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1
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Boyde A. Scanning Electron Microscopy and Bone. Methods Mol Biol 2025; 2885:621-670. [PMID: 40448783 DOI: 10.1007/978-1-0716-4306-8_31] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/02/2025]
Abstract
This chapter discusses methods for preparing samples of bone and bone cells for scanning electron microscopy (SEM). Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. Samples may have detail in a 3D surface, or be topography-free, polished or micromilled, resin-embedded block surfaces, or resin casts of space compartments surrounded by bone matrix. Shipping wet bone samples between labs is best done in glycerol. Methods for cells include fixation, drying, looking at undersides of bone cells, and metallic conductive coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralized matrix is described in detail. Attention is given especially to methods for 3D BSE-SEM imaging of bone samples. PMMA is recommended for embedding because it is easily micromilled or polished. Iodine vapor staining of PMMA blocks gives excellent histology for cells and unmineralized matrix with BSE-SEM. Making spatial casts from PMMA or other resin embedded samples is an important use of this material. Correlation with other imaging means, including confocal scanning light microscopy, point projection X-ray microscopy, microradiography and microtomography, is important. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualizing fluorescent mineralizing front labels such as calcein and tetracyclines. Laser ablation microtomy allows preparation of samples as slides for any method of light microscopy combined with SEM. Control of the vacuum pressure in the SEM sample chamber can be used to eliminate "charging" problems without the need to apply a surface conductive coating, making SEM quick and easy to use.
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Affiliation(s)
- Alan Boyde
- DPSU Queen Mary University of London, Mile End Campus, London, UK.
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2
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Fu YF, Shi SW, Wu JJ, Yuan ZD, Wang LS, Nie H, Zhang ZY, Wu X, Chen YC, Ti HB, Zhang KY, Mao D, Ye JX, Li X, Yuan FL. Osteoclast Secretes Stage-Specific Key Molecules for Modulating Osteoclast-Osteoblast Communication. J Cell Physiol 2025; 240:e31484. [PMID: 39606839 DOI: 10.1002/jcp.31484] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2024] [Revised: 10/22/2024] [Accepted: 10/30/2024] [Indexed: 11/29/2024]
Abstract
In most cases of bone metabolic disorders, such as osteoporosis and osteomalacia, these conditions are often attributed to dysfunctional osteoclasts, leading to their common characterization as "destructors." In addition to the widely documented regulatory process where osteoblasts direct osteoclastic bone resorption, there is increasing evidence suggesting that osteoclasts also in turn influence osteoblastic bone formation through direct and indirect mechanisms. It is well-known that differentiation of osteoclasts involves several stages, each characterized by specific cellular features and functions. Stage-specific key molecules secreted during these stages play a critical role in mediating osteoclast-osteoblast communication. In this review, we described the different stages of osteoclast differentiation and reviewed stage-specific key molecules involved in osteoclasts-osteoblasts communication. We highlighted that a detailed understanding of these processes and molecular mechanism could facilitate the development of novel treatments for bone metabolic disorders.
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Affiliation(s)
- Yi-Fei Fu
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Shu-Wen Shi
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Jun-Jie Wu
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
| | - Zheng-Dong Yuan
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
| | - Lei-Sheng Wang
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Hao Nie
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
| | - Zheng-Yu Zhang
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Xian Wu
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Yue-Chun Chen
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Hui-Bo Ti
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Ke-Yue Zhang
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Dong Mao
- Orthopaedic Institute, Wuxi Ninth People's Hospital Affiliated to Soochow University, Wuxi, Jiangsu, China
| | - Jun-Xing Ye
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Xia Li
- Wuxi Medical College, Jiangnan University, Wuxi, China
| | - Feng-Lai Yuan
- Institute of Integrated Chinese and Western Medicine, Affiliated to Jiangnan University, Wuxi, China
- Wuxi Medical College, Jiangnan University, Wuxi, China
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Grass DM, Malek G, Taïeb HM, Ittah E, Richard H, Reznikov N, Laverty S. Characterization and quantification of in-vitro equine bone resorption in 3D using μCT and deep learning-aided feature segmentation. Bone 2024; 185:117131. [PMID: 38777311 DOI: 10.1016/j.bone.2024.117131] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/08/2024] [Revised: 05/18/2024] [Accepted: 05/19/2024] [Indexed: 05/25/2024]
Abstract
High cyclic strains induce formation of microcracks in bone, triggering targeted bone remodeling, which entails osteoclastic resorption. Racehorse bone is an ideal model for studying the effects of high-intensity loading, as it is subject to focal formation of microcracks and subsequent bone resorption. The volume of resorption in vitro is considered a direct indicator of osteoclast activity but indirect 2D measurements are used more often. Our objective was to develop an accurate, high-throughput method to quantify equine osteoclast resorption volume in μCT 3D images. Here, equine osteoclasts were cultured on equine bone slices and imaged with μCT pre- and postculture. Individual resorption events were then isolated and analyzed in 3D. Modal volume, maximum depth, and aspect ratio of resorption events were calculated. A convolutional neural network (CNN U-Net-like) was subsequently trained to identify resorption events on post-culture μCT images alone, without the need for pre-culture imaging, using archival bone slices with known resorption areas and paired CTX-I biomarker levels in culture media. 3D resorption volume measurements strongly correlated with both the CTX-I levels (p < 0.001) and area measurements (p < 0.001). Our 3D analysis shows that the shapes of resorption events form a continuous spectrum, rather than previously reported pit and trench categories. With more extensive resorption, shapes of increasing complexity appear, although simpler resorption cavity morphologies (small, rounded) remain most common, in acord with the left-hand limit paradigm. Finally, we show that 2D measurements of in vitro osteoclastic resorption are a robust and reliable proxy.
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Affiliation(s)
- Debora M Grass
- Comparative Orthopaedic Research Laboratory, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte, St-Hyacinthe, QC J2S 2M2, Canada
| | - Gwladys Malek
- Comparative Orthopaedic Research Laboratory, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte, St-Hyacinthe, QC J2S 2M2, Canada
| | - Hubert M Taïeb
- Department of Bioengineering, Faculty of Engineering, McGill University, 3480 University Street, Montreal, Quebec H3A 0E9, Canada
| | - Eran Ittah
- Department of Bioengineering, Faculty of Engineering, McGill University, 3480 University Street, Montreal, Quebec H3A 0E9, Canada
| | - Hélène Richard
- Comparative Orthopaedic Research Laboratory, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte, St-Hyacinthe, QC J2S 2M2, Canada
| | - Natalie Reznikov
- Department of Bioengineering, Faculty of Engineering, McGill University, 3480 University Street, Montreal, Quebec H3A 0E9, Canada
| | - Sheila Laverty
- Comparative Orthopaedic Research Laboratory, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte, St-Hyacinthe, QC J2S 2M2, Canada.
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4
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Liu Y, Wang W, Zeng Y, Zeng H. Transcriptome analysis of hydrogen inhibits osteoclastogenesis of mouse bone marrow mononuclear cells. Exp Ther Med 2023; 26:436. [PMID: 37614423 PMCID: PMC10443061 DOI: 10.3892/etm.2023.12135] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2022] [Accepted: 09/01/2022] [Indexed: 08/25/2023] Open
Abstract
Hydrogen (H2) is a major biodegradation product of implanted magnesium (Mg) alloys that are commonly used in the healing of bone fractures. Our earlier study showed that H2 can inhibit mouse bone marrow mononuclear cell (BMMC) osteoclastogenesis during the differentiation of these cells into osteoclasts, thereby facilitating fracture healing. However, the way by which H2 inhibits osteoclastogenesis remains to be elucidated. The present study used RNA-sequencing to study the transcriptome of H2-exposed BMMCs in an osteoclast-induced environment and identified the target genes and signaling pathways through which H2 exerts its biological effects. Several upregulated genes were identified: Fos, Dusp1, Cxcl1, Reln, Itga2b, Plin2, Lif, Thbs1, Vegfa and Gadd45a. Several downregulated genes were also revealed: Hspa1b, Gm4951, F830016B08Rik, Fads2, Hspa1a, Slc27a6, Cacna1b, Scd2, Lama3 and Col4a5. These differentially expressed genes were mainly involved in osteoclast differentiation cascades, as well as PI3K-AKT, Forkhead box O (FoxO), MAPK, peroxisome proliferator-activated receptor (PPAR), TNF, TGF-β, JAK-STAT, RAS, VEGF, hypoxia-inducible factor (HIF-1) and AMPK signaling pathways. In summary, the present study revealed the key genes and signaling pathways involved in the H2-mediated inhibition of osteoclastogenesis, thereby providing a theoretical basis for the significance of H2 and an experimental basis for the application of Mg alloys in the treatment of osteoporosis.
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Affiliation(s)
- Yong Liu
- Department of Orthopedics, Chengdu Second People's Hospital, Chengdu, Sichuan 610017, P.R. China
| | - Wei Wang
- Department of Human Anatomy and Histoembryology, Zhuhai Campus of Zunyi Medical University, Zhuhai, Guangdong 519041, P.R. China
| | - Yong Zeng
- Department of Orthopedics, Chengdu Second People's Hospital, Chengdu, Sichuan 610017, P.R. China
| | - Hui Zeng
- Department of Bone and Joint Surgery, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, P.R. China
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Thurner GC, Haybaeck J, Debbage P. Targeting Drug Delivery in the Elderly: Are Nanoparticles an Option for Treating Osteoporosis? Int J Mol Sci 2021; 22:8932. [PMID: 34445639 PMCID: PMC8396227 DOI: 10.3390/ijms22168932] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Revised: 08/14/2021] [Accepted: 08/16/2021] [Indexed: 12/12/2022] Open
Abstract
Nanoparticles bearing specific targeting groups can, in principle, accumulate exclusively at lesion sites bearing target molecules, and release therapeutic agents there. However, practical application of targeted nanoparticles in the living organism presents challenges. In particular, intravasally applied nanoparticles encounter physical and physiological barriers located in blood vessel walls, blocking passage from the blood into tissue compartments. Whereas small molecules can pass out of the blood, nanoparticles are too large and need to utilize physiological carriers enabling passage across endothelial walls. The issues associated with crossing blood-tissue barriers have limited the usefulness of nanoparticles in clinical applications. However, nanoparticles do not encounter blood-tissue barriers if their targets are directly accessible from the blood. This review focuses on osteoporosis, a disabling and common disease for which therapeutic strategies are limited. The target sites for therapeutic agents in osteoporosis are located in bone resorption pits, and these are in immediate contact with the blood. There are specific targetable biomarkers within bone resorption pits. These present nanomedicine with the opportunity to treat a major disease by use of simple nanoparticles loaded with any of several available effective therapeutics that, at present, cannot be used due to their associated side effects.
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Affiliation(s)
- Gudrun C. Thurner
- Institute of Pathology, Neuropathology and Molecular Pathology, Medical University of Innsbruck, Müllerstraße 44, 6020 Innsbruck, Austria;
| | - Johannes Haybaeck
- Institute of Pathology, Neuropathology and Molecular Pathology, Medical University of Innsbruck, Müllerstraße 44, 6020 Innsbruck, Austria;
- Diagnostic & Research Center for Molecular BioMedicine, Institute of Pathology, Medical University Graz, Neue Stiftingtalstraße 6, 8010 Graz, Austria
| | - Paul Debbage
- Department of Anatomy, Histology and Embryology, Medical University of Innsbruck, Müllerstraße 59, 6020 Innsbruck, Austria
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6
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Kwak EJ, Oh KY, Perinpanayagam H, Kum KY. Internal Resorption of Multiple Posterior Teeth in a Patient Diagnosed with Hyperparathyroidism: A Case Report. J Endod 2021; 47:1321-1327. [DOI: 10.1016/j.joen.2021.04.015] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2021] [Revised: 04/06/2021] [Accepted: 04/12/2021] [Indexed: 02/07/2023]
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Galler KM, Grätz EM, Widbiller M, Buchalla W, Knüttel H. Pathophysiological mechanisms of root resorption after dental trauma: a systematic scoping review. BMC Oral Health 2021; 21:163. [PMID: 33771147 PMCID: PMC7995728 DOI: 10.1186/s12903-021-01510-6] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Accepted: 03/11/2021] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND The objective of this scoping review was to systematically explore the current knowledge of cellular and molecular processes that drive and control trauma-associated root resorption, to identify research gaps and to provide a basis for improved prevention and therapy. METHODS Four major bibliographic databases were searched according to the research question up to February 2021 and supplemented manually. Reports on physiologic, histologic, anatomic and clinical aspects of root resorption following dental trauma were included. Duplicates were removed, the collected material was screened by title/abstract and assessed for eligibility based on the full text. Relevant aspects were extracted, organized and summarized. RESULTS 846 papers were identified as relevant for a qualitative summary. Consideration of pathophysiological mechanisms concerning trauma-related root resorption in the literature is sparse. Whereas some forms of resorption have been explored thoroughly, the etiology of others, particularly invasive cervical resorption, is still under debate, resulting in inadequate diagnostics and heterogeneous clinical recommendations. Effective therapies for progressive replacement resorptions have not been established. Whereas the discovery of the RANKL/RANK/OPG system is essential to our understanding of resorptive processes, many questions regarding the functional regulation of osteo-/odontoclasts remain unanswered. CONCLUSIONS This scoping review provides an overview of existing evidence, but also identifies knowledge gaps that need to be addressed by continued laboratory and clinical research.
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Affiliation(s)
- Kerstin M Galler
- Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Franz-Josef-Strauß Allee 11, 93053, Regensburg, Germany.
| | - Eva-Maria Grätz
- Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Franz-Josef-Strauß Allee 11, 93053, Regensburg, Germany
| | - Matthias Widbiller
- Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Franz-Josef-Strauß Allee 11, 93053, Regensburg, Germany
| | - Wolfgang Buchalla
- Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Franz-Josef-Strauß Allee 11, 93053, Regensburg, Germany
| | - Helge Knüttel
- University Library, University of Regensburg, Regensburg, Germany
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Kolb AD, Dai J, Keller ET, Bussard KM. 'Educated' Osteoblasts Reduce Osteoclastogenesis in a Bone-Tumor Mimetic Microenvironment. Cancers (Basel) 2021; 13:cancers13020263. [PMID: 33445695 PMCID: PMC7828118 DOI: 10.3390/cancers13020263] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2020] [Revised: 01/01/2021] [Accepted: 01/08/2021] [Indexed: 12/31/2022] Open
Abstract
Breast cancer (BC) metastases to bone disrupt the balance between osteoblasts and osteoclasts, leading to excessive bone resorption. We identified a novel subpopulation of osteoblasts with tumor-inhibitory properties, called educated osteoblasts (EOs). Here we sought to examine the effect of EOs on osteoclastogenesis during tumor progression. We hypothesized that EOs affect osteoclast development in the bone-tumor niche, leading to suppressed pre-osteoclast fusion and bone resorption. Conditioned media (CM) was analyzed for protein expression of osteoclast factors receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) via ELISA. EOs were co-cultured with pre-osteoclasts on a bone mimetic matrix to assess osteoclast resorption. Pre-osteoclasts were tri-cultured with EOs plus metastatic BC cells and assessed for tartrate-resistance acid phosphatase (TRAP)-positive, multinucleated (≥3 nuclei), mature osteoclasts. Tumor-bearing murine tibias were stained for TRAP to determine osteoclast number in-vivo. EO CM expressed reduced amounts of soluble TNFα and OPG compared to naïve osteoblast CM. Osteoclasts formed in the presence of EOs were smaller and less in number. Upon co-culture on a mimetic bone matrix, a 50% reduction in the number of TRAP-positive osteoclasts formed in the presence of EOs was observed. The tibia of mice inoculated with BC cells had less osteoclasts per bone surface in bones with increased numbers of EO cells. These data suggest EOs reduce osteoclastogenesis and bone resorption. The data imply EOs provide a protective effect against bone resorption in bone metastatic BC.
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Affiliation(s)
- Alexus D. Kolb
- Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA;
| | - Jinlu Dai
- Department of Urology and Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA; (J.D.); (E.T.K.)
| | - Evan T. Keller
- Department of Urology and Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA; (J.D.); (E.T.K.)
| | - Karen M. Bussard
- Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA;
- Correspondence:
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Akisaka T, Yoshida A. Surface distribution of heterogenous clathrin assemblies in resorbing osteoclasts. Exp Cell Res 2020; 399:112433. [PMID: 33359468 DOI: 10.1016/j.yexcr.2020.112433] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2020] [Revised: 12/06/2020] [Accepted: 12/12/2020] [Indexed: 01/04/2023]
Abstract
Osteoclasts seeded on either glass coverslips or apatite pellets have at least two morphologically distinct substrate adhesion sites: actin-based adhesion structures including podosome belts and sealing zones, and adjacent clathrin sheets. Clathrin-coated structures are exclusively localized at the podosome belts and sealing zone, in both of which the plasma membrane forms a tight attachment to the substrate surface. When cultured on apatite osteoclasts can degrade the apatite leading to the formation of resorption lacunae. The sealing zone divides the ventral membrane into different domains, outside and inside of the sealing zones. The former facing the smooth-surfaced intact apatite contains relatively solitary or networks of larger flat clathrin structures; and the latter, facing the rough-surfaced degraded apatite in the resorption lacunae contain clathrin in various shapes and sizes. Clathrin assemblies on the membrane domain facing not only a resorption lacuna, or trails but also intact apatite indeed were observed to be heterogeneous in size and intensity, suggesting that they appeared to follow variations in the surface topography of the apatite surface. These results provide a detailed insight into the flat clathrin sheets that have been suggested to be the sites of adhesion and mechanosensing in co-operation with podosomes.
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Affiliation(s)
- Toshitaka Akisaka
- Department of Oral Anatomy and Neurobiology, Graduate School of Dentistry, Osaka University, Japan.
| | - Atsushi Yoshida
- Department of Oral Anatomy and Neurobiology, Graduate School of Dentistry, Osaka University, Japan.
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10
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Liu W, Le CC, Wang D, Ran D, Wang Y, Zhao H, Gu J, Zou H, Yuan Y, Bian J, Liu Z. Ca 2+/CaM/CaMK signaling is involved in cadmium-induced osteoclast differentiation. Toxicology 2020; 441:152520. [PMID: 32522522 DOI: 10.1016/j.tox.2020.152520] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2019] [Revised: 06/04/2020] [Accepted: 06/04/2020] [Indexed: 10/24/2022]
Abstract
Environmental cadmium (Cd) pollution can ultimately lead to chronic toxicity via food consumption. Previous studies have demonstrated that long-term low-dose Cd exposure decreases bone mineral density and bone mineralization. Cd may increase receptor activator of nuclear factor-κ B ligand (RANKL) expression by osteoclasts, and inhibit the expression of osteoprotegerin. However, the molecular mechanism underlying Cd toxicity toward osteoclasts is unclear. In this study, bone marrow monocytes were isolated from C57BL/6 mice and treated with macrophage colony-stimulating factor and RANKL to induce the formation of osteoclasts. The results show that low-dose Cd exposure induced osteoclast differentiation. Cd also increased the intracellular calcium concentration of osteoclasts by triggering release of calcium ions from the endoplasmic reticulum into the cytoplasm. Furthermore, the elevation of intracellular calcium levels was shown to activate the calmodulin (CaM)/calmodulin-dependent protein kinase (CaMK) pathway. NFATc1 is a downstream protein of CaM/CaMK signaling, as well as a key player in osteoclast differentiation. Overall, we conclude that Cd activates the CaM/CaMK/NFATc1 pathway and regulates osteoclast differentiation by increasing intracellular calcium concentration. Our data provide new insights into the mechanisms underlying osteoclast differentiation following Cd exposure. This study provides a theoretical basis for future investigations into the therapeutic application of CaMK inhibitors in osteoporosis induced by Cd exposure.
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Affiliation(s)
- Wei Liu
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Chung Chi Le
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Dong Wang
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Di Ran
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Yi Wang
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Hongyan Zhao
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Jianhong Gu
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Hui Zou
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Yan Yuan
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Jianchun Bian
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China
| | - Zongping Liu
- College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009 Jiangsu, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 Jiangsu, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009 Jiangsu, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China.
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11
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Cai B, Tan P, Jiang N, Guo Z, Ay B, Li S, Hou Y, Li Y, You Y, Zhang L, Zhu S. Bioinspired Fabrication of Calcium-Doped TiP Coating with Nanofibrous Microstructure to Accelerate Osseointegration. Bioconjug Chem 2020; 31:1641-1650. [PMID: 32426977 DOI: 10.1021/acs.bioconjchem.0c00201] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Bioinspired by the morphology of osteoclast-resorbed bone surfaces, we prepared a calcium-doped titanium phosphate (Ca-TiP) coating, which consists of a nanofibrous network, on titanium (Ti) substrate via a simple two-step hydrothermal method, trying to mimic natural bone compositionally and microstructurally. The in vitro studies show that the Ca-TiP coating with synergistic features of nanofibrous biomimetic topography and surface chemistry could elicit intensively osteogenic behavior and responses including enhanced cell adhesion, spreading, and proliferation as well as alkaline phosphatase (ALP) activity and up-regulated expression of bone-related genes, which inevitably benefit the formation of new bone and the quality of osseointegration. When the two control groups are compared in vivo, the significantly improved new bone formation in the early stage and the much stronger interfacial bonding with the surrounding bone for Ca-TiP coating suggest that Ca-TiP coating modified Ti implants hold great potential for orthopedic and dental applications.
