Expression Of Osteoblastic Markers Research Articles

Nfil3, a target of the NACA transcriptional coregulator, affects osteoblast and osteocyte gene expression differentially.

Intermittent administration of PTH(1-34) has a profound osteoanabolic effect on the skeleton. At the cellular level, osteoblasts and osteocytes are two crucial cell types that respond to PTH stimulation in bone. The transcriptional cofactor Nascent polypeptide Associated Complex and coregulator alpha (NACA) is a downstream target of the PTH-Gαs-PKA axis in osteoblasts. NACA functions as a transcriptional cofactor affecting bZIP factor-mediated transcription of target promoters in osteoblasts, such as Osteocalcin (Bglap2). Here, we used RNA-Seq and ChIP-Seq against NACA in PTH-treated MC3T3-E1 osteoblastic cells to identify novel targets of the PTH-activated NACA. Our approach identified Nuclear factor interleukin-3-regulated (Nfil3) as a target promoter of this pathway. Knockdown of Naca reduced the response of Nfil3 to PTH(1-34) stimulation. In silico analysis of the Nfil3 promoter revealed potential binding sites for NACA (located within the ChIP fragment) and CREB. We show that following PTH stimulation, phosphorylated-CREB binds the proximal promoter of Nfil3 in osteoblasts. The activity of the Nfil3 promoter (-818/+182bp) is regulated by CREB and this activation relies on the presence of NACA. In addition, we show that knockdown of Nfil3 enhances the expression of osteoblastic differentiation markers in MC3T3-E1 cells while it represses osteocytic marker gene expression in IDG-SW3 cells. These results show that the PTH-induced NACA axis regulates Nfil3 expression and suggest that NFIL3 acts as a transcriptional repressor in osteoblasts while it exhibits differential activity as an activator in osteocytes.

Active sites of human MEPE-ASARM regulating bone matrix mineralization

The proteolytic fragment ASARM (acidic serine- and aspartate-rich motif) of MEPE (matrix extracellular phosphoglycoprotein) (MEPE-ASARM) may act as an endogenous anti-mineralization factor involved in X-linked hypophosphatemic rickets/osteomalacia (XLH). We synthesized MEPE-ASARM peptides and relevant peptide fragments with or without phosphorylated Ser residues (pSer) to determine the active site(s) of MEPE-ASARM in a rat calvaria cell culture model. None of the synthetic peptides elicited changes in cell death, proliferation or differentiation, but the peptide (pASARM) with three pSer residues inhibited mineralization without causing changes in gene expression of osteoblast markers tested. The anti-mineralization effect was maintained in peptides in which any one of three pSer residues was deleted. Polyclonal antibodies recognizing pASARM but not ASARM abolished the pASARM effect. Deletion of six N-terminal residues but leaving the recognition sites for PHEX (phosphate regulating endopeptidase homolog, X-linked), a membrane endopeptidase responsible for XLH, intact and two C-terminal amino acid residues did not alter the anti-mineralization activity of pASARM. Our results strengthen understanding of the active sites of MEPE-pASARM and allowed us to identify a shorter more stable sequence with fewer pSer residues still exhibiting hypomineralization activity, reducing peptide synthesis cost and increasing reliability for exploring biological and potential therapeutic effects.

Effects of the Sintering Process on Nacre-Derived Hydroxyapatite Scaffolds for Bone Engineering.

A hydroxyapatite scaffold is a suitable biomaterial for bone tissue engineering due to its chemical component which mimics native bone. Electronic states which present on the surface of hydroxyapatite have the potential to be used to promote the adsorption or transduction of biomolecules such as protein or DNA. This study aimed to compare the morphology and bioactivity of sinter and nonsinter marine-based hydroxyapatite scaffolds. Field emission scanning electron microscopy (FESEM) and micro-computed tomography (microCT) were used to characterize the morphology of both scaffolds. Scaffolds were co-cultured with 5 × 104/cm2 of MC3T3-E1 preosteoblast cells for 7, 14, and 21 days. FESEM was used to observe the cell morphology, and MTT and alkaline phosphatase (ALP) assays were conducted to determine the cell viability and differentiation capacity of cells on both scaffolds. Real-time polymerase chain reaction (rtPCR) was used to identify the expression of osteoblast markers. The sinter scaffold had a porous microstructure with the presence of interconnected pores as compared with the nonsinter scaffold. This sinter scaffold also significantly supported viability and differentiation of the MC3T3-E1 preosteoblast cells (p < 0.05). The marked expression of Col1α1 and osteocalcin (OCN) osteoblast markers were also observed after 14 days of incubation (p < 0.05). The sinter scaffold supported attachment, viability, and differentiation of preosteoblast cells. Hence, sinter hydroxyapatite scaffold from nacreous layer is a promising biomaterial for bone tissue engineering.

Gemigliptin Inhibits Interleukin-1β-Induced Endothelial-Mesenchymal Transition via Canonical-Bone Morphogenetic Protein Pathway.

BackgroundEndothelial-to-mesenchymal transition (EndMT) contributes to inflammatory conditions inducing conversion of endothelial cells (ECs) into activated fibroblasts, promoting fibrotic diseases. Pro-inflammatory cytokine is the most potent inducer of EndMT. We investigated inhibition of interleukin-1β (IL-1β)-induced EndMT by gemigliptin, a dipeptidyl peptidase-IV inhibitor.MethodsWe exposed human umbilical vein endothelial cells (HUVECs) to 10 ng/mL IL-1β/20 μM gemigliptin and analyzed the expression of endothelial, smooth muscle, mesenchymal, and osteoblastic markers, bone morphogenetic protein (BMP), Smad, and non-Smad signaling pathway proteins.ResultsMorphological changes showed gemigliptin blocked IL-1β-induced EndMT, upregulated EC markers, and downregulated smooth muscle and mesenchymal markers. IL-1β activation of HUVECs is initiated by the BMP/Smad and non-smad BMP signaling pathways. Gemigliptin inhibited IL-1β induction of BMP2 and 7, activin receptor type IA, BMP receptor type IA, and BMP receptor type II. Reversal of IL-1β-mediated inhibition of BMP-induced Smad1/5/8, Smad2, and Smad3 phosphorylation by gemigliptin suggests involvement of the Smad pathway in gemigliptin action. In the non-Smad BMP pathway, gemigliptin treatment significantly increased the deactivation of extracellular regulated protein kinase (ERK), p38, and JNK by IL-1β. Gemigliptin treatment suppressed BMP-2-induced expression of key osteoblastic markers including osterix, runt-related transcription factor 2, and hepcidin during IL-1β-induced EndMT.ConclusionWe demonstrated a novel protective mechanism of gemigliptin against fibrosis by suppressing IL-1β-induced EndMT.

Possible function of GDNF and Schwann cells in wound healing of periodontal tissue.

The purpose of this study was to evaluate the function of Schwann cells in wound healing of periodontal tissue. In our previous study, glial cell line-derived neurotrophic factor (GDNF) promoted the migration of human periodontal ligament (PDL) cells and that GDNF expression increased in wounded periodontal tissue. GDNF reportedly induces the migration of Schwann cell precursors. Schwann cells play a crucial role in the regeneration of peripheral tissues, including bone tissue. However, the role of Schwann cells on periodontal tissue regeneration remains unclear. A transwell assay and a WST-1 (water-soluble tetrazolium compound-1) proliferation assay were used to determine whether GDNF promotes the migration and proliferation of Schwann cells, respectively. Quantitative RT-PCR and Alizarin Red S staining were performed to examine the effect of these cells on the differentiation of human preosteoblast (Saos2 cells) using conditioned medium from YST-1 (YST-1-CM). Western blotting analysis was performed to determine whether YST-1-CM activates ERK signaling pathway in Saos2 cells. The expression of Schwann cell markers, S100 calcium-binding protein B (S100-B) and growth associated protein 43 (GAP-43), was determined in normal and wounded periodontal tissue by immunofluorescent staining. Glial cell line-derived neurotrophic factor promoted the migration of YST-1 cells but did not affect the proliferation of YST-1 cells. Saos2 cells cultured with YST-1-CM increased the expression of osteoblastic markers and mineralization. YST-1-CM also induced phosphorylation of ERK1/2 in Saos2 cells. The number of S100-B-immunoreactive cells which also expressed GAP-43 was increased in rat wounded periodontal tissue during healing process. The accumulation of Schwann cells in wounded periodontal tissue suggests that they play a significant role in wound healing of this tissue, especially alveolar bone tissue.

