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Frontiers in Bioscience (Landmark... Oct 2022Recently, single-cell RNA sequencing (scRNA-seq) technology was increasingly used to study transcriptomics at a single-cell resolution, scRNA-seq analysis was...
BACKGROUND
Recently, single-cell RNA sequencing (scRNA-seq) technology was increasingly used to study transcriptomics at a single-cell resolution, scRNA-seq analysis was complicated by the "dropout", where the data only captures a small fraction of the transcriptome. This phenomenon can lead to the fact that the actual expressed transcript may not be detected. We previously performed osteoblast subtypes classification and dissection on freshly isolated human osteoblasts.
MATERIALS AND METHODS
Here, we used the scImpute method to impute the missing values of dropout genes from a scRNA-seq dataset generated on freshly isolated human osteoblasts.
RESULTS
Based on the imputed gene expression patterns, we discovered three new osteoblast subtypes. Specifically, these newfound osteoblast subtypes are osteoblast progenitors, and two undetermined osteoblasts. Osteoblast progenitors showed significantly high expression of proliferation related genes ( and ). Analysis of each subtype showed that in addition to bone formation, these undetermined osteoblasts may involve osteoclast and adipocyte differentiation and have the potential function of regulate immune activation.
CONCLUSIONS
Our findings provided a new perspective for studying the osteoblast heterogeneity and potential biological functions of these freshly isolated human osteoblasts at the single-cell level, which provides further insight into osteoblasts subtypes under various (pathological) physiological conditions.
Topics: Humans; RNA-Seq; Osteoblasts; Transcriptome; Cell Differentiation; Osteogenesis; Gene Expression Profiling
PubMed: 36336853
DOI: 10.31083/j.fbl2710295 -
Archivum Immunologiae Et Therapiae... Oct 2014Vascular endothelial growth factor-A (VEGF) is a key regulator of physiological hemangiogenesis during development, postnatal growth, and homeostasis. It is well known... (Review)
Review
Vascular endothelial growth factor-A (VEGF) is a key regulator of physiological hemangiogenesis during development, postnatal growth, and homeostasis. It is well known that VEGF is required for effective coupling of angiogenesis to endochondral and membranous bone formation during skeletal development. However, less well known are the roles of VEGF in regulating the differentiation and/or functions of skeletal cells such as chondrocytes, osteoblasts, and osteoclasts. In this review, we discuss some of these functions. During early skeletal development, VEGF is important for the survival of chondrocytes in the hypoxic regions of the cartilage models of future bones, the vascularization of developing bones and proliferation and differentiation of osteoblastic cells. Postnatally, osteoblast-derived VEGF is critical for maintaining bone homeostasis by stimulating the differentiation of mesenchymal stem cells to osteoblasts and repressing their differentiation to adipocytes. Recent data indicate that these effects of VEGF on osteogenic/adipogenic stem cell fates are based on an intracellular (intracrine) mechanism. In contrast, osteoblast-derived VEGF is also known to stimulate the differentiation of monocytes to osteoclasts by a paracrine mechanism. Mice with VEGF-deficient osteoblastic lineage cells exhibit age-dependent loss of bone mass and an increase in bone marrow fat. These changes are similar to the changes associated with osteoporosis in humans. Thus, a better understanding of the intracellular mechanisms by which VEGF regulates osteoblastic and adipogenic differentiation may lead to the identification of new targets for therapies to prevent osteoporotic bone loss.
Topics: Animals; Cell Differentiation; Chondrocytes; Homeostasis; Humans; Mesenchymal Stem Cells; Mice; Neovascularization, Physiologic; Osteoblasts; Osteoclasts; Osteogenesis; Vascular Endothelial Growth Factor A
PubMed: 24699630
DOI: 10.1007/s00005-014-0285-y -
Physiological Research Jul 2021The objective of this study was to evaluate and compare titanium surfaces: machined (MA); sintered ceramic-blasted (HAS); sintered ceramic-blasted and acid-etched (HAS...
The objective of this study was to evaluate and compare titanium surfaces: machined (MA); sintered ceramic-blasted (HAS); sintered ceramic-blasted and acid-etched (HAS DE) and to determine the effects of surface topography, roughness and chemical composition on human osteoblast cell reaction. Titanium surface samples were analyzed with respect to surface chemical composition, topography, and roughness. The effects of material surface characteristics on osteoblasts was examined by analyzing osteoblast morphology, viability and differentiation. Osteoblasts cultured on these materials had attached, spread and proliferated on every sample. The viability of osteoblasts cultured on HAS and HAS DE samples increased more intensively in time comparing to MA sample. The viability of osteoblast cultured on HAS samples increased more intensively in the early phases of culture while for cells cultured on HAS DE the cells viability increased later in time. Alkaline phosphate activity was the highest for the cells cultured on HAS sample and statistically higher than for the MA sample. The least activity occurred on the smooth MA sample along with the rougher HAS DE samples. All the examined samples were found to be biocompatible, as indicated by cell attachment, proliferation, and differentiation. Titanium surfaces modification improved the dynamics of osteoblast viability increase. Osteoblast differentiation was found to be affected by the etching procedure and presence of Ca and P on the surface.
Topics: Alkaline Phosphatase; Calcium; Cell Differentiation; Cell Line; Cell Proliferation; Cell Survival; Cells, Cultured; Ceramics; Humans; Osteoblasts; Osteogenesis; Phosphorus; Surface Properties; Titanium
PubMed: 33982574
DOI: 10.33549/physiolres.934582 -
Bone May 2020Non-traditional bisphosphonates, that is, bisphosphonates that do not inhibit osteoclast viability or function, were initially reported in the 1990s by Socrates...
Non-traditional bisphosphonates, that is, bisphosphonates that do not inhibit osteoclast viability or function, were initially reported in the 1990s by Socrates Papapoulos' group. Originally designed to study the role of the R1 residue of aminobisphosphonates on bisphosphonate affinity for hydroxyapatite, these modified bisphosphonates retained similar affinity for mineralized bone as their parent compounds, but they lacked the potential to inhibit the mevalonate pathway or bone resorption. We found that, similar to classical bisphosphonates, these non-traditional compounds prevented osteoblast and osteocyte apoptosis in vitro through a pathway that requires the expression of the gap junction protein connexin 43, and the activation of the Src/MEK/ERK signaling pathway. Furthermore, one of those compounds named IG9402 (also known as amino-olpadronate or lidadronate), was able to inhibit osteoblast and osteocyte apoptosis, without affecting osteoclast number or bone resorption in vivo in a model of glucocorticoid-induced osteoporosis. IG9402 administration also ameliorated the decrease in bone mass and in bone mechanical properties induced by glucocorticoids. Similarly, IG9402 prevented apoptosis of osteoblastic cells in a model of immobilization due to hindlimb unloading. However, in this case, the bisphosphonate was not able to preserve the bone mass, and only partially prevented the decrease in bone mechanical properties induced by immobilization. The effect of IG9402 administration was also tested in a mouse model of masticatory hypofunction through the induction of masseter muscle atrophy by unilateral injection of botulinum toxin type A (BoNTA). IG9402 partially inhibited the loss of trabecular bone microstructure in the mandibular condyle, but not the decrease in masseter muscle mass induced by BoNTA administration. In summary, these non-traditional bisphosphonates that lack anti-resorptive activity but are able to preserve osteoblast and osteocyte viability could constitute useful tools to study the consequences of preventing apoptosis of osteoblastic cells in animal models. Furthermore, they could be used to treat conditions associated with reduced bone mass and increased bone fragility in which a reduction of bone remodeling is not desirable.
Topics: Animals; Apoptosis; Bone Resorption; Diphosphonates; Mice; Osteoblasts; Osteoclasts; Osteocytes
PubMed: 32112989
DOI: 10.1016/j.bone.2020.115301 -
Nutrients Jul 2016Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by... (Comparative Study)
Comparative Study
Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further, these regulatory actions are independent of Nrf2.
Topics: Animals; Antioxidants; Apoptosis; Avena; Cell Differentiation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression Regulation; Mice, Inbred C57BL; Mice, Knockout; NF-E2-Related Factor 2; Osteoblasts; Osteoclasts; Osteocytes; Osteoprotegerin; RANK Ligand; Signal Transduction; ortho-Aminobenzoates
PubMed: 27409635
DOI: 10.3390/nu8070423 -
Journal of Biomedical Optics Jul 2018The objective of this study was to evaluate the effects of varying light doses on the viability and cellular activity of osteoblasts, osteocytes, and osteoclasts. A...
The objective of this study was to evaluate the effects of varying light doses on the viability and cellular activity of osteoblasts, osteocytes, and osteoclasts. A light application device was developed to apply 940-nm wavelength light from light-emitting diodes on three cultured cells, MC3T3-E1, MLO-A5, and RANKL-treated RAW264.7 cells. The doses (energy density) on cells were 0, 1, 5, and 7.5 J / cm2. The corresponding light power densities at the cell site were 0, 1.67, 8.33, and 12.5 mW / cm2, respectively, and the duration was 10 min. The results showed that the three cell types respond differently to light and their responses were dose dependent. Low-dose treatment (1 J / cm2) enhanced osteoblast proliferation, osteoclast differentiation, and osteoclastic bone resorption activity. Osteocyte proliferation was not affected by both low- and high-dose (5 J / cm2) treatments. While 1 J / cm2 did not affect viability of all three cell types, 5 J / cm2 significantly decreased viability of osteocytes and osteoclasts. Osteoblast viability was negatively impacted by the higher dose (7.5 J / cm2). The findings suggest that optimal doses exist for osteoblast and osteoclast, which can stimulate cell activities, and there is a safe dose range for each type of cell tested.
Topics: Animals; Cell Line; Cell Physiological Phenomena; Low-Level Light Therapy; Mice; Osteoblasts; Osteoclasts; RAW 264.7 Cells
PubMed: 30030913
DOI: 10.1117/1.JBO.23.7.075008 -
Advances in Dental Research Mar 2016Changes in dental implant materials, structural design, and surface properties can all affect biological response. While bulk properties are important for mechanical... (Review)
Review
Changes in dental implant materials, structural design, and surface properties can all affect biological response. While bulk properties are important for mechanical stability of the implant, surface design ultimately contributes to osseointegration. This article reviews the surface parameters of dental implant materials that contribute to improved cell response and osseointegration. In particular, we focus on how surface design affects mesenchymal cell response and differentiation into the osteoblast lineage. Surface roughness has been largely studied at the microscale, but recent studies have highlighted the importance of hierarchical micron/submicron/nanosurface roughness, as well as surface roughness in combination with surface wettability. Integrins are transmembrane receptors that recognize changes in the surface and mediate downstream signaling pathways. Specifically, the noncanonical Wnt5a pathway has been implicated in osteoblastic differentiation of cells on titanium implant surfaces. However, much remains to be elucidated. Only recently have studies been conducted on the differences in biological response to implants based on sex, age, and clinical factors; these all point toward differences that advocate for patient-specific implant design. Finally, challenges in implant surface characterization must be addressed to optimize and compare data across studies. An understanding of both the science and the biology of the materials is crucial for developing novel dental implant materials and surface modifications for improved osseointegration.
Topics: Cell Differentiation; Dental Implants; Dental Materials; Dental Prosthesis Design; Humans; Mesenchymal Stem Cells; Nanotechnology; Osseointegration; Osteoblasts; Signal Transduction; Surface Properties; Wettability; Wnt-5a Protein
PubMed: 26927483
DOI: 10.1177/0022034515624444 -
International Journal of Molecular... Mar 2019Bone remodelling is a tightly-coordinated and lifelong process of replacing old damaged bone with newly-synthesized healthy bone. In the bone remodelling cycle, bone... (Review)
Review
Bone remodelling is a tightly-coordinated and lifelong process of replacing old damaged bone with newly-synthesized healthy bone. In the bone remodelling cycle, bone resorption is coupled with bone formation to maintain the bone volume and microarchitecture. This process is a result of communication between bone cells (osteoclasts, osteoblasts, and osteocytes) with paracrine and endocrine regulators, such as cytokines, reactive oxygen species, growth factors, and hormones. The essential signalling pathways responsible for osteoclastic bone resorption and osteoblastic bone formation include the receptor activator of nuclear factor kappa-B (RANK)/receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG), Wnt/β-catenin, and oxidative stress signalling. The imbalance between bone formation and degradation, in favour of resorption, leads to the occurrence of osteoporosis. Intriguingly, vitamin E has been extensively reported for its anti-osteoporotic properties using various male and female animal models. Thus, understanding the underlying cellular and molecular mechanisms contributing to the skeletal action of vitamin E is vital to promote its use as a potential bone-protecting agent. This review aims to summarize the current evidence elucidating the molecular actions of vitamin E in regulating the bone remodelling cycle.
