Preparation method and application of bisphosphonate functionalized orthopedic implant material

Abstract

The present invention discloses a preparation method and application of a bisphosphonate-functionalized orthopedic implant material. The bisphosphonate-functionalized orthopedic implant material comprises a base material, and a bisphosphonate-functionalized layer is formed on the surface of the base material. The preparation method of the bisphosphonate-functionalized orthopedic implant material comprises the following steps: immersing the base material in an alkaline solution of a phenolamine compound, and reacting for a period of time to obtain a base material functionalized with a polyphenolamine compound; and immersing the base material functionalized with a polyphenolamine compound in an alkaline solution of a bisphosphonate, and reacting for a period of time to obtain a base material functionalized with a bisphosphonate. The present invention chemically modifies the surface of the base material and applies it to orthopedic implants, thereby achieving positive regulation of the early inflammatory immune response and subsequent bone regeneration after implantation of the orthopedic implant material in the body, thereby improving the bone integration performance of the orthopedic implant material.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials, and in particular relates to a preparation method and application of a bisphosphonate-functionalized orthopedic implant material. Background Art

[0002] With the accelerated aging of the population, the increase in traffic accidents and diseases such as bone tumors, the incidence of bone defects has been rising year by year, resulting in an increasing demand for orthopedic implant materials in clinical practice. At present, the orthopedic implant materials widely used in clinical practice mainly include two categories: metal orthopedic implant materials such as titanium and titanium alloys, and polymer orthopedic implant materials such as polyetheretherketone. However, both types of materials are bioinert materials. After implantation into the body, on the one hand, they cause severe inflammatory immune responses in the early stage, prompting fibrous connective tissue to wrap around the orthopedic implant materials; on the other hand, their surface is not conducive to the adhesion, proliferation and differentiation of bone-forming cells, and cannot stimulate bone formation. Ultimately, the orthopedic implant materials cannot directly form a strong bone integration with the surrounding bone tissue, and thus cannot function for a long time. Orthopedic implant materials interact with the surrounding bone tissue through their surface. Therefore, the surface physical and chemical properties of orthopedic implant materials are key factors in regulating the inflammatory immune response in the early stage of implantation and the subsequent bone regeneration, and ultimately determine bone integration.

[0003] Currently, various methods are used to modify the surface morphology and chemical state of orthopedic implant materials to improve the adhesion, growth, and differentiation of bone-forming cells on their surfaces and promote osseointegration. Osseointegration is a complex physiological process that requires the spatiotemporal regulation of inflammatory immune responses and osteogenesis. However, existing technologies often only regulate osteogenesis while ignoring the early inflammatory immune response, resulting in strong osteogenesis in vitro but poor in vivo osseointegration. The early inflammatory immune response is primarily regulated by macrophages. If macrophages can effectively and timely transform from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, inflammation can be eliminated and an immune microenvironment conducive to osteogenesis can be created. Otherwise, the orthopedic implant will be encapsulated by fibrous connective tissue. Therefore, the timely resolution of early inflammation and the timely transformation of immune cells from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype are key factors in determining the robust osseointegration of orthopedic implants. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a preparation method and application of a bisphosphonate-functionalized orthopedic implant material. By chemically modifying the surface of the base material, the early inflammatory immune response and subsequent bone regeneration after the orthopedic implant material is implanted in the body are positively regulated, thereby improving the bone integration performance of the orthopedic implant material.

[0005] The object of the present invention is achieved through the following technical solutions: a method for preparing a bisphosphonate-functionalized orthopedic implant material, wherein the bisphosphonate-functionalized orthopedic implant material comprises a base material, and a bisphosphonate-functional layer is formed on the surface of the base material;

[0006] The preparation method of the bisphosphonate functionalized orthopedic implant material comprises the following steps:

[0007] S1, immersing the base material in an alkaline solution of a phenolamine compound, and reacting for a period of time to obtain a base material functionalized with a polyphenolamine compound;

[0008] S2. Immersing the matrix material functionalized with the polyphenolamine compound in a bisphosphonate alkaline solution, and reacting for a period of time to obtain a bisphosphonate functionalized matrix material.

