Anti-Fibronectin Aptamer Modifies Blood Clot Pattern and Stimulates Osteogenesis: An Ex Vivo Study.

BACKGROUND: Scaffold (SCA) functionalization with aptamers (APT) provides adsorption of specific bioactive molecules on biomaterial surfaces. The aim of this study was to observe if SCA enriched with anti-fibronectin APT can favor coagulum (PhC) and osteoblasts (OSB) differentiation. METHODS: 20 µg of APT was functionalized on SCA by simple adsorption. For PhC formation, SCAs were inserted into rat calvaria defects for 17 h. Following proper transportation (buffer solution PB), OSBs (UMR-106 lineage) were seeded over PhC + SCAs with and without APT. Cells and PhC morphology, PhC cell population, protein labeling and gene expression were observed in different time points. RESULTS: The APT induced higher alkaline phosphatase and bone sialoprotein immunolabeling in OSB. Mesenchymal stem cells, leukocytes and lymphocytes cells were detected more in the APT group than when scaffolds were not functionalized. Additionally, an enriched and dense fibrin network and different cell types were observed, with more OSB and white blood cells in PhC formed on SCA with APT. The gene expression showed higher transforming growth factor beta 1 (TGF-b1) detection in SCA with APT. CONCLUSIONS: The SCA functionalization with fibronectin aptamers may alter key morphological and functional features of blood clot formation, and provides a selective expression of proteins related to osteo differentiation. Additionally, aptamers increase TGF-b1 gene expression, which is highly associated with improvements in regenerative therapies.

Comparison of the effects of platelet concentrates produced by high and low-speed centrifugation protocols on the healing of critical-size defects in rat calvaria: a microtomographic and histomorphometric study.

The current study evaluated the healing of critical-size defects (CSD) created in rat calvaria treated with platelet concentrates produced by high-speed (Leukocyte- and Platelet-Rich Fibrin - L-PRF) and low-speed (Advanced Platelet-Rich Fibrin - A-PRF) protocols of centrifugation. Twenty-four rats were distributed into three groups: Control, L-PRF, and A-PRF. Five mm diameter CSD were created on the animals' calvaria. The defects of the L-PRF and A-PRF groups were filled with 0.01 ml of L-PRF and A-PRF, respectively. The control group defects were filled with a blood clot only. All animals were euthanized on the 35th postoperative day. Histomorphometric and microtomographic analyses were then performed. The L-PRF and A-PRF groups had significantly higher bone volume and neoformed bone area than those of the control group and lowered bone porosity values (p < .05). No significant differences were observed between A-PRF and L-PRF groups for the analyzed parameters. Therefore, it can be concluded that i) L-PRF and A-PRF potentiated the healing of CSD in rat calvaria; ii) high and low-speed centrifugation protocols did not produce PRF matrices with different biological impacts on the amount of bone neoformation.

Role of hypoxia-related proteins in adenoid cystic carcinoma invasion.

BACKGROUND: Among cancers affecting the oral cavity, adenoid cystic carcinoma (ACC) is a relatively common malignant neoplasm. It has high rates of metastasis and recurrence and is associated with significant morbidity. During the progression of ACC, the oxygen concentration is reduced in specific areas of the tumour microenvironment, leading to intratumoural hypoxia. The expression of NOTCH1, a disintegrin and metalloproteinase 12 (ADAM-12), hypoxia-inducible factor 1 alpha (HIF-1α), and heparin-binding epidermal growth factor (HB-EGF) under hypoxic conditions has been implicated in invadopodia formation, tumour invasiveness, and metastasis. The aim of this study was to analyse the expression of these proteins to elucidate the mechanisms underlying ACC invasiveness. METHODS: Fifteen ACC samples and 10 normal-looking salivary gland (SG) samples were used to investigate the expression of these proteins by immunohistochemistry. Primary antibodies against NOTCH1, ADAM-12, HIF-1α, and HB-EGF were used. RESULTS: The immunoexpression of all proteins was higher in ACC samples than in SG samples (p < 0.05). CONCLUSIONS: There was increased expression of proteins associated with hypoxia and tumour invasiveness in ACC samples, which indicates a possible role of these proteins in the biological behaviour of this tumour.

HIF-1α, NOTCH1, ADAM12, and HB-EGF are overexpressed in mucoepidermoid carcinoma.

OBJECTIVE: Intratumoral hypoxia (IH) occurs during cellular proliferation of malignant tumors. This phenomenon is characterized by a decrease in oxygen levels in the neoplastic microenvironment. Throughout this condition, the proteins HIF-1α, NOTCH1, ADAM12, and HB-EGF can be activated, triggering signaling pathways associated with tumor invasiveness through invadopodia formation. This study aimed to evaluate the immunostaining of HIF-1α, NOTCH1, ADAM12, and HBEGF in 19 cases of mucoepidermoid carcinoma (MEC) and 10 samples of salivary glands (control group). STUDY DESIGN: The immunoperoxidase technique was employed to detect the proteins of interest. The Student t test was used to compare immunoexpression between MEC samples and the control group. RESULTS: Protein immunostaining was statistically significantly higher in MEC samples than in the control group (P < .01), and the proteins were especially overexpressed in epidermoid cells of MEC. CONCLUSIONS: We suggest that there is an association between the NOTCH1 signaling pathway activated by IH and the biologic behavior of MEC.

