Mechanism of Chir99021 regulating Wnt / β⁃catenin signaling pathway inhibiting osteogenic differentiation of rat dental pulp stem cells / 解剖学报

Objective To explore the effect and mechanism of Chir99021 on osteogenic differentiation of rat dental pulp stem cells. Methods Primary rat dental pulp stem cells were isolated from rat dental pulp and verified by fluorescence immunoassay. Different concentrations of Chir99021 were set, and the cell proliferation was detected by CCK⁃8 to select the optimal concentration. Osteogenic differentiation was detected by alizarin red staining. The expression of osteogenic differentiation related genes and proteins recombinant wingless type MMTV integration site famity member 1 (Wnt1), Wnt3a and Wnt3a β⁃expression of catenin, axis inhibition protein 2(Axin 2), dentin sialophosphoprotein(OCN) and dentin matrix acidic phosphoprotein 1(DMP1) was detected by Real⁃time PCR and Western blotting. Results The positive expression of dentin sialophosphoprotein (DSPP) and vimentin indicated that rat dental pulp stem cells were successfully isolated. After osteogenic induction of rat dental pulp stem cells, calcium deposits significantly increased with the addition of glycogen synthase kinase⁃3β(GSK⁃3β) inhibitor Chir99021, calcium deposits were significanted reduced. After osteogenic differentiation of rat dental pulp stem cells, the expression of Wnt1, Wnt3a, β⁃catenin, Axin2, OCN and DMP1 increased, while the expression of Wnt1, Axin2, OCN and DMP1 decreased with the addition of Chir99021. Conclusion Chir99021 can inhibit the osteogenic differentiation of rat dental pulp stem cells after 7 days of induction.

Assessment of impact of varied preconditioning factors, used in isolation and combination, for enhancement of cytokine secretions in stem cell conditioned medium

Stem cell conditioned medium exhibits a huge regenerative potential but low concentration of cytokines, highdevelopment cost, and scalability challenges are major deterrents to product advances. This research studiedimpact on vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) secretions afterpreconditioning human dental pulp stem cells (HDPSCs) with diverse factors, viz., Deferoxamine (250 µM),Epidermal Growth Factor (EGF) (2 ng/ml), Angiotensin I (5mM) and Insulin Transferrin Selenium (ITS) (1%),and Niacinamide (5 mM). Additionally, advantage of using “pooled population” of HDPSCs was ascertained.HDPSCs were incubated under standard culture conditions using optimized growth media. On reaching 80%–85% confluency, media was discarded and fresh serum free media with preconditioning factors, initially oneat a time and subsequently in combination, was added. Spent media, collected after 48 hours of incubation,was used for enzyme-linked immunosorbent assay testing of selected cytokines. In comparison to control,preconditioning factors, used in isolation showed varied but significant upregulation in the secretion of VEGFbut down regulation of HGF. When used in combination, VEGF secretion increased almost 10 times of controlwith no significant change in HGF level. Results demonstrated importance of choosing right preconditioningfactors and right interplay of preconditioning factors to enhance secretions. This research paves the path todevelop an effective and commercially viable conditioned medium that can be used as “biological active” forpharmaceuticals and for developing “customized” serum free medium enriched with selected cytokines

Transcriptome sequencing technology and its application in oral diseases, dental implants and regeneration / 中国组织工程研究

BACKGROUND: Since the application of transcriptome sequencing technology has made remarkable achievements in the research of melanoma and breast cancer, transcriptome sequencing technology has become a hot research method in scientific research and widely used in the field of stomatology. OBJECTIVE: To review the development of transcriptome sequencing technology and its application in various disciplines of stomatology through searching, screening and reading literature. METHODS: A search of CNKI, CBM and PubMed was performed for relevant literature published from 2015 through 2020. The search terms were “transcriptome, sequencing technology, RNA-seq, microarray, oral cancers, OSCC, periodontal, caries, pulp disease, tooth development, DPSCs, PDLSCs, orthodontics, implant” in English and Chinese, respectively. According to the inclusion and exclusion criteria, 72 literatures were included to review the development of transcriptome sequencing technology and its application in the field of stomatology. RESULTS AND CONCLUSION: In the development of transcriptome sequencing technology, RNA-seq technology has the greatest advantage in scientific research, because of its high accuracy, high throughput and low price. In the field of stomatology, this technology has been used in the research of oral squamous cell carcinoma, periodontitis, dental pulp disease, tooth regeneration, orthodontics and dental implantation, and has achieved some achievements. However, the current research on the same kind of disease does not reflect the relationship between the various studies, and the research content is limited. It is believed that more discoveries can be yielded in stomatology by exploring the relationship between different studies and expanding the research content.

