A method for preparing a medical material of a mineralized collagen scaffold by using a polymer-induced liquid precursor

Abstract

The application provides a preparation method of a mineralized collagen scaffold medical material prepared by a polymer-induced liquid precursor, and phosphine polyethyleneimine and polyacrylic acid are used in a high-concentration calcium phosphate solution to prepare a polymer-induced liquid precursor; collagen fiber scaffolds are immersed in the polymer-induced liquid precursor to be mineralized to obtain the mineralized collagen scaffold medical material; the polymer-induced liquid precursor comprises the following components in percentage by mass: 1-3% of phosphine polyethyleneimine, 4-6% of polyacrylic acid, 0.5-1% of calcium chloride, 2.5-3% of disodium hydrogen phosphate and the balance of water. The PILP in the application can penetrate into the inside of the collagen scaffold to be mineralized, the mineralization effect of the collagen scaffold is enhanced, the biocompatibility and osteogenic activity of the collagen scaffold are improved, and the application prospect of the PILP in the field of bone repair materials is wide.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to a method for preparing mineralized collagen scaffolds using polymer-induced liquid precursors. Background Technology

[0002] Trauma, surgery, and osteoporosis can all lead to bone defects. Most bone defects can be repaired through the body's self-healing process. However, when the bone defect is large enough to exceed the body's self-repair capabilities, suitable bone repair materials must be used to replace the bone and reconstruct the defect. Autologous bone grafting is a commonly used clinical method, but it has drawbacks such as infection, donor site limitations, and the risk of new trauma, making it unsuitable for widespread application. The characteristics and manufacturing techniques of collagen / inorganic materials for constructing tissue-engineered bone necessitate the development of new materials for bone repair. Bone repair materials can generally be categorized into medical-grade metallic materials, bioceramic materials, and medical-grade polymer materials.

[0003] Polymer bone repair materials are divided into two categories: synthetic and natural polymer bone repair materials. Synthetic polymer bone repair materials mainly include polyamide, polyethylene, and polylactic acid. If the polymer material has a certain degree of degradability, it will degrade into harmless small molecule products after implantation into the human body, without affecting the normal growth of human tissues. However, synthetic polymer bone repair materials have poor mechanical properties. Natural polymer bone repair materials mainly include chitosan, fibrin, gelatin, and collagen, which have good biocompatibility. Among them, collagen is a large molecular structural protein widely present in organisms and is the most important organic component of bone tissue. It has good biocompatibility, good degradability, and low immunogenicity. It has been widely used in biomedical materials, but due to the rapid degradation and absorption rate of collagen, coupled with its limitations in mechanical and expansion properties, it is difficult to use it alone as a bone repair material for clinical bone defect repair.

[0004] In recent years, biomimetic methods have been developed, and the use of biomimetic methods to manufacture biomimetic mineralized collagen fibers has attracted widespread attention and has become a new method for achieving mineralization within collagen fibers. This method introduces hydroxyapatite, which can improve the mechanical properties of collagen fibers to meet biomedical requirements. The most common method for preparing biomimetic mineralized collagen fibers is to use the traditional nucleation and growth method of hydroxyapatite to mineralize recombinant collagen scaffolds. Specifically, a collagen matrix is ​​placed in a mineralization solution (such as simulated body fluid SBF or modified SBF) to initiate the nucleation and growth of hydroxyapatite. However, this traditional biomimetic mineralization method produces large calcium phosphate crystals that cannot penetrate into the collagen fiber for mineralization, resulting in low mineralization levels and low mineral content in the prepared mineralized collagen fibers, which is insufficient to meet the requirements of medical mineralized collagen scaffolds. Applying polymer-induced liquid precursors (PILPs) to collagen fibers can achieve mineralization within the collagen fibers, resulting in higher mineralization and a greater mineral content. The precursor is formed by the combination of anionic polymers and the ionic components of hydroxyapatite. It is attracted into the grooves of the collagen fibers through capillary action. After water is removed from the metastable amorphous precursor, the precursor can solidify and crystallize into hydroxyapatite inside the collagen fibers. Summary of the Invention

