CN112494712A - Absorbable spongy bone wax with hemostatic and bone healing promoting functions and preparation method thereof - Google Patents

Abstract

The invention discloses a preparation method of absorbable spongy bone wax with hemostasis and bone healing promotion functions, which comprises the following steps: uniformly mixing the quaternized chitosan/modified starch composite microspheres, beta-tricalcium phosphate and silk fibroin solution, and freeze-drying to obtain the absorbable spongy bone wax. The invention combines the specific raw material proportion and the preparation process to prepare the absorbable spongy bone wax with the functions of stopping bleeding and promoting bone healing. The raw materials used in the invention have good biocompatibility and can be absorbed by human body, so the prepared spongy bone wax can be absorbed by human body without residue, and is beneficial to healing of bone wound. And secondly, due to the action of the quaternized chitosan/modified starch composite microspheres, the bone wax has a good hemostatic effect, has a certain antibacterial and anti-inflammatory effect, and has a good effect of promoting the healing of a bone wound. The bone wax is also added with beta-TCP with osteoinduction and osteoinduction functions, so that the bone wax can promote the repair of bone defect parts in the bone wound surface.

Description

Absorbable spongy bone wax with hemostatic and bone healing promoting functions and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical engineering materials, and particularly relates to an absorbable spongy bone wax with the functions of stopping bleeding and promoting bone healing and a preparation method thereof.

Background

The bleeding of the cancellous bone wound surface in the orthopedic surgery is not only easy or difficult to completely stop bleeding, but also is a troublesome problem frequently encountered by bone surgeons. Cancellous bone is loose in structure, blood circulation is rich to form densely distributed blood sinuses, vasoconstriction in tissues is poor, wound surfaces are mostly caused by sharp instrument cutting and violent striking, bleeding is mostly oozing blood, bleeding is different from bleeding of other tissues, self-hemostasis by vasoconstriction is difficult, hemostasis is difficult to be realized in operations by conventional methods such as electric coagulation, clamping, hemostatic gauze and collodion sponge filling. The most effective hemostasis method of the cancellous bone is to quickly fill cracks and blood sinuses on the surface of the cancellous bone, stop the process of bleeding and further activate the blood coagulation ways inside and outside the blood vessel, thereby achieving the purpose of hemostasis.

The bone wax is utilized to rapidly fill surface cracks and blood sinuses of the cancellous bone, and the aim of stopping bleeding can be achieved. The bone wax is the most common and effective hemostatic material for orthopedics hemostasis, and has the advantages of exact hemostatic effect, simplicity, easy use, convenient storage and the like. The main components of the bone wax commonly used at present are beeswax, sesame oil, vaseline and the like. However, the bone wax has poor adhesiveness, high brittleness and poor biocompatibility, is difficult to degrade and absorb by organisms, remains in local parts and causes great obstacle to bone healing, on one hand, the bone wax can reduce the anti-infection capability of tissues and increase the risk of postoperative infection, on the other hand, the bone wax hemostatic mechanism is only the mechanical physical barrier function, prevents the aggregation of blood components (fibrin and platelets), is not beneficial to the formation of original callus and hinders the repair of bone wound surfaces. In addition, local pain and exudation caused by granuloma of bone wax forming foreign body are also common in clinic. A great deal of documents at home and abroad report a plurality of cases of fracture nonunion caused by applying traditional bone wax to stop bleeding in the operation; especially in spinal surgery, the application of bone wax inevitably affects the fusion effect after bone grafting, and the traditional bone wax is applied to hemostasis in most cases of poor fusion effect in spinal surgery reported in literature. Therefore, there is a need to develop a new absorbable bone wax with good hemostatic effect and capable of promoting the healing of bone wound.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide an absorbable spongy bone wax with the functions of stopping bleeding and promoting bone healing and a preparation method thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for preparing absorbable spongy bone wax with hemostatic and bone healing promoting functions, which comprises the following steps: uniformly mixing the quaternized chitosan/modified starch composite microspheres, beta-tricalcium phosphate and silk fibroin solution, and freeze-drying to obtain the absorbable spongy bone wax.

