Effects of aerobic exercise on the regulation of mitochondrial carrier homolog-2 and its influence on the catabolic and anabolic activity of lipids in the mesenteric adipose tissue of obese mice.

The aim was to understand the direct impact of aerobic short-term exercise on lipid metabolism, specifically in regulating the mitochondrial carrier homolog 2 (MTCH2) and how it interferes with lipid metabolism in mesenteric adipose tissue. Swiss mice were divided into three groups: control, sedentary obese, and exercised obese. The obese groups were induced into obesity for fourteen weeks of a high-fat diet, and the trained submitted to seven aerobic exercise sessions. The exercise proved the significant increase of the pPerilipin-1, a hormone-sensitive lipase gene, and modulates lipid metabolism by increasing the expression of Mtch2 and acetyl Co-A carboxylase, perhaps occurring as feedback to regulate lipid metabolism in adipose tissue. In conclusion, we demonstrate, for the first time, how aerobic physical exercise increases Mtch2 transcription in mesenteric adipose tissue. This increase was due to changes in energy demand caused by exercise, confirmed by observing the significant reduction in mesenteric adipose tissue mass in the exercised group. Also, we showed that physical exercise increased the phosphorylative capacity of PLIN1, a protein responsible for the degradation of fatty acids in the lipid droplet, providing acyl and glycerol for cellular metabolism. Although our findings demonstrate evidence of MTCH2 as a protein that regulates lipid homeostasis, scant knowledge exists concerning the signaling of the MTCH2 pathway in regulatingfatty acid metabolism. Therefore, unveiling the means of molecular signaling of MTCH2 demonstrates excellent potential for treating obesity.

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.

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.

Short-term strength exercise reduces the macrophage M1/M2 ratio in white adipose tissue of obese animals.

Obesity can exacerbate the systemic inflammatory process, leading to increased infiltration of monocytes in white adipose tissue (WAT) and polarization of these cells into pro-inflammatory M1 macrophages, while reducing the population of anti-inflammatory M2 macrophages. Aerobic exercise has been shown to be effective in reducing the pro-inflammatory profile. However, the impact of strength training and the duration of training on macrophage polarization in the WAT of obese individuals have not been widely studied. Therefore, our aim was to investigate the effects of resistance exercise on macrophage infiltration and polarization in the epididymal and subcutaneous adipose tissue of obese mice. We compared the following groups: Control (CT), Obese (OB), Obese 7-day strength training (STO7d), and Obese 15-day strength training (STO15d). Macrophage populations were evaluated by flow cytometry: total macrophages (F4/80+), M1 (CD11c), and M2 (CD206) macrophages. Our results demonstrated that both training protocols improved peripheral insulin sensitivity by increasing AKT phosphorylation (Ser473). Specifically, the 7-day training regimen reduced total macrophage infiltration and M2 macrophage levels without altering M1 levels. In the STO15d group, significant differences were observed in total macrophage levels, M1 macrophages, and the M1/M2 ratio compared to the OB group. In the epididymal tissue, a reduction in the M1/M2 ratio was observed in the STO7d group. Overall, our data demonstrate that 15 days of strength exercise can reduce the M1/M2 ratio of macrophages in white adipose tissue.

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.

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.

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.

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.

The role of apoptosis associated speck-like protein containing a caspase-1 recruitment domain (ASC) in response to bone substitutes.

The apoptosis-associated Speck-like protein containing a caspase-1 recruitment domain (ASC), present in inflammasomes, regulates inflammation events and is involved in osteogenic phenotype. Nevertheless, its function in bone repair induced by bone substitute biomaterials is unclear. This study aimed to unveil the role of ASC on osteoprogenitor and tissue response to stoichiometric-hydroxyapatite (HA), nanostructured carbonated-hydroxyapatite (CHA), and CHA containing 5% Strontium (SrCHA), characterized previously by XRD, uXRF-SR, and FTIR spectroscopy implants. Thereafter, conditioned media by the biomaterials were used later to treat pre-osteoblasts and an osteogenic stimulus was shown in response to the materials, with higher expression of Runx2, Osterix, ALP, and Collagen 1a1 genes, with significant involvement of inflammatory-related genes. Thus, to better address the involvement of inflammasome, primary cells obtained from both genotypes [Wild-Type (WT) and ASC Knockout (ASC-KO) mice] were subjected to conditioned media up to 7 days, and our data reinforces both HA and CHA induces lower levels of alkaline phosphatase (ALP) than SrCHA, considering both genotypes (p < 0.01), and ASC seems contribute with osteogenic stimulus promoted by SrCHA. Complimentarily, the biomaterials were implanted into both subcutaneous and bone defects in tibia. Histological analysis on 28 days after implantation of biomaterials into mice's subcutaneous tissue revealed moderate inflammatory response to them. Both histomorphometry and µCT analysis of tibias indicated that the biomaterials did not reverse the delay in bone repair of ASC KO, reinforcing the involvement of ASC on bone regeneration and bone de novo deposition. Also, the bone density in CHA was >2-fold higher in WT than ASC-KO samples. HA was virtually not resorbed throughout the experimental periods, in opposition to CHA in the WT group. CHA reduced to half-area after 28 days, and the bone deposition was higher in CHA for WT mice than HA. Taken together, our results show that biomaterials did not interfere with the healing pattern of the ASC KO, but CHA promoted higher bone deposition in the WT group, probably due to its greater biodegradability. These results reinforce the importance of ASC during bone de novo deposition and healing.

