Osteoblast Marker Genes Research Articles

Modulation of osteoblast differentiation and function by the P2X4 receptor

Bone cells are known to express multiple P2 receptor subtypes, and the functional effects of receptor activation have been described for many of these. One exception is the P2X4 receptor, which despite strong expression in osteoblasts and osteoclasts, has no defined functional activity. This study used the selective P2X4 receptor antagonists, 5-BDBD and PSB-12062, to investigate the role of this receptor in bone. Both antagonists (≥ 0.1 μM) dose-dependently decreased bone formation by 60–100%. This was accompanied by a ≤ 70% decrease in alkaline phosphatase activity, a ≤ 40% reduction in cell number, and a ≤ 80% increase in the number of adipocytes present in the culture. The analysis of gene expression showed that levels of osteoblast marker genes (e.g. Alpl, Bglap) were decreased in 5-BDBD treated cells. Conversely, expression of the adipogenic transcription factor PPARG was increased 10-fold. In osteoclasts, high doses of both antagonists were associated with a reduction in osteoclast formation and resorptive activity by ≤ 95% and ≤ 90%, respectively. Taken together, these data suggest that the P2X4 receptor plays a role in modulating bone cell function. In particular, it appears to influence osteoblast differentiation favouring the osteogenic lineage over the adipogenic lineage.

A Highly Selective GSK-3β Inhibitor CHIR99021 Promotes Osteogenesis by Activating Canonical and Autophagy-Mediated Wnt Signaling.

The discovery and application of small molecules is one of the practical strategies of safe osteogenic drugs. The small molecule CHIR99021 (C91) is a highly specific, safe, and most effective GSK-3β Inhibitor. This study found that it efficiently activates the canonical Wnt signaling of bone marrow stromal cell ST2 and promotes osteoblast differentiation and mineralization. C91 increases the production and biochemical activity of osteoblast marker alkaline phosphatase, the expression of osteoblast marker genes Alpl, Bglap, Runx2, and Sp7, and the formation of bone nodules. Triptonide is a transcription inhibitor of Wnt target gene, which diminishes C91-induced osteoblast differentiation in a dose-dependent manner. Meanwhile, C91 also induces autophagy through autophagosome formation and conversion of autophagy biomarker LC-3I into LC-3II. Autophagy inhibitor 3MA partially reduces C91-induced osteoblast differentiation and mineralization; autophagy inducer Rapamycin increases the expression of β-catenin to promote osteogenic differentiation, but cannot alleviate the inhibition of Triptonide on C91-induced osteogenic differentiation, indicating the crosstalk of canonical Wnt signaling and autophagy regulates C91-induced osteoblast differentiation. Furthermore, in order to simulate the in vivo detection of C91 in osteogenesis process, we made a C91 slow-release hydrogel with our newly established polycaprolactone and cell-integrated 3D printing system (PCCI3D module). The sustained release C91 promotes the differentiation and mineralization of ST2 cells. C91 can also enhance the proliferative activity of ST2 cells. The release rate of C91 from hydrogel gradually decreases within 7 days. During this period, the C91 is released by 83.0% and the cell viability maintained at 96.4%. Therefore, the small molecule Wnt agonist C91 promotes osteogenesis through caonical and autophagy-mediated Wnt signaling pathway with an option for translational application.

Enhancement of critical-sized bone defect regeneration using UiO-66 nanomaterial in rabbit femurs

