CN113061255B - Polyoxyethylene polyoxypropylene-chitosan segmented copolymer, bone hemostatic material and preparation method thereof - Google Patents

Polyoxyethylene polyoxypropylene-chitosan segmented copolymer, bone hemostatic material and preparation method thereof Download PDF

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CN113061255B
CN113061255B CN202110316641.8A CN202110316641A CN113061255B CN 113061255 B CN113061255 B CN 113061255B CN 202110316641 A CN202110316641 A CN 202110316641A CN 113061255 B CN113061255 B CN 113061255B
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polyoxyethylene polyoxypropylene
chitosan
bone
block copolymer
hemostatic material
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CN113061255A (en
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石锐
薛芸
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BEIJING RESEARCH INSTITUTE OF TRAUMATOLOGY AND ORTHOPAEDICS
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BEIJING RESEARCH INSTITUTE OF TRAUMATOLOGY AND ORTHOPAEDICS
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Abstract

The invention provides a polyoxyethylene polyoxypropylene-chitosan block copolymer, a bone hemostatic material and a preparation method thereof, and relates to the technical field of medicines. The polyoxyethylene polyoxypropylene-chitosan segmented copolymer can be used as a raw material for preparing a bone hemostatic material, can improve the biocompatibility and the bacterial inhibition of the bone hemostatic material, can prolong the hemostatic time, reduce the dissolution rate and improve the water absorption.

Description

Polyoxyethylene polyoxypropylene-chitosan segmented copolymer, bone hemostatic material and preparation method thereof
Technical Field
The invention relates to the technical field of medicines, in particular to a polyoxyethylene polyoxypropylene-chitosan segmented copolymer, a bone hemostatic material and a preparation method thereof.
Background
Clinically, the bone destruction problem is often involved in the operative procedures of orthopedics, neurosurgery, thoracic surgery and the like, and the cancellous bone wound surface oozes blood. Cancellous bone is loose in structure and rich in blood circulation, bleeding of wound surface is mainly caused by blood seepage, self-hemostasis by vasoconstriction is difficult, and hemostasis is also difficult to be carried out by conventional methods such as hemostatic gauze, electric coagulation, gelatin sponge filling, forceps and the like. Currently, cancellous bone wound hemostasis is generally performed by bone wax clinically. The traditional bone wax mainly comprises beeswax and vaseline, and is used for stopping bleeding of a cancellous bone wound surface through physical filling, is high in bleeding stopping speed, and is the most widely used bleeding stopping mode for surgical bone bleeding at present. However, bone wax is not degradable, can not be absorbed by the body, has poor biocompatibility, is easy to be retained in the body as foreign matter for a long time, hinders bone repair, and is easy to cause foreign matter reactions such as infection, pain and the like.
In addition, the existing bone hemostatic material product is a completely water-soluble component, and the surface is rapidly liquefied after meeting blood to cause the reduction of the plugging strength, so the hemostatic effect is poor.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
One of the objects of the present invention is to provide a method for preparing a polyoxyethylene polyoxypropylene-chitosan block copolymer, which has mild reaction conditions, is easy to operate, has a high reaction conversion rate, and can increase the molecular weight of the polyoxyethylene polyoxypropylene-chitosan block copolymer.
It is another object of the present invention to provide a polyoxyethylene polyoxypropylene-chitosan block copolymer having a relatively high molecular weight.
The bone hemostatic material has high biocompatibility and bacteriostasis, long hemostatic time and low dissolution rate, and has high adhesion to bone and excellent plasticity.
The fourth purpose of the invention is to provide a preparation method of the bone hemostatic material, which has simple and convenient process and is suitable for large-scale production in factories.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
in a first aspect, a method for preparing a polyoxyethylene polyoxypropylene-chitosan block copolymer, comprising the steps of:
(a) the polyoxyethylene polyoxypropylene segmented copolymer and an activating agent react in a solvent, and the activated polyoxyethylene polyoxypropylene segmented copolymer is obtained through precipitation and solid-liquid separation;
(b) carrying out graft copolymerization reaction on chitosan and the activated polyoxyethylene polyoxypropylene block copolymer obtained in the step (a) in acid liquor, precipitating, and carrying out solid-liquid separation to obtain a polyoxyethylene polyoxypropylene-chitosan block copolymer;
wherein the activator comprises N, N' -carbonyldiimidazole;
further, the polyoxyethylene polyoxypropylene block copolymer before activation has a polyoxyethylene molar content of 10-45%;
further, the reaction temperature in the step (a) is 37-40 ℃, and the reaction time is 8-16 h;
preferably, the mass fraction of the added activating agent is 5-15%;
preferably, the solvent comprises a mixed solvent of 1, 4-dioxane and dimethyl sulfoxide;
preferably, the volume ratio of the 1, 4-dioxane to the dimethyl sulfoxide in the mixed solvent of the 1, 4-dioxane and the dimethyl sulfoxide is 1.8-2.2: 0.8 to 1.2, more preferably 2: 1;
preferably, the precipitating agent comprises anhydrous diethyl ether;
further, the time of the graft copolymerization in the step (b) is 10-18h, and the temperature of the graft copolymerization is 37-40 ℃;
preferably, the deacetylation degree of the chitosan is 90-99%;
preferably, the mass fraction of the added chitosan is 3-10%;
preferably, the acid solution comprises a hydrochloric acid solution;
preferably, the pH value of the graft copolymerization reaction is 7-8;
preferably, the precipitated precipitating agent comprises a mixed reagent of anhydrous diethyl ether and acetone;
preferably, the volume ratio of the anhydrous ether to the acetone in the mixed reagent of the anhydrous ether and the acetone is 0.8-1.2: 1.8 to 2.2, more preferably 1: 2.
