CN114699561B - Calcium-doped material, bone repair material and preparation method thereof - Google Patents

Abstract

The invention provides a doped calcium-based material, which comprises a calcium-based material and Cu and Gd doped in the calcium-based material, wherein the molar ratio of the Cu to the Gd to Ca in the calcium-based material is 0.1-2. The invention also provides a preparation method of the doped calcium-based material and a bone repair material. According to the invention, cu and Gd are simultaneously added into the calcium-based material, wherein the Cu element has remarkable antibacterial property, anti-inflammatory property and immunoregulation property, and the doping of the Gd element is beneficial to improving the Magnetic Resonance Imaging (MRI) and CT imaging capability of the composite material, so that the nano particles and the composite material thereof have the imaging observation characteristic, and the observation of the material after bone implantation is facilitated. More importantly, the simultaneous doping of Cu and Gd can promote the adhesion of cells, is beneficial to the generation of calcium nodules of the cells and the osteogenic differentiation of the cells, and is further beneficial to bone repair.

Description

A calcium-doped material, bone repair material and preparation method thereof

technical field

The invention belongs to the technical field of materials, and in particular relates to a calcium-doped material, a bone repair material and a preparation method thereof.

Background technique

Inorganic substances such as hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and biphasic calcium phosphate (BCP) are widely used alone or as composite materials because of their good biocompatibility and osteogenic properties. Applied in the field of bone repair bracket. However, in terms of infectious bone injury, drug-resistant infectious bone injury, and immunomodulatory treatment of some complex bone injuries, these materials often show relatively single-function defects, and can only provide structural support and simple osteogenesis. .

The prior art discloses a variety of technologies for improving the performance of bone repair materials by modifying and doping hydroxyapatite, β-tricalcium phosphate, biphasic calcium phosphate, etc., for example, doping hydroxyapatite Rare earth ions to improve the mechanical strength and toughness of hydroxyapatite; or deposit nano-copper nano-zinc oxide in nano-hydroxyapatite to enhance its antibacterial activity, etc.

Contents of the invention

In view of this, the purpose of this application is to provide a calcium-doped material, a bone repair material and a preparation method thereof. The bone repair material provided by the present invention has the ability of immunity, antibacterial and imaging, and can promote cell adhesion at the same time. It is conducive to the generation of calcium nodules and osteogenic differentiation of cells, which is more conducive to bone repair.

The present invention provides a doped calcium-based material, comprising calcium-based material and Cu and Gd doped in the calcium-based material, wherein the molar ratio of Cu, Gd and Ca in the calcium-based material is 0.1-2 :0.1～2:6～9.8.

In the present invention, Cu and Gd are simultaneously added to the calcium-based material, wherein the Cu element has significant antibacterial properties, anti-inflammatory properties and immune regulation properties, and the doping of the Gd element helps to improve the nuclear magnetic (MRI) and CT imaging of the composite material The ability to make nanoparticles and their composite materials have imaging observation characteristics, which is conducive to the observation of materials after bone implantation. More importantly, the simultaneous doping of Cu and Gd can promote the adhesion of cells, which is beneficial to the generation of calcium nodules and osteogenic differentiation of cells, which is more conducive to bone repair.

In one embodiment, the calcium-based material is selected from one or more of hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) and biphasic calcium phosphate (BCP).

In one embodiment, the molar ratio of Cu, Gd and Ca in the calcium-based material is 0.2-1.5:0.2-1.5:7-9.6.

In one embodiment, the molar ratio of Cu, Gd and Ca in the calcium-based material is 0.5˜1:0.5˜1:8˜9.

Specifically, Cu and Gd partially replace the calcium atoms in the calcium-based material. When the calcium-based material is hydroxyapatite, the molecular formula of the doped material obtained is:

Ca _10-xy Cu _x Gd _y (PO ₄ ) ₆ (OH) ₂ ;

Among them, 0.2≤x≤1.5, 0.2≤y≤1.5.

When the calcium-based material is β-tricalcium phosphate, the molecular formula of the obtained doping material is:

Ca _1.5-x-1.5y Cu _x Gd _y (PO ₄ ) ₂ ;

Among them, 0.2≤x≤1.5, 0.2≤y≤1.5.

When the calcium-based material is a biphasic calcium phosphate or biphasic calcium phosphate, Cu and Gd still replace Ca.

The present invention also provides a preparation method for doped calcium-based materials, including:

Calcium salt, copper salt, gadolinium salt and (NH ₄ ) ₂ HPO ₃ are mixed and then reacted, and the obtained reaction product is calcined to obtain doped calcium-based nanomaterials;

Wherein, the molar ratio of Cu, Gd and Ca is 0.1-2:0.1-2:6-9.8.

