Soh Sato

Dedifferentiated fat cells： an alternative source of adult multipotent cells from the adipose tissues

When adipose‐derived stem cells (ASCs) are retrieved from the stromal vascular portion of adipose tissue, a large amount of mature adipocytes are often discarded. However, by modified ceiling culture technique based on their buoyancy, mature adipocytes can be easily isolated from the adipose cell suspension and dedifferentiated into lipid‐free fibroblast‐like cells, named dedifferentiated fat (DFAT) cells. DFAT cells re‐establish active proliferation ability and undertake multipotent capacities. Compared with ASCs and other adult stem cells, DFAT cells showed unique advantages in their abundance, isolation and homogeneity. In this concise review, the establishment and culture methods of DFAT cells are introduced and the current profiles of their cellular nature are summarized. Under proper induction culture in vitro or environment in vivo, DFAT cells could demonstrate adipogenic, osteogenic, chondrogenic and myogenic potentials. In angiogenic conditions, DFAT cells could exhibit perivascular characteristics and elicit neovascularization. Our preliminary findings also suggested the pericyte phenotype underlying such cell lineage, which supported a novel interpretation about the common origin of mesenchymal stem cells and tissue‐specific stem cells within blood vessel walls. Current research on DFAT cells indicated that this alternative source of adult multipotent cells has great potential in tissue engineering and regenerative medicine.

The relationship between physiologic halitosis and periodontopathic bacteria of the tongue and gingival sulcus

To determine the influence of oral status on halitosis, the relationship between halitosis and periodontopathic bacteria present in plaque on the tongue and the subgingival sulcus was examined in 62 periodontally healthy adults. Halitosis indicators used were the organoleptic score; gas chromatography results [total volatile sulfur compounds (VSCs) = H2S + CH3SH + (CH3)2S]; Halimeter values; and the results of three clinical tests, plaque control record (PlCR), plaque index (PlI), and tongue coat status. Significant correlations with organoleptic scores was observed for PlCR, PlI, tongue coat status, VSC amounts, and Halimeter values, indicating that halitosis in periodontally healthy subjects tended to originate from tongue plaque deposits. Polymerase chain reaction analysis was used to detect six periodontopathic bacteria (Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Treponema denticola) from the tongue and subgingival plaque. Significant effects on the organoleptic scores, tongue coat status, total VSC, H2S and CH3SH amounts, and Halimeter values were observed only for T. denticola and F. nucleatum and only in the tongue plaque, not in the subgingival plaque. Thus, therapies developed to inhibit the growth of these bacteria may lead to future treatments of halitosis.

Effect of particle diameter on air polishing of dentin surfaces

We examined the abrasiveness of glycine powders with particle diameters of 63 and 100 μm by measuring the depth and volume of defects produced during air polishing of human dentin. A total of 36 extracted human teeth were embedded in acrylic resin. The resin blocks were polished until the dentin surfaces were exposed. The nozzle of an air polisher was mounted 4 mm from the dentin surface, and the dentin surface was treated for 5 s at one of two angles of incidence (45° or 90°). Three materials were used in the polishing process: NaHCO3 powder with a mean particle diameter of 100 μm (Handy Jet Powder), glycine powder with a mean particle diameter of 63 μm (Handy Jet Powder PMTC), and glycine powder with a mean particle diameter of 100 μm (Handy Jet Powder Recall). The defect depth at both angles was significantly deeper after treatment with Handy Jet Powder or Handy Jet Powder PMTC. The defect volume was the greatest with Handy Jet Powder, followed by Handy Jet Powder PMTC, and Handy Jet Powder Recall. The larger diameter glycine powder resulted in less damage to the dentin.

Salivary pathogen and serum antibody to assess the progression of chronic periodontitis: a 24-mo prospective multicenter cohort study

BACKGROUND AND OBJECTIVE A diagnosis of periodontitis progression is presently limited to clinical parameters such as attachment loss and radiographic imaging. The aim of this multicenter study was to monitor disease progression in patients with chronic periodontitis during a 24-mo follow-up program and to evaluate the amount of bacteria in saliva and corresponding IgG titers in serum for determining the diagnostic usefulness of each in indicating disease progression and stability. MATERIAL AND METHODS A total of 163 patients with chronic periodontitis who received trimonthly follow-up care were observed for 24 mo. The clinical parameters and salivary content of Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were assessed using the modified Invader PLUS assay, and the corresponding serum IgG titers were measured using ELISA. The changes through 24 mo were analyzed using cut-off values calculated for each factor. One-way ANOVA or Fishers exact test was used to perform between-group comparison for the data collected. Diagnostic values were calculated using Fishers exact test. RESULTS Of the 124 individuals who completed the 24-mo monitoring phase, 62 exhibited periodontitis progression, whereas 62 demonstrated stable disease. Seven patients withdrew because of acute periodontal abscess. The ratio of P. gingivalis to total bacteria and the combination of P. gingivalis counts and IgG titers against P. gingivalis were significantly related to the progression of periodontitis. The combination of P. gingivalis ratio and P. gingivalis IgG titers was significantly associated with the progression of periodontitis (p = 0.001, sensitivity = 0.339, specificity = 0.790). CONCLUSIONS It is suggested that the combination of P. gingivalis ratio in saliva and serum IgG titers against P. gingivalis may be associated with the progression of periodontitis.