Preferably, the mass volume concentration of the silk fibroin solution is 4%.

Preferably, the mass-to-volume ratio of the quaternized chitosan/modified starch composite microspheres to the beta-tricalcium phosphate to the silk fibroin solution is 50mg to 10mg to 1 mL. The prepared absorbable spongy bone wax has excellent hemostatic effect, can promote the repair of bone defect parts in the bone wound surface, has good biocompatibility, can be absorbed by human bodies, and has no cytotoxicity.

Preferably, the freeze-drying conditions are-80 ℃ freeze-drying for 24 h.

Preferably, the preparation method of the quaternized chitosan/modified starch composite microsphere comprises the following steps:

(1) adding soluble starch and quaternized chitosan into water, heating to gelatinize, and adjusting the pH value to be more than or equal to 10 to obtain a gelatinized starch/quaternized chitosan compound;

(2) dissolving sodium trimetaphosphate and polyethylene glycol in water, adding the gelatinized starch/quaternized chitosan compound obtained in the step (1), and uniformly stirring to obtain a mixed solution;

(3) and (3) dissolving span80 in olive oil, then dripping the mixed solution obtained in the step (2), after complete reaction, freezing and standing overnight, and finally centrifuging, washing and freeze-drying to obtain the quaternized chitosan/modified starch composite microspheres.

Preferably, the mass ratio of the soluble starch to the quaternized chitosan to the sodium trimetaphosphate to the polyethylene glycol to the span80 is 10:10:0.8:0.8: 2. Under the condition, the quaternized chitosan/modified starch composite microspheres with better biocompatibility, hemostatic effect and bacteriostatic action can be successfully prepared.

Preferably, the washing is alternating washing with absolute ethanol and purified water.

The invention also provides an absorbable spongy bone wax with the functions of stopping bleeding and promoting bone healing, which is prepared by the preparation method of the absorbable spongy bone wax with the functions of stopping bleeding and promoting bone healing.

The quaternized chitosan/modified starch composite microsphere is prepared by an emulsification method, the production process is simple, and the prepared quaternized chitosan/modified starch composite microsphere has the advantages of both quaternized chitosan and modified starch. The quaternized chitosan does not produce toxicity, irritation and immunity antigenicity, does not cause hemolysis, is a degradable bio-based material with good biocompatibility, and also has certain functions of hemostasis, bacteriostasis and the like. The quaternized chitosan has a certain coagulation effect on blood cells, because the negative charges of muramic acid on the surfaces of the red blood cells and the positive charges of nitrogen atoms in the quaternized chitosan units generate electrostatic attraction, so that the red blood cells are aggregated, and finally, the blood is coagulated into blood clots to achieve the hemostatic effect; the hemostasis effect is independent of the human coagulation process, and is particularly suitable for the bleeding condition of patients with the coagulation disorder. The modified starch endows the composite microspheres with the following characteristics: the adsorbent has a porous structure and strong adsorbability; has certain deformability in organisms; in the human body, it is susceptible to degradation by the relevant enzymes. Meanwhile, the modified starch has good biocompatibility, no toxicity, biodegradability, no immunogenicity, wide raw material source, low price and far better comprehensive performance than the early raw materials such as synthetic liposome, protein, high molecular material and the like. Tests prove that the quaternized chitosan/modified starch composite microsphere has an excellent hemostatic effect, can quickly absorb water in blood due to a microporous structure on the surface and has certain water absorption, and simultaneously plays a role of a molecular sieve, so that visible components such as red blood cells, thrombin, platelets, fibrin and the like in the blood are gathered on the surface of particles to form a gelatinous mixture, thereby achieving the effect of quickly stopping bleeding; also has antibacterial and anti-inflammatory effects.

