A collagen-based pickering emulsion 3D-printed cartilage regeneration scaffold and a preparation method thereof

Abstract

The application discloses a kind of collagen Pickering emulsion 3D printing-based cartilage regeneration scaffold and preparation method thereof.Collagen is extracted from fresh collagen extraction raw material, and purified collagen sponge is obtained, the collagen sponge is dissolved in acetic acid, and high-concentration collagen solution is obtained, then oil phase is added to the collagen solution, and high-speed emulsification is carried out using high-speed homogenizer, to obtain collagen-based Pickering emulsion, finally, computer digital modeling and low-temperature hydrogel forming equipment are used, and collagen-based Pickering emulsion is used as biomaterial ink to 3D print cartilage regeneration hydrogel scaffold, the printed scaffold is quickly frozen in liquid nitrogen, and vacuum drying is carried out, to obtain cartilage regeneration scaffold.The application develops collagen Pickering emulsion biomaterial ink with excellent printability, and through 3D printing, collagen-based cartilage regeneration scaffold with good quick customization forming property, adjustable pore structure and good biological activity is obtained.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a cartilage regeneration scaffold based on collagen Pickering emulsion 3D printing and its preparation method. Background Technology

[0002] Cartilage is a special type of connective tissue, mainly distributed in various parts of the human body, such as joints, nose, ears, and trachea. Chondrocytes have a low density (composed of approximately 1% chondrocytes and 99% extracellular matrix) and lack blood vessels, nerves, or lymphatic tissue, resulting in weak self-repair capabilities. Various diseases resulting from cartilage defects (arthritis, sinusitis, or bronchitis, etc.) can significantly impact a patient's daily life and personal health, severely limiting the function of damaged organs and individual mobility. Therefore, tissue repair after cartilage defects is a long-standing clinical challenge and a global problem troubling clinicians. Currently, common clinical treatments for cartilage defects mainly include microfracture surgery, autologous cartilage or chondrocyte transplantation, and implantation of non-degradable prostheses. While these treatments have achieved some efficacy, problems remain, such as donor loss, long treatment cycles, and implant infection. Tissue engineering technology offers a promising clinical option for cartilage repair; implanting cell-loaded cartilage tissue-engineered regenerative scaffolds into the defect site for wound repair is an ideal clinical treatment method.

[0003] An ideal cartilage tissue-engineered regenerative scaffold must perfectly mimic the chondrocyte survival environment and possess excellent bioactivity, biocompatibility, and appropriate biodegradability. Simultaneously, the scaffold should have both high porosity and an interconnected multi-level pore structure; larger pores provide an ideal environment for cell proliferation, migration, and extracellular matrix secretion, while smaller pores provide channels for nutrient transport and metabolic waste removal. Furthermore, cartilage has an extremely rich and intricate three-dimensional structure, exhibiting significant individual differences across age and sex, and varying degrees of cartilage defect among patients. Therefore, constructing highly porous, refined, and customized, bioactive cartilage regenerative tissue-engineered scaffolds presents a significant challenge.

[0004] In recent years, 3D bioprinting technology has been widely used in the rapid prototyping of tissue engineering. It allows for the customization of high-precision scaffold contours and internal pore structures through digital modeling, making it particularly suitable for preparing cartilage regeneration scaffold materials with complex internal and external structures. The extracellular matrix of chondrocytes is mainly composed of abundant collagen and a small amount of glycosaminoglycans. Collagen possesses excellent biocompatibility and biodegradability, while also exhibiting bioactivity that promotes cell proliferation, differentiation, and extracellular matrix secretion. Therefore, collagen used in cartilage regeneration scaffolds can perfectly simulate the survival environment of chondrocytes, making it an ideal tissue engineering material. Developing collagen-based biomaterial inks not only enables the high-value utilization of biomass polymers but also aligns with my country's significant strategic needs for biomedical materials in developing the health industry and transforming its economic development model.

