Clinical efficacy and safety of zoledronic acid in osteoporosis and Paget's disease

Osteoporosis and Paget’s bone disease are the most common diseases of the bone. In addition to glucocorticoid treatment, there are many other secondary causes of osteoporosis. Bisphosphonates are used to treat these bone conditions. Zoledronic acid is the most potent bisphosphonate at inhibiting bone resorption. In osteoporosis, zoledronic acid increases bone mineral density for at least 1 year following a single intravenous administration. The efficacy and safety of zoledronic acid in the treatment of osteoporosis and Paget’s bone disease are reviewed. This article also covers the studies of the effects of zoledronic acid in the bone loss associated with the secondary osteoporosis.

Ovine bone and marrow derived progenitor cells : isolation, characterization, and osteogenic potential

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) for their potential clinical use in bone engineering. Prior to clinical application, MPC-based treatment concepts need to be evaluated in preclinical, immunocompetent, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, ovine MPC and their osteogenic potential remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than osteoblasts (OB) derived from tibial compact bone as assessed using standard 2D culture. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14-/CD31-/CD45- /CD29+/CD44+/CD166+) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed >90%. Cells displayed a spindle-like morphology and formed an interconnected network. Implanted subcutaneously into NOD/SCID mice on type I collagen coated polycaprolactone-tricalciumphosphate (mPCL-TCP) scaffolds, MPC presented a higher developmental potential than osteoblasts. In summary, this study provides a detailed in vitro characterisation of ovine MPC from a bone engineering perspective and suggests that MPC provide promising means for future bone disease related treatment applications.

Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds

Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.

Application of a human bone engineering platform to an in vitro and in vivo breast cancer metastasis model

Breast cancer in its advanced stage has a high predilection to the skeleton. Currently, treatment options of breast cancer-related bone metastasis are restricted to only palliative therapeutic modalities. This is due to the fact that mechanisms regarding the breast cancer celI-bone colonisation as well as the interactions of breast cancer cells with the bone microenvironment are not fully understood, yet. This might be explained through a lack of appropriate in vitro and in vivo models that are currently addressing the above mentioned issue. Hence the hypothesis that the translation of a bone tissue engineering platform could lead to improved and more physiological in vitro and in vivo model systems in order to investigate breast cancer related bone colonisation was embraced in this PhD thesis. Therefore the first objective was to develop an in vitro model system that mimics human mineralised bone matrix to the highest possible extent to examine the specific biological question, how the human bone matrix influences breast cancer cell behaviour. Thus, primary human osteoblasts were isolated from human bone and cultured under osteogenic conditions. Upon ammonium hydroxide treatment, a cell-free intact mineralised human bone matrix was left behind. Analyses revealed a similar protein and mineral composition of the decellularised osteoblast matrix to human bone. Seeding of a panel of breast cancer cells onto the bone mimicking matrix as well as reference substrates like standard tissue culture plastic and collagen coated tissue culture plastic revealed substrate specific differences of cellular behaviour. Analyses of attachment, alignment, migration, proliferation, invasion, as well as downstream signalling pathways showed that these cellular properties were influenced through the osteoblast matrix. The second objective of this PhD project was the development of a human ectopic bone model in NOD/SCID mice using medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold. Human osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP scaffold, fabricated using a fused deposition modelling technique. After subcutaneous implantation in conjunction with the bone morphogenetic protein 7, limited bone formation was observed due to the mechanical properties of the applied scaffold and restricted integration into the soft tissue of flank of NOD/SCID mice. Thus, a different scaffold fabrication technique was chosen using the same polymer. Electrospun tubular scaffolds were seeded with human osteoblasts, as they showed previously the highest amount of bone formation and implanted into the flanks of NOD/SCID mice. Ectopic bone formation with sufficient vascularisation could be observed. After implantation of breast cancer cells using a polyethylene glycol hydrogel in close proximity to the newly formed bone, macroscopic communication between the newly formed bone and the tumour could be observed. Taken together, this PhD project showed that bone tissue engineering platforms could be used to develop an in vitro and in vivo model system to study cancer cell colonisation in the bone microenvironment.

Significant deterioration in nanomechanical quality occurs through incomplete extrafibrillar mineralization in rachitic bone: Evidence from in-situ synchrotron X-ray scattering and backscattered electron imaging

Bone diseases such as rickets and osteoporosis cause significant reduction in bone quantity and quality, which leads to mechanical abnormalities. However, the precise ultrastructural mechanism by which altered bone quality affects mechanical properties is not clearly understood. Here we demonstrate the functional link between altered bone quality (reduced mineralization) and abnormal fibrillar-level mechanics using a novel, real-time synchrotron X-ray nanomechanical imaging method to study a mouse model with rickets due to reduced extrafibrillar mineralization. A previously unreported N-ethyl-N-nitrosourea (ENU) mouse model for hypophosphatemic rickets (Hpr), as a result of missense Trp314Arg mutation of the phosphate regulating gene with homologies to endopeptidase on the X chromosome (Phex) and with features consistent with X-linked hypophosphatemic rickets (XLHR) in man, was investigated using in situ synchrotron small angle X-ray scattering to measure real-time changes in axial periodicity of the nanoscale mineralized fibrils in bone during tensile loading. These determine nanomechanical parameters including fibril elastic modulus and maximum fibril strain. Mineral content was estimated using backscattered electron imaging. A significant reduction of effective fibril modulus and enhancement of maximum fibril strain was found in Hpr mice. Effective fibril modulus and maximum fibril strain in the elastic region increased consistently with age in Hpr and wild-type mice. However, the mean mineral content was ∼21% lower in Hpr mice and was more heterogeneous in its distribution. Our results are consistent with a nanostructural mechanism in which incompletely mineralized fibrils show greater extensibility and lower stiffness, leading to macroscopic outcomes such as greater bone flexibility. Our study demonstrates the value of in situ X-ray nanomechanical imaging in linking the alterations in bone nanostructure to nanoscale mechanical deterioration in a metabolic bone disease. Copyright

Zoledronic acid once-yearly : what role in the prevention of non-vertebral osteoporotic fractures?

Osteoporosis is the most common bone disease. Low levels of oestrogens or testosterone are risk factors for primary osteoporosis. The most common cause of secondary osteoporosis is glucocorticoid treatment, but there are many other secondary causes of osteoporosis. Osteoporosis can be secondary to anti-oestrogen treatment for hormone-sensitive breast cancer and to androgen-deprivation therapy for prostate cancer. Zoledronic is the most potent bisphosphonate at inhibiting bone resorption. In osteoporosis, zoledronic acid increases bone mineral density for at least a year after a single intravenous administration. The efficacy and safety of extended release (once-yearly) zoledronic acid in the treatment of osteoporosis is reviewed.

Vitamin D intake, sun exposure and 25-hydroxy vitamin D status in Peritoneal dialysis (PD) and Haemodialysis (HD) patients

Inadequate vitamin D levels have been linked to bone disease but more recently have been associated with wider health implications. Limited studies suggest a high prevalence of Vitamin D deficiency in dialysis patients, although evidence is lacking on whether this is due to dietary restrictions, limited mobility and time outdoors or a combination of these. The aim of this study was to assess the contributions of diet, supplements and sunlight exposure to serum Vitamin D (25(OH)D) levels in dialysis patients. Cross-sectional data were obtained from 30 PD (Mean±SD age 56.9±16.2 y; n=13 male) and 22 HD (Mean±SD age 65.4±14.0 y; n=18 male) patients between 2009 and 2010. Serum 25(OH)D was measured and oral vitamin D intake estimated through a food-frequency-questionnaire and quantifying inactive supplementation. Sunlight exposure was assessed using a validated questionnaire. Prevalence of inadequate/insufficient vitamin D differed between dialysis modality (31% and 43% insufficient (<50nmol/L); 4% and 34% deficient (<25nmol/L) in HD and PD patients respectively (p=0.002)). In HD patients, there was a significant correlation between diet plus supplemental vitamin D intake and 25(OH)D (ρ=0.84, p<0.001). Results suggest a higher frequency of 25(OH)D inadequacy/deficiency in PD compared to HD patients. No other relationships between intake, sun exposure and 25(OH)D were seen. This could reflect limitations of the study design or the importance of other factors such as age, ethnicity and sun protection as interactions in the analysis. Understanding these factors is important given Vitamin D’s emerging status as a biomarker of systemic ill health.

Genetic disorders of the LRP5-Wnt signalling pathway affecting the skeleton

Osteoporosis is a common, increasingly prevalent and potentially debilitating condition of men and women. Genetic factors are major determinants of bone mass and the risk of fracture, but few genes have been definitively demonstrated to be involved. The identification of these factors will provide novel insights into the processes of bone formation and loss and thus the pathogenesis of osteoporosis, enabling the rational development of novel therapies. In this article, we present the extensive genetic and functional data indicating that the LRP5 gene and the Wnt signalling pathway are key players in bone formation and the risk of osteoporosis, and that LRP5 signalling is essential for normal morphology, developmental processes and bone health.