[0009] The present invention utilizes phenolamine compounds to self-polymerize on the surface of a matrix material in advance to prepare a matrix material functionalized with a polyphenolamine compound. The phenolamine compound is chemically reacted with the amino group in the bisphosphonate to chemically bond and graft the bisphosphonate groups, thereby exposing the bisphosphonate groups to obtain a bisphosphonate-functionalized bone implant. This allows the bone implant to positively regulate early inflammatory immune responses and subsequent bone regeneration after implantation in the body, thereby improving the bone integration performance of the orthopedic implant material.

[0010] Furthermore, the bisphosphonate is any one or more of alendronate sodium, pamidronate sodium, neridronate sodium, olpadronate sodium, risedronate sodium, ibandronate sodium, zoledronic acid, tiludronate sodium or their derivatives with amino or thiol groups.

[0011] Furthermore, the phenolamine compound is any one or more of dopamine, dopamine hydrochloride, norepinephrine, levodopa, and 6-nitrodopamine.

[0012] Furthermore, the matrix material is a combination of any one or more of metal, polymer, and inorganic non-metal orthopedic implant materials.

[0013] Furthermore, the metal orthopedic implant material is selected from any one or more of titanium, titanium alloy, stainless steel, cobalt alloy, zinc alloy, and magnesium alloy.

[0014] Furthermore, the polymer orthopedic implant material is selected from any one or more of polyetheretherketone, ultra-high molecular weight polyethylene, polyurethane, polylactic acid-glycolic acid copolymer, polylactic acid and polyacrylate.

[0015] Furthermore, the inorganic non-metallic orthopedic implant material is selected from any one or more of alumina ceramics, zirconia ceramics, hydroxyapatite, bioactive glass, and tricalcium phosphate.

[0016] The invention discloses an application of a bisphosphonate-functionalized orthopedic implant material, wherein the bisphosphonate-functionalized orthopedic implant material is used to improve the bone integration performance of the orthopedic implant material.

[0017] The beneficial effects of the present invention are:

[0018] (1) The orthopedic implant material prepared by the present invention can not only promote the osteogenesis of osteoblasts in the long term, but also can clear the inflammatory immune response in time at an early stage, create a beneficial bone immune regulation microenvironment, and enhance the bone integration effect of the orthopedic implant material in the body.

[0019] (2) The bisphosphonate functionalization method is carried out through a solution phase reaction, making it suitable for preparing a bisphosphonate functional layer on the surface of orthopedic implant materials with complex shapes.

[0020] (3) Dopamine self-polymerization can form a polydopamine functional layer on the surface of various matrix materials, making it applicable to various orthopedic implant materials.

[0021] (4) The process of the present invention is simple, easy to operate, low in cost, and suitable for industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scanning electron microscope photo of the untreated PEEK surface in the embodiment, and the upper right corner is a scanning electron microscope photo with a higher magnification;

[0023] Figure 2 This is a scanning electron microscope photo of the substrate material after the polydopamine functional layer is formed on the surface of the substrate in the embodiment. The upper right corner is a scanning electron microscope photo at a higher magnification.

[0024] Figure 3 This is a scanning electron microscope photograph of the substrate material after the bisphosphonic acid functional layer is formed on the surface of the substrate in the embodiment, and the upper right corner is a scanning electron microscope photograph at a higher magnification;

[0025] Figure 4 The following is a full spectrum of X-ray photoelectron spectroscopy of the surface of each group of treated samples in the embodiment. From top to bottom, the spectrum lines are PEEK-ALN, PEEK-PDA and PEEK;

[0026] Figure 5 is the atomic percentage of each treated sample surface in the embodiment;

[0027] Figure 6 is the hydrophilicity and hydrophobicity of the surface of each treated sample in the embodiment;