In addition, the silk fibroin adopted by the invention has good biocompatibility, easy processability and degradability, and is beneficial to tissue vascularization. Beta-tricalcium phosphate (beta-TCP) has good biocompatibility, bone conduction function and biodegradability, the components of the beta-tricalcium phosphate are similar to inorganic components in biological bones, the beta-tricalcium phosphate can be dissolved after being implanted into a body, and Ca and P dissolved out enter a living body circulatory system to form living new bones.

Compared with the prior art, the invention has the beneficial effects that: the invention prepares the absorbable spongy bone wax with the functions of stopping bleeding and promoting bone healing by using silk fibroin, quaternized chitosan/modified starch composite microspheres and beta-TCP as matrixes through a freeze drying method and combining a specific raw material ratio and a preparation process. The raw materials used in the invention have good biocompatibility and can be absorbed by human body, so the prepared spongy bone wax can be absorbed by human body without residue, and is beneficial to healing of bone wound. And secondly, due to the action of the quaternized chitosan/modified starch composite microspheres, the bone wax has a good hemostatic effect, has a certain antibacterial and anti-inflammatory effect, and has a good effect of promoting the healing of a bone wound. In addition, the bone wax is also added with beta-TCP with bone induction and bone guiding functions, so that the bone wax can promote the repair of bone defect parts in the bone wound surface. The invention has the advantages of easily obtained raw materials, simple production process, high economic value and clinical value, and is beneficial to industrialization and marketization.

Drawings

FIG. 1 is a scanning electron microscope image of the quaternized chitosan/modified starch composite microspheres prepared in example 2;

FIG. 2 is a scanning electron micrograph of absorbable cancellous bone wax prepared in examples 3-4 and cancellous bone wax prepared in comparative examples 1-4;

FIG. 3 is a graph showing the results of cytotoxicity tests on absorbable cancellous bone waxes prepared in examples 3-4 and cancellous bone waxes prepared in comparative examples 1-3;

FIG. 4 is an ALP test result of the absorbable cancellous bone waxes prepared in examples 3-4 and the cancellous bone waxes prepared in comparative examples 1-3;

FIG. 5 is a process of transplanting each cancellous bone wax sample to a bleeding defect of a tibia of a rabbit, wherein A is a defect made by the tibia of the rabbit, and B is a process of transplanting cancellous bone wax to a defect made by the tibia of the rabbit;

FIG. 6 is a general graph of the repair of a wound bed of bone with an absorbable cancellous bone wax prepared in example 3 and cancellous bone waxes prepared in comparative examples 1-4;

FIG. 7 is a 3D reconstruction of the repair of a wound bed of bone from an absorbable cancellous bone wax prepared in example 3 and cancellous bone waxes prepared in comparative examples 1-4.

Detailed Description

The technical solutions of the present invention will be further described with reference to the following embodiments and the accompanying drawings, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. 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. The methods used in the examples are conventional in the art unless otherwise specified.

Example 1

The preparation method of the silk fibroin solution comprises the following steps:

(1) using 0.5% of Na by mass fraction₂CO₃Boiling the solution for 5 times, each time for 1 hr, at 100 deg.C, at bath ratio of l:100, washing with distilled water, and naturally drying to obtain pure silk cellulose fiber;

(2) using CaCl with a molar ratio of 1:2:8₂、CH₃CH₂OH and H₂Dissolving the pure silk cellulose fiber prepared in the step (1) by using a ternary solvent formed by mixing O, wherein the mass ratio of the ternary solvent to the silk cellulose fiber is 10:1, stirring and dissolving for 1-2 hours at 75-80 ℃ to obtain a mixed solution;

(3) and (3) cooling the mixed solution prepared in the step (2), injecting the cooled mixed solution into a cellulose dialysis membrane, dialyzing the mixed solution in running water for 2-4 days, and storing the dialyzed solution at 4 ℃ to obtain the silk fibroin solution with the mass volume concentration of 4%.

