A method for preparing a high osteogenic activity of sisal fiber reinforced chitosan-based antibacterial porous material

Abstract

The application discloses a kind of sisal fiber reinforced high osteogenic activity chitosan-based antibacterial porous material and preparation method thereof, the porous material refers to the compound of carboxylated modified sisal fiber and chitosan, multifunctional hybrid nanometer apatite and sodium agastache rugosus in aqueous solution is uniformly mixed after ultrasonic stirring, then add a small amount of glacial acetic acid, i.e. composite gel material is obtained, it is placed in pre-cooled after freeze-drying in petri dish, finally with NaOH solution immersion washes to neutral, drying can be used.The porous material in poly anion sisal fiber and cation chitosan acid solution have strong ionic crosslinking effect, so that the mechanical properties of the porous material are greatly improved;And added hybrid nanometer apatite is strontium doped and contains a small amount of auren phosphate, and its osteogenic activity is better;In addition, hybrid nanometer apatite also introduces phytic acid to chelate, and sodium agastache rugosus is also loaded in the porous material, so it can coordinate to play the slow and fast dual release antibacterial effect.The raw material used in the new porous material is rich in source, and the preparation method is simple and easy to operate, and its mechanical properties, degradation performance, osteogenic activity and antibacterial property can be regulated by changing the content of each component of the porous material, and an excellent bone tissue engineering scaffold material is expected to be obtained.

Description

Technical Field

[0001] This invention relates to a chitosan-based antibacterial porous material with high osteogenic activity reinforced by sisal fibers and its preparation method, belonging to the field of biomedical materials. Background Technology

[0002] Porous materials, acting as carriers of cells and signaling molecules, provide unique microstructures and microenvironments for bone tissue growth, playing a crucial role in new bone development and thus representing a key issue in bone tissue engineering research. Clinically, bone tissue engineering scaffold materials must meet several criteria: a porous structure with high mechanical strength, good osteoconductivity and osteoinductive properties, a suitable degradation rate, and good antibacterial activity. Based on biomimetic principles, the preparation of porous materials by combining the inorganic component of natural bone, nano-hydroxyapatite (n-HA), with biocompatible chitosan (CS) polymers is considered the preferred research subject for bone tissue engineering scaffolds. However, in clinical applications, the poor mechanical properties of the chitosan matrix and the tendency of n-HA nanoparticles to aggregate make it difficult to obtain high-strength n-HA / CS composite materials, especially the highly porous, interconnected structure required for tissue engineering scaffolds. Furthermore, the mechanical properties are insufficient to maintain the stress required for bone tissue regeneration. Additionally, since synthesized n-HA structural components typically lack trace components such as carbonate, fluorine, silicon, magnesium, and sodium found in bioapatite, their osteogenic activity is lower than that of bioapatite, making it difficult to meet the needs of vascularized bone formation. Simultaneously, traditional n-HA / CS composite scaffolds lack sufficient antibacterial properties, making it difficult to prevent recurrent and delayed bacterial infections in orthopedic surgical areas, which can directly lead to bone tissue repair failure. Therefore, the shortcomings in mechanical properties, osteogenic activity, and antibacterial properties of traditional n-HA / CS composite scaffolds are multifaceted problems that urgently need to be addressed in the development of bone tissue engineering scaffold materials.

[0003] To improve the mechanical properties of n-HA / CS, metal ions, aldehydes, or crown ether crosslinking agents are commonly used to crosslink chitosan, but residual toxic crosslinking agents are harmful to human health. Ionic crosslinking of chitosan with a polyanionic polymer can effectively improve its mechanical properties, especially using carboxylated modified natural fibers for chitosan crosslinking, which is even more beneficial for improving the mechanical properties of CS-based composites. Sisal fibers possess high mechanical strength, abrasion resistance, corrosion resistance, and are green and renewable, and have been widely used in polymer-reinforced materials. Therefore, if sisal fibers are carboxylated into polyanionic polymers and used for ionic crosslinking with chitosan, it is expected to improve the mechanical properties of n-HA / CS porous composites.

