Copper-deposited Diatom-biosilica Enhanced Osteogenic Potential In Periodontal Ligament Stem Cells And Rat Cranium

Objective:The treatment of bone defects,especially the repair of alveolar bone defects caused by periodontitis,is a major challenge for clinicians.Currently,bioactive porous materials for the regeneration of missing bone have been extensively studied in clinical disciplines.For better repair results,the material should not only be bone conductive（to guide new bone growth）,but also have the ability to stimulate osteogenesis（to promote new bone formation）and angiogenesis（to induce blood vessel formation）.In addition,the specificity of the oral environment requires the antibacterial properties of the material as well as the built-in drug delivery capability.Cu²⁺loaded mineralized diatom（Cu-DBs）was synthesized by hydrothermal processing of diatom shell（DBs）.Copper and silicon ions in the form of basic copper silicates have been shown to promote osteogenesis and promote angiogenesis.At the same time,Cu²⁺also has a great antibacterial effect,meeting the need for developing versatile bioactive materials.Therefore,the purpose of our study is to investigate whether Cu-DBs could promote the osteogenic differentiation of human periodontal ligament stem cells（PDLSCs）in vitro and promote the repair of rat skull limit bone defects in vivo.Methods:1.Observation and treatment of the material:The surface properties of DBs and Cu-DBs were observed using scanning electron microscopy.Experimental DBs and Cu-DBs were sterilized by Co60 irradiation.The extract concentration of 2μg/m L DBs and the extract concentration of 1,2,2.5μg/m L Cu-DBs were prepared in vitro.2.Isolation and culture of PDLSCs:Primary PDLSCs were obtained by a limited dilution method,the cells were directly observed by optical microscopy,and second to fourth generation cells were selected for subsequent cell experiments.3.Identification of PDLSCs:Cell monoclonal formation units obtained from PDLSCs clone formation experiments.Osteogenic differentiation of PDLSCs was induced by osteogenic induction solution,and the results of osteogenic differentiation were observed by alizarin red staining.PDLSCs were induced by a lipid-induced solution,and lipid differentiation was observed by oil-red O-staining.PDLSCs marked with different antibody markers（CD29,CD73,CD90,and CD45）were analyzed and identified by flow cytometry.4.Cytotoxicity evaluation:The CCK-8 method was used to detect the effect of DBs and Cu-DBs on PDLSC activity.5.In vitro osteogenesis experiment:the experimental cells were grouped:Control group,2μg/m L DBs（2 DBs）,1μg/m L Cu-DBs（1 Cu-DBs）,2μg/m L Cu-DBs（2 Cu-DBs）,2.5μg/m L Cu-DBs（2.5 Cu-DBs）.The expressions of bone-forming genes alkaline phosphatase（ALP）,Runt-associated transcription factor 2（RUNX-2）,bone sialate protein（BSP）,osteocalcin（OCN）and osteopontin（OPN）were detected by alkaline phosphatase staining,alizarin red staining,and real-time quantitative PCR（q RT-PCR）.The expression of the osteogenic proteins ALP,RUNX-2,and OPN were detected by Western Blot and the effect of Cu-DBs on the osteogenic differentiation of PDLSCs was validated.6.Animal osteogenic experiment:A rat skull limit bone defect model and a full-layer skin incision model were established,and the in vivo osteogenic properties of Cu-DBs were detected by Micro-CT analysis,hematoxylin-eosin（HE）staining,Masson staining and CD31 immunohistochemical analysis.Results:1.Observations of the material:The surface of DBs shows a porous structure,and the surface of Cu-DBs shows a bristle-like structure according to scanning electron microscopy.2.Isolation and culture of PDLSCs:Microscopically,primary cells were observed to emerge from the edges of tissue clumps and take on a fibroblast-like morphology.3.Identification of PDLSCs:Monoclonal colonies were observed microscopically,calcified nodules were observed after alizarin staining,and lipid droplets were observed after oil-red O-staining after lipid induction.Positive expression of mesenchymal stem cell specific markers CD29,CD73 and CD90 and negative expression of leukocyte marker CD45.5.Cytotoxicity evaluation:Cu-DBs did not have a significant effect on cell proliferation and activity compared to the DBs extract and control groups.6.In vitro osteogenesis experiments:alkaline phosphatase staining,alizarine red staining,q RT-PCR and western blotting showed that PDLSCs ALP and calcified nodules were significantly increased upon treatment with Cu-DBs extract,the expression of osteogenic genes and proteins were increased,and the cells showed stronger osteogenic differentiation.7.Animal osteogenic experiment:The Cu-DBs group showed better bone defect repair dynamics compared to the control and DBs groups,with fresh bone growing from the defect edge to the defect center,which was most evident in micro-CT imaging.HE staining was used to assess tissue inflammation.The results showed that after 12 weeks of treatment,the number of inflammatory cells in the Cu-DBs treatment group was significantly lower than in the control and DBs treatment groups,and nearly no vacuolar cells were observed histologically,suggesting the potential of Cu-DBs to inhibit inflammation.At the same time,immunohistochemical experiments confirmed that Cu-DBs promote microangiogenesis and express more CD31 positives.Conclusions:In summary,Cu²⁺loaded copper diatoms can combine the chemical properties of Si⁴⁺and Cu²⁺to promote bone tissue regeneration and angiogenesis in vivo,promote osteogenic differentiation of PDLSCs in vitro,and enhance their osteogenic potential.Combined with the results of previous studies on promoting healing of infected wounds,we can conclude that Cu-DBs are an innovative,safe and effective biomaterial with great potential to improve the efficacy of bone defect repair in periodontitis patients.