Histologic and histobacteriologic observations of failed revascularization/revitalization therapy: a case report
INTRODUCTION: Mechanical debridement plays an important role in eliminating intracanal bacteria, such as biofilm on the canal walls and bacteria in the dentinal tubules. Mechanical debridement is not recommended for root canal disinfection in revascularization/revitalization therapy. Here we report a failed revascularization/revitalization case, which could be due to inadequate root canal disinfection without mechanical removal of biofilm and bacteria in dentinal tubules. METHODS: A 6-year-old boy had a traumatic injury to tooth #9, which was avulsed and replanted within 40 minutes. The tooth subsequently developed a local swelling in the periapical area. The patient was referred to the Postgraduate Endodontic Clinic for revascularization/revitalization therapy on tooth #9. The treated tooth remained asymptomatic for 16 months and then developed pain and local periapical swelling. The oral surgeon extracted the revascularized/revitalized tooth. On request, the extracted tooth was processed for histologic and histobacteriologic examination. RESULTS: The tissue in the canal was completely destroyed. Most bacteria were observed in the apical portion and not in the coronal portion of the canal and formed biofilm on the canal walls and penetrated into the dentinal tubules. CONCLUSIONS: On the basis of histobacteriologic observations, the failure of revascularized/revitalized tooth could be due to inadequate root canal disinfection without mechanical debridement. It may be important to perform mechanical debridement as part of the revascularization/revitalization therapy to disrupt the biofilm on the canal walls and remove bacteria in the dentinal tubules because revascularization/revitalization therapy is able to increase thickening of the canal walls.
Triclosan Blocks Mmp 13 Expression in Hormone-Stimulated Osteoblasts
Background: Matrix metalloproteinase-13 (Mmp-13) is an important enzyme for the modulation of bone turnover and gingival recession. Elevated levels of Mmp-13 are associated with alveolar bone resorption, periodontal ligament destruction, and gingival attachment loss, which are the clinical symptoms of periodontal disease. Continued evidence suggests periodontal disease contributes to oral tissue destruction and is linked to numerous systemic conditions. Triclosan is a long standing, proven antibacterial and anti-inflammatory agent found in the only FDA-approved dentifrice for the treatment of plaque and gingivitis. Methods: This study examined the inhibitory effects of triclosan on lipopolysaccharide (LPS), parathyroid hormone (PTH) and prostaglandin E(2) (PGE(2)) induced expression of Mmp-13 in UMR 106-01 cells, an osteoblastic osteosarcoma cell line. The cells were stimulated with PTH or PGE(2) to induce Mmp-13 mRNA expression and Real Time RT-PCR was performed to determine gene expression levels. Western blot analysis assessed the presence or absence of protein degradation or inhibition of protein synthesis. Mmp-13 Promoter Reporter Assay was utilized to explore possible direct effects of triclosan on the Mmp-13 promoter. Results: Triclosan significantly reduced PTH or PGE(2) elevated expression of Mmp-13 in osteoblastic cells without affecting basal levels of the mRNA. Surprisingly, triclosan enhanced the expression of c-fos and amphiregulin mRNA. A promoter assay indicated triclosan directly inhibits the activation of the PTH-responsive minimal promoter of Mmp-13. Conclusion: Our data appear to have identified a nuclear mechanism of action of triclosan which accounts for triclosan's ability to inhibit PTH or PGE(2) induced Mmp-13 expression in osteoblastic cells.
