Cordycepin-loaded, mineralized, angiogenic hydrogel microspheres, preparation method, and application thereof

Abstract

The present invention relates to the technical field of biomaterials, specifically to a cordycepin-loaded, mineralized, angiogenic hydrogel microspheres, and a preparation method and application thereof. The present invention combines immersion adsorption and intermittent ultrasonic method, uses dopamine-modified biomineralized hydrogel microspheres to adsorb cordycepin solution for 12-24 hours, washes, freeze-dries, and obtains cordycepin-loaded, mineralized, angiogenic hydrogel microspheres. In vitro experiments and rat experiments have shown that the cordycepin-loaded, mineralized, angiogenic hydrogel microspheres have extremely low stem cell toxicity, can be well adhered to the alveolar bone surface, can effectively promote the osteogenic differentiation of periodontal ligament stem cells, inhibit osteoclast differentiation, and promote cell angiogenic differentiation. Local injection of cordycepin-loaded, mineralized, angiogenic hydrogel microspheres in a rat silk thread ligature-induced periodontitis model can effectively delay bone loss caused by periodontitis.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to cordycepin-loaded, adherent, mineralized, and angiogenic hydrogel microspheres, and a preparation method and application thereof. Background Art

[0002] Chronic periodontitis is a chronic infectious disease, which is determined by bacteria, host, and environment. The antigenic components of dental plaque bacteria and the toxins and enzymes they produce cause damage to periodontal tissues, triggering host immune and inflammatory responses, leading to periodontal tissue destruction. Its pathological essence is an imbalance in periodontal bone homeostasis, which is mainly manifested as dysfunction between osteoblasts and osteoclasts, resulting in reduced bone density and bone regeneration capacity, affecting the self-repair of the jawbone, resulting in a decrease in the success rate of dental implants and slower healing of tooth extraction wounds. The simple use of treatments that promote bone formation (addition of BMP2 active protein, etc.) or inhibit bone resorption also has the bottleneck problem of short-lasting efficacy and inability to build a healthy periodontal ecological microenvironment.

[0003] At present, the clinical treatment of periodontal disease is still mainly based on traditional mechanical scaling (removal of bacteria) and drug treatment (metronidazole, tetracycline drugs). Drug treatment has poor effect, short treatment duration, and cannot reshape the periodontal immune microenvironment, stimulate the potential for local periodontal new bone regeneration, and cannot restore damaged periodontal tissues. The stability and aesthetic issues of periodontal treatment need to be solved urgently. In addition, long-term use of such drugs is prone to drug resistance and hepato-renal toxicity side effects. Compared with the liver, kidneys and other organs, the periodontal bone tissue has a low degree of vascularization, poor drug penetration, and is not easy to stay after local application.

[0004] Cordycepin is also known as cordyceps sinensis, cordycepin, and cordycepin. Its alias is 3′-deoxyadenosine, and its chemical formula is C 10 H 13 N5O3 is the first nucleoside antibiotic isolated from fungi. As a traditional Chinese medicine compound, cordycepin has biological activities such as lung and kidney protection, anti-three highs, anti-tumor, neuroprotection, anti-inflammatory, antioxidant and immunomodulatory. Therefore, cordycepin has attracted much attention from scholars in the fields of anti-aging, health care, and new drug development. However, no research has reported that it can be used in periodontal ligament stem cells and periodontitis treatment.

[0005] Hydrogel is a polymer material with three-dimensional porosity, good hydrophilicity, high viscoelasticity, and good biocompatibility. In recent years, hydrogel microspheres have gradually emerged. They have a three-dimensional network structure similar to that of large bulk hydrogels, and their unique micron-scale size (usually between 1-1000μm) gives them modular functional properties. Compared with macroscopic hydrogels, hydrogel microspheres can improve the response speed of smart hydrogels, and have the ability to protect, deliver and locally release drugs and growth factors. Therefore, they have broad application prospects for the local delivery of bioactive substances. The emulsification-solvent extraction method is to add the hydrogel prepolymer solution to a container containing an appropriate volume of oil and stir it. Through simple mechanical mixing, the hydrogel prepolymer will be dispersed into droplets of different sizes, and then the droplets are cross-linked, washed, and centrifuged to remove the oil phase. This preparation method has low equipment dependence, simple operation, and is easy to mass produce.

