HIF-1α Overexpression Using a Protein Transduction Domain to Increase the Osteogenic Potential of SHED

Background: Hypoxia-Inducible Factor 1 (HIF-1) alpha (HIF-1α) is known to be expressed in hypoxia and affects stemness and bone differentiation. Protein Transduction Domains (PTDs) safely and effectively deliver proteins into cells. This study combined HIF-1α delta Oxygen-Dependent Degradation Domain–Transcription Modulation Domain (ntHIF-1α-ΔODD-TMD) with the PTD and then used it to treat Stem cells from Human Exfoliated Deciduous teeth (SHED) to determine its effect on stemness and bone differentiation. Methods: The SHED were treated using PDT-conjugated ntHIF-1α-ΔODD-TMD, and its effects on STRO-1 expression, cytotoxicity, stemness-related gene expression, and osteogenic differentiation were confirmed. These were also transferred to 5-week-old mice in order to confirm the effects in vivo after recellularization of SHED treated using ntHIF-1α-ΔODD-TMD on decellularized tooth specimens. After 9 weeks, they were sacrificed to confirm the expression of genes related to bone differentiation and angiogenesis. Results: ntHIF-1α-ΔODD-TMD increased the expression levels of STRO-1, HIF-1α-related genes, and stemness-related genes in SHED. Osteogenic differentiation was also increased both in vitro and in vivo. Conclusion: These results suggest that ntHIF-1α-ΔODD-TMD maintains or increases the stemness of SHED and also increases the osteogenic potential. SHED have previously been found to have excellent proliferative but low differentiation capacities compared with other cells. However, properly using ntHIF-1α-ΔODD-TMD increases the osteogenic potential of SHED, indicating the potential of using SHED as a useful source for hard-tissue regeneration.ope

분리방법에 따른 유치 줄기세포의 특성

Dept. of Dental Science/석사Stem 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. Dental pulp stem cells were isolated from 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, 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 HE staining, immunohistochemical staining, qPCR, and alkaline phosphatase analysis. The cell-proliferation potential, colony-forming ability, and Stro-1 and CD146 expression were higher in e-SHED. While the in vitro adipogenic differentiation potential was greater in e-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. 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.ope

Decellularized human periodontal ligament for periodontium regeneration

Regenerating the periodontal ligament (PDL) is a crucial factor for periodontal tissue regeneration in the presence of traumatized and periodontally damaged teeth. Various methods have been applied for periodontal regeneration, including tissue substitutes, bioactive materials, and synthetic scaffolds. However, all of these treatments have had limited success in structural and functional periodontal tissue regeneration. To achieve the goal of complete periodontal regeneration, many studies have evaluated the effectiveness of decellularized scaffolds fabricated via tissue engineering. The aim of this study was to fabricate a decellularized periodontal scaffold of human tooth slices and determine its regeneration potential. We evaluated two different protocols applied to tooth slices obtained from human healthy third molars. The extracellular matrix scaffold decellularized using sodium dodecyl sulfate and Triton X-100, which are effective in removing nuclear components, was demonstrated to preserve an intact structure and composition. Furthermore, the decellularized scaffold could support repopulation of PDL stem cells near the cementum and expressed cementum and periodontal-ligament-related genes. These results show that decellularized PDL scaffolds of human teeth are capable of inducing the proliferation and differentiation of mesenchymal stem cells, thus having regeneration potential for use in future periodontal regenerative tissue engineering.ope

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

If it is possible to preserve and culture cells from exfoliated deciduous teeth in a readily available storage medium within each family, more stem cells would be obtained. This research is about the effect of storage media and time on pulpal cell viability of exfoliated deciduous teeth. 330 exfoliated deciduous teeth were randomly divided into 11 groups; fresh group, dry group, groups stored in cell culture medium (2, 4, 7 days each), in milk (2, 4, 7 days each), and in saline (2, 4, 7 days each). Primary culture of pulpal cells was conducted in each group and the success rates were compared by calculating the number of teeth with viable cells. The result of primary culture shows that the success rate decreases as the time of storage gets longer. There was no statistical difference between groups stored in the cell culture medium, milk, and saline for 2 and 4 days. However, the groups stored in milk and saline for 7 days showed dramatic decrease in success rate compared to the group stored in the cell culture medium. In conclusion, exfoliated or extracted deciduous teeth can be used to culture pulpal cells when they are stored in milk and saline for a certain period of time; however obtaining viable pulpal cells becomes harder as the storage time gets longer.ope

