Yunpeng Zhang

Platelet-rich plasma enhanced umbilical cord mesenchymal stem cells-based bone tissue regeneration.

OBJECTIVES To evaluate the effects of platelet-rich plasma (PRP) on the proliferation and differentiation of umbilical cord mesenchymal stem cells (UC-MSCs) and explore the possibility that PRP combined with UC-MSCs may be useful for bone tissue regeneration in vivo. METHODS The proliferation potential of UC-MSCs was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The pluripotent differentiation capacity and alkaline phosphatase (ALP) expression were further determined by ALP staining. The expression of osteoblast-associated genes was evaluated by real-time PCR. In addition, rat critical-sized calvarial defects were examined to evaluate bone regeneration in vivo. RESULTS PRP enhanced UC-MSC proliferation, and 10% PRP caused the strongest ALP and Alizarin red staining. At 7 days, the expression levels of ALP, Collagen 1 (COL-1) and Runt-related transcription factor 2 (RUNX2) in the PRP group were higher than those in the FBS group. Newly regenerated bone was observed in the defect areas, and PRP combined with UC-MSCs can accelerate bone regeneration at an early stage. CONCLUSIONS Our current data suggest that UC-MSCs may be utilized in alternative stem cell-based approaches for the reconstruction and regeneration of bone defects, and PRP combined with UC-MSCs can enhance bone regeneration in vivo.

CTHRC1 promotes osteogenic differentiation of periodontal ligament stem cells by regulating TAZ

Collagen triple helix repeat containing 1 (CTHRC1) is associated with bone metabolism. Alveolar bone has an ability to rapidly remodel itself to adapt its biomechanical environment and function. However, whether CTHRC1 is expressed in alveolar bone tissue and the role of CTHRC1 in alveolar bone remodeling remain unclear. We used orthodontic tooth movement (OTM) rat model to study the effects of CHTRC1 in alveolar bone remodeling in vivo. We found that CTHRC1 was expressed in normal physiological condition of osteocytes, bone matrix, and periodontal ligament cells in rat. During the OTM, the expression of CTHRC1, Runx2 and TAZ were increased. We further studied the effects of CTHRC1 on osteogenic differentiation of human periodontal ligament stem cells in vitro. CTHRC1 can positively regulate the expression of TAZ and osteogenic differentiation markers like Col1, ALP, Runx2 and OCN. Overexpression of CHTRC1 increased osteogenic differentiation of PDLSCs, which could be abolished by TAZ siRNA. Our results suggest that CTHRC1 plays an important role in alveolar bone remodeling and osteogenic differentiation of PDLSCs.

Nicotine promotes cervical carcinoma cell line HeLa migration and invasion by activating PI3k/Akt/NF-κB pathway in vitro

Cigarette smoking is one of highly risk factors of cervical cancer. Recently nicotine has been reported to increase proliferation and invasion in some smoking related cancers, like non-small cell lung cancer and esophageal squamous cell cancer. However, the effects and mechanisms of nicotine stimulation on cervical cancer cells are not clear. Here, we investigated the effects and mechanisms of nicotine stimulation on HeLa cells in vitro. In our study, we found that nicotine could accelerate HeLa cells migration and invasion, activate PI3K/Akt and NF-κB pathways and increase the expression of Vimentin in vitro. Moreover, we demonstrated that the specific PI3K inhibitor LY294002 could reverse nicotine-induced cell migration and invasion, NF-κB activation and up-regulation of Vimentin. Inhibition of NF-κB by Pyrrolidine dithiocarbamate (PDTC) also antagonized nicotine-induced cell migration, invasion and up-regulation of Vimentin. Simply put, these findings suggest that nicotine promotes cervical carcinoma cell line HeLa migration and invasion by activating PI3k/Akt/NF-κB pathway in vitro.

Knockdown of Yes-Associated Protein Induces the Apoptosis While Inhibits the Proliferation of Human Periodontal Ligament Stem Cells through Crosstalk between Erk and Bcl-2 Signaling Pathways