Hepatoma-derived growth factor enhances osteoblastic transformation of rat aortic vascular smooth muscle cells in vitro

AimsVascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells. Materials and methodsCell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively. Key findingsRecombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression. SignificanceIn conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis. Chemical compounds studied in this studyHDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).

Transforming Growth Factor-Beta and Sonic Hedgehog Signaling in Palatal Epithelium Regulate Tenascin-C Expression in Palatal Mesenchyme During Soft Palate Development.

During palatogenesis, the palatal shelves first grow vertically on either side of the tongue before changing their direction of growth to horizontal. The extracellular matrix (ECM) plays an important role in these dynamic changes in palatal shelf morphology. Tenascin-C (TNC) is an ECM glycoprotein that shows unique expression in the posterior part of the palatal shelf, but little is known about the regulation of TNC expression. Since transforming growth factor-beta-3 (TGF-β3) and sonic hedgehog (SHH) signaling are known to play important roles in palatogenesis, we investigated whether TGF-β3 and SHH are involved in the regulation of TNC expression in the developing palate. TGF-β3 increased the expression of TNC mRNA and protein in primary mouse embryonic palatal mesenchymal cells (MEPM) obtained from palatal mesenchyme dissected at embryonic day 13.5–14.0. Interestingly, immunohistochemistry experiments revealed that TNC expression was diminished in K14-cre;Tgfbr2fl/fl mice that lack the TGF-β type II receptor in palatal epithelial cells and exhibit cleft soft palate, whereas TNC expression was maintained in Wnt1-cre;Tgfbr2fl/fl mice that lack the TGF-β type II receptor in palatal mesenchymal cells and exhibit a complete cleft palate. SHH also increased the expression of TNC mRNA and protein in MEPM cells. However, although TGF-β3 up-regulated TNC mRNA and protein expression in O9-1 cells (a cranial neural crest cell line), SHH did not. Furthermore, TGF-β inhibited the expression of osteoblastic differentiation markers (osterix and alkaline phosphatase) and induced the expression of fibroblastic markers (fibronectin and periostin) in O9-1 cells, whereas SHH did not affect the expression of osteoblastic and fibroblastic markers in O9-1 cells. However, immunohistochemistry experiments showed that TNC expression was diminished in the posterior palatal shelves of Shh–/+;MFCS4+/– mice, which have deficient SHH signaling in the posterior palatal epithelium. Taken together, our findings support the proposal that TGF-β and SHH signaling in palatal epithelium co-ordinate the expression of TNC in the posterior palatal mesenchyme through a paracrine mechanism. This signal cascade may work in the later stage of palatogenesis when cranial neural crest cells have differentiated into fibroblast-like cells. The spatiotemporal regulation of ECM-related proteins by TGF-β and SHH signaling may contribute not only to tissue construction but also to cell differentiation or determination along the anterior–posterior axis of the palatal shelves.

Osteogenic and Chondrogenic Potential of the Supramolecular Aggregate T-LysYal®.

