Modified GelMA microspheres for oral maxillofacial cavity sinus administration and preparation method thereof

Abstract

The invention is suitable for the technical field of biological materials, and provides a modified GelMA microsphere for oral maxillofacial cavity sinus administration and a preparation method, and the preparation method comprises the following steps: synthesizing GelMA; carrying out grafting modification to obtain GelMA-beta-CD; the preparation method comprises the following steps: dissolving GelMA-beta-CD in a PBS solution to prepare a GelMA-beta-CD solution, dissolving coenzyme Q10 in acetone, slowly dropwise adding the dissolved coenzyme Q10 into the GelMA-beta-CD solution, carrying out constant temperature stirring overnight, and filtering out non-clathrated coenzyme Q10 to obtain a GelMA-beta-CD / CoQ10 clathrate compound, the concentration of the GelMA-beta-CD solution is 20%, and the GelMA-beta-CD solution loads 500 [mu] mol / L of CoQ10; and preparing the GelMA-beta-CD / CoQ10 microspheres by an emulsion cross-linking method. The texture flexibility of the microspheres can be adjusted according to the concentration of a GelMA-beta-CD solution and the grafting degree of cyclodextrin, the microspheres have good fluidity and spatial distribution compliance, the problem that traditional treatment materials such as artificially calcined bone meal and blocky hydrogel are uneven in distribution after being implanted into micro cavity sinus is solved, iatrogenic injury is avoided, and the treatment effect is good. The solubility, the stability and the bioavailability of the fat-soluble medicine in a water-soluble medium are improved, and the purpose of slow release is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a modified GelMA microsphere for intracavitary administration in the oral and maxillofacial cavity and a preparation method thereof. BACKGROUND

[0002] The size of the cavity space of the human maxillofacial part is small, the individual difference of the anatomical structure is large, the adjacent structure is complex, and important nerves and blood vessels are often accompanied, so there are serious challenges in the treatment of related diseases and drug delivery.

[0003] The existing drug delivery devices have problems such as being unable to effectively reach the target organ and easily causing secondary damage to the above-mentioned anatomical structure. For example, when the maxillary sinus is filled with artificial bone powder, the thin maxillary sinus mucosa is often perforated due to the hard and rough material. In addition, the anatomical form of the cavity is greatly different among individuals, and traditional treatment materials such as artificial calcined bone powder and block hydrogel cannot ensure the distribution state of the filling material in the cavity after being implanted into the small cavity. In addition, in tissue engineering and clinical practice, whether the drug can be slowly released at a speed that is suitable for the period of tissue regeneration is one of the key factors that directly affect the effect of tissue regeneration.

[0004] Therefore, it is urgent to improve the traditional drug delivery system in view of the problems of drug burst release, uneven distribution of filling therapeutic materials, and iatrogenic secondary damage (such as perforation of the maxillary sinus mucosa). SUMMARY

[0005] The purpose of the embodiment of the present application is to provide a preparation method of a modified GelMA microsphere for intracavitary administration in the oral and maxillofacial cavity, which aims to solve the problems raised in the background art.

[0006] The embodiment of the present application is implemented in the following way: a preparation method of a modified GelMA microsphere for intracavitary administration in the oral and maxillofacial cavity, comprising the following steps: (1) GelMA synthesis: add type A gelatin to a preheated PBS solution, stir until there is no floating and caking gelatin in the solution, drop methacrylic anhydride into the gelatin solution, and continuously stir at a constant temperature and speed, then perform dialysis treatment, filter the dialysis product and collect it in a culture dish, freeze-dry, and obtain a light yellow porous solid, i.e. GelMA; (2) GelMA modification: dissolve GelMA in a PBS solution, add carboxymethyl-β-cyclodextrin to an equal volume of MES buffer with the PBS solution, then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, after adding the dissolved GelMA solution, adjust the pH value to 8-9, constant temperature stirring, then perform dialysis treatment, collect the product in a culture dish, freeze-dry, and obtain modified GelMA-β-CD; (3) Drug loading: GelMA-β-CD is dissolved in PBS solution to prepare a GelMA-β-CD solution, and coenzyme Q10 is dissolved in acetone and slowly dripped into the GelMA-β-CD solution, constant temperature stirring overnight, filtering out the un-included coenzyme Q10, to obtain a GelMA-β-CD / CoQ10 inclusion compound, the concentration of the GelMA-β-CD solution is 20%, and the GelMA-β-CD solution loads 500 μmol / L of CoQ10; (4) Emulsification cross-linking method for preparing hydrogel microspheres: a. Oil phase system preparation: mix span 80 and liquid paraffin and stir well, and heat for standby; b. Water phase system preparation: add twin 60 and photo-crosslinking agent LAP to the GelMA-β-CD / CoQ10 inclusion compound and mix well; c. In the constant stirring condition of the oil phase system, the water phase system is uniformly and continuously dripped into the oil phase system to stir to obtain an oil-water mixed phase system, and then it is moved to an ice bath for stirring; d. Use a power of 10 W / cm 2 Blue light source for photo-crosslinking of water phase droplets in the oil-water mixed phase system to form hydrogel microspheres; e. Washing microspheres: add an equal volume of acetone to the oil-water mixed phase system, stir, stand, and then remove the supernatant after the microspheres are precipitated to wash; f. Drying: avoid light and dry the microspheres precipitate to obtain light yellow sand-like GelMA-β-CD / CoQ10 microspheres.

