Exosomes from dental pulp cells attenuate bone loss in mouse experimental periodontitis.

BACKGROUND AND OBJECTIVE: Exosomes are small vesicles secreted from many cell types. Their biological effects largely depend on their cellular origin and the physiological state of the originating cells. Exosomes secreted by mesenchymal stem cells exert therapeutic effects against multiple diseases and may serve as potential alternatives to stem cell therapies. We previously established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this study, we aimed to investigate the effect of local administration of HHH-DPC exosomes in a mouse model of periodontitis. METHODS: Exosomes purified from HHH-DPCs were subjected to particle size analysis, and expression of exosome markers was confirmed by western blotting. We also confirmed the effect of exosomes on the migration of both HHH-DPCs and mouse osteoblastic MC3T3-E1 cells. A mouse experimental periodontitis model was used to evaluate the effect of exosomes in vivo. The morphology of alveolar bone was assessed by micro-computed tomography (µCT) and histological analysis. The effect of exosomes on osteoclastogenesis was evaluated using a co-culture system. RESULTS: The exosomes purified from HHH-DPCs were homogeneous and had a spherical membrane structure. HHH-DPC exosomes promoted the migration of both human DPCs and mouse osteoblastic cells. The MTT assay showed a positive effect on the proliferation of human DPCs, but not on mouse osteoblastic cells. Treatment with HHH-DPC exosomes did not alter the differentiation of osteoblastic cells. Imaging with µCT revealed that the exosomes suppressed alveolar bone resorption in the mouse model of periodontitis. Although no change was apparent in the dominance of TRAP-positive osteoclast-like cells in decalcified tissue sections upon exosome treatment, HHH-DPC exosomes significantly suppressed osteoclast formation in vitro. CONCLUSIONS: HHH-DPC exosomes stimulated the migration of human DPCs and mouse osteoblastic cells and effectively attenuated bone loss due to periodontitis.

Effects of periodontal treatment on the medical status of patients with type 2 diabetes mellitus: a pilot study.

BACKGROUND: Studies have demonstrated that periodontal disease is associated with the development of systemic complications in patients with type 2 diabetes mellitus (T2DM). The purpose of this pilot study was to investigate which markers among various systemic disease parameters are affected by periodontal treatment in patients with T2DM. METHODS: Twelve patients with T2DM were given oral hygiene instructions and subsequent subgingival scaling and root planing. The periodontal status was recorded, and blood and urine samples were taken to measure various parameters of glucose control and systemic status at baseline and 1 month following the periodontal treatment. Serum concentrations of tumor necrosis factor-α and high-sensitivity C-reactive protein were measured by enzyme-linked immunosorbent assay. RESULTS: After the periodontal treatment, the glycated hemoglobin value was significantly improved. The levels of urinary N-acetyl-ß-D-glucosaminidase and albumin, which are markers of renal dysfunction, also decreased significantly after treatment. Among the parameters measured in serum, the γ-glutamyl transpeptidase level, which is usually interpreted as a marker of liver dysfunction, was significantly reduced. The serum concentrations of tumor necrosis factor-α and high-sensitivity C-reactive protein were also significantly reduced by periodontal treatment. CONCLUSION: Within the limitations of this pilot study, periodontal treatment may be effective not only in improving metabolic control, but also in reducing the risk of diabetic kidney and liver disease in patients with T2DM.

Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients.

We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.

Lysine-specific proteolytic activity responsible for forsythia detaching factor modification.

OBJECTIVES: The objective of the present study was to clarify the lysine-specific proteolytic activity derived from periodontal pathogens responsible for Forsythia detaching factor (FDF) modification. DESIGN: The activity responsible for FDF modification in Tannerella forsythia and Porphyromonas gingivalis were evaluated by colorimetric assay using Ac-Arg-Ala-Lys-p-nitroaniline as a substrate. FDF modification in T. forsythia and P. gingivalis were evaluated by Western blotting using recombinant FDF (rFDF) as a substrate. Furthermore, the activity in GCF of 20 patients with periodontitis and 10 healthy subjects was also evaluated by colorimetric assay. Bacteria in subgingival plaque were detected using polymerase chain reaction. RESULTS: The activity of both bacteria in colorimetric assay were 21.35 unit (P. gingivalis) and 3.61 unit (T. forsythia), respectively. Western blot analysis revealed that P. gingivalis was found to efficiently degrade rFDF and T. forsythia partially cleaved rFDF. The activity in GCF from patients with periodontitis (clinically healthy sites: CH, deep bleeding sites: DB and deep non-bleeding sites: DNB) was significantly higher than those from healthy subjects (healthy sites: H). Among the patients with periodontitis, the activity from CH was significantly lower than those from DB and DNB. T. forsythia was detected in 68.4% of DNB, in 78.4% of DB and in none of CH. P. gingivalis was detected in 63.2% of DNB, in 84.0% of DB and in 10.5% of CH. No bacterium was detected in healthy subjects. CONCLUSION: The lysine-specific proteolytic activity responsible for FDF modification correlates with the presence of major periodontal pathogens.