[0005] This invention proposes to use polymer-induced liquid precursors (PILPs) to mineralize collagen scaffolds, obtaining mineralized collagen scaffolds with internal mineralization, enhancing the mineralization effect of collagen fibers, and improving the biocompatibility and osteogenic activity of the collagen scaffolds. The purpose of this invention is to provide a method for preparing mineralized collagen scaffold medical materials using polymer-induced liquid precursors. The method employs phosphoethyleneimine (PPEI) and polyacrylic acid (PAA) to induce the mineralization of amorphous calcium phosphate precursors in the collagen scaffolds: First, polyethyleneimine (PEI) is phosphonated to impart phosphonic acid groups, enhancing the polymer's calcium chelating ability. Then, it is combined with PAA in a calcium phosphate solution to prepare PILPs. The collagen scaffolds are then placed in the PILPs for mineralization, resulting in high mineralization levels, cell compatibility, and osteogenic activity in the collagen scaffolds.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing a mineralized collagen scaffold medical material using a polymer-induced liquid precursor includes the following steps:

[0008] S1 added phosphine-modified polyethyleneimine (PPEI) and polyacrylic acid (PAA) to a solution of calcium chloride and disodium hydrogen phosphate to prepare a polymer-induced liquid precursor (PILP);

[0009] S2 involves immersing a collagen fiber scaffold in PILP and then mineralizing it in an oven at 35-40°C for 1-7 days to obtain a mineralized collagen scaffold medical material.

[0010] The PILP comprises the following components by mass percentage: PPEI 1-3%; PAA 4-6%; CaCl2 0.8%; Na2HPO4 2.8%; water balance.

[0011] After PILP mineralization treatment, the collagen fiber scaffold achieves mineralization inside the collagen fibers, resulting in a higher degree of mineralization and good biocompatibility and osteogenic activity.

[0012] The phosphoethyleneimine was obtained by phosphoacidification of polyethyleneimine with phosphorous acid.

[0013] The preparation method of the phosphoethyleneimine includes the following steps: 2-4g of polyethyleneimine is dissolved in deionized water and placed in a three-necked flask equipped with a reflux condenser. 4-6g of dissolved phosphorous acid solution and 6-8g of hydrochloric acid solution are added to the three-necked flask in sequence. The mixture is heated to 60°C in a nitrogen atmosphere and reacted for 30 minutes. Then, 4-6g of formaldehyde solution is added dropwise to the three-necked flask. The mixture is heated to 100°C and reacted for 72 hours. The reaction solution is extracted and separated, and then rotary evaporated to remove the residual aqueous phase in the solution to obtain PPEI.

[0014] The preparation method of the polymer-induced liquid precursor specifically includes the following steps: dissolving CaCl2 in deionized water to obtain a CaCl2 solution, denoted as solution A; dissolving phosphine-modified polyethyleneimine and polyacrylic acid in Na2HPO4 solution, denoted as solution B; adding solutions A and B sequentially to a three-necked flask, mixing thoroughly, and adding sodium hydroxide solution to adjust its pH to 7-8 to obtain the polymer-induced liquid precursor (PILP).

[0015] The collagen fiber scaffold was prepared in a mold using a freeze-drying method.

[0016] The preparation method of the collagen fiber scaffold includes the following steps: Bovine Achilles tendon type I collagen powder is dissolved in a 0.05–1 mol / L acetic acid solution and stirred thoroughly in an ice-water bath for 3 days to ensure complete dissolution, yielding a collagen fiber solution. The prepared collagen fiber solution is placed in a high-speed centrifuge, centrifuged and filtered to remove the white solid, obtaining the supernatant. This supernatant is placed in a mold, and the pH is adjusted to 7–8 using concentrated ammonia. Then, it is cross-linked using glutaraldehyde solution. The sample mold is frozen and then freeze-dried in a freeze dryer to remove excess moisture, yielding the collagen fiber scaffold.

[0017] S2 also includes washing and freeze-drying the mineralized collagen fiber scaffold.