[0004] To enhance the osteogenic activity of sisal fiber-reinforced n-HA / CS materials, researchers, based on the principle of component biomimicry, doped n-HA with various trace elements such as carbonate, silicon, and strontium from bioapatite, which can improve its biological properties, especially strontium doping. Additionally, alendronate sodium is an anti-osteoporosis drug with strong anti-bone resorption capabilities. The two terminal groups of its bisphosphate structure have a special affinity for n-HA, and it is usually chemically bonded to the n-HA surface to exert a sustained osteogenic promoting effect. [Reference] ACS Appl. Mater. & Inter. [2018, 10: 25547-25560] confirmed that alendronate and Fe3O4 can self-assemble on the surface of nano-apatite, which has a significant promoting effect on osteoblasts. Therefore, if alendronate and trace element strontium are introduced together to prepare hybrid nano-apatite, it is expected to endow sisal fiber-reinforced n-HA / CS materials with higher osteogenic activity.

[0005] To improve the antibacterial properties of sisal fiber-reinforced n-HA / CS materials, phytic acid, a naturally occurring, non-toxic small molecule with unique biological activities, is widely found in plant seeds and fruits and vegetables. Phytic acid's unique structure (a cyclic molecule containing six phosphate groups and 12 free hydrogens) determines its superior chelating properties. Studies have also found that it possesses antioxidant, anticancer, and antibacterial effects. The antibacterial and anti-biofilm properties of phytic acid can not only be used in dentistry but also provide new strategies for other fields requiring resistance to antimicrobial resistance. [References] Inter J Appl Ceram Tech [2022, 19:1498-1510] It is reported that the strong chelating properties of phytic acid can be used to prepare hydroxyapatite, while also ensuring high antibacterial activity. Therefore, if phytic acid is introduced together with alendronate and strontium, it is expected to obtain hybrid nanoapatite with high osteogenic activity and long-lasting antibacterial properties.

[0006] To further enhance the antibacterial properties of sisal fiber-reinforced hybrid nano-apatite / CS porous materials, directly loading antibacterial drugs onto the porous material to achieve rapid initial release and exert antibacterial effects is an effective method. Houttuynia cordata, with its main active ingredient sodium houttuynia cordata, has a stable chemical structure. Besides its in vivo antibacterial effects, it also exhibits inhibitory effects against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Candida albicans, with the most significant effects against Staphylococcus aureus and Candida albicans. With further advancements in medicine, researchers have also discovered that sodium houttuynia cordata has some effect in treating osteoporosis. Therefore, loading houttuynia cordata onto sisal fiber-reinforced hybrid nano-apatite / CS porous materials can work synergistically with the phytic acid chelated in the hybrid nano-apatite structure to achieve both rapid and sustained-release antibacterial effects, better meeting the antibacterial requirements of bone tissue engineering scaffolds. Summary of the Invention

[0007] To address the above issues, the present invention aims to provide a chitosan-based antibacterial porous material with high osteogenic activity reinforced with sisal fibers and its preparation method. The porous material prepared by this invention possesses good mechanical properties, osteogenic activity, antibacterial activity, and biocompatibility, and is a novel biodegradable porous material that meets the basic performance requirements of bone tissue engineering scaffold materials.

[0008] This invention employs the following technical solution: sisal fiber refers to sisal fiber that has undergone carboxylation modification; chitosan is... The degree of deacetylation is above 90%, and the viscosity-average molecular weight is around 400,000; phytic acid is a commercially available 70% aqueous solution; hybrid nano-apatite refers to hybrid nano-apatite prepared by introducing strontium, alendronate, and phytic acid together; houttuynin refers to commercially available sodium houttuynin powder.

[0009] The surface carboxylated sisal fiber of this invention is characterized by pretreating sisal fiber (average length 0.5~1 mm) with 8% NaOH solution and H2O2 at room temperature and stirring for 2 hours, washing with water until neutral and drying, and then adding it to a 10% (m / v) citric acid solution, wherein the citric acid content is 4 times the mass of the sisal fiber, and adding potassium dihydrogen phosphate at 30% mass of the sisal fiber. After stirring at room temperature for 1 hour, the temperature is raised to 120℃ and reacted for 3 hours. After washing with water until neutral and drying, the surface carboxylated sisal fiber is obtained.

