CN111012946A - Thermo-sensitive calcium phosphate bone cement and preparation method thereof - Google Patents
Thermo-sensitive calcium phosphate bone cement and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/12—Phosphorus-containing materials, e.g. apatite
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
Abstract
The invention discloses a temperature-sensitive calcium phosphate bone cement and a preparation method thereof, wherein the temperature-sensitive calcium phosphate bone cement comprises solid-phase powder and liquid-phase components, wherein the solid-phase powder is calcium salt, and the liquid-phase components are formed by mixing gamma-glycidyl ether oxypropyl trimethoxy silane aqueous solution, chitosan acetic acid aqueous solution, sodium glycerophosphate aqueous solution and bletilla striata polysaccharide aqueous solution.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and particularly relates to temperature-sensitive calcium phosphate cement and a preparation method thereof.
Background
The calcium phosphate bone cement forms hydroxyapatite when meeting water, has biocompatibility, forms a block mass with enough strength after self-curing, can be subjected to plasticity according to requirements, can be applied to orthopedics and dentistry, is used for filling bone defects, filling tooth root canals and the like, can be absorbed and replaced by newly-generated bone after the bone cement is implanted, does not degrade the volume of the implant, and keeps the integrity of the implanted material.
The prior art uses chitosan/β -disodium glycerophosphate hydrogel to prepare the temperature-sensitive smooth cement, but has some defects, such as lower mechanical property and poorer degradation resistance in vivo, thereby limiting the application of the temperature-sensitive smooth cement, and β -disodium glycerophosphate has cytotoxicity.
Disclosure of Invention
The invention aims to provide the temperature-sensitive calcium phosphate bone cement which is low in toxicity, shortens the gelation time of hydrogel, enhances the mechanical property of the bone cement and improves the anti-collapsibility.
Further, it is necessary to provide a method for preparing the temperature-sensitive calcium phosphate cement.
The temperature-sensitive calcium phosphate cement comprises solid-phase powder and liquid-phase components, and is characterized in that:
the solid phase powder is a calcium salt;
the liquid phase component is formed by mixing the following components: gamma-glycidoxypropyltrimethoxysilane, chitosan-acetic acid, sodium glycerophosphate and bletilla polyose.
Wherein the mass ratio of the solid-phase powder to the liquid-phase component is (1.5-2.7):1, more preferably, the mass ratio of the solid-phase powder to the liquid-phase component is 2: 1.
Wherein the solid phase powder is selected from one or more of α -tricalcium phosphate, apatite, tetracalcium phosphate and dicalcium phosphate, preferably, the solid phase powder is α -tricalcium phosphate, and the particle size of α -tricalcium phosphate is 1-200 μm.
Wherein, the liquid phase component is formed by mixing the following components: 4-6 wt% of gamma-glycidyl ether oxypropyl trimethoxy silane aqueous solution; chitosan acetic acid aqueous solution with the mass fraction of 3-5 wt%; sodium glycerophosphate aqueous solution with the mass fraction of 40-60 wt%; 10-20 wt% of bletilla striata polysaccharide aqueous solution;
wherein, the mass ratio of the bletilla striata polysaccharide aqueous solution, the gamma-glycidyl ether oxygen propyl trimethoxy silane aqueous solution, the chitosan aqueous solution and the sodium glycerophosphate aqueous solution is (20-30): 40-60): (5-10).
Preferably, the liquid phase component is formed by mixing the following components: 4-6 wt% of gamma-glycidyl ether oxypropyl trimethoxy silane aqueous solution; chitosan acetic acid aqueous solution with the mass fraction of 3-5 wt%; sodium glycerophosphate aqueous solution with the mass fraction of 40-60 wt%; 10-20 wt% of bletilla striata polysaccharide aqueous solution;
wherein the mass ratio of the bletilla polysaccharide aqueous solution, the gamma-glycidyl ether oxygen propyl trimethoxy silane aqueous solution, the chitosan aqueous solution and the sodium glycerophosphate aqueous solution is 25: 50: 50: 8.
