Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes : role of integrinβ1 and focal adhesion kinase signaling cues

INTRODUCTION: Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function. METHODS: De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies. RESULTS: The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell's attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression. CONCLUSION: This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.

Breast cancer cells induce osteolytic bone lesions in vivo through a reduction in osteoblast activity in mice

Bone metastases are severely debilitating and have a significant impact on the quality of life of women with metastatic breast cancer. Treatment options are limited and in order to develop more targeted therapies, improved understanding of the complex mechanisms that lead to bone lesion development are warranted. Interestingly, whilst prostate-derived bone metastases are characterised by mixed or osteoblastic lesions, breast-derived bone metastases are characterised by osteolytic lesions, suggesting unique regulatory patterns. This study aimed to measure the changes in bone formation and bone resorption activity at two time-points (18 and 36 days) during development of the bone lesion following intratibial injection of MDA-MB-231 human breast cancer cells into the left tibiae of Severely Combined Immuno-Deficient (SCID) mice. The contralateral tibia was used as a control. Tibiae were extracted and processed for undecalcified histomorphometric analysis. We provide evidence that the early bone loss observed following exposure to MDA-MB-231 cells was due to a significant reduction in mineral apposition rate, rather than increased levels of bone resorption. This suggests that osteoblast activity was impaired in the presence of breast cancer cells, contrary to previous reports of osteoclast-dependent bone loss. Furthermore mRNA expression of Dickkopf Homolog 1 (DKK-1) and Noggin were confirmed in the MDA-MB-231 cell line, both of which antagonise osteoblast regulatory pathways. The observed bone loss following injection of cancer cells was due to an overall thinning of the trabecular bone struts rather than perforation of the bone tissue matrix (as measured by trabecular width and trabecular separation, respectively), suggesting an opportunity to reverse the cancer-induced bone changes. These novel insights into the mechanisms through which osteolytic bone lesions develop may be important in the development of new treatment strategies for metastatic breast cancer patients.

Bone regeneration based on tissue engineering conceptions – a 21st century perspective

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteo- conductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineer- ing and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.

A soluble activin Type IIA receptor induces bone formation and improves skeletal integrity

Diseases that affect the regulation of bone turnover can lead to skeletal fragility and increased fracture risk. Members of the TGF-superfamily have been shown to be involved in the regulation of bone mass. Activin A, a TGF-� signaling ligand, is present at high levels in bone and may play a role in the regulation of bone metabolism. Here we demonstrate that pharmacological blockade of ligand signaling through the high affinity receptor for activin, type II activin receptor (ActRIIA), by administration of the soluble extracellular domain of ActRIIA fused to a murine IgG2a-Fc, increases bone formation, bone mass, and bone strength in normal mice and in ovariectomized mice with established bone loss. These observations support the development of this pharmacological strategy for the treatment of diseases with skeletal fragility.

A prospective model of care for breast cancer rehabilitation : bone health and arthralgias

Musculoskeletal health can be compromised by breast cancer treatment. In particular, bone loss and arthralgias are prevalent side effects experienced by women treated with chemotherapy and/or adjuvant endocrine therapy. Bone loss leads to osteoporosis and related fractures, while arthralgias threaten quality of life and compliance to treatment. Because the processes that lead to these musculoskeletal problems are initiated when treatment begins, early identification of women who may be at higher risk of developing problems, routine monitoring of bone density and pain at certain stages of treatment, and prudent application of therapeutic interventions are key to preventing and/or minimizing musculoskeletal sequelae. Exercise may be a particularly suitable intervention strategy because of its potential to address a number of impairments; it may slow bone loss, appears to reduce joint pain in noncancer conditions, and improves other breast cancer outcomes. Research efforts continue in the areas of etiology, measurement, and treatment of bone loss and arthralgias. The purpose of this review is to provide an overview of the current knowledge on the management and treatment of bone loss and arthralgias in breast cancer survivors and to present a framework for rehabilitation care to preserve musculoskeletal health in women treated for breast cancer.

