CN114949342A - Composite monetite/amorphous calcium phosphate bone cement for promoting bone regeneration - Google Patents

Abstract

The invention provides a composite triclinite/amorphous calcium phosphate bone cement for promoting bone regeneration. In the present invention, triclinite and amorphous calcium phosphate powder are mixed according to different mass ratios, and then deionized water is added to prepare composite triclinite/amorphous calcium phosphate bone cement, and the optimal triclinite/amorphous calcium phosphate bone cement is explored. Proportion of shaped calcium phosphate composite bone cement. The composite triclinite/amorphous calcium phosphate bone cement of the present invention has the advantages of short setting time, no exothermic reaction and slow degradation speed, can promote the proliferation, migration and osteogenesis of bone marrow mesenchymal stem cells, and is not conducive to osteoclasts The proliferation, migration and osteoclast of osteoclasts can inhibit the effect of osteoclasts on bone and CPC resorption in the early stage of bone defect to a certain extent. The injectability, setting time and mechanical properties of the triclinite/amorphous calcium phosphate composite bone cement of the present invention all meet the clinical application requirements.

Description

A composite triclinite/amorphous calcium phosphate bone cement for promoting bone regeneration

technical field

The invention belongs to the technical field of bone cement, and relates to a composite triclinite/amorphous calcium phosphate bone cement and a preparation method thereof.

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Large-scale bone defects caused by bone diseases such as trauma, tumors, and inflammation are often critical-sized bone defects, which cannot be repaired solely by the regeneration ability of bone tissue. Since the extraction of autologous bone will bring additional damage to the patient, and the xenogeneic bone can easily cause immune rejection, the application of artificial bone repair materials is increasing. Calcium phosphate materials such as hydroxyapatite and tricalcium phosphate have been widely studied and applied in orthopedics. Compared with the above calcium phosphate materials, although monetite (MC) has excellent self-curing ability and biological properties, the synthesis process is not stable enough, and the research on its biological properties is not deep enough. Triclinic worfite exhibits a continuous degradation profile and exhibits sustained in vivo absorption behavior. This offers the possibility to form new bone tissue by implant resorption without affecting the mechanical stability of the implant. Synthetic bone repair materials based on triclinite have been implanted into animal models of bone defects in different parts of the body, and these studies confirmed the osteoconductive and even osteoinductive potential of triclinite. The research on amorphous calcium phosphate (ACP) is relatively mature. Due to the amorphous and unstable nature of amorphous calcium phosphate, it has higher degradability and richer microscopic appearance compared to other calcium phosphate-based materials. Amorphous calcium phosphate crystallizes spontaneously in the presence of water to form calcium phosphate-based materials (usually hydroxyapatite nanoparticles) that closely resemble the inorganic components of human bone. At the same time, the clustered, non-rigid structure of amorphous calcium phosphate can accommodate a variety of foreign ions, which are more numerous and species than other calcium phosphate-based material lattices. Amorphous calcium phosphate is unstable, easy to transform under humid conditions, and degrades rapidly, which is its disadvantage.

As mentioned above, both triclinite and amorphous calcium phosphate have their own advantages, and their weaknesses can complement each other, but there is no research on the combination of triclinite and amorphous calcium phosphate as a new calcium phosphate bone cement, And the effect of triclinite/amorphous calcium phosphate bone cement on the proliferation, migration and osteogenic differentiation of mouse bone marrow mesenchymal stem cells and the proliferation, migration and osteoclast differentiation of RAW 264.7 cells have not been studied.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the present invention provides a new type of calcium phosphate bone cement, that is, composite triclinite/amorphous calcium phosphate bone cement, and finds the most suitable triclinite/amorphous calcium phosphate bone cement for practical application. Composition of calcium phosphate composite bone cement. In the present invention, the triclinite and amorphous calcium phosphate powder are mixed in different proportions, and then deionized water is added to prepare the composite triclinite/amorphous calcium phosphate bone cement. The composite triclinite/amorphous calcium phosphate bone cement of the invention has the advantages of short setting time, no exothermic reaction and slow degradation speed, can promote the proliferation, migration and osteogenesis of mouse bone marrow mesenchymal stem cells, and is not conducive to the destruction of bone marrow. The proliferation, migration and osteoclast of osteocytes can inhibit the effect of osteoclasts on bone and calcium phosphate bone cement absorption in the early stage of bone defect to a certain extent. The injectability, setting time and mechanical properties of the triclinite/amorphous calcium phosphate composite bone cement of the present invention all meet the clinical application requirements.

Specifically, the implementation technical scheme of the present invention is as follows:

In the first aspect of the present invention, a composite triclinite/amorphous calcium phosphate bone cement is proposed. The components of the composite triclinite/amorphous calcium phosphate bone cement include triclinite and amorphous calcium phosphate. Further, the mass fraction of the triclinic wornetite is 25-75%, preferably 75%.

The second aspect of the present invention proposes the preparation method of the above-mentioned composite triclinic fendrite/amorphous calcium phosphate bone cement, comprising the following steps:

(1) Weighing triclinic folite and amorphous calcium phosphate powder according to a certain mass ratio;

(2) Mixing the above-mentioned powders uniformly by a non-intrusive homogenizer;

(3) Mix the uniformly mixed powder with deionized water according to a certain solid-liquid ratio.