[0028] Figure 7 The embodiment shows the expression of M1 marker genes in RAW264.7 cells after culturing on the surface of each group of materials for 3 days by real-time fluorescence quantitative PCR;

[0029] Figure 8 The embodiment shows the expression of M2 marker genes in RAW264.7 cells after culturing on the surface of each group of materials for 3 days by real-time fluorescence quantitative PCR;

[0030] Figure 9 The concentration of TNF-α in the culture supernatant of RAW264.7 cells after culturing on the surface of each material group for 3 days in the example;

[0031] Figure 10 The concentration of IL-10 in the culture supernatant of RAW264.7 cells after culturing on the surface of each material group for 3 days in the example;

[0032] Figure 11 The proliferation of MC3T3-E1 osteoblasts after culture on the surface of each group of materials in the example;

[0033] Figure 12 The expression of osteoblast-related genes in MC3T3-E1 osteoblasts after 7 days of culture on the surface of each material group detected by real-time fluorescence quantitative PCR in the embodiment;

[0034] Figure 13 The expression of osteoblast-related genes in MC3T3-E1 osteoblasts after 14 days of culture on the surface of each material group detected by real-time fluorescence quantitative PCR in the embodiment;

[0035] Figure 14 The alkaline phosphatase activity results of MC3T3-E1 osteoblasts after being cultured on the surfaces of various materials for 7 and 14 days in the examples;

[0036] Figure 15 The extracellular matrix mineralization of MC3T3-E1 osteoblasts after culture on the surface of each group of materials for 7 days and 14 days in the example. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the following. Example

[0038] S1. Preparation of a polydopamine functional layer on the surface of the substrate

[0039] S11. Polyetheretherketone (PEEK) was selected as the matrix material of orthopedic implant materials; the PEEK sheet was polished with 1000, 2000, and 3000 grit sandpaper in sequence, and then ultrasonically cleaned with acetone, ethanol, and deionized water in sequence; the PEEK sample was obtained and its surface morphology (such as Figure 1 : The surface is relatively smooth, and the scratches left by sandpaper polishing are clearly visible);

[0040] S12, dissolving dopamine hydrochloride in 10 mM Tris buffer solution (pH 8.5) to a concentration of 2 mg / L to obtain soaking solution A;

[0041] S13, immersing the PEEK sample in the immersion solution A, taking out the PEEK sample after 24 hours, and performing ultrasonic cleaning; obtaining PEEK-PDA, and observing the surface of the PEEK-PDA sample using a scanning electron microscope (such as Figure 2 : The surface morphology of the PEEK-PDA sample is not much different from that of PEEK, but polydopamine particles formed by dopamine self-polymerization can be clearly seen deposited on the surface).

[0042] S2. Preparing a bisphosphonic acid functional layer on the surface of the substrate

[0043] S21, dissolving alendronate sodium in 10 mM Tris buffer solution (pH 8.5) to a concentration of 1.0 mg / L to obtain soaking solution B;

[0044] S22, immersing the PEEK-PDA sheet in the immersion solution B, taking out the sample after 24 hours, and performing ultrasonic cleaning. PEEK-ALN is obtained, and the surface of the PEEK-ALN sample is observed using a scanning electron microscope (eg, Figure 3 As shown in Figure 2, there is no significant difference in the surface morphology of the PEEK-ALN sample compared to that of PEEK-PDA).

[0045] (1) XPS full spectrum

[0046] The surfaces of the samples obtained in the examples (samples PEEK, PEEK-PDA and PEEK-ALN) were scanned by X-ray photoelectron spectroscopy (XPS) wide field, and the following results were obtained: Figure 4 The full XPS spectrum is shown. Comparing PEEK-PDA with PEEK reveals that the presence of the N1s peak indicates the successful formation of a polydopamine functional layer on the PEEK surface. Comparing PEEK-ALN with PEEK-PDA reveals that the presence of the P2p and P2s peaks indicates the successful formation of a bisphosphonate functional layer on the PEEK surface.

