Arginine carbon dot photoinitiated hydrogel pre-polymer, preparation method and application thereof

Abstract

This invention belongs to the field of guided bone regeneration materials technology, specifically relating to an arginine carbon dot photoinitiated hydrogel prepolymer, its preparation method, and its application. The arginine carbon dot photoinitiated hydrogel prepolymer of this invention, when used for alveolar bone regeneration, inhibits the proliferation / migration of gingival fibroblasts (hGFs) while simultaneously inducing and promoting the proliferation / migration and osteogenic differentiation of bone marrow mesenchymal stem cells (hBMSCs), thus creating a "reverse-response" cell-selective regulatory effect.

Description

Technical Field

[0001] This invention belongs to the field of guided bone regeneration materials technology, specifically relating to an arginine carbon dot photoinitiated hydrogel prepolymer, its preparation method, and its application. Background Technology

[0002] Dental implantology has become an important means of restoring missing teeth, but ideal implant sites depend on sufficient alveolar bone volume and stable bone quality. Due to factors such as periodontitis, trauma, infection, and bone resorption after tooth extraction, a significant proportion of patients in clinical practice have alveolar ridge atrophy or bone defects before implantation, requiring bone augmentation to achieve implantability. Therefore, how to achieve predictable, stable, and less complication-prone new bone formation in the defect area is a key issue affecting the long-term success rate of implantation.

[0003] Guided bone regeneration (GBR) surgery, based on the core concept of "barrier membranes blocking soft tissue and providing a closed space," can increase bone mass, but significant technical challenges remain in clinical practice: First, commonly used bone substitutes are primarily osteoconductive, lacking sufficient osteoinductive activity, and have loose interparticle connections and limited overall mechanical support. Under oral movement and soft tissue pressure, they are prone to insufficient space maintenance, affecting the stability of bone regeneration. Second, the barrier membrane system itself has limitations: absorbable membranes are limited in terms of shaping stability and barrier persistence; non-absorbable membranes, while improving barrier and space maintenance, increase the risk of membrane exposure and often require secondary surgery for removal, leading to additional trauma and infection risks. Third, from a histological perspective, GBR restoration involves competitive healing between bone tissue and gingival soft tissue within a limited space: rapid soft tissue encroachment inhibits new bone formation, leading to failed augmentation or unstable results. Therefore, relying solely on the mechanical barrier of an "external barrier membrane" is still insufficient to solve the delicate balance between "promoting bone formation and inhibiting gingival tissue formation" from a biological perspective, and the clinical complications and technical sensitivity remain high.

[0004] Carbon dots, as carbon-based nanomaterials, possess excellent water solubility and biocompatibility. Their tunable surface functional groups have been reported to participate in the regulation of biological processes such as cell proliferation, differentiation, and stress responses. Carbon dots prepared using arginine as a precursor are rich in nitrogen- and oxygen-containing functional groups, potentially exhibiting differential responses to different cell lineages: promoting bone-related cells while potentially inhibiting or remodeling fibroblast proliferation / migration. This provides a new materials science pathway for constructing "selective regulation" within the GBR microenvironment. On the other hand, GelMA-like hydrogels, due to their biocompatibility, biodegradability, and adhesion motifs similar to the extracellular matrix, are suitable as tissue repair scaffolds, enabling injectable filling and in-situ gelation to match irregular bone defect morphologies and improve graft integrity. However, traditional GelMA often uses UV light to induce cross-linking, posing potential phototoxicity and tissue / cell damage risks, limiting its safe operating window in oral clinical settings. Furthermore, pure GelMA lacks a clear "directional biological function," making it difficult to simultaneously meet the dual requirements of promoting ossification and inhibiting soft tissue lesions. Summary of the Invention

[0005] In view of this, the present invention provides an arginine carbon dot photoinitiated hydrogel for alveolar bone guided bone regeneration, its preparation method and application. The arginine carbon dot photoinitiated hydrogel prepolymer of the present invention has an inhibitory effect on the proliferation / migration of gingival fibroblasts (hGFs) during alveolar bone guided regeneration, while having an inducing and promoting effect on the proliferation / migration and osteogenic differentiation of bone marrow mesenchymal stem cells (hBMSCs), which can form a cell-selective regulatory effect of "one increasing while the other decreases".

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing an arginine carbon dot photoinitiated hydrogel prepolymer, comprising the following steps: Arginine powder is heat-treated to obtain intermediate powder; the heat treatment includes a sequential heating process and a calcination process at a calcination temperature; the calcination is carried out under ventilation conditions; The aqueous dispersion of the intermediate powder was allowed to stand and centrifuged, and the resulting supernatant was dialyzed and dried sequentially to obtain Arg-CDs powder. A DPBS solution of methacrylamide gelatin, Arg-CDs powder, and a photoinitiator system were mixed to obtain an arginine carbon dot photoinitiated hydrogel prepolymer; the photoinitiator system included triethanolamine, N-vinylcaprolactam, and riboflavin.

[0007] Preferably, the heating process involves heating to the calcination temperature at a heating rate of 3.5~4.5 °C / min.

[0008] Preferably, the calcination temperature is 235~245 ℃, and the calcination time is 175~185 min.

[0009] Preferably, the Arg-CDs powder has a particle size of 1.74 ± 0.31 nm.

[0010] Preferably, the concentration of the intermediate powder in the aqueous dispersion of the intermediate powder is 0.1~0.2 g / mL; the standing time is 10~15 min; and the molecular weight cutoff of the dialysis bag is 1000 Da.

[0011] Preferably, the concentration of triethanolamine in the Arg-CDs photoinitiated hydrogel prepolymer solution is 145~155 mM; the concentration of N-vinylcaprolactam in the Arg-CDs photoinitiated hydrogel prepolymer solution is 155~165 mM; and the concentration of riboflavin in the Arg-CDs photoinitiated hydrogel prepolymer solution is 0.61~0.71 mM.

[0012] Preferably, the mass concentration of methacrylamide gelatin in the DPBS solution of the methacrylamide gelatin is 14.5-15.5%.

[0013] Preferably, the concentration of Arg-CDs in the Arg-CDs photoinitiated hydrogel prepolymer solution is 230~270 mg / mL.

[0014] The present invention also provides an arginine carbon dot photoinitiated hydrogel prepolymer prepared by the preparation method described above.

[0015] This invention also provides the application of the arginine carbon dot photoinitiated hydrogel prepolymer prepared by the above-described preparation method in guided bone regeneration. The method of guided bone regeneration is to inject the arginine carbon dot photoinitiated hydrogel prepolymer into the bone defect site and then irradiate it with blue light to obtain an arginine carbon dot photoinitiated hydrogel scaffold and release active ingredients.

[0016] Compared with the prior art, the present invention has the following advantages: (1) The present invention uses a malleable hydrogel as a material for filling and maintaining space in the defect area, which can effectively reduce the total volume of the implant while meeting the bone increase requirements, reduce the need for suture tension reduction, thereby reducing the risk of early postoperative exposure; and can avoid the trauma of secondary surgical removal caused by the use of non-absorbable barrier membrane.