more preferably, the liquid phase component is formed by mixing the following components: 5 wt% of gamma-glycidoxypropyltrimethoxysilane aqueous solution; a chitosan acetic acid aqueous solution with the mass fraction of 4 wt%; a sodium glycerophosphate aqueous solution with the mass fraction of 40 wt%; 15 wt% of bletilla striata polysaccharide aqueous solution;
wherein the mass ratio of the bletilla polysaccharide aqueous solution, the gamma-glycidyl ether oxygen propyl trimethoxy silane aqueous solution, the chitosan aqueous solution and the sodium glycerophosphate aqueous solution is 25: 50: 50: 8.
the bletilla striata polysaccharide is prepared by the following method:
freezing dried bletilla tuber at-20 deg.C for 2 hr, pulverizing to obtain bletilla powder, mixing with distilled water at a mass ratio of 1: 4-1: 8, stirring at the temperature of between 60 and 100 ℃ for 0.5 to 2 hours at the speed of between 300 and 700r/min, filtering, and collecting the bletilla striata polysaccharide filtrate.
Wherein, the chitosan acetic acid aqueous solution is prepared by the following method:
dissolving chitosan in 1 wt% acetic acid solution to prepare 4 wt% chitosan acetic acid aqueous solution, wherein the chitosan is chitosan powder with deacetylation degree of 85-100% and molecular weight of 70-80 ten thousand.
A method for preparing the temperature-sensitive calcium phosphate cement comprises the following steps of respectively obtaining solid-phase powder and liquid-phase components:
1) preparing 4-6 wt% of gamma-glycidoxypropyltrimethoxysilane aqueous solution;
preparing a sodium glycerophosphate aqueous solution with the mass fraction of 40-60 wt%;
preparing 10-20 wt% of bletilla striata polysaccharide aqueous solution:
freezing dried bletilla striata stem blocks at-20 ℃ for 2 hours, crushing and sieving by a 60-mesh sieve to obtain bletilla striata powder, mixing the powder with distilled water in a mass ratio of 1: 4-1: 8, stirring at the temperature of between 60 and 100 ℃ for 0.5 to 2 hours at the speed of between 300 and 700r/min, filtering, and collecting bletilla striata polysaccharide filtrate; continuing soaking and filtering the filtered bletilla striata powder for 4 times, combining and collecting bletilla striata polysaccharide filtrates, combining the bletilla striata polysaccharide filtrates collected in a grading manner, concentrating the filtrate by rotary evaporation, adding absolute ethyl alcohol into the bletilla striata polysaccharide concentrated solution, stirring, carrying out alcohol precipitation, filtering to obtain a precipitate, washing the precipitate obtained by filtering by using the absolute ethyl alcohol, carrying out vacuum freeze drying on the precipitate, adding deionized water into the dried bletilla striata polysaccharide to dissolve the dried bletilla striata polysaccharide, centrifuging, passing the supernatant through a 0.45-micrometer microporous filter membrane, carrying out ultrafiltration at 40 ℃ under 0.10MPa to obtain an ultrafiltrate with a filtering section ratio of 7:3, concentrating the ultrafiltrate to 1g/L, adding the absolute ethyl alcohol to carry out alcohol precipitation, carrying out vacuum freeze drying on the precipitate to obtain bletilla striata polysaccharide, dissolving the bletilla striata polysaccharide in an aqueous solution, and preparing a 10-20 wt% bletilla striata polysaccharide aqueous solution;
preparing 3-5 wt% chitosan acetic acid aqueous solution: dissolving chitosan in 1 wt% of acetic acid solution to prepare solution with the mass fraction of 3-5 wt%, wherein the chitosan is chitosan powder with the deacetylation degree of 90% and the molecular weight of 70-80 ten thousand;
2) the bletilla polysaccharide aqueous solution, the gamma-glycidyl ether oxygen propyl trimethoxy silane aqueous solution, the chitosan aqueous solution and the sodium glycerophosphate aqueous solution are mixed according to the proportion of (20-30) to (40-60): (5-10) mixing the components in a mass ratio to obtain a liquid phase component;
3) the solid phase powder is calcium salt selected from one or more of apatite, tetracalcium phosphate, and dicalcium phosphate.
