Mijeong Jeon

The effect of epigallocatechin-3-gallate (EGCG) on human alveolar bone cells both in vitro and in vivo

OBJECTIVE The effects of epigallocatechin-3-gallate (EGCG), a major catechin in green tea, on human and mouse osteoblasts remain controversial. This study investigated the direct effects of EGCG on human alveolar bone-derived cells (hABCs) both in vitro and in vivo. DESIGN hABCs which were collected from eight children (aged 7-9 years, seven males and one female) were treated with EGCG at various concentrations (1, 5, 10, 25, and 50μM), and a proliferation assay, flow cytometric analysis for apoptosis evaluation, migration assay, and in vitro osteogenic differentiation were performed. hABCs that were pretreated with 10μM EGCG and mixed with calcium phosphate carrier combined with EGCG (0.1, 0.5, or 1.5mg) in vivo were transplanted into immunodeficient mouse. Histological staining, quantitative gene expressions, and alkaline phosphatase activity were evaluated in the retrieved transplants. RESULTS The proliferation and migration were decreased when EGCG was present at over 25μM. The osteogenic differentiation increased slightly when EGCG was present at up to 10μM, and clearly decreased for higher concentrations of EGCG. In vivo, the potential for hard-tissue formation was slightly higher for the group with 0.1mg of EGCG than for the control group, and decreased sharply for higher concentrations of EGCG. CONCLUSION The present observations suggest that EGCG at a low concentration can slightly enhance the osteogenic effect in vivo, whereas at a higher concentration it can prevent the osteogenic differentiation of hABCs both in vitro and in vivo.

In vitro and in vivo characteristics of stem cells from human exfoliated deciduous teeth obtained by enzymatic disaggregation and outgrowth

OBJECTIVE Stem cells from human exfoliated deciduous teeth (SHED) are a good source of dental tissue for regeneration therapy, and can be obtained using different primary culture methods. The aim of this study was to determine the differences in the in vitro and in vivo characteristics between SHED isolated via enzymatic disaggregation (e-SHED) and outgrowth (o-SHED) primary culture methods. DESIGN Dental pulp stem cells were isolated from 14 exfoliated deciduous teeth by enzymatic disaggregation (n=7) and outgrowth (n=7). Their proliferation potential and colony-forming ability were evaluated in vitro, as was their mesenchymal stem-cell-marker expression (using flow cytometry), and their differentiation was verified using quantitative real-time PCR (qPCR) and histochemical staining. In addition, the qualitative and quantitative characteristics of the hard tissue that was generated after in vivo transplantation were compared using haematoxylin and eosin staining, immunohistochemical staining, qPCR, and quantitative alkaline phosphatase analysis. RESULTS The cell-proliferation potential, colony-forming ability, and Stro-1 and CD146 expression were higher in e-SHED than in o-SHED. While the in vitro adipogenic differentiation potential was greater in e-SHED than in o-SHED, the in vitro osteogenic differentiation did not differ significantly between the two cell types. Although in vivo hard tissue formation was greater following transplantation of o-SHED into mice, there was no difference in the quality of hard tissue generated by e-SHED and o-SHED. CONCLUSION The findings of this study indicate that e-SHED exhibit stronger stemness characteristics, but that o-SHED are more suitable for hard-tissue regeneration therapy in teeth.

Comparative gene expression analysis of the human periodontal ligament in deciduous and permanent teeth.

