Fábio Bezerra

Clinical analysis of the soft tissue integration of non-submerged (ITI) and submerged (3i) implants: a prospective- controlled cohort study

AIM The aim of this study was to compare the soft tissue integration of submerged and non-submerged implants by means of periodontal parameter assessments and analysis. MATERIAL AND METHODS Thirty-one patients, who received 42 non-submerged implants (ITI) and 48 submerged implants (3i), participated in the study. There was no significant difference (P>0.05) between both groups considering gender; educational level; handedness; toothbrushing frequency; the number of auxiliary devices used; and smoking habits. The parameters assessed were gingival index (GI), plaque index (PII), retention index (RI), pocket probing depth (PPD) and keratinized mucosa index. RESULTS At evaluation, 66.67% of all sites showed a GI of 0; 72.22% a PI of 0, and 93.33% the absence of calculus. The average PPD was 2.56 mm in the non-submerged and 2.70 mm in the submerged group. With regard to the width of keratinized mucosa, 100% of the ITI implants showed a band of keratinized gingiva around the implant, whereas 14.58% in the 3i group showed a complete absence of keratinized mucosa. The intra-examiner reproducibility was 90.96% for all parameters and the Kendall tau-b analysis showed a powerless correlation between the chosen parameters for both studied groups. CONCLUSIONS The study material showed no major differences between submerged and non-submerged dental implants regarding GI, PII, RI and PPD, except the width of keratinized mucosa. Regarding the presence of keratinized mucosa, there is a need for further longitudinal studies to elucidate a possible benefit of one implant system over the other.

Nano hydroxyapatite-blasted titanium surface affects pre-osteoblast morphology by modulating critical intracellular pathways

Although, intracellular signaling pathways are proposed to predict the quality of cell‐surface relationship, this study addressed pre‐osteoblast behavior in response to nano hydroxyapatite (HA)‐blasted titanium (Ti) surface by exploring critical intracellular pathways and pre‐osteoblast morphological change. Physicochemical properties were evaluated by atomic force microscopy (AFM) and wettability considering water contact angle of three differently texturized Ti surfaces: Machined (Mac), Dual acid‐etching (DAE), and nano hydroxyapatite‐blasted (nHA). The results revealed critical differences in surface topography, impacting the water contact angle and later the osteoblast performance. In order to evaluate the effect of those topographical characteristics on biological responses, we have seeded pre‐osteoblast cells on the Ti discs for up to 4 h and subjected the cultures to biological analysis. First, we have observed pre‐osteoblasts morphological changes resulting from the interaction with the Ti texturized surfaces whereas the cells cultured on nHA presented a more advanced spreading process when compared with the cells cultured on the other surfaces. These results argued us for analyzing the molecular machinery and thus, we have shown that nHA promoted a lower Bax/Bcl2 ratio, suggesting an interesting anti‐apoptotic effect, maybe explained by the fact that HA is a natural element present in bone composition. Thereafter, we investigated the potential effect of those surfaces on promoting pre‐osteoblast adhesion and survival signaling by performing crystal violet and immunoblotting approaches, respectively. Our results showed that nHA promoted a higher pre‐osteoblast adhesion supported by up‐modulating FAK and Src activations, both signaling transducers involved during eukaryotic cell adhesion. Also, we have shown Ras‐Erk stimulation by the all evaluated surfaces. Finally, we showed that all Ti‐texturing surfaces were able to promote osteoblast differentiation up to 10 days, when alkaline phosphatase (ALP) activity and osteogenic transcription factors were up‐modulated. Altogether, our results showed for the first time that nano hydroxyapatite‐blasted titanium surface promotes crucial intracellular signaling network responsible for cell adapting on the Ti‐surface.Biotechnol. Bioeng. 2017;114: 1888–1898.

Titanium-released from dental implant enhances pre-osteoblast adhesion by ROS modulating crucial intracellular pathways

It is important to understand the cellular and molecular events that occur at the cell-material interface of implants used for bone repair. The mechanisms involved in the initial stages of osteoblast interactions with the surface of the implant material must be decisive for cell fating surrounding them. In order to address this issue, we decided to investigate if conditioned medium for dental implants was able to modulate murine pre-osteoblast metabolism. First, we determined the concentration of titanium (Ti)-containing conditioned medium and found that it was 2-fold increased (p < 0.0001). We have reported that this conditioned medium significantly up-modulated pre-osteoblast adhesion up to 24 h (p < 0.0001). In parallel, our results showed that both phosphorylations of FAK (focal adhesion kinase) at Y397 (p < 0.0011) and Cofilin at Ser03 (p < 0.0053) were also up-modulated, as well as for Rac1 expression (p < 0.0175); both of them are involved with cell adaptation by rearranging cytoskeleton actin filaments. Thereafter, Ti-containing medium stimulated ROS (reactive oxygen species) production by pre-osteoblast cells, and it is very possible that ROS compromised PTP-1B (protein tyrosine phosphatase 1B) activation since PTP1B was down-phosphorylated (p < 0.0148). The low PTP activity guarantees the phosphorylation of FAK at Y-residue, causing better pre-osteoblast adhesion in response to Ti-containing medium. Altogether, these data indicate that ROS indirectly modulate FAK phosphorylation in response to Ti-released from dental implants. Taken the results in account, these data showed for the first time that the implanted dental device is able to dynamically affect surrounding tissues, mainly by promoting a better performance of the pre-osteoblast cells.