BackgroundRepair of large-sized bone defects is a challengeable obstacle in orthopedics and evoked the demand for the development of biomaterials that could induce bone repair in such defects. Recently, UiO-66 has emerged as an attractive metal–organic framework (MOF) nanostructure that is incorporated in biomedical applications due to its biocompatibility, porosity, and stability. In addition, its osteogenic properties have earned a great interest as a promising field of research. Thus, the UiO-66 was prepared in this study and assessed for its potential to stimulate and support osteogenesis in vitro and in vivo in a rabbit femoral condyle defect model. The nanomaterial was fabricated and characterized using x-ray diffraction (XRD) and transmission electron microscopy (TEM). Afterward, in vitro cytotoxicity and hemolysis assays were performed to investigate UiO-66 biocompatibility. Furthermore, the material in vitro capability to upregulate osteoblast marker genes was assessed using qPCR. Next, the in vivo new bone formation potential of the UiO-66 nanomaterial was evaluated after induction of bone defects in rabbit femoral condyles. These defects were left empty or filled with UiO-66 nanomaterial and monitored at weeks 4, 8, and 12 after bone defect induction using x-ray, computed tomography (CT), histological examinations, and qPCR analysis of osteocalcin (OC) and osteopontin (OP) expressions.ResultsThe designed UiO-66 nanomaterial showed excellent cytocompatibility and hemocompatibility and stimulated the in vitro osteoblast functions. The in vivo osteogenesis was enhanced in the UiO-66 treated group compared to the control group, whereas evidence of healing of the treated bone defects was observed grossly and histologically. Interestingly, UiO-66 implanted defects displayed a significant osteoid tissue and collagen deposition compared to control defects. Moreover, the UiO-66 nanomaterial demonstrated the potential to upregulate OC and OP in vivo.ConclusionsThe UiO-66 nanomaterial implantation possesses a stimulatory impact on the healing process of critical-sized bone defects indicating that UiO-66 is a promising biomaterial for application in bone tissue engineering.

DKK-1 Is Underexpressed in Mesenchymal Stem Cells from Patients with Ankylosing Spondylitis and Further Downregulated by IL-17

Dickkopf-1 (Dkk-1) is a key regulator of bone remodeling in spondyloarthropathies. Nevertheless, data regarding its expression in cells of pathophysiologic relevance, such as mesenchymal stem cells (MSCs), are lacking. Herein, we aimed to address DKK1 gene expression and Wnt pathway activation in MSCs from patients with ankylosing spondylitis (AS) and explore the effect of IL-17 on MSCs with respect to DKK-1 expression and Wnt pathway activation. Primary MSCs were isolated from the bone marrow of the femoral head of two patients with AS and two healthy controls undergoing orthopedic surgery. MSCs were cultured for 7 days in expansion medium and for 21 days in osteogenic medium in the presence or absence of IL-17A. Gene expression of DKK-1 and osteoblastic markers was determined by RT-PCR. Alkaline phosphatase activity, alizarin red and Van Kossa staining were used to assess osteoblastic function and mineralization capacity. DKK-1 was significantly downregulated in MSCs and osteoblasts from patients with AS compared to controls. Moreover, MSCs and osteoblasts from AS patients displayed increased Wnt pathway activation and enhanced osteoblastic activity, as indicated by increased expression of osteoblast marker genes and alkaline phosphatase activity. IL-17 downregulated DKK-1 expression and increased osteoblastic activity and mineralization capacity. DKK-1 is underexpressed in MSCs from AS patients compared to controls, whereas IL-17 has an inhibitory effect on DKK-1 expression and stimulates osteoblastic function. These data may have pathogenetic and clinical implications in AS.

Evaluation on antiosteoporosis of collagen peptides prepared by immobilized protease with eggshell membrane.

Collagen peptides are a potential treatment for osteoporosis due to their antiosteoporosis activity. In this study, we prepared immobilized protease with eggshell membrane as carrier, and then hydrolyzed collagen to obtain collagen peptide. The antiosteoporosis of collagen peptides was confirmed by hBMSC osteogenic differentiation and bone mineralization improvement results. Surprisingly, antiosteoporosis of collagen peptides was related to the molecular weight of collagen peptides. This was derived from the osteoblast marker gene expressions, and mineral elements in P1 treatment were higher than those in P3 treatment. Consequently, these results confirmed that antiosteoporosis of low molecular weight collagen peptides is higher than that of higher molecular weight collagen peptides. Furthermore, the antiosteoporosis activity of P1 was due to its peptide sequences with known antiosteoporosis activity in P1. PRACTICAL APPLICATION: Using eggshell membrane as carrier to prepare immobilized protease was meaningful for solving the problem of resource waste. In addition, the results showed that collagen peptides possessed antiosteoporosis, and the effect of low molecular weight collagen peptides was better. This study provides a theoretical basis for developing high antiosteoporosis collagen peptides able to treat osteoporosis.

