A composite artificial tissue microparticle scaffold inner and outer layer filling material and its preparation method

Abstract

This invention relates to the field of medical materials technology, and more particularly to an inner and outer layer filling material for a composite artificial tissue microparticle scaffold and its preparation method. The method includes: first, modifying bovine Achilles tendon type I collagen and chondroitin sulfate with thiolation, then mixing and reacting them with corresponding components to granulate, thus obtaining an inner layer of injectable microparticle gel; then preparing boric acid-modified ε-polylysine, separately preparing a dense moisturizing gel membrane and a gradient porous drainage sponge, bonding the two together at the interface, and drying to obtain the final product. This invention, through the dual cross-linking structure design of the inner layer of injectable microparticle gel and the synergistic construction with the outer double-layer membrane, significantly improves the mechanical strength, degradation rate, matching with new tissue growth, and biocompatibility of the composite material.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to an inner and outer layer filling material for a composite artificial tissue microparticle scaffold and its preparation method. Background Technology

[0002] Composite artificial tissue microparticle scaffolds are core carriers in tissue engineering used to biomimetically construct three-dimensional cell growth microenvironments and facilitate the regeneration and repair of damaged tissues. Their core design concept involves the collaborative construction of inner and outer layers with differentiated filling materials to adapt to the hierarchical structural characteristics of natural human tissues. Because natural soft and hard tissues, blood vessels, and cartilage all exhibit distinct layered structures, a single homogeneous material scaffold cannot simultaneously meet the differentiated needs of the tissue's internal and external layers for mechanical support, interfacial wetting, cell adhesion and proliferation, and the time-sequential release of biological factors. Therefore, the academic community generally employs a combination of different filling materials for the inner and outer layers to construct microparticle scaffolds. The inner layer filling material often uses synthetic polymers or cross-linked hydrogels such as polycaprolactone and polylactic acid-glycolic acid, primarily providing a stable three-dimensional porous framework, a controllable degradation cycle, and sufficient space for internal cell colonization and nutrient transport. The outer layer filling material often uses natural hydrophilic polymer composite fiber materials such as gelatin, chitosan, and silk fibroin to optimize the hydrophilic wettability of the scaffold surface, improve interfacial compatibility for cell adhesion, and simultaneously simulate the matrix barrier structure of the tissue's outer layer. Relying on mature fabrication processes such as coaxial microfluidics, electrospinning, and 3D coaxial printing, these inner and outer layer filling materials can achieve ordered composite molding, enabling the scaffold to balance overall structural stability and surface biocompatibility. Currently, they are widely used in the regeneration and repair research of various types of defective tissues such as bone, cartilage, blood vessels, and skin. The related combination schemes of natural polymers, synthetic biodegradable polymers, and bioactive composite media have formed a systematic research system, which can control the degradation rate, pore connectivity characteristics, and biological functional response of the inner and outer layer materials as needed, thereby highly mimicking the layered physicochemical and biological characteristics of human native tissues.

[0003] In existing technologies, the inner and outer layer filling materials of composite artificial tissue microparticle scaffolds still have many substantial drawbacks. Commonly used synthetic polymers for the inner layer generally suffer from insufficient bioactivity and the potential accumulation of acidic products during degradation, leading to aseptic inflammation. Some synthetic polymers, such as polycaprolactone, have excessively slow degradation rates, making it difficult to match the growth rhythm of newly formed tissues. Cross-linked hydrogels, on the other hand, suffer from insufficient mechanical strength and poor structural stability. Commonly used natural hydrophilic polymers for the outer layer often exhibit weak mechanical properties and high brittleness. Materials such as gelatin also suffer from unstable degradation rates, potential toxicity of cross-linking agents, and poor thermal stability. Chitosan requires combination with other materials to meet the scaffold performance requirements, while silk fibroin not only has low bioactivity but also lacks direct signal induction for specific cells. Furthermore, the interfacial bonding between the inner and outer filling materials is generally weak, easily leading to delamination. Existing materials also lack precise temporal control over degradation rates, making it difficult to dynamically match degradation behavior with the growth rate of newly formed tissues. Some materials also exhibit limited cell adhesion and proliferation promotion effects and limited biological functional responses. Summary of the Invention

[0004] To address the problems mentioned in the background section, this invention provides an inner and outer layer filling material for a composite artificial tissue microparticle scaffold and its preparation method.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A composite artificial tissue microparticle scaffold has inner and outer layer filling materials, comprising an inner filling material and an outer covering material; the inner filling material is an injectable microparticle gel containing bovine Achilles tendon type I collagen and chondroitin sulfate (mucopolysaccharide), wherein the purity of bovine Achilles tendon type I collagen is ≥95%, the molecular weight of chondroitin sulfate is 5000-20000 Da, and the mass ratio of bovine Achilles tendon type I collagen to chondroitin sulfate is 1:0.2-1:1; the outer covering material is a bilayer membrane, consisting of a dense moisturizing layer and a gradient porous drainage layer bonded together by interfacial chemical bonding; the dense moisturizing layer... The product comprises a gel formed by cross-linking Artemisia argyi seed gum and boric acid-modified ε-polylysine via dynamic borate ester bonds, wherein the purity of Artemisia argyi seed gum is ≥95% and the viscosity-average molecular weight is 1800-2200 kDa, and the weight-average molecular weight of boric acid-modified ε-polylysine is 4.0-5.0 kDa; the gradient porous drainage layer comprises a blend of Artemisia argyi seed gum and polylactic acid, wherein the mass ratio of Artemisia argyi seed gum to polylactic acid is 1:(0.05-0.2), and the side of the gradient porous drainage layer adjacent to the dense moisturizing layer is bridged by Artemisia argyi seed gum and boric acid-modified ε-polylysine via borate ester bonds.