Further, the amorphous calcium phosphate powder was synthesized by a co-precipitation method using CaCl ₂ and (NH 4 ) ₂ HPO ₄ .

Further, amorphous calcium phosphate powder was synthesized by co-precipitation method using CaCl ₂ and (NH 4 ) ₂ HPO ₄ . Among them, the molar ratio of calcium to phosphorus is 1.5:1. The CaCl ₂ solution and the (NH 4 ) ₂ HPO ₄ solution were mixed with magnetic stirring at 37° C. for 10 min in a collector magnetic stirrer to obtain a white emulsion. The emulsion was filtered, followed by the addition of a certain amount of water and a large amount of ethanol. The resulting white solid was air-dried at room temperature.

Further, the triclinic wornetite is synthesized by hydrothermal method using Ca(NO ₃ ) ₂ ·4H ₂ O and (NH ₄ ) ₂ HPO ₄ .

Furthermore, the triclinite nanofibers were synthesized by a two-step hydrothermal method of Ca(NO ₃ ) ₂ ·4H ₂ O and (NH ₄ ) ₂ HPO ₄ . The molar ratio of calcium to phosphorus is 1:1. The Ca(NO ₃ ) ₂ .4H ₂ O solution and the (NH ₄ ) ₂ HPO ₄ solution were mixed by fast mechanical stirring for 5 min to obtain a white emulsion. The emulsion was put into a reaction kettle lined with polytetrafluoroethylene and reacted at 90°C for 18h. After the first reaction was completed, it was heated twice at 60 °C for 24 h to obtain a suspension. The suspension was filtered, followed by the addition of appropriate amounts of water and ethanol. The resulting white solid was dried in an oven at 65°C.

Further, in step (2), the mixing conditions are: the rotation speed is 800r/min, the time is 60s, and the number of times is 6-9 times.

Further, the solid-liquid ratio is 0.6-1.0, preferably the solid-liquid ratio is 0.5.

The third aspect of the present invention proposes the application method of the above-mentioned composite triclinic folite/amorphous calcium phosphate bone cement, comprising the following steps:

(1) injecting the composite triclinite/amorphous calcium phosphate bone cement into a cylindrical mold with a needle to obtain a bone cement block;

(2) then placing the block in a constant temperature incubator to maintain the bone cement block;

(3) The cured bone cement block is taken out and dried for later use.

Further, the size of the cylindrical mold is 10mm×10mm.

Further, the curing temperature was 37° C., the curing humidity was 100%, and the curing time was 72 h.

Generally, polymethyl methacrylate bone cement sets very quickly, but after injection, there is a large amount of exothermic reaction in the body, which will damage the surrounding tissue. After cooling, the bone cement block will shrink, and it will not fit closely with the surrounding bone. The triclinite/amorphous calcium phosphate composite bone cement, as a new type of calcium phosphate bone cement, has a more suitable setting time for clinical applications, and has no exothermic reaction. The bone cement block degrades slowly, and it fits better with the surrounding bone. Tighter, more conducive to bone growth.

Ordinary calcium phosphate self-curing properties are generally poor, β-tricalcium phosphate will not self-curing, α-tricalcium phosphate self-curing performance is worse than triclinic calcium phosphate, while amorphous calcium phosphate and triclinic calcium phosphate are phosphoric acid. Calcium has the strongest self-curing performance and the highest injectability, and the triclinic calcium phosphate/amorphous calcium phosphate composite bone cement has better performance than the former two, indicating that their injectability and self-curing performance are better than ordinary phosphoric acid. Calcium bone cement is better. Moreover, the triclinite/amorphous calcium phosphate composite bone cement has a shorter setting time than the bone cement in the prior art, and is more suitable for use in clinical operations. At the same time, the degradation characteristics of triclinite/amorphous calcium phosphate composite bone cement are more conducive to bone regeneration after repairing bone defects.

One or more embodiments of the present invention have the following beneficial effects:

The setting time of the triclinite/amorphous calcium phosphate composite bone cement of the present invention is more suitable for clinical application, and the porosity is lower than that of the commonly used calcium phosphate bone cements such as amorphous calcium phosphate and tricalcium phosphate, which makes its compressive strength higher. Strong, its in vitro degradation is also sustainable and slow degradation, and biological experiments have also proved that no matter from cell proliferation, cell migration, or the effect of this composite bone cement on osteogenic and osteoclastic activities, triclinite/amorphous Compared with other calcium phosphate bone cements, calcium phosphate composite bone cements, especially triclinic calcium phosphate 75, have better mechanical and degradable properties, especially excellent biological properties, so they can be used as existing calcium phosphates. A better alternative to bone cement. The injectability, setting time and mechanical properties of the triclinite/amorphous calcium phosphate composite bone cement of the present invention all meet the clinical application requirements.

Description of drawings

The accompanying drawings that form a part of the present application are used to provide further understanding of the present application, and the schematic embodiments and descriptions of the present application are used to explain the present application and do not constitute improper limitations on the present application.

Figure 1 shows the XRD composition analysis and SEM morphology of triclinite/amorphous calcium phosphate composite bone cement.

Figure 2 is a graph showing the test results of the initial setting time, final setting time, injectability and compressive strength of the triclinite/amorphous calcium phosphate composite bone cement.