Example 2

The preparation method of the quaternized chitosan/modified starch composite microsphere comprises the following steps:

(1) weighing 10g of soluble starch and 10g of quaternized chitosan, placing the soluble starch and the quaternized chitosan in a beaker, adding 100mL of purified water, heating the mixture to 95 ℃ in a water bath kettle under magnetic stirring until gelatinization, and adjusting the pH to be more than or equal to 10 by using 1% of sodium hydroxide solution to obtain a gelatinized starch/quaternized chitosan compound;

(2) weighing 0.8g of sodium trimetaphosphate and 0.8g of PEG (polyethylene glycol) in a beaker, adding 30mL of purified water, stirring for dissolving, adding the gelatinized starch/quaternized chitosan compound obtained in the step (1), and uniformly stirring to obtain a mixed solution;

(3) measuring 150mL of olive oil, adding 2g of span80, stirring and dissolving in a water bath at 60 ℃, cooling to 50 ℃, then slowly dripping the mixed solution obtained in the step (2), continuing to react for 6 hours after dripping is finished, then standing overnight in a refrigerator at 4 ℃, then centrifuging at the rotating speed of 5000RPM, taking the precipitate, alternately washing with absolute ethyl alcohol and purified water for 3 times, and freeze-drying to obtain the quaternized chitosan/modified starch composite microspheres.

Example 3

The preparation method of the absorbable spongy bone wax comprises the following steps:

the silk fibroin solution prepared in example 1, the quaternized chitosan/modified starch composite microspheres prepared in example 2, and beta-TCP were stirred at 400RPM for 24 hours to mix them uniformly, and then freeze-dried at-80 ℃ for 24 hours to obtain absorbable cancellous bone wax. In this example, the mass-to-volume ratio of the quaternized chitosan/modified starch composite microspheres, the β -tricalcium phosphate, and the silk fibroin solution is 50mg, 10mg, and 1 mL.

Example 4

The preparation method of the absorbable spongy bone wax comprises the following steps:

the silk fibroin solution prepared in example 1, the quaternized chitosan/modified starch composite microspheres prepared in example 2, and beta-TCP were stirred at 400RPM for 24 hours to mix them uniformly, and then freeze-dried at-80 ℃ for 24 hours to obtain absorbable cancellous bone wax. In this example, the mass-to-volume ratio of the quaternized chitosan/modified starch composite microspheres, the β -tricalcium phosphate, and the silk fibroin solution is 100mg, 10mg, and 1 mL.

Comparative example 1

Preparation of a cancellous bone wax comprising the steps of: the silk fibroin solution prepared in example 1 was freeze-dried at-80 ℃ for 24 hours to obtain cancellous bone wax.

Comparative example 2

Preparation of a cancellous bone wax comprising the steps of:

the silk fibroin solution prepared in example 1 and β -TCP were stirred at 400RPM for 24 hours to mix them uniformly, and then freeze-dried at-80 ℃ for 24 hours to obtain spongy bone wax. The mass-volume ratio of the beta-tricalcium phosphate to the silk fibroin solution is 10mg to 1 mL.

Comparative example 3

Preparation of a cancellous bone wax comprising the steps of:

the silk fibroin solution prepared in example 1 and the quaternized chitosan/modified starch composite microspheres prepared in example 2 were stirred at 400RPM for 24 hours to mix them uniformly, and then freeze-dried at-80 ℃ for 24 hours to obtain the spongy bone wax. The mass-volume ratio of the quaternized chitosan/modified starch composite microspheres to the silk fibroin solution is that the quaternized chitosan/modified starch composite microspheres and the silk fibroin solution account for 20mg to 1 mL.

