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.

BMP7-induced osteoblast differentiation requires hedgehog signaling and involves nuclear mechanisms of gene expression control.

During the morphological changes occurring in osteoblast differentiation, Sonic hedgehog (Shh) plays a crucial role. While some progress has been made in understanding this process, the epigenetic mechanisms governing the expression of Hh signaling members in response to bone morphogenetic protein 7 (BMP7) signaling in osteoblasts remain poorly understood. To delve deeper into this issue, we treated pre-osteoblasts (pObs) with 100 ng/mL of BMP7 for up to 21 days. Initially, we validated the osteogenic phenotype by confirming elevated expression of well-defined gene biomarkers, including Runx2, Osterix, Alkaline Phosphatase (Alp), and bone sialoprotein (Bsp). Simultaneously, Hh signaling-related members Sonic (Shh), Indian (Ihh), and Desert (Dhh) Hedgehog (Hh) exhibited nuanced modulation over the 21 days in vitro period. Subsequently, we evaluated epigenetic markers, and our data revealed a notable change in the CpG methylation profile, considering the methylation/hydroxymethylation ratio. CpG methylation is a reversible process regulated by DNA methyltransferases and demethylases, including Ten-eleven translocation (Tets), which also exhibited changes during the acquisition of the osteogenic phenotype. Specifically, we measured the methylation pattern of Shh-related genes and demonstrated a positive Pearson correlation for GLI Family Zinc Finger 1 (Gli1) and Patched (Ptch1). This data underscores the significance of the epigenetic machinery in modulating the BMP7-induced osteogenic phenotype by influencing the activity of Shh-related genes. In conclusion, this study highlights the positive impact of epigenetic control on the expression of genes related to hedgehog signaling during the morphogenetic changes induced by BMP7 signaling in osteoblasts.

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.

Epigenetic changes in shear-stressed endothelial cells.

Epigenetic changes, particularly histone compaction modifications, have emerged as critical regulators in the epigenetic pathway driving endothelial cell phenotype under constant exposure to laminar forces induced by blood flow. However, the underlying epigenetic mechanisms governing endothelial cell behavior in this context remain poorly understood. To address this knowledge gap, we conducted in vitro experiments using human umbilical vein endothelial cells subjected to various tensional forces simulating pathophysiological blood flow shear stress conditions, ranging from normotensive to hypertensive forces. Our study uncovers a noteworthy observation wherein endothelial cells exposed to high shear stress demonstrate a decrease in the epigenetic marks H3K4ac and H3K27ac, accompanied by significant alterations in the levels of HDAC (histone deacetylase) proteins. Moreover, we demonstrate a negative regulatory effect of increased shear stress on HOXA13 gene expression and a concomitant increase in the expression of the long noncoding RNA, HOTTIP, suggesting a direct association with the suppression of HOXA13. Collectively, these findings represent the first evidence of the role of histone-related epigenetic modifications in modulating chromatin compaction during mechanosignaling of endothelial cells in response to elevated shear stress forces. Additionally, our results highlight the importance of understanding the physiological role of HOXA13 in vascular biology and hypertensive patients, emphasizing the potential for developing small molecules to modulate its activity. These findings warrant further preclinical investigations and open new avenues for therapeutic interventions targeting epigenetic mechanisms in hypertensive conditions.

JARID1B represses the osteogenic potential of human periodontal ligament mesenchymal cells.

BACKGROUND: Here, we evaluated whether the histone lysine demethylase 5B (JARID1B), is involved in osteogenic phenotype commitment of periodontal ligament cells (PDLCs), by considering their heterogeneity for osteoblast differentiation. MATERIALS AND METHODS: Epigenetic, transcriptional, and protein levels of a gene set, involved in the osteogenesis, were investigated by performing genome-wide DNA (hydroxy)methylation, mRNA expression, and western blotting analysis at basal (without osteogenic induction), and at the 3rd and 10th days of osteogenic stimulus, in vitro, using PDLCs with low (l) and high (h) osteogenic potential as biological models. RESULTS: h-PDLCs showed reduced levels of JARID1B, compared to l-PDLCs, with significant inversely proportional correlations between RUNX2 and RUNX2/p57. Epigenetically, a significant reduction in the global H3K4me3 content was observed only in h-PDLCs. Immunoblotting data reveal a significant reduction in the global H3K4me3 content, at 3 days of induction only in h-PDLCs, while an increase in the global H3K4me3 content was observed at 10 days for both PDLCs. Additionally, positive correlations were found between global H3K4me3 levels and JARID1B gene expression. CONCLUSIONS: Altogether, our results show the crucial role of JARID1B in repressing PDLCs osteogenic phenotype and this claims to pre-clinical protocols proposing JARID1B as a potential therapeutic target.