Deferoxamine Reduces Inflammation and Osteoclastogenesis in Avulsed Teeth

Replacement and inflammatory resorption are serious complications associated with the delayed replantation of avulsed teeth. In this study, we aimed to assess whether deferoxamine (DFO) can suppress inflammation and osteoclastogenesis in vitro and attenuate inflammation and bone resorption in a replanted rat tooth model. Cell viability and inflammation were evaluated in RAW264.7 cells. Osteoclastogenesis was confirmed by tartrate-resistant acid phosphatase staining, reactive oxygen species (ROS) measurement, and quantitative reverse transcriptase-polymerase chain reaction in teeth exposed to different concentrations of DFO. In vivo, molars of 31 six-week-old male Sprague-Dawley rats were extracted and stored in saline (n = 10) or DFO solution (n = 21) before replantation. Micro-computed tomography (micro-CT) imaging and histological analysis were performed to evaluate inflammation and root and alveolar bone resorption. DFO downregulated the genes related to inflammation and osteoclastogenesis. DFO also reduced ROS production and regulated specific pathways. Furthermore, the results of the micro-CT and histological analyses provided evidence of the decrease in inflammation and hard tissue resorption in the DFO group. Overall, these results suggest that DFO reduces inflammation and osteoclastogenesis in a tooth replantation model, and thus, it has to be further investigated as a root surface treatment option for an avulsed tooth.ope

