Multiple verification in computational modeling of bone pathologies

We introduce a model checking approach to diagnose the emerging of bone pathologies. The implementation of a new model of bone remodeling in PRISM has led to an interesting characterization of osteoporosis as a defective bone remodeling dynamics with respect to other bone pathologies. Our approach allows to derive three types of model checking-based diagnostic estimators. The first diagnostic measure focuses on the level of bone mineral density, which is currently used in medical practice. In addition, we have introduced a novel diagnostic estimator which uses the full patient clinical record, here simulated using the modeling framework. This estimator detects rapid (months) negative changes in bone mineral density. Independently of the actual bone mineral density, when the decrease occurs rapidly it is important to alarm the patient and monitor him/her more closely to detect insurgence of other bone co-morbidities. A third estimator takes into account the variance of the bone density, which could address the investigation of metabolic syndromes, diabetes and cancer. Our implementation could make use of different logical combinations of these statistical estimators and could incorporate other biomarkers for other systemic co-morbidities (for example diabetes and thalassemia). We are delighted to report that the combination of stochastic modeling with formal methods motivate new diagnostic framework for complex pathologies. In particular our approach takes into consideration important properties of biosystems such as multiscale and self-adaptiveness. The multi-diagnosis could be further expanded, inching towards the complexity of human diseases. Finally, we briefly introduce self-adaptiveness in formal methods which is a key property in the regulative mechanisms of biological systems and well known in other mathematical and engineering areas.Comment: In Proceedings CompMod 2011, arXiv:1109.104

Recruitment, augmentation and apoptosis of rat osteoclasts in 1,25-(OH)2D3 response to short-term treatment with 1,25-dihydroxyvitamin D3in vivo

Background Although much is known about the regulation of osteoclast (OC) formation and activity, little is known about OC senescence. In particular, the fate of of OC seen after 1,25-(OH)2D3 administration in vivo is unclear. There is evidence that the normal fate of OC is to undergo apoptosis (programmed cell death). We have investigated the effect of short-term application of high dose 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on OC apoptosis in an experimental rat model. Methods OC recruitment, augmentation and apoptosis was visualised and quantitated by staining histochemically for tartrate resistant acid phosphatase (TRAP), double staining for TRAP/ED1 or TRAP/DAPI, in situ DNA fragmentation end labelling and histomorphometric analysis. Results Short-term treatment with high-dose 1,25-(OH)2D3 increased the recruitment of OC precursors in the bone marrow resulting in a short-lived increase in OC numbers. This was rapidly followed by an increase in the number of apoptotic OC and their subsequent removal. The response of OC to 1,25-(OH)2D3 treatment was dose and site dependent; higher doses producing stronger, more rapid responses and the response in the tibiae being consistently stronger and more rapid than in the vertebrae. Conclusions This study demonstrates that (1) after recruitment, OC are removed from the resorption site by apoptosis (2) the combined use of TRAP and ED1 can be used to identify OC and their precursors in vivo (3) double staining for TRAP and DAPI or in situ DNA fragmentation end labelling can be used to identify apoptotic OC in vivo

Estrogens Attenuate Oxidative Stress and the Differentiation and Apoptosis of Osteoblasts by DNA-Binding-Independent Actions of the ERα

Estrogens diminish oxidative stress in bone and bone marrow, attenuate the generation of osteoblasts, and decrease the prevalence of mature osteoblast apoptosis. We have searched for the molecular mechanism of these effects using as tools a mouse model bearing an estrogen receptor α (ERα) knock-in mutation that prevents binding to DNA (ERαNERKI/−) and several osteoblast progenitor cell models expressing the wild-type ERα or the ERαNERKI/−. We report that the ability of estrogens to diminish the generation of reactive oxygen species, stimulate the activity of glutathione reductase, and decrease the phosphorylation of p66shc, as well as osteoblastogenesis and osteoblast number and apoptosis, were fully preserved in ERαNERKI/− mice, indicating that the DNA-binding function of the ERα is dispensable for all these effects. Consistent with the attenuation of osteoblastogenesis in this animal model, 17β-estradiol attenuated bone morphogenetic protein 2 (BMP-2)–induced gene transcription and osteoblast commitment and differentiation in murine and human osteoblastic cell lines. Moreover, 17β-estradiol attenuated BMP-2-induced differentiation of primary cultures of calvaria- or bone marrow–derived osteoblastic cells from ERαNERKI/− mice as effectively as in cells from wild-type littermates. The inhibitory effect of the hormone on BMP-2 signaling resulted from an ERα-mediated activation of ERKs and the phosphorylation of Smad1 at the linker region of the protein, which leads to proteasomal degradation. These results illustrate that the effects of estrogens on oxidative stress and the birth and death of osteoblasts do not require the binding of ERα to DNA response elements, but instead they result from the activation of cytoplasmic kinases. © 2010 American Society for Bone and Mineral Researc