[0006] Furthermore, the bovine Achilles tendon type I collagen and chondroitin sulfate in the inner filling material are modified with thiol groups by 2-iminothione hydrochloride. The modification reaction temperature is 25-37°C, the reaction time is 1-3 hours, and the mass ratio of 2-iminothione hydrochloride to the total mass of bovine Achilles tendon type I collagen and chondroitin sulfate is 0.00044:1 to 0.00133:1. The injectable microparticle gel also contains methacrylic anhydride gelatin and 4-arm-polyethylene glycol-maleimide. The degree of substitution of methacrylic anhydride-modified gelatin is 20%-40%, and the molecular weight of 4-arm-polyethylene glycol-maleimide is 2000-10000 Da. The mass ratio of methacrylic anhydride-modified gelatin to 4-arm-polyethylene glycol-maleimide is (5-10):1. Primary crosslinking is formed through the Michael addition reaction of mercapto groups with maleimide, and further crosslinking and curing can be achieved under ultraviolet light irradiation. The ultraviolet light wavelength is 365-405 nm, and the irradiation intensity is 10-30 mW / cm. 2 The irradiation time is 10-60 seconds.

[0007] Furthermore, the inner filling material also contains at least one of recombinant human fibroblast growth factor, human granulocyte-macrophage stimulating factor, human serum albumin, glycerol, and sodium carboxymethyl cellulose, wherein the concentration of recombinant human fibroblast growth factor is 10-50 ng / mL, the concentration of human granulocyte-macrophage stimulating factor is 5-20 ng / mL, the mass fraction of human serum albumin is 0.5%-2%, the volume fraction of glycerol is 1%-5%, and the mass fraction of sodium carboxymethyl cellulose is 0.1%-1%.

[0008] Furthermore, the boric acid-modified ε-polylysine is a graft copolymer formed by linking ε-polylysine (molecular weight approximately 3000) and 3-carboxy-5-fluorophenylboronic acid (molecular weight 184.02) through amide bonds, wherein the mass ratio of ε-polylysine to 3-carboxy-5-fluorophenylboronic acid is (2-4):1.

[0009] Furthermore, the pore size of the gradient porous drainage layer increases from the small pores facing the dense moisturizing layer to the large pores moving away from it. The average pore size of the small pores is 30-50 μm, the average pore size of the large pores is 150-250 μm, the thickness of the gradient porous drainage layer is 0.5-2 mm, and the porosity is 70%-90%.

[0010] Furthermore, the dense moisturizing layer has a water content of 20%-40%, a thickness of 50-150μm, a tensile strength of 0.1-0.5MPa, and an elongation at break of 100%-300%.

[0011] According to another aspect of the present invention, a method for using the above-described inner and outer layer filling materials is provided, comprising the following steps: S1. Bovine Achilles tendon type I collagen and chondroitin sulfate are dissolved in phosphate buffer solution at pH 7.0-7.8 at a dissolution temperature of 4-25℃ and a stirring time of 2-6 hours to achieve a concentration of 1-5 mg / mL. The solution is then modified with thiol and mixed with methacrylic anhydride gelatin at a mixing temperature of 25-37℃ and a stirring time of 30-60 minutes. The mixture is then reacted with 4-arm-polyethylene glycol-maleimide at a reaction temperature of 25-37℃ and a reaction time of 1-2 hours. The mixture is then granulated to obtain injectable microparticle gel as an inner layer filling material. S2. Preparation of boric acid-modified ε-polylysine: ε-polylysine is subjected to an amidation reaction with 3-carboxy-5-fluorophenylboronic acid under the catalysis of carbodiimide and N-hydroxysuccinimide. The reaction pH is 5.0-6.0, the reaction temperature is 4-10℃, and the reaction time is 10-14 hours. The mass ratio of carbodiimide to 3-carboxy-5-fluorophenylboronic acid is 1.24:1 to 1.55:1; the mass ratio of N-hydroxysuccinimide to carbodiimide is 0.60:1 to 0.72:1. Purification and lyophilization are carried out by dialyzing with a dialysis bag with a molecular weight cutoff of 3000-4000 Da for 48-72 hours. The lyophilization temperature is -50 to -60℃, the vacuum degree is <10 Pa, and the lyophilization time is 24-48 hours. S3. Prepare an aqueous solution of Artemisia argyi seed gum with a concentration of 3%-7% (mass volume fraction), a dissolution temperature of 70-90℃, and a stirring time of 1-3 hours. Dissolve the product obtained in step S2 in a buffer solution containing magnesium ions with a concentration of 2%-4% (mass volume fraction) and a stirring time of 30-60 minutes. Obtain a dense moisturizing gel film by contact crosslinking at a temperature of 20-25℃ and a crosslinking time of 5-15 minutes. S4. Mix and emulsify a polylactic acid organic solution, with chloroform or dichloromethane as the solvent, at a concentration of 15%-25% (mass-volume fraction), with an Artemisia argyi seed gum aqueous solution. The mixing volume ratio is 1:40-1:60, the emulsification temperature is 40-60℃, the stirring speed is 1000-2000 r / min, and the emulsification time is 10-20 minutes. Spread a pore-forming agent with a particle size gradient distribution to a thickness of 0.5-1.5 mm. Freeze-dry the mixture. The pre-freezing temperature is -40-60℃, the pre-freezing time is 2-6 hours, the freeze-drying temperature is -50-60℃, the vacuum degree is <10 Pa, and the freeze-drying time is 20-30 hours. Then wash the mixture with salt at a temperature of 35-39℃ for 20-28 hours, changing the water every 6-8 hours to obtain a gradient porous drainage sponge. S5. Lay a dense moisturizing film onto the pore surface of a gradient porous drainage sponge, ensuring the interface is moistened. Then, spray with a magnesium ion-containing phosphate buffer solution at a spray volume of 0.1-0.3 mL / cm³. 2Then, it is kept at 35-39℃ and 5-15kPa pressure for 2-5 hours, and then composited through borate ester bonds. After drying, the drying temperature is 35-45℃ and the drying time is 4-8 hours. The moisture content after drying is controlled at 5%-10%.

