CN113694258A

CN113694258A - Bioactive bone cement composite material and preparation method and application thereof

Info

Publication number: CN113694258A
Application number: CN202111003286.5A
Authority: CN
Inventors: 翁习生; 梁瑞政; 王英杰; 胡婷婷
Original assignee: Beijing University of Chemical Technology; Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Beijing University of Chemical Technology; Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2021-11-26

Abstract

The invention provides a bioactive bone cement composite material, a preparation method and application thereof, and belongs to the technical field of preparation of medical bone tissue regeneration materials. The invention firstly adopts a hydrothermal coprecipitation method to prepare the magnesium-aluminum hydrotalcite microchip, then the magnesium-aluminum hydrotalcite microchip is used as an improved doping agent, methyl methacrylate and polymethyl methacrylate bone cement are polymerized and are simultaneously combined with magnesium-aluminum hydrotalcite and I type collagen to prepare the magnesium-aluminum hydrotalcite modified polymethyl methacrylate bone cement composite material, the magnesium-aluminum hydrotalcite microchip can continuously release magnesium ions in the bone regeneration process, the formation of calcium nodules in extracellular matrix is promoted, and the regeneration of bones at bone defect positions can be promoted by influencing various key osteogenesis signal paths, so that the magnesium-aluminum hydrotalcite microchip has excellent osteogenesis performance.

Description

Bioactive bone cement composite material and preparation method and application thereof

Technical Field

The invention relates to the technical field of preparation of medical bone tissue regeneration materials, in particular to a bioactive bone cement composite material and a preparation method and application thereof.

Background

Although bone tissue has sufficient natural regenerative capacity to repair small injury sites, such as cracks and certain types of fractures, bone defects exceeding a critical size threshold (typically >2 cm, depending on the anatomical site) will not heal without assistance. Surgical removal of wounds, degenerative diseases, congenital defects or tumors may result in large bone defects or deletions, requiring clinical intervention if functional recovery and complete healing is to be achieved, bone fixation using bioinert metal devices or autologous bone grafts.

At present, the bone cement is a good material for clinically filling and repairing bone defects, and the bone cement which is mainly used clinically worldwide is polymethyl methacrylate (PMMA). However, the polymethylmethacrylate bone cement has some limitations, such as the limitation of bone regrowth due to the biological inertness, non-degradability and potential cytotoxicity of the polymethylmethacrylate bone cement, and the death of the surrounding associated osteoblasts due to the higher polymerization temperature, and the subsequent formation of a fibrotic membrane that prevents the polymethylmethacrylate bone cement from bonding to bone. Therefore, polymethylmethacrylate bone cement cannot form biological osseointegration at the implant site due to the above disadvantages and also causes aseptic loosening of the bone cement, which has become a major factor in more than 75% of total hip/knee replacement surgery failures.

To date, materials such as hydroxyapatite or strontium-doped hydroxyapatite, biodegradable chitosan and sodium hyaluronate have been added in order to improve the biosafety and osteogenic properties of polymethylmethacrylate bone cement. However, the addition of the above materials does not bring about effective osteogenic properties.

Disclosure of Invention

The invention aims to provide a bioactive bone cement composite material, and a preparation method and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of a bioactive bone cement composite material, which comprises the following steps:

mixing magnesium salt, aluminum salt, urea and water, and carrying out hydrothermal coprecipitation on the obtained mixed solution to obtain magnesium-aluminum hydrotalcite micro-tablets;

and mixing the magnesium-aluminum hydrotalcite microchip, type I collagen, polymethyl methacrylate bone cement and methyl methacrylate monomer, and carrying out polymerization reaction to obtain the bioactive bone cement composite material.

Preferably, the magnesium salt comprises magnesium nitrate, magnesium sulfate or magnesium chloride; the aluminum salt comprises aluminum nitrate, aluminum chloride, or aluminum sulfate; the molar ratio of magnesium ions in the magnesium salt to aluminum ions in the aluminum salt is (2-4): 1.

Preferably, the molar ratio of the urea to the aluminum ions in the aluminum salt is 12: 1; the total concentration of the magnesium salt and the aluminum salt in the mixed solution is 0.04-0.5 mol/L.

Preferably, the temperature of the hydrothermal coprecipitation is 80-120 ℃, and the reaction time is 24-48 h.

Preferably, the diameter of the magnalium hydrotalcite microchip is 6 +/-2 μm, and the morphology of the magnalium hydrotalcite microchip is a hexagonal nanosheet.

Preferably, the weight percentage of the magnesium aluminum hydrotalcite microchip, the type I collagen and the polymethyl methacrylate bone cement is 10-20%, 70%, calculated by the total weight percentage of the magnesium aluminum hydrotalcite microchip, the type I collagen and the polymethyl methacrylate bone cement being 100%.

Preferably, the dosage ratio of the methyl methacrylate monomer to the total mass of the magnesium aluminum hydrotalcite microchip, the type I collagen and the polymethyl methacrylate bone cement is 5mL:10 g.

Preferably, the temperature of the polymerization reaction is room temperature, and the time is 15-25 min.