[0018] The beneficial effects of this invention are as follows:

[0019] 1. The method for preparing mineralized collagen scaffolds using polymer-induced liquid precursors is simple. Compared with traditional biomimetic mineralization methods, this method is fast and efficient, and can achieve internal mineralization of collagen fibers, resulting in scaffold samples with higher mineralization.

[0020] 2. Phosphoethyleneimine and polyacrylic acid work synergistically to achieve higher stability and higher calcium ion chelation. The same mass of polymer can stabilize more calcium and phosphorus ions, thereby achieving a better mineralization effect on collagen fibers, and further improving the cell compatibility and osteogenic activity of mineralized collagen fibers.

[0021] 3. The mineralized collagen scaffold prepared by this invention has good biocompatibility and osteogenic activity, and has broad application prospects in bone repair materials. Attached Figure Description

[0022] Figure 1 The Fourier transform infrared (FT-IR) spectrum of phosphonate-modified polyethyleneimine (PPEI) is shown.

[0023] Figure 2 The 1H NMR spectrum of phosphonomodified polyethyleneimine (PPEI) 1 1H-NMR spectrum.

[0024] Figure 3 (a) is the X-ray photoelectron spectroscopy (XPS) spectrum of calcium atoms in PILP, and (b) is the X-ray photoelectron spectroscopy (XPS) spectrum of phosphorus atoms in PILP.

[0025] Figure 4 (a) is the frequency scan rheological test diagram of PILP, and (b) is the strain scan rheological test diagram of PILP.

[0026] Figure 5 Transmission electron microscopy (TEM) images of PILP crystallization at different times: (a) 1 day, (b) 3 days, and (c) 7 days.

[0027] Figure 6 X-ray diffraction (XRD) spectra of PILP crystallization at different times.

[0028] Figure 7 (a) Thermogravimetric analysis (TGA) plots of NMC, TMC and BMC, and (b) Thermogravimetric analysis (DTG) plots of NMC, TMC and BMC.

[0029] Figure 8 Transmission electron microscopy (TEM) images of (a) NMC, (b) TMC and (c) BMC seven days after mineralization.

[0030] Figure 9The X-ray diffraction (XRD) spectra of NMC, TMC and BMC seven days after mineralization.

[0031] Figure 10 Cytotoxicity assays for NMC, TMC, and BMC.

[0032] Figure 11 Osteogenic activity testing for NMC, TMC, and BMC. Detailed Implementation

[0033] The following specific embodiments further illustrate the essential features and significant advancements of the present invention. However, the content of the present invention is not limited to the following embodiments and can be adjusted according to actual circumstances.

[0034] Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (NMR) were used to analyze the phosphonic acid groups in phosphonate-modified polyethyleneimine (PPEI). The calcium chelating ability of the polymer was determined by EDTA complexometric titration. X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental content of the prepared PILP, rheological properties were analyzed by rheometer, and crystallinity was analyzed by transmission electron microscopy (TEM) and X-ray diffraction (XRD). Thermogravimetric analysis (TGA) was used to analyze the mineral content of the mineralized collagen scaffold, and TEM and XRD were used to analyze the degree of mineralization of the collagen fiber scaffold.

[0035] The polymer's calcium chelating ability was tested as follows: 0.2 g of dried polymer was weighed into a 150 mL three-necked flask, dissolved in 10 mL of deionized water, and the pH was adjusted to neutral using sodium hydroxide (NaOH) solution. Then, 10 mL of ammonia and 40 mL of 0.05 M CaCl2 solution were added, and the mixture was allowed to stand at room temperature for 30 minutes before filtration to remove insoluble matter. 10 mL of the filtrate was then placed in a three-necked flask, and 12.5 mL of deionized water, 5 mL of NH3-NH4Cl buffer solution, and 0.1 g of EBT indicator were added sequentially. The mixture was shaken until the solid was completely dissolved. The mixture was titrated with 0.01 M EDTA-2Na standard solution until the solution changed abruptly from wine red to blue. The volume of standard solution used was recorded as V1 mL. A blank control experiment was conducted with no polymer, and the volume of standard solution consumed was recorded as V2 mL. The chelating amount of calcium ions by the polymer, W mg, can be calculated using the following formula.

