CN114832155B - Preparation and application of biomineralization-based hydroxyapatite with controllable hydrophilicity and hydrophobicity - Google Patents
Preparation and application of biomineralization-based hydroxyapatite with controllable hydrophilicity and hydrophobicity Download PDFInfo
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Abstract
The invention relates to the field of nano biological materials, and particularly discloses a preparation method of a hydroxyapatite nano material with controllable hydrophilicity and hydrophobicity based on a biomineralization method. The nano hydroxyapatite material prepared by the biomineralization method has a plate-shaped structure which is similar to the shape of a natural bone, uniformly covers the surface of a substrate, has good biocompatibility and has good application prospect in the field of bone tissue engineering. The hydroxyapatite is compounded with the polypeptide with different functions, so that the advantages of the hydroxyapatite and the functions of the peptide are combined, for example, the H12 peptide shown in the invention, and the possibility of antibiosis of the hydroxyapatite is given. The nano-sized hydroxyapatite material which is wrapped with functional peptides is synthesized in a bionic environment, so that the advantages of the hydroxyapatite are combined with the functions of the peptides, the physicochemical properties of the material are further changed by the influence of the polypeptide on the crystal structure of the nano-hydroxyapatite, and the nano-sized hydroxyapatite material has a good prospect in the field of bone tissue engineering.
Description
Technical Field
The invention relates to the technical field of nano biological materials, in particular to a preparation method of a hydroxyapatite nano material with controllable hydrophilicity and hydrophobicity based on a biomineralization method and potential application thereof in bone tissue engineering.
Background
The natural bone is the main mechanical support of the human skeletal system, is a natural composite material and shows a unique hierarchical structure from the molecular scale to the macroscopic scale. For a long time, diseases such as osteoporosis, osteoarthritis, osteomyelitis, myeloma and bone defects seriously threaten human health.
Among various bone materials, hydroxyapatite (HAP) is most closely related to natural bone components, has excellent osteoconductivity and good biocompatibility, and can promote osteogenesis, and thus has been widely studied and applied. Among many antibacterial coatings, the antibacterial peptide biological coating has the characteristics of wide antibacterial spectrum, small molecular weight, good thermal stability, broad-spectrum antibacterial activity on clinical drug-resistant bacteria, difficulty in inducing the bacteria to generate drug resistance and the like.
Disclosure of Invention
The invention selects an amino acid sequence MLPHHGA with the bone-promoting function at the C end of amelogenin 1 derived peptide (Human cemepin 1, CEMP1), and the amino acid sequence is named as C7 peptide; selecting an amino acid sequence GKCSTRGRKCCRRKK with an antibacterial function at the C-terminal of Human beta-defensin 3 (Human beta-defensin-3, hBD3) and naming the amino acid sequence as H12 peptide; on the basis, H12-C7 peptide is designed by combining C7 peptide with H12 peptide, the amino acid sequence of the H12-C7 peptide is GKCSTRGRKCCRRKKMLPHHGA, and three kinds of titanium can be combined on the surface of HAP with higher combination efficiency. Tortoise polypeptide bioengineering (Ningbo) Ltd prepared 15mg of C7 peptide, 15mg of H12 peptide and 15mg of H12-C7 peptide by solid phase synthesis, respectively.
The crystal structure of the HAP nano material is regulated and controlled from a molecular level by combining the HAP nano material with the functional polypeptide, so that the hydrophilicity and hydrophobicity of the material are changed, and the material has antibacterial capability.
Against the background above, the main contents of the present invention are as follows:
(1) Preparing supersaturated mineral solution with inorganic mineral salt;
(2) Synthesizing a compact flaky hydroxyapatite nano material attached to the surface of the glass by a biomineralization method;
(3) Design of peptide: 3 novel short peptides are designed based on the C-terminal mineralization function sequence of CEMP1 and the C-terminal antibacterial function sequence of hBD3, and are respectively named as C7 peptide (amino acid sequence: MLPHHGA), H12 peptide (amino acid sequence: GKCSTRGRKCCRRKK) and H12-C7 peptide (amino acid sequence: GKCSTRGRKCCRRKKMLPHHGA);
(4) The self-assembly immobilization of the peptide on the surface of the hydroxyapatite is researched, and the regulation and control effects of different polypeptides on the appearance and the crystal structure of the HAP crystal are researched;
(5) The original morphology and the crystal structure of HAP are changed based on the induction of the peptide, and the hydrophilicity and hydrophobicity of different peptide-doped HAP nano materials are further researched;
(5) Escherichia coli and staphylococcus aureus are used as bacterial experimental models, the in-vitro antibacterial effect of the synthesized hydroxylapatite nano material is explored, and the physicochemical property-dependent antibacterial capacity and related structure-effect relationship are confirmed.