Intravenous injection of leconotide, an omega conotoxin : synergistic antihyperalgesic effects with morphine in a rat model of bone cancer pain

Objective.  Leconotide (CVID, AM336, CNSB004) is an omega conopeptide similar to ziconotide, which blocks voltage sensitive calcium channels. However, unlike ziconotide, which must be administered intrathecally, leconotide can be given intravenously because it is less toxic. This study investigated the antihyperalgesic potency of leconotide given intravenously alone and in combinations with morphine-administered intraperitoneally, in a rat model of bone cancer pain. Design.  Syngeneic rat prostate cancer cells AT3B-1 were injected into one tibia of male Wistar rats. The tumor expanded within the bone causing hyperalgesia to heat applied to the ipsilateral hind paw. Measurements were made of the maximum dose (MD) of morphine and leconotide given alone and in combinations that caused no effect in an open-field activity monitor, rotarod, and blood pressure and heart rate measurements. Paw withdrawal thresholds from noxious heat were measured. Dose response curves for morphine (0.312–5.0 mg/kg intraperitoneal) and leconotide (0.002–200 µg/kg intravenous) given alone were plotted and responses compared with those caused by morphine and leconotide in combinations. Results.  Leconotide caused minimal antihyperalgesic effects when administered alone. Morphine given alone intraperitoneally caused dose-related antihyperalgesic effects (ED50 = 2.40 ± 1.24 mg/kg), which were increased by coadministration of leconotide 20 µg/kg (morphine ED50 = 0.16 ± 1.30 mg/kg); 0.2 µg/kg (morphine ED50 = 0.39 ± 1.27 mg/kg); and 0.02 µg/kg (morphine ED50 = 1.24 ± 1.30 mg/kg). Conclusions.  Leconotide caused a significant increase in reversal by morphine of the bone cancer-induced hyperalgesia without increasing the side effect profile of either drug. Clinical Implication.  Translation into clinical practice of the method of analgesia described here will improve the quantity and quality of analgesia in patients with bone metastases. The use of an ordinary parenteral route for administration of the calcium channel blocker (leconotide) at low dose opens up the technique to large numbers of patients who could not have an intrathecal catheter for drug administration. Furthermore, the potentiating synergistic effect with morphine on hyperalgesia without increased side effects will lead to greater analgesia with improved quality of life.

One step closer into the design of fall protective device for individuals fitted with a bone-anchored prosthesis

The consequences of falls are often dreadful for individuals with lower limb amputation using bone-anchored prosthesis.[1-5] Typically, the impact on the fixation is responsible for bending the intercutaneous piece that could lead to a complete breakage over time. .[3, 5-8] The surgical replacement of this piece is possible but complex and expensive. Clearly, there is a need for solid data enabling an evidence-based design of protective devices limiting impact forces and torsion applied during a fall. The impact on the fixation during an actual fall is obviously difficult to record during a scientific experiment.[6, 8-13] Consequently, Schwartze and colleagues opted for one of the next best options science has to offer: simulation with an able-bodied participant. They recorded body movements and knee impacts on the floor while mimicking several plausible falling scenarios. Then, they calculated the forces and moments that would be applied at four levels along the femur corresponding to amputation heights.[6, 8-11, 14-25] The overall forces applied during the falls were similar regardless of the amputation height indicating that the impact forces were simply translated along the femur. As expected, they showed that overall moments generally increased with amputation height due to changes in lever arm. This work demonstrates that devices preventing only against force overload do not require considering amputation height while those protecting against bending moments should. Another significant contribution is to provide, for the time, the magnitude of the impact load during different falls. This loading range is crucial to the overall design and, more precisely, the triggering threshold of protective devices. Unfortunately, the analysis of only a single able-bodied participant replicating falls limits greatly the generalisation of the findings. Nonetheless, this case study is an important milestone contributing to a better understanding of load impact during a fall. This new knowledge will improve the treatment, the safe ambulation and, ultimately, the quality of life of individuals fitted with bone-anchored prosthesis.