Keratocystic odontogenic tumor overexpresses invadopodia-related proteins, suggesting invadopodia formation.

OBJECTIVE: Keratocystic odontogenic tumor (KOT) is an odontogenic neoplasm that shows aggressive clinical behavior and local invasiveness. Invadopodia are actin-rich cellular protrusions exhibiting proteolytic pericellular activity, thereby inducing focal invasion in neoplastic cells and increasing neoplasms aggressiveness. Thus, this study aimed to evaluate immunoexpression of invadopodia-related proteins, cortactin, MT1-MMP, Tks4, and Tks5, in KOT. STUDY DESIGN: Immunohistochemistry of 16 cases of KOT, eight cases of calcifying cystic odontogenic tumor (CCOT), and eight samples of the oral mucosa (OM) was carried out to assess the expression of the above described invadopodia-related proteins in the basal and suprabasal layer. RESULTS: KOT samples showed higher and significant immunoexpression of cortactin, MT1-MMP, TKs4, and TKs5 compared with the CCOT and OM samples. Significant expression of all these proteins was observed in the basal layer compared with the suprabasal layer in KOT. CONCLUSIONS: Overexpression of cortactin, MT1-MMP, TKs4, and TKs5 was observed in KOT compared with samples of CCOT and OM. These proteins were also overexpressed in the basal over the suprabasal layer of KOT samples. Taken together, these results suggest the participation of invadopodia-related proteins on the pathogenesis of this lesion.

Role of hypoxia-related proteins in invasion of ameloblastoma cells: crosstalk between NOTCH1, hypoxia-inducible factor 1α, a disintegrin and metalloproteinase 12, and heparin-binding epidermal growth factor.

AIMS: Ameloblastoma AME is a benign tumour characterized by local invasiveness, high recurrence rates, and diverse histological patterns. The oxygen concentration is reduced in specific areas of the tumour microenvironment, which leads to intratumoral hypoxia. Crosstalk between NOTCH1, a disintegrin and metalloproteinase 12 (ADAM-12), hypoxia-inducible factor 1α (HIF-1α) and heparin-binding epidermal growth factor (HB-EGF) under hypoxic conditions has been implicated in invadopodia formation, tumour invasiveness, and metastasis development. The aim of this study was to analyse the expression of these proteins, in order to further elucidate the mechanisms underlying AME invasiveness. METHODS AND RESULTS: Twenty cases of AME, eight calcifying cystic odontogenic tumours CCOTs and 10 samples of dental follicle were used to investigate the expression of these proteins by immunohistochemistry with the primary antibodies anti-NOTCH1, anti-ADAM-12, anti-HIF-1α, and anti-HB-EGF. Immunostaining results were expressed as the percentage of stained area in images acquired in an AxioScope microscope equipped with an AxioCamHRc camera and a × 40 objective. The results showed that immunoexpression of all proteins was higher in the AME samples than in the CCOT and dental follicle samples (P < 0.05). CONCLUSIONS: AME showed an increased presence of proteins associated with tumour invasiveness, which indicates a possible role of these proteins in the biological behaviour of this tumour.

Morphological aspects and physical properties of enamel and dentine of Sus domesticus: A tooth model in laboratory research.

This study aims to describe and analyze morphological and physical properties of deciduous teeth of Sus domesticus. Ultrastructural analysis, mineral composition and microhardness of enamel and dentine tissues were performed on 10 skulls of S. domesticus. External anatomic characteristics and the internal anatomy of the teeth were also described. Data regarding microhardness and ultrastructural analysis were subjected to statistical tests. For ultrastructural analysis, we used the analysis of variance (ANOVA) with Tukey's post hoc (p≤0.05) test. In the analysis of microhardness, the difference between the enamel and dentine tissues was analyzed by a Student's t test. Values were expressed as mean with standard error. The results of ultrastructural analysis showed the presence of an enamel prism pattern. A dentinal tubule pattern was also observed, with a larger diameter in the pulp chamber and the cervical third, in comparison to middle and apical thirds. We observed an average microhardness of 259.2kgf/mm(2) for enamel and 55.17kgf/mm(2) for dentine. In porcine enamel and dentine, the chemical elements Ca and P showed the highest concentration. The analysis of internal anatomy revealed the presence of a simple root canal system and the occurrence of main canals in the roots. The observed features are compatible with the functional demand of these animals, following a pattern very similar to that seen in other groups of mammals, which can encourage the development of research using dental elements from the pig as a substitute for human teeth in laboratory research.