PTEN regulates proliferation and osteogenesis of dental pulp cells and adipogenesis of human adipose-derived stem cells.

OBJECTIVE: To investigate the role of phosphatase and tensin homolog (PTEN) in dental pulp cells (hDPs) and adipose-derived mesenchymal stem cells (hADSCs). MATERIALS AND METHODS: Genetic variant was identified with exome sequencing. The hDPs isolated from a patient with Cowden syndrome were investigated for their proliferation, osteogenesis, adipogenesis, and gene expression compared with controls. The normal hDPs and hADSCs were treated with the PTEN inhibitor, VO-OHpic trihydrate (VOT), to investigate the effect of PTEN inhibition. RESULTS: A heterozygous nonsense PTEN variant, c.289C>T (p.Gln97*), was identified in the Cowden patient's blood and intraoral lipomas. The mutated hDPs showed significantly decreased proliferation, but significantly upregulated RUNX2 and OSX expression and mineralization, indicating enhanced osteogenic ability in mutated cells. The normal hDPs treated with VOT showed the decreases in proliferation, colony formation, osteogenic marker genes, alkaline phosphatase activity, and mineral deposition, suggesting that PTEN inhibition diminishes proliferation and osteogenic potential of hDPs. Regarding adipogenesis, the VOT-treated hADSCs showed a reduced number of cells containing lipid droplets, suggesting that PTEN inhibition might compromise adipogenic ability of hADSCs. CONCLUSIONS: PTEN regulates proliferation, enhances osteogenesis of hDPs, and induces adipogenesis of hADSCs. The gain-of-function PTEN variant, p.Gln97*, enhances osteogenic ability of PTEN in hDPs.

The Prostacyclin Analog Iloprost Promotes Cementum Formation and Collagen Reattachment of Replanted Molars and Up-regulates Mineralization by Human Periodontal Ligament Cells.

INTRODUCTION: This study evaluated the use of the prostacyclin analog iloprost as a root surface treatment agent in promoting acellular cementum (AC) formation and collagen reattachment after tooth replantation in vivo. In addition, its effect on human periodontal ligament cell (hPDLC) mineralization was assessed in vitro. METHODS: First molars of 8-week-old Wistar rats were extracted. In 1 group, the root surfaces were treated with Hank's balanced salt solution (HBSS), and the other group's root surfaces were treated with 10-6 mol/L iloprost before replantation. At day 30, maxillae were prepared for micro-computed tomographic imaging and histomorphometric analysis. The effect of iloprost on mineralization by hPDLCs was analyzed by mineralized nodule formation and quantitative polymerase chain reaction at 7 and 14 days. RESULTS: Micro-computed tomographic imaging demonstrated a significant higher bone volume in the iloprost groups, whereas the HBSS groups had extensive bone and root resorption. Histologic analysis revealed deposition of a thick AC layer along the root in the iloprost group with well-organized periodontal ligament fibers inserted into the cementum. The HBSS group demonstrated more osteoclasts than the iloprost group. In vitro, iloprost-treated hPDLCs had a significantly increased RUNX2, OSX, BSP, and ALP gene expression that coincided with an increased deposition of mineralized nodules. These effects were abrogated by a PGI2 receptor inhibitor. CONCLUSIONS: Our results revealed that iloprost promoted PDL regeneration in replanted molars. Furthermore, resorption of the roots was decreased, whereas AC deposition was stimulated. Iloprost-treatment increased hPDLC mineralization and was mediated by PGI2 receptor activation. These observations indicate that iloprost may be a promising root surface treatment agent.

