Preparation method and application of drug-loaded metformin carbon dots

Abstract

The invention discloses a preparation method and application of drug-loaded metformin carbon dots, and the preparation method comprises the following steps: synthesizing fluorescent carbon MCDs by using metformin hydrochloride and glycine as precursors through a hydrothermal method, further carrying out PDA wrapping, and realizing loading of DEX through PDA to prepare a nano system MCDs-PDA (at) DEX. The nano material can be taken by hiPSCs and has good biocompatibility, experimental results prove that the prepared nano material can induce osteogenic differentiation of the hiPSCs, and an MCDs-PDA (at) DEX / GG hydrogel system prepared on the basis of the material has a remarkable bone defect repair effect, so that a new direction and basis are provided for development of bone tissue repair related materials.

Description

Technical Field

[0001] This invention relates to the field of bone repair technology, and in particular to a method for preparing drug-loaded metformin carbon dots and their application. Background Technology

[0002] In recent years, carbon-based nanomaterials have attracted considerable attention from scholars due to their excellent drug delivery performance and biocompatibility, showing broad research and application prospects in biomedical fields such as biosensing, antibacterial, antitumor, and bone and cartilage formation. Among them, carbon dots (CDs) are fluorescent nanomaterials with a size of less than 20 nm, possessing advantages such as simple synthesis, good fluorescence properties, good biocompatibility, and ease of functional group modification. Based on this, CDs have been widely used in biosensing, drug delivery, antitumor, and antibacterial therapy. However, there are currently few studies applying CDs to osteogenic differentiation of hiPSCs. Summary of the Invention

[0003] Therefore, based on the above background, this invention provides a method for preparing metformin hydrochloride carbon dots and their applications. This invention prepares metformin hydrochloride carbon dots (MCDs) with good biocompatibility and can be taken up by hiPSCs, and uses polydopamine (PDA) to modify the surface of MCDs to carry the commonly used stem cell osteogenic factor dexamethasone (DEX), thus preparing the nanosystem MCDs-PDA@DEX. Applying CDs to the osteogenic differentiation of hiPSCs provides a new direction and basis for the development of bone tissue repair-related materials.

[0004] The technical solution of this invention is as follows:

[0005] A method for preparing drug-loaded metformin carbon dots, comprising the following steps:

[0006] 1) Preparation of MCDS

[0007] MCDS powder was synthesized using metformin hydrochloride and glycine as raw materials via a one-step hydrothermal method.

[0008] 2) Preparation of MCDs-PDA

[0009] After mixing Tris·HCl solution, dopamine hydrochloride and the MCDS prepared in step 1), the mixture was reacted, filtered, and the filtrate was washed and freeze-dried to obtain MCDs-PDA.

[0010] 3) Preparation of MCDs-PDA@DEX

[0011] After reacting the MCDs-PDA prepared in step 2) with dexamethasone in a solvent, the mixture was filtered, and the filtered solid was washed and freeze-dried to obtain drug-loaded metformin carbon dots MCDs-PDA@DEX.

[0012] Based on the same inventive concept, the present invention also provides the application of the drug-loaded metformin carbon dots prepared above in promoting the in vitro osteogenic differentiation cell culture of hiPSCs for non-therapeutic and diagnostic purposes.

[0013] Preferably, during cell culture, the concentration of the MCDs-PDA@DEX is 12.5 μg / mL.

[0014] Based on the same inventive concept, the present invention also provides the application of the drug-loaded metformin carbon dots prepared above in the preparation of bone injury repair materials.

[0015] Furthermore, the bone injury repair material includes a bone injury repair gel material.

[0016] Based on the same inventive concept, the present invention also provides a method for preparing a bone injury repair gel, comprising the following steps:

[0017] ① Prepare genipin ethanol solution and gelatin aqueous solution separately;

[0018] ② Take the drug-loaded metformin carbon dots prepared according to claim 1 and add water to prepare MCDs-PDA@DEX solution;

[0019] ③ After mixing genipin ethanol solution, gelatin aqueous solution and MCDs-PDA@DEX solution, the gel is prepared for bone injury repair after reaction.

[0020] Based on the same inventive concept, the present invention also provides a bone injury repair gel prepared by the above-described preparation method.

[0021] Based on the same inventive concept, the present invention also provides the application of the bone injury repair gel prepared by the above preparation method in the preparation of bone injury repair scaffolds.

[0022] The beneficial effects achieved by adopting this invention are as follows:

[0023] This invention utilizes metformin hydrochloride and glycine as precursors to synthesize fluorescent carbon MCDs via a hydrothermal method. These MCDs are then encapsulated with PDA, and DEX is loaded onto the PDA to prepare the MCDs-PDA@DEX nanosystem. This nanomaterial exhibits good biocompatibility while being taken up by hiPSCs. Experimental results demonstrate that the prepared nanomaterial can induce osteogenic differentiation in hiPSCs. Furthermore, the MCDs-PDA@DEX / GG hydrogel system prepared based on this material exhibits significant bone defect repair effects, providing a new direction and basis for the development of bone tissue repair-related materials. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the synthesis of MCDs-PDA@DEX according to the present invention.

[0025] Figure 2 The optical performance of the MCDs in Example 1 of this invention is characterized as follows: (a) UV-Vis absorption spectrum of the MCDs (illustrated in the figure of MCDs in solution under natural light and 365 nm UV light); (b) Fluorescence spectrum of the MCDs under excitation light of 370-430 nm.

[0026] Figure 3 TEM images of the materials in Embodiment 1 of the present invention: (ab) MCDs; (a) Particle size distribution of MCDs in the upper right inset; (c) MCDs-PDA; (d) MCDs-PDA@DEX.

[0027] Figure 4 Fourier transform infrared spectra of the materials in Example 1 of the present invention: (a) glycine, metformin hydrochloride and MCDs; (b) MCDs, MCDs-PDA and MCDs-PDA@DEX.

[0028] Figure 5 The ultraviolet-visible absorption spectra of MCDs, MCDs-PDA, and MCDs-PDA@DEX in Embodiment 1 of the present invention are shown.

[0029] Figure 6 (a) is the standard curve of DEX in Embodiment 1 of the present invention; Figure 6 (b) is the DEX sustained-release curve of MCDs-PDA@DEX according to an embodiment of the present invention.

[0030] Figure 7 The effect of 0-400 μg / mMLMCDs, MCDs-PDA, and MCDs-PDA@DEX on the activity of hiPSCs in Example 1 of this invention is shown in ***, where p < 0.001.