Cost-effectiveness of bone-anchored prosthesis: the Queensland experience

Individuals with limb amputation fitted with conventional socket-suspended prostheses often experience socket-related discomfort leading to a significant decrease in quality of life. Bone-anchored prostheses are increasingly acknowledged as viable alternative method of attachment of artificial limb. In this case, the prosthesis is attached directly to the residual skeleton through a percutaneous fixation. To date, a few osseointegration fixations are commercially available. Several devices are at different stages of development particularly in Europe and the US. [1-15] Clearly, surgical procedures are currently blooming worldwide. Indeed, Australia and Queensland, in particular, have one of the fastest growing populations. Previous studies involving either screw-type implants or press-fit fixations for bone-anchorage have focused on biomechanics aspects as well as the clinical benefits and safety of the procedure. In principle, bone-anchored prostheses should eliminate lifetime expenses associated with sockets and, consequently, potentially alleviate the financial burden of amputation for governmental organizations. Unfortunately, publications focusing on cost-effectiveness are sparse. In fact, only one study published by Haggstrom et al (2012), reported that “despite significantly fewer visits for prosthetic service the annual mean costs for osseointegrated prostheses were comparable with socket-suspended prostheses”. Consequently, governmental organizations such as Queensland Artificial Limb Services (QALS) are facing a number of challenges while adjusting financial assistance schemes that should be fair and equitable to their clients fitted with bone-anchored prostheses. Clearly, more scientific evidence extracted from governmental databases is needed to further consolidate the analyses of financial burden associated with both methods of attachment (i.e., conventional sockets prostheses, bone-anchored prostheses). The purpose of the presentation will be to share the current outcomes of a cost-analysis study lead by QALS. The specific objectives will be: • To outline methodological avenues to assess the cost-effectiveness of bone-anchored prostheses compared to conventional sockets prostheses, • To highlight the potential obstacles and limitations in cost-effectiveness analyses of bone-anchored prostheses, • To present cohort results of a cost-effectiveness (QALY vs cost) including the determination of fair Incremental cost-effectiveness Ratios (ICER) as well as cost-benefit analysis focusing on the comparing costs and key outcome indicators (e.g., QTFA, TUG, 6MWT, activities of daily living) over QALS funding cycles for both methods of attachment.

Meta-analysis of genome-wide studies identifies MEF2C SNPs associated with bone mineral density at forearm

Background Forearm fractures affect 1.7 million individuals worldwide each year and most occur earlier in life than hip fractures. While the heritability of forearm bone mineral density (BMD) and fracture is high, their genetic determinants are largely unknown. Aim To identify genetic variants associated with forearm BMD and forearm fractures. Methods BMD at distal radius, measured by dualenergy x-ray absorptiometry, was tested for association with common genetic variants. We conducted a metaanalysis of genome-wide association studies for BMD in 5866 subjects of European descent and then selected the variants for replication in 715 Mexican American samples. Gene-based association was carried out to supplement the single-nucleotide polymorphism (SNP) association test. We then tested the BMD-associated SNPs for association with forearm fracture in 2023 cases and 3740 controls. Results We found that five SNPs in the introns of MEF2C were associated with forearm BMD at a genome-wide significance level (p<5×10-8) in meta-analysis (lead SNP, rs11951031[T] -0.20 SDs per allele, p=9.01×10-9). The gene-based association test suggested an association between MEF2C and forearm BMD ( p=0.003). The association between MEF2C variants and risk of fracture did not achieve statistical significance (SNP rs12521522[A]: OR=1.14 (95% CI 0.92 to 1.35), p=0.14). Meta-analysis also revealed two genome-wide suggestive loci at CTNNA2 and 6q23.2. Conclusions These findings demonstrate that variants at MEF2C were associated with forearm BMD, implicating this gene in the determination of BMD at forearm.

The development of a component to improve the loading safety of bone-anchored prostheses

