Bioactive dental implant with coating capable of promoting regeneration of surrounding tissues and preparation method of bioactive dental implant

Abstract

The invention discloses a bioactive dental implant with a coating capable of promoting regeneration of surrounding tissues and a preparation method of the bioactive dental implant. The titanium implant of the bioactive dental implant is sequentially subjected to acid etching and alkali treatment, and the surface of the titanium implant is of a nano honeycomb network structure; the coating comprises multiple layers of biomimetic mineralized calcium phosphate coatings uniformly loaded with protein-loaded calcium phosphate nanoparticles; the protein-loaded calcium phosphate nanoparticles are loaded into the biomimetic mineralized calcium phosphate coating in a self-assembly manner in the nucleation and mineralization crystallization process of calcium phosphate. According to the invention, a multi-layer biomimetic mineralized calcium phosphate coating is constructed on the surface of the titanium implant through a biomimetic mineralization technology, protein-loaded calcium phosphate nanoparticles are uniformly assembled into the coating, the binding force between the coating and a titanium substrate is strong, and the release of protein in the protein-loaded calcium phosphate nanoparticles is matched with the regeneration stage of tissues around the implant, so that the biomimetic mineralized calcium phosphate coating has good biocompatibility. The activity loss of the active factors can be effectively avoided, the controllable release of the active factors is realized, the preparation process is simple and the cost is low.

Description

Technical Field

[0001] This invention relates to the field of dental implant technology, and more specifically, to a bioactive dental implant with a coating that promotes the regeneration of surrounding tissues and a method for preparing the same. Background Technology

[0002] Currently, the mainstream commercially available dental implants are titanium implants, primarily manufactured using a sandblasting and acid etching process. In the initial implantation stage, the implant surface is in direct contact with the surrounding alveolar bone. However, the high-temperature, high-acid process means that the surface lacks nerve and blood vessel attachments, is biologically inactive, and has no sensory function. Due to the lack of the ability to actively promote the regeneration of surrounding tissues, the implant does not easily form a strong bond with the alveolar bone, resulting in a long osseointegration time. Clinically, weight-bearing is only permitted after 3-6 months, and this process is highly prone to occlusal trauma, leading to a reduced implant survival rate.

[0003] To enhance the bioactivity of titanium implant surfaces and improve their integration with surrounding bone tissue, researchers have explored loading bioactive molecules onto the implant surface. Bioactive protein nanocarriers can protect protein activity while extending its half-life, effectively avoiding adverse reactions caused by repeated injections or oral administration. Currently, the most studied protein nanocarrier materials include graphene oxide, magnetic nanoparticles, gold nanoparticles, liposomes, and polymer nanoparticles. However, determining which bioactive factors to load to promote bone and angiogenesis in surrounding tissues after implantation, how to effectively protect protein activity, and how to achieve effective protein loading and release remain key research areas. Calcium phosphate nanoparticles have attracted significant attention. Calcium phosphate materials not only possess excellent biocompatibility and osteoconductivity but also degrade in vivo, releasing ions to create a weakly alkaline environment that enhances cell activity and accelerates bone repair.

[0004] Currently, methods for loading bioactive molecules mainly include direct adsorption, layer-by-layer self-assembly, and biomimetic mineralization deposition. Direct adsorption is simple to operate, but its loading capacity is limited, its loading strength is insufficient, and its short half-life makes it difficult to maintain bioactivity for extended periods. Layer-by-layer self-assembly involves alternating deposition of polyelectrolyte ions with opposite charges on the implant surface to construct a multi-molecular bioactive coating; however, this method suffers from drawbacks such as long preparation cycles, insufficient loading strength, and inadequate mechanical properties. Several existing technologies are used to generate calcium phosphate coatings, such as electrophoretic deposition and plasma spraying. Electrophoretic deposition requires high-temperature sintering to achieve high density, and thermal expansion during cooling can cause the coating to easily detach. Plasma spraying can efficiently and rapidly form coatings, but it still has some problems, such as the tendency to generate residual stress in the coating during the spraying process, which reduces the bonding strength between the coating and the substrate, and the difficulty in achieving a uniform coating.

[0005] Biomimetic mineralization deposition technology on implant surfaces mimics the composition and structure of natural bone matrix to construct a bioactive mineralized layer on the implant surface, thereby accelerating osseointegration and improving long-term stability. However, technical challenges remain regarding the uniform assembly of protein-loaded nanoparticles into the coating and the mismatch between the release of these nanoparticles and the regeneration stage of the surrounding tissues.

[0006] Therefore, it is essential to develop a dental implant active coating that can promote the regeneration of surrounding tissues, effectively avoid the loss of active factors, enable the controlled release of active factors, and has a simple and low-cost preparation process.