The present invention prepares Cu and Gd-doped calcium-based materials by a hydrothermal method, mixes calcium salts, copper salts, gadolinium salts and (NH ₄ ) ₂ HPO ₃ and reacts, and calcines the obtained reaction products to obtain doped Calcium-based materials.

In one embodiment, the calcium salt is selected from one or more of calcium nitrate and calcium chloride.

In one embodiment, the copper salt is selected from one or more of copper nitrate and copper chloride.

In one embodiment, the gadolinium salt is selected from one or more of gadolinium nitrate and gadolinium chloride.

In one embodiment, the reaction is carried out in a water bath, the reaction temperature is 40-80°C, and the reaction time is 0.5-2h. In one embodiment, the pH of the reaction is 9-11.

After the reaction is completed, the reaction product is calcined to obtain a doped calcium-based material. In one embodiment, the temperature of the calcination is 180-200° C., and the time is 10-20 hours.

The doped calcium-based material provided by the invention is a nano particle, which can be used to prepare a bone repair material.

The present invention also provides a bone repair material, comprising the doped calcium-based material and the polymer material described in the above technical solution.

In one embodiment, the mass ratio of the doped calcium-based material to the polymer material is 5-25:75-95. In one embodiment, the mass ratio of the doped calcium-based material to the polymer material is 10-20:80-90.

In one embodiment, the polymer material is selected from one or more of polylactic acid, polylactide-glycolide (PLGA) or polycaprolactone.

In one embodiment, the bone repair material is prepared according to the following method:

mixing the doped calcium-based material with a first organic solvent to obtain a first mixed solution;

Dissolving the polymer material in a second solvent to obtain a second mixed solution;

After the first mixed liquid and the second mixed liquid are mixed, they are frozen in a mold, the frozen bone repair material is replaced with solvent in water, and the bone repair material with a specific shape is obtained after freeze-drying.

In one embodiment, the first organic solvent is acetone, and the second organic solvent is N-methylpyrrolidone (NMP).

In one embodiment, the freezing temperature is -20 to -80° C., and the freezing time is 6 to 24 hours.

In one embodiment, the solvent replacement specifically includes: soaking the frozen bone repair material in pre-cooled water, replacing the solvent and water for 3-5 days, and changing the water 2-3 times a day. After the replacement, The obtained bone repair material can be freeze-dried and shaped.

The bone repair material provided by the invention has the ability of immunity, antibacterial and imaging, and can promote the adhesion of cells, which is beneficial to the generation of calcium nodules of cells and the osteogenic differentiation of cells, thereby being more beneficial to bone repair.

Description of drawings

Fig. 1 is the SEM photograph of the nanoparticle that the embodiment of the present invention 1 prepares;

Fig. 2 obtains SEM photograph for the nanoparticle prepared by Comparative Example 1 of the present invention;

Fig. 3 obtains the SEM photo for the nanoparticles prepared by Comparative Example 2 of the present invention;

Fig. 4 obtains SEM photo for the nanoparticle prepared by Comparative Example 3 of the present invention;

Fig. 5 is the XRD diffraction pattern of the nanoparticle that the embodiment of the present invention 1 and comparative example prepare;

Fig. 6 is the ICP analysis result of the nanoparticle that the embodiment of the present invention 1 prepares;

Fig. 7 is the ICP analysis result of the nanoparticle that comparative example 1 of the present invention prepares;

Fig. 8 is the ICP analysis result of the nanoparticle that comparative example 2 of the present invention prepares;

Fig. 9 is the ICP analysis result of the nanoparticle that comparative example 3 of the present invention prepares;

Fig. 10 is the FTIR spectrum of the nanoparticles prepared by Example 2 and Comparative Example of the present invention;

Fig. 11 is the nuclear magnetic imaging photograph of the composite material stent provided by the embodiment of the present invention and comparative example;

Fig. 12 is the nuclear magnetic imaging photograph when the composite material stent adopted in Example 2 of the present application has different dosages;

Figure 13 is the dyed cell adhesion result of the composite material prepared in Example 1 of the present invention;

Fig. 14 is the dyed cell adhesion result of the composite material prepared in Comparative Example 1 of the present invention;

Fig. 15 is the dyed cell adhesion result of the composite material prepared in Comparative Example 2 of the present invention;

Fig. 16 is the dyed cell adhesion result of the composite material prepared in Comparative Example 3 of the present invention;

Fig. 17 is the staining result of the alizarin erythrocyte calcium nodule of the composite material prepared in Example 1 of the present invention;

Fig. 18 is the staining result of the alizarin erythrocyte calcium nodule of the composite material prepared in Comparative Example 1 of the present invention;

Fig. 19 is the staining result of the alizarin erythrocyte calcium nodule of the composite material prepared in comparative example 2 of the present invention;

Fig. 20 is the staining result of alizarin erythrocyte calcium nodules of the composite material prepared in Comparative Example 3 of the present invention.

