CN114642772B - Tragacanth/chitosan/hybrid nano apatite ternary composite porous material and preparation method thereof - Google Patents
Tragacanth/chitosan/hybrid nano apatite ternary composite porous material and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a tragacanth/chitosan/hybrid nano apatite ternary composite porous material and a preparation method thereof, wherein the porous material is prepared by ultrasonically stirring and uniformly mixing three compounds of tragacanth, chitosan and hybrid nano apatite in an aqueous solution, adding a small amount of glacial acetic acid to obtain a composite gel, placing the composite gel in a culture dish for precooling, freeze-drying, finally soaking and washing the composite gel in a NaOH solution to be neutral, and drying. The polyanionic tragacanth and the cationic chitosan acid solution have strong ionic crosslinking, so that the mechanical property and the degradation property of the porous material are greatly improved, and the added hybrid nano apatite contains a small amount of diphosphate, so that the osteogenic activity is better. The novel porous material is simple in preparation method, the sources of the used raw materials are rich, and the mechanical property, the degradation property and the osteogenic activity of the novel porous material can be regulated and controlled through the change of the content of each component of the porous material, so that the novel porous material is expected to obtain a bone tissue engineering scaffold material with excellent performance.
Description
Technical Field
The invention relates to a tragacanth/chitosan/hybrid nano apatite ternary composite porous material and a preparation method thereof, belonging to the field of biomedical materials.
Background
The bone tissue engineering scaffold material is a key element for bone tissue engineering research, and an ideal bone tissue engineering scaffold has good osteoconductivity, high porosity, excellent mechanical strength and a proper degradation rate. The bone tissue engineering scaffold comprises a degradable polymer, bioactive ceramics and an inorganic/organic composite scaffold, wherein the inorganic/organic composite scaffold combines the advantages of the polymer and the bioactive ceramics, so that the nano-hydroxyapatite/chitosan (n-HA/CS) composite porous material becomes a research hotspot of the bone tissue engineering scaffold. However, n-HA/CS HAs the problems of insufficient mechanical property and osteogenic activity in application. In previous researches, the applicant introduces carboxylated bamboo fibers into a polycationic chitosan system by using the electrostatic self-assembly principle of a high polymer to prepare a porous ternary composite material with higher strength. However, the modification steps of the carboxylated bamboo fiber are complicated, and the osteogenic activity of the nano hydroxyapatite itself needs to be improved, so that other polyanionic high polymers and porous materials compounded by nano apatite and chitosan with high osteogenic activity need to be explored.
Tragacanth, a natural polyanionic polysaccharide, has a backbone composed of partially methoxylated D-galacturonic acid units linked by α -1,4 linkages and side chains composed of many different polysaccharides. The tragacanth is a colloidal secretion from plants of the genus Dolabra, has good biocompatibility, degradability, non-toxicity and acid resistance, has been used in the fields of food additives, thickeners, stabilizers, emulsifiers, hydrogels and pharmaceuticals, and has also been widely used in the pharmaceutical fields of drug carriers, wound healing dressings, skin regeneration and the like. However, no report is found on the research on whether the tragacanth gum can be prepared into a porous material for a bone tissue engineering scaffold by the ionic crosslinking effect with chitosan.
In addition, in order to improve the osteogenic activity of the nano hydroxyapatite, various trace elements such as strontium, silicon and the like are doped into the n-HA, so that the biological performance of the nano hydroxyapatite can be improved, but the effective doping amount is difficult to control. The loaded bone growth factor is beneficial to promoting the growth of bone tissues, but the growth factor has short half-life period, is easy to rapidly degrade in vivo and dilute by body fluid, is easy to cause local toxicity and angiomatous lesion when loaded in large dose, and is expensive. The diphosphonate is used for treating osteoporosis, but can be introduced into the preparation process of n-HA to prepare hybrid nano apatite with high osteogenic activity, and is used for preparing a porous composite material with a polymer to be used for a bone tissue engineering scaffold, and the report is not shown, so that the diphosphonate is worthy of being deeply explored.
