Altered cell interactions of subchondral bone osteoblasts and articular chondrocytes in osteoarthritis is through the mediation of ERK1/2 phosphorylation

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo a typical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. However, the mechanism(s) by which these changes occur during the OA development are not completely understood. Materials and Methods: ACCs and subchondral bone osteoblasts (SBOs) were harvested from OA and healthy patients for the cross-talk studies between normal and OA ACCs and SBOs. The involvement of mitogen activated protein kinase (MAPK) signalling pathway during the cell-cell interactions was analysed by zymography, ELISA and western blotting methods. Results: The direct and in-direct co-culture studies showed that OA (ACCs and SBOs) cells induced osteoarthritic changes of normal (ACC and SBOs) cells. This altered cell interaction induced by OA cells significantly aggravated the proteolytic activity, which resulted cartilage degeneration. The altered cell interaction appeared to significantly activate ERK 1/2 phosphorylation and inhibition of MAPK-ERK 1/2 pathway reversed the osteoarthrtitic phenotypic changes. Discussion and Conclusion: Our study has demonstrated that the altered bi-directional communication of SBOs and ACCs are critical for initiation and progression of OA related changes and that this process is mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA related disorders.

Bioactive SrO–SiO2 glass with well-ordered mesopores : characterization, physiochemistry and biological properties

For a biomaterial to be considered suitable for bone repair it should ideally be both bioactive and have a capacity for controllable drug delivery; as such, mesoporous SiO2 glass has been proposed as a new class of bone regeneration material by virtue of its high drug-loading ability and generally good biocompatibility. It does, however, have less than optimum bioactivity and controllable drug delivery properties. In this study, we incorporated strontium (Sr) into mesoporous SiO2 in an effort to develop a bioactive mesoporous SrO–SiO2 (Sr–Si) glass with the capacity to deliver Sr2+ ions, as well as a drug, at a controlled rate, thereby producing a material better suited for bone repair. The effects of Sr2+ on the structure, physiochemistry, drug delivery and biological properties of mesoporous Sr–Si glass were investigated. The prepared mesoporous Sr–Si glass was found to have an excellent release profile of bioactive Sr2+ ions and dexamethasone, and the incorporation of Sr2+ improved structural properties, such as mesopore size, pore volume and specific surface area, as well as rate of dissolution and protein adsorption. The mesoporous Sr–Si glass had no cytotoxic effects and its release of Sr2+ and SiO44− ions enhanced alkaline phosphatase activity – a marker of osteogenic cell differentiation – in human bone mesenchymal stem cells. Mesoporous Sr–Si glasses can be prepared to porous scaffolds which show a more sustained drug release. This study suggests that incorporating Sr2+ into mesoporous SiO2 glass produces a material with a more optimal drug delivery profile coupled with improved bioactivity, making it an excellent material for bone repair applications. Keywords: Mesoporous Sr–Si glass; Drug delivery; Bioactivity; Bone repair; Scaffolds

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40 ng/ml in the culture medium, but decreased at 80 ng/ml. Under CoCl2- induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or antiangiogenic activities of BMSCs.

Can the contra-lateral limb be used as a control with respect to analyses of bone remodelling?

Bone loss may result from remodelling initiated by implant stress protection. Quantifying remodelling requires bone density distributions which can be obtained from computed tomography scans. Pre-operative scans of large animals however are rarely possible. This study aimed to determine if the contra-lateral bone is a suitable control for the purpose of quantifying bone remodelling. CT scans of 8 pairs of ovine tibia were used to determine the likeness of left and right bones. The deviation between the outer surfaces of the bone pairs was used to quantify geometric similarity. The density differences were determined by dividing the bones into discrete volumes along the shaft of the tibia. Density differences were also determined for fractured and contra-lateral bone pairs to determine the magnitude of implant related remodelling. Left and right ovine tibiae were found to have a high degree of similarity with differences of less than 1.0 mm in the outer surface deviation and density difference of less than 5% in over 90% of the shaft region. The density differences (10–40%) as a result of implant related bone remodelling were greater than left-right differences. Therefore, for the purpose of quantifying bone remodelling in sheep, the contra-lateral tibia may be considered an alternative to a pre-operative control.