Compared with the prior art, the temperature-sensitive calcium phosphate bone cement provided by the invention adopts a temperature-sensitive bone cement system prepared from chitosan, β -disodium glycerophosphate and a-tricalcium phosphate, realizes temperature sensitivity of sol-gel transformation by regulating and controlling interaction with water molecules, and optimizes the components, so that β -sodium glycerophosphate is reduced, toxicity is reduced, gelation time of hydrogel is shortened, mechanical properties of the bone cement are enhanced, anti-collapsibility is improved, and the defect of poor mechanical properties of the hydrogel is overcome.
Detailed Description
The core of the invention is to provide a calcium phosphate cement and a preparation method thereof. The present invention will be described in further detail with reference to specific embodiments in order to make the technical field better understand the scheme of the present invention.
Description of the raw materials
Chitosan, degree of deacetylation 85% -100%, molecular weight: 70-80 ten thousand, manufacturer: shanghai blue season science development Co., Ltd.
Description of self-made raw materials:
freezing the dried bletilla striata stem blocks at-20 ℃ for 2 hours, crushing and sieving by a 60-mesh sieve to obtain bletilla striata powder, mixing the powder with distilled water in a mass ratio of 1: 6, stirring at 70 ℃ at 500r/min for 1h, soaking for 0.8h, filtering, and collecting bletilla polysaccharide filtrate.
Continuing soaking and filtering the filtered bletilla striata powder for 4 times, combining and collecting bletilla striata polysaccharide filtrates, combining the bletilla striata polysaccharide filtrates collected in a plurality of times, concentrating the filtrate by rotary evaporation, adding absolute ethyl alcohol into the bletilla striata polysaccharide concentrated solution, stirring, precipitating with ethanol, filtering to obtain a precipitate, washing the precipitate obtained by filtering with absolute ethyl alcohol, freeze-drying the precipitate in vacuum, adding deionized water into the dried bletilla striata polysaccharide to dissolve the dried bletilla striata polysaccharide, centrifuging, passing the supernatant through a 0.45 mu m microporous filter membrane, performing ultrafiltration at 40 ℃ under 0.10MPa to obtain ultrafiltrate with a filtering ratio of 7:3, concentrating the ultrafiltrate to 1g/L, adding absolute ethyl alcohol to perform ethanol precipitation, and freeze-drying the precipitate in vacuum to obtain the bletilla striata polysaccharide.
Example 1
The thermo-sensitive calcium phosphate cement comprises solid-phase powder and liquid-phase components, wherein the mass ratio of the solid-phase powder to the liquid-phase components is as follows: 1.5: 1;
the solid phase powder is α -tricalcium phosphate;
the liquid phase component is prepared by the following method:
preparing 4 mass percent of gamma-glycidoxypropyltrimethoxysilane;
preparing a chitosan acetic acid aqueous solution with the mass fraction of 3%: dissolving chitosan in 1 wt% of acetic acid solution to prepare 3% of chitosan acetic acid aqueous solution, wherein the chitosan is chitosan powder with deacetylation degree of 90%, and the molecular weight is 70 ten thousand;
preparing a sodium glycerophosphate solution with the mass fraction of 50%;
preparing a bletilla striata polysaccharide solution with the mass fraction of 10%;
mixing rhizoma bletilla polysaccharide solution, gamma-glycidyl ether oxygen propyl trimethoxy silane, chitosan acetic acid water solution and sodium glycerophosphate solution according to the mass ratio of 20:40:40:5, adjusting the system temperature to 15 ℃, and stirring for 30 minutes to prepare a liquid phase component.