Specificity of Antimicrobial Peptide LL-37 to Neutralize Periodontopathogenic Lipopolysaccharide Activity in Human Oral Fibroblasts

BACKGROUND The antimicrobial peptide LL-37 is known to have a potent lipopolysaccharide (LPS)-neutralizing activity in various cell types. Because of observed heterogeneity within periodontopathogenic LPS, the authors hypothesized that LL-37 had specificity to neutralize such LPS activity. The present study, therefore, aims to investigate the LPS-neutralizing activity of LL-37 to various periodontopathogenic LPS in interleukin-8 (IL-8) production after challenging them in human oral fibroblasts. METHODS Human periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) were cultured from biopsies of periodontal ligament and gingival tissues. After cell confluence in 24-well plates, LPS (10 μg/mL) from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were added with or without LL-37 (10 μg/mL). After 18 hours, the supernatant was collected and analyzed in IL-8 production by enzyme-linked immunosorbent assay. RESULTS All periodontopathogenic LPS statistically significantly induced IL-8 production in both PDLF and GF (P <0.01). After neutralization with LL-37, both PDLF and GF showed a statistically significant reduction in IL-8 production compared with LPS-treated groups without LL-37 (P <0.01), and the percentage of reduction in IL-8 production in PDLF appeared to be higher than in GF. In addition, the percentage of reduction in IL-8 production varied considerably according to each periodontopathogenic LPS. CONCLUSIONS The antimicrobial peptide LL-37 had an ability to suppress periodontopathogenic LPS-induced IL-8 production in both PDLF and GF. Its LPS-neutralizing activity revealed specificity to periodontopathogenic LPS and seemed to be dependent on the heterogeneity within LPS between different genera.

The Efficacy of Povidone-Iodine Products against Periodontopathic Bacteria

A total of 8 strains of 6 bacterial species, Porphyromonas gingivalis ATCC33277 and TDC286, Actinobacillus actinomycetemcomitans ATCC29523 and JP2, Fusobacterium nucleatum No. 2, Tannerella forsythensis ATCC43937, Prevotella intermedia ATCC25611 and Streptococcus anginosus ATCC33397, were treated with povidone-iodine (PVP-I) gargle (PVP-I: 0.47 and 0.23% w/v) or chlorhexidine gluconate (CHG) gargle (CHG: 0.002% w/v) for 15, 30 or 60 s, after which they were inoculated into various media, cultured and counted for residual bacteria. At both concentrations, PVP-I gargle reduced the viable cell count of all 8 bacterial strains to below the measurable limit within 15 s. By contrast, there were more than 1,000 viable colonies 60 s following treatment with the CHG gargle. The results demonstrate that povidone-iodine gargle has rapid bactericidal activity against the causative bacteria of periodontal disease.

Novel amelanotic and melanotic cell lines NM78-AM and NM78-MM derived from a human oral malignant melanoma

Novel cell lines, designated NM78-AM and NM78-MM, have been established from a malignant melanoma of the cheek oral mucosa. NM78-AM cells were spherical, grew in suspension as clusters, and produced no melanin. In contrast, NM78-MM cells were adherent and produced melanin granules. Initially, NM78-AM cells were grown on fibroblast feeder cells or in growth media supplemented with 10% conditioned medium from fibroblasts, but eventually grew in standard growth media alone. NM78-AM cells had interdigitating microvilli and formed cell clusters. They had large nucleoli, desmosomes, lipid droplets, and well-developed Golgi apparatuses. In contrast, NM78-MM cells grew as adherent neuron-like cells. They had large prominent nucleoli, irregular nuclear membranes, a number of mitochondria, well-developed Golgi apparatuses, melanosomes at various stages of development in the cytoplasm, and the cells secreted melanin granules. Projections from these melanotic cells formed anastomoses with each other. NM78-MM cells stained immunofluorescently for internexin, neuron specific enolase, NF-200, and glial fibrillary acidic protein. These cells were severely aneuploid, approximating to triploidy, and had many marker chromosomes. We used a real-time monitoring system to evaluate oxygen concentrations in culture medium to investigate the susceptibility of both cell lines to various anti-cancer drugs. NM78-AM cells were slightly sensitive to actinomycin D, but not to cisplatin, irinotecan, the irinotecan metabolite SN-38, taxol, taxotere, bleomycin and methotrexate; NM78-MM cells were sensitive to cisplatin, and not to taxol, taxotere, carboplatin, and irinotecan. These new cell lines, NM78-AM and NM78-MM, will be very important for the development of new chemotherapeutics for oral malignant melanoma.

Assessing the progression of chronic periodontitis using subgingival pathogen levels: a 24-month prospective multicenter cohort study

BackgroundThe diagnosis of the progression of periodontitis presently depends on the use of clinical symptoms (such as attachment loss) and radiographic imaging. The aim of the multicenter study described here was to evaluate the diagnostic use of the bacterial content of subgingival plaque recovered from the deepest pockets in assessing disease progression in chronic periodontitis patients.MethodsThis study consisted of a 24-month investigation of a total of 163 patients with chronic periodontitis who received trimonthly follow-up care. Subgingival plaque from the deepest pockets was recovered and assessed for bacterial content of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans using the modified Invader PLUS assay. The corresponding serum IgG titers were measured using ELISA. Changes in clinical parameters were evaluated over the course of 24 months. The sensitivity, specificity, and prediction values were calculated and used to determine cutoff points for prediction of the progression of chronic periodontitis.ResultsOf the 124 individuals who completed the 24-month monitoring phase, 62 exhibited progression of periodontitis, whereas 62 demonstrated stable disease. The P. gingivalis counts of subgingival plaque from the deepest pockets was significantly associated with the progression of periodontitis (p < 0.001, positive predictive value = 0.708).ConclusionsThe P. gingivalis counts of subgingival plaque from the deepest pockets may be associated with the progression of periodontitis.

In vitro study on regeneration of periodontal tissue microvasculature using human dedifferentiated fat cells

BACKGROUND Human dedifferentiated fat cells (HDFATs) may be a new cell type suitable for regenerative therapies. The aim of this study is to assess the potential of HDFATs for vascular regeneration of periodontal tissue. To do this, HDFATs and human gingival endothelial cells (HGECs) were cocultivated, and vascular regeneration was examined in vitro. METHODS HDFATs were isolated from subcutaneous adipose tissue, and HGECs were isolated from gingival cells using anti-cluster of differentiation 31 antibody-coated magnetic beads. HDFATs were cocultured with HGECs in microvascular endothelial cell growth medium-2 (EGM-2MV) for 7 days. Expression of endothelial cell (EC) markers, the formation of capillary-like tubes, and the expression of pericyte markers were determined. RESULTS HDFATs, cultured in EGM-2MV or cocultured with HGECs, expressed EC markers. HDFATs in both conditions initiated tube formation within 5 hours of seeding and formed extensive capillary-like structures within 12 hours. These structures disintegrated within 24 hours when cells were cultured in EGM-2MV alone, whereas cocultured HDFATs maintained tubes for >24 hours. Cocultured HDFATs significantly increased expression of pericyte markers, a cell type associated with microvasculature. CONCLUSION HDFATs possess the ability to express EC markers, and coculture with HGECs promotes differentiation into pericytes involved in the maturation and stabilization of the microvasculature.

The perivascular phenotype and behaviors of dedifferentiated cells derived from human mature adipocytes

Derived from mature adipocytes, dedifferentiated fat (DFAT) cells represent a special group of multipotent cells. However, their phenotype and cellular nature remain unclear. Our study found that human DFAT cells adopted perivascular characteristics and behaviors. Flow cytometry and immunofluorescent staining revealed that human DFAT cells positively expressed markers highly related to perivascular cell lineages, such as CD140b, NG2 and desmin, but were negative for common endothelial markers, including CD31, CD34, and CD309. Furthermore, DFAT cells displayed vascular network formation ability in Matrigel, and they noticeably promoted and stabilized the vessel structures formed by human umbilical vascular endothelial cells (HUVECs) in vitro. These results provide novel evidence on the pericyte nature of human DFAT cells, further supporting the recent model for the perivascular origin of adult stem cells, in which tissue-specific progenitor cells in mesenchymal tissues associate with blood vessels, exhibiting perivascular characteristics and functions.