A bi-phasic scaffold consisting of a columnar formaldehyde-acetalized polyvinyl alcohol (PVF) sponge and a cylindrical porous hydroxyapatite (HA) with a hollow center was devised. Rat bone marrow cells (rBMCs) were seeded into the sponge placed in the hollow center of the cylindrical porous HA. The bi-phasic scaffold, a cylindrical porous HA and a PVF sponge separated from a bi-phasic scaffold after rBMC seeding, and a PVF sponge without rBMCs as a negative control, were implanted for 6 weeks into rat dorsal subcutaneous tissue. In each construct, bone formation was examined histologically and osteocalcin was measured immunochemically. Bone formation was observed in the bi-phasic scaffold and also in the cylindrical porous HA isolated from the bi-phasic scaffold. A significant difference in the quantity of osteocalcin was observed between the bi-phasic scaffold and the isolated cylindrical porous HA. No bone formation was found in the isolated PVF sponge. The bi-phasic scaffold as an outer layer of the scaffold seemed to inhibit the outflow of rBMCs from the PVF sponge. This type of bi-phasic scaffold may have two specific characteristics: Attachment of cells both in PVF sponge and cylindrical porous HA.
Enterococcus faecalis is an etiological agent of endodontic infections. The present study was performed to investigate the gene profiles of E. faecalis induced by type I collagen stimulation. E. faecalis ATCC 19433 was cultivated with [collagen (+)] or without type I collagen [collagen (−)], and transcriptome analysis was performed using high-throughput sequencing technology. A total of 3.6 gb of information was obtained by sequence analysis and 77 differentially expressed genes (DEGs) between the two culture conditions were identified. Among the 77 DEGs, 35 genes were upregulated in collagen (+) E. faecalis, whereas 42 genes were downregulated. Gene Ontology (GO) enrichment analysis was performed and 11 GO terms, including metalloendopeptidase activity (GO:0004222) and two related GO terms (GO:0031012, GO:0044421), were significantly enriched in the set of upregulated genes. We focused on an upregulated DEG belonging to the matrixin metalloprotease gene family, and matrix metalloprotease (MMP) activities of the bacterial cell were examined. The generic MMP, MMP-8, and MMP-9 activities of collagen (+) E. faecalis were significantly higher than those of collagen (−) E. faecalis. These results suggested that contact with type I collagen may alter the gene expression profile of E. faecalis, and upregulation of metalloprotease genes may result in enhanced MMP activities in E. faecalis.
This study was performed to apply denaturing gradient gel electrophoresis to microbiological examination of endodontic infections. The method was used to assess the bacterial communities in dentinal tubules. Samples were collected using #15, #35, and #60 K-type files from five infected root canals during treatment. Deoxyribonucleic acid(DNA) was extracted from the samples, and 16S ribosomal DNA was amplified by PCR using universal primers. The polymerase chain reaction(PCR) products were separated in the denaturing gel and band patterns were compared between the deep (#60 file) and superficial layers (#15 file) of the dentin. The major bands were then excised and DNA fragments in the gel were cloned and sequenced. The sequence data were subjected to BLAST search in the GenBank database for determination of bacterial species or closest relatives. In three root canals, similar band patterns were observed in both superficial and deep layers of the dentin, while several specific bands remained in the deep layer in two roots. The bacteria isolated from the deep layers were Porphyromonas gingivalis, Olsenella profuse, Atopobium rimae, and Prevotella, Flexistipes, Firmicutes, Peptostreptococcus, Dialister, and Eubacterium spp. Unlike previous studies, gram-negative anaerobic rods were isolated from the deep layers. Clone library analysis was simultaneously performed and similar results were obtained. The method utilized here will be useful for microbiological examination of endodontic infections. In addition, although it is still unknown whether they were viable, this study demonstrated the presence of gram-negative rods in dentinal tubules.
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