[0007] Another purpose of the embodiment of the present application is to provide a modified GelMA microsphere for oral and maxillofacial cavity sinus administration, which is prepared by the above preparation method.

[0008] Another purpose of the embodiment of the present application is to provide a modified GelMA microsphere for oral and maxillofacial cavity sinus administration, which is prepared by the above preparation method.

[0009] The embodiment of the application utilizes GelMA modified by carboxymethyl-beta-cyclodextrin as a main molecule, and a fat-soluble drug as a guest molecule, which can be wrapped and sealed into the hydrophobic cavity of carboxymethyl-beta-cyclodextrin. Because of the flexible texture and good flowability and spatial distribution compliance, it can not only solve the uneven distribution of traditional treatment materials such as artificial calcined bone powder and block hydrogel after implantation into a small cavity sinus, but also effectively avoid iatrogenic injury caused by the hard texture and rough surface of the implanted material itself and the incommensurability of the delivery instrument and the anatomical structure, greatly increase the solubility, stability and bioavailability of the fat-soluble drug in the water-soluble medium, and achieve the purpose of preventing drug burst release and achieving sustained release. It can be applied in the preparation of biomaterials for related diseases of the human small cavity sinus, such as maxillary sinus bottom area, paranasal sinus, temporomandibular joint cavity, middle ear tympanic cavity and other related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 A preparation flowchart of a modified GelMA microsphere for oral and maxillofacial cavity sinus administration is provided for the embodiment of the application. Figure 2 A physical map of GelMA-beta-CD and freeze-dried GelMA is provided for the embodiment of the application. Figure 3 A molecular mode diagram of GelMA-beta-CD / CoQ10 inclusion complex is provided for the embodiment of the application. Figure 4 Different administration dosage forms of GelMA-beta-CD / CoQ10 microspheres are provided for the embodiment of the application. Figure 5 Gelation and ball formation experimental results are provided for the embodiment of the application, wherein A is the gelation result, and B is the ball formation result. Figure 6 A nuclear magnetic resonance hydrogen spectrum is provided for the embodiment of the application. Figure 7 Microscope diagrams of GelMA-beta-CD and GelMA-beta-CD / CoQ10 microspheres are provided for the embodiment of the application. Figure 8 Scanning electron microscope images of GelMA-beta-CD and GelMA-beta-CD / CoQ10 microspheres are provided for the embodiment of the application. Figure 9 Encapsulation efficiency results are provided for the embodiment of the application. Figure 10 Degradation curves of microspheres are provided for the embodiment of the application. Figure 11 Drug cumulative release amount curves are provided for the embodiment of the application. Figure 12The cell compatibility experiment results provided in the embodiments of the present invention are shown in Figure A, where A represents the results of DAY1, B represents the results of DAY4, and C represents the results of DAY7. Figure 13 The cell activity results provided in the embodiments of the present invention; Figure 14 The staining results of the osteogenic differentiation experiment provided in the embodiments of the present invention; Figure 15 The expression levels of osteogenic and metabolic genes related to early osteogenic formation provided in the embodiments of the present invention are shown in the figures, where A is the ALP gene, B is RUNX2, C is the PGC-1α gene, and D is the NRF-1 gene. Figure 16 The expression levels of osteogenic and metabolic genes related to mid-stage osteoogenesis provided in this embodiment of the invention are: A is ALP gene, B is RUNX2, C is PGC-1α gene, and D is NRF-1 gene. Figure 17 The immunofluorescence experiment results provided in the embodiments of the present invention; Figure 18 This is a schematic diagram of the molding process provided in an embodiment of the present invention; Figure 19 The soft tissue staining results provided in the embodiments of the present invention; Figure 20 The microCT scan results provided in the embodiments of the present invention; Figure 21 The hard tissue staining results provided in the embodiments of the present invention. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0012] The hydrogel microspheres can form a spherical hydrogel structure based on a microemulsion cross-linking method, a micro-flow control technology, etc., and by adjusting various parameters of the preparation process of the above method, the diameter of the microspheres can be made into any size in theory, and the commonly used particle size is 50-1000 μm. The hydrogel microspheres have the advantages of small volume, large specific surface area, high fluidity and strong spatial compliance, and are suitable for being used as a drug delivery system for treating microcavities and sinus-related diseases; the methacrylic acid acylated gelatin (GelMA) is rich in tripeptide sequences, i.e., arginyl-glycyl-aspartic acid sequences (RGD sequences) and matrix metalloproteinase (MMP) binding sites. The former is helpful for cell adhesion, proliferation and differentiation, and the latter can degrade the hydrogel; in addition, the GelMA hydrogel has the characteristics of light curing cross-linking, does not need to introduce additional chemical cross-linking agents to increase the risk of biological toxicity, and can be cross-linked under light irradiation of a specific wavelength and power. Therefore, the GelMA hydrogel has good biocompatibility and extremely low biological toxicity.

