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1.  Collecting a comprehensive evidence base to monitor fracture rehabilitation: A case study 
World Journal of Orthopedics  2013;4(4):259-266.
AIM: To determine the feasibility and potential role of combining radiostereometric analysis (RSA), gait analysis and activity monitoring in the follow-up of fracture patients.
METHODS: Two patients with similar 41B3 tibial plateau fractures were treated by open reduction internal fixation augmented with impaction bone grafting and were instructed to partial weight bear to 10 kg for the first six postoperative weeks. Fracture reduction and fixation were assessed by postoperative computer tomographic (CT) scanning. Both patients had tantalum markers inserted intra-operatively to monitor their fracture stability during healing using RSA and differentially loaded RSA (DLRSA) at 6 and 12 wk postoperatively. Gait analyses were performed at 1, 2, 6, and 12 wk postoperatively. Activity monitors were worn for 4 wk between the 2 and 6 wk appointments. In addition to gait analysis, knee function was assessed using the patient reported Lysholm scores, and doctor reported knee range of motion and stability, at 6 and 12 wk postoperatively.
RESULTS: There were no complications. CT demonstrated that both fractures were reduced anatomically. Gait analysis indicated that Patient 1 bore weight to 60% of body weight at 2 wk postoperative and 100% at 6 wk. Patient 2 bore weight at 10% of body weight to 6 wk and had very low joint contact forces to that time. At 12 wk however, there was no difference between the gait patterns in the two patients. Patient 1 increased activities of moderate-vigorous intensity from 20 to 60 min/d between 2 and 6 postoperative weeks, whereas Patient 2 remained more stable at 20-30 min/d. The Lysholm scores were similar for both patients and did not improve between 6 and 12 wk postoperatively. DLRSA examination at 12 wk showed that both patients were comfortable to weight bear to 80 kg and under this weight the fractures displaced less than 0.4 mm. RSA measurements demonstrated over time fracture migrations of less than 2 mm in both cases. However, Patient 2, who followed the postoperative weight bearing instructions most closely, displaced less (0.3 mm vs 1.6 mm).
CONCLUSION: This study demonstrates the potential of using a combination of RSA, gait analysis and activity monitoring to obtain a comprehensive evidence base for postoperative weight bearing schedules during fracture healing.
doi:10.5312/wjo.v4.i4.259
PMCID: PMC3801245  PMID: 24147261
Radiostereometric analysis; Gait analysis; Activity monitoring; Rehabilitation protocols; Lower limb trauma
2.  Osteoblast-Chondrocyte Interactions in Osteoarthritis 
Current Osteoporosis Reports  2014;12:127-134.
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.
doi:10.1007/s11914-014-0192-5
PMCID: PMC3933767  PMID: 24458429
Osteoarthritis; Articular; Cartilage; Subchondral; Bone; Osteoblasts; Chondrocytes; Gene expression; Communication; Pathogenesis; Transforming growth factor; Sclerostin
3.  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.
doi:10.5312/wjo.v2.i10.93
PMCID: PMC3302032  PMID: 22474627
Polyethylene; Aseptic loosening; Osteolysis; Wear particle; Peri-prosthetic
4.  Critical role of p38 MAPK for regeneration of the sciatic nerve following crush injury in vivo 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1742-2094-10-1
PMCID: PMC3541116  PMID: 23282009
P38 MAPK; Nerve regeneration; Crush injury; Inflammatory cytokines; TNF-α; IL-1β; Mutant mice; In vivo
5.  Characterization of drug-release kinetics in trabecular bone from titania nanotube implants 
Purpose
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
Methods
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.
Results
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.
Conclusion
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.
doi:10.2147/IJN.S33655
PMCID: PMC3446838  PMID: 23028217
local drug delivery; Zetos bone bioreactor; drug-releasing implant; drug diffusion
6.  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.
doi:10.1002/jbmr.345
PMCID: PMC3358926  PMID: 21312267
OSTEOCYTE; SCLEROSTIN; SOST; MEPE; ASARM
7.  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.
doi:10.1371/journal.pone.0025900
PMCID: PMC3186800  PMID: 21991382
8.  If good things come from above, do bad things come from below? 
Factors in the synovial fluid that maintain healthy articular cartilage, such as hyaluronic acid and lubricin, come from above. Is it possible that factors which lead to the destruction of cartilage come from below in the subchondral bone? The recent acquisition of tools to probe early events in osteoarthritis is shedding new light on possible contributions from this compartment on the initiation and progression of the disease. Tanamas and co-workers now provide evidence that bone marrow lesions in the subchondral bone are predictive, both of loss of cartilage and of formation of subchondral cysts. These data provoke questions about the nature and role of bone marrow lesions.
doi:10.1186/ar3007
PMCID: PMC2911864  PMID: 20519029
9.  Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium 
Introduction
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types.
Methods
Using immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied.
Results
Significantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels.
Conclusions
This study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.
doi:10.1186/ar2603
PMCID: PMC2688245  PMID: 19171073
10.  Microarray gene expression profiling of osteoarthritic bone suggests altered bone remodelling, WNT and transforming growth factor-β/bone morphogenic protein signalling 
Osteoarthritis (OA) is characterized by alterations to subchondral bone as well as articular cartilage. Changes to bone in OA have also been identified at sites distal to the affected joint, which include increased bone volume fraction and reduced bone mineralization. Altered bone remodelling has been proposed to underlie these bone changes in OA. To investigate the molecular basis for these changes, we performed microarray gene expression profiling of bone obtained at autopsy from individuals with no evidence of joint disease (control) and from individuals undergoing joint replacement surgery for either degenerative hip OA, or fractured neck of femur (osteoporosis [OP]). The OP sample set was included because an inverse association, with respect to bone density, has been observed between OA and the low bone density disease OP. Compugen human 19K-oligo microarray slides were used to compare the gene expression profiles of OA, control and OP bone samples. Four sets of samples were analyzed, comprising 10 OA-control female, 10 OA-control male, 10 OA-OP female and 9 OP-control female sample pairs. Print tip Lowess normalization and Bayesian statistical analyses were carried out using linear models for microarray analysis, which identified 150 differentially expressed genes in OA bone with t scores above 4. Twenty-five of these genes were then confirmed to be differentially expressed (P < 0.01) by real-time PCR analysis. A substantial number of the top-ranking differentially expressed genes identified in OA bone are known to play roles in osteoblasts, osteocytes and osteoclasts. Many of these genes are targets of either the WNT (wingless MMTV integration) signalling pathway (TWIST1, IBSP, S100A4, MMP25, RUNX2 and CD14) or the transforming growth factor (TGF)-β/bone morphogenic protein (BMP) signalling pathway (ADAMTS4, ADM, MEPE, GADD45B, COL4A1 and FST). Other differentially expressed genes included WNT (WNT5B, NHERF1, CTNNB1 and PTEN) and TGF-β/BMP (TGFB1, SMAD3, BMP5 and INHBA) signalling pathway component or modulating genes. In addition a subset of genes involved in osteoclast function (GSN, PTK9, VCAM1, ITGB2, ANXA2, GRN, PDE4A and FOXP1) was identified as being differentially expressed in OA bone between females and males. Altered expression of these sets of genes suggests altered bone remodelling and may in part explain the sex disparity observed in OA.
doi:10.1186/ar2301
PMCID: PMC2212557  PMID: 17900349
11.  TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis 
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/ar3294
PMCID: PMC3132040  PMID: 21435232

Results 1-11 (11)