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Affiliation(s)
- Bianyun Cai
- Analytical & Testing Center, Sichuan University, Chengdu 610065, China
| | - Peijie Tan
- State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Disease & West China Hospital of Stomatology, Sichuan University, Chengdu 610065, China
| | - Nan Jiang
- State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Disease & West China Hospital of Stomatology, Sichuan University, Chengdu 610065, China
| | - Zhijun Guo
- School of Materials Science and Physics, China University of Mining and Technology, Xuzhou 221116, China
| | - Birol Ay
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada
| | - Shujun Li
- Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang 110016, China
| | - Yi Hou
- Analytical & Testing Center, Sichuan University, Chengdu 610065, China
| | - Yubao Li
- Analytical & Testing Center, Sichuan University, Chengdu 610065, China
| | - Yanjun You
- Sichuan Institute for Food and Drug Control, Chengdu 610065, China
| | - Li Zhang
- Analytical & Testing Center, Sichuan University, Chengdu 610065, China.,State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Disease & West China Hospital of Stomatology, Sichuan University, Chengdu 610065, China
| | - Songsong Zhu
- Analytical & Testing Center, Sichuan University, Chengdu 610065, China.,State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Disease & West China Hospital of Stomatology, Sichuan University, Chengdu 610065, China
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12
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Liu Y, Wang DL, Huang YC, Wang TB, Zeng H. Hydrogen inhibits the osteoclastogenesis of mouse bone marrow mononuclear cells. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2020; 110:110640. [PMID: 32204074 DOI: 10.1016/j.msec.2020.110640] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/08/2019] [Revised: 11/05/2019] [Accepted: 01/03/2020] [Indexed: 12/16/2022]
Abstract
Hydrogen (H2) is one of the major biodegradation products of magnesium (Mg) alloys implanted for bony fracture healing and reconstruction; H2 thus plays a significant role in the regulation of local microenvironment and the biology of resident cells. The interactions between the H2 and the local cells are of great interest, and a full understanding of the effect of H2 on bone marrow mononuclear cells (BMMCs) would accelerate the development of effective strategies for successful bony healing. This study investigates how H2, with different concentrations and durations, regulates the osteoclastogenesis of mouse BMMCs. First, using H2 with five concentrations (0%, 2%, 25%, 50% and 75%) and three durations (5, 7 and 10 days), the osteoclastogenesis of mouse BMMCs in these H2 conditions were measured using TRAP staining, F-actin ring formation assay, pit formation assay and RT-qPCR analysis. Based on these findings, the proliferation assay, apoptosis assay, western blot analysis and ELISA assay of BMMCs after osteoclast induction were performed. The findings showed that H2 (especially the 50% and 75% H2) obviously inhibited the osteoclast formation, function and osteoclast-related genes expression of osteoclast-induced BMMCs; additionally, H2 (50%) was found to reduce the proliferation, promote the apoptosis and inhibit the expression of osteoclast-related proteins of BMMCs with the presence of osteoclast-induced medium. Therefore, H2 significantly inhibited the osteoclastogenesis of mouse BMMCs, which may become a new therapeutic agent for anti-bony resorption and open new avenues for the translational research of Mg alloys.
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Affiliation(s)
- Yong Liu
- Department of Bone & Joint Surgery, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong 518036, China; National & Local Joint Engineering Research Center of Orthopaedic Biomaterials, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, China
| | - De-Li Wang
- Department of Bone & Joint Surgery, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong 518036, China; Shenzhen Engineering Laboratory of Orthopaedic Regenerative Technologies, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, China
| | - Yong-Can Huang
- National & Local Joint Engineering Research Center of Orthopaedic Biomaterials, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, China; Shenzhen Engineering Laboratory of Orthopaedic Regenerative Technologies, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, China.
| | - Tian-Bing Wang
- Department of Orthopaedics, Peking University People's Hospital, Beijing 100044, China.
| | - Hui Zeng
- Department of Bone & Joint Surgery, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong 518036, China; Shenzhen Engineering Laboratory of Orthopaedic Regenerative Technologies, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, China.
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13
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Rao SR, Edwards CM, Edwards JR. Modeling the Human Bone-Tumor Niche: Reducing and Replacing the Need for Animal Data. JBMR Plus 2020; 4:e10356. [PMID: 32258970 PMCID: PMC7117847 DOI: 10.1002/jbm4.10356] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/25/2019] [Revised: 03/04/2020] [Accepted: 03/05/2020] [Indexed: 12/17/2022] Open
Abstract
Bone is the most common site for cancer metastasis. Understanding the interactions within the complex, heterogeneous bone-tumor microenvironment is essential for the development of new therapeutics. Various animal models of tumor-induced bone disease are routinely used to provide valuable information on the relationship between cancer cells and the skeleton. However, new model systems exist that offer an alternative approach to the use of animals and might more accurately reveal the cellular interactions occurring within the human bone-tumor niche. This review highlights replacement models that mimic the bone microenvironment and where cancer metastases and tumor growth might be assessed alongside bone turnover. Such culture models include the use of calcified regions of animal tissue and scaffolds made from bone mineral hydroxyapatite, synthetic polymers that can be manipulated during manufacture to create structures resembling trabecular bone surfaces, gel composites that can be modified for stiffness and porosity to resemble conditions in the tumor-bone microenvironment. Possibly the most accurate model system involves the use of fresh human bone samples, which can be cultured ex vivo in the presence of human tumor cells and demonstrate similar cancer cell-bone cell interactions as described in vivo. In addition, the use of mathematical modeling and computational biology approaches provide an alternative to preliminary animal testing. The use of such models offers the capacity to mimic significant elements of the human bone-tumor environment, and complement, refine, or replace the use of preclinical models. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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Affiliation(s)
- Srinivasa R Rao
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences University of Oxford Oxford UK.,Nuffield Department of Surgical Sciences University of Oxford Oxford UK
| | - Claire M Edwards
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences University of Oxford Oxford UK.,Nuffield Department of Surgical Sciences University of Oxford Oxford UK
| | - James R Edwards
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences University of Oxford Oxford UK
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14
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Kolb AD, Bussard KM. The Bone Extracellular Matrix as an Ideal Milieu for Cancer Cell Metastases. Cancers (Basel) 2019; 11:cancers11071020. [PMID: 31330786 PMCID: PMC6678871 DOI: 10.3390/cancers11071020] [Citation(s) in RCA: 51] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2019] [Revised: 07/01/2019] [Accepted: 07/18/2019] [Indexed: 12/12/2022] Open
Abstract
Bone is a preferential site for cancer metastases, including multiple myeloma, prostate, and breast cancers.The composition of bone, especially the extracellular matrix (ECM), make it an attractive site for cancer cell colonization and survival. The bone ECM is composed of living cells embedded within a matrix composed of both organic and inorganic components. Among the organic components, type I collagen provides the tensile strength of bone. Inorganic components, including hydroxyapatite crystals, are an integral component of bone and provide bone with its rigidity. Under normal circumstances, two of the main cell types in bone, the osteoblasts and osteoclasts, help to maintain bone homeostasis and remodeling through cellular communication and response to biophysical signals from the ECM. However, under pathological conditions, including osteoporosis and cancer, bone remodeling is dysregulated. Once in the bone matrix, disseminated tumor cells utilize normal products of bone remodeling, such as collagen type I, to fuel cancer cell proliferation and lesion outgrowth. Models to study the complex interactions between the bone matrix and metastatic cancer cells are limited. Advances in understanding the interactions between the bone ECM and bone metastatic cancer cells are necessary in order to both regulate and prevent metastatic cancer cell growth in bone.
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Affiliation(s)
- Alexus D Kolb
- Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA
| | - Karen M Bussard
- Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
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15
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Plotkin LI, Bruzzaniti A. Molecular signaling in bone cells: Regulation of cell differentiation and survival. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2019; 116:237-281. [PMID: 31036293 PMCID: PMC7416488 DOI: 10.1016/bs.apcsb.2019.01.002] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The achievement of proper bone mass and architecture, and their maintenance throughout life requires the concerted actions of osteoblasts, the bone forming cells, and osteoclasts, the bone resorbing cells. The differentiation and activity of osteoblasts and osteoclasts are regulated by molecules produced by matrix-embedded osteocytes, as well as by cross talk between osteoblasts and osteoclasts through secreted factors. In addition, it is likely that direct contact between osteoblast and osteoclast precursors, and the contact of these cells with osteocytes and cells in the bone marrow, also modulates bone cell differentiation and function. With the advancement of molecular and genetic tools, our comprehension of the intracellular signals activated in bone cells has evolved significantly, from early suggestions that osteoblasts and osteoclasts have common precursors and that osteocytes are inert cells in the bone matrix, to the very sophisticated understanding of a network of receptors, ligands, intracellular kinases/phosphatases, transcription factors, and cell-specific genes that are known today. These advances have allowed the design and FDA-approval of new therapies to preserve and increase bone mass and strength in a wide variety of pathological conditions, improving bone health from early childhood to the elderly. We have summarized here the current knowledge on selected intracellular signal pathways activated in osteoblasts, osteocytes, and osteoclasts.
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Affiliation(s)
- Lilian I Plotkin
- Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States; Indiana Center for Musculoskeletal Health, Indianapolis, IN, United States; Roudebush Veterans Administration Medical Center, Indianapolis, IN, United States.
| | - Angela Bruzzaniti
- Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States; Indiana Center for Musculoskeletal Health, Indianapolis, IN, United States; Department of Biomedical and Applied Sciences, Indiana University School of Dentistry, Indianapolis, IN, United States
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16
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Ay B, Mendes VC, Zhang L, Davies JE. A "best fit" approach for synergistic surface parameters to guide the design of candidate implant surfaces. J Biomed Mater Res B Appl Biomater 2019; 107:2165-2177. [PMID: 30677220 DOI: 10.1002/jbm.b.34312] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2018] [Revised: 11/23/2018] [Accepted: 12/19/2018] [Indexed: 11/07/2022]
Abstract
Human bone resorption surfaces can provide a template for endosseous implant surface design. We characterized the topography of such sites using four synergistic parameters (fractal dimension, lacunarity, porosity, and surface roughness) and compared the generated values with those obtained from two groups of candidate titanium implant surfaces. For the first group (n = 5/group): grit-blasted acid etched (BAE), BAE with either discrete calcium phosphate crystal deposition or nanotube formation, machined titanium with nanotubes, or a nanofiber surface; each measured synergistic parameter was statistically compared with that of the resorbed bone surface and scored for inclusion in a "best fit" analysis. The analysis informed changes that could be made to a candidate implant surface to render it a closer "best fit" to that of the resorbed bone surface. In a second group of either titanium or titanium alloy implants their micro-topography, created by dual acid etching, was the same for each material substrate; but their nanotopographic complexity was changed by varying the degree of calcium phosphate crystalline deposits. These implants were also used in vivo where bone anchorage was tested using a tensile disruption test; and the "best fit" of synergistic parameters coincided with the best biological outcome for both titanium and titanium alloy implants. In conclusion, the four chosen synergistic parameters can be used to guide the sub-micron surface design of candidate implants, and our "best fit" approach is capable of identifying the surfaces with the best biological outcomes. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2165-2177, 2019.
