Dominique Heymann

Osteoclastic resorption of calcium phosphate ceramics with different hydroxyapatite/β-tricalcium phosphate ratios

To study the influence of calcium phosphate ceramic solubility on osteoclastic resorption, neonatal rabbit bone cells were cultured for 2 days on hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP) and two types of biphasic calcium phosphate (BCP) with HA/beta-TCP ratios of 25/75 and 75/25. Solubility was regulated by varying the ratio of less-soluble HA and more-soluble beta-TCP. After removal of stromal cells by pronase E treatment, ceramic surfaces were observed by scanning electron microscopy. Osteoclasts resorbed BCP most extensively, with an HA/beta-TCP ratio of 25/75, producing typical lobulated, zig-zag track-like resorption lacunae. On pure beta-TCP, which had the highest solubility in acid, osteoclasts formed smaller discontinuous island-like lacunae. The resorption pattern may have been modified by the large number of calcium ions released into the acidic microenvironment at the osteoclast-ceramic interface. No resorption lacunae were found on the other specimens. The extent of osteoclastic resorption of calcium phosphate ceramics might, to a certain degree, be proportional to solubility, although this was not the case when solubility was very high. It would appear that ceramic solubility influences osteoclast resorption activity.

Receptor Activator of Nuclear Factor κB Ligand (RANKL)/Osteoprotegerin (OPG) Ratio Is Increased in Severe Osteolysis

Pathological osteolyses are considered a consequence of a disturbance in the mechanisms that govern the bone remodeling, mainly the communication between osteoclasts and osteoblasts. Osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) are newly discovered molecules that play a key role in these communications. RANKL is essential for osteoclast differentiation via its receptor RANK located on the osteoclast membrane. OPG is a soluble decoy receptor that inhibits osteoclast differentiation through its binding to RANKL. The aim of this study is the analysis of the RANKL/OPG balance by complementary methods (semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and enzyme-linked immunosorbent assay) in human osteolysis associated to various bone etiologies (n = 60), tumoral (primitive, secondary) or not, compared to healthy tissues (n = 16). Results demonstrated that RANKL/OPG ratio was significantly increased in patients suffering from severe osteolysis compared to the control group and that this imbalance is involved in bone resorption mechanisms. In this study, OPG expression appears to reflect a protective mechanism of the skeleton to compensate increased bone resorption by inhibiting osteoclast formation and bone resorbing activity. Moreover, as revealed by immunohistochemistry, RANKL and OPG were colocalized in all of the tissues analyzed. To define the veracity of RANKL/OPG index in assessing and managing patients with severe osteolysis, an extended population of patients suffering from severe osteolysis must be now monitored.

Recent advances in TGF-beta effects on chondrocyte metabolism. Potential therapeutic roles of TGF-beta in cartilage disorders.

Novel approaches to treat osteoarthritis are required and progress in understanding the biology of cartilage disorders has led to the use of genes whose products stimulate cartilage repair or inhibit breakdown of the cartilaginous matrix. Among them, transforming growth factor-β (TGF-β) plays a significant role in promoting chondrocyte anabolism in vitro (enhancing matrix production, cell proliferation, osteochondrogenic differentiation) and in vivo (short-term intra-articular injections lead to increased bone formation and subsequent cartilage formation, beneficial effects on osteochondrogenesis). In vivo induction of the expression of TGF-β and the use of gene transfer may provide a new approach for treatment of osteoarthritic lesions.

Mechanisms of bone repair and regeneration.

Bone problems can have a highly deleterious impact on life and society, therefore understanding the mechanisms of bone repair is important. In vivo studies show that bone repair processes in adults resemble normal development of the skeleton during embryogenesis, which can thus be used as a model. In addition, recent studies of skeletal stem cell biology have underlined several crucial molecular and cellular processes in bone formation. Hedgehog, parathyroid hormone-related protein, Wnt, bone morphogenetic proteins and mitogen-activated protein kinases are the main molecular players, and osteoclasts and mesenchymal stem cells are the main cells involved in these processes. However, questions remain regarding the precise mechanisms of bone formation, how the different molecular processes interact, and the real identity of regenerative cells. Here, we review recent studies of bone regeneration and repair. A better understanding of the underlying mechanisms is expected to facilitate the development of new strategies for improving bone repair.