[0012] Further, in step S1, the thiolization modification uses 2-iminothione hydrochloride (molecular weight 138.62), the reaction pH is 7.5-8.5, the reaction temperature is 25-37℃, and the reaction time is 1-3 hours; the granulation is carried out by a microdroplet generator and sieving to obtain particles with a particle size of 150-350μm, the flow rate of the microdroplet generator is 1-5mL / h, and the sieve mesh is 40-100 mesh.

[0013] Further, the magnesium-containing buffer solution mentioned in step S3 is a phosphate buffer solution containing 0.005%-0.02% magnesium sulfate by mass / volume, with a pH of 7.4 and a concentration of 0.01-0.1 mol / L. The buffer solution may also contain 0.1-0.5 mol / L sodium chloride.

[0014] Further, the pore-forming agent in step S4 is sodium chloride crystals, with crystals of less than 50 μm in diameter laid on the small pore side and a thickness of 0.2-0.5 mm, and crystals of 150-250 μm in diameter laid on the large pore side and a thickness of 0.3-1.0 mm; the polylactic acid is low molecular weight poly-L-lactic acid with an intrinsic viscosity of 0.2-0.5 dL / g.

[0015] The beneficial effects of this invention are: 1. The inner layer filling material of this invention is an injectable microparticle gel, which is made by dissolving and thiolizing natural bovine Achilles tendon type I collagen and chondroitin sulfate, then reacting them with methacrylic anhydride-modified gelatin and 4-arm-polyethylene glycol-maleimide and granulating the mixture. This fundamentally solves the problems of insufficient bioactivity of existing synthetic polymers in the inner layer, degradation and acid production leading to aseptic inflammation, and insufficient mechanical strength of cross-linked hydrogels. Bovine Achilles tendon type I collagen can mimic the structure of human extracellular matrix, exhibiting excellent bioactivity and compatibility. Its fibrous structure provides natural binding sites for cell adhesion and proliferation. Chondroitin sulfate works synergistically with collagen to enhance the stability of the gel network through intermolecular hydrogen bonds, compensating for the mechanical shortcomings of existing cross-linked hydrogels. After thiolization modification, the thiol groups formed by collagen and chondroitin sulfate and related components form a primary cross-linked network through Michael addition reaction. This network is then further cross-linked and cured by ultraviolet light to form a double cross-linked structure, balancing injectability and mechanical strength, avoiding the problem of degradation and acid production of synthetic polymers. Furthermore, the degradation rate can be controlled by the degree of cross-linking, achieving dynamic matching with the growth of new tissue. The bioactive factors added to the inner layer can be uniformly dispersed in the gel network and slowly released, providing continuous signal induction for cell growth, making up for the shortcomings of existing materials with single biological functions, and improving tissue repair efficiency.