Fig. 3 is the XRD result and porosity diagram of the biodegradation of triclinite/amorphous calcium phosphate composite bone cement.

Figure 4 shows the changes in surface morphology and degradation of triclinite/amorphous calcium phosphate composite bone cement soaked in SBF.

Figures 5 and 6 show the effects of triclinite/amorphous calcium phosphate composite bone cement on the adhesion, proliferation, migration and osteogenic differentiation of mouse bone marrow mesenchymal stem cells

Figures 7 and 8 show the effects of triclinite/amorphous calcium phosphate composite bone cement on the adhesion, proliferation, migration and osteoclast differentiation of mouse RAW264.7 cells

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1

Preparation of triclinite/amorphous calcium phosphate composite bone cement

The calcium phosphate composite bone cement is prepared by using triclinite and amorphous calcium phosphate powder as raw materials, and the specific steps are as follows:

(1) Amorphous calcium phosphate powder was synthesized by a coprecipitation method using CaCl ₂ and (NH 4 ) ₂ HPO ₄ . Among them, the molar ratio of calcium to phosphorus is 1.5:1. The CaCl ₂ solution and the (NH 4 ) ₂ HPO ₄ solution were mixed with magnetic stirring at 37° C. for 10 min in a collector magnetic stirrer to obtain a white emulsion. The emulsion was filtered, followed by the addition of a certain amount of water and a large amount of ethanol. The resulting white solid was air-dried at room temperature.

(2) Triclinic worfite nanofibers were synthesized by a two-step hydrothermal method of Ca(NO ₃ ) ₂ ·4H ₂ O and (NH ₄ ) ₂ HPO ₄ . The molar ratio of calcium to phosphorus is 1:1. The Ca(NO ₃ ) ₂ .4H ₂ O solution and the (NH ₄ ) ₂ HPO ₄ solution were mixed by fast mechanical stirring for 5 min to obtain a white emulsion. The emulsion was put into a reaction kettle lined with polytetrafluoroethylene and reacted at 90°C for 18h. After the first reaction was completed, it was heated twice at 60 °C for 24 h to obtain a suspension. The suspension was filtered, followed by the addition of appropriate amounts of water and ethanol. The resulting white solid was dried in an oven at 65°C.

(3) According to different mass ratios (the mass fraction of amorphous calcium phosphate is 0%, 25%, 50%, 75%, 100%), weigh triclinite and amorphous calcium phosphate powder to prepare the composite bone The cements are named triclinic 100 (containing 0% amorphous calcium phosphate) (MC100), triclinic 75 (containing 25% amorphous calcium phosphate) (MC75), triclinic 50 (containing 25% amorphous calcium phosphate) (MC75) 50% amorphous calcium phosphate) (MC50), triclinic calcium phosphate 25 (containing 75% amorphous calcium phosphate) (MC25), amorphous calcium phosphate (containing 100% amorphous calcium phosphate) (ACP);

(4) Mix the above-mentioned powder uniformly by a non-intrusive homogenizer, and the mixing conditions are: the rotating speed is 800r/min, the time is 60s, and the number of times is 6-9 times;

(5) Mix the uniformly mixed bone cement powder with deionized water according to a certain solid-liquid ratio, and inject it into a 10mm×10mm cylindrical mold with a needle to obtain a bone cement block;

(6) The block is then placed in a constant temperature incubator, and the bone cement block is cured under the conditions of a temperature of 37°C and a humidity of 100%, and the curing time is 72h;

(7) The cured bone cement block is taken out and dried for later use.

Phase Characterization Methods

The present invention uses X-ray diffractometer (X-ray Diffraction, XRD, DMAX-2500PC) to analyze the phase composition of the sample. Test conditions: The samples were fully ground, sieved, and dried before the test. CuKα (λ=0.1542nm) was used as the radiation source, the acceleration voltage was 45kv, the current was 150mA, and the scanning speed was 10°/min.

The samples were analyzed by Fourier Transform Infrared Spectroscopy (FTIR, Nicolet is 50). Test conditions: The sample should be fully ground, sieved, and dried before the test. The ratio of the sample and potassium bromide is 1:100. The two are fully mixed and pressed into a transparent sheet, which is put into the machine for testing.

A thermogravimetric analyzer (Thermogravimetric Analysis, TG, HTC-4) was used to analyze the weight change of the sample during the heating process to study the phase change of the sample. Test conditions: the heating interval is 20-1000°C, the heating rate is 10°C/min, and the atmosphere is air.

Topographic Characterization Methods

In the present invention, a scanning electron microscope (Scanning Electron Microscope, SEM, SU-70) is used to observe the surface morphology of the sample. Test conditions: The sample should be fully dried before the test, the powder should be fully ground, pasted on the sample table with conductive adhesive, sprayed with gold to solve its conductivity problem, and then observed, the luminescent single crystal is tungsten single crystal, and the test voltage is 15kV.

The nanoscale morphology of the samples was observed by transmission electron microscope (Transmission Electron Microscope, TEM, Talos F200S). Test conditions: before the sample is tested, it needs to be ultrasonicated in absolute ethanol for 1h. After the dispersion is complete, the supernatant is sucked and dropped on the copper mesh. After it is completely dried, it is moved to a transmission electron microscope for observation.