The invention provides a bioactive bone cement composite material prepared by the preparation method in the technical scheme, which comprises blended polymethyl methacrylate bone cement, magnesium aluminum hydrotalcite micro-sheets and I type collagen.

The invention provides application of the bioactive bone cement composite material in the technical scheme in preparation of a bone defect regeneration and repair material.

The invention provides a preparation method of a bioactive bone cement composite material, which comprises the steps of firstly preparing a magnesium-aluminum hydrotalcite microchip by a hydrothermal coprecipitation method, then using the magnesium-aluminum hydrotalcite microchip as an improved doping agent, polymerizing methyl methacrylate and polymethyl methacrylate bone cement, and simultaneously combining the methyl methacrylate with the magnesium-aluminum hydrotalcite microchip and I-type collagen to prepare the magnesium-aluminum hydrotalcite modified polymethyl methacrylate bone cement composite material, wherein the magnesium-aluminum hydrotalcite microchip can continuously release magnesium ions in the bone regeneration process, promote the formation of calcium nodules in an extracellular matrix, and promote the regeneration of bones at bone defect positions by influencing various key osteogenic signal paths, so that the composite material has excellent osteogenesis performance. The results of the examples show that the bioactive bone cement composite material prepared by the invention can activate the osteogenesis signal pathways of P38MAPK, ERK/MAPK, FGF-18 and TGF-beta, and remarkably promote the expression of osteogenesis genes P-P38/P38, Runx2 and Alp.

The preparation method is simple and easy to operate, the required raw materials are cheap and easy to obtain, and the biological active bone cement composite material has good biocompatibility and degradation performance, so that the prepared biological active bone cement composite material has good biocompatibility, has more excellent bone formation performance compared with pure polymethyl methacrylate bone cement, and has potential application prospects in related orthopedic surgeries.

The prepared bioactive bone cement composite material comprises magnesium-aluminum hydrotalcite micro-sheets, wherein a magnesium-rich microenvironment can stimulate osteogenic differentiation of stem cells, promote vascularized bone regeneration and enhance tissue regeneration, proper release of magnesium ions in the magnesium-aluminum hydrotalcite can activate an osteogenic signal channel, promote expression of osteogenic genes, promote protein adsorption, osteoblast attachment, spreading and subsequent proliferation, thereby improving osteogenic differentiation of mesenchymal stem cells, improving the bioactivity of PMMA, effectively endowing the bioactive polymethyl methacrylate bone cement with bioactivity, promoting in vivo bone formation, improving the interfacial bone combination of bone and bone cement, and being expected to become a promising biomaterial for orthopedic surgery.

Drawings

FIG. 1 is an SEM image of materials prepared in example 1 and comparative examples 1 to 3, a physical image of the material prepared in example 1, and an atomic force microscope image of the materials prepared in example 1 and comparative examples 1 to 3;

FIG. 2 is a biocompatibility chart of the composite material prepared in example 1 and comparative examples 1 to 3 and a control group detected by a cell counting kit-8 (CCK-8);

FIG. 3 is a graph showing cell alkaline phosphatase (ALP) staining patterns of the composite materials prepared in example 1 and comparative examples 1 to 3;

FIG. 4 is a staining pattern of alizarin red S (Alizarin red S) obtained by culturing the composite materials prepared in example 1 and comparative examples 1 to 3 in different forms;

FIG. 5 is a volcano plot visually showing the number of up-regulated genes (red dots) and down-regulated genes (green dots);

FIG. 6 is a Gene Ontology (GO) enrichment analysis diagram;

FIG. 7 is a diagram of genome encyclopedia (KEGG) analysis;

FIG. 8 is a diagram of the first 18 exemplary paths generated by the inventive path analysis software;

FIG. 9 is a graph of the molecular interaction network and subcellular localization of differentially expressed genes associated with osteogenesis-related signaling pathways generated by the Inventity Path Analysis software;

FIG. 10 is a graph showing the results of verifying transcriptome sequencing and IPA of the materials prepared in example 1 and comparative examples 1-3 using quantitative polymerase chain reaction (qPCR) and Western Blotting (WB);

FIG. 11 is an operation view of the skull of a rabbit implanted with the composite material prepared in example 1 and comparative examples 1 to 3 and Micro-CT views of the skull after 0 week, 4 weeks and 8 weeks;

FIG. 12 is a graph of acid fuchsin staining of the composite prepared in example 1 and comparative examples 1-3 at 0, 4 and 8 weeks after implantation into a rabbit skull;

FIG. 13 is a graph showing the results of measuring the polymerization temperature of the composite materials prepared in example 1 and comparative examples 1 to 3;

FIG. 14 is a graph showing the results of measuring the concentration of released magnesium ions on

days

1, 3, 5, 7, 9, 11, and 13 of culture in the composite osteogenesis medium prepared in example 1 and comparative example 2.

Detailed Description

In the present invention, unless otherwise specified, all the starting materials required for the preparation are commercially available products well known to those skilled in the art.