[0016] 2. The outer covering material of this invention is a bilayer structure formed by interfacial chemical bonding of a dense moisturizing layer and a gradient porous drainage layer. Through stepwise preparation and precise construction, it effectively solves the problems of weak mechanical properties, high brittleness, unstable degradation, and toxicity of crosslinking agents in existing natural hydrophilic polymer outer layers. The dense moisturizing layer is prepared by contact crosslinking of *Artemisia argyi* seed gum aqueous solution with boric acid-modified ε-polylysine in a magnesium ion buffer solution. *Artemisia argyi* seed gum has good hydrophilicity and compatibility, and its mechanical properties are superior to gelatin and chitosan, avoiding the performance defects of the latter. The boric acid-modified ε-polylysine is prepared by amidation reaction, purification, and freeze-drying, and crosslinks with *Artemisia argyi* seed gum through dynamic borate ester bonds to form a dense network, possessing both mechanical strength and flexibility, without the need for toxic crosslinking agents, thus avoiding toxicity risks. The gradient porous drainage layer is made by emulsifying polylactic acid organic solution and Artemisia argyi seed gum aqueous solution, laying gradient pore-forming agent, and then freeze-drying and washing with salt. The hydrophilicity of Artemisia argyi seed gum and the mechanical strength and degradability of polylactic acid work synergistically to improve the wettability and structural stability of the porous layer. Its degradability enables the scaffold degradation and new tissue growth to be synchronized, thus optimizing the degradation regulation effect. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0018] A method for preparing inner and outer layer filling materials for a composite artificial tissue microparticle scaffold includes the following steps: S1. Take 1.0g of bovine Achilles tendon type I collagen and 0.2g of chondroitin sulfate, and dissolve them together in 1000mL of pH 7.0 phosphate buffer at 4℃ for 2 hours. Take 0.00064g of 2-iminothione hydrochloride and add it to the above solution for thiolation modification at pH 7.5, 25℃ for 1 hour. After modification, add 0.5g of methacrylic anhydride gelatin and stir at 25℃ for 30 minutes to mix evenly. Then add 0.1g of 4-arm-polyethylene glycol-maleimide and react at 25℃ for 1 hour. Granulate the mixture using a microdroplet generator and a 40-mesh sieve to obtain injectable microparticle gel with a particle size of 150-250μm as the inner layer filling material. Add 0.001mg of recombinant human fibroblast growth factor and 1mL of glycerol to the inner layer filling material. S2. Preparation of boric acid-modified ε-polylysine: 2.0 g of ε-polylysine and 1.0 g of 3-carboxy-5-fluorophenylboronic acid were added to a reaction vessel. 1.24 g of carbodiimide and 0.74 g of N-hydroxysuccinimide were added as catalysts. The pH of the reaction was adjusted to 5.0, and the amidation reaction was carried out at 4 °C for 10 hours. After the reaction was completed, the mixture was purified by dialyzing with deionized water for 48 hours using a dialysis bag with a molecular weight cutoff of 3000 Da. Subsequently, it was freeze-dried at -50 °C and a vacuum degree <10 Pa for 24 hours to obtain approximately 2.8 g of boric acid-modified ε-polylysine. S3. Take 3g of Artemisia argyi seed gum and add it to 100mL of deionized water to prepare a 3% (w / v) Artemisia argyi seed gum aqueous solution. Stir at 70℃ for 1 hour until completely dissolved. Take 2g of boric acid-modified ε-polylysine obtained in step S2 and dissolve it in 100mL of phosphate buffer (pH 7.4, concentration 0.01mol / L, containing 0.005g magnesium sulfate) containing 0.005% (w / v) magnesium sulfate. Stir for 30 minutes until dissolved. Mix the two solutions and crosslink them at 20℃ for 5 minutes to obtain a dense moisturizing gel film. S4. Take 1.5g of low molecular weight poly-L-lactic acid and dissolve it in 8.5mL of chloroform to prepare a 15% (w / v) polylactic acid chloroform solution. Take 40mL of Artemisia argyi seed gum aqueous solution from step S3 and mix it with 1mL of the above polylactic acid solution at a volume ratio of 1:40. Emulsify at 40℃ and 1000r / min for 10 minutes. Take 0.5g of sodium chloride crystals with a gradient particle size distribution as a pore-forming agent, wherein 0.2g of sodium chloride crystals with a particle size less than 50μm are laid on the small pore side and 0.3g of sodium chloride crystals with a particle size of 150-200μm are laid on the large pore side, and spread evenly on the surface of the emulsion. Pre-freeze the sample at -40℃ for 2 hours, then freeze-dry it at -50℃ and vacuum degree <10Pa for 20 hours. Then wash it in deionized water at 35℃ for 20 hours, changing the water every 6 hours to remove the sodium chloride pore-forming agent, and obtain a gradient porous drainage sponge. S5. Lay a dense moisturizing film on the pore surface of a gradient porous drainage sponge, spray it with a magnesium ion-containing phosphate buffer solution, and maintain it at 35°C and 5kPa pressure for 2 hours to bridge the composite through borate ester bonds; dry the composite film at 35°C for 4 hours, and control the water content after drying to 5% to obtain the inner and outer layer filling materials. Example 2