Induction of Osteogenesis in Mesenchymal Stem Cells by Activated Monocytes/Macrophages Depends on Oncostatin M Signaling†‡§

Bone resorption by osteoclasts and bone formation by osteoblasts are tightly coupled processes implicating factors in TNF, bone morphogenetic protein, and Wnt families. In osteoimmunology, macrophages were described as another critical cell population regulating bone formation by osteoblasts but the coupling factors were not identified. Using a high‐throughput approach, we identified here Oncostatin M (OSM), a cytokine of the IL‐6 family, as a major coupling factor produced by activated circulating CD14+ or bone marrow CD11b+ monocytes/macrophages that induce osteoblast differentiation and matrix mineralization from human mesenchymal stem cells while inhibiting adipogenesis. Upon activation of toll‐like receptors (TLRs) by lipopolysaccharide or endogenous ligands, OSM was produced in classically activated inflammatory M1 and not M2 macrophages, through a cyclooxygenase‐2 and prostaglandin‐E2 regulatory loop. Stimulation of osteogenesis by activated monocytes/macrophages was prevented using neutralizing antibodies or siRNA to OSM, OSM receptor subunits gp130 and OSMR, or to the downstream transcription factor STAT3. The induced osteoblast differentiation program culminated with enhanced expression of CCAAT‐enhancer‐binding protein δ, Cbfa1, and alkaline phosphatase. Overexpression of OSM in the tibia of mice has led to new bone apposition with no sign of bone resorption. Two other cytokines have also a potent role in bone formation induced by monocytes/macrophages and activation of TLRs: IL‐6 and leukemia inhibitory factor. We propose that during bone inflammation, infection, or injury, the IL‐6 family signaling network activated by macrophages and TLR ligands stimulates bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies. STEM CELLS 2012; 30:762–772

Osteoclastic acidification pathways during bone resorption.

Osteoclasts resorb bone by attaching to the surface and then secreting protons into an extracellular compartment formed between osteoclast and bone surface. This secretion is necessary for bone mineral solubilization and the digestion of organic bone matrix by acid proteases. This study summarizes the characterization and role of each type of ion transport and defines the main biochemical mechanisms involved in the dissolution of bone mineral during bone resorption. The primary mechanism responsible for acidification of the osteoclast-bone interface is vacuolar H+-adenosine triphosphatase (ATPase) coupled with Cl- conductance localized to the ruffled membrane. Carbonic anhydrase II (CAII) provides the proton source for extracellular acidification by H+-ATPase and the HCO3- source for the HCO3-/Cl- exchanger. Whereas some transporters are responsible for the bone resorption process, others are essential for pH regulation in the osteoclast. The HCO3-/Cl- exchanger, in association with CAII, is the major transporter for maintenance of normal intracellular pH. An Na+/H+ antiporter may also contribute to the recovery of intracellular pH during early osteoclast activation. Once this mechanism has been rendered inoperative, another conductive pathway translocates the protons and modulates cytoplasmic pH. Inward-rectifying K+ channels may also be involved by compensating for the external acidification due to H+ transport. These different effects of transport processes, either on bone resorption or pH homeostasis, increase the number of possible sites for pharmacological intervention in the treatment of metabolic bone diseases.