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Affiliation(s)
- Birol Ay
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
| | - Vanessa C Mendes
- Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
| | - Li Zhang
- Analytical & Testing Center, Sichuan University, Chengdu, China
| | - John E Davies
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.,Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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17
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Abstract
Osteoclasts are highly specialized multinucleated cells derived from the monocyte/macrophage hematopoietic lineage that are uniquely capable of adhering to bone matrix and resorbing bone. The tartrate-resistant acid phosphatase (TRAP) assay is the most common method to detect osteoclasts population in vitro. Here we described a general protocol of inducing osteoclast differentiation from the murine macrophage cell line, RAW264.7, and identification of osteoclasts with the classical TRAP assay.
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Affiliation(s)
- Jingxuan Yang
- Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
- Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
| | - Xiaohong Bi
- Department of Nanomedicine and Biomedical Engineering, The University of Texas Medical School at Houston, Houston, TX, USA.
| | - Min Li
- Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
- Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
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18
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Abstract
This chapter describes methods for preparing samples of bone and bone cells for scanning electron microscopy (SEM). Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. Samples may have 3D detail in a 3D surface, or be topography-free, polished or micromilled, resin-embedded block surfaces, or resin casts of space compartments surrounded by bone matrix. Methods for cells include fixation, drying, looking at undersides of bone cells, and metallic conductive coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralized matrix is described in detail. Attention is given especially to methods for 3D BSE SEM imaging of bone samples. Recommendations are made for the types of resin embedding for BSE SEM imaging. Correlated confocal and SEM imaging of PMMA embedded bone requires the use of glycerol to coverslip. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualizing fluorescent mineralizing front labels such as calcein and tetracyclines. Making spatial casts from PMMA or other resin-embedded samples is an important use of this material. Correlation with other imaging means, including microradiography and microtomography is important. Shipping wet bone samples between labs is best done in glycerol. Control of the vacuum pressure in the SEM sample chamber (now generally available) can be used to eliminate "charging" problems which were common, for example, with large, complex, cancellous bone samples.
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MESH Headings
- Animals
- Bone and Bones/diagnostic imaging
- Bone and Bones/ultrastructure
- Cells, Cultured
- Histocytological Preparation Techniques/instrumentation
- Histocytological Preparation Techniques/methods
- Humans
- Imaging, Three-Dimensional/instrumentation
- Imaging, Three-Dimensional/methods
- Microradiography/instrumentation
- Microradiography/methods
- Microscopy, Confocal/instrumentation
- Microscopy, Confocal/methods
- Microscopy, Electron, Scanning/instrumentation
- Microscopy, Electron, Scanning/methods
- Microscopy, Fluorescence/instrumentation
- Microscopy, Fluorescence/methods
- Multimodal Imaging/instrumentation
- Multimodal Imaging/methods
- Osteoclasts
- Osteocytes
- Software
- X-Ray Microtomography/instrumentation
- X-Ray Microtomography/methods
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Affiliation(s)
- Alan Boyde
- Dental Physical Sciences, Biophysics Section, Oral Growth and Development, Dental Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
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19
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Matsumoto Y, Sringkarnboriboon S, Ono T. Effects of continuous force application for extrusive tipping movement on periapical root resorption in the rat mandibular first molar. Korean J Orthod 2018; 48:339-345. [PMID: 30206533 PMCID: PMC6123074 DOI: 10.4041/kjod.2018.48.5.339] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2016] [Revised: 09/12/2017] [Accepted: 09/26/2017] [Indexed: 11/16/2022] Open
Abstract
Objective The purpose of this study was to clarify the effects of continuous force application for extrusive tipping movement and occlusal interference on periapical root resorption in the rat mandibular first molar. Methods We constructed an appliance comprising a titanium screw implant with a cobalt-chromium post as the anchorage unit and a nickel-titanium closed coil spring (50 cN) as the active unit. Force was applied on the mandibular left first molar of rats for 8 (n = 10) and 15 days (n = 10; experimental groups), with the tooth in occlusion. Five rats were included as a non-treated control group to examine the body effect of the appliance. Active root resorption lacunae, identified using tartrate-resistant acid phosphatase, were evaluated in terms of the length, depth, and area. Results The rat mandibular first molars were mesially tipped and extruded in the occlusal direction. This mesio-occlusal tipping movement and occlusion resulted in the formation of a compression zone and active root resorption lacunae in the distoapical third of the distal roots. However, there was no significant difference in the amount of root resorption between the two experimental groups. The control group did not exhibit any active root resorption lacunae. Conclusions Periapical root resorption was induced by continuous extrusive tipping force and occlusal interference in rat mandibular molars. These data suggest that we orthodontists had better take care not to induce occlusal interference during our orthodontic treatment.
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Affiliation(s)
- Yoshiro Matsumoto
- Department of Orthodontic Science, Division of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Siripen Sringkarnboriboon
- Department of Orthodontic Science, Division of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
- Private Practice, Bangkok, Thailand
| | - Takashi Ono
- Department of Orthodontic Science, Division of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
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20
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Clarke SA, Martin J, Nelson J, Hornez JC, Bohner M, Dunne N, Buchanan F. Surrogate Outcome Measures of In Vitro Osteoclast Resorption of β Tricalcium Phosphate. Adv Healthc Mater 2017; 6. [PMID: 27930865 DOI: 10.1002/adhm.201600947] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2016] [Revised: 11/01/2016] [Indexed: 01/12/2023]
Abstract
Introduction of porosity to calcium phosphate scaffolds for bone repair has created a new challenge when measuring bioresorption in vitro, rendering traditional outcome measures redundant. The aim of this study is to identify a surrogate endpoint for use with 3D scaffolds. Murine RAW 264.7 cells are cultured on dense discs of β-tricalcium phosphate in conditions to stimulate osteoclast (OC) formation. Multinucleated OCs are visible from day 6 with increases at days 8 and 10. Resorption pits are first observed at day 6 with much larger pits visible at days 8, 10, and 12. The concentration of calcium ions in the presence of cells is significantly higher than cell-free cultures at days 3 and 9. Using linear regression analysis, Ca ion release could account for 35.9% of any subsequent change in resorption area. The results suggest that Ca ion release is suitable to measure resorption of a beta-tricalcium phosphate ceramic substrate in vitro. This model could replace the more accepted resorption pit assay in circumstances where quantification of pits is not possible, e.g., when characterizing 3D tissue engineered bone scaffolds.
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Affiliation(s)
- Susan A. Clarke
- School of Nursing and Midwifery; Medical Biology Centre; 97, Lisburn Road Belfast BT9 7BL UK
| | - Joanne Martin
- School of Mechanical and Aerospace Engineering; Queen's University Belfast; Ashby Building, Stranmillis Rd Belfast BT9 5AH UK
| | - John Nelson
- School of Biological Sciences; Queens University Belfast; MBC, 97 Lisburn Rd Belfast BT9 7BL UK
| | | | - Marc Bohner
- Skeletal Substitutes Group; RMS Foundation; Bischmattstr. 12 CH-2544 Bettlach Switzerland
| | - Nicholas Dunne
- School of Mechanical and Aerospace Engineering; Queen's University Belfast; Ashby Building, Stranmillis Rd Belfast BT9 5AH UK
| | - Fraser Buchanan
- School of Mechanical and Aerospace Engineering; Queen's University Belfast; Ashby Building, Stranmillis Rd Belfast BT9 5AH UK
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21
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Almeida M, Laurent MR, Dubois V, Claessens F, O'Brien CA, Bouillon R, Vanderschueren D, Manolagas SC. Estrogens and Androgens in Skeletal Physiology and Pathophysiology. Physiol Rev 2017; 97:135-187. [PMID: 27807202 PMCID: PMC5539371 DOI: 10.1152/physrev.00033.2015] [Citation(s) in RCA: 547] [Impact Index Per Article: 68.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Estrogens and androgens influence the growth and maintenance of the mammalian skeleton and are responsible for its sexual dimorphism. Estrogen deficiency at menopause or loss of both estrogens and androgens in elderly men contribute to the development of osteoporosis, one of the most common and impactful metabolic diseases of old age. In the last 20 years, basic and clinical research advances, genetic insights from humans and rodents, and newer imaging technologies have changed considerably the landscape of our understanding of bone biology as well as the relationship between sex steroids and the physiology and pathophysiology of bone metabolism. Together with the appreciation of the side effects of estrogen-related therapies on breast cancer and cardiovascular diseases, these advances have also drastically altered the treatment of osteoporosis. In this article, we provide a comprehensive review of the molecular and cellular mechanisms of action of estrogens and androgens on bone, their influences on skeletal homeostasis during growth and adulthood, the pathogenetic mechanisms of the adverse effects of their deficiency on the female and male skeleton, as well as the role of natural and synthetic estrogenic or androgenic compounds in the pharmacotherapy of osteoporosis. We highlight latest advances on the crosstalk between hormonal and mechanical signals, the relevance of the antioxidant properties of estrogens and androgens, the difference of their cellular targets in different bone envelopes, the role of estrogen deficiency in male osteoporosis, and the contribution of estrogen or androgen deficiency to the monomorphic effects of aging on skeletal involution.
