Bifunctional treatment reagent for promoting osteogenesis and inhibiting osteoclast on titanium surface and application

Abstract

The invention discloses a bifunctional treatment reagent for promoting osteogenesis and inhibiting osteoclast on a titanium surface and application, and belongs to the field of biomedical engineering. The bifunctional treatment reagent is prepared from a phosphate buffer solution and 1 to 20 mmol / L of N-acetylcysteine, the mass ratio of dipotassium phosphate to monopotassium phosphate in the phosphate buffer solution is (4: 1)-(3: 1). The difunctional treatment reagent is an injection preparation, and the use method of the difunctional treatment reagent is as follows: the difunctional treatment reagent is locally injected at the implant position of an implant once every 3-7 days, and 5-20 [mu] L / site is injected each time. The bifunctional treatment reagent provided by the invention can improve the bone combination effect of the implant by adjusting osteogenesis-osteoclast balance on the surface of the implant and remodeling a local bone metabolism microenvironment, and a one-stop solution is provided for a patient suffering from systemic systematic diseases, so that the bifunctional treatment reagent is suitable for dentality repair of the patient suffering from systemic systematic diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to a dual-function therapeutic agent for promoting osteogenesis and inhibiting osteoclastosis on a titanium surface and its application, wherein a modified PBS buffer is used in combination with NAC to promote osteogenesis and reduce osteoclastosis to enhance implant bone integration. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Implant restoration is a common method of oral clinical treatment for various tooth defects and missing teeth. It is increasingly used due to its beautiful appearance and excellent retention. Good bone integration determines the success of implant restoration treatment, while various systemic diseases and chronic diseases are adverse factors that endanger the success rate of implant restoration treatment.

[0004] After implantation, the surrounding bone tissue will undergo a long-term bone remodeling process. In the process of early woven bone formation and mid-to-late lamellar bone formation, physiological osteoclast activity is accompanied to maintain normal bone remodeling. However, the latest studies have shown that common systemic diseases such as diabetes and hyperlipidemia can significantly enhance local osteoclast activity and inhibit osteoblastic activity due to their easy occurrence of systemic low-grade inflammation in patients, thus seriously affecting the effect of peri-implant bone integration. Therefore, for patients with systemic diseases, developing a dual-function treatment system that effectively promotes osteoblastic activity and inhibits osteoclast activity, and improving the implant bone integration effect by regulating the osteoblast-osteoclast balance on the implant surface is the frontier direction and urgent problem to be solved in the current research on implant restoration treatment. The remodeling of the physiological osteoblast-osteoclast balance in the peri-implant tissue will provide a good local microenvironment for the formation and remodeling of woven bone and lamellar bone, and ultimately greatly improve the implant restoration success rate and clinical treatment effect of patients with systemic diseases.

[0005] The article "ALDH6A1 improves implant bone integration in hyperlipidemic mice by regulating the ROS / Wnt / β-catenin pathway and its mechanism" records the role of N-acetylcysteine ​​(NAC) in improving implant bone integration and remodeling osteoblast activity in hyperlipidemic mice. NAC effectively restored the Wnt / β-catenin pathway that was inhibited by osteoblasts under high-fat stimulation, significantly increased the expression of Non-p-β-catenin and ALP in the peri-implant tissue of hyperlipidemic mice, remodeled the osteogenic activity of osteoblasts, and significantly improved implant bone integration in hyperlipidemic mice. However, due to its strong reducing properties, NAC will quickly lose its activity if exposed to an aerobic environment for a long time, and will lose 50% of its reducing ability within a few hours even in a neutral solution. Therefore, it is urgent to extend the half-life of NAC to improve its effect in improving implant bone integration. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a dual-function therapeutic agent and application thereof for promoting osteogenesis and inhibiting osteogenesis on a titanium surface. The dual-function therapeutic agent provided by the present invention can improve the bone bonding effect of the implant by adjusting the osteogenesis-osteogenesis balance on the implant surface, thereby being suitable for tooth restoration in patients with systemic diseases. In order to achieve the above purpose, the technical scheme of the present invention is as follows:

[0007] The first aspect of the present invention provides a dual-function therapeutic agent for promoting osteogenesis and inhibiting osteoclastosis on a titanium surface, wherein the dual-function therapeutic agent is composed of a phosphate buffer and 1 to 20 mmol / L N-acetylcysteine;

[0008] The mass ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate in the phosphate buffer is 4:1 to 3:1.