in a second aspect, a polyoxyethylene polyoxypropylene-chitosan block copolymer, which has a number average molecular weight of 9000-.
In a third aspect, the bone hemostatic material is mainly prepared from the following raw materials in parts by weight:
10-50 parts of polyoxyethylene polyoxypropylene-chitosan block copolymer, 25-80 parts of polyoxypropylene polyoxyethylene random copolymer, 1-5 parts of optional bone repair material and 2-20 parts of optional hemostatic component;
preferably, the bone repair material comprises at least one of hydroxyapatite, beta-tricalcium phosphate, calcium carbonate, biphasic calcium phosphate and bioglass;
preferably, the particle size of the hydroxyapatite is less than 150 μm;
preferably, the hemostatic composition comprises at least one of thrombin and starch microspheres;
further, the bone hemostatic material is wax-like.
In a fourth aspect, a method for preparing a bone hemostatic material comprises the following steps:
mixing the polyoxyethylene polyoxypropylene-chitosan block copolymer, the polyoxyethylene polyoxypropylene random copolymer, an optional bone repair material and an optional hemostatic component to obtain a bone hemostatic material;
further, the preparation method comprises the following steps:
heating and melting the polyoxyethylene polyoxypropylene-chitosan segmented copolymer and the polyoxyethylene polyoxypropylene random copolymer, adding an optional bone repair material and an optional hemostatic component, mixing, curing and forming to obtain a bone hemostatic material;
further, the curing and molding time is 3-4h, and preferably 3 h.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a preparation method of a polyoxyethylene polyoxypropylene-chitosan block copolymer, which adopts a two-step method to synthesize the polyoxyethylene polyoxypropylene-chitosan block copolymer, firstly utilizes N, N' -carbonyl diimidazole to activate the polyoxyethylene polyoxypropylene block copolymer with hydroxyl at two ends to obtain the activated polyoxyethylene polyoxypropylene block copolymer, and then carries out graft copolymerization with chitosan to generate the polyoxyethylene polyoxypropylene-chitosan block copolymer.
(2) The polyoxyethylene polyoxypropylene-chitosan block copolymer provided by the invention has higher molecular weight, and the average molecular weight is 9000-20000.
(3) According to the bone hemostatic material provided by the invention, the polyoxyethylene polyoxypropylene-chitosan segmented copolymer can not only improve the biocompatibility and the antibacterial activity of the bone hemostatic material, but also prolong the hemostatic time, reduce the dissolution rate and improve the water absorption of the bone hemostatic material; meanwhile, the addition of the polyoxyethylene polyoxypropylene random copolymer enhances the adhesiveness of the bone hemostatic material and the bone, and improves the plasticity of the bone hemostatic material.
(4) The preparation method of the bone hemostatic material provided by the invention is simple and convenient in process, high in product excellent rate and suitable for large-scale production in factories.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a reaction scheme of a polyoxyethylene polyoxypropylene-chitosan block copolymer according to one embodiment of the present invention;
FIG. 2 is a graph showing the results of absorbance measurements obtained in the cell viability test in the experimental examples of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to a first aspect of the present invention, there is provided a method for preparing a polyoxyethylene polyoxypropylene-chitosan block copolymer, comprising the steps of:
(a) the polyoxyethylene polyoxypropylene segmented copolymer and an activating agent react in a solvent, and the activated polyoxyethylene polyoxypropylene segmented copolymer is obtained through precipitation and solid-liquid separation;
(b) carrying out graft copolymerization reaction on chitosan and the activated polyoxyethylene polyoxypropylene block copolymer obtained in the step (a) in acid liquor, precipitating, and carrying out solid-liquid separation to obtain a polyoxyethylene polyoxypropylene-chitosan block copolymer;
wherein the activator comprises N, N' -carbonyldiimidazole.
The invention adopts a two-step method to synthesize the polyoxyethylene polyoxypropylene-chitosan block copolymer, firstly utilizes N, N' -carbonyl diimidazole to activate the polyoxyethylene polyoxypropylene block copolymer with hydroxyl at two ends to obtain the activated polyoxyethylene polyoxypropylene block copolymer, and then carries out graft copolymerization reaction with chitosan to generate the polyoxyethylene polyoxypropylene-chitosan block copolymer.
The polyoxyethylene polyoxypropylene-chitosan block copolymer refers to a block copolymer comprising polyoxyethylene polyoxypropylene and chitosan, and may also be referred to as chitosan-polyoxyethylene polyoxypropylene block copolymer, wherein polyoxyethylene polyoxypropylene serves as a main chain of the block copolymer, and chitosan serves as a side chain of the block copolymer.
In a preferred embodiment, the polyoxyethylene polyoxypropylene block copolymer prior to activation has a polyoxyethylene molar content of 10% to 45%, with typical but non-limiting molar contents being, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%;
step (a)
In a preferred embodiment, the temperature of the activation reaction is 37-40 deg.C, typical but not limiting reaction temperatures are for example 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C; the time of the activation reaction is 8-16h, and typical but non-limiting reaction times are 8h, 10h, 12h, 14h and 16 h;
in a preferred embodiment, the activator is added in a mass fraction of 5 to 15%, typically but not limited to a mass fraction of 5%, 10%, 15%, based on the total weight of the polyoxyethylene polyoxypropylene block copolymer and the N, N' -carbonyldiimidazole;
in a preferred embodiment, the solvent for the activation reaction includes, but is not limited to, a mixed solvent of 1, 4-dioxane and dimethyl sulfoxide, wherein the volume ratio of 1, 4-dioxane to dimethyl sulfoxide is 1.8-2.2: 0.8 to 1.2, more preferably 2: 1;
in a preferred embodiment, after the activation reaction is finished, the precipitant for the precipitation treatment includes, but is not limited to, anhydrous ethyl ether, wherein the precipitation treatment is to drop anhydrous ethyl ether under the condition of ice bath stirring, after the precipitation treatment, solid-liquid separation is performed, the solid-liquid separation manner includes, but is not limited to, filtration, washing the precipitate with anhydrous ethyl ether, and vacuum drying to obtain a white solid, i.e., the activated polyoxyethylene polyoxypropylene block copolymer.
Step (b)
In a preferred embodiment, the time of the graft copolymerization reaction is 10 to 18 hours, typical but not limiting reaction times being, for example, 10 hours, 14 hours, 18 hours; the temperature of the graft copolymerization is 37 to 40 ℃ and typical but not limiting reaction temperatures are, for example, 37 ℃, 38 ℃, 39 ℃, 40 ℃;
in a preferred embodiment, the degree of deacetylation of chitosan in the graft copolymerization reaction is between 90% and 99%, with typical but non-limiting degrees of deacetylation being, for example, 90%, 93%, 96%, 99%;
chitin is called chitosan after deacetylation, the greatest benefit of deacetylation is that the chitin can be dissolved in some diluted acids, such as acetic acid, hydrochloric acid and the like, and the higher the deacetylation degree is, the better the solubility is.
In a preferred embodiment, the chitosan is added in a mass fraction of 3% to 10%, typically but not limited to 3%, 5%, 8%, 10% based on the total weight of the chitosan and the activated polyoxyethylene polyoxypropylene block copolymer in the graft copolymerization reaction;
in a preferred embodiment, the acid solution of the graft copolymerization reaction includes, but is not limited to, hydrochloric acid solution;
in a preferred embodiment, the graft copolymerization reaction has a pH of 7 to 8, which is typically, but not limited to, pH 7, 7.5, 8;
in a preferred embodiment, after the graft copolymerization reaction is finished, the precipitating agent for precipitation treatment includes but is not limited to a mixed reagent of anhydrous ether and acetone, wherein the precipitation treatment is to dropwise add the mixed reagent of anhydrous ether and acetone under the condition of ice bath stirring, and the volume ratio of the anhydrous ether to the acetone is 0.8-1.2: 1.8 to 2.2, more preferably 1: and 2, after precipitation treatment, carrying out solid-liquid separation in a manner including but not limited to filtration, washing the precipitate with acetone, and drying in vacuum to obtain the polyoxyethylene polyoxypropylene-chitosan segmented copolymer.
A typical preparation method of a polyoxyethylene polyoxypropylene-chitosan block copolymer, as shown in fig. 1, comprises the following steps:
dissolving a certain amount of polyoxyethylene polyoxypropylene block copolymer and N, N ' -carbonyl diimidazole in a proper amount of mixed reagent of 1, 4-dioxane and dimethyl sulfoxide (the volume ratio of 1, 4-dioxane to dimethyl sulfoxide is 2: 1), stirring and reacting on a magnetic stirrer at the reaction temperature of 37-40 ℃ for 8-16h, wherein the weight of an activating agent N, N ' -carbonyl diimidazole is 5-15% based on the total weight of the polyoxyethylene polyoxypropylene block copolymer and the N, N ' -carbonyl diimidazole, the molar content of polyoxyethylene in the polyoxyethylene polyoxypropylene block copolymer before activation is 10-45%, stirring and dripping anhydrous ether after the reaction is finished, precipitating the polymer, filtering to obtain a precipitate, washing with the anhydrous ether, the white precipitate obtained after vacuum drying is the activated polyoxyethylene polyoxypropylene segmented copolymer;
weighing a certain amount of chitosan and activated polyoxyethylene polyoxypropylene block copolymer, dissolving the chitosan and activated polyoxyethylene polyoxypropylene block copolymer in a proper amount of hydrochloric acid solution, placing the mixture on a magnetic stirrer for stirring, adjusting the pH value to 7-8, carrying out graft copolymerization reaction, wherein the time of the graft copolymerization reaction is 10-18h, the reaction temperature is 37-40 ℃, the deacetylation degree of the used chitosan is 90-99%, the weight of the added chitosan is 3-10%, after the reaction is finished, dropwise adding a mixed reagent of anhydrous ether and acetone (the volume ratio of the anhydrous ether to the acetone is 1:2) under the condition of ice-bath stirring, carrying out suction filtration, washing and precipitating with acetone, and carrying out vacuum drying to obtain the polyoxyethylene polyoxypropylene block copolymer.
According to a second aspect of the present invention, there is provided a polyoxyethylene polyoxypropylene-chitosan block copolymer prepared by the above method, having a number average molecular weight of 9000-.
According to a third aspect of the present invention, there is provided a bone hemostatic material, which is mainly prepared from the following raw materials by weight:
10-50 parts of polyoxyethylene polyoxypropylene-chitosan block copolymer, 25-80 parts of polyoxypropylene polyoxyethylene random copolymer, 1-5 parts of optional bone repair materials and 2-20 parts of optional hemostatic components.
The protocol that can be implemented is as follows:
1. 10-50 parts of polyoxyethylene polyoxypropylene-chitosan segmented copolymer and 25-80 parts of polyoxypropylene polyoxyethylene random copolymer are taken as raw materials to prepare the bone hemostatic material;
2. 10-50 parts of polyoxyethylene polyoxypropylene-chitosan segmented copolymer, 25-80 parts of polyoxypropylene polyoxyethylene random copolymer and 1-5 parts of bone repair material are taken as raw materials to prepare the bone hemostatic material;
3. 10-50 parts of polyoxyethylene polyoxypropylene-chitosan segmented copolymer, 25-80 parts of polyoxypropylene polyoxyethylene random copolymer and 2-20 parts of hemostatic components are taken as raw materials to prepare the bone hemostatic material;
4. 10-50 parts of polyoxyethylene polyoxypropylene-chitosan segmented copolymer, 25-80 parts of polyoxypropylene polyoxyethylene random copolymer, 1-5 parts of bone repair material and 2-20 parts of hemostatic component.
According to the bone hemostatic material provided by the invention, the polyoxyethylene polyoxypropylene-chitosan segmented copolymer can not only improve the biocompatibility and the antibacterial activity of the bone hemostatic material, but also prolong the hemostatic time, reduce the dissolution rate and improve the water absorption of the bone hemostatic material; meanwhile, the addition of the polyoxyethylene polyoxypropylene random copolymer enhances the adhesiveness of the bone hemostatic material and the bone, and improves the plasticity of the bone hemostatic material.
In addition, the bone repair material, such as hydroxyapatite, beta-tricalcium phosphate, calcium carbonate, biphasic calcium phosphate, bioglass and the like, is added into the bone hemostatic material, so that mineral substances formed by bones are provided during bone defect repair, osteoblasts are promoted to proliferate, and hemostasis can be stimulated by releasing calcium ions in the bone repair material; the hemostatic components added in the invention, such as thrombin, starch microspheres and the like, achieve the aim of rapid hemostasis.
In a preferred embodiment, the bone repair material includes, but is not limited to, at least one of hydroxyapatite, β -tricalcium phosphate, calcium carbonate, biphasic calcium phosphate, and bioglass;
in a preferred embodiment, the hydroxyapatite has a particle size of less than 150 μm;
the bone repair material added into the bone hemostatic material provides mineral substances formed by bones during bone defect repair, promotes osteoblast proliferation, and can also stimulate hemostasis by releasing calcium ions in the bone repair material.
In a preferred embodiment, the hemostatic composition includes, but is not limited to, at least one of thrombin and starch microspheres;
the hemostatic components added in the bone hemostatic material, such as thrombin and starch microspheres, have the function of rapidly stopping bleeding on the injured part.
In a preferred embodiment, the bone hemostatic material of the present invention is wax-like for ease of use and storage.
According to a fourth aspect of the present invention, there is provided a method for preparing a bone hemostatic material, comprising the steps of:
mixing the polyoxyethylene polyoxypropylene-chitosan block copolymer, the polyoxyethylene polyoxypropylene random copolymer, the optional bone repair material and the optional hemostatic component to obtain the bone hemostatic material.
In a preferred embodiment, the preparation method of the bone hemostatic material of the present invention comprises the following steps:
heating and melting the polyoxyethylene polyoxypropylene-chitosan segmented copolymer and the polyoxyethylene polyoxypropylene random copolymer, adding an optional bone repair material and an optional hemostatic component, mixing, curing and forming to obtain the bone hemostatic material.
In a preferred embodiment, the curing time is 3 to 4 hours, preferably 3 hours.
The preparation method of the bone hemostatic material provided by the invention is simple and convenient in process, high in product excellent rate and suitable for large-scale production in factories.
A typical preparation method of the bone hemostatic material comprises the following steps:
a proper amount of a polyoxyethylene polyoxypropylene-chitosan block copolymer and a polyoxyethylene polyoxypropylene random copolymer are mixed, and the mixture is stirred and heated to be liquid. Adding appropriate amount of bone repair material hydroxyapatite, and mixing. Pouring the mixture into a mold, and standing at room temperature for 3h for curing and molding. Packaging and sealing the product, and using after gamma ray or high pressure sterilization, wherein the particle diameter of the hydroxyapatite is less than 150 μm, the hydroxyapatite accounts for 1-5% of the weight, and the bone repair material can also be beta-tricalcium phosphate, calcium carbonate, biphase calcium phosphate, bioglass and the like.
A typical preparation method of the bone hemostatic material comprises the following steps:
mixing appropriate amount of chitosan-polyoxyethylene polyoxypropylene block copolymer and polyoxyethylene polyoxypropylene random copolymer, heating to melt, heating and mechanically stirring to obtain liquid, adding thrombin, and mixing. Pouring the mixture into a mold, and standing at room temperature for 3h for curing and molding. Packaging and sealing the product, and sterilizing with gamma ray or high pressure, wherein the hemostatic components can be starch microsphere.
The invention is further illustrated by the following examples. The materials in the examples are prepared according to known methods or are directly commercially available, unless otherwise specified.
Example 1
A preparation method of polyoxyethylene polyoxypropylene-chitosan segmented copolymer comprises the following steps:
(1) synthesis of activated polyoxyethylene polyoxypropylene block copolymer:
30g of polyoxyethylene polyoxypropylene block copolymer and 5.89g of N, N' -carbonyldiimidazole are dissolved in 150ml of a mixed reagent of 1, 4-dioxane and dimethyl sulfoxide in a volume ratio of 2:1, and the mixture is stirred and reacted at 37 ℃ for 12 hours. After the reaction is finished, stirring and dropwise adding anhydrous ether under the ice bath condition, filtering to obtain a precipitate, washing three times with the anhydrous ether, and drying in vacuum to obtain the activated polyoxyethylene polyoxypropylene segmented copolymer which is a white precipitate.
(2) Synthesis of polyoxyethylene polyoxypropylene-chitosan block copolymer:
dissolving 4g of chitosan and 8g of activated polyoxyethylene polyoxypropylene block copolymer in 0.1mol/L hydrochloric acid solution, stirring for 12h at the temperature of 37 ℃, adjusting the pH value to 7-8, stirring under the ice bath condition, dropwise adding a mixed reagent of anhydrous ether and acetone (the volume ratio of the anhydrous ether to the acetone is 1:2), after the reaction is finished, carrying out suction filtration, washing and precipitating with acetone, and carrying out vacuum drying to obtain the polyoxyethylene polyoxypropylene block copolymer, wherein the number average molecular weight of the polyoxyethylene polyoxypropylene block copolymer is 9000-20000.
A preparation method of a bone hemostatic material comprises the following steps:
mixing the above 20g polyoxyethylene polyoxypropylene-chitosan block copolymer and 80g polyoxyethylene polyoxypropylene random copolymer, heating to melt, mechanically stirring under heating for 8 hr to obtain liquid, pouring into a mold, standing at room temperature for 3 hr for curing molding to obtain bone hemostatic material, packaging and sealing, and sterilizing with gamma ray or high pressure.
Example 2
A preparation method of polyoxyethylene polyoxypropylene-chitosan segmented copolymer comprises the following steps:
(1) synthesis of activated polyoxyethylene polyoxypropylene block copolymer:
50g of polyoxyethylene polyoxypropylene block copolymer and 9.75g of N, N' -carbonyldiimidazole are dissolved in 200ml of a mixed reagent of 1, 4-dioxane and dimethyl sulfoxide in a volume ratio of 2:1, and the mixture is stirred and reacted for 12 hours at the temperature of 37 ℃. After the reaction is finished, stirring and dropwise adding anhydrous ether under the ice bath condition, filtering to obtain a precipitate, washing three times with the anhydrous ether, and drying in vacuum to obtain the activated polyoxyethylene polyoxypropylene segmented copolymer which is a white precipitate.
