Medical modified titanium material and preparation method and application thereof

Abstract

The invention discloses a medical modified titanium material as well as a preparation method and application thereof, belongs to the technical field of medical materials, and solves the problem that a titanium implant cannot have the effects of removing aged MSCs and promoting osteogenic differentiation of the MSCs at the same time. The preparation method of the medical modified titanium material comprises the following steps that after a titanium implant is cleaned and subjected to an acid etching reaction, the titanium implant reacts in a silane coupling agent, then cleaning and drying are conducted in sequence, and the amino-modified micron-structure titanium implant is obtained; the preparation method comprises the following steps: mixing dopamine, calcium ions and a glutaminase inhibitor in a solvent to obtain a mixed solution; reacting the amino-modified micron-structure titanium implant in the mixed solution, cleaning again, and drying; and alternately immersing the titanium powder into chitosan and a polyanion glutamic acid donor to obtain the medical modified titanium material. Aiming at the metabolism difference of senescent and non-senescent MSCs, the osteogenic differentiation of the MSCs is remarkably promoted while the senescent MSCs are specifically removed, so that the osseointegration of the senescent patient is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and more specifically to a medical modified titanium material and a preparation method and application thereof. Background Art

[0002] Existing technologies often improve the surface roughness of titanium by simulating the hierarchical micro-nano composite structure of bone tissue to promote bone integration, and a series of surface-modified titanium developed for this purpose has also been commercialized in clinical practice. Higher roughness greatly affects the material's ability to adsorb proteins in the physiological environment, thereby promoting the adhesion of mesenchymal stem cells (MSCs). In addition, the topological structure of the material has also been shown to be recognized by MSCs, thereby affecting multiple signaling pathways (such as Wnt signaling, MAPK signaling, etc.) and promoting osteogenic differentiation. However, the regeneration and repair of bone tissue are severely affected by aging, and ignoring the existence of aging MSCs may lead to misjudgment of titanium implants. For example: micro-nanostructured titanium implants can also promote the occurrence of senescence-associated secretory phenotype (SASP) in aging MSCs through the activation of the p38MAPK signaling pathway (a non-classical osteogenic pathway), thereby accelerating tissue aging. This complicates the bone integration of titanium implants in elderly patients.

[0003] Existing technologies often enhance the osteogenic differentiation of MSCs by constructing different micro-nano composite structures and loading different metal ions or drugs (CN118345352A, CN117414468A, CN115501387A, CN114129773A, etc.). Some cutting-edge studies also attempt to promote bone repair in elderly patients by inhibiting the secretion of SASP factors or eliminating senescent cells (loading anti-aging drugs).

[0004] As for the existing technology to promote osteogenic differentiation of MSCs, it is difficult to comprehensively and even correctly evaluate the bone integration efficiency of implants due to the neglect of aging MSCs and their special physiological characteristics, which may lead to implant failure. On the other hand, the use of anti-aging drugs (or SASP factor inhibitors) in some cutting-edge research is also difficult to avoid its negative impact on normal cell function (known factors include anemia, thrombocytopenia and cytotoxicity), which limits the bone repair process under injury. Titanium implants in existing technologies cannot simultaneously remove aging MSCs and promote osteogenic differentiation of MSCs. Summary of the invention

[0005] The present invention provides a medical modified titanium material and a preparation method and application thereof, so as to solve the problem that titanium implants in the prior art cannot simultaneously remove aged MSCs and promote the osteogenic differentiation of MSCs.

[0006] In a first aspect, the present invention provides a method for preparing a medical modified titanium material, comprising the following steps: cleaning a titanium implant and then subjecting it to an acid etching reaction to obtain a microstructured titanium implant; reacting the microstructured titanium implant in a silane coupling agent and then washing and drying the microstructured titanium implant in sequence to obtain an amino-modified microstructured titanium implant; mixing dopamine, calcium ions and a glutaminase inhibitor in a solvent to obtain a mixed solution; reacting the amino-modified microstructured titanium implant in the mixed solution, and then washing and drying the mixed solution again to obtain a micro-nano composite structured titanium implant loaded with a glutaminase inhibitor; alternately immersing the micro-nano composite structured titanium implant loaded with a glutaminase inhibitor in chitosan and a polyanion glutamate donor to obtain the medical modified titanium material.