[0031] Figure 8The images show fluorescence images of 12.5 μg / mLMCDs, MCDs-PDA, and MCDs-PDA@DEX from Example 1 of this invention after being taken up by hNF-C1hiPSCs for different times (6 h, 12 h, 24 h, 48 h). Scale bar: 100 μm.

[0032] Figure 9 The relative fluorescence intensities are those of 12.5 μg / mMLMCDs, MCDs-PDA, and MCDs-PDA@DEX from Example 1 of this invention after uptake by hNF-C1hiPSCs for different times (6 h, 12 h, 24 h, 48 h). ** indicates p < 0.01, *** indicates p < 0.001.

[0033] Figure 10 The gene expression of hiPSCs in Example 2 of this invention at 3 and 7 days under different experimental intervention conditions (endoderm: AFP, GATA4; mesoderm: MSX1, T; ectoderm: PAX6, SOX1). ** indicates p < 0.01, *** indicates p < 0.001, n = 3.

[0034] Figure 11 The expression of osteogenic-related genes (a) ALP, (b) RUNX2, (c) COL1A1, and (d) OCN in hiPSCs of Example 2 of this invention after osteogenic induction and differentiation at different times (7, 14, 21, and 28 days) under different experimental intervention conditions is shown. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and n = 3.

[0035] Figure 12 The image shows the expression of the pluripotency protein OCT-4 in hiPSCs of Example 2 of this invention after osteogenic induction and differentiation at different times (7, 14, 21, 28 days) under different experimental conditions. Scale bar: 100 μm.

[0036] Figure 13 The image shows the expression of the osteogenic-related protein RUNX2 in hiPSCs of Example 2 of this invention after different osteogenic induction differentiation times (7, 14, 21, and 28 days) under different experimental interventions. Scale bar: 100 μm.

[0037] Figure 14 The image shows the expression of osteogenic-related protein COL1A1 in hiPSCs of Example 2 of this invention after different osteogenic induction differentiation times (7, 14, 21, 28 days) under different experimental interventions. Scale bar: 100 μm.

[0038] Figure 15The image shows the expression of osteogenic-related protein OCN in hiPSCs of Example 2 of this invention after different osteogenic induction differentiation times (7, 14, 21, 28 days) under different experimental interventions. Scale bar: 100 μm.

[0039] Figure 16 The images show the cell morphology of hiPSCs from Example 2 of this invention after osteogenic induction and differentiation at different times (7, 14, 21, and 28 days) under different experimental conditions. Scale bar: 100 μm.

[0040] Figure 17 These are photographs of cell culture plates stained with Alizarin Red after osteogenic induction and differentiation of hiPSCs in Example 2 of the present invention at different times (7, 14, 21, 28 days) under different experimental interventions.

[0041] Figure 18 The images show the morphology of hiPSCs from Example 2 of this invention after osteogenic induction and differentiation at different times (7, 14, 21, and 28 days) under different experimental conditions, stained with Alizarin Red. Scale bar: 100 μm.

[0042] Figure 19 Alizarin Red staining quantitative results of hiPSCs from Example 2 of this invention after osteogenic induction differentiation at different times (7, 14, 21, 28 days) under different experimental intervention conditions. ** indicates p < 0.01, *** indicates p < 0.001, n = 3.

[0043] Figure 20 The images shown are of GG, MCDs / GG, and MCDs-PDA@DEX / GG before and after crosslinking in Example 3 of the present invention: (a) before GG crosslinking, (bc) after GG crosslinking, (d) before MCDs / GG crosslinking, (ef) after MCDs / GG crosslinking, (g) before MCDs-PDA@DEX / GG crosslinking, and (hi) after MCDs-PDA@DEX / GG crosslinking.

[0044] Figure 21 Scanning electron microscope (SEM) images of the hydrogel prepared in Example 3 of this invention: (a) GG, (b) MCDs / GG, (c) MCDsPDA@DEX / GG. Scale bar: 50 μm.

[0045] Figure 22 The effects of GG, MCDs / GG, and MCDs-PDA@DEX / GG on the activity of hiPSCs in Example 3 of this invention are shown. ** indicates p < 0.01, *** indicates p < 0.001.

[0046] Figure 23MicroCT images of rabbit skull defects 4 and 8 weeks after implantation of GG, MCDs / GG, and MCDs-PDA@DEX / GG in Example 3 of this invention: (a) 3D reconstructed image, (b) coronal view. Scale bar: 1 mm.

[0047] Figure 24 The quantitative analysis results of new bone formation at 4 and 8 weeks after implantation of GG, MCDs / GG, and MCDs-PDA@DEX / GG into rabbit skull defects in Example 3 of this invention are as follows: (a) BV represents the volume of new bone, (b) BV / TV represents the percentage of bone mass to total volume, and (c) BS represents the surface area of ​​new bone. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0048] Figure 25 The following images show the in vivo biocompatibility assessment of GG, MCDs / GG, and MCDs-PDA@DEX / GG implanted into rabbit skull defects at 4 and 8 weeks after implantation: (a) H&E staining images of major rabbit organs (heart, liver, spleen, lung, and kidney) at 4 weeks; (b) rabbit body weight change curves within 4 weeks; (c) H&E staining images at 8 weeks; and (d) body weight change curves within 8 weeks. Scale bar: 100 μm. Detailed Implementation

[0049] To better understand the above-mentioned objectives, features and advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments. The following embodiments are implemented based on the technical solutions of the present invention, and detailed implementation methods and specific operation processes are given. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited to the following embodiments.

[0050] The meanings of the abbreviations used in the following examples are as follows:

[0051] MSCs: Mesenchymal stem cells; hESCs: Human embryonic stem cells; hiPSCs: Human induced pluripotent stem cells;

[0052] BMSCs: Bone marrow mesenchymal stem cells; CDs: Carbon quantum dots; DEX: Dexamethasone; PDA: Polydopamine;

[0053] COL1A1: Type I collagen; RUNX2: Runt-related transcription factor 2.

[0054] Example 1: Preparation of MCDs-PDA@DEX

[0055] The main experimental reagents prepared in this embodiment are shown in Table 1.

[0056] Table 1: Main Reagents and Manufacturers

[0057]

[0058] The preparation of MCDs-PDA@DEX includes the following steps:

[0059] 1) Preparation of MCDS

[0060] Metformin hydrochloride carbon quantum dots (MCDs) are synthesized via a one-step hydrothermal method:

[0061] Specifically, 0.5 g of metformin hydrochloride and 0.5 g of glycine (Gly) were dissolved in 10 mL of deionized water and placed in a 25 mL hydrothermal reactor. The reactor was then placed in a forced-air dryer and heated at 200 °C for 6 h. After the reaction was complete, the resulting liquid was centrifuged at 12000 rpm for 10 min. The supernatant, clear liquid, was filtered through a 0.22 μm filter membrane and dialyzed in pure water for 24 h using a dialysis bag (Mw = 3500 Da). Finally, the solid powder of MCDs was obtained by freeze-drying.