The hydroxyapatite nano material with controllable hydrophilicity and hydrophobicity based on the biomineralization method is prepared in a bionic physical environment, and the shape, crystal structure and phase purity of the material are closer to those of calcium phosphate components in natural bones; has antibacterial property; has the application potential of guiding the bone regeneration and rehabilitation.
A preparation method of a hydroxyapatite nano material with controllable hydrophilicity and hydrophobicity based on a biomineralization method comprises the following steps:
1) Preparing supersaturated mineral solution with inorganic mineral salt, adjusting pH of the supersaturated mineral solution to 7.4 with hydrochloride buffer solution, and filtering;
2) Synthesizing a compact sheet hydroxyapatite nano material attached to the surface of the substrate by a biomineralization method;
3) The functional polypeptide is combined with the hydroxyapatite nano material surface in the step 2) in a self-assembly way, and the method comprises the following steps: uniformly spin-coating the functional polypeptide on the surface of the hydroxyapatite nano material, incubating, washing and drying to obtain the hydroxyapatite nano material with the surface combined with the functional polypeptide;
4) Placing the hydroxyapatite nano material with the surface combined with the functional polypeptide into the supersaturated mineral solution obtained in the step 1) for incubation to carry out mineralization reaction, separating the supernatant after the reaction, washing and drying to obtain the flake hydroxyapatite nano material loaded with the functional polypeptide.
Further, the preparation method of the supersaturated mineral solution in the step 1) comprises the following steps: firstly, preparing potassium dihydrogen phosphate, sodium chloride, potassium chloride and sodium bicarbonate into an aqueous solution, mixing to obtain a mixed solution, then dropwise adding the aqueous solution of calcium chloride dihydrate into the mixed solution by using an automatic pipettor, and continuously stirring and uniformly stirring by using a magnetic stirrer in the dropwise adding process to obtain a supersaturated mineral solution.
Further, the supersaturated mineral solution comprises: 2.5mM calcium chloride dihydrate, 1mM potassium dihydrogen phosphate, 141mM sodium chloride, 4mM potassium chloride and 4.2mM sodium bicarbonate.
Further, the filtering operation of step 1) is: carrying out primary filtration operation on the saturated mineral solution by using a microporous filter membrane with the pore diameter of 0.22-0.45 mu m; the hydrochloride buffer solution is TRIS hydrochloride buffer solution with the concentration of 0.5-3M.
Further, the step 2) of preparing the hydroxyapatite nano material by using a biomineralization method comprises the following steps: firstly, an ion cleaner is used for treating the surface of a substrate in a pure oxygen mode, and the preferable treatment conditions are that the oxygen flow is 0.5-3sccm and the treatment time is 1-10min; the hydroxyapatite has stronger adsorption force on hydroxyapatite precipitate; adding 1-3mL of mineralized liquid into each hole of a 24-hole culture plate, adding a processed round glass sheet with a proper size for precipitation growth, sealing the hole plate, placing in a constant-temperature incubator, and performing incubation culture;
further, the incubation condition in the step 2) is incubation for 18-48h at 37 ℃, and the drying temperature is room temperature; the substrate is a gold sheet, a glass sheet or a silicon wafer.
Further, the spin coating conditions in the step 3) are that the rotating speed is 200-1000rpm, and the spin coating time is 1-3min; the incubation time is 1-3H, the functional polypeptide is C7 peptide, H12 peptide and H12-C7 peptide, the concentration is 30-1000 mu g/mL, and the spin coating amount is 50 mu L.
Further, the incubation condition in the step 4) is incubation at 37 ℃ for 24h; the drying temperature is 20-37 ℃, and the drying time is 1-3h.
The verification method for the combination of the polypeptide in the step 4) on the surface of the hydroxyapatite is to modify a Fluorescein Isothiocyanate (FITC) label at the N-terminal of the amino acid sequence of the polypeptide, observe the fluorescence intensity of the surface of the material by using an inverted fluorescence microscope, and quantitatively analyze the fluorescence intensity of the surface of the hydroxyapatite by using ImageJ software so as to quantitatively analyze the combined peptide;
the invention provides a hydroxyapatite nano material with controllable hydrophilicity and hydrophobicity based on a biomineralization method, which is prepared by the preparation method.
The invention provides application of a hydroxyapatite nano material with controllable hydrophilicity and hydrophobicity based on a biomineralization method in an antibacterial and anti-infection material.
In addition, the invention also discloses a research on the antibacterial effect of the polypeptide-loaded hydroxyapatite nano material in an in vitro experiment, and the specific method comprises the following steps:
1) Ability of material to resist gram-negative bacteria adhesion: selecting escherichia coli as a gram-negative bacteria representative strain, inoculating quantitative escherichia coli into a sterile culture medium, putting the prepared material into the sterile culture medium, taking out the material after shaking culture for a period of time, placing the material into a sterile buffer solution, performing ultrasonic treatment for 5-10min to separate bacteria adhered to the surface of the material, performing quantitative analysis on the number of live bacteria adhered to the surface of the material by using a dilution plate counting method, and reflecting the capability of the material for inhibiting the bacteria from adhering by measuring the number of the reduced live bacteria; meanwhile, the antibacterial capacity of the material is calculated by using a calculation formula of the antibacterial rate;
2) Capacity of the material to resist adhesion of gram-positive bacteria: selecting staphylococcus aureus as a gram-positive bacteria representative strain, inoculating quantitative staphylococcus aureus into a sterile culture medium, putting the prepared material into the sterile culture medium, taking out the material after shaking culture for a period of time, placing the material into a sterile buffer solution, separating bacteria adhered to the surface of the material by ultrasonic treatment for 5-10min, quantitatively analyzing the number of the live bacteria adhered to the surface of the material by using a dilution plate counting method, and reflecting the capability of the material for inhibiting the bacteria from adhering by measuring the reduced number of the live bacteria; meanwhile, the antibacterial capacity of the material is calculated by using a calculation formula of the antibacterial rate;
for step 1) and step 2), it is recommended that the bacteria be inoculated at a density of 10 6 ～10 8 CFU/mL；
For step 1) and step 2), the co-culture time of the bacteria and the material is recommended to be 6-48h, and in order to ensure that the bacteria are uniformly contacted with the surface of the material, the reaction condition of the shaking culture is optimized to be 37 ℃ and 160rmp. The control group for experiment is glass surface with the same size or substrate made of other materials with the same size;
the invention has the advantages and positive effects that: the lamellar hydroxyapatite nano material similar to a natural bone structure and synthesized on the basis of a biomineralization method is synthesized into the nano hydroxyapatite material with the nano-size encapsulated functional peptide in a bionic environment by utilizing the self-assembly binding capacity of the micromolecule polypeptide with different functions and the hydroxyapatite, so that the advantages of the hydroxyapatite and the functions of the peptide are combined, the physicochemical properties of the material are further changed by the influence of the polypeptide on the crystal structure of the nano hydroxyapatite, and the lamellar hydroxyapatite nano material has a good prospect in the field of bone tissue engineering.
Drawings
FIG. 1 shows the analysis of the binding efficiency of three polypeptides to hydroxyapatite by relative fluorescence intensity quantification using an inverted fluorescence microscope using Fluorescein Isothiocyanate (FITC) to label energetic peptides.
FIG. 2 is a scanning electron microscope characterization result diagram of the peptide-loaded hydroxyapatite nano-material.
Fig. 3 is an X-ray diffraction characterization result of peptide-loaded hydroxyapatite nanomaterial.
FIG. 4 is the infrared spectrum characterization result of peptide-loaded hydroxyapatite nano-material.
FIG. 5 shows the results of a test of hydrophilicity and hydrophobicity of the surface of a material measured by a dye adsorption method using the hydrophilic dye Naerlan (NB). a) Linear relationship of NB partition to material surface area; b) Dye distribution method for determining adsorption relationship of different materials to NB (hydrophilicity of corresponding material)
Fig. 6 shows the results of the surface contact angle test of the synthesized hydroxyapatite Dan Na m material using deionized water and ethylene glycol as standard liquids.
FIG. 7 is a graph of the surface free energy results of materials calculated by the Owens and Wemit equations based on the contact angle data experimentally measured in FIG. 5.