Hard tissue regeneration represents a challenge for the Regenerative Medicine and Mesenchymal stem cells (MSCs) could be a successful therapeutic strategy. T-LysYal® (T-Lys), a new derivative of Hyaluronic Acid (HA) possessing a superior stability, has already been proved efficient in repairing corneal epithelial cells damaged by dry conditions in vitro. We investigated the regenerative potential of T-Lys in the hard tissues bone and cartilage. We have previously demonstrated that cells isolated from the tooth germ, Dental Bud Stem Cells (DBSCs), differentiate into osteoblast-like cells, representing a promising source of MSCs for bone regeneration. Herewith, we show that T-Lys treatment stimulates the expression of typical osteoblastic markers, such as Runx-2, Collagen I (Col1) and Alkaline Phosphatase (ALP), determining a higher production of mineralized matrix nodules. In addition, we found that T-Lys treatment positively affects αVβ3 integrin expression, key integrin in the osteoblastic commitment, leading to the formation of focal adhesions (FAs). The efficacy of T-Lys was also tested on chondrogenic differentiation starting from human articular chondrocytes (HACs) resulting in an increase of differentiation markers and cell number.

Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation

Application of messenger RNA (mRNA) for bone regeneration is a promising alternative to DNA, recombinant proteins and peptides. However, exogenous in vitro transcribed mRNA (IVT mRNA) triggers innate immune response resulting in mRNA degradation and translation inhibition. Inspired by the ability of viral immune evasion proteins to inhibit host cell responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A virus (A/Texas/36/1991) as an IVT mRNA enhancer. We evidenced a dose-dependent blocking of RNA sensors by NS1 expression. The co-delivery of NS1 mRNA with mRNA of reporter genes significantly increased the translation efficiency. Interestingly, unlike the use of nucleosides modification, NS1-mediated mRNA translation enhancement does not dependent to cell type. Dual delivery of NS1 mRNA and BMP-2 mRNA to murine pluripotent stem cells (C3H10T1/2), promoted osteogenic differentiation evidenced by enhanced expression of osteoblastic markers (e.g. alkaline phosphatase, type I collagen, osteopontin, and osteocalcin), and extracellular mineralization. Overall, these results support the adjuvant potentiality of NS1 for mRNA-based regenerative therapies. Statement of SignificancemRNA therapy has the potential to improve the efficiency of nucleic acid based regenerative medicine. Up to now, the incorporation of expensive modified nucleotides is a common way to avoid IVT mRNA-induced detrimental immunogenicity. We here introduce co-delivery of Influenza virus immune evasion protein-NS1 coding mRNA as a strategy to suppress RNA sensors for maximizing IVT mRNA expression. An increased osteogenic commitment of pluripotent stem cells was observed after BMP2 mRNA and NS1 mRNA delivery. This study revealed how applying non-modified mRNA with NS1 could be a promising alternative as a therapeutic in bone regeneration.

Optimisation of cell and ex vivo culture conditions to study vascular calcification.

Medial vascular calcification (MVC) is a highly prevalent disease associated with a high risk of severe, potentially lethal, complications. While animal studies may not systematically be circumvented, in vitro systems have been proven useful to study disease physiopathology. In the context of MVC, the absence of a clinically relevant standardized in vitro method prevents the appropriate comparison and overall interpretation of results originating from different experiments. The aim of our study is to establish in vitro models mimicking in vivo vascular calcification and to select the best methods to unravel the mechanisms involved in MVC. Human aortic smooth muscle cells and rat aortic rings were cultured in different conditions. The influence of fetal calf serum (FCS), alkaline phosphatase, phosphate and calcium concentrations in the medium were evaluated. We identified culture conditions, including the herein reported Aorta Calcifying Medium (ACM), which allowed a reproducible and specific medial calcification of aortic explants. Studying cells and aortic explants cultured, the involvement of bone morphogenetic protein 2 (BMP2) pathway, fibrosis and apoptosis processes in in vitro MVC were demonstrated. Expression of osteoblastic markers was also observed suggesting the occurrence of transdifferentiation of smooth muscle cells to osteoblasts in our models. The use of these models will help researchers in the field of vascular calcification to achieve reproducible results and allow result comparison in a more consistent way.

Effect of interleukin-22 on osteogenic differentiation and the osteoclastogenic response of human periodontal ligament fibroblasts in vitro.