MicroRNA biogenesis machinery activation and lncRNA and REST overexpression as neuroprotective responses to fight inflammation in the hippocampus.

Brain Long non-coding RNA (lncRNA) and microRNAs (miRs) play essential roles in the regulation of several important biological processes, including neuronal activity, cognitive processes, neurogenesis, angiogenesis, and neuroinflammation. In this context, the transcriptional repressor, RE1 silencing transcription factor (Rest), acts regulating the expression of neuronal genes as well as of lncRNAs and multiple miRNAs in the central nervous system. Nevertheless, its role in neuroinflammation was less explored. Here, we demonstrate, using an in vivo model of neuroinflammation induced by i.p. injection of LPS (0.33 mg/kg), that neuroinflammation increases gene expression of pro-inflammatory cytokines concomitant with the native and truncated forms of Rest and of non-coding RNAs. Additionally, the increased expression of enzymes Drosha ribonuclease III) (Drosha), Exportin 5 (Xpo5) and Endoribonuclease dicer (Dicer), associated with high expression of neuroprotective miRs 22 and 132 are indicative that the activation of biogenesis of miRs in the hippocampal region is a Central Nervous System (CNS) protective mechanism for the deleterious effects of neuroinflammation. Our results indicate that positive regulation of Rest gene expression in the hippocampal region by neuroinflammation correlates directly with the expression of miRs 22 and 132 and inversely with miR 335. In parallel, the confirmation of the possible alignment between the lncRNAs with miR 335 by bioinformatics corroborates with the sponge effect of Hottip and Hotair hybridizing and inhibiting the pro-inflammatory action of miR 335. This suggests the existence of a possible correlation between the activation of miR biogenesis machinery with increased expression of the transcription factor Rest, contributing to neuroprotection.

Hyperglycemic microenvironment compromises the homeostasis of communication between the bone-brain axis by the epigenetic repression of the osteocalcin receptor, Gpr158 in the hippocampus.

Diabetes mellitus (DM) is a chronic metabolic disease, mainly characterized by increased blood glucose and insulin dysfunction. In response to the persistent systemic hyperglycemic state, numerous metabolic and physiological complications have already been well characterized. However, its relationship to bone fragility, cognitive deficits and increased risk of dementia still needs to be better understood. The impact of chronic hyperglycemia on bone physiology and architecture was assessed in a model of chronic hyperglycemia induced by a single intraperitoneal administration of streptozotocin (STZ; 55 mg/kg) in Wistar rats. In addition, the bone-to-brain communication was investigated by analyzing the gene expression and methylation status of genes that encode the main osteokines released by the bone [Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) and their receptors in both, the bone and the brain [Fgfr1 (fibroblast growth factor receptor 1), Gpr6A (G-protein coupled receptor family C group 6 member A), Gpr158 (G protein-coupled receptor 158) and Slc22a17 (Solute carrier family 22 member 17)]. It was observed that chronic hyperglycemia negatively impacted on bone biology and compromised the balance of the bone-brain endocrine axis. Ultrastructural disorganization was accompanied by global DNA hypomethylation and changes in gene expression of DNA-modifying enzymes that were accompanied by changes in the methylation status of the osteokine promoter region Bglap and Lcn2 (lipocalin 2) in the femur. Additionally, the chronic hyperglycemic state was accompanied by modulation of gene expression of the osteokines Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) in the different brain regions. However, transcriptional regulation mediated by DNA methylation was observed only for the osteokine receptors, Fgfr1(fibroblast growth factor receptor 1) in the striatum and Gpr158 (G protein-coupled receptor 158) in the hippocampus. This is a pioneer study demonstrating that the chronic hyperglycemic state compromises the crosstalk between bone tissue and the brain, mainly affecting the hippocampus, through transcriptional silencing of the Bglap receptor by hypermethylation of Gpr158 gene.

