CN114699561B

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

Info

Publication number: CN114699561B
Application number: CN202111659702.7A
Authority: CN
Inventors: 郭跃明; 王宗良; 章培标; 刘香笈
Original assignee: FOSHAN HOSPITAL OF TCM; Changchun Institute of Applied Chemistry of CAS
Current assignee: FOSHAN HOSPITAL OF TCM; Changchun Institute of Applied Chemistry of CAS
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-03-31
Anticipated expiration: 2041-12-30
Also published as: CN114699561A

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

Calcium-doped material, bone repair material and preparation method thereof

Technical Field

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

Background

Inorganic substances such as Hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP), biphase Calcium Phosphate (BCP) and the like are independently or as composite materials to be widely applied to the field of bone repair scaffolds due to good biocompatibility and osteogenic property. However, in the field of immune-modulated treatment of infectious bone lesions, drug-resistant infectious bone lesions, and some complex bone lesions, these materials often exhibit the drawback of relatively single function, being only capable of structural support and simple osteogenesis.

The prior art discloses various techniques for improving the performance of bone repair materials by modifying and doping hydroxyapatite, beta-tricalcium phosphate, biphasic calcium phosphate and the like, for example, rare earth ions are doped in the hydroxyapatite to improve the mechanical strength and toughness of the hydroxyapatite; or the nano-copper nano-zinc oxide is deposited in the nano-hydroxyapatite to enhance the antibacterial activity and the like.

Disclosure of Invention

In view of the above, the present application aims to provide a calcium-doped material, a bone repair material and a preparation method thereof, and the bone repair material provided by the present invention has immune antibacterial and imaging capabilities, can promote cell adhesion, and is favorable for cell calcium nodule generation and cell osteogenic differentiation, so as to be more favorable for bone repair.

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.

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 nuclear 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 the 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.

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 to 1.5.

In one embodiment, the molar ratio of Cu, gd, and Ca in the calcium-based material is 0.5 to 1.

Specifically, cu and Gd partially replace calcium atoms in the calcium-based material, and when the calcium-based material is hydroxyapatite, the molecular formula of the obtained doped material is as follows:

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

wherein x is more than or equal to 0.2 and less than or equal to 1.5,0.2 and less than or equal to 1.5.

When the calcium-based material is beta-tricalcium phosphate, the molecular formula of the obtained doped material is as follows:

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

When the calcium-based material is a biphasic calcium phosphate or a biphasic calcium phosphate, cu and Gd remain as Ca substituted therein.

The invention also provides a preparation method of the doped calcium-based material, which comprises the following steps:

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

The invention prepares Cu and Gd-doped calcium-based material by a hydrothermal method, and calcium salt, copper salt, gadolinium salt and (NH) ₄ ) ₂ HPO ₃ And mixing, reacting, and calcining the obtained reaction product to obtain the doped calcium-based material.

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, copper chloride.

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

In one embodiment, the reaction is carried out in a water bath at a temperature of 40-80 ℃ for 0.5-2 h. In one embodiment, the reaction has a pH of 9 to 11.

And after the reaction is finished, calcining the reaction product to obtain the doped calcium-based material. In one embodiment, the calcination temperature is 180-200 ℃ and the time is 10-20 h.

The doped calcium-based material provided by the invention is a nano particle and can be used for preparing a bone repair material.

The invention also provides a bone repair material, which comprises the calcium-doped material and the high polymer material in the technical scheme.

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

In one embodiment the polymeric material is selected from one or more of polylactic acid, polylactide-co-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 a high polymer material in a second solvent to obtain a second mixed solution;

and mixing the first mixed solution and the second mixed solution, freezing in a mold, performing solvent replacement on the frozen bone repair material in water, and performing freeze drying to obtain the bone repair material with a specific shape.

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 ℃, and the freezing time is 6 to 24 hours.

In one embodiment, the solvent displacement is specifically: and (3) soaking the frozen bone repair material in pre-cooled water, replacing the solvent with the water for 3-5 days, changing the water 2-3 times every day, and after replacement is finished, freezing, drying and molding the obtained bone repair material.