Use of socket prostheses Currently, for individuals with limb loss, the conventional method of attaching a prosthetic limb relies on a socket that fits over the residual limb. However, there are a number of issues concerning the use of a socket (e.g., blisters, irritation, and discomfort) that result in dissatisfaction with socket prostheses, and these lead ultimately a significant decrease in quality of life. Bone-anchored prosthesis Alternatively, the concept of attaching artificial limbs directly to the skeletal system has been developed (bone anchored prostheses), as it alleviates many of the issues surrounding the conventional socket interface.Bone anchored prostheses rely on two critical components: the implant, and the percutaneous abutment or adapter, which forms the connection for the external prosthetic system (Figure 1). To date, an implant that screws into the long bone of the residual limb has been the most common intervention. However, more recently, press-fit implants have been introduced and their use is increasing. Several other devices are currently at various stages of development, particularly in Europe and the United States. Benefits of bone-anchored prostheses Several key studies have demonstrated that bone-anchored prostheses have major clinical benefits when compared to socket prostheses (e.g., quality of life, prosthetic use, body image, hip range of motion, sitting comfort, ease of donning and doffing, osseoperception (proprioception), walking ability) and acceptable safety, in terms of implant stability and infection. Additionally, this method of attachment allows amputees to participate in a wide range of daily activities for a substantially longer duration. Overall, the system has demonstrated a significant enhancement to quality of life. Challenges of direct skeletal attachment However, due to the direct skeletal attachment, serious injury and damage can occur through excessive loading events such as during a fall (e.g., component damage, peri-prosthetic fracture, hip dislocation, and femoral head fracture). These incidents are costly (e.g., replacement of components) and could require further surgical interventions. Currently, these risks are limiting the acceptance of bone-anchored technology and the substantial improvement to quality of life that this treatment offers. An in-depth investigation into these risks highlighted a clear need to re-design and improve the componentry in the system (Figure 2), to improve the overall safety during excessive loading events. Aim and purposes The ultimate aim of this doctoral research is to improve the loading safety of bone-anchored prostheses, to reduce the incidence of injury and damage through the design of load restricting components, enabling individuals fitted with the system to partake in everyday activities, with increased security and self-assurance. The safety component will be designed to release or ‘fail’ external to the limb, in a way that protects the internal bone-implant interface, thus removing the need for restorative surgery and potential damage to the bone. This requires detailed knowledge of the loads typically experienced by the limb and an understanding of potential overload situations that might occur. Hence, a comprehensive review of the loading literature surrounding bone anchored prostheses will be conducted as part of this project, with the potential for additional experimental studies of the loads during normal activities to fill in gaps in the literature. This information will be pivotal in determining the specifications for the properties of the safety component, and the bone-implant system. The project will follow the Stanford Biodesign process for the development of the safety component.

A 3D in vitro model reflecting the androgen deprivation state in prostate cancer bone metastasis

In castrate-resistant prostate cancer (CRPC), the prevailing organ for metastasis is bone, where the survival of cancer cells is regulated by the permissive metastatic niche offered by the bone marrow. The tumour microenvironment and cellular interactions with the matrix and bone cells enable metastasis and lead to cancer cells becoming androgen resistant. Hence, 3D models that mimic CRPC in terms of an androgen deprivation state (ADS) are needed to identify the mechanisms for CPRC growth in bone and further develop therapeutic strategies.

Pannus invasion into cartilage and bone in rheumatoid arthritis

Rheumatoid arthritis is the most common of all types of arthritis and despite of intensive research etiology of the disease remains unclear. Distinctive features of rheumatic arthritis comprise continuous inflammation of synovium, in which synovial membrane expands on cartilage leading to pannus tissue formation. Pannus formation, appearance of proteolytic enzymes and osteoclast formation cause articular cartilage and bone destruction, which lead to erosions and permanent joint damage. Proteolytic pathways play major roles in the development of tissue lesions in rheumatoid arthritis. Degradation of extracellular matrix proteins is essential to pannus formation and invasion. Matrix metalloproteinases (MMP) form a large proteolytic enzyme family and in rheumatoid arthritis they contribute to pannus invasion by degrading extracellular matrix and to joint destruction by directly degrading the cartilage. MMP-1 and MMP-3 are shown to be increased during cell invasion and also involved in cartilage destruction. Increase of many cytokines has been observed in rheumatoid arthritis, especially TNF-α and IL-1β are studied in synovial tissue and are involved in rheumatoid inflammation and degradation of cartilage. Underlying bone resorption requires first demineralization of bone matrix with acid secreted by osteoclasts, which exposes the collagen-rich matrix for degradation. Cathepsin K is the best known enzyme involved in bone matrix degradation, however deficiency of this protein in pycnodysostosis patient did not prevent bone erosion and on the contrary pannus tissue invading to bone did not expressed much cathepsin K. These indicate that other proteinases are involved in bone degradation, perhaps also via their capability to replace the role of other enzymes especially in diseases like pycnodysostosis or during medication e.g. using cathepsin K inhibitors. Multinuclear osteoclasts are formed also in pannus tissue, which enable the invasion into underlying bone matrix. Pannus tissue express a receptor activator of nuclear factor kappa B ligand (RANKL), an essential factor for osteoclast differentiation and a disintegrin and a metalloproteinase 8 (ADAM8), an osteoclast-activating factors, involved in formation of osteoclast-like giant cells by promoting fusion of mononuclear precursor cells. The understanding of pannus invasion and degradation of extracellular matrix in rheumatic arthritis will open us new more specific methods to prevent this destructive joint disease.