Powder performance characterization method

The present invention utilizes a nano-particle size analyzer (Nano-particle Analyzer, ZS90) to measure the particle size and particle size distribution of the powder. Test conditions: The samples should be fully ground and sieved before the test. According to the solid-liquid ratio of 1:5000, the samples should be dispersed in absolute ethanol, fully ultrasonically shaken, centrifuged, and 1-2ml of the supernatant should be drawn for testing.

The specific surface area, pore diameter and pore volume of the sample powder were tested by the specific surface area and micropore analyzer (BSD-PM1). Test conditions: Before the test, the sample needs to be fully ground and sieved, dried at 60°C for 24h, and 0.1-0.2g of the sample is weighed for testing.

Mechanical Properties Characterization Method

The present invention mainly measures the compressive strength of the sample to characterize its mechanical properties. The compressive strength of the samples was tested by a universal testing machine (CMT5105). Test conditions: The sample needs to be smoothed before the test, and the aspect ratio is ≥2. At the same time, measure the actual stress area of the sample, and calculate its compressive strength according to formula (1):

In the formula, σ represents the compressive strength, F represents the maximum load value, and S represents the actual stress area.

The present invention utilizes the Archimedes drainage method to measure the porosity of the sample, and the specific steps are as follows:

(1) Put the sample into a vacuum drying oven, dry it at 60°C for 24 hours, weigh its weight, and record it as m ₁ ;

(2) Put the sample into a beaker containing deionized water, use an oil bath to heat it to boiling, keep it boiling for 1 hour, quickly put it into another beaker containing deionized water, and weigh its content in water. weight, recorded as m ₂ ;

(3) Take out the sample, gently wipe off its surface moisture, weigh its weight, and record it as m ₃ ;

(4) Finally, calculate the porosity of the sample according to formula (2).

In the formula, P is the porosity of the sample, and m1, m2, and m3 are given.

Bone cement performance characterization method

The invention utilizes the Gilmore double-needle method to measure the initial setting time and final setting time of calcium phosphate bone cement with different liquid phase contents. The initial setting time T1 of bone cement was measured by light needle (m=113.4±0.5g), and the final setting time Tf of bone cement was measured by heavy needle (m=453.6±0.5g). The test makes the needle tip at a certain height, and then falls freely perpendicular to the surface of the sample. When the needle tip cannot leave obvious traces on the surface of the sample, record the initial setting time and final setting time of the sample.

cell culture

Primary bone marrow mesenchymal stem cells (mBMMSCs) of C57 mice were purchased from Stem Cell Company, and were passaged and cultured in DMEM/F12 complete medium containing 10% FBS and 1% penicillin/streptomycin dual antibody. The cells were cultured in a 37°C, 5% CO2 incubator, and the cells were passaged every three days, and the medium was changed every other day.

The primary macrophage-like cell line (RAW264.7) of C57 mice was purchased from the cell company. This cell line can differentiate into osteoclasts, which are the precursor cells of osteoclasts. Passage and culture in DMEM high glucose complete medium/streptomycin double antibody. The cells were cultured in a 37°C, 5% CO2 incubator, and the cells were passaged every two days.

Cell morphology

The samples used for co-culture with mouse bone marrow mesenchymal stem cells were first soaked in simulated body fluids in vitro for 14 days, and the samples used for co-culture with RAW264.7 cells did not need to be soaked. In the well plate, mouse bone marrow mesenchymal stem cells RAW264.7 were digested, centrifuged, resuspended, and cell counted, and then seeded at a density of 1×10 ⁴ cells/mL, and the medium was changed every other day. After 7 days of cell seeding, the samples were transferred to a new 24-well plate, and the surface of the samples was rinsed with PBS for 3 minutes each time, for a total of 3 times. Sample surface cells were fixed with 4% paraformaldehyde at 4°C for 25 minutes. It was then dehydrated with 30%, 45%, 60%, 75%, 90% and 100% ethanol in sequence for 10 minutes at room temperature. Finally, cells and samples were dried with hexamethyldisilane (HMDS), gold sprayed on the samples under vacuum, and cell morphology (Hitachi, Japan) was observed under SEM (Hitachi, Japan).

Cell proliferation assay

The proliferation levels of mouse bone marrow mesenchymal stem cells and RAW264.7 were determined using CCK-8 reagent. The samples of each group after ultraviolet disinfection were placed in a 24-well plate, and a blank control group without samples was set. After the cells were digested, centrifuged, resuspended, and counted, the cells were seeded at a density of 1×10 ⁴ cells/mL, and the medium was changed every other day. 1, 4, and 7 days after cell seeding, aspirate all the medium in each well, add 500 μL of fresh complete medium, and add 50 μL of CCK-8 solution to each sample well, mix well, and place at 37 Incubate for 1.5 hours in a 5% CO2 incubator. Then, 100 μL of the mixture was aspirated from each sample well and placed in a 96-well plate. Three replicate controls were set in each sample well, and the absorbance (OD) at 450 nm was measured using a microplate reader (Thermo Labsysterms, USA). The whole experiment was carried out in a dark environment.