[0010] The hybrid nano-apatite of this invention is characterized by dissolving strontium nitrate and calcium nitrate in an aqueous solution, wherein the strontium nitrate content is 2-10% (Sr / (Sr+Ca) molar ratio), adding phytic acid and reacting for 1 hour, designated as solution A, wherein the phosphate content in the phytic acid accounts for 10-40% of the total phosphate molar ratio; separately, 5-10 wt% alendronate is added to sodium phosphate solution and reacted for 1 hour, designated as solution B, solution B is slowly added dropwise to solution A, maintaining a Ca / P molar ratio of 1.67, finally adjusting the pH value to above 10 with 1 mol / L sodium hydroxide, heating and stirring at 70 ℃ for 5 hours, aging for 48 hours, washing with deionized water until neutral, drying and grinding into powder.

[0011] The chitosan-based antibacterial composite porous material with high osteogenic activity, reinforced with hemp fiber, provided by this invention is achieved by the following technical solution, characterized by the following process steps: A certain amount of hemp fiber was ultrasonically dispersed with water, and then ultrasonically dispersed hybrid nano-apatite slurry was slowly added dropwise. A certain amount of chitosan powder and sodium houttuynia cordata powder were added, and the mixture was mechanically stirred at high speed for 4 hours. After adding 2% glacial acetic acid, a composite gel was obtained. The gel was then frozen at -20 ℃ for 12 hours and then freeze-dried. The dried porous material was then immersed in 10% NaOH solution for 30 minutes, washed with deionized water until neutral, and vacuum-dried at 40 ℃ to constant weight.

[0012] The porous material of the present invention has the following advantages over existing porous materials: (1) The natural hemp fiber and chitosan used in this invention are both biocompatible natural biodegradable polymers with wide availability and low price. The natural hemp fiber is negatively charged after carboxylation modification, which can generate ionic crosslinks with the positively charged chitosan solution, thereby improving the mechanical properties of the composite porous material and providing support stress for cell adhesion and proliferation. In addition, the added hybrid nano-apatite is a hybrid nano-apatite doped with strontium, alenium phosphate and phytic acid. The trace elements strontium and alenium phosphate can give the nano-hydroxyapatite better osteoconductivity and can release slowly and continuously. More importantly, the introduced phytic acid has antibacterial properties and can work synergistically with the sodium houttuynia cordata loaded in the porous material to exert a dual-bed antibacterial effect of slow and rapid release. In addition, the hybrid nano-apatite structure has a certain steric hindrance effect due to the introduction of alenium phosphate and phytic acid molecules, which makes the nano-hydroxyapatite particles more dispersed, so as to have good interfacial compatibility with the sisal fiber-chitosan matrix and make its mechanical properties better. In summary, the natural sisal fiber and hybrid nano-apatite selected in this invention are beneficial to improving the mechanical properties, osteogenic activity, and antibacterial properties of the n-HA / CS composite porous material.

[0013] (2) The sisal fiber reinforced chitosan-based antibacterial composite porous material with high osteogenic activity provided by the present invention has a simple and easy preparation process, low production cost, and is environmentally friendly and suitable for mass production; and its mechanical properties, degradation properties, osteoconductivity and antibacterial properties can be controlled by adjusting the content of each component, so as to obtain bone tissue engineering scaffold materials that meet various performance requirements. Attached Figure Description

[0014] Figure 1 SEM images of a chitosan-based antibacterial composite porous material reinforced with sisal fiber and exhibiting high osteogenic activity. (a) Internal microstructure of the porous material under low magnification, (b) Internal microstructure of the porous material under high magnification, (c) Surface microstructure of the porous material under low magnification, (d) Surface microstructure of the porous material under high magnification. Implementation