[0006] The present invention is based on emulsion-solvent extraction technology and utilizes biomineralization and polydopamine modification to prepare injectable, adherent mineralized hydrogel microspheres loaded with cordycepin compounds, which can promote osteogenesis of periodontal ligament stem cells, inhibit osteoclast differentiation, promote cell angiogenesis, and promote the treatment of periodontitis. Summary of the invention

[0007] The purpose of the present invention is to provide a cordycepin-loaded, mineralized, angiogenic hydrogel microsphere and a preparation method and application thereof.

[0008] The cordycepin-loaded, adherent, mineralized, and angiogenic hydrogel microspheres prepared by the present invention combine the advantages of hydrogel microspheres such as three-dimensional porosity, good hydrophilicity, high viscoelasticity, and good biocompatibility with the high adhesion and biomineralization characteristics of the dopamine coating, and can effectively load the compound cordycepin and increase the unit load, thereby preparing an injectable preparation with jawbone adhesion and drug carrying capacity, which can be used to promote the osteogenic differentiation of periodontal ligament stem cells, inhibit osteoclast differentiation, promote cell angiogenesis, and achieve the treatment of periodontitis.

[0009] In order to solve the above technical problems, this application provides the following technical solutions:

[0010] The invention discloses a cordycepin-loaded, adherent, mineralized, and angiogenic hydrogel microsphere, comprising dopamine-modified biomineralized hydrogel microspheres and cordycepin loaded on the surface thereof.

[0011] Furthermore, the dopamine-modified biomineralized hydrogel microspheres include a microporous hydrogel and a dopamine self-polymerized coating and a calcium-phosphate mineralization layer covering the surface of the microporous hydrogel. The microporous hydrogel and the dopamine self-polymerized coating are chemically connected by covalent and non-covalent bonding methods, and a calcium-phosphate biomineralization layer is deposited.

[0012] The method for preparing the cordycepin-loaded, mineralized, angiogenic hydrogel microspheres of the present invention is specifically as follows: combining immersion adsorption and intermittent ultrasonic method, using dopamine-modified biomineralized hydrogel microspheres to adsorb cordycepin solution for 12-24 hours, washing, and freeze-drying to obtain the cordycepin-loaded, mineralized, angiogenic hydrogel microspheres of the present invention.

[0013] Furthermore, the preparation method of dopamine-modified biomineralized hydrogel microspheres comprises the following steps:

[0014] (1) Preparation of methacryloyl gelatin (GelMA) hydrogel;

[0015] Add methacrylic anhydride to the gelatin solution for reaction. After the reaction is completed, remove the free methacrylic anhydride to obtain methacrylic gelatin. After freeze-drying, obtain a white foamy freeze-dried gel, namely methacrylic gelatin (GelMA) hydrogel.

[0016] (2) Preparation of microporous GelMA hydrogel microspheres;

[0017] The GelMA hydrogel with temporary physical crosslinking and photoinitiator was exposed to ultraviolet light to induce photocrosslinking, and then subjected to gradient freeze-drying to obtain microporous GelMA hydrogel microspheres;

[0018] (3) Preparation of dopamine-modified microporous GelMA hydrogel microspheres;

[0019] The microporous GelMA hydrogel microspheres were immersed in a dopamine alkaline solution by an immersion method, and then washed and freeze-dried to obtain dopamine-modified microporous GelMA hydrogel microspheres.

[0020] (4) Preparation of dopamine-modified microporous biomineralized GelMA hydrogel microspheres

[0021] The construction of the biomineralization layer of hydrogel microspheres is achieved by simulating the gradient infiltration of body fluids.