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.

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.

Hydrogen peroxide-mediated oxidative stress induces inflammasome activation in term human placental explants.

BACKGROUND: The placenta is a multifunctional organ that can suffer with imbalances between pro- and antioxidant molecules, contributing for inflammatory imbalance. The inflammation generated by oxidative stress may induce inflammasome activation, an essential complex for pro-inflammatory cytokine production. OBJECTIVE: The aim of this study was to evaluate whether hydrogen peroxide (H2O2) mediated oxidative stress induces inflammasome activation on placental explants. STUDY DESIGN: Tissue cultures of placental explants obtained from normotensive pregnant women were performed in different concentrations of H2O2. Gene expressions of NLRP3, caspase-1, IL-1ß, TNF-α and IL-10 were evaluated by qPCR. Superoxide dismutase (SOD), catalase, Heat shock protein 70 (Hsp70), Caspase-1, TNF-α, IL-1ß, IL-10 and human Chorionic Gonadotropin (hCG) were determined by ELISA. RESULTS: Concentrations of catalase, Hsp70, hCG and SOD were higher in cultures with 100 and 1000 µM H2O2 compared to controls. Gene and protein expressions of TNF-α and IL-1ß were elevated in cultures with 1000 µM H2O2 compared to controls. This concentration led to inflammasome activation, by increasing gene expressions of NLRP3, caspase-1 and IL-1ß. In contrast, gene and protein expressions of IL-10 were reduced at 100 and 1000 µM H2O2. Protein expression of caspase-1 was higher in cultures of 100 µM H2O2 compared to controls. Treatment with Glybenclamide at 200 µM was used to prevent NLRP3 inflammasome activation. This concentration reduced protein expression of caspase-1 compared to culture with only H2O2 and control cultures. CONCLUSIONS: Our results confirm that H2O2 induces oxidative stress on placental explants and demonstrate that cell responses to this stress involve inflammasome activation.

Mg-Al and Zn-Al Layered Double Hydroxides Promote Dynamic Expression of Marker Genes in Osteogenic Differentiation by Modulating Mitogen-Activated Protein Kinases.

The effect of LDH samples comprised of chloride anions intercalated between positive layers of magnesium/aluminum (Mg-Al LDH) or zinc/aluminum (Zn-Al LDH) chemical composition on pre-osteoblast performance is investigated. Non-cytotoxic concentrations of both LDHs modulated pre-osteoblast adhesion by triggering cytoskeleton rearrangement dependent on recruiting of Cofilin, which is modulated by the inhibition of the Protein Phosphatase 2A (PP2A), culminating in osteoblast differentiation with a significant increase of osteogenic marker genes. The alkaline phosphatase (ALP) and bone sialoprotein (BSP) are significantly up-modulated by both LDHs; however, Mg-Al LDH nanomaterial promoted even more significance than both experimental controls, while the phosphorylations of mitogen-activated protein kinase (MAPKs)- extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) significantly increased. MAPK signaling is necessary to activate Runt-related transcription factor 2 (RUNX2) gene. Concomitantly, it is also investigated whether challenged osteoblasts are able to modulate osteoclastogenesis by investigating both osteoprotegerin (OPG) and Receptor activator of nuclear factor kappa-ligand (RANKL) in this model; a dynamic reprogramming of both these genes is found, suggesting LDHs in modulating osteoclastogenesis. These results suggest that LDHs interfere in bone remodeling, and they can be considered as nanomaterials in graft-based bone healing or drug-delivery materials for bone disorders.

Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions.

The reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions.