Cephaeline is an inductor of histone H3 acetylation and inhibitor of mucoepidermoid carcinoma cancer stem cells.

AIM: To evaluate the potential use of Cephaeline as a therapeutic strategy to manage mucoepidermoid carcinomas (MEC) of the salivary glands. MATERIAL AND METHODS: UM-HMC-1, UM-HMC-2, and UM-HMC-3A MEC cell lines were used to establish the effects of Cephaeline over tumor viability determined by MTT assay. In vitro wound healing scratch assays were performed to address cellular migration while immunofluorescence staining for histone H3 lysine 9 (H3k9ac) was used to identify the acetylation status of tumor cells upon Cephaeline administration. The presence of cancer stem cells was evaluated by the identification of ALDH enzymatic activity by flow cytometry and through functional assays using in vitro tumorsphere formation. RESULTS: A single administration of Cephaeline resulted in reduced viability of MEC cells along with the halt on tumor growth and cellular migration potential. Administration of Cephaeline resulted in chromatin histone acetylation as judged by the increased levels of H3K9ac and disruption of tumorspheres formation. Interestingly, ALDH levels were increased in UM-HMC-1 and UM-HMC-3A cell lines, while UM-HMC-2 showed a reduced enzymatic activity. CONCLUSION: Cephaeline has shown anti-cancer properties in all MEC cell lines tested by regulating tumor cells' viability, migration, proliferation, and disrupting the ability of cancer cells to generate tumorspheres.

Propolis reduces the stemness of head and neck squamous cell carcinoma.

OBJECTIVE: To evaluate the effect of Brazilian propolis on head and neck cancer stem cells in vitro. METHODS: Head and neck squamous cell carcinoma (HNSCC) cell lines (UM-SCC-17B and UM-SCC-74A), human keratinocytes (HK), and primary human dermal microvascular endothelial cells (HDMEC) were treated with 0.5, 5.0, or 50 µg/mL green, brown or red Brazilian propolis or vehicle control for 24, 36, and 72 h. Cell viability was evaluated by Sulforhodamine B assay. Western blots evaluated expression of cancer stem cell (CSC) markers (i.e. ALDH, CD44, Oct-4, Bmi-1) and flow cytometry was performed to determine the impact of propolis in the fraction of CSC, defined as ALDHhighCD44high cells. RESULTS: propolis significantly reduced cell viability of HNSCC and HDMEC cells, but not HK. Notably, red propolis caused a significant reduction in the percentage of CSC, reduced the number of orospheres, and downregulated the expression of stem cell markers. CONCLUSIONS: Collectively, our data demonstrate an anti-CSC effect of propolis, and suggest that propolis (i.e. red propolis) might be beneficial for patients with head and neck cancer.

Venous Blood Derivatives as FBS-Substitutes for Mesenchymal Stem Cells: A Systematic Scoping Review.

Although the biological properties of mesenchymal stem cells (MSC) are well-characterized in vitro, MSC clinical application is still far away to be achieved, mainly due to the need of xenogeneic substances for cell expansion, such as fetal bovine serum (FBS). FBS presents risks regarding pathogens transmissions and internalization of animal's proteins, which can unleash antigenic responses in patients after MSC implantation. A wide range of venous blood derivatives (VBD) has been reported as FBS substitutes showing promising results. Thus, the aim of this study was to conduct a systematic scoping review to analyze whether VBD are effective FBS substitutes for MSC ex vivo expansion. The search was performed in SciVerse ScopusTM, PubMed, Web of ScienceTM, BIREME, Cochrane library up to January 2016. The keywords were selected using MeSH and entry terms. Two independent reviewers scrutinized the records obtained considering specific inclusion criteria. The included studies were evaluated in accordance with a modified Arksey and O' Malley's framework. From 184 found studies, 90 were included. Bone marrow mesenchymal stem cells (BMMSC) were presented in most of these studies. Overall, VBD allowed for either, maintenance of MCS's fibroblast-like morphology, high proliferation, high colony-formation ability and maintenance of multipotency. Besides. MSC expanded in VBD supplements presented higher mitogen activity than FBS. VBD seems to be excellent xeno-free serum for ex vivo expansion of mesenchymal stem cells. However, an accentuated heterogeneity was observed between the carried out protocols for VBD isolation did not allowing for direct comparisons between the included studies.

Venous Blood Derivatives as FBS-Substitutes for Mesenchymal Stem Cells: A Systematic Scoping Review

Abstract Although the biological properties of mesenchymal stem cells (MSC) are well-characterized in vitro, MSC clinical application is still far away to be achieved, mainly due to the need of xenogeneic substances for cell expansion, such as fetal bovine serum (FBS). FBS presents risks regarding pathogens transmissions and internalization of animal's proteins, which can unleash antigenic responses in patients after MSC implantation. A wide range of venous blood derivatives (VBD) has been reported as FBS substitutes showing promising results. Thus, the aim of this study was to conduct a systematic scoping review to analyze whether VBD are effective FBS substitutes for MSC ex vivo expansion. The search was performed in SciVerse ScopusTM, PubMed, Web of ScienceTM, BIREME, Cochrane library up to January 2016. The keywords were selected using MeSH and entry terms. Two independent reviewers scrutinized the records obtained considering specific inclusion criteria. The included studies were evaluated in accordance with a modified Arksey and O' Malley's framework. From 184 found studies, 90 were included. Bone marrow mesenchymal stem cells (BMMSC) were presented in most of these studies. Overall, VBD allowed for either, maintenance of MCS's fibroblast-like morphology, high proliferation, high colony-formation ability and maintenance of multipotency. Besides. MSC expanded in VBD supplements presented higher mitogen activity than FBS. VBD seems to be excellent xeno-free serum for ex vivo expansion of mesenchymal stem cells. However, an accentuated heterogeneity was observed between the carried out protocols for VBD isolation did not allowing for direct comparisons between the included studies.

Resumo Embora as propriedades biológicas das células-tronco mesenquimais (MSC) sejam bem caracterizadas in vitro, a aplicação clínica das MSC ainda está longe de ser alcançada, principalmente devido à necessidade de substâncias xenogênicas para expansão celular, como o soro fetal bovino (FBS). O FBS apresenta riscos quanto às transmissões de patógenos e à internalização de proteínas animais, o que pode desencadear respostas antigênicas em pacientes após a implantação das MSC. Uma vasta gama de derivados do sangue venoso (VBD) têm sido relatada como substitutos do FBS mostrando resultados promissores. Assim, o objetivo deste estudo foi conduzir uma revisão de escopo sistemática para analisar se VBD poderiam ser substitutos do FBS eficazes para expansão das MSC em condições ex vivo. A pesquisa foi realizada no SciVerse Scopus, PubMed, Web of Science, BIREME e biblioteca Cochrane até janeiro de 2016. As palavras-chave foram selecionadas usando MeSH e entre termos. Dois revisores independentes examinaram os registros obtidos considerando critérios de inclusão específicos. Os estudos incluídos foram avaliados de acordo com uma estrutura modificada de Arksey e O 'Malley. Dos 184 estudos encontrados, 90 foram incluídos. As células-tronco da medula óssea (BMMSC) foram utilizadas na maior parte destes estudos. Em geral, o VBD permitiu tanto a manutenção da morfologia semelhante a fibroblastos das MCS, alta proliferação, alta capacidade de formação de colônias e manutenção de multipotêncialidade. Além disso, as MSC expandidas em suplementos derivados do sangue venoso apresentaram uma maior atividade mitogênica do que as expandidas em FBS. Os VBD parecem ser excelentes soro livres de agentes xenogênicos para expansão ex vivo de MSC. Entretanto, observou-se uma heterogeneidade acentuada entre os protocolos realizados para o isolamento VBD, não permitindo assim comparações diretas entre os estudos incluídos.

Lipoprotein Receptor-related Protein 6 Signaling is Necessary for Vasculogenic Differentiation of Human Dental Pulp Stem Cells.