EphB-EphrinB Interaction Controls Odontogenic/Osteogenic Differentiation with Calcium Hydroxide
INTRODUCTION: Calcium hydroxide is used in direct pulp capping of uncontaminated exposed vital pulps caused by mechanical or traumatic injury. Calcium hydroxide creates a high alkaline pH environment and initiates a mineralized tissue formation in the pulp. The exact mechanism by which calcium hydroxide induces the reparative dentin formation is unknown. Because Eph receptors and ephrin ligands play a role in pulp stem cell migration and proliferation, our hypothesis is that calcium hydroxide-related odontogenic/osteogenic differentiation may be associated with Eph-ephrin interaction. The aim of this study was to investigate whether Eph-ephrin interaction regulates odontogenic/osteogenic differentiation with calcium hydroxide. METHODS: Primary pulp cells were harvested from the molars of C57BL/6 mice. The cells were treated with calcium hydroxide. Immunofluorescence was used to detect protein expression. A knockout of the ephrinB1 or EphB2 gene was performed with short hairpin RNAs. Cell migration, proliferation, and gene expression were then analyzed. RESULTS: Calcium hydroxide stimulated EphB2 gene expression but suppressed ephrinB1 gene expression at the proliferation stage. However, calcium hydroxide stimulated both ephrinB1 and EphB2 gene expression at the differentiation stage. In addition, EphB2 localized at ephrinB1-positive cells at the area of Dentin sialoprotein (DSP) staining, which increased with calcium hydroxide treatment. Knockdown of ephrinB1-EphB2 significantly suppressed cell proliferation. Additionally, knockdown of the ephrinB1 gene caused cell migration, whereas a lack of the EphB2 gene suppressed calcium hydroxide-induced mineralization from primary pulp cells. CONCLUSIONS: EphrinB1-EphB2 interaction contributes to calcium hydroxide-induced odontogenic/osteogenic differentiation. This observation is the first finding of the mechanism of calcium hydroxide-induced odontogenic/osteogenic differentiation.
Monocyte chemoattractant protein-1 is a mediator of the anabolic action of parathyroid hormone on bone
Parathyroid hormone (PTH) has a significant role as an anabolic hormone in bone when administered by intermittent injection. Previous microarray studies in our laboratory have shown that the most highly regulated gene, monocyte chemoattractant protein-1 (MCP-1), is rapidly and transiently induced when hPTH(1-34) is injected intermittently in rats. Through further in vivo studies, we found that rats treated with hPTH(1-34) showed a significant increase in serum MCP-1 levels 2 hours after PTH injection compared with basal levels. Using immunohistochemistry, increased MCP-1 expression in osteoblasts and osteocytes is evident after PTH treatment. PTH also increased the number of marrow macrophages. MCP-1 knockout mice injected daily with hPTH(1-34) showed less trabecular bone mineral density and bone volume compared with wild-type mice as measured by peripheral quantitative computed tomography (pQCT) and micro-computed tomography (microCT). Histomorphometric analysis revealed that the increase in osteoclast surface and osteoclast number observed with intermittent PTH treatment in the wild-type mice was completely eliminated in the MCP-1 null mice, as well as much lower numbers of macrophages. Consequently, the lack of osteoclast and macrophage activity in the MCP-1 null mice was paralleled by a reduction in bone formation. We conclude that osteoblast and osteocyte MCP-1 expression is an important mediator for the anabolic effects of PTH on bone.
Repulsive guidance molecule a regulates hippocampal mossy fiber branching in vitro
Repulsive guidance molecule a (RGMa), which binds to its receptor neogenin, has been well determined as a repulsive axon guidance molecule. However, whether RGMa affects the growth of hippocampal mossy fibers, the axons of dentate granule cells, has been unknown. In the present study, we found that the primary neurons in the hippocampus express both RGMa and neogenin in the postnatal rats. To examine the role of RGMa in the mossy fiber growth, the morphology of granule cells was clearly visualized by transfecting membrane-targeted green fluorescent protein using a single-cell electroporation method in cultured hippocampal slices. In the slice cultures, we found that intrinsic RGMa is required to inhibit excess branching of the hippocampal mossy fibers. Furthermore, hyperexcitability-induced aberrant branching of the mossy fibers in the cultured slices was blocked by applying the recombinant RGMa protein. Therefore, this study suggests that RGMa regulates the proper axonal branching of hippocampal mossy fibers.