Interleukin-22 (IL-22) exerts extensive biological effects, playing both protective and pathological roles in autoimmune and infectious diseases. However, the specific role and mechanism of IL-22 in the pathogenesis of periodontitis have not been clarified. The aim of this study was to analyze the possible roles of IL-22 in the osteoclastogenesis and osteogenesis of periodontitis. Human periodontal ligament fibroblasts (hPDLFs) were treated with IL-22 and/or lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS), and the mRNA and protein expression of RANKL and OPG were measured by qRT-PCR and Western blotting, respectively. Western blotting was also used to examine the phosphorylated and total protein expression of MAPK signaling molecules. The role of the MAPK pathway in osteoclastogenesis marker expression was further confirmed by inhibition assays. For osteogenic assays, the mRNA expression of osteoblastic markers was quantified by qRT-PCR, the alkaline phosphatase (ALP) activity of hPDLFs was measured by an ALP assay, and the mineralized nodules formed by hPDLFs were determined by Alizarin Red S staining. IL-22 promoted the expression of RANKL in hPDLFs via the MAPK signaling pathway and further upregulated RANKL expression together with Pg-LPS via the p38 MAPK pathway. IL-22 could enhance the ALP activity and mineralized nodule formation of hPDLFs in the early period of osteogenic induction, while exhibiting no profound effect on the expression of osteoblastic markers. IL-22 plays regulatory roles in bone homeostasis, and it is likely to contribute to osteoclastogenesis as a proinflammatory cytokine in the pathogenesis of periodontitis.

Cell immaturity and white/beige adipocyte potential of primary human adipose-derived stromal cells are restrained by culture-medium TGFβ1.

Human adipose-derived stem/stromal cells (hASCs) can differentiate into specialized cell types and thereby contribute to tissue regeneration. As such, hASCs have drawn increasing attention in cell therapy and regenerative medicine, not to mention the ease to isolate them from donors. Culture conditions are critical for expanding hASCs while maintaining optimal therapeutic capabilities. Here, we identified a role for transforming growth factor β1 (TGFβ1) in culture medium in influencing the fate of hASCs during in vitro cell expansion. Human ASCs obtained after expansion in standard culture medium (Standard-hASCs) and in endothelial cell growth medium 2 (EGM2-hASCs) were characterized by high-throughput transcriptional studies, gene set enrichment analysis and functional properties. EGM2-hASCs exhibited enhanced multipotency capabilities and an immature phenotype compared with Standard-hASCs. Moreover, the adipogenic potential of EGM2-hASCs was enhanced, including toward beige adipogenesis, compared with Standard-hASCs. In these conditions, TGFβ1 acts as a critical factor affecting the immaturity and multipotency of Standard-hASCs, as suggested by small mother of decapentaplegic homolog 3 (SMAD3) nuclear localization and phosphorylation in Standard-hASCs vs EGM2-hASCs. Finally, the typical priming of Standard-hASCs into osteoblast, chondroblast, and vascular smooth muscle cell (VSMC) lineages was counteracted by pharmacological inhibition of the TGFβ1 receptor, which allowed retention of SMAD3 into the cytoplasm and a decrease in expression of osteoblast and VSMC lineage markers. Overall, the TGFβ1 pathway appears critical in influencing the commitment of hASCs toward osteoblast, chondroblast, and VSMC lineages, thus reducing their adipogenic potential. These effects can be counteracted by using EGM2 culture medium or chemical inhibition of the TGFβ1 pathway.