[0036]

[0037] The cytotoxicity test was performed as follows: First, a 5 mg / mL MTT solution was prepared and stored at 4°C in the dark. 10 mg of mineralized collagen scaffold samples (NMC, TMC, and BMC) were each immersed in 3 mL of α-MEM medium and incubated at 37°C for 24 h to obtain the extract. The extract was then diluted using a two-fold dilution method. MC3T3-E1 cells were introduced at a density of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μl per well in 96-well plates, with three replicates. After cell attachment, the culture medium was aspirated, and 100 μl of a gradient concentration of extraction buffer was added to each well. After incubation at 37°C for 1 and 3 days, 10 μl of MTT solution was added to each well, and the cells were incubated at 37°C for 4 hours. After aspirating the extraction buffer, 100 μl of DMSO solution was added to each well and mixed. The OD value of each well was measured at 490 nm using a microplate reader, and the relative cell proliferation rate of each well was calculated.

[0038] Alkaline phosphatase (ALP) and staining assays in MC3T3-E1 cells: MC3T3-E1 cells were seeded in 24-well plates at a density of 1×10⁻⁶ cells / well. 5 Three scaffold samples were placed in each well and co-cultured with cells, with the cell culture medium changed every two days. Alkaline phosphatase staining was performed on days 7 and 14 of co-culture, and the stained samples were observed under an optical microscope. The specific procedure was as follows: after removing the scaffold sample from each well, adherent cells were washed three times with PBS buffer, then fixed with 4% paraformaldehyde solution (m / v) for 30 minutes, followed by three washes with PBS buffer. An appropriate amount of BCIP / NBT staining working solution (10 mL alkaline phosphatase chromogenic buffer, 33 μL BCIP solution, and 66 μL NBT solution) was added to ensure adequate coverage of the sample. After incubation in the dark for 30 minutes, the staining working solution was removed, and the cells were washed three times with PBS buffer to terminate the chromogenic reaction.

[0039] Example

[0040] The first step involved introducing phosphonic acid groups onto polyethyleneimine to prepare phosphonated polyethyleneimine (PPEI), as shown in reaction formula (Ⅰ). Polyethyleneimine (PEI, 3 g) was dispersed in 10 mL of water and added to a three-necked flask connected to a reflux condenser. Dissolved phosphorous acid (H3PO3, 5.72 g) and 37% hydrochloric acid (HCl, 6.86 g) were added dropwise to the flask, and the mixture was heated to 60 °C under nitrogen for 30 minutes. A 37% formaldehyde solution (HCHO, 5.66 g) was slowly added to the flask at 100 °C, and the reaction was carried out under a nitrogen atmosphere for 72 hours. The resulting sample was washed with diethyl ether and separated using a separating funnel to obtain an aqueous solution of PPEI. Water was then removed under vacuum to obtain the PPEI polymer, which was a viscous, brownish-yellow oily substance.

[0041]

[0042] The second step involved preparing a polymer-induced liquid precursor (PILP). The calcium chelating ability of the phosphonated polyethyleneimine prepared in the first step with polyacrylic acid was determined, and the results are shown in Table 1. 2.0 mL of 0.05 M CaCl2 solution was designated as solution A. 2.0 mL of 0.05 M Na2HPO4 solution and 0.05 g of PPEI were mixed with 1 mL of solution containing 0.3 g of PPEI. -1 The solution mixture of PAA is denoted as solution B. 3 mL of solution B is gradually added to 2.0 mL of solution A, and after vigorous stirring, the pH is adjusted to 7.4 with NaOH solution to prepare PILP.

