A covalently bound inhibitor triggers EZH2 degradation through CHIP-mediated ubiquitination.

Enhancer of zeste homolog 2 (EZH2) has been characterized as a critical oncogene and a promising drug target in human malignant tumors. The current EZH2 inhibitors strongly suppress the enhanced enzymatic function of mutant EZH2 in some lymphomas. However, the recent identification of a PRC2- and methyltransferase-independent role of EZH2 indicates that a complete suppression of all oncogenic functions of EZH2 is needed. Here, we report a unique EZH2-targeting strategy by identifying a gambogenic acid (GNA) derivative as a novel agent that specifically and covalently bound to Cys668 within the EZH2-SET domain, triggering EZH2 degradation through COOH terminus of Hsp70-interacting protein (CHIP)-mediated ubiquitination. This class of inhibitors significantly suppressed H3K27Me3 and effectively reactivated polycomb repressor complex 2 (PRC2)-silenced tumor suppressor genes. Moreover, the novel inhibitors significantly suppressed tumor growth in an EZH2-dependent manner, and tumors bearing a non-GNA-interacting C668S-EZH2 mutation exhibited resistance to the inhibitors. Together, our results identify the inhibition of the signaling pathway that governs GNA-mediated destruction of EZH2 as a promising anti-cancer strategy.

Systemic biomarkers of inflammation, oxidative stress and tissue injury and repair among waterpipe, cigarette and dual tobacco smokers.

BACKGROUND: Waterpipe tobacco (WPT) smoking is associated with deleterious effects on cardio-pulmonary systems which may have adverse repercussions in pathophysiology and progression of chronic lung and cardiovascular diseases. We compared the biomarkers of systemic inflammation, lipid mediators, injury/repair and oxidative stress between groups of non-smokers (NS), exclusive WPT smokers (WPS), exclusive cigarette smokers (CS) and dual WPS and CS (DS). METHODS: Two cohorts were recruited. Cohort I consisted of WPS (n=12), CS (n=26), DS (n=10) and NS (n=25). Cohort II consisted of WPS (n=33) and NS (n=24). Plasma and urine samples were collected and analysed for various systemic biomarkers. RESULTS: Compared with NS, plasma levels of inflammatory mediators (interleukin (IL)-6, IL-8, IL1ß and tumor necrosis factor-α) were significantly higher in WPS and CS, and were further augmented in DS. Endothelial biomarkers (intracellular adhesion molecule-1, prostaglandin E-2 and metalloproteinase-9) were significantly higher in CS. Most notably, pro-resolving lipid mediator (resolvin E1) and biomarkers of immunity, tissue injury, and repair were significantly lower in WPS and CS. Urinary levels of 8-isoprostane were significantly higher in all smoking groups in cohort I, while 8-isoprostane, myeloperoxidase, receptor for advanced glycation end products (RAGE), En-RAGE and matrix metalloproteinase-9 were significantly higher in all smoking groups in cohort II. CONCLUSIONS: Biomarkers of inflammation, oxidative stress, immunity, tissue injury and repair were elevated in WPS and CS groups. Furthermore, concurrent use of WPT and cigarettes is more harmful than cigarette or WPT smoking alone. These data may help inform the public and policy-makers about the dangers of WPT smoking and dual use of tobacco products.

Stabilization of Slug by NF-κB is Essential for TNF-α -Induced Migration and Epithelial-Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma Cells.