(2) Synthesis of polyoxyethylene polyoxypropylene-chitosan block copolymer:
dissolving 6g of chitosan and 8g of activated polyoxyethylene polyoxypropylene block copolymer in 0.1mol/L hydrochloric acid solution, stirring for 12h at the temperature of 37 ℃, adjusting the pH value to 7-8, stirring under the ice bath condition, dropwise adding a mixed reagent of anhydrous ether and acetone (the volume ratio of the anhydrous ether to the acetone is 1:2), after the reaction is finished, carrying out suction filtration, washing and precipitating with acetone, and carrying out vacuum drying to obtain the polyoxyethylene polyoxypropylene block copolymer, wherein the number average molecular weight of the polyoxyethylene polyoxypropylene block copolymer is 9000-20000.
A preparation method of a bone hemostatic material comprises the following steps:
mixing the 35g of polyoxyethylene polyoxypropylene-chitosan segmented copolymer and 65g of polyoxyethylene polyoxypropylene random copolymer, heating to melt, heating and mechanically stirring for 8 hours to obtain liquid, adding 1g of thrombin, uniformly mixing, pouring into a mould, standing at room temperature for 3 hours, curing and forming to obtain the bone hemostatic material, packaging and sealing the bone hemostatic material, and using the bone hemostatic material after gamma-ray or high-pressure sterilization.
Example 3
A preparation method of polyoxyethylene polyoxypropylene-chitosan segmented copolymer comprises the following steps:
(1) synthesis of activated polyoxyethylene polyoxypropylene block copolymer:
dissolving 40g of polyoxyethylene polyoxypropylene block copolymer and 8.46g of N, N' -carbonyl diimidazole in 180ml of mixed reagent of 1, 4-dioxane and dimethyl sulfoxide with the volume ratio of 2:1, stirring and reacting for 12 hours at the temperature of 37 ℃, stirring and dripping anhydrous ether under the ice bath condition after the reaction is finished, filtering to obtain precipitate, washing with the anhydrous ether for three times, and drying in vacuum to obtain activated polyoxyethylene polyoxypropylene block copolymer which is white precipitate;
(2) synthesis of polyoxyethylene polyoxypropylene-chitosan block copolymer:
5.5g of chitosan and 8g of activated polyoxyethylene polyoxypropylene block copolymer are dissolved in 0.1mol/L hydrochloric acid solution, stirred for 12 hours at the temperature of 37 ℃, the pH value is adjusted to 7-8, stirring is carried out under ice bath conditions, a mixed reagent of anhydrous ether and acetone (the volume ratio of the anhydrous ether to the acetone is 1:2) is added dropwise, suction filtration is carried out, acetone washing and precipitation are carried out, vacuum drying is carried out, and the polyoxyethylene polyoxypropylene-chitosan block copolymer with the number average molecular weight of 9000-20000 is obtained.
A preparation method of a bone hemostatic material comprises the following steps:
mixing the 19g of the polyoxyethylene polyoxypropylene-chitosan segmented copolymer and 80g of the polyoxyethylene polyoxypropylene random copolymer, heating to melt, heating and mechanically stirring for 8 hours to obtain liquid, adding 1g of hydroxyapatite, uniformly mixing, pouring into a mould, standing at room temperature for 3 hours, curing and forming to obtain the bone hemostatic material, packaging and sealing the product, and using the product after gamma-ray or high-pressure sterilization.
Example 4
A preparation method of polyoxyethylene polyoxypropylene-chitosan segmented copolymer comprises the following steps:
(1) synthesis of activated polyoxyethylene polyoxypropylene block copolymer:
dissolving 45g of polyoxyethylene polyoxypropylene block copolymer and 9.25g of N, N' -carbonyl diimidazole in 190ml of mixed reagent of 1, 4-dioxane and dimethyl sulfoxide with the volume ratio of 2:1, stirring and reacting for 12h at 37 ℃, stirring and dripping anhydrous ether after the reaction is finished under the ice bath condition, filtering to obtain precipitate, washing with the anhydrous ether for three times, and drying in vacuum to obtain the activated polyoxyethylene polyoxypropylene block copolymer which is white precipitate.
(2) Synthesis of polyoxyethylene polyoxypropylene-chitosan block copolymer:
dissolving 7g of chitosan and 9.5g of activated polyoxyethylene polyoxypropylene block copolymer in 0.1mol/L hydrochloric acid solution, stirring at 37 ℃ for 12h, adjusting the pH to 7-8, dropwise adding a mixed reagent of anhydrous ether and acetone (the volume ratio of the anhydrous ether to the acetone is 1:2) under the ice-bath stirring condition, carrying out suction filtration, washing and precipitating with acetone, and carrying out vacuum drying to obtain the polyoxyethylene polyoxypropylene-chitosan block copolymer, wherein the number average molecular weight of the block copolymer is 9000-20000.
A preparation method of a bone hemostatic material comprises the following steps:
mixing 29g of the polyoxyethylene polyoxypropylene-chitosan segmented copolymer and 69g of the polyoxyethylene polyoxypropylene random copolymer, heating to melt, heating and mechanically stirring for 8 hours to obtain liquid, adding 1g of hydroxyapatite and 1g of thrombin, uniformly mixing, pouring into a mould, standing at room temperature for 3 hours, curing and molding to obtain the bone hemostatic material, packaging and sealing the bone hemostatic material, and using the bone hemostatic material after gamma-ray or high-pressure sterilization.
