Activated Yes-Associated Protein Accelerates Cell Cycle, Inhibits Apoptosis, and Delays Senescence in Human Periodontal Ligament Stem Cells.

Objectives: To provide insight into the biological effects of activated Yes-associated protein (YAP) on the proliferation, apoptosis, and senescence of human periodontal ligament stem cells (h-PDLSCs). Methods: h-PDLSCs were isolated by the limiting dilution method, and their surface markers were quantified by flow cytometry. Enhanced green fluorescence protein (EGFP)-labeled lentiviral vector was used to activate YAP in h-PDLSCs, then qRT-PCR and Western blotting were used to evaluate the expression level of YAP. Immunofluorescence was used to detect the location of YAP in h-PDLSCs. The proliferation activity was detected by cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU), and the cell cycle was determined by flow cytometry. Apoptosis was analyzed by Annexin V-APC staining. Cell senescence was detected by ß-galactosidase staining. Proteins in ERK, Bcl-2, and p53 signaling pathways were detected by Western blotting. Results: h-PDLSCs were isolated successfully and were positive for human mesenchymal stem cell surface markers. After YAP was activated by lentiviral vector, the mRNA and protein of YAP were highly expressed, and more YAP translocated into the nucleus. When YAP was overexpressed in h-PDLSCs, proliferation activity was improved; early and late apoptosis rates decreased (P<0.05); the proportion of cells in G2/M phases increased (P<0.05), while that in G0/G1 phase decreased (P<0.05); cellular senescence was delayed (P<0.01); the expression of P-MEK, P-ERK, P-P90RSK and P-Msk increased, while the expression of Bcl-2 family members (Bak, Bid and Bik) decreased. Conclusions: Activated YAP promotes proliferation, inhibits apoptosis, and delays senescence of h-PDLSCs. The Hippo-YAP signaling pathway can influence ERK and Bcl-2 signaling pathways.

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 ß-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.

[Relationships among the periodontal biotype characteristics in the maxillary anterior].

OBJECTIVE: To explore the correlation among gingival thickness (GT), underlying alveolar bone thickness (BT), and other periodontal biotype characteristics in the maxillary anterior. METHODS: A total of 40 young volunteers with healthy periodontal were involved in this research. The periodontal probe was previously used to divide the gingiva from thick to thin. Two records were measured by cone beam CT (CBCT) GT, which was measured at the cement-enamel junction level; and BT, which was measured at 3 locations: 1, 3, 5 mm below the alveolar crest. Oral and gypsum measurements were used to analyze the associations of the crown width/crown length ratio (CW/CL), the keratinized mucosa width (KM), and the free gingival margin curvature. RESULTS: Significant difference in the GT was observed between the thick and thin biotypes, which were divided by periodontal probe (P<0.01). Difference was observed in each periodontal biotype characteristic between the thick (GT≥1 mm) and thin biotypes (GT<1 mm) (P<0.05). BT was positively associated with GT (r=0.293, P=0.001), CW/CL (r=0.273, P=0.003), KM (r=0.291, P=0.001), and free gingival margin curvature (r=0.290, P=0.001). CONCLUSIONS: The transparency of the probing in the sulcus could analyze the GT qualitatively. The thick and thin biotypes have different periodontal biotype characteristics. Compared with individuals with thick biotype, those with thin biotype are susceptible to risk dental aesthetic.

[Relationship among gingival thickness, underlying alveolar bone thickness, and sagittal root position in the maxillary anterior].

OBJECTIVE: This study aimed to explore the relationship among gingival thickness (GT), underlying alveolar bone thickness (BT), and sagittal root position in the maxillary anterior measured by cone-beam computed tomography (CBCT). The theoretical foundation was applied to aesthetic dentistry, implant treatment planning, and therapeutic effect assessment. METHODS: A total of 40 young volunteers with healthy periodontal status were involved in this research [16 males and 24 females aged 23-34 years with a mean age of (26.30±2.29) years]. Three records were measured by CBCT. GT was measured at the cemento-enamel junction level. Buccal BT was measured at three locations: 1, 3, and 5 mm below the alveolar crest, along the sagittal angle between the long axis of teeth, and along the long axis of the respective alveolar bone. RESULTS: The average GT and alveolar BT thicknesses were (1.08±0.34) mm and (0.79±0.29) mm, respectively. The average angle between teeth and alveolar bone was 18.01°±8.96°. BT was positively associated with GT (r=0.293, P=0.001). The BT of canine was positively associated with the angle between teeth and alveolar bone (r=0.457, P=0.003). CONCLUSIONS: BT was relatively thin. An angle was found between the long axis of teeth and that of the alveolar bone. BT was positively associated with GT. An accurate diagnosis of GT, underlying alveolar BT, and sagittal root position in the maxillary anterior is necessary before implant surgery to devise an appropriate implant treatment plan and achieve a predictable esthetic outcome.

[Short-term evaluation of clinical effect of bone ring grafting and immediate insertion].