[0009] It should be noted that the dual-function therapeutic agent is an injectable preparation, and its method of use is: locally inject the dual-function therapeutic agent at the implantation site, once every 3 to 7 days, 5 to 20 μL / site each time.

[0010] It should be noted that the pH of conventional phosphate buffer is usually between 7.0 and 7.4. The present invention uses modified phosphate buffer solution (mPBS) and N-acetylcysteine ​​(NAC) to form a dual-functional therapeutic agent, namely mPBS@NAC.

[0011] First, NAC can effectively regulate the local tissue microenvironment, reduce the level of inflammatory response and oxidative stress, thereby promoting bone formation and inhibiting bone resorption at the same time. However, due to its strong reducing property, NAC will quickly lose its activity when exposed to an aerobic environment for a long time, and even in a neutral solution (such as PBS solution, pH = 7.0-7.4), it will lose 50% of its reducing ability within a few hours. Therefore, the present invention innovatively proposes to use weakly acidic conditions to stabilize the reducing ability of NAC for a long time, thereby effectively extending the half-life of its efficacy. A large number of experiments have shown that compared with ordinary PBS solution (pH = 7.0-7.4), the mPBS solution prepared by adding distilled water to potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a mass ratio of 4:1-3:1 has a 36-fold increase in the half-life of NAC dissolved inside.

[0012] Secondly, although NAC has a good regulatory effect on the surge in local oxidative stress levels caused by systemic diseases (such as diabetes and hyperlipidemia), and can reshape the microenvironment by promoting osteogenesis and inhibiting osteoclasts, excessive local application of NAC will also lead to the occurrence of reductive stress reactions, thereby causing a large number of cell apoptosis. Therefore, in order to have good biocompatibility and eliminate possible cytotoxicity, the present invention has been verified through a large number of experiments to determine that the local safe application concentration of mPBS@NAC solution in the tissue around the implant is 1-20mmol / L.

[0013] Finally, since the mPBS solution containing NAC is a flowing liquid, there is still no appropriate operation method and technical standard for local application. The present invention, after comprehensive consideration of factors such as drug concentration, absorption rate, patient comfort and compliance with treatment, proposes an operation method of local injection in the tissue around the implant after a large number of experimental demonstrations, and clarifies that the injection frequency should be once every 3 to 7 days, 5 to 20 μL / site each time.

[0014] The above-mentioned dual-functional therapeutic reagent and its use method constitute a dual-functional therapeutic system, which is safe, non-toxic, simple and easy to operate, and can bring patients a good treatment experience and comfort, thereby improving the clinical implant restoration treatment effect of patients with systemic diseases.

[0015] In some embodiments of the present invention, the pH of the phosphate buffer is between 6.0 and 6.6. At room temperature, the pH of the phosphate buffer is 6.2.

[0016] In some embodiments of the present invention, the concentration of N-acetylcysteine ​​in the dual-functional therapeutic agent is 10 mmol / L.

[0017] In some embodiments of the present invention, the phosphate buffer consists of potassium dihydrogen phosphate, dipotassium hydrogen phosphate and water.

[0018] In some embodiments of the present invention, in the phosphate buffer, the concentration ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate is 8 g / L:2 g / L to 6 g / L:2 g / L.

[0019] In some embodiments of the present invention, in the phosphate buffer, the concentration of dipotassium hydrogen phosphate is 6.6 g / L, and the concentration of potassium dihydrogen phosphate is 2 g / L.

[0020] The second aspect of the present invention provides a use of the dual-function therapeutic agent in improving the bone integration effect of implants.

[0021] In some embodiments of the present invention, the implant is a titanium implant.

[0022] In some embodiments of the present invention, the application includes improving the implant restoration treatment effect of patients suffering from systemic diseases.

[0023] In some embodiments of the present invention, the patient suffering from a systemic disease includes an edentulous patient suffering from a systemic disease.

[0024] In some embodiments of the present invention, the systemic disease causes systemic low-grade inflammation in the patient, including hyperlipidemia and diabetes.