[0022] The step (1) is as follows: dissolving gelatin at 1 g: 8-12 mL in PBS at 40-60° C., adding an equal volume of methacrylic anhydride, stirring, and then adding PBS to dilute the reaction system by 3-10 times to terminate the reaction; dialyzing with a dialysis bag, replacing double distilled water every day to completely remove free methacrylic anhydride until the liquid in the dialysis bag is clear, and the product is GelMA with a methacryloyl substitution degree of 100%, and freeze-drying to obtain a white foamy freeze-dried gel, namely, GelMA hydrogel.

[0023] Specifically, step (2) comprises: preparing a GelMA hydrogel into a GelMA solution with a mass volume concentration of 3-8%, dropping the solution into liquid paraffin containing a lipophilic nonionic surfactant, stirring the solution at room temperature to form an oil-in-water emulsion, and forming a plurality of aqueous microspheres in the oil phase; continuously stirring the solution in an ice bath to generate temporary physical crosslinks between the microspheres, and then exposing the solution to ultraviolet light (405 nm) to induce photocrosslinking, and then removing the oil phase with acetone, and subjecting the separated microspheres to gradient cooling and freeze drying to form microporous GelMA hydrogel microspheres.

[0024] Wherein, step (3) specifically comprises: immersing the microporous GelMA hydrogel microspheres in a dopamine alkaline solution for more than 4 hours, taking them out, washing the unbound dopamine with PBS, and freeze-drying them.

[0025] In step (1) and step (3), the prepared products are stored at -20°C for future use.

[0026] Wherein, in step (2), the lipophilic nonionic surfactant is Span80; in step (3), the dopamine solution contains 2-4 mg / ml dopamine, 10 mM Tris-HCL, pH 8-9.

[0027] Specifically, step (4) comprises placing the microporous GelMA hydrogel microspheres in a 5x simulated body fluid solution for more than 12-24 hours, during which time ultrasonic treatment is performed at a frequency of 35 kHz every 2 hours for 12 hours, and then taking out the microspheres, washing the unbound simulated body fluid with PBS, and freeze-drying the microspheres.

[0028] The cordycepin-loaded, mineralized, angiogenic hydrogel microspheres prepared by the invention can be used to prepare drugs for promoting osteogenesis of periodontal ligament stem cells, inhibiting osteoclast differentiation, promoting cell angiogenesis, and promoting the treatment of periodontitis.

[0029] Furthermore, the periodontal ligament stem cells are periodontal ligament stem cells derived from adult individuals.

[0030] Furthermore, the medicine is an injection.

[0031] Compared with the prior art, the cordycepin-loaded, mineralized, angiogenic hydrogel microspheres of the present invention and their preparation method and application have at least the following beneficial effects:

[0032] (1) The present invention uses dopamine-modified adherent mineralized angiogenic hydrogel microspheres loaded with the compound cordycepin. The composite material combines the advantages of hydrogel microspheres such as three-dimensional porosity, good hydrophilicity, high viscoelasticity, and good biocompatibility with the high adhesion and biomimetic mineralization characteristics of the dopamine coating to improve cell adhesion performance and material osteogenic activity. It has very low cytotoxicity and can improve the cell's ability to transport microspheres and drugs.

[0033] (2) The present invention adopts emulsion-solvent extraction technology to prepare hydrogel porous microspheres, which are then placed in a dopamine PDA solution. Dopamine self-polymerizes to form a dopamine coating, which is covalently and non-covalently connected to the hydrogel porous microspheres. The porous hydrogel microspheres have a large specific surface area due to their spherical shape. The porosity of the hydrogel further increases the specific surface area and adsorption (adsorption of adherent cells) of the microspheres. Dopamine can serve as a bridge for loading compounds to increase the unit loading capacity of the compounds. On the other hand, dopamine has high adhesion, which is conducive to the adhesion and transportation of cells and improves the bioavailability of the compounds. The biomineralization layer can well neutralize the acidic ions in the periodontitis microenvironment. The hydrogel porous microspheres are generally uniformly round, regular in shape, and evenly covered with honeycomb-shaped micropores on the surface.