[0007] As a possible implementation, the acid etching reaction includes the steps of: reacting the titanium implant in hydrofluoric acid and then reacting in a sulfuric acid solution to obtain the microstructured titanium implant; and / or, in the mixed solution, the molar ratio of the dopamine, the calcium chloride and the glutaminase inhibitor is 10:1:0.1-0.2; and / or, the solvent of the mixed solution is: a mixed solution of trimethylammonium hydrochloride buffer, dimethyl sulfoxide and a polymerization initiator in a volume ratio of 50:45-55:0.5-1.5; wherein the polymerization initiator includes a component of persulfate.

[0008] As a possible implementation manner, the mass percentage of the titanium implant in the hydrofluoric acid is 0.2wt% to 3wt%; and / or the mass percentage of the titanium implant in the sulfuric acid is 65wt% to 70wt%.

[0009] As a possible implementation, the calcium ion is one or a combination of calcium chloride, calcium nitrate, and calcium gluconate; and / or the glutaminase inhibitor is one or a combination of BPTES, Telaglenastat, L-Methionine-DL-sulfoximine, and Trivalent hydroxyarsinothricin; and / or the silane coupling agent is one or a combination of 3-aminopropyltriethoxysilane solution, γ-aminopropylmethyldiethoxysilane, silane-polyethylene glycol-amino, and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane; and / or the polyanionic glutamate donor is one or a combination of polyglutamic acid, polyglutamic acid-γ-benzyl ester, polyglutamic acid-γ-methyl ester, polyglutamic acid-aspartic acid copolymer, and polyglutamic acid-γ-benzyl ester-polyethylene glycol copolymer.

[0010] As a possible implementation, the cleaning process includes the steps of: ultrasonic cleaning, drying; and / or, the cleaning is all ultrasonic treatment.

[0011] In a second aspect, the present invention provides a medical modified titanium material prepared by the preparation method described in any possible implementation of the first aspect.

[0012] As a possible implementation manner, it is loaded with a glutaminase inhibitor and a polyanionic glutamate donor; the in vivo degradation product of the polyanionic glutamate donor contains glutamate.

[0013] In a third aspect, the present invention provides a use of a medical modified titanium material prepared by the preparation method described in any possible implementation of the first aspect or a medical modified titanium material described in any possible implementation of the second aspect in preparing a titanium implant.

[0014] As a possible implementation method, the titanium implant is used in orthopedic implant surgery for patients with degenerative diseases caused by aging.

[0015] As a possible implementation, the degenerative diseases caused by aging include diabetes and / or osteoporosis.

[0016] Compared with the prior art, the medical modified titanium material provided by the present invention targets the metabolic differences between aged and non-aged MSCs, achieves specific removal of aged MSCs and significantly promotes the osteogenic differentiation of MSCs, thereby promoting bone integration in elderly patients.

[0017] Based on the basic differences in metabolism between senescent and non-senescent MSCs, the present invention affects the intracellular pH stability of senescent MSCs by inhibiting glutamine hydrolysis, and specifically induces their apoptosis; at the same time, by supplementing its downstream hydrolysis product glutamate, without affecting the specific removal of senescent MSCs by the implant, it significantly promotes the anabolism of non-senescent MSCs and promotes their osteogenic differentiation. The present invention simultaneously achieves the dual purpose of removing senescent cells and promoting osteogenic differentiation, and is expected to promote the bone integration of implants in elderly patients, and has broad application prospects in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1Schematic diagram of the preparation process of the medical modified titanium material provided in the embodiment of the present invention, wherein Ti represents titanium, Acid etching represents acid etching reaction, MT represents microstructure, APTES represents 3-aminopropyltriethoxysilane solution, MT-NH2 represents amino-modified microstructure titanium implant, APS represents ammonium persulfate, Tris-HCl represents trimethylamine hydrochloride, Dopamine represents dopamine, Ca represents 2+ represents calcium ion, BPTES represents glutaminase inhibitor, chi / PGA represents chitosan / polyglutamic acid, LBL represents layer-by-layer self-assembly, and Polydopamine represents polydopamine.

[0020] Figure 2 SEM spectra of Ti, MT, BPTES@MNT and BPTES@MNT / PGA provided in the embodiments of the present invention, wherein the first row is the SEM spectrum at 2000 times (scale 40 μm), and the second row is the SEM spectrum at 10000 times (scale 8 μm).

[0021] Figure 3 AFM images of Ti, MT, BPTES@MNT and BPTES@MNT / PGA provided in the embodiments of the present invention. The surface Ra roughness data of different samples are shown at the bottom of the corresponding image (n=3).

[0022] Figure 4 Water contact angles of Ti, MT, BPTES@MNT and BPTES@MNT / PGA provided in the embodiments of the present invention (n=3).

[0023] Figure 5 High-resolution XPS spectra of the N spectrum (left) and C spectrum (right) of BPTES@MNT / PGA provided in the embodiments of the present invention.

[0024] Figure 6 The release profile results of the main components of BPTES@MNT / PGA provided in the embodiment of the present invention at different pH values ​​(n=3), wherein Ca 2+ represents calcium ion, BPTES represents glutaminase inhibitor, and Glu represents glutamate.

[0025] Figure 7 Annexin V-FITC analysis results of Ti, MT, BPTES@MNT and BPTES@MNT / PGA provided in the embodiments of the present invention, wherein the first row is senescent MSCs and the second row is non-senescent MSCs.

[0026] Figure 8Cell viability results of Ti, MT, BPTES@MNT and BPTES@MNT / PGA provided in the examples of the present invention (n=6), wherein the upper figure is for aged MSCs and the lower figure is for non-aged MSCs.

[0027] Fig. 9 Representative images of Ti, MT, BPTES@MNT and BPTES@MNT / PGA Alizarin Red staining of non-senescent MSCs after 14 days of incubation provided in the embodiments of the present invention.

[0028] Fig.10 The embodiment of the present invention provides Fig. 9 Quantitative analysis of (n=6).

[0029] Fig.11 The present invention is a schematic diagram of the experimental process of implantation of the medical modified titanium material in rats according to an embodiment of the present invention, wherein 18-month-old means 18 months old, Day 0 means day 0, Week 6 means week 6, Surgery means surgical operation, micro-CT means micro-CT, and Masson means Masson staining.

[0030] Fig.12 The three-dimensional μ-CT reconstructed images of the surroundings of Ti, MT, BPTES@MNT and BPTES@MNT / PGA provided in the embodiments of the present invention are respectively used as implants.

[0031] Fig.13 Representative images of Masson's staining around Ti, MT, BPTES@MNT and BPTES@MNT / PGA implants provided in the embodiments of the present invention.

[0032] Fig.14 The quantitative results of histological staining of Ti, MT, BPTES@MNT and BPTES@MNT / PGA as implants provided in the embodiments of the present invention, wherein the left figure is the BIC quantitative result (n=6), and the right figure is the new bone thickness quantitative result (n=12) DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In order to solve the problem that titanium implants in the prior art cannot simultaneously remove aged MSCs and promote osteogenic differentiation of MSCs, the present invention provides a preparation experiment of a medical modified titanium material; Figure 1 The schematic diagram of the preparation process of medical modified titanium materials is given as an example. Figure 1 The process shown is used to prepare medical modified titanium material.

[0035] The embodiment of the present invention provides the performance characterization of the medical modified titanium material. It can be seen that in the medical modified titanium material prepared by the present invention, polydopamine and the titanium substrate are covalently cross-linked through chemical bonds, and BPTES is loaded on the implant surface by π-π stacking. This covalent cross-linking method improves the stability of the coating, and the non-covalent drug loading method ensures the smooth release of the drug.

[0036] Furthermore, the present invention embodiment tested the effect of medical modified titanium materials, and it was found that the medical modified titanium material BPTES@MNT / PGA provided by the present invention can significantly induce apoptosis of senescent MSCs, and has no obvious cytotoxicity to non-senescent MSCs, and has a significant effect of promoting the proliferation of non-senescent MSCs; this confirms that BPTES@MNT / PGA can effectively remove senescent cells while maintaining the normal function of non-senescent cells. Furthermore, BPTES@MNT / PGA showed the strongest ability to promote osteogenic differentiation in non-senescent MSCs, which ensured the osteointegration effect of BPTES@MNT / PGA.

[0037] Furthermore, the present invention provides a rat implantation experiment, and it can be seen that the medical modified titanium material BPTES@MNT / PGA provided by the present invention exhibits the strongest bone integration ability in aged rats, and the new bone tissue around the implant is significantly thicker and the bone-implant contact rate is significantly higher. This shows that BPTES@MNT / PGA is expected to promote bone integration in elderly patients, confirming its broad application prospects in the biomedical field.

[0038] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0039] Example 1

[0040] This embodiment provides a preparation experiment of a medical modified titanium material.

[0041] The titanium implant (Ti, Alfa Aesar, 013975, 10 mm×10 mm) was ultrasonically cleaned and dried, and then immersed in a hydrofluoric acid solution (3 wt%, 10 mL, 2 min, RT) and a sulfuric acid solution (66 wt%, 10 mL, 5 min, 80°C) for acid etching. After the reaction, a medical modified titanium material MT was obtained. The medical modified titanium material MT was placed in a 3-aminopropyltriethoxysilane solution (3 wt%, 10 mL, 24 h, RT) for reaction to obtain a medical modified titanium material MT-NH2. The medical modified titanium material MT-NH2 was thoroughly cleaned and dried by ultrasound, and then placed in a mixed solution (the solvent is V Tris-HCl buffer (10 mM, pH 8.5):V DMSO =1:1) to obtain the medical modified titanium material BPTES@MNT; the medical modified titanium material BPTES@MNT was alternately immersed in chitosan (0.5wv%, solvent is 0.5% glacial acetic acid aqueous solution) and polyglutamic acid solution (1% (w / v), solvent is deionized water) to obtain BPTES@MNT / PGA.

[0042] Example 2

[0043] This embodiment provides a performance characterization experiment of a medical modified titanium material.

[0044] The titanium implant (Ti), medical modified titanium material MT, medical modified titanium material BPTES@MNT and BPTES@MNT / PGA in Example 1 were subjected to SEM analysis, surface roughness analysis and hydrophilicity analysis, respectively, and the following results were obtained: Figure 2 to Figure 4 The results shown in the figure are as follows; and the surface interface XPS analysis of BPTES@MNT / PGA and the release analysis of its main components are performed, and the results are as follows Figure 5-6 The results are shown in Table 1. The data are expressed as mean ± standard deviation (*p<0.05, **p<0.01, ***p<0.001).

[0045] Depend on Figure 2 It can be seen that BPTES@MNT presents a significant micro-nano composite structure, and the self-assembly of the multilayer film forms a visible coating on the micro-nano structure of BPTES@MNT / PGA, which indicates the success of the surface modification.

[0046] Depend on Figure 3 It can be seen that the sample roughness measurement is consistent with the observed morphological features, confirming the establishment of the micro-nano composite structure and the coverage of the self-assembled coating.

[0047] Depend on Figure 4It can be seen that BPTES@MNT / PGA exhibits significant hydrophilic characteristics, and the average water contact angle of its surface interface is about 14.3°, which is conducive to the rapid contact between the implant and the body fluid.

[0048] Depend on Figure 5 It can be seen that polydopamine and the titanium substrate are covalently cross-linked through chemical bonds, and BPTES is loaded on the implant surface through π-π stacking. This covalent cross-linking method improves the stability of the coating, and the non-covalent drug loading method ensures the smooth release of the drug.

[0049] Depend on Figure 6 It can be seen that calcium ions, BPTES and glutamate can be effectively released from the implant interface, and a lower pH environment can accelerate the release of calcium ions and BPTES, but does not affect the release of glutamate.

[0050] Example 3

[0051] This embodiment provides an application effect testing experiment of a medical modified titanium material.

[0052] 2×10 4 Senescent MSCs and 2×10 4 Non-senescent MSCs were seeded on the surface of the sample to be tested and cultured in an incubator. The culture medium was changed every 3 days and the cells were taken out at the designated time for apoptosis (Annexin V-FITC method), cell viability (CCK8 method) and Alizarin red staining experiments.

[0053] In this example, the titanium implant (Ti), medical modified titanium material MT, medical modified titanium material BPTES@MNT and BPTES@MNT / PGA in Example 1 were used as test samples, and apoptosis analysis and cell viability detection at different time points were performed on the aged MSCs and non-aged MSCs. The results are as follows: Figure 7 AnnexinV-FITC analysis results shown and Figure 8 The cell viability results are shown and provided as Fig. 9 Representative images of non-senescent MSCs stained with Alizarin red in each group after 14 days of incubation are shown, and quantitative analysis was performed. Fig.10 The data are shown as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

[0054] Depend on Figure 7 and Figure 8It can be seen that BPTES@MNT / PGA can significantly induce apoptosis of senescent MSCs, and has no obvious cytotoxicity to non-senescent MSCs. Compared with BPTES@MNT, it can significantly promote the proliferation of non-senescent MSCs; this confirms that BPTES@MNT / PGA can achieve effective clearance of senescent cells while maintaining the normal functions of non-senescent cells.

[0055] Depend on Fig. 9 and Fig.10 It can be seen that BPTES@MNT / PGA exhibits the strongest osteogenic differentiation ability on non-aging MSCs, which ensures the osteointegration-promoting effect of BPTES@MNT / PGA.

[0056] Example 4

[0057] This embodiment provides a rat implantation experiment of a medical modified titanium material.

[0058] The aging model was established using 18-month-old rats with similar growth conditions. Fig.11 As shown in the schematic diagram of the implantation experiment process, the titanium implant (Ti), medical modified titanium material MT, medical modified titanium material BPTES@MNT and BPTES@MNT / PGA in Example 1 are respectively implanted into rats, and the rats are cultured under the same conditions.

[0059] Six weeks after implantation, three-dimensional μ-CT reconstruction of the implant area was performed to obtain the following Fig.12 The results shown in the figure were obtained by performing Masson staining around the implant. Fig.13 The representative images of Masson staining are shown in Figure 2. The staining results were quantitatively analyzed to obtain Fig.14 The quantitative results of BIC and new bone thickness are shown, where the data are expressed as mean ± standard deviation (*p<0.05, **p<0.01, ***p<0.001).

[0060] Depend on Figure 12 to Figure 14 It can be seen that compared with other groups, BPTES@MNT / PGA showed the strongest bone integration ability in aged rats, with significantly thicker new bone tissue around the implant and significantly higher bone-implant contact rate. This shows that BPTES@MNT / PGA is expected to promote bone integration in elderly patients, confirming its broad application prospects in the biomedical field.

[0061] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0062] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a medical modified titanium material, characterized in that: The following steps are involved: The titanium implant is cleaned and then subjected to acid etching reaction to obtain a titanium implant with a micron structure; The microstructured titanium implant is reacted in a silane coupling agent, and then washed and dried in sequence to obtain an amino-modified microstructured titanium implant; mixing dopamine, calcium ions and a glutaminase inhibitor in a solvent to obtain a mixed solution; reacting the amino-modified microstructured titanium implant in the mixed solution, washing and drying again to obtain a micro-nano composite structured titanium implant loaded with a glutaminase inhibitor; The micro-nano composite structure titanium implant loaded with glutaminase inhibitor is alternately immersed in chitosan and polyanion glutamate donor to obtain the medical modified titanium material.

2. The preparation method according to claim 1, characterized in that: The acid etching reaction comprises the steps of: immersing the titanium implant in hydrofluoric acid for reaction, and then immersing the titanium implant in sulfuric acid for reaction to obtain the microstructured titanium implant; and / or, in the mixed solution, the molar ratio of the dopamine, the calcium chloride and the glutaminase inhibitor is 10:0.5-1:0.1-0.2; And / or, the solvent of the mixed solution is: a mixed solution of trimethylammonium hydrochloride buffer, dimethyl sulfoxide and a polymerization initiator in a volume ratio of 50:45-55:0.5-1.5; wherein the polymerization initiator includes a persulfate component.

3. The preparation method according to claim 2, characterized in that: The mass percentage of the titanium implant in the hydrofluoric acid is 0.2wt% to 3wt%; And / or, the mass percentage of the titanium implant in the sulfuric acid is 65wt% to 70wt%.

4. The preparation method according to claim 1, characterized in that: The calcium ion is one or a combination of calcium chloride, calcium nitrate and calcium gluconate; And / or, the glutaminase inhibitor is one or a combination of BPTES, Telaglenastat, L-Methionine-DL-sulfoximine, Trivalent hydroxyarsinothricin; And / or, the silane coupling agent is one or a combination of 3-aminopropyltriethoxysilane solution, γ-aminopropylmethyldiethoxysilane, silane-polyethylene glycol-amino, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane; And / or, the polyanionic glutamate donor is one or a combination of polyglutamic acid, polyglutamic acid-γ-benzyl ester, polyglutamic acid-γ-methyl ester, polyglutamic acid-aspartic acid copolymer, polyglutamic acid-γ-benzyl ester-polyethylene glycol copolymer.

5. The preparation method according to claim 1, characterized in that: The cleaning process comprises the steps of: ultrasonic cleaning and drying; And / or, the cleaning is all carried out by ultrasonic treatment.

6. A medical modified titanium material, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 5.

7. The medical modified titanium material according to claim 6, characterized in that: It is loaded with a glutaminase inhibitor and a polyanionic glutamate donor; The in vivo degradation product of the polyanionic glutamate donor comprises glutamate.

8. Use of the medical modified titanium material obtained by the preparation method according to any one of claims 1 to 5 or the medical modified titanium material according to any one of claims 6 to 7 in the preparation of titanium implants.

9. The use according to claim 8, characterized in that: The titanium implant is used for orthopedic implant surgery for patients with degenerative diseases caused by aging.

10. The use according to claim 9, characterized in that: The degenerative diseases caused by aging include diabetes and / or osteoporosis.

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