Association Between PD-L1 and Histatin1, 3 Expression in Advanced Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: The prognosis of advanced stage head and neck squamous cell carcinoma (HNSCC) has remained unimproved for the past decades. Therefore, novel diagnostic markers and treatment options are required. Recently, an inhibitor for immune checkpoint program death ligand-1 (PD-L1), was approved by the FDA, and used in HNSCC patients. Histatins (HTNs), one of the common antimicrobial peptides in saliva, have demonstrated wound healing and antifungal capabilities and other functions on the oral epithelium. Dysregulation of HTN1 and HTN3 has also been reported in HNSCC through genomic and proteomic studies. This study aimed to investigate the association between histatins (HTN1 and HTN3) and PD-L1 in advanced HNSCC. PATIENTS AND METHODS: Data of gene expression in HNSCC were collected from TCGA and analyzed using a data-mining platform website (https://ualcan.path.uab.edu/). Tissue microarrays containing 98 samples of HNSCC patients and non-neoplastic controls were immunolabeled against PD-L1, HTN1, and HTN3. The immunohistochemistry results were quantified using ImageJ. RESULTS: The expression of PD-L1 and HTN1 was significantly higher in tumors than normal tissues (p<0.001), but no significant difference was found regarding HTN3. Metastatic HNSCC samples exhibited significantly higher expression of PD-L1 (p<0.018), compared to the non-metastatic group. Association between HTN1 and HTN3 was found using Pearson correlation coefficient (r=0.603, p<0.001). No overall survival difference was evident among our samples. CONCLUSION: PD-L1 and HTN1 are associated with the progression of HNSCC. PD-L1 expression correlated with that of HTN3.

The impact of deproteinized bovine bone particle size on histological and clinical bone healing outcomes in the augmented sinus: A randomized controlled clinical trial.

OBJECTIVE: The effect of different deproteinized bovine bone mineral (DBBM) particle sizes on bone healing in maxillary sinus floor augmentation remains unclear. This study compared the newly formed tissue and angiogenesis-related bone healing after sinus floor augmentation using large or small DBBM particles. MATERIALS AND METHODS: Overall 32 patients were randomly divided into two groups using either large (1-2 mm) or small (0.25-1 mm) DBBM particles for sinus floor augmentation. After 6 months, the mineralized tissue volume was calculated using micro-computed tomography (micro-CT) analysis. The newly formed tissue composition was histomorphometrically analyzed. Angiogenesis was also examined by means of vascular endothelial growth factor (VEGF) expression. Implant failure and marginal bone loss were measured at a 1-year follow-up. Statistical analysis was performed using independent samples t-test. RESULTS: Micro-CT analysis demonstrated that grafting with large particles resulted in higher bone volume (6.99 ± 2.72 mm3 , p = 0.002) and Bone Volume/Tissue Volume (0.25 ± 0.1, p = 0.03) compared with small particles (3.76 ± 1.83 mm3 and 0.14 ± 0.13, respectively). Small particles showed higher non-mineralized tissue volume (26.31 mm3 ) compared with large particle group (17.4 ± 5.34 mm3 ) with p = 0.001. The histological data revealed significantly higher area of newly formed bone (32.15% ± 14.04% for the large particle and 15.99% ± 14.12% for the small particle groups, p = 0.004). Likewise, non-mineralized tissue was significantly greater in the small particle group (66.48% ± 20.97%) compared with the large particle group (44.36%, p = 0.016). Moreover, use of large particles resulted in a significantly higher VEGF staining intensity score and VEFG positive cells. No implant failure was recorded in both groups, while no difference was found in terms of marginal bone loss at the 1-year follow-up. CONCLUSIONS: Sinus floor augmentation using large DBBM particles resulted in more angiogenesis expression, higher bone volume, and new bone formation at 6 months after sinus augmentation. However, clinical outcomes with regards to implant placement were similar in both groups.

A novel mutation in COL1A2 leads to osteogenesis imperfecta/Ehlers-Danlos overlap syndrome with brachydactyly.

Osteogenesis imperfecta (OI) is mainly characterized by bone fragility and Ehlers-Danlos syndrome (EDS) by connective tissue defects. Mutations in COL1A1 or COL1A2 can lead to both syndromes. OI/EDS overlap syndrome is mostly caused by helical mutations near the amino-proteinase cleavage site of type I procollagen. In this study, we identified a Thai patient having OI type III, EDS, brachydactyly, and dentinogenesis imperfecta. His dentition showed delayed eruption, early exfoliation, and severe malocclusion. For the first time, ultrastructural analysis of the tooth affected with OI/EDS showed that the tooth had enamel inversion, bone-like dentin, loss of dentinal tubules, and reduction in hardness and elasticity, suggesting severe developmental disturbance. These severe dental defects have never been reported in OI or EDS. Exome sequencing identified a novel de novo heterozygous glycine substitution, c.3296G > A, p.Gly1099Glu, in exon 49 of COL1A2. Three patients with mutations in the exon 49 of COL1A2 were previously reported to have OI with brachydactyly and intracranial hemorrhage. Notably, two of these three patients did not show hyperextensible joints and hypermobile skin, while our patient at the age of 5 years had not developed intracranial hemorrhage. Here, we demonstrate that the novel glycine substitution in the carboxyl region of alpha2(I) collagen triple helix leads to OI/EDS with brachydactyly and severe tooth defects, expanding the genotypic and phenotypic spectra of OI/EDS overlap syndrome.

The Akt/mTOR pathway is activated in verrucous carcinoma of the oral cavity.

BACKGROUND: The Akt/mTOR pathway is activated in many malignancies, including oral squamous cell carcinoma (OSCC). However, the role of the Akt/mTOR pathway in oral verrucous carcinoma (OVC), a low-grade variant of OSCC, remains unknown. Thus, the objective of this study was to investigate the activation level of important markers of the Akt/mTOR pathway in OVC and to compare the results with OSCC samples. METHODS: The expression of p-Akt (Thr308), p-Akt (Ser473), and p-RPS6 was evaluated by immunohistochemistry in 30 OSCC cases, 18 OVC cases, and 30 control cases (normal epithelium overlying fibromas). Statistical analysis was performed to determine the differences in protein expression between samples. RESULTS: All OVC cases were positive for p-Akt (Thr308), p-Akt (Ser473), and p-RPS6. There were significant differences in expression level of all studied proteins between OVC and control, as well as between OVC and OSCC. However, OVC showed significant lower staining scores than OSCC. CONCLUSIONS: Our findings demonstrate that the Akt/mTOR pathway is upregulated in OVC, indicating a role for this pathway in the development and progression of this malignancy.

Activation of the Akt/mTOR pathway in dentigerous cysts, odontogenic keratocysts, and ameloblastomas.

OBJECTIVE: The aim of this study was to investigate the Akt/mTOR pathway in dentigerous cysts (DCs), odontogenic keratocysts (OKCs), and ameloblastomas. STUDY DESIGN: A total of 90 cases were studied (30 DCs, 30 OKCs, and 30 ameloblastomas). Patient records on age, sex, lesion location, symptoms, and radiographic and histopathologic features were collected. The phosphorylation of components of the Akt/mTOR pathway [p-Akt (Ser473), p-Akt (Thr308), and phosphorylated-ribosomal protein S6 (p-RPS6)] was studied using immunohistochemistry. Correlations with clinical features were analyzed using the Spearman rank test. RESULTS: Over 90% of OKCs and ameloblastomas and 60% of DCs stained positive for p-Akt (Ser473). Phospho-Akt (Thr308) was positive in 73% of ameloblastomas, 40% of OKCs, and 20% of DCs. Phospho-RPS6 was detected most frequently in OKCs (83%), followed by ameloblastomas (76%) and DCs (53%). No correlations were noted between the immunohistochemical findings and the clinicopathologic parameters. CONCLUSIONS: The Akt/mTOR pathway is upregulated in DCs, OKCs, and ameloblastomas. This pathway may be involved in the development of these lesions.

An analysis of microvessel density in salivary gland tumours: a single centre study.

BACKGROUNDS: Microvessel density (MVD) can be used for determining neoplastic neovascularisation. Tumour angiogenesis correlates with prognosis of cancers in many organs. The aims of this study were to evaluate MVD as demonstrated by CD31 and CD105 in salivary gland tumours (SGTs), and to correlate the MVD results with clinicopathological characteristics of the tumours. MATERIALS AND METHODS: Using a retrospective cohort study design, we enrolled SGTs patients at the Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand, over the 22-year period. The predictor variables included demographic, anatomic and histopathological parameters. The outcome measure was average CD31-MVD and CD105-MVD counted by the "hot spot" method. Descriptive, uni- and bivariate statistics were computed, and P <0.05 was considered statistically significant. RESULTS: The study sample consisted of 43 subjects with a mean age of 39.6 ± 17.8 years (range, 9-82), including 26 females (60.5%), diagnosed with SGTs. In this cohort, 58.1% of the cases were benign, and 83.7% were minor SGTs. There was a significant correlation between CD31-MVD and CD105-MVD (r = 0.8, P < 0.001), but mean CD31-MVD and CD105-MVD were 17.7 ± 9.3 and 12.8 ± 7.4, respectively (P = 0.009). Age, gender and tumour site were not individually associated with significant differences between CD31-MVD and CD105-MVD. Tumours with myoepithelial cells had lower MVD than those without myoepithelial cells (P = 0.04 for CD31; P = 0.03 for CD105). Only CD105-MVD showed statistical difference between benign and malignant SGTs (P = 0.01). CONCLUSIONS: These results suggest that MVD in SGTs can be demonstrated by CD31 and CD105. Despite a strong correlation, CD31-MVD is always higher than CD105-MVD and cannot differentiate between benign and malignant SGTs. The presence of myoepithelial cells within SGTs affects the MVD analysis using either CD31 or CD105, while age, gender and tumour location do not.

Ribosomal protein S6 phosphorylation is associated with epithelial dysplasia and squamous cell carcinoma of the oral cavity.

Ribosomal protein S6 (RPS6), a downstream effector of the mammalian target of rapamycin pathway (mTOR), is activated in many cancers including oral squamous cell carcinoma (OSCC). However, the role of RPS6 in the progression of potentially malignant disorders (or premalignant lesions) to OSCC is unknown. The purpose of this study was to examine the expression of RPS6 in epithelial dysplasia and OSCC to determine the association of RPS6 in tumor progression. In our study, an immunohistochemical analysis of RPS6 was performed on tissue microarrays containing 30 control samples, 15 epithelial dysplasia cases, and 53 OSCC cases. Correlations between the clinicopathologic features of OSCC and RPS6 expression were analyzed using the Chi-square test. We found RPS6 phosphorylation (p-RPS6) in 15/30 (50 %) control normal oral mucosa samples, 15/15 (100 %) epithelial dysplasia cases, and 47/53 (88.68 %) OSCC cases. The frequency of p-RPS6 in epithelial dysplasia or OSCC showed a statistically significant difference compared to control (P < 0.001). However, there were no significant correlations between p-RPS6 and the clinicopathologic features of OSCC. Our findings suggest that RPS6 activation is associated with the early events of tumor progression, suggesting p-RPS6 as a potential marker for early detection of oral cancer.

Esthetic alveolar ridge preservation with calcium phosphate and collagen membrane: preliminary report.

OBJECTIVE: The objective of this study was to evaluate clinically, histologically and radiographically a ridge preservation technique used on extraction sockets grafted with biphasic calcium phosphate (BCP) and a resorbable collagen membrane. MATERIAL AND METHODS: Patients having a labial socket wall defect more than one-third in mesio-distal socket width after maxillary central incisor tooth extraction were included. The labial defect was sealed with resorbable collagen membrane and the defect filled with BCP. The grafted socket was covered with a resorbable collagen wound dressing material. The treated sockets were evaluated after a 4-month healing period when implants were placed and followed for up to 12 months. RESULTS: There were 8 subjects enrolled in this study. A statistical difference was found only in ridge width reduction at 3 mm below the cement-enamel junction of the adjacent teeth (P < .05) with 1 mm widening at 8 mm. The amount of new bone formation was extensively varied with diminutive graft remnants. Most cells in the connective tissue were osteopontin positive indicating they were osteoblast-like cells. A declination in the radiodensity of the grafted socket was observed during the analyzed period. CONCLUSION: Ridge preservation with BCP with collagen membrane can be used as an alternative treatment for maintaining ridge dimension before implant placement.

Osteopontin expression in normal skin and non-melanoma skin tumors.

Osteopontin (OPN) is an adhesive, matricellular glycoprotein, whose expression is elevated in many types of cancer and has been shown to facilitate tumorigenesis in vivo. To understand the role of OPN in human skin cancer, this study is designed to determine whether OPN is expressed in premalignant [solar/actinic keratosis (AK)] and in malignant skin lesions such as squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), as well as in normal skin exposed or not exposed to sunlight. Immunohistochemical analyses showed that OPN is expressed in SCC (20/20 cases) and in AK (16/16 cases), which are precursors to SCC, but is absent or minimally expressed in solid BCC (17 cases). However, positive staining for OPN was observed in those BCC that manifest differentiation toward epidermal appendages such as keratotic BCC. In sunlight-exposed normal skin, OPN is minimally expressed in the basal cell layer, but in contrast to those not exposed to sunlight, OPN is more prominent in the spinous cell layer with increasing intensity toward the granular cell layer. Additionally, OPN is expressed in the hair follicles, sebaceous glands, and sweat glands of normal skin. In conclusion, these data suggest that OPN is associated with keratinocyte differentiation and that it is expressed in AK and SCC, which have metastatic potential, but minimally expressed in solid BCC.