[0013] Cyclodextrin (CD) is a cyclic oligosaccharide formed by connecting glucose monomers through α-1,4-glycosidic bonds. Common types include three cyclodextrin molecules containing 6, 7, and 8 monomer glucose molecules, respectively, referred to as α, β, and γ. The hydrophobic cavities formed by the three cyclodextrins increase in size in turn. As a drug inclusion medium, cyclodextrin, as a host molecule, can form an inclusion compound by wrapping the hydrophobic drug as a guest molecule in its hydrophobic cavity, thereby improving the solubility, stability, and bioavailability of the liposoluble drug in the water-soluble medium. Different types of cyclodextrins can be selected to adapt the size of the guest molecule to the host molecule according to the spatial structure and molecular weight of the guest molecule. To enable cyclodextrin to be covalently grafted onto other molecules, carboxymethyl, acetyl, and other functional group modified cyclodextrins can be used. The molecular plane and stereostructure of the three cyclodextrin molecules are shown in the following formula (from left to right, α, β, and γ-CD): 、 、

[0014] In the embodiments of the present application, a covalent amide bond is formed between the amino group of the side chain of GelMA and the carboxymethyl group of carboxymethyl-β-cyclodextrin (hereinafter referred to as cyclodextrin), and then the GelMA hydrogel grafted with carboxymethyl-β-cyclodextrin forms an amphiphilic molecule, which can load a liposoluble drug. The liposoluble drug, as a guest molecule, is combined with the cavity bowl structure of the cyclodextrin as a host molecule through a non-covalent bond, greatly improving the solubility and stability of the water-insoluble drug in the hydrophilic material. The drug loading capacity and drug release rate can be adjusted by the proportion of grafted cyclodextrin.

[0015] The specific implementation of the present application is described in detail below in conjunction with specific embodiments.