Disclosure of Invention
In view of the above situation, the present invention aims to provide a tragacanth/chitosan/hybrid nano apatite ternary composite porous material and a preparation method thereof. The composite porous material prepared by the invention has higher mechanical property, degradation property and biocompatibility, is a novel degradable porous material with high strength and high osteogenic activity, and can meet the basic requirements of the performance of a bone tissue engineering scaffold material.
The invention adopts the following technical scheme: tragacanth gum is commercially available with a viscosity of greater than 1000; the hybrid nano apatite is characterized in that the nano apatite contains a small amount of bisphosphates such as zoledronate, alendronate and pamidronate; the chitosan has a deacetylation degree of more than 90% and a viscosity-average molecular weight of 20 to 40 ten thousand.
The invention relates to hybrid nano apatite, which is characterized in that diphosphate is dissolved in water and slowly dripped into a calcium nitrate solution to react for 1 hour, a sodium phosphate solution is slowly dripped into the mixed solution, the molar ratio of Ca to P is kept to be 1.67, the adding content of the diphosphate is 0.1-1% of the mass of the nano apatite, the pH value is adjusted to be more than 10 by 1 mol/L sodium hydroxide, the mixture is heated and stirred for 4 hours at 70 ℃, the mixture is aged for 48 hours, washed to be neutral by deionized water, dried and ground into powder.
The invention provides a tragacanth/chitosan/hybrid nano apatite ternary composite porous material, which is realized by the following technical scheme and is characterized by adopting the following process steps:
ultrasonically dispersing a certain amount of hybridized nanometer apatite slurry, slowly adding a certain amount of mixed powder of chitosan and tragacanth, mechanically stirring at a high speed for 2 to 4 hours, and adding glacial acetic acid with the concentration of 2 to 4 percent to obtain compound gel; freezing at-20 deg.C for 12 hr, and freeze drying; and soaking the dried porous material in a 5-10% NaOH solution for 30 minutes, then washing the porous material to be neutral by using deionized water, and drying the porous material in vacuum at 40 ℃ to constant weight.
Compared with the existing porous material, the porous material of the invention has the advantages that:
(1) The chitosan and the tragacanth used in the invention are natural degradable polymers with good biocompatibility, and the raw materials have wide sources and low price; the tragacanth tape has negative electricity and can generate ion crosslinking with a chitosan solution with positive electricity, so that the mechanical property of the composite porous material is improved, the degradation performance of the composite porous material is greatly slowed down, and the composite porous material can provide a longer-time supporting effect; more importantly, the chitosan and the tragacanth have natural antibacterial property and can continuously and slowly prevent infection; in addition, the added hybrid nano apatite containing diphosphonate has better bone conductivity than the traditional nano hydroxyapatite and can be released slowly and continuously. In conclusion, the selected tragacanth and the hybrid nano apatite are beneficial to improving the mechanical property, the degradation property and the bone conductivity of the n-HA/CS composite porous material, and can also improve the antibacterial property.
(2) The tragacanth/chitosan/hybrid nano apatite ternary composite porous material provided by the invention has the advantages of simple and feasible preparation process, low production cost, greenness and environmental protection, and is suitable for mass production; the mechanical property, the degradation property, the osteoconductivity and the antibacterial property of the porous composite material can be regulated and controlled by adjusting the content of each component, and meanwhile, other medicines can be added to obtain a bone tissue engineering scaffold material with better performance.
Drawings
FIG. 1 is SEM photograph of a tragacanth/chitosan/hybrid nano apatite ternary composite porous material and a simulated body fluid soaked for 8 weeks.
Detailed Description
Example 1: weighing 4.0 g of chitosan (with deacetylation degree of 90% and molecular weight of 20 ten thousand), adding 100 ml of deionized water, then adding 2.0 g of tragacanth, simultaneously dispersing 2.0 g of hybridized nano apatite containing 0.5 wt% of zoledronate in 100 ml of deionized water by using ultrasonic, then dropwise adding the dispersed mixture into the tragacanth/chitosan mixed solution, continuing to stir by using ultrasonic magnetic force for 3 hours, and then adding 5 ml of glacial acetic acid to obtain the tragacanth/chitosan/hybridized nano apatite ternary composite gel. Freezing in a refrigerator at-20 deg.C for 12 hr, and freeze drying with a freeze dryer to constant weight. And finally, soaking the mixture in 8 percent NaOH solution for 30 minutes, washing the mixture to be neutral, and drying the mixture to obtain the finished product. Cutting the material into blocks of 10 mm multiplied by 10 mm, and measuring that the compression strength is about 0.8 MPa, the porosity is 81 percent and the average aperture is 200 um; the in vitro simulated body fluid is soaked by 8 w, a great amount of bone-like apatite is deposited on the surface, and the compressive strength can still maintain about 0.3 MPa.
Example 2: weighing 4.0 g of chitosan (deacetylation degree is 95%, molecular weight is 30 ten thousand), adding 100 ml of deionized water, adding 1.0 g of tragacanth, simultaneously dispersing 2.5 g of hybridized nano apatite containing 0.3 wt% of alendronate in 100 ml of deionized water by ultrasonic, dropwise adding into the tragacanth/chitosan mixed solution, continuing to stir by ultrasonic magnetic force for 3 hours, and adding 6 ml of glacial acetic acid to obtain the tragacanth/chitosan/hybridized nano apatite ternary composite gel. Freezing in a refrigerator at-20 deg.C for 12 hr, and freeze drying with a freeze dryer to constant weight. Then soaking the mixture in 10 percent NaOH solution for 30 minutes, washing the mixture to be neutral, and drying the mixture to obtain the finished product. Cutting the material into blocks of 10 mm multiplied by 10 mm, and measuring that the compression strength is about 1.0 MPa, the porosity is 79 percent, and the average pore diameter is 220 um; the in vitro simulated body fluid is soaked by 8 w, a great amount of bone-like apatite is deposited on the surface, and the compressive strength can still maintain about 0.4 MPa.
Example 3: weighing 4.5 g of chitosan (deacetylation degree is 95%, molecular weight is 20 ten thousand), adding 100 ml of deionized water, adding 1.5 g of tragacanth, simultaneously dispersing 4.0 g of hybridized nano apatite containing 1.0 wt% of alendronate in 100 ml of deionized water by ultrasonic, dropwise adding into the tragacanth/chitosan mixed solution, continuing ultrasonic magnetic stirring for 4 hours, and adding 8 ml of glacial acetic acid to obtain the tragacanth/chitosan/hybridized nano apatite ternary composite gel. Freezing in a refrigerator at-20 deg.C for 12 hr, and freeze drying to constant weight with a freeze dryer. Then soaking the mixture in 10% NaOH solution for 30 minutes, washing the mixture to be neutral, and drying the mixture. Cutting the material into blocks of 10 mm multiplied by 10 mm, and measuring that the compression strength is about 0.6 MPa, the porosity is 78 percent and the average aperture is 150 um; the in vitro simulated body fluid is soaked in 8 w, a large amount of bone-like apatite is deposited on the surface, and the compressive strength can still maintain about 0.1 MPa.
Comparative example 1: weighing 4.0 g of chitosan (deacetylation degree is 95%, molecular weight is 30 ten thousand), adding 200 ml of deionized water, adding 6 ml of glacial acetic acid, stirring until the chitosan is dissolved, dropwise adding a dispersion liquid prepared by ultrasonically dispersing 2.0 g of nano-hydroxyapatite in 100 ml of water into the chitosan solution, and continuously carrying out ultrasonic magnetic stirring for 2 hours to obtain the nano-hydroxyapatite/chitosan binary composite gel. Freezing in a refrigerator at-20 deg.C for 48 hr, and freeze drying to constant weight with a freeze dryer. Then soaking the mixture in 10% NaOH solution for 30 minutes, washing the mixture to be neutral, and drying the mixture. Cutting the material into blocks of 10 mm multiplied by 10 mm, and measuring that the compression strength is about 0.4 MPa, the porosity is 73 percent, and the average pore diameter is 150 um; the porous material is degraded into powder after being soaked in simulated body fluid in vitro for 8 w.