Multi-scale permeability of murine bone measured by nanoindentation

The determination of lacunar-canalicular permeability is essential to understand the mechano-transduction mechanism of bone. Murine models are widely used to investigate skeletal growth and regulation, but the value of lacunar-canalicular permeability is still unclear. To address this question, a poroelastic analysis based on nanoindentation data was used to calculate the lacunar-canalicular permeability of wild type C57BL/6 mice of 12 months. Cross-sections of three tibiae were indented using spherical fluid cell indenter tips of two sizes. Results suggest that the value of lacunar-canalicular intrinsic permeability of B6 female murine tibia is in the order of 10 -24 m2. The distribution of the values of intrinsic permeability suggests that with larger contact sizes, nanoindentation alone is capable of capturing the multi-scale permeability of bone. Multi-scale permeability of bone measured by nanoindentation will lead to a better understanding of the role of fluid flow in mechano-transduction. © 2013 American Society of Civil Engineers.

Age-related changes in mouse bone permeability.

The determination of lacunar-canalicular permeability is essential for understanding local fluid flow in bone, which may indicate how bone senses changes in the mechanical environment to regulate mechano-adaptation. The estimates of lacunar-canalicular permeability found in the literature vary by up to eight orders of magnitude, and age-related permeability changes have not been measured in non-osteonal mouse bone. The objective of this study is to use a poroelastic approach based on nanoindentation data to characterize lacunar-canalicular permeability in murine bone as a function of age. Nine wild type C57BL/6 mice of different ages (2, 7 and 12 months) were used. Three tibiae from each age group were embedded in epoxy resin, cut in half and indented in the longitudinal direction in the mid-cortex using two spherical fluid indenter tips (R=238 μm and 500 μm). Results suggest that the lacunar-canalicular intrinsic permeability of mouse bone decreases from 2 to 7 months, with no significant changes from 7 to 12 months. The large indenter tip imposed larger contact sizes and sampled larger ranges of permeabilities, particularly for the old bone. This age-related difference in the distribution was not seen for indents with the smaller radius tip. We conclude that the small tip effectively measured lacunar-canalicular permeability, while larger tip indents were influenced by vascular permeability. Exploring the age-related changes in permeability of bone measured by nanoindentation will lead to a better understanding of the role of fluid flow in mechano-transduction. This understanding may help indicate alterations in bone adaptation and remodeling.

Age-related changes in mouse bone permeability

The determination of lacunar-canalicular permeability is essential for understanding local fluid flow in bone, which may indicate how bone senses changes in the mechanical environment to regulate mechano-adaptation. The estimates of lacunar-canalicular permeability found in the literature vary by up to eight orders of magnitude, and age-related permeability changes have not been measured in non-osteonal mouse bone. The objective of this study is to use a poroelastic approach based on nanoindentation data to characterize lacunar-canalicular permeability in murine bone as a function of age. Nine wild type C57BL/6 mice of different ages (2, 7 and 12 months) were used. Three tibiae from each age group were embedded in epoxy resin, cut in half and indented in the longitudinal direction in the mid-cortex using two spherical fluid indenter tips (R=238 μm and 500 μm). Results suggest that the lacunar-canalicular intrinsic permeability of mouse bone decreases from 2 to 7 months, with no significant changes from 7 to 12 months. The large indenter tip imposed larger contact sizes and sampled larger ranges of permeabilities, particularly for the old bone. This age-related difference in the distribution was not seen for indents with the smaller radius tip. We conclude that the small tip effectively measured lacunar-canalicular permeability, while larger tip indents were influenced by vascular permeability. Exploring the age-related changes in permeability of bone measured by nanoindentation will lead to a better understanding of the role of fluid flow in mechano-transduction. This understanding may help indicate alterations in bone adaptation and remodeling. © 2013 Elsevier Ltd.