FIG. 8 shows the results of experiments on the antibacterial ability of materials tested by co-culturing Escherichia coli and Staphylococcus aureus as representatives of gram-negative bacteria and gram-positive bacteria with the materials for a period of time.
Detailed Description
The present invention will be more fully understood by those of ordinary skill in the art in view of the following detailed description of the present invention, which is provided in connection with the examples, but the embodiments of the method of the present invention are not limited thereto.
It is to be noted that the method is also applicable to the combination of the hydroxyapatite nanomaterial and other functional peptides, and it is known to those skilled in the art that the present application only uses the functional peptides mentioned in the present invention, and the combination of the hydroxyapatite nanomaterial and other functional peptides belongs to many possible variations and modifications of the technical solution of the present invention, which should fall within the protection scope of the present invention.
Example 1
68mg of monopotassium phosphate, 4.124g of sodium chloride, 149mg of potassium chloride and 176.4 mg of sodium bicarbonate are accurately weighed and dissolved in 450mL of deionized water to prepare 450mL of aqueous solution, 183.75mg of calcium chloride dihydrate is dissolved in 50mL of deionized water to prepare 50mL of aqueous solution, an automatic pipettor is used for dropwise adding the calcium chloride solution into 450mL of mixed solution to avoid precipitation, a magnetic stirrer is used for continuous stirring in the dropwise adding process, and then Tris-HCl buffer solution with the concentration of 1M is used for regulating the pH value to 7.4. Table 1 shows the formulation of supersaturated mineral solutions according to the invention.
TABLE 1
Prior to the experiment, the glass sheet used as the substrate was previously subjected to Pure O using a Tereo plasma cleaner 2 The oxygen flow is 0.5-3sccm, and the treatment is carried out for 2min, so that the hydroxyapatite precipitate has stronger adsorption force; the synthesis of hydroxyapatite was subsequently carried out in 24-well culture plates: adding 1mL of mineralized liquid into each hole, adding the treated glass sheet for precipitation growth, placing the glass sheet in a constant temperature incubator for incubation at 37 ℃ for 24 hours, sucking the residual liquid in the holes, carefully taking out the glass sheet, washing the glass sheet with absolute ethyl alcohol to remove impurities attached to the upper side, and drying at room temperature to obtain hydroxyapatite attached to the glass sheet. Respectively spin-coating 30 microgram/mL 50 microliter C7 peptide, H12 peptide and H12-C7 peptide on the surface of hydroxyapatite under the conditions of rotation speed of 200-1000rpm, spin-coating time of 1-3min, incubation at 37 ℃ for 1-3H, washing and drying, then placing into mineralized liquid for culturing at 37 ℃ for 24H to continue precipitation reaction, separating the reacted supernatant, washing and drying at room temperature for 1-3H to respectively obtain hydroxyapatite nano materials loaded with different polypeptides, which are named HAP, HAP/C7/HAP, HAP/H12/HAP and HAP/H12-C7/HAP in sequence.
Example 2
The presence of functional peptide immobilized on hydroxyapatite was confirmed by experiment, and glass of the same size was used as a control group. The following table 2 shows the names, amino acid sequences, isoelectric points and main functions of the polypeptides designed by the present invention. Based on the C-terminal 12 amino acid sequence of the antimicrobial peptide (hBD 3) and the C-terminal 7 amino acid sequence of the bone peptide (CEMP 1), a peptide sequence having both antimicrobial property and HAP growth binding ability was designed.
TABLE 2
The functional peptide marked by Fluorescein Isothiocyanate (FITC) is adopted, the binding efficiency of the three polypeptides is compared through relative fluorescence intensity, and an experimental result proves that the hydroxyapatite is beneficial to the immobilization of the peptide on the surface of a substrate (the result is shown in figure 1). The hydroxyapatite nano material synthesized by the method is characterized, and Scanning Electron Microscopy (SEM) results show that the nano hydroxyapatite composite material with the plate-shaped appearance is obtained, and hydroxyapatite crystals induced to grow by different peptides all have a sheet-shaped structure (the results are shown in figure 2). The chemical components and the crystal structure of the synthesized composite nano hydroxyapatite material are analyzed by a characterization means, and the Fourier infrared spectrum result is shown in a 4,X ray diffraction spectrum result in a figure 3.