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.ope

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.ope

Phosphocholine derivative stimulates megakaryopoiesis

Platelets are produced from megakaryocytes through complex events, megakaryopoiesis which includes increased nucleus numbers through the endomitosis (up to 128N) and cytoplasmic maturation leading to the production of proplatelets and release of platelets. A new substance analogously related to the compounds in deer antler, phosphocholine derivative, was synthesized. I investigated the effect of phosphocholine derivative on the signal pathways in megakaryopoiesis of human leukemic cell lines K562 and HEL cells. To enhance the megakaryopoiesis by phosphocholine derivative, we used co-culture system using OP9 stromal cells. Treatment of phosphocholine derivative in K562 and HEL cells induced morphological and biochemical changes, which are characteristic for megakaryocytes. Phosphocholine derivative-treated cells resulted in increased megakaryocyte markers, CD41, CD61, CD42a and CD42b compared to control cells. The cell size and nucleus number were increased in phosphocholine derivative-treated cells. To elucidate mechanism underlying megakaryopoiesis, I performed two inhibitory experiments. In phosphocholine derivative-treated K562 cells were inhibited PI3K inhibitor, LY294002 and mTOR inhibitor, rapamycin. In phosphocholine derivative-treated HEL cells were inhibited PI3K inhibitor, LY294002. These results suggest that megakaryopoiesis in phosphocholine derivative-treated K562 and HEL cells went through PI3K and mTOR signaling pathway. To understand the genetic events during megakaryopoiesis, I carried out Real-time PCR analysis in differentiated K562 cells differentiated. In previous studies, 10 genes were chosen as genes whore expression change more than 10 fold by a gene chip analysis. These genes, geneD, geneE, geneG, geneI, geneK, geneL, geneP2, geneP3, geneT and geneU were shown similar pattern between a gene chip data and Real-time PCR data. Among these genes, geneE, geneL, geneP3, geneU were chosen for silencing experiments. Megakaryocytic development, such as polyploidization and expressions of megakaryocytic specific markers, CD41, were suppressed when geneL and geneP3 genes were knock-downed by shRNA. To investigate gene overexpression in megakaryopoiesis, geneL gene was overexpressed by overexpression DNA. ;혈소판은 megakaryopoiesis라는 복잡한 과정을 통해 거핵구에 의해서 생성된다. Megakaryopoiesis는 핵 내 유사분열을 통한 핵의 수 증가, 전-혈소판의 형성을 위한 세포질의 성숙으로 이루어진다. 녹용으로부터 추출된 물질의 유도체인 새로운 합성 화합물 phosphocholine 유도체로 인간 백혈구 세포주인 K562, HEL 세포에서의 거핵구 발달 작용을 밝히고자 하였다. 거핵구로의 분화와 성숙을 증진시키기 위해 기질 세포인 OP9 세포를 사용하여 함께 배양하였다. Phosphocholine 유도체에 의해 분화된 K562, HEL 세포는 거핵구의 특징적인 형태적 변화와 생화학적 변화를 보였다. Phosphocholine 유도체가 처리된 세포는 거핵구 표지인자인 CD41, CD61, CD42a, CD42b를 발현하였고, 세포 크기와 핵 수도 증가하였다. 거핵구 분화 과정에서의 신호 전달과정을 밝히기 위해 두 종류의 저해제를 사용하여 세포신호 전달 저해 실험을 수행하였다. Phosphocholine 유도체를 처리한 K562 세포에서는 PI3K 저해제인 LY294002와 mTOR 저해제인 rapamycin에 의해 명확하게 저해되었다. Phosphocholine 유도체를 처리한 HEL 세포에서는 PI3K 저해제인 LY294002에 의해 명확하게 저해되었고, mTOR 저해제인 rapamycin에 의해서는 저해 정도가 약하게 나타났다. 거핵구 발달 과정에서의 유전자 변화를 조사하기 위해 phosphocholine 유도체를 처리하여 분화한 K562 세포의 유전자 분석을 수행한 결과를 바탕으로 10배 이상 증가한 유전자 분석을 수행하였다. 10배 이상 증가한 유전자D, 유전자E, 유전자G, 유전자I, 유전자K, 유전자L, 유전자P2, 유전자P3, 유전자T, 유전자U를 선택하여 실시간 PCR을 수행하였다. 우리는 실시간 PCR을 통해서도 이들 유전자가 유사한 발현양상을 보인다는 것을 확인하였다. 그리고 shRNA를 이용하여 이 중 4가지 유전자인 유전자E, 유전자L, 유전자P3, 유전자U의 발현을 억제함으로써, 거핵구 발달 과정에서 거핵구의 생성과 표지인자 발현이 감소되는 것을 확인하였다. 특히 유전자L, 유전자P3는 거핵구 발달과정에 지배적인 영향을 준다는 것을 확인할 수 있었다.1. Introduction 1 2. Materials and Methods 6 2.1. Reagents 6 2.2. In vivo megakaryopoiesis 7 2.3. Growth assay 8 2.4. Flowcytometry 8 2.5. RNA isolation 8 2.6. Quantitative Real-time PCR 9 2.7. Synthesis of shRNA(small hairpin RNA) 10 2.8. Transfection of shRNA(small hairpin RNA) 12 2.9. Synthesis of overexpression DNA 13 2.10. Transfection of overexpression DNA 14 3. Results 15 3.1. Growth of phosphocholine derivative-treated K562 and HEL cell lines 15 3.2. Morphological changes of phosphocholine derivative-treated leukemic cell lines 15 3.3. Time-dependent CD41, CD61, CD42a and CD42b expression of phosphocholine derivative-treated K562 and HEL cells 15 3.4. Polyploidization of phosphocholine derivative-treated K562 and HEL cell lines 18 3.5. Inhibition of differentiation by two inhibitors 22 3.6. Expression of genes in megakaryocytic development 27 3.7. Inhibition of gene in megakaryocytic development by shRNA 28 3.8. Overexpression of genes in megakaryocytic development by overexpression DNA 32 4. Discussion 38 5. References 41 논문개요 4

Analysis of anti-arthritic effect of ursolic acid using acute and chronic inflammation animal models

학위논문(석사) --서울대학교 대학원 :수의학과,2007.Maste