[0043] The third step is the preparation of mineralized collagen scaffolds. Type I bovine Achilles tendon collagen powder was dissolved in a 0.05 mol / L acetic acid solution and stirred thoroughly for 3 days at 0°C to ensure complete dissolution, yielding a collagen fiber solution. The prepared collagen fiber solution was placed in a high-speed centrifuge, centrifuged, filtered to remove the white solid, and the supernatant was placed in a mold. The pH was adjusted to 7 using concentrated ammonia, and then cross-linked with glutaraldehyde solution. The sample mold was frozen and then freeze-dried to remove excess moisture, yielding the collagen fiber scaffolds. The prepared collagen scaffolds were then immersed in deionized water, SBF solution, and the PILP prepared in the second step, respectively. After mineralization at 37°C for 7 days, the samples were removed, washed with deionized water, and freeze-dried to obtain non-mineralized (NMC), conventionally mineralized (TMC), and biomimetic mineralized (BMC) collagen scaffolds.

[0044] The properties of PPEI prepared by the above method are as follows:

[0045] Fourier transform infrared spectroscopy (e.g.) Figure 1 As shown in the figure, the characteristic absorption peaks of NH2 and NH association are located at 3361 cm⁻¹. -1 and 3280cm -1 The characteristic absorption peak of the methylene stretching vibration is at 2813 cm⁻¹. -1 The characteristic absorption peak of the NH bending vibration is at 1621 cm⁻¹, while the characteristic absorption peaks of PO and P=O are located at 1178 cm⁻¹. -1 and 993cm -1 Location. Analyzed using nuclear magnetic resonance hydrogen spectroscopy (e.g., ... Figure 2 As shown in the figure, a characteristic doublet of -N-CH2-PO3H2 is observed at δ = 3.26 ppm and 3.17 ppm. This demonstrates the introduction of phosphonic acid groups onto polyethyleneimine.

[0046] Table 1 Polymer Chelating Ability

[0047]

[0048] The calcium chelating ability of PPEI and PAA was determined (as shown in Table 1). It was found that using 0.3g PAA and 0.05g PPEI can achieve a high calcium chelating ability with a relatively small amount of polymer.

[0049] The properties of the PILP prepared by the above method are as follows:

[0050] Through XPS spectra (e.g.) Figure 3 As shown, the peaks at electron binding energies of 345.5 eV and 349.2 eV are characteristic peaks of Ca, and the peak at electron binding energy of 132.1 eV is a characteristic peak of P, proving that the prepared PILP successfully chelates calcium atoms and phosphate ions.

[0051] Rheological testing (e.g.) Figure 4 As shown, the prepared PILP is a viscous, flowable gel-like material exhibiting linear viscoelasticity within a strain range of 0.01% to 100%, indicating a wide processing range within this range. At low scanning frequencies, the PILP material is a fluid, gradually transforming into a solid at higher scanning frequencies.

[0052] The crystallization of PILP at different time points was studied using transmission electron microscopy (e.g., Figure 5 As shown, copper mesh with carbon support mold was immersed in PILP and incubated at 37°C for 1, 3 and 7 days. The sample gradually transformed from a clustered morphology to nanorod-shaped crystals, indicating that amorphous calcium phosphate in PILP can gradually transform into hydroxyapatite crystals over time.

[0053] X-ray diffraction analysis was performed on PILP at 1, 3, and 7 days (e.g. Figure 6 As shown in the figure, the peak intensities detected at 2θ = 25.8°, 31.7°, and 34° gradually increased with the extension of incubation time. These peaks correspond to the crystal planes (211), (300), and (002) of HAP, further confirming the transformation of amorphous calcium phosphate into HAP in the PILP material.

[0054] The mineralized collagen scaffold prepared by the above method has the following properties:

[0055] Thermogravimetric analysis was used to analyze the mineral content of three types of mineralized scaffolds (e.g., Figure 7 As shown in the figure, the results indicate that the mineral content of the BMC stent is much higher than that of the NMC and TMC stents.

[0056] like Figure 8 As shown, PILP can achieve intracellular mineralization of collagen fibers, resulting in a better mineralization effect on collagen scaffolds.

[0057] like Figure 9 As shown, the BMC scaffold has obvious crystallization peaks at 2θ = 27° and 31°, which correspond to the characteristic peaks of hydroxyapatite, proving that the collagen scaffold obtained by mineralization of PILP material has a higher degree of mineralization compared with the other two types of scaffolds.