[0017] (2) Existing photocurable hydrogel systems often rely on ultraviolet (UV) light to trigger crosslinking, which may pose a risk of phototoxicity and DNA damage to tissue cells. This invention uses GelMA as a carrier and constructs a photoinitiation system and crosslinking network through riboflavin (RF), N-vinylcaprolactam (NVC), triethanolamine (TEOA), and arginine carbon dots (Arg-CDs). This allows the hydrogel prepolymer to achieve photoinitiated crosslinking under visible blue light irradiation, thereby significantly reducing UV-related biosafety risks and improving clinical usability.

[0018] (3) Existing materials often fail to simultaneously address the contradiction between "rapid soft tissue occlusion" and "insufficient bone regeneration" in periodontal / alveolar bone defect repair. The arginine carbon dot photoinitiated hydrogel of this invention has an inhibitory effect on the proliferation / migration of gingival fibroblasts (hGFs) and an inducing and promoting effect on the proliferation / migration and osteogenic differentiation of bone marrow mesenchymal stem cells (hBMSCs), thereby forming a "give and take" cell-selective regulatory effect, which is beneficial for preventing gingival tissue occlusion, creating a window period for bone regeneration, and improving the repair effect of alveolar bone defects.

[0019] (4) Arg-CDs have good antibacterial properties against Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus sanguinis, which can effectively reduce the impact of surgical site infection on the effect of guided bone regeneration. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 Figure showing the establishment and grouping of a rabbit mandibular alveolar bone defect model; Figure 2 Image showing the results of staining live / dead cells; Figure 3 The results are for evaluating the cytotoxicity / proliferation of CCK-8 cells. Figure 3 Figure A shows the effect of Arg-CDs on hBMSCs cell viability under CCK8 assay, and Figure B shows the effect of Arg-CDs on hGFs cell viability under CCK8 assay. Figure 4 Figure showing the results of a cell scratch migration experiment; Figure 5 Figure showing the results of a cell scratch migration experiment; Figure 6 In the diagram, C represents the effect of different concentrations of Arg-CDs on the wound healing rate of hBMSCs cells, and D represents the effect of different concentrations of Arg-CDs on the wound healing rate of hGFs cells. Figure 7In the diagram, A represents the effect of Arg-CDs on hBMSCs and hGFs within the concentration gradient range, B represents the effect of Arg-CDs on the relative mobility of hBMSCs within the concentration gradient range, and C represents the effect of Arg-CDs on the relative mobility of hGFs within the concentration gradient range. Figure 8 In the middle section, A represents the ALP staining diagram of arginine carbon dot photoinitiated hydrogel promoting early osteogenic differentiation of hBMSCs, and B is a semi-quantitative statistical diagram of ALP staining. Figure 9 In the middle section, A represents the ARS staining diagram of arginine carbon dot photoinitiated hydrogel promoting mid-to-late osteogenic differentiation of hBMSCs, and B is a semi-quantitative statistical diagram of ARS staining. Figure 10 In the diagram, A represents the effect of arginine carbon dot hydrogel on the cell cycle distribution of hBMSCs and hGFs, B represents the changes in the cell cycle distribution of hBMSCs under the action of arginine carbon dot hydrogel, and C represents the changes in the cell cycle distribution of hGFs under the action of arginine carbon dot hydrogel. Figure 11 This is a map showing the colocalization of Arg-CDs and mitochondria in hBMSCs and hGFs. Figure 12 This is a map showing the colocalization of Arg-CDs and lysosomes in hBMSCs and hGFs. Figure 13 Transcriptomic quality control images of two cell samples; Figure 14 Volcano plot showing the differential gene expression in two cell groups under Arg-CDs photoinitiated hydrogel treatment; Figure 15 GO analysis diagram for differential gene enrichment; Figure 16 KEGG analysis diagram for differential gene enrichment; Figure 17 Proteomics quality control diagram of hBMSCs and hGFs cell samples; Figure 18 Volcano plot showing the differential protein expression in two cell groups under Arg-CDs photoinitiated hydrogel treatment; Figure 19 GO analysis plot for differentially expressed proteins; Figure 20 KEGG analysis plot for differential protein enrichment; Figure 21 Figure showing the qPCR validation results for hBMSCs cells; Figure 22 Figure showing the qPCR validation results for hGFs cells; Figure 23Western blot validation results and semi-quantitative statistical plots for hBMSCs cells; Figure 24 Western blot validation results and semi-quantitative statistical plots for hGFs cells; Figure 25 The image shows the results of the Arg-CDs photoinitiated hydrogel antibacterial loop test. Detailed Implementation

[0022] This invention also provides a method for preparing an arginine carbon dot photoinitiated hydrogel prepolymer, comprising the following steps: Arginine powder is heat-treated to obtain intermediate powder; the heat treatment includes a sequential heating process and a calcination process at a calcination temperature; the calcination is carried out under ventilation conditions; The aqueous dispersion of the intermediate powder was allowed to stand and centrifuged, and the resulting supernatant was dialyzed and dried sequentially to obtain Arg-CDs powder. A DPBS solution of methacrylamide gelatin, Arg-CDs powder, and a photoinitiator system were mixed to obtain an arginine carbon dot photoinitiated hydrogel prepolymer (denoted as Arg-CDs photoinitiated hydrogel prepolymer); the photoinitiator system included triethanolamine, N-vinylcaprolactam, and riboflavin.

[0023] The present invention involves heat-treating arginine powder to obtain intermediate powder; the heat treatment includes a sequential heating process and a calcination process at a calcination temperature; the calcination is carried out under ventilated conditions.

[0024] In this invention, the heat treatment can be carried out in a tube furnace; the heating process involves heating to the calcination temperature at a rate of 3.5~4.5 °C / min, preferably 4 °C / min; the calcination temperature can be 235~245 °C, specifically 240 °C; and the calcination time can be 175~185 min, specifically 180 min. In this invention, the tube furnace is kept open during the calcination process, and ventilation is assisted by a fan.

[0025] In this invention, the function of opening the tubular furnace and using a fan for ventilation during the calcination process is as follows: (1) Ventilation allows the reaction system to be in continuous contact with air, providing a suitable amount of oxygen for the heat treatment process, which is conducive to the moderate surface oxidation of the precursor during carbonization, thereby promoting the formation of oxygen-containing functional groups such as hydroxyl, carboxyl, and carbonyl groups. (2) During the pyrolysis of arginine, water vapor, ammonia and other volatile cracking small molecules will be generated. Assisted ventilation helps to remove the above by-products in a timely manner, avoiding their local accumulation and causing secondary condensation, deposition or excessive carbonization. (3) Ventilation helps maintain a relatively uniform atmosphere in the furnace, reducing the uneven reaction caused by local oxygen deficiency or volatile enrichment, thereby improving the uniformity and batch stability of the obtained intermediates and subsequent carbon dot products. (4) Moderate ventilation also helps to avoid excessive carbonization of the system and is conducive to retaining the nitrogen- and oxygen-containing active functional groups on the surface of the product. Therefore, the ventilation conditions in this step play a positive role in regulating the heat treatment process of arginine precursor and obtaining carbon dot intermediates with suitable surface functional group composition.

[0026] (5) During the heat treatment of arginine, water vapor, ammonia and other volatile cracking products are generated. Under non-ventilated conditions, these small molecules are prone to local accumulation, which may induce secondary condensation, redeposition or further aggregation between particles, thereby promoting the continued growth of carbon nuclei. Ventilation can remove the above-mentioned volatile by-products from the reaction zone in a timely manner, reduce secondary reactions, and thus be more conducive to the formation of carbon dots with smaller particle sizes.