[0025] In some embodiments of the present invention, the application is to improve the effect of tooth restoration in toothless patients suffering from systemic diseases.

[0026] The third aspect of the present invention provides the use of phosphate buffer in prolonging the half-life of N-acetylcysteine;

[0027] The phosphate buffer consists of potassium dihydrogen phosphate, dipotassium hydrogen phosphate and water, and the mass ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate is 4:1 to 3:1.

[0028] The present invention innovatively proposes to use weakly acidic conditions to stabilize the reducing ability of NAC for a long time, thereby effectively extending the half-life of its efficacy. A large number of experiments have shown that compared with ordinary PBS solution (pH = 7.0-7.4), the half-life of NAC dissolved in mPBS solution prepared by adding dipotassium hydrogen phosphate and potassium dihydrogen phosphate to distilled water in a mass ratio of 4:1-3:1 is extended by 36 times.

[0029] The beneficial effects of the present invention are:

[0030] After the dual-function therapeutic agent provided by the present invention is injected into the implantation site, a good local microenvironment is provided for implant bone integration in a systemic low-grade inflammatory environment. The mPBS in the dual-function therapeutic agent mPBS@NAC can maintain the reducing activity of NAC within 72h to 168h, promote osteogenic activity on the titanium surface and inhibit osteoclast activity, thereby reshaping the osteogenic-osteoclast balance in the tissue around the implant; local microinjection can significantly improve the patient's comfort and compliance in receiving treatment, and maximize the biological safety of the treatment, providing the possibility for safe and efficient clinical treatment. The dual-function therapeutic agent for promoting osteogenesis and inhibiting osteoclasts on the titanium surface provided by the present invention can provide a one-stop solution for toothless patients suffering from systemic diseases, improve implant bone integration, and ultimately greatly improve the clinical implant restoration treatment effect.

[0031] Experiments have shown that the dual-function therapeutic agent provided by the present invention is safe and comfortable, and can significantly improve the bone bonding level of implants under hyperlipidemia, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0033] Figure 1 The present invention is a method for determining the reducing activity of NAC and detecting the biosafety concentration of NAC in Example 1; wherein A shows the changes in the reducing activity of NAC within 72 hours in a conventional PBS solution, an αMEM medium, and a high-fat medium determined by a DPPH free radical scavenging experimental method; B shows the changes in the reducing activity of NAC within 72 hours in a conventional PBS solution and a modified PBS solution determined by a DPPH free radical scavenging experimental method; C shows the effect of the mPBS@NAC solution on the proliferation activity of BMMSCs when the concentration is 0 to 1000 mmol / L; D shows the effect of the locally injected mPBS@NAC solution on the body weight of mice when the concentration is 0 to 50 mmol / L;

[0034] Figure 2 The figure is a schematic diagram of the setting and operation instructions for the in vivo animal experiment grouping in Example 1 of the present invention; wherein, the in vivo animal experiment is divided into 3 groups: a negative control group (NC group) formed by normal feeding of mice, a hyperlipidemia mouse group (HF group) formed by feeding mice with a high-fat diet, and a hyperlipidemia mouse group (HF+NAC group) formed by feeding mice with a high-fat diet and then locally injected with mPBS@NAC solution. Each group of mice is implanted with a titanium implant with a length of 3 mm and a diameter of 1 mm at the distal end of the femur, and receives an injection treatment every 3 days until the mice are killed 30 days later and the corresponding specimens are obtained;

[0035] Figure 3 The results of H&E staining of tissue sections of the liver and spleen of each group of mice receiving injection treatment every 3 days in the in vivo animal experiment in Example 1 of the present invention; the H&E staining results of the two main detoxification organs both show that the injection treatment in the example of the present invention has no adverse effects on the mice, that is, the dual-function treatment system (including the treatment reagent and the operation method) used in the present invention has good biosafety;

[0036] Figure 4 The present invention is about the bone integration of mice around the implant and the bone density of the new bone in each group after implantation in Example 1 of the present invention; wherein A shows the bone integration effect of the implant in mice in the negative control group (NC group) and the experimental group (HF group and HF+NAC group); B shows Figure 4 The percentage of new bone in the total volume around the implant in A, as well as the number of trabeculae, trabecular spacing, and trabecular thickness of the new bone;