Genome-wide DNA (hydroxy) methylation reveals the individual epigenetic landscape importance on osteogenic phenotype acquisition in periodontal ligament cells.

BACKGROUND: Mesenchymal cells' biology has been an important investigative tool to maximize bone regeneration through tissue engineering. Here we used mesenchymal cells from periodontal ligament (PDLCs) with high (h-) and low (l-) osteogenic potential, isolated from different donors, to investigate the impact of the individual epigenetic and transcriptional profiles on the osteogenic potential. METHODS: Genome-wide and gene-specific DNA (hydroxy) methylation, mRNA expression and immunofluorescence analysis were carried out in h- and l-PDLCs at DMEM (non-induced to osteogenesis) and OM (induced-3rd and 10th days of osteogenic differentiation) groups in vitro. RESULTS: Genome-wide results showed distinct epigenetic profile among PDLCs with most of the differences on 10th day of OM; DMEMs showed higher concentrations (xOM) of differentially methylated probes in gene body, intronic and open sea (3rd day), increasing this concentration in TSS200 and island regions, at 10 days. At basal levels, h- and l-PDLCs showed different transcriptional profiles; l-PDLCs demonstrated higher levels of NANOG/OCT4/SOX2, BAPX1, DNMT3A, TET1/3, and lower levels of RUNX2 transcripts, confirmed by NANOG/OCT4 and RUNX2 immunofluorescence. After osteogenic induction, the distinct transcriptional profile of multipotentiality genes was maintained among PDLCs. In l-PDLCs, the anti-correlation between DNA methylation and gene expression in RUNX2 and NANOG indicates methylation could play a role in modulating both transcripts. CONCLUSIONS: Epigenetic and transcriptional distinct profiles detected at basal levels among PDLCs were maintained after osteogenic induction. We cannot discard the existence of a complex that represses osteogenesis, suggesting the individual donors' characteristics have significant impact on the osteogenic phenotype acquisition.

Propolis from southeastern Brazil produced by Apis mellifera affects innate immunity by modulating cell marker expression, cytokine production and intracellular pathways in human monocytes.

OBJECTIVES: Propolis is a bee-made product used for centuries due to its diverse biological properties, including its immunomodulatory action. This work aimed at investigating whether propolis may affect monocyte functions challenged with retinoic acid (RA), B subunit of Escherichia coli heat-labile enterotoxin (EtxB), human melanoma-associated antigen-1 (MAGE-1) and lipopolysaccharide (LPS). METHODS: Monocytes from healthy donors were treated with the stimuli separately or in the presence of propolis. Cell viability was evaluated by MTT assay, cell marker expression was assessed by flow cytometry, cytokine production by ELISA, gene expression by RT-qPCR. KEY FINDINGS: Propolis alone maintained TLR-2, TLR-4, HLA-DR, CD40 and CD80 expression in the monocytes; however, its combination with either MAGE-1 or LPS decreased CD40 expression triggered by the stimuli. Propolis maintained RA action on cell marker expression. Propolis inhibited TNF-α (with either EtxB or MAGE-1) and IL-6 (with either RA or MAGE-1), and increased IL-10 (with MAGE-1) production. Propolis downmodulated LC3 expression induced by LPS. It also induced a lower NF-kB expression than control cells and its combination with RA induced a higher expression than the stimulus alone. CONCLUSIONS: Propolis potentially affected innate immunity by downmodulating the monocytes pro-inflammatory activity.

Non-coding RNAs repressive role in post-transcriptional processing of RUNX2 during the acquisition of the osteogenic phenotype of periodontal ligament mesenchymal stem cells.

Mesenchymal stem cells are candidates for therapeutic strategies in periodontal repair due to their osteogenic potential. In this study, we identified epigenetic markers during osteogenic differentiation, taking advantage of the individual pattern of mesenchymal cells of the periodontal ligament with high (h-PDLCs) and low (l-PDLCs) osteogenic capacity. We found that the involvement of non-coding RNAs in the regulation of the RUNX2 gene is strongly associated with high osteogenic potential. Moreover, we evaluated miRs and genes that encode enzymes to process miRs and their biogenesis. Our data show the high expression of the XPO5 gene, and miRs 7 and 22 observed in the l-PDLCs might be involved in acquiring osteogenic potential, suppressing RUNX2 gene expression. Further, an inversely proportional correlation between lncRNAs (HOTAIR and HOTTIP) and RUNX2 gene expression was observed in both l- and h-PDLCs, and it was also related to the distinct osteogenic phenotypes. Thus, our results indicate the low expression of XPO5 in h-PDLC might be the limiting point for blocking the miRs biogenesis, allowing the high gene expression of RUNX2. In accordance, the low expression of miRs, HOTAIR, and HOTTIP could be a prerequisite for increased osteogenic potential in h-PDLCs. These results will help us to better understand the underlying mechanisms of osteogenesis, considering the heterogeneity in the osteogenic potential of PDLCs that might be related to a distinct transcriptional profile of lncRNAs and the biogenesis machinery.