The magnesium salt, the aluminum salt, the urea and the water are mixed, and the obtained mixed solution is subjected to hydrothermal coprecipitation to obtain the magnesium-aluminum hydrotalcite microchip. In the present invention, the magnesium salt preferably includes magnesium nitrate, magnesium sulfate or magnesium chloride; in the examples of the present invention, Mg (NO) is specifically mentioned₃)₂·6H₂O; the aluminium salt preferably comprises aluminium nitrate, aluminium chloride or aluminium sulphate, in particular Al (NO) in embodiments of the invention₃)₃·9H₂O; the molar ratio of magnesium ions in the magnesium salt to aluminum ions in the aluminum salt is preferably (2-4): 1, and more preferably (2.5-3.5): 1. In the present invention, the molar ratio of urea to aluminum salt is preferably 12: 1; the total concentration of magnesium salt and aluminum salt in the mixed solution is 0.04-0.5 mol/L, more preferably 0.1-0.4 mol/L, and further preferably 0.2-0.3 mol/L。

The process of mixing the magnesium salt, the aluminum salt, the urea and the water is not particularly limited in the invention, and the materials are uniformly mixed according to the process well known in the field.

In the invention, the hydrothermal coprecipitation is preferably carried out in a 100mL stainless steel autoclave with a Teflon lining, the temperature of the hydrothermal coprecipitation is preferably 80-120 ℃, more preferably 100 ℃, and the reaction time is preferably 24-48 h, more preferably 36 h.

After the hydrothermal coprecipitation is finished, the obtained product is preferably naturally cooled to room temperature, centrifuged, collected and washed with ethanol and deionized water for three times, and dried to obtain the magnesium-aluminum hydrotalcite microchip; the drying is preferably carried out in an oven, the temperature of the drying is preferably 60 ℃, the time of the drying is not particularly limited in the present invention, and the drying is carried out according to a process well known in the art. The cooling and centrifuging process is not particularly limited in the present invention and may be performed according to a process well known in the art.

In the present invention, the composition of the hydrotalcite microchip is M²⁺ _1-xM³⁺ _x(OH)₂·A^n- _x/n·zH₂O, wherein M²⁺Represents Mg²⁺，M³⁺Represents Al³⁺，A^n-Is an interlayer anion; a is CO₃ ^2-、NO₃ ^-、Cl^-、OH-、SO₄ ^2-Or PO₄ ^3-；x＝0.17～0.33，n＝1～3。

In the invention, the diameter of the magnalium hydrotalcite microchip is preferably 6 +/-2 μm, and the morphology of the magnalium hydrotalcite microchip is a hexagonal nanosheet. The magnesium-aluminum hydrotalcite synthesized by the urea method has lower supersaturation degree, the magnesium-aluminum hydrotalcite nanosheet grows completely, the crystallinity is higher, the size of the obtained crystal grain is larger, a certain number of pores are formed on the surface of the polymethyl methacrylate, and the bone repair is facilitated.

After the magnesium aluminum hydrotalcite microchip is obtained, the magnesium aluminum hydrotalcite microchip, type I collagen, polymethyl methacrylate bone cement and methyl methacrylate monomer are mixed for polymerization reaction to obtain the bioactive bone cement composite material (PMMA & COL-I & LDH).

In the invention, the total mass percentage of the magnesium aluminum hydrotalcite microchip, the type I collagen and the polymethyl methacrylate bone cement is 100%, and the mass percentage of the magnesium aluminum hydrotalcite microchip, the type I collagen and the polymethyl methacrylate bone cement is preferably 10-20% to 70%, more preferably 15% to 70%.

The invention utilizes the type I collagen to repair the damaged tissue, has good in vitro biocompatibility with adipose-derived stem cells, provides a suitable three-dimensional space for the growth of the tissue engineering seed cells, and is used as a carrier material of the adipose tissue engineering seed cells.

In the invention, the dosage ratio of the methyl methacrylate monomer to the total mass of the magnesium aluminum hydrotalcite microchip, the type I collagen and the polymethyl methacrylate bone cement is preferably 5mL to 10 g.

In the present invention, the process of mixing the magnesium aluminum hydrotalcite microchip, the type I collagen, the polymethylmethacrylate bone cement and the polymethylmethacrylate monomer is preferably to mix the magnesium aluminum hydrotalcite microchip, the type I collagen and the polymethylmethacrylate bone cement (powder), and then add the polymethylmethacrylate monomer (liquid state) to the mixture.

In the invention, the temperature of the polymerization reaction is preferably room temperature, and the time is preferably 15-25 min, and more preferably 20 min. In the polymerization reaction process, the polymethyl methacrylate bone cement powder and a methyl methacrylate monomer are subjected to polymerization reaction to generate polymethyl methacrylate, and the polymethyl methacrylate is mixed with the powdery magnesium aluminum hydrotalcite microchip and the I type collagen to form the composite material.

After the polymerization reaction is completed, the bioactive bone cement composite material can be obtained without post-treatment.

The invention provides a bioactive bone cement composite material prepared by the preparation method in the technical scheme, which comprises blended polymethyl methacrylate bone cement, magnesium aluminum hydrotalcite micro-sheets and I type collagen. In the invention, the blended polymethyl methacrylate bone cement, the magnalium hydrotalcite microchip and the type I collagen exist in a physical blending mode.

