Effect of SHP2 knockdown on the proliferation and osteogenic differentiation of human periodontal ligament stem cells under inflammatory environment / 口腔疾病防治

Objective @# The purpose of this study was to clarify the regulatory effect and mechanism of Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) on human periodontal ligament stem cell (hPDLSC) proliferation and osteogenic differentiation under inflammatory environment and to provide a new target for the treatment of periodontitis. @*Methods@#SHP2 was knocked down in hPDLSCs, and the transfection efficiency of SHP2 was detected by RT-qPCR and Western blot. An in vitro inflammatory environment was created using tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The effect of SHP2 knockdown on hPDLSC viability under normal and inflammatory conditions was detected by CCK-8, and the osteogenic capacity of hPDLSCs under normal and inflammatory conditions was detected by ALP staining, ALP activity, ARS staining, RT-qPCR and Western blot. The mechanism by which SHP2 knockdown affected the MAPK pathway and its downstream NF-κB pathway under inflammatory conditions was assessed by Western blot. @*Results@# Green fluorescence was observed after transfection for 72 h, and the titer of SHP2 shRNA recombinant lentivirus was 2.9×108 TU/mL. SHP2 expression was significantly downregulated in lentivirus-transfected cells, as demonstrated by Western blot and RT-qPCR (P<0.001). SHP2 knockdown inhibited hPDLSC proliferation to a certain extent and increased the expression of early osteogenic markers under normal conditions, including increased ALP activity and increased ALP and COL-1 expression (P<0.05). However, SHP2 knockdown exerted no effect on mineralized nodule formation. In the TNF-α- and IL-1β-induced inflammatory environment, SHP2 knockdown exerted no effect on hPDLSC proliferation (P>0.05). Osteogenic markers were upregulated (P<0.05), and mineralized nodules were significantly increased (P<0.05) after SHP2 knockdown. Western blot analysis showed that p65 phosphorylation and IκB-α degradation were reduced in SHP2-knockdown hPDLSCs in the inflammatory environment. Moreover, SHP2 knockdown significantly inhibited the expression of p-p38 and p-JNK MAPK, which represent pathways upstream of the NF-κB pathway (P<0.05). @*Conclusion @# SHP2 knockdown did not affect cell viability but promoted the osteogenic potential of hPDLSCs by inhibiting the MAPK/NF-κB-mediated signaling pathway under inflammatory environment.

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.

Effects of bacterial lipopolysaccharide on the mineralization and differentiation of periodontal ligament stem cells / 医学研究生学报

Objective Periodontal tissue engineering has shown a highlight prospect in the treatment of periodontitis,but the related clinical experiments have not achieved the predetermined goal. In this study,we analyzed the reasons for the limited clinical efficacy of periodontal tissue engineering. Methods We primarily cultured the periodontal tissue from the young permanent teeth extracted for or-thodontic treatment,isolated periodontal ligament stem cells (PDLSCs),and transplanted the well-grown third-generation PDLSCs onto the fluorapatite-polycaprolactone (FA-PCL) nanofiber scaffolds and PCL nanofiber scaffolds. We randomly divided the cells into groups A (cultured with 10 ng/mL porphyromonas gingivalis lipopo-lysaccharide (Pg-LPS)+FA-PCL),B (cultured with 10 ng/mL PG-LPS+PCL),C (cultured with 10 μg/mL PG-LPS+FA-PCL),D (cultured with 10 μg/mL PG-LPS+PCL),E (cultured with FA-PCL),and F (cultured with PCL),and observed their proliferation,differentiation and mineralization. Results The PDLSCs adhered and grew well after transplanted onto the nanofiber scaffolds and their proliferation significantly increased in groups A and B but decreased in C and D as compared with E and F. At 7 days,the expres-sions of ALP and mineralization-related genes runx2 and SPP1 in the PDLSCs were significantly higher in group E than in the other five groups (P<0.05),but higher groups A and C than in B and D as well as in A than in C. At 28 days,alizarin red and Von Kossa stai-ning showed a higher positivity in group E than in the other five groups,but higher groups A and C than in B and D as well as in A than in C. Conclusion The inflammatory environment not only affects the proliferation of PDLSCs,but also inhibits their differentiation and mineralization. The FA-PCL scaffold can reduce the cytotoxic effect of PG-LPS.