PCL nanofibrous incorporating unique matrix fusion protein adsorbed mesoporous bioactive glass for bone tissue engineering

Mesoporous bioactive glass (MBG) is a potential biomedical material in bone defect repairment because of its bioactivity, biocompatibility, and osteoinduction properties. Here we report that Mg-doped MBG scaffold with 3:1 Ca/Mg ratio (MBG-Ca/Mg-3) is good for MC3T3-E1 osteoblast differentiation and mineralization. Mimicking bone extracellular matrix structure by electrospinning, we used MBG-Ca/Mg-3 adsorbed with Osteocalcin-Osteopontin-Biglycan (OOB), a new unique matrix fusion protein, to form OOB@MBG-Ca/Mg-3 scaffold, which has multifunctional ability in calvarial bone defect repairment in vivo. Intriguingly, we found that OOB@MBG-Ca/Mg-3 scaffold increases the expression of osteoblastic marker genes, including bone morphogenetic protein (Bmp2), osteopontin (Opn), Osterix, Runx2 through activation of ERK1/2. We concluded that OOB@MBG-Ca/Mg-3 scaffold promotes osteoblast differentiation and mineralization through ERK1/2 pathway and it can also enhance bone formation in vivo, which provides a new biomaterial in bone tissue engineering.

Titanium with nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by regulating histone methylation

The bone remodeling process is crucial for titanium (Ti) osseointegration and involves the crosstalk between osteoclasts and osteoblasts. Considering the high osteogenic potential of Ti with nanotopography (Ti Nano) and that osteoclasts inhibit osteoblast differentiation, we hypothesized that nanotopography attenuate the osteoclast-induced disruption of osteoblast differentiation. Osteoblasts were co-cultured with osteoclasts on Ti Nano and Ti Control and non-co-cultured osteoblasts were used as control. Gene expression analysis using RNAseq showed that osteoclasts downregulated the expression of osteoblast marker genes and upregulated genes related to histone modification and chromatin organization in osteoblasts grown on both Ti surfaces. Osteoclasts also inhibited the mRNA and protein expression of osteoblast markers, and such effect was attenuated by Ti Nano. Also, osteoclasts increased the protein expression of H3K9me2, H3K27me3 and EZH2 in osteoblasts grown on both Ti surfaces. ChIP assay revealed that osteoclasts increased accumulation of H3K27me3 that represses the promoter regions of Runx2 and Alpl in osteoblasts grown on Ti Control, which was reduced by Ti Nano. In conclusion, these data show that despite osteoclast inhibition of osteoblasts grown on both Ti Control and Ti Nano, the nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by preventing the increase of H3K27me3 accumulation that represses the promoter regions of some key osteoblast marker genes. These findings highlight the epigenetic mechanisms triggered by nanotopography to protect osteoblasts from the deleterious effects of osteoclasts, which modulate the process of bone remodeling and may benefit the osseointegration of Ti implants.

Inhibition of TET-mediated DNA demethylation suppresses osteoblast differentiation.

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.

Factors secreted by monosodium urate crystal-stimulated macrophages promote a proinflammatory state in osteoblasts: a potential indirect mechanism of bone erosion in gout

BackgroundTophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts.MethodsConditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay.ResultsIn MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1, Runx2, Sp7, Bglap, Ibsp, and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE2 and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE2 secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs.ConclusionsFactors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.

UPLC-ESI-Q-TOF-MSE-based metabolomics analysis of Acer mono sap and evaluation of osteogenic activity in mouse osteoblast cells.