The invention provides application of the bioactive bone cement composite material in the technical scheme in preparation of a bone defect regeneration and repair material. The method of the present invention is not particularly limited, and the method may be applied according to a method known in the art. In the present invention, the bioactive bone cement composite material is preferably a material that can be compressed into a specific shape, facilitating its application. The compressing method preferably comprises the steps of filling the bioactive bone cement composite material into a mould with a corresponding shape, and compressing; the mold and the pressing process are not particularly limited in the present invention, and the mold known in the art may be selected and pressed according to the process known in the art. In the present invention, the mold is preferably a cylindrical test piece having a diameter of 6mm and a height of 12mm, or preferably a flat test piece having a length of 75 mm, a width of 10 mm and a thickness of 3.3 mm.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Magnesium and aluminum (the molar ratio of magnesium to aluminum elements is 2:1) hydrotalcite (MgAl-LDH) synthesis:

2mmol of Mg (NO)₃)₂·6H₂O、1mmol Al(NO₃)₃·9H₂Dissolving O and 12mmol of urea in 70mL of deionized water to obtain a mixed solution;

transferring 70mL of the mixed solution into a 100mL stainless steel autoclave with a teflon lining, and carrying out hydrothermal coprecipitation for 24h at 100 ℃; naturally cooling the obtained product system to room temperature, centrifuging and collecting the obtained product, then washing the product with ethanol and deionized water for three times respectively, and drying the product in a 60 ℃ oven to obtain magnesium-aluminum hydrotalcite microchip powder which is recorded as MgAl-LDH;

mixing polymethyl methacrylate bone cement powder (7g), type I collagen powder (1.5g) and magnesium aluminum hydrotalcite microchip powder (1.5g), adding 5mL of liquid methyl methacrylate monomer into the obtained mixture, fully mixing, and carrying out polymerization reaction at room temperature (25 ℃) for 20min to obtain the bioactive bone cement composite material, wherein the total mass is 10g and is recorded as PMMA & COL-I & LDH.

Comparative example 1

Adding polymethyl methacrylate bone cement powder (8.5g) and type I collagen powder (1.5g) into 5mL of liquid methyl methacrylate monomer, fully mixing, and carrying out polymerization reaction for 20min at room temperature (25 ℃) to obtain a mixed material of polymethyl methacrylate bone cement and type I collagen, wherein the mass of the mixed material is 10g and is marked as PMMA & COL-I.

Comparative example 2

Preparing magnesium aluminum hydrotalcite micro-flake powder according to the method of example 1;

adding polymethyl methacrylate bone cement powder (8.5g) and magnesium aluminum hydrotalcite microchip powder (1.5g) into 5mL of liquid methyl methacrylate monomer, fully mixing, and initiating methyl methacrylate polymerization reaction at room temperature (25 ℃) for 20min to obtain a mixed material of polymethyl methacrylate bone cement and magnesium aluminum hydrotalcite microchip, wherein the mass of the mixed material is 10g and is recorded as PMMA & LDH.

Comparative example 3

Polymethyl methacrylate bone cement powder (10g) was added to 5mL of liquid methyl methacrylate monomer, and after thorough mixing, polymerization was carried out at room temperature (25 ℃) for 20min to obtain polymethyl methacrylate bone cement, the mass of which was 10g and was recorded as PMMA.

Characterization and testing

1) SEM tests are respectively carried out on the magnesium-aluminum hydrotalcite prepared in example 1, the composite material and the materials prepared in comparative examples 1 to 3, and transmission electron microscope tests are carried out on the materials prepared in example 1 and comparative examples 1 to 3, and the results are shown in figure 1, wherein a to e are SEM images of the LDH prepared in example 1, the materials prepared in comparative examples 1 to 3 and the materials prepared in example 1, and g is an atomic force microscope image of the materials prepared in example 1 and the materials prepared in comparative examples 1 to 3; as can be seen from FIG. 1, MgAl-LDHs consists of hexagonal nanosheets with diameters distributed as 6 +/-2 μm; micropores exist on the surfaces of PMMA & LDH and PMMA & COL-I & LDH, which is beneficial to bone formation.

F in fig. 1 is a photograph showing a real object of the bioactive bone cement composite prepared in example 1, and as can be seen from f in fig. 1, the bioactive bone cement composite can be compressed into a circular material having a diameter of 6 mm.

2) The materials prepared in example 1 and comparative examples 1 to 3 were tested for in vitro biocompatibility by the following method: PMMA, PMMA & Col-I, PMMA & LDH and PMMA & Col-I & LDH were prepared into disk-shaped samples having a diameter of 6mm and a thickness of 2.5mm, respectively, and all of the samples were sterilized with ethylene oxide before cell testing. First, 10000 human mesenchymal stem cells were seeded in a 24-well plate having a pore size of 3 μm for 12 hours to completely adhere the cells, and then a disc-shaped sample was added to the upper chamber. In addition, a disc sample with collagen fixed in the 24 hole plate bottom 1h, 10000 cells were directly inoculated on the disc sample surface, each group is provided with 3 parallel hole. These orifice plates were placed in an atmosphere of 37 deg.C, 5% carbon dioxide and 95% relative humidity. The blank contained cells only. CCK-8 measurements were carried out on

days

1, 3, 5 and 7, respectively, and OD values at 450. + -. 5nm were measured with a multifunctional full-wavelength microplate reader (Varioskan Flash; Thermo Fisher Science, USA), and flow cytometry was carried out on day 3 with a FACSCAnto plus from BD Biosciences, USA; the results are shown in FIG. 2, wherein a is the cell viability of the materials prepared in example 1 and comparative examples 1-3 after 1, 3, 5 and 7 days of indirect contact culture; b is the cell viability of the material prepared in example 1 and comparative examples 1-3 after 1, 3, 5 and 7 days of direct contact culture.