[0016] Example 1, a modified GelMA microsphere for oral and maxillofacial cavity sinus administration, its preparation method is as shown in Figure 1 Specifically, it includes the following steps: (1) GelMA synthesis: accurately weigh 10 g of type A gelatin (Gelatin Type A) and slowly add it to 100 mL of 0.1M PBS solution previously filtered with a 0.22μm filter and preheated at 50℃, and continuously stir under a magnetic stirrer until there is no floating and clumping gelatin in the solution, then keep the temperature at 50℃, and slowly drop 250μL of methacrylic anhydride (MA) into the above 100mL gelatin solution at a speed of 500 revolutions / min (0.25%, MA:Gelatin solution=v:v), and continuously stir at constant temperature and speed for 3 hours; put it into a dialysis bag with a molecular weight cut-off of 8-14kD and dialyze in double deionized water at a constant temperature of 50℃ for 3-5 days to remove unreacted MA and other impurities, filter the dialysis product and collect it in a culture dish with a diameter of 100mm, each dish contains 15-20mL of dialysis product, and then freeze-dry for 48 hours, obtain a light yellow porous solid GelMA, as shown in Figure 2 , and store at -20℃ for later use; (2) GelMA modification: accurately weigh 5g of synthesized GelMA and dissolve it in 50mL of 0.1M sterile PBS (pH=7.4) to make a solution with a mass volume ratio (w:v) of 10%, and keep it at a constant temperature of 50℃ for complete dissolution; add 10 g of carboxymethyl-β-cyclodextrin (CM-β-CD) to an equal volume (50mL) of 0.1mol / L MES buffer (2- (N-morpholino) ethanesulfonic acid, pH=6) as above, then add 4g of EDC and 6g of NHS to activate the carboxyl group for 30 minutes; add the above dissolved GelMA solution, adjust the pH value to 8-9, and stir overnight at a constant temperature of 50℃ under a magnetic stirrer at a speed of 500 revolutions / min; dialyze in distilled water using a dialysis bag with a molecular weight cut-off of 8-14kDa for 3-5 days to remove unreacted CM-β-CD and impurities, collect the product in a 100mm culture dish, each dish contains 15-20mL of dialysis product, then pre-freeze overnight in a -80℃ refrigerator, and obtain a white sponge-like solid after vacuum freeze-drying at a freeze-drying temperature of -100℃ and a vacuum degree of 5 or less for 48 hours, as shown in Figure 2 , that is, modified GelMA-β-CD, stored at -20℃ for later use; (3) Drug loading: Accurately weigh 200 mg of GelMA-β-CD and completely dissolve it in 1000 μL of PBS solution at a constant temperature of 40°C (mass-volume ratio g / mL is 20%). Weigh out 5 mg of excess coenzyme Q10 (CoQ10), dissolve it completely in 200 μL of acetone, and slowly add it dropwise to the above GelMA-β-CD solution to prepare a mixed solution. Maintain a constant temperature of 40°C and a stirring speed of 400 rpm overnight in the dark using magnetic stirring. Filter out the unencapsulated CoQ10 to obtain the GelMA-β-CD / CoQ10 inclusion complex (hereinafter referred to as the inclusion complex). Figure 3 As shown; (4) Preparation of hydrogel microspheres by emulsification crosslinking method: a. Preparation of oil phase system: Add 40mg of Span80 to 40mL of liquid paraffin, stir the mixture evenly with a constant temperature magnetic stirrer and heat it to 40℃ for later use. b. Preparation of aqueous system: Add 4 mg of twin60 and 2 mg of photocrosslinking agent LAP to each 1 mL of inclusion complex and mix well using a constant temperature magnetic stirrer; c. Using a magnetic stirrer, at a constant temperature and constant stirring speed (100 rpm), uniformly and continuously drop the aqueous phase system into the oil phase system (hereinafter referred to as the oil-water mixed phase system) and stir for 10-15 minutes. d. Transfer the oil-water mixture to an ice bath and stir at a constant speed (100 rpm) for 5 minutes; e. Power consumption is 10W / cm 2 Blue light source is used to photocrosslink aqueous droplets in an oil-water mixed system (photocrosslinking time is 5 min) to form hydrogel microspheres (hereinafter referred to as microspheres). f. Cleaning the microspheres: Add an equal volume of acetone to the oil-water mixture and stir at a constant speed of 100 rpm for 5 minutes on a magnetic stirrer. After standing in the dark for 1-2 hours, allow the microspheres to precipitate. Remove the supernatant and wash with anhydrous ethanol 2-3 times. Remove the ethanol each time after the upper layer becomes clear. g. Drying: Dry the above precipitate at no more than 40°C in the dark until it becomes a light yellow granular substance to obtain GelMA-β-CD / CoQ10 microspheres.

[0017] These GelMA-β-CD / CoQ10 microspheres can be formulated into different dosage forms, such as... Figure 4 As shown.

[0018] Performance testing: I. Gel formation and sphericity test of GelMA modified with carboxymethyl-β-cyclodextrin: GelMA alone can undergo photocrosslinking and be formed into hydrogel microspheres. This invention verifies whether GelMA hydrogel modified with cyclodextrin can be photocrosslinked and formed into hydrogel microspheres, verifying the photocrosslinking and sphericity of the modified hydrogel. The results are as follows: Figure 5 As shown.

[0019] II. Successful grafting of carboxymethyl-β-cyclodextrin and proton nuclear magnetic resonance spectroscopy: The proton NMR spectrum is as follows Figure 6 As shown: the characteristic peak of the CM-β-CD molecule is the proton on the carboxymethyl group (-O-CH2-COOH), and its chemical shift is usually at 3.4-4.5 ppm. The characteristic peak of the pure GelMA molecule is the olefin proton at the end of the methacrylamide chain (-NH-C(CH3)=CH2), and the two chemically inequivalent protons on =CH2 appear in the ranges of 5.6-5.8 ppm and 5.0-5.3 ppm. The GelMA-β-CD molecule shows the same characteristic peak as the CM-β-CD molecule at the corresponding chemical shift, which indicates that the CM-β-CD molecule has been successfully grafted onto the GelMA molecule. Calculate the molar grafting degree (GD) of CM-β-CD to GelMA: GD = [(Ic / Nc) / (Ig / Ng)] × 100% (Ic represents the integrated area of ​​the characteristic proton peak of CM-β-CD; Nc represents the number of protons represented by the characteristic peak of CM-β-CD; Ig represents the integrated area of ​​the characteristic proton peak of GelMA; Ng represents the number of protons represented by the characteristic peak of GelMA); calculated by measuring the area under the curve of the characteristic peak of the proton NMR spectrum of the grafted molecule (GelMA-β-CD) obtained by different mass ratios of the two molecules: The data obtained by proton nuclear magnetic resonance spectroscopy for different mass ratios (GelMA:CM-β-CD w:w) are as follows: ①1:1 (Ic=1.01, Nc=2; lg=0.17, Ng=2); ②1: 1.5 (Ic=1.11, Nc=2; lg=0.15, Ng=2); ③1:2 (Ic=1.41, Nc=2; lg=0.09, Ng=2); The above formula yields molar grafting rates of 594%, 740%, and 1567% for different mass ratios, which means that 1 mol of GelMA molecules can be grafted with 5.94 mol, 7.4 mol, and 15.67 mol of CM-β-CD molecules at different mass ratios of CM-β-CD.