[0037] Figure 5 The analysis of the osteoclast activity intensity in the bone tissue around the implants of mice in each group after implantation in Example 1 of the present invention; wherein A shows the number of osteoclasts and bone pits in the bone tissue around the implants; B shows Figure 5 Statistical analysis of the number of osteoclasts and bone pits in A; C shows the level of oxidative stress (8-OHdG, green) and the expression of osteoclast activity markers (RANKL, red) in peri-implant tissues; D shows Figure 5 Statistical analysis of the mean fluorescence intensity of 8-OHdG and RANKL in C. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0039] The dual-function therapeutic system described in the following embodiments consists of a dual-function therapeutic agent and an operation method. The operation method is a method for using the dual-function therapeutic agent, specifically, locally injecting the above-mentioned therapeutic agent mPBS@NAC at the implantation site, once every 3 to 7 days, 5 to 20 μL / site each time.

[0040] Example 1

[0041] Experimental materials and methods:

[0042] (I) Determine the optimal formula of the solvent mPBS for preparing the therapeutic reagent mPBS@NAC 1. Determination of the reducing ability of NAC in different buffers:

[0043] First, PBS (pH = 7.4), αMEM medium, and HF medium were used to prepare NAC solutions with a concentration of 10 mmol / L. Secondly, 50 μg / mL of DPPH ethanol solution was prepared, and after sufficient ultrasonic dispersion, it was kept still and protected from light. Again, 100 μL of NAC solution was fully mixed with 100 μL of DPPH solution and kept at room temperature in the dark for 30 minutes. Finally, the absorbance of each well was measured at 517 nm using a spectrophotometer. According to the measured absorbance value, the DPPH clearance rate of each concentration was calculated, and the clearance rates of different NAC solutions were plotted as a line graph to facilitate the observation and analysis of the experimental results.

[0044] The formula of HF culture medium is as follows: DMEM finished culture medium + 500 μM oleic acid + 500 μM palmitic acid + 10% FBS. The DMEM culture medium is: Dulbecco's modified eagle medium (DMEM, Biological Industries, Israel).

[0045] 2. Preparation of modified PBS solution (mPBS):

[0046] According to the test results of "NAC reducing ability determination in different buffers", a weakly acidic modified PBS solution was prepared. First, multiple gradient PBS aqueous solutions were prepared by adjusting the mass ratio between potassium dihydrogen phosphate and dipotassium hydrogen phosphate. Secondly, the pH value and buffer range of PBS aqueous solutions with different ratios were determined using a pH agent. Finally, the formula that can make the pH buffer range between 6.0 and 6.6 and the pH value of 6.2 at room temperature was determined to be the optimal formula ratio of mPBS.

[0047] 3. Determination of reducing ability of NAC in PBS and mPBS:

[0048] First, PBS and mPBS were used as solvents to prepare 10 mmol / L NAC solutions, which were placed at room temperature and exposed to air. Secondly, 100 μL of samples were taken at 1, 2, 6, 12, 24, 48, and 72 hours, mixed with 100 μL of the previously prepared DPPH ethanol solution, and incubated at room temperature for 30 minutes in the dark. Finally, the absorbance of each well was measured at 517 nm using a spectrophotometer. Based on the measured absorbance values, the DPPH clearance rate of each concentration was calculated, and the clearance rates of the NAC solutions sampled at different time points were plotted as line graphs to facilitate the observation and analysis of the changes in the reducing ability of NAC in different solvents when exposed to an oxygen-containing environment.

[0049] (II) NAC biosafety concentration detection

[0050] 1. In vitro biosafety concentration detection of NAC: First, mouse femoral bone marrow mesenchymal stem cells (BMMSCs) were extracted and serially passaged to P3-P6 for subsequent use; secondly, mPBS was used as a solvent to prepare mPBS@NAC solutions with concentrations of 0, 1, 10, 100, and 1000 mmol / L, respectively, and stored in a sealed container at room temperature; thirdly, the culture medium of BMMSCs was replaced with mPBS@NAC solutions of different concentrations and incubated at 37°C for 1 hour; finally, the proliferation activity of BMMSCs was detected by the CCK8 method and compared with the experimental group with 0 mmol / L to reflect the cytotoxicity of mPBS@NAC solutions of different concentrations.