[0062] 2) Preparation of MCDs-PDA

[0063] Prepare 30 mL of 0.1 mM Tris·HCl solution (pH = 8.5), add 30 mg of dopamine hydrochloride and 10 mg of MCDs, stir at 650 rpm for 6 h at room temperature using a magnetic stirrer, remove impurities by centrifugation (12000 rpm, 10 min) and washing three times with ultrapure water, collect the obtained solid product, and freeze-dry to obtain MCDs-PDA.

[0064] 3) Preparation of MCDs-PDA@DEX

[0065] Add 15 mg MCDs-PDA and 5 mg dexamethasone (DEX) to 20 mL of pure water, stir at 650 rpm for 24 h at room temperature to ensure effective reaction, centrifuge and wash three times at 12000 rpm, freeze dry the product to obtain MCDs-PDA@DEX.

[0066] The prepared MCDs-PDA@DEX were characterized.

[0067] (1) The characterization operations are as follows:

[0068] 1) Photoluminescence characteristics analysis of MCDs

[0069] MCD solid powder was fully dissolved in deionized water, and the photoluminescence characteristics of MCDs were investigated using fluorescence spectrophotometry. The excitation wavelength range was 370-430 nm, with the wavelength increasing by 10 nm each time.

[0070] 2) Transmission electron microscopy (TEM) analysis of materials

[0071] Take 100 μg each of MCDs, MCDs-PDA, and MCDs-PDA@DEX and add them to 1 mL of pure water. Sonicate for 20 min to disperse the materials evenly. Take 10 μL of the liquid and drop it onto an ultrathin carbon film copper grid. Let it stand for 10 min, absorb the excess liquid with filter paper, and air dry naturally. Use a Tecnai F30 transmission electron microscope (TEM) to observe the morphology and dispersibility of the materials.

[0072] 3) Fourier transform infrared (FT-IR) spectroscopy analysis of materials

[0073] To demonstrate the synthesis of MCDs-PDA and the successful loading of DEX, the functional groups of MCDs, MCDs-PDA, and MCDs-PDA@DEX were detected using Fourier transform infrared spectroscopy.

[0074] 4) Ultraviolet-Vis (UV-Vis) absorption spectroscopy analysis of materials

[0075] MCDs, MCDs-PDA, and MCDs-PDA@DEX were prepared into 100 μg / mL aqueous solutions, dispersed by ultrasonic vibration for 20 min, and then placed in cuvettes. The absorption curves of the aqueous solutions of different materials in the wavelength range of 200 nm to 700 nm were measured using a UV spectrophotometer.

[0076] 5) Drug loading rate, encapsulation efficiency, and DEX sustained-release analysis of MCDs-PDA@DEX

[0077] Different concentrations (10, 20, 40, 50, 70 μg / mL) of DEXPBS solutions were prepared, and their absorbance at 242 nm was measured using UVvis to plot a DEX solution standard curve. The supernatant and washing buffer from three washes after centrifugation during the MCDsPDA@DEX synthesis process were used to calculate the drug loading and encapsulation efficiency using the following formulas.

[0078] Drug encapsulation rate (%) = Mass of DEX in MCDs-PDA@DEX / Amount of DEX added × 100%.

[0079] Drug loading (%) = Mass of DEX in MCDs-PDA@DEX / Mass of MCDs-PDA@DEX × 100%.

[0080] 4 mg MCDs-PDA@DEX was dispersed in 4 mL of PBS solution and then added to a dialysis bag (Mw = 3500 Da). The dialysis bag was placed in 16 mL of PBS solution for dialysis at room temperature. 1 mL of dialysis solution was aspirated at 3 h, 6 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d to measure the absorbance at 242 nm using UV-Vis. 1 mL of PBS was added after each measurement, and the DEX release rate was calculated according to the standard curve.

[0081] (2) Evaluation of biological applications

[0082] 1) Cell Culture

[0083] The hNF-C1hiPSCs cell line was provided by the Guangzhou Institute of Biomedical and Health. Cells were cultured in PSC medium with daily medium changes. 1 mL of Matrigel (1:80, diluted with DPBS, operated on ice) was pre-seeded into 6-well cell culture plates planned for hNF-C1hiPSCs, shaken well, and incubated overnight at 37°C. Cells with approximately 80% confluence were washed with DPBS, digested with 0.5 mmol / L EDTA, and then cultured at 2.5 × 10⁻⁶ cells / well. 4 Inoculate at a density of cells / cm² onto the surface of a culture plate pre-coated with Matrigel.

[0084] 2) Biocompatibility test

[0085] hNF-C1 hiPSCs grown on Matrigel surface were digested with EDTA after reaching 80% confluence, and the cells were counted and stored at 2.5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of cells / cm² in 48-well plates. On the second day, the culture medium was replaced with PSC medium containing different concentrations of materials MCDs, MCDs-PDA, and MCDs-PDA@DEX (0 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL). Three parallel controls were set up for each group. Cell viability was measured 1 day and 3 days after the addition of materials using CCK8 assay.

[0086] 3) Cell uptake experiment

[0087] hNF-C1 hiPSCs grown on Matrigel surface were digested with EDTA after reaching 80% confluence, and the cells were counted and then stored at 1.5 × 10⁻⁶ cells / year. 4Inoculate the medium into 24-well plates at a density of 12.5 μg / mL MCDs. The next day, replace the medium with PSC medium containing 12.5 μg / mL MCDs. Four groups were established based on the co-culture time of MCDs and materials: 6 h, 12 h, 24 h, and 48 h, with 3 parallel control wells in each group. After 6 h, 12 h, 24 h, and 48 h, samples were collected from the corresponding experimental groups. Before collection, the plates were washed three times with PBS solution, then 95% anhydrous ethanol was added to the plates, aspirated after 30 min, and washed three more times. The samples were observed using an inverted fluorescence microscope and photographed.

[0088] Statistical analysis: SPSS 13.0 statistical software was used to analyze the experimental results. One-way ANOVA was used to compare the differences between groups, and Tukey's method was used for multiple comparisons. p < 0.05 was considered statistically significant.