[0020] III. Comparison between modified GelMA hydrogel microspheres and drug-loaded hydrogel microspheres: Unloaded and drug-loaded hydrogel microspheres were immersed in 0.1% lipid-soluble Nile Red fluorescent dye and stained overnight. After rinsing with PBS, the microspheres were photographed under a light microscope and a fluorescence microscope to observe their morphology. Figure 7 As shown, unloaded hydrogel microspheres showed no red fluorescence under mercury lamp excitation, while drug-loaded hydrogel microspheres showed red fluorescence after excitation, with a relatively uniform red light distribution, proving successful drug loading; scanning electron microscope images are shown below. Figure 8 As shown.

[0021] Example 2: Analysis of the effect of different concentrations of GelMA-β-CD on the encapsulation efficiency of the same mass of CoQ10: Referring to the preparation method of Example 1, four portions of GelMA-β-CD obtained in step (2), each 300 mg, were weighed and prepared into 10%, 15%, 20%, and 25% solutions with 0.1 mol / L PBS. Four portions of 5 mg CoQ10 were accurately weighed in the dark, and each portion was dissolved in 200 μL of acetone and then added dropwise to the four concentrations of GelMA-β-CD solution. The mixture was stirred magnetically overnight at 400 rpm in the dark. After filtering out unencapsulated CoQ10, the drug encapsulation efficiency was measured. The results are as follows: Figure 9 As shown, the encapsulation efficiency exhibits a concentration dependence on GelMA-β-CD.

[0022] Example 3, Degradation test of hydrogel microspheres: Following the preparation method of Example 1, the inclusion complex GelMA-β-CD / CoQ10 obtained in Example 2 was used to prepare GelMA-β-CD / CoQ10 microspheres of four concentrations by emulsification crosslinking method; Microspheres of each concentration were weighed at 20 mg ± 2.5 mg, with three parallel samples for each concentration. The microspheres were redispersed using 1 mL of sterile 0.1 mol / L PBS. Degradation characteristics were observed at a constant temperature of 37℃ and pH 7.2-7.4. Degradation curves of the four different concentrations of hydrogel microspheres were obtained by lyophilization and weighing at time points of 0, 3, 7, 14, 21, 28, and 35 days. The resulting degradation curves are shown below. Figure 10 As shown.

[0023] Example 4: Determination of drug release characteristics of hydrogel microspheres: Because the drug inclusion capacity of four different concentrations (10%, 15%, 20%, and 25%) of GelMA-β-CD differs, in order to measure the cumulative drug release, hydrogel microspheres encapsulating 3 mg of CoQ10 in each of the four concentrations were prepared with the lowest drug inclusion rate (63.46% for 5 mg CoQ10 at a 10% concentration) as 3 mg. Four portions of lyophilized GelMA-β-CD, each 300 mg, were accurately weighed and prepared into four different concentrations of GelMA-β-CD (10%, 15%, 20%, and 25%) using sterile 0.1 mol / L PBS. Four portions of CoQ10, each 3 mg, were accurately weighed and prepared into drug-loaded hydrogel microspheres according to the preparation method in Example 1. Weigh 20 mg of each of the four types of hydrogel microspheres. The total amount of CoQ10 loaded on the 20 mg microspheres is 0.2 mg. Place the weighed microspheres into four dialysis bags with a cutoff of 8000-14000 kD and seal them. Preparation of drug release medium: sterile 0.1 mol / L PBS and anhydrous ethanol in a volume ratio of 1:1 (v:v=1:1). Four dialysis bags containing microspheres were placed into four 15 mL centrifuge tubes, with 10 mL of release medium added to each tube. The tubes were incubated at a constant temperature of 37℃ and pH 7.2-7.4 for 35 days. Drug release was measured using a CoQ10 assay kit, measuring the absorbance (OD value) at 620 nm. OD values ​​were monitored at time points of 1, 3, 7, 14, 21, 28, and 35 days to calculate the cumulative drug release. Figure 11 As shown (horizontal axis: detection time point; vertical axis: cumulative release amount, unit: mg); It can be seen that the drug release characteristics are different at different concentrations: Rapid and complete release: 10% GelMA-β-CD, which can completely release the drug within approximately 14 days; Stable and sustained release: 15% GelMA-β-CD, which exhibits optimal zero-order release kinetics, with a stable rate and eventual complete release; Long-lasting release: 20% GelMA-β-CD, which provides the slowest release process, with 95% release in 35 days; Lowest release efficiency: 25% GelMA-β-CD has a slow release rate; As a drug delivery system applied in bone tissue engineering, the long-acting sustained-release 20% GelMA-β-CD loaded with CoQ10 is a good match for the bone regeneration process.