[0051] 2. Detection of biosafety concentration of NAC in vivo: First, mPBS was used as solvent (pH buffer range was between 6.0 and 6.6 and pH value was 6.2 at room temperature) to prepare mPBS@NAC solutions with concentrations of 0, 1, 10, 20, and 50 mmol / L, respectively, and stored in a sealed container at room temperature; secondly, 30 8-week-old C57 male mice were selected, randomly divided into 5 groups and the initial body weight was weighed one by one, which was the body weight on the first day; thirdly, the above solutions were injected into the knee joint capsule of C57 mice, and the mice were fed continuously for 6 days according to the uniform standard, and the body weight was weighed again on the 4th and 7th days; finally, the above body weight data of different groups were compared to reflect the effects of different concentrations of mPBS@NAC solutions injected into mice on their physiological activity, so as to determine the biosafe application concentration of NAC in vivo.

[0052] (III) In vivo experiments to test the effect of the dual-function treatment system on the osseointegration of titanium implants and its role in regulating the osteoblastic-osteoblastic balance on titanium surfaces

[0053] 1. In vivo experimental grouping and operation procedures Figure 2 As shown:

[0054] First, C57 mice were fed with a high-fat diet for 30 days until their blood lipid levels significantly increased to 3 times that of mice fed with a normal diet, which defined them as hyperlipidemic mice. Secondly, C57 mice with a normal diet and the above-mentioned high-fat diet-induced hyperlipidemia mice were taken for animal experiments and divided into 3 groups: a negative control group (NC group) formed by normal feeding of mice, a hyperlipidemia group (HF group) formed by mice fed with a high-fat diet, and a hyperlipidemia group (HF+NAC group) formed by mice fed with a high-fat diet and locally injected with mPBS@NAC solution. Finally, each group of mice was implanted with a titanium implant with a length of 3 mm and a diameter of 1 mm at the distal end of the femur, and received an injection treatment every 3 days until the mice were killed 30 days later and the corresponding specimens were obtained. Among them, the mice in the NC group and HF group were locally injected with 5 μL of mPBS solution (6.6 g / L dipotassium hydrogen phosphate + 2 g / L potassium dihydrogen phosphate) each time, and the mice in the HF+NAC group were locally injected with 5 μL of mPBS@NAC solution (6.6 g / L dipotassium hydrogen phosphate + 2 g / L potassium dihydrogen phosphate + 10 mmol / L NAC, wherein the pH buffer range of the mPBS solution was between 6.0 and 6.6 and the pH value was 6.2 at room temperature) each time.

[0055] 2. In vivo toxicity testing of the dual-function therapeutic system of local injection of NAC:

[0056] First, after 30 days of local injection every 3 days, the mice were killed and their liver and kidney, two major detoxification organs, were obtained. Secondly, the above organ tissues were fixed with paraformaldehyde, dehydrated and paraffin-embedded to make 5μm thick tissue sections. Finally, the paraffin tissue sections were stained with H&E staining and the tissue morphology was observed under an optical microscope to determine the in vivo toxicity of the dual-function therapeutic system of local injection of NAC.

[0057] 3. Detection and evaluation of the dual-function treatment system to improve the bone integration effect of titanium implants in hyperlipidemia:

[0058] Micro computed tomography (Micro-CT) was used to evaluate the implant bone integration effect and the proportion of new bone around the titanium implants, trabecular density, trabecular width, trabecular spacing and other indicators of each group of mice, and then to analyze the effect of the dual-function treatment system on the osteogenic activity around titanium implants under hyperlipidemia and its role in improving the bone integration level.

[0059] 4. Detection and evaluation of the dual-function treatment system to inhibit the level of osteoclast activity in the tissue around titanium implants under hyperlipidemia:

[0060] First, the titanium implant in the femoral tissue of mice was removed after paraformaldehyde fixation and ethanol gradient dehydration, and paraffin-embedded and tissue sections were obtained. Secondly, the above tissue sections were stained with the TRAP staining kit to observe the number of osteoclasts and the intensity of osteoclast activity in the tissue around the titanium implant. Finally, the above tissue sections were double-stained with immunofluorescence to observe the level of oxidative stress damage (8-OHdG, green) and the expression of osteoclast marker protein (RANKL, red) in the tissue around the titanium implant, and then analyze the inhibitory effect of the dual-function treatment system on the level of local inflammatory damage and the intensity of osteoclast activity under hyperlipidemia.

