RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation.

BACKGROUND: Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and ß-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. AIM: To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. METHODS: We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0), TATA-binding protein (TBP), GAPDH, actin beta (ACTB), tubulin (TUB), aminolevulinic acid synthase 1 (ALAS1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), eukaryotic translational elongation factor 1 alpha (EF1a), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), and beta-2-microglobulin (B2M)] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. RESULTS: All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. CONCLUSION: For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

Differential expression of Cadherins switch and Caveolin-2 during stages of oral carcinogenesis.

Background: Oral squamous cell carcinoma (OSCC) accounts for 90% of oral malignancies, which may be preceded by oral potentially malignant disorders (OPMDs). Cancer progression involves the downregulation of epithelial markers (E-cadherin) and the upregulation of mesenchymal markers (N-cadherin), which together characterise the epithelial-mesenchymal transition (EMT). Furthermore, caveolin can act on cell adhesion and migration events that regulate the expression of the E-cadherin/α-ß-catenin complex, thus favouring aggressive biological behaviour. This study aimed to analyse the immunoexpression of E-cadherin, N-cadherin and caveolin-2 at different stages of oral carcinogenesis to identify reliable biomarkers to predict malignant potential. Methods: Expressions of E-cadherin and N-cadherin in 14 normal oral mucosae (NOM), 14 OPMD and 33 OSCC specimens were evaluated using immunohistochemistry. Clinicopathological parameters were also assessed. Results: E-cadherin immunoexpression was significantly reduced during the progression of oral carcinogenesis (P = 0.0018). N-cadherin immunoexpression did not show any statistical differences between these groups. However, a representative number of N-cadherin-positive OSCC cases did not express E-cadherin. The expression of caveolin-2 increased significantly with the progression of the disease, from NOM to OSCC (P value: 0.0028). Conclusion: These findings indicate that cadherin switch and caveolin-2 immunoexpression may be regulatory events in oral carcinogenesis.

Matrix Metalloproteinases in Bone Resorption, Remodeling, and Repair.

Matrix metalloproteinases (MMPs) are the major protease family responsible for the cleavage of the matrisome (global composition of the extracellular matrix (ECM) proteome) and proteins unrelated to the ECM, generating bioactive molecules. These proteins drive ECM remodeling, in association with tissue-specific and cell-anchored inhibitors (TIMPs and RECK, respectively). In the bone, the ECM mediates cell adhesion, mechanotransduction, nucleation of mineralization, and the immobilization of growth factors to protect them from damage or degradation. Since the first description of an MMP in bone tissue, many other MMPs have been identified, as well as their inhibitors. Numerous functions have been assigned to these proteins, including osteoblast/osteocyte differentiation, bone formation, solubilization of the osteoid during bone resorption, osteoclast recruitment and migration, and as a coupling factor in bone remodeling under physiological conditions. In turn, a number of pathologies, associated with imbalanced bone remodeling, arise mainly from MMP overexpression and abnormalities of the ECM, leading to bone osteolysis or bone formation. In this review, we will discuss the functions of MMPs and their inhibitors in bone cells, during bone remodeling, pathological bone resorption (osteoporosis and bone metastasis), bone repair/regeneration, and emergent roles in bone bioengineering.

Cellular behavior as a dynamic field for exploring bone bioengineering: a closer look at cell-biomaterial interface.

Bone is a highly dynamic and specialized tissue, capable of regenerating itself spontaneously when afflicted by minor injuries. Nevertheless, when major lesions occur, it becomes necessary to use biomaterials, which are not only able to endure the cellular proliferation and migration, but also to substitute the original tissue or integrate itself to it. With the life expectancy growth, regenerative medicine has been gaining constant attention in the reconstructive field of dentistry and orthopedy. Focusing on broadening the therapeutic possibilities for the regeneration of injured organs, the development of biomaterials allied with the applicability of gene therapy and bone bioengineering has been receiving vast attention over the recent years. The progress of cellular and molecular biology techniques gave way to new-guided therapy possibilities. Supported by multidisciplinary activities, tissue engineering combines the interaction of physicists, chemists, biologists, engineers, biotechnologist, dentists and physicians with common goals: the search for materials that could promote and lead cell activity. A well-oriented combining of scaffolds, promoting factors, cells, together with gene therapy advances may open new avenues to bone healing in the near future. In this review, our target was to write a report bringing overall concepts on tissue bioengineering, with a special attention to decisive biological parameters for the development of biomaterials, as well as to discuss known intracellular signal transduction as a new manner to be explored within this field, aiming to predict in vitro the quality of the host cell/material and thus contributing with the development of regenerative medicine.