Example 3
The hydrophilicity and hydrophobicity of a material are key physicochemical properties that determine its state and application under various environmental and biological conditions. The hydrophilicity and hydrophobicity of the synthesized nano-hydroxyapatite encapsulating different polypeptides are determined by using a hydrophilic dye nai erlan (NB) reagent and adopting a dye adsorption method, and the experimental result is shown in figure 5. Degree of hydrophilicity comparison (hydrophilic → hydrophobic): HAP/H12-C7/HAP > HAP/H12/HAP > HAP/C7/HAP. The static contact angle of the surface of the material with water and glycol is measured by using a contact angle measuring instrument, and the surface free energy of the material is calculated by Owens and Wemit equations (the result is shown in figure 7), and the surface energy is compared in size (small → large): HAP/C7/HAP < HAP/H12/HAP < HAP/H12-C7/HAP; calculation of the surface energy of the binding material: the HAP materials induced by C7 peptide have the lowest surface energy and are relatively most hydrophobic.
Example 4
Referring to fig. 8, in the present example, escherichia coli and staphylococcus aureus were used as bacterial experimental models to verify the antibacterial ability of the synthesized material. The synthesized HAP, HAP/C7, HAP/H12-C7, HAP/C7/HAP, HAP/H12/HAP and HAP/H12-C7/HAP materials are co-cultured with escherichia coli and staphylococcus aureus respectively for a period of time, and then the number of the live bacteria adhered to the surface of the material is calculated by a dilution plate technology method, and the antibacterial rate is calculated. Experimental results show that the combination of the H12 peptide on the surface of the nano-hydroxyapatite and the combination of the H12-C7 peptide on the surface of the material have good antibacterial effect, and the antibacterial reason of the nano-hydroxyapatite is probably the bactericidal effect of the peptide; when the functional peptide is fixed and then mineralized for the second time, the synthesized HAP/H12/HAP and HAP/H12-C7/HAP materials have higher antibacterial rate, which indicates that the synthesized materials have the function of inhibiting the adhesion of gram-positive bacteria and gram-negative bacteria. The influence of the polypeptide on the crystal structure of the nano-hydroxyapatite further changes the physicochemical properties of the material, particularly reflects the change of the surface energy of the material, and further enables the material to have the function of inhibiting the adhesion of bacteria.
It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
Claims (10)
1. A preparation method of a hydroxyapatite nano material with controllable hydrophilicity and hydrophobicity based on a biomineralization method is characterized by comprising the following steps: the method comprises the following steps:
1) Preparing supersaturated mineral solution with inorganic mineral salt, adjusting pH of the supersaturated mineral solution to 7.4 with hydrochloride buffer solution, and filtering;
2) Synthesizing a compact sheet hydroxyapatite nano material attached to the surface of the substrate by a biomineralization method;
3) The functional polypeptide is combined with the hydroxyapatite nano material surface in the step 2) in a self-assembly way, and the method comprises the following steps: uniformly spin-coating the aqueous solution of the functional polypeptide on the surface of the hydroxyapatite nano material, incubating, washing and drying to obtain the hydroxyapatite nano material with the surface combined with the functional polypeptide;
4) Placing the hydroxyapatite nano material with the surface combined with the functional polypeptide into the supersaturated mineral solution obtained in the step 1) for incubation to carry out mineralization reaction, separating the supernatant after the reaction, washing and drying to obtain a flaky hydroxyapatite nano material loaded with the functional polypeptide;
the functional polypeptide is C7 peptide, H12-C7 peptide, the C7 peptide is selected from an amino acid sequence MLPHHGA of enamel protein 1 derived peptide (Human cemepin 1, CEMP1) with bone promoting function at the C end; the H12 peptide selects an amino acid sequence GKCSTRGRKCCRRKK with an antibacterial function at the C end of Human beta-defensin 3 (Human beta-defensin-3, hBD3); the H12-C7 peptide is a functional polypeptide designed by combining C7 peptide with H12 peptide, and the amino acid sequence of the functional polypeptide is GKCSTRGRKCCRRKKMLPHHGA;
the hydroxyapatite nano material can realize the regulation and control of the hydrophilicity and the hydrophobicity of the HAP nano material by combining the functional polypeptide.
2. The method of claim 1, wherein: the preparation method of the supersaturated mineral solution in the step 1) comprises the following steps: firstly, preparing potassium dihydrogen phosphate, sodium chloride, potassium chloride and sodium bicarbonate into an aqueous solution, mixing to obtain a mixed solution, then dropwise adding the aqueous solution of calcium chloride dihydrate into the mixed solution by using an automatic pipettor, continuously stirring by using a magnetic stirrer in the dropwise adding process, and uniformly stirring to obtain a supersaturated mineral solution.
