It was reported that postnatal stem cells are present in adult tissues such as bone marrow, liver, muscle, dental pulp, and periodontal ligament. We isolated postnatal stem cells from human dental tissues such as dental pulp (DPSC), periodontal ligament (PDLSC), periapical follicle (PAFSC), and the surrounding mandibular bone marrow (MBMSC) to ascertain their properties. Immunocytochemistry proved the existence of stem cells in these cell populations using STRO-1 as a stem cell marker. These cells also expressed the mesenchymal stem cell (MSC) markers CD29 and CD44. The isolated cells showed self-renewal capabilities and colony-forming efficiency. Almost all of the dental stem cells showed optimal growth when they were cultured in alpha modification of Eagle's medium (alpha-MEM) supplemented with 10% fetal calf serum (FCS) and 100 microM ascorbic acid. Only the PAFSC showed increased proliferation in 20% FCS and 50 microM ascorbic acid. All of the dental stem cells were capable of differentiating into adipocytes and mineral nodule forming cells. MBMSC, in particular, showed much better mineralization compared to the others. These results indicate that MSCs exist in various tissues of the teeth and can differentiate into osteoblasts, adipocytes, and other kinds of cells with varying efficiency.

Angiogenesis plays an important role in active inflammation and wound healing. Our results showed that silk sericin and 4-hexylresorcinol (4HR) increased vascular endothelial growth factor (VEGF) expression in a dose-dependent manner in RAW264.7 cells. Unlike 4HR, silk sericin increased the expression of hypoxia inducible factor-1α (HIF-1α) and HIF-2α. Pretreatment with an HIF inhibitor decreased the sericin-induced increase in VEGF expression. However, the HIF inhibitor did not affect the 4HR-induced increase in VEGF expression. An inhibitor of matrix metalloproteinase (MMP) declined the 4HR-induced increase in VEGF expression. Silk sericin increased production of reactive oxygen species (ROS), whereas 4HR decreased ROS. M1 markers were increased by silk sericin treatment, and M2 markers were increased by 4HR treatment. VEGF and angiogenin expression were higher in rats treated with a 4HR-incorporated silk mat than in rats treated with a silk mat alone. In conclusion, silk sericin and 4HR increased VEGF expression in RAW264.7 cells via HIF-mediated and MMP-mediated pathways, respectively. Silk sericin exerted like pro-oxidant effects and 4HR exerted anti-oxidant effects. Rats treated with a 4HR-incorporated silk mat showed higher levels of VEGF and angiogenin than those treated with a silk mat alone.

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To understand the osteogenic effect of the middle layer of the silk cocoon, sericin was examined for its cellular effects associated with tumor necrosis factor-α (TNF-α) signaling in this study. The fragmented sericin proteins in the silk mat were evaluated for the TNF-α expression level in murine macrophages. The concentration of protein released from silk mats was higher in the outermost and the innermost layers than in the middle layers, and the protein released from the silk mat was identified as sericin. The level of TNF-α in murine macrophages was dependent on the applied concentration of sericin, and the expression of genes associated with osteogenesis in osteoblast-like cells was dependent on the applied concentration of TNF-α. In animal experiments, silk mats from the middle layers led to a higher regenerated bone volume than silk mats from the innermost layer or the outermost layer. If TNF-α protein was incorporated into the silk mats from the middle layers, bone regeneration was suppressed compared with unloaded silk mats from the middle layers. Accordingly, silk mats from the silk cocoon can be considered to be a fragmented sericin-secreting carrier, and the level of sericin secretion is associated with TNF-α induction and bone regeneration.

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The effect of S-adenosylmethionine (SAM) on the production of various antibiotics was investigated to determine whether SAM-dependent methylation is required in biosynthetic pathways of antibiotics. Pristinamycin II(B) and granaticin do not require SAM-dependent methylation in their biosynthesis pathways, and production of these two antibiotics was increased about 2-fold when a low concentration (50 and 10 microM, respectively) of SAM was treated; in contrast, oleandomycin and avermectin B1a require SAM as a methyl donor in their biosynthesis, and production of these two antibiotics was increased 5-fold and 6-fold, depending on the SAM concentration within a certain range. We also found that the transcription of a pathway-specific regulator, gra-ORF9, was activated by exogenous SAM treatment. Production of oleandomycin and avermectin B1a was decreased by using a methyltransferase inhibitor, sinefungin, but the production levels of these antibiotics were restored to the control level by simultaneously adding SAM and sinefungin. Interestingly, we have found a similar stimulatory effect of S-adenosylhomocysteine (SAH), the methylation product of SAM, on antibiotic production in the four strains. Our results clearly demonstrate the widespread activation of antibiotic production using SAM in streptomycetes.

The aim of this study was to evaluate the in vivo bone regeneration capability of alginate (AL), AL/hydroxyapatite (HA), and AL/HA/silk fibroin (SF) composites. Forty Sprague Dawley rats were used for the animal experiments. Central calvarial bone (diameter: 8.0 mm) defects were grafted with AL, AL/HA, or AL/HA/SF. New bone formation was evaluated by histomorphometric analysis. To demonstrate the immunocompatibility of each group, the level of tumor necrosis factor (TNF)-α expression was studied by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) at eight weeks post implantation. Additionally, osteogenic markers, such as fibroblast growth factor (FGF)-23, osteoprotegerin (OPG), and Runt-related transcription factor (Runx2) were evaluated by qPCR or IHC at eight weeks post implantation. The AL/HA/SF group showed significantly higher new bone formation than did the control group (p = 0.044) and the AL group (p = 0.035) at four weeks post implantation. Additionally, the AL/HA/SF group showed lower relative TNF-α mRNA levels and higher FGF-23 mRNA levels than the other groups did at eight weeks post implantation. IHC results demonstrated that the AL/HA/SF group had lower TNF-α expression and higher OPG and Runx2 expression at eight weeks post implantation. Additionally, no evidence of the inflammatory reaction or giant cell formation was observed around the residual graft material. We concluded that the AL/HA/SF composite could be effective as a scaffold for bone tissue engineering.

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