Calcifying Cystic Odontogenic Tumour: immunohistochemical expression of matrix metalloproteinases, their inhibitors (TIMPs and RECK) and inducer (EMMPRIN).

BACKGROUND: Calcifying cyst odontogenic tumour (CCOT) is a rare benign cystic neoplasm of odontogenic origin. MMPs are responsible for extracellular matrix remodelling and, together their inhibitors and inducer, determinate the level of its turnover in pathological processes, leading to an auspicious microenvironment for tumour development. Thus, our goal was to evaluate matrix metalloproteinases (MMPs-2, -7, -9 and -14), their inhibitors (TIMPs-2, -3, -4 and RECK) and its inductor (EMMPRIN) expression in CCOT. MATERIALS AND METHODS: We used 18 cases of CCOT submitted to immunolocalization of the target proteins and analysed in both neoplastic odontogenic epithelial and stromal compartments. RESULTS: All molecules evaluated were expressed in both compartments in CCOT. In epithelial layer, immunostaining for MMPs, TIMPs, RECK and EMMPRIN was found in basal, suprabasal spindle and stellate cells surrounding ghost cells and ghost cells themselves, except for MMP-9 and TIMP-2 which were only expressed by ghost cells. In stromal compartment, extracellular matrix, mesenchymal (MC) and endothelial cells (EC) were positive for MMP-2, -7, TIMP-3 and -4, while MMP-9, TIMP-2 and RECK were positive only in MC and MMP-14 only in EC. Statistical significance difference was found between both compartments for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001), being more expressed in epithelium than in stroma. Positive correlation between both stromal EMMPRIN and RECK expression was found (R = 0.661, P = 0.003). CONCLUSIONS: We concluded that these proteins/enzymes are differentially expressed in both epithelium and stroma of CCOT, suggesting an imbalance between MMPs and their inducer/inhibitors may contribute on the tumour behaviour.

MMP1 and MMP20 contribute to tooth agenesis in humans.

OBJECTIVE: Variations in genes that are critical for tooth formation may contribute to the tooth agenesis. MMPs are potential candidate genes for dental alterations based on the roles they play during embryogenesis. The aim of this study was to investigate the possible association between MMP1, MMP3, and MMP20 and tooth agenesis. METHODS: One hundred sixty-seven nuclear families from two different populations were analysed, 116 from Brazil and 51 from Turkey. Probands had at least one congenitally missing tooth. DNA samples were obtained from blood or saliva samples and genotyping was performed using TaqMan chemistry. In addition, Mmp20 was selected for quantitative real-time polymerase chain reaction analysis with SYBR Green I Dye in mouse tooth development. RESULTS: Associations between tooth agenesis and MMP1 (p=0.007), and MMP20 (p=0.03) were found in Brazilian families. In the total dataset, MMP20 continued to be associated with tooth agenesis (p=0.01). Mmp20 was not expressed during the initial stages of tooth development. CONCLUSION: Our findings provide evidence that MMP1 and MMP20 play a role in human tooth agenesis.

Studies with Wnt genes and nonsyndromic cleft lip and palate.