Example 2
The thermo-sensitive calcium phosphate cement comprises solid-phase powder and liquid-phase components, wherein the mass ratio of the solid-phase powder to the liquid-phase components is as follows: 2: 1;
the solid phase powder is α -tricalcium phosphate;
the liquid phase component is prepared by the following method:
preparing gamma-glycidoxypropyltrimethoxysilane with the mass fraction of 6%;
preparing a 5% chitosan acetic acid aqueous solution by mass: dissolving chitosan in 1 wt% of acetic acid solution to prepare 5% of chitosan acetic acid aqueous solution, wherein the chitosan is chitosan powder with deacetylation degree of 90%, and the molecular weight is 70 ten thousand;
preparing a sodium glycerophosphate solution with the mass fraction of 60%;
preparing bletilla striata polysaccharide solution with the mass fraction of 20%;
mixing rhizoma bletilla polysaccharide solution, gamma-glycidyl ether oxygen propyl trimethoxy silane, chitosan acetic acid water solution and sodium glycerophosphate solution according to the mass ratio of 30:60:60:10, adjusting the system temperature to 15 ℃, and stirring for 30 minutes to prepare a liquid phase component.
Example 3
The thermo-sensitive calcium phosphate cement comprises solid-phase powder and liquid-phase components, wherein the mass ratio of the solid-phase powder to the liquid-phase components is as follows: 2.7: 1;
the solid phase powder is α -tricalcium phosphate;
the liquid phase component is prepared by the following method:
preparing 5 mass percent of gamma-glycidoxypropyltrimethoxysilane;
preparing a chitosan acetic acid aqueous solution with the mass fraction of 4%: dissolving chitosan in 1 wt% of acetic acid solution to prepare 4% of chitosan acetic acid aqueous solution by mass fraction, wherein the chitosan is chitosan powder with deacetylation degree of 90%, and the molecular weight is 80 ten thousand;
preparing a sodium glycerophosphate solution with the mass fraction of 40%;
preparing a bletilla striata polysaccharide solution with the mass fraction of 15%;
mixing bletilla striata polysaccharide solution, gamma-glycidoxypropyltrimethoxysilane, chitosan acetic acid aqueous solution and sodium glycerophosphate solution according to the mass ratio of 25: 50: 50: 8, mixing, adjusting the temperature of the system to 15 ℃, and stirring for 30 minutes to prepare the liquid phase component.
Example 4
The thermo-sensitive calcium phosphate cement comprises solid-phase powder and liquid-phase components, wherein the mass ratio of the solid-phase powder to the liquid-phase components is as follows: 2: 1;
the solid phase powder is α -tricalcium phosphate;
the liquid phase component is prepared by the following method:
preparing 5 mass percent of gamma-glycidoxypropyltrimethoxysilane;
preparing a chitosan acetic acid aqueous solution with the mass fraction of 4%: dissolving chitosan in 1 wt% of acetic acid solution to prepare 4% of chitosan acetic acid aqueous solution, wherein the chitosan is chitosan powder with deacetylation degree of 90%, and the molecular weight is 70 ten thousand;
preparing a sodium glycerophosphate solution with the mass fraction of 40%;
preparing a bletilla striata polysaccharide solution with the mass fraction of 15%;
mixing bletilla striata polysaccharide solution, gamma-glycidoxypropyltrimethoxysilane, chitosan acetic acid aqueous solution and sodium glycerophosphate solution according to the mass ratio of 25: 50: 50: 30, adjusting the temperature of the system to 15 ℃, and stirring for 30 minutes to prepare the liquid phase component.
Comparative example 1:
the thermo-sensitive calcium phosphate cement comprises solid-phase powder and liquid-phase components, wherein the mass ratio of the solid-phase powder to the liquid-phase components is as follows: 2: 1;
the solid phase powder is α -tricalcium phosphate;
the liquid phase component is prepared by the following method:
preparing a 5% chitosan acetic acid aqueous solution by mass: dissolving chitosan in 1 wt% of acetic acid solution to prepare 5% of chitosan acetic acid aqueous solution, wherein the chitosan is chitosan powder with deacetylation degree of 90%, and the molecular weight is 70 ten thousand;
preparing a sodium glycerophosphate solution with the mass fraction of 60%;
mixing chitosan acetic acid water solution and sodium glycerophosphate solution according to the mass ratio of 60:10, adjusting the system temperature to 15 ℃, and stirring for 30 minutes to prepare a liquid phase component.