[0027] (6) Ventilation keeps the system in an open air atmosphere, where oxygen can participate in the mild surface oxidation during the heat treatment process, which helps to introduce oxygen-containing functional groups during carbonization and, to a certain extent, inhibits the continuous accumulation and excessive growth of carbon nuclei, so that the particles can be stabilized at a smaller scale earlier.

[0028] (7) Ventilation also helps to maintain a relatively uniform atmosphere inside the furnace, reduce uneven reaction caused by local oxygen deficiency or volatile enrichment, thereby reducing the probability of local over-carbonization and abnormal growth.

[0029] As one embodiment of the present invention, after the heat treatment, the product is further cooled naturally to room temperature to obtain an intermediate powder.

[0030] After obtaining the intermediate powder, the present invention allows the aqueous dispersion of the intermediate powder to stand and centrifuge, and the resulting supernatant is dialyzed and dried sequentially to obtain Arg-CDs powder.

[0031] In this invention, the concentration of the intermediate powder in the aqueous dispersion of the intermediate powder can be 0.1~0.2 g / mL, specifically 0.1 g / mL, 0.15 g / mL or 0.2 g / mL; the aqueous dispersion of the intermediate powder can be obtained by dispersing the intermediate powder in water; the dispersion can be ultrasonic dispersion; the ultrasonic dispersion can be carried out under water bath heating conditions; the water bath heating temperature can be 50℃.

[0032] In this invention, the settling time can be 10-15 min, specifically 10 min, 11 min, 12 min, 13 min, 14 min or 15 min; the centrifugation speed can be 15000 rpm, and the centrifugation time can be 30 min.

[0033] In this invention, the molecular weight cutoff of the dialysis bag can be 1000 Da; the drying can be a combination of pre-freezing and freeze-drying; the pre-freezing temperature can be -20 ℃ and the time can be 2 h; the freeze-drying temperature can be -80 ℃ and the time can be 72 h.

[0034] This invention mixes a DPBS solution of methacrylamide gelatin, Arg-CDs powder, and a photoinitiation system to obtain an Arg-CDs photoinitiated hydrogel prepolymer.

[0035] In this invention, the photoinitiating system comprises triethanolamine, N-vinylcaprolactam, and riboflavin. In this invention, the concentration of triethanolamine in the Arg-CDs photoinitiating hydrogel prepolymer solution can be 145-155 mM, specifically 150 mM; the concentration of N-vinylcaprolactam in the Arg-CDs photoinitiating hydrogel prepolymer solution can be 155-165 mM, specifically 160 mM; and the concentration of riboflavin in the Arg-CDs photoinitiating hydrogel prepolymer solution can be 0.61-0.71 mM, specifically 0.66 mM. In this invention, the mass concentration of methacrylamide gelatin in the DPBS solution of methacrylamide gelatin can be 14.5-15.5% (w / v), specifically 15% (w / v). In this invention, the concentration of Arg-CDs in the Arg-CDs photoinitiated hydrogel prepolymer solution can be 230~270 mg / mL, specifically 250 mg / mL.

[0036] The present invention also provides an arginine carbon dot photoinitiated hydrogel prepolymer prepared by the preparation method described above.

[0037] This invention also provides the application of the arginine carbon dot photoinitiated hydrogel prepolymer prepared by the above-described preparation method in guided bone regeneration. The method of guided bone regeneration is to inject the arginine carbon dot photoinitiated hydrogel prepolymer into the bone defect site and then irradiate it with blue light to obtain an arginine carbon dot photoinitiated hydrogel scaffold and release active ingredients.

[0038] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0039] The main materials and equipment used in this embodiment are as follows: Materials: Arg-CDs photoinitiated hydrogel, human gingival fibroblasts (hGFs), human bone marrow mesenchymal stem cells (hBMSCs), live / dead cell double staining kit, CCK-8 kit, Transwell chambers and nested 24-well plates, alkaline phosphatase staining kit, human bone marrow mesenchymal stem cell osteogenic induction differentiation medium, Mito Tracker Red mitochondrial red fluorescent probe, Lyso-Tracker Red lysosomal red fluorescent probe, anti-fluorescence quenching blocking agent, TriZol lysis buffer, protein lysis buffer, protease inhibitor, reverse transcription kit, whole cell lysis buffer, BCA protein quantification kit, BMP2 monoclonal antibody, WNT4, P2RX1, P2RX5, CDH1, ADCY4, CDK1, RRM2 polyclonal antibody, GDF5, anti-rabbit IgG-HRP secondary antibody, ECL chemiluminescent substrate.

[0040] Equipment: Inverted fluorescence microscope, flow cytometer, laser confocal microscope, real-time quantitative PCR instrument, gel imaging system.

[0041] Example 1 Preparation of Arg-CDs powder: Arginine powder was placed in a corundum crucible and heat-treated in a tube furnace at a heating rate of 4 °C / min for 1 h, followed by constant-temperature calcination at 240 °C for 3 h. During calcination, the tube furnace was kept open with ventilation aided by a fan. After heat treatment, the powder was allowed to cool naturally to room temperature, yielding a dark brown intermediate powder, which was then sealed, dried, and stored for later use.

[0042] The intermediate was dispersed at a ratio of 5 g to 50 mL of ultrapure water, and dissolved by ultrasonication for 30 min in a 50 ℃ water bath. After standing for 10 min, the supernatant was collected and centrifuged at 15000 rpm for 30 min. The supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 1000 Da. Dialysis was performed for 12 h at 25 ℃ and 40 rpm using ultrapure water as the external dialysis fluid. The external dialysis fluid was changed once in the first h and then once every 4 h thereafter. After dialysis, the solution in the bag was collected, pre-frozen at -20 ℃ for 2 h, and then freeze-dried at -80 ℃ for 72 h to obtain Arg-CDs powder. The powder was placed in a desiccant container and stored at 4 ℃ in the dark for later use.

[0043] Preparation of methacrylamide gelatin (GelMA) and construction of Arg-CDs photoinitiated hydrogels Preparation was carried out under light-protected conditions: Gelatin was dissolved in PBS at pH 7.4 and magnetically stirred at 50 °C until it reached a mass fraction of 10% (w / v) and was completely dissolved. Methacrylic anhydride was slowly added dropwise in portions with continuous stirring, the amount added determined by 0.8 mL MA / g gelatin. Simultaneously, the pH of the reaction system was maintained at 7.5-8.0 using NaOH solution. The reaction was allowed to proceed for 2 h to ensure complete reaction between the methacryloyl groups and the gelatin amino groups. Subsequently, pre-cooled PBS at 4 °C was added to dilute the mixture to twice its original volume to terminate the reaction. After cooling the reaction solution to room temperature, it was transferred to a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed against deionized water at 40 °C for 7 days to remove unreacted MA and low-molecular-weight byproducts (water changed for the first 2 h, then twice daily thereafter). After dialysis, the solution was frozen and lyophilized to obtain white, porous, sponge-like GelMA, which was then stored at -20 °C in the dark.