The genetics of osteoporosis

Introduction: Osteoporosis is the commonest metabolic bone disease worldwide. The clinical hallmark of osteoporosis is low trauma fracture, with the most devastating being hip fracture, resulting in significant effects on both morbidity and mortality. Sources of data: Data for this review have been gathered from the published literature and from a range of web resources. Areas of agreement: Genome-wide association studies in the field of osteoporosis have led to the identification of a number of loci associated with both bone mineral density and fracture risk and further increased our understanding of disease. Areas of controversy: The early strategies for mapping osteoporosis disease genes reported only isolated associations, with replication in independent cohorts proving difficult. Neither candidate gene or linkage studies showed association at genome-wide level of significance. Growing points: The advent of massive parallel sequencing technologies has proved extremely successful in mapping monogenic diseases and thus leading to the utilization of this new technology in complex disease genetics. Areas timely for developing research: The identification of novel genes and pathways will potentially lead to the identification of novel therapeutic options for patients with osteoporosis. © 2014 The Author.

Bone health in children with inflammatory bowel disease : adjusting for bone age

OBJECTIVES: Clinical results of bone mineral density for children with inflammatory bowel disease are commonly reported using reference data for chronological age. It is known that these children, particularly those with Crohn disease, experience delayed growth and maturation. Therefore, it is more appropriate to compare clinical results with bone age rather than chronological age. MATERIALS AND METHODS: Areal bone mineral density (aBMD) was measured using dual energy x-ray absorptiometry, and bone age was assessed using the Tanner-Whitehouse 3 method from a standard hand/wrist radiograph. Results were available for 44 children ages 7.99 to 16.89 years. Areal bone mineral density measurements were converted to z scores using both chronological and bone ages for each subject. RESULTS: Areal bone mineral density z scores calculated using bone age, as opposed to chronological age, were significantly improved for both the total body and lumbar spine regions of interest. When subjects were grouped according to diagnosis, bone age generated z scores remained significantly improved for those with Crohn disease but not for those diagnosed with ulcerative colitis. Grouping of children with Crohn disease into younger and older ages produced significantly higher z scores using bone age compared with chronological for the older age group, but not the younger age group. CONCLUSIONS: Our findings, in accordance with those presented in the literature, suggest that aBMD results in children with Crohn disease should include the consideration of bone age, rather than merely chronological age. Bone size, although not as easily available, would also be an important consideration for interpreting results in paediatric populations. © 2009 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.

The measurement of broadband ultrasonic attenuation in cancellous bone-a review of the science and technology

The measurement of broadband ultrasonic attenuation (BUA) in cancellous bone at the calcaneus was first described in 1984. The assessment of osteoporosis by BUA has recently been recognized by Universities UK, within its EurekaUK book, as being one of the “100 discoveries and developments in UK Universities that have changed the world” over the past 50 years, covering the whole academic spectrum from the arts and humanities to science and technology. Indeed, BUA technique has been clinically validated and is utilized worldwide, with at least seven commercial systems providing calcaneal BUA measurement. However, a fundamental understanding of the dependence of BUA upon the material and structural properties of cancellous bone is still lacking. This review aims to provide a science- and technology-orientated perspective on the application of BUA to the medical disease of osteoporosis.

Vitamin D in health and disease : an insight into traditional functions and new roles for the 'sunshine vitamin'

Vitamin D is unique among the vitamins in that humans can synthesize it via the action of UV radiation upon the skin. This combined with its ability to act on specific target tissues via Vitamin D Receptor’s (VDR) make its classification as a steroid hormone more appropriate. While Vitamin D deficiency is a recognized problem in some northern latitude countries, recent studies have shown even in sunny countries such as Australia, vitamin D deficiency may be more prevalent than first thought. Vitamin D is most well known for its role in bone health, however, the discovery of VDR’s on a wide variety of tissue types has also opened up roles for vitamin D far beyond traditional bone health. These include possible associations with autoimmune diseases such as multiple sclerosis and inflammatory bowel diseases, cancer, cardiovascular diseases and muscle strength. Firstly, this paper presents an overview of the two sources of vitamin D: exposure to ultraviolet-B radiation and food sources of vitamin D, with particular focus on both Australian and international studies on dietary vitamin D intake and national fortification strategies. Secondly, the paper reviews recent epidemiological and experimental evidence linking vitamin D and its role in health and disease for the major conditions linked to suboptimal vitamin D, while identifying significant gaps in the research and possible future directions for research.

Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment

Prostate cancer metastasis is reliant on the reciprocal interactions between cancer cells and the bone niche/micro-environment. The production of suitable matrices to study metastasis, carcinogenesis and in particular prostate cancer/bone micro-environment interaction has been limited to specific protein matrices or matrix secreted by immortalised cell lines that may have undergone transformation processes altering signaling pathways and modifying gene or receptor expression. We hypothesize that matrices produced by primary human osteoblasts are a suitable means to develop an in vitro model system for bone metastasis research mimicking in vivo conditions. We have used a decellularized matrix secreted from primary human osteoblasts as a model for prostate cancer function in the bone micro-environment. We show that this collagen I rich matrix is of fibrillar appearance, highly mineralized, and contains proteins, such as osteocalcin, osteonectin and osteopontin, and growth factors characteristic of bone extracellular matrix (ECM). LNCaP and PC3 cells grown on this matrix, adhere strongly, proliferate, and express markers consistent with a loss of epithelial phenotype. Moreover, growth of these cells on the matrix is accompanied by the induction of genes associated with attachment, migration, increased invasive potential, Ca2+ signaling and osteolysis. In summary, we show that growth of prostate cancer cells on matrices produced by primary human osteoblasts mimics key features of prostate cancer bone metastases and thus is a suitable model system to study the tumor/bone micro-environment interaction in this disease.

Identification of bone morphogenetic proteins 2 and 4 in commercial demineralized freeze-dried bone allograft preparations : pilot study

BACKGROUND: Demineralized freeze-dried bone allografts (DFDBAs) have been proposed as a useful adjunct in periodontal therapy to induce periodontal regeneration through the induction of new bone formation. The presence of bone morphogenetic proteins (BMPs) within the demineralized matrix has been proposed as a possible mechanism through which DFDBA may exert its biologic effect. However, in recent years, the predictability of results using DFDBA has been variable and has led to its use being questioned. One reason for the variability in tissue response may be attributed to differences in the processing of DFDBA, which may lead to loss of activity of any bioactive substances within the DFDBA matrix. Therefore, the purpose of this investigation was to determine whether there are detectable levels of bone morphogenetic proteins in commercial DFDBA preparations. METHODS: A single preparation of DFDBA was obtained from three commercial sources. Each preparation was studied in triplicate. Proteins within the DFDBA samples were first extracted with 4M guanidinium HCI for seven days at 40 degrees celsius and the residue was further extracted with 4M guanidinium HCL/EDTA for seven days at 40 degrees celsius. Two anti-human BMP-2 and -4 antibodies were used for the detection of the presence of BMP's in the extracts. RESULTS: Neither BMP-2 nor BMP-4 was detected in any of the extracts. When recombinant human BMP-2 and -4 were added throughout the extraction process of DFDBA extraction, not only were intact proteins detected but smaller molecular weight fragments were also noted in the extract. CONCLUSIONS: These results indicate that all of the DFDBA samples tested had no detectable amounts of BMP-2 and -4. In addition, an unknown substance present in the DFDBA may be responsible for degradation of whatever BMPs might be present.

Humanised xenograft models of bone metastasis revisited : novel insights into species-specific mechanisms of cancer cell osteotropism

The determinants and key mechanisms of cancer cell osteotropism have not been identified, mainly due to the lack of reproducible animal models representing the biological, genetic and clinical features seen in humans. An ideal model should be capable of recapitulating as many steps of the metastatic cascade as possible, thus facilitating the development of prognostic markers and novel therapeutic strategies. Most animal models of bone metastasis still have to be derived experimentally as most syngeneic and transgeneic approaches do not provide a robust skeletal phenotype and do not recapitulate the biological processes seen in humans. The xenotransplantation of human cancer cells or tumour tissue into immunocompromised murine hosts provides the possibility to simulate early and late stages of the human disease. Human bone or tissue-engineered human bone constructs can be implanted into the animal to recapitulate more subtle, species-specific aspects of the mutual interaction between human cancer cells and the human bone microenvironment. Moreover, the replication of the entire "organ" bone makes it possible to analyse the interaction between cancer cells and the haematopoietic niche and to confer at least a partial human immunity to the murine host. This process of humanisation is facilitated by novel immunocompromised mouse strains that allow a high engraftment rate of human cells or tissue. These humanised xenograft models provide an important research tool to study human biological processes of bone metastasis.