Comparative example 2
The thermo-sensitive calcium phosphate cement comprises solid-phase powder and liquid-phase components, wherein the mass ratio of the solid-phase powder to the liquid-phase components is as follows: 2: 1;
the solid phase powder is α -tricalcium phosphate;
the liquid phase component is prepared by the following method:
preparing gamma-glycidoxypropyltrimethoxysilane with the mass fraction of 6%;
preparing a 5% chitosan acetic acid aqueous solution by mass: dissolving chitosan in 1 wt% of acetic acid solution to prepare 5% of chitosan acetic acid aqueous solution, wherein the chitosan is chitosan powder with deacetylation degree of 90%, and the molecular weight is 70 ten thousand;
preparing a sodium glycerophosphate solution with the mass fraction of 60%;
mixing gamma-glycidoxypropyltrimethoxysilane, chitosan acetic acid water solution and sodium glycerophosphate solution in a mass ratio of 60:60:10, adjusting the system temperature to 15 ℃, and stirring for 30 minutes to prepare a liquid phase component.
Comparative example 3
The thermo-sensitive calcium phosphate cement comprises solid-phase powder and liquid-phase components, wherein the mass ratio of the solid-phase powder to the liquid-phase components is as follows: 2: 1;
the solid phase powder is α -tricalcium phosphate;
the liquid phase component is prepared by the following method:
preparing gamma-glycidoxypropyltrimethoxysilane with the mass fraction of 6%;
preparing a 5% chitosan acetic acid aqueous solution by mass: dissolving chitosan in 1 wt% of acetic acid solution to prepare 5% of chitosan acetic acid aqueous solution, wherein the chitosan is chitosan powder with deacetylation degree of 90%, and the molecular weight is 70 ten thousand;
preparing a sodium glycerophosphate solution with the mass fraction of 60%;
preparing bletilla striata polysaccharide solution with the mass fraction of 20%;
mixing the bletilla striata polysaccharide solution, the chitosan acetic acid aqueous solution and the sodium glycerophosphate solution according to the mass ratio of 30:60:10, adjusting the system temperature to 15 ℃, and stirring for 30 minutes to prepare a liquid phase component.
The following performance tests were performed on the calcium phosphate cements prepared in examples 1 to 4 and comparative examples 1 to 3.
1. Measurement of gelation time
The gelation formation of the liquid phase components was tested by an inverted bottle experiment, the liquid phase components of examples 1 to 4 and comparative examples 1 to 3 were placed in test tubes, respectively, placed in a thermostatic water bath at 37.5 ℃ to gel, the test tubes were tilted every 2min, and it was observed whether the liquid level of the sample in the test tube would be changed by the tilting of the test tube, and if not, it would have been gelled, and recorded as the gelation time.
TABLE 1 gelation time
| Group of | Example 1 | Example 2 | Example 3 | Example 4 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Gelation time (min) | 12 | 16 | 16 | 22 | 42 | 32 | 34 |
The gelation time of examples 1 to 4 is shorter than that of comparative examples 1 to 3, the gelation time of example 4 is longer than that of example 3, the sodium glycerophosphate is added in an amount larger than that of example 3, the gelation phenomenon of example 4 occurs, the liquid phase component of examples 1 to 3 is added with bletilla polysaccharide and gamma-glycidoxypropyltrimethoxysilane in addition to chitosan and sodium glycerophosphate, the gelation time of the liquid phase component can be reduced, and the gelation time of examples 1 to 3 is shortened and the gel is stable compared with that of comparative examples 1 to 3.