3. The production method according to claim 2, characterized in that: the supersaturated mineral solution comprises the following components: 2.5 Calcium chloride dihydrate, 1mM potassium dihydrogen phosphate, 141mM sodium chloride, 4mM potassium chloride and 4.2mM sodium bicarbonate in mM.
4. The method of claim 1, wherein: the filtering operation of the step 1) is as follows: carrying out primary filtration operation on the saturated mineral solution by using a microporous filter membrane with the aperture of 0.22-0.45 mu m; the hydrochloride buffer solution is a Tris-HCl buffer solution with the concentration of 1M.
5. The production method according to claim 1, characterized in that: the step 2) of preparing the hydroxyapatite nano material by a biomineralization method comprises the following steps: firstly, treating the surface of a substrate by using an ion cleaner in a pure oxygen mode to ensure that the substrate has stronger adsorption force on hydroxyapatite precipitate; the treated substrate is then added to a supersaturated mineral solution, incubated, washed and dried.
6. The production method according to claim 5, characterized in that: the incubation condition in the step 2) is incubation for 18-48h at 37 ℃, and the drying temperature is room temperature; the substrate is a gold sheet, a glass sheet or a silicon wafer.
7. The method of claim 1, wherein: the spin coating conditions in the step 3) are that the rotating speed is 200-1000rpm, and the spin coating time is 1-3min; the incubation time is 1-3h, the concentration of the functional polypeptide is 30-1000 mu g/mL, and the spin coating amount is 50 mu L.
8. The method of claim 1, wherein: the incubation condition in the step 4) is to culture 24h at 37 ℃; the drying temperature is 20-37 ℃, and the drying time is 1-3h.
9. The hydroxyapatite nano material with controllable hydrophilicity and hydrophobicity based on the biomineralization method, which is prepared by the preparation method of any one of claims 1 to 8.
10. The use of the hydroxyapatite nano material with controllable hydrophilicity and hydrophobicity based on the biomineralization method of claim 9 in the antibacterial and anti-infection material.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101214394A (en) * | 2008-01-10 | 2008-07-09 | 中国人民解放军第三军医大学第三附属医院 | Bone plate with sustained-release antimicrobial peptide |
| CN102241738A (en) * | 2011-06-03 | 2011-11-16 | 清华大学 | Mimic polypeptide of non-collagen protein and application of the mimic peptide in biomimetic mineralization |
| CN104817629A (en) * | 2015-05-05 | 2015-08-05 | 华中科技大学同济医学院附属同济医院 | Sequence of anti-bacterial peptide capable of being specially combined with hydroxylapatite crystal on tooth surface and application thereof |
| CN113321710A (en) * | 2021-05-27 | 2021-08-31 | 吉林大学 | Polypeptide with function of promoting enamel structural mineralization and regeneration |
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| KR101979932B1 (en) * | 2017-08-18 | 2019-05-21 | 서울대학교산학협력단 | Peptide for Inhibiting Bone Resorption |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101214394A (en) * | 2008-01-10 | 2008-07-09 | 中国人民解放军第三军医大学第三附属医院 | Bone plate with sustained-release antimicrobial peptide |
| CN102241738A (en) * | 2011-06-03 | 2011-11-16 | 清华大学 | Mimic polypeptide of non-collagen protein and application of the mimic peptide in biomimetic mineralization |
| CN104817629A (en) * | 2015-05-05 | 2015-08-05 | 华中科技大学同济医学院附属同济医院 | Sequence of anti-bacterial peptide capable of being specially combined with hydroxylapatite crystal on tooth surface and application thereof |
| CN113321710A (en) * | 2021-05-27 | 2021-08-31 | 吉林大学 | Polypeptide with function of promoting enamel structural mineralization and regeneration |
Non-Patent Citations (2)
| Title |
|---|
| Regulation of in vitro Calcium Phosphate Mineralization by Combinatorially Selected Hydroxyapatite-Binding Peptides;Mustafa Gungormus，et al.;《Biomacromolecules》;20080214;第9卷;第966–973页 * |
| Self-assembling antimicrobial peptides on nanotubular titanium surfaces coated with calcium phosphate for local therapy;Hilal Yazici，et al.;《Materials Science and Engineering: C》;20190101;第94卷;第333-343页 * |
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