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

Drawings

FIG. 1 is an SEM photograph of nanoparticles prepared in example 1 of the present invention;

FIG. 2 is an SEM photograph of nanoparticles prepared in comparative example 1 of the present invention;

FIG. 3 is an SEM photograph of nanoparticles prepared in comparative example 2 of the present invention;

FIG. 4 is an SEM photograph of nanoparticles prepared in comparative example 3 of the present invention;

FIG. 5 is an XRD diffraction pattern of nanoparticles prepared in example 1 of the present invention and comparative example;

FIG. 6 shows the ICP analysis of nanoparticles prepared in example 1 of the present invention;

FIG. 7 is an ICP analysis result of nanoparticles prepared in comparative example 1 of the present invention;

FIG. 8 is the ICP analysis result of nanoparticles prepared in comparative example 2 of the present invention;

FIG. 9 shows the result of ICP analysis of nanoparticles prepared in comparative example 3 of the present invention;

FIG. 10 is an FTIR spectrum of nanoparticles prepared in example 2 of the present invention and comparative example;

FIG. 11 is a nuclear magnetic image of the composite material scaffolds provided by the examples and comparative examples of the present invention;

FIG. 12 is a photograph of nuclear magnetic images of composite stents passed through in example 2 of the present application at different dosages;

FIG. 13 shows the stained cell adhesion results of the composite material prepared in example 1 of the present invention;

FIG. 14 shows stained cell adhesion results for composites prepared in comparative example 1 of the present invention;

FIG. 15 is a result of stained cell adhesion of the composite material prepared in comparative example 2 of the present invention;

FIG. 16 is a result of stained cell adhesion of the composite material prepared in comparative example 3 of the present invention;

FIG. 17 shows the results of staining alizarin red cell calcium nodules of the composite material prepared in example 1 of the present invention;

FIG. 18 shows the results of staining alizarin red cell calcium nodules of the composite material prepared in comparative example 1 of the present invention;

FIG. 19 shows the results of staining alizarin red cell calcium nodules of the composite material prepared in comparative example 2 of the present invention;

FIG. 20 shows the results of staining alizarin red cell calcium nodules of the composite material prepared in comparative example 3 of the present invention.

Detailed Description

The doped calcium-based material, bone repair material and methods of making the same are further illustrated by the examples herein, however, it is to be understood that these examples are not limiting of the invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and claimed below.

Example 1

Synthesizing Cu/Gd-codoped hydroxyapatite nanoparticles: ca (NO) at 1M/L ₃ ) ₂ 、CuCl ₂ 、Gd(NO ₃ ) ₃ ˙H ₂ And (4) O liquid. Adjusting the molar ratio of Ca/Cu/Gd to be 9.5 ₄ ) ₂ HPO ₃ In a 60 ℃ water bath, stirring was performed, pH =10 was adjusted, and the reaction was continued for 1 hour. And after the reaction is finished, adding the reaction product into a reaction kettle, heating at 180 ℃ for 12 hours, naturally cooling to room temperature after the heating is finished, washing, centrifuging and drying to obtain the nano particles (0.5 Cu-0.5 Gd-HA).

Preparing a composite material of double-doped 0.5Cu-0.5Gd-HA and PLGA (the mass ratio of 0.5Cu-0.5Gd-HA to PLGA is 10: weighing Cu-Gd-HA, adding into acetone, and ultrasonically stirring and uniformly mixing; weighing PLGA, fully dissolving with N-methylpyrrolidone (NMP), mixing the two solutions, transferring the mixed solution into a syringe, and freezing at-80 ℃ for 20h. Cutting off the needle part at the front section, injecting the material into pre-cooled deionized water, and replacing solvent and water for 3-5 days with water for 2-3 times per day. And after the replacement of the scaffold is finished, taking out the scaffold, and forming after freeze drying to obtain the scaffold-like bone repair material.

Example 2

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

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 Ca/Cu molar ratio was adjusted to 9.5.

Comparative example 3

The difference from example 1 is that the Ca/Gd molar ratio was adjusted to 9.5, cu was not doped.

Comparative example 4

The difference from example 2 is that the Ca/Cu molar ratio was adjusted to 9:1 without doping with Gd.

Comparative example 5

The difference from example 1 is that the Ca/Gd molar ratio was adjusted to 9:1, without doping with Cu.