[0015] Example 1: 1.0 g of carboxylated modified hemp fiber was dispersed in 100 ml of deionized water. 3.0 g of chitosan was added. Simultaneously, 0.5 g of hybrid nano-apatite containing 5 wt% alendronate, 2% Sr, and 10% phytic acid was ultrasonically dispersed in 100 ml of deionized water and then added dropwise to the above hemp fiber / chitosan mixed solution. 0.2 g of sodium houttuynia cordata was added, and the mixture was ultrasonically stirred magnetically for 4 h. Then, 4 ml of glacial acetic acid was added to obtain a ternary composite gel of hemp fiber / chitosan / hybrid nano-apatite. The gel was frozen at -20 ℃ for 24 h and freeze-dried to constant weight using a freeze dryer. Finally, it was immersed in 10% NaOH solution for 30 min, washed until neutral, and then dried. It was cut into 10 mm × 10 mm × 10 mm blocks, and the compressive strength was measured to be about 0.5 MPa, the porosity was 78%, and the average pore size was 300 μm. After being immersed in simulated body fluid for 8 weeks, a large amount of bone-like apatite was deposited on the surface, and the compressive strength could still be maintained at about 0.3 MPa. The antibacterial rate was 90%.

[0016] Example 2: 1.5 g of carboxylated modified hemp fiber was dispersed in 100 ml of deionized water, and 2.0 g of chitosan was added. Simultaneously, 1.0 g of hybrid nano-apatite containing 10 wt% alendronate, 10% Sr, and 30% phytic acid was ultrasonically dispersed in 150 ml of deionized water and then added dropwise to the hemp fiber / chitosan mixed solution. 0.4 g of sodium houttuynia cordata was added, and the mixture was ultrasonically stirred magnetically for 4 h. Then, 5 ml of glacial acetic acid was added to obtain a ternary composite gel of hemp fiber / chitosan / hybrid nano-apatite. The gel was frozen at -20 ℃ for 24 h and then freeze-dried to constant weight. Finally, it was immersed in 10% NaOH solution for 30 min, washed until neutral, and then dried. It was cut into 10 mm × 10 mm × 10 mm blocks, and the compressive strength was measured to be about 0.6 MPa, the porosity was 74%, and the average pore size was 260 μm. After being immersed in simulated body fluid for 8 weeks, a large amount of bone-like apatite was deposited on the surface, and the compressive strength could still be maintained at about 0.4 MPa. The antibacterial rate was 93%.

[0017] Example 3: 2.0 g of carboxylated modified hemp fiber was dispersed in 200 ml of deionized water, and 2.0 g of chitosan was added. Simultaneously, 1.0 g of hybrid nano-apatite containing 5 wt% alendronate, 10% Sr molar content, and 40% phytic acid molar content was ultrasonically dispersed in 150 ml of deionized water and then added dropwise to the above hemp fiber / chitosan mixed solution. 0.6 g of sodium houttuynia cordata was added, and the mixture was ultrasonically stirred magnetically for 4 h. Then, 7 ml of glacial acetic acid was added to obtain a ternary composite gel of hemp fiber / chitosan / hybrid nano-apatite. The gel was frozen at -20 ℃ for 24 h and then freeze-dried to constant weight using a freeze dryer. Finally, it was immersed in a 10% NaOH solution for 30 min, washed until neutral, and then dried. It was cut into 10 mm × 10 mm × 10 mm blocks, and the compressive strength was measured to be about 0.8 MPa, the porosity was 75%, and the average pore size was 250 μm. After being immersed in simulated body fluid for 8 weeks, a large amount of bone-like apatite was deposited on the surface, and the compressive strength could still be maintained at about 0.4 MPa. The antibacterial rate was 95%.

[0018] Example 4: 1.0 g of carboxylated modified hemp fiber was dispersed in 100 ml of deionized water, and 2.0 g of chitosan was added. Simultaneously, 1.0 g of hybrid nano-apatite containing 5 wt% alendronate, 10% Sr molar content, and 40% phytic acid molar content was ultrasonically dispersed in 150 ml of deionized water and then added dropwise to the above hemp fiber / chitosan mixed solution. 0.4 g of sodium houttuynia cordata was added, and the mixture was continued to be ultrasonically stirred magnetically for 4 h. Then, 5 ml of glacial acetic acid was added to obtain a ternary composite gel of hemp fiber / chitosan / hybrid nano-apatite. The gel was frozen at -20 ℃ for 24 h and then freeze-dried to constant weight using a freeze dryer. Finally, it was immersed in a 10% NaOH solution for 30 min, washed until neutral, and then dried. It was cut into 10 mm × 10 mm × 10 mm blocks, and the compressive strength was measured to be about 0.7 MPa, the porosity was 76%, and the average pore size was 320 μm. After being immersed in simulated body fluid for 8 weeks, a large amount of bone-like apatite was deposited on the surface, and the compressive strength could still be maintained at about 0.4 MPa. The antibacterial rate was 94%.