[0005] Collagen solutions exhibit poor plasticity during 3D printing. Low-concentration collagen solutions show immediate leveling after extrusion, while high-concentration solutions have numerous entanglement sites in the collagen molecular chains, resulting in higher yield stress and difficulty in extruding smoothly from the needle. Increasing the extrusion pressure further complicates maintaining the continuity and uniformity of the extrusion. Furthermore, collagen solutions exhibit the extrusion swell effect of polymers, which becomes more pronounced with increased extrusion pressure, thus affecting the shape retention of the scaffold. To address the difficulty in molding low-concentration collagen, researchers have directly extruded the collagen solution into a high-viscosity support bath (gelatin, carbomer, etc.) to aid in filament formation. After printing, the viscosity of the support bath is reduced by altering the temperature, pH, or ionic strength, allowing for material separation. However, lines extruded using fine needles with high printing precision are difficult to maintain their shape in a high-viscosity support bath. Therefore, this printing method typically sacrifices printing precision by using coarser needles, failing to meet the highly refined requirements of cartilage regeneration scaffolds. Summary of the Invention

[0006] The purpose of this invention is to provide a cartilage regeneration scaffold based on collagen Pickering emulsion 3D printing and its preparation method. First, a collagen Pickering emulsion biomaterial ink with excellent printability is prepared. Then, a collagen-based cartilage regeneration scaffold with good formability, adjustable pore structure, and good bioactivity is rapidly customized via 3D printing.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing a cartilage regeneration scaffold based on collagen Pickering emulsion 3D printing includes the following steps:

[0009] 1) Collagen extraction

[0010] Fresh collagen extract raw materials are washed, chopped, and added to sodium carbonate solution. The mixture is shaken at 4°C for 10-14 hours to remove impurities. Acetic acid is added at a liquid-to-material ratio of 30-50:1. Then, pepsin at 1-3% of the raw material mass is added. After stirring at 4°C for 72-80 hours, the supernatant is collected by centrifugation at 8000-9000 r / min. Sodium chloride is continuously added to the supernatant to precipitate collagen solid precipitate. The final concentration of sodium chloride is 0.1-1 mol / L. Acetic acid is used to dissolve the collagen solid precipitate. After complete dissolution, the solution is dialyzed for more than 72 hours. The retentate is collected and freeze-dried at -50°C to obtain purified collagen sponge. The collagen sponge is then stored in a constant temperature, dry, and light-protected place at 4°C.

[0011] 2) Preparation of collagen-based Pickering emulsion

[0012] Collagen sponge was dissolved in acetic acid to obtain a high-concentration collagen solution. The solution was then stirred at a high speed of 5000~10000 r / min for 1~3 min to aid in uniform dissolution. Then, an oil phase was added to the collagen solution and emulsified at a high speed of 8000~20000 r / min for 2~5 min to obtain a collagen-based Pickering emulsion.

[0013] 3) Preparation of collagen-based cartilage regeneration scaffolds

[0014] Using computer digital modeling and low-temperature hydrogel molding equipment, a cartilage regeneration hydrogel scaffold was 3D printed using collagen-based Pickering emulsion as the biomaterial ink. The printed scaffold was then rapidly frozen in liquid nitrogen and vacuum dried at -50 to -150°C to obtain the cartilage regeneration scaffold.

[0015] Furthermore, the collagen extraction raw material mentioned in step 1) is animal skin, tendons, or bones, etc.

[0016] Furthermore, the concentration of the sodium carbonate solution in step 1) is 1-5%.

[0017] Further, the concentration of acetic acid in step 1) is 0.1~0.7 mol / L, and the concentration of pepsin is 1~3%.

[0018] Further, the concentration of acetic acid in step 2) is 0.1~0.7 mol / L, and the concentration of the obtained collagen solution is 40~80 mg / mL.

[0019] Furthermore, the oil phase mentioned in step 2) is soybean oil, tea tree oil, cyclohexane, dichloromethane, toluene, etc.

[0020] Further, the mass ratio of the collagen solution to the oil phase in step 2) is 3:7 to 9:1, such as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6 or 3:7, etc.

[0021] Furthermore, the cartilage regeneration hydrogel scaffold mentioned in step 3) is a meniscus regeneration hydrogel, a nose regeneration hydrogel, an auricle regeneration hydrogel, etc.

[0022] In recent years, collagen molecular chains in collagen solutions have been proven to be stable at the water-oil interface, forming Pickering emulsions. This invention uses collagen Pickering emulsions as biomaterial inks to print cartilage regeneration scaffolds, offering the following advantages: 1. Due to the close proximity of high-concentration collagen molecular chains and high intermolecular friction, this invention emulsifies the collagen solution into a collagen Pickering emulsion. Emulsification allows collagen molecules to encapsulate on the droplet surface, transforming the sliding friction between molecules into rolling friction of the emulsion microspheres. This improves the poor printability and weak shape retention of collagen after extrusion, overcoming the application limitations of poor printability of collagen solutions. 2. During extrusion, the emulsion microspheres can adaptively rearrange to adapt to different shear stresses, resulting in good thixotropy and excellent shape retention after extrusion, leading to high-precision scaffolds. Therefore, using collagen Pickering emulsions as inks, through 3D printing and freeze-drying (Pickering emulsion template method), customized macroporous structures can be formed, and rich and interconnected microporous structures can be formed within the printed filaments. 3. After chondrocytes are inoculated into the collagen-based cartilage regeneration scaffold formed by this method, the collagen can act as an artificial extracellular matrix to provide bioactivity for chondrocytes; the rich and multi-level interconnected macroporous-microporous structure provides an ideal living environment for chondrocytes and improves the efficiency of nutrient supply and waste removal. Attached Figure Description

[0023] Figure 1 Printed line images (top) and optical microscope images (bottom) of collagen Pickering emulsion and collagen solution; where (a) is collagen Pickering emulsion; (b) is collagen solution with the same solid content.

[0024] Figure 2 Printed models of the meniscus, nose, and auricle using collagen Pickering emulsion and collagen solution; where (a) is collagen Pickering emulsion and (b) is collagen solution with the same solid content. Detailed Implementation Example 1

[0025] (1) Wash and chop fresh cowhide, add 1% sodium carbonate solution, shake at 4℃ for 12h to remove impurities, and add 0.1 mol / L acetic acid solution at a liquid-to-material ratio of 30:1. Then add pepsin at 3% of the raw material mass. After stirring at 4℃ for 72h, collect the supernatant by centrifugation at 8000 r / min, and continuously add sodium chloride to the supernatant to precipitate collagen. The final concentration of sodium chloride is 1 mol / L. Dissolve the collagen solid precipitate with 0.5 mol / L acetic acid. After complete dissolution, dialyze the solution for more than 72h. Collect the retentate and freeze-dry it under vacuum at -50℃ to obtain purified collagen sponge. Store the extracted collagen sponge in a constant temperature, dry and dark place at 4℃.

[0026] (2) Dissolve the collagen sponge in 0.1 mol / L acetic acid to obtain a high-concentration collagen solution with a concentration of 60 mg / mL. Stir the solution at high speed for 1 min using a high-speed homogenizer to aid in uniform dissolution. Add cyclohexane to the collagen solution at a water-to-oil ratio of 7:3 and emulsify it at high speed for 2 min using a high-speed homogenizer at a speed of 15000 r / min to obtain a collagen-based Pickering emulsion.

