| Periodontitis is a highly prevalent inflammatory disease characterized by the formation of periodontal pockets and the resorption of alveolar bone,ultimately leading to tooth loss.In the progression of periodontal disease,inflammatory mediators they induce cancirculatory system,potentially triggering systemic immune responses throughout the body.This type of systemic immunological activation is considered to be associated with an increased risk of various systemic diseases.Consequently,periodontitis has come to be recognized as a key factor affecting overall health.Currently,primary treatment strategies include supragingival plaque removal,subgingival curettage,and scaling and root planing（SRP）.These interventions are designed to significantly reduce the accumulation of dental plaque and calculus,thereby improving the overall periodontal health status.However,when dealing with deep periodontal pockets,furcation involvement,and complex subgingival defects in patients with severe periodontitis,it’s challenging for instruments to thoroughly clean these deep structures.Moreover,susceptible bacteria or microorganisms from other areas in the mouth can recolonize the deep pockets,leading to disease recurrence.Therefore,periodontal initial therapy needs to be complemented by certain pharmacological treatments to achieve better outcomes,which depend on further research into the pathogenesis of periodontal diseases.Current studies indicate that many inflammatory diseases are closely associated with ROS（reactive oxygen species）,and the oxidative stress that accompanies the inflammatory process can exacerbate tissue damage.Hence,antioxidants represent an important strategy in treating inflammation-related diseases associated with oxidative stress.Melatonin（MT）,an antioxidant with multiple physiological functions,not only regulates circadian rhythms,improves sleep quality,and has anti-aging effects but also enhances the body’s antioxidant capacity by efficiently scavenging free radicals and increasing the activity of antioxidant enzymes.However,due to melatonin’s poor water solubility and low bioavailability,its further application in the field of periodontics is limited.There is an urgent need for new delivery systems or processing methods to optimize or improve its use.In recent years,nanomaterials with ROS scavenging capabilities have rapidly advanced in the treatment of periodontal diseases.Among them,carbon dots have attracted widespread attention in academia due to their good water solubility,low toxicity,and high biocompatibility.Additionally,their advantages of having a wide range of raw material sources and low cost make them the materials of choice in the field of bio-nanotechnology.Against this backdrop,in our study,we synthesized melatonin-derived carbon dots with free radical scavenging abilities,preliminarily assessed their biocompatibility and antioxidant capacity,observed their anti-inflammatory and antioxidant effects both in vitro and in vivo,and further explored their potential to inhibit bone resorption and underlying mechanisms in a periodontitis model.This research aims to provide new experimental evidence and therapeutic strategies for the treatment of periodontal and other inflammatory diseases.In chapter two,we used melatonin as a precursor to synthesize Melatonin-derived carbon dots（MT-CDs）via a hydrothermal method.After purification and lyophilization,the MT-CDs were characterized and then evaluated for their in vitro and in vivo biosafety.The MT-CDs appeared as well-dispersed dots with a lattice spacing of 0.18nm and an average diameter of 2.5 nm.Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analyses indicate that MT-CDs are primarily composed of elements such as carbon,nitrogen,and oxygen,and are rich in functional groups.The total antioxidant capacity and KMn O4 redox reaction experiments confirmed that MT-CDs possessed good antioxidant abilities.The results from the DPPH radical scavenging assay and hydroxyl radical test kit showed that MT-CDs could effectively scavenge free radicals,and the radical scavenging ability was concentration-dependent.In vitro biosafety tests were conducted using MC3T3-E1 and Raw 264.7 cells to assess the cytotoxicity of MT and MT-CDs.CCK-8 results indicated that the toxicity of MT-CDs was significantly lower than that of MT at the same concentration,and within the range of 50μg/m L,MT-CDs did not have a noticeable impact on cell cycle and apoptosis.In chapter three,we assessed the protective effects of MT-CDs on cells under oxidative stress and preliminarily explored regulatory mechanisms on mitochondrial homeostasis.By establishing an H2O2-induced oxidative stress model in MC3T3-E1cells,we observed the recovery function of MT-CDs after addition.The results indicated that MT-CDs could effectively alleviate cell damage and morphological changes caused by oxidative stress,reduce intracellular ROS levels and mitochondrial-derived ROS,and enhance mitochondrial membrane potential.Building on this,we further explored the mechanisms of MT-CDs’antioxidant properties and inhibition of cell apoptosis,examining the gene and protein expression levels of factors related to oxidative stress and apoptosis.The findings revealed that melatonin carbon dots significantly regulated the antioxidant genes and proteins,effectively suppressing the expression of apoptosis-related factors.In chapter four,we investigated the osteogenic and anti-inflammatory capabilities of MT-CDs.Using ALP and ARS staining,we evaluated the ability of MT-CDs to promote osteogenic differentiation under oxidative stress,which showed a positive impact on osteogenic differentiation at different stages.Additionally,MT-CDs demonstrated a promotional effect on osteogenic factors BMP-2 and Runx2 under oxidative stress.To further assess the anti-inflammatory effects of MT-CDs,we used lipopolysaccharide-induced Raw 264.7 cells as an inflammatory cell model and examined the impact of MT-CDs on relevant inflammatory factors.Real-time quantitative PCR and immunofluorescence assays revealed that MT-CDs could effectively suppressed the expression of inflammatory factors at both the gene and protein levels,exhibiting good anti-inflammatory effects.In chapter five,we evaluated the therapeutic effects and in vivo biosafety of MT-CDs by constructing a small animal periodontitis model.The experiment used 5-0 silk suture ligation to induce periodontitis in mice,assessing the inhibitory effect of MT-CDs on bone resorption.Micro-CT results showed that MT-CDs could effectively inhibit alveolar bone resorption,with bone tissue parameters significantly improved compared to the periodontitis group.Histological examination also indicated that MT-CDs could effectively reduce inflammatory responses in periodontal tissues,alleviate tissue damage,and improve the arrangement of collagen fibers.Immunohistochemical results further proved that MT-CDs could effectively promote the expression of Nrf2 and HO-1 in periodontal tissues while reducing the levels of IL-1βand TNF-α,elucidating the anti-inflammatory and antioxidant mechanisms of MT-CDs in vivo.Moreover,blood routine and serum oxidative stress index results further verified the systemic anti-inflammatory and antioxidant effects of MT-CDs through the bloodstream.Additionally,we conducted a biosafety assessment of vital organs in experimental animals,finding no significant pathological changes among all groups,indicating good in vivo biosafety of MT-CDs.In summary,we synthesized a novel type of carbon dot using melatonin as a precursor that demonstrated anti-inflammatory and antioxidant properties both in vitro and in vivo.The resulting carbon dots exhibited good water solubility,uniform size,and excellent biosafety both in vitro and in vivo.They effectively ameliorated cellular oxidative stress and inhibited apoptosis through the Nrf2/HO-1 axis,and significantly reduced alveolar bone resorption and periodontal tissue damage in mice with periodontitis.This discovery holds significant promise for the development of novel nanomedicines aimed at treating periodontitis and offers empirical support for the clinical management of inflammatory diseases triggered by ROS. |
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