Valproic Acid Modulates the Multipotency in Periodontal Ligament Stem Cells via p53-Mediated Cell Cycle

Human periodontal ligament stem cells (PDLSCs), a type of mesenchymal stem cell, are a promising source for dental regeneration and are identified in human periodontal ligaments from extracted third molars. Valproic acid (VPA) is a histone deacetylase inhibitor that has been used as a wide-spectrum antiepileptic drug and a medication for mood disorders. VPA has shown several effects on increasing the pluripotency of embryonic stem cells and controlling osteogenic differentiation, besides the prevention of seizures. However, its effect on proliferation and osteogenesis depends on the cell type and concentration. The aim of this study was to investigate the effects of cyclic and constant VPA treatment on PDLSCs. Proliferation and apoptosis of PDLSCs were determined with cyclic and constant VPA treatment. In cemento/ osteogenic differentiation, osteogenic markers decreased significantly after cyclic treatment with 0.5 mM VPA. In contrast, VPA enhanced osteogenic differentiation after constant treatment. With cyclic VPA treatment, p53 levels related to apoptotic pathway decreased to induce proliferation. These findings indicated that VPA has different roles in proliferation and differentiation of PDLSCs in vitro and in vivo via p53-related pathway.

Methods of Isolation and Characterization of Stem Cells from Different Regions of Oral Cavity Using Markers: A Systematic Review

BACKGROUND: Periodontitis is a destructive inflammatory disorder of the periodontium caused by the destruction of periodontal tissues namely the PDL, cementum, alveolar bone, and gingiva. Once these tissues are lost, the foremost goal of periodontal therapy is to regenerate the diseased tissues if possible to their original form, architecture, and function. Various regenerative procedures were employed and still a gap was found in achieving the goal. As stem cells are characterized by their ability to self-renew and differentiate to produce specialized cells, there could be a possibility of using them for regenerative therapy. Recently, dental tissues such as the PDL, the dental pulp and the tooth follicle have been recognized as readily available sources of adult stem cells. AIM: The aim was to identify the various sources and methodologies in isolation of stem cells from human oral cavity and its differentiation into various lineages using markers. MATERIALS AND METHODS: The electronic databases PUBMED, GOOGLE SCHOLAR, SCIENCE DIRECT, COCHRANE LIBRARY along with a complimentary manual search of all periodontics journal till the year 2016. Thirteen articles were selected on the basis of the inclusion criteria. Isolation of stem cells from oral cavity through various methods has been evaluated and similarly characterization to different lineages were tabulated as variables of interest. They included human in-vitro and ex-vivo studies. RESULTS: The results showed that PDLSC's and pulpal stem cells are the most common source from where stem cells were isolated. Each source has used different methodology in isolating the stem cells and it was found that STRO-1 was the commonly used marker in all the studies mentioned. CONCLUSIONS: The studies showed that there is no standard protocol existed in isolating the stem cells from different sources of oral cavity. Moreover, there was no standard marker or methodology used in characterization.

Effects of ginsenoside Rg-1 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate the effects of ginsenoside Rg-1 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and to explore the possible application on the alveolar bone regeneration.</p><p><b>METHODS</b>To determine the optimum concentration, the effects of ginsenoside Rg-1 ranging from 10 to 100 μmol/L were evaluated by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, alkaline phosphatase activity and calcium deposition. Expressions of runt-related transcription factor 2, collagen alpha-2(I) chain, osteopontin, osteocalcin protein were examined using real-time polymerase chain reaction.</p><p><b>RESULTS</b>Compared with the control group, a certain concentration (10 μmol/L) of the Rg-1 solution significantly enhanced the proliferation and osteogenic differentiation of hPDLSCs (P<0.05). However, concentrations that exceeds 100 μmol/L led to cytotoxicity whereas concentrations below 10 nmol/L showed no significant effect as compared with the control.</p><p><b>CONCLUSION</b>Ginsenoside Rg-1 can enhance the proliferation and osteogenic differentiation of hPDLSCs at an optimal concentration of 10 μmol/L.</p>

Effects of TGF-β1 on the migration,adhesion and proliferation of periodontal ligament stem cells / 西安交通大学学报(医学版)

Objective To evaluate the effects of transforming growth factor β1 (TGF-β1 )on migration, adhesion and proliferation of periodontal ligament stem cells (PDLSCs)and explore the mechanisms of PDLSCs-induced periodontal remodeling.Methods PDLSCs were isolated and identified from human teeth.The effect of TGF-β1 on migration of PDLSCs was evaluated using transwell migration assay.Cells attachment assay was used to test the effect of TGF-β1 on the adhesion of PDLSCs.In addition,the effect of TGF-β1 on the proliferation of PDLSCs was evaluated by MTT and cell growth rate assay.Results The results showed that TGF-β1 induced the migration of PDLSCs in a dose-dependent manner,improved the adhesion and proliferation of PDLSCs.So we propose that TGF-β1 may promote periodium remodeling by inducing PDLSCs migration,following adhesion and proliferation in these areas.Conclusion This study demonstrated for the first time that TGF-β1 increases the adhesion and migration of PDLSCs in vitro .The signal pathway is involved in the TGF-β1-induced migration of PDLSCs and the mechanical-chemical interaction during the orthodontic periodontal remodeling will be researched in our further studies.

Skeletal myogenic differentiation of human periodontal ligament stromal cells isolated from orthodontically extracted premolars / 대한치과교정학회지

OBJECTIVE: To investigate the stem cell-like characteristics of human periodontal ligament (PDL) stromal cells outgrown from orthodontically extracted premolars and to evaluate the potential for myogenic differentiation. METHODS: PDL stromal cells were obtained from extracted premolars by using the outgrowth method. Cell morphological features, self-replication capability, and the presence of cell-surface markers, along with osteogenic, adipogenic, and chondrogenic differentiation, were confirmed. In addition, myogenic differentiation was induced by the use of 5-aza-2'-deoxycytidine (5-Aza) for DNA demethylation. RESULTS: PDL stromal cells showed growth patterns and morphological features similar to those of fibroblasts. In contrast, the proliferation rates of premolar PDL stromal cells were similar to those of bone marrow and adipogenic stem cells. PDL stromal cells expressed surface markers of human mesenchymal stem cells (i.e., CD90 and CD105), but not those of hematopoietic stem cells (i.e., CD31 and CD34). PDL stromal cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages. Myotube structures were induced in PDL stromal cells after 5-Aza pretreatment, but not in the absence of 5-Aza pretreatment. CONCLUSIONS: PDL stromal cells isolated from extracted premolars can potentially be a good source of postnatal stem cells for oromaxillofacial regeneration in bone and muscle.

Isolation,purification and identification of human periodontal ligament stem cells / 实用口腔医学杂志

Objective:To isolate and identify the periodontal ligament stem cells.Methods:Periodontal ligament tissues were digested with a mixture of collagenase and Dispase,then the periodontal ligament stem cell clones were isolated by limited dilution method.The transmission electron microscopy(TEM),flow cytometery and immunohistochemistry procedure were employed to study the ultrastructure,cell cycle and surface marker of the cloned cells.Results:The obtained cells showed the characteristics of undifferentiated morphology at ultrastructural level.94.4% of the cells were in phase G_0/G_1.The cells were Vimentin,STRO-1,ON and BSP positive.Conclusion:Cloning incubation may be the effective way to isolate and purify the periodontal ligament stem cells.