[0027] (3) Using computer digital modeling and low-temperature hydrogel molding equipment, 3D printing of cartilage regeneration hydrogel scaffolds was carried out using collagen-based Pickering emulsion as biomaterial ink (the meniscus, nose and auricle were printed respectively). The printed scaffolds were frozen in liquid nitrogen and then vacuum dried at -50℃ to obtain cartilage regeneration scaffolds. Example 2

[0028] (1) Wash and chop fresh fish skin, add 5% sodium carbonate solution, shake at 4℃ for 12h to remove impurities, and add 0.7 mol / L acetic acid solution at a liquid-to-material mass ratio of 50:1. Then, add pepsin at 1% of the raw material mass. After stirring at 4℃ for 72h, collect the supernatant by centrifugation at 8000 r / min, and continuously add sodium chloride to the supernatant to precipitate collagen. The final concentration of sodium chloride is 0.2 mol / L. Dissolve the collagen solid precipitate with 0.7 mol / L acetic acid. After complete dissolution, dialyze the solution for more than 72h, collect the threshold, freeze-dry at -50℃ to obtain purified collagen sponge, and store the extracted collagen sponge in a constant temperature, dry and dark place at 4℃.

[0029] (2) Dissolve the collagen sponge in 0.3 mol / L acetic acid to obtain a high-concentration collagen solution with a concentration of 70 mg / mL. Stir the solution at high speed for 2 min using a high-speed homogenizer at a speed of 8000 r / min to aid in uniform dissolution. Add tea tree oil to the collagen solution at a water-to-oil ratio of 6:4 and emulsify it at high speed for 4 min using a high-speed homogenizer at a speed of 18000 r / min to obtain a collagen-based Pickering emulsion.

[0030] (3) Using computer digital modeling and low-temperature hydrogel molding equipment, auricular cartilage regeneration hydrogel scaffolds were 3D printed using collagen-based Pickering emulsion as biomaterial ink. The printed scaffolds were frozen in liquid nitrogen and then vacuum dried at -150℃ to obtain cartilage regeneration scaffolds. Example 3

[0031] (1) Wash and chop fresh beef Achilles tendon, add 2% sodium carbonate solution, shake at 4℃ for 12h to remove impurities, add 0.4 mol / L acetic acid solution at a liquid-to-material mass ratio of 50:1. Then add pepsin at 2% of the raw material mass. After stirring at 4℃ for 72h, collect the supernatant by centrifugation at 8000 r / min, and continuously add sodium chloride to the supernatant to precipitate collagen. The final concentration of sodium chloride is 0.4 mol / L. Dissolve the collagen solid precipitate with 0.3 mol / L acetic acid. After complete dissolution, dialyze the solution for more than 72h, collect the retentate, freeze-dry at -50℃ to obtain purified collagen sponge, and store the extracted collagen sponge in a constant temperature, dry and dark place at 4℃.

[0032] (2) Dissolve the collagen sponge in 0.5 mol / L acetic acid to obtain a high-concentration collagen solution with a concentration of 80 mg / mL. Stir the solution at high speed for 3 min using a high-speed homogenizer at a speed of 10000 r / min to aid in uniform dissolution. Add dichloromethane to the collagen solution at a water-to-oil ratio of 3:7 and emulsify it at high speed for 2 min using a high-speed homogenizer at a speed of 20000 r / min to obtain a collagen-based Pickering emulsion.

[0033] (3) Using computer digital modeling and low-temperature hydrogel molding equipment, a 3D-printed hydrogel scaffold for tracheal cartilage regeneration was prepared using collagen-based Pickering emulsion as the biomaterial ink. The printed scaffold was frozen in liquid nitrogen and then vacuum-dried at -50°C to obtain the cartilage regeneration scaffold. Comparative Example 1

[0034] Using computer digital modeling and a low-temperature hydrogel molding device, the collagen solution (i.e., unemulsified) from step (2) of Example 1 was used as the biomaterial ink to 3D print auricular cartilage regeneration hydrogel scaffolds (the meniscus, nose, and auricle were printed separately). The printed scaffolds were frozen in liquid nitrogen and then vacuum dried at -150°C to obtain the cartilage regeneration scaffolds.

