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1.  Role of polyethylene particles in peri-prosthetic osteolysis: A review 
World Journal of Orthopedics  2011;2(10):93-101.
There is convincing evidence that particles produced by the wear of joint prostheses are causal in the peri-prosthetic loss of bone, or osteolysis, which, if it progresses, leads to the phenomenon of aseptic loosening. It is important to fully understand the biology of this bone loss because it threatens prosthesis survival, and loosened implants can result in peri-prosthetic fracture, which is disastrous for the patient and presents a difficult surgical scenario. The focus of this review is the bioactivity of polyethylene (PE) particles, since there is evidence that these are major players in the development and progression of osteolysis around prostheses which use PE as the bearing surface. The review describes the biological consequences of interaction of PE particles with macrophages, osteoclasts and cells of the osteoblast lineage, including osteocytes. It explores the possible cellular mechanisms of action of PE and seeks to use the findings to date to propose potential non-surgical treatments for osteolysis. In particular, a non-surgical approach is likely to be applicable to implants containing newer, highly cross-linked PEs (HXLPEs), for which osteolysis seems to occur with much reduced PE wear compared with conventional PEs. The caveat here is that we know little as yet about the bioactivity of HXLPE particles and addressing this constitutes our next challenge.
PMCID: PMC3302032  PMID: 22474627
Polyethylene; Aseptic loosening; Osteolysis; Wear particle; Peri-prosthetic
2.  Pharmacologic inhibition of bone resorption prevents cancer-induced osteolysis but enhances soft tissue metastasis in a mouse model of osteolytic breast cancer 
International Journal of Oncology  2014;45(2):532-540.
Osteoprotegerin (OPG) is a secreted member of the TNF receptor superfamily, which binds to the receptor activator of nuclear factor κB ligand (RANKL) and inhibits osteoclast activity and bone resorption. Systemic administration of recombinant OPG was previously shown to inhibit tumor growth in bone and to prevent cancer-induced osteolysis. In this study, we examined the effect of OPG, when produced locally by breast cancer cells located within bone, using a mouse model of osteolytic breast cancer. MDA-MB-231-TXSA breast cancer cells, tagged with a luciferase reporter gene construct and engineered to overexpress full-length human OPG, were transplanted directly into the tibial marrow cavity of nude mice. Overexpression of OPG by breast cancer cells protected the bone from breast cancer-induced osteolysis and diminished intra-osseous tumor growth but had no effect on extra-skeletal tumor growth. This effect was associated with a significant reduction in the number of osteoclasts that lined the bone surface, resulting in a net increase in bone volume. Despite limiting breast cancer-mediated bone loss, OPG overexpression resulted in a significant increase in the incidence of pulmonary metastasis. Our results demonstrate that inhibition of osteoclastic bone resorption by OPG when secreted locally by tumors in bone may affect the behaviour of cancer cells within the bone microenvironment and their likelihood of spreading and establishing metastasis elsewhere in the body.
PMCID: PMC4091969  PMID: 24865346
osteoprotegerin; bone metastasis; bone resorption; antiresorptive agents; bisphosphonates
3.  Sclerostin Is a Locally Acting Regulator of Late-Osteoblast/Preosteocyte Differentiation and Regulates Mineralization Through a MEPE-ASARM-Dependent Mechanism 
Journal of Bone and Mineral Research  2011;26(7):1425-1436.
The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unknown. Since sclerostin is expressed in vivo by mineral-embedded osteocytes, we tested the hypothesis that sclerostin would regulate the behavior of cells actively involved in mineralization in adult bone, the preosteocyte. Differentiating cultures of human primary osteoblasts exposed to recombinant human sclerostin (rhSCL) for 35 days displayed dose- and time-dependent inhibition of in vitro mineralization, with late cultures being most responsive in terms of mineralization and gene expression. Treatment of advanced (day 35) cultures with rhSCL markedly increased the expression of the preosteocyte marker E11 and decreased the expression of mature markers DMP1 and SOST. Concomitantly, matrix extracellular phosphoglycoprotein (MEPE) expression was increased by rhSCL at both the mRNA and protein levels, whereas PHEX was decreased, implying regulation through the MEPE-ASARM axis. We confirmed that mineralization by human osteoblasts is exquisitely sensitive to the triphosphorylated ASARM-PO4 peptide. Immunostaining revealed that rhSCL increased the endogenous levels of MEPE-ASARM. Importantly, antibody-mediated neutralization of endogenous MEPE-ASARM antagonized the effect of rhSCL on mineralization, as did the PHEX synthetic peptide SPR4. Finally, we found elevated Sost mRNA expression in the long bones of HYP mice, suggesting that sclerostin may drive the increased MEPE-ASARM levels and mineralization defect in this genotype. Our results suggest that sclerostin acts through regulation of the PHEX/MEPE axis at the preosteocyte stage and serves as a master regulator of physiologic bone mineralization, consistent with its localization in vivo and its established role in the inhibition of bone formation.