Detailed ways

The present application further illustrates the doped calcium-based material, bone repair material and preparation method thereof through examples, however, it should be understood that these examples do not limit the present invention. Variations of the invention now known or further developed are considered to fall within the scope of the invention described herein and claimed below.

Example 1

Synthesis of Cu/Gd co-doped hydroxyapatite nanoparticles: Prepare 1M/L Ca(NO ₃ ) ₂ , CuCl ₂ , Gd(NO ₃ ) ₃ ˙H ₂ O solution. Adjust the molar ratio of Ca/Cu/Gd to 9:0.5:0.5, add the above solution to (NH ₄ ) ₂ HPO ₃ , stir in a water bath at 60°C, adjust the pH to 10, and continue the reaction for 1 hour. After the reaction, the reaction product was added into the reaction kettle, heated at 180° C. for 12 hours, naturally cooled to room temperature after the heating, washed, centrifuged and dried to obtain nanoparticles (0.5Cu-0.5Gd-HA).

Preparation of double-doped 0.5Cu-0.5Gd-HA and PLGA composite material (the mass ratio of 0.5Cu-0.5Gd-HA to PLGA is 10:90): Weigh Cu-Gd-HA, add it into acetone and mix it with ultrasonic ; Weigh PLGA, fully dissolve with N-methylpyrrolidone (NMP), mix the above two solutions, transfer the mixture into a syringe, and freeze at -80°C for 20h. Cut off the front section with needles, push the material into pre-cooled deionized water, replace the solvent and water for 3-5 days, and change the water 2-3 times a day. After the replacement of the scaffold, it can be molded after being taken out and freeze-dried to obtain a scaffold-like bone repair material.

Example 2

The difference from Example 1 is that the molar ratio of Ca/Cu/Gd is adjusted to be 8:1:1.

Comparative example 1

The difference from Example 1 is that Cu and Gd are not doped.

Comparative example 2

The difference from Example 1 is that the molar ratio of Ca/Cu is adjusted to 9.5:0.5, and Gd is not doped.

Comparative example 3

The difference from Example 1 is that the molar ratio of Ca/Gd is adjusted to 9.5:0.5, and Cu is not doped.

Comparative example 4

The difference from Example 2 is that the molar ratio of Ca/Cu is adjusted to 9:1, and Gd is not doped.

Comparative Example 5

The difference from Example 1 is that the molar ratio of Ca/Gd is adjusted to 9:1, and Cu is not doped.

The nanoparticles prepared in Example 1 and Comparative Examples 1 to 3 were characterized by SEM, the results are shown in Fig. 1, Fig. 2, Fig. 3 and Fig. 4, and Fig. 1 is a SEM photo of the nanoparticles prepared in Example 1 of the present invention, wherein, Fig. 1 (A) is the SEM photograph under the 500nm scale, and Fig. 1 (B) is the SEM photograph under the 200nm scale; Fig. 2 obtains the SEM photograph for the nanoparticle prepared by Comparative Example 1 of the present invention, wherein, Fig. 2 (A) is The SEM photograph under the 500nm scale, Fig. 2 (B) is the SEM photograph under the 200nm scale; Fig. 3 obtains the SEM photograph for the nanoparticles prepared in Comparative Example 2 of the present invention, wherein, Fig. 3 (A) is the SEM photograph under the 500nm scale , Fig. 3 (B) is the SEM photo under the 200nm scale; Fig. 4 obtains the SEM photo for the nanoparticles prepared in Comparative Example 3 of the present invention, wherein, Fig. 4 (A) is the SEM photo under the 500nm scale, Fig. 4 (B) It is the SEM picture under 200nm scale. From Figures 1 to 4, it can be seen that the prepared HA and Cu-HA, Gd-HA, and Cu-Gd-HA doped with Cu or/and Gd are all short rod-shaped structures, indicating that element doping does not change the nanoparticle microscopic morphology.