[0019] A method for preparing inner and outer layer filling materials for a composite artificial tissue microparticle scaffold includes the following steps: S1. Take 1.0g of bovine Achilles tendon type I collagen and 0.6g of chondroitin sulfate, and dissolve them together in 500mL of phosphate buffer solution with pH 7.4 at 15℃ for 4 hours. Take 0.00141g of 2-iminothione hydrochloride and add it to the above solution for thiolation modification at pH 8.0, 30℃ for 2 hours. After modification, add 0.7g of methacrylic anhydride gelatin and stir at 30℃ for 45 minutes to mix evenly. Then add 0.1g of 4-arm-polyethylene glycol-maleimide and react at 30℃ for 1.5 hours. Granulate the gel using a microdroplet generator and a 70-mesh sieve to obtain injectable microparticle gel with a particle size of 200-300μm as the inner layer filling material. Add 0.006mg of human granulocyte-macrophage stimulating factor, 0.018g of human serum albumin and 0.0075g of sodium carboxymethyl cellulose to the inner layer filling material. S2. Preparation of boric acid-modified ε-polylysine: 3.0 g of ε-polylysine and 1.0 g of 3-carboxy-5-fluorophenylboronic acid were added to a reaction vessel. 1.40 g of carbodiimide and 0.92 g of N-hydroxysuccinimide were added as catalysts. The pH of the reaction was adjusted to 5.5, and the amidation reaction was carried out at 7 °C for 12 hours. After the reaction was completed, the mixture was purified by dialyzing with deionized water for 60 hours using a dialysis bag with a molecular weight cutoff of 3500 Da. Subsequently, it was freeze-dried at -55 °C and a vacuum degree <10 Pa for 36 hours to obtain approximately 3.7 g of boric acid-modified ε-polylysine. S3. Take 5g of Artemisia argyi seed gum and add it to 100mL of deionized water to prepare a 3% (w / v) Artemisia argyi seed gum aqueous solution. Stir at 80℃ for 2 hours until completely dissolved. Take 3g of boric acid-modified ε-polylysine obtained in step S2 and dissolve it in 100mL of phosphate buffer containing 0.012% (w / v) magnesium sulfate (pH 7.4, concentration 0.05mol / L, containing 0.012g magnesium sulfate and 0.292g sodium chloride). Stir for 45 minutes until dissolved. Mix the two solutions and crosslink them at 22℃ for 10 minutes to obtain a dense moisturizing gel film. S4. Take 2.0 g of low molecular weight poly-L-lactic acid and dissolve it in 8.0 mL of dichloromethane to prepare a 20% (w / v) polylactic acid dichloromethane solution. Take 50 mL of the Artemisia argyi seed gum aqueous solution from step S3 and mix it with 1 mL of the above polylactic acid solution at a volume ratio of 1:50. Emulsify at 50°C and 1500 r / min for 15 minutes. Take 1.0 g of sodium chloride crystals with a gradient particle size distribution as a pore-forming agent. 0.35 g of sodium chloride crystals with a particle size less than 50 μm are laid on the small pore side, and 0.65 g of sodium chloride crystals with a particle size of 200-220 μm are laid on the large pore side, and evenly spread on the surface of the emulsion. Pre-freeze the sample at -50°C for 4 hours, then freeze-dry it at -55°C and a vacuum degree <10 Pa for 25 hours. Then, wash it in deionized water at 37°C for 24 hours, changing the water every 7 hours to remove the sodium chloride pore-forming agent, and obtain a gradient porous drainage sponge. S5. Lay a dense moisturizing film on the pore surface of a gradient porous drainage sponge, spray it with a magnesium ion-containing phosphate buffer solution, and maintain it at 37℃ and 10kPa pressure for 3.5 hours to bridge the composite through borate ester bonds; dry the composite film at 40℃ for 6 hours, and control the water content after drying to 7.5% to obtain the inner and outer layer filling materials. Example 3

[0020] A method for preparing inner and outer layer filling materials for a composite artificial tissue microparticle scaffold includes the following steps: S1. Take 1.0g of bovine Achilles tendon type I collagen and 1.0g of chondroitin sulfate, and dissolve them together in 400mL of phosphate buffer solution at pH 7.8. The dissolution temperature is 25℃, and the stirring time is 6 hours. Take 0.00266g of 2-iminothione hydrochloride and add it to the above solution for thiolation modification. The reaction pH is 8.5, the reaction temperature is 37℃, and the reaction time is 3 hours. After modification, add 1.0g of methacrylic anhydride gelatin and stir at 37℃ for 60 minutes to mix evenly. Then add 0.1g of 4-arm-polyethylene glycol-maleimide and react at 37℃ for 2 hours. Granulate the gel using a microdroplet generator and a 100-mesh sieve to obtain injectable microparticle gel with a particle size of 250-350μm as the inner layer filling material. Add 0.025mg of recombinant human fibroblast growth factor, 0.04g of human serum albumin, 5mL of glycerol, and 0.02g of sodium carboxymethyl cellulose to the inner layer filling material. S2. Preparation of boric acid-modified ε-polylysine: 4.0 g of ε-polylysine and 1.0 g of 3-carboxy-5-fluorophenylboronic acid were added to a reaction vessel. 1.55 g of carbodiimide and 1.12 g of N-hydroxysuccinimide were added as catalysts. The pH of the reaction was adjusted to 6.0, and the amidation reaction was carried out at 10 °C for 14 hours. After the reaction was completed, the mixture was purified by dialyzing with deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 4000 Da. Subsequently, it was freeze-dried at -60 °C and a vacuum degree <10 Pa for 48 hours to obtain approximately 4.6 g of boric acid-modified ε-polylysine. S3. Take 7g of Artemisia argyi seed gum and add it to 100mL of deionized water to prepare a 7% (w / v) Artemisia argyi seed gum aqueous solution. Stir at 90℃ for 3 hours until completely dissolved. Take 4g of boric acid-modified ε-polylysine obtained in step S2 and dissolve it in 100mL of phosphate buffer (pH 7.4, concentration 0.1mol / L, containing 0.02g magnesium sulfate and 2.92g sodium chloride) containing 0.02% (w / v) magnesium sulfate. Stir for 60 minutes until dissolved. Mix the two solutions and crosslink them at 25℃ for 15 minutes to obtain a dense moisturizing gel film. S4. Take 2.5g of low molecular weight poly-L-lactic acid and dissolve it in 7.5mL of chloroform to prepare a 25% (w / v) polylactic acid chloroform solution. Take 60mL of the Artemisia argyi seed gum aqueous solution from step S3 and mix it with 1mL of the above polylactic acid solution at a volume ratio of 1:60. Emulsify at 60℃ and 2000r / min for 20 minutes. Take 1.5g of sodium chloride crystals with a gradient particle size distribution as a pore-forming agent. 0.5g of sodium chloride crystals with a particle size less than 50μm are laid on the small pore side, and 1.0g of sodium chloride crystals with a particle size of 220-250μm are laid on the large pore side, and evenly spread on the surface of the emulsion. Pre-freeze the sample at -60℃ for 6 hours, then freeze-dry it at -60℃ and a vacuum degree <10Pa for 30 hours. Then, wash it in deionized water at 39℃ for 28 hours, changing the water every 8 hours to remove the sodium chloride pore-forming agent, and obtain a gradient porous drainage sponge. S5. Lay a dense moisturizing film on the pore surface of a gradient porous drainage sponge, spray it with a magnesium ion-containing phosphate buffer solution, and maintain it at 39℃ and 15kPa pressure for 5 hours to bridge the composite through borate ester bonds; dry the composite film at 45℃ for 8 hours, and control the water content after drying to 10% to obtain the inner and outer layer filling materials.