Effect of mechanical stimulation on dental pulp stem cells / 中国组织工程研究

BACKGROUND: Mechanical stimulation plays a necessary regulatory role in developing and repairing many organs and tissues in the human body. Except for biochemical factors, mechanical factors are considered as key regulatory factors that affect the behavior and function of dental pulp stem cells. OBJECTIVE: To review the role and effect of cellular mechanical stimulation on the biological behavior of dental pulp stem cells. METHODS: PubMed, Embase, Medline and CNKI databases were searched for relevant literatures using the keywords of “human dental pulp stem cells (hDPSCs), mechanical strain, mechanical stretch, mechanical tension, shear stress, cell proliferation, osteogenesis differentiation” in English and Chinese, respectively. Fifty-six articles were finally eligible for review, which were closely related to the proliferation and differentiation of dental pulp stem cells under cellular mechanical stimulation. RESULTS AND CONCLUSION: Cellular mechanical stimulation is an important biological factor affecting cell proliferation, differentiation, apoptosis and protein expression. Dental pulp stem cells are mesenchymal stem cells derived from the dental pulp tissue, and their biological behaviors are also affected by cellular mechanical stimulation. Cellular mechanical stimulation is involved in the proliferation, odontogenesis/osteogenesis of dental pulp stem cells. When the dentin is subjected to fluid flow forces, mechanoreceptors are activated to regulate and maintain the integrity of tooth structure. Signal pathways that mediate the biological behavior of dental pulp stem cells include MAPK, Wnt, Akt, BMP-7, and Nrf2/HO-1, which are involved in promoting and inhibiting the proliferation and odontogenesis/osteogenesis of dental pulp stem cells to varying degrees.

Prostaglandin e2 induces expression of mineralization genes by undifferentiated dental pulp cells

Abstract Prostaglandin E2 (PGE2) is a lipid mediator usually released during inflammation. This study aimed to investigate the potential of soluble or microsphere-loaded PGE2 on inducing differentiation of dental pulp stem cells. PGE2-loaded microspheres (MS) were prepared using an oil-in-water emulsion solvent extraction-evaporation process and were characterized. Mouse dental pulp stem cells (OD-21) were stimulated with soluble or PGE2-loaded MS (0.01 and 0.1 µM). Cell viability was determined by MTT colorimetric assay. Ibsp, Bmp2 and Runx2 expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after 3, 6, and 24 h. The results showed that the soluble PGE2 reduced dental pulp stem cells viability after 24 h of stimulation whereas PGE2-loaded MS did not. Soluble PGE2 up-regulated Ibsp and Bmp2 at 3 h, differently from PGE2-loaded MS. On the other hand, PGE2-MS induced Bmp2 and Runx2 at 6 h and Ibsp at 24 h. In conclusion, our in vitro results show that PGE2, soluble or loaded in MS are not cytotoxic and modulateIbsp,Bmp2, andRunx2gene expression in cultured OD-21 cells.

Resumo A Prostaglandina E2 (PGE2) é um mediador lipídico comumente liberado durante a inflamação. Este estudo teve como objetivo investigar o potencial da PGE2, solúvel ou na forma de microesferas, na diferenciação de células-tronco de polpa dentária. Microesferas de PGE2 (MS) foram preparadas por meio do processo de extração/evaporação de solvente em emulsão óleo-em-água e foram caracterizadas. Células-tronco de linhagem derivadas da polpa dentária de camundongos (OD-21) foram estimuladas com PGE2 solúvel ou na forma de MS (0,01 e 0,1 µM). A citotoxicidade foi determinada por ensaio colorimétrico MTT. A expressão gênica de Ibsp, Bmp2 e Runx2 foi avaliada por meio de reação em cadeia da polimerase em tempo real (qRT-PCR) após 3, 6 e 24 h. Os resultados mostraram que as MS contendo PGE2 não foram citotóxicas para células-tronco da polpa dentária, enquanto MS vazias ou PGE2 solúvel reduziram a viabilidade celular após 24 h de estimulação. PGE2 solúvel aumentou a expressão de Ibsp e Bmp2 após 3 h, diferentemente da PGE2 em MS. Por outro lado, PGE2-MS induziram a expressão de Bmp2 e Runx2 após 6h de estímulo e Ibsp após 24h. Em conclusão, nossos resultados in vitro demonstram que a PGE2, solúvel ou em microesferas não são citotóxicas e modulam a expressão gênica deIbsp,Bmp2 eRunx2em células OD-21.

