Mechanosignaling-related angiocrine factors drive osteoblastic phenotype in response to zirconia.

BACKGROUND: The growing use of zirconia as a ceramic material in dentistry is attributed to its biocompatibility, mechanical properties, esthetic appearance, and reduced bacterial adhesion. These favorable properties make ceramic materials a viable alternative to commonly used titanium alloys. Mimicking the physiological properties of blood flow, particularly the mechanosignaling in endothelial cells (ECs), is crucial for enhancing our understanding of their role in the response to zirconia exposure. METHODS: In this study, EC cultures were subjected to shear stress while being exposed to zirconia for up to 3 days. The conditioned medium obtained from these cultures was then used to expose osteoblasts for a duration of 7 days. To investigate the effects of zirconia on osteoblasts, we examined the expression of genes associated with osteoblast differentiation, including Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Additionally, we assessed the impact of mechanosignaling-related angiocrine factors on extracellular matrix (ECM) remodeling by measuring the activities of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) during the acquisition of the osteogenic phenotype, which precedes mineralization. RESULTS: Our data revealed that mechanosignaling-related angiocrine factors play a crucial role in promoting an osteoblastic phenotype in response to zirconia exposure. Specifically, exposed osteoblasts exhibited significantly higher expression levels of genes associated with osteoblast differentiation, such as Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Furthermore, the activities of MMP2 and MMP9, which are involved in ECM remodeling, were modulated by mechanosignaling-related angiocrine factors. This modulation is likely an initial event preceding the mineralization phase. CONCLUSION: Based on our findings, we propose that mechanosignaling drives the release of angiocrine factors capable of modulating the osteogenic phenotype at the biointerface with zirconia. This process creates a microenvironment that promotes wound healing and osseointegration. Moreover, these results highlight the importance of considering the mechanosignaling of endothelial cells in the modulation of bone healing and osseointegration in the context of blood vessel effects. Our data provide new insights and open avenues for further investigation into the influence of mechanosignaling on bone healing and the osseointegration of dental devices.

Nanohydroxyapatite-Coated Titanium Surface Increases Vascular Endothelial Cells Distinct Signaling Responding to High Glucose Concentration.

AIM: The success of dental implants depends on osseointegration can be compromised by well-known related adverse biological processes, such as infection and diabetes. Previously, nanohydroxyapatite-coated titanium surfaces (nHA_DAE) have been shown to contain properties that promote osteogenesis by enhancing osteoblast differentiation. In addition, it was hypothesized to drive angiogenesis in high-glucose microenvironments, mimicking diabetes mellitus (DM). On the other hand, the null hypothesis would be confirmed if no effect was observed in endothelial cells (ECs). MATERIALS AND METHODS: Titanium discs presenting the differential surfaces were previously incubated in an FBS-free cell culture medium for up to 24 h, which was, thereafter, supplemented with 30.5 mM of glucose to expose human umbilical vein endothelial cells (HUVECs, ECs) for 72 h. They were then harvested, and the sample was processed to provide molecular activity of specific genes related to EC survival and activity by using qPCR, and the conditioned medium by ECs was used to evaluate the activity of matrix metalloproteinases (MMPs). RESULTS: Our data guaranteed better performance of this nanotechnology-involved titanium surface to this end once the adhesion and survival characteristics were ameliorated by promoting a higher involvement of ß1-Integrin (~1.5-fold changes), Focal Adhesion Kinases (FAK; ~1.5-fold changes) and SRC (~2-fold changes) genes. This signaling pathway culminated with the cofilin involvement (~1.5-fold changes), which guaranteed cytoskeleton rearrangement. Furthermore, nHA_DAE triggered signaling that was able to drive the proliferation of endothelial cells once the cyclin-dependent kinase gene was higher in response to it, while the P15 gene was significantly down-regulated with an impact on the statement of angiogenesis. CONCLUSIONS: Altogether, our data show that a nanohydroxyapatite-coated titanium surface ameliorates the EC performance in a high-glucose model in vitro, suggesting its potential application in DM patients.

Titanium-Enriched Medium Promotes Environment-Induced Epigenetic Machinery Changes in Human Endothelial Cells.