[0047] Figure 5 is the atomic percentage of each element on the material surface obtained from XPS analysis. This result shows that the nitrogen content on the material surface increases after the polydopamine functional layer is formed. The phosphorus content on the material surface increases after the bisphosphonate functional layer is formed.

[0048] (2) Surface hydrophilicity

[0049] The hydrophilicity of the material surface was tested using a static water contact angle tester. A 4μL drop of deionized water was vertically dropped onto the sample surface using a syringe. The instrument's built-in imaging system was used to capture a photo of the droplet and analyze the contact angle.

[0050] Figure 6 The static water contact angle results of the samples (PEEK, PEEK-PDA and PEEK-ALN) obtained in the examples are shown in Table 1. Figure 6 As can be seen, the contact angle of the untreated PEEK sample is 80.7°; the contact angle of the PEEK-PDA sample after forming a polydopamine functional layer on the PEEK is 69.6°; and the contact angle of the PEEK-ALN sample after forming a bisphosphonate functional layer on the surface is 64°. This indicates that the series of treatments of the present invention gradually improve the hydrophilicity of the material surface. This improved surface hydrophilicity helps to achieve a better tissue response in vivo.

[0051] (3) Experiment on the effect of reducing inflammation in the early stage

[0052] Mouse RAW264.7 macrophages were inoculated and cultured on the surface of each group of treated samples in the example and cultured for 3 days. Real-time fluorescence quantitative PCR was then used to detect the expression of M1 marker genes and M2 marker genes in the RAW264.7 cells, as well as the concentrations of the inflammatory factor TNF-α and the anti-inflammatory factor IL-10 in the cell culture supernatant, thereby evaluating the effect of the material in reducing inflammation. Figure 7 The horizontal axis represents the name of the M1 marker gene of RAW264.7 cells, and the vertical axis represents the expression of each marker gene. Figure 8 The horizontal axis represents the name of the M2 marker gene of RAW264.7 cells, and the vertical axis represents the expression of each marker gene. Figure 9 The vertical axis represents the concentration of TNF-α in the cell culture supernatant. Figure 10 The vertical axis represents the concentration of IL-10 in the cell culture supernatant. The results show that the expression of M1 marker genes is the lowest on the surface of PEEK-ALN samples ( Figure 7 ), M2 marker genes were most highly expressed on the surface of PEEK-ALN samples ( Figure 8 TNF-α was detected in the cell culture supernatant ( Figure 9 ) and IL-10 ( Figure 10 ) concentration, cells cultured on the PEEK-ALN sample released the least pro-inflammatory factors and secreted the most anti-inflammatory factors. These results demonstrate that the PEEK-ALN sample prepared by this invention can promptly eliminate inflammation early after implantation, inhibiting the transformation of macrophages into the pro-inflammatory M1 phenotype while promoting their transformation into the anti-inflammatory M2 phenotype.

[0053] (4) Promoting bone formation

[0054] Mouse MC3T3-E1 osteoblasts were seeded and cultured on the surfaces of the treated samples in each group described in the examples. Figure 11 Cell proliferation activity was measured using a CCK-8 assay kit after 1, 3, and 5 days of culture on the sample surface. The horizontal axis represents the number of days the cells were cultured on the material surface, while the vertical axis represents the absorbance at 450 nm of the corresponding wells during detection using the assay kit. Higher absorbance values ​​indicate a greater number of cells and greater cell proliferation activity. These results indicate that cells proliferated most rapidly on the PEEK-ALN sample surface. Figure 12 and Figure 13 These are the results of using real-time fluorescence quantitative PCR to detect the expression of osteoblast-related genes in cells after the cells were cultured on the material surface for 7 days and 14 days, respectively. Figure 12 and Figure 13 The horizontal axis represents the name of the osteogenesis-related gene, and the vertical axis represents the expression of each osteogenesis-related gene. The results show that the expression of osteogenesis-related genes is the best on the surface of PEEK-ALN samples. Figure 14 and Figure 15 The results are respectively the activity of alkaline phosphatase (ALP), a marker of early osteogenic differentiation, and the mineralization of the extracellular matrix after cells were cultured on the material surface for 7 days and 14 days. Figure 14 The horizontal axis represents the number of days the cells were cultured on the material surface, and the vertical axis represents the ALP activity, which was normalized using the total amount of intracellular protein. Figure 15 The horizontal axis represents the number of days cells were cultured on the material surface, while the vertical axis represents the absorbance at 620 nm in the corresponding wells containing the detection reagent. A higher absorbance value indicates a higher degree of cell mineralization. The results show that cells on the PEEK-ALN surface exhibited the highest ALP activity and the highest degree of mineralization. These results demonstrate that PEEK-ALN samples not only stimulate osteoblast proliferation and osteogenic differentiation in the middle and late stages of implantation but also promote extracellular matrix mineralization, a process known as osteogenesis.