Carry out X-ray diffraction pattern analysis to the nanoparticles prepared in Example 1 and Comparative Examples 1 to 3, the results are shown in Fig. 5, Fig. 5 is the XRD diffraction pattern of the nanoparticles prepared in Example 1 and Comparative Example of the present invention, wherein, from the following The above are the XRD diffraction patterns of the nanoparticles prepared in Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 1 respectively. It can be seen from Figure 5 that HA has typical crystal characteristic peaks, and the positions of the three strong peaks are at 25.8°, 31.7°, and 32.9° respectively. The introduction of foreign ions in doped HA will cause the crystal lattice of HA to change, and the results shown in XRD are slight changes in peak position, peak width, and peak size.

For the analysis of the elemental composition of the nanoparticles prepared in Example 1 and Comparative Examples 1 to 3, the results are shown in Fig. 6, Fig. 7, Fig. 8, and Fig. 9. Fig. 6 is the ICP analysis result of the nanoparticles prepared in Example 1 of the present invention, wherein , the Ca content is 60.9%, the P content is 29.4%, the Gd content is 9%, and the Cu content is 0.7%. The P content is 29.4%, the Gd content is 0%, and the Cu content is 0%. content is 0%, Cu content is 1.8%; Fig. 9 is the ICP analysis result of the nanoparticle prepared in Comparative Example 3 of the present invention, wherein, Ca content is 60.2%, P content is 30.3%, Gd content is 9.5%, Cu content is 0%. It can be known from FIGS. 6 to 9 that the method provided by the present invention obtains hydroxyapatite nanoparticles doped with copper and gadolinium.

Carry out infrared analysis to the composite material of embodiment 2, comparative example 1, comparative example 4 and 5, see Fig. 10 for the result, Fig. 10 is the FTIR collection of illustrative plates of the nanoparticles prepared by embodiment 2 of the present invention and comparative example, wherein, from top to bottom The following are the FTIR spectra of the nanoparticles prepared in Comparative Example 1, Comparative Example 5, Comparative Example 4 and Example 2 respectively. It can be seen from Figure 10 that the sample has absorption peaks of different groups at different wavelengths, and the absorption peaks appear around 3445cm ^-1 and 1420cm ^-1 , corresponding to the stretching vibration and bending vibration of the -OH hydroxyl group, indicating the presence of -OH in the sample . The absorption peaks at 565 cm ^-1 and 607 cm ^-1 fit the bending vibration mode of OPO, proving the presence of phosphate groups in the material. And the strong absorption peak at 1038cm ^-1 originates from the PO antisymmetric stretching vibration mode. The position of these functional groups is consistent with the infrared absorption peak wavelength of HA reported in the literature, and there are no other miscellaneous peaks, indicating that the main groups in the product are hydroxyl and phosphate groups, which is consistent with the infrared absorption spectrum results of HA. There was no significant change after doping.

The composite material brackets prepared in Examples 1-2, Comparative Examples 1-3, and Comparative Example 5 were subjected to NMR imaging tests, and each group of bracket materials was put into a sample tube containing PBS, and the addition amount was 20%. 1.2T nuclear magnetic developing apparatus, take pictures, the result is referring to Fig. 11, and Fig. 11 is the nuclear magnetic developing photograph of the composite material support that the embodiment of the present invention and comparative example provide; 10%, 20% and 40% of the composite material support were added to the sample tube containing PBS, placed in a 1.2T nuclear magnetic imaging apparatus, and photographed, the results are shown in Figure 12, Figure 12 shows the different dosages of the composite material provided in Example 2 of the present application MRI photographs at the time. Figure 11 shows that doping 0.5Gd-HA and 1Gd-HA can significantly improve the nuclear magnetic MRI imaging ability of the material, and Figure 12 shows that when 5%, 10%, 20% and 40% of 1Cu-1Gd-HA with different percentages are added The imaging effect has obvious gradient difference, indicating that the prepared composite material has potential advantages in nuclear magnetic MRI imaging and continuous observation.

Example 3

First prepare the composite film for cell culture:

Take a certain amount of PLGA and be dissolved in chloroform, be mixed with a 10% (w/v) solution, after fully dissolving, the nanoparticles prepared in Examples 1～2 and Comparative Examples 1～5 are according to the nanoparticle: PLGA= The ratio of 10:90 is dispersed in the solution, magnetic stirring is supplemented by ultrasonic treatment. Take the cover glass that has been siliconized with dimethyldichlorosilane (DMDC), apply the HA/PLGA composite solution on the glass slide, place it on a cell culture plate, and dry it in vacuum to remove the solvent to form a HA/PLGA composite film, 75 Sterilize with % ethanol and wash with PBS for later use.