It is important to understand whether endothelial cells are epigenetically affected by titanium-enriched media when angiogenesis is required during bone development and it is expected to be recapitulated during osseointegration of biomaterials. To better address this issue, titanium-enriched medium was obtained from incubation of titanium discs for up to 24 h as recommended by ISO 10993-5:2016, and further used to expose human umbilical vein endothelial cells (HUVECs) for up to 72 h, when the samples were properly harvested to allow molecular analysis and epigenetics. In general, our data show an important repertoire of epigenetic players in endothelial cells responding to titanium, reinforcing protein related to the metabolism of acetyl and methyl groups, as follows: Histone deacetylases (HDACs) and NAD-dependent deacetylase sirtuin-1 (Sirt1), DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) methylcytosine dioxygenases, which in conjunction culminate in driving chromatin condensation and the methylation profile of DNA strands, respectively. Taking our data into consideration, HDAC6 emerges as important player of this environment-induced epigenetic mechanism in endothelial cells, while Sirt1 is required in response to stimulation of reactive oxygen species (ROS) production, as its modulation is relevant to vasculature surrounding implanted devices. Collectively, all these findings support the hypothesis that titanium keeps the surrounding microenvironment dynamically active and so affects the performance of endothelial cells by modulating epigenetics. Specifically, this study shows the relevance of HDAC6 as a player in this process, possibly correlated with the cytoskeleton rearrangement of those cells. Furthermore, as those enzymes are druggable, it opens new perspectives to consider the use of small molecules to modulate their activities as a biotechnological tool in order to improve angiogenesis and accelerate bone growth with benefits of a fast recovery time for patients.

Evaluation of Bone Response to a Nano HA Implant Surface on Sinus Lifting Procedures: Study in Rabbits.

The aim of this study was to evaluate the bone response to two different implant surfaces on sinus lift procedures in rabbits. Bilateral sinus lifting with inorganic bovine bone associated with collagen membrane and immediate implantation were performed in 16 rabbits. Custom mini-implants were randomly installed in the prepared sites: one side received a double acid-etched (DAE) surface and the other a nano-hydroxyapatite (NHA) surface. The animals were euthanized 30 and 60 days after surgery, and biopsies were collected for microtomographic and histomorphometric analysis. After 30 days, no intra- and inter-group statistical differences were observed in microtomographic analysis, while at 60 days, bone analysis showed statistically significant differences between groups (p < 0.05) for all the evaluated parameters. Histomorphometric analysis showed, after 30 days, mean % of Bone-to-Implant Contact (BIC) for DAE and NHA of 31.70 ± 10.42% vs. 40.60 ± 10.22% (p > 0.05), respectively; for % of Bone Area Fraction Occupancy (BAFO), mean values were 45.43 ± 3.597% for DAE and 57.04 ± 5.537% for NHA (p < 0.05). After 60 days, mean %BIC and %BAFO for DAE and NHA implants were statistically significant (p < 0.05). The NHA surface showed superior biological features compared to the DAE treatment, promoting higher bone formation around the implants in an experimental model of bone repair in a grafted area.

Metabolic effects of CoCr-enriched medium on shear-stressed endothelial cell and osteoblasts: A possible mechanism involving a hypoxic condition on bone healing.