Clinical, radiographic, and histological observation of a human immature permanent tooth with chronic apical abscess after revitalization treatment
INTRODUCTION: Revitalization procedures have been widely used for the treatment of immature permanent teeth with apical periodontitis. The treatment procedures appear to be capable of encouraging continued root development and thickening of the canal walls. The nature of tissues formed in the canal space and at the root apex after revitalization has been shown histologically in several animal studies; similar studies in humans were recently reported. METHODS: A 9-year-old boy had a traumatic injury to his upper anterior teeth. Tooth #9 suffered a complicated crown fracture with a pulp exposure, which was restored with a composite resin. The tooth developed a chronic apical abscess. Revitalization procedures were performed on tooth #9 because it was an immature permanent tooth with an open apex and thin canal walls. Twenty-six months after revitalization, the tooth had a horizontal crown fracture at the cervical level and could not be restored. The tooth was extracted and processed for routine histological and immunohistochemical examination to identify the nature of tissues formed in the canal space. RESULTS: Clinically and radiographically, the revitalization of the present case was successful because of the absence of signs and symptoms and the resolution of periapical lesion as well as thickening of the canal walls and continued root development. The tissue formed in the canal was well-mineralized cementum- or bone-like tissue identified by routine histology and immunohistochemistry. No pulp-like tissue characterized by the presence of polarized odontoblast-like cells aligning dentin-like hard tissue was observed. CONCLUSIONS: The tissues formed in the canal of revitalized human tooth are similar to cementum- or bone-like tissue and fibrous connective tissue.
Role of Sirtuin 1 in the Function of PTH in Osteoblasts [Meeting Abstract]
Histologic observation of a human immature permanent tooth with irreversible pulpitis after revascularization/regeneration procedure
INTRODUCTION: Histological studies of immature human permanent necrotic teeth with or without apical periodontitis after revascularization have not been reported. This case report describes the histological findings of tissue formed in the canal space of an immature permanent tooth #9 with irreversible pulpitis without apical periodontitis after revascularization. METHODS: An immature human permanent tooth #9 was fractured 3.5 weeks after revascularization and could not be retained. The tooth was extracted and prepared for routine histological and immunohistochemical evaluation in order to examine the nature of tissue formed in the root canal following the revascularization procedure. RESULTS: At 3.5 weeks after revascularization, more than one half of the canal was filled with loose connective tissue similar to the pulp tissue. A layer of flattened odontoblast-like cells lined along the predentin. Layers of epithelial-like cells, similar to the Hertwig's epithelial root sheath, surrounded the root apex. No hard tissue was formed in the canal. CONCLUSIONS: Based on the histological findings in the present case, regeneration of pulp-like tissue is possible after revascularization. In this case, both the apical papilla and the Hertwig's epithelial root sheath survived in an immature permanent tooth despite irreversible pulpitis but without apical periodontitis.
Erratum: EGFR signaling suppresses osteoblast differentiation and inhibits expression of master osteoblastic transcription factors Runx2 and osterix (Journal of Cellular Biochemistry (2011) 112 (1749-1760) DOI: 10.1002/jcb.23094) [Correction]
Alendronate Affects Osteoblast Functions by Crosstalk through EphrinB1-EphB
Bisphosphonates are therapeutic agents in the treatment of post-menopausal osteoporosis. Although they have been associated with delayed healing in injured tissues, inappropriate femoral fractures, and osteonecrosis of the jaw (ONJ), the pathophysiological mechanisms involved are not clear. Our hypothesis is that alendronate, a member of the N-containing bisphosphonates, indirectly inhibits osteoblast function through the coupling of osteoclasts to osteoblasts by ephrinB-EphB interaction. We found that alendronate increased gene and protein expression of ephrinB1 and EphB1, as well as B3, in femurs of adult mice injected with alendronate (10 microg/100 g/wk) for 8 weeks. Alendronate suppressed the expression of bone sialoprotein (BSP) and osteonectin in both femurs and bone marrow osteoblastic cells of mice. After elimination of pre-osteoclasts from bone marrow cells, alendronate did not affect osteoblast differentiation, indicating the need for pre-osteoclasts for alendronate's effects. Alendronate stimulated EphB1 and EphB3 protein expression in osteoblasts, whereas it enhanced ephrinB1 protein in pre-osteoclasts. In addition, a reverse signal by ephrinB1 inhibited osteoblast differentiation and suppressed BSP gene expression. Thus, alendronate, through its direct effects on the pre-osteoclast, appears to regulate expression of ephrinB1, which regulates and acts through the EphB1, B3 receptors on the osteoblast to suppress osteoblast differentiation.