[0021] Comparative Example 1 The difference between this comparative example and Example 1 is that chondroitin sulfate was removed from the inner filling material, and only 1.0g of bovine Achilles tendon type I collagen was dissolved in 1000mL of pH 7.0 phosphate buffer. The rest of the preparation steps and dosages were completely consistent with Example 1.

[0022] Comparative Example 2 The difference between this comparative example and Example 1 is that the boric acid-modified ε-polylysine in step S2 is replaced with unmodified ε-polylysine (the amount is still 2g), while the remaining preparation steps and amounts are completely consistent with Example 1.

[0023] Comparative Example 3 The difference between this comparative example and Example 2 is that polylactic acid was omitted in the preparation of the gradient porous drainage sponge, and only 50 mL of the Artemisia argyi seed gum aqueous solution in step S3 was directly emulsified without adding polylactic acid dichloromethane solution. The remaining preparation steps and dosages are completely consistent with Example 2.

[0024] Comparative Example 4 The difference between this comparative example and Example 3 is that 2-iminothione hydrochloride was removed from the inner layer filling material, and no thiolation modification was performed. Instead, a mixed solution of bovine Achilles tendon type I collagen and chondroitin sulfate was directly mixed with methacrylic anhydride gelatin and 4-arm-polyethylene glycol-maleimide. The remaining preparation steps and dosages were completely consistent with those in Example 3.

[0025] (I) Mechanical property testing: Take the outer composite film samples (10mm×10mm×2mm) prepared in Examples 1-3 and Comparative Examples 1-4, weigh each sample using an electronic balance, and record the data. Fix the samples on the fixtures of the universal testing machine, adjust the fixture spacing to ensure uniform force on the samples, set the tensile rate to 1mm / min, and test the tensile strength and elongation at break of the samples. Each parallel sample is tested 3 times, and the average value is taken. After the test, record the tensile strength (MPa) and elongation at break (%) of each group of samples.

[0026] (II) Degradation Performance Testing: Take samples of the inner and outer composite materials from each group, with an outer composite membrane of 10mm×10mm×2mm and an inner microparticle gel of 1mL, and place them in sterile centrifuge tubes. Add 20mL of PBS buffer at pH 7.4 and label the groups accordingly. Place the centrifuge tubes in a 37℃ constant temperature incubator to simulate the physiological environment of the human body. Take out the samples at 1d, 3d, 7d, 14d, 21d, and 28d. Gently rinse the samples three times with deionized water, absorb the surface moisture, and dry them in a 40℃ vacuum drying oven to constant weight. Weigh the remaining mass using an electronic balance and calculate the degradation rate: Degradation rate = (initial mass - remaining mass) / initial mass × 100%).

[0027] (III) Biocompatibility Testing: Sample Extract Preparation: Take 1 mL of the inner layer microparticle gel and 0.1 g of the outer layer composite membrane from each group, place them separately in sterile centrifuge tubes, add 10 mL of culture medium containing 10% fetal bovine serum, incubate at 37℃ for 24 hours, centrifuge, and collect the supernatant as the sample extract. Filter and sterilize before use. Seed L929 fibroblasts in 96-well culture plates at a seeding density of 1×10⁻⁶. 4Add 100 μL of culture medium to each well and incubate at 37°C and 5% CO2 for 24 hours to allow cell adhesion. Discard the old culture medium from the 96-well plate and add 100 μL of extract from each group of samples. Set up a blank control group (culture medium only). Each group has 3 parallel wells. Continue incubation for 24, 48, and 72 hours. At each incubation time point, discard the extract and add 10 μL of CCK-8 reagent and 90 μL of fresh culture medium to each well. Continue incubation for 2 hours, and measure the absorbance (OD value) of each well at 450 nm using a microplate reader. Calculate cell viability: Cell viability = (OD value of sample group / OD value of blank control group) × 100%.