Cobalt-chromium (CoCr)-based alloys have emerged as an interesting biomaterial within biomedical field, mainly considering their biocompatibility, resistance to corrosion and absence of magnetism; however, its effect on cell metabolism is barely known and this prompted us better evaluating whether CoCr-enriched medium affects the metabolism of both osteoblast and endothelial cells, and also if there is a coupling between them. This is also considered here the already-known effect of Cobalt (Co) as a hypoxic element. Firstly, discs of CoCr [subjecting (W) or not (Wo) to dual acid-etched (DAE)] were incubated into FBS-free cell culture medium up to 24 h (37 °C). This CoCr-enriched medium was further used to treat shear-stressed endothelial cells cultures up to 72 h. Thereafter, the conditioned medium containing metabolites of shear-stressed endothelial cells in response to CoCr-enriched medium was further used to subject osteoblast's cultures, when the samples were properly harvested to allow the analysis of the molecular issues. Our data shows that CoCr-enriched medium contains 1.5 ng-2.0 ng/mL of Co, which was captured by endothelial cells and osteoblasts in about 30% in amount and it seems modulate their metabolic pathways: shear-stressed endothelial cells expressed higher profile of HIF1α, VEGF and nNOS genes, while their global profile of protein carbonylation was lower than the control cultures, suggesting lower oxidative stress commitment. Additionally, osteoblasts responding to metabolites of CoCr-challenged endothelial cells show dynamic expression of marker genes in osteogenic differentiation, with alkaline phosphatase (ALP), osteocalcin, and bone sialoprotein (BSP) genes being significantly increased. Additionally, tensional shear-stress forces decrease the stimulus for ColA1gene expression in osteoblasts responding to endothelial cells metabolites, as well as modifying the extracellular matrix remodeling related genes. Analyzing the activities of matrix metalloproteinases (MMPs), the data shows that shear-stressed endothelial cells metabolites increase the activities of both MMP9 and MMP2 in osteoblasts. Altogether, our data shows for the first time that shear-stressed endothelial metabolites responding to CoCr discs contribute to osteogenic phenotype in vitro, and this predicts an active crosstalk between angiogenesis and osteogenesis during osseointegration of CoCr alloy and bone healing, maybe guided by the Co-induced hypoxic condition.

Cobalt-chromium-enriched medium ameliorates shear-stressed endothelial cell performance.

Angiogenesis is a relevant mechanism to be considered for the success of bone healing, even considering endosseous implantable devices, providing adequate delivery of substances necessaries for the cell viability and bone de novo deposition. Within of the repertory of metal-based implantable alloys, cobalt-chromium (CoCr) has emerged with very interesting properties for biomedical applications. Additionally, we have shown that released molecules from implants devices are able to modulate cells away and because that we hypothesized these released molecules might act on endothelial cells. In order to better address this issue, we investigated the effect of Co-Cr-enriched medium on endothelial cells (HUVECs), considering a biological model subjecting those cells to shear-stress to partially mimic the physiological environment and further allow investigating intracellular pathways responsible to drive cytoskeletal rearrangement, cell viability and extracellular matrix (ECM) remodeling processes. Considering the analysis of the metalloproteinases (MMPs) activities, our data indicates an intense ECM remodeling in response to CoCr-enriched medium suggesting some role on angiogenesis once ECM remodeling is prerequisite to cell growth. This was better addressed by revealing its involvement on modifying both mRNA expression and protein levels of members of the MAPK family. Additionally, the expression of CDK4 gene was modulated within the cell response to Co-Cr-enriched medium, while the modulation in the expression of P15 and P21 indicates an important regulatory mechanism required. Overall, our results demonstrate that trace of CoCr elements triggers decisive intracellular signaling in shear-stressed endothelial cells, suggesting influence on angiogenesis-related mechanism and they bring novel insights to explain the biological activity of CoCr as it has been emerged as interesting biomedical materials within the medical and dentistry fields.

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.

Bone Remodeling Around Implants with External Hexagon and Morse-Taper Connections: A Randomized, Controlled, Split-Mouth, Clinical Trial.

OBJECTIVES: To evaluate clinical, radiographic, microbiologic, and biomechanical parameters related to bone remodeling around implants with external hexagon (EH) and Morse-taper (MT) connections. MATERIALS AND METHODS: Twelve totally edentulous patients received four custom-made implants in the interforaminal region of the mandible. Two of those implants had the same macroscopic design, but different prosthetic connections. All patients received an immediate implant-supported prosthesis. Clinical parameters (periimplant probing pocket depth (PPD), modified gingival index (mGI), and mucosal thickness (MTh)) were evaluated at 12 months follow-up. The distance between the top of the implant and the first bone-to-implant contact (IT-FBIC) was evaluated on standardized digital peri-apical radiographs acquired at 1, 3, 6, and 12 months follow-up. Samples of the subgingival microbiota were collected 1, 3, and 6 months after implant loading and used for the quantification of Tanerella forsythia, Porphyromonas gingivalis, Aggragatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum. Further, 36 computerized-tomography based finite element (FE) models were accomplished, simulating each patient under three loading conditions. RESULTS: The evaluated clinical parameters were equal for EH and MT implants. Mean IT-FBIC was significantly different between the tested connections (1.17 ± 0.44 mm for EH, and 0.17 ± 0.54 mm for MT, considering all evaluated time periods). No significant microbiological differences could be observed between tested connections. FE analysis showed a significantly higher peak of equivalent (EQV) strain (p = 0.005) for EH (mean 3,438.65 µÎµ) compared to MT (mean 840.98 µÎµ) connection. CONCLUSIONS: Radiographic periimplant bone loss depends on the implant connection type. MT connections showed less periimplant bone loss, compared to EH connections.