[0061] Experimental results:

[0062] (I) In vitro experiments to determine the formula and biocompatibility of the dual-functional therapeutic agent mPBS@NAC

[0063] 1. As Figure 1 As shown in A, the DPPH free radical scavenging test method was used to determine the reduction activity changes of NAC in conventional PBS solution, αMEM medium and HF medium within 72 hours. Among them, when conventional PBS solution and αMEM medium were used as solvents, the reduction activity of NAC exposed to aerobic environment decayed rapidly, and the reduction capacity was reduced to 49% of the initial state after 2 hours; when HF medium was used as solvent, the reduction activity of NAC exposed to aerobic environment decayed rapidly at a slower rate, and the reduction capacity decayed to 46% of the initial state after 12 hours.

[0064] The above results indicate that oxidation does lead to a rapid loss of the reducing activity of NAC in neutral or weakly alkaline solutions, while a weakly acidic environment (HF medium) has a certain protective effect on the reducing ability of NAC.

[0065] 2. If Figure 1 As shown in Figure B, the DPPH free radical scavenging experimental method was used to determine the changes in the reducing activity of NAC within 72 hours in conventional PBS solution (PBS group) and modified PBS solution (mPBS group). Since mPBS has a pH of 6.2 at room temperature and has a good buffering capacity in the range of pH 6.0 to 6.6, it can protect the reducing ability of NAC. In mPBS, the reducing activity of NAC will not drop to 47% of the initial state until 72 hours, that is, compared with conventional PBS as a solvent, mPBS as a solvent can extend the half-life of NAC reducing ability by 36 times.

[0066] 3. Such as Figure 1As shown in Figure C, when the concentration of NAC was between 0 and 1000mmol / L, its effect on the proliferation activity of BMMSCs was significantly different. 1mmol / L and 10mmol / L NAC could significantly increase the proliferation activity of BMMSCs, while 100mmol / L and 1000mmol / L NAC had obvious cytotoxicity to BMMSCs. Figure 1 As shown in D, when the concentration of the mPBS@NAC solution injected locally was 0-20mmol / L, it had no significant effect on the weight of mice, while when the concentration of the mPBS@NAC solution injected locally was 50mmol / L, it could induce weight loss in mice. The above in vitro and in vivo evaluation results on the biosafety of NAC showed that 10mmol / L is the optimal concentration for local application and is safe and non-toxic.

[0067] (II) In vivo experiments to examine the effects of the dual-function treatment system on the osseointegration of titanium implants and its role in regulating the osteoblastic-osteoblastic balance on titanium surfaces

[0068] 1. The in vivo animal experiment was divided into three groups: a negative control group (NC group) formed by normal feeding of mice, a group of mice with hyperlipidemia formed by high-fat feeding of mice (HF group), and a group of mice with hyperlipidemia formed by high-fat feeding of mice and then locally injected with mPBS@NAC solution (HF+NAC group). Mice in each group were implanted with a titanium implant with a length of 3 mm and a diameter of 1 mm at the distal end of the femur, and received an injection treatment every 3 days until the mice were killed 30 days later and the corresponding specimens were obtained ( Figure 2 Among them, the mice in the NC group and the HF group were locally injected with 5 μL of mPBS solution (6.6 g / L dipotassium hydrogen phosphate + 2 g / L potassium dihydrogen phosphate), and the mice in the HF+NAC group were locally injected with 5 μL of mPBS@NAC solution (6.6 g / L dipotassium hydrogen phosphate + 2 g / L potassium dihydrogen phosphate + 10 mmol / L NAC).

[0069] like Figure 3 As shown, the H&E staining results of the liver and spleen tissue sections of each group of mice showed that the injection of the dual-function therapeutic agent mPBS@NAC had no adverse effects on the mice, that is, the dual-function therapeutic system (including therapeutic agents and operation methods) used in the present invention has good biosafety.

