Vascular smooth muscle cells exhibit elevated hypoxia-inducible Factor-1α expression in human blood vessel organoids, influencing osteogenic performance.

Considering the importance of alternative methodologies to animal experimentation, we propose an organoid-based biological model for in vitro blood vessel generation, achieved through co-culturing endothelial and vascular smooth muscle cells (VSMCs). Initially, the organoids underwent comprehensive characterization, revealing VSMCs (α-SMA + cells) at the periphery and endothelial cells (CD31+ cells) at the core. Additionally, ephrin B2 and ephrin B4, genes implicated in arterial and venous formation respectively, were used to validate the obtained organoid. Moreover, the data indicates exclusive HIF-1α expression in VSMCs, identified through various methodologies. Subsequently, we tested the hypothesis that the generated blood vessels have the capacity to modulate the osteogenic phenotype, demonstrating the ability of HIF-1α to promote osteogenic signals, primarily by influencing Runx2 expression. Overall, this study underscores that the methodology employed to create blood vessel organoids establishes an experimental framework capable of producing a 3D culture model of both venous and arterial endothelial tissues. This model effectively guides morphogenesis from mesenchymal stem cells through paracrine signaling, ultimately leading to an osteogenic acquisition phenotype, with the dynamic involvement of HIF-1α.

PI3K/AKT signaling drives titanium-induced angiogenic stimulus.

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.

Cystatin-like protein of sweet orange (CsinCPI-2) modulates pre-osteoblast differentiation via ß-Catenin involvement.

Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.

Bisphosphonate-based surface biofunctionalization improves titanium biocompatibility.

Novel-biofunctionalized surfaces are required to improve the performance of endosseous implants, which are mainly related to the resistance against biocorrosion, as well as for the consideration of osteoinductive phenomena. Among different strategies, the use of bisphosphonate molecules as linkers between titanium dioxide (TiO2) surfaces and proteins is a distinctive approach, one in which bisphosphonate could play a role in the osseointegration. Thus, to address this issue, we proposed a novel biofunctionalization of TiO2 surfaces using sodium alendronate (ALN) as a linker and bovine serum albumin as the protein. Physicochemical analysis of the functionalized surfaces was performed using contact angle analyses and surface roughness measurements, which indicated an efficient functionalization. The biocompatibility of the functionalized surfaces was analyzed through the adhesion behavior of the pre-osteoblasts onto the samples. Overall, our data showed a significant improvement concerning the cell adhesion by modulating the adhesion cell-related set of genes. The obtained results show that for modified surfaces there is an increase of up to 100 times in the percentage of cells adhered when compared to the control, besides the extracellular matrix remodeling seemed to be an essential prerequisite for the early stages of cell adhesion on to the biomaterials, which was assayed by evaluating the matrix metalloproteinase activities as well as the gene activations. In the expressions of the Bsp and Bglap2 genes, for the group containing ALN (TiO2 + ALN), it was observed an increase in expression (approximately sixfold change) when compared to the control. Altogether, our data clearly showed that the bisphosphonate-biofunctionalized surface enhanced the biocompatibility of titanium and claims to further progress preclinical in vivo experimentation.

Publisher Correction: Bisphosphonate-based surface biofunctionalization improves titanium biocompatibility.

A Correction to this paper has been published: https://doi.org/10.1007/s10856-020-06470-x.

In vivo and in vitro anti-inflammatory and pro-osteogenic effects of citrus cystatin CsinCPI-2.

Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (p < 0.05) on cathepsin K, cathepsin B, IL-1ß, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (p < 0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.

Improvement of lipase obtaining system by orange waste-based solid-state fermentation: production, characterization and application.