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.

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.

The Impact of Bioactive Surfaces in the Early Stages of Osseointegration: An In Vitro Comparative Study Evaluating the HAnano® and SLActive® Super Hydrophilic Surfaces.

There is an increased effort on developing novel and active surfaces in order to accelerate their osteointegration, such as nanosized crystalline hydroxyapatite coating (HAnano®). To better understand the biological behavior of osteoblasts grown on HAnano® surface, the set of data was compared with SLActive®, a hydrophilic sandblasted titanium surface. Methodologically, osteoblasts were seeded on both surfaces up to 72 hours, to allow evaluating cell adhesion, viability, and set of genes encoding proteins related with adhesion, proliferation, and differentiation. Our data shows HAnano® displays an interesting substrate to support cell adhesion with typical spread morphologic cells, while SLActive®-adhering cells presented fusiform morphology. Our data shows that the cellular adhesion mechanism was accompanied with upexpression of integrin ß1, Fak, and Src, favoring the assembling of focal adhesion platforms and coupling cell cycle progression (upmodulating of Cdk2, Cdk4, and Cdk6 genes) in response to HAnano®. Additionally, both bioactive surfaces promoted osteoblast differentiation stimulus, by activating Runx2, Osterix, and Alp genes. Although both surfaces promoted Rankl gene expression, Opg gene expression was higher in SLActive® and this difference reflected on the Rankl/Opg ratio. Finally, Caspase1 gene was significantly upmodulated in response to HAnano® and it suggests an involvement of the inflammasome complex. Collectively, this study provides enough evidences to support that the nanohydroxyapatite-coated surface provides the necessary microenvironment to drive osteoblast performance on dental implants and these stages of osteogenesis are expected during the early stages of osseointegration.

Infraphysiological 17ß-estradiol (E2) concentration compromises osteoblast differentiation through Src stimulation of cell proliferation and ECM remodeling stimulus.

It has been shown that 17ß-estradiol (E2) helps to prevent bone loss. This study was undertaken to verify whether E2 action in human osteoblasts involves changes in the transcriptional profile of the TNF-α, IFN-γ, NF-κB, TRAIL, TGF-ß, MMP2, MMP9, RECK, TIMP1, TIMP2, CDK2, CDK4, SRC, RUNX2, and SHH genes. Infraphysiological doses of E2 elevated mRNAs in all genes except for INF-γ, TRAIL, and TGF-ß. Importantly, a significant increase in the CDKs -2 and -4 genes was found, which strongly suggests cell cycle progression, with a potential dependency of Src involvement, as well as a suppression of the osteoblast differentiation machinery, with ECM remodeling being involved. These data suggest that E2 plays an important role in bone formation and remodeling, and Src seems to play a pivotal role in driving cell proliferation and ECM remodeling. Taken together, these findings contribute to an understanding of the effects of infraphysiological E2 on modulating bone homeostasis, favoring bone resorption, and leading to osteoporosis.

Biofunctionalization of titanium surfaces with alendronate and albumin modulates osteoblast performance.