BACKGROUND/AIMS: Slug protein, a transcription factor for the induction of epithelial-mesenchymal transition (EMT) and cancer cell invasion and metastasis, is frequently upregulated in human epithelial cancers. However, mutation of this gene in cancer is rare, and the mechanism of its dysregulation remains unknown, especially in head and neck squamous cell carcinoma (HNSCC). METHODS: We examined the role of TNF-α in the stabilization of Slug by immunoprecipitation-westernblot analysis. Migration of HNSCC cells with or without knockdown of Slug gene expression was assayed by a wound healing assay. Immunohistochemical staining analysis was used to measurement Slug levels in both normal and HNSCC tumor tissues. RESULTS: The inflammatory cytokine TNF-α stabilized Slug protein by inhibiting its ubiquitination through the NF-κB pathway. Inhibition of NF-κB or knockdown of p65 abrogated the TNF-α-induced stabilization of Slug. Knockdown of Slug expression inhibited cancer cell migration and EMT characteristics induced by TNF-α. Moreover, increased levels of Slug were found to correlate with lymph node metastasis and predict poor prognosis in patients with HNSCC. CONCLUSIONS: NF-κB-mediated stabilization of Slug underlies the inflammation-induced EMT and metastasis in HNSCC, which may serve as a therapeutic target for metastatic HNSCC.

Repression of GPRC5A is associated with activated STAT3, which contributes to tumor progression of head and neck squamous cell carcinoma.

BACKGROUND: G protein-coupled receptor family C group 5 member A (GPRC5A), a retinoic acid-inducible gene, is a lung tumor suppressor. Previously, we showed that repression of GPRC5A expression was associated with pathologic differentiation grade of oral squamous cell carcinomas (OSCC) and overexpression of GPRC5A gene inhibited the malignant phenotype in OSCC cells, suggesting that GPRC5A also functions as a tumor suppressor in oral cancer. However, the molecular mechanisms underlying GPRC5A deficiency in head and neck squamous cell carcinoma (HNSCC) are still unclear. METHODS: In this study, we used Western blot analysis and immunohistochemical (IHC) staining to investigate the expression of GPRC5A in both HNSCC cell lines and clinical samples. GPRC5A stable transfectants and their parental HNSCC cells were characterized for their biological activities in anchorage-independent growth. RESULTS: IHC analysis showed that, GPRC5A expression was high in normal tissue, but gradually decreased in oral leukoplakia, a precancerous stage, and greatly suppressed in primary cancer. Repression of GPRC5A was correlated with activated STAT3, which associates with aggressive clinicopathological features in HNSCC patients. Moreover, overexpression of GPRC5A suppressed IL-6-induced-STAT3 activation and inhibited anchorage-independent growth in HNSCC cells. CONCLUSIONS: Repressed GPRC5A associates with increased tumor grade and activated STAT3, which may be used as a prognostic marker for tumor progression of HNSCC.

Overexpression of c-fos promotes cell invasion and migration via CD44 pathway in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the most deadly malignant tumors with high invasive potential and frequently cervical lymph node metastasis. AP-1 plays a critical role in tumor invasion and metastasis, but there are few reports on the role of c-Fos in OSCC carcinogenesis and metastasis. METHODS: Investigate c-Fos expression in clinical samples from 58 primary patients with OSCC by immunohistochemistry. c-Fos knockdown stable cell lines were established by lentiviral infection and transwell cell invasion assay to detect the effects of c-Fos knockdown on tumor cell invasion. RESULTS: Nuclear and cytoplasmic c-Fos protein were both overexpression in cancerous tissues compared with adjacent non-malignant epithelia (nuclear: P < 0.001, cytoplasmic: P = 0.005). Higher level nuclear c-Fos expression was found in the tumor samples of patients with lymph node metastasis than those without lymph node metastasis (4.85 ± 1.43 vs. 3.61 ± 1.28, P = 0.002). Higher level of c-Fos expression was also found in tumor invasive front margin than tumor center and high nuclear expression of c-Fos indicated poor survival. Knockdown of c-Fos greatly suppressed tumor cell proliferation and invasion and downregulated CD44 and CyclinD1 expression in HN6 and SCC9 cells. However, CyclinD3, c-myc, and Erk1/2 were found no changes to c-Fos depletion. CONCLUSIONS: c-Fos promoted cell invasion and migration via CD44 pathway in OSCC. c-Fos could be used as a potential therapeutic target gene and an additional marker for evaluation of lymph node metastasis.