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Affiliation(s)
- Maria Almeida
- Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; Departments of Cellular and Molecular Medicine and Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium; Center for Metabolic Bone Diseases, University Hospitals Leuven, Leuven, Belgium; and Institut National de la Santé et de la Recherche Médicale UMR1011, University of Lille and Institut Pasteur de Lille, Lille, France
| | - Michaël R Laurent
- Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; Departments of Cellular and Molecular Medicine and Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium; Center for Metabolic Bone Diseases, University Hospitals Leuven, Leuven, Belgium; and Institut National de la Santé et de la Recherche Médicale UMR1011, University of Lille and Institut Pasteur de Lille, Lille, France
| | - Vanessa Dubois
- Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; Departments of Cellular and Molecular Medicine and Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium; Center for Metabolic Bone Diseases, University Hospitals Leuven, Leuven, Belgium; and Institut National de la Santé et de la Recherche Médicale UMR1011, University of Lille and Institut Pasteur de Lille, Lille, France
| | - Frank Claessens
- Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; Departments of Cellular and Molecular Medicine and Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium; Center for Metabolic Bone Diseases, University Hospitals Leuven, Leuven, Belgium; and Institut National de la Santé et de la Recherche Médicale UMR1011, University of Lille and Institut Pasteur de Lille, Lille, France
| | - Charles A O'Brien
- Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; Departments of Cellular and Molecular Medicine and Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium; Center for Metabolic Bone Diseases, University Hospitals Leuven, Leuven, Belgium; and Institut National de la Santé et de la Recherche Médicale UMR1011, University of Lille and Institut Pasteur de Lille, Lille, France
| | - Roger Bouillon
- Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; Departments of Cellular and Molecular Medicine and Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium; Center for Metabolic Bone Diseases, University Hospitals Leuven, Leuven, Belgium; and Institut National de la Santé et de la Recherche Médicale UMR1011, University of Lille and Institut Pasteur de Lille, Lille, France
| | - Dirk Vanderschueren
- Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; Departments of Cellular and Molecular Medicine and Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium; Center for Metabolic Bone Diseases, University Hospitals Leuven, Leuven, Belgium; and Institut National de la Santé et de la Recherche Médicale UMR1011, University of Lille and Institut Pasteur de Lille, Lille, France
| | - Stavros C Manolagas
- Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; Departments of Cellular and Molecular Medicine and Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium; Center for Metabolic Bone Diseases, University Hospitals Leuven, Leuven, Belgium; and Institut National de la Santé et de la Recherche Médicale UMR1011, University of Lille and Institut Pasteur de Lille, Lille, France
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22
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Wu VM, Uskoković V. Is there a relationship between solubility and resorbability of different calcium phosphate phases in vitro? BIOCHIMICA ET BIOPHYSICA ACTA 2016; 1860:2157-68. [PMID: 27212690 PMCID: PMC4961619 DOI: 10.1016/j.bbagen.2016.05.022] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/19/2016] [Revised: 05/01/2016] [Accepted: 05/17/2016] [Indexed: 10/21/2022]
Abstract
BACKGROUND Does chemistry govern biology or it is the other way around - that is a broad connotation of the question that this study attempted to answer. METHOD Comparison was made between the solubility and osteoclastic resorbability of four fundamentally different monophasic calcium phosphate (CP) powders with monodisperse particle size distributions: alkaline hydroxyapatite (HAP), acidic monetite (DCP), β-calcium pyrophosphate (CPP), and amorphous CP (ACP). Results With the exception of CPP, the difference in solubility between different CP phases became neither mitigated nor reversed, but augmented in the resorptive osteoclastic milieu. Thus, DCP, a phase with the highest solubility, was also resorbed more intensely than any other CP phase, whereas HAP, a phase with the lowest solubility, was resorbed least. CPP becomes retained inside the cells for the longest period of time, indicating hindered digestion of only this particular type of CP. Osteoclastogenesis was mildly hindered in the presence of HAP, ACP and DCP, but not in the presence of CPP. The most viable CP powder with respect to the mitochondrial succinic dehydrogenase activity was the one present in natural biological bone tissues: HAP. CONCLUSION Chemistry in this case does have a direct effect on biology. Biology neither overrides nor reverses the chemical propensities of inorganics with which it interacts, but rather augments and takes a direct advantage of them. SIGNIFICANCE These findings set the fundamental basis for designing the chemical makeup of CP and other biosoluble components of tissue engineering constructs for their most optimal resorption and tissue regeneration response.
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Affiliation(s)
- Victoria M Wu
- Advanced Materials and Nanobiotechnology Laboratory, Department of Bioengineering, University of Illinois, Chicago, IL, USA
| | - Vuk Uskoković
- Advanced Materials and Nanobiotechnology Laboratory, Department of Bioengineering, University of Illinois, Chicago, IL, USA
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Maria SM, Prukner C, Sheikh Z, Müller FA, Komarova SV, Barralet JE. Characterization of biomimetic calcium phosphate labeled with fluorescent dextran for quantification of osteoclastic activity. Acta Biomater 2015; 20:140-146. [PMID: 25829107 DOI: 10.1016/j.actbio.2015.03.026] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2014] [Revised: 02/21/2015] [Accepted: 03/23/2015] [Indexed: 01/03/2023]
Abstract
Bone resorbing osteoclasts represent an important therapeutic target for diseases associated with bone and joint destruction, such as rheumatoid arthritis, periodontitis, and osteoporosis. The quantification of osteoclast resorptive activity in vitro is widely used for screening new anti-resorptive medications. The aim of this paper was to develop a simplified semi-automated method for the quantification of osteoclastic resorption using fluorescently labeled biomimetic mineral layers which can replace time intensive, often subjective and clearly non-sustainable use of translucent slices of tusks from vulnerable or endangered species such as the elephant. Osteoclasts were formed from RAW 264.7 mouse monocyte cell line using the pro-resorptive cytokine receptor activator of nuclear factor kappa-B ligand (RANKL). We confirmed that fluorescent labeling did not interfere with the biomimetic features of hydroxyapatite, and developed an automated method for quantifying osteoclastic resorption. Correlation between our assay and traditional manual measurement techniques was found to be very strong (R(2)=0.99). In addition, we modified the technique to provide depth and volume data of the resorption pits by confocal imaging at defined depths. Thus, our method allows automatic quantification of total osteoclastic resorption as well as additional data not obtainable by the current tusk slice technique offering a better alternative for high throughput screening of potential antiresorptives.
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Generation and culture of osteoclasts. BONEKEY REPORTS 2014; 3:570. [PMID: 25228983 DOI: 10.1038/bonekey.2014.65] [Citation(s) in RCA: 86] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/26/2014] [Accepted: 07/08/2014] [Indexed: 01/09/2023]
Abstract
Osteoclasts are highly specialized cells of haematopoietic lineage that are uniquely responsible for bone resorption. In the past, osteoclasts were isolated as mature cells from chicken long bones, or were generated using osteoblasts or stromal cells to induce osteoclast formation in total bone marrow from mice or rabbits. The Copernican revolution in osteoclast biology began with the identification of macrophage-colony stimulating factor (M-CSF) and receptor activator NFκB-ligand (RANKL ) as the key regulators of osteoclast formation, fusion and function. The availability of recombinant human and mouse M-CSF and RANKL has enabled researchers to reliably generate osteoclasts from primary monocyte/macrophage cells as well as from cell lines such as RAW 264.7. This article summarizes the most commonly used procedures for the isolation, generation and characterization of human, rodent and chicken osteoclasts in vitro. Lists of further reading and recommendations are included to facilitate a successful application by the reader.
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Téletchéa S, Stresing V, Hervouet S, Baud'huin M, Heymann MF, Bertho G, Charrier C, Ando K, Heymann D. Novel RANK antagonists for the treatment of bone-resorptive disease: theoretical predictions and experimental validation. J Bone Miner Res 2014; 29:1466-77. [PMID: 24390798 DOI: 10.1002/jbmr.2170] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2013] [Revised: 12/17/2013] [Accepted: 01/01/2013] [Indexed: 12/15/2022]
Abstract
Receptor activator of nuclear factor-κB (RANK) and RANK ligand (RANKL) play a pivotal role in bone metabolism, and selective targeting of RANK signaling has become a promising therapeutic strategy in the management of resorptive bone diseases. Existing antibody-based therapies and novel inhibitors currently in development were designed to target the ligand, rather than the membrane receptor expressed on osteoclast precursors. We describe here an alternative approach to designing small peptides able to specifically bind to the hinge region of membrane RANK responsible for the conformational change upon RANKL association. A nonapeptide generated by this method was validated for its biological activity in vitro and in vivo and served as a lead compound for the generation of a series of peptide RANK antagonists derived from the original sequence. Our study presents a structure- and knowledge-based strategy for the design of novel effective and affordable small peptide inhibitors specifically targeting the receptor RANK and opens a new therapeutic opportunity for the treatment of resorptive bone disease.
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Affiliation(s)
- Stéphane Téletchéa
- INSERM, UMR 957, Equipe labellisée LIGUE 2012, Université de Nantes, Laboratory of the Physiopathology of Bone Resorption and Therapy of Primary Bone Tumors (LPRO), Nantes, France
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FoxO proteins restrain osteoclastogenesis and bone resorption by attenuating H2O2 accumulation. Nat Commun 2014; 5:3773. [PMID: 24781012 PMCID: PMC4015330 DOI: 10.1038/ncomms4773] [Citation(s) in RCA: 198] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2013] [Accepted: 04/02/2014] [Indexed: 12/26/2022] Open
Abstract
Besides their cell-damaging effects in the setting of oxidative stress, reactive oxygen species (ROS) play an important role in physiological intracellular signalling by triggering proliferation and survival. FoxO transcription factors counteract ROS generation by upregulating antioxidant enzymes. Here we show that intracellular H2O2 accumulation is a critical and purposeful adaptation for the differentiation and survival of osteoclasts, the bone cells responsible for the resorption of mineralized bone matrix. Using mice with conditional loss or gain of FoxO transcription factor function, or mitochondria-targeted catalase in osteoclasts, we demonstrate this is achieved, at least in part, by downregulating the H2O2-inactivating enzyme catalase. Catalase downregulation results from the repression of the transcriptional activity of FoxO1, 3 and 4 by RANKL, the indispensable signal for the generation of osteoclasts, via an Akt-mediated mechanism. Notably, mitochondria-targeted catalase prevented the loss of bone caused by loss of oestrogens, suggesting that decreasing H2O2 production in mitochondria may represent a rational pharmacotherapeutic approach to diseases with increased bone resorption.
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Tsukiboshi M, Tsukiboshi T. Bone morphology after delayed tooth replantation - case series. Dent Traumatol 2014; 30:477-83. [DOI: 10.1111/edt.12111] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/20/2014] [Indexed: 12/31/2022]
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Detsch R, Boccaccini AR. The role of osteoclasts in bone tissue engineering. J Tissue Eng Regen Med 2014; 9:1133-49. [PMID: 24478169 DOI: 10.1002/term.1851] [Citation(s) in RCA: 95] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2013] [Revised: 09/18/2013] [Accepted: 10/20/2013] [Indexed: 12/13/2022]
Abstract
The success of scaffold-based bone regeneration approaches strongly depends on the performance of the biomaterial utilized. Within the efforts of regenerative medicine towards a restitutio ad integrum (i.e. complete reconstruction of a diseased tissue), scaffolds should be completely degraded within an adequate period of time. The degradation of synthetic bone substitute materials involves both chemical dissolution (physicochemical degradation) and resorption (cellular degradation by osteoclasts). Responsible for bone resorption are osteoclasts, cells of haematopoietic origin. Osteoclasts play also a crucial role in bone remodelling, which is essential for the regeneration of bone defects. There is, however, surprisingly limited knowledge about the detailed effects of osteoclasts on biomaterials degradation behaviour. This review covers the relevant fundamental knowledge and progress made in the field of osteoclast activity related to biomaterials used for bone regeneration. In vitro studies with osteoclastic precursor cells on synthetic bone substitute materials show that there are specific parameters that inhibit or enhance resorption. Moreover, analyses of the bone-material interface reveal that biomaterials composition has a significant influence on their degradation in contact with osteoclasts. Crystallinity, grain size, surface bioactivity and density of the surface seem to have a less significant effect on osteoclastic activity. In addition, the topography of the scaffold surface can be tailored to affect the development and spreading of osteoclast cells. The present review also highlights possible areas on which future research is needed and which are relevant to enhance our understanding of the complex role of osteoclasts in bone tissue engineering.