Lipases are an economic important group of biocatalysts that can be produced by some fungal under solid-state fermentation. Orange wastes are source of lipases and potential substrates for lipases production. This work assessed 19 fugal strains cultivated in Citrus sinensis cv. Hamlin orange wastes (peel, frit and core) for production of lipases in order to generate compounds with antioxidant, antimicrobial and cytotoxic properties. Fifteen of those fungi grew and produced lipases, mainly the Aspergillus brasiliensis [National Institute of Quality Control (INCQS) 40036]/frit system, which showed 99.58 U/g total lipase. The substrate with the highest production of lipase was frit with 26.67 and 78.91 U/g of total lipases produced on average by the 15 microorganisms. Aspergillus niger 01/frit (33.53 U/g) and Aspergillus niger (INCQS 40015)/frit (34.76 U/g) systems showed the highest specificity values in all the herein tested synthetic substrates with 4, 12 and 16 carbons. Analysis of the fatty acid profile of hydrolysis products obtained in the most prominent systems applied to corn and sunflower oils showed: palmitic acid, linoleic acid, oleic acid, and stearic acid. These acids showed antioxidant capacity of up to 58% DPPH (2,2-diphenyl-1-pierylhydrazyl) radical reduction and antibacterial activity against Escherichia coli, Listeria monocytogenes, Pseudomonas aureginosa, Salmonella Enteritidis and Staphylococcus aureus, as well as cytotoxicity to SCC9 cells (squamous cancer cells).

Cellular behavior as a dynamic field for exploring bone bioengineering: a closer look at cell-biomaterial interface.

Bone is a highly dynamic and specialized tissue, capable of regenerating itself spontaneously when afflicted by minor injuries. Nevertheless, when major lesions occur, it becomes necessary to use biomaterials, which are not only able to endure the cellular proliferation and migration, but also to substitute the original tissue or integrate itself to it. With the life expectancy growth, regenerative medicine has been gaining constant attention in the reconstructive field of dentistry and orthopedy. Focusing on broadening the therapeutic possibilities for the regeneration of injured organs, the development of biomaterials allied with the applicability of gene therapy and bone bioengineering has been receiving vast attention over the recent years. The progress of cellular and molecular biology techniques gave way to new-guided therapy possibilities. Supported by multidisciplinary activities, tissue engineering combines the interaction of physicists, chemists, biologists, engineers, biotechnologist, dentists and physicians with common goals: the search for materials that could promote and lead cell activity. A well-oriented combining of scaffolds, promoting factors, cells, together with gene therapy advances may open new avenues to bone healing in the near future. In this review, our target was to write a report bringing overall concepts on tissue bioengineering, with a special attention to decisive biological parameters for the development of biomaterials, as well as to discuss known intracellular signal transduction as a new manner to be explored within this field, aiming to predict in vitro the quality of the host cell/material and thus contributing with the development of regenerative medicine.

Hypoxia modulates the phenotype of mechanically stressed endothelial cells responding to CoCr-enriched medium.

Given the importance of the endothelial cell phenotype in dental peri-implant healing processes, the aim of this study was to better assess the involvement of endothelial cells responding to cobalt-chromium (CoCr)-enriched medium. Biologically, cobalt is widely used molecule to induce chemical experimental hypoxia because it stabilizes hypoxia inducible factors (HIF1α). The aplication of hypoxia models provides better experimental condition to allow its impact on cellular metabolism, by looking for biochemical and molecular issues. Thus, this study looks for understaing whether CoCr-based materials are able to modulate endothelial cells considering the hypoxic effect prmoted by cobalt. Firstly, our data shows there is a siginificant effect on endothelial phenotype by modulating the expression of VEGF and eNOS genes, whith low requirement of genes related with proteasome intracellular complex. Importantly, the data were validated using classical chemical modulators of hypoxia signaling [chrysin (5,7-dihydroxyflavone) and Dimethyloxalylglycine (DMOG)] in functional assays. Altogether, these data validate the hypothesis that hipoxya is important to maintain the phenotype of endothelial cells, and it is properly interesting during the tissue regeneration surrounding implants and so compromising osseointegration process. Finally, it is important to mention that the cobalt released from CoCr devices might contribute with an sufficient microenvironment surrounding implanted devices and it paviments new roads looking for more bioactive surfaces of implantable materials in human health.