Resveratrol promotes osteogenesis via activating SIRT1/FoxO1 pathway in osteoporosis mice

AimsThis study aimed to investigate the effects of resveratrol (3, 4′, 5-trihydroxystilbene, RES) on osteoporosis and the role of SIRT1/FoxO1 pathway in the process. Main methodsIn vivo, mice were divided into 3 groups, Sham, ovariectomized (OVX) and OVX-RES group. Micro-CT, histology and histomorphometry were conducted to detect details of bone mass and microstructure. The expression of osteoblast markers was tested by Real-time qPCR and serum markers which reflected bone formation and resorption were analyzed by enzyme-linked immunosorbent assay (ELISA). Besides, we assayed sirtuin 1 (SIRT1) expression and the concentration of serum superoxide dismutase (SOD). In vitro, osteoblasts were seperated into 3 groups: control, H2O2 (hydrogen peroxide, H2O2) and H2O2-RES group. Cell proliferation, differentiation and apoptosis were detected. In addition, we tested intracellular reactive oxygen species (ROS) formation and SOD activity detection of osteoblasts. The SIRT1, acetylated FoxO1 (Ac-FoxO1) and nuclear FoxO1 (Nu-FoxO1) expression were detected by western blot. Key findingsResults revealed that RES could ameliorate bone loss and promote osteogenesis by reinforcing resistance of oxidative stress in OVX mice. RES enhanced proliferation, differentiation and suppressed apoptosis of H2O2-treated osteoblasts. In this process, SIRT1 was upregulated and the level of Nu-FoxO1, which had high transcriptional activity to regulate redox balance, significantly increased. SignificanceOxidative stress plays a crucial role in osteoporosis. RES can reinforce resistance to oxidative damage and hence promote osteogenesis via the activation of SIRT1/FoxO1 signaling pathway, which provides a new idea for the prevention and treatment of osteoporosis.

Neuromedin U (NMU) regulates osteoblast differentiation and activity

Osteoporosis is a disease of low bone mass that places individuals at enhanced risk for fracture, disability, and death. Osteoporosis rates are expected to rise significantly in the coming decades yet there are limited pharmacological treatment options, particularly for long-term management of this chronic condition. The drug development pipeline is relatively bereft of new strategies, causing an urgent and unmet need for developing new strategies and targets for treating osteoporosis. Here, we examine a lesser-studied bone remodeling pathway, Neuromedin U (NMU), which is expressed in the bone microenvironment along with its cognate receptors NMU receptor 1 (NMUR1) and 2 (NMUR2). We independently corroborate a prior report that global loss of NMU expression leads to high bone mass and test the hypothesis that NMU negatively regulates osteoblast differentiation. Consistent with this, in vitro studies reveal NMU represses osteoblastic differentiation of osteogenic precursors but, in contrast, promotes osteoblastic marker expression, proliferation and activity of osteoblast-like cells. Phospho-profiling arrays were used to detail differential signaling outcomes that may underlie the opposite responses of these cell types. Collectively, our findings indicate that NMU exerts cell-type-specific responses to regulate osteoblast differentiation and activity.

Tissue-nonspecific alkaline phosphatase promotes the osteogenic differentiation of osteoprogenitor cells

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in the calcification sites of the skeletal tissue. It promotes hydroxyapatite crystal formation by degrading inorganic pyrophosphate (PPi) and increasing inorganic phosphate (Pi) concentration. However, abnormalities in Alpl−/− mouse-derived osteoblasts are poorly understood, and the involvement of TNAP in osteoblast differentiation remains unclear. Therefore, in this study, we aimed to investigate the precise role of TNAP in osteoblast differentiation. TNAP inhibition by levamisole, a reversible TNAP inhibitor, suppressed the expression of osteoblast differentiation marker genes in wild-type osteoblastic cells. Alpl overexpression increased the expression of master osteoblast transcription factor genes runt-related transcription factor 2 (Runx2) and Sp7 and the mature osteoblast and osteocyte marker genes, bone γ-carboxyglutamate protein 2 (Bglap2) and dentin matrix protein 1 (Dmp1), respectively in Alpl-deficient osteoblastic cells. TNAP regulated Runx2 expression, which in turn regulated the expression of all other osteoblast markers, except Dmp1. Dmp1 expression was independent of RUNX2 but was dependent on extracellular Pi concentration in Runx2-deficient osteogenic cells. These results suggest that TNAP functions as an osteogenic differentiation regulator either by regulating Runx2 expression or by controlling extracellular Pi concentration.