PMCID: PMC3358926  PMID: 21312267
4.  Osteoblast-Chondrocyte Interactions in Osteoarthritis 
Current Osteoporosis Reports  2014;12(1):127-134.
There is now general agreement that osteoarthritis (OA) involves all structures in the affected joint, culminating in the degradation of the articular cartilage. It is appropriate to focus particularly on the subchondral bone because characteristic changes occur in this tissue with disease progression, either in parallel, or contributing to, the loss of cartilage volume and quality. Changes in both the articular cartilage and the subchondral bone are mediated by the cells in these two compartments, chondrocytes and cells of the osteoblast lineage, respectively, whose primary roles are to maintain the integrity and function of these tissues. In addition, altered rates of bone remodeling across the disease process are due to increased or decreased osteoclastic bone resorption. In the altered mechanical and biochemical environment of a progressively diseased joint, the cells function differently and show a different profile of gene expression, suggesting direct effects of these external influences. There is also ex vivo and in vitro evidence of chemical crosstalk between the cells in cartilage and subchondral bone, suggesting an interdependence of events in the two compartments and therefore indirect effects of, for example, altered loading of the joint. It is ultimately these cellular changes that explain the altered morphology of the cartilage and subchondral bone. With respect to crosstalk between the cells in cartilage and bone, there is evidence that small molecules can transit between these tissues. For larger molecules, such as inflammatory mediators, this is an intriguing possibility but remains to be demonstrated. The cellular changes during the progression of OA almost certainly need to be considered in a temporal and spatial manner, since it is important when and where observations are made in either human disease or animal models of OA. Until recently, comparisons have been made with the assumption, for example, that the subchondral bone is behaviorally uniform, but this is not the case in OA, where regional differences of the bone are evident using magnetic resonance imaging (MRI). Nevertheless, an appreciation of the altered cell function during the progression of OA will identify new disease modifying targets. If, indeed, the cartilage and subchondral bone behave as an interconnected functional unit, normalization of cell behavior in one compartment may have benefits in both tissues.
PMCID: PMC3933767  PMID: 24458429
Osteoarthritis; Articular; Cartilage; Subchondral; Bone; Osteoblasts; Chondrocytes; Gene expression; Communication; Pathogenesis; Transforming growth factor; Sclerostin
5.  Critical role of p38 MAPK for regeneration of the sciatic nerve following crush injury in vivo 
The physiological function of p38α, which is an isoform of p38 MAPK, has been investigated previously in several studies using pharmacological inhibitors. However, the results regarding whether p38α promotes or inhibits nerve regeneration in vivo have been controversial.
We generated novel p38α mutant mice (sem mice) with a point mutation in the region encoding the p38α substrate-docking-site, which serves as a limited loss-of-function model of p38α. In the present study, we utilized sem mice and wild-type littermates (wt mice) to investigate the physiological role of p38α in nerve regeneration following crush injuries.
At four weeks after crush injury, the average axon diameter and the average axon area in sem mice were significantly smaller than those in wt mice. The average myelin sheath thickness in sem mice was reduced compared to wt mice, but no significant difference was observed in the G-ratio between the two groups. The sciatic functional index value demonstrated that functional nerve recovery in sem mice following crush injury was delayed, which is consistent with the histological findings. To investigate the underlying mechanisms of these findings, we examined inflammatory responses of the sciatic nerve by immunohistochemistry and western blotting. At an early phase following crush injury, sem mice showed remarkably lower expression of inflammatory cytokines, such as TNF-α and IL-1β, than wt mice. The expression of Caspase-3 and Tenascin-C were also lower in sem mice. Conversely, at a late phase of the response, sem mice showed considerably higher expression of TNF-α and of IL-1β with lower expression of S-100 than wt mice.
This is the first study of the physiological role of p38 MAPK in nerve regeneration that does not rely on the use of pharmacological inhibitors. Our results indicate that p38α insufficiency may cause an inflammatory disorder, resulting in a delay of histological and functional nerve recovery following crush injury. We conclude that p38 MAPK has an important physiological role in nerve regeneration and may be important for controlling both initiation of inflammation and recovery from nerve injury.
PMCID: PMC3541116  PMID: 23282009
P38 MAPK; Nerve regeneration; Crush injury; Inflammatory cytokines; TNF-α; IL-1β; Mutant mice; In vivo
6.  Characterization of drug-release kinetics in trabecular bone from titania nanotube implants 
The aim of this study was to investigate the application of the three-dimensional bone bioreactor for studying drug-release kinetics and distribution of drugs in the ex vivo cancellous bone environment, and to demonstrate the application of nanoengineered titanium (Ti) wires generated with titania nanotube (TNT) arrays as drug-releasing implants for local drug delivery
Nanoengineered Ti wires covered with a layer of TNT arrays implanted in bone were used as a drug-releasing implant. Viable bovine trabecular bone was used as the ex vivo bone substrate embedded with the implants and placed in the bone reactor. A hydrophilic fluorescent dye (rhodamine B) was used as the model drug, loaded inside the TNT–Ti implants, to monitor drug release and transport in trabecular bone. The distribution of released model drug in the bone was monitored throughout the bone structure, and concentration profiles at different vertical (0–5 mm) and horizontal (0–10 mm) distances from the implant surface were obtained at a range of release times from 1 hour to 5 days.
