Over the last several years, we have focused on the importance of intracellular signaling pathways in dynamically governing the biointerface between pre-osteoblast and surface of biomaterial. Thus, this study investigates the molecular hallmarks involved in the pre-osteoblast relationship with different topography considering Machined (Mc), Dual Acid-Etching (DAE), and nano hydroxyapatite-blasted (nHA) groups. There was substantial differences in topography of titanium surface, considering Atomic Force Microscopy and water contact angle (Mc = 81.41 ± 0.01; DAE = 97.18 ± 0.01; nHA = 40.95 ± 0.02). Later, to investigate their topography differences on biological responses, pre-osteoblast was seeded on the different surfaces and biological samples were collected after 24 h (to consider adhesion signaling) and 10 days (to consider differentiation signaling). Preliminary results evidenced significant differences in morphological changes of pre-osteoblasts mainly resulting from the interaction with the DAE and nHA, distinguishing cellular adaptation. These results pushed us to analyze activation of specific genes by exploring qPCR technology. In sequence, we showed that Src performs crucial roles during cell adhesion and later differentiation of the pre-osteoblast in relationship with titanium-based biomaterials, as our results confirmed strong feedback of the Src activity on the integrin-based pathway, because integrin-ß1 (∼5-fold changes), FAK (∼12-fold changes), and Src (∼3.5-fold changes) were significantly up-expressed when Src was chemically inhibited by PP1 (5 μM). Moreover, ECM-related genes were rigorously reprogrammed in response to the different surfaces, resulting on Matrix Metalloproteinase (MMP) activities concomitant to a significant decrease of MMP inhibitors. In parallel, we showed PP1-based Src inhibition promotes a significant increase of MMP activity. Taking all our results into account, we showed for the first time nano hydroxyapatite-blasted titanium surface creates a biointerface able to govern Src-dependent osteoblast metabolism as pre-requisite to ECM remodeling.

The performance of the dentine bonding systems was material dependent. CHX did not improve immediate bond strength; however, CHX negatively affected the bond strength of the self-etching system, especially in the third apical.

Although osseointegration and clinical success of titanium (Ti)-implanted materials depend on neovascularization in the reactional peri-implant tissue, very little has been achieved considering the Ti-molecules release on the behavior of endothelial cells. To address this issue, we challenged endothelial cells (HUVECs) with Ti-enriched medium obtained from two types of commercial titanium surfaces [presenting or not dual-acid etching (DAE)] up to 72 h to allow molecular machinery analysis. Our data show that the Ti-enriched medium provokes significant stimulus of angiogenesis-related machinery in endothelial cells by upexpressing VEGFR1, VEGFR2, VEGF, eNOS, and iNOS genes, while the PI3K/Akt signaling pathway was also significantly enhanced. As PI3K/AKT signaling was related to angiogenesis in response to vascular endothelial growth factor (VEGF), we addressed the importance of PI3K/Akt upon Ti-enriched medium responses by concomitantly treating the cells with wortmannin, a well-known PI3K inhibitor. Wortmannin suppressed the angiogenic factors, because VEGF, VEGFR1, and eNOS genes were downregulated in those cells, highlighting the importance of PI3K/AKT signaling on driving angiogenic phenotype and angiogenesis performance within the peri-implant tissue reaction. In conjunction, these data reinforce that titanium-implantable devices modify the metabolism of surrounding cells, such as endothelial cells, probably coupling osteogenesis and angiogenesis processes in peri-implant tissue and then contributing to successfully osseointegration of biomedical titanium-based devices.

Nanosized crystalline hydroxyapatite coating (HAnano®) accelerates the osteointegration of dental implants which is hypothesized to drive angiogenesis. In order to test this hypothesis, we have subjected shear-stressed human umbilical vein endothelial cells (HUVECs) to a HAnano®-enriched medium, as well as to surface presenting dual acid etching (DAE) as a control. To note, the titanium implants were coated with 10 nm in diameter HA particles using the Promimic HAnano method. Our data reveals that HAnano® modulates higher expression of genes related with endothelial cell performance and viability, such as VEGF, eNOS, and AKT, and further angiogenesis in vitro by promoting endothelial cell migration. Additionally, the data shows a significant extracellular matrix (ECM) remodeling, and this finding seems developing a dual role in promoting the expression of VEGF and control endothelial cell growth during angiogenesis. Altogether, these data prompted us to further validate this phenomenon by exploring genes related with the control of cell cycle and in fact our data shows that HAnano® promotes higher expression of CDK4 gene, while p21 and p15 genes (suppressor genes) were significantly lower. In conjunction, our data shows for the first time that HAnano®-coated surfaces drive angiogenesis by stimulating a proliferative and migration phenotype of endothelial cells, and this finding opens novel comprehension about osseointegration mechanism considering nanosized hydroxyapatite coating dental implants.

Modifications on shear stress‐based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1–4 Pa. First, our data shows there is a significant up‐expression of phosphatidylinositol 3‐kinase (PI3K) in shear‐stressed cells culminating downstream with an up‐phosphorylation of AKT and up‐expression of MAPK‐ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear‐stressed EC, while cell cycle‐related events seem to being changed. Additionally, although diminution of 5‐hydroxymethylcytosine in shear‐stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear‐stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear‐stressed cells, once wortmannin promotes a significant suppression of ten‐11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET‐related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system.