Comparative example 2: weighing 4.5 g of chitosan (the deacetylation degree is 95%, the molecular weight is 20 ten thousand), adding 100 ml of deionized water, adding 1.5 g of tragacanth, simultaneously dropwise adding a dispersion liquid in which 2.0 g of nano-hydroxyapatite is ultrasonically dispersed in 300 ml of water into the tragacanth/chitosan mixed solution, continuing ultrasonic magnetic stirring for 4 hours, and adding 8 ml of glacial acetic acid to obtain the tragacanth/chitosan/nano-apatite ternary composite gel. Freezing in a refrigerator at-20 deg.C for 12 hr, and freeze drying to constant weight with a freeze dryer. Then soaking the mixture in 10% NaOH solution for 30 minutes, washing the mixture to be neutral, and drying the mixture. Cutting the material into blocks of 10 mm multiplied by 10 mm, and measuring that the compression strength is about 0.7 MPa, the porosity is 80 percent, and the average pore diameter is 150 um; the in vitro simulated body fluid is soaked by 8 w, a small amount of bone-like apatite is deposited on the surface, and the compressive strength can still maintain about 0.2 MPa.
The test conditions of the compressive strength are as follows: the compression property of the cut 10 mm × 10 mm × 10 mm bulk material was tested by a universal material testing machine (SANSCMT 4503, SANS, shenzhen, china) at a compression deformation of 40%. The test temperature was 20 ℃ 2 ℃ and the loading speed was 1 mm/min. Five replicates were tested per group and the results averaged.
Determination of porosity: adding a proper amount of absolute ethyl alcohol into the vector cylinder, and recording the dry weight of the cut 10 mm multiplied by 10 mm block-shaped material as m 1 Put into absolute ethyl alcohol, weigh the ethyl alcohol and record the initial volume of the sample as V 1 . Soaking at room temperature for 1 week, and collecting the materialThe wet weight of the weighing material is m 2 Volume of remaining ethanol is V 2 . Then porosity θ = [ (m) 2 -m 1 )/ρ]/(V 1 -V 2 ). Three replicates of each sample were measured and averaged.
Claims (1)
1. A tragacanth/chitosan/hybrid nanometer apatite ternary composite porous material is characterized by being prepared by the following method: ultrasonically dispersing a certain amount of hybridized nanometer apatite slurry, slowly adding a certain amount of mixed powder of chitosan and tragacanth, mechanically stirring at a high speed for 2 to 4 hours, adding glacial acetic acid with the concentration of 2 to 4 percent to obtain compound gel, freezing at the temperature of minus 20 ℃ for 12 hours, then carrying out freeze drying, soaking the dried porous material in a NaOH solution with the concentration of 5 to 10 percent for 30 minutes, then washing with deionized water to be neutral, and carrying out vacuum drying at the temperature of 40 ℃ to constant weight; wherein the viscosity of the tragacanth is more than 1000, the deacetylation degree of the chitosan is more than 90 percent, and the viscosity-average molecular weight is 20 to 40 ten thousand; the mass ratio of the tragacanth to the chitosan is 1/5 to 5/1, and the mass ratio of the hybrid nano apatite to the composite porous material is 10-60%; the hybrid nano apatite is prepared by the following method: dissolving the bisphosphate in water, slowly and dropwise adding the bisphosphate into a calcium nitrate solution, reacting for 1 hour, then slowly and dropwise adding a sodium phosphate solution, keeping the Ca/P molar ratio at 1.67, wherein the adding content of the bisphosphate is 0.1-1% of the mass of the nano apatite, adjusting the pH value to be more than 10 by using 1 mol/L sodium hydroxide, heating and stirring at 70 ℃ for 4 hours, aging for 48 hours, washing with deionized water to be neutral, drying and grinding into powder.
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| Hydroxyapatite mineralization of chitosan-tragacanth blend/ZnO/Ag nanocomposite films with enhanced antibacterial activity;Shadpour Mallakpour，等;《International Journal of Biological Macromolecules》;20210205;第175卷;第330-332、338页 * |
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