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.

Green tea and hyaluronic acid gel enhance fibroblast activation and improves the gingival healing post-third molar extraction.

This study evaluates the effects of a green tea (Camellia sinensis) and hyaluronic acid gel on fibroblast activity and alveolar bone repair following third molar extractions. By examining the gene expression related to cell survival, proliferation, and angiogenesis, the study bridges in vitro findings with clinical outcomes in a split-mouth randomized trial. Human fibroblasts were exposed to the treatment gel, analysing gene expression through RT-qPCR. Twenty participants undergoing bilateral third molar extractions received the test gel on one side and a placebo on the other. Assessments included patient-reported outcomes, professional evaluations, and radiographic analyses at multiple postoperative intervals. The test gel significantly enhanced AKT, CDKs, and VEGF gene expressions, indicating a positive effect on angiogenesis and cell proliferation. Clinically, it resulted in reduced exudate, swelling, and secondary interventions, with radiographs showing improved alveolar bone density after 90 days. The green tea and hyaluronic acid gel significantly improves soft tissue and bone healing post-extraction, offering a promising adjunctive therapy for enhancing postoperative recovery. This gel represents a novel adjuvant treatment option for facilitating improved healing outcomes after third molar extractions, highlighting its potential utility in clinical dental practice.

Influence of inflammation on the expression of microRNA-140 in extracellular vesicles from 2D and 3D culture models of synovial-membrane-derived stem cells.

Background: In osteoarthritis (OA), articular homeostasis is regulated by microRNA-140 that inhibits ADAMTS-5, an enzyme that cleaves aggrecan and stimulates the synthesis of other inflammatory mediators. This study aims to evaluate the expression of microRNA-140 in extracellular vesicles (EVs) derived from equine synovial-membrane-derived mesenchymal stem cells (eqSMMSCs) cultured in monolayer (2D) and three-dimensional (3D) culture models under an in vitro inflammatory environment. Methods: Four experimental groups of eqSMMSC cultures were defined for isolation of the EVs. The 2D and 3D control groups were cultured in a conventional cell culture medium, while the 2D-OA and 3D-OA treatment groups were exposed to an OA-like medium containing IL-1ß and TNFα. The culture media samples were collected at 24 h, 72 h, and 120 h time points for EV isolation and characterization using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Reverse transcription quantitative polymerase chain reaction was employed to assess the expressions of microRNA-140 in both the cells and EVs. All statistical analyses were conducted at the 5% significance level. Results: Encapsulation of the eqSMMSCs protected the cells from the inflammatory media compared to the monolayer cultures. EVs were found in higher concentrations in the 3D-OA cultures. Additionally, higher expressions of microRNA-140 were observed in the cells of the 3D-OA group at 24 and 72 h, whereas microRNA-140 expressions in the EVs were higher in the 3D group at 72 h and in the 2D-OA group at 120 h (p < 0.001). However, the 3D-OA culture showed higher expression of the mRNA Adamts5 in the EVs at 120 h. Conclusion: The responses of the eqSMMSCs to inflammatory stimuli involve intracellular expression of microRNA-140 and its subsequent transportation via the EVs, with quicker responses observed in the 3D than 2D cultures. This study sheds light on the behaviors of stem cells in restoring homeostasis in osteoarthritic joints.

Cyclopamine targeting hedgehog modulates nuclear control of the osteoblast activity.