2. Measurement of collapse resistance
Taking 1g of solid-phase powder, mixing the solid-phase powder and the liquid-phase components in proportion to obtain uniform paste, injecting the paste into a beaker filled with 30mL of SBF solution through an injector, shaking the mixture for 1 hour in a constant-temperature shaking table at 60rpm and 37 ℃, and calculating the disintegration rate of the CPC. The collapsibility is the percentage of the mass remaining after the calcium phosphate cement slurry was cured in SBF to the total mass of calcium phosphate cement before curing, and the results are shown in table 2.
3. Injectability
Pouring the calcium phosphate cement paste into a 10mL disposable syringe, vertically placing the syringe on a flat plate of a mechanical testing machine, pushing out the cement paste at the speed of 15mm/min, and stopping until the maximum pushing force is 100N. Injectability is the percentage of the mass of calcium phosphate cement after extrusion injection to the total mass of cement before injection, and the results are shown in table 2.
4. Compressive strength
Mixing solid-phase powder of calcium phosphate cement with liquid-phase components, blending with a spoon for 30s to form uniform cement slurry, filling into a stainless steel cylinder mold with the diameter of 6mm and the height of 12mm, applying pressure of 0.7MPa for compaction, plugging the prepared cylinder sample into a glass tube with the diameter of 6.5mm and the height of 13mm, placing the cylinder sample into an environment with the temperature of 37 ℃ and the relative humidity of 100% for solidification for 3d, testing the average compression strength of the sample by a universal material testing machine, wherein the loading rate is 1mm/min, and the results are shown in Table 2.
5. Coagulation time
The setting time of the calcium phosphate cement was measured by means of a Gilromson according to the standard ASTM C191-13, and the results are shown in Table 2.
6. Degradability in vitro
Mixing solid-phase powder of calcium phosphate cement with liquid-phase components, blending for 30s by using a spoon to form uniform cement slurry, filling the uniform cement slurry into a stainless steel cylinder mould with the diameter of 6mm and the height of 12mm, applying pressure of 0.7MPa for compaction, plugging a prepared cylinder sample into a glass tube with the diameter of 6.5mm and the height of 13mm, then placing the cylinder sample into an environment with the temperature of 37 ℃ and the relative humidity of 100% for solidification for 3d, drying and weighing, soaking the cylinder sample into 30mL of PBS (phosphate buffer solution), placing a bone cement sample in a constant-temperature shaking table with the rotation speed of 120rpm and the temperature of 37 ℃ for 4 weeks, taking out and drying the bone cement, and calculating the mass loss rate of the bone cement sample, wherein the results are shown in Table 2.
TABLE 2
From the results of examples 1-4 and comparative examples 1-3, it can be seen that by adding gamma-glycidoxypropyltrimethoxysilane, bletilla polysaccharide, chitosan and sodium glycerophosphate to the liquid phase component, the prepared liquid phase component is mixed with the solid phase powder at 37 ℃, the liquid phase component in the bone cement is converted into hydrogel, and the chitosan is grafted with the gamma-glycidoxypropyltrimethoxysilane and interacts with the bletilla polysaccharide to form a multi-layer network structure, so that the collapsibility can be reduced, the collapsibility of examples 1-3 is significantly lower than that of comparative examples 1-3, the setting time can be shortened, and the mechanical properties of the bone cement can be improved.
7. Cell proliferation
The calcium phosphate cement samples of examples 1 to 4 were used as experimental groups, the samples of comparative examples 1 to 3 were used as control groups, the proliferation of cells (mouse mesenchymal stem cells, ATCC CRL-12424) was detected using CCK-8 kit, the samples were placed in 48-well plates, the number of cells was 1 × 104 cells/well, the solution was changed every other day, after culturing 1, 3, 7 days in a 37 ℃, 5% CO2 incubator, absorbance values at 450nm by microplate reader were obtained, and the results are shown in table 3;
TABLE 3 absorbance values
As shown by the results in Table 3, it can be seen that the absorbance values increased with the increase of the culture time, the absorbance values of examples 1 to 3 were higher than those of comparative examples 1 to 3, and the cell growth-promoting effects of examples 1 to 3 were superior to those of comparative examples 1 to 3.