[0024] Based on the above conclusions, subsequent experiments were conducted with a fixed 20% GelMA-β-CD content.

[0025] Example 5: In vitro experiments observing bone regeneration with different concentrations of CoQ10: CoQ10 concentrations were grouped: Control group (only 20%) GelMA-β-CD (without CoQ10 loading), 10 μmol / L, 50 μmol / L, 100 μmol / L, 500 μmol / L, and 1000 μmol / L (the molecular weight of CoQ10 is numerically expressed as a molar mass of 863.34 g / mol; the above concentrations are equivalent to adding 0.086 mg, 0.43 mg, 0.86 mg, 4.3 mg, and 8.6 mg of CoQ10 to each mL of GelMA-β-CD PBS solution, respectively. Each portion was dissolved in 200 μL of acetone and then added dropwise to the GelMA-β-CD PBS solution to prepare inclusion complex precursor solutions containing CoQ10 at concentrations of 10 μmol / L, 50 μmol / L, 100 μmol / L, 500 μmol / L, and 1000 μmol / L). Drug-loaded microspheres GelMA-β-CD / CoQ10 with different concentrations were synthesized according to the preparation method in Example 1. 1. Cell compatibility experiment of co-culturing mesenchymal stem cells with hydrogel microspheres loaded with different concentrations of coenzyme Q10 (hereinafter referred to as microspheres): Cell source: Mesenchymal stem cells derived from the mucosa of the maxillary sinus floor (collected from patients aged 18-30 years (including males and females, with an average age of 24 years) undergoing maxillary orthognathic surgery at the Department of Oral and Maxillofacial Surgery, School of Stomatology, Jilin University, with approval from the Medical Ethics Committee of the School of Stomatology, Jilin University (2022-62). All patients signed written informed consent. The collected tissue was healthy mucosal tissue. Smokers or patients with diseases such as maxillary sinusitis were not included in the collection. These are referred to as stem cells). Cells were co-cultured with 20 mg of microspheres at different drug loading concentrations for 1, 4, and 7 days. Cell viability was detected using a CCK-8 assay kit. Figure 12 As shown in the figure, the GelMA-β-CD / CoQ10 microspheres with a drug loading concentration of 500 μmol / L exhibited the best cell activity.

[0026] 2. Experiment on early osteogenic differentiation promotion by co-culturing microspheres loaded with different concentrations of coenzyme Q10 with mesenchymal stem cells: After co-culturing mesenchymal stem cells with 20 mg of microspheres at different drug loading concentrations for 7 days, the cellular synthesis of alkaline phosphatase (ALP), an early marker of bone regeneration, was measured. A deeper blue-purple color indicated a greater synthesis amount, suggesting higher osteogenic activity. The results were as follows: Figure 13 As shown, the GelMA-β-CD / CoQ10 microspheres loaded with CoQ10 at a concentration of 500 μmol / L exhibited the highest osteogenic activity.

[0027] 3. Co-culturing microspheres loaded with different concentrations of coenzyme Q10 with mesenchymal stem cells to promote osteogenic differentiation in the late stage: After co-culturing mesenchymal stem cells with 20 mg of microspheres at different drug loading concentrations for 30 days, the cells were stained with Alizarin Red S (ARS). Alizarin Red specifically binds to calcium nodules secreted by osteogenic differentiated cells, appearing as red granular or patchy calcium nodule images under a light microscope. Figure 14 As shown, the deeper the red staining and the larger the red image area, the better the bone regeneration activity. To better observe the red staining trend, 1% hexadecyl chloride monohydrate was added to the red-stained area. This compound specifically binds to alizarin red and calcium nodules to produce a blue substance. The absorbance of the blue product was measured at 562 nm using an ELISA reader, and the trend of OD value is shown in the figure. The results also show that the osteogenic activity of GelMA-β-CD / CoQ10 microspheres loaded with CoQ10 at a concentration of 500 μmol / L is the highest.