Modification of forsythia detaching factor by gingival crevicular fluid in periodontitis.

OBJECTIVES: Forsythia detaching factor (FDF) is a virulence factor of Tannerella forsythia detected as a mixture of the 60-kDa form of FDF and the 28-kDa C-terminal fragment (FDFc). The objective of the present study was to clarify the proteolytic activity of gingival crevicular fluid (GCF) from patients with periodontitis and healthy subjects using recombinant FDF (rFDF) as substrate. DESIGN: Eleven patients with periodontitis and 6 healthy subjects were recruited. Modification of rFDF and subsequent production of rFDFc by proteolytic activity of GCF was determined by Western blotting. Proteolytic activity of GCF was evaluated using an Ac-Arg-Ala-Lys-p-nitroaniline substrate. Correlation analysis between two different sets of variables was performed. Variables used in this analysis were proteolytic activity, clinical parameters, relative band density of rFDFc and those of rFDF. RESULTS: Proteolytic activity in GCF was significantly higher in patients with periodontitis than in healthy subjects. Production of rFDFc was determined by treatment of rFDF with GCF from patients with periodontitis and with GCF from healthy subjects. Correlations between clinical parameters and proteolytic activity in GCF were significantly positive. On the other hand, correlations between relative band density of rFDFc or rFDF on Western blot and cleaving activity or clinical parameters were significantly negative. CONCLUSION: The detected extend of GCF-activity generating rFDFc from rFDF and/or even further degrading rFDF correlates with severity of periodontitis.

Hypoxia and reoxygenation augment bone-resorbing factor production from human periodontal ligament cells.

BACKGROUND: Oxygen deficiency caused by occlusal trauma and smoking may be associated with bone resorption in periodontitis. In the present study, the effects of hypoxia and reoxygenation on the production of bone-resorbing factors by cultured human periodontal ligament (PDL) cells were examined. METHODS: Human PDL cells were cultured in 1% O(2) (hypoxia), 20% O(2) (normal oxygen tension [normoxia]), or an oxygen concentration that went from 1% to 20% (reoxygenation). The concentrations of bone-resorbing factors, i.e., vascular endothelial growth factor (VEGF), interleukin (IL)-6 and -1beta, tumor necrosis factor-alpha (TNF-alpha), and prostaglandin E(2) (PGE(2)), in the cell culture supernatants were determined by enzyme-linked immunosorbent assay. Expression of the corresponding mRNAs was detected by reverse transcription-polymerase chain reaction. RESULTS: Significantly higher extracellular concentrations of VEGF and IL-6 were detected along with greater corresponding mRNA expression in the hypoxia group compared to the normoxia group. The protein production and mRNA expression of IL-1beta were observed only in the hypoxia group. Neither TNF-alpha nor PGE(2) was detectable in samples from either group, whereas cyclooxygenase-2 mRNA was detected. However, PGE(2) was detected after reoxygenation. Furthermore, VEGF and IL-6 and -1beta production also tended to increase in extracellular concentration and mRNA level after reoxygenation. CONCLUSION: Hypoxia and reoxygenation may stimulate the PDL to produce VEGF, IL-6 and -1beta, and PGE2, which could result in the resorption of alveolar bone in periodontitis.

Osteocalcin, deoxypyridinoline and interleukin-1beta in peri-implant crevicular fluid of patients with peri-implantitis.

The aim of the present study was to analyze the levels of osteocalcin, deoxypyridinoline (Dpd) and interleukin-1beta as markers of bone metabolism in peri-implant crevicular fluid (PICF) from peri-implantitis patients. PICF was sampled from a total of 34 endosseous titanium implants from 16 patients; nine females (mean age 52.8, range 40-62 years) and seven males (mean age 56.0, range 36-66 years). The implants had been in place for a period of 9-112 months (mean; 35.8 months) since the loading. These sites were categorized as six peri-implantitis, eight peri-implant mucositis and 20 healthy implant. PICF volume from peri-implantitis sites was significantly higher than mucositis and healthy implant sites (P < 0.01). Osteocalcin levels in PICF from mucositis sites were significantly higher than healthy implants (P < 0.05), whereas peri-implantitis sites were not significantly different from either mucositis or healthy implant sites. Dpd could not be detected in any of the samples examined. IL-1beta levels in PICF from peri-implantitis sites were significantly higher than levels from peri-implant mucositis (P < 0.05) and healthy implant sites (P < 0.01). In conclusion, osteocalcin in PICF may reflect increased local bone turnover around implants. Further, IL-1beta should be a useful marker for peri-implant inflammation.