The aim of this study was to evaluate the effects of Wnt signaling through lipoprotein receptor-related protein 6 (LRP6) and Frizzled6 on the endothelial differentiation of dental pulp stem cells (DPSCs). DPSCs were stably transduced with enhanced green fluorescent protein (EGFP)-tagged lentiviral vectors (short hairpin RNA-LRP6, short hairpin RNA-Frizzled6, or empty vector controls). We evaluated the effects of LRP6 and Frizzled6 on expression of endothelial markers and on capillary tube formation mediated by DPSCs induced with recombinant human Wnt1 (rhWnt1) and/or recombinant human vascular endothelial growth factor165 (rhVEGF165). In vivo, tooth slices/scaffolds were seeded with LRP6-silenced, Frizzled6-silenced, or vector control DPSC cells and transplanted into immunodeficient mice. The density of blood vessels generated by DPSCs differentiated into vascular endothelial cells was analyzed by immunohistochemistry for EGFP. The rhWnt1 and rhVEGF165 induced expression of active ß-catenin in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. Furthermore, VEGF and interleukin-8 were downregulated in LRP6-silenced DPSCs, but not in control DPSCs or in Frizzled6-silenced DPSCs (P < .05). Likewise, rhWnt1 and rhVEGF165 induced expression of the endothelial marker VEGF receptor-2 in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. These data correlated with a trend for lower density of capillary sprouts generated by LRP6-silenced DPSCs when compared with control DPSCs in Matrigel. In vivo, tooth slice/scaffolds seeded with DPSC-short hairpinRNA-LRP6 cells showed lower density of human blood vessels (ie, EGFP-positive blood vessels), when compared with tooth slice/scaffolds seeded with vector control cells (P < .05). Collectively, these data demonstrated that LRP6 signaling is necessary for the vasculogenic differentiation of human DPSCs.

BH3-mimetic small molecule inhibits the growth and recurrence of adenoid cystic carcinoma.

OBJECTIVES: To evaluate the anti-tumor effect of BM-1197, a new potent and highly specific small molecule inhibitor of Bcl-2/Bcl-xL, in preclinical models of human adenoid cystic carcinoma (ACC). METHODS: Low passage primary human adenoid cystic carcinoma cells (UM-HACC-2A,-2B,-5,-6) and patient-derived xenograft (PDX) models (UM-PDX-HACC) were developed from surgical specimens obtained from 4 patients. The effect of BM-1197 on cell viability and cell cycle were evaluated in vitro using this panel of low passage ACC cells. The effect of BM-1197 on tumor growth, recurrence and tumor cell apoptosis in vivo was evaluated with the PDX model of ACC (UM-PDX-HACC-5). RESULTS: Exposure of low passage primary human ACC cells to BM-1197 mediated an IC50 of 0.92-2.82 µM. This correlated with an increase in the fraction of apoptotic cells (p<0.0001) and an increase in caspase-3 activity (p<0.0001), but no noticeable differences in cell cycle (p>0.05). In vivo, BM-1197 inhibited tumor growth (p=0.0256) and induced tumor cell apoptosis (p=0.0165) without causing significant systemic toxicities, as determined by mouse weight over time. Surprisingly, weekly BM-1197 decreased the incidence of tumor recurrence (p=0.0297), as determined by Kaplan-Meier analysis. CONCLUSION: These data demonstrated that single agent BM-1197 induces apoptosis and inhibits tumor growth in preclinical models of adenoid cystic carcinoma. Notably, single agent BM-1197 inhibited tumor recurrence, which is considered a major clinical challenge in the clinical management of adenoid cystic carcinoma. Collectively, these results suggest that patients with adenoid cystic carcinoma might benefit from therapy with a BH3-mimetic small molecule.

Glucose-regulated protein 78 (Grp78) confers chemoresistance to tumor endothelial cells under acidic stress.

