Development of cobalt (Co)-doped monetites for bone regeneration.

Cobalt-doped monetite powders were synthesized by coprecipitation method under a cobalt nominal content between 2 and 20 mol % of total cation. Structural characterization of samples was performed by using X-ray diffraction (XRD), Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy. XRD results indicated that the Co-doped samples exhibited a monetite single-phase with the cell parameters and crystallite size dependent on the amount of substitutional element incorporated into the triclinic crystalline structure. Cell viability and adhesion assays using pre-osteoblastic cells showed there is no toxicity and the RTqPCR analysis showed significant differences in the expression for osteoblastic phenotype genes, showing a potential material for the bone regeneration.

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.

The Action of Angiocrine Molecules Sourced from Mechanotransduction-Related Endothelial Cell Partially Explain the Successful of Titanium in Osseointegration.

Since Branemark's findings, titanium-based alloys have been widely used in implantology. However, their success in dental implants is not known when considering the heterogenicity of housing cells surrounding the peri-implant microenvironment. Additionally, they are expected to recapitulate the physiological coupling between endothelial cells and osteoblasts during appositional bone growth during osseointegration. To investigate whether this crosstalk was happening in this context, we considered the mechanotransduction-related endothelial cell signaling underlying laminar shear stress (up to 3 days), and this angiocrine factor-enriched medium was harvested further to use exposing pre-osteoblasts (pOb) for up to 7 days in vitro. Two titanium surfaces were considered, as follows: double acid etching treatment (w_DAE) and machined surfaces (wo_DAE). These surfaces were used to conditionate the cell culture medium as recommended by ISO10993-5:2016, and this titanium-enriched medium was later used to expose ECs. First, our data showed that there is a difference between the surfaces in releasing Ti molecules to the medium, providing very dynamic surfaces, where the w_DAE was around 25% higher (4 ng/mL) in comparison to the wo_DAE (3 ng/mL). Importantly, the ECs took up some of this titanium content for up to 3 days in culture. However, when this conditioned medium was used to expose pOb for up to 7 days, considering the angiocrine factors released from ECs, the concentration of Ti was lesser than previously reported, reaching around 1 ng/mL and 2 ng/mL, respectively. Thereafter, pOb exposed to this angiocrine factor-enriched medium presented a significant difference when considering the mechanosignaling subjected to the ECs. Shear-stressed ECs showed adequate crosstalk with osteoblasts, stimulating the higher expression of the Runx2 gene and driving higher expressions of Alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin. Mechanotransduction-related endothelial cell signaling as a source of angiocrine molecules also stimulated the higher expression of the Col3A1 gene in osteoblasts, which suggests it is a relevant protagonist during trabecular bone growth. In fact, we investigated ECM remodeling by first evaluating the expression of genes related to it, and our data showed a higher expression of matrix metalloproteinase (MMP) 2 and MMP9 in response to mechanosignaling-based angiocrine molecules, independent of considering w_DAE or the wo_DAE, and this profile reflected on the MMP2 and MMP9 activities evaluated via gelatin-based zymography. Complimentarily, the ECM remodeling seemed to be a very regulated mechanism in mature osteoblasts during the mineralization process once both TIMP metallopeptidase inhibitor 1 and 2 (TIMP1 and TIMP2, respectively) genes were significantly higher in response to mechanotransduction-related endothelial cell signaling as a source of angiocrine molecules. Altogether, our data show the relevance of mechanosignaling in favoring ECs' release of bioactive factors peri-implant, which is responsible for creating an osteogenic microenvironment able to drive osteoblast differentiation and modulate ECM remodeling. Taking this into account, it seems that mechanotransduction-based angiocrine molecules explain the successful use of titanium during osseointegration.

Epigenetic Differences Arise in Endothelial Cells Responding to Cobalt-Chromium.

Cobalt-chromium (Co-Cr)-based alloys are emerging with important characteristics for use in dentistry, but the knowledge of epigenetic mechanisms in endothelial cells has barely been achieved. In order to address this issue, we have prepared a previously Co-Cr-enriched medium to further treat endothelial cells (HUVEC) for up to 72 h. Our data show there is important involvement with epigenetic machinery. Based on the data, it is believed that methylation balance in response to Co-Cr is finely modulated by DNMTs (DNA methyltransferases) and TETs (Tet methylcytosine dioxygenases), especially DNMT3B and both TET1 and TET2. Additionally, histone compaction HDAC6 (histone deacetylase 6) seems to develop a significant effect in endothelial cells. The requirement of SIRT1 seems to have a crucial role in this scenario. SIRT1 is associated with a capacity to modulate the expression of HIF-1α in response to hypoxia microenvironments, thus presenting a protective effect. As mentioned previously, cobalt is able to prevent HIF1A degradation and maintain hypoxia-related signaling in eukaryotic cells. Together, our results show, for the first time, a descriptive study reporting the relevance of epigenetic machinery in endothelial cells responding to cobalt-chromium, and it opens new perspectives to better understand their repercussions as prerequisites for driving cell adhesion, cell cycle progression, and angiogenesis surrounding this Co-Cr-based implantable device.

Preparation and characterization of novel as-cast Ti-Mo-Nb alloys for biomedical applications.

Ti and its alloys are the most used metallic biomaterials devices due to their excellent combination of chemical and mechanical properties, biocompatibility, and non-toxicity to the human body. However, the current alloys available still have several issues, such as cytotoxicity of Al and V and high elastic modulus values, compared to human bone. ß-type alloys, compared to α-type and (α + ß)-type Ti alloys, have lower elastic modulus and higher mechanical strength. Then, new biomedical ß-type alloys are being developed with non-cytotoxic alloying elements, such as Mo and Nb. Therefore, Ti-5Mo-xNb system alloys were prepared by argon arc melting. Chemical composition was evaluated by EDS analysis, and the density measurements were performed by Archimedes' method. The structure and microstructure of the alloys were obtained by X-ray diffraction and optical and scanning electron microscopy. Microhardness values were analyzed, and MTT and crystal violet tests were performed to assess their cytotoxicity. As the Nb concentration increases, the presence of the ß-Ti phase also grows, with the Ti-5Mo-30Nb alloy presenting a single ß-Ti phase. In contrast, the microhardness of the alloys decreases with the addition of Nb, except the Ti-5Mo-10Nb alloy, which has its microhardness increased probably due to the ω phase precipitation. Biological in-vitro tests showed that the alloys are not cytotoxic.

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.