CoCr enriched medium modulates integrin‐based downstream signaling and requires a set of inflammatory genes reprograming in vitro

Significant health concerns have been raised by the high levels of Cr and Co ions into whole blood as resulted of corrosion process released from biomedical implants, but very little is known about their biological behavior in governing cell metabolism. Thus, we prompted to address this issue by exploring the effects of CoCr enriched medium on both fibroblast and preosteoblast (pre-Ob) cells. First, we showed there is a significant difference in Co and Cr releasing dependent on engineered surface, it being even more released in dual acid-etching treating surface (named w/DAE) than the machined surfaces (named wo/DAE). Thereafter, we showed CoCr affects pre-osteoblast and fibroblast metabolism by dynamically modulating integrin-based downstream signaling (FAK, Src, Rac1, and Cofilin). Specifically on this matter, we have shown there is dynamic β1-integrin gene activation up 24 h in both preosteoblast and fibroblast. Our analysis showed also that both pre-Ob and fibroblast are important resource of proinflammatory cytokines when responding to CoCr enriched medium. In addition, survival-related signaling pathway was also affected interfering on survival and proliferating signal, mainly affecting CDK2, mapk-Erk and mapk-p38 phosphorylations, while AKT/PKB-related gene remained active. In addition, during cell adhesion PP2A (an important Ser/Thr phosphatase) was inactive in both cell lineages and it seems be a CoCrs molecular fingerprint, regulating specific metabolic pathways involved with cytoskeleton rearrangement. Altogether, our results showed for the first time CoCr affects cellular performance in vitro by modulating integrin activation-based downstream signaling and requiring a reprograming of inflammatory genes activations in vitro.

Nano hydroxyapatite-blasted titanium surface creates a biointerface able to govern Src-dependent osteoblast metabolism as prerequisite to ECM remodeling

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.

Titanium-enriched medium drives low profile of ECM remodeling as a pre-requisite to pre-osteoblast viability and proliferative phenotype

Titanium is widely used for biomedical applications, but little information is being delivered regarding the cellular/molecular mechanisms explaining their efficacy, mainly considering the effects of the Ti-released trace elements on pre-osteoblasts. We addressed this issue by investigating decisive intracellular signal transduction able to modulate cytoskeleton rearrangement, proliferative phenotype and extracellular matrix (ECM) remodeling. We considered titanium grades IV and V, submitted or not to dual acid-etching (w/DAE or wo/DAE, respectively). Our results showed there is no cytotoxicity, preserving AKT involvement. Additionally, Ti-enriched medium promoted a diminution of the downstream signaling upon integrin activation (phosphorylating Rac1 and cofilin), guaranteeing a dynamic cytoskeleton rearrangement. Moreover, the low profile of ECM remodeling obtained in response to trace molecules released by Ti-based devices seems contributing to the osteoblast performance in mediating extracellular support to cell anchorage. This hypothesis was validated by the up-expression of ß1-integrin, src and Focal adhesion kinase (fak) genes, mainly in response to titanium grade V. Proliferative phenotype showed an unbalance between cyclin-dependent kinases (CDKs) and p15INK4b/p21Cip1. In conjunction, we showed for the first time that trace elements from Ti-based biomedical devices provoke important modulation of the osteoblast biology, driving cell anchoring, viability, and proliferative phenotype. Certainly, these biological outcomes compromise implant osseointegration.

Zirconia stimulates ECM-remodeling as a prerequisite to pre-osteoblast adhesion/proliferation by possible interference with cellular anchorage

The biological response to zirconia (ZrO2) is not completely understood, which prompted us to address its effect on pre-osteoblastic cells in both direct and indirect manner. Our results showed that zirconia triggers important intracellular signaling mainly by governing survival signals which leads to cell adhesion and proliferation by modulating signaling cascade responsible for dynamic cytoskeleton rearrangement, as observed by fluorescence microscopy. The phosphorylations of Focal Adhesion Kinase (FAK) and Rac1 decreased in response to ZrO2 enriched medium. This corroborates the result of the crystal violet assay, which indicated a significant decrease of pre-osteoblast adhesion in responding to ZrO2 enriched medium. However, we credit this decrease on pre-osteoblast adhesion to the need to govern intracellular repertory of intracellular pathways involved with cell cycle progression, because we found a significant up-phosphorylation of Mitogen-Activated Protein Kinase (MAPK)-p38 and Cyclin-dependent kinase 2 (CDK2), while p15 (a cell cycle suppressor) decreased. Importantly, Protein phosphatase 2 A (PP2A) activity decreased, guaranteeing the significant up-phosphorylation of MAPK -p38 in response to ZrO2 enriched medium. Complementarily, there was a regulation of Matrix Metalloproteinases (MMPs) in response to Zirconia and this remodeling could affect cell phenotype by interfering on cell anchorage. Altogether, our results show a repertory of signaling molecules, which suggests that ECM remodel as a pre-requisite to pre-osteoblast phenotype by affecting their anchoring in responding to zirconia.