Objective: The purpose of this study was to provide an insight into the biological effects of knockdown Yes-associated protein (YAP) on the proliferation and apoptosis of human periodontal ligament stem cells (h-PDLSCs). Methods: Immunofluorescence and Western blot were used to evaluate Hippo-YAP signaling expression level. Enhanced green fluorescence protein lentiviral vector was constructed to down-regulate YAP in h-PDLSCs. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect the interfering efficiency of YAP expression. The proliferation activity was detected by EdU staining. Analysis of apoptosis in h-PDLSCs was done through Annexin V-APC staining, while cell cycle analysis was detected by flow cytometry. Cellular senescence was analyzed by β-galactosidase activity detection. The expression of elements in signaling pathways related with proliferation and apoptosis was detected by Western blot. Results: YAP was located in nucleus and cytoplasm. After the lentivirus transfection, the expression of YAP mRNA and protein was significantly reduced (P<0.001). When YAP was knocked down, the proliferation activity of h-PDLSCs was inhibited; the early & late apoptosis rates increased; the proportion of cells in G1 phases increased (P<0.05), while that in G2 and S phase decreased (P<0.05); cellular senescence was accelerated (P<0.01); ERK and its target proteins P-P90RSK and P-MEK were reduced while Bcl-2 family members increased. Conclusion: Knockdown of YAP inhibits the proliferation activity and induces apoptosis of h-PDLSCs with the involvement of Hippo pathway and has a crosstalk between Erk and Bcl-2 signaling pathways.

Expression and localization of Yap and Taz during development of the mandibular first molar in rats

Abstract Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz) are two downstream factors in the Hippo signaling pathway. Yap and Taz participate in regulating organ size, stem cell self-renewal, proliferation and differentiation. We investigated the spatial-temporal expression and relative expression levels of Yap and Taz using immunohistochemistry and real-time polymerase chain reaction. We found Yap and Taz in the oral epithelium and mesenchyme at embryonic (E) day 14.5 (E14.5) and E16.5. By E18.5, Yap and Taz were detected in the dental papilla and the entire enamel organ. At postnatal (P) day 0 (PN0), PN3 and PN7, Yap and Taz expression was localized in ameloblasts, odontoblasts and stratum intermedium. Yap and Taz were expressed in Hertwig’s epithelial root sheath (HERS) at PN7. At PN3, PN7 and PN14, Yap was detected in the enamel matrix. From PN21 to PN28, Yap and Taz were absent from differentiated ameloblasts, but they were expressed in odontoblasts. From PN0 to PN10, the Yap and Taz mRNA expression increased, then decreased. We found that Yap and Taz may influence the differentiation of ameloblasts and odontoblasts; they also may contribute to enamel mineralization, crown morphogenesis and root formation.

A new biosafe reactive oxygen species (ROS)-responsive nanoplatform for drug delivery

A new ROS-cleavable diblock copolymer (Meo-PEG-TK-PLGA) was designed and prepared. This polymer can self-assemble into small-sized (<100 nm) nanoparticles (NPs), with hydrophilic PEG shells and hydrophobic PLGA cores. This new nanoplatform shows high stability, satisfactory drug loading ability, excellent ROS sensitivity and good compatibility resulting from the use of FDA-approved PEG and PLGA. When using these new NPs to load the anticancer drug doxorubicin (DOX) and incubate with oral cancer cell line (Cal27 cells), the NPs can efficiently escape from endosomes and enter into the cytoplasm. With the presence of intracelluar ROS to cleave TK-containing linker, the loaded DOX could be rapidly released and induce the apoptosis of Cal27 cells. The polymer presents great potential as a novel drug delivery platform for cancer chemotherapy.

Expression pattern of YAP and TAZ during orthodontic tooth movement in rats

Orthodontic tooth movement (OTM) is a periodontal tissue remodeling and regeneration process that is caused by bio-mechanical stimulation. This mechanical–chemical transduction process involves a variety of biological factors and signaling pathways. It has been shown that the Hippo-YAP/TAZ signaling pathway plays a pivotal role in the mechanical–chemical signal transduction process. Moreover, YAP and TAZ proteins interact with RUNX family proteins via different mechanisms. To explore the regulation of the Hippo signaling pathway during periodontal tissue remodeling, we examined the upper first molar OTM model in rats. We examined YAP, TAZ and RUNX2 expression at 12 hours, 24 hours, 2 days (2d), 4 days, 7 days (7d) and 14 days (14d) after force application. Haemotoxylin and eosin staining, immunohistochemical staining and western blot analysis were used to examine the expression level and localization of these proteins. We found that YAP, TAZ and RUNX2 expression started increasing at 2d, YAP and TAZ expression was proportional to the orthodontic force applied until peaking at 7d, and at 14d the expression started to decrease. YAP and TAZ were observed in osteocytes, bone matrix and periodontal ligament cells during OTM. Furthermore, using double labeling immunofluorescence staining, we found that the increase in TAZ expression was associated with RUNX2 expression, however, YAP and RUNX2 showed different expression patterns. These results suggest that the Hippo-YAP/TAZ signaling pathway participates in periodontal tissue remodeling through various mechanisms; TAZ may adjust bone tissue remodeling through RUNX2 during OTM, while YAP may regulate periodontal cell proliferation and differentiation.

An in vitro comparative study of multi-sources derived mesenchymal stem cells for bone tissue engineering

Mesenchymal stem cells (MSCs) have been considered promising tools for tissue engineering and regenerative medicine. However, the optimal cell source for bone regeneration remains controversial. To better identify seed cells for bone tissue engineering, we compared MSCs from seven different tissues, including four from dental origins, dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), gingival MSCs (GMSCs), and dental follicle stem cells (DFSCs); two from somatic origins, bone marrow-derived MSCs (BM-MSCs) and adipose-derived stem cells (ADSCs); and one from birth-associated perinatal tissue umbilical cord (UCMSCs). We cultured the cells under a standardized culture condition and studied their biological characteristics. According to our results, these cells exhibited similar immunophenotype and had potential for multilineage differentiation. MSCs from dental and perinatal tissues proliferated more rapidly than those from somatic origins. Simultaneously, DPSCs and PDLSCs owned stronger antiapoptotic ability under the microenvironment of oxidative stress combined with serum deprivation. In respect to osteogenic differentiation, the two somatic MSCs, BM-MSCs and ADSCs, demonstrated the strongest ability for osteogenesis compared to PDLSCs and DFSCs, which were just a little bit weaker than the formers. However, GMSCs and UCMSCs were the most pertinacious ones to differentiate to osteoblasts. We also revealed that the canonical intracellular protein kinase-based cascade signaling pathways, including PI3K/AKT, MAPK/ERK, and p38 MAPK, possessed different levels of activation in different MSCs after osteoblast induction. Our conclusions suggest that PDLSCs might be a good potential alternative to BM-MSCs for bone tissue engineering.

Effect of 1α, 25-dihydroxyvitamin D3 on the osteogenic differentiation of human periodontal ligament stem cells and the underlying regulatory mechanism

1α, 25-dihydroxyvitamin D3 (1,25-D3), an active vitamin D metabolite, is a well-known regulator of osteogenic differentiation. However, how 1,25-D3 regulates osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs) remains to be fully elucidated. The present study aimed to clarify this issue through well-controlled in vitro experiments. After hPDLSCs were treated with 1,25-D3, immunofluorescence and western blotting were used to detect the expression of vitamin D receptor; Cell Counting Kit-8 and western blotting were used to assay the cell proliferation ability. Alkaline phosphatase staining, Alizarin Red staining and western blotting were used to detect the osteogenic differentiation. It was found that treating hPDLSCs with 1,25-D3: i) Inhibited cell proliferation; ii) promoted osteogenic differentiation; iii) upregulated the expression of transcriptional coactivator with PDZ-binding motif (TAZ), an important downstream effector of Hippo signaling that has been demonstrated to be involved in the osteogenic differentiation of stem/progenitor cells; and iv) that co-treatment of TAZ-overexpressing hPDLSCs with 1,25-D3 synergistically stimulated the expression of osteogenic markers. These results suggested that the induction of osteogenic differentiation promoted by 1,25-D3 in hPDLSCs involves, at least in part, the action of TAZ.

The Crosstalk between HDPSCs and HUCMSCs on Proliferation and Osteogenic Genes Expression in Coculture System

Objectives: The present study established a non-contact coculture system in vitro, aiming to investigate the crosstalk between human dental pulp stem cells (hDPSCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) on proliferation activity and osteogenic genes expression through paracrine. Materials and methods: The stemness of hDPSCs and hUCMSCs were identified by flow cytometric analysis and multipotential differentiation assays. With the help of transwell inserts, the non-contact coculture system in vitro was established between hDPSCs and hUCMSCs. EdU labeling analysis and Western Blot were used to detect the proliferation activity. The mRNA and protein levels of osteogenic genes were evaluated by RT-PCR and Western Blot. The expression of elements in Akt/mTOR signaling pathway were detected by Western Blot. Results: Both hDPSCs and hUCMSCs were positive to MSCs specific surface markers and had multi-differentiation potential. The proportion of EdU-positive cells increased and the expression of CDK6 and CYCLIN A were up-regulated in cocultured hDPSCs. Both prior coculture and persistent coculture improved mRNA and protein levels of osteogenic genes in hDPSCs. While in cocultured hUCMSCs, no statistical differences were observed on proliferation and osteogenesis. The phosphorylation of Akt and mTOR was up-regulated in cocultured hDPSCs. Conclusions: The crosstalk between hDPSCs and hUCMSCs in coculture system increased the proliferation activity and enhanced osteogenic genes expression in hDPSCs. Akt/mTOR signaling pathway might take part in the enhancing effects in both cell proliferation and gene expression.