As can be seen from FIG. 2, after 1, 3, 5, 7 days of culture, the survival rates of human bone marrow mesenchymal stem cells treated with PMMA, PMMA & COL-I, PMMA & LDH and PMMA & COL-I & LDH indirectly (a in FIG. 2) and directly contacted (b in FIG. 2) were measured using the cytometric kit-8 (CCK-8) colorimetry. As shown in fig. 2, the Optical Density (OD) values of the four groups of cells gradually increased with the increase of the incubation time, and showed a similar trend to that of the blank control group. Furthermore, the cell viability of PMMA & COL-I, PMMA & LDH and PMMA & COL-I & LDH was 1.55 times, 1.43 times and 1.48 times that of PMMA (a in fig. 2), respectively, and c in fig. 2 was the cell viability of the four groups of materials as determined by flow cytometry, and it can be seen from c in fig. 2 that the percentage of viable cells of PMMA, PMMA & COL-I, PMMA & LDH and PMMA & COL-I & LDH was 80.6% and 90.5% and 84.6% and 89.9%, respectively, and the survival rate of viable cells of PMMA & COL-I & LDH group was higher than that of PMMA & LDH group, which indicates that the biocompatibility of PMMA & COL-I, PMMA & LDH and PMMA & COL-I & LDH was much improved compared with PMMA alone, improving the bio-inertness of PMMA. The biocompatibility of the material is good, which indicates that the toxicity of the material is low, but the mechanical property and the osteogenesis capacity of the material are considered besides the biocompatibility, so that the expression of bone genes is promoted, and the osteogenesis capacity of PMMA & COL-I & LDH is comprehensively considered to be more excellent.

3) Alkaline phosphatase (ALP) staining experiments are carried out on the materials prepared in example 1 and comparative examples 1-3, and the condition that different materials induce osteogenic differentiation of human mesenchymal stem cells is researched, wherein the method comprises the following steps: PMMA, PMMA & Col-I, PMMA & LDH and PMMA & Col-I & LDH were prepared into disk-shaped samples having a diameter of 6mm and a thickness of 2.5mm, respectively, and all of the samples were sterilized with ethylene oxide before cell testing. Osteogenic differentiation medium and 6-well transwell plate with a pore diameter of 3 μm were selected for indirect culture. 3 ten thousand human mesenchymal stem cells were seeded into the lower chamber, and after 12 hours, PMMA & Col-I, PMMA & LDH and PMMA & Col-I & LDH samples (3 samples per upper chamber) were added to each upper chamber, respectively. In addition, 3 specimens per set were fixed to the bottom of a 6-well plate, and then 3 ten thousand cells were directly seeded on the surface of the specimens. On day 14, the upper chamber and ODM (osteogenic differentiation medium) were removed in their entirety, and the cells were washed 2 times with 2m phosphate per well. Then, fixing the cells with 2mL of paraformaldehyde with a mass concentration of 4% for 15min, washing each well with distilled water for 3 times, and then incubating with 2mL of alkaline phosphatase (ALP) staining solution for 20 min; subsequently, all cells were washed 3 times with PBS and observed with Eclipse80i microscope, and the results are shown in fig. 3.

As shown in FIG. 3, the osteogenic differentiation capacity of the different materials was assessed by alkaline phosphatase (ALP) staining assay on day 14, with the ALP positive cells of the PMMA & LDH and PMMA & COL-I & LDH groups being significantly larger than the PMMA and PMMA & COL-I groups.

When the three-dimensional reconstruction and the quantitative analysis of the new bone volume of different materials in fig. 3 are carried out, the bone formation volumes of PMMA & LDH and PMMA & Col-I & LDH are respectively 18.34 times and 17.29 times of the PMMA group 2 months after the operation, because the presence of Col-I occupies partial surface of PMMA & Col-I & LDH, compared with PMMA & LDH, the number of LDH particles exposed on the surface is reduced, thereby weakening the bone formation effect induced by LDH micro-sheets. The LDH modified PMMA and the Col-I & LDH modified PMMA bone cement have obvious osteogenesis functions in vivo. As the Col-I occupies part of the surface of the PMMA & Col-I & LDH, compared with the PMMA & LDH, the number of LDH particles exposed on the surface is reduced, so that the osteogenesis effect induced by LDH micro-sheets is weakened, and the osteogenic volume of the PMMA & Col-I & LDH is smaller than that of the PMMA & LDH.