[0058] To demonstrate the biocompatibility of the prepared material, the MTT test results are as follows: Figure 10 As shown, after co-culturing with MC3T3-E1 cells for one day and three days, the cell survival rate was above 80%, proving that the prepared collagen scaffold had no obvious cytotoxicity.

[0059] The osteogenic capacity of the collagen scaffold was characterized, and the results are as follows: Figure 11 As shown, ALP activity was highest in the BMC scaffold, which can promote osteogenic differentiation of pre-osteoblasts.

Claims

1. A method for preparing mineralized collagen scaffold medical materials using polymer-induced liquid precursors, characterized in that: Includes the following steps: S1 adds phosphine-modified polyethyleneimine and polyacrylic acid to a solution of calcium chloride and disodium hydrogen phosphate to prepare a polymer-induced liquid precursor; S2 involves immersing a collagen fiber scaffold in a polymer-induced liquid precursor and then mineralizing it in an oven at 35-40°C for 1-7 days to obtain a mineralized collagen scaffold medical material. The polymer-induced liquid precursor comprises the following components by mass percentage: phosphoethyleneimine 1-3%; polyacrylic acid 4-6%; calcium chloride 0.5-1%; disodium hydrogen phosphate 2.5-3%; water balance; the phosphoethyleneimine is obtained by phosphoacidification of polyethyleneimine with phosphorous acid, and the reaction formula is (I): （Ⅰ）。 2. The method for preparing mineralized collagen scaffold medical materials using polymer-induced liquid precursors as described in claim 1, characterized in that: The preparation method of the phosphoethyleneimine includes the following steps: 2-4 g of polyethyleneimine is dissolved in deionized water and placed in a three-necked flask equipped with a reflux condenser. 4-6 g of dissolved phosphorous acid solution and 6-8 g of hydrochloric acid solution are added to the three-necked flask sequentially. The mixture is heated to 60 °C in a nitrogen atmosphere and reacted for 30 minutes. Then, 4-6 g of formaldehyde solution is added dropwise to the three-necked flask. The mixture is heated to 100 °C and reacted for 72 hours. The reaction solution is extracted and separated, and then rotary evaporated to remove the residual aqueous phase in the solution to obtain phosphoethyleneimine.

3. The method for preparing mineralized collagen scaffold medical materials using polymer-induced liquid precursors as described in claim 1, characterized in that: The preparation method of the polymer-induced liquid precursor described in S1 specifically includes the following steps: dissolving calcium chloride in deionized water to obtain a calcium chloride solution, denoted as solution A; dissolving phosphoethyleneimine and polyacrylic acid in disodium hydrogen phosphate solution, denoted as solution B; adding solutions A and B sequentially to a three-necked flask, mixing thoroughly, and adding sodium hydroxide solution to adjust its pH to 7-8 to obtain the polymer-induced liquid precursor.

4. The method for preparing mineralized collagen scaffold medical materials using polymer-induced liquid precursors as described in claim 1, characterized in that: The collagen fiber scaffold was prepared in a mold using a freeze-drying method.

5. The method for preparing mineralized collagen scaffold medical materials using polymer-induced liquid precursors as described in claim 4, characterized in that: The preparation method of the collagen fiber scaffold includes the following steps: 300~600 mg of bovine Achilles tendon type I collagen powder is dissolved in 100 mL of 0.05~1 mol / L acetic acid solution, and stirred thoroughly for 3 days in an ice-water bath to ensure complete dissolution, thereby obtaining a collagen fiber solution; the prepared collagen fiber solution is placed in a high-speed centrifuge, centrifuged and filtered to remove the white solid, and the supernatant is obtained. The supernatant is placed in a mold, and the pH value is adjusted to 7 using concentrated ammonia water. Then, it is cross-linked with glutaraldehyde solution. The sample mold is frozen and then placed in a freeze dryer for freeze drying to remove excess water, thereby obtaining the collagen fiber scaffold.

6. The method for preparing mineralized collagen scaffold medical materials using polymer-induced liquid precursors as described in claim 1, characterized in that: S2 also includes washing and freeze-drying the mineralized collagen fiber scaffold.

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