Apoptosis detection

After culturing the cells for 7 days in the 6-well plate in which the samples were placed, the cells in the medium and the adherent cells were collected. Cell apoptosis was detected by annexin V-FITC/PI apoptosis detection kit (Vazyme, Nanjing, China). The collected cells were washed with PBS and centrifuged twice at 1000 rpm, 5 min, 4°C. 100 μL of 1× binding buffer was then added to each tube to resuspend the cells. Additionally, 5 μL of V-FITC and 5 μL of PI were added to each tube, followed by incubation at room temperature for 10 minutes in the dark. Finally, 400 μL of 1× binding buffer was added to each tube prior to assay. Detection was performed with a flow cytometer (cytoflex, USA).

Immunofluorescence staining detection

The osteogenic differentiation performance of mouse bone marrow mesenchymal stem cells and the osteoclastic differentiation performance of RAW264.7 were evaluated by immunofluorescence method. RAW 264.7 cells and 5 groups of samples were cultured for 7 days under the induction of nuclear factor-κB ligand receptor activator (RANKL), and mouse bone marrow mesenchymal stem cells and 5 groups of samples were cultured for 14 days under the induction of osteogenic induction medium . Fixed with 4% paraformaldehyde at 4°C for 20 minutes, and permeabilized with 0.2% Triton X-100 at room temperature for 20 minutes. Samples were blocked in goat serum for 30 minutes at room temperature. Subsequently, the cells were incubated with 2 μg·mL-1 of OCN primary antibody (1:100) at 4°C overnight. The next day, soaked in FITC-labeled goat anti-rabbit secondary antibody solution for 1 hour, then stained with TRITC-labeled phalloidin for 20 minutes at room temperature, washed 3 times with PBS, and then stained with DAPI (0.5 μg·ml- 1) Stain for 20 minutes and wash three times with PBS. Finally, it was observed under a high-speed spinning disk confocal microscope (Dragonfly 200, USA).

Transwell cell migration ability assay

Cells were re-digested, centrifuged, and suspended in serum-free medium, and seeded at 8 x 103 cells per well into the upper chamber of a Transwell chamber containing an 8 μm pore filter. A medium containing 10% FBS was added to the lower chamber, and each group of samples was placed at the bottom. After 24 hours of culture, cells on the upper surface of the filter were washed with PBS and wiped with cotton swabs, and cells on the lower surface were fixed with methanol and stained with 0.5% crystal violet. Cell migration was observed under a light microscope (Lecia, Germany) and pictures were taken at high power. Cell migration capacity was estimated by the average number of cells per high power field.

Cell gene expression assay

In order to detect the effects of the components and micro-nano composite structures of different groups of samples on the expression levels of osteogenic differentiation-related genes and RAW264.7 osteoclastic differentiation-related genes of mouse bone marrow mesenchymal stem cells, real-time fluorescence quantitative polymerase chain reaction ( RT-qPCR) to detect the expression of Ctsk and c-Fos in COL-1, Runx-2, OCN and RAW264.7 in mouse bone marrow mesenchymal stem cells. RAW 264.7 cells and 5 groups of samples were cultured under RANKL induction for 7 days, and mouse bone marrow mesenchymal stem cells and 5 groups of samples were cultured under the induction of osteogenic induction medium for 14 days. After 7 days of culture, firstly, the total RNA was extracted from the cells using the RNA rapid extraction kit. After the concentration was determined, the liquid corresponding to 1 μg RNA was quantitatively drawn from the RNA extracted from each group of samples, and then the ReverTra Ace qPCR RT kit was used. (TOYOBO, Japan) RNA was reverse transcribed into cDNA, and finally, SYBR Green Realtime PCR Master Mix (TOYOBO, Japan) was used as the dye, and the PCR reaction and real-time monitoring were carried out in a rapid real-time PCR reaction system (Biorad, USA), and read fetch data. The obtained results were processed and analyzed by the relative quantitative comparison method (2-ΔΔCT). The sequences of primers used are shown in the table.

Primer sequences used in Table 1

Cellular protein expression detection

RAW264.7 cells and mouse bone marrow mesenchymal stem cells were inoculated on the surface of 5 groups of samples, respectively, and the cell proteins were extracted after adding inducer for 7 days. First, a lysate of mixed RIPA and PMSF was prepared, added to the cells, and then lysed on ice for 30 min. Cell lysates were collected into EP tubes and centrifuged at 12,000 rpm for 20 min at 4 °C. Afterwards, protein concentration was measured using a BCA kit (Beyotime, Shanghai, China), and an equal amount of protein was mixed with loading buffer. Proteins were transferred to polyvinylidene fluoride membranes (PVDF membranes, Millipore, Germany) by electrophoresis and membrane transfer. The PVDF membrane was then blocked with Quick block solution (Beyotime, Shanghai, China) for 15 minutes. After that, PVDF membranes were incubated with RUNX2 and Ctsk primary antibodies overnight at 4 °C, respectively, followed by secondary antibodies for 1 h at room temperature. Finally, protein levels were visualized by chemiluminescent HRP substrate (Millipore, Germany) and analyzed by ImageJ. The gray value of the target protein corresponding to each group and the internal reference GAPDH were measured by ImageJ, three times. The ratio of the gray value of the target protein to the gray value of GAPDH is the relative expression level of the target protein in this group.

data analysis

At least three independent replicates were set for each variable in all experiments, and experimental data were expressed as mean ± standard deviation (SD). Each group of Transwell took 5 high-magnification fields and counted the cells. For the cell proliferation detection, 3 absorbance values were taken for each group. RT-qPCR took 3 Ct values for each group. One-way analysis of variance was used, using GraphPad Prism 9 software. Analyze the differences. The significance level of statistical analysis was set as *P<0.05, and the difference was considered very significant when **P<0.01.