[0089] Characterization results:

[0090] 1) Material characterization results

[0091] In this embodiment, metformin hydrochloride and glycine were used as precursors to synthesize MCDs via a one-step hydrothermal method. Impurities were removed by centrifugation, filtration, and dialysis to obtain relatively pure MCD materials. Figure 2 As shown in (a), the UV-Vis absorption spectrum of visible MCDs, as measured by a UV spectrophotometer, exhibits a significant absorption peak at 230 nm. Figure 2 As shown in the inset of (a), the prepared MCDs exhibit a clear, transparent yellow color under natural light, while displaying bright blue fluorescence under ultraviolet light. To investigate the photoluminescence properties of the MCDs, fluorescence spectroscopy was used to analyze the emission light of the material under laser excitation in the wavelength range of 370-430 nm, such as... Figure 2 (b) shows that in the range of 370-400 nm, the fluorescence intensity of the emitted light increases with every 10 nm increase in the excitation wavelength, and the strongest fluorescence emission intensity can be observed with 400 nm excitation light. In addition, it can be observed that the emission wavelength of MCDs exhibits an excitation light-dependent characteristic similar to that of most carbon dots, that is, the emission wavelength of carbon dots changes accordingly with the change of excitation wavelength.

[0092] The morphology of MCDs, MCDs-PDA, and MCDs-PDA@DEX was observed using transmission electron microscopy (TEM), such as... Figure 3 As shown in (a)-(b), MCDs exhibit a uniform spherical shape and good dispersibility. Figure 3 (a) The inset shows the particle size distribution obtained by statistical analysis of MCD nanoparticles. The particle size of MCDs is mainly distributed in the range of 5.5-10.0 nm, with an average particle size of 8.21 nm. The results are consistent with the characteristics of MCDs as nanoscale materials. Figure 3 (c) shows that after encapsulating the PDA, larger aggregates of the PDA are visible, suggesting that the PDA aggregates on the outer layer of the MCDs and encapsulates the MCDs inside. Figure 3 (d) is a TEM image of MCDsPDA@DEX. Since the DEX is small in size, it is difficult to clearly show in the TEM image. Furthermore, since the outer layer of MCDs is almost completely wrapped by PDA, it is inferred that the DEX is combined with MCDs by being loaded by PDA.

[0093] In this embodiment, Fourier transform infrared spectroscopy (FT-IR) was used to analyze the surface functional groups of metformin hydrochloride, glycine, and MCDs. The results are as follows: Figure 4 As shown, in the range of 3400-2800cm -1 The broad absorption peak at 1565 cm⁻¹ is the stretching vibration peak of NH₃. -1 The spectral peak at 1367 cm⁻¹ is the stretching vibration peak of C=N. -1 and 1065cm -1 The spectral peaks were generated by the vibrations of CO and CN, respectively, indicating that the synthesized MCDs possess amino groups and proving the successful synthesis of MCDs. After being mounted on a PDA, the FT-IR spectrum of the MCDs-PDA is visible in the 3600-3200 cm⁻¹ range. -1 The broad absorption peak at 1626 cm⁻¹ is caused by the OH stretching vibration. -1 The peak at 1050 cm⁻¹ is attributed to the overlap of the C=C resonance in the aromatic ring of PDA, indicating that PDA has been successfully encapsulated on the surface of MCDs. After loading DEX, the FT-IR spectrum of MCDs-PDA@DEX shows a peak at 1050 cm⁻¹. -1 The spectral peaks were generated by CF stretching vibrations, confirming the successful loading of DEX. The UV-Vis absorption spectra of MCDs-PDA and MCDs-PDA@DEX were evaluated using a UV-Vis spectrophotometer, as shown below. Figure 5 MCDs exhibit an absorption peak at 230 nm. Literature review indicates that the UV-Vis absorption spectrum of PDA has an absorption peak at 280 nm. The modified and synthesized MCDs-PDA shows a typical absorption peak at 280 nm, confirming the successful synthesis of MCDs-PDA. Subsequently, the integration of DEX was evaluated. Literature review indicates that DEX has an absorption peak at 242 nm. MCDs-PDA@DEX exhibits broad absorption in the 225-310 nm range, with a typical absorption peak at 242 nm, suggesting the successful synthesis of MCDs-PDA@DEX.

[0094] like Figure 6As shown in (a), the absorbance of DEX at 242 nm was measured within the concentration range of 0-80 μg / mL, and fitting verification showed a linear relationship between the concentration and absorbance of DEX within this range (R² = 0.99854). Based on the DEX standard curve, the drug loading and encapsulation efficiency of DEX were calculated, resulting in a drug loading of 23.97% and an encapsulation efficiency of 87.40%. The release of DEX was calculated based on the standard curve, and a sustained-release curve was plotted. Figure 6 (b) shows that MCDs-PDA@DE X releases approximately saturation around day 5, with 78.27% of the drug load being released, while DEX releases approximately 78.52% of the drug load around day 7.

[0095] 2) Evaluation of biological applications

[0096] ① Before conducting further in vitro osteogenic experiments with the material, it is essential to confirm its safety to normal cells. If the viability of normal cells treated with the nanomaterial remains above 80%, the material can be considered to have good biocompatibility. Different concentrations of MCDs, MCDs-PDA, and MCDs-PDA@DEX were co-cultured with hNF-C1 cells for 1 and 3 days, and cell viability was assessed using CCK8 assay. Results are as follows... Figure 7 As shown, MCDs in the concentration range of 0-400 μg / mL promoted the proliferation of hNF-C1 cells. Within this concentration range, the higher the concentration of MCDs, the stronger the promoting effect on hNF-C1 cell proliferation. For the MCDsPDA and MCDs-PDA@DEX groups, the cell activity showed a trend of decreasing with increasing material concentration. In the concentration range of 0-12.5 μg / mL, the activity of the 3-day treatment group was greater than that of the 1-day group at the same concentration of the same material, which is considered to be due to the proliferation of previously surviving cells. When the concentration increased to 25-400 μg / mL, even if some cell proliferation occurred, the cell activity of the 3-day group was still lower than that of the 1-day group, possibly because the toxicity of the material at this concentration was not low. Specifically, after treating hNF-C1 cells with 25 μg / mL MCDs-PDA for 1 day, cell viability was only 72.9%. When the MCDs-PDA concentration decreased to 12.5 μg / mL, the cell viability of hNF-C1 cells treated for 1 day was 83.4%, and the cell viability after 3 days was 88.4%, indicating ideal cell viability. For the MCDs-PDA@DEX group, the cell viability was 82.3% after 1 day of treatment with a concentration of 25 μg / mL, but when the treatment period increased to 3 days, the cell viability decreased to 77.1%. In contrast, the viability of the 12.5 μg / mL concentration groups for 1 day and 3 days were 88.7% and 89.3%, respectively, both higher than 80%. Based on these results, the optimal concentration of 12.5 μg / mL for cell culture was selected.