[0007] The inventors' technical team disclosed an amorphous-crystalline calcium phosphate composite material based on biomimetic mineralization, its preparation method, and its application in patent application CN113304318A. This material involves forming an amorphous calcium phosphate mineralization layer and a crystalline hydroxyapatite mineralization layer on the surface of a matrix such as titanium through collagen-induced biomimetic mineralization. In patent application CN113368302A, they disclosed protein-loaded bioactive calcium phosphate nanoparticles and their preparation method. However, how to utilize biomimetic mineralization technology to construct a novel implant coating with strong adhesion to the matrix, enabling the protein-loaded nanoparticles to assemble uniformly into the coating, and matching the release of the protein-loaded nanoparticles with the regeneration stage of the tissue surrounding the implant, remains a pressing technical problem to be solved. Summary of the Invention

[0008] The purpose of this invention is to address the current problem that dental implants are difficult to effectively promote the regeneration of surrounding tissues. It proposes a bioactive dental implant with a coating that promotes the regeneration of surrounding tissues and its preparation method. Through biomimetic mineralization technology, a multi-layer biomimetic mineralized calcium phosphate coating is constructed on the surface of a titanium implant. Protein-loaded calcium phosphate nanoparticles are uniformly assembled into the coating, resulting in strong adhesion between the coating and the titanium substrate. Furthermore, the release of proteins from the protein-loaded calcium phosphate nanoparticles matches the regeneration stage of the tissues surrounding the implant, effectively preventing the loss of active factors and achieving controllable release of active factors. The preparation process is simple and low-cost.

[0009] To achieve the above objectives, in a first aspect, the present invention provides a bioactive dental implant with a coating that promotes the regeneration of surrounding tissues, comprising a titanium implant and a coating on its surface; the titanium implant is subjected to acid etching and alkali treatment in sequence, resulting in a nano-honeycomb mesh structure on its surface; the coating comprises a multi-layered biomimetic mineralized calcium phosphate coating uniformly loaded with protein-loaded calcium phosphate nanoparticles; the protein-loaded calcium phosphate nanoparticles are loaded into the biomimetic mineralized calcium phosphate coating through self-assembly during the nucleation, mineralization, and crystallization of calcium phosphate; the thickness of each coating layer is 100~300 nm; the protein is a bioactive protein that promotes angiogenesis or bone formation.

[0010] The above technical solution introduces a nanocarrier to load multiple bioactive factors, protects their activity, and achieves controlled release, actively promoting the regeneration of tissues around the implant; it uses biomimetic mineralization technology to construct a novel implant coating, while modifying the surface of the titanium implant to enhance the interfacial bonding strength between the coating and the implant; it assembles nanoparticles loaded with bioactive factors into the bioactive coating, and disperses them uniformly through self-assembly to achieve phased and controlled release, promoting the regeneration of tissues around the implant at different stages.

[0011] Further, the coating comprises an inner coating and an outer coating. The inner coating is a first biomimetic mineralized calcium phosphate coating loaded with a first protein-loaded calcium phosphate nanoparticle; the outer coating is a second biomimetic mineralized calcium phosphate coating loaded with a second protein-loaded calcium phosphate nanoparticle; the first protein-loaded calcium phosphate nanoparticle is a calcium phosphate nanoparticle loaded with bone morphogenetic protein 2 (BMP-2); the second protein-loaded calcium phosphate nanoparticle is a calcium phosphate nanoparticle loaded with vascular endothelial growth factor (VEGF); the thickness of the first biomimetic mineralized calcium phosphate coating is 200-300 nm; and the thickness of the second biomimetic mineralized calcium phosphate coating is 100-200 nm.

[0012] To further match the different needs for bioactive factors at different stages of peri-implant tissue regeneration, the above-mentioned technical solution releases vascular endothelial growth factor (VEGF) after implantation to promote angiogenesis, followed by the release of bone morphogenetic protein 2 (BMP-2) to promote bone formation, ensuring the phased and controllable release of the biomimetic mineralization coating. This "adjustable and controllable" approach allows nanoparticles loaded with different functional proteins such as angiogenesis-promoting and osteogenic growth factors to be released from the coating in stages. This biomimetic release of appropriate amounts of growth factors at different times and spaces is beneficial for improving tissue repair, accelerating bone integration, and avoiding adverse reactions from excessive dosage. The thickness of each coating layer ensures high bonding strength between the coating and the implant interface.

[0013] Secondly, the present invention provides a method for preparing a bioactive dental implant, comprising the following steps:

[0014] Step S1: Prepare various calcium phosphate nanoparticles loaded with different proteins using a co-precipitation method: Dissolve the protein in a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, then add calcium chloride, magnesium chloride and disodium hydrogen phosphate solution dropwise until the reaction is complete, then add polyaspartic acid (pASP) aqueous solution, and obtain polyaspartic acid-modified protein-loaded calcium phosphate nanoparticles by centrifugation and freeze-drying; the protein is a bioactive protein that promotes angiogenesis or bone formation; Step S1 prepares bioactive calcium phosphate nanoparticles by simply mixing the protein and inorganic salt components and reacting rapidly in an open environment.

[0015] Step S2, Pre-treatment of titanium implants: The titanium implants are cleaned, polished, acid-etched and alkaline-treated in sequence, then dried, and then treated with plasma oxygen. Finally, collagen solution is spin-coated onto the surface.

[0016] Step S3: Prepare a mineralization solution for a biomimetic mineralized calcium phosphate coating; the mineralization solution contains 0.075~2 g / L of polyaspartic acid;

[0017] Step S4: Preparation of a multi-layer biomimetic mineralized calcium phosphate coating on the surface of the titanium implant: The single-layer biomimetic mineralized calcium phosphate coating is prepared by placing the titanium implant treated in step S2 into a mineralization solution, adding a protein-carrying calcium phosphate nanoparticle obtained in step S1, and then performing biomimetic mineralization; after each biomimetic mineralization and before the next biomimetic mineralization, the titanium implant is cleaned, dried, and coated with a collagen solution.