Investigation of phytochemicals and bioactive molecules is tremendously vital for the applications of new plant resources in chemistry, food, and medicine. In this study, the chemical profiling of sap of Acer mono (SAM), a Korean syrup known for its anti-osteoporosis effect, was performed using UPLC-ESI-Q-TOF-MSE analysis. A total of 23 compounds were identified based on the mass and fragmentation characteristics and most of the compounds have significant biomedical applications. The in vitro antioxidant assessment of SAM indicated excellent activity by scavenging DPPH and ABTS-free radicals and were found to be 23.35 mg mL-1 and 29.33 mg mL-1, respectively, as IC50 concentrations. As well, the in vitro proliferation effect of the SAM was assessed against mouse MC3T3-E1 cells, and the results showed that the SAM enhanced the proliferation of the cells, and 12.5 mg mL-1 and 25 mg mL-1 of SAM were selected for osteogenic differentiation. The morphological analysis clearly evidenced the SAM enhanced the osteogenic activity in MC3T3-E1 cells by the increased deposition of extracellular calcium and nodule formation. Moreover, the qRT-PCR analysis confirmed the increased expression of osteoblast marker gene expression including ALP, osteocalcin, osteopontin, collagen1α1, Runx2, and osterix in SAM-treated MC3T3-E1 cells. Together, these results suggest that SAM possesses osteogenic effects and can be used for bone regeneration and bone loss-associated diseases such as osteoporosis.

Effects of the Lysine Methyltransferase Inhibitor AZ505 on Bone Metabolism.

BackgroundProtein methylation has important role in regulating diverse cellular responses, including differentiation, by affecting protein activity, stability, and interactions. AZ505 is an inhibitor of the SET and MYND domain-containing protein 2 lysine methylase. In this study, we investigated the effect of AZ505 on osteoblast and osteoclast differentiation in vitro and evaluated the effect of AZ505 in vivo on the long bones in mice.MethodsOsteoblast differentiation was assessed by alkaline phosphatase (ALP) and Alizarin red staining after culturing calvarial preosteoblasts in an osteogenic medium. Osteoclast differentiation was analyzed by tartrate-resistant acid phosphatase (TRAP) staining in bone marrow-derived macrophages cultured with macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). For in vivo experiments, mice were intraperitoneally injected with AZ505 and femurs were examined by micro-computed tomography.ResultsAZ505 increased ALP and Alizarin red staining in cultured osteoblasts and the expression of osteoblast marker genes, including Runx2 and osteocalcin. AZ505 resulted in decreased TRAP-staining of osteoclasts and expression of c-Fos and nuclear factor of activated T cells transcription factors and osteoclast marker genes, including cathepsin K and dendritic cell-specific transmembrane protein. Unexpectedly, in vivo administration of AZ505 markedly decreased the trabecular bone mass of femurs. In support of this catabolic result, AZ505 strongly upregulated RANKL expression in osteoblasts.ConclusionsThe results indicate that AZ505 has a catabolic effect on bone metabolism in vivo despite its anabolic effect in bone cell cultures. The findings indicate that cell culture data should be extrapolated cautiously to in vivo outcomes for studying bone metabolism.

Nanog/NFATc1/Osterix signaling pathway-mediated promotion of bone formation at the tendon\u2013bone interface after ACL reconstruction with De-BMSCs transplantation

BackgroundBone formation plays an important role in early tendon–bone healing after anterior cruciate ligament reconstruction (ACLR). Dedifferentiated osteogenic bone marrow mesenchymal stem cells (De-BMSCs) have enhanced osteogenic potential. This study aimed to investigate the effect of De-BMSCs transplantation on the promotion of bone formation at the tendon–bone interface after ACLR and to further explore the molecular mechanism of the enhanced osteogenic potential of De-BMSCs.MethodsBMSCs from the femurs and tibias of New Zealand white rabbits were subjected to osteogenic induction and then cultured in medium without osteogenic factors; the obtained cell population was termed De-BMSCs. De-BMSCs were induced to undergo osteo-, chondro- and adipo-differentiation in vitro to examine the characteristics of primitive stem cells. An ACLR model with a semitendinosus tendon was established in rabbits, and the animals were divided into a control group, BMSCs group, and De-BMSCs group. At 12 weeks after surgery, the rabbits in each group were sacrificed to evaluate tendon–bone healing by histologic staining, micro-computed tomography (micro-CT) examination, and biomechanical testing. During osteogenic differentiation of De-BMSCs, an siRNA targeting nuclear factor of activated T-cells 1 (NFATc1) was used to verify the molecular mechanism of the enhanced osteogenic potential of De-BMSCs.ResultsDe-BMSCs exhibited some properties similar to BMSCs, including multiple differentiation potential and cell surface markers. Bone formation at the tendon–bone interface in the De-BMSCs group was significantly increased, and biomechanical strength was significantly improved. During the osteogenic differentiation of De-BMSCs, the expression of Nanog and NFATc1 was synergistically increased, which promoted the interaction of NFATc1 and Osterix, resulting in increased expression of osteoblast marker genes such as COL1A, OCN, and OPN.ConclusionsDe-BMSCs transplantation could promote bone formation at the tendon–bone interface after ACLR and improve the biomechanical strength of the reconstruction. The Nanog/NFATc1/Osterix signaling pathway mediated the enhanced osteogenic differentiation efficiency of De-BMSCs.

Design of novel functionalized collagen-chitosan-MBG scaffolds for enhancing osteoblast differentiation in BMSCs

Collagen and chitosan are two different kinds of natural biodegradable polymers commonly used in the regeneration of bone defects. Mesoporous bioactive glass (MBG) is a type of favorable bone filler which can effectively constitute an enlarged microenvironment to facilitate an exchange of important factors between the cells and scaffolds. Here we prepared a collagen-chitosan-MBG (C-C-MBG) scaffold which displayed significantly increased proliferation, differentiation and mineralization in bone mesenchymal stem cells (BMSCs). Additionally, we found that the scaffold can stimulate extra-cellular signal regulated kinase 1/2 (Erk1/2) activated Runx2 pathway, which is the predominant signaling pathway involved in osteoblast differentiation. Consistently, we observed that the scaffold can markedly enhance the expression of Type I collagen, Osteopontin (Opn), and Runx2, which are important osteoblastic marker genes implicated in the process of osteoblast differentiation. Therefore, we conclude that the composite scaffold can significantly promote the differentiation of BMSCs into osteoblasts by activating Erk1/2-Runx2 pathway. Our finding thereby implies that the C-C-MBG scaffold can possibly act as a potential biomaterial in the bone regeneration.

MiR-452-3p inhibited osteoblast differentiation by targeting Smad4

Osteoblast differentiation is a complex process that is essential for normal bone formation. A growing number of studies have shown that microRNAs (miRNAs) are key regulators in a variety of physiological and pathological processes, including osteogenesis. In this study, BMP2 was used to induce MC3T3-E1 cells to construct osteoblast differentiation cell model. Then, we investigated the effect of miR-452-3p on osteoblast differentiation and the related molecular mechanism by RT-PCR analysis, Western blot analysis, ALP activity, and Alizarin Red Staining. We found that miR-452-3p was significantly downregulated in osteoblast differentiation. Overexpression miR-452-3p (miR-452-3p mimic) significantly inhibited the expression of osteoblast marker genes RUNX2, osteopontin (OPN), and collagen type 1 a1 chain (Col1A1), and decreased the number of calcium nodules and ALP activity. In contrast, knockdown miR-452-3p (miR-452-3p inhibitor) produced the opposite effect. In terms of mechanism, we found that Smad4 may be the target of miR-452-3p, and knockdown Smad4 (si-Smad4) partially inhibited the osteoblast differentiation enhanced by miR-452-3p. Our results suggested that miR-452-3p plays an important role in osteoblast differentiation by targeting Smad4. Therefore, miR-452-3p is expected to be used in the treatment of bone formation and regeneration.