SEM characterization was performed on the nanoparticles prepared in example 1 and comparative examples 1 to 3, and the results are shown in fig. 1, fig. 2, fig. 3 and fig. 4, wherein fig. 1 is an SEM photograph of the nanoparticles prepared in example 1 of the present invention, wherein fig. 1 (a) is an SEM photograph on a 500nm scale, and fig. 1 (B) is an SEM photograph on a 200nm scale; FIG. 2 is an SEM photograph of nanoparticles prepared in comparative example 1 of the present invention, wherein FIG. 2 (A) is an SEM photograph on a scale of 500nm and FIG. 2 (B) is an SEM photograph on a scale of 200 nm; FIG. 3 is an SEM photograph of nanoparticles prepared in comparative example 2 of the present invention, wherein FIG. 3 (A) is an SEM photograph on a scale of 500nm and FIG. 3 (B) is an SEM photograph on a scale of 200 nm; FIG. 4 is an SEM photograph of nanoparticles prepared in comparative example 3 of the present invention, wherein FIG. 4 (A) is an SEM photograph on a scale of 500nm, and FIG. 4 (B) is an SEM photograph on a scale of 200 nm. As can be seen from FIGS. 1 to 4, the prepared HA and Cu-HA, gd-HA and Cu-Gd-HA doped with Cu or/and Gd are all short rod-shaped structures, which indicates that element doping does not change the micro morphology of the nanoparticles.

The nanoparticles prepared in example 1 and comparative examples 1 to 3 were subjected to X-ray diffraction pattern analysis, and as a result, referring to fig. 5, fig. 5 is an XRD diffraction pattern of the nanoparticles prepared in example 1 and comparative example of the present invention, wherein, XRD diffraction patterns of the nanoparticles prepared in comparative example 1, comparative example 2, comparative example 3 and example 1 are from bottom to top, respectively. As can be seen from FIG. 5, HA HAs typical characteristic crystal peaks, and the positions of the three strong peaks are at three positions of 25.8 °, 31.7 ° and 32.9 °, respectively. The doped HA is introduced with foreign ions, so that the crystal lattice of the HA is transformed, and the result of XRD is slight changes of peak position, peak width, peak size and the like.

The results of the elemental composition analysis of the nanoparticles prepared in example 1 and comparative examples 1 to 3 are shown in fig. 6, 7, 8 and 9, and fig. 6 is the ICP analysis result of the nanoparticles prepared in example 1 of the present invention, in which the Ca content is 60.9%, the P content is 29.4%, the Gd content is 9% and the Cu content is 0.7%; FIG. 7 is an ICP analysis result of nanoparticles prepared in comparative example 1 of the present invention, in which the Ca content is 70.6%, the P content is 29.4%, the Gd content is 0%, and the Cu content is 0%; FIG. 8 is an ICP analysis result of nanoparticles prepared in comparative example 2 of the present invention, in which the Ca content was 67.1%, the P content was 31.1%, the Gd content was 0%, and the Cu content was 1.8%; fig. 9 shows ICP analysis results of nanoparticles prepared in comparative example 3 of the present invention, in which the Ca content was 60.2%, the P content was 30.3%, the Gd content was 9.5%, and the Cu content was 0%. As can be seen from fig. 6 to 9, the hydroxyapatite nanoparticles doped with copper and gadolinium are obtained by the method provided by the present invention.

The infrared analysis of the composite materials of example 2, comparative example 1, comparative example 4 and 5 is performed, and as a result, referring to fig. 10, fig. 10 is a FTIR spectrum of the nanoparticles prepared in example 2 and comparative example of the present invention, wherein the FTIR spectra of the nanoparticles prepared in comparative example 1, comparative example 5, comparative example 4 and example 2 are respectively from top to bottom. As can be seen from FIG. 10, the sample shows absorption peaks at 3445cm for different groups at different wavelengths ^-1 And 1420cm ^-1 An absorption peak corresponding to stretching vibration and bending vibration of the-OH hydroxyl group appears in the vicinity, indicating the presence of-OH in the sample. At 565cm ^-1 And 607cm ^-1 The absorption peak at (A) is consistent with the bending vibration mode of O-P-O, and the existence of phosphate radicals in the material is proved. And at 1038cm ^-1 The strong absorption peak occurs due to the P-O antisymmetric stretching vibration mode. The positions of the functional groups are consistent with the infrared absorption peak wavelength of HA reported in the literature, and no other miscellaneous peaks exist, which indicates that the main groups in the product are hydroxyl groups and phosphate groups, and the infrared absorption spectrum result of HA is met. There was no significant change after doping.

Performing Magnetic Resonance Imaging (MRI) imaging test on the composite material scaffolds prepared in the examples 1-2, the comparative examples 1-3 and the comparative example 5, putting each group of scaffold materials into a sample tube containing PBS (phosphate buffer solution), adding the materials into the sample tube with the addition of 20%, putting the sample tube into a 1.2T nuclear magnetic imaging instrument, and taking a picture, wherein the result is shown in figure 11, and figure 11 is a nuclear magnetic imaging picture of the composite material scaffolds provided by the examples and the comparative examples of the invention; the composite material scaffolds prepared in example 2 and having the contents of 1Cu-1Gd-HA of 5%, 10%, 20% and 40% respectively were added into a sample tube containing PBS, and placed into a 1.2T nuclear magnetic imaging apparatus, and photographs were taken, and as a result, see fig. 12, and fig. 12 are nuclear magnetic imaging photographs of the composite material provided in example 2 of the present application at different dosages. Fig. 11 shows that the nuclear Magnetic Resonance Imaging (MRI) capability of the material can be obviously improved by doping 0.5Gd-HA and 1Gd-HA, and fig. 12 shows that the imaging effect HAs obvious gradient difference when 5%, 10%, 20% and 40% of 1Cu-1Gd-HA with different percentage contents are added, which shows that the prepared composite material HAs potential advantages in the aspects of nuclear Magnetic Resonance Imaging (MRI) and continuous observation.

Example 3

Firstly, preparing a composite material film for cell culture:

weighing a certain amount of PLGA, dissolving the PLGA in chloroform to prepare a solution of 10% (w/v), and after fully dissolving, mixing the nanoparticles prepared in the examples 1-2 and the comparative examples 1-5 according to the following ratio: PLGA =10:90 in proportion, and magnetically stirring with ultrasonic treatment. Taking a cover glass which is siliconized by dimethyldichlorosilane (DMDC), coating the HA/PLGA composite material solution on the glass, placing the glass on a cell culture plate, drying in vacuum to remove the solvent to form an HA/PLGA composite material film, disinfecting with 75% ethanol, and washing with PBS for later use.

Mouse osteoblast precursor cell MC3T3-E1 at 2.0X 10 ⁴ Density of/mL on composite film, 5% CO at 37% ₂ And (5) incubator culture. After 24h of culture, PBS was washed three times; fixing 4% paraformaldehyde for 10min, and washing with PBS for three times; FITC (fluorescein isothiocyanate) staining for 10min, PBS washing three times. And (5) observing and photographing by using a fluorescence microscope. Results referring to fig. 13, 14, 15 and 16, fig. 13 is a result of stained cell adhesion of the composite material prepared in example 1 of the present invention; FIG. 14 is a result of stained cell adhesion of the composite material prepared in comparative example 1 of the present invention; FIG. 15 is the bookStained cell adhesion results for the composite material prepared in inventive comparative example 2; FIG. 16 is a result of stained cell adhesion of the composite material prepared in comparative example 3 of the present invention. As can be seen from fig. 13 to 16, the composite material prepared according to the present invention can promote cell adhesion.

Cells were cultured for 14 and 21 days, washed three times with PBS, fixed 10min with 4% paraformaldehyde at room temperature, washed three times with PBS, incubated for 30min with Alizarin Red (ARS) stain (0.1% ARS in Tris HCl, pH = 8.0), and washed three more times. And observing and photographing by using a microscope. The results are shown in fig. 17 to 20, wherein fig. 17 is a result of staining alizarin red cell calcium nodules of the composite material prepared in example 1 of the present invention, wherein fig. 17 (a) is a result of staining for 14 days, and fig. 17 (B) is a result of staining for 21 days; FIG. 18 is a graph showing the results of staining alizarin red blood cell calcium nodules of the composite material prepared in comparative example 1 of the present invention, wherein FIG. 18 (A) is the result of staining for 14 days, and FIG. 18 (B) is the result of staining for 21 days; FIG. 19 is a graph showing the results of staining alizarin red blood cell calcium nodules of the composite material prepared in comparative example 2 of the present invention, wherein FIG. 19 (A) is the result of staining for 14 days, and FIG. 19 (B) is the result of staining for 21 days; fig. 20 is a result of staining of alizarin red blood cell calcium nodule by the composite material prepared in comparative example 3 of the present invention, wherein fig. 20 (a) is a result of staining for 14 days and fig. 20 (B) is a result of staining for 21 days. As can be seen from fig. 17 to 20, the doping element favors the generation of calcium nodules in the cells, i.e., mineralization, indicating that it favors the osteogenic differentiation of the cells.

This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the claims, and such combined features are also within the scope of the present disclosure as if they were specifically recited therein.

Claims

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:

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.