[0019] Comparative Example 1: 2.0 g of chitosan was weighed, added to 100 ml of deionized water, and 2 ml of glacial acetic acid was added. After stirring until dissolved, 1.0 g of nano-hydroxyapatite was ultrasonically dispersed in 100 ml of water and added dropwise to the above chitosan solution. After continuing ultrasonic magnetic stirring for 2 h, a nano-hydroxyapatite / chitosan binary composite gel was obtained. It was frozen at -20 ℃ for 24 h and then freeze-dried to constant weight using a freeze dryer. Then, it was immersed in a 10% NaOH solution for 30 min, washed until neutral, and dried. It was cut into 10 mm × 10 mm × 10 mm blocks, and the compressive strength was measured to be approximately 0.3 MPa, the porosity was 75%, and the average pore size was 160 μm. After immersion in simulated body fluid for 4 w, the porous material degraded into powder; the antibacterial rate was 60%.

[0020] Comparative Example 2: 1.0 g of unmodified hemp fiber was dispersed in 100 ml of deionized water, and 2.0 g of chitosan was added. Simultaneously, hybrid nano-apatite containing 5 wt% alendronate, 10% Sr molar content, and 40% phytic acid molar content was ultrasonically dispersed in 150 ml of deionized water and then added dropwise to the above hemp fiber / chitosan mixed solution. 0.4 g of sodium houttuynia cordata was added, and the mixture was ultrasonically stirred magnetically for 4 h. Then, 5 ml of glacial acetic acid was added to obtain a ternary composite gel of hemp fiber / chitosan / hybrid nano-apatite. The gel was frozen at -20 ℃ for 24 h and then freeze-dried to constant weight. Finally, it was immersed in 10% NaOH solution for 30 min, washed until neutral, and then dried. It was cut into 10 mm × 10 mm × 10 mm blocks, and the compressive strength was measured to be about 0.5 MPa, the porosity was 75%, and the average pore size was 280 μm. After being immersed in simulated body fluid for 8 weeks, a large amount of bone-like apatite was deposited on the surface, and the compressive strength could still be maintained at about 0.2 MPa. The antibacterial rate was 93%.

[0021] Comparative Example 3: 1.0 g of carboxylated modified hemp fiber was dispersed in 100 ml of deionized water, and 2.0 g of chitosan was added. Simultaneously, 1.0 g of nano-hydroxyapatite was ultrasonically dispersed in 150 ml of deionized water and then added dropwise to the above hemp fiber / chitosan mixed solution. 0.4 g of sodium houttuynia cordata was added, and the mixture was ultrasonically and magnetically stirred for 4 h. Then, 5 ml of glacial acetic acid was added to obtain a ternary composite gel of hemp fiber / chitosan / hybrid nano-hydroxyapatite. The gel was frozen at -20 ℃ for 24 h and then freeze-dried to constant weight. Finally, it was immersed in 10% NaOH solution for 30 min, washed until neutral, and then dried. It was cut into 10 mm × 10 mm × 10 mm blocks, and the compressive strength was measured to be about 0.5 MPa, the porosity was 77%, and the average pore size was 320 μm. After being immersed in simulated body fluid for 8 weeks, a small amount of bone-like apatite was deposited on the surface, and the compressive strength was still maintained at about 0.3 MPa. The antibacterial rate was 94%.