[0034] (3) The present invention has verified through cell level experiments and rat experiments that the adherent mineralized angiogenic hydrogel microspheres loaded with cordycepin can significantly promote the osteogenic differentiation potential of periodontal ligament stem cells under inflammatory conditions, inhibit osteoclast differentiation, and promote cell angiogenic differentiation. The injection of cordycepin-loaded adherent mineralized angiogenic hydrogel microspheres into the silk thread-ligated rat periodontitis model confirmed that the alveolar bone loss caused by periodontitis can be slowed down. Therefore, the adherent mineralized angiogenic hydrogel microspheres loaded with cordycepin can be used to prepare drugs that promote the osteogenic differentiation potential of periodontal ligament stem cells under inflammatory conditions, inhibit osteoclast differentiation, cell angiogenic differentiation, and treat bone loss caused by periodontitis.

[0035] (4) The present invention has confirmed through in vitro studies that cordycepin-loaded adherent mineralized angiogenic hydrogel microspheres can significantly promote the osteogenic differentiation potential of periodontal ligament stem cells under inflammatory conditions, inhibit osteoclast differentiation, and promote the angiogenic ability of cells. Injection of cordycepin-loaded adherent mineralized angiogenic hydrogel microspheres into a silk thread-ligated rat periodontitis model has been shown to slow down alveolar bone loss in periodontitis.

[0036] (5) The cordycepin-loaded mineralized angiogenic hydrogel microspheres of the present invention are simple and convenient to prepare, have strong operability, and can be effectively and long-term used to prepare drugs for treating periodontitis-related diseases, and the drug is preferably an injection.

[0037] The following is a further description of the cordycepin-loaded, mineralized, angiogenic hydrogel microspheres of the present invention and their preparation method and application in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 In the middle, the left picture shows the macroscopic and scanning electron microscope images of the primary microporous hydrogel microspheres (GM) extracted by emulsion-solvent extraction and the biomineralized modified hydrogel microspheres (PGM@CAP) (where a is a macroscopic image, b is an inverted microscope image, and c is a scanning electron microscope image), and the right picture shows the bonding performance of PGM@CAP hydrogel microspheres on the alveolar bone.

[0039] Figure 2 EDS elemental analysis diagram of biomineralized modified hydrogel microspheres (PGM@CAP).

[0040] Figure 3 Immunofluorescence image of biomineralized hydrogel microspheres loaded with cordycepin (PGM@CAP &CY).

[0041] Figure 4 Live / dead cell immunofluorescence staining of cordycepin-loaded biomineralized hydrogel microspheres (PGM@CAP &CY) and periodontal ligament stem cells after 3 days of co-culture.

[0042] Figure 5 In the middle, the left picture shows the Alizarin red staining of biomineralized hydrogel microspheres loaded with cordycepin (PGM@CAP &CY) after stimulating periodontal ligament stem cells in osteogenic induction medium (the darker areas in the picture are positive staining areas), and the right picture is a statistical chart of the corresponding staining intensity.

[0043] Figure 6 RT-qPCR statistics of osteogenic-related genes OSX and RUNX2 after periodontal ligament stem cells were stimulated by biomineralized hydrogel microspheres loaded with cordycepin nanoparticles (PGM@CAP &CY) in osteogenic induction medium.

[0044] Figure 7 In the middle, the left figure is a Transwell diagram of angiogenesis of human umbilical vein endothelial cells treated with biomineralized hydrogel microspheres loaded with cordycepin (PGM@CAP &CY), and the right figure is a statistical diagram of the corresponding number of migrating cells.

[0045] Figure 8 RT-qPCR statistical graph of angiogenesis-related genes VEGF and HIF-1 in human umbilical vein endothelial cells treated with cordycepin-loaded biomineralized hydrogel microspheres (PGM@CAP &CY).

[0046] Fig. 9This is the Trap staining image of biomineralized hydrogel microspheres (PGM@CAP &CY) loaded with cordycepin under osteoclast differentiation induction (the cell fusion vacuolar area in the image is the positive expression area).

