| Part 1:The Periodontal Regeneration Potential of Decellularized Periodontal Ligament Cell SheetsObjective:This study aims to construct decellularized periodontal ligament cell sheets,PCL/GE electrospun nanofibers and 15d-PGJ2 nanoparticles,and to investigate the regeneration ability of this complex in a rat model of periodontal bone defectsMethods:Primary human periodontal ligament cells were cultured by tissue block-enzyme digestion method.And the cell source was identified by HE staining,immunohistochemical staining and ALP staining.The periodontal ligament cell sheets were induced by ascorbic acid and the decellularization was implemented by static chemical method.Then,the change of the expression of collagen I,fibronectin was assessed by immunofluorescence.The content of DNA,collagen and growth factos(bFGF,HGF and VEGF)was measured though PicoGreen kits,hydroxyproline kits and ELISA kits,respectively.Besides,scanning electron microscope was used to evaluate the structural integrity of cell sheets after decellularization.In vitro,human periodontal ligament stem cells were inoculated into the decellularized periodontal ligament cell sheets.After recellularization,the expression and distribution of fibronectin were detected by immunofluorescence and the osteogenic induction potential were detected by ALP.A model of periodontal bone defects was constructed in rat mandibular bone(length × height × depth,4 mm×2 mm×1mm).The gelatin sponge containing different doses(0,1.5 mg,2.5?g,3.5?g)of 15d-PGJ2 nanoparticles was implanted.Soft tissue around the defects was collected on 3,7 and 14 days after surgery.The expressions of inflammatory factors IL-1?,IL-6 and TNF-? were detected by Western blot to investigate the anti-inflammatory effect of 15d-PGJ2 nanoparticles on periodontal bone defects in rats.The same periodontal bone defects model of rat was constructed again The defects were divided into three groups,including control group,decellularized cell sheets group and combined PCL/GE decellularized cell sheets group.The only difference between these groups was the decellularized cell sheets type.Gelatin sponges containing 1.5?g of 15d-pgj2 nanoparticles were implanted in all three groups.The mandibular bone was collected in 2 and 4 weeks after surgery to evaluate the healing of defects by Micro-CTResults:(1)Spindle primary human periodontal ligament cells were seen crawling out after 5 days of tissue block-enzyme digestion culture.Immunohistochemistry showed positive staining of vimentin,negative staining of cytokeratin,and positive staining of ALP.(2)Ascorbic acid successfully induced the formation of human periodontal ligament cell sheets.And decellularized periodontal ligament cell sheets was successfully constructed by static chemical method.After decellularization,the DNA content was removed by 96.6%.The total collagen content of decellularized cell sheets was 113.6±1.804 mg/mL and increased by 6 times compared to cell sheets.Before decellularization,the contents of bFGF,HGF and VEGF were 8.494 ±3.608 pg/mL,2590 ± 2.374 pg/mL and 231.3 ± 48.71 pg/mL,respectively.After decellularization,they were reduced to 0.344 ± 0.875 pg/mL(p=0.1062),594.5 ± 47.4 pg/mL(p<0.0001)and 30.97 ± 5.43 pg/mL(p<0.05),respectively.Immunofluorescence staining and scanning electron microscopy showed the structure of decellularized cell sheets was still intact.Collagen I and fibronectin were expressed and distributed similarly between decellularized cell sheets and cell sheets(3)The allogeneic human periodontal ligament cells were successfully inoculated into the decellularized periodontal ligament cell sheets.After 14 days'culture,ALP staining of cells inoculated on cell sheets was positive in both normal medium and osteogenic induction medium,while ALP staining of cells inoculated at the bottom of plate was positive only in osteogenic induction medium(4)A rat model of periodontal bone defects was successfully constructed.Western blot showed that the expressions of IL-1? and TNF-? in the mandibular bone defects treated with low dose of 15d-PGJ2 nanoparticles decreased compared with the control group on 3 days after surgery(p<0.05),and there was no significant difference in IL-6 between the four groups.The expression of IL-1?,IL-6 and TNF-? in the four groups decreased on 7 days after surgery compared with that on 3 days,with no significant difference between the groups(p>0.05).Those still decreased on 14 days after surgery,compared with that on 7 days,with no significant difference between the groups(p>0.05)Micro-CT images showed that defects in decellularized cell sheets group began to heal at 2 weeks,while that in other groups did not heal.At 4 weeks,part of the root surface of bone defects in the control group was still exposed,and the defects in the other two groups were almost healed.At 2 weeks,the bone volume fraction of the control group,decellularized cell sheets group and combined PCL/GE decellularized cell sheets group were 26.15%,52.94%and 27.23%,and increased to 53.23%,62.21%and 60.39%at 4 weeks,respectivelyConclusion:(1)Human periodontal ligament cells were successfully cultured,and decellularized periodontal ligament cell sheets were constructed.In vivo and in vitro experiments verified the role of decellularized periodontal ligament cell sheets in promoting the regeneration of periodontal bone defects(2)Low-dose 15d-PGJ2 nanoparticles have a good local anti-inflammatory effectPart 2:The Efficacy of Augmentation of Keratinized Tissue Around Dental Implant with Free Gingival GraftObjective:The purpose of this study was to investigate the efficacy of augmentation of keratinized tissue around dental implant with free gingival graft in the posterior tooth region,and to evaluate the peri-implant condition,aesthetics and patients' satisfactionMethods:This study is a case-series study.The participants of this study were the patients who received implantation in the posterior tooth area.And the width of keratinized tissue around implant was within 2 mm.Patients with PPD no more than 5 mm,FMBS less than 15%,and the average GI of implant no more than 2 were included After excluding contraindications,free gingival flap surgery was performed.Then,regular follow-up visits will be conducted in 2 weeks,2 months and 6 months.The clinical intraoral images,clinical parameters and visual analogue scales were collected and analyzed.The clinical parameters included keratinized tissue width and thickness,gingival recession depth and width,GI,PPD,and aesthetic evaluation index to assess the condition of implant.And the patient-centered parameters included pain VAS and satisfaction VAS to evaluate patient satisfactionResults:In this study,8 patients and 11 implants were included.The keratinized tissues with width of 4.5±1.60 mm and thickness of 1.8±0.70 mm were obtained at 6 months after the surgery,and all the PPD and GI were normal.None of patients suffered peri-implant mucositis or peri-implantitis.Only one implant developed gingival recession at 6 months after surgery.The average aesthetic evaluation index was 4.5.The average VAS score of pain was 4.7 and the average VAS score of satisfaction was 9.4(2 months)and 9.7(6 months)Conclusion:Free gingival flap is efficient in augmentation of keratinized tissue around dental implant.FGG chould obtain sufficient stable peri-implant keratinized tissues and maintain the health condition of peri-implant.Within limits,the aesthetic effects of FGG is satisfied and the pain of patients is mild.But further research is needed to evaluate the long-term stability of free gingival flap in augmentation of keratinized tissue around dental implant. |
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