Cellular biology of fracture healing

The biology of bone healing is a rapidly developing science. Advances in transgenic and gene‐targeted mice have enabled tissue and cell‐specific investigations of skeletal regeneration. As an example, only recently has it been recognized that chondrocytes convert to osteoblasts during healing bone, and only several years prior, seminal publications reported definitively that the primary tissues contributing bone forming cells during regeneration were the periosteum and endosteum. While genetically modified animals offer incredible insights into the temporal and spatial importance of various gene products, the complexity and rapidity of healing—coupled with the heterogeneity of animal models—renders studies of regenerative biology challenging. Herein, cells that play a key role in bone healing will be reviewed and extracellular mediators regulating their behavior discussed. We will focus on recent studies that explore novel roles of inflammation in bone healing, and the origins and fates of various cells in the fracture environment. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop ResAdvances in transgenic and gene‐targeted mice have enabled tissue and cell‐specific investigation of skeletal regeneration. While genetically modified animals offer incredible insights into the temporal and spatial importance of various molecules, the complexity and rapidity of healing renders studies of regenerative biology challenging. Herein, cells and extracellular mediators that play a key role in bone healing are reviewed. We will focus on recent studies that explore the origins and fates of various cells in the fracture environment.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148261/1/jor24170_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148261/2/jor24170-sup-0002-SuppTab-S2.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148261/3/jor24170-sup-0001-SuppTab-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148261/4/jor24170.pd

The Architecture of the Adhesive Apparatus of Cultured Osteoclasts: From Podosome Formation to Sealing Zone Assembly

BACKGROUND: Osteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood. PRINCIPAL FINDINGS: Combining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers. SIGNIFICANCE: The sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions

Substrate Adhesion Regulates Sealing Zone Architecture and Dynamics in Cultured Osteoclasts

The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data

Genome-Wide Profiling of MicroRNAs in Adipose Mesenchymal Stem Cell Differentiation and Mouse Models of Obesity

In recent years, there has been accumulating evidence that microRNAs are key regulator molecules of gene expression. The cellular processes that are regulated by microRNAs include e.g. cell proliferation, programmed cell death and cell differentiation. Adipocyte differentiation is a highly regulated cellular process for which several important regulating factors have been discovered, but still not all are known to fully understand the underlying mechanisms. In the present study, we analyzed the expression of 597 microRNAs during the differentiation of mouse mesenchymal stem cells into terminally differentiated adipocytes by real-time RT-PCR. In total, 66 miRNAs were differentially expressed in mesenchymal stem cell-derived adipocytes compared to the undifferentiated progenitor cells. To further study the regulation of these 66 miRNAs in white adipose tissue in vivo and their dependence on PPARγ activity, mouse models of genetically or diet induced obesity as well as a mouse line expressing a dominant negative PPARγ mutant were employed

Bone Is Not Essential for Osteoclast Activation

Background: The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that avb3 ligands are important, because blockade of avb3 receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether avb3 ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption. Methodology/Principal Findings: Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading. Conclusions/Significance: Our results show that ligands avb3 are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative ‘mineral receptor’

The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Prevention of Wear Particle-Induced Osteolysis by a Novel V-ATPase Inhibitor Saliphenylhalamide through Inhibition of Osteoclast Bone Resorption

Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis

Distinctive subdomains in the resorbing surface of osteoclasts.

We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a 'sealing zone', encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but also to facilitate resorption by acting as a 'functional sealing zone'

Dasatinib as a Bone-Modifying Agent: Anabolic and Anti-Resorptive Effects

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function. [Methods]: For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model. [Results]: Dasatinib inhibited the platelet derived growth factor receptor-β (PDGFR-β), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2-5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1-2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVβ3 integrin and CCR1 expression. [Conclusions]: Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease. © 2012 Garcia-Gomez et al.This work was supported by grants from the Spanish Ministry of Science and Innovation – ISCIII (PI081825); Mutua Madrileña Medical Research Foundation (AP27262008); Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León, Consejería de Sanidad JCyL – ISCIII; the Cooperative Research Thematic Network in Cancer (RTICC; RD06/0020/0006 and RD03/0020/0041); and Spanish FIS (PS09/01897). AG-G and CS are supported by the Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León Project.Peer Reviewe