Characterization of triclinite/amorphous calcium phosphate cement

After hydration of calcium phosphate bone cement, its composition and morphology will change greatly due to its self-curing properties. Therefore, the present invention explores the composition and morphology of the bone cement block samples before and after hydration respectively. Figure 1A shows the XRD compositional analysis of triclinite/amorphous calcium phosphate bone cement blocks with different raw material powder ratios before hydration (Figure 1A(a)) and after hydration (Figure 1A(b)). The XRD patterns of pure triclinite and triclinite 75 before and after hydration have little change, and triclinite 50 and triclinite 25 also have slight changes. However, the XRD patterns of amorphous calcium phosphate before and after hydration changed significantly, characterized by the presence of broad crystalline peaks after hydration.

Figure 1B shows the surface morphologies of the bone cement block before and after hydration observed by SEM, in which (a) and (b) represent the surface and cross-sectional morphologies before hydration, and (c) and (d) represent the morphologies after hydration. Surface and cross-sectional topography. The surface morphology and cross-sectional morphology of each group changed before and after hydration. Long fibers appeared on the surface of triclinite 100, triclinite 75 and triclinite 25 after hydration, and the long fibers of triclinite 25 were connected into sheets. The surface of triclinite 50 is mostly short rod-like fibers. The cross-section of triclinite 100 before hydration is distributed as nanorods, while the nanorods after hydration transform into rods and flakes. The cross section of the amorphous calcium phosphate before hydration is in the form of nanoparticles, and after hydration, the nanoparticles are bound together to form a relatively dense internal morphology, but compared with the other four groups, both the surface and the cross section are relatively loose. Before hydration, triclinite 25, triclinite 50, and triclinite 75 are mixed forms of nanorods and nanoparticles. With the increase of amorphous calcium phosphate, the nanorods gradually decrease. , the nanoparticles gradually increased. Similarly, with the increase of amorphous calcium phosphate, the cross-sections of triclinite 75, triclinite 50 and triclinite 25 after hydration become denser and denser, among which triclinite 25 The section is the smoothest and densest of the 5 groups.

Figures 2A, B, and C show the initial and final setting times of triclinite 100, triclinite 50, and amorphous calcium phosphate at different liquid-to-solid ratios. When the liquid-solid ratio is 0.8, 0.9, and 1, the average initial setting time of triclinic calcium phosphate 100 is 15min, 11min, and 9min, which are all greater than 8min, and the average final setting time is 35min, 27min, and 21min, which are all greater than 15min. When the liquid-solid ratio reaches 0.7, the initial setting time of triclinic 100 bone cement is 6 minutes, and the final setting time is 15 minutes, which can meet the practical application requirements. When the liquid-solid ratio drops to 0.5 and 0.6, the initial setting time of bone cement is less than 3min, and the final setting time is less than 15min, and the setting is too fast at this time.

Figure 2D shows the injectability of triclinite 100, triclinite 50 and amorphous calcium phosphate. From the overall trend, under different liquid-solid ratios, amorphous calcium phosphate has the best injectability, followed by triclinite 50, and triclinite 100 has the worst injectability. When the liquid-solid ratio is 0.7, the injectability of triclinite 50 is comparable to that of amorphous calcium phosphate.

Figure 2E shows the test results of the compressive strength of apatite-based bone cement with different addition amounts of amorphous calcium phosphate before (a) and after (b) hydration. The compressive strengths before hydration of triclinite 100, triclinite 75, triclinite 50, triclinite 25, and amorphous calcium phosphate are 1.08MPa, 1.10MPa, 1.01MPa, 1.00MPa and 0.77MPa. The compressive strengths after hydration of triclinite 100, triclinite 75, triclinite 50, triclinite 25 and amorphous calcium phosphate are 0.8MPa, 1.29MPa, 1.4MPa, 1.67MPa and 1.19MPa.

The porosity of different liquid-solid ratios and different groups is shown in Fig. 3B. Compared with different liquid-solid ratios, as the liquid-solid ratio increases from 0.6 to 0.8, the porosity of the samples increases gradually, reaching a maximum of 1. In the case of the same liquid-solid ratio, comparing the porosity between different groups, it can be found that the porosity from low to high is triclinite 25, triclinite 50, triclinite 75, amorphous Calcium phosphate, triclinite 100. The porosity of triclinite 75 is lower than that of amorphous calcium phosphate and triclinite 100, but higher than that of triclinite 50 and triclinite 25.

In vitro degradation characterization

Figure 3A shows the XRD results of the biodegradation of triclinite/amorphous calcium phosphate bone cement soaked in simulated body fluids in vitro. In the XRD pattern of triclinic worfite 100, the strongest peak is the (020) crystal plane of the triclinic worfite phase, and there are also (030) crystal face peaks on the 7th, 14th, and 21st days. There was no difference from day 28, nor did the characteristic diffraction peaks of hydroxyapatite appear (Fig. 3A(a)). The XRD pattern of amorphous calcium phosphate is shown in Fig. 3A(e), showing the characteristic diffraction peaks (211) and (300) of hydroxyapatite. However, only the (020) and (030) diffraction peaks of triclinite in the bone cement composited with triclinite and amorphous calcium phosphate were only the (020) and (030) diffraction peaks of the triclinite 75 and 50, but did not appear Diffraction peaks of hydroxyapatite.