OBJECTIVE: To observe the short-term clinical effectiveness of bone ring graft technique and to summarize the key points of related surgical operation to provide comprehensive clinical guidelines. METHODS: Fifteen patients with severe alveolar bone absorption were selected to receive bone ring grafting and immediate dental implant. Final fixed prostheses were cemented five months after initial implantation. Cone beam CT scans were conducted on all subjects before the procedure, as well as four months post-operation to evaluate alveolar bone height and level of bone height and absorption around the implants. Four to six months after prosthesis installation, each implant's Jemt classification, gingiva attachment, and probing depth (PD) were analyzed. The difference of PD between implants and adjacent teeth, as well as the difference of the bone absorption between labial and lingual sides, was compared. The survival rate of the bone ring and the retention rate of implants were calculated. Complications and patient satisfaction were also investigated. RESULTS: Bone graft survival rate was 94.4% and dental implantation retention rate was 100% four months post-operation. Average bone level increase was (6.06 +/- 1.06) mm, average bone absorption was (1.33 +/- 0.84) mm, and average bone thickness at the neck of the dental implant body was (6.94 +/- 0.73) mm. Approximately 4 to 6 months after crown restoration, average bone level increase was (5.62 +/- 1.03) mm, average bone absorption was (1.51 +/- 1.02) mm, and average bone thickness at the neck of the dental implant body was (6.77 +/- 0.72) mm. The PD around the implant body and the adjacent teeth was statistically insignificant. No major post-operative complication was observed, restorations were successful, and patient satisfaction level was high. CONCLUSION: Bone ring graft technique and immediate dental implantation are relatively simple to perform, and these techniques facilitate reduction in required treatment time. Short-term effect is reliable and satisfactory, whereas long-term outcomes require further follow up and study.

[Application of attached gingiva reconstruction in implant-supported dental prosthesis].

PURPOSE: To explore a method of attached gingiva reconstruction around dental implants and to evaluate its clinical outcomes and technical characters. METHODS: From January 2011 to August 2011, a total of 10 implants were included in this study from 8 patients who had inadequate attached gingiva and needed to undergo secondary operation. A modified apically positioned flap technique was applied to reconstruct attached gingiva at the time of secondary operation. Crown restoration was finished 4 weeks after the operation and the patients were recalled 6 months after restoration. Effective keratinized mucosal width was recorded separately before the operation, 4 weeks after the operation and 6 months after restoration. Clinical examination was conducted 6 months after restoration including modified plaque index(mPI), probing depth(PD), bleeding index(BI) and percentage of bleeding on probing. Statistical analysis was performed using Wilcoxon signed rank test with SPSS18.0 software package. RESULTS: Four weeks after the operation, there was an average increase of 2.41 mm in effective keratinized mucosal width compared with the preoperative value (P<0.05). After 6 months of function, the newly formed keratinized mucosa remained stable and attached to the crown tightly without obvious inflammation. The mean width was (2.64±0.53) mm. Clinical examinations showed a mean mPI of 0.70±0.82, a mean PD of (1.80±0.36) mm, and a mean BI of 0.73±0.64. In terms of bleeding on probing, 23.3% of sites showed positive outcome. CONCLUSIONS: By using the modified apically positioned flap technique at the time of secondary operation, the attached gingiva around implants can be effectively reconstructed. The short-term effects are satisfactory.

Superior osteogenic capacity of different mesenchymal stem cells for bone tissue engineering.

OBJECTIVE: We evaluated the effect of human bone marrow stromal cells (hBMSCs), human adipose tissue-derived mesenchymal stem cells (hAD-MSCs), and umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in bone tissue engineering and identified a reliable cell source. STUDY DESIGN: Alkaline phosphatase (ALP) activity and quantitative polymerase chain reaction were used to evaluate osteogenic in vitro, X-ray and histologic analysis in vivo. RESULTS: hBMSCs exhibited strongest ALP staining, followed by hAD-MSCs and hUC-MSCs. At 7 days, hUC-MSCs and hAD-MSCs had higher expression of collagen type I and Runt-related transcription factor 2 than hBMSCs, and hUC-MSCs showed higher osteopontin expression. Bone structure was observed in the hUC-MSC group. Defects showed good healing in the hBMSC and hAD-MSC groups. Enhanced green fluorescent protein and osteopontin were detected in newly formed bone at 8 weeks. CONCLUSIONS: Our results suggested that hUC-MSCs and hAD-MSCs could be used for bone tissue engineering effectively; hUC-MSCs could serve as a new alternative cell source.

[Differential expression genes of bone tissues surrounding implants in diabetic rats by gene chip].

PURPOSE: To compare mRNA expression profiles of bone tissues surrounding implants between normal rats and rats with diabetes using microarray technology. METHODS: Six Wistar rats were randomly selected and divided into normal model group and diabetic group. Diabetic model condition was established by injecting Streptozotocin into peritoneal space. Titanium implants were implanted into the epiphyseal end of the rats' tibia. Bone tissues surrounding implant were harvested and sampled after 3 months to perform comprehensive RNA gene expression profiling, including 17983 for genome-wide association study.GO analysis was used to compare different gene expression and real-time PCR was used to confirm the results on core samples. RESULTS: The results indicated that there were 1084 differential gene expression. In the diabetic model, there were 352 enhanced expression genes, 732 suppressed expression genes. GO analysis involved 1154 different functional type. CONCLUSIONS: Osteoblast related gene expressions in bone tissue samples of diabetic rats were decreased, and lipid metabolism pathway related gene expression was increased.

Application of eGFP to label human periodontal ligament stem cells in periodontal tissue engineering.

OBJECTIVES: To establish human periodontal ligament stem cells (hPDLSC) with high and stable expression of enhanced green fluorescent protein (eGFP) and to obtain an ideal vector expression system that suitable for gene therapy in periodontal tissue engineering. METHODS: hPDLSCs were transfected with eGFP for 48h via different MOI (25, 50, 100, 200 and 400) by lentiviral vector, the transfection efficiency was evaluated by fluorescent microscopy and flow cytometry, and transfected hPDLSCs proliferation was evaluated by MTT. Pluripotent, differentiation capacity and ALP expression status were determined further. Osteoblast-associated genes expressions for osteogenesis were evaluated by quantitative-PCR. In addition, rat molar periodontal fenestration defect model was used for evaluating periodontal tissue engineering. RESULTS: The transfection efficiency after 48h were 44.7%, 60.9%, 71.7%, 85.8%, and 86.9% respectively. There was no significant effect of transfection (at different MOI levels of 25, 50, 100, and 200) on the proliferation of hPDLSCs (designated as eGFP-hPDLSCs) compared with hPDLSCs (P>0.05). However, proliferation of eGFP hPDLSCs at MOI 400 became slower (P<0.05). Both eGFP hPDLSCs and hPDLSCs were able to differentiate into osteocytes and adipocytes under certain conditioned media. At 7 days, expression levels of COL-1, RUNX2 in hPDLSCS were higher than those in eGFP hPDLSCs (P<0.05); expression levels of ALP and OPN in eGFP hPDLSCs were similar to those in hPDLSCs (P>0.05). Newly regenerated bone formation was observed in the defect model used. CONCLUSIONS: Among the transfection conditions, 48h transfection at MOI 200 is optimal for labelling hPDLSCs with eGFP in a lentiviral vector. There is no change in capability of the eGFP hPDLSCs osteogenesis. The lentiviral vector with eGFP is an appropriate expression vector system and hPDLSCs are ideal seeding cells for gene therapy in periodontal tissue engineering.

[Study of labeling human periodontal ligament stem cells with enhanced green fluorescent protein by lentivirus vector infection].

OBJECTIVE: The aim of this study is to optimize conditions for labeling human periodontal ligament stem cells (PDLSCs) using enhanced green fluorescent protein (eGFP) infected by lentivirus vector and to obtain PDLSCs with high stable expressed eGFP. METHODS: PDLSCs were transfected with eGFP by lentivirus vector for 48 h via different multiplicity of infection (MOI) (25, 50, 100, 200 and 400) and the infection efficiency were analyzed by both fluorescent microscope and flow cytometry. The proliferation rate of infected PDLSCs was evaluated by MTT. The infected PDLSCs were further for detection of pluripotent, differentiation ability and alkaline phosphatase (ALP) expression ability. RESULTS: The infection efficiency for each group were 44.7%, 60.9%, 71.7%, 85.8% and 86.9% respectively. Proliferation of PDLSCs was not affected when MOI was below 200; however, at MOI 400, the proliferation ability was affected compared with control group. The pluripotent and ALP abilities of PDLSCs were not changed by the infection. CONCLUSION: Infection for 48 h at MOI 200 is optimal for labeling PDLSCs with eGFP using lentivirus vector, and the proliferation and differentiation abilities of PDLSCs are not affected obviously.

[Clinical retrospective study on membrane guided bone tissue regeneration technique in dental implants in the anterior maxilla].

PURPOSE: The purpose of this study was to investigate the effect of Bio-Gide in MGBR (membrane guided bone regeneration ) in the anterior maxilla dental implant therapy. METHODS: Fifty five cases underwent dental implant therapy in the anterior maxilla with insufficient maxillary anterior bone, including 29 patients with embedded 40 implants using MGBR and 26 patients with embedded 40 implants without using MGBR. The condition of bone around implant before implantation and before final restoration was observed and recorded. The thickness of the bone was measured with vernier caliper, the density and the bone loss were determined by X-ray examination. The results were analyzed with X-ray radiographs image analysis system (Sidexis) and SPSS16.0 software package. RESULTS: Univariate analysis showed that there was significant difference in the thickness and the density of the bone between before implant operation and before restoration(P<0.05). There was significant difference between the thickness and the density of the bone between with and without MGBR(P<0.05). CONCLUSIONS: These results suggest that the thickness and the density of the bone in the group with MGBR is better than the group without MGBR. It is recommended to use MGBR in dental implant therapy in the anterior maxilla with insufficient bone.