Shaomian Yao

Differentiation of Stem Cells in the Dental Follicle

The dental follicle (DF) differentiates into the periodontal ligament. In addition, it may be the precursor of other cells of the periodontium, including osteoblasts and cementoblasts. We hypothesized that stem cells may be present in the DF and be capable of differentiating into cells of the periodontium. Stem cells were identified in the DF of the rat first mandibular molar by Hoechst staining, alkaline phosphatase staining, and expression of side-population stem cell markers. These cells were shown to be able to differentiate into osteoblasts/cementoblasts, adipocytes, and neurons. Treating the DF cell population with doxorubicin, followed by incubation in an adipogenesis medium, suggested that the adipocytes originated from stem cells. Thus, a possibly puripotent stem cell population is present in the rat DF.

CSF-1 Regulation of Osteoclastogenesis for Tooth Eruption

The dental follicle regulates the alveolar bone resorption needed for tooth eruption. In the rat first mandibular molar, a decrease in the expression of osteoprotegerin (OPG) in the dental follicle at day 3 enables the osteoclastogenesis needed for eruption to occur. Because colony-stimulating factor-1 (CSF-1) is maximally expressed in the dental follicle at day 3, it was hypothesized that CSF-1 down-regulates OPG gene expression in the dental follicle in vivo. To test this, we compared the expression of OPG in osteopetrotic toothless (tl/tl) rats deficient in CSF-1 with expression in their normal littermates for given ages. OPG gene expression was found to be higher in the dental follicle of the tl/tl mutants than in normals. Transfecting short interfering RNA specific for CSF-1 mRNA into dental follicle cells resulted in an up-regulation of OPG expression. Thus, these studies support our hypothesis that the down-regulation of OPG needed for tooth eruption is mediated by CSF-1.

Inhibition of osteoclastogenesis by the secretion of osteoprotegerin in vitro by rat dental follicle cells and its implications for tooth eruption.

Tooth eruption requires the presence of the dental follicle, a loose connective tissue sac that surrounds each unerupted tooth. Early postnatally in the rat, the follicle secretes colony-stimulating factor-1 (CSF-1) and monocyte chemotactic protein-1 (MCP-1), chemotactic molecules that are probably responsible for the recruitment of mononuclear cells. These cells, in turn, fuse to form osteoclasts, which are required for alveolar bone resorption to form an eruption pathway. Recent studies have shown that the osteoprotegerin (OPG) gene is expressed in the dental follicle, but in the first mandibular molar of the rat, that expression is reduced at day 3, the time of maximal osteoclast numbers on the alveolar bone. Inhibition of OPG expression at this time would allow osteoclast formation/activation. To determine if the dental follicle cells do secrete OPG that inhibits osteoclastogenesis, spleen cell cultures were established and soluble osteoclast differentiation factor (ODF) and CSF-1 added to some of them to promote osteoclast formation. In other cultures, dental follicle cells were added in an insert, such that they did not touch the spleen cells. Using a quantitative, tartrate-resistant acid phosphatase (TRAP) assay, it was shown that ODF and CSF-1 promoted osteoclastogenesis in the spleen cell cultures, but the addition of the follicle cells inhibited this and returned the TRAP activities to those seen in cultures of spleen cells only. Adding anti-OPG to these cultures, however, negated the effect of the follicle cells, demonstrating that OPG was the inhibitory molecule secreted by those cells. The follicle cells also immunostained for OPG, confirming that they synthesize OPG. These findings, coupled with those of other studies which show that the periodontal ligament (a derivative of the dental follicle) also secretes OPG, indicate that, except for the period of time in tooth eruption, where osteoclast formation is needed to form an eruption pathway, secretion of OPG would be the norm, presumably to prevent resorption of alveolar bone and subsequent disruption of the periodontal ligament.