4) Alizarin red S staining experiments were performed on the materials prepared in example 1 and comparative examples 1 to 3, and the method was: PMMA, PMMA & Col-I, PMMA & LDH and PMMA & Col-I & LDH were respectively prepared into disc-shaped samples having a diameter of 6mm and a thickness of 2.5 mm. All samples were sterilized with ethylene oxide prior to cell testing. Selecting osteogenic differentiation medium and a 6-hole cross-well plate with the aperture of 3 mu m for indirect culture: 3 ten thousand human mesenchymal stem cells were seeded into the lower chamber, and after 12 hours, PMMA & Col-I, PMMA & LDH and PMMA & Col-I & LDH samples (3 samples per upper chamber) were added to each upper chamber, respectively. In addition, 3 specimens per set were fixed to the bottom of a 6-well plate, and then 3 ten thousand cells were directly seeded on the surface of the specimens. On day 14, the upper chamber and ODM were removed in their entirety and the cells were washed 2 times with 2m phosphate per well. Then, the cells were fixed with 2mL of paraformaldehyde having a mass concentration of 4% for 15 min. After washing each well 3 times with distilled water, incubation was carried out for 15min with 2mL alizarin Red S staining solution. Subsequently, all cells were washed 3 times with PBS and observed with Eclipse80i microscope, and the results are shown in fig. 4; wherein a is a dyeing effect graph of the materials of the example 1 and the comparative examples 1 to 3 under the condition of 500 μm, and b is a dyeing effect graph of the materials of the example 1 and the comparative examples 1 to 3 under the condition of 200 μm.

As can be seen from FIG. 4, the red regions of extracellular matrix indirectly treated with PMMA & LDH and PMMA & COL-I & LDH (alizarin Red S-stained calcium nodules) are more pronounced than the PMMA and PMMA & COL-I groups (a in FIG. 4). In addition, calcium nodules in the extracellular matrix of human bone marrow mesenchymal stem cells cultured directly on the surface of PMMA & LDH and PMMA & COL-I & LDH were also more pronounced than in the PMMA and PMMA & COL-I groups (b in FIG. 4). Therefore, calcium nodules in the extracellular matrix of hBMSCs directly and indirectly cultured by the PMMA & LDH group and the PMMA & Col-I & LDH group are obvious, which indicates that the number of osteoblasts is large, namely LDH modified PMMA has good osteogenesis capacity.

The surface roughness of the materials of example 1 and comparative examples 1 to 3 was quantitatively analyzed by atomic force microscopy, and the results are shown in fig. 4 c. As shown in c of FIG. 4, the surface roughness of PMMA, PMMA & COL-I, PMMA & LDH and PMMA & COL-I & LDH was 0.38. + -. 0.02. mu.m, 0.82. + -. 0.02. mu.m, 1.57. + -. 0.09. mu.m and 1.93. + -. 0.07. mu.m, respectively, where PMMA & COL-I & LDH had the highest surface roughness and was more favorable for osteogenesis.

The flexural strength of the materials prepared in example 1 and comparative examples 1 to 3 was tested according to the method of ISO 5833-. As shown in d in FIG. 4, the addition of COL-I or LDH reduced the flexural strength of PMMA by 17.72% and 23.66%, which did not meet the IOS 5833 criteria. Thus, addition of COL-I or LDH may reduce stress-shielding osteolysis and indirectly promote osteointegration.

4) Carrying out transcriptome gene sequencing on the material prepared in the comparative ratio 2-3, analyzing a sequencing result and an original approach, revealing a mechanism of inducing osteogenic differentiation by the polymethyl methacrylate bone cement and the magnesium-aluminum hydrotalcite microchip, and comparing the mechanism with the polymethyl methacrylate bone cement, wherein the obtained result is shown in the figures 5-9; FIG. 5 is a volcano plot visually showing the number of up-regulated genes (red dots) and down-regulated genes (green dots); FIG. 6 is a Gene Ontology (GO) enrichment analysis diagram; FIG. 7 is a diagram of genome encyclopedia (KEGG) analysis; FIG. 8 is a diagram of the first 18 exemplary paths generated by the inventive path analysis software; FIG. 9 is a diagram of the molecular interaction network and subcellular localization of differentially expressed genes associated with osteogenesis-related signaling pathways generated by the Inventity Path Analysis software.

As can be seen from fig. 5, the total number of differentially expressed genes was 739, with 367 up-regulated genes and 372 down-regulated genes; analysis of the differentially expressed genes by Gene Ontology (GO) enrichment, most of which are involved in osteogenesis and angiogenesis, including extracellular matrix organization, cell adhesion, collagen catabolic processes, collagen fiber organization, vascular development, cellular regions, extracellular matrix structural components, nuclear receptor activity, and E-box binding (fig. 6); of the first 20 signal paths, 4 are associated with osteogenesis (fig. 7); FIG. 8 shows that PMMA & LDH activates the p38MAPK, extracellular signal-regulated kinase (ERK)/MAPK, FGF and transforming growth factor-beta (TGF-beta) signaling pathways, all of which promote osteogenic differentiation of human mesenchymal stem cells; in addition, fig. 9 shows the molecular interaction network and subcellular localization of differentially expressed genes involved in osteogenesis-related signaling pathways.