Reduced Reactivation from Dormancy but Maintained Lineage Choice of Human Mesenchymal Stem Cells with Donor Age

Mesenchymal stem cells (MSC) are promising for cell-based regeneration therapies but up to date it is still controversial whether their function is maintained throughout ageing. Aim of this study was to address whether frequency, activation in vitro, replicative function, and in vitro lineage choice of MSC is maintained throughout ageing to answer the question whether MSC-based regeneration strategies should be restricted to younger individuals. MSC from bone marrow aspirates of 28 donors (5–80 years) were characterized regarding colony-forming unit-fibroblast (CFU-F) numbers, single cell cloning efficiency (SSCE), osteogenic, adipogenic and chondrogenic differentiation capacity in vitro. Alkaline phosphatase (ALP) activity, mineralization, Oil Red O content, proteoglycan- and collagen type II deposition were quantified. While CFU-F frequency was maintained, SSCE and early proliferation rate decreased significantly with advanced donor age. MSC with higher proliferation rate before start of induction showed stronger osteogenic, adipogenic and chondrogenic differentiation. MSC with high osteogenic capacity underwent better chondrogenesis and showed a trend to better adipogenesis. Lineage choice was, however, unaltered with age. Conclusion: Ageing influenced activation from dormancy and replicative function of MSC in a way that it may be more demanding to mobilize MSC to fast cell growth at advanced age. Since fast proliferation came along with high multilineage capacity, the proliferation status of expanded MSC rather than donor age may provide an argument to restrict MSC-based therapies to certain individuals

Sex-related variation in compact bone microstructure of the femoral diaphysis in juvenile rabbits

<p>Abstract</p> <p>Background</p> <p>While gross morphological changes in the skeleton between males and females are well know, differences between sexes in the histomorphology are less known. It is important to have knowledge on the bone structure of rabbits, as this is a widely used species in biomedical research. A study was performed to evaluate the association between sex and the compact bone morphology of the femoral diaphysis in juvenile rabbits.</p> <p>Methods</p> <p>Seventeen clinically healthy 2–3 month-old rabbits (9 females, 8 males) were included in the study. The rabbits were euthanized and the right femur was sampled for analysis. 70–80 microns thick bone sections of the femoral diaphysis were prepared using standard histological equipment. The qualitative histological characteristics were determined according to internationally accepted classification systems while the quantitative parameters were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of primary osteons' vascular canals, Haversian canals and secondary osteons were measured. Additionally, blood plasma concentrations of progesterone, corticosterone, IGF-I, testosterone and estradiol were analyzed.</p> <p>Results</p> <p>Qualitative histological characteristics were similar for both sexes. However, variations of certain quantitative histological characteristics were identified. Measured parameters of the primary osteons' vascular canals were higher in males than for females. On the other hand, females had significant higher values of secondary osteons parameters. Differences in Haversian canals parameters were only significant for minimum diameter.</p> <p>Conclusion</p> <p>The study demonstrated that quantitative histological characteristics of compact bone tissue of the femoral diaphysis in juvenile rabbits were sex dependent. The variations may be associated with different growth and modeling of the femur through influence by sex-specific steroids, mechanical loads, genetic factors and a multitude of other sources. The results can be applied in experimental studies focusing on comparison of the skeletal biology of the sexes.</p

Feeding Blueberry Diets in Early Life Prevent Senescence of Osteoblasts and Bone Loss in Ovariectomized Adult Female Rats

Appropriate nutrition during early development is essential for maximal bone mass accretion; however, linkage between early nutrition, childhood bone mass, peak bone mass in adulthood, and prevention of bone loss later in life has not been studied.In this report, we show that feeding a high quality diet supplemented with blueberries (BB) to pre-pubertal rats throughout development or only between postnatal day 20 (PND20) and PND34 prevented ovariectomy (OVX)-induced bone loss in adult life. This protective effect of BB is due to suppression of osteoblastic cell senescence associated with acute loss of myosin expression after OVX. Early exposure of pre-osteoblasts to serum from BB-fed rats was found to consistently increase myosin expression. This led to maintenance osteoblastic cell development and differentiation and delay of cellular entrance into senescence through regulation of the Runx2 gene. High bone turnover after OVX results in insufficient collagenous matrix support for new osteoblasts and their precursors to express myosin and other cytoskeletal elements required for osteoblast activity and differentiation.These results indicate: 1) a significant prevention of OVX-induced bone loss from adult rats can occur with only 14 days consumption of a BB-containing diet immediately prior to puberty; and 2) the molecular mechanisms underlying these effects involves increased myosin production which stimulates osteoblast differentiation and reduces mesenchymal stromal cell senescence