In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for BMP-7 gene delivery

Introduction and aims: For a scaffold material to be considered effective and efficient for tissue engineering it must be biocompatible as well as bioinductive. Silk fiber is a natural biocompatible material suitable for scaffold fabrication; however, silk is tissue-conductive and lacks tissue-inductive properties. One proposed method to make the scaffold tissue-inductive is to introduce plasmids or viruses encoding a specific growth factor into the scaffold. In this study, we constructed adenoviruses encoding bone morphogenetic protein-7 (BMP-7) and incorporated these into silk scaffolds. The osteo-inductive and new bone formation properties of these constructs were assessed in vivo in a critical-sized skull defect animal model. Materials and methods: Silk fibroin scaffolds containing adenovirus particles coding BMP-7 were prepared. The release of the adenovirus particles from the scaffolds was quantified by tissue-culture infective dose (TCID50) and the bioactivity of the released viruses was evaluated on human bone marrow mesenchymal stromal cells (BMSCs). To demonstrate the in vivo bone forming ability of the virus-carrying silk fibroin scaffold, the scaffold constructs were implanted into calvarial defects in SCID mice. Results: In vitro studies demonstrated that the virus-carrying silk fibroin scaffold released virus particles over a 3 week period while preserving their bioactivity. In vivo test of the scaffold constructs in critical-sized skull defect areas revealed that silk scaffolds were capable of delivering the adenovirus encoding BMP-7, resulting significantly enhanced new bone formation. Conclusions: Silk scaffolds carrying BMP-7 encoding adenoviruses can effectively transfect cells and enhance both in vitro and in vivo osteogenesis. The findings of this study indicate silk fibroin is a promising biomaterial for gene delivery to repair critical-sized bone defects.

Preclinical animal models for segmental bone defect research and tissue engineering

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. Analysing the tissue engineering literature it can be concluded that bone regeneration has become a focus area in the field. Hence, a considerable number of research groups and commercial entities work on the development of tissue engineered constructs for bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. In translational orthopaedic research, the utilisation of large preclinical animal models is a conditio sine qua non. Consequently, to allow comparison between different studies and their outcomes, it is essential that animal models, fixation devices, surgical procedures and methods of taking measurements are well standardized to produce reliable data pools as a base for further research directions. The following chapter reviews animal models of the weight-bearing lower extremity utilized in the field which include representations of fracture-healing, segmental bone defects, and fracture non-unions.

Waist circumference cut-off values for the prediction of cardiovascular risk factors clustering in Chinese school-aged children : a cross-sectional study

Background: Waist circumference has been identified as a valuable predictor of cardiovascular risk in children. The development of waist circumference percentiles and cut-offs for various ethnic groups are necessary because of differences in body composition. The purpose of this study was to develop waist circumference percentiles for Chinese children and to explore optimal waist circumference cut-off values for predicting cardiovascular risk factors clustering in this population.----- ----- Methods: Height, weight, and waist circumference were measured in 5529 children (2830 boys and 2699 girls) aged 6-12 years randomly selected from southern and northern China. Blood pressure, fasting triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and glucose were obtained in a subsample (n = 1845). Smoothed percentile curves were produced using the LMS method. Receiver-operating characteristic analysis was used to derive the optimal age- and gender-specific waist circumference thresholds for predicting the clustering of cardiovascular risk factors.----- ----- Results: Gender-specific waist circumference percentiles were constructed. The waist circumference thresholds were at the 90th and 84th percentiles for Chinese boys and girls respectively, with sensitivity and specificity ranging from 67% to 83%. The odds ratio of a clustering of cardiovascular risk factors among boys and girls with a higher value than cut-off points was 10.349 (95% confidence interval 4.466 to 23.979) and 8.084 (95% confidence interval 3.147 to 20.767) compared with their counterparts.----- ----- Conclusions: Percentile curves for waist circumference of Chinese children are provided. The cut-off point for waist circumference to predict cardiovascular risk factors clustering is at the 90th and 84th percentiles for Chinese boys and girls, respectively.

Extending constructability concept to include operation and maintenance issues

The concept of constructability uses integration art of individual functions through a valuable and timely construction inputs into planning and design development stages. It results in significant savings in cost and time needed to finalize infrastructure projects. However, available constructability principles, developed by CII Australia (1993), do not cover Operation and Maintenance (O&M) phases of projects, whilst major cost and time in multifaceted infrastructure projects are spent in post-occupancy stages. This paper discusses the need to extend the constructability concept by examining current O&M issues in the provision of multifaceted building projects. It highlights available O&M problems and shortcomings of building projects, as well as their causes and reasons in different categories. This initial categorization is an efficient start point for testing probable present O&M issues in various cases of complex infrastructure building projects. This preliminary categorization serve as a benchmark to develop an extended constructability model that considers the whole project life cycle phases rather than a specific phase. It anticipates that the development of an extended constructability model can reduce significant number of reworks, mistakes, extra costs and time wasted during delivery stages of multifaceted building projects.

Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in Vitro redifferentiation

Background: If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. ----- ----- Hypothesis: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture).----- ----- Study Design: Controlled laboratory study.----- ----- Methods: Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes).----- ----- Results: Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone.----- ----- Conclusion: Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion.

The molecular mechanisms utilised by mesenchymal stem cells in migration to acute myocardial infarction

Heart damage caused by acute myocardial infarction (AMI) is a leading cause of death and disability in Australia. Novel therapies are still required for the treatment of this condition due to the poor reparative ability of the heart. As such, cellular therapies that assist in the recovery of heart muscle are of great current interest. Culture expanded mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical studies of AMI. For MSC-based therapies in the clinic, an intravenous route of administration would ideally be used due to the low cost, ease of delivery and relative safety. The study of MSC migration is therefore clinically relevant for a minimally invasive cell therapy to promote regeneration of damaged tissue. C57BL/6, UBI-GFP-BL/6 and CD44-/-/GFP+/+ mice were utilised to investigate mMSC migration. To assist in murine models of MSC migration, a novel method was used for the isolation of murine MSC (mMSC). These mMSC were then expanded in culture and putative mMSC were positive for Sca-1, CD90.2, and CD44 and were negative for CD45 and CD11b. Furthermore, mMSC from C57BL/6 and UBI-GFP-BL/6 mice were shown to differentiate into cells of the mesodermal lineage. Cells from CD44-/-/GFP+/+ mice were positive for Sca-1 and CD90.2, and negative for CD44, CD45 and CD11b however, these cells were unable to differentiate into adipocytes and chondrocytes and express lineage specific genes, PLIN and ACAN. Analysis of mMSC chemokine receptor (CR) expression showed that although mMSC do express chemokine receptors, (including those specific for chemokines released after AMI), these were low or undetectable by mRNA. However, protein expression could be detected, which was predominantly cytoplasmic. It was further shown that in both healthy (unperturbed) and inflamed tissues, mMSC had very little specific migration and engraftment after intravenous injection. To determine if poor mMSC migration was due to the inability of mMSC to respond to chemotactic stimuli, chemokine expression in bone marrow, skin injury and hearts (healthy and after AMI) was analysed at various time points by quantitative real-time PCR (qRT PCR). Many chemokines were up-regulated after skin biopsy and AMI, but the highest acute levels were found for CXCL12 and CCL7. Due to their high expression in infarcted hearts, the chemokines CXCL12 and CCL7 were tested for their effect on mMSC migration. Despite CR expression at both protein and mRNA levels, migration in response to CXCL12 and CCL7 was low in mMSC cultured on Nunclon plastic. A novel tissue culture plastic technology (UpCellTM) was then used that allowed gentle non-enzymatic dissociation of mMSC, thus preserving surface expression of the CRs. Despite this the in vitro data indicated that CXCL12 fails to induce significant migration ability of mMSC, while CCL7 induces significant, but low-level migration. We speculated this may be because of low levels of surface expression of chemokine receptors. In a strategy to increase cell surface expression of mMSC chemokine receptors and enhance their in vitro and in vivo migration capacity, mMSC were pre-treated with pro-inflammatory cytokines. Increased levels of both mRNA and surface protein expression were found for CRs by pre-treating mMSC with pro-inflammatory cytokines including TNF-á, IFN-ã, IL-1á and IL-6. Furthermore, the chemotactic response of mMSC to CXCL12 and CCL7 was significantly higher with these pretreated cells. Finally, the effectiveness of this type of cell manipulation was demonstrated in vivo, where mMSC pre-treated with TNF-á and IFN-ã showed significantly increased migration in skin injury and AMI models. Therefore this thesis has demonstrated, using in vitro and in vivo models, the potential for prior manipulation of MSC as a possible means for increasing the utility of intravenously delivery for MSC-based cellular therapies.

Similarity metrics within a point of view

In vector space based approaches to natural language processing, similarity is commonly measured by taking the angle between two vectors representing words or documents in a semantic space. This is natural from a mathematical point of view, as the angle between unit vectors is, up to constant scaling, the only unitarily invariant metric on the unit sphere. However, similarity judgement tasks reveal that human subjects fail to produce data which satisfies the symmetry and triangle inequality requirements for a metric space. A possible conclusion, reached in particular by Tversky et al., is that some of the most basic assumptions of geometric models are unwarranted in the case of psychological similarity, a result which would impose strong limits on the validity and applicability vector space based (and hence also quantum inspired) approaches to the modelling of cognitive processes. This paper proposes a resolution to this fundamental criticism of of the applicability of vector space models of cognition. We argue that pairs of words imply a context which in turn induces a point of view, allowing a subject to estimate semantic similarity. Context is here introduced as a point of view vector (POVV) and the expected similarity is derived as a measure over the POVV's. Different pairs of words will invoke different contexts and different POVV's. Hence the triangle inequality ceases to be a valid constraint on the angles. We test the proposal on a few triples of words and outline further research.