Comparative example 4

Preparation of a cancellous bone wax comprising the steps of:

the silk fibroin solution prepared in example 1 and the quaternized chitosan/modified starch composite microspheres prepared in example 2 were stirred at 400RPM for 24 hours to mix them uniformly, and then freeze-dried at-80 ℃ for 24 hours to obtain the spongy bone wax. The mass-volume ratio of the quaternized chitosan/modified starch composite microspheres to the silk fibroin solution is that the quaternized chitosan/modified starch composite microspheres and the silk fibroin solution are 50mg to 1 mL.

Test examples

Firstly, after spraying gold on the quaternized chitosan/modified starch composite microspheres prepared in example 2, observing the microspheres under a scanning electron microscope, wherein the test conditions are as follows: 5kV electron beam.

Fig. 1 is a scanning electron microscope image of the quaternized chitosan/modified starch composite microsphere, and as can be seen from fig. 1, the quaternized chitosan/modified starch composite microsphere prepared by the invention is oval, and the particle size is 20-50 microns.

Secondly, after spraying gold on the absorbable cancellous bone wax prepared in the examples 3-4 and the cancellous bone wax prepared in the comparative examples 1-4, observing under a scanning electron microscope under the test conditions that: 5kV electron beam.

FIG. 2 is a scanning electron micrograph of absorbable cancellous bone wax prepared in examples 3-4 and cancellous bone wax prepared in comparative examples 1-4. In the drawing, a is a spongy bone wax of comparative example 1, B is a spongy bone wax of comparative example 2, C is a spongy bone wax of comparative example 3, D is a spongy bone wax of comparative example 4, E is a spongy bone wax of example 3, and F is a spongy bone wax of example 4.

As can be seen from FIG. 2, the spongy bone wax of group 6 was porous, and the powder filling in the porous structure was not seen in Panel A, while the powder filling in the porous structure was clearly seen in the remaining panels, and the amount of powder filled in the pores of the porous structure was continuously increased as the amount of powder added was increased. Wherein a large amount of powder is uniformly distributed in the porous structure in the spongy bone waxes D and E, and the porous structure is not damaged. While the powder can be seen layered in the porous structure in the cancellous bone wax F, the porous structure of the cancellous bone wax has been destroyed. Therefore, the absorbable spongy bone wax with a porous structure, an excellent hemostatic function and a bone repair promoting function can be obtained only by mixing the silk fibroin solution, the quaternized chitosan/modified starch composite microspheres and the beta-TCP in a specific ratio to prepare the spongy bone wax.

Third, cytotoxicity

Leaching standard of leaching liquor of medical apparatus according to national standard GB/T16886.12, and surface area is 1.25cm²Leach liquor at a ratio of/mL. Adding the leaching solution according to national standard, culturing in shaking table at 37 deg.C for 24 + -2 h, and storing the rest in refrigerator at 4 deg.C for use. Will contain 100uL, 1X 10⁴Media of mouse bone marrow mesenchymal stem cells (BMSCs) per mL were seeded in 96-well plates. After 12h of inoculation, the original culture solution was taken out, and 100. mu.L of the test material leaching solution was added to each well dish. Each group is provided with at least 5 holes. The culture medium was changed once every 2 days. After the cells were cultured to the set time point, the culture broth was removed and the cells were washed 2 times with PBS. A CCK8 solution was added to each well in an amount of 50uL medium, and a negative control (blank medium) was set and cultured in a cell incubator for 1 to 2 hours. According to the color change judgment, the culture plate is taken out, and the liquid in the corresponding hole is sucked into a 96-well plate. And detecting the absorbance value (OD value) under the wavelength of 450nm of a microplate reader, recording and calculating data. Samples of absorbable cancellous bone wax prepared in examples 3-4 and cancellous bone wax prepared in comparative examples 1-3 were subjected to a cell compatibility test-cytotoxicity, and the results are shown in FIG. 3. In FIG. 3, a drawing A shows a cancellous bone wax of comparative example 1, a drawing B shows a cancellous bone wax of comparative example 2, a drawing C shows a cancellous bone wax of comparative example 3, a drawing E shows a cancellous bone wax of example 3, and a drawing F shows a cancellous bone wax of example 4.

As can be seen from FIG. 3, the absorbable cancellous bone waxes prepared in examples 3-4 and the cancellous bone wax samples prepared in comparative examples 1-3 all have good cell compatibility, and the cell survival rates are all more than 80%. Among them, the spongy bone wax E has the highest cell survival rate because: (1) the spongy bone wax E contains beta-TCP which has the functions of bone induction and bone induction, thereby promoting the proliferation and differentiation of BMSCs cells; (2) the spongy bone wax E maintains a porous scaffold structure, is favorable for cell adhesion and has a promoting effect on the proliferation and differentiation of BMSCs cells.

Tetra, alkaline phosphatase Activity (ALP)

Samples of the absorbable cancellous bone wax prepared in examples 3-4 and the cancellous bone wax prepared in comparative examples 1-3 were placed in 96-well culture plates. Mouse bone marrow mesenchymal stem cells (BMSCs) were digested from the medium with 0.25% pancreatin/PBS solution, centrifuged at 1500RPM for 5min, the supernatant removed, fresh low sugar DMEM medium containing serum was added, and the cell suspension concentration was adjusted to 5X 10⁶and/mL. The cell planting density is 1 multiplied by 10⁵A support. After incubation for 2h, 500. mu.L of medium was added to each well and 24h replaced with dexamethasone medium (high-sugar DMEM containing 10% FCS, 50mM ascorbic acid, 10mM disodium beta-phosphate and 100nM dexamethasone). 5% CO at 37 deg.C₂The culture was continued in an incubator for the required time, and the medium was changed every other day in order to maintain the nutrient supply to the cells.

The scaffolds were removed from the plates, washed 3 times with PBS solution, added with 500. mu.L of cell lysate, subsequently lysed at 4 ℃ overnight, and sonicated. Adding 500 μ L ALP substrate reaction solution into a pore plate, heating in water bath at 37 deg.C for reaction for 30min, adding 500 μ L NaOH solution with concentration of 0.1M to terminate the reaction, measuring the absorbance at 405nm with an ultraviolet-visible spectrophotometer (UV-Probe 2550, Shimadzu), and calculating ALP according to the instruction. Each set of samples was tested in parallel 3 times and the results are shown in figure 4.

Alkaline phosphatase (ALP) is a membrane-bound protein, an early marker of osteoblast differentiation, involved in mineral deposition processes. Therefore, we investigated the differentiation of mouse bone marrow mesenchymal stem cells (BMSCs) by spongiform bone wax in an in vitro experiment by investigating the measurement of alkaline phosphatase (ALP) activity on scaffolds.

As can be seen in FIG. 4, the ALP activity of the cells of each of the cancellous bone waxes increased with the increase of the culture time, indicating that five cancellous bone waxes were able to support osteogenic differentiation of BMSCs. In addition, as shown in FIG. 4, the spongy bone wax E has a higher ALP activity in particular, which is consistent with the result of cell compatibility, also because the spongy bone wax E contains β -TCP and maintains a porous scaffold structure.

Fifthly, the hemostatic effect of the spongy bone wax and the repair condition of the bone defect

The new Zealand rabbits which are qualified by quarantine are 40, males are randomly divided into 5 groups according to the weight of the animals.

After the ear margin of a New Zealand rabbit is anesthetized by injecting a 3% sodium pentobarbital solution (40mg/kg) intravenously, the right hind limb of the animal is shaved and cleaned, the animal is deiodinated by alcohol after iodophor disinfection, skin and subcutaneous tissues are cut along the midline of the right hind limb tibia of the animal, and the lateral surface of the right hind limb tibia is exposed. A bone defect region of 4.2mm in diameter and 3mm in depth was drilled 3cm below the knee joint using a ZAZJ-I battery-operated medical electric drill. And taking a picture after the model is successful. The defect sites were immediately filled with the absorbable cancellous bone wax prepared in example 3 and the cancellous bone waxes prepared in comparative examples 1-4, respectively, and the time taken for the cancellous bone wax to stop bleeding from the defect sites was recorded (at most 5min for observation), followed by suturing the incisions. The experimental procedure is shown in FIG. 5. The results are shown in Table 1 and FIGS. 6 to 7.

In table 1 and fig. 6 to 7, the cancellous bone wax a is the cancellous bone wax of comparative example 1, the cancellous bone wax B is the cancellous bone wax of comparative example 2, the cancellous bone wax C is the cancellous bone wax of comparative example 3, the cancellous bone wax D is the cancellous bone wax of comparative example 4, and the cancellous bone wax E is the cancellous bone wax of example 3.

TABLE 1 hemostasis time for cancellous bone wax

As can be seen from Table 1, the bleeding times for both cancellous bone waxes A and B were greater than 5min, with no hemostatic effect. The hemostasis time for cancellous bone wax C was 30 + -5S, while the hemostasis time for cancellous bone waxes D and E was immediate. The experimental result shows that the spongy bone wax containing SF (silk fibroin) only or SF and beta-TCP has no hemostatic effect, while the spongy bone wax containing the quaternized chitosan/modified starch composite microspheres has the hemostatic effect, which shows that the quaternized chitosan/modified starch composite microspheres are main components of the hemostatic function of the spongy bone wax, and the higher the content of the quaternized chitosan/modified starch composite microspheres is, the better the hemostatic effect is.

It can be seen from a combination of fig. 6 and 7 that over time, five groups of cancellous bone waxes had a repairing effect on the site of the bone defect. Among them, the spongy bone waxes B and E have the best repairing effect because they contain β -TCP having osteoinductive and osteoinductive effects.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (8)

1. A method for preparing absorbable spongy bone wax with hemostatic and bone healing promoting functions, which comprises the following steps: uniformly mixing the quaternized chitosan/modified starch composite microspheres, beta-tricalcium phosphate and silk fibroin solution, and freeze-drying to obtain the absorbable spongy bone wax.

2. The method of claim 1, wherein the silk fibroin solution is provided at a concentration of 4% by mass/volume.

3. The method of claim 1, wherein the mass-to-volume ratio of the quaternized chitosan/modified starch composite microspheres, β -tricalcium phosphate, and silk fibroin solution is 50mg:10mg:1 mL.

4. The method of preparing an absorbable cancellous bone wax according to claim 1 wherein said lyophilization conditions are-80 ℃ lyophilization for 24 hours.

5. The method of preparing an absorbable cancellous bone wax according to claim 1 wherein said method of preparing said quaternized chitosan/modified starch composite microspheres comprises:

(1) adding soluble starch and quaternized chitosan into water, heating to gelatinize, and adjusting the pH value to be more than or equal to 10 to obtain a gelatinized starch/quaternized chitosan compound;

(2) dissolving sodium trimetaphosphate and polyethylene glycol in water, adding the gelatinized starch/quaternized chitosan compound obtained in the step (1), and uniformly stirring to obtain a mixed solution;

(3) and (3) dissolving span80 in olive oil, then dripping the mixed solution obtained in the step (2), after complete reaction, freezing and standing overnight, and finally centrifuging, washing and freeze-drying to obtain the quaternized chitosan/modified starch composite microspheres.

6. The method for preparing the absorbable spongy bone wax according to claim 5, wherein the mass ratio of the soluble starch, the quaternized chitosan, the sodium trimetaphosphate, the polyethylene glycol and the span-80 is 10:10:0.8:0.8: 2.

7. The method of claim 5, wherein the washing is performed by alternately washing with absolute ethanol and purified water.

8. An absorbable spongy bone wax with hemostatic and bone healing promoting functions, which is prepared by the preparation method of the absorbable spongy bone wax with hemostatic and bone healing promoting functions as claimed in any one of claims 1-7.

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