[0055] The foregoing description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein and should not be construed as excluding other embodiments. Rather, the present invention can be used in various other combinations, modifications, and environments and can be modified within the scope of the concept described herein through the above teachings or techniques or knowledge in the relevant field. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention are intended to be protected by the appended claims.

Claims

1. A method for preparing a bisphosphonate functionalized orthopedic implant material, characterized in that: The bisphosphonic acid functionalized orthopedic implant material comprises a base material, and a bisphosphonic acid functional layer is formed on the surface of the base material; The preparation method of the bisphosphonate functionalized orthopedic implant material comprises the following steps: S1, immersing the base material in an alkaline solution of a phenolamine compound, and reacting for a period of time to obtain a base material functionalized with a polyphenolamine compound; S2. Immersing the matrix material functionalized with the polyphenolamine compound in a bisphosphonate alkaline solution, reacting for a period of time, and then ultrasonically cleaning the matrix material to obtain a bisphosphonate functionalized matrix material.

2. The method for preparing a bisphosphonate functionalized orthopedic implant material according to claim 1, characterized in that: The bisphosphonate is any one or more of alendronate sodium, pamidronate sodium, neridronate sodium, olpadronate sodium, risedronate sodium, ibandronate sodium, zoledronic acid, tiludronate sodium or their derivatives with amino or thiol groups.

3. The method for preparing a bisphosphonate functionalized orthopedic implant material according to claim 1, characterized in that: The phenolamine compound is any one or more of dopamine, dopamine hydrochloride, norepinephrine, levodopa, and 6-nitrodopamine.

4. The method for preparing a bisphosphonate functionalized orthopedic implant material according to claim 1, wherein: The matrix material is a combination of any one or more of metal, polymer, and inorganic non-metal orthopedic implant materials.

5. The method for preparing a bisphosphonate functionalized orthopedic implant material according to claim 4, characterized in that: The metal orthopedic implant material is selected from any one or more of titanium, titanium alloy, stainless steel, cobalt alloy, zinc alloy, and magnesium alloy.

6. The method for preparing a bisphosphonate functionalized orthopedic implant material according to claim 4, characterized in that: The polymer orthopedic implant material is selected from any one or more of polyetheretherketone, ultra-high molecular weight polyethylene, polyurethane, polylactic acid-glycolic acid copolymer, polylactic acid and polyacrylate.

7. The method for preparing a bisphosphonate functionalized orthopedic implant material according to claim 4, characterized in that: The inorganic non-metallic orthopedic implant material is selected from any one or more of alumina ceramics, zirconia ceramics, hydroxyapatite, bioactive glass, and tricalcium phosphate.

8. Use of a bisphosphonate-functionalized orthopedic implant material obtained by the preparation method according to any one of claims 1 to 7 in the preparation of an orthopedic implant material for inhibiting the transformation of macrophages to the pro-inflammatory M1 phenotype and promoting the transformation of macrophages to the anti-inflammatory M2 phenotype, characterized in that: Bisphosphonate-functionalized orthopedic implant materials are used to improve the bone integration performance of orthopedic implant materials.

Images