Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (cemp1-p4) Promotes Mineralization through wnt/ß-catenin Signaling in Human Oral Mucosa Stem Cells.

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1's secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1's short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1's C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/ß-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/ß-catenin pathway. CEMP1-p4 stimulation upregulated the expression of ß-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of ß-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a "mineralizing-like" phenotype by activating the ß-catenin signaling cascade.

Viability and Osteogenic Differentiation of Human Periodontal Ligament Progenitor Cells Are Maintained After Incubation With Porphyromonas gingivalis Protein Extract.

BACKGROUND: Porphyromonas gingivalis (Pg) is a major periodontal pathogen that contains immunostimulatory components. Periodontal ligament mesenchymal stem cells (PDLMSCs) are responsible for regeneration of the periodontium that is lost due to periodontitis. Pathologic factors within the microenvironment that impair resident PDLMSCs are not well understood. The present study investigates in vitro the effects of Pg protein extract (PgPE) on biologic properties of CD105-enriched PDL progenitor cell populations (PDL-CD105+). METHODS: Five populations of PDL-CD105+ cells were exposed to PgPE and assessed for cell viability, apoptosis, and proinflammatory gene expression (interleukin-1ß [IL-1ß], tumor necrosis factor-alpha [TNF-α], and IL-6) by quantitative reverse transcription polymerase chain reaction, IL-6 immunostaining, activation of IL-6/signal transducer and activator of transcription (STAT) 3 signaling pathway, and osteogenic differentiation potential. RESULTS: PgPE treatment (2 µg/mL) did not affect cell viability or survival but induced a significant increase in IL-1ß, TNF-α, and IL-6 messenger RNA (mRNA) expression and positive staining for IL-6. A total of 29 genes from the IL-6/STAT3 pathway were upregulated on PgPE stimulation. These genes are related to biologic processes involved in the control of cell survival (B-cell lymphoma 2 [BCL2]), cell proliferation (hepatocytehepatocyte growth factor), cytokine-mediated signaling pathway (suppressor of cytokine signaling 3, C-X-C ligand 8 [CXCL8]), and response to stress (CXCL8, mitogen-activated protein kinase 3, BCL2-associated X protein, and BCL2). Additionally, PgPE treatment caused an increase in alkaline phosphatase mRNA expression in PDL-CD105+ cells after 7 days of osteogenic induction, although mineral nodule formation was comparable to the control group. CONCLUSIONS: These results suggest that the inflammatory profile induced by PgPE treatment in PDL-CD105+ cells did not affect cell viability, apoptosis, or osteogenic differentiation, perhaps due to increased expression of genes involved in the control of cell proliferation and protection against cell death.

Fluoxetine effects on periodontogenesis: histomorphometrical and immunohistochemical analyses in rats.

OBJECTIVE: This study aimed to assess the effects of fluoxetine, a selective serotonin reuptake inhibitor, on the formation of the periodontal ligament during pregnancy and lactation in rat pups. MATERIAL AND METHODS: Twelve pregnant rats of Wistar lineage were divided into four study groups. In the control group, 0.9% sodium chloride solution was administered orally, throughout the entire period of the 21 days of pregnancy (CG group) and in the CGL group, it was administrated during pregnancy and lactation (from day 1 of pregnancy to the 21st day after birth). Fluoxetine was administered orally at the dose of 20 mg/kg in a group treated during pregnancy only (FG group), and during pregnancy and lactation (FGL group). Histometrical, histochemical and immunohistochemical analysis of the maxillary first molar periodontium region of the 24 rat pups was made under light microscopy, and periodontal ligament collagen was qualitatively evaluated under a polarizing light microscope. RESULTS: The quantity of fibroblasts (p=0.006), osteoblasts (p=0.027) and cementoblasts (p=0.001) was reduced in pups from the rats that received fluoxetine during pregnancy and lactation. No alterations were seen in the collagen fibers. CONCLUSION: These findings suggest that periodontal tissue may be sensitive to fluoxetine, and its interference in reducing periodontal cells depends on exposure time during lactation.

Fluoxetine effects on periodontogenesis: histomorphometrical and immunohistochemical analyses in rats

Abstract Reports have indicated that serotonin plays an important role in cell migration and differentiation during the organogenesis of several tissues, including the oral types. Administration of selective serotonin reuptake inhibitor (SSRI) drugs during pregnancy could affect the delivery of serotonin to embryonic tissues altering its development. Objective This study aimed to assess the effects of fluoxetine, a selective serotonin reuptake inhibitor, on the formation of the periodontal ligament during pregnancy and lactation in rat pups. Material and Methods Twelve pregnant rats of Wistar lineage were divided into four study groups. In the control group, 0.9% sodium chloride solution was administered orally, throughout the entire period of the 21 days of pregnancy (CG group) and in the CGL group, it was administrated during pregnancy and lactation (from day 1 of pregnancy to the 21st day after birth). Fluoxetine was administered orally at the dose of 20 mg/kg in a group treated during pregnancy only (FG group), and during pregnancy and lactation (FGL group). Histometrical, histochemical and immunohistochemical analysis of the maxillary first molar periodontium region of the 24 rat pups was made under light microscopy, and periodontal ligament collagen was qualitatively evaluated under a polarizing light microscope. Results The quantity of fibroblasts (p=0.006), osteoblasts (p=0.027) and cementoblasts (p=0.001) was reduced in pups from the rats that received fluoxetine during pregnancy and lactation. No alterations were seen in the collagen fibers. Conclusion These findings suggest that periodontal tissue may be sensitive to fluoxetine, and its interference in reducing periodontal cells depends on exposure time during lactation.

Area of rests of Malassez in young and adult rat molars: evidences in the formation of large rests.

Recently, we have demonstrated that the reduction in the number of rests of Malassez (RM) according to age may be due to epithelial cell death by apoptosis. It is known that, under experimental or pathological conditions, the epithelial cells of RM can proliferate, resulting in the formation of odontogenic cysts and tumors. In this study, the area of RM profiles and the number of profiles of epithelial cell nuclei/RM were investigated in young and adult rats. Fragments containing upper molars of rats aged 29, 45, and 120 days were fixed, decalcified, and embedded for light microscopy. In the sections stained by hematoxylin and eosin, the numerical density of RM profiles/mm(2) of periodontal ligament, the area of each RM profile and the number of epithelial cell nuclei profile/RM were obtained. Moreover, detection of cytokeratin by immunohistochemistry and ultrastructural analysis of RM were carried out. Smaller RM were found in all groups, but the very large RM were only observed in the 120-day-old rats. The presence of cells in close juxtaposition exhibiting cytokeratin filaments in their cytoplasm, including in the large RM, characterized their epithelial nature. The number of epithelial cells/RM varied proportionally to the area. Moreover, epithelial cells in mitosis were found in small and large RM, indicating that cellular proliferation can be responsible for the enlargement of RM, under physiological conditions. The results suggest that a possible disequilibrium between the proliferative and apoptotic processes can be related to the enlargement of some RM in the 120-day-old rats.

Development of functional dentin incisors after a partial resection of the odontogenic organ of rat incisors.

The resection of the labial half of the odontogenic organ of rat incisors resulted in the development of teeth without enamel. Ten out of 26 operated rats developed a functional dentin incisor, i.e. a continuously growing and erupting tooth. These teeth were a little shorter and much thinner than normal incisors. The dentin and pulp presented a normal structure. Periodontal ligament and cement started to develop at the lingual face and gradually all tooth faces were invested by these tissues. The original socket space, to accommodate a thinner tooth, was narrowed by newly formed bone around the inner face of the socket. Eleven rats developed defective dentin incisors; these teeth showed signs of growth, however, their eruption was impaired. The operation failed in five rats. The odontogenic organ of the dentin incisor presented islands of epithelial cells at the labial aspect of a dense mass of mesenchyme cells. These islands, formed by densely packed, dark-staining cells encircling a few pale-staining cells, merged gradually, forming a root sheath and a cervical loop limiting a long apical foramen. The bulk of the bulbous part (apical bud) was absent; thus, there was no differentiation of ameloblasts and of the crown-analogue part of the incisor. The growth and eruptive behaviour of the dentin incisor, similar to that of a normal incisor, indicates that it has to bear a stem cell niche to retain its regenerative capacity. As in the apical bud, this niche is apparently located at the stellate reticulum of the cervical loop. The putative molecular mechanisms related to either the maintenance of the stem cell niche or the differentiation of the enamel organ and the root sheath are discussed. These data and our results, showing the development of a functional dentin incisor, suggest that the root-analogue part of the rodent incisor is an anatomic-physiological entity.