There are histological and functional differences between human deciduous and permanent periodontal ligament (PDL) tissues. The aim of this study was to determine the differences between these two types of tissue at the molecular level by comparing their gene expression patterns. PDL samples were obtained from permanent premolars (n = 38) and anterior deciduous teeth (n = 31) extracted from 40 healthy persons. Comparative cDNA microarray analysis revealed several differences in gene expression between the deciduous and permanent PDL tissues. These findings were verified by qRT-PCR (quantitative reverse-transcription–polymerase chain reaction) analysis, and the areas where genes are expressed were revealed by immunohistochemical staining. The expressions of 21 genes were up-regulated in deciduous relative to PDL tissues, and those of 30 genes were up-regulated in permanent relative to deciduous PDL tissues. The genes that were up-regulated in deciduous PDL tissues were those involved in the formation of the extracellular matrix (LAMC2, LAMB3, and COMP), tissue development (IGF2BP, MAB21L2, and PAX3), and inflammatory or immune reactions leading to tissue degradation (IL1A, CCL21, and CCL18). The up-regulated genes in permanent PDL tissues were related to tissue degradation (IL6 and ADAMTS18), myocontraction (PDE3B, CASQ2, and MYH10), and neurological responses (FOS, NCAM2, SYT1, SLC22A3, DOCK3, LRRTM1, LRRTM3, PRSS12, and ARPP21). The analysis of differential gene expressions between deciduous and permanent PDL tissues aids our understanding of histological and functional differences between them at the molecular level.

Decellularized Human Dental Pulp as a Scaffold for Regenerative Endodontics

Teeth undergo postnatal organogenesis relatively late in life and only complete full maturation a few years after the crown first erupts in the oral cavity. At this stage, development can be arrested if the tooth organ is damaged by either trauma or caries. Regenerative endodontic procedures (REPs) are a treatment alternative to conventional root canal treatment for immature teeth. These procedures rely on the transfer of apically positioned stem cells, including stem cells of the apical papilla (SCAP), into the root canal system. Although clinical success has been reported for these procedures, the predictability of expected outcomes and the organization of the newly formed tissues are affected by the lack of an available suitable scaffold that mimics the complexity of the dental pulp extracellular matrix (ECM). In this study, we evaluated 3 methods of decellularization of human dental pulp to be used as a potential autograft scaffold. Tooth slices of human healthy extracted third molars were decellularized by 3 different methods. One of the methods generated the maximum observed decellularization with minimal impact on the ECM composition and organization. Furthermore, recellularization of the scaffold supported the proliferation of SCAP throughout the scaffold with differentiation into odontoblast-like cells near the dentinal walls. Thus, this study reports that human dental pulp from healthy extracted teeth can be successfully decellularized, and the resulting scaffold supports the proliferation and differentiation of SCAP. The future application of this form of an autograft in REPs can fulfill a yet unmet need for a suitable scaffold, potentially improving clinical outcomes and ultimately promoting the survival and function of teeth with otherwise poor prognosis.

Distinctive Genetic Activity Pattern of the Human Dental Pulp between Deciduous and Permanent Teeth

Human deciduous and permanent teeth exhibit different developmental processes, morphologies, histological characteristics and life cycles. In addition, their pulp tissues react differently to external stimuli, such as the pulp sensitivity test, dental trauma and pulp therapy materials. These suggest differences in gene expression and regulation, and in this study we compared gene-expression profiles of the human dental pulp from deciduous and permanent teeth. Pulp tissues from permanent premolars and deciduous molars aged 11–14 years were extirpated and mRNA was isolated for cDNA microarray analysis, and quantitative real-time PCR (qPCR). Other teeth were used for immunohistochemical analysis (IHC). Microarray analysis identified 263 genes with a twofold or greater difference in expression level between the two types of pulp tissue, 43 and 220 of which were more abundant in deciduous and permanent pulp tissues, respectively. qPCR analysis was conducted for eight randomly selected genes, and the findings were consistent with the cDNA microarray results. IHC confirmed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was broadly expressed in deciduous dental pulp tissue, but minimally expressed in permanent dental pulp tissue. Immunohistochemical analysis showed that calbindin 1 (CALB1), leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and gamma-aminobutyric acid A receptor beta 1 (GABRB1) were abundantly expressed in permanent predentin/odontoblasts, but only minimally expressed in deciduous dental pulp tissue. These results show that deciduous and permanent pulp tissues have different characteristics and gene expression, suggesting that they may have different functions and responses to therapies focused on pulp or dentin regeneration.