[0028] The results are shown in Table 1: Table 1. Experimental results of different embodiments and comparative examples As shown in Table 1, in Examples 1-3, the inner layer consisted of bovine Achilles tendon type I collagen and chondroitin sulfate, which were dissolved, thiolated, and then reacted with methacrylic anhydride-modified gelatin and 4-arm polyethylene glycol-maleimide before granulation to form injectable microparticle gels. Bovine Achilles tendon type I collagen mimics the structure of the human extracellular matrix. Chondroitin sulfate and collagen synergistically enhance the stability of the gel network through intermolecular hydrogen bonds, and the thiolation modification forms a double cross-linked structure, balancing injectability and mechanical strength, providing good mechanical support for the inner layer. In the outer layer, the dense moisturizing layer is formed by cross-linking Artemisia annua seed gum and boric acid-modified ε-polylysine through dynamic borate ester bonds to form a dense network, possessing both mechanical strength and flexibility. In the gradient porous drainage layer, the hydrophilicity of Artemisia annua seed gum and the mechanical strength of polylactic acid synergistically enhance the stability of the porous layer structure. The combined effect of the inner and outer layers gives the outer composite film high tensile strength and elongation at break. For example, in Example 3, the tensile strength reaches 0.48 MPa and the elongation at break reaches 296.4%.

[0029] In Comparative Example 1, chondroitin sulfate was omitted from the inner layer filling material, and only bovine Achilles tendon type I collagen was used. Due to the lack of the synergistic effect of chondroitin sulfate and collagen in enhancing the stability of the gel network through intermolecular hydrogen bonds, the mechanical properties of the inner layer decreased, leading to a reduction in the overall mechanical properties of the outer composite membrane. The tensile strength was only 0.07 MPa, and the elongation at break was 72.3%, both lower than in Example 1. In Comparative Example 2, boric acid-modified ε-polylysine was replaced with unmodified ε-polylysine. Unmodified ε-polylysine could not crosslink with Artemisia argyi seed gum through dynamic borate ester bonds to form a dense network, resulting in a decrease in the mechanical strength and flexibility of the dense moisturizing layer, affecting the overall mechanical properties of the outer composite membrane. The tensile strength was 0.08 MPa, and the elongation at break was 81.6%, lower than in Example 1. In Comparative Example 3, polylactic acid was omitted from the preparation of the gradient porous drainage sponge, and only Artemisia argyi seed gum aqueous solution was used. Although Artemisia argyi seed gum has good hydrophilicity, its mechanical strength is relatively weaker than that of polylactic acid (PLA). Lacking the synergistic effect of PLA, the stability of the porous layer structure deteriorates, leading to a decrease in the tensile strength and elongation at break of the outer composite membrane, which are 0.15 MPa and 123.8%, respectively, lower than in Example 2. In Comparative Example 4, 2-iminothione hydrochloride was removed from the inner layer filler material, and no thiolization modification was performed. Without thiolization modification, the primary cross-linking network and double cross-linking structure cannot be formed, affecting the mechanical properties of the inner layer, which in turn reduces the mechanical properties of the outer composite membrane, with a tensile strength of 0.22 MPa and an elongation at break of 167.5%, lower than in Example 3.

[0030] In Examples 1-3, the degradation rate of the inner injectable microparticle gel can be controlled by the degree of cross-linking to achieve dynamic matching with the growth of new tissue. The polylactic acid in the outer gradient porous drainage layer is degradable, and the synergistic effect of Artemisia argyi seed gum and polylactic acid optimizes the degradation control effect, so that the degradation rate of the entire composite material is moderate at 28 days, such as 82.4% in Example 3. This ensures that the material can play a sufficient role in the tissue repair process and gradually degrades as new tissue grows.

[0031] Comparative Example 1 lacked chondroitin sulfate in its inner layer, which may have affected the formation and stability of the cross-linked structure, leading to changes in degradation performance. The 28-day degradation rate was 59.8%, lower than Example 1, possibly due to a change in the degree of cross-linking causing a slower degradation rate. Comparative Example 2 did not use boric acid-modified ε-polylysine, affecting the structure and performance of the dense moisturizing layer, and consequently affecting the degradation performance of the entire outer composite film. The 28-day degradation rate was 61.2%, lower than Example 1. Comparative Example 3 lacked polylactic acid in its gradient porous drainage sponge. The degradation characteristics of Artemisia annua seed gum alone differed from the synergistic effect of polylactic acid, resulting in a 28-day degradation rate of 88.7%, higher than Example 2, possibly due to a worsening degradation control effect. Comparative Example 4 did not undergo thiol modification in its inner layer, altering the cross-linked structure and affecting degradation performance. The 28-day degradation rate was 70.1%, lower than Example 3.

[0032] In Examples 1-3, the bioactive factors added to the inner layer can be uniformly dispersed in the gel network and slowly released, providing continuous signal induction for cell growth; the outer layer material also has good hydrophilicity and biocompatibility, resulting in a high cell survival rate after contact with the material extract. For example, in Example 3, the cell survival rate reached 98.2% after 72 hours, indicating that the material has no adverse effect on cell growth and has good biocompatibility.