It is known that cellular events underlying the processes of bone maintenance, remodeling, and repair have their basis in the embryonic production of bone. Shh signaling is widely described developing important morphogenetic control in bone by modifying the activity of osteoblast. Furthermore, identifying whether it is associated with the modulation of nuclear control is very important to be the basis for further applications. Experimentally, osteoblasts were exposed with cyclopamine (CICLOP) considering up to 1 day and 7 days, here considered an acute and chronic responses respectively. Firstly, we have validated the osteogenic model in vitro by exposing the osteoblasts to classical differentiating solution up to 7 days to allow the analysis of alkaline phosphatase and mineralization. Conversely, our data shows that differentiating osteoblasts present higher activity of inflammasome-related genes, while Shh signaling members were lower, suggesting a negative feedback between them. Thereafter, to better know about the role of Shh signaling on this manner, functional assays using CICLOP (5 µM) were performed and the data validates the previously hypothesis that Shh represses inflammasome related genes activities. Altogether, our data supports the anti-inflammatory effect of Shh signaling by suppressing Tnfα, Tgfß and inflammasome related genes during osteoblast differentiation, and this comprehension might support the understanding the molecular and cellular mechanisms related in bone regeneration by reporting molecular-related osteoblast differentiation.

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.

Recombinant sugarcane cystatin CaneCPI-5 promotes osteogenic differentiation.

Cysteine proteases orchestrate bone remodeling, and are inhibited by cystatins. In reinforcing our hypothesis that exogenous and naturally obtained inhibitors of cysteine proteases (cystatins) act on bone remodeling, we decided to challenge osteoblasts with sugarcane-derived cystatin (CaneCPI-5) for up to 7 days. To this end, we investigated molecular issues related to the decisive, preliminary stages of osteoblast biology, such as adhesion, migration, proliferation, and differentiation. Our data showed that CaneCPI-5 negatively modulates both cofilin phosphorylation at Ser03, and the increase in cytoskeleton remodeling during the adhesion mechanism, possibly as a prerequisite to controlling cell proliferation and migration. This is mainly because CaneCPI-5 also caused the overexpression of the CDK2 gene, and greater migration of osteoblasts. Extracellular matrix remodeling was also evaluated in this study by investigating matrix metalloproteinase (MMP) activities. Our data showed that CaneCPI-5 overstimulates both MMP-2 and MMP-9 activities, and suggested that this cellular event could be related to osteoblast differentiation. Additionally, differentiation mechanisms were better evaluated by investigating Osterix and alkaline phosphatase (ALP) genes, and bone morphogenetic protein (BMP) signaling members. Altogether, our data showed that CaneCPI-5 can trigger biological mechanisms related to osteoblast differentiation, and broaden the perspectives for better exploring biotechnological approaches for bone disorders.

Biological monitoring of a promissory xenogenic pin for biomedical applications: a preliminary intraosseous study in rats.

OBJECTIVES: Over the last years, it is known that in some cases metal devices for biomedical applications present some disadvantages suggesting absorbable materials (natural or synthetic) as an alternative of choice. Here, our goal was to evaluate the biological response of a xenogenic pin, derived from bovine cortical bone, intraosseously implanted in the femur of rats. MATERIAL AND METHODS: After 10, 14, 30 and 60 days from implantation, the animals (n=5/period) were killed and the femurs carefully collected and dissected out under histological demands. For identifying the osteoclastogenesis level at 60 days, we performed the immunohistochemisty approach using antibody against RANKL. RESULTS: Interestingly, our results showed that the incidence of neutrophils and leukocytes was observed only at the beginning (10 days). Clear evidences of pin degradation by host cells started at 14 days and it was more intensive at 60 days, when we detected the majority of the presence of giant multinucleated cells, which were very similar to osteoclast cells contacting the implanted pin. To check osteoclastogenesis at 60 days, we evaluated RANKL expression and it was positive for those resident multinucleated cells while a new bone deposition was verified surrounding the pins in all evaluated periods. CONCLUSIONS: Altogether, our results showed that pins from fully processed bovine bone are biocompatible and absorbable, allowing bone neoformation and it is a promissory device for biomedical applications.

Periosteal-derived cells for bone bioengineering: a promising candidate.