Comparative Gene Expression Analysis of the Coronal Pulp and Apical Pulp Complex in Human Immature Teeth

INTRODUCTION This study determined the gene expression profiles of the human coronal pulp (CP) and apical pulp complex (APC) with the aim of explaining differences in their functions. METHODS Total RNA was isolated from the CP and APC, and gene expression was analyzed using complementary DNA microarray technology. Gene ontology analysis was used to classify the biological function. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining were performed to verify microarray data. RESULTS In the microarray analyses, expression increases of at least 2-fold were present in 125 genes in the APC and 139 genes in the CP out of a total of 33,297 genes. Gene ontology class processes found more genes related to immune responses, cell growth and maintenance, and cell adhesion in the APC, whereas transport and neurogenesis genes predominated in the CP. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining confirmed the microarray results, with DMP1, CALB1, and GABRB1 strongly expressed in the CP, whereas SMOC2, SHH, BARX1, CX3CR1, SPP1, COL XII, and LAMC2 were strongly expressed in the APC. CONCLUSIONS The expression levels of genes related to dentin mineralization, neurogenesis, and neurotransmission are higher in the CP in human immature teeth, whereas those of immune-related and tooth development-related genes are higher in the APC.

Comparative Gene-Expression Analysis of Periodontal Ligament and Dental Pulp in the Human Permanent Teeth

There is no genetic activity information with the functions of dental pulp and periodontal ligament in human. The purpose of this study was to identify the gene-expression profiles of, and the molecular biological differences between periodontal ligament and dental pulp obtained from human permanent teeth. cDNA microarray analysis identified 347 genes with a fourfold or greater difference in expression level between the two tissue types 83 and 264, of which were more plentiful in periodontal ligament and dental pulp, respectively. Periodontal ligament exhibited strong expression of genes related to collagen synthesis (FAP), collagen degradation (MMP3, MMP9, and MMP13), and bone development and remodeling (SSP1, BMP3, ACP5, CTSK, and PTHLH). Pulp exhibited strong expression of genes associated with calcium ions (CALB1, SCIN, and CDH12) and the mineralization and formation of enamel and dentin (SPARC/SPOCK3, PHEX, AMBN, and DSPP). Among these genes, SPP1, SPARC/SPOCK3, AMBN, and DSPP were well known in dental research. However, the other genes are the newly found and it may help to find a good source of regenerative therapy if further study is performed.

Effect of Storage Media and Duration on Pulpal Cell Viability in Exfoliated Deciduous Teeth

Abstract Corresponding author : Prof. Heungkyu Son Department of Pediatric Dentistry, Yonsei University College of Dentistry, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, KoreaTel: +82-2-2228-3172 / Fax: +82-2-392-7420 / E-mail: HGSON@yuhs.ac Received July 3, 2013 / Revised November 14, 2013 / Accepted November 14, 2013 Ⅰ. 서 론흡수탈락된유치의치수조직 1) , 발거된선천치의치수 2) 및유치의치주인대 3) 에서줄기세포를얻을수있다고보고되었다. 탈락된유치에서얻은줄기세포는비침습적으로얻을수있으며,영구치의치수에서얻은줄기세포와비교하였을때더높은증식률, 분화능을보였다는보고 4) 가있었다. 이러한유치의장점때문에최근유치에서얻은조직을간엽성줄기세포의원천으로사용하는연구가활발하게이루어지고있다 1,5-7) .치아를용액에보관하는것에대한연구는영구치의이식이나재식을위한치아주변인대세포의생존 8-12) 과관련된연구가선행되었으며, 유치의경우에는재식이나이식하는경우가없어현재까지탈락유치의보관용액및조건에따른추출세포의생존률이나특성에대한보고는없는상태이다. 그러나유치줄기세포에대한관심이증가하면서치과및집에서발거된유치추출세포의생존률을높이는방법에대한연구가필요하다고판단되고, 가정에서불시에유치가탈락하였을경우주변에서쉽게구할수있는저장용액에유치를임시로보관한후줄기세포를추출할수있다면지금보다더많은유치를줄기세포연구에이용할수있을것이라고예상된다. ※This study was supported by a faculty research grant of Yonsei University College of Dentistry for 2013 (No. 6-2013-0096).�These authors equally contributed to this work.J Korean Acad Pediatr Dent 41(1) 2014ISSN (print) 1226-8496 ISSN (online) 2288-3819http://dx.doi.org/10.5933/JKAPD.2014.41.1.1