Milk peptide induced miRNA-369 facilitates bone functions in the rat osteoblasts

The animal’s health and productivity is chiefly influenced by its anatomy and physiology. Moreover, bone growth in animals is also associated to overall health and production. Besides, bone fractures accounts to constrained food intake, reduced immune function and general productivity losses. In poultry animals, bone deformities, renders severe costs in the meat production and egg quality. The treatment for various bone related issues is both time and capital seeking. Besides, currently available therapies, i.e. bisphosphonates, raloxifene are associated with various side effects like jaw osteonecrosis, vaginal bleeding etc. The present study reports the bone promoting effect in milk peptide induced miRNA, miR369. Findings represents an enhanced bone proliferation and differentiation under the influence of miR369 in in vitro rat osteoblasts culture. The bone marker enzyme alkaline phosphatase and osteoblast specific protein expression were tested using ELISA. miR369 significantly stimulated the marker protein levels. Additionally, miR369 when injected to neonatal rat pups, showed improved osteoblast marker gene(s) expression. The study demonstrates the positive role of miR369 in the bone functions and hence, advocates its functional utility for the management of various bone ailments prevailing in animals and/or humans. Besides miR369, a putative molecular gene therapeutic agent can prove revolutionary in controlling the animal health losses ascribed to deteriorating bone health.

Identification of candidate regulators of mandibular bone loss in Fc\u03b3RIIB-/- Mice

Patients with systemic lupus erythematosus (SLE) have increased inflammatory cytokines, leading to periodontitis and alveolar bone loss. However, the mechanisms driving this phenomenon are still unknown. Here, we have identified novel therapeutic targets for and mediators of lupus-mediated bone loss using RNA-sequencing (RNA-seq) in a FcγRIIB-/- mouse model of lupus associated osteopenia. A total of 2,710 upregulated and 3,252 downregulated DEGs were identified. The GO and KEGG annotations revealed that osteoclast differentiation, bone mineralization, ossification, and myeloid cell development were downregulated. WikiPathways indicated that Hedgehog, TNFα NF-κB and Notch signaling pathway were also decreased. We identified downregulated targets, Sufu and Serpina12, that have important roles in bone homeostasis. Sufu and Serpina12 were related to Hedgehog signaling proteins, including Gli1, Gli2, Gli3, Ptch1, and Ptch2. Gene knockdown analysis demonstrated that Sufu, and Serpina12 contributed to osteoclastogenesis and osteoblastogenesis, respectively. Osteoclast and osteoblast marker genes were significantly decreased in Sufu-deficient and Serpina12-deficient cells, respectively. Our results suggest that alterations in Hedgehog signaling play an important role in the pathogenesis of osteopenia in FcγRIIB-/- mice. The novel DEGs and pathways identified in this study provide new insight into the underlying mechanisms of mandibular bone loss during lupus development.

Four and a Half LIM Domains Protein 2 Mediates Bortezomib-Induced Osteogenic Differentiation of Mesenchymal Stem Cells in Multiple Myeloma Through p53 Signaling and β-Catenin Nuclear Enrichment.

Myeloma bone disease (MBD), caused by the inhibition of osteoblast activity and the activation of osteoclast in the bone marrow environment, is the most frequent and life-threatening complication in multiple myeloma (MM) patients. Bortezomib (Bzb) was shown to promote MM-derived mesenchymal stem cells (MM-MSCs) differentiation to osteoblast in vitro and in animal models, promoting the bone formation and regeneration, may be mediated via β-catenin/T-cell factor (TCF) pathway. Further defining molecular mechanism of Bzb-enhanced bone formation in MM will be beneficial for the treatment of myeloma patients. The present study has identified for the first time four and a half LIM domains protein 2 (FHL2), a tissue-specific coregulator that interacts with many osteogenic marker molecules, as a therapeutic target to ameliorate MM bone disease. First, increased messenger RNA (mRNA) and protein levels of FHL2, and the mRNA level of main osteoblast markers (including Runx2, ALP, and Col1A1), were found in MM-patients-derived MSCs after Bzb treatment. FHL2 KD with short hairpin RNA (shRNA) reduced the expression of osteoblast marker genes and blocked the osteogenic differentiation of MM-MSCs regardless of the presence or absence of Bzb, implying that FHL2 is an important activator of the osteogenic differentiation of human MSCs under a proteasome inhibition condition. Molecular analysis showed that the enhanced expression of FHL2 was associated with the Bzb-induced upregulation of p53. No significant change at protein level of total β-catenin was observed with or without Bzb treatment. However, it was mostly enriched to nuclei in MSCs after Bzb treatment. Moreover, β-catenin was restricted to the perinuclear region in FHL2 KD cells. These data provide evidence that FHL2 is essential for promoting β-catenin nuclear enrichment in MM-MSCs. In conclusion, FHL2 is critical for Bzb-induced osteoblast differentiation of MM-MSCs and promotes the osteogenesis, through p53 signaling and β-catenin activation. Targeting FHL2 in MM may provide a new therapeutic strategy for treating MBD.