Semaphorin 3A-hypoxia inducible factor 1 subunit alpha co-overexpression enhances the osteogenic differentiation of induced pluripotent stem cells-derived mesenchymal stem cells in vitro.

Background:Mesenchymal stem or stromal cells (MSCs) derived from the induced pluripotent stem cells (iPSCs) have uniform biological activity, which makes the clinical application of MSCs in bone repair possible. Culturing the iPSC-MSCs onto osteoconductive materials is a promising tissue engineering-based strategy in bone regeneration. The aim of this work was to evaluate the effects of semaphorin 3A (Sema3A) and hypoxia inducible factor 1 subunit alpha (HIF1α) co-overexpression on the survival and osteogenic differentiation of iPSC-MSCs.Methods:Sema3A and HIF1α were linked together with the three (GGGGS; G, glycine; S, serine) peptide fragment, and their co-expression in iPSC-MSCs was mediated by a lentiviral vector. The fusion protein retained the immune reactivity for both Sema3A and HIF1α as determined with Western blotting. iPSC-MSCs were infected with overexpression lentivirus (oeLenti) as negative control, oeLenti-Sema3A, oeLenti-HIF1α or oeLenti-Sema3A-HIF1α lentiviruses.Results:Sema3A overexpression alone promoted the osteogenic differentiation of iPSC-MSCs (the activity and/or expression of osteoblast markers, such as alkaline phosphatase, osteopontin, and osteocalcin, were upregulated), and suppressed cell survival. The Sema3A-HIF1α fusion protein showed a comparable osteoconductive effect to that of Sema3A without reducing cell survival. We further seeded iPSC-MSCs modified by SemaA-HIF1α overexpression onto hydroxyapatite (HA) scaffolds, and evaluated their growth and differentiation on this three-dimensional material. Additional data indicated that, as compared to iPSC-MSCs cultured in ordinary two-dimensional dishes, cells cultured in HA scaffolds grew (blank vs. HA scaffolds: 0.83 vs. 1.39 for survival) and differentiated better (blank vs. HA scaffolds: 11.29 vs. 16.62 for alkaline phosphatase activity).Conclusion:Modifying iPSC-MSCs with pro-osteogenic (Sema3A) and pro-survival (HIF1α) factors may represent a promising strategy to optimize tissue engineering-based strategy in bone repair.

Lack of the Thyroid Hormone Transporter Mct8 in Osteoblast and Osteoclast Progenitors Increases Trabecular Bone in Male Mice

Background: Bone is an important target of thyroid hormones (THs), which require transport into target cells to exert their actions. Recently, the TH-specific monocarboxylate transporter 8 (Mct8) was reported as a regulator of bone mass in male mice. However, its global deletion leads to high 3,3',5-L-triiodothyronine (T3) serum concentrations that may mask direct effects of Mct8-deficiency on bone. In this study, we assessed the bone cell intrinsic function of Mct8 ex vivo and in vivo using conditional Mct8-knockout lines specifically targeting osteoclast and osteoblast progenitors, as well as mature osteoblasts and osteocytes. Materials and Methods: Twelve-week-old male mice with a global Mct8-deficiency or a conditional Mct8-knockout in osteoclast precursors, osteoprogenitors, or mature osteoblasts/osteocytes were analyzed regarding their bone microarchitecture, turnover, and strength. Furthermore, ex vivo studies were conducted to investigate the role of Mct8 in bone cell differentiation and functionality, as well as TH uptake. Results: Global Mct8-knockout mice demonstrated 1.7-fold higher T3 serum concentrations and trabecular bone loss (-28%) likely due to an increased bone turnover as shown by increased osteoblast (+45%) and osteoclast numbers (+41%). However, cortical bone mineral density was increased. Ex vivo cultures of bone marrow-derived osteoblasts and osteoclasts revealed highest expression of Mct8 in mature bone cells. In addition, Mct8-deficiency resulted in a lower mRNA expression of osteoblast and osteoclast differentiation markers, as well as a reduced mineralization capacity and osteoclast numbers, respectively, indicating a bone cell intrinsic role of Mct8. In fact, conditional Mct8-knockout and inhibition of Mct8 in osteoblasts led to an attenuated T3 uptake ex vivo. In vivo, osteoprogenitor-specific Mct8-knockout enhanced trabecular bone volume (+16%) with osteoblast numbers being increased 3.7 fold. Interestingly, Mct8-deficiency in osteoprogenitors and late osteoblasts/osteocytes both resulted in cortical bone loss. Finally, Mct8-deletion in osteoclast progenitors increased trabecular bone volume (+20%) due to reduced osteoclast numbers (-32%), whereas osteoblast numbers were enhanced (+25%). Conclusions: This study confirms that high systemic T3 in global Mct8-knockout mice masks the direct effect of Mct8. Moreover, it identifies Mct8 as a critical regulator of trabecular vs. cortical bone by regulating T3 uptake and highlights its cell intrinsic role in osteoclast and osteoblast progenitors.