[0097] ②Results of material cell uptake

[0098] To evaluate the uptake of MCDs, MCDs-PDA, and MCDs-PDA@DEX by hiPSCs, hiPSCs were co-cultured with MCDs, MCDs-PDA, and MCDs-PDA@DEX at a safe concentration of 12.5 μg / mL determined by cytotoxicity assays for 6 h, 12 h, 24 h, and 48 h. Intracellular fluorescence was observed using an inverted fluorescence microscope. The results are as follows: Figure 8 and Figure 9 As shown, cell fluorescence increases with time from 6h to 24h, reaching its peak at 24h, and gradually weakens at 48h. This indicates that hiPSCs cells have reached maximum uptake of MCDs, MCDs-PDA, and MCDs-PDA@DEX at 24h, and after 24 hours, due to cell metabolism, MCDs, MCDs-PDA, and MCDs-PDA@DEX are gradually expelled from the cell.

[0099] Example 2: Regulation of osteogenic differentiation of hiPSCs by the nanoparticles MCDs-PDA@DEX prepared in Example 1

[0100] The main experimental reagents used in this embodiment are shown in Table 2.

[0101] Table 2: Main Reagents and Manufacturers

[0102]

[0103]

[0104] Experimental procedure:

[0105] 1) Experiment on differentiation of hiPSCs under the action of MCDs-PDA@DEX

[0106] hNF-C1hiPSCs cells were cultured using PSC medium with medium changes. Once the cells reached 80% confluence, the medium was replaced with serum-containing growth medium GM, consisting of DMEM / high glucose + 15% fetal bovine serum (FBS) + 1% non-essential amino acids (NEAA) + 1% L-alanyl-glutamine (LGlutaMax) + 1% penicillin / streptomycin (double antibiotic). This day was designated as day 0 (D0). Materials were added to the medium at a concentration of 12.5 μg / mL, and the medium was changed every other day. The specific experimental groups were: 1) Control GM; 2) GM + MCDs; 3) GM + MCDs-PDA; 4) GM + MCDs-PDA@DEX; 5) GM + DEX, with three replicates for each group. On days D3 and D7 of culture, samples were collected and RT-PCR was used to detect the expression of genes related to the three germ layers (endodermis: AFP, GATA4; mesoderm: MSX1, T; ectoderm: PAX6, SOX1).

[0107] 2) Experiment on osteogenic differentiation of hiPSCs under the action of MCDs-PDA@DEX

[0108] hNF-C1hiPSCs cells were cultured using PSC medium with medium changes. Once the cell confluence reached 80%, the medium was replaced with serum-containing growth medium GM, whose composition was DMEM / high glucose + 15% FBS + 1% non-essential amino acids (NEAA) + 1% L-alanyl-glutamine (L-GlutaMax) + 1% penicillin / streptomycin (double antibiotic). This day was designated as day 0 (D0). Materials were added to the medium at a concentration of 12.5 μg / mL and the medium was changed every other day. The specific experimental groups were: 1) Control GM; 2) GM + MCDs; 3) GM + MCDs PDA; 4) GM + MCDs-PDA@DEX; 5) GM + DEX, with three replicates for each experimental group. At days 7, 14, 21 and 28 of osteogenic induction culture, samples were collected for osteogenic-related assays, including RT-PCR to detect the expression of osteogenic-related genes (ALP, RUNX2, COL1A1 and OCN), qualitative and quantitative assays using Alizarin Red staining to detect calcium nodule formation, and immunofluorescence assays to detect the expression of osteogenic-related proteins (RUNX2, COL1A1 and OCN) and stem protein OCT-4.

[0109] 3) RT-PCR detection

[0110] Cells were washed with DPBS on days 7, 14, 21, and 28 of osteogenic induction culture. Then, Trizol lysis buffer was added for 15 min to lyse the cells. The cells were then pipetted to evenly disperse them in the lysis buffer, and the lysis buffer was collected in a 1.5 mL EP tube. Chloroform was added to the cell lysis buffer and the mixture was vortexed to mix. After standing for 5 min, the liquid was centrifuged at 12000 rpm for 15 min at 4°C. The liquid separated into three layers: RNA on top, DNA in the middle, and protein on the bottom. Approximately 200 μL of the upper RNA layer was placed in an EP tube, isopropanol was added, and the mixture was inverted for 15 s. After standing for 10 min, it was centrifuged again for 15 min. The supernatant was discarded, and the cells were washed twice with 75% ethanol. Finally, after centrifugation for 2 min, the EP tube was opened to air dry the remaining liquid, and 20 μL of LEPC water was added to dissolve the RNA. The RNA concentration of each sample was measured and recorded using a spectrophotometer. The RNA from each sample was reverse transcribed into 20 μl cDNA using the kit, and then diluted with ddH2O for later use. A 20 μl reaction mixture was then prepared using SYBR Green dye to detect the target gene, and the cycle threshold (Ct value) of the target gene was detected using an ABIQ5 real-time quantitative PCR instrument. The expression of the target gene was assessed using the 2-ΔΔCt method, with ACTB as an internal control gene, and three replicates were performed for each sample. Primer sequences are shown in Table 3 below.

[0111] Table 3: Primer sequences

[0112]

[0113]

[0114] 4) Alizarin Red staining and quantification

[0115] Cells were washed three times with DPBS buffer at days 7, 14, 21, and 28 of osteogenic induction culture, and then fixed with 4% paraformaldehyde for 15 minutes at room temperature. Subsequently, the cells were washed three more times with DPBS buffer to obtain fixed cell samples. A 0.5% alizarin red solution was prepared using 0.01 M Tris-HCl buffer, and after adjusting the pH to 4.2, it was filtered through slow-speed neutral filter paper to obtain the prepared alizarin red staining solution. The fixed cells were observed and photographed under an inverted microscope to obtain cell morphology images of different intervention groups. The prepared alizarin red solution was added to the wells of the cell culture plates for staining. After 10 minutes, the plates were washed with pure water until the washing solution was clear and transparent. Calcium nodules in the wells were observed and photographed using a camera and an inverted microscope to observe and analyze the formation of calcium nodules. To quantify the formation of calcium nodules, an alizarin red staining quantitative experiment was performed. A 1% (w / v) hexadecylpyrimidine solution was added to the wells that had been stained with alizarin red. After reacting at room temperature for 24 hours, the supernatant in the wells was aspirated and the absorbance at 490 nm was measured using an ELISA reader.