[0044] Preparation of Arg-CDs visible light curable hydrogel: Based on a prepolymer solution volume of 10 mL, a photoinitiation system solution was first prepared by adding 150 mM triethanolamine (TEA), 160 mM N-vinylcaprolactam (NVC), and 0.66 mM riboflavin (RF) sequentially and dissolving them thoroughly to obtain the RF / TEA / NVC photoinitiation system for later use. Then, 15% (w / v) GelMA was added to preheated DPBS and magnetically stirred in a water bath at 37 ℃ until completely dissolved. Arg-CDs powder was added to make the final concentration of Arg-CDs in the prepolymer solution 250 mg / mL. The photoinitiation system solution was then added, and the volume was made up with DPBS (to 10 mL). The mixture was thoroughly mixed and allowed to stand for several minutes to remove gas, resulting in the Arg-CDs photoinitiated hydrogel prepolymer solution.

[0045] Test 1: (1) The particle size distribution of Arg-CDs in the aqueous phase was characterized by dynamic light scattering (DLS). The test steps are as follows: 100 mg of Arg-CDs sample powder was weighed and dispersed in 50 mL of ultrapure water. The Arg-CDs suspension was sonicated for 5 min to ensure sufficient dispersion of the sample while avoiding agglomeration and excessive temperature. 50 μL of the solution was injected into a special quartz cuvette and placed in the instrument for dynamic light scattering measurement. Results: The intrinsic nanoparticle size of the carbon dots was 1.74 ± 0.31 nm; the hydration dynamic particle size / aggregate size of the carbon dots in water due to the formation of the hydration layer and the interaction of surface functional groups was 79.3 ± 38.2 nm.

[0046] (2) Surface electrical properties and charge heterogeneity of Arg-CDs (Zeta potential) Zeta potential tests were performed on Arg-CDs suspensions at pH 7. Peak results showed that the zeta potential of the main particle population was concentrated in the range of -25 to -5 mV, corresponding to an area of ​​approximately 60% to 70%. At the same time, strong positive peaks (+40 to +110 mV) were observed in each measurement, accounting for 6% to 30%, with an overall mean of -2.01 ± 0.45 mV.

[0047] (3) XPS Survey full spectrum shows that, except for the undisplayed H element, Arg-CDs are mainly composed of C, N, and O elements, with corresponding characteristic peaks at approximately 285 eV (C 1s), 400 eV (N 1s), and 531 eV (O 1s). The O 1s high-resolution spectrum shows a relatively symmetrical single main peak at approximately 531 eV, which can be attributed to oxygen in the carbonyl (C=O) or carboxyl (O–C=O) groups. The N 1s shows a distinct peak shape at approximately 399-400 eV, mainly corresponding to amide or amino nitrogen, confirming that Arg-CDs are nitrogen-doped carbon points. The C 1s is dominated by the CC / C=C peak at approximately 284.8 eV, with a related component at approximately 288 eV (C=O / OC=O) on the high binding energy side. There is an overlap between the CO and CN components at approximately 286 eV. In summary, Arg-CDs possess a carbonized covalent carbon skeleton (CC / C=C), and their surface is enriched with carbonyl / carboxyl groups, as well as oxygen- and nitrogen-containing functional groups such as amino / amide groups.

[0048] Test 2 (1) pH and Zeta potential The Arg-CDs photoinitiated hydrogel was determined to be weakly acidic and close to physiological neutral using an extraction method, with the extract pH being 6.75 ± 0.29 (n = 3). Further zeta potential tests were performed on the hydrogel solution at pH 6, with an average value of 1.43 ± 1.85 mV (n = 3).

[0049] (2) FTIR detection of functional group changes before and after crosslinking The uncrosslinked Arg-CDs photoinitiated hydrogel prepolymer film and the photoinitiated crosslinked hydrogel were characterized by FTIR contrast. Both were characterized at 3300 cm⁻¹. -1 A broad and strong absorption band (-OH / -NH stretching) appears nearby, but the peak intensity decreases significantly after photocuring, suggesting that some hydroxyl / amino groups participate in cross-linking or secondary reactions during irradiation with 445 nm visible light. Simultaneously, the amount of adsorbed water and hydrogen bond network decreases, reflecting a denser gel network and a reduced number of free hydrophilic groups. The peak intensity at 2930 cm⁻¹ after curing... -1 More distinct -CH2- symmetric / asymmetric stretching vibration peaks appear nearby; and at 1630-1640 cm⁻¹. -1The changes in regional peak shape and relative intensity both suggest that the methacryloyl C=C group partially transforms into a saturated -CH2- structure during the crosslinking process. Simultaneously, at 1634 cm⁻¹... -1 The strength of the amide I (C=O stretching) on ​​both sides did not change significantly before and after curing, indicating that the overall structure of the GelMA polypeptide backbone remained stable; while at 1450 cm -1 Near (-CH2-bending / amide II) and 1240 cm -1 The absorption of the left and right (amide III: CN stretching and NH bending) groups was slightly enhanced and the peak shape was adjusted after curing, suggesting that the CN / NH vibrational environment around the side chains was constrained and rearranged, and some amino groups may have participated in crosslinking or formed new hydrogen bond networks. Furthermore, the photocured spectra showed improved absorption at 1583, 1401, 1336, 1286, 1161, and 1080 cm⁻¹. -1 The presence or enhancement of absorption peaks at locations such as Arg-CDs and photoinitiation system can be attributed to the aromatic C=C, CN, and CO / COC vibrations associated with these systems. This indicates that nitrogen- and oxygen-containing aromatic groups are introduced and participate in network construction, thereby enriching the functional group composition and hydrogen bonding modes of the hydrogel surface.

[0050] (3) Chemical state of elements and surface functional groups of Arg-CDs photoinitiated hydrogels (XPS) Except for the undisplayed hydrogen element, the XPS Survey spectrum of the composite hydrogel shows that it is mainly composed of C, N, O, and P. The O 1s high-resolution spectrum shows a single main peak at approximately 531 eV, attributed to C=O / OC=O oxygen species such as amide carbonyl and carboxyl groups, indicating that the hydrogel is rich in oxygen-containing functional groups such as amides, carboxylic acids / esters. The N 1s spectrum shows a strong peak at approximately 399-400 eV, mainly corresponding to amide / amine nitrogen, reflecting a significant nitrogen-containing characteristic of the composite hydrogel as a whole. The C 1s spectrum is composed of multiple superimposed components: the main peak at approximately 284.8 eV is CC / C=C, with shoulder peaks at approximately 286 eV and 288 eV on its high binding energy side, corresponding to nitrogen-containing and oxygen-containing functional groups such as CN / CO and C=O / OC=O, respectively. In summary, the hydrogel surface is simultaneously rich in a CC / C=C backbone, amides, carboxyl groups, and multiple polar functional groups such as CN / CO.

[0051] (4) Rheological properties Rheological tests on Arg-CDs photoinitiated hydrogels, specifically the viscosity-shear rate curves, revealed significant shear-thinning properties: at low shear rates of approximately 0.1 s⁻¹... -1 At high shear rates, the viscosity can reach the order of 10⁶ mPa·s, while at high shear rates of approximately 1000 s⁻¹... -1 At that time, the viscosity decreased to approximately 10. 2The viscosity is on the order of mPa·s. This indicates that the hydrogel has high viscosity under low shear conditions, which is beneficial for shaping and retention, and the viscosity decreases significantly under high shear conditions such as injection / extrusion, which is beneficial for achieving the injectability and spreadability required for clinical operations.