Avaliação da estabilidade dos tecidos duros e moles em implantes imediatos com carga imediata em área estética: estudo clínico prospectivo longitudinal / Soft and hard tissue maintenance following placement of immediate-loaded implants in the aesthetic zone: a prospective longitudinal clinical trial

O objetivo desse trabalho foi avaliar, clínica e radiograficamente (bi/tridimensional), as alterações em tecidos moles e duros encontradas após a colocação de implantes imediatos com carga imediata em alvéolos pós-extração. Dez pacientes tratados com implantes imediatos com carga imediata nos incisivos central e lateral superiores foram incluídos. Parâmetros clínicos foram avaliados em fotografias padronizadas tiradas após a instalação do implante ( baseline ) e 1, 3 e 6 meses após. Parâmetros bi- e tridimensionais foram medidos em tomografias Cone-Beam, para avaliar a estabilidade do tecido ósseo na região vestibular. Também foram realizadas análises clínicas e fotográficas dos tecidos moles, para avaliar a sua estabilidade durante o período de avaliação. Não houve variações estatisticamente significativas nos parâmetros clínicos e radiográficos avaliados, tendo sido demonstrada uma manutenção de 94% do volume do osso vestibular. Implantes imediatos com carga imediata apresentam boa estabilidade dos tecidos moles e duros peri-implantares...

To evaluate by clinical and radiographic bi- and tridimensional means the soft and hard tissues alterations following immediate implant placement and loading in postextraction sockets in theanterior maxilla. Ten patients, treated with immediate-loaded implants in the maxillary central or lateral incisors, were evaluated in this study. Clinical parameters were evaluated in standardized pictures taken at baseline (immediately after), and 1, 3, and 6 months after provisional implant-supported single crown placement. Bi- and tridimensional radiographic parameters were evaluated from standardized digital periapical radiographies and from CBCT images. The volume of the buccal bone wall covering the central millimeter of the implant was also assessed in the CBCT images. The variation for all clinical, bi- and tridimensional parameters assessed was non-statistically significant. There was 94% maintenance of bone volume. The parameters assessed showed good clinical, bi- and tridimensional radiographic stability of soft and hard tissues for implants immediately placed and loaded in aesthetic zones...

Avaliação funcional de implantes por mineralização óssea in vitro / Functional evaluation of implants by bone mineralization in vitrot

A evolução do projeto dos implantes osseointegráveis é resultado do desenvolvimento de diferentes tipos de estruturas em sua superfície. No entanto, ainda existe a necessidade de estudos para definir o tipo de superfície ideal. Esse trabalho discute métodos de avaliação da superfície de implantes que mostram o potencial de determinadas superfícies para induzir mineralização óssea in vitro, partir do uso de células mesenquimais progenitoras. Foram realizadas análises comparativas entre a topografia de implantes com e sem rugosidades nanométricas e o tipo de interação entre pré-osteoblastos semeados diretamente nesses implantes. Características distintas foram observadas em cada superfície.

Improvements in dental implants structure is the result of development of different types of geometrically intelligent surfaces, provided by the emergence of companies interested in innovation of these materials, however, there is still a need for studies to define the type of ideal surface. This work addresses an unprecedented discussion regarding implant surface evaluation methods, able to show the potential of certain areas to induce bone mineralization in vitro. From the use of mesenchymal progenitor cells, which have the capacity to respond to stimuli surface, comparative tests were performed between the topography implants with and without nano-roughness and the type of functional interaction between pre-osteoblasts seeded directly into these implants. Different characteristics of coating cells and mineralization niches on different surfaces were found.

Biomechanical evaluation of platform switching: different mismatch sizes, connection types, and implant protocols.