[0116] 5) Immunofluorescence

[0117] Cell samples from osteogenic induction culture at days D7, D14, D21, and D28 were fixed using the same steps described above. The fixed cells were washed three times with DPBS buffer, with the wells gently shaken on a shaker for 2 minutes during washing. Then, 0.2% Triton-X100 (prepared with PBS) was added to the wells, and the plates were permeated at room temperature for 30 minutes. The cells were then washed three more times with DPBS on a shaker. Next, 3% BSA solution (prepared with PBS) was added to the wells, and the plates were blocked at room temperature for 2 hours. The plates were then washed three more times with DPBS. Finally, 3% BSA and 0.2% Triton-X100 were mixed and diluted 1:100 to obtain the primary antibody solution. This solution was added to the wells, and each well was covered with a sealing film slightly smaller than the well diameter. The plates were then placed in a humidified chamber and incubated at 4°C for 12 hours. The sealing film was then removed with a needle to remove the primary antibody solution from the wells, and the plates were washed three times with DPBS. Under light-protected conditions, the secondary antibody was diluted 1:50 with DPBS. The diluted solution was added to the wells of the plate, and the reaction was carried out at room temperature for 1 hour. After incubation, the secondary antibody was removed, and the plate was washed three times with DPBS. DAPI was diluted 1:5000 with DPBS, and the diluted solution was added to the wells of the plate. The plate was stained at room temperature for 10 minutes, washed once with DPBS, and 500 μL DPBS was added to each well. Cells were observed and photographed using an inverted fluorescence microscope.

[0118] Statistical analysis: SPSS 13.0 statistical software was used to analyze the experimental results. One-way ANOVA was used to compare the differences between groups, and Tukey's method was used for multiple comparisons. p < 0.05 was considered statistically significant.

[0119] result:

[0120] 1) Effect of MCDs-PDA@DEX on spontaneous differentiation of hiPSCs

[0121] like Figure 10 As shown, after adding MCDs, MCDs-PDA, and MCDs-PDA@DEX to the culture medium of hiPSCs, samples were collected on days 3 and 7, and the effect of the materials on the spontaneous differentiation of hiPSCs was detected by RT-PCR. The expression of endoderm genes AFP and GATA4 is shown in Figures 10(a) and (b). It can be seen that the expression levels of AFP and GATA4 in hiPSCs decreased after the addition of materials, and their expression further decreased on day 7 with the increase of culture time, suggesting that the addition of MCDs, MCDs-PDA, and MCDs-PDA@DEX inhibited endoderm differentiation. Figure 10 (c) and (d) show the expression of mesodermal genes MSX1 and T. It is evident that the expression of mesodermal genes significantly increased after the addition of materials. Furthermore, all experimental groups showed an increase in expression levels from D3 to D7, with a trend of MCDs-PDA@DEX group > MCDs-PDA group > MCDs group > DEX group, indicating that the addition of materials in each group promoted mesodermal differentiation of hiPSCs. The expression results of ectoderm genes PAX6 and SOX1 are shown below. Figure 10 As shown in (e) and (f), their expression trends are similar to those of mesodermal gene expression levels. All groups showed an increase in expression levels from D3 to D7, and the MCDs-PDA@DEX group had the most significant effect on promoting hiPSC ectoderm differentiation.

[0122] 2) Effects of MCDs-PDA@DEX on osteogenic genes in osteogenic differentiation of hiPSCs

[0123] To evaluate the effect of nanomaterials on osteogenic differentiation of stem cells, RT-PCR was used to detect the expression levels of osteogenic-related genes. Alkaline phosphatase (ALP) is an important protein formed during osteoblast differentiation and is a marker of early osteoblasts. ALP expression results are shown in 11(a). ALP expression reached its highest value at 14 days of osteogenic induction and gradually decreased with increasing induction time. Furthermore, except for the D7 group with lower ALP expression, the ALP expression levels in the MCDs, MCDs-PDA, and MCDs-PDA@DEX experimental groups were higher than those in the control group at other time points. The MCDs-PDA@DEX group showed the highest ALP expression, indicating that the prepared MCDs themselves possess a certain function in inducing osteogenic differentiation of hiPS Cs. The nanosystem MCDs-PDA@DEX, prepared after loading with the osteogenic drug DEX, exhibited a significantly higher osteogenic differentiation-promoting effect than DEX.

[0124] Runt-related transcription factor 2 (RUNX2) is a key transcription factor in osteoblast differentiation and bone formation, playing a crucial role in bone formation. It upregulates bone morphogenetic protein 2 (BMP), an early marker of osteogenic differentiation, and enhances the expression of late-stage osteogenic marker genes. RUNX2 expression results are as follows: Figure 11 As shown in (b), its expression began to increase significantly at 14 days of osteogenic induction and continued to rise at 21 and 28 days. In addition, the expression level of RUNX2 in each time group showed the trend of MCDs-PDA@DEX>MCDs-PDA>MCDs>DEX>Control, which also indicates that carbon dot MCDs themselves have the ability to promote the osteogenic formation of hiPSCs. After modifying their surface with PDA and DEX, their osteogenic induction function of stem cells was further enhanced.

[0125] Type I collagen (COL1A1) is one of the main collagen components in the bone matrix, secreted by osteoblasts, and participates in bone tissue formation and maturation. Osteocalcin (OCN) is an important bone matrix protein in the bone formation process and is considered a marker of the terminal stage of osteoblast differentiation. The expression results of COL1A1 and OCN are as follows: Figure 11 As shown in (c) and (d), COL1A1 and OCN showed similar expression patterns to RUNX2 in each experimental group, and both showed a trend of gradually increasing expression with differentiation. At 28 days, they showed a significant increase in expression level, suggesting that osteogenic differentiation entered the mature stage at this time point.

[0126] 3) Effects of MCDs-PDA@DEX on osteogenic methyl-associated proteins in the osteogenic differentiation of hiPSCs

[0127] After using RT-PCR to examine the expression levels of osteogenic-related genes in hiPSCs induced by various materials, this study used immunofluorescence to detect the expression of osteogenic-related proteins in hiPSCs at 7, 14, 21, and 28 days after osteogenic differentiation induced by MCDs, MCDs-PDA, and MCDsPDA@DEX. The results are as follows: Figures 12 to 15 As shown, the expression of the stem protein OCT-4 gradually decreased with increasing culture days, which is due to the differentiation of hiPSCs during the induction process. Figure 13 As observed, osteogenic-associated protein RUNX2 showed no significant expression in group D7 of all materials, gradually began to be expressed in group D14, and further increased expression levels were observed in groups D21 and D28. Osteogenic-associated proteins COL1A1 and OCN showed the same trend, and both showed the highest expression levels in the MCDs-PDA@DEX group, demonstrating the ideal effect of this material in promoting osteogenic differentiation of hiPSCs.