LncRNA TUG1 promotes proliferation and differentiation of osteoblasts by regulating the miR-545-3p/CNR2 axis.

Osteoblast differentiation is an effective way to promote bone formation. Long non-coding RNA taurine upregulated 1 (TUG1) has been identified as a crucial modulator of multiple biological processes. This study was designed to investigate the function of TUG1 in the proliferation and differentiation of osteoblast precursor cells hFOB1.19. In this study, we found that TUG1 promoted hFOB1.19 cell proliferation, while TUG1 knockdown hindered cell proliferation. TUG1 and cannabinoid receptor 2 (CNR2) were upregulated, while miR-545-3p was down-regulated in hFOB1.19 cells undergoing osteoblastic differentiation. TUG1 induced osteoblast differentiation by increasing alkaline phosphatase (ALP) activity and the expression of osteoblastic differentiation markers. TUG1 was a sponge of miR-545-3p and regulated osteoblastic differentiation by modulating miR-545-3p. Moreover, miR-545-3p directly targeted CNR2 and restored the effect of CNR2 on osteoblastic differentiation. In conclusion, TUG1 accelerated the proliferation and differentiation of osteoblasts by sponging miR-545-3p and increasing CNR2 expression, which might provide a new biomarker for bone diseases.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies.

Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS).Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively.ResultsFAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces.Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

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Application of messenger RNA (mRNA) for bone regeneration is a promising alternative to DNA, recombinant proteins and peptides. However, exogenous in vitro transcribed mRNA (IVT mRNA) triggers innate immune response resulting in mRNA degradation and translation inhibition. Inspired by the ability of viral immune evasion proteins to inhibit host cell responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A virus (A/Texas/36/1991) as an IVT mRNA enhancer. We evidenced a dose-dependent blocking of RNA sensors by NS1 expression. The co-delivery of NS1 mRNA with mRNA of reporter genes significantly increased the translation efficiency. Interestingly, unlike the use of nucleosides modification, NS1-mediated mRNA translation enhancement does not dependent to cell type. Dual delivery of NS1 mRNA and BMP-2 mRNA to murine pluripotent stem cells (C3H10T1/2), promoted osteogenic differentiation evidenced by enhanced expression of osteoblastic markers (e.g. alkaline phosphatase, type I collagen, osteopontin, and osteocalcin), and extracellular mineralization. Overall, these results support the adjuvant potentiality of NS1 for mRNA-based regenerative therapies.mRNA therapy has the potential to improve the efficiency of nucleic acid based regenerative medicine. Up to now, the incorporation of expensive modified nucleotides is a common way to avoid IVT mRNA-induced detrimental immunogenicity. We here introduce co-delivery of Influenza virus immune evasion protein-NS1 coding mRNA as a strategy to suppress RNA sensors for maximizing IVT mRNA expression. An increased osteogenic commitment of pluripotent stem cells was observed after BMP2 mRNA and NS1 mRNA delivery. This study revealed how applying non-modified mRNA with NS1 could be a promising alternative as a therapeutic in bone regeneration.