BACKGROUND: It is not yet well understood to what extent different implant-abutment mismatch sizes and implant-abutment connection types may influence the peri-implant biomechanical environment of implants in different clinical situations. METHODS: Computed tomography-based finite element models comprising a maxillary central incisor socket and 4.5 × 13 mm outer-diameter implants with external and internal hex connection types were constructed. The abutments were designed with diameters of 3.5 mm (platform switching [PS] with 1 mm of diametral mismatch [PS - 1]), 4.0 mm (PS with 0.5 mm of diametral mismatch [PS - 0.5]), and 4.5 mm (conventional matching implant-abutment design [CD]). Analysis of variance at the 95% confidence interval was used to evaluate peak equivalent strain (EQV strain) in the bone, bone volume affected by a strain >4,000 µÎµ (EQV strain >4,000 µÎµ), the peak von Mises stress (EQV stress) in abutment screw, and the bone-implant relative displacement. RESULTS: Similar bone strain levels (EQV strain and EQV strain >4,000 µÎµ) were encountered in PS - 1, PS - 0.5, and CD models for immediately placed implants, independent of the connection type. For immediately loaded implants, slightly smaller peak EQV strain and EQV strain >4,000 µÎµ were found for PS - 1. However, for both connection types in osseointegrated models, the higher the mismatch size, the lesser the amount of strain found. CONCLUSIONS: The increase in mismatch size of PS configuration results in a significant decrease of strain levels in bone for osseointegrated implants, principally for external hex connections. No significant effect of PS could be noted in immediately placed implants.

Intrusão de molares superiores utilizando microparafusos ortodônticos de titânio autoperfurantes / Maxillary molar instrusion using self-drilling titanium orthodontic microscrews

Os microsparafusos ortodônticos de titânio fornecem ancoragem esquelética para os tratamentos ortodônticos, viabilizando movimentações dentárias de forma mais controlada, minimizando os efeitos colaterias e tornando a mecânica ortodôntica independente da colaboração do paciente. Este novo dispositivo tem grande aplicabilidade clínica, com destaque para a intrusão de molares, devido à eficiência e simplicidade da mecânica, quando comparada à mecânica ortodôntica convencional. Este artigo tem como objetivo abordar pontos relevantes que norteiam o sucesso desta nova terapia de ancoragem esquelética na execução da mecânica de intrusão de molares superiores. Dentre os pontos discutidos na abordagem ortodôntica se destaca o planejamento do movimento a ser executado, a escolha, a seleção dos microparafusos e a ativação ortodôntica. Serão apresentados casos clínicos para exemplificar suas aplicações.

Evaluation of the relationship between interleukin-1 gene cluster polymorphisms and early implant failure in non-smoking patients.

OBJECTIVE: The aim of the present study was to investigate the relationship between specific polymorphisms of the interleukin-1 gene cluster and the early failure of osseointegrated implants. MATERIAL AND METHODS: The subject population was composed by a test group comprising 28 non-smoking patients (mean age 52.7) that had suffered one or more early implant failures and by a control group consisting of 34 individuals (mean age 43.3) with one or more healthy implants. Genomic DNA from buccal mucosa was amplified by the polymerase chain reaction (PCR), followed by restriction fragment length polymorphism (RFLP) and submitted to polyacrylamide gel electrophoresis to distinguish the alleles of the interleukin-1A (-889), interleukin-1B (+3953), interleukin-1B (-511) and interleukin-RN (intron 2) gene polymorphisms. Differences in the allele and genotype frequencies between control and test groups were assessed by chi(2) test or by Monte Carlo simulations (P<0.05). Haplotype frequencies, linkage disequilibrium and Hardy-Weinberg equilibrium were also estimated. RESULTS: No statistically significant differences were found in the genotype distribution or allelic frequencies of the polymorphisms. No differences were observed between control and test groups when different interleukin-1 gene cluster haplotypes were compared. Nevertheless, the interleukin-1A (-889) and interleukin-1B (+3953) polymorphic sites were in strong linkage disequilibrium (P=0.00014 for control group and P=0.0238 for the test group). CONCLUSION: This study suggests that polymorphisms in the interleukin-1 gene cluster are not associated with early implant failure in a non-smoking Brazilian population.