[0018] The above technical solution improves the dispersion stability of protein-loaded calcium phosphate nanoparticles by surface modification, thus solving the problem of poor dispersion of protein-loaded nanoparticles. At the same time, the introduction of polyaspartic acid into the mineralization solution makes it possible for the protein-loaded nanoparticles to be uniformly distributed in the mineralization coating through biomimetic mineralization.

[0019] Further, in step S1, the Ca in the reaction solution 2+ With Mg 2+ The molar ratio is 1:1; Ca 2+ With P 2- The molar ratio is 1.67; the concentration of the polyaspartic acid aqueous solution is 10 mg / mL; the protein concentration in the reaction solution is 0.01~5 mg / mL; the total volume ratio of the tris(hydroxymethyl)aminomethane hydrochloride buffer solution, calcium chloride, magnesium chloride and disodium hydrogen phosphate solution to the volume ratio of the polyaspartic acid aqueous solution is 9:1.

[0020] Furthermore, the concentration of protein dissolved in tris(hydroxymethyl)aminomethane hydrochloride buffer solution was 0.01–5 mg / mL; the size of the polyaspartic acid-modified protein-loaded calcium phosphate nanoparticles was 51.90 ± 9.47 nm. Appropriate size of the protein-loaded calcium phosphate nanoparticles facilitates highly dispersed self-assembly of these nanoparticles in the biomimetic mineralized calcium phosphate coating.

[0021] Further, in step S2, the acid etching process is as follows: a dual-acid etching solution is prepared by mixing 49wt% sulfuric acid, 36% hydrochloric acid, and pure water in a volume ratio of 2:3:3, and the titanium implant is etched at a constant temperature of 60°C. After the acid etching is completed, the implant is cleaned. The alkaline solution treatment process is as follows: a 5M sodium hydroxide aqueous solution is prepared, and the titanium implant is sealed in the sodium hydroxide aqueous solution and treated at a constant temperature of 60°C for 6-7 hours.

[0022] Furthermore, the collagen solution is a 1 mg / mL type I collagen solution.

[0023] Further, in step S4, a double-layer biomimetic mineralized calcium phosphate coating is prepared on the surface of the titanium implant. The inner coating is a first biomimetic mineralized calcium phosphate coating loaded with bone morphogenetic protein 2 calcium phosphate nanoparticles; the outer coating is a second biomimetic mineralized calcium phosphate coating loaded with vascular endothelial growth factor calcium phosphate nanoparticles. The bone morphogenetic protein 2 calcium phosphate nanoparticles and the vascular endothelial growth factor calcium phosphate nanoparticles are prepared in step S1.

[0024] Furthermore, the mineralization solution used to prepare the first biomimetic mineralized calcium phosphate coating consisted of 423 mM Na + 18 mM Mg 2+ 7.5mM Ca 2+ 443.4 mM Cl - 3 mM HPO4 2- 12.6 mM HCO3 - The solution for preparing the second biomimetic mineralized calcium phosphate coating consisted of 2 g / L polyaspartic acid aqueous solution; the mineralization solution for preparing the second biomimetic mineralized calcium phosphate coating consisted of 213 mM Na + 9mM Mg 2+ 3.75mM Ca 2+ 221.7mM Cl - 1.5mM HPO4 2- 6.3 mM HCO3 - It consists of a 0.075 g / L polyaspartic acid aqueous solution. During the mineralization process, the thickness of the mineralized coating can be adjusted by regulating the parameters of each formulation in the mineralization solution.

[0025] Finally, the present invention provides a bioactive dental implant, which is prepared by the method for preparing a bioactive dental implant as described above.

[0026] Compared with the prior art, the present invention has the following technical effects:

[0027] (1) This invention modifies the surface of protein-carrying nanoparticles and constructs a biomimetic mineralized calcium phosphate coating on the surface of titanium implants by combining biomimetic mineralization technology. This allows the protein-carrying nanoparticles to be loaded into the calcium phosphate coating through self-assembly while the calcium phosphate nucleates, mineralizes and crystallizes. This results in the protein-carrying nanoparticles being evenly distributed in the biomimetic mineralized calcium phosphate coating, thereby achieving controlled protein release.

[0028] (2) To further match the different needs of bioactive factors at different stages of peri-implant tissue regeneration, this invention selects to construct a double-layer calcium phosphate mineralization coating. The outermost mineralization layer is loaded with VEGF nanoparticles that promote angiogenesis, and the inner mineralization layer is loaded with BMP-2 nanoparticles that promote osteogenic growth, ensuring the phased and controllable release of the biomimetic mineralization coating. This "adjustable and controllable" approach allows nanoparticles loaded with different functional proteins such as angiogenesis and osteogenic growth factors to be released from the coating in stages. It can biomimeticly release appropriate amounts of growth factors at different times and spaces, which is beneficial to improving the effect of tissue repair, accelerating bone integration, and avoiding adverse reactions from excessive dosage.

[0029] (3) By etching the surface of the titanium implant and controlling the thickness of the coating, the present invention improves the interfacial bonding force between the titanium implant and the coating, so that the bioactive dental implant containing the coating that promotes the regeneration of surrounding tissues has high clinical application value. Attached Figure Description

[0030] The invention, its features and advantages will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings.