Therapeutic Relevance of Osteoprotegerin Gene Therapy in Osteosarcoma: Blockade of the Vicious Cycle between Tumor Cell Proliferation and Bone Resorption

Osteosarcoma is the most frequent primary bone tumor that develops mainly in the young, the median age of diagnosis being 18 years. Despite improvement in osteosarcoma treatment, survival rate is only 30% at 5 years for patients with pulmonary metastases at diagnosis. This warrants exploration of new therapeutic options, and among them, osteoprotegerin (OPG), a naturally occurring protein that inhibits bone resorption, is very promising in blocking the vicious cycle between bone resorption and tumor proliferation that takes place during tumor development in bone site. As OPG binds and inhibits the activity of tumor necrosis factor-related apoptosis-inducing ligand, the truncated form of murine OPG 1-194 was used. The cDNA encoding OPG was administered by gene transfer using replication-defective adenoviral vector or was associated with an amphiphilic polymer in two models of rodent osteosarcoma. In both models, OPG gene transfer was effective in preventing the formation of osteolytic lesions associated with osteosarcoma development, in reducing the tumor incidence and the local tumor growth, leading to a 4-fold augmentation of mice survival 28 days postimplantation. On the contrary, OPG did not prevent the development of pulmonary metastasis alone, suggesting that bone environment is necessary for OPG therapeutic efficacy. Because OPG has no direct activity on osteosarcoma cells in vitro (cell binding, cell proliferation, apoptosis, or cell cycle distribution), we show that OPG exerts indirect inhibitory effect on tumor progression through the inhibition of RANKL whose production is enhanced in bone tumor environment, leading to osteolysis inhibition as reflected by osteoclast number decrease.

RANKL, RANK, osteoprotegerin : key partners of osteoimmunology and vascular diseases

Abstract.1997 saw the identification of a novel set of proteins within the tumor necrosis factor (TNF)/TNF receptor families that are required for the control of bone remodeling. Therefore, these receptors, receptor activator of nuclear factor kappa B (RANK), osteoprotegerin (OPG) and their ligand RANK ligand (RANKL) became the critical molecular triad controlling osteoclastogenesis and pathophysiologic bone remodeling. However, the establishment of the corresponding knock-out and transgenic mice revealed unexpected results, most particularly, the involvement of these factors in the vascular system and immunity. Thus, the OPG/RANK/RANKL molecular triad appears to be associated with vascular calcifications and plays a pivotal function in the development of the immune system through dendritic cells. OPG/RANK/RANKL thus constitute a molecular bridge spanning bone metabolism, vascular biology and immunity. This review summarizes recent knowledge of OPG/RANK/RANKL interactions and activities as well as the current evidence for their participation in osteoimmunology and vascular diseases. In fine, the targeting of the OPG/RANK/RANKL axis as novel therapeutic approaches will be discussed.

Zoledronic Acid Suppresses Lung Metastases and Prolongs Overall Survival of Osteosarcoma-Bearing Mice

Although there is no doubt that bisphosphonates (BPs), specific inhibitors of osteoclasts, are beneficial for the treatment of bone metastases, their effects on visceral metastases are unclear. The effect of zoledronic acid (ZOL) was examined in vivo on lung metastasis progression and animal survival, and in vitro on the cellular mechanisms involved.

Influence of biphasic calcium phosphate granulometry on bone ingrowth, ceramic resorption, and inflammatory reactions: preliminary in vitro and in vivo study.

Calcium-phosphate ceramics used in surgery, as bone-bonding materials, are currently available in different forms (blocks, granules, etc.). However, progress in noninvasive surgery has favored the development of injectable composite materials associating a polymeric and a dusty mineral phase. The purpose of this study was the in vivo evaluation of biphasic calcium phosphate of various grains sizes, to elucidate the role of granulometries in ceramic degradation/resorption, bone ingrowth, and inflammatory reactions. Three particle sizes were compared: 10-20, 80-100, and 200-400 microm. The 10-20-microm powders provided the best bone ingrowth, with a higher resorption/degradation rate in conjunction with stronger early inflammatory reactions. The 200-400-microm powders showed higher bone ingrowth than 80-100-microm ones, indicating that properties of cell recruitment for osseous apposition and mechanical support for bone bonding may both play a role in both ingrowth mechanisms. Our results suggest that the strong inflammatory reaction in 10-20-microm granulated powders was due to a faster reversal of the resorption/apposition sequence in bone. This may have resulted from massive release of bone ingrowth factors, which implies that the brief inflammatory process observed in the early stages of implantation was favorable to the osteoconduction process.