Plasma membrane proteomics of tumor spheres identify CD166 as a novel marker for cancer stem-like cells in head and neck squamous cell carcinoma.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) have a poor prognosis with the currently available therapy, and tumor recurrence is frequently observed. The discovery of specific membrane-associated cancer stem cell (CSC) markers is crucial for the development of novel therapeutic strategies to target these CSCs. To address this issue, we established sphere cultures to enrich CSCs and used them for plasma membrane proteomics to identify specific membrane signatures of the HNSCC spheres. Of a dataset that included a total of 376 identified proteins, 200 were bona fide membrane proteins. Among them, 123 proteins were at least 1.5-fold up- or down-regulated in the spheres relative to the adherent cultures. These proteins included cell adhesion molecules, receptors, and transporter proteins. Some of them play key roles in wnt, integrin, and TGFß signaling pathways. When we compared our dataset with two published hESC membrane protein signatures, we found 18 proteins common to all three of the databases. CD166 and CD44 were two such proteins. Interestingly, the expression of CD166, rather than that of the well-established HNSCC CSC marker CD44, was significantly related to the malignant behavior of HNSCC. Relative to CD166(low) HNSCC cells, CD166(high) HNSCC cells had a greater sphere-formation ability in vitro and tumor formation ability in vivo. Patients whose tumors expressed high levels of CD166 had a significantly poorer clinical outcome than those whose tumors expressed low levels of CD166 (cohort 1: 96 cases, p = 0.040), whereas the level of CD44 expression had only a marginal influence on the clinical outcome of patients with HNSCC (p = 0.078). The level of CD166 expression in HNSCC tumors was also associated with the tumor recurrence rate (cohort 2: 104 cases, p = 0.016). This study demonstrates that CD166 is a valuable cell surface marker for the enrichment of HNSCC stem cells and that plasma membrane proteomics is a promising biological tool for investigating the membrane proteins of CSCs.

Implementing tissue engineering and regenerative medicine solutions in medical implants.

BACKGROUND: Surgical implants are widely used in the medical field but their long-term performance is limited due to failure of integration with tissues. This manuscript describes very well-known problems associated with implants and discusses novel solutions used in tissue engineering and regenerative medicine that can be implemented in this uncommonly discussed medical area. SOURCES OF DATA: General and medical literature describing modifications of medical and surgical implants, biofunctionalization, tissue engineering and regenerative medicine. AREAS OF AGREEMENT: Procedures for surgical implantation have grown substantially in the last few decades and provided improved quality of life for patients, regardless of area of implantation and device type and purpose. AREAS OF CONTROVERSY: In general, implants fail because of lack of long-term integration with the surrounding tissues. Implant manufacturers have not addressed implant failure from the point of view of biointegration. In addition, some medical practitioners are inclined to treat implant failure by using anti-infection methods to prevent bacterial adhesion. However, both approaches are conceptually limited, as discussed in this manuscript. GROWING POINTS: Implantation in the future will not be limited to medically needed procedures but also to a growing number of cosmetic body transformation procedures, which may include perceived 'improved implant functions' over natural tissues or organs. An additional trend is that implant procedures are being progressively performed in younger individuals. AREAS TIMELY FOR DEVELOPING RESEARCH: Current implants generally do not allow the physician to have controlled long-term access to internal tissues in contact with the implants, for example to release specific compounds when medically needed to the problem area.

A novel electrospun nerve conduit enhanced by carbon nanotubes for peripheral nerve regeneration.