[0022] Comparative Example 4: 1.0 g of carboxylated modified hemp fiber was dispersed in 100 ml of deionized water, and 2.0 g of chitosan was added. Simultaneously, hybrid nano-apatite containing 5 wt% alendronate phosphate, 10% Sr molar content, and 40% phytic acid molar content was ultrasonically dispersed in 150 ml of deionized water and then added dropwise to the above hemp fiber / chitosan mixed solution. After continuing ultrasonic magnetic stirring for 4 h, 5 ml of glacial acetic acid was added to obtain a ternary composite gel of hemp fiber / chitosan / hybrid nano-apatite. The gel was frozen at -20 ℃ for 24 h and then freeze-dried to constant weight using a freeze dryer. Finally, it was immersed in a 10% NaOH solution for 30 min, washed until neutral, and then dried. It was cut into 10 mm × 10 mm × 10 mm blocks, and the compressive strength was measured to be about 0.4 MPa, the porosity was 76%, and the average pore size was 300 μm. After being immersed in simulated body fluid for 8 weeks, a large amount of bone-like apatite was deposited on the surface, and the compressive strength could still be maintained at about 0.2 MPa. The antibacterial rate was 65%.

[0023] Compressive strength test conditions: Cut 10 mm × 10 mm × 10 mm blocks were tested using a universal testing machine (SANSCMT4503, SANS Company, Shenzhen, China) to determine their compressive properties at 40% compression deformation. The test temperature was 20℃ ± 2℃, and the loading speed was 1 mm / min. Five parallel samples were tested in each group, and the average value was taken.

[0024] Porosity determination: Add an appropriate amount of anhydrous ethanol to a vector cylinder. Weigh the dry weight of a 10 mm × 10 mm × 10 mm block of material (recorded as m1). Place the block in anhydrous ethanol and weigh the initial volume of the ethanol and sample (recorded as V1). After soaking at room temperature for one week, remove the material and weigh its wet weight (recorded as m2). The remaining ethanol volume is V2. The porosity is then:

[0025] Three parallel samples were measured for each sample, and the average value was taken.

[0026] Antibacterial activity assay: Escherichia coli (DH5α) was incubated in lysogenic broth (LB) in an incubator for 3 hours. At this time, 5 mL of bacterial suspension (10...) was added... 5 The CFU / mL and a quarter-disc sterile plate were incubated in a shaker incubator at 37°C for 24 hours. Finally, the bacteria remaining in the suspension were counted. The sterilization rate was calculated using the following formula.

[0027]

Claims

1. A sisal fiber / chitosan / hybrid nano-apatite composite porous material, characterized in that, It is prepared by the following method: (1) After the carboxyl-modified sisal fiber is ultrasonically dispersed with water, ultrasonically dispersed hybrid nano-apatite slurry is slowly added dropwise. (2) Add chitosan powder and sodium houttuynia cordata powder, stir mechanically at high speed for 4 hours, add 2% glacial acetic acid solution to obtain complex gel; (3) Freeze-dry the complex gel, then soak it in 10% NaOH solution for 30 minutes, wash it with deionized water until neutral, and vacuum dry it at 40°C to constant weight to obtain the product; The sisal fiber refers to carboxylated modified sisal fiber with an average length of 0.5-1 mm and a diameter of 0.1-0.2 mm; the mass ratio of sisal fiber to chitosan is 1 / 3 to 1 / 1; the hybrid nano-apatite accounts for 10% to 25% of the mass of the composite porous material; and the sodium houttuynia cordata accounts for 4% to 12% of the mass of the composite porous material. The hybrid nano-apatite is prepared by the following method: Strontium nitrate and calcium nitrate are dissolved in an aqueous solution, wherein the Strontium nitrate content is 2-10% (Sr / (Sr+Ca) molar ratio), phytic acid is added and reacted for 1 hour, which is set as solution A, wherein the phosphate content in the phytic acid accounts for 10-40% of the total phosphate molar ratio; 5-10 wt% alendronate is added to sodium phosphate solution and reacted for 1 hour, which is set as solution B. Solution B is slowly added dropwise to solution A, maintaining the Ca / P molar ratio at 1.

67. Finally, the pH value is adjusted to above 10 with 1 mol / L sodium hydroxide, heated and stirred at 70 ℃ for 5 hours, aged for 48 hours, washed with deionized water until neutral, dried and ground into powder.

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