Comparative example 1
The present comparative example is different from example 1 in that it does not use N, N' -carbonyldiimidazole as an activator, it uses sodium hydride as an activator, and the other steps are the same as example 1, and the number average molecular weight of the polyoxyethylene polyoxypropylene-chitosan block copolymer obtained in the present comparative example is 5000-20000, which is significantly lower than the number average molecular weight of 9000-20000 of the examples of the present invention.
Comparative example 2
The difference between the comparative example and the example 1 is that the bone hemostatic material in the comparative example does not contain the polyoxyethylene polyoxypropylene random copolymer, and is replaced by the polyethylene glycol 10000 copolymer, and other components and contents are the same as those of the bone hemostatic material in the example 1.
Examples of the experiments
And (3) performance testing:
1. disintegration time test
The bone hemostatic materials obtained in Experimental examples 1-4 and comparative example 2 were placed in a phosphate buffer at a ratio of 1:50(w/v), and their disintegration time was observed. The evaluation principle of disintegration time is the time without influence on occupation and incomplete form. Example 1 is labeled a1, example 2 is labeled a2, example 3 is labeled A3, example 4 is labeled a4, and comparative example 2 is labeled a 5. The test results are given in table 1 below:
TABLE 1
Test sample A1 A2 A3 A4 A5
Disintegration time 5d 9d 6d 9d 3d
2. Mechanical compression strength test
The bone hemostatic materials obtained in examples 1 to 4 and comparative example 2 were prepared into a cylinder having a diameter of 6cm and a height of 11cm, and the diameter and height of each set of samples were measured with a vernier caliper. And (3) putting the prepared bone hemostatic material into a universal mechanical testing machine, and performing axial mechanics according to the speed of 3 mm/min. And recording a mechanical compression curve and compression strength, carrying out 5 times of experiments in each group, and taking an average value. Calculating the compressive strength according to the formula:
ρ＝4P/πD2
ρ is the compressive strength, P is the peak load value, and D is the sample diameter.
The example 1 is marked as A1, the example 2 is marked as A2, the example 3 is marked as A3, the example 4 is marked as A4, the comparative example 2 is marked as A5, compared with the polyoxyethylene polypropylene-chitosan block copolymer, the hydroxyapatite can enhance the compression strength of the polyoxyethylene polyoxypropylene-chitosan block copolymer bone hemostatic material together with thrombin, and the test results are shown in Table 2.
TABLE 2
Figure BDA0002989892230000151
3. Cell viability assay
Preparing a leaching solution: the serum-containing medium was added to the bone hemostatic materials obtained in examples 1 to 4 and comparative example 2, respectively, to prepare a 0.2g/ml solution, and the solution was subjected to shaking extraction at 37 ℃ for 24 hours.
The viability of the bone wax cells of examples 1-4 and comparative example 2 was tested using CCK-8, example 1 being labelled A1, example 2 being labelled A2, example 3 being labelled A3, example 4 being labelled A4, comparative example 2 being labelled A5, 3T3 cells were used, the medium in which the 3T3 cells were cultured was high-sugar DMEM medium containing 10% foetal calf serum and 1% diabody (containing 100IU/ml penicillin and 100. mu.g/ml streptomycin), 5% CO at 37 ℃ with 5% CO23T3 cells were cultured in a thermostatted incubator. The 3T3 cells were subsequently diluted to 3X 104One/ml, inoculated in 96-well plates, 100. mu.l of suspension per well, placed in an incubator and incubated overnight. Changing culture medium to obtain leaching solution, placing in incubator, incubating for 1, 3, 7 and 10 days, adding CCK-8 reagent, 37 deg.C, 5% CO2Incubate the incubator for 3h, mix the 96 wellsThe plate was shaken on a shaker for 1min and the absorbance was measured on a microplate reader at a wavelength of 450nm, as shown in FIG. 2, showing that the cell proliferation was significantly increased on days 3, 7 and 10 as compared with the first day.
Therefore, the absorbable bone hemostatic material has good biocompatibility, and the proliferation of cells is increased by adding the hydroxyapatite.
4. Test of antibacterial Property
The bone hemostatic materials obtained in examples 1 to 4 and comparative example 2 were placed on solid medium plates coated with staphylococcus aureus and escherichia coli, respectively, and the antibacterial effect of absorbable bone wax was observed after incubation at 37 ℃ overnight, with the label of example 1 being a1, the label of example 2 being a2, the label of example 3 being A3, the label of example 4 being a4, and the label of comparative example 2 being a 5. The experiment was repeated 3 times for 5 different samples for bacteriostatic testing. The obtained results showed antibacterial properties with a bacteriostatic diameter (mm), and the test results are shown in table 3.