[0070] 2. If Figure 4As shown in the figure, the results of evaluating the bone integration effect of implants in each group of mice by micro computed tomography (Micro-CT) showed that compared with the simple hyperlipidemia mice (HF group), the bone integration rate of mice receiving local injection of dual-function therapeutic reagent mPBS@NAC injection treatment (HF+NAC group) was significantly improved, and the new bone ratio, trabecular density, trabecular width, trabecular spacing and other indicators around the titanium implants were significantly improved, which were closer to the normal group of mice (NC group). The above results show that receiving local injection of dual-function therapeutic reagent mPBS@NAC injection treatment can effectively enhance the intensity of osteogenic activity on the surface of titanium implants under systemic diseases (such as hyperlipidemia), and ultimately significantly improve the bone integration effect.

[0071] 3. Such as Figure 5 As shown in the figure, the results of using TRAP staining to observe the number of osteoclasts and the intensity of osteoclast activity in the tissue around titanium implants showed that compared with the simple hyperlipidemia mice (HF group), the number of osteoclasts and the number of bone pits in the bone tissue around titanium implants of mice receiving local injection of the dual-function therapeutic agent mPBS@NAC injection treatment (HF+NAC group) were significantly reduced, that is, the NAC in the dual-function therapeutic agent mPBS@NAC effectively played a role in inhibiting local osteoclast activity. At the same time, the results of using immunofluorescence double staining to observe the level of oxidative stress damage (8-OHdG, green) and the expression of osteoclast marker protein (RANKL, red) in the tissue around titanium implants showed that after repeated injections of NAC treatment, the level of oxidative stress in the tissue around titanium implants was downregulated, thereby inhibiting the expression of osteoclast-related proteins.

[0072] In summary, the bifunctional therapeutic agent mPBS@NAC was successfully prepared and optimized in this embodiment, which has good biocompatibility. After being applied to the tissue around the implant by local injection, it can significantly promote osteogenic activity on the titanium surface and inhibit osteoclastic activity, ultimately significantly improving the implant bone integration effect under hyperlipidemia.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A dual-function therapeutic agent for promoting osteogenesis and inhibiting osteoclastosis on a titanium surface, characterized in that: The dual-function therapeutic reagent is composed of phosphate buffer and 1-20 mmol / L N-acetylcysteine; The mass ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate in the phosphate buffer is 4:1 to 3:

1.

2. The dual-function therapeutic agent according to claim 1, characterized in that: The pH of the phosphate buffer is between 6.0 and 6.

6. At room temperature, the pH of the phosphate buffer is 6.

2.

3. The dual-function therapeutic agent according to claim 1, characterized in that: The concentration of N-acetylcysteine ​​in the dual-functional therapeutic agent is 10 mmol / L.

4. The dual-function therapeutic agent according to claim 1, characterized in that: The phosphate buffer consists of potassium dihydrogen phosphate, dipotassium hydrogen phosphate and water.

5. The bifunctional therapeutic agent according to claim 1, characterized in that: In the phosphate buffer, the concentration ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate is 8g / L:2g / L to 6g / L:2g / L.

6. The bifunctional therapeutic agent according to claim 1, characterized in that: In the phosphate buffer, the concentration of dipotassium hydrogen phosphate is 6.6 g / L, and the concentration of potassium dihydrogen phosphate is 2 g / L.

7. Use of the dual-function therapeutic agent according to any one of claims 1 to 6 in improving the bone integration effect of implants.

8. The use according to claim 7, characterized in that The implant is a titanium implant.

9. The use according to claim 7, characterized in that The application includes improving the treatment effect of implant restoration for patients suffering from systemic diseases; Preferably, the patients suffering from systemic diseases include toothless patients suffering from systemic diseases; Preferably, the systemic disease causes low-grade inflammation in the patient's whole body, including hyperlipidemia and diabetes; Preferably, the application is to improve the effect of tooth restoration in toothless patients suffering from systemic diseases.

10. Application of phosphate buffer in prolonging the half-life of N-acetylcysteine; The phosphate buffer consists of potassium dihydrogen phosphate, dipotassium hydrogen phosphate and water, and the mass ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate is 4:1 to 3:1.

Images