[0128] 4) Effects of MCDs-PDA@DEX on cell morphology during osteogenic differentiation of hiPSCs

[0129] To observe the morphological changes of hiPSCs during osteogenic differentiation, inverted microscopes were used to observe and photograph cell morphology images on days 7, 14, 21, and 28 of different intervention groups. The results are as follows: Figure 16 As shown in the diagram, at day 7, the number of hiPS Cs cells in each experimental group was relatively large, and the cell clones were densely arranged. As the cells continued to differentiate, by day 14, the number of cells decreased and the cell volume increased. In the day 21 group, the cell volume further increased, and the matrix increased significantly, with the cell morphology of the MCDs-PDA@DEX group being typical. By day 28, the collagen fibers in the MCDs-PDA@DEX group had further increased, and a large number of "dendritic" and "vacuolar" structures appeared, indicating that the cells in the MCDs-PDA@DEX group underwent osteogenic differentiation, and that MCDs-PDA@DEX played a role in promoting collagen secretion.

[0130] 5) Effects of MCDs-PDA@DEX on calcium nodules in osteogenic differentiation of hiPSCs

[0131] In the osteogenic differentiation of stem cells, mineralized nodules are important indicators of terminal differentiation and maturation of osteoblasts. The mineral structure and distribution can be observed under a microscope using alizarin red staining. hNF-C1 hiPSCs were cultured in media containing 12.5 μg / mL MCDs, MCDs-PDA, MCDs-PDA@DEX, and DEX for different times (7, 14, 21, and 28 days). The osteogenic effect was evaluated using alizarin red staining. Figure 22-24 The results of cell well plate imaging are as follows: Figure 17 As shown in Figure 3-9, cell morphology under an inverted microscope reveals no obvious calcium nodules in the 7-day and 14-day groups. With increasing culture time, the number of calcium nodules formed in the same experimental group gradually increased, indicating a significant effect of osteoblast maturation and osteogenic induction culture. Under the same culture time conditions, the MCDs-PDA@DEX experimental group showed a significantly higher amount of calcium nodules than other groups. To further visualize the formation of calcium nodules, a quantitative assay using hexadecylpyrimidine was performed to analyze the amount of minerals visible after Alizarin Red staining. The results are shown in Figure 3-9. Figure 19 As shown, the quantification results of alizarin red, consistent with the cell morphology diagram, gradually increased over time. In the 21-day and 28-day groups, the quantification results of the MCDs-PDA@DEX group were significantly higher than those of other groups, indicating that MCDs-PDA@DEX has a good effect on inducing osteogenic differentiation of MCDs-PDA@DEX.

[0132] Example 3: Preparation of bone injury repair gel

[0133] The main experimental reagents used in this embodiment are shown in Table 4.

[0134] Table 4: Major Reagents and Manufacturers

[0135]

[0136] The MCDs-PDA@DEX used was prepared in Example 1.

[0137] The preparation of bone injury repair gel (MCDs-PDA@DEX genipin / gelatin hydrogel) includes the following steps:

[0138] 10 mg of genipin was dissolved in 500 μl of anhydrous ethanol to prepare a genipin solution. 1 g of gelatin was added to 9.5 mL of deionized water (heated and stirred at 50 °C to dissolve). 500 μl of the genipin ethanol solution and 12.5 μL of MCDs-PDA@DEX solution (10 mg / mL, aqueous solution) were added to the gelatin solution. The mixture was stirred at 650 rpm for 30 min at 50 °C using a magnetic stirrer, and then allowed to stand for 24 h to allow cross-linking, yielding a deep blue hydrogel MCDs-PDA@DEX / GG. Removing MCDs-PDA@DEX from the experimental protocol and following the same steps yielded a genipin / gelatin hydrogel GG without nanomaterials. Replacing MCDs-PDA@DEX with MCDs and following the same experimental protocol yielded a hydrogel MCDs / GG containing MCDs. Solid powders were obtained by freeze-drying GG, MCDs / GG, and MCDs-PDA@DEX / GG hydrogels using a freeze dryer, and the surface morphology of the solid powders was characterized by scanning electron microscopy (SEM).

[0139] Characterization operations:

[0140] 1) Cytotoxicity assay of MCDs-PDA@DEX / GG hydrogel

[0141] 0.2 g of hydrogel (GG, MCDs / GG, MCDs-PDA@DEX / GG) was added to 1 mL LPSC medium and left for 24 h. The hydrogel was then removed from the medium, and the extract was obtained by filtration. hNF-C1 hiPSCs cells grown on the Matrigel surface were digested with EDTA after reaching 80% confluence. Cells were counted and then stored at 2.5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 cell / cm² in 48-well plates. After 24 hours of culture, the medium was replaced with medium containing hydrogel extract, while the control group continued to use medium without extract. Three parallel control wells were set up for each group. Cell viability was measured 1 day and 3 days after material addition using a CCK8 assay.

[0142] 2) Establish a rabbit skull defect animal model

[0143] Thirty-two male New Zealand white rabbits, weighing (2.5±0.5) kg, were randomly divided into eight groups (see Table 5). After general anesthesia with sodium pentobarbital via the marginal ear vein, the rabbits were fixed in a prone position. Routine skin preparation and iodine disinfection were performed on the skull area. A 3 cm longitudinal incision was made along the midline of the skull, and the skull was exposed by blunt dissection. A circular defect approximately 5 mm in diameter was created on each side of the parietal bone using a trephine drill, while simultaneously dripping physiological saline to cool the area. The prepared hydrogel was then placed into the skull defect of the corresponding group of rabbits. In the blank control group, no implantation was performed, only defect treatment. Postoperatively, the wound was sutured in layers, and penicillin (1.0×10⁻⁶) was injected for three consecutive days.4 Rabbits were fed a normal diet (U / kg). Postoperative recovery was monitored, and weight was recorded. Animals were euthanized at 4 and 8 weeks post-surgery, and the parietal skull and major organs (heart, liver, spleen, lungs, and kidneys) were collected intact, fixed in 4% paraformaldehyde for 48 hours, and then processed.

[0144] Table 5: Experimental Groups of the Effects of Biomaterials on Bone Regeneration and Repair of Rabbit Skull Defects

[0145]

[0146] The male New Zealand white rabbits used in this experiment were all obtained from the Experimental Animal Center of the School of Basic Medical Sciences, Lanzhou University, Gansu Province. The experimental procedures followed the "Regulations on the Management of Experimental Animals". The experimental protocol has been approved by the Ethics Committee of the School of Stomatology, Lanzhou University (LZU KQ-2025-029).