Cytokine Expression of Stem Cells Originating from the Apical Complex and Coronal Pulp of Immature Teeth

Introduction: The aim of this study was to measure and compare the expression levels of cytokines from developing apical complex cells (DACCs) and dental pulp stem cells (DPSCs) of the immature tooth. Methods: DPSC‐conditioned medium (CM) and DACCs‐CM were obtained from human young teeth, and 174 cytokines secreted from each CM were identified and compared. A cytokine membrane array and enzyme‐linked immunosorbent assay were used to measure and compare the expression levels of the cytokines. Immunocytochemistry targeting insulin‐like growth factor‐1 and neurotrophin‐3 was additionally performed. Results: There were statistically significant differences in the expression levels of 25 cytokines: 22 and 3 were expressed more strongly in DPSCs‐CM and DACCs‐CM, respectively. Odontoblast differentiation‐related cytokines were more strongly expressed in DPSCs‐CM, while cell‐proliferation–related cytokines were more strongly expressed in DACCs‐CM. Proinflammatory and anti‐inflammatory cytokines were predominantly expressed in DPSCs‐CM and DACCs‐CM, respectively. Conclusions: DPSCs may exert a stronger paracrine effect than DACCs on regeneration of the dentin–pulp complex, in terms of odontoblast differentiation. HighlightsThis study compared the expression levels of different cytokines using a cytokine membrane array, with additional enzyme‐linked immunosorbent assay and immunocytochemistry (ICC) tests performed to elucidate the paracrine effects of DPSCs and DACCs.Cytokines associated with odontoblast differentiation (NT‐3, BMP‐4, TGF‐&bgr;1, and TGF‐&bgr;3) were expressed more strongly in DPSCs than in DACCs. It could be suggested that cytokines expressed in DPSCs provide a more suitable environment for odontoblast differentiation for the same epigenetic signals.The secretion level of cell‐proliferation–related cytokines was higher for DACCs than for DPSCs. The stronger expression of proliferation cytokines observed in this study corroborates the high proliferation activity of the DACCs found in previous in vitro studies. Thus, the developing apical complex of the young tooth acts as a growth center via the proliferation ability of cytokines expressed in the periphery.This study is the first to attempt to measure and compare the expression levels of cytokines from DACCs and DPSCs of immature teeth. The findings have demonstrated that DPSCs may exert a stronger paracrine effect than DACCs on regeneration of the dentin–pulp complex, in terms of odontoblast differentiation.

Effect of Hypoxia-Inducible Factor 1α on Early Healing in Extraction Sockets

The aim of the present study was to investigate the effect of hypoxia-inducible factor 1α (HIF1A) on the early healing (4 weeks) of extraction sockets exhibiting partial loss of the labial bone. Two extraction sockets of the maxillary incisors from each of six dogs were assigned to two treatment modalities: deproteinized bovine bone mineral (i) with 10% collagen (DBBM-C) soaked with HIF1A and covered by a collagen membrane (CM) (HIF group) or (ii) treated with DBBM-C only and covered by a CM (control group). Microcomputed tomography revealed some degree of collapse of the labial contour. The totally augmented volume and new bone volume did not differ significantly between two groups (P > 0.05). The histological analysis revealed that the apical area of the socket was mostly filled with newly formed bone, while there was less newly formed bone in the coronal area and incomplete cortex formation. The histomorphometric analysis revealed that the area of newly formed bone was significantly larger in the HIF group than the control group (12.16 ± 3.04 versus 9.48 ± 2.01 mm2, P < 0.05), while there was no significant intergroup difference in the total augmented area. In conclusion, even though DBBM-C soaked with HIF1A enhanced histomorphometric bone formation, this intervention did not demonstrate superiority in preventing ridge shrinkage compared to DBBM-C alone. Clinical relevance of these findings should be further studied.