[0047] Fig.10 RT-qPCR statistics of osteoclast-related genes Nfatc and CTSK in biomineralized hydrogel microspheres loaded with cordycepin (PGM@CAP &CY) under osteoclast differentiation induction.

[0048] Fig.11 Micro-CT images of cordycepin-loaded biomineralized hydrogel microspheres (PGM@CAP &CY) formulation for the treatment of periodontitis in aged rats.

[0049] Fig.12 HE-stained tissue image of aged rats with periodontitis treated with cordycepin-loaded biomineralized hydrogel microspheres (PGM@CAP &CY) formulation. DETAILED DESCRIPTION

[0050] The principles and features of the present invention are described below in conjunction with the accompanying drawings and specific embodiments. The examples are only used to explain the present invention and are not used to limit the scope of the embodiments of the present invention. The experimental methods in the following examples that do not specify specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The reagents and raw materials used in the present invention are all commercially available.

[0051] Hydrogel is a polymer material with three-dimensional porosity, which has the advantages of good hydrophilicity, high viscoelasticity and good biocompatibility. Hydrogel microspheres have a three-dimensional network structure similar to bulk hydrogels, and their unique micron-scale size (usually 1-1000μm) gives them modular functional properties. Compared with macroscopic hydrogels, hydrogel microspheres can improve the response speed of smart hydrogels and have the ability to protect, deliver and locally release drugs and growth factors. The emulsification-solvent extraction method is to add the hydrogel prepolymer solution to a container containing an appropriate volume of oil and stir it. Through simple mechanical mixing, an oil-in-water emulsion is obtained. The hydrogel prepolymer will be dispersed into droplets of different sizes (the prototype of hydrogel microspheres), and then the droplets are cross-linked, washed, and centrifuged to remove the oil phase to obtain the hydrogel. This preparation method has low equipment dependence, simple operation, and easy large-scale production. The hydrogel is temporarily physically cross-linked and photo-induced cross-linked to finally obtain hydrogel microspheres with a large number of microporous structures. Dopamine is a compound containing catechol and amino groups. Under alkaline conditions, catechol will be converted into quinone and self-polymerize to form polydopamine, which gives the material extremely strong cell adhesion ability. In recent years, polydopamine coatings have been widely used in the biological field due to their extremely strong adhesion properties, but there are relatively few reports on coating polydopamine on the surface of highly uniform hydrogel microspheres to improve the cell carrying capacity of microspheres. Simulated body fluid (SBF) is a metastable solution, which is a highly supersaturated solution relative to apatite. It is a simulated body fluid designed with the concentration of various ions in human plasma as a template. After the microspheres are treated, an apatite coating can be formed on the surface.

[0052] The present invention adopts dopamine-modified biomineralized hydrogel microspheres to load the compound cordycepin. The composite material combines the advantages of hydrogel microspheres such as three-dimensional porosity, good hydrophilicity, high viscoelasticity, and good biocompatibility with the high adhesion and biomineralization performance of the dopamine coating to improve cell adhesion performance, has very low cytotoxicity, and can improve the cell's carrying capacity for microspheres and their drugs.

[0053] Example 1

[0054] This example is the preparation of microporous hydrogel microspheres (GM), dopamine-modified hydrogel microspheres (GM@PDA), and microporous biomineralized GelMA hydrogel microspheres (PGM@CAP). The preparation steps are as follows:

[0055] (1) Preparation of GelMA hydrogel:

[0056] ① Dissolve gelatin at 1g / 10mL in 50℃ PBS, add an equal volume of methacrylic anhydride, pump in a small amount while stirring, and then dilute 5 times with PBS to terminate the reaction;

[0057] ② Use dialysis tape for 2 weeks, replace double distilled water every day, and completely remove free methacrylic anhydride until the liquid is clear. The reaction product is GelMA with a methacryloyl substitution degree of 100%.