[0035] Printing Results: By comparing Example 1 and Comparative Example 1, it can be seen that compared with the unemulsified collagen solution, the collagen Pickering emulsion significantly enhances the uniformity of printed lines and provides stronger line size fidelity. Figure 1 Under the same printing conditions, when printing the meniscus, nose, and auricle as a whole, collagen Pickering emulsion exhibits excellent self-supporting ability, forming ideal stacked structures and distinct hollow structures. In contrast, collagen solution suffers from poor shape retention due to extrusion swelling, leveling phenomena, and gravity, resulting in the eventual collapse of the printed structure. (See...) Figure 2 .

Claims

1. A method for preparing a cartilage regeneration scaffold based on collagen Pickering emulsion 3D printing, characterized in that, Includes the following steps: 1) Collagen extraction Fresh collagen extract raw material was washed and chopped, and sodium carbonate solution was added. The mixture was shaken to remove impurities. Acetic acid solution was added at a liquid-to-material mass ratio of 30-50:

1. Then, pepsin was added. After stirring at 4°C for 72-80 hours, the supernatant was collected by centrifugation. Sodium chloride was continuously added to the supernatant to precipitate collagen solid precipitate. The final concentration of sodium chloride was 0.1-1 mol / L. Acetic acid was used to dissolve the collagen solid precipitate. After complete dissolution, the solution was dialyzed for more than 72 hours. The retentate was collected and freeze-dried under vacuum to obtain purified collagen sponge. 2) Preparation of collagen-based Pickering emulsion Collagen sponges were dissolved in acetic acid with a concentration of 0.1–0.7 mol / L to obtain a collagen solution with a concentration of 40–80 mg / mL. The solution was then stirred using a high-speed homogenizer at a speed of 5000–10000 r / min for 1–3 min to aid in uniform dissolution. The oil phase was then added to the collagen solution and emulsified at high speed using a high-speed homogenizer at a speed of 8000–20000 r / min for 2–5 min to obtain a collagen-based Pickering emulsion. The oil phase is soybean oil, tea tree oil, cyclohexane, dichloromethane or toluene, and the mass ratio of the collagen solution to the oil phase is 3:7 to 9:1; 3) Preparation of collagen-based cartilage regeneration scaffolds Using computer digital modeling and low-temperature hydrogel molding equipment, a cartilage regeneration hydrogel scaffold was 3D printed using collagen-based Pickering emulsion as the biomaterial ink. The printed scaffold was then rapidly frozen in liquid nitrogen and vacuum dried at -50 to -150°C to obtain the cartilage regeneration scaffold.

2. The method for preparing a cartilage regeneration scaffold based on collagen Pickering emulsion 3D printing according to claim 1, characterized in that, The collagen extraction raw materials mentioned in step 1) are animal skin, tendons or bones.

3. The method for preparing a cartilage regeneration scaffold based on collagen Pickering emulsion 3D printing according to claim 1, characterized in that, The concentration of the sodium carbonate solution mentioned in step 1) is 1~5%.

4. The method for preparing a cartilage regeneration scaffold based on collagen Pickering emulsion 3D printing according to claim 1, characterized in that, The concentration of acetic acid in step 1) is 0.1~0.7 mol / L, and the amount of pepsin added is 1~3% of the raw material mass.

5. The method for preparing a cartilage regeneration scaffold based on collagen Pickering emulsion 3D printing according to claim 1, characterized in that, The oscillation temperature in step 1) is 4℃, the oscillation time is 10~14h, and the vacuum freeze-drying temperature is -50℃.

6. The method for preparing a cartilage regeneration scaffold based on collagen Pickering emulsion 3D printing according to claim 1, characterized in that, Step 3) The cartilage regeneration hydrogel scaffold is one of the following: meniscus regeneration hydrogel, nasal regeneration hydrogel, and auricle regeneration hydrogel.

7. A cartilage regeneration scaffold obtained by the preparation method according to any one of claims 1 to 6.

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