Scanning electron microscopy confirmed that well-ordered, vertically aligned nanotube arrays were formed on the surface of prepared TNT–Ti wires. Thermogravimetric analysis proved loading of the model drug and fluorescence spectroscopy was used to show drug-release characteristics in-vitro. The drug release from implants inserted into bone ex vivo showed a consistent gradual release of model drug from the TNT–Ti implants, with a characteristic three-dimensional distribution into the surrounding bone, over a period of 5 days. The parameters including the flow rate of bone culture medium, differences in trabecular microarchitecture between bone samples, and mechanical loading were found to have the most significant influence on drug distribution in the bone.
These results demonstrate the utility of the Zetos™ system for ex vivo drug-release studies in bone, which can be applied to optimize the delivery of specific therapies and to assist in the design of new drug delivery systems. This method has the potential to provide new knowledge to understand drug distribution in the bone environment and to considerably improve existing technologies for local administration in bone, including solving some critical problems in bone therapy and orthopedic implants.
PMCID: PMC3446838  PMID: 23028217
local drug delivery; Zetos bone bioreactor; drug-releasing implant; drug diffusion
7.  A Bioinformatics Resource for TWEAK-Fn14 Signaling Pathway 
Journal of Signal Transduction  2012;2012:376470.
TNF-related weak inducer of apoptosis (TWEAK) is a new member of the TNF superfamily. It signals through TNFRSF12A, commonly known as Fn14. The TWEAK-Fn14 interaction regulates cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis, tissue remodeling and inflammation. Although TWEAK has been reported to be associated with autoimmune diseases, cancers, stroke, and kidney-related disorders, the downstream molecular events of TWEAK-Fn14 signaling are yet not available in any signaling pathway repository. In this paper, we manually compiled from the literature, in particular those reported in human systems, the downstream reactions stimulated by TWEAK-Fn14 interactions. Our manual amassment of the TWEAK-Fn14 pathway has resulted in cataloging of 46 proteins involved in various biochemical reactions and TWEAK-Fn14 induced expression of 28 genes. We have enabled the availability of data in various standard exchange formats from NetPath, a repository for signaling pathways. We believe that this composite molecular interaction pathway will enable identification of new signaling components in TWEAK signaling pathway. This in turn may lead to the identification of potential therapeutic targets in TWEAK-associated disorders.
PMCID: PMC3357548  PMID: 22649723
8.  Sclerostin Stimulates Osteocyte Support of Osteoclast Activity by a RANKL-Dependent Pathway 
PLoS ONE  2011;6(10):e25900.
Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regulator of bone mass and osteoblast differentiation. While evidence suggests that sclerostin has an anti-anabolic role, the possibility also exists that sclerostin has catabolic activity. To test this we treated human primary pre-osteocyte cultures, cells we have found are exquisitely sensitive to sclerostin, or mouse osteocyte-like MLO-Y4 cells, with recombinant human sclerostin (rhSCL) and measured effects on pro-catabolic gene expression. Sclerostin dose-dependently up-regulated the expression of receptor activator of nuclear factor kappa B (RANKL) mRNA and down-regulated that of osteoprotegerin (OPG) mRNA, causing an increase in the RANKL∶OPG mRNA ratio. To examine the effects of rhSCL on resulting osteoclastic activity, MLO-Y4 cells plated onto a bone-like substrate were primed with rhSCL for 3 days and then either mouse splenocytes or human peripheral blood mononuclear cells (PBMC) were added. This resulted in cultures with elevated osteoclastic resorption (approximately 7-fold) compared to untreated co-cultures. The increased resorption was abolished by co-addition of recombinant OPG. In co-cultures of MLO-Y4 cells with PBMC, SCL also increased the number and size of the TRAP-positive multinucleated cells formed. Importantly, rhSCL had no effect on TRAP-positive cell formation from monocultures of either splenocytes or PBMC. Further, rhSCL did not induce apoptosis of MLO-Y4 cells, as determined by caspase activity assays, demonstrating that the osteoclastic response was not driven by dying osteocytes. Together, these results suggest that sclerostin may have a catabolic action through promotion of osteoclast formation and activity by osteocytes, in a RANKL-dependent manner.
PMCID: PMC3186800  PMID: 21991382
9.  TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis 
TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.
TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.
TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts.
The expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.
PMCID: PMC3132040  PMID: 21435232

Results 1-9 (9)