[0058] ③ After freeze-drying, a white foamy freeze-dried gel was obtained and stored at -20°C for later use.

[0059] (2) Preparation of primary microporous hydrogel microspheres (GM)

[0060] ① Add 5% w / v GelMA aqueous solution dropwise into liquid paraffin containing Span80 and stir at room temperature for 30 minutes to form a W / O (water-in-oil) emulsion.

[0061] ② The W / O emulsion was stirred continuously in an ice bath for 30 minutes to cause temporary physical cross-linking of the GelMA spheres.

[0062] ③ Expose the GelMA spheres with photoinitiator (methacryloyl) to 405nm UV light to initiate photocrosslinking.

[0063] ④ After removing the oil phase by acetone separation, the separated GelMA spheres were subjected to gradient cooling and freeze-drying to form microporous GelMA hydrogel microspheres.

[0064] (3) Preparation of dopamine modified hydrogel microspheres (GM@PDA)

[0065] ① Immerse the porous GelMA hydrogel microspheres in dopamine solution (2 mg / mL, 10 mM Tris-HCL, pH 8.5) for 6 hours.

[0066] ② Wash the sample three times with 10 mM PBS solution by shaking thoroughly to remove excess dopamine solution, freeze-dry it, and store it at -20°C for later use.

[0067] (4) Preparation of dopamine-modified microporous biomineralized GelMA hydrogel microspheres (PGM@CAP)

[0068] The GM@PDA was fully immersed in 5x SBF solution for 12-24 hours, during which ultrasonic treatment was performed every 2 hours at a frequency of 35 kHz for 12 hours. After that, it was fully shaken and washed 3 times in 10 mM PBS solution to remove excess SBF solution. After that, it was freeze-dried and placed at -20°C for use.

[0069] The freeze-dried PGM@CAP microspheres were placed under a volume microscope for photography. The freeze-dried microporous PGM@CAP hydrogel microspheres were further sprayed with gold and photographed under a scanning electron microscope. Figure 1As shown in Figure c on the left, the PGM@CAP hydrogel microspheres are evenly and densely covered with holes, making the microspheres present a honeycomb structure. The right picture shows the bonding of the microspheres on the surface of the mandibular alveolar bone, showing that the microspheres can be well bonded to the alveolar bone surface.

[0070] like Figure 2 As shown, EDS spectrum analysis shows that the surface of PGM@CAP microspheres presents a uniform distribution of Ca, C, O, and N elements.

[0071] Example 2

[0072] This example is the preparation of biomineralized hydrogel microspheres (PGM@CAP &CY) loaded with the compound cordycepin, and the preparation steps are as follows:

[0073] (1) Preparation of cordycepin compounds

[0074] The nanoparticle solution was prepared by using PLGA nanoparticles (PLGA is polylactic acid-glycolic acid copolymer) through a rotary evaporation method and stirring for 12 hours. After completion, the solution was centrifuged and washed three times at 10000 rpm for 10 minutes for use.

[0075] (2) Take an appropriate amount of cordycepin aqueous solution, mix the freeze-dried drug with the microporous PGM@CAP hydrogel microspheres, soak for 24 hours until swelling equilibrium, wash three times with deionized water to remove excess unbound nanodrugs, and detect the drug loading by laser confocal microscopy. Figure 3 .like Figure 3 The results showed that cordycepin had been loaded onto the surface of dopamine-modified mineralized hydrogel microspheres.

[0076] Example 3

[0077] This example is a biological performance test of PGM@CAP microspheres (i.e., simple mineralized microspheres) and cordycepin-loaded PGM@CAP microspheres (i.e., PGM@CAP &CY). The test method is as follows:

[0078] This project intends to select 5 patients aged 18-25 years old, with no history of systemic diseases, caries and periodontal diseases, who underwent orthodontic reduction teeth extraction in the Department of Oral and Maxillofacial Surgery. After obtaining informed consent from the patients and their families, their orthodontic reduction teeth will be collected, and periodontal ligament stem cells (PDLSCs) from the teeth will be extracted and cultured using the collagenase-neutral dispase digestion method. Subsequent experiments will be carried out using cells from the third (P3) to fifth (P5) generations to verify whether cordycepin-loaded PGM@CAP microspheres are toxic to periodontal ligament stem cells.

[0079] The control group was treated with equal volumes of PBS solution and PGM@CAP microspheres; the experimental group was treated with PGM@CAP &CY microspheres added to periodontal ligament stem cells and the following treatments were performed:

[0080] After culturing under the same conditions, live / dead cell staining was performed to detect the live / dead status of periodontal ligament stem cells under a microscope. Figure 4 As shown in the figure, compared with the control group and PGM@CAP microsphere group, the number of live cells in the PGM@CAP &CY microsphere group was similar, and no obvious distribution of dead cells was shown.

[0081] Example 4

[0082] This example is a verification experiment of the biological function of PGM@CAP &CY microspheres in promoting osteogenic differentiation of periodontal ligament stem cells.

[0083] In order to verify that PGM@CAP &CY microspheres have the effect of promoting osteogenic differentiation, Alizarin Red S (ARS, a marker of osteoblast differentiation or osteocytes) and RT-qPCR experiments were used for verification.

[0084] The control group added PBS and PGM@CAP microspheres to the periodontal ligament stem cell culture dish, and the experimental group added PGM@CAP&CY microspheres to the periodontal ligament stem cell culture dish. For the osteogenic function test, the control group and the experimental group added fresh osteogenic induction medium every 2-3 days and performed the following treatments:

[0085] (1) After 14 days of continuous culture, ARS staining was performed, random photos were taken under the microscope, and the staining positive intensity was counted; (2) After 14 days of continuous culture under the same conditions, RT-qPCR technology was used to count the expression of osteogenic related genes RUNX2 and OSX. The osteogenic differentiation ability of periodontal ligament stem cells was reflected by detecting the expression levels of RUNX2 and OSX in osteoblasts. The experimental results are as follows:

[0086] Among them, Figure 5 It can be seen that the proportion of positive staining area in the control group is lower, while the proportion of positive staining area in the experimental group is higher, and there is statistical significance between the experimental group and the control group. Figure 6 It can be seen that the expression of osteogenic-related genes RUNX2 and OSX in the experimental group was significantly increased compared with the control group, and the difference between the experimental group and the control group was statistically significant. The experimental results show that the osteogenic differentiation ability of periodontal ligament stem cells can be significantly promoted after the treatment of periodontal ligament stem cells with cordycepin PGM@CAP microspheres osteogenic induction medium.

[0087] Example 5

[0088] This example is a verification experiment on the biological function of PGM@CAP &CY microspheres in promoting cell angiogenic differentiation.

[0089] In order to verify that cordycepin-loaded PGM@CAP microspheres have the effect of promoting cell angiogenesis, the length of blood vessel formation was observed by microscopy and the expression of blood vessel formation-related genes was detected by qRT-PCR.

[0090] The control group added PBS to the human umbilical vein endothelial cell culture dish, and the experimental group added PGM@CAP &CY microspheres to the human umbilical vein endothelial cell culture dish. Fresh cell induction culture medium was added to the control group and the experimental group every 2-3 days, and the following treatments were performed:.

[0091] The experimental results are as follows Figure 7 As shown: the length of blood vessels formed in the experimental group (PGM@CAP &CY microspheres) was longer than that in the control group (PBS and PGM@CAP microspheres); Figure 8 The expression of angiogenesis-related markers VEGF and HIF1 was significantly increased.

[0092] From the above experimental results, it can be seen that the angiogenesis experiment and qRT-PCR detection proved that PGM@CAP &CY microspheres promote the angiogenic differentiation of human umbilical vein endothelial cells.

[0093] Example 6

[0094] This example is a verification experiment on the biological function of PGM@CAP &CY microspheres in inhibiting osteoclast differentiation.

[0095] In order to verify that PGM@CAP &CY microspheres have the effect of inhibiting osteoclast differentiation, TRAP staining and RT-qPCR experiments were used to demonstrate this.

[0096] The control group added PBS and PGM@CAP microspheres to the Raw264.7 cell culture dish, and the experimental group added PGM@CAP&CY microspheres to the Raw264.7 cell culture dish. Fresh osteoclast induction medium was added to the control and experimental groups every 2-3 days, and the following treatments were performed:

[0097] After 7 days of continuous culture under the same conditions, the TRAP staining technique was used. Fig. 9 The expression of osteoclastogenesis-related markers NFATC and CTSK is statistically shown. The osteoclast differentiation ability is reflected by detecting the expression levels of NFATC and CTSK in osteoclasts.

[0098] The experimental results are as follows Fig.10 As shown: the expression of osteoclastogenesis-related markers NFATC and CTSK in the experimental group (PGM@CAP &CY) was significantly reduced compared with the control group PBS and PGM@CAP microspheres.

[0099] From the above experimental results, it can be seen that the TRAP staining experiment proves that the use of cordycepin-loaded PGM@CAP microspheres to treat Raw264.7 cells under osteoclast induction medium conditions can significantly inhibit the osteoclast differentiation ability.

[0100] Example 7

[0101] This example is a verification experiment of the biological function of PGM@CAP &CY microspheres in promoting periodontitis induced by silk ligation in rats.

[0102] In order to verify that PGM@CAP &CY microspheres have the biological function of promoting silk ligation-induced periodontitis in rats, Micro-CT imaging and HE staining experiments were used to verify it. HE staining facilitates the observation of the morphology of cell tissues.

[0103] Treatment group (PGM@CAP & CY microspheres): (1) Animal treatment experiment of rat periodontitis model: Sprague Dawley rats weighing 250-300g were selected and anesthetized by intraperitoneal injection of 0.1% sodium pentobarbital solution at a dose of 40mg / kg. The maxillary / mandibular first molars were exposed with a retractor. A 2-0 orthodontic wire was passed through the gap between the first and second molars to ligate the first molar, and placed as close to the bottom of the gingival sulcus as possible. The injection was performed every two days at three sites on the palatal side of the affected tooth: mesial, middle, and distal. The rats were killed after 2 weeks to collect tissues; the mouse maxilla was fixed with 4% paraformaldehyde, and then subjected to Micro-CT scanning, and then used for HE staining experiments.

[0104] Control group (PBS group, PGM@CAP group): pure PBS solution and PGM@CAP solution of equal concentration.

[0105] Experimental group (PGM@CAP &Cy group): PGM@CAP &CY solution of equal concentration.

[0106] Perform the following processing:

[0107] Eight weeks after surgery, Micro-CT scanning was performed, followed by HE staining to detect the height of the alveolar bone. Fig.11 As shown: Micro-CT images showed that the alveolar bone loss of the second molar in the treatment group was significantly lower than that in the control group. HE staining histological results (such as Fig.12 The results of this experiment show that the alveolar bone loss in the treatment group was significantly reduced, and the alveolar bone height in the control group was significantly reduced. The results of this experiment prove that the injection of cordycepin PGM@CAP microspheres can delay the bone loss in periodontitis.

[0108] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of cordycepin-loaded adherent mineralized angiogenic hydrogel microspheres in the preparation of drugs for promoting the treatment of periodontitis, characterized in that: The cordycepin-loaded, mineralized, angiogenic hydrogel microspheres include dopamine-modified biomineralized hydrogel microspheres and cordycepin loaded on the surface thereof; Specifically, the dopamine-modified biomineralized hydrogel microspheres were combined with cordycepin solution for 12-24 hours by immersion adsorption and intermittent ultrasound, and then washed and freeze-dried to obtain cordycepin-loaded adherent mineralized angiogenic hydrogel microspheres.

2. The use of the cordycepin-loaded mineralized angiogenic hydrogel microspheres according to claim 1 in the preparation of a drug for promoting the treatment of periodontitis, characterized in that: The medicine is an injection.

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