TABLE 3
Test bacterium A1 A2 A3 A4 A5
Staphylococcus aureus 9.2 10.2 9.3 10.5 3.2
Escherichia coli 9 10 9.1 10.5 3.5
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (26)

1. A preparation method of a polyoxyethylene polyoxypropylene-chitosan segmented copolymer is characterized by comprising the following steps:
(a) the polyoxyethylene polyoxypropylene segmented copolymer and an activating agent react in a solvent, and the activated polyoxyethylene polyoxypropylene segmented copolymer is obtained through precipitation and solid-liquid separation;
(b) carrying out graft copolymerization reaction on chitosan and the activated polyoxyethylene polyoxypropylene block copolymer obtained in the step (a) in acid liquor, precipitating, and carrying out solid-liquid separation to obtain a polyoxyethylene polyoxypropylene-chitosan block copolymer;
wherein the activator comprises N, N' -carbonyldiimidazole.
2. The method according to claim 1, wherein the polyoxyethylene polyoxypropylene block copolymer before activation has a polyoxyethylene content of 10 to 45% by mole.
3. The method according to claim 1, wherein the reaction temperature in the step (a) is 37 to 40 ℃ and the reaction time is 8 to 16 hours.
4. The method according to claim 1, wherein the activator is added in the step (a) in an amount of 5 to 15% by mass.
5. The method according to claim 1, wherein the solvent in the step (a) comprises a mixed solvent of 1, 4-dioxane and dimethyl sulfoxide.
6. The method according to claim 5, wherein the volume ratio of 1, 4-dioxane to dimethyl sulfoxide in the mixed solvent of 1, 4-dioxane and dimethyl sulfoxide is 1.8-2.2: 0.8-1.2.
7. The method according to claim 6, wherein the volume ratio of 1, 4-dioxane to dimethyl sulfoxide in the mixed solvent of 1, 4-dioxane and dimethyl sulfoxide is 2: 1.
8. the method of claim 1, wherein the precipitating agent in step (a) comprises anhydrous diethyl ether.
9. The method according to claim 1, wherein the graft copolymerization in the step (b) is carried out for 10 to 18 hours at a temperature of 37 to 40 ℃.
10. The method of claim 1, wherein the degree of deacetylation of chitosan in step (b) is 90-99%.
11. The method according to claim 1, wherein the chitosan is added in the step (b) in a mass fraction of 3 to 10%.
12. The method according to claim 1, wherein the acid solution in the step (b) comprises a hydrochloric acid solution.
13. The method according to claim 1, wherein the pH of the graft copolymerization reaction in the step (b) is 7 to 8.
14. The method of claim 1, wherein the precipitating agent in step (b) comprises a mixed reagent of anhydrous ethyl ether and acetone.
15. The method according to claim 14, wherein the volume ratio of the anhydrous diethyl ether to the acetone in the mixed reagent of the anhydrous diethyl ether and the acetone in the step (b) is 0.8 to 1.2: 1.8-2.2.
16. The method according to claim 14, wherein the volume ratio of the anhydrous diethyl ether to the acetone in the mixed reagent of the anhydrous diethyl ether and the acetone in step (b) is 1: 2.
17. a polyoxyethylene polyoxypropylene-chitosan block copolymer prepared by the method of any one of claims 1-16, having a number average molecular weight of 9000-.
18. The bone hemostatic material is characterized by being mainly prepared from the following raw materials in parts by weight:
10-50 parts of the polyoxyethylene polyoxypropylene-chitosan block copolymer, 25-80 parts of the polyoxypropylene polyoxyethylene random copolymer, 1-5 parts of a bone repair material and 2-20 parts of a hemostatic component according to claim 17.
19. The bone hemostatic material of claim 18, wherein the bone repair material comprises at least one of hydroxyapatite, β -tricalcium phosphate, calcium carbonate, biphasic calcium phosphate, and bioglass.
20. A bone haemostatic material according to claim 19, wherein the particle size of said hydroxyapatite is less than 150 μm.
21. The bone hemostatic material of claim 18, wherein the hemostatic composition comprises at least one of thrombin and starch microspheres.
22. The bone hemostatic material of claim 18, wherein the bone hemostatic material is wax-like.
23. A method of preparing a bone hemostatic material according to any one of claims 18-22, comprising the steps of:
and mixing the polyoxyethylene polyoxypropylene-chitosan segmented copolymer, the polyoxyethylene polyoxypropylene random copolymer, the bone repair material and the hemostatic components to obtain the bone hemostatic material.
24. The method of manufacturing of claim 23, comprising the steps of:
heating and melting the polyoxyethylene polyoxypropylene-chitosan segmented copolymer and the polyoxyethylene polyoxypropylene random copolymer, adding an optional bone repair material and an optional hemostatic component, mixing, curing and forming to obtain the bone hemostatic material.
25. The method for preparing the composite material as claimed in claim 24, wherein the curing and molding time is 3-4 h.
26. The method according to claim 25, wherein the curing time is 3 hours.
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