6) The results of transcriptome sequencing and the creative pathway analysis (IPA) of the materials prepared in example 1 and comparative examples 1 to 3 were verified by using quantitative polymerase chain reaction (qPCR) and Western Blotting (WB) tests, and the obtained results are shown in fig. 10, wherein a is the relative gene transcription level of the human mesenchymal stem cell osteogenic markers (Runx2, ALP, P38, and P-P38) of day 7 by qPCR; b is the expression of the gene Runx2 and ALP by Western blot analysis, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is used as a control gene; c is ImageJ 1.52q 1.52v software analysis of expression of Runx2 gene; d is ImageJ 1.52q 1.52v software analysis of ALP gene expression; e is the expression of P38 and P-P38 genes analyzed by Western blotting, GAPDH as a control gene; f ImageJ 1.52q 1.52v software analysis of the expression of the P38/P-P38 ratio.

As can be seen from a in fig. 10, the RUNX2 transcript levels of PMMA & COL-I, PMMA & LDH and PMMA & coi & LDH treated human mesenchymal stem cells were 1.16 times, 2.01 times and 2.06 times respectively that of the PMMA treated group. ALP transcript levels of PMMA & COL-I, PMMA & LDH, PMMA & COL-I & LDH treated human mesenchymal stem cells were 1.51-fold, 2.40-fold, and 2.55-fold for the PMMA group, respectively. In addition, the P-P38 to P-38 ratios of the PMMA & COL-I, PMMA & LDH, and PMMA & COL-I & LDH groups were 2.17 times, 7.37 times, and 7.46 times, respectively, that of the PMMA group. In addition to the transcription level, the translation level was also detected by the WB method; the translation levels of RUNX2 of human bone marrow mesenchymal stem cells in the PMMA & COL-I, PMMA & LDH, and PMMA & COL-I & LDH groups were 1.99-fold, 3.00-fold, and 3.01-fold higher than those in the PMMA group, respectively (b and c in FIG. 10). ALP translation levels of human bone marrow mesenchymal stem cells of PMMA & COL-I group, PMMA & LDH group, and PMMA & COL-I & LDH group were 2.01-fold, 2.95-fold, and 3.23-fold, respectively, that of PMMA group (b and d in FIG. 10). FIG. 10 e determines the expression levels of P-P38 and P-38 genes by Western blot analysis, and P-P38/P-38 translational levels of human mesenchymal stem cells were 2.62-fold, 6.34-fold and 7.66-fold higher for the PMMA & COL-I, PMMA & LDH and PMMA & COL-I & LDH groups, respectively (e and f in FIG. 10). The above results indicate that LDH significantly promotes the expression of osteogenic genes by activating p38MAPK, ERK/MAPK, FGF18 and transforming growth factor-beta signaling pathways.

7) The materials prepared in example 1 and comparative examples 1 to 3 were implanted into rabbit cranium bones, and the regeneration state of the cranium bones at 0 week, 4 weeks and 8 weeks was measured by Micro-CT, and the implantation process is shown in fig. 11, wherein a to d are implantation processes: the operation area was sterilized, rabbit skin was incised, 4 bone defects having a diameter of 6mm were made, different test materials were put in the respective positions (e in fig. 11 is the corresponding implant position of the material of example 1 and comparative examples 1 to 3), and representative cross-sectional views of the three-dimensional structure (left), sagittal plane (upper right) and coronal plane (lower right) at 0 month, 1 month and 2 months after the micro CT scanning, and the results are shown in fig. 11. From the Micro-CT images at 1 month and 2 months in fig. 11, it can be seen that the osseointegration between the bone and the implant was significantly better for the PMMA & LDH group and the PMMA & COL-I & LDH group than for the PMMA group and the PMMA & COL-I group, and that the osseointegration increased with time in both the sagittal plane and the coronal plane.

8) The acid fuchsin staining was performed on the skull of a rabbit implanted with different materials at the corresponding positions in 7) at 0, 4 and 8 weeks, respectively, and the results are shown in fig. 12. As can be seen in FIG. 12, acid fuchsin staining analyzed the extent of implant bone growth, and the results were similar to Micro-CT results. Two reasons why PMMA & LDH has a stronger bone-promoting ability than PMMA & COL-I and PMMA & COL-I & LDH are: for PMMA & COL-I and PMMA & LDH, LDH releases magnesium ions that promote osteogenesis more than COL-I. For PMMA & LDH and PMMA & COL-I & LDH, the presence of COL-I occupies part of the surface of PMMA & COL-I & LDH, and compared with PMMA & LDH, the number of LDH particles exposed on the surface is reduced, thereby weakening the LDH micro-flake induced osteogenesis effect.

Through transcriptome sequencing and osteoblast gene expression determination, results show that the addition of hydrotalcite and Col-I in PMMA & Col-I & LDH activates p38MAPK, ERK/MAPK, FGF18 and a transforming growth factor-beta signal pathway compared with single PMMA, the expression level of related bone genes is higher, and the osteogenic performance of PMMA & Col-I & LDH is better under comprehensive consideration.