[0033] Comparative Examples 1-4, due to deletions or alterations in material composition or structure, may have affected the release of bioactive factors or the biocompatibility of the materials, resulting in lower cell survival rates compared to the corresponding embodiments. For example, the cell survival rate of Comparative Example 1 at 72 hours was 88.9%, lower than that of Example 1. This indicates that the synergistic effect of the components in the invention plays an important role in improving the biocompatibility of the materials.

[0034] In summary, this invention, through the dual cross-linking structure design of the inner injectable microparticle gel and the synergistic construction of the outer double-layer membrane, relies on the intermolecular interactions and interfacial bonding of each core component to significantly improve the mechanical strength, degradation rate, matching with new tissue growth, and biocompatibility of the composite material, effectively solving the problems of weak mechanical properties, uncontrolled degradation, and insufficient biocompatibility of existing tissue scaffold materials.

[0035] In the description of this specification, the reference to terms such as "embodiment," "various embodiments," etc., indicates that a specific feature, structure, material, or characteristic described in connection with that embodiment or preparation example is included in at least one embodiment of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments.

[0036] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An inner and outer layer filling material for a composite artificial tissue microparticle scaffold, characterized in that, The material comprises an inner filling material and an outer covering material. The inner filling material is an injectable microparticle gel containing bovine Achilles tendon type I collagen and chondroitin sulfate. The outer covering material is a bilayer membrane composed of a dense moisturizing layer and a gradient porous drainage layer bonded together at the interface. The dense moisturizing layer contains a gel formed by cross-linking Artemisia argyi seed gum and boric acid-modified ε-polylysine through dynamic borate ester bonds. The gradient porous drainage layer contains a blend of Artemisia argyi seed gum and polylactic acid, and on the side of the gradient porous drainage layer adjacent to the dense moisturizing layer, Artemisia argyi seed gum and boric acid-modified ε-polylysine are bridged by borate ester bonds.

2. The inner and outer layer filling material according to claim 1, characterized in that, The bovine Achilles tendon type I collagen and chondroitin sulfate in the inner filling material are modified with thiol groups by 2-iminothione hydrochloride; the injectable microparticle gel also contains methacrylic anhydride gelatin and 4-arm-polyethylene glycol-maleimide, which form primary crosslinks through Michael addition reaction of thiol groups with maleimide, and can be further crosslinked and cured under ultraviolet light irradiation.

3. The inner and outer layer filling material according to claim 2, characterized in that, The inner filling material also contains at least one of recombinant human fibroblast growth factor, human granulocyte-macrophage stimulating factor, human serum albumin, glycerol, and sodium carboxymethyl cellulose.

4. The inner and outer layer filling material according to claim 1, characterized in that, The boric acid-modified ε-polylysine is a graft copolymer formed by linking ε-polylysine and 3-carboxy-5-fluorophenylboronic acid through amide bonds.

5. The inner and outer layer filling material according to claim 1, characterized in that, The pore size of the gradient porous drainage layer increases from the small pores towards the dense moisturizing layer to the large pores away from it. The average pore size of the small pores is 30-50 μm, and the average pore size of the large pores is 150-250 μm.

6. The inner and outer layer filling material according to claim 1, characterized in that, The dense moisturizing layer has a water content of 20%-40% and a thickness of 50-150μm.

7. A method for preparing the inner and outer layer filling material according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Bovine Achilles tendon type I collagen and chondroitin sulfate are dissolved and modified with thiol, mixed with methacrylic anhydride gelatin, and then reacted with 4-arm-polyethylene glycol-maleimide and granulated to obtain injectable microparticle gel as an inner layer filling material. S2. Preparation of boric acid-modified ε-polylysine: ε-polylysine is subjected to amidation reaction with 3-carboxy-5-fluorophenylboronic acid under the catalysis of carbodiimide and N-hydroxysuccinimide, and then purified and lyophilized. S3. Prepare an aqueous solution of Artemisia argyi seed gum, dissolve the product obtained in step S2 in a buffer solution containing magnesium ions, and obtain a dense moisturizing gel film by contact cross-linking. S4. Polylactic acid organic solution and Artemisia argyi seed gum aqueous solution are mixed and emulsified, and a pore-forming agent with a particle size gradient is spread on it. After freeze drying and salt washing, a gradient porous drainage sponge is obtained. S5. Lay the dense moisturizing film on the pore surface of the gradient porous drainage sponge. After the interface is moistened, keep it at 35-39℃ and 5-15kPa pressure for 2-5 hours. The composite is then bridged by borate ester bonds and dried to obtain the final product.

8. The preparation method according to claim 7, characterized in that, The thiolization modification in step S1 uses 2-iminothione hydrochloride, and the reaction pH is 7.5-8.5; the granulation is carried out by a microdroplet generator and sieving to obtain particles with a particle size of 150-350 μm.

9. The preparation method according to claim 7, characterized in that, The magnesium-containing buffer solution mentioned in step S3 is a phosphate buffer solution containing 0.005%-0.02% magnesium sulfate by mass volume, with a pH of 7.2-7.

6.

10. The preparation method according to claim 7, characterized in that, The pore-forming agent mentioned in step S4 is sodium chloride crystals, with crystals with a particle size of less than 50 μm laid on the small pore side and crystals with a particle size of 150-250 μm laid on the large pore side.

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