The surface topography of the five groups of samples immersed in SBF for 7, 14, 21 and 28 days changed significantly (Fig. 4A). The nanorods of triclinite 100 gradually transformed into massive particles, and the surface gradually flattened. After soaking for 28 days, the massive particles disappeared and were replaced by tiny particles. The surface morphology of triclinite 75 was very dense on the 7th and 14th days. On the 21st day, the surface of triclinite 75 showed a grid-like morphology formed by irregular arrangement of slender fibers, with different sizes. pore structure. This morphology makes the surface of triclinite 75 rough. Continuing to soak for the 28th day, it was found that the slender fibers of triclinite 75 gradually grew into thin sheets. Although small pores were retained, the large pores basically disappeared. The surface of triclinite 50 was flatter than the other four groups on the 7th day, and then a sheet-like morphology with larger holes appeared on the 14th day. With the increase of soaking time, the flaky fibers and holes disappeared, while the surface gradually became flat and compact. On the 7th day, tiny rod-like fibers were formed on the surface of triclinite 25. After soaking for 21 days, macropores appeared on the surface of triclinite 25, and micropores were distributed on the inner wall of the macropore, which was conducive to the induction of osteogenesis. On the 28th day , forming a flat surface composed of small particles. Amorphous calcium phosphate formed a grid-like structure with small pores on day 7, but this morphology disappeared on days 14 and 21, and finally a massive sheet-like morphology was formed on day 28.

Biocompatibility of triclinite/amorphous calcium phosphate bone cement

The absorbance value at 450 nm of the mixture added with CCK-8 solution and incubated for 2 hours represents the cell proliferation activity, and the higher the absorbance value, the stronger the cell proliferation ability. The absorbance of each group showed an upward trend from day 1, day 4 to day 7 (Fig. 5D and Fig. 7D). For mouse bone marrow mesenchymal stem cells, the absorbance of each group was higher than that of the control group without cell culture samples, indicating that none of the samples in each group had cytotoxicity to mouse bone marrow mesenchymal stem cells. The proliferation ability of mouse bone marrow mesenchymal stem cells of triclinite 75 was the highest on the 4th and 7th day, followed by triclinite 50 and triclinite 25, and amorphous calcium phosphate was slightly lower than that of triclinite 50 and triclinite 25. Tween 50 and 25, triclinite 100 are similar to amorphous calcium phosphates (FIG. 5D). For RAW264.7 cells, the absorbance value of triclinite 100 was the highest in the control and other groups. The absorbance values of triclinite 50 and triclinite 25 are close and not statistically significant, and triclinite 75 is slightly lower than them. The absorbance value of amorphous calcium phosphate was lower than that of the control group, indicating that the microscopic morphology or chemical composition of amorphous calcium phosphate could inhibit the proliferation of RAW264.7 cells (Fig. 7D).

After alcohol gradient dehydration treatment, SEM images were obtained to observe cell adhesion and morphology in different samples. Large numbers of cells and abundant pseudopodia indicate that the sample promotes cell adhesion. As shown in Figure 6A, the mouse bone marrow mesenchymal stem cells on triclinite 75 and triclinite 50 were polygonal with more pseudopodia, and the pseudopodia of amorphous calcium phosphate were less than those of the former two. However, mouse BMSCs on triclinite 100 are spindle-shaped and lack pseudopodia. As shown in Figure 8A,

The numbers of RAW 264.7 cells in triclinite 75 and amorphous calcium phosphate were less and the cells were not plump. The number of triclinite 50 and triclinite 25 cells gradually increased, the spreading area of the cells increased, and the pseudopodia contacted and fused with each other. The number of triclinite 100 cells continued to increase, the area of cell spreading increased, and intercellular fusion occurred, indicating that it was more conducive to the adhesion, proliferation and osteoclast differentiation of RAW264.7 cells, thereby accelerating the osteolysis process.

Effects of triclinite/amorphous calcium phosphate cement on cell migration ability

Figures 5E and 7E show images and average numbers of cells passing through the membrane to the lower surface of the Transwell chamber membrane. The number of mouse bone marrow mesenchymal stem cells in the triclinite 100 group was the least among the 5 groups, and the cell morphology was not plump and not fully extended. Tinclinite 75, amorphous calcium phosphate, and Ticlinite 50 had more cells than Ticlinite 100, and the cell spread area was larger. Ticlinite 75 was the best in each group in terms of cell number and cell morphology, indicating that it had the strongest effect on the migratory ability of mouse BMSCs (Fig. 5E). The number of RAW 264.7 cells in the triclinite 75 group was the least among the 5 groups, indicating that the triclinite 75 was the least favorable for the migration of RAW 264.7 cells among the 5 groups (Fig. 7E).