9) Measurement of polymerization reaction temperature:

the materials tested were respectively: PMMA (100%), PMMA and COL-I (85% and 15%), PMMA and LDH (85% and 15%) and PMMA, COL-I and LDH (70% and 15%) respectively, the total weight of the material is 10g, and the specific preparation process of the material is as follows: according to the schemes of example 1 and comparative examples 1 to 3, after the polymethyl methacrylate powder and COL-I and/or LDH are sufficiently mixed, a liquid methyl methacrylate monomer is added to the polymethyl methacrylate powder to initiate polymerization of methyl methacrylate. A20 mL syringe barrel was used as a reaction vessel for the polymerization of methyl methacrylate. The results of the FLUKE infrared imager (FLUKE, Washington, USA) for measuring the temperature change of the polymerization reaction of different materials are shown in FIG. 13.

As can be seen from fig. 13, the maximum temperature of LDH-modified PMMA (PMMA & LDH) was reduced by 7.0 ℃ compared to PMMA, which is attributable to some thermal insulation effect of LDH, and the maximum temperature of MMA polymerization reaction was reduced after adding LDH, thereby reducing thermal damage to osteogenesis-related cells around LDH-modified PMMA; second, LDH addition will reduce stress-shielding osteolysis and indirectly promote osteointegration.

10) The PMMA & COL-I & LDH and PMMA & LDH prepared in example 1 and comparative example 2 were cultured in an osteogenic medium for 1, 3, 5, 7, 9, 11, 13 days, and their magnesium ion release concentrations were measured, and the results are shown in fig. 14; as can be seen from FIG. 14, the concentrations of magnesium ions released by PMMA & LDH and PMMA & COL-I & LDH were about 1.60 and 1.31. mu. mol/mL, respectively, less than the total magnesium ion concentration in the synthetic hydrotalcite, and the concentrations were substantially constant, indicating that the material prepared in example 1 moderately released magnesium ions in the osteogenesis medium.

As can be seen from the above examples and comparative examples, since the presence of Col-I occupies a part of the surface of PMMA & Col-I & LDH, the number of LDH particles exposed on the surface is reduced as compared with PMMA & LDH, thereby weakening LDH micro-flake induced osteogenesis and making the osteogenic volume of PMMA & Col-I & LDH smaller than that of PMMA & LDH. However, compared with PMMA & LDH, MMA & Col-I & LDH has higher surface roughness and is more beneficial to bone formation; the addition of Col-I reduces the bending strength of PMMA, relieves stress shielding osteolysis and indirectly promotes osseointegration; the flow cytometry detection can find that the PMMA & Col-I & LDH group has higher living cell survival rate and better biocompatibility than the PMMA & LDH group; through transcriptome sequencing and osteoblast gene expression determination, the addition of PMMA & Col-I & LDH activates p38MAPK, ERK/MAPK, FGF18 and a transforming growth factor-beta signal channel, the expression level of related osteocytes is higher, the osteogenic differentiation is promoted by comprehensively considering various aspects such as mechanical property, biocompatibility, gene expression and the like, and the osteogenic property of PMMA & Col-I & LDH is better.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a bioactive bone cement composite material comprises the following steps:

2. The method of claim 1, wherein the magnesium salt comprises magnesium nitrate, magnesium sulfate, or magnesium chloride; the aluminum salt comprises aluminum nitrate, aluminum chloride, or aluminum sulfate; the molar ratio of magnesium ions in the magnesium salt to aluminum ions in the aluminum salt is (2-4): 1.

3. The method according to claim 1, wherein the molar ratio of urea to aluminum ions in the aluminum salt is 12: 1; the total concentration of the magnesium salt and the aluminum salt in the mixed solution is 0.04-0.5 mol/L.

4. The preparation method according to claim 1, wherein the temperature of the hydrothermal coprecipitation is 80-120 ℃ and the reaction time is 24-48 h.

5. The preparation method of claim 1, wherein the diameter of the magnesium-aluminum hydrotalcite micro-flake is 6 +/-2 μm, and the morphology of the micro-flake is hexagonal nano-flake.

6. The preparation method of claim 1, wherein the weight percentage of the magnesium aluminum hydrotalcite microchip, the type I collagen and the polymethyl methacrylate bone cement is 10-20% to 70% based on 100% of the total weight percentage of the magnesium aluminum hydrotalcite microchip, the type I collagen and the polymethyl methacrylate bone cement.

7. The preparation method of claim 1 or 6, wherein the dosage ratio of the methyl methacrylate monomer to the total mass of the magnesium aluminum hydrotalcite micro-sheets, the type I collagen and the polymethyl methacrylate bone cement is 5mL:10 g.

8. The method according to claim 1, wherein the polymerization reaction is carried out at room temperature for 15-25 min.

9. The bioactive bone cement composite material prepared by the preparation method of any one of claims 1 to 8, which comprises the blended polymethyl methacrylate bone cement, the magnesium aluminum hydrotalcite microchip and the type I collagen.

10. Use of the bioactive bone cement composite of claim 9 in the preparation of a bone defect regenerative repair material.