BACKGROUND: Biofunctionalization of titanium surfaces can improve host responses, especially considering the time for osteointegration and patient recovery. This prompted us to modify titanium surfaces with alendronate and albumin and to investigate the behavior of osteoblasts on these surfaces. METHODS: The biofunctionalization of titanium surfaces was characterized using classical physicochemical approaches and later used to challenge pre-osteoblast cells up to 24 h. Then their viability and molecular behavior were investigated using mitochondrial dehydrogenase activity and RTq-PCR technologies, respectively. Potential stimulus of extracellular remodeling was also investigated by zymography. RESULTS: Our data indicates a differential behavior of cells responding to the surfaces, considering the activity of mitochondrial dehydrogenases. Molecularly, the differential expression of genes related with cell adhesion highlighted the importance of Integrin-ß1, Fak, and Src. These 3 genes were significantly decreased in response to titanium surfaces modified with alendronate, but this behavior was reverted when alendronate was associated with albumin. Alendronate-modified surfaces promoted a significant increase on ECM remodeling, as well as culminating with greater gene activity related to the osteogenic phenotype (Runx2, Alp, Bsp). CONCLUSION: Altogether, our study found interesting osteogenic behavior of cells in response to alendronate and albumin surfaces, which indicates the need for in vivo analyses to better consider these surfaces before clinical trials within the biomedical field.

Osteogenic gene markers are epigenetically reprogrammed during contractile-to-calcifying vascular smooth muscle cell phenotype transition.

The understanding of vascular calcification-based mechanism is an urgent pending task in vascular biology and this prompted us to better address this issue by investigating whether DNA methylation mechanism might drive osteogenic marker genes modulation in primary human vascular smooth muscle cells (VSMCs) responding to calcium and phosphate levels overload up to 72 h. Firstly, our data shows this calcifying process recapitulates the molecular repertory of osteogenic biomarkers and specifically requiring RUNX2, Osterix and ALP, BSP genes activations along 72 h in vitro, and this behavior was validated here using other lineages. Conversely, both BMPs 4 and 7 were significantly overexpressed, maybe already as a mechanism in response to RUNX2 and Osterix genes activities identified earlier in response to the calcifying condition, and taken into maintain the calcifying phenotype of VSMCs. Additionally, survival signaling was maintained active and accompanied by a dynamic cytoskeleton rearrangement signaling requiring MAPK and AKT phosphorylations. Moreover, during the contractile-to-calcifying transition phenotype of VSMCs, epigenetic machinery was finely modulated, requiring the translocation of DNMT3B and TET2 into nucleus and this prompted us evaluating whether the profile of osteogenic-related gene promoters' methylation might contribute with this process. By firstly estimating 5meC/5 hmeC ratio changes, we further specifically show the significance of the epigenetic modulation of Osterix and Bone sialoprotein related gene promoters, presenting a positive correlation between the epigenetic signature of their gene promoters and transcriptional patterns. Altogether, our results show for the first time the importance of epigenetic mechanism on modulating osteogenic gene markers reprogramming during calcifying VSMCs phenotype acquisition, which might drive the genesis of vascular ectopic calcification.

HOXA cluster gene expression during osteoblast differentiation involves epigenetic control.

The HOXA gene cluster is generally recognized as a pivotal mediator of positional identity in the skeletal system, expression of different orthologues conferring alternative locational phenotype of the vertebrate bone. Strikingly, however, the molecular mechanisms that regulate orthologue-specific expression of different HOXA cluster members in gestating osteoblasts remain largely obscure, but in analogy to the processes observed in acute lymphatic leukemia it is assumed that alternative methylation of HOXA promoter regions drives position specific expression patterns. In an effort to understand HOXA cluster gene expression in osteogenesis we characterize both expression and the epigenetic landscape of the HOXA gene cluster during in vitro osteoblast formation from mesenchymal precursors. We observe that osteoblast formation per se provokes strong upregulation of HOXA gene cluster expression, in particular of midcluster genes, and paradoxal downregulation of HOXA7 and HOXA10. These differences in expression appear related to promoter methylation. LnRNAs HOTAIR and HOTTIP, known to modulate HOXA expression, are also regulated by their promoter methylation processing, but do not correlate with HOXA cluster expression profile. We thus conclude that HOXA expression is profoundly regulated during osteoblast differentiation through canonical methylation-dependent mechanisms but not through the flanking lnRNAs.

Differential inflammatory landscape stimulus during titanium surfaces obtained osteogenic phenotype.

Molecular mechanism governing inflammatory scenario in response to titanium (Ti)-nanotexturing surfaces needs to be better addressed. Thus, we subjected pre-osteoblast to different Ti-texturing surfaces, as follows: machined (Mac), double acid-etching (DAE), and nanoscaled hydroxyapatite-blasted titanium surface (nHA), considering the cells chronically responding either directly (when the cells were cultured onto the surfaces) or indirectly (when the cells were challenged with the conditioned medium by the surfaces), up to 10 days. Our results showed that there is a dynamic requirement of inflammatory-related genes activation in response to nHA by up expressing IL1ß, IL6, IL10, and IL33 (direct condition) and IL6, IL10, IL18 (indirect condition). Importantly, our data show that there is inflammasome involvement, once NLRP3, ASC1, and CASP1 genes were also required. As we found a strong signal of IL10, an anti-inflammatory cytokine, we further investigated Sonic Hedgehog (Shh) signaling cascade. Surprisingly, Shh ligand and Smoothened (Smo) genes were up-modulated in response to nHA, while Patched (Ptc) was down-modulated. Finally, an interactome was built using bioinformatics reinforcing Shh signaling cascade on modulating IL10 transcripts by Src mediating this process and this prevalence of anti-inflammatory picture might explain the low profile of RANKL transcripts in response to nHA, compromising the osteoclastogenesis surrounding the implants. Taking our results into account, our data show that the inflammatory landscape promoted by nHA is strictly modulated by Shh signaling promoted anti-inflammatory pathways. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1597-1604, 2019.

Sonic hedgehog drives layered double hydroxides-induced acute inflammatory landscape.

Although layered double hydroxides (LDH) have been listed as promising nanomaterials in human healthcare, very little has been achieved on osteoblast inflammatory signaling. Thus, osteoblasts were challenged with two LDHs (Mg2Al-Cl and Zn2Al-Cl, at 0.002 mg/mL) up to 24 h, establishing an acute inflammatory mechanism, as well as identifying whether Sonic hedgehog (Shh) signaling has an influence. Functional experiments were performed by previously treating (2 h) semiconfluent osteoblast cultures with cyclopamine molecule (cyc), a widely used Shh inhibitor. Considering inflammasome complex, the asc1 gene was significantly up-expressed in response to Zn2Al-Cl - LDHs, as well as the nrlp3 gene. By treating the osteoblast with cyc, the asc1 gene presented an even higher profile. Our results found a down-modulation of major pro-inflammatory cytokines-related genes, when tnfα and il1ß were significantly down-modulated in response to LDHs. Conversely, anti-inflammatory cytokines were up-modulated considering the same experimental procedures. Except the il6, the other il13, il10, and tgfß genes were up modulated. Additionally, Shh signaling seems to modulate this repertory as both the il13 and il10 genes were significantly up-modulated when the Shh signaling was inhibited. Altogether, our results reveal for the first time the exigency of Shh-dependent anti-inflammatory signals in LDH-induced osteoblast responses.

Laminar shear stress-provoked cytoskeletal changes are mediated by epigenetic reprogramming of TIMP1 in human primary smooth muscle cells.

Whereas endothelial responses to shear stress are well-characterized, the cell physiological effects of shear stress in smooth muscle cells (SMCs) remain largely obscure. As SMCs are directly challenged by shear stress after endothelial denuding injury following procedures such as angioplasty or endarterectomy, characterization of these responses represents an important scientific question. Hence we decided to contrast cytoskeletal reorganization, epigenetic reprogramming, signaling transduction, and changes in miRNA (miRs) profiles in primary human aortic smooth muscle cells (AoSMCs) between unstressed cells and cells exposed to shear stress. We observed that shear stress-provoked reorganization of the actin cytoskeleton in an apparently Cofilin-dependent fashion and which related to altered integrin signaling, apparently caused by remodeling of the extracellular matrix. The latter appeared a downstream effect of increased expression of matrix metalloproteinases and downregulation of tissue metalloproteinase inhibitor 1 (TIMP1) protein levels. In turn, these effects related to shear stress-provoked changes in expression and nuclear localization of the epigenetic regulators demethylases TET1, TET2, DNMT1, DNMT3A and DNMT3B, HDAC6, and SIRT1. Accordingly, TIMP1 promotor CpG hypomethylation was a prominent effect, and resulted in a significant increase in TIMP1 transcription, which may also have related increased expression of miRs involved in modulating TIMP1 translation. Thus epigenetic-reprogramming of TIMP1 emerges as critical element in smooth muscle responses to mechanical signals and as epigenetic machinery is amendable to pharmacological manipulation, this pathway may have important clinical consequences.