Characterization of Human Dental Pulp Cells from Supernumerary Teeth by Using Flow Cytometry Analysis / 대한소아치과학회지

The aim of this study was to analyze cells from human dental pulp tissue of impacted supernumerary teeth as stem cells with flow cytometry. Human dental pulp cells from 15 supernumerary teeth were identified their characteristics as stem cells by expression of mesenchymal stem cell markers through flow cytometry analysis at passage 3 and passage 10. Cluster of differentiation (CD) 73, CD 90, CD 34, CD 45 and STRO-1 cell surface markers were used to figure out characteristics of dental pulp stem cells from supernumerary teeth. At passage 3, the cell population showed positive expression of CD 73, CD90 and STRO-1, lacked expression of CD 34 and CD 45. At passage 10, CD 73, CD 90 and STRO-1 showed positive expression while CD 34 and CD 45 showed negative expression. This study indicated that dental pulp stem cells of supernumerary teeth had the properties of mesenchymal stem cells at both early and late passage. Impacted supernumerary teeth could be considered as a noble source of stem cells because of rapid growth and maintaining characteristics of stem cells until late passage.

Differentiation of human dental pulp stem cells into corneal epithelial-like cells by conditioned medium / 解剖学报

Objective: To investigate the feasibility of inducing human dental pulp stem cells (DPSCs) to differentiate into corneal epithelial-like cells by conditioned medium (CM). Methods: DPSCs were isolated and identified by flow cytometry. The effect of basal medium (BM) and different CM on the proliferation activity of DPSCs was detected by cell counting kit-8(CCK-8) assay. DPSCs were induced by 30%CM, 60%CM, 90%CM. The cells cultured in BM were negative control group. Corneal epithelial cells markers cytokeratin 3 (CK3) and cytokeratin 12 (CK12) were detected by immunofluorescence assay. Results: There was no significant difference in the proliferation activity of DPSCs between BM group and different CM group (P > 0. 05). Cells in the 30%, 60%, 90% CM group did not express CK3 after 3 days induction, cells in the 60%and the 90%CM group began to express CK12; CK3 and CK12 were expressed in the 30%, 60%, 90%CM group after 7 days; At the 11th and 14th day, cells continued to express CK3 and CK12 in the 30%, 60%, and 90% CM groups. No expression of CK3 and CK12 was observed in the BM group. Conclusion: DPSCs are capable of differentiating into corneal epithelial-like cells under the induction of CM.

Research progress of pulpotomy and crown pulp regeneration / 口腔疾病防治

@#Crown pulp regeneration is a method to replace the pulp-capping agent with stem cells and scaffold structures, which are placed on the section of healthy root pulp tissue after pulpotomy to regenerate the pulp-dentin complex in the crown. This paper reviews the significance, physiological basis, application and challenges of crown pulp regeneration to provide new ideas for the study of pulp regeneration. The results of a literature review show that the combination of traditional pulpotomy, stem cell biology and tissue engineering, as well as the regeneration of crowns and pulp by using the tissue repair potential of healthy root pulp can effectively promote the regeneration of dentin and parts of pulp. In recent years, with the development of research on pulp regeneration, many challenges have been gradually revealed. It is necessary not only to select the treatment methods that can promote the proliferation and differentiation of dental pulp stem cells safely and effectively but also to continue to explore the scaffold materials suitable for the structural and functional diversity of the pulp chamber.

Differentiation of CD31-Positive Vascular Endothelial Cells from Organoid Culture of Dental Pulp Stem Cells

The mesenchymal stem cells (MSCs) that reside in dental tissues hold a great potential for future applications in regenerative dentistry. In this study, we used human dental pulp cells, isolated from the molars (DPCs), in order to establish the organoid culture. DPCs were established after growing pulp cells in an MSC expansion media (MSC-EM). DPCs were subjected to organoid growth media (OGM) in comparison with human dental pulp stem cells (DPSCs). Inside the extracellular matrix in the OGM, the DPCs and DPSCs readily formed vessel-like structures, which were not observed in the MSC-EM. Immunocytochemistry analysis and flow cytometry analysis showed the elevated expression of CD31 in the DPCs and DPSCs cultured in the OGM. These results suggest endothelial cell-prone differentiation of the DPCs and DPSCs in organoid culture condition.

Characteristics of Supernumerary Tooth-derived Pulp Cells during Long-term Culture / 대한소아치과학회지

This study was conducted to investigate the characteristics of subculture times in the early, middle, and late passages by measuring the time under subculture until it was judged that the supernumerary tooth-derived pulp stem cells (sDPSCS) were no longer proliferating. Three supernumerary teeth from two healthy six-years old boys were extracted and stem cells were obtained from the pulp tissue. This was called SNT1 (supernumerary tooth 1), SNT2, and the supernumerary tooth from another child was named SNT3. SNT1 and 2 were subcultured at the same time and SNT3 was subcultured a little faster. The mean time of complete subculture was 3.6 ± 1.1 days. Total passages were cultured up to 23.3 ± 0.6 and took 83 days. These were divided into three groups based on the passage. The increase rate of time taken in subculture between group I and group II was 11.9%, but the rate between group II and group III was 28.6%, which was 2.4 times increased. The time taken between passages during long-term subculture up to 22 passages shows a regressive pattern y = 0.1169x + 2.25 and y = 0.1169x + 2.0. In conclusion, the passage time of SPSCs increased in late passages, and it shows a similar pattern.

Effects of nanoscale ridge/groovepattern arrayed surface on in vitro differentiation of multi-potent pulp cells derived from human supernumerary teeth

Human dental pulp stem cells (DPSCs) are multi-potent mesenchymal stem cells that have several differentiation potentials. An understanding of the tissues that differentiate from these cells can provide insights for future regenerative therapeutics and tissue engineering strategies. The mesiodens is the most frequent form of supernumerary tooth from which DPSCs can differentiate into several lineages similar to cells from normal deciduous teeth. Recently, it has been shown that nanoscale structures can affect stem cell differentiation. In our presentstudy, we investigated the effects of a 250-nm nanoscale ridge/groove pattern array on the osteogenic and adipogenic differentiation of dental pulp cells from mesiodenscontaining human DPSCs. To this end, the expression of lineage specific markers after differentiation induction was analyzed by lineage specific staining and RT-PCR. The nanoscale pattern arrayed surface showed apositive effect on the adipogenic differentiation of DPSCs. There was no difference between nanoscale pattern arrayed surface and conventional surface groups onosteogenic differentiation. In conclusion, the nanoscale ridge/groove pattern arrayed surface can be used to enhance the adipogenic differentiation of DPSCs derived from mesiodens. This finding provides an improved understanding of the effects of topography on cell differentiation as well as the potential use of supernumerary tooth in regenerative dental medicine.

Morphological evaluation during in vitro chondrogenesis of dental pulp stromal cells

OBJECTIVES: The aim was to confirm the stem cell-like properties of the dental pulp stromal cells and to evaluate the morphologic changes during in vitro chondrogenesis. MATERIALS AND METHODS: Stromal cells were outgrown from the dental pulp tissue of the premolars. Surface markers were investigated and cell proliferation rate was compared to other mesenchymal stem cells. Multipotency of the pulp cells was confirmed by inducing osteogenesis, adipogenesis and chondrogenesis. The morphologic changes in the chondrogenic pellet during the 21 day of induction were evaluated under light microscope and transmission electron microscope. TUNEL assay was used to evaluate apoptosis within the chondrogenic pellets. RESULTS: Pulp cells were CD90, 105 positive and CD31, 34 negative. They showed similar proliferation rate to other stem cells. Pulp cells differentiated to osteogenic, adipogenic and chondrogenic tissues. During chondrogenesis, 3-dimensional pellet was created with multi-layers, hypertrophic chondrocyte-like cells and cartilage-like extracellular matrix. However, cell morphology became irregular and apoptotic cells were increased after 7 day of chondrogenic induction. CONCLUSIONS: Pulp cells indicated mesenchymal stem cell-like characteristics. During the in vitro chondrogenesis, cellular activity was superior during the earlier phase (within 7 day) of differentiation.

Expression of DSP,DMP1,CBFA1,BMP2 in rat dental pulp stem cells / 实用口腔医学杂志

Objective:To examine the expression pattern of DSP,DMP1,CBFA1,BMP2 in rat dental pulp stem cells(RDPSCs).Methods:Immunohistochemical staining was carried out using antibodies against DSP,DMP1, CBFA1 ,BMP2 in rat dental pulp stem cells. Mineralization was induced in the RDPSCs and expression of DSP and DMP1 was measured after induction.Results:CBFA1 and BMP2 were positive in RDPSCs. Only a few RDPSCs were stained positive for DMP1. DSP expression was observed in the minority of these cells. However, the majority of the RDPSCs were found strongly positive for DSP and DMP1 after mineralization induction.Conclusion:Positive expression of CBFA1 and BMP2 indicates the premature nature of RDPSCs. The dentin-specific expression of DSP demonstrates that the RDPSCs can differentiate along odontoblastic lineage.