[0028] 4. Osteogenesis-metabolism coupled qPCR experiment: 4.1 After co-culturing mesenchymal stem cells with 20 mg of microspheres at different drug loading concentrations for 7 days (early osteogenic stage), the mRNA of the stem cells was extracted and reverse transcribed into a cDNA library. Under the induction of primers for early osteogenic markers, relevant osteogenic and metabolic genes were amplified, such as... Figure 15 As shown: ALP and Runx2 are genes actively expressed in early bone regeneration, while PGC-1α and NRF-1 are genes related to mitochondrial energy metabolism. Compared with the control group (empty vector concentration of 0), the gene expression levels of microspheres loaded with CoQ10 at a concentration of 500 μM were significantly different (*p<0.01, **0.001). <p<0.01、***p<0.001); 4.2 After co-culturing mesenchymal stem cells with 20 mg of microspheres at different drug loading concentrations for 14 days (mid-osteogenic stage), mRNA from the stem cells was extracted and reverse transcribed into a cDNA library. Under the induction of primers for early osteogenic markers, relevant osteogenic and metabolic genes were amplified, such as... Figure 16 As shown: ALP and Col-1 are genes actively expressed during mid-stage bone regeneration, while PGC-1α and NRF-1 are genes related to mitochondrial energy metabolism. Compared with the control group (empty vector concentration of 0), the gene expression levels of microspheres loaded with CoQ10 at a concentration of 500 μM were significantly different (*p<0.01, **0.001). <p<0.01、***p<0.001)。

[0029] 5. Immunofluorescence experiment: The above experimental results indicate that GelMA-β-CD / CoQ10 microspheres loaded with 500 μmol / L CoQ10 exhibit the best osteogenic activity and cellular energy metabolism. Therefore, the following immunofluorescence experiments were conducted in two groups: one group consisted of unloaded hydrogel microspheres, and the other group consisted of microspheres loaded with 500 μmol / L (500 μM) CoQ10. Immunofluorescence staining was performed after co-culturing with stem cells for 14 days, and the results are as follows. Figure 17 As shown, the fluorescence intensity of the microspheres with a drug loading concentration of 500 μmol / L (500 μM) was significantly higher than that of the unloaded control group.

[0030] In summary, the in vitro experiments show that microspheres with a GelMA-β-CD concentration of 20% and a CoQ10 loading concentration of 500 μmol / L exhibit the best performance.

[0031] Example 6, In vivo experiment: Model animal: Male 8-week-old, 2.5kg long-eared white rabbits were selected (purchased from the Animal Experiment Center of Jilin University, fed for 7 days before surgery, and the feeding and operation procedures were strictly in accordance with ethical requirements): 1. Modeling: The specific procedures are as follows: Under intravenous anesthesia (1% sodium pentobarbital sulfate, 3 mL / kg) and local anesthesia (1% lidocaine hydrochloride, 0.5 mL / animal) in the rabbit's ear margin, the hair in the nasal bone area is shaved. After strict disinfection with iodine, a longitudinal incision of about 4-5 cm is made in the middle of the nasal bone area. The skin is separated to expose the bone surface of the rabbit's maxillary sinus. Using an implantation machine and a trephine drill at a speed not exceeding 800 rpm, and after thorough cooling with sterile saline at 4°C, the implant is carefully drilled at a distance of 0.5 cm from the nasal bone suture. A bone defect with a diameter of 0.3-0.4 cm, located 1.0 cm from the nasofrontal suture, was treated by using a lifting tool to push up the mucosa at the bone defect site. The bone wall was then lifted to its full thickness to separate the maxilla and maxillary sinus floor mucosa in rabbits. Lyophilized microspheres were then placed between the maxilla and maxillary sinus floor mucosa. A self-controlled method was used (20 mg of unloaded microspheres were placed on the left side of the rabbits, designated as the Control group; 20 mg of microspheres loaded with a drug concentration of 500 μmol / L were placed on the right side, designated as the Treatment group). Figure 18 As shown, patients received intramuscular injections of sodium penicillin (800,000 U / day) for 5 days post-surgery to prevent wound infection. 2. All rabbits recovered well after surgery and were kept in individual cages with sufficient food and water for 8 weeks. After 8 weeks, the animals were euthanized under deep anesthesia, and the maxillary sinus tissue and important organs were harvested and placed in 4% paraformaldehyde or liquid nitrogen. Soft tissue analysis: Tissue sections of important organs (heart, liver, spleen, lung, and kidney) from rabbits were taken and stained with eosin-hematoxylin (HE staining) to observe the biocompatibility of the drug-loaded hydrogel microspheres. The results are as follows: Figure 19 As shown, no obvious abnormalities were observed in the HE staining results of sections of important rabbit organs, indicating that the hydrogel microspheres of the experimental dose have good biocompatibility and no obvious biotoxicity. Hard tissue analysis (osteogenesis between the maxillary sinus floor mucosa and the maxilla in rabbits): microCT scan results are as follows Figure 20 As shown, the osteogenic effect of the CoQ10-loaded hydrogel microspheres on the right side is better than that of the control group; After decalcifying the rabbit maxilla in a decalcifying solution for two months, sections of the hard tissue were stained with hematoxylin and eosin (HE) and Masson's stain. The results are as follows: Figure 21 As shown, the red-stained areas of HE staining and the blue-stained areas of Masson staining represent the matrix of regenerated bone, with the treatment group showing superiority over the control group.