Removing a 48-hole plate from an incubator, sucking out a culture medium, washing for 2 times by using PBS (phosphate buffer solution), adding 4% paraformaldehyde for fixing for 1h, sucking out the paraformaldehyde solution, performing gradient dehydration by using ethanol with multi-level concentration gradient, freezing the dehydrated material in a refrigerator at the temperature of-20 ℃ for 24h, performing surface gold spraying treatment after vacuum freeze-drying for 24h, and observing the adhesion growth condition of cells on bone cement by using a scanning electron microscope, wherein the cell morphology is circular and the cell morphology is normal in examples 1-3 and comparative examples 1-3. The kit of example 4 is expected to inhibit cells by inducing cytotoxicity such as partial cell death after 7 days of culture, and therefore, it is necessary to control the amount of sodium glycerophosphate since it is estimated that the concentration of sodium glycerophosphate is too high.
In conclusion, the gamma-glycidoxypropyltrimethoxysilane, the bletilla striata polysaccharide, the chitosan and the sodium glycerophosphate are added into the liquid phase component, the prepared liquid phase component is mixed with the solid phase powder at 37 ℃, the liquid phase component in the bone cement is converted into hydrogel, and the chitosan is grafted with the gamma-glycidoxypropyltrimethoxysilane and interacts with the bletilla striata polysaccharide to form a multilayer network structure, so that the collapsibility can be reduced, the coagulation time can be shortened, the mechanical property of the bone cement can be improved, the good biocompatibility can be realized, the growth of cells can be promoted, the using amount of the sodium glycerophosphate can be reduced, and the cytotoxicity can be reduced.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The temperature-sensitive calcium phosphate cement comprises solid-phase powder and liquid-phase components, and is characterized in that:
the solid phase powder is a calcium salt;
the liquid phase component is formed by mixing the following components: gamma-glycidoxypropyltrimethoxysilane, chitosan-acetic acid, sodium glycerophosphate and bletilla polyose.
2. The thermo-sensitive calcium phosphate cement according to claim 1, wherein:
the mass ratio of the solid-phase powder to the liquid-phase component is (1.5-2.7): 1.
3. The thermo-sensitive calcium phosphate cement according to claim 1, wherein:
the solid phase powder is selected from one or more of α -tricalcium phosphate, apatite, tetracalcium phosphate, and dicalcium phosphate.
4. The thermo-sensitive calcium phosphate cement according to claim 1, wherein:
the liquid phase component is formed by mixing the following components: 4-6 wt% of gamma-glycidyl ether oxypropyl trimethoxy silane aqueous solution; chitosan acetic acid aqueous solution with the mass fraction of 3-5 wt%; sodium glycerophosphate aqueous solution with the mass fraction of 40-60 wt%; 10-20 wt% of bletilla striata polysaccharide aqueous solution.
5. The thermo-sensitive calcium phosphate cement according to claim 1, wherein:
the mass ratio of the bletilla polysaccharide aqueous solution, the gamma-glycidyl ether oxygen propyl trimethoxy silane aqueous solution, the chitosan aqueous solution and the sodium glycerophosphate aqueous solution is (20-30): (40-60): (5-10).
6. The thermo-sensitive calcium phosphate cement according to claim 1, wherein:
the particle size of the solid phase powder is 1-200 mu m.
7. The thermo-sensitive calcium phosphate cement according to claim 1, wherein:
the bletilla striata polysaccharide is prepared by the following method:
freezing dried bletilla tuber at-20 deg.C for 2 hr, pulverizing to obtain bletilla powder, mixing with distilled water at a mass ratio of 1: 4-1: 8, stirring at the temperature of between 60 and 100 ℃ for 0.5 to 2 hours at the speed of between 300 and 700r/min, filtering, and collecting the bletilla striata polysaccharide filtrate.