Expression of tumour necrosis factor-alpha in the rat dental follicle

Tooth eruption requires the presence of the dental follicle, a loose connective tissue sac that surrounds each unerupted tooth. The follicle appears to regulate many of the cellular and molecular events of eruption, including the formation of osteoclasts needed to resorb alveolar bone to form an eruption pathway. To that end, the expression of the tumour necrosis factor-alpha (TNF-alpha) gene was examined in the dental follicle as a possible regulator of osteoclastogenesis. TNF-alpha was expressed slightly in the dental follicle of the first mandibular molar of the rat beginning at day 3 postnatally, but maximal expression was seen at day 9, a time that correlates with a slight burst of osteoclast formation seen at day 10 postnatally. In vitro, TNF-alpha was not expressed constitutively in the follicle cells but incubating them with interleukin 1alpha resulted in a strong expression of TNF-alpha after only 0.5h. TNF-alpha itself enhanced monocyte chemotactic protein 1 (MCP-1) and vascular endothelial growth factor (VEGF) gene expression. It also slightly decreased the expression of osteoprotegerin after 3-h incubation but this returned to the control level at 6h. MCP-1 and VEGF could aid in recruiting mononuclear cells (osteoclast precursors) to the dental follicle. In addition to the potential role of TNF-alpha in tooth eruption, this study suggests that the periodontal ligament derived from the dental follicle might have the capacity to synthesize TNF-alpha, and thereby contribute to the destructive events of periodontitis.

Requirement of alveolar bone formation for eruption of rat molars

Tooth eruption is a localized event that requires a dental follicle (DF) to regulate the resorption of alveolar bone to form an eruption pathway. During the intra-osseous phase of eruption, the tooth moves through this pathway. The mechanism or motive force that propels the tooth through this pathway is controversial but many studies have shown that alveolar bone growth at the base of the crypt occurs during eruption. To determine if this bone growth (osteogenesis) was causal, experiments were designed in which the expression of an osteogenic gene in the DF, bone morphogenetic protein-6 (Bmp6), was inhibited by injection of the first mandibular molar of the rat with a small interfering RNA (siRNA) targeted against Bmp6. The injection was followed by electroporation to promote uptake of the siRNA. In 45 first molars injected, eruption was either delayed or completely inhibited (seven molars). In the impacted molars, an eruption pathway formed but bone growth at the base of the crypt was greatly reduced compared with the erupted first-molar controls. These studies show that alveolar bone growth at the base of the crypt is required for tooth eruption and that Bmp6 may be essential for promoting this growth.

MicroRNA biogenesis and their functions in regulating stem cell potency and differentiation.

Stem cells are unspecialized/undifferentiated cells that exist in embryos and adult tissues or can be converted from somatic differentiated cells. Use of stem cells for tissue regeneration and tissue engineering has been a cornerstone of the regenerative medicine. Stem cells are also believed to exist in cancer tissues, namely cancer stem cells (CSCs). Growing evidence suggests that CSCs are the culprit of cancer dormancy, progression and recurrence, and thus have recently received great attention. MicroRNAs (miRNAs) are a group of short, non-coding RNAs that regulate expression of a wide range of genes at a post-transcriptional manner. They are emerging as key regulators of stem cell behaviors. This mini review summarizes the basic biogenesis and mode of actions of miRNAs, recent progress and discoveries of miRNAs in cellular reprogramming, stem cell differentiation and cellular communication, as well as miRNAs in CSCs. Some potential of miRNAs in future biomedical applications and research pertaining to stem cells are briefly discussed.

TNF-α Upregulates Expression of BMP-2 and BMP-3 Genes in the Rat Dental Follicle – Implications for Tooth Eruption

The dental follicle appears to regulate both the alveolar bone resorption and bone formation needed for tooth eruption. Tumor necrosis factor-alpha (TNF-α) gene expression is maximally upregulated at postnatal day 9 in the rat dental follicle of the first mandibular molar, a time that correlates with rapid bone growth at the base of the tooth crypt, as well as a minor burst of osteoclastogenesis. TNF-α expression is correlated with the expression of bone morphogenetic protein-2 (BMP-2), a molecule expressed in the dental follicle that can promote bone formation. Because BMP-2 signaling may be augmented by bone morphogenetic protein-3 (BMP-3), our objective in this study was to determine if the dental follicle expresses BMP-3 and if TNF-α stimulates the dental follicle cells to express BMP-2 and BMP-3. Dental follicles were collected from different postnatal ages of rat pups. Dental follicle cells were incubated with TNF-α to study its dosage and time-course effects on gene expression of BMP-2 and BMP-3, as determined by real-time RT-PCR. Next, immunostaining was conducted to confirm if the protein was synthesized and ELISA of the conditioned medium was conducted to determine if BMP-2 was secreted. We found that BMP-3 expression is correlated with the expression of TNF-α in the dental follicle and TNF-α significantly increased BMP-2 and BMP-3 expression in vitro. Immunostaining and ELISA showed that BMP-2 and BMP-3 were synthesized and secreted. This study suggests that TNF-α can upregulate the expression of bone formation genes that may be needed for tooth eruption.

Regulation of Osteoprotegerin Gene Expression in Dental Follicle Cells

Colony-stimulating factor-one (CSF-1) and parathyroid-hormone-related protein (PTHrP) down-regulate osteoprotegerin (OPG) gene expression in the dental follicle of the rat first mandibular molar. To examine this regulation at the signal transduction level, we treated cultured dental follicle cells with either phorbolmyristate acetate (PMA) or dibutyryl cyclic AMP (dbcAMP) to activate either protein kinase C (PKC) or protein kinase A (PKA). Our results demonstrate that PMA up-regulates OPG gene expression and down-regulates the expression of CSF-1 and the PTHrP receptor (PTHrP-R). Conversely, dbcAMP down-regulates OPG expression and up-regulates CSF-1 and PTHrP-R expression. Immunostaining shows that PMA also increases the steady-state levels of protein. Thus, treatment with agents that affect protein kinase activity also enhance the steady-state mRNA and protein levels of OPG, as well as decreasing the mRNA levels of CSF-1 and PTHrP-R. The PKC-α isoform may be critical in OPG regulation because PKC-α gene expression is enhanced by PMA and reduced by either CSF-1 or PTHrP.

Evaluation of bone regeneration potential of dental follicle stem cells for treatment of craniofacial defects

BACKGROUND AIMS Stem cell-based tissue regeneration offers potential for treatment of craniofacial bone defects. The dental follicle, a loose connective tissue surrounding the unerupted tooth, has been shown to contain progenitor/stem cells. Dental follicle stem cells (DFSCs) have strong osteogenesis capability, which makes them suitable for repairing skeletal defects. The objective of this study was to evaluate bone regeneration capability of DFSCs loaded into polycaprolactone (PCL) scaffold for treatment of craniofacial defects. METHODS DFSCs were isolated from the first mandibular molars of postnatal Sprague-Dawley rats and seeded into the PCL scaffold. Cell attachment and cell viability on the scaffold were examined with the use of scanning electron microscopy and alamar blue reduction assay. For in vivo transplantation, critical-size defects were created on the skulls of 5-month-old immunocompetent rats, and the cell-scaffold constructs were transplanted into the defects. RESULTS Skulls were collected at 4 and 8 weeks after transplantation, and bone regeneration in the defects was evaluated with the use of micro-computed tomography and histological analysis. Scanning electron microscopy and Alamar blue assay demonstrated attachment and proliferation of DFSCs in the PCL scaffold. Bone regeneration was observed in the defects treated with DFSC transplantation but not in the controls without DFSC transplant. Transplanting DFSC-PCL with or without osteogenic induction before transplantation achieved approximately 50% bone regeneration at 8 weeks. Formation of woven bone was observed in the DFSC-PCL treatment group. Similar results were seen when osteogenic-induced DFSC-PCL was transplanted to the critical-size defects. CONCLUSIONS This study demonstrated that transplantation of DFSCs seeded into PCL scaffolds can be used to repair craniofacial defects.

Activation of proliferation and differentiation of dental follicle stem cells (DFSCs) by heat stress.

Adult stem cells (ASCs) remain in a slowly cycling/quiescent state under normal physiological conditions, but they can be awakened from this by certain factors, such as injury signals. Previously, our group has shown that dental follicle stem cells (DFSCs) appear to proliferate more rapidly than their non‐stem cell counterparts at elevated temperatures. The study described here has aimed to (i) elucidate optimal temperature in which to culture DFSCs, (ii) determine whether elevated temperatures could enhance differentiation capability of DFSCs and (iii) characterize stem cell and osteogenic marker expression of DFSCs at elevated temperatures.