[0032] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing modified GelMA microspheres for intra-sinus drug delivery in the oral and maxillofacial region, characterized in that, Includes the following steps: (1) GelMA synthesis: Type A gelatin was added to a preheated PBS solution and stirred until there was no floating or clumping gelatin in the solution. Methacrylic anhydride was added dropwise to the gelatin solution and stirred continuously at a constant temperature and speed. Then, dialysis was performed, the dialysis product was filtered and collected in a petri dish, and freeze-dried to obtain a light yellow porous solid, namely GelMA. (2) GelMA modification: GelMA was dissolved in PBS solution, carboxymethyl-β-cyclodextrin was added to MES buffer with an equal volume of PBS solution, then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, and then the dissolved GelMA solution was added. The pH value was adjusted to 8-9, and the mixture was stirred at a constant temperature. Then, the mixture was dialyzed, and the product was collected in a petri dish and freeze-dried to obtain modified GelMA-β-CD. (3) Drug loading: GelMA-β-CD was dissolved in PBS solution to prepare GelMA-β-CD solution. Coenzyme Q10 was dissolved in acetone and slowly added dropwise to GelMA-β-CD solution. The mixture was stirred at constant temperature overnight. Unencapsulated coenzyme Q10 was filtered out to obtain GelMA-β-CD / CoQ10 inclusion complex. The concentration of GelMA-β-CD solution was 20%, and the GelMA-β-CD solution was loaded with 500 μmol / L CoQ10. (4) Preparation of hydrogel microspheres by emulsification crosslinking method: a. Preparation of oil phase system: Mix Span80 and liquid paraffin thoroughly and heat until ready for use; b. Preparation of aqueous system: Add twin60 and photocrosslinking agent LAP to the GelMA-β-CD / CoQ10 inclusion complex and mix well; c. While the oil phase system is being stirred at a constant temperature, the aqueous phase system is continuously and uniformly added dropwise to the oil phase system and stirred to obtain an oil-water mixed phase system, which is then transferred to an ice bath and stirred. d. Power consumption is 10W / cm 2 Blue light source photocrosslinks aqueous droplets in an oil-water mixed system to form hydrogel microspheres; e. Cleaning the microspheres: Add an equal volume of acetone to the oil-water mixed phase system, stir, let stand, and wait for the microspheres to precipitate. Remove the supernatant and then wash the microsphere precipitate three times with anhydrous ethanol. f. Drying: Dry the microspheres in the dark to obtain a light yellow granular substance, GelMA-β-CD / CoQ10 microspheres.

2. The method for preparing modified GelMA microspheres for intra-sinus drug delivery in the oral and maxillofacial region according to claim 1, characterized in that, The grafting rate of methacrylic anhydride in the GelMA is 20-25%.

3. The method for preparing modified GelMA microspheres for intra-sinus drug delivery in the oral and maxillofacial region according to claim 1, characterized in that, The molar grafting rate of carboxymethyl-β-cyclodextrin in the GelMA-β-CD was 1567%.

4. A modified GelMA microsphere for intra-sinus drug delivery in the oral and maxillofacial region, characterized in that, It is prepared using the preparation method described in any one of claims 1-3.

5. The use of the modified GelMA microspheres as described in claim 4 for intra-sinus administration in the oral and maxillofacial region in the preparation of biomaterials that promote osteogenic differentiation.

6. The application according to claim 5, characterized in that, The osteogenic differentiation-promoting biomaterial is either an injectable formulation or a lyophilized formulation.

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