Constructing validity evidence from a pilot key-features assessment of clinical decision-making in cerebral palsy diagnosis: application of Kane's validity framework to implementation evaluations.

BACKGROUND: Physician decision-making skills training is a priority to improve adoption of the cerebral palsy (CP) clinical guideline and, through this, lower the age of CP diagnosis. Clinical guideline implementation aims to improve physician practice, but evaluating meaningful change is complex. Limitations in the validity evidence of evaluation instruments impact the evidence base. Validity frameworks, such as Kane's, enable a targeted process to gather evidence for instrument scores, congruent to context and purpose. Yet, application of argument-based methodology to implementation validation is rare. Key-features examination methodology has established validity evidence supporting its use to measure decision-making skills, with potential to predict performance. We aimed to apply Kane's framework to evaluate a pilot key-features examination on physician decision-making in early CP diagnosis. METHODS: Following Kane's framework, we evaluated evidence across inferences of scoring, generalisation, extrapolation and implications in a study design describing the development and pilot of a CP diagnosis key-features examination for practising physicians. If found to be valid, we proposed to use the key-feature scores as an outcome measure of decision-making post education intervention to expedite CP diagnosis and to correlate with real-world performance data to predict physician practice. RESULTS: Supporting evidence for acceptance of scoring inferences was achieved through examination development with an expert group (n = 10) and pilot results (n = 10): (1) high internal consistency (0.82); (2) acceptable mean item-discrimination (0.34); and (3) acceptable reliability of examination scorers (95.2% congruence). Decreased physician acceptance of examination time (70%) was identified as a threat and prioritised in case reduction processes. Partial acceptance of generalisation, extrapolation and implications inferences were defensible with: (1) accumulated development evidence following established key-features methodology; (2) high pilot acceptance for authenticity (90%); and (3) plausibility of assumptions of score correlation with population register data. CONCLUSIONS: Kane's approach is beneficial for prioritising sources of validity evidence alongside the iterative development of a key-features examination in the CP field. The validity argument supports scoring assumptions and use of scores as an outcome measure of physician decision-making for CP guideline education implementation interventions. Scoring evidence provides the foundation to direct future studies exploring association of key-feature scores with real-world performance.

Temporal changes in cortical microporosity during estrogen deficiency associated with perilacunar resorption and osteocyte apoptosis: A pilot study.

Osteocytes can actively regulate bone microporosity, through either perilacunar resorption or micropetrosis following apoptosis. Osteocyte apoptosis is more prevalent in estrogen deficiency and changes in the lacunar-canalicular network of osteocytes have been reported. Temporal changes in bone mineralisation and osteocytes cellular strains occur, which might be associated with osteocyte-driven microporosity changes, although time dependant changes in bone microporosity are not yet fully understood. In this pilot study we conducted micro-CT analysis, backscatter electron imaging and histological analysis of femoral cortical bone form an ovariectomized rat model of osteoporosis to investigate whether estrogen deficiency causes temporal changes in lacunar and vascular porosity. We also assessed MMP14 expression, lacunar occupancy and mineral infilling, as indicators of perilacunar resorption and micropetrosis. We report temporal changes in cortical microporosity in estrogen deficiency. Specifically, canalicular and vascular porosity initially increased (4 weeks post-OVX), coinciding with the period of rapid bone loss, whereas in the longer term (14 weeks post-OVX) lacunar and canalicular diameter decreased. Interestingly, these changes coincided with an increased prevalence of empty lacunae and osteocyte lacunae were observed to be more circular with a mineralised border around the lacunar space. In addition we report an increase in MMP14+ osteocytes, which also suggests active matrix degradation by these cells. Together these results provide an insight into the temporal changes in cortical microporosity during estrogen deficiency and suggest the likelihood of occurrence of both perilacunar resorption and osteocyte apoptosis leading to micropetrosis. We propose that microporosity changes arise due to processes driven by distinct populations of osteocytes, which are either actively resorbing their matrix or have undergone apoptosis and are infilling lacunae by micropetrosis.

Estrogen depletion alters osteogenic differentiation and matrix production by osteoblasts in vitro.

Recent studies have revealed that the effects of estrogen deficiency are not restricted to osteoclasts and bone resorption, but that bone matrix composition is altered and osteoblasts exhibit an impaired response to mechanical stimulation. In this study, we test the hypothesis that estrogen depletion alters osteogenic differentiation and matrix production by mechanically stimulated osteoblasts in vitro. MC3T3-E1 cells were pre-treated with estrogen for 14 days, after which estrogen was withdrawn or inhibited with Fulvestrant up to 14 days. Fluid shear stress (FSS) was applied using an orbital shaker. Under estrogen depletion in static culture, osteogenic marker (ALP) and gene expression (Runx2) were decreased at 2 and after 7 days of estrogen depletion, respectively. In addition, up to 7 day the inhibition of the estrogen receptor significantly decreased fibronectin expression (FN1) under static conditions. Under estrogen depletion and daily mechanical stimulation, changes in expression of Runx2 occurred earlier (4 days) and by 14 days, changes in matrix production (Col1a1) were reported. We propose that changes in osteoblast differentiation and impaired matrix production during estrogen depletion may contribute to the altered quality of the bone and act as a contributing factor to increased bone fragility in postmenopausal osteoporosis.

Stem Cell Mechanobiology and the Role of Biomaterials in Governing Mechanotransduction and Matrix Production for Tissue Regeneration.

Mechanobiology has underpinned many scientific advances in understanding how biophysical and biomechanical cues regulate cell behavior by identifying mechanosensitive proteins and specific signaling pathways within the cell that govern the production of proteins necessary for cell-based tissue