[0147] Micro-CT was used to assess new bone formation in collected skull specimens. Analysis of three-dimensional reconstructed CT images and coronal images of the skull showed that different implants promoted new bone formation at the defect site. HiscanAnalyzer software was used to quantitatively analyze bone tissue parameters, including bone volume (BV), bone volume fraction (BV / TV), and bone surface area (BS), in the regenerated area.

[0148] Histological analysis and in vivo biocompatibility assessment: Fixed rabbit hearts, livers, spleens, lungs, and kidneys were prepared into paraffin sections and stained with Hematoxylin & Eosin (H&E). Observation and photography were performed under a microscope to verify the in vivo biocompatibility of GG, MCDs / GG, and MCDs-PDA@DEX / GG. The weight of each rabbit was recorded every 3 days, and weight change curves were plotted for the 4-week and 8-week groups.

[0149] Statistical analysis: SPSS 13.0 statistical software was used to analyze the experimental results. One-way ANOVA was used to compare the differences between groups, and Tukey's method was used for multiple comparisons. p < 0.05 was considered statistically significant.

[0150] result:

[0151] 1) Stent morphology of GG, MCDs / GG, and MCDs-PDA@DEX / GG

[0152] The morphology of GG, MCDs / GG, and MCDs-PDA@DEX / GG hydrogels before and after crosslinking is as follows: Figure 20As shown, the hydrogel is a dark liquid before crosslinking and becomes a solid with a deep blue color after crosslinking. This is due to the blue pigment produced by the amino crosslinking of genipin and gelatin. The surface morphology of GG, MCDs / GG, and MCDs-PDA@DEX / GG hydrogels was analyzed by SEM, and the results are as follows: Figure 21 As shown, the three types of hydrogels obtained exhibit a regular porous structure, and the loading of MCD and MCDs-PDA@DEX has no significant effect on the pore size of the hydrogels.

[0153] 2) In vitro toxicity test results of GG, MCDs / GG, and MCDs-PDA@DEX / GG

[0154] Before conducting further in vivo osteogenic biological experiments on the material, the in vitro biocompatibility of the hydrogel was first tested using an extract of the hydrogel. The results were as follows: Figure 22 As shown, hiPSCs in the GG, MCDs / GG, and MCDs-PDA@DEX / GG groups all exhibited good cell viability of more than 80% under conditions of 1 day and 3 days of culture.

[0155] 3) Effects of GG, MCDs / GG, and MCDs-PDA@DEX / GG on the repair of skull defects in rabbits

[0156] Rabbit skull samples were collected at 4 and 8 weeks after the hydrogel was implanted into the skull defect. Micro-CT scan results are as follows: Figure 23 As shown in the figure, the skull in the control group (without implanted material) showed no significant changes at 4 weeks, but some new bone formation was observed at 8 weeks. The GG group showed a larger area of ​​new bone formation compared to the control group. In contrast, the MCDs / GG group and the MCDs-PDA@DEX / GG group showed more significant bone repair effects. Among them, the MCDs-PDA@DEX / GG group showed the most significant repair effect at 8 weeks, almost completely covering the original round bone defect. This indicates that MCDs / GG and MCDs-PDA@DEX / GG can effectively promote bone regeneration in the skull defect area. Quantitative analysis results of new bone formation are shown below. Figure 24 As shown, consistent with the results of micro-CT scans, this fully demonstrates the excellent bone regeneration promoting effect of MCDs / GG and MCDs-PDA@DEX / GG.

[0157] 4) In vivo biocompatibility of GG, MCDs / GG, and MCDs-PDA@DEX / GG

[0158] Four and eight weeks after implantation of GG, MCDs / GG, and MCDs-PDA@DEX / GG hydrogels, H&E staining results of major rabbit organs (heart, liver, spleen, lungs, and kidneys) were collected. Figure 25As shown in (a) and (c), compared with the blank control group, no obvious pathological abnormalities or damage were observed in the major organs of rabbits in the GG group, MCDs / GG group, and MCDs-PDA@DEX / GG group, demonstrating that the prepared hydrogel material did not exhibit obvious toxic reactions in animals and had good biocompatibility. The weight change curves of rabbits in the 4-week and 8-week groups are shown in... Figure 25 As shown in (b) and (d), the animals' body weight decreased during the first weighing after modeling. Apart from that, the body weight of each group of animals showed a steady increase, further confirming the good in vivo safety of GG, MCDs / GG and MCDs-PDA@DEX / GG.

[0159] The foregoing has shown and described the main features, methods of use, basic principles, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only for illustrating the principles of the invention. Without departing from the spirit and scope of the invention, corresponding changes and modifications may be made according to actual circumstances, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing drug-loaded metformin carbon dots, characterized in that, It includes the following steps: 1) Preparation of MCDS MCDS powder was synthesized using metformin hydrochloride and glycine as raw materials via a one-step hydrothermal method. 2) Preparation of MCDs-PDA After mixing Tris·HCl solution, dopamine hydrochloride and the MCDS prepared in step 1) and reacting, the mixture was filtered, the filtrate was collected, washed and freeze-dried to obtain MCDs-PDA; 3) Preparation of MCDs-PDA@DEX Take the MCDs-PDA prepared in step 2) and dexamethasone, add them to a solvent for reaction, filter, take the filter solid, wash and freeze-dry to obtain drug-loaded metformin carbon dots MCDs-PDA@DEX.

2. The application of the drug-loaded metformin carbon dots prepared according to claim 1 in promoting the in vitro osteogenic differentiation cell culture of hiPSCs for non-therapeutic and diagnostic purposes.

3. The application according to claim 2, characterized in that, During cell culture, the concentration of the MCDs-PDA@DEX was 12.5 μg / mL.

4. The application of the drug-loaded metformin carbon dots prepared according to claim 1 in the preparation of bone injury repair materials.

5. The application according to claim 4, characterized in that, The bone injury repair material includes a bone injury repair gel material.

6. A method for preparing a gel for bone injury repair, characterized in that, Includes the following steps: ① Prepare genipin ethanol solution and gelatin aqueous solution separately; ② Take the drug-loaded metformin carbon dots prepared according to claim 1 and add water to prepare MCDs-PDA@DEX solution; ③ After mixing genipin ethanol solution, gelatin aqueous solution and MCDs-PDA@DEX solution, the gel is prepared for bone injury repair after reaction.

7. The bone injury repair gel prepared by the method of claim 6.

8. The use of the bone injury repair gel prepared by the method of claim 6 in the preparation of bone injury repair scaffolds.

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