Mouse osteoblast precursor cells MC3T3-E1 were seeded on the composite film at a density of 2.0×10 ⁴ /mL, and cultured in a 5% CO ₂ incubator at 37°C. After culturing for 24 hours, wash with PBS three times; fix with 4% paraformaldehyde for 10 minutes, wash with PBS three times; stain with FITC (fluorescein isothiocyanate) for 10 minutes, and wash with PBS for three times. Fluorescence microscope observation and photographing. Refer to Figure 13, Figure 14, Figure 15 and Figure 16 for the results, Figure 13 is the stained cell adhesion result of the composite material prepared in Example 1 of the present invention; Figure 14 is the stained cell adhesion result of the composite material prepared in Comparative Example 1 of the present invention Results; Figure 15 is the stained cell adhesion result of the composite material prepared in Comparative Example 2 of the present invention; Figure 16 is the stained cell adhesion result of the composite material prepared in Comparative Example 3 of the present invention. It can be known from Figures 13-16 that the composite material prepared by the present invention can promote cell adhesion.

After the cells were cultured for 14 and 21 days, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 10 min, washed three times with PBS, and incubated with Alizarin Red (ARS) staining solution (0.1% ARS in Tris·HCl solution, pH=8.0) 30min, and then washed three times. Use a microscope to observe and take pictures. The results are shown in Figures 17 to 20, wherein Figure 17 shows the staining results of alizarin erythrocyte calcium nodules of the composite material prepared in Example 1 of the present invention, wherein Figure 17(A) is the staining result after 14 days, and Figure 17(B) It is the dyeing result in 21 days; Fig. 18 is the alizarin erythrocyte calcium nodule staining result of the composite material prepared in Comparative Example 1 of the present invention, wherein, Fig. 18 (A) is the staining result in 14 days, and Fig. 18 (B) is the staining result in 21 days Result; Fig. 19 is the alizarin erythrocyte calcium nodule staining result of the composite material prepared in comparative example 2 of the present invention, wherein, Fig. 19 (A) is the staining result in 14 days, and Fig. 19 (B) is the staining result in 21 days; Fig. 20 Alizarin erythrocyte calcium nodule staining results of the composite material prepared for Comparative Example 3 of the present invention, wherein, Fig. 20(A) is the staining result on day 14, and Fig. 20(B) is the staining result on day 21. It can be known from Figures 17 to 20 that doping elements are beneficial to the generation of calcium nodules in cells, that is, beneficial to mineralization, indicating that they are beneficial to osteogenic differentiation of cells.

The summary of the present invention only exemplifies some specific embodiments of the claims, wherein the technical features recorded in one or more technical solutions can be combined with any one or more technical solutions, and the technical solutions obtained by these combinations It is also within the scope of protection of the present application, just as these combined technical solutions have been specifically recorded in the disclosure content of the present invention.

Claims (10)

1. A doped calcium-based material comprises a calcium-based material and Cu and Gd doped in the calcium-based material, wherein the charging molar ratio of the Cu to the Gd to Ca in the calcium-based material is 0.1-2, and the charging molar ratio of the Cu to the Gd to Ca in the calcium-based material is 0.1-2:6-9.8.

2. The doped calcium-based material according to claim 1, wherein said calcium-based material is selected from one or more of hydroxyapatite, β -tricalcium phosphate and biphasic calcium phosphate.

3. The calcium-doped base material according to claim 2, wherein the molar ratio of Cu, gd, and Ca in the calcium-based material is 0.2 to 1.5.

4. The doped calcium-based material of claim 3, wherein the molar ratio of Cu, gd, and Ca in the calcium-based material is from 0.5 to 1.

5. A bone repair material comprising the doped calcium-based material of any one of claims 1 to 4 and a polymeric material.

6. The bone repair material according to claim 5, wherein the mass ratio of the doped calcium-based material to the polymer material is 5 to 25.

7. The bone repair material according to claim 6, wherein the polymeric material is selected from one or more of polylactic acid, polylactide-glycolide or polycaprolactone.

8. A method of preparing a doped calcium-based material, comprising:

mixing calcium salt, copper salt, gadolinium salt and (NH) ₄ ) ₂ HPO ₃ Mixing, reacting, and calcining the obtained reaction product to obtain the doped calcium-based nano material;

wherein the molar ratio of Cu, gd and Ca is 0.1-2:6-9.8.

9. The preparation method according to claim 8, wherein the reaction temperature is 40-80 ℃ and the reaction time is 0.5-2 h.

10. The preparation method according to claim 9, wherein the calcination is carried out at a temperature of 180 to 200 ℃ for 10 to 20 hours.

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