Analysis of Object-Specific Authorization Protocol (OSAP) using coloured petri nets - Version 1.0

Analyzing security protocols is an ongoing research in the last years. Different types of tools are developed to make the analysis process more precise, fast and easy. These tools consider security protocols as black boxes that can not easily be composed. It is difficult or impossible to do a low-level analysis or combine different tools with each other using these tools. This research uses Coloured Petri Nets (CPN) to analyze OSAP trusted computing protocol. The OSAP protocol is modeled in different levels and it is analyzed using state space method. The produced model can be combined with other trusted computing protocols in future works.

Ontology-based specific and exhaustive user profiles for constraint information fusion for multi-agents

Intelligent agents are an advanced technology utilized in Web Intelligence. When searching information from a distributed Web environment, information is retrieved by multi-agents on the client site and fused on the broker site. The current information fusion techniques rely on cooperation of agents to provide statistics. Such techniques are computationally expensive and unrealistic in the real world. In this paper, we introduce a model that uses a world ontology constructed from the Dewey Decimal Classification to acquire user profiles. By search using specific and exhaustive user profiles, information fusion techniques no longer rely on the statistics provided by agents. The model has been successfully evaluated using the large INEX data set simulating the distributed Web environment.

A biomechanical investigation of vertebral staples for fusionless scoliosis correction

Background: Fusionless scoliosis surgery is an early-stage treatment for idiopathic scoliosis which claims potential advantages over current fusion-based surgical procedures. Anterior vertebral stapling using a shape memory alloy staple is one such approach. Despite increasing interest in this technique, little is known about the effects on the spine following insertion, or the mechanism of action of the staple. The purpose of this study was to investigate the biomechanical consequences of staple insertion in the anterior thoracic spine, using in vitro experiments on an immature bovine model. Methods: Individual calf spine thoracic motion segments were tested in flexion, extension, lateral bending and axial rotation. Changes in motion segment rotational stiffness following staple insertion were measured on a series of 14 specimens. Strain gauges were attached to three of the staples in the series to measure forces transmitted through the staple during loading. A micro-CT scan of a single specimen was performed after loading to qualitatively examine damage to the vertebral bone caused by the staple. Findings: Small but statistically significant decreases in bending stiffness occurred in flexion,extension, lateral bending away from the staple, and axial rotation away from the staple. Each strain-gauged staple showed a baseline compressive loading following insertion which was seen to gradually decrease during testing. Post-test micro-CT showed substantial bone and growth plate damage near the staple. Interpretation: Based on our findings it is possible that growth modulation following staple insertion is due to tissue damage rather than sustained mechanical compression of the motion segment.

The effect of friction on indenter force and pile-up in numerical simulations of bone nanoindentation

Nanoindentation is a useful technique for probing the mechanical properties of bone, and finite element (FE) modeling of the indentation allows inverse determination of elasto-plastic constitutive properties. However, all but one FE study to date have assumed frictionless contact between indenter and bone. The aim of this study was to explore the effect of friction in simulations of bone nanoindentation. Two dimensional axisymmetric FE simulations were performed using a spheroconical indenter of tip radius 0.6 m and angle 90°. The coefficient of friction between indenter and bone was varied between 0.0 (frictionless) and 0.3. Isotropic linear elasticity was used in all simulations, with bone elastic modulus E=13.56GPa and Poisson‟s ratio f 0.3. Plasticity was incorporated using both Drucker-Prager and von Mises yield surfaces. Friction had a modest effect on the predicted force-indentation curve for both von Mises and Drucker-Prager plasticity, reducing maximum indenter displacement by 10% and 20% respectively as friction coefficient was increased from zero to 0.3 (at a maximum indenter force of 5mN). However, friction has a much greater effect on predicted pile-up after indentation, reducing predicted pile-up from 0.27 to 0.11 m with a von Mises model, and from 0.09 to 0.02 m with Drucker-Prager plasticity. We conclude that it is potentially important to include friction in nanoindentation simulations of bone if pile-up is used to compare simulation results with experiment.