OBJECTIVES: This study was designed to investigate the activation of the unfolded protein response (UPR) in tumor associated endothelial cells (TECs) and its association with chemoresistance during acidic pH stress. MATERIALS AND METHODS: Endothelial cells from human oral squamous cell carcinomas (OSCC) were excised by laser capture microdissection (LCM) followed by analysis of UPR markers (Grp78, ATF4 and CHOP) using quantitative PCR. Grp78 expression was also determined by immunostaining. Acidic stress was induced in primary human dermal microvascular endothelial cells (HDMECs) by treatment with conditioned medium (CM) from tumor cells grown under hypoxic conditions or by adjusting medium pH to 6.4 or 7.0 using lactic acid or hydrochloric acid (HCl). HDMEC resistance to the anti-angiogenic drug Sunitinib was assessed with SRB assay. RESULTS: UPR markers, Grp78, ATF4 and CHOP were significantly upregulated in TECs from OSCC compared to HDMECs. HDMECs cultured in acidic CM (pH 6.0-6.4) showed increased expression of the UPR markers. However, severe acidosis led to marked cell death in HDMECs. Alternatively, HDMECs were able to adapt when exposed to chronic acidosis at pH 7.0 for 7 days, with concomittant increase in Grp78 expression. Chronic acidosis also confers drug resistance to HDMECs against Sunitinib. Knockdown of Grp78 using shRNA resensitizes HDMECs to drug treatment. CONCLUSIONS: UPR induction in ECs under acidic pH conditions is related to chemoresistance and may contribute to therapeutic failures in response to chemotherapy. Targeting Grp78, the key component of the UPR pathway, may provide a promising approach to overcome ECs resistance in cancer therapy.

Spatial distribution of cancer stem cells in head and neck squamous cell carcinomas.

BACKGROUND: CD44 and aldehyde dehydrogenase 1 (ALDH1) are considered putative markers of highly tumorigenic cells (i.e., cancer stem-like cells) in head and neck squamous cell carcinomas. This small subset of cells is believed to be the primary responsible for tumor initiation and progression. The objectives of this study were (i) to evaluate the patterns of CD44 and ALDH1 expression in the tumor center and in the invasive front, as well as in adjacent non-tumor epithelium, and (ii) to correlate these findings with clinical parameters. MATERIALS AND METHODS: The sample comprised 44 patients with primary head and neck squamous cell carcinomas. Hematoxylin and eosin (HE) staining was used for histopathological tumor grading and for morphological analysis of adjacent non-tumor epithelium. Semiquantitative analysis was performed in histological sections immunostained for CD44 and ALDH1. RESULTS: ALDH1 immunostaining in the invasive front showed positive association with tumor size, regional metastasis, tumor histopathological grading, and disease progression. Moreover, expression of this marker in both tumor invasive front and adjacent non-tumor epithelium was related with more aggressive tumors. CD44 immunostaining was heterogeneous in all areas evaluated and did not show association with clinical data. CONCLUSION: Collectively, these data suggest that ALDH1 immunostaining in the invasive front and in adjacent non-tumor epithelium may help identify tumors with a more aggressive behavior, potentially contributing to improving treatment customization and the monitoring of patients with head and neck cancer.

Telomeres and tissue engineering: the potential roles of TERT in VEGF-mediated angiogenesis.

Telomeres are protective structures located at the end of eukaryotic chromosomes which are shortened after each cell division, leading to senescence. Telomerase activity prevents telomere shortening by reverse transcription catalyzed by the subunit called TERT (telomerase reverse transcriptase). TERT expression has shown interesting cellular properties, which may be appealing for tissue engineering, such as the enhancement of cell proliferation and differentiation abilities in vitro. Despite some evidence for playing these roles in VEGF (vascular endothelial growth factor)-mediated angiogenesis, it is still unclear whether TERT can contribute to this essential event to generate functional organs. This review suggests a hypothesis that TERT and VEGF potentially regulates the transcriptional expression of each other, which would give new perspectives in the roles of telomerase in regulating several cellular processes, and also contributing for a better comprehension of the molecular mechanisms underlying VEGF signaling (both paracrine and autocrine). In general, based on the literature revised, it is possible to conclude that TERT is a potential VEGF enhancer; however, it is necessary to elaborate methodological approaches to explore this potential and to assess the potential benefits on tissue engineering approaches.

Endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma.

BACKGROUND: Loco-regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl-2 expression in lymphatic endothelial cells. METHODS: Endothelial cells were selectively retrieved from paraffin-embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection. RT-PCR was used to evaluate Bcl-2 expression in tumor-associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)-C on the expression of Bcl-2 in primary human lymphatic endothelial cells. RESULTS: We observed that Bcl-2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage-matched tumors without metastasis. VEGF-C induced Bcl-2 expression in lymphatic endothelial cells via VEGFR-3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF-C and induce Bcl-2 in lymphatic endothelial cells. CONCLUSIONS: Collectively, this work unveiled a mechanism for the induction of Bcl-2 in lymphatic endothelial cells and suggested that endothelial cell Bcl-2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma.

Células-tronco na saúde e na doença / Stem cells in health and disease

Células-tronco foram recentemente caracterizadas como componentes fundamentais parao desenvolvimento e função de órgãos e tecidos do corpo humano. O descontrole nos processos de diferenciação e sobrevivência das células-tronco tem sido caracterizado como fator etiológico importante em alterações de desenvolvimento do complexo crânio-facial. O conhecimento gerado através de estudos feitos nesta área levantaram a possibilidade de se utilizar células-tronco terapeuticamente para regeneração de tecidos da cavidade oral. Por outro lado, células-tronco também foram caracterizadas em patologias importantes como o câncer. Em câncer, células-tronco participam dos processos de progressão tumoral, bem como na resistência a terapias uma vez que estas células são resistentes a quimioterapia convencional. Tanto em regeneração de tecidos quanto em câncer, é fundamental que se tenha uma compreensão profunda dos mecanismos que governam o comportamento das células-tronco para que se possa desenvolver terapias que sejam seguras e eficazes para os pacientes. O objetivo principal desta revisão é discutir o papel das células tronco em saúde e na doença, usando a regeneração de tecido pulpar e o câncer oral como exemplos de relevância no contexto de Odontologia.

Stem cells were recently characterized as critical components of the development and function of most tissues and organs in humans. Indeed, deregulated survival and differentiation of stem cells have been characterized as important etiological factors of developmental anomalies in the craniofacial region. This knowledge raised the possibility that stem cells could be useful for the therapeutic regeneration of oral tissues. On the other hand, stem cells appear to have an important role in tumor progression, as well as in the resistance of cancer to therapy. This might be due to the fact that stem cells are resilient, long-lived, and tend to survive to conventional chemotherapeutic drugs. Both, in tissue regeneration and in cancer, it is critical to have a profound understanding of the mechanisms that regulate the behavior of stem cells to develop therapies that are safe and effective for patients. The main objective of this review is to discuss the role of stem cells in health and disease, using dental pulp tissue regeneration and oral cancer as examples of relevance to Dentistry.

Effect of adhesive restorations over incomplete dentin caries removal: 5-year follow-up study in primary teeth.

PURPOSE: The purpose of this study was to evaluate the effect of the materials used for indirect pulp treatment (IPT) on the long-term outcome of primary molar teeth. METHODS: Forty-eight teeth with deep carious lesions, but without signs and symptoms of irreversible pulpitis, were randomly divided into 2 groups, according to the material placed on the demineralized dentin remain: (1) experimental group, adhesive system (Scotchbond Multipurpose); and (2) control group, calcium hydroxide liner (Dycal). Both groups were followed by a resin restoration application. RESULTS: After 4 to 5 years, the clinical and radiographic success rates between groups were similar (group 1=14 of 15; group 2=8 of 10; P=0.350). Subsequent to exfoliation, scanning electron microscopy revealed the presence of a hybrid layer at the resin-dentin interface and a microtensile bond strength of 9.63 MPa (group 1). Histological analysis showed that the pulp health status was similar in both groups. CONCLUSIONS: Indirect pulp treatment has a high clinical and radiographic long-term success rate in primary teeth and is not material-dependent.

Vascular endothelial growth factor receptor-2 expression in the pulp of human primary and young permanent teeth.

The purpose of this study was to evaluate the expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in endothelial cells within the dental pulp of human primary and young permanent teeth and the spatial distribution of VEGFR-2-positive cells. Nine sound primary teeth and 4 sound young permanent teeth were evaluated by immunohistochemistry with a human anti-VEGFR-2 antibody. Stained tissue sections were analyzed qualitatively under light microscopy. Here we observed that endothelial cells of both primary and permanent teeth showed positive immunostaining for VEGFR-2. Notably, VEGFR-2-positive cells in the primary teeth tended to be found close to the subodontoblastic layer, whereas the spatial distribution of VEGFR-2 immunostaining in the permanent teeth was more uniform. In conclusion, VEGFR-2 was expressed in the microvascular endothelial cells of both primary and young permanent teeth, which suggests that these cells are capable of responding to the morphogenetic and survival signals mediated by VEGF.

Indirect pulp capping in the primary dentition: a 4 year follow-up study.

PURPOSE: Evaluate clinical and radiographic changes in primary teeth submitted to indirect pulp capping (IPC) over a 48-month-period. METHODS: Twenty seven primary molars with deep caries, but without preoperative signs of irreversible pulpits, were treated with IPC. The teeth were randomly divided into two groups, according to the material used for protection of the dentin-pulp complex. (1) a calcium hydroxide liner (Dycal) and (2) glass ionomer cement (Vitremer). RESULTS: After 48 months, Group-1 showed a success rate of 88.8% and Group-2 of 93%. No statistical significant difference between the groups was observed (P = 0.62). CLINICAL SIGNIFICANCE: The results of this study suggested that indirect pulp capping in primary teeth arrests the progression of the underlying caries, regardless of the material used as a liner.

Indirect pulp treatment: in vivo outcomes of an adhesive resin system vs calcium hydroxide for protection of the dentin-pulp complex.

PURPOSE: The purpose of this prospective and randomized in vivo study was to compare the clinical and radiographic outcomes of an adhesive resin system vs a calcium hydroxide liner for protection of the dentin-pulp complex of primary molars treated with indirect pulp treatment. METHODS: Forty-eight primary molars with deep occlusal caries, but without preoperative signs and symptoms of irreversible pulpitis, received indirect pulp treatment and were restored with a composite resin (Z100). The teeth were randomly divided into 2 groups according to the material used for protection of the dentin-pulp complex: (1) adhesive resin system (Scotchbond MultiPurpose); and (2) calcium hydroxide liner (Dycal). These teeth were evaluated clinically and radiographicaly for 2 years. RESULTS: After 2 years, 83% (19/23) of the teeth treated with calcium hydroxide and 96% (24/25) of teeth treated with only the adhesive resin system presented a successful outcome, as determined by clinical and radiographic examination. Interradicular and/or periapical lesions were the most predominant signs of treatment failure, since 3 out of 23 teeth treated with calcium hydroxide and 1 out of 25 teeth treated with only adhesive resin presented this outcome. One tooth treated with the calcium hydroxide liner was diagnosed with internal root resorption at the 18-month examination. Of the 5 teeth diagnosed from radiographs as a failure of the indirect pulp treatment, none presented clinical signs/symptoms of pulpitis or necrosis such as the presence of fistula, enhanced tooth mobility, or pain. CONCLUSIONS: This study demonstrates that protection of the dentin-pulp complex of primary molars with an adhesive resin system results in similar clinical and radiographic 2-year outcomes as compared to calcium hydroxide when indirect pulp treatment is performed in Class I composite restorations.

Dentin bonding: SEM evaluation of the glass ionomer-dentim interface

The purpose of this study was to evaluate the microstructure of the glass ionomerdentin interface after various dentin surface treatments, and to determine which treatment established a hybrid layer at this interface. Thirty sound extracted human teeth (permanent molars) were stored in a disinfectant solution (0.2 percent sodium azide). The occlusal enamel was removed, the dentin exposed and divided into two halves allowing two different dentin treatments in the same tooth. All teeth were mounted in a hydrostatic intrapulpal pressure apparatus, to simulate the in vivo conditions, and removed 12 hours after restorations had been completed. Groups were studied with regards to dentin surface conditioning: 1) no dentin conditioning; 2) GC Conditioner; 3) Vitremer Primer; 4) 35 percent Phosphoric Acid + Vitremer Primer; 5) 35 percent Phosphoric Acid + Scotchbond Multi-Purpose Primer + Scotchbond Multi-Purpose Adhesive Resin. Standard procedures for preparing these specimens for SEM were utilized and the glass ionomer-dentin interface was evaluated under three different magnifications (300, 3000 and 13000 x). The presence and thickness of the hybrid layer was recorded for each specimen and a photomicrograph of a representative area was taken. The results indicated that the interfacial seal was more homogeneous when the dentin was conditioned with 35 percent Phosphoric Acid and either Vitremer Primer or Scotchbond Multi-Purpose was applied prior to the glass ionomer. This suggests that these two dentin treatments may provide better adhesion and less microleakage for glass ionomer restorations.