Epo/EpoR signaling in osteoprogenitor cells is essential for bone homeostasis and Epo-induced bone loss

High erythropoietin (Epo) levels are detrimental to bone health in adult organisms. Adult mice receiving high doses of Epo lose bone mass due to suppressed bone formation and increased bone resorption. In humans, high serum Epo levels are linked to fractures in elderly men. Our earlier studies indicated that Epo modulates osteoblast activity; however, direct evidence that Epo acts via its receptor (EpoR) on osteoblasts in vivo is still missing. Here, we created mice lacking EpoR in osteoprogenitor cells to specifically address this gap. Deletion of EpoR in osteoprogenitors (EpoR:Osx-cre, cKO) starting at 5 weeks of age did not alter red blood cell parameters but increased vertebral bone volume by 25% in 12-week-old female mice. This was associated with low bone turnover. Histological (osteoblast number, bone formation rate) and serum (P1NP, osteocalcin) bone formation parameters were all reduced, as were the number of osteoclasts and TRAP serum level. Differentiation of osteoblast precursors isolated from cKO versus control mice resulted in lower expression of osteoblast marker genes including Runx2, Alp, and Col1a1 on day 21, whereas the mineralization capacity was similar. Moreover, the RANKL/OPG ratio, which determines the osteoclast-supporting potential of osteoblasts, was substantially decreased by 50%. Similarly, coculturing cKO osteoblasts with control or cKO osteoclast precursors produced significantly fewer osteoclasts than coculture with control osteoblasts. Finally, exposing female mice to Epo pumps (10 U·d−1) for 4 weeks resulted in trabecular bone loss (−25%) and increased osteoclast numbers (1.7-fold) in control mice only, not in cKO mice. Our data show that EpoR in osteoprogenitors is essential in regulating osteoblast function and osteoblast-mediated osteoclastogenesis via the RANKL/OPG axis. Thus, osteogenic Epo/EpoR signaling controls bone mass maintenance and contributes to Epo-induced bone loss.

Immobilization of bioactive glass ceramics @ 2D and 3D polyamide polymer substrates for bone tissue regeneration

The osteoinductivity of bioactive glass ceramics (BGCs) is dependent on their ability to effectively interact with the surrounding physiological environment and influence the fate of target cells; however, masking and poor dispersion can negatively influence bioactivity. Here, we address this problem via a simple fabrication method of assembling highly ordered and aligned arrays of BGC nanoneedle-like structures (@) on the outer surface of various polyamide 6 (PA6) substrates (casted film, 3D printing filaments, electrospun fibre mats) by ion dissociation. The staggered-like nanoneedles of BGC@PA6 substrates with excellent uniform dispersion showed favourable cell viability, proliferation, and spreading of MC3T3 osteoblast-like cells. Moreover, the immobilized BGC nanoneedles induced osteogenic differentiation and accelerated the expression of late osteoblast marker genes, compared to the control group. Furthermore, the BGC@PA6 composite showed high affinity for bone-like apatite formation when incubated in physiological body fluids. These findings suggest that the unique dispersion of BGCs@PA6 substrates with nanostructure features make them attractive candidates for bone tissue regeneration and open avenues for future investigation into exploiting these properties for bone tissue engineering. Importantly, this work provides a novel concept for in-situ immobilization of BGC with distinctive topographical features onto polymer substrates simulating natural bone structure.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

SUMMARYTeriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.