8. The thermo-sensitive calcium phosphate cement according to claim 1, wherein:
the aqueous solution of chitosan acetic acid is prepared by the following method:
dissolving chitosan in 1 wt% acetic acid solution to prepare 4 wt% chitosan acetic acid aqueous solution, wherein the chitosan is chitosan powder with deacetylation degree of 85-100% and molecular weight of 70-80 ten thousand.
9. The thermo-sensitive calcium phosphate cement according to claim 1, wherein:
the liquid phase component is formed by mixing the following components: 5 wt% of gamma-glycidoxypropyltrimethoxysilane aqueous solution; a chitosan acetic acid aqueous solution with the mass fraction of 4 wt%; a sodium glycerophosphate aqueous solution with the mass fraction of 40 wt%; 15 wt% of bletilla striata polysaccharide aqueous solution;
wherein the mass ratio of the bletilla polysaccharide aqueous solution, the gamma-glycidyl ether oxygen propyl trimethoxy silane aqueous solution, the chitosan aqueous solution and the sodium glycerophosphate aqueous solution is 25: 50: 50: 8.
10. a process for the preparation of a temperature sensitive calcium phosphate cement according to any of claims 1-9, characterized in that a solid phase powder and a liquid phase component are obtained by the following process:
1) preparing 4-6 wt% of gamma-glycidoxypropyltrimethoxysilane aqueous solution;
preparing a sodium glycerophosphate aqueous solution with the mass fraction of 40-60 wt%;
preparing 10-20 wt% of bletilla striata polysaccharide aqueous solution:
freezing dried bletilla striata stem blocks at-20 ℃ for 2 hours, crushing and sieving by a 60-mesh sieve to obtain bletilla striata powder, mixing the powder with distilled water in a mass ratio of 1: 4-1: 8, stirring at the temperature of between 60 and 100 ℃ for 0.5 to 2 hours at the speed of between 300 and 700r/min, filtering, and collecting bletilla striata polysaccharide filtrate; continuing soaking and filtering the filtered bletilla striata powder for 4 times, combining and collecting bletilla striata polysaccharide filtrates, combining the bletilla striata polysaccharide filtrates collected in a grading manner, concentrating the filtrate by rotary evaporation, adding absolute ethyl alcohol into the bletilla striata polysaccharide concentrated solution, stirring, carrying out alcohol precipitation, filtering to obtain a precipitate, washing the precipitate obtained by filtering by using the absolute ethyl alcohol, carrying out vacuum freeze drying on the precipitate, adding deionized water into the dried bletilla striata polysaccharide to dissolve the dried bletilla striata polysaccharide, centrifuging, passing the supernatant through a 0.45-micrometer microporous filter membrane, carrying out ultrafiltration at 40 ℃ under 0.10MPa to obtain an ultrafiltrate with a filtering section ratio of 7:3, concentrating the ultrafiltrate to 1g/L, adding the absolute ethyl alcohol to carry out alcohol precipitation, carrying out vacuum freeze drying on the precipitate to obtain bletilla striata polysaccharide, dissolving the bletilla striata polysaccharide in an aqueous solution, and preparing a 10-20 wt% bletilla striata polysaccharide aqueous solution;
preparing 3-5 wt% chitosan acetic acid aqueous solution: dissolving chitosan in 1 wt% of acetic acid solution to prepare solution with the mass fraction of 3-5 wt%, wherein the chitosan is chitosan powder with the deacetylation degree of 90% and the molecular weight of 70-80 ten thousand;
2) the bletilla polysaccharide aqueous solution, the gamma-glycidyl ether oxygen propyl trimethoxy silane aqueous solution, the chitosan aqueous solution and the sodium glycerophosphate aqueous solution are mixed according to the ratio of (20-30) to (40-60): (5-10) mixing the components in a mass ratio to obtain a liquid phase component;
3) the solid phase powder is calcium salt selected from one or more of apatite, tetracalcium phosphate, and dicalcium phosphate.
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