PURPOSE: Over the last years so many efforts have been made in order to indentify natural sources of osteogenic cells for the success of bone bioengineering. Among them, periosteum tissue has emerged as an interesting candidate. Thus, we decided to evaluate the osteogenic potential of periosteal-derived cells by describing a sequence of biological events since initial morphological changes to mineralization of extracellular matrix (ECM). METHODS: Periosteal-derived cells were obtained from calvarial of adult rats. After the primary culture and expansion, the adherent cells were cultured at 7, 14, 21 and 28 days under a classical osteogenic culture medium in order to evaluate the differentiation of those cells in mature osteoblast. It was monitored by evaluating a time-line of alkaline phosphatase (ALP) activity (biomarker of osteoblast differentiation) and afterwards nodules of mineralization (measured by von Kossa staining and calcium content). RESULTS: Analysis from phase-contrast microscopy revealed mainly morphological changes ranging since fibroblast-shaped (7 days, semi-confluent culture at exponential growth) to polyhedral-shaped cells (14-28 days, confluent culture during differentiation process). ALP activity was linearly increased since 14-28 days while amount of protein remained unchanged. Interesting, our data from von Kossa staining reveled a highest incidence of mineralization nodules at 28 days. CONCLUSION: Taken our results together, we can suggest that periosteal-derived cells present an interesting potential to differentiate in mature osteoblast able to promote mineralization in vitro by incorporating to ECM circulating calcium from extracellular compartment. From our point of view, this source of osteogenic cells can be explored by bioengineers in order to advance therapeutic protocols able to solve bone degenerative lesions.

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.

Biological monitoring of a xenomaterial for grafting: an evaluation in critical-size calvarial defects.

Our purpose was to evaluate the osteoconduction potential of mixed bovine bone (MBB) xenografts as an alternative for bone grafting of critical-size defects in the calvaria of rats. After surgery, in the time intervals of 1, 3, 6, and 9 months, rats were killed and their skulls collected, radiographed and histologically prepared for analysis. The data obtained from histological analysis reported that the particles of MBB did not promote an intense immunological response, evidencing its biocompatibility in rats. Our results clearly showed the interesting evidence that MBB was not completely reabsorbed at 9 months while a small amount of newly formed bone was deposited by osteoprogenitor cells bordering the defect. However, this discrete bone-forming stimulation was unable to regenerate the bone defect. Overall, our results suggest that the properties of MBB are not suitable for stimulating intense bone regeneration in critical bone defects in rats.

Platelet microparticles load a repertory of miRNAs programmed to drive osteogenic phenotype.

Autologous platelet-rich plasma accelerates bone healing by releasing biomolecules during their degranulation process, which are transported by vesicle-like structures called platelet microparticles (PMPs). However, the underlying mechanisms regulating the osteogenic differentiation by PMP-released miRs remain poorly understood and this prompted us to better address this issue. Thus, miRNAseq expression profiles (E-GEOD-76789) were downloaded from ArrayExpress database. GEO2R was performed to evaluate the differential expression, and mirnatap R package was used to find targets for differentially expressed miRNAs. An extend protein-protein (ePPI) network for osteogenic marker proteins was generated using String, and DAVID tools were used to perform gene ontology and KEGG pathway analysis from ePPI and miRNAs targets. Our data show that ePPI network was composed by 232 nodes and 2,175 edges, with a clustering coefficient of 0.546. MCODE was able to identify seven clusters contained in the ePPI network, and the two that presented a score above 10 were used in further analysis. Conversely, 15,944 different targets were found as down-expressed while 5,715 different targets were up-expressed. Among the downregulated 75 miRNAs, 70 have predicted targets present in the ePPI network, while the 21 upregulated miRNAs have 19 predicted targets in the ePPI network. Our study provides a registry of miRNAs that play a central role in regulating osteogenic phenotype, which might have potential therapeutic applications in bone regeneration and bone tissue engineering.