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Affiliation(s)
- Rainer Detsch
- Institute of Biomaterials, University of Erlangen-Nuremberg, Germany
| | - Aldo R Boccaccini
- Institute of Biomaterials, University of Erlangen-Nuremberg, Germany
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Rementer CW, Wu M, Buranaphatthana W, Yang HYL, Scatena M, Giachelli CM. An inducible, ligand-independent receptor activator of NF-κB gene to control osteoclast differentiation from monocytic precursors. PLoS One 2013; 8:e84465. [PMID: 24386387 PMCID: PMC3874012 DOI: 10.1371/journal.pone.0084465] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2013] [Accepted: 11/22/2013] [Indexed: 12/28/2022] Open
Abstract
Osteoclasts are bone-resorbing cells that are critical for the normal formation and maintenance of teeth and skeleton. Osteoclast deficiency can contribute to heterotopic ossification (HO), a pathology that is particularly detrimental to the mechanical functions of joints, valves and blood vessels. On the other hand, osteoclast over-activity is a major cause of osteoporosis. A reliable method for controlled generation of osteoclasts would be useful as a potential autologous cell therapy for HO, as well as high-throughput drug screening for anti-osteoporotic drugs. In this report, we describe the development of a cell engineering approach to control monocytic precursor cell differentiation to osteoclasts. Oligomerization of receptor activator of nuclear factor κB (RANK) is known to be essential for osteoclast differentiation from monocyte/macrophage precursors. We engineered a murine monocytic cell line, RAW264.7 to express a fusion protein comprising the intracellular RANK signaling domain and FK506-derived dimerization domains that bind to a small molecule chemical inducer of dimerization (CID). Virally infected cells expressing this fusion protein were treated with CID and dose-dependent induction of tartrate-resistant acid phosphatase activity, as well as multinucleated osteoclast formation were observed. Furthermore, NF-κB signaling was upregulated in a CID-dependent fashion, demonstrating effective RANK intracellular signaling. Functionally CID-induced osteoclasts had robust mineral resorptive activity in both two-dimensional and three-dimensional in vitro resorption assays. In addition, the CID-induced osteoclasts have the same life span as native RANKL-induced osteoclasts. Most importantly and crucially, the engineered cells differentiated into osteoclasts that were resistant to the potent osteoclast inhibitor, osteoprotegerin. Taken together, these studies are the first to describe a method for inducible control of monocytic precursor differentiation to osteoclasts that may be useful for future development of an engineered autologous cell therapy as well as high-throughput drug testing systems to treat diseases of osteoclast over-activity that are independent of osteoprotegerin.
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Affiliation(s)
- Cameron W. Rementer
- Department of Bioengineering, University of Washington, Seattle, Washington, United States of America
| | - Meiting Wu
- Department of Bioengineering, University of Washington, Seattle, Washington, United States of America
| | - Worakanya Buranaphatthana
- Department of Oral Health Sciences, University of Washington, Seattle, Washington, United States of America
| | - Hsueh-Ying L. Yang
- Department of Bioengineering, University of Washington, Seattle, Washington, United States of America
| | - Marta Scatena
- Department of Bioengineering, University of Washington, Seattle, Washington, United States of America
| | - Cecilia M. Giachelli
- Department of Bioengineering, University of Washington, Seattle, Washington, United States of America
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Sriarj W, Aoki K, Ohya K, Takahashi M, Takagi Y, Shimokawa H. TGF-β in dentin matrix extract induces osteoclastogenesis in vitro. Odontology 2013; 103:9-18. [PMID: 24366403 DOI: 10.1007/s10266-013-0140-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2013] [Accepted: 11/20/2013] [Indexed: 01/03/2023]
Abstract
Previously, we have demonstrated that the extracellular matrix from dentin affects osteoclastic activity in co-culture between osteoclast and osteoblast-rich fraction from mouse marrow cells. In the present study, we aimed to investigate the mechanisms of dentin matrix extract-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs). Dentin proteins were extracted from bovine incisor root dentin using 0.6 M HCl. BMMs were cultured in α-MEM containing macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-B ligand in the presence or absence of dentin matrix extract. Tartrate-resistant acid phosphatase (TRAP)-positive cell number, total TRAP activity, and the mRNA levels of osteoclast-related genes, assayed by real-time RT-PCR, were determined as markers of osteoclastogenesis. A neutralizing antibody against transforming growth factor-β1 (TGF-β1), SB431542, a TGF-β receptor inhibitor, and ELISA were used to determine the role of TGF-β1. We observed increases in TRAP-positive cell number, TRAP activity, and the mRNA levels of osteoclast-related genes of BMMs cultured with dentin extract. The use of a neutralizing antibody against TGF-β1 or SB431542 inhibited the inductive effect of dentin extract, suggesting TGF-β1 involvement. The addition of exogenous TGF-β1, but not bone morphogenic protein-2, also increased osteoclastogenesis, corresponding to the ELISA determination of TGF-β1 in the dentin extract. In conclusion, our results indicate that proteins from dentin matrix have an inductive effect in osteoclastogenesis, which is mediated, in part, by TGF-β1.
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Affiliation(s)
- Wannakorn Sriarj
- Department of Pediatric Dentistry, Faculty of Dentistry, Chulalongkorn University, 34, Henri-Dunant Road, Pathumwan, Bangkok, 10330, Thailand,
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Martin TJ. Historically significant events in the discovery of RANK/RANKL/OPG. World J Orthop 2013; 4:186-197. [PMID: 24147254 PMCID: PMC3801238 DOI: 10.5312/wjo.v4.i4.186] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/04/2012] [Revised: 01/07/2013] [Accepted: 03/23/2013] [Indexed: 02/06/2023] Open
Abstract
After it was suggested 30 years ago that the osteoblast lineage controlled the formation of osteoclasts, methods were developed that established this to be the case, but the molecular controls were elusive. Over more than a decade much evidence was obtained for signaling mechanisms that regulated the production of a membrane - bound regulator of osteoclastogenesis, in the course of which intercellular communication in bone was revealed in its complexity. The discovery of regulation by tumor necrosis factor ligand and receptor families was made in the last few years of the twentieth century, leading since then to a new physiology of bone, and to exciting drug development.
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32
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Davies JE, Mendes VC, Ko JCH, Ajami E. Topographic scale-range synergy at the functional bone/implant interface. Biomaterials 2013; 35:25-35. [PMID: 24099707 DOI: 10.1016/j.biomaterials.2013.09.072] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2013] [Accepted: 09/23/2013] [Indexed: 10/26/2022]
Abstract
We sought to explore the biological mechanisms by which endosseous implant surface topography contributes to bone anchorage. To address this experimentally, we implanted five groups of custom-made commercially pure titanium implants of varying surface topographical complexity in rat femora for 9 days; subjected them to mechanical testing; and then examined the interfacial bone matrix by electron microscopy. The five implant surfaces were prepared by combinations of dual acid etching and grit blasting the titanium substrates and, in some cases, modifying the created surfaces with the deposition of nanocrystals of calcium phosphate, which resulted in 10 samples per group. In parallel, we cultured rat bone marrow cells on surrogate implants constructed from polymer resin coated with the same calcium phosphate nanocrystals, and monitored the deposition of bone sialoprotein by transmission electron immunohisto-micrography. We found that implant samples modified with sub-micron scale crystals were bone-bonding, as described by the interdigitation of a mineralized cement line matrix with the underlying implant surface. The in vitro assay showed that bone sialoprotein could be deposited in the interstices between, and undercuts below, the nanocrystals. In addition, when mineralized, the cement line matrix globules occupied micron-sized pits in the implant surfaces, and in part obliterated them, creating an additional form of anchorage. Our results also showed that collagen, elaborated by the osteogenic cells, wrapped around the coarse-micron features, and became mineralized in the normal course of bone formation. This provided a mechanism by which coarse-micron implant features contributed to a functional interface, which we have previously described, that is capable of resisting the mechanical loading that increases as peri-implant bone matures. Thus, our findings provide mechanistic explanations for the biologically-relevant criteria that can be employed to assess the importance of implant surface topography at different scale-ranges.
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Affiliation(s)
- John E Davies
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada; Dental Research Institute, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario M5G 1G6, Canada.
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Torres A, Santos S, Oliveira M, Barbosa M. Fibrinogen promotes resorption of chitosan by human osteoclasts. Acta Biomater 2013; 9:6553-62. [PMID: 23376128 DOI: 10.1016/j.actbio.2013.01.015] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2012] [Revised: 01/12/2013] [Accepted: 01/18/2013] [Indexed: 02/07/2023]
Abstract
The osteoconductive and osteoinductive properties of materials intended for bone regeneration have been extensively tested, but the resorbability of these materials is often overlooked. Osteoclasts are responsible for bone resorption and play a crucial role in bone remodeling, which is essential for complete regeneration of bone tissue following injury. In this study we compare, for the first time, the ability of unmodified and fibrinogen (Fg)-modified chitosan (Ch) substrates to support the formation of multinucleated osteoclasts, and the potential of these cells to resorb the two substrates in vitro. Osteoclasts were differentiated from primary human peripheral blood monocytes directly on the substrates being investigated. Our results showed similar cell adhesion to unmodified and Fg-modified Ch substrates. Although the number of multinucleated osteoclasts on both Ch substrates increased throughout the culture period, by 21 days of culture significantly more highly multinucleated osteoclasts (>10 nuclei per cell) were observed on Fg-modified Ch, when compared to Ch alone. In addition, cells were tartrate-resistant acid phosphatase positive and secreted significantly more enzyme on Ch-based substrates than in control conditions. Unmodified and Fg-modified Ch resorption was investigated by fluorescence microscopy and confirmed by electron microscopy. Quantification of results obtained by fluorescence microscopy shows that Fg modification led to significantly higher substrate resorption by 17 days of culture. Our results show that osteoclasts, beyond resorbing mineralized substrates, successfully resorb a polymeric substrate (Ch), with Fg accelerating this process. Thus, in bone tissue regeneration strategies employing polymeric biomaterials, resorption may depend not only on macrophages, but also on osteoclasts.
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Davies JE, Ajami E, Moineddin R, Mendes VC. The roles of different scale ranges of surface implant topography on the stability of the bone/implant interface. Biomaterials 2013; 34:3535-46. [PMID: 23415644 DOI: 10.1016/j.biomaterials.2013.01.024] [Citation(s) in RCA: 65] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2012] [Accepted: 01/04/2013] [Indexed: 10/27/2022]
Abstract
We sought to deconvolute the effects of sub-micron topography and microtopography on the phenomena of bone bonding and interfacial stability of endosseous implants. To address this experimentally, we implanted custom-made titanium alloy implants of varying surface topographical complexity in rat femora, for 6, 9 or 12 days. The five surfaces were polished, machined, dual acid etched, and two forms of grit blasted and acid etched; each surface type was further modified with the deposition of nanocrystals of calcium phosphate to make a total of 10 materials groups (n = 10 for each time point; total 300 implants). At sacrifice, we subjected the bone-implant interface to a mechanical disruption test. We found that even the smoothest surfaces, when modified with sub-micron scale crystals, could be bone-bonding. However, as locomotor loading through bone to the implant increased with time of healing, such interfaces failed while others, with sub-micron features superimposed on surfaces of increasing microtopographical complexity remained intact under loading. We demonstrate here that higher order, micron or coarse-micron, topography is a requirement for longer-term interfacial stability. We show that each of these topographical scale-ranges represents a scale-range seen in natural bone tissue. Thus, what emerges from an analysis of our findings is a new means by which biologically-relevant criteria can be employed to assess the importance of implant surface topography at different scale-ranges.