Topics: Animals; Biomarkers; Bone and Bones; Humans; Osteoblasts; Osteoclasts; Oxidative Stress; Protective Agents; Reactive Oxygen Species; Signal Transduction; Vitamin E
PubMed: 30909398
DOI: 10.3390/ijms20061453 -
Journal of Cellular and Molecular... Feb 2018We recently showed that patients with primary Sjögren Syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of...
We recently showed that patients with primary Sjögren Syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favourable effects on BMD. To study the direct effects of HCQ on human MSC-derived osteoblast activity. Osteoblasts were cultured from human mesenchymal stromal cells (hMSCs). Cultures were treated with different HCQ doses (control, 1 and 5 µg/ml). Alkaline phosphatase activity and calcium measurements were performed to evaluate osteoblast differentiation and activity, respectively. Detailed microarray analysis was performed in 5 µg/ml HCQ-treated cells and controls followed by qPCR validation. Additional cultures were performed using the cholesterol synthesis inhibitor simvastatin (SIM) to evaluate a potential mechanism of action. We showed that HCQ inhibits both MSC-derived osteoblast differentiation and mineralization in vitro. Microarray analysis and additional PCR validation revealed a highly significant up-regulation of the cholesterol biosynthesis, lysosomal and extracellular matrix pathways in the 5 µg/ml HCQ-treated cells compared to controls. Besides, we demonstrated that 1 µM SIM also decreases MSC-derived osteoblast differentiation and mineralization compared to controls. It appears that the positive effect of HCQ on BMD cannot be explained by a stimulating effect on the MSC-derived osteoblast. The discrepancy between high BMD and decreased MSC-derived osteoblast function due to HCQ treatment might be caused by systemic factors that stimulate bone formation and/or local factors that reduce bone resorption, which is lacking in cell cultures.
Topics: Calcification, Physiologic; Cell Differentiation; Cells, Cultured; Gene Expression Regulation; Gene Ontology; Humans; Hydroxychloroquine; Mesenchymal Stem Cells; Osteoblasts; Reproducibility of Results; Simvastatin
PubMed: 28975700
DOI: 10.1111/jcmm.13373 -
European Review For Medical and... Jun 2019Long non-coding ribonucleic acids (lncRNAs) play a vital role in bone development, but the function of lncRNA taurine up-regulated gene 1 (TUG1) in osteoblast...
OBJECTIVE
Long non-coding ribonucleic acids (lncRNAs) play a vital role in bone development, but the function of lncRNA taurine up-regulated gene 1 (TUG1) in osteoblast proliferation and differentiation is still unknown.
MATERIALS AND METHODS
The expression of TUG1 and the messenger RNA (mRNA) expressions of the Wnt/β-catenin signaling pathway markers [Runt-related transcription factor 2 (Runx2), Frizzled-2, axis inhibition protein 2 (Axin 2) and β-catenin] at 0 d, 1 d, 7 d and 14 d after in vitro culture of osteoblasts were detected, respectively, by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The effects of TUG1 on the Wnt/β-catenin signaling pathway markers and osteoblast proliferation and differentiation were studied through the silencing of TUG1 by short hairpin TUG1 (shTUG1). Furthermore, the effects of the Wnt/β-catenin signal on osteoblast proliferation and differentiation was verified by Wnt/β-catenin signal inhibitors.
RESULTS
With the continuous differentiation of osteoblasts, the level of TUG1 was significantly increased. The mRNA levels of the Wnt/β-catenin signaling pathway markers (Runx2, Frizzled-2, Axin 2 and β-catenin) also showed the same increasing trend. ShTUG1 notably reduced the activity of alkaline phosphatases (ALPs), the levels of osteocalcin and osteopontin and osteoblast proliferation activity. In addition, the silencing of TUG1 by shTUG1 resulted in significant reductions in the proteins of the Wnt/β-catenin signaling pathway markers (Runx2, Frizzled-2, Axin 2 and β-catenin), and Wnt/β-catenin inhibitors markedly reduced osteoblast proliferation and differentiation activity.
CONCLUSIONS
LncRNA TUG1 inhibition can suppress the Wnt/β-catenin signaling pathway and reduce osteoblast proliferation and differentiation.
Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Osteoblasts; Osteogenesis; Phosphorylation; Primary Cell Culture; RNA, Long Noncoding; Rats; Up-Regulation; Wnt Signaling Pathway
PubMed: 31210284
DOI: 10.26355/eurrev_201906_18035