[0031] Figure 1 This is a schematic diagram of the structure of a bioactive dental implant and its surface coating in one embodiment of the present invention.

[0032] Figure 2 The images shown are transmission electron microscopy (TEM) images and mapping elemental distribution diagrams of CaP-BSA-pASP nanoparticles in this embodiment of the invention.

[0033] Figure 3 The images shown are scanning electron microscope (SEM) images of CaP-BMP2-pAsp and CaP-VEGF-pAsp nanoparticles in the embodiments of the present invention.

[0034] Figure 4 The infrared spectra of CaP-BMP2-pAsp and CaP-VEGF-pAsp nanoparticles in the embodiments of the present invention are shown.

[0035] Figure 5 The figure shows the dispersion stability of protein-loaded calcium phosphate nanoparticles in the mineralization solution before and after pASP modification in the example.

[0036] Figure 6 This is a schematic diagram illustrating the synthesis process of the titanium implant coating in an embodiment of the present invention.

[0037] Figure 7This is a confocal fluorescence microscopy z-stack image showing the distribution of protein-loaded nanoparticles within the coating of a titanium implant with a double-layer coating in an embodiment of the present invention. The first layer is a biomimetic mineralized calcium phosphate coating loaded with CaP-BSA-pAsp nanoparticles labeled with Alexa Fluor 488 fluorescent dye; the second layer is a biomimetic mineralized calcium phosphate coating loaded with CaP-My-pASP nanoparticles labeled with Alexa Fluor 568 fluorescent dye.

[0038] Figure 8 The images shown are test results of the bonding strength in the coated titanium implants in this embodiment of the invention; wherein, Figure A is a nano-scratch detection image; and Figure B is a Raman element distribution image at each scratch segment.

[0039] Figure 9 The figures shown are in vivo experimental results of titanium implants with double coatings in the embodiments of the present invention; Figure A is a flowchart of the immediate implantation surgery after extraction of the maxillary first molar in the rat's oral cavity, Figure B is a histological section and magnified view of the titanium implant after osseointegration, the first column is a pure titanium implant (without coating), the second column is a titanium implant surface loaded with a calcium phosphate coating (without bioactive protein), and the third column is a calcium phosphate coating loaded with BMP-2-loaded calcium phosphate nanoparticles and VEGF-loaded calcium phosphate nanoparticles loaded with protein nanoparticles on the surface of the titanium implant.

[0040] Figure 10 for Figure 9 Laser scanning confocal microscope images of histological sections after osseointegration of three types of titanium implants. White dots and lines indicate the implant-bone interface, red fluorescence indicates CD31 (vascular endothelial marker), green fluorescence indicates COL1A1 (osteogenic marker), and white fluorescence indicates beta III Tubulin (neurogenic marker). Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but these are not intended to limit the scope of the invention.

[0042] The reaction apparatus, monomer compounds, solutes, and solvents involved in the following examples are all commercially available. The detection instruments and reagents involved in the following effect examples are all commercially available, and the detection methods used are existing technologies that can be found online.

[0043] Furthermore, the execution order of actions, steps, etc. in the apparatus and methods shown in the claims, specification, and drawings can be implemented in any order, unless a specific order is explicitly specified, and as long as the output of the preceding processing is not used in the subsequent processing.

[0044] See Figure 1This invention provides a bioactive dental implant with a coating that promotes the regeneration of surrounding tissues, comprising a titanium implant and a coating on its surface; the titanium implant is subjected to acid etching and alkali treatment in sequence, resulting in a nano-honeycomb mesh structure on its surface; the coating comprises a multi-layered biomimetic mineralized calcium phosphate coating uniformly loaded with protein-loaded calcium phosphate nanoparticles; the protein-loaded calcium phosphate nanoparticles are loaded into the biomimetic mineralized calcium phosphate coating through self-assembly during the nucleation, mineralization, and crystallization of calcium phosphate; the thickness of each coating layer is 100~300 nm; the protein is a bioactive protein that promotes angiogenesis or bone formation.

[0045] In traditional techniques, the poor dispersion of protein-loaded nanoparticles in the coating results in suboptimal controlled protein release. Therefore, the uniform assembly of protein-loaded nanoparticles into a bioactive coating is a key technical challenge this invention aims to overcome. To ensure the bonding strength between the coating and the titanium substrate, not only is surface structure design of the titanium substrate necessary, but the thickness of the mineralized coating also needs to be controlled. Thus, the challenge of this invention also lies in controlling the thickness of the mineralized coating. This invention achieves adjustment of the mineralized coating thickness by regulating the parameters of each formulation in the mineralization solution during the mineralization process. The advantage of preparing multilayer calcium phosphate mineralized coatings is that different bioactive factors can be loaded separately, further matching the different needs of peri-implant tissue regeneration at different stages for bioactive factors. It can biomimeticly release appropriate amounts of growth factors at different times and spaces, which is beneficial for improving tissue repair, accelerating bone integration, and avoiding adverse reactions from excessive dosage.