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Affiliation(s)
- John E Davies
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario, Canada.
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35
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García-López S, Villanueva R, Meikle MC. Alterations in the Synthesis of IL-1β, TNF-α, IL-6, and Their Downstream Targets RANKL and OPG by Mouse Calvarial Osteoblasts In vitro: Inhibition of Bone Resorption by Cyclic Mechanical Strain. Front Endocrinol (Lausanne) 2013; 4:160. [PMID: 24194731 PMCID: PMC3809383 DOI: 10.3389/fendo.2013.00160] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/07/2013] [Accepted: 10/11/2013] [Indexed: 01/01/2023] Open
Abstract
Mechanical strain is an important determinant of bone mass and architecture, and the aim of this investigation was to further understand the role of the cell-cell signaling molecules, IL-1β, TNF-α, and IL-6 in the mechanobiology of bone. Mouse calvarial osteoblasts in monolayer culture were subjected to a cyclic out-of-plane deformation of 0.69% for 6 s, every 90 s for 2-48 h, and the levels of each cytokine plus their downstream targets RANKL and OPG measured in culture supernatants by ELISAs. Mouse osteoblasts constitutively synthesized IL-1β, TNF-α, and IL-6, the production of which was significantly up-regulated in all three by cyclic mechanical strain. RANKL and OPG were also constitutively synthesized; mechanical deformation however, resulted in a down-regulation of RANKL and an up-regulation OPG synthesis. We next tested whether the immunoreactive RANKL and OPG were biologically active in an isolated osteoclast resorption pit assay - this showed that culture supernatants from mechanically deformed cells significantly inhibited osteoclast-mediated resorptive activity across the 48 h time-course. These findings are counterintuitive, because IL-1β, TNF-α, and IL-6 have well-established reputations as bone resorptive agents. Nevertheless, they are pleiotropic molecules with multiple biological activities, underlining the complexity of the biological response of osteoblasts to mechanical deformation, and the need to understand cell-cell signaling in terms of cytokine networks. It is also important to recognize that osteoblasts cultured in vitro are deprived of the mechanical stimuli to which they are exposed in vivo - in other words, the cells are in a physiological default state that in the intact skeleton leads to decreased bone strains below the critical threshold required to maintain normal bone structure.
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Affiliation(s)
- Salvador García-López
- Health Science Department/Cell Biology and Immunology Laboratory, Universidad Autónoma Metropolitana-Xochimilco, Mexico City, Mexico
- Orthodontic Department, General Hospital “Dr. Manuel Gea González”, Universidad Nacional Autónoma de México, Mexico City, Mexico
- Orthodontic Department, Universidad Intercontinental, Mexico City, Mexico
| | - Rosina Villanueva
- Health Science Department/Cell Biology and Immunology Laboratory, Universidad Autónoma Metropolitana-Xochimilco, Mexico City, Mexico
| | - Murray C. Meikle
- Faculty of Dentistry, National University of Singapore, Singapore
- *Correspondence: Murray C. Meikle, Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, 119083 Singapore e-mail:
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Zhao H. Membrane trafficking in osteoblasts and osteoclasts: new avenues for understanding and treating skeletal diseases. Traffic 2012; 13:1307-14. [PMID: 22759194 DOI: 10.1111/j.1600-0854.2012.01395.x] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2012] [Revised: 06/28/2012] [Accepted: 07/03/2012] [Indexed: 12/21/2022]
Abstract
The endocytic and exocytic/secretory pathways are two major intracellular membrane trafficking routes that regulate numerous cellular functions in a variety of cell types. Osteoblasts and osteoclasts, two major bone cells responsible for bone remodeling and homeostasis, are no exceptions. During the past few years, emerging evidence has pinpointed a critical role for endocytic and secretory pathways in osteoblast and osteoclast differentiation and function. The endosomal membrane provides a platform to integrate bone tropic signals of hormones and growth factors in osteoblasts. In osteoclasts, endocytosis, followed by transcytosis, of degraded bone matrix promotes bone resorption. Secretory pathways, especially lysosome secretion, not only participate in bone matrix deposition by osteoblasts and degradation of mineralized bone matrix by osteoclasts; they may also be involved in the coupling of bone resorption and bone formation during bone remodeling. More importantly, mutations in genes encoding regulatory factors within the endocytic and secretory pathways have been identified as causes for bone diseases. Identification of the molecular mechanisms of these genes in bone cells may provide new therapeutic targets for skeletal disorders.
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Affiliation(s)
- Haibo Zhao
- Department of Internal Medicine, Center for Osteoporosis and Bone Metabolic Diseases, College of Medicine, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR, 72205, USA.
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Abstract
This chapter describes quantitative methods for isolating and culturing rodent osteoclasts on dentine, a bone-like, resorbable substrate. These techniques generate relatively large numbers of osteoclasts and allow the key processes of osteoclast formation and activation to be studied independently. A special focus will be on the role of extracellular pH, a critical factor in the control of osteoclast function.
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Affiliation(s)
- Isabel R Orriss
- Department of Cell and Developmental Biology, University College London, London, UK.
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Abstract
This chapter described methods for Scanning Electron Microscopical imaging of bone and bone cells. Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. This chapter considers preparing and imaging samples of unembedded bone having 3D detail in a 3D surface, topography-free, polished or micromilled, resin-embedded block surfaces, and resin casts of space in bone matrix. The chapter considers methods for fixation, drying, looking at undersides of bone cells, and coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralised matrix is described in detail. Attention is given especially to methods for 3D BSE SEM imaging of bone samples and recommendations for the types of resin embedding of bone for BSE imaging are given. Correlated confocal and SEM imaging of PMMA-embedded bone requires the use of glycerol to coverslip. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualising fluorescent mineralising front labels such as calcein and tetracyclines. Making spatial casts from PMMA or other resin embedded samples is an important use of this material. Correlation with other imaging means, including microradiography and microtomography is important. Shipping wet bone samples between labs is best done in glycerol. Environmental SEM (ESEM, controlled vacuum mode) is valuable in eliminating -"charging" problems which are common with complex, cancellous bone samples.
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Affiliation(s)
- Alan Boyde
- Biophysics Section, Oral Growth and Development, Dental Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
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Zhang Z, Egaña JT, Reckhenrich AK, Schenck TL, Lohmeyer JA, Schantz JT, Machens HG, Schilling AF. Cell-based resorption assays for bone graft substitutes. Acta Biomater 2012; 8:13-9. [PMID: 21971416 DOI: 10.1016/j.actbio.2011.09.020] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2011] [Revised: 09/16/2011] [Accepted: 09/20/2011] [Indexed: 01/28/2023]
Abstract
The clinical utilization of resorbable bone substitutes has been growing rapidly during the last decade, creating a rising demand for new resorbable biomaterials. An ideal resorbable bone substitute should not only function as a load-bearing material but also integrate into the local bone remodeling process. This means that these bone substitutes need to undergo controlled resorption and then be replaced by newly formed bone structures. Thus the assessment of resorbability is an important first step in predicting the in vivo clinical function of bone substitute biomaterials. Compared with in vivo assays, cell-based assays are relatively easy, reproducible, inexpensive and do not involve the suffering of animals. Moreover, the discovery of RANKL and M-CSF for osteoclastic differentiation has made the differentiation and cultivation of human osteoclasts possible and, as a result, human cell-based bone substitute resorption assays have been developed. In addition, the evolution of microscopy technology allows advanced analyses of the resorption pits on biomaterials. The aim of the current review is to give a concise update on in vitro cell-based resorption assays for analyzing bone substitute resorption. For this purpose models using different cells from different species are compared. Several popular two-dimensional and three-dimensional optical methods used for resorption assays are described. The limitations and advantages of the current ISO degradation assay in comparison with cell-based assays are discussed.
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Bozec L, Odlyha M. Thermal denaturation studies of collagen by microthermal analysis and atomic force microscopy. Biophys J 2011; 101:228-36. [PMID: 21723833 PMCID: PMC3127184 DOI: 10.1016/j.bpj.2011.04.033] [Citation(s) in RCA: 157] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2010] [Revised: 03/30/2011] [Accepted: 04/14/2011] [Indexed: 10/18/2022] Open
Abstract
The structural properties of collagen have been the subject of numerous studies over past decades, but with the arrival of new technologies, such as the atomic force microscope and related techniques, a new era of research has emerged. Using microthermal analysis, it is now possible to image samples as well as performing localized thermal measurements without damaging or destroying the sample itself. This technique was successfully applied to characterize the thermal response between native collagen fibrils and their denatured form, gelatin. Thermal transitions identified at (150 ± 10)°C and (220 ± 10)°C can be related to the process of gelatinization of the collagen fibrils, whereas at higher temperatures, both the gelatin and collagen samples underwent two-stage transitions with a common initial degradation temperature at (300 ± 10)°C and a secondary degradation temperature of (340 ± 10)°C for the collagen and of (420 ± 10)°C for the gelatin, respectively. The broadening and shift in the secondary degradation temperature was linked to the spread of thermal degradation within the gelatin and collagen fibrils matrix further away from the point of contact between probe and sample. Finally, similar measurements were performed inside a bone resorption lacuna, suggesting that microthermal analysis is a viable technique for investigating the thermomechanical response of collagen for in situ samples that would be, otherwise, too challenging or not possible using bulk techniques.
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Affiliation(s)
- Laurent Bozec
- Biomaterials and Tissue Engineering, Eastman Dental Institute, University College London, London, United Kingdom.
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Hou L, Hou J, Yao J, Zhou Z. Effects of osteoprotegerin from transfection of pcDNA3.1(+)/chOPG on bioactivity of chicken osteoclasts. Acta Vet Scand 2011; 53:21. [PMID: 21435269 PMCID: PMC3076242 DOI: 10.1186/1751-0147-53-21] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2010] [Accepted: 03/24/2011] [Indexed: 12/02/2022] Open
Abstract
Background Osteoprotegerin (OPG) has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models. However, the effects of OPG on bone metabolism in avian species have not been described. The objective of this study was to investigate the effects of chicken OPG (chOPG) expressed in chicken embryo fibroblasts (CEFs) on chicken osteoclast function in vitro. Methods The chOPG sequence containing the open reading frame (ORF) was amplified from chicken embryo frontal bone and inserted into the pcDNA3.1 (+) vector. PcDNA3.1 (+)/chOPG was transiently transfected into CEFs by lipofectamine 2000. Transcription of OPG mRNA and expression of chOPG recombinant protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence. The level of chOPG recombinant protein was detected by enzyme-linked immunosorbent assay. The suspension of osteoclasts was separated from chicken embryos and divided into three groups (control group, pcDNA3.1 (+) group and pcDNA3.1 (+)/chOPG group). The percentage of osteoclast apoptosis was detected by flow cytometry. The tartrate-resistant acid phosphatase (TRAP) secreted by osteoclasts was measured by the diazol method. The resorbing activity of osteoclasts was evaluated by the area of lacunae on bone flaps and the concentration of calcium in the supernatant liquid of osteoclasts. Results 48 h after transfection, the exogenous OPG gene transcription was detected by RT-PCR. After 72 h, the CEFs transfected from pcDNA3.1 (+)/chOPG displayed green fluorescence and the concentration of chOPG protein was 15.78 ± 0.22 ng/mL. After chicken osteoclasts were cultured for 5 d in a medium containing supernatant from transfected CEFs, the percentage of osteoclast apoptosis was increased significantly, the concentration of TRAP, the area of lacunae on bone flaps and calcium concentration were decreased significantly in the pcDNA3.1(+)/OPG group compared to the control group and the pcDNA3.1 (+) group. Conclusion Constructed pcDNA3.1 (+)/chOPG transfected into CEFs expressed bioactive OPG protein that was able to inhibit osteoclast function.
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PASCARETTI-GRIZON F, MABILLEAU G, BASLE M, CHAPPARD D. Measurement by vertical scanning profilometry of resorption volume and lacunae depth caused by osteoclasts on dentine slices. J Microsc 2011; 241:147-52. [DOI: 10.1111/j.1365-2818.2010.03410.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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Asano M, Yamaguchi M, Nakajima R, Fujita S, Utsunomiya T, Yamamoto H, Kasai K. IL-8 and MCP-1 induced by excessive orthodontic force mediates odontoclastogenesis in periodontal tissues. Oral Dis 2010; 17:489-98. [PMID: 21496183 DOI: 10.1111/j.1601-0825.2010.01780.x] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
OBJECTIVE The aim of this study was to investigate how interleukin (IL)-8 (cytokine-induced neutrophil chemoattractant; CINC-1) and monocyte chemotactic protein (MCP)-1/CCL2 contribute to root resorption during orthodontic tooth movement. MATERIALS AND METHODS Forty 6-week-old male Wistar rats were subjected to orthodontic force of 10 or 50 g to induce a mesially tipping movement of the upper first molars for 7 days. We determined the expressions of CINC-1, CXCR2, and MCP-1 proteins in root resorption area using immunohistochemistry. Furthermore, we investigated the effects of compression forces (CF) on IL-8 and MCP-1 production by human periodontal ligament (hPDL) cells. We observed an effect of chemokine treatment on rat odonto/osteoclasts in dentin slices that recapitulated root resorption. RESULTS The immunoreactivity for CINC-1/CXCR2 and MCP-1 was detected in odontoclasts and PDL fibroblasts by the orthodontic force of 50 g on day 7. CF increased the secretion and the expression of mRNA of IL-8 and MCP-1 from PDL cells in a magnitude-dependent manner. Moreover, CINC-1 and MCP-1 stimulated osteoclastogenesis from rat osteoclast precursor cells. CONCLUSION IL-8 (CINC-1) and MCP-1 may therefore facilitate the process of root resorption because of excessive orthodontic force.
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Affiliation(s)
- M Asano
- Department of Orthodontics, Nihon University School of Dentistry at Matsudo, Chiba, Japan
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Rissanen JP, Halleen JM. Models and screening assays for drug discovery in osteoporosis. Expert Opin Drug Discov 2010; 5:1163-74. [DOI: 10.1517/17460441.2010.532484] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Rissanen JP, Ylipahkala H, Fagerlund KM, Long C, Väänänen HK, Halleen JM. Improved methods for testing antiresorptive compounds in human osteoclast cultures. J Bone Miner Metab 2009; 27:105-9. [PMID: 19018457 DOI: 10.1007/s00774-008-0002-1] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/02/2007] [Accepted: 02/11/2008] [Indexed: 10/21/2022]
Abstract
We cultured human bone marrow-derived stem cells on bovine bone slices in 96-well plates in the presence of M-CSF and RANKL, allowing them to differentiate into osteoclasts. Secreted TRACP 5b was a useful endpoint measurement to demonstrate effects of inhibitors of osteoclast differentiation in the culture system, reflecting accurately the number of formed osteoclasts. Inhibitors of osteoclast activity were added into the cultures after the differentiation period, and the cultures were continued to allow the formed osteoclasts to resorb bone. CTX values obtained after the resorption period were normalized with TRACP 5b values obtained after the differentiation period, before adding the inhibitors. This normalization prevents false results that could be obtained from the presence of different amounts of osteoclasts in different wells before adding the inhibitors. These results demonstrate that the use of TRACP 5b and CTX allows rapid and reliable testing of antiresorptive compounds in human osteoclast cultures.
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Affiliation(s)
- Jukka P Rissanen
- Pharmatest Services Ltd., Itäinen Pitkäkatu 4 C, 20520 Turku, Finland
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Sriarj W, Aoki K, Ohya K, Takagi Y, Shimokawa H. Bovine dentine organic matrix down-regulates osteoclast activity. J Bone Miner Metab 2009; 27:315-23. [PMID: 19296049 DOI: 10.1007/s00774-009-0063-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/11/2008] [Accepted: 11/13/2008] [Indexed: 01/24/2023]
Abstract
Physiological root resorption is a phenomenon that normally takes place in deciduous teeth; root resorption of permanent teeth occurs only under pathological conditions. The molecular mechanisms underlying these processes are still unclear. Our previous study showed that osteoclasts cultured on deciduous dentine exhibited a higher degree of resorption and higher levels of cathepsin K and MMP-9 mRNA than osteoclasts cultured on permanent dentine. These results could be because of different susceptibilities to acid and the different organic matrices between deciduous and permanent dentine. Thus, the purpose of this study was to investigate the effect of dentine extracts from bovine deciduous and permanent dentine on osteoclast activity. Osteoclasts, obtained from mouse bone marrow cells co-cultured with an osteoblast-rich fraction in the presence of 1,25-(OH)(2)-vitamin D3 and PGE2, were incubated with or without 0.6 M HCl extracts from bovine deciduous or permanent dentine for 48 h. TRAP positive cell number, TRAP activity, the areas of resorption pits, and mRNA levels of TRAP, v-ATPase, calcitonin receptor, cathepsin K, and MMP-9 were examined. The results illustrated that TRAP activity, the resorbed area, and the mRNA levels of osteoclast marker genes seemed to be suppressed by both deciduous and permanent dentine extracts. These findings indicate that some factors that suppress osteoclast activity are contained in both deciduous and permanent dentine extracts. Although there was no significant difference in osteoclast activity between deciduous and permanent dentine extracts, osteoclasts incubated with permanent dentine extracts tend to exhibit less resorption activity than those incubated with deciduous dentine extracts. However, we could not clearly explain the causes of this.
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Affiliation(s)
- Wantida Sriarj
- Section of Developmental Oral Health Science, Department of Orofacial Development and Function, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
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Greiner S, Kadow-Romacker A, Wildemann B, Schwabe P, Schmidmaier G. Bisphosphonates incorporated in a poly(D,L-lactide) implant coating inhibit osteoclast like cells in vitro. J Biomed Mater Res A 2008; 83:1184-1191. [PMID: 17595027 DOI: 10.1002/jbm.a.31444] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Nitrogen containing bisphosphonates such as zoledronic acid (ZOL) are clinically used to prevent osteoclast induced bone loss. Previous studies indicated that bisphosphonates prevent osteoclast formation, decreases their resorption activity and lead to osteoclast apoptosis. Aim of the study was to investigate the effect of ZOL on fusion and resorption activity of osteoclast like cells (OLC) derived from human peripheral blood mononuclear cells (PBMNC) in vitro. For application of ZOL a local drug delivery system based on a coating for medical devices was used. ZOL was incorporated in the coating based on Poly(D,L-Lactide) (PDLLA) in different concentrations (10-50 microM). Control groups were treated without ZOL or ZOL pure substance in corresponding concentrations. Human PBMNCs were isolated and stimulated to form OLCs. After an experimental period of 144 h, TRAP staining of polynucleated cells was performed and TRAP positive cells were counted. A pit formation assay was performed and resorption lacunas on dentin chips were counted. Results showed a significant dose dependent decrease in the number of TRAP positive cells after exposure to ZOL incorporated in the drug delivery system or applied as pure substance. The amount of resorption lacunas was also dose dependent decreased using both application methods. In conclusion, exposure to specific concentrations of ZOL incorporated in a drug delivery system showed a significant decrease in OLC formation and activity comparable to the effect of pure substance. The effect on osteoclasts might be of clinical benefit to reduce orthopedic implant loosening and to support fracture healing.
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Affiliation(s)
- Stefan Greiner
- Center for Musculoskeletal Surgery, Charité-Universitätsmedizin Berlin, Germany
| | - Anke Kadow-Romacker
- Center for Musculoskeletal Surgery, Charité-Universitätsmedizin Berlin, Germany
| | - Britt Wildemann
- Center for Musculoskeletal Surgery, Charité-Universitätsmedizin Berlin, Germany
| | - Philipp Schwabe
- Center for Musculoskeletal Surgery, Charité-Universitätsmedizin Berlin, Germany
| | - Gerhard Schmidmaier
- Center for Musculoskeletal Surgery, Charité-Universitätsmedizin Berlin, Germany
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Apoptotic bodies convey activity capable of initiating osteoclastogenesis and localized bone destruction. J Bone Miner Res 2008; 23:915-27. [PMID: 18435576 DOI: 10.1359/jbmr.080207] [Citation(s) in RCA: 157] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
INTRODUCTION Osteocyte apoptosis co-localizes with sites of osteoclastic bone resorption in vivo, but to date, no causal molecular or signaling link has been identified between these two processes. MATERIALS AND METHODS Osteocyte apoptotic bodies (OABs) derived from the MLO-Y4 osteocyte-like cell line and primary murine osteocytes and apoptotic bodies (ABs) derived from primary murine osteoblasts were introduced onto the right parietal bone of murine calvariae, and osteoclastic bone resorption was examined 5 days after treatment. In addition, the ability of primary murine and cell line-derived OABs to support osteoclastogenesis was examined in vitro in co-culture with murine bone marrow hematopoietic progenitors in the absence of RANKL or macrophage-colony stimulating factor. RESULTS For the first time, we show that OABs are capable of initiating de novo osteoclastic bone resorption on quiescent bone surfaces in vivo. Furthermore, the addition of OABs to mononuclear osteoclast precursors (OPs) in vitro resulted in the maintenance of OP cell numbers and an increase in the proportion and activity of TRACP(+) cells. In contrast, application of ABs from osteoblasts showed no osteoclastogenic activity either in vivo or in vitro. The osteoclastogenic capacity of OABs was shown to be independent of the known osteoclastogenic factor RANKL but dependent on the induction of TNF-alpha production by OP. CONCLUSIONS These data point to a mechanism by which dying osteocytes might target bone destruction through the distribution of OAB-associated signals and give further physiological meaning to the apoptotic process in bone.
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Mulari MT, Nars M, Laitala-Leinonen T, Kaisto T, Metsikkö K, Sun Y, Väänänen HK. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts. Exp Cell Res 2008; 314:1641-51. [DOI: 10.1016/j.yexcr.2008.02.011] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2007] [Revised: 02/10/2008] [Accepted: 02/15/2008] [Indexed: 10/22/2022]
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