BACKGROUND: Clefts of the lip and/or palate (cleft lip/palate) are notable for their complex etiology. The WNT pathway regulates multiple developmental processes including craniofacial development and may play a role in cleft lip/palate and other defects of craniofacial development such as tooth agenesis. Variations in WNT genes have been recently associated with cleft lip/palate in humans. In addition, two WNT genes, Wnt3 and Wnt9B, are located in the clf1 cleft locus in mice. METHODS: We investigated 13 SNPs located in Wnt3A, Wnt5A, Wnt8A, Wnt11, Wnt3, and Wnt9B genes for association with cleft lip/palate subphenotypes in 463 cleft cases and 303 unrelated controls. Genotyping of selected polymorphisms was carried out using Taqman assays. PLINK 1.06 software was used to test for differences in allele frequencies of each polymorphism between affected and unaffected individuals. Haplotype analysis was also performed. RESULTS: Individuals carrying variant alleles in WNT3 presented an increased risk for cleft lip/palate (p = 0.0003; OR, 1.61; 95% CI, 1.29-2.02) in the population studied. CONCLUSION: Our results continue to support a role for WNT genes in the pathogenesis of cleft lip/palate. Although much remains to be learned about the function of individual WNT genes during craniofacial development, additional studies should focus on the identification of potentially functional variants in these genes as contributors to human clefting. Birth Defects Research (Part A), 2010. © 2010 Wiley-Liss, Inc.

MMP-9 and CD68(+) cells are required for tissue remodeling in response to natural hydroxyapatite.

Large bone defects represent major clinical problems in the practice of reconstructive orthopedic and craniofacial surgery. The aim of this study was to examine, through immunohistochemistry approach, the involvement of MMP-9 and CD68(+) cells during tissue remodeling in response to natural hydroxyapatite (HA) implanted in rat subcutaneous tissue. Before experimentation, forty animals were randomly distributed into two experimental groups: Group-I (Gen-Ox micro-granules) and Group-II (Gen-Ox macro-granules). Afterwards, the biopsies were collected after 10, 20, 30, and 60 days post-implantation. Our results showed that at 10 days, a low-renewal foreign body type granuloma formation was observed in most of the cases. Macrophage- and fibroblast-like cells were the predominant type of cells positively stained for MMP-9 in both groups. Once macrophage-like cells seemed to be the major source of MMP9, antibody against pan-CD68 epitope was used to correlate these findings. In agreement, MMP-9 and CD68(+) cells were distributed at the periphery and the central region of the granuloma in all experimental periods, however no staining was observed in cell contacting to material. Besides macrophages, the lysosomal glycoprotein epitope recognized by CD68 antibodies can be expressed by mast cell granules and sometimes by fibroblasts. Taken together, our results suggest that xenogenic HA promotes extracellular matrix remodeling through induction of MMP-9 activity and presence of CD68(+) cells.

Immunolocalization of matrix metalloproteinases-2 and -9 during apical periodontitis development.

OBJECTIVE: The objective of this study was to determine the expression of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) during apical periodontitis development. METHODS: Using an experimental design of induced periapical lesions in rats and immunohistochemistry assay as investigative tool, the MMP-2 and MMP-9 expression and distribution were evaluated at 3, 7, 14, 21, 30, 60 and 90 days after coronary access and pulp exposure of the first left mandibular molar to the oral environment. Two blind observers scored the immunoreactivity. A semi-quantitative analysis was performed. RESULTS: Except at day 3, MMP-2 and MMP-9 immunostaining was observed in all experimental periods. The MMP-2 (p=0.004) and MMP-9 (p=0.005) immunostaining was higher in the period between 7 and 21 days. They were mainly observed in cells surrounding the apical foramen and adjacent periapical areas. Cells into the hypercementosis areas were strongly stained while both osteoblasts and osteoclasts presented discrete staining along of this study. No staining was observed on epithelial walls. At 30, 60 and 90 days, the subjacent connective tissue presented intense MMP-2 and MMP-9 immunostaining in mononuclear cells (suggestive of fibroblasts, macrophages, infiltrating neutrophils and lymphocytes). CONCLUSION: The results observed in this study suggest that MMP-2 and MMP-9 play a critical role in the development of inflammatory periapical lesions, probably involved in the extracellular matrix (ECM) degradation during the initial phase of the lesion development.