[0052] Strain scanning of the hydrogel at 1 Hz showed that as the shear strain increased from approximately 0.08% to approximately 1000%, the storage modulus G′ and loss modulus G″ exhibited typical gel viscoelastic behavior. In the low strain range (approximately 0.1%–10%), G′ maintained a stable plateau of approximately 2.8–2.9 kPa, significantly higher than G″ (approximately 0.2 kPa), corresponding to a loss tangent tanδ(G″ / G′) ≈ 0.07, indicating that the material exhibited predominantly elastic response and remained in a well-defined linear viscoelastic region. When the strain increased to approximately 20% or more, G′ began to decrease slightly while G″ gradually increased, with tanδ increasing accordingly, suggesting that the cross-linked network underwent nonlinear deformation and entered the yielding stage. In the high strain region (>100%), G′ decreased significantly while the relative proportion of G″ increased, and tanδ approached or exceeded 1, indicating partial disruption of the network structure and a gradual shift from elastic dominance to viscoelastic dominance.

[0053] (5) Compression performance The hydrogel was subjected to compression mechanics tests, and its average compressive strength was 53.3±7.8 kPa and its average compressive modulus was 3.4±1.0 kPa.

[0054] Test 3: 1. Cell experiments 1.1 Main Materials and Equipment Materials: Arg-CDs photoinitiated hydrogel, human gingival fibroblasts (hGFs), human bone marrow mesenchymal stem cells (hBMSCs), live / dead cell double staining kit, CCK-8 kit, Transwell chambers and nested 24-well plates, alkaline phosphatase staining kit, human bone marrow mesenchymal stem cell osteogenic induction differentiation medium, Mito Tracker Red mitochondrial red fluorescent probe, Lyso-Tracker Red lysosomal red fluorescent probe, anti-fluorescence quenching blocking agent, TriZol lysis buffer, protein lysis buffer, protease inhibitor, reverse transcription kit, whole cell lysis buffer, BCA protein quantification kit, BMP2 monoclonal antibody, WNT4, P2RX1, P2RX5, CDH1, ADCY4, CDK1, RRM2 polyclonal antibody, GDF5, anti-rabbit IgG-HRP secondary antibody, ECL chemiluminescent substrate.

[0055] Equipment: Inverted fluorescence microscope, flow cytometer, laser confocal microscope, real-time quantitative PCR instrument, gel imaging system.

[0056] 1.2 Live / Dead Cell Staining Human bone marrow mesenchymal stem cells (hBMSCs) and human gingival fibroblasts (hGFs) from passages 3 to 5 were selected and cultured in DMEM / F12 (hBMSCs) or DMEM (hGFs) containing 10% fetal bovine serum and 1% penicillin / streptomycin, respectively. Treatment medium containing arginine carbon dots (Arg-CDs) was prepared at final concentrations of 0 (control), 62.5, 125, 250, and 500 μg / mL. Cells were seeded in 24-well plates and cultured until adherence and the logarithmic growth phase. The medium was then replaced with the aforementioned treatment medium and incubated for 24 h. After washing with PBS, Calcein-AM / PI double staining working solution was added and incubated at 37 °C in the dark for approximately 30 min. Following gentle washing with PBS, green (live cells) and red (dead cells) fluorescence images were acquired under an inverted fluorescence microscope. The number of live / dead cells was counted and the survival rate was calculated using image analysis software. Three parallel wells were set up for each group.

[0057] The test results are shown in Figure 2 ,from Figure 2 It can be seen that within the Arg-CDs range of 62.5~500 μg / mL, almost no PI-positive dead cell signals were observed in any of the hBMSCs groups, and the viable cell rate can be considered as... ≈100%; a small number of dead cells appeared at hGFs concentrations of 250–500 μg / mL, but the overall survival rate remained >90%. Furthermore, there was no significant difference between the two high concentrations (p>0.05). These results indicate that Arg-CDs generally exhibit good in vitro biocompatibility within the aforementioned concentration range.

[0058] 1.3 Evaluation of CCK-8 Cytotoxicity / Proliferation Third-generation hBMSCs and hGFs were seeded in 96-well plates. Arg-CDs treatment concentrations were consistent with those in Experiment 1 (0, 62.5, 125, 250, 500 μg / mL). After incubation for 24 h and 48 h, respectively, fresh medium containing CCK-8 was added for further incubation at appropriate times. Absorbance (OD) was measured at 450 nm. Background subtraction was performed using blank wells, and the control group (0 μg / mL) was normalized to 100% to calculate relative cell viability for each group. Three parallel wells were set up for each group. The experimental results are shown below. Figure 3 ,in Figure 3 Figure A shows the effect of Arg-CDs on hBMSCs cell viability under CCK8 assay, and Figure B shows the effect of Arg-CDs on hGFs cell viability under CCK8 assay. Figure 3It was found that Arg-CDs selectively regulated two types of cells. Specifically, for hBMSCs, cell viability was increased at concentrations of 62.5–250 μg / mL, reaching approximately 110–130% of the control at 24 h, and maintained at a significantly higher level (approximately ~150%) at 48 h. At 500 μg / mL, cell viability was slightly lower than the control, but the difference was not significant.

[0059] hGFs: Within the range of 62.5~500 μg / mL, proliferation / activity decreased at both 24 h and 48 h, with the overall decrease being approximately 70~90% of the control, and no significant difference was observed between different concentrations.

[0060] 1.4 Cell scratch migration assay (inhibited proliferation state) Third-generation hBMSCs and hGFs were seeded in 24-well plates and cultured until the monolayer confluence was approximately 90%. After pretreatment with low-serum medium containing proliferation-inhibiting conditions, straight scratches were made on the monolayer cells using a sterile pipette tip. After washing with PBS to remove free cells, the cells were further cultured in medium containing Arg-CDs (0, 62.5, 125, 250, 500 μg / mL). Images were acquired in the same field of view at 0, 6, 12, and 24 h, and changes in scratch width or area were measured. Migration rates were calculated and normalized to 0 h for comparison of differences between groups. Each group had ≥3 parallel wells.

[0061] The experimental results for hBMSCs and hGFs are shown in the following figures. Figures 4-5 ,from Figures 4-5 It was found that hBMSCs:Arg-CDs significantly promoted migration / healing at concentrations of 62.5–250 μg / mL. At 12 h, the healing rates at 62.5 and 125 μg / mL were significantly higher than the control (p<0.001, p<0.01); at 24 h, the rates at 62.5, 125, and 250 μg / mL were all significantly higher than the control (p<0.0001, p<0.0001, p<0.05, respectively). The promoting effect disappeared in the 500 μg / mL group.

[0062] hGFs: There was no significant difference between 6 h and 12 h; at 24 h, the healing rate of each Arg-CDs concentration group was significantly lower than that of the control (p<0.01~0.0001), and showed a relatively independent inhibitory trend.

[0063] 1.5 Transwell migration / chemotaxis experiments Cells were pretreated under low serum conditions to reduce proliferation interference, digested, and resuspended in low serum-free Arg-CD medium with adjusted cell density. Transwell chambers with approximately 8 μm pores were used: cell suspension was added to the upper chamber, and complete medium containing Arg-CDs (0, 62.5, 125, 250, 500 μg / mL) was added to the lower chamber to create a concentration gradient. Incubation was performed at 37 °C and 5% CO2 for approximately 4 h. After incubation, unmigrated cells from the upper chamber were removed, and migrating cells on the lower membrane surface were fixed and stained. Microscopic imaging and cell counting were performed to evaluate migration ability. Three parallel wells were used for each group.

[0064] Transwell migration results are shown below Figures 6-7 The results were compared with the migration numbers normalized to 100%. Figure 6 ,in Figure 6 In the diagram, C represents the effect of different concentrations of Arg-CDs on the wound healing rate of hBMSCs cells, and D represents the effect of different concentrations of Arg-CDs on the wound healing rate of hGFs cells. Figure 7 In the diagram, A represents the effect of Arg-CDs on hBMSCs and hGFs within the concentration gradient range, B represents the effect of Arg-CDs on the relative mobility of hBMSCs within the concentration gradient range, and C represents the effect of Arg-CDs on the relative mobility of hGFs within the concentration gradient range. Figures 6-7 The results showed that: hBMSCs migration was significantly increased in the lower chamber when Arg-CDs were present at concentrations of 125 and 250 μg / mL (p<0.001), while the migration was similar to that of the control at 500 μg / mL. hGFs migration decreased in all concentration groups, with an overall migration rate of approximately 59-87% of the control; the differences between 250 μg / mL (p<0.05) and 500 μg / mL (p<0.01) were significant.

[0065] Overall, consistent with the scratch assay, Arg-CDs promote the migration of hBMSCs and inhibit the migration of hGFs.

[0066] 1.6 ALP staining (early osteogenic differentiation) Based on cell compatibility and migration assays, a final Arg-CD concentration of 250 μg / mL was selected for preparing Arg-CD photoinitiated hydrogel membranes. The prepolymer solution was filtered through a 0.22 μm filter and added to a 12-well plate (approximately 200 μL / well) to form a thin layer. The membrane was cured by irradiation with 445 nm visible blue light for approximately 2 min; washed with PBS and equilibrated with culture medium for 1 h. Third- to fifth-generation hBMSCs were then cultured at approximately 2 × 10⁻⁶... 5Cells / mL were seeded on gel membranes (the control group was without gel membranes or Arg-CDs, and the concentration of Arg-CDs in the liquid phase was kept consistent with that in the gel membrane), and cultured in osteogenic induction medium. On day 7, the cells were fixed and stained with ALP. Microscopic imaging was performed, and the staining intensity was semi-quantitatively compared using image software.

[0067] Early osteogenic ALP results are shown in Figure 8 ,in Figure 8 In the image, A represents the ALP staining pattern of arginine carbon dot photoinitiated hydrogel promoting early osteogenic differentiation of hBMSCs, and B is a semi-quantitative statistical diagram of ALP staining. On day 7 of osteogenic induction, ALP staining in the Arg-CDs photoinitiated hydrogel group was significantly enhanced. The semi-quantitative results showed that the staining intensity was about 1.5–2 times that of the control (p<0.05), suggesting that the hydrogel promotes early osteogenic differentiation of hBMSCs.

[0068] 1.7 Alizarin Red staining of ARS (intermediate to late mineralization) The preparation, pretreatment, and cell seeding methods for the gel membrane were the same as in Experiment 5. hBMSCs were cultured in osteogenic induction medium and fixed on days 14 and 21. They were then stained with 0.1% Alizarin Red S (pH approximately 4.1–4.3), thoroughly washed to remove background, and then imaged under a microscope. The area of ​​mineralized nodules and staining intensity were semi-quantitatively compared using image analysis software to evaluate differences in mineralization capacity.

[0069] The results of the mid-to-late stage mineralization ARS are shown in Figure 9 ,in Figure 9 Image A represents the ARS staining map of arginine carbon dot photoinitiated hydrogel promoting mid-to-late-stage osteogenic differentiation of hBMSCs, and image B is a semi-quantitative statistical map of ARS staining. The Arg-CDs photoinitiated hydrogel group showed large-area calcification and mineralized nodules by day 14; semi-quantitative analysis showed a significantly higher level on day 14 compared to the control group (p<0.001). Both groups showed increases by day 21, but the experimental group remained significantly higher than the control group (p<0.01), and the control group's level on day 21 was still lower than the experimental group's level on day 14. These results indicate that the hydrogel can accelerate and enhance the formation of mineralized nodules.

[0070] 1.8 Cell Cycle Flow Cytometry Analysis hBMSCs and hGFs were cultured for 24 h in the control and material treatment groups, respectively. Cells were then collected, washed with PBS, and single-cell suspensions were prepared. Cells were fixed overnight at 4 °C with 70% ethanol. Before detection, cells were treated with RNase and stained with PI. After filtering to remove cell aggregates, cells were detected by flow cytometry (488 nm excitation). Flow cytometry analysis software was used to fit and calculate the G0 / G1, S, and G2 / M phase ratios to evaluate the effect of the materials on cell cycle progression. Three replicates were set up for each group.

[0071] Test results are available Figure 10 ,in Figure 10 In diagram A, arginine carbon dot hydrogels are used to influence the cell cycle distribution of hBMSCs and hGFs. Diagram B shows the changes in the cell cycle distribution of hBMSCs under the influence of arginine carbon dot hydrogels. Diagram C shows the changes in the cell cycle distribution of hGFs under the influence of arginine carbon dot hydrogels. Figure 10 The results showed that Arg-CDs photoinitiated hydrogels exerted opposite regulatory effects on the two cell types. Specifically, in hBMSCs, the G0 / G1 ratio significantly decreased (p<0.0001), the S phase increased (p<0.001), and the G2 / M ratio increased (p<0.0001), indicating accelerated cell cycle progression and enhanced proliferation. In hGFs, the G0 / G1 ratio significantly increased (p<0.0001), the S phase significantly decreased (p<0.0001), and there was an accompanying change in the G2 / M ratio (p<0.0001), suggesting suppressed G1→S transition and restricted cell cycle progression.

[0072] 1.9 Confocal observation of Arg-CDs entry into cells and subcellular localization hBMSCs and hGFs were seeded in 24-well plates containing climbing slides. When the cell confluence reached approximately 70%–80%, the medium was replaced with complete medium containing Arg-CDs and incubated for approximately 2 hours. Subsequently, mitochondrial or lysosomal fluorescent probes were added and incubated for another 25–30 minutes. After fixation and mounting, Arg-CD autofluorescence and probe signals were acquired using laser confocal microscopy, and colocalization analysis was performed to assess the cellular and subcellular distribution characteristics of Arg-CDs.

[0073] Test results are available Figures 11-12 ,in Figure 11 This is a map showing the colocalization of Arg-CDs and mitochondria in hBMSCs and hGFs. Figure 12 This is a colocalization map of Arg-CDs and lysosomes in hBMSCs and hGFs. From... Figures 11-12 It can be seen that Arg-CDs autofluorescence signals can be detected in both types of cells, mainly distributed in the cytoplasm, with weak signals visible in the nuclear region; there is obvious colocalization with mitochondrial and lysosomal probes, suggesting that Arg-CDs can efficiently enter cells and associate with key organelles.

[0074] 1.10 Reference Transcriptomics Detection hBMSCs / hGFs cultured in Arg-CDs photoinitiated hydrogel membranes served as the experimental group, while conventionally cultured cells served as the control group. After cells achieved good adhesion and near-confluence, they were washed with PBS, lysed with TRIzol, and the lysate was collected and stored at -80°C. RNA extraction and quality control were performed by a testing institution (cells meeting concentration and integrity standards were used for library construction). Reference transcriptome sequencing and differential gene analysis were conducted. Results are shown below. Figures 13-16 ,in Figure 13Transcriptomic quality control images of two cell samples. Figure 14 Volcano plot showing differential gene expression in two cell groups under Arg-CDs photoinitiated hydrogel treatment. Figure 15 GO analysis diagram for differential gene enrichment. Figure 16 KEGG analysis diagram for differential gene enrichment.

[0075] 1.11 DIA Proteomics Detection Cells from both the experimental and control groups were collected at predetermined time points. After washing with PBS, cells were scraped, centrifuged, and the precipitate was collected. The samples were then flash-frozen in liquid nitrogen and stored at -80 °C. Samples underwent lysis, sonication, and centrifugation to collect the supernatant. After protein quantification and electrophoresis integrity control, samples were sent for DIA quantitative proteomics detection and differential protein analysis. Results are shown below. Figures 17-20 ,in Figure 17 These are proteomic quality control images of two cell samples, among which... Figure 18 Volcano plot showing the differential protein expression in two cell groups under Arg-CDs photoinitiated hydrogel treatment. Figure 19 GO analysis plot for differentially expressed proteins. Figure 20 KEGG analysis diagram for differential protein enrichment.

[0076] 1.12 qPCR Validation Based on transcriptome differential results, representative target genes were screened (hBMSCs focused on osteogenic and ion / Ca). 2+ Related signals and adhesion migration; hGFs focus on genes related to cell cycle and stress / repair. Cells were treated in groups, and total RNA was extracted and reverse transcribed to obtain cDNA. qPCR was performed using the SYBR Green assay, with GAPDH as an internal control. Relative expression levels were calculated as 2^-ΔΔCt (control group normalized to 1). Each sample was divided into three replicate wells, and a template-free control was included.

[0077] Test results are available Figures 21-22 ,from Figures 21-22 It can be seen that the qPCR results are consistent with the transcriptome trend, and hBMSCs are associated with osteogenic, migration, and calcium metabolism. 2+ / Purinergic signaling-related genes (such as BMP2, WNT4, AREG, ADORA2A, SIPA1L2, P2RX1, SLC8A2, etc.) were significantly upregulated, while adhesion / receptor-related genes (such as CDH1, P2RX5, etc.) were downregulated (most p<0.001).

[0078] hGFs: Cell cycle and DNA replication-related genes (such as CDK1, RRM2, GTSE1, BUB1B, etc.) were significantly downregulated; repair / regulation and stress defense-related genes (such as GDF5, INHBB, MT1G, MT1X, etc.) were upregulated, and IL21R was downregulated (most p<0.001).

[0079] 1.13 Western blot validation Based on proteomics results, representative differentially expressed proteins (hBMSCs, focusing on osteogenic signals and ion channels / Ca) were screened. 2+ -cAMP-related and adhesion-related; hGFs focus on cell cycle and stress / repair). After cell collection, total protein was extracted and quantified, separated by SDS-PAGE electrophoresis, transferred to a membrane, blocked, incubated with primary antibody, and then subjected to HRP secondary antibody for colorimetric imaging. Grayscale analysis was used and normalized with GAPDH to calculate the relative expression level of the target protein (normalized to 1 for the control group), and differences between groups were compared.

[0080] Figure 23 The images show the Western blot validation results and semi-quantitative statistical plots for hBMSCs cells. Figure 24 for Figure 24 These are the Western blot validation results and semi-quantitative statistical graphs of hGFs cells, from... Figures 23-24 The results showed that protein level validation was consistent with omics findings. In hBMSCs, BMP2 and WNT4 were upregulated (p<0.001); P2RX1 and ADCY4 were upregulated (p<0.01); P2RX5 and CDH1 were decreased (p<0.01), supporting the pattern of "enhanced Ca-cAMP-related axis and osteogenic signaling, and moderate downregulation of adhesion." In hGFs, CDK1 and RRM2 were significantly downregulated (p<0.001); GDF5 was upregulated (p<0.05), supporting enhanced cell cycle inhibition and repair regulatory signals.

[0081] 1.14 Overview of Multi-omics Results Transcriptomics and proteomics revealed cell type differences in the molecular responses generated by Arg-CD photoinduced hydrogels. Specifically, in hBMSCs, differentially expressed genes and proteins were enriched in pathways related to cell activity / regeneration (such as Ca signaling, Rap1, Hippo / Wnt / BMP, as well as nucleotide metabolism, DNA replication / repair, transcription, and splicing), generally indicating enhanced proliferation and osteogenic-related programs. In hGFs, differentially expressed genes were enriched in pathways related to negative regulation of cell cycle / mitosis and p53 / Cell cycle; accompanied by stress defense and cytokine network remodeling, generally indicating a "slowdown" of proliferation and migration drives, while homeostasis / repair-related programs were enhanced.

[0082] 2. Animal experiments: 2.1 Main Materials and Equipment Materials: Arg-CDs photoinitiated hydrogel, iodine tincture, sodium pentobarbital, Sutacetin 50, veterinary lidocaine hydrochloride, 4% paraformaldehyde general tissue fixative, EDTA decalcification solution, H&E staining kit, Masson staining kit, Sirius red staining kit.

[0083] Equipment: implantation machine, bone harvesting drill, stainless steel surgical instrument set for animal experiments, stepless dimming flashlight, Micro-CT, upright microscope, imaging system.

[0084] 2.2 In vivo biosafety evaluation Healthy adult New Zealand white rabbits were selected and randomly divided into experimental and control groups after acclimatization. The experimental group was administered a pre-prepared Arg-CDs photoinitiated hydrogel suspension orally by gavage, while the control group was administered an equal volume of physiological saline by gavage. The dosage was 1 mL / kg. Animals were fasted appropriately before gavage but had free access to water. Twenty-four hours after administration, the animals were sacrificed, and major organs such as the liver, heart, spleen, lungs, and kidneys were harvested. After washing with PBS, the organs were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to approximately 4 μm for H&E staining. The histological morphology of the organs was observed under a light microscope, focusing on assessing the presence of pathological changes such as cell swelling / attachment, necrosis, hemorrhage / edema, and inflammatory cell infiltration to determine the potential systemic toxicity risk after oral exposure.

[0085] Establishment and grouping of rabbit mandibular alveolar bone defect model Healthy adult New Zealand white rabbits (male or female, weighing approximately 3 kg) were selected, and a standardized alveolar bone defect model was established on the buccal side of both mandibles. After general anesthesia and local infiltration anesthesia in the surgical area, the mandibular region was incised and flapped to expose the bone surface. Two cylindrical bone defects were prepared on each mandible using a trephine, with a diameter of 3 mm, a depth of 2 mm, and a spacing of approximately 5 mm between the defects, resulting in a total of 4 defect sites per animal. Different defect sites of the same animal were randomly assigned to treatment groups, and the following groups were set up: (1) Blank control group: only defects were established, without implantation of materials; (2) Bone graft negative control group: bone substitute material was filled into the defect, without covering with a membrane; (3) Classic GBR positive control group: bone substitute material was filled into the defect and covered with a barrier membrane, and sutured and fixed if necessary; (4) Experimental group: bone substitute material was filled into the defect and pre-prepared Arg-CDs photoinitiated hydrogel prepolymer solution was added, and the gel was cured in situ by irradiation with 445 nm blue light for about 2 min. Each group was fully filled according to the defect volume, and postoperative layered suturing and routine postoperative care were performed. Grouping details are as follows: Figure 1 .

[0086] 2.3 Sampling Micro-CT Scanning and Three-Dimensional Quantitative Analysis Based on the bone formation time window, animals were sacrificed and samples were collected at 2 and 4 weeks post-surgery. When collecting mandibular bone specimens, the defect area and surrounding bone tissue were preserved. After rinsing with PBS, the specimens were fixed in 4% paraformaldehyde for approximately 72 hours. After fixation, the samples were transferred to 70% ethanol for preservation.

[0087] Before scanning, mandibular specimens fixed and preserved in ethanol were rinsed with PBS to remove surface air bubbles and placed in a Micro-CT system for in vitro high-resolution scanning. After obtaining the raw data, cross-sectional images were reconstructed and DICOM data were exported. Regions of interest (ROIs) in the defect area were selected using 3D reconstruction software, thresholding and 3D model reconstruction were performed, and bone regeneration-related structural parameters (such as new bone volume / bone volume fraction, trabecular bone structure parameters, etc.) were calculated for comparison between different treatment groups.

[0088] 2.4 Decalcification, Paraffin Embedding and Sectioning After micro-CT, bone blocks containing the defect area and adjacent normal bone were excised and decalcified with EDTA (the decalcification solution was changed regularly) until there was no significant resistance when punctured. Subsequently, the bone was dehydrated, cleared, and embedded in paraffin according to standard histological procedures. After uniformly oriented the sections, approximately 4 μm serial sections were prepared for histological staining analysis.

[0089] 2.5 H&E staining After dewaxing and hydration, sections were stained with H&E, dehydrated, cleared, and mounted. Microscopic observation of histological changes in the defect area, including new bone formation, fibrous tissue proliferation, and inflammatory cell infiltration, was used to evaluate the repair quality and soft tissue response of different treatment groups.

[0090] 2.6 Masson trichrome staining After dewaxing and hydration, the sections were stained with Masson's trichrome staining. The distribution and proportion of collagen fibers / fibrous connective tissue and newly formed bone matrix in the defect area were observed and compared under a microscope to assess the degree of fibrosis and the formation of bone matrix.

[0091] 2.7 Sirius Red staining After dewaxing and hydration, the sections were stained with Sirius Red and mounted. The birefringence signal of collagen fibers was observed under a polarized light microscope to compare the differences in collagen deposition, remodeling and fiber orientation in different treatment groups at the defect area and soft tissue interface.

[0092] 3. Antibacterial test: 3.1 Main materials and equipment: Arg-CDs and Arg-CDs photoinitiated hydrogels, Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus sanguinis, bacterial incubator, and bacterial concentration turbidimeter.

[0093] 3.2 MIC / MBC Detection Bacterial suspensions were prepared and cultured to the logarithmic growth phase under corresponding culture media and gas conditions: Staphylococcus aureus and Streptococcus sanguinis were cultured at 37 °C under conventional aerobic / 5% CO2 conditions; Porphyromonas gingivalis was cultured under strictly anaerobic conditions at 37 °C. The logarithmic growth phase suspensions were diluted with sterile culture medium to achieve an initial bacterial concentration of approximately 1 × 10⁻⁶. 6 CFU / mL, the bacterial solution was inoculated into Arg-CDs medium with concentration gradient at a volume ratio of 1:1, and cultured for 24 h under the corresponding bacterial culture conditions.

[0094] The OD values ​​of each well were measured at 600 nm using a bacterial turbidimeter. After subtracting the background control from the OD values ​​of each well, the values ​​were compared with the growth control. MIC determination: the OD growth after background subtraction showed an inhibition rate of ≥90%–100% relative to the growth control (preferably ≥95%). MBC detection (plate double-cropping method). Samples were taken from the MIC wells and wells of higher concentrations (as well as adjacent wells of one lower concentration for comparison) for replication: 10–100 μL of culture medium was taken from each selected well, serially diluted, and then spread onto the corresponding solid medium plates. MBC determination: The lowest concentration of the test solution at which no visible colony growth was observed after plate replication was defined as the MBC. The results are shown in Table 1.

[0095] Table 1 Arg-CDs MIC / MBC Measurement

[0096] 3.3 Arg-CDs photoinitiated hydrogel antibacterial loop test Prepare agar medium, make 5 mm pores in it using a punch, add hydrogel and allow it to solidify, then add the initial bacterial concentration of approximately 1 × 10⁻⁶ cells. 6 Three types of bacteria at CFU / mL were inoculated into their respective culture media and cultured under appropriate conditions for 24 h before observation. Results are shown below. Figure 25 .

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing arginine carbon dot photo-initiated hydrogel pre-polymer solution, characterized in that, Includes the following steps: Arginine powder is heat-treated to obtain intermediate powder; the heat treatment includes a sequential heating process and a calcination process at a calcination temperature; the calcination is carried out under ventilation conditions; The aqueous dispersion of the intermediate powder was allowed to stand and centrifuged, and the resulting supernatant was dialyzed and dried sequentially to obtain Arg-CDs powder. A DPBS solution of methacrylamide gelatin, Arg-CDs powder, and a photoinitiator system were mixed to obtain an arginine carbon dot photoinitiated hydrogel prepolymer; the photoinitiator system included triethanolamine, N-vinylcaprolactam, and riboflavin.

2. The production method according to claim 1, wherein The heating process involves heating to the calcination temperature at a rate of 3.5~4.5 °C / min.

3. The preparation method according to claim 1 or 2, characterized in that, The calcination temperature is 235~245 ℃, and the calcination time is 175~185 min.

4. The preparation method according to claim 1, characterized in that, The Arg-CDs powder has a particle size of 1.74 ± 0.31 nm.

5. The preparation method according to claim 1, characterized in that, The concentration of the intermediate powder in the aqueous dispersion is 0.1~0.2 g / mL; the standing time is 10~15 min; and the molecular weight cutoff of the dialysis bag is 1000 Da.

6. The preparation method according to claim 1, characterized in that, The concentration of triethanolamine in the Arg-CDs photoinitiated hydrogel prepolymer solution is 145~155 mM; the concentration of N-vinylcaprolactam in the Arg-CDs photoinitiated hydrogel prepolymer solution is 155~165 mM; and the concentration of riboflavin in the Arg-CDs photoinitiated hydrogel prepolymer solution is 0.61~0.71 mM.

7. The preparation method according to claim 1, characterized in that, The mass concentration of methacrylated gelatin in the DPBS solution of the methacrylated gelatin is 14.5-15.5%.

8. The preparation method according to claim 1, characterized in that, The concentration of Arg-CDs in the Arg-CDs photoinitiated hydrogel prepolymer solution is 230~270 mg / mL.

9. The arginine carbon dot photoinitiated hydrogel prepolymer prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the arginine carbon dot photoinitiated hydrogel prepolymer prepared by the preparation method according to any one of claims 1 to 8, or the arginine carbon dot photoinitiated hydrogel prepolymer according to claim 9, in guided bone regeneration, characterized in that, The method of guiding bone regeneration involves injecting arginine carbon dot photoinitiated hydrogel prepolymer into the bone defect site and then irradiating it with blue light to obtain an arginine carbon dot photoinitiated hydrogel scaffold, which releases active ingredients.

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