For artificial nerve conduits, great improvements have been achieved in mimicking the structures and components of autologous nerves. However, there are still some problems in conduit construction, especially in terms of mechanical properties, biomimetic surface tomography, electrical conductivity and sustained release of neurotrophic factors or cells. In this study, we designed and fabricated a novel electrospun nerve conduit enhanced by multi-walled carbon nanotubes (MWNTs) on the basis of a collagen/poly(ε-caprolactone) (collagen/PCL) fibrous scaffold. Our aim was to provide further knowledge about the mechanical effects and efficacy of MWNTs on nerve conduits as well as the biocompatibility and toxicology of MWNTs when applied in vivo.The results showed that as one component, carboxyl MWNTs could greatly alter the composite scaffold's hydrophilicity, mechanical properties and degradability. The electrospun fibers enhanced by MWNTs could support Schwann cell adhesion and elongation as a substrate in vitro. In vivo animal studies demonstrated that the MWNT-enhanced collagen/PCL conduit could effectively promote nerve regeneration of sciatic nerve defect in rats and prevent muscle atrophy without invoking body rejection or serious chronic inflammation. All of these results showed that this MWNT-enhanced scaffold possesses good biocompatibility and MWNTs might be excellent candidates as engineered nanocarriers for further neurotrophic factor delivery research.

Vacuum extraction enhances rhPDGF-BB immobilization on nanotubes to improve implant osseointegration in ovariectomized rats.

Nanotube morphology has been previously applied to improve osseointegration in osteoporosis, but the osteogenic capability of the technique requires further improvements. This study aimed to investigate the effects of vacuum extraction on the loading of rhPDGF-BB on nanotube arrays as well as its effects on the osseointegration of ovariectomized (OVX) rats. More rhPDGF-BB protein particles aggregated on the nanotube surface and into the nanotube after vacuum extraction for 10 min. The immobilized protein could be slowly released for at least 14 days and still kept its biological activity. In vitro, the immobilized rhPDGF-BB enhanced cell adhesion, proliferation and osteogenic differentiation. In vivo, more rhPDGF-BB immobilized on the nanotube surface also promoted the osseointegration. These results suggest that the enhanced immobilization of rhPDGF-BB on nanotube arrays can potentially be used in the future as an implant surface modification strategy in dental and orthopedic applications in osteoporotic patients. FROM THE CLINICAL EDITOR: This study presents convincing evidence that enhanced immobilization of recombinant human PDGF-BB protein particles on nanotubes lead to improved osteogenic differentiation in an experimental system. When used as a surface modification strategy for dental or orthopedic implants, this method was able to promote osseointegration even in an osteoporotic animal model, raising the likelihood for potential future clinical applications.

A Systematic Review of the Use of Intraoral Scanning for Human Identification Based on Palatal Morphology.

A common application for intraoral scanners is the digitization of the morphology of teeth and palatal rugae. Palatal scans are most commonly required to fabricate complete dentures and immediate transitional dentures and serve as a reference point for assessing orthodontic results. However, they are also frequently included by accident, even though the main purpose of intraoral scanning is to reconstruct dentition using computer-aided manufacturing (CAM). The literature shows that the identification of disaster victims has frequently involved palatal rugae impressions. As the skull provides sound insulation, the rugae are resistant to heat, chemicals, and stress. Antemortem data might be difficult to find during a forensic inquiry, particularly in disaster victim identification cases. In contrast with DNA and fingerprints, there is a greater likelihood of having a dental record that contains palatal scans. With specialized software, the scans can be exported as open stereolithography (STL) files. Considering that a full case consumes up to about 100 MB of hard drive space, long-term storage should not be an issue compared to a plaster model. Additionally, dentists widely use online databases to exchange data for smile design, implant registration, and orthodontic purposes. This will produce a digital database that grows quickly and is readily usable for forensic investigations. The uniqueness of forensic features is frequently challenged; however, palatal morphology's unique trait could make it possible as it is characteristic of individuals as well as the most distinguishing factor. This review will highlight how rugae, palatal morphology, mirroring, superimposition, and geometrics can serve in forensic identification.

Repression of G protein-coupled receptor family C group 5 member A is associated with pathologic differentiation grade of oral squamous cell carcinoma.

BACKGROUND: G protein-coupled receptor family C group 5 member A (GPRC5A), a member of G protein-coupled receptor family, has been shown to function as a tumor suppressor in lung tissue. The biological functions of GPRC5A have therefore been linked to lung tissue. However, the biological significance of this gene product remains obscure. In this study, we investigated the expression of GPRC5A proteins in normal oral tissue and oral squamous cell carcinoma (OSCC), and we characterized its biological activity in OSCC cell lines. METHODS: Western blot analysis and immunohistochemical staining were used to investigate the expression of GPRC5A in both OSCC cell lines and clinical samples. GPRC5A stable transfectants and their parental OSCC cells were characterized for their biological activities in anchorage-independent growth. RESULTS: High levels of immunohistochemical GPRC5A expression were detected in normal oral tissue, especially differentiated area. In contrast, GPRC5A expression was dramatically repressed in OSCCs (P < 0.01). The immunohistochemical GPRC5A expression was moderately well differentiated, but greatly repressed in moderately differentiated OSCCs and completely repressed in poorly differentiated OSCCs. Overexpression of GPRC5A in OSCC CAL27 cells resulted in a suppressed anchorage-independent growth activity, a transforming phenotype. CONCLUSIONS: GPRC5A is expressed in normal oral epithelium. Repression of GPRC5A is associated with poorly differential grade of OSCCs. Overexpression of GPRC5A in OSCC cell line reversed the malignant phenotype. Thus, GPRC5A is important for homeostasis in oral tissue, and deletion or repression of this gene may involve in tumorigenesis of OSCCs and may serve as a prognostic marker for malignant type of OSCCs.

Emerging Oral Nicotine Products and Periodontal Diseases.

Oral nicotine pouches are emerging as a new "modern oral" nicotine product. These prefilled pouches contain nicotine, flavorings, and filling agents that dissolve in the mouth. Nicotine can be derived from tobacco leaf or chemical synthesis. Traces of TSNAs and toxic chromium were detected in the pouch products. This raises the concern about general and periodontal health. This review aims to update the current oral nicotine products research relating to periodontal disease and its relevance in periodontal inflammation. Nicotine interacts with host cells and affects inflammatory responses to microbial challenges. It may directly or indirectly deteriorate periodontal tissues by activating nicotinic acetylcholine receptors, repressing PDL fibroblasts cells, increasing cellular ROS and cytokines/chemokines, growth factors, breaking microbiota balance, and dysregulating miRNAs expression. Studies show that appealing flavorings contained in nicotine pouches pose harm to periodontal innate immune responses and increase penetration of nitrosamines. In addition, flavored ONPs increase the risk of dual or poly-tobacco products among young adults, stacking up detrimental effects on the periodontium. Given the recent growth of users, further studies are needed to elucidate the impact of ONPs, even poly-tobacco use, on systemic and periodontal health. Moreover, policymakers should ensure to avoid generating a new wave of nicotine addiction among youths in the U.S.

The Relationship between Oral Health and Schizophrenia in Advanced Age-A Narrative Review in the Context of the Current Literature.

Schizophrenia is a psychiatric disorder that makes patients incompetent to perform day-to-day activities due to their progressing mental illness. In addition to disturbances with thoughts, behavioral changes, and impaired cognitive functions, oro-systemic health also becomes compromised. Even though the population with schizophrenia is primarily made up of older people, little is known about this group's oral health treatment. The present review explores the relationship between oral healthcare and elderly patients with schizophrenia. Our literature search included databases, like PubMed, Embase, and Google Scholar, for appropriate and evidence-based information. Preventive and management strategies outlined in the included articles and future research perspectives in this field are discussed. To the best of our knowledge, this is the first review that looked at dental care and related characteristics in older schizophrenia patients. The findings highlight the necessity for targeted dental interventions to address the dental health challenges faced by this vulnerable population. Integrating dental health into the overall medical management of elderly individuals with schizophrenia is crucial. Although specific therapies remain limited, the emphasis is on preventive dentistry to reduce the occurrence and progression of oral diseases in this group.

Role of toll-like receptor 4 on the immune escape of human oral squamous cell carcinoma and resistance of cisplatin-induced apoptosis.

BACKGROUND: Toll-like receptor 4 (TLR4) is expressed on immune cells as a sensor that recognizes lipopolysaccharide (LPS), a microbial conserved component. It has recently been determined that the expression of TLR4 is also found in various types of tumor cells. Cisplatin is a widely used chemotherapeutic agent for oral squamous cell carcinoma (OSCC) treatment. However, the mechanisms responsible for cisplatin resistance are not well understood. RESULTS: The present study was designed to elucidate the role of TLR4 expression in human OSCC regarding immune escape and apoptotic resistance to cisplatin. TLR4 and the myeloid differentiation primary response gene 88 (MyD88) were highly expressed in OSCC cell lines. Upon LPS stimulation both NF-κB and p38 MAPK pathways were activated in OSCC cell lines, followed by the production of large quantities of IL-6, IL-8 and VEGF compared with human immortalized oral epithelia cells (HIOECs). OSCC cell lines were found to be resistant to cisplatin-mediated apoptosis after pretreatment with LPS. CONCLUSIONS: Our results suggested that TLR4 was functionally expressed in human OSCC cells and development of resistance to cisplatin in human OSCC might occur through the mechanism involving TLR4 and its signaling pathway. Suppression of TLR4 and its signaling pathway might thus elevate sensitivity to cisplatin and potentially help improve the prognosis of patients with OSCC.

Repair of critical-sized rat calvarial defects using genetically engineered bone marrow-derived mesenchymal stem cells overexpressing hypoxia-inducible factor-1α.

The processes of angiogenesis and bone formation are coupled both temporally and spatially during bone repair. Bone marrow-derived mesenchymal stem cells (BMSCs) have been effectively used to heal critical-size bone defects. Enhancing their ability to undergo angiogenic and osteogenic differentiation will enhance their potential use in bone regeneration. Hypoxia-inducible factor-1α (HIF-1α) has recently been identified as a major regulator of angiogenic-osteogenic coupling. In this study, we tested the hypothesis that HIF-1α gene therapy could be used to promote the repair of critical-sized bone defects. Using lentivirus-mediated delivery of wild-type (HIF) or constitutively active HIF-1α (cHIF), we found that in cultured BMSCs in vitro, HIF and cHIF significantly enhanced osteogenic and angiogenic mRNA and protein expression when compared with the LacZ group. We found that HIF-1α-overexpressing BMSCs dramatically improved the repair of critical-sized calvarial defects, including increased bone volume, bone mineral density, blood vessel number, and blood vessel area in vivo. These data confirm the essential role of HIF-1α modified BMSCs in angiogenesis and osteogenesis in vitro and in vivo.

Sciatic nerve regeneration in rats by a promising electrospun collagen/poly(ε-caprolactone) nerve conduit with tailored degradation rate.

BACKGROUND: To cope with the limitations faced by autograft acquisitions particularly for multiple nerve injuries, artificial nerve conduit has been introduced by researchers as a substitute for autologous nerve graft for the easy specification and availability for mass production. In order to best mimic the structures and components of autologous nerve, great efforts have been made to improve the designation of nerve conduits either from materials or fabrication techniques. Electrospinning is an easy and versatile technique that has recently been used to fabricate fibrous tissue-engineered scaffolds which have great similarity to the extracellular matrix on fiber structure. RESULTS: In this study we fabricated a collagen/poly(ε-caprolactone) (collagen/PCL) fibrous scaffold by electrospinning and explored its application as nerve guide substrate or conduit in vitro and in vivo. Material characterizations showed this electrospun composite material which was made of submicron fibers possessed good hydrophilicity and flexibility. In vitro study indicated electrospun collagen/PCL fibrous meshes promoted Schwann cell adhesion, elongation and proliferation. In vivo test showed electrospun collagen/PCL porous nerve conduits successfully supported nerve regeneration through an 8 mm sciatic nerve gap in adult rats, achieving similar electrophysiological and muscle reinnervation results as autografts. Although regenerated nerve fibers were still in a pre-mature stage 4 months postoperatively, the implanted collagen/PCL nerve conduits facilitated more axons regenerating through the conduit lumen and gradually degraded which well matched the nerve regeneration rate. CONCLUSIONS: All the results demonstrated this collagen/PCL nerve conduit with tailored degradation rate fabricated by electrospinning could be an efficient alternative to autograft for peripheral nerve regeneration research. Due to its advantage of high surface area for cell attachment, it is believed that this electrospun nerve conduit could find more application in cell therapy for nerve regeneration in future, to further improve functional regeneration outcome especially for longer nerve defect restoration.

Maxillary sinus floor elevation using a tissue-engineered bone with rhBMP-2-loaded porous calcium phosphate cement scaffold and bone marrow stromal cells in rabbits.

The aim of this study was to evaluate the effects of maxillary sinus floor elevation by a tissue-engineered bone complex with recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded porous calcium phosphate cement (CPC) scaffold and bone marrow stromal cells (bMSCs) in rabbits. bMSCs were cultured and osteogenically induced. The osteoblastic differentiation of expanded bMSCs was detected by alkaline phosphatase activity, and calcium deposits in vitro. Thirty-six rabbits were randomly allocated into week 2, 4 and 8 observation groups. At each time point, 24 maxillary sinus floor elevation surgeries in 12 rabbits were performed bilaterally and randomly implanted by (1) CPC materials alone (group A, n = 6), (2) rhBMP-2/CPC composite materials alone (group B, n = 6), (3) CPC/bMSCs complex (group C, n = 6) and (4) rhBMP-2/CPC/bMSCs complex (group D, n = 6). As for maxillary sinus floor elevation, rhBMP-2-loaded CPC could promote new bone formation as compared to CPC, while addition of bMSCs could further enhance its new bone formation and maturity significantly, as detected by histological findings, and fluorochrome labeling. Our data suggested that rhBMP-2/CPC possessed excellent osteoinductive ability, while combining with bMSCs could further promote new bone formation and maturation in maxillary sinus elevation.

Downregulation of tapasin expression in primary human oral squamous cell carcinoma: association with clinical outcome.

MHC class I peptide loading complex defects are frequently observed in tumor cells which facilitate tumor cells escaping from immune surveillance. Tapasin plays an important role in the assembly of MHC class I molecules with peptides in the endoplasmic reticulum. The aim of this study was to investigate the expression of tapasin in primary oral squamous cell carcinoma (OSCC) and its potential clinical implication. Formalin-fixed, paraffin-embedded tumor biopsies from 67 patients with primary OSCC were analyzed for tapasin expression using immunohistochemistry. Tapasin promoter methylation status in OSCC cell lines was determined using ethylation-specific polymerase chain reaction, bisulphate genomic sequencing, and expression reactivation assay. Lack of tapasin expression was observed in 30 of 67 (43%) tumors and was significantly associated with poor pathologic differentiation grade of OSCC (P = 0.028). The cumulative 5-year survival rate was also significantly correlated with pathologic differentiation grade (P = 0.001) and tapasin expression level (P = 0.015). Decreased tapasin expression was an indicator of poor survival (P = 0.048). Tapasin promoter methylation was observed in all three OSCC cell lines examined, and the mRNA and protein levels of tapasin increased markedly after treatment with 5-aza-2'-deoxycytidine. Downregulation of tapasin is associated with a poor clinical outcome for OSCC patients and may serve as a prognostic biomarker. The promoter methylation may contribute to the tapasin downregulation in OSCC.

Decreased Osteogenic Ability of Periodontal Ligament Stem Cells Leading to Impaired Periodontal Tissue Repair in BRONJ Patients.

Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is a severe adverse reaction, which results in progressive bone destruction in the maxillofacial region of patients. To date, the pathological mechanisms remain largely unclear. Recently, we found that BRONJ patient had significantly deep periodontal pockets and severe periodontal bone defects before the exposed necrotic bone. Human periodontal ligament stem cells (hPDLSCs) play key roles in physiological maintenance and regeneration of periodontal tissues. However, the activities of hPDLSCs derived from BRONJ lesions and the role of hPDLSCs in BRONJ periodontal defect repair remain poorly understood. The aim of the present study was to elucidate the role of hPDLSCs in BRONJ. In this study, we found that the capacities of cell proliferation, adhesion, and migration of hPDLSCs derived from BRONJ lesions (BRONJ-hPDLSCs) were significantly decreased compared with control-hPDLSCs. BRONJ-hPDLSCs underwent early apoptosis compared with control-hPDLSCs. Importantly, we first demonstrated that BRONJ-hPDLSCs exhibited impaired osteogenic differentiation abilities in ectopic osteogenesis of nude mice. The above results suggested that the impaired BRONJ-hPDLSCs may be an important factor in deficient periodontal repair of BRONJ lesions and provide new insight into the underlying mechanism of BRONJ.

Activation of mesenchymal stem cells promotes new bone formation within dentigerous cyst.

BACKGROUND: Dentigerous cyst (DC) is a bone destructive disease and remains a challenge for clinicians. Marsupialization enables the bone to regenerate with capsule maintaining, making it a preferred therapeutic means for DC adjacent to vital anatomical structures. Given that capsules of DC are derived from odontogenic epithelium remnants at the embryonic stage, we investigated whether there were mesenchymal stem cells (MSCs) located in DC capsules and the role that they played in the bone regeneration after marsupialization. METHODS: Samples obtained before and after marsupialization were used for histological detection and cell culture. The stemness of cells isolated from fresh tissues was analyzed by morphology, surface marker, and multi-differentiation assays. Comparison of proliferation ability between MSCs isolated from DC capsules before (Bm-DCSCs) and after (Am-DCSCs) marsupialization was evaluated by Cell Counting Kit-8 (CCK-8), fibroblast colony-forming units (CFU-F), and 5'-ethynyl-2'-deoxyuridine (EdU) assay. Their osteogenic capacity in vitro was detected by alkaline phosphatase (ALP) and Alizarin Red staining (ARS), combined with real-time polymerase chain reaction (RT-PCR) and immunofluorescence (IF) staining. Subcutaneous ectopic osteogenesis as well as cranial bone defect model in nude mice was performed to detect their bone regeneration and bone defect repairability. RESULTS: Bone tissue and strong ALP activity were detected in the capsule of DC after marsupialization. Two types of MSCs were isolated from fibrous capsules of DC both before (Bm-DCSCs) and after (Am-DCSCs) marsupialization. These fibroblast-like, colony-forming cells expressed MSC markers (CD44+, CD90+, CD31-, CD34-, CD45-), and they could differentiate into osteoblast-, adipocyte-, and chondrocyte-like cells under induction. Notably, Am-DCSCs performed better in cell proliferation and self-renewal. Moreover, Am-DCSCs showed a greater osteogenic capacity both in vitro and in vivo compared with Bm-DCSCs. CONCLUSIONS: There are MSCs residing in capsules of DC, and the cell viability as well as the osteogenic capacity of them is largely enhanced after marsupialization. Our findings suggested that MSCs might play a crucial role in the healing process of DC after marsupialization, thus providing new insight into the treatment for DC by promoting the osteogenic differentiation of MSCs inside capsules.