing the 3D printing composite bio-ink for repairing cartilage defects according to claim 6, characterized in that, In step S2, the rotor is added and mixed with a magnetic stirrer.

8. The application of the 3D printing composite bio-ink for repairing cartilage defects according to any one of claims 1-5 or the 3D printing composite bio-ink prepared by the preparation method according to any one of claims 6-7 in the preparation of cartilage tissue engineering scaffolds.

9. The application according to claim 8, characterized in that, The cartilage tissue engineering scaffold is in the shape of a cylindrical mesh cage.

10. The application according to claim 9, characterized in that, The cartilage tissue engineering scaffold is in the shape of a multi-layered cylindrical mesh cage.

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