[0046] As a preferred technical solution, see Figure 1 The coating comprises an inner coating and an outer coating. The inner coating is a first biomimetic mineralized calcium phosphate coating loaded with first protein-loaded calcium phosphate nanoparticles; the outer coating is a second biomimetic mineralized calcium phosphate coating loaded with second protein-loaded calcium phosphate nanoparticles. The first protein-loaded calcium phosphate nanoparticles are calcium phosphate nanoparticles loaded with BMP-2; the second protein-loaded calcium phosphate nanoparticles are calcium phosphate nanoparticles loaded with VEGF. The thickness of the first biomimetic mineralized calcium phosphate coating is 200-300 nm; the thickness of the second biomimetic mineralized calcium phosphate coating is 100-200 nm. To ensure that the novel implant has multiple functions, such as promoting bone formation and angiogenesis, and to match the regeneration stage of the tissue surrounding the implant, this invention preferably uses two types of protein-loaded nanoparticles: vascular endothelial growth factor (VEGF) nanoparticles for promoting angiogenesis, and bone morphogenetic protein 2 (BMP-2) nanoparticles for promoting bone formation.

[0047] The above-mentioned bioactive dental implant with a coating that promotes the regeneration of surrounding tissues is prepared by the following method, including the following steps:

[0048] Step S1: Prepare various calcium phosphate nanoparticles loaded with different proteins using a co-precipitation method: Dissolve the protein in a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, then add calcium chloride, magnesium chloride and disodium hydrogen phosphate solution dropwise until the reaction is complete, then add an aqueous solution of polyaspartic acid, centrifuge and freeze-dry to obtain polyaspartic acid-modified protein-loaded calcium phosphate nanoparticles; the protein is a bioactive protein that promotes angiogenesis or bone formation;

[0049] Step S2, Pre-treatment of titanium implants: The titanium implants are cleaned, polished, acid-etched and alkaline-treated in sequence, then dried, and then treated with plasma oxygen. Finally, collagen solution is spin-coated onto the surface.

[0050] Step S3: Prepare a mineralization solution for a biomimetic mineralized calcium phosphate coating; the mineralization solution contains 0.075~2 g / L of polyaspartic acid;

[0051] Step S4: Preparation of a multi-layer biomimetic mineralized calcium phosphate coating on the surface of the titanium implant: The single-layer biomimetic mineralized calcium phosphate coating is prepared by placing the titanium implant treated in step S2 into a mineralization solution, adding a protein-carrying calcium phosphate nanoparticle obtained in step S1, and then performing biomimetic mineralization; after each biomimetic mineralization and before the next biomimetic mineralization, the titanium implant is cleaned, dried, and coated with a collagen solution.

[0052] The above preparation method modifies the surface of protein-loaded nanoparticles and combines it with biomimetic mineralization technology to construct a biomimetic mineralized calcium phosphate coating on the surface of titanium implants. This allows the protein-loaded nanoparticles to be loaded into the calcium phosphate coating through self-assembly during the nucleation and mineralization crystallization of calcium phosphate, resulting in a uniform distribution of the protein-loaded nanoparticles within the biomimetic mineralized calcium phosphate coating. This method for preparing protein-loaded bioactive calcium phosphate nanoparticles avoids harsh conditions such as high temperature and high pressure, does not introduce other toxic components, is simple to operate, and has a rapid reaction. It can effectively protect the activity of growth factors and meet the requirements for effective loading and release of growth factors.

[0053] To further aid in understanding the technical solution of this invention, the technical solution of this invention will be described in more detail below through specific implementation examples.

[0054] Example

[0055] This embodiment takes a bioactive dental implant with two layers of coating that promote the regeneration of surrounding tissues as an example to describe its preparation process and excellent performance.

[0056] 1. Preparation of bioactive calcium phosphate nanoparticles loaded with multiple active factors

[0057] A co-precipitation method was used to prepare protein-loadable calcium phosphate nanoparticles by controlling the calcium-to-phosphorus molar ratio (Ca / P) and the pH of the reaction solution. The preparation method involves dissolving the protein in a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, then adding calcium chloride, magnesium chloride, and disodium hydrogen phosphate solutions dropwise until homogeneous, followed by centrifugation and lyophilization. This simple mixing of protein and inorganic salts allows for rapid preparation of bioactive calcium phosphate nanoparticles in an open environment.

[0058] The protein to be loaded (VEGF, BMP-2, bovine serum albumin (BSA), or myoglobin (My)) was dissolved in a buffer solution of tris(hydroxymethyl)aminomethane hydrochloride at a concentration of 0.01 mg / mL. This concentration range can also be 0.01–5 mg / mL. Referring to Table 1, BSA and My are model proteins with similar physicochemical properties to BMP-2 and VEGF. The inventors utilized these two model proteins with similar physicochemical properties for materials detection.

[0059] Add 0.25 mL of 0.5 M calcium chloride solution and 0.25 mL of 0.5 M magnesium chloride solution dropwise, and stir for 15 min.

[0060] Add 0.15 mL of 0.5 M disodium hydrogen phosphate solution (calcium-to-phosphorus ratio 1.67) dropwise while stirring vigorously.

[0061] After the reaction was complete, an aqueous solution of polyaspartic acid (pASP) was added and mixed thoroughly. The concentration of the polyaspartic acid aqueous solution was 10 mg / mL. The total volume ratio of the tris(hydroxymethyl)aminomethane hydrochloride buffer solution, calcium chloride, magnesium chloride, and disodium hydrogen phosphate solution to the volume of the polyaspartic acid aqueous solution was 9:1. Then, the mixture was centrifuged at 8000 rpm for 15 min, the supernatant was removed, and the collected precipitate was washed three times with deionized water. The precipitate was then freeze-dried for at least 10 h to obtain four types of protein-loaded bioactive calcium phosphate nanoparticles, which were designated as CaP-VEGF-pASP, CaP-BMP2-pASP, CaP-BSA-pASP, and CaP-My-pASP, respectively.

[0062] Table 1 Physicochemical properties of various growth factors and model proteins

[0063]

[0064] Figure 2 Transmission electron microscopy (TEM) images and energy-dispersive X-ray spectroscopy (EDS) elemental distribution maps of CaP-BSA-pAsp are shown. The calcium phosphate nanoparticles have a size of approximately 40 nm, exhibit good biocompatibility, and can effectively retain growth factor activity. BSA, as a model protein, is uniformly loaded onto the calcium phosphate nanoparticles, verifying that protein molecules can be well loaded into calcium phosphate nanoparticles.

[0065] See Figure 3 CaP-VEGF-pASP and CaP-BMP2-pASP are nanoparticles with a size of less than 100 nm. See also Figure 4 The infrared spectra of CaP-BSA-pASP, CaP-VEGF-pASP, and CaP-BMP2-pASP are shown at 3406 cm⁻¹. -1 This is the stretching vibration of -OH, 1658 cm⁻¹ -1 It is an amide I band (C=O stretching vibration), 1533 cm⁻¹ -1 The peaks of the protein in the amide II band (-NH bending vibration and -CN stretching vibration) were observed to decrease significantly after the synthesis of protein-loaded calcium phosphate nanoparticles, indicating that the protein and mineral reacted and confirming its effective loading in the nanoparticles.

[0066] See Figure 5 After surface modification of nanoparticles with anionic polymer polyaspartic acid, they can achieve dispersion stability in solution for up to 72 hours, thus uniformly assembling in the coating.

[0067] 2. Pre-treatment of titanium implants

[0068] 2.1 Titanium implant cleaning: Prepare titanium implants of the required size (designed according to specific needs) and clean the substrate by ultrasonic cleaning in acetone (10-15 min), ethanol (10-20 min) and deionized water (20-30 min) continuously.

[0069] 2.2 Polishing of titanium implants: Sandpaper grinding and diamond rough polishing are used. Final polishing is performed using a cotton cloth wheel combined with silica suspension, with a pressure of 27N and a rotation speed of 120rpm in the opposite direction for 10 minutes. The sample is rotated 90°-180° every 2 minutes to avoid hard phase tailing. Then, it is ultrasonically cleaned with anhydrous ethanol for 15 minutes and dried in an oven for later use.

[0070] 2.3 Acid etching of titanium implants: Prepare a dual-acid etching solution by mixing 49wt% sulfuric acid, 36% hydrochloric acid and pure water in a volume ratio of 2:3:3. Etch for 30 minutes in a 60℃ water bath. After etching, ultrasonically clean three times.

[0071] 2.4 Alkali Treatment of Titanium Implants: Prepare a 5M sodium hydroxide solution using double-distilled water. Seal the titanium implants tightly in the sodium hydroxide solution and treat at a constant temperature of 60℃ for 6 hours. After treatment, remove the implants, rinse with double-distilled water for 5 minutes, dry with nitrogen, and then proceed with subsequent heat treatment. Place the alkali-treated titanium implants in a muffle furnace, starting at 37℃, increasing the temperature to 600℃ after 1 hour and treating for another hour, then allowing them to cool naturally to 37℃.

[0072] 2.5 Coating of titanium implant surface with collagen: After the titanium implant treated as described above is subjected to plasma oxygen treatment, the surface is coated with type I collagen solution (1 mg / mL, 0.1%).

[0073] 3. See Figure 6 Biomimetic mineralized calcium phosphate coating on titanium implants

[0074] 3.1 Preparation of mineralization solution 1 for biomimetic mineralized calcium phosphate coating: Take 450 mL of deionized water, 11.9049 g of sodium chloride, 0.5292 g of sodium bicarbonate, 0.5373 g of disodium hydrogen phosphate dodecahydrate, 1.83 g of magnesium chloride hexahydrate, and 1 g of pASP. Purge carbon dioxide to pH 5.5, add 3.75 mL of calcium chloride, add tris(hydroxymethyl)aminomethane hydrochloride buffer solution with pH=9 to adjust the pH of the mineralization solution to 6.5, add deionized water to 500 mL, and continue to purge carbon dioxide to adjust the final pH of the mineralization solution to 6.2.

[0075] 3.2 Biomimetic mineralization of BMP-2-loaded calcium phosphate nanoparticles: 30 mL of mineralization solution 1 was placed in a beaker, with two titanium implants placed in each beaker. Protein A-loaded nanoparticles (CaP-BMP2-pASP, 1-5 mg / mL) were added and dispersed evenly. The beaker was then sealed with plastic wrap and placed in a 37°C oven for mineralization for 2 days. See also... Figure 5 The surface-modified pASP-loaded protein-loaded calcium phosphate nanoparticles can be uniformly dispersed in the mineralization solution and incorporated into the calcium phosphate coating through self-assembly during the process of calcium phosphate nucleation, mineralization, and crystallization.

[0076] 3.3 Coating collagen onto the surface of titanium implants: Remove the mineralized titanium implants, wash them three times with deionized water, and after drying, spin-coat the surface with collagen (as before).

[0077] 3.4 Preparation of mineralization solution 2: Take 450 mL of deionized water, 5.9524 g of sodium chloride, 0.2646 g of sodium bicarbonate, 0.2686 g of disodium hydrogen phosphate dodecahydrate, 0.9148 g of magnesium chloride hexahydrate, and 3.75 mL of pASP stock solution (10 mg / mL). Purge carbon dioxide to pH 5.5, add 1.875 mL of calcium chloride, and add tris(hydroxymethyl)aminomethane hydrochloride buffer solution (pH=9) to adjust the pH of the mineralization solution to 6.5. Add deionized water to 500 mL, and continue to purge carbon dioxide to adjust the final pH of the mineralization solution to 6.2.

[0078] 3.5 Biomimetic mineralization of VEGF-loaded calcium phosphate nanoparticles: Take 30 mL of mineralization solution 2 and place it in a beaker. Place 2 titanium implants in each beaker, add protein B-loaded nanoparticles CaP-VEGF-pASP (1-5 mg / mL), disperse evenly, seal with plastic wrap, and place in a 37-degree oven for mineralization for 2 days.

[0079] 3.6 Drying and Preparing for Use: After 2 days, remove the mineralized titanium implant, wash it 3 times with deionized water, and then dry it.

[0080] To facilitate material testing, biomimetic mineralized titanium implants loaded with BSA-containing calcium phosphate nanoparticles, biomimetic mineralized titanium implants loaded with My-containing calcium phosphate nanoparticles, and biomimetic mineralized titanium implants sequentially loaded with BSA-containing calcium phosphate nanoparticles and My-containing calcium phosphate nanoparticles were also prepared.

[0081] See Figure 7 Two types of protein-carrying nanoparticles were successfully assembled into the coating, and a bilayer calcium phosphate coating was successfully prepared according to the pre-design.

[0082] See Figure 8 The mechanical properties of the coating were tested using nano-scratch assays, and the scratches were characterized using Raman spectroscopy. The results showed that the coating remained under the scratches, verifying that the coating and the substrate had good adhesion.

[0083] In vivo experiments were conducted using the novel bioactive dental implant with a coating that promotes the regeneration of surrounding tissues, obtained in the examples. See also Figure 9 and Figure 10 The results confirmed that: 1) the novel implant successfully achieved osseointegration. HE staining showed that the peri-implant bone volume was higher in the coated group with active factor nanocarriers, suggesting that the coating constructed in this invention has a promoting effect on peri-implant bone tissue remodeling. 2) Immunofluorescence staining results showed that the expression of osteogenic markers COL1A1, vascular endothelial marker CD31, and neurogenic marker beta III Tubulin in the peri-implant bone tissue of the drug-loaded coating group was upregulated, suggesting that the coating in this invention has a biological function of actively promoting peri-implant tissue regeneration, mainly manifested in promoting angiogenesis, promoting neurogenesis, and promoting osseointegration.

[0084] As can be seen from the above results, this invention has developed a bioactive dental implant with a coating that promotes the regeneration of surrounding tissues. The advantages are: (1) By modifying the surface of protein-carrying nanoparticles and combining them with biomimetic mineralization technology, a biomimetic mineralized calcium phosphate coating is constructed on the surface of the titanium implant. This allows the protein-carrying nanoparticles to be loaded into the calcium phosphate coating through self-assembly while the calcium phosphate nucleates and crystallizes, resulting in a uniform distribution of the protein-carrying nanoparticles in the biomimetic mineralized calcium phosphate coating, thereby achieving controlled protein release. (2) The above technology can be used to prepare double or more layers of coatings, loaded with different bioactive factors, to achieve controlled degradation of the coating and phased release of the active factors to match the peri-implant tissue regeneration process. (3) An innovative alkaline etching design of a nano-honeycomb mesh structure is used on the titanium substrate, combined with the control of the coating thickness, to enhance the bonding strength between the coating and the substrate. The novel implant provided by this invention can achieve peri-implant bone, nerve, and blood vessel regeneration, immediate loading in the shortest time, and ensure the success rate of the implant.

[0085] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above. Systems and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention are still within the scope of protection of the present invention.

Claims

1. A bioactive dental implant with a coating capable of promoting peripheral tissue regeneration, comprising a titanium implant and a coating on the surface of the implant; characterized in that the titanium implant is sequentially treated by acid etching and alkali treatment, and the surface has a nano-honeycomb network structure; the coating comprises a plurality of layers of biomimetic mineralized calcium phosphate coating uniformly loaded with protein-loaded calcium phosphate nanoparticles; the protein-loaded calcium phosphate nanoparticles are loaded into the biomimetic mineralized calcium phosphate coating by self-assembly during the process of calcium phosphate nucleation and mineralized crystallization; the thickness of each layer of coating is 100-300 nm; and the protein is a bioactive protein capable of promoting angiogenesis or osteogenesis. The coating comprises an inner layer coating and an outer layer coating, the inner layer coating is a first biomimetic mineralized calcium phosphate coating loaded with first protein-loaded calcium phosphate nanoparticles, and the outer layer coating is a second biomimetic mineralized calcium phosphate coating loaded with second protein-loaded calcium phosphate nanoparticles; the first protein-loaded calcium phosphate nanoparticles are bone morphogenetic protein 2-loaded calcium phosphate nanoparticles; the second protein-loaded calcium phosphate nanoparticles are vascular endothelial growth factor-loaded calcium phosphate nanoparticles; the thickness of the first biomimetic mineralized calcium phosphate coating is 200-300 nm; and the thickness of the second biomimetic mineralized calcium phosphate coating is 100-200 nm. The preparation of the bioactive dental implant with a coating capable of promoting peripheral tissue regeneration according to claim 1 or 2 comprises the following steps:

2. The bioactive dental implant with a coating for promoting the regeneration of surrounding tissue according to claim 1, characterized in that, Step S1: preparing a plurality of protein-loaded calcium phosphate nanoparticles loaded with different proteins by a coprecipitation method: dissolving the protein in a tris-hydroxymethyl aminomethane hydrochloride buffer solution, then adding calcium chloride, magnesium chloride and disodium hydrogen phosphate solution drop by drop until the reaction is complete, then adding a polyaspartic acid aqueous solution, and obtaining the polyaspartic acid surface-modified protein-loaded calcium phosphate nanoparticles by centrifugation and freeze-drying; the protein is a bioactive protein capable of promoting angiogenesis or osteogenesis; 3. A method of producing a bioactive dental implant, characterized by, Step S2: pretreating the titanium implant: after the titanium implant is sequentially cleaned, polished, acid-etched and treated with an alkali solution, it is cleaned and dried, and then treated with plasma oxygen, and the surface is spin-coated with a collagen solution; Step S3: preparing a mineralization solution for the biomimetic mineralized calcium phosphate coating; the mineralization solution contains 0.075-2 g / L polyaspartic acid; Step S4: preparing a plurality of layers of biomimetic mineralized calcium phosphate coating on the surface of the titanium implant: a single layer of biomimetic mineralized calcium phosphate coating is prepared by placing the titanium implant treated in step S2 into the mineralization solution and adding one kind of protein-loaded calcium phosphate nanoparticles obtained in step S1 for biomimetic mineralization; after each biomimetic mineralization, the titanium implant is cleaned and dried and the surface is coated with a collagen solution before the next biomimetic mineralization. The concentration of the protein dissolved in the tris-hydroxymethyl aminomethane hydrochloride buffer solution is 0.1-5 mg / mL; and the size of the polyaspartic acid surface-modified protein-loaded calcium phosphate nanoparticles is 51.90±9.47 nm. ​ 4. The method of claim 3, wherein the bioactive dental implant is prepared by the steps of: Ca 2+ and Mg 2+ in a molar ratio of 1:1; Ca 2+ and P 2- in a molar ratio of 1.67; the concentration of the aqueous polyaspartic acid solution is 10 mg / mL; the protein concentration in the reaction solution is 0.01-5 mg / mL; and the total volume of the Tris-HCl buffer solution, the calcium chloride, the magnesium chloride, and the disodium hydrogen phosphate solution to the volume of the aqueous polyaspartic acid solution is 9:

1.

5. The method for preparing a bioactive dental implant according to claim 4, characterized in that, ​ 6. The method for preparing a bioactive dental implant according to claim 3, characterized in that, In the step S2, the acid etching process is as follows: a double acid etching solution is prepared by mixing 49wt% sulfuric acid, 36% hydrochloric acid and pure water at a volume ratio of 2:3:3, and the titanium implant is etched at 60℃ under constant temperature condition, and then cleaned after the etching; the alkali solution treatment process is as follows: a 5M sodium hydroxide aqueous solution is prepared, and the titanium implant is sealed in the sodium hydroxide aqueous solution and treated at 60℃ under constant temperature for 6-7 hours.

7. The method for preparing a bioactive dental implant according to claim 3, characterized in that, The collagen solution is a 1mg / mL type I collagen solution.

8. The method for preparing a bioactive dental implant according to claim 3, characterized in that, In the step S4, a double-layer biomimetic mineralized calcium phosphate coating is prepared on the surface of the titanium implant, the inner layer coating is a first biomimetic mineralized calcium phosphate coating loaded with bone morphogenetic protein 2 loaded calcium phosphate nanoparticles, and the outer layer coating is a second biomimetic mineralized calcium phosphate coating loaded with vascular endothelial growth factor loaded calcium phosphate nanoparticles; the bone morphogenetic protein 2 loaded calcium phosphate nanoparticles and the vascular endothelial growth factor loaded calcium phosphate nanoparticles are prepared by the step S1.

9. The method for preparing a bioactive dental implant according to claim 8, characterized in that, The mineralization solution for preparing the first biomimetic mineralized calcium phosphate coating consists of 423 mM Na + , 18 mM Mg 2+ , 7.5 mM Ca 2+ , 443.4 mM Cl - , 3 mM HPO4 2- , 12.6 mM HCO3 - , 2 g / L polyaspartic acid in water; the mineralization solution for preparing the second biomimetic mineralized calcium phosphate coating consists of 213 mM Na + , 9 mM Mg 2+ , 3.75 mM Ca 2+ , 221.7 mM Cl - , 1.5 mM HPO4 2- , 6.3 mM HCO3 - , 0.075 g / L polyaspartic acid in water.

10. A bioactive dental implant, characterized by It is prepared by the preparation method of the bioactive dental implant according to any one of claims 3 to 8.

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