Effects of triclinite/amorphous calcium phosphate bone cement on osteogenic differentiation of mouse bone marrow mesenchymal stem cells

In Figure 5A, osteogenesis-related genes such as collagen 1 (COL-1), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2) ( The expressions in Table 1) were significantly higher than those in the control group and the other 4 groups. The mRNA expressions of triclinite 100, triclinite 50, triclinite 25, and amorphous calcium phosphate were higher than those in the control group to varying degrees. The results of IF staining to detect OCN protein expression in mouse bone marrow mesenchymal stem cells cultured in osteogenic induction medium for 14 days also supported the above view (Fig. 6B). The OCN fluorescence intensity of triclinite 75 was the strongest, and the cell number and cell spreading area of triclinite 75 were the largest among the five groups. This indicated that triclinite 75 was more favorable for the growth and osteogenic differentiation of mouse bone marrow mesenchymal stem cells under the induction of osteogenic induction medium.

Effects of triclinite/amorphous calcium phosphate cement on osteoclast differentiation of RAW 264.7 cells

The relative expression levels of osteoclast differentiation-related genes cathepsin K (Ctsk) and c-Fos (Tab.1) in RAW 264.7 cells are shown in Figure 7A. The mRNA expression level of triclinite 100 was significantly the highest, and the relative expression level was more than 1.5 times that of the control group. The relative mRNA expression of triclinite 75 was the lowest, which was 0.75 times less than that of the control group. Ticlinite 50 and Ticlinite 25 are close to the control group, and the amorphous calcium phosphate is slightly lower than the control group, but there is no statistical difference between the three and the control group. The results of the detection of Ctsk protein expression in RAW 264.7 cells cultured for 7 days by IF staining also supported the above notion (Fig. 8B). The Ctsk of triclinite 100, triclinite 50 and triclinite 25 have stronger fluorescence intensity than triclinite 75 and amorphous calcium phosphate, and the number of cells is more than that of triclinite 25 Stone 75 and amorphous calcium phosphate.

From the above experiments, it can be seen that the triclinite/amorphous calcium phosphate composite bone cement, as a new type of composite bone cement, is more efficient than amorphous calcium phosphate, tricalcium phosphate and triclinic calcium phosphate which have been used in clinical practice. It has better self-curing properties, mechanical properties, and degradation properties, especially biological properties that are more conducive to bone regeneration. This study shows that triclinite 75 complexed with 75 wt.% triclinite and 25 wt.% amorphous calcium phosphate has the best surface morphology and material composition, and can promote the proliferation of mouse bone marrow mesenchymal stem cells It is not conducive to the proliferation, migration and osteoclast of osteoclasts, and to a certain extent, it can inhibit the effect of osteoclasts on bone and calcium phosphate bone cement absorption in the early stage of bone defect. The injectability, setting time and mechanical properties of triclinite 75 all meet the clinical application requirements, but there are still some deficiencies compared with triclinite 25. Considering the properties and biological properties of the material itself, triclinite 75 is more suitable as an ideal composite bone cement.

The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

Claims (10)

1. a composite triclinite/amorphous calcium phosphate bone cement, is characterized in that, the component of described composite triclinite/amorphous calcium phosphate bone cement comprises triclinite and amorphous calcium phosphate . 2. according to the described composite triclinite/amorphous calcium phosphate bone cement, it is characterized in that, based on the mass summation of triclinite and amorphous calcium phosphate, the triclinite massfraction is 25- 75%, preferably 75%. 3. The compound triclinite/amorphous calcium phosphate bone cement according to claim 1, wherein the liquid phase of the composite triclinite/amorphous calcium phosphate bone cement is deionized water. 4. a preparation method of composite triclinic hefite/amorphous calcium phosphate bone cement as described in any one of claims 1-3, is characterized in that, comprises the steps: (1) Weighing triclinic folite and amorphous calcium phosphate powder according to a certain mass ratio; (2) Mixing the above-mentioned powders uniformly by a non-intrusive homogenizer; (3) Mix the uniformly mixed powder with deionized water according to a certain solid-liquid ratio. 5. The preparation method according to claim 4, characterized in that, in step (1), the mass fraction of the triclinic worfite is 75-25%, preferably 75%. 6. preparation method according to claim 4 is characterized in that, in step (2), mixing condition is: rotating speed is 800r/min, time is 60s, and times are 6-9 times; Described solid-liquid ratio is 0.6- 1.0, preferably a solid-liquid ratio of 0.5. 7. preparation method according to claim 4 is characterized in that, described amorphous calcium phosphate powder is by using CaCl ₂ and (NH ) ₂ HPO _The co-precipitation method synthesis; NO ₃ ) ₂ ·4H ₂ O and (NH ₄ ) ₂ HPO ₄ were synthesized by hydrothermal method. 8. the application method of composite triclinic folite/amorphous calcium phosphate bone cement as described in any one of claim 1-3, is characterized in that, comprises the steps: (1) injecting the composite triclinite/amorphous calcium phosphate bone cement into a cylindrical mold with a needle to obtain a bone cement block; (2) then placing the block in a constant temperature incubator to maintain the bone cement block; (3) The cured bone cement block is taken out and dried for later use. 9 . The application method according to claim 8 , wherein the size of the cylindrical mold is 10 mm×10 mm. 10 . 10 . The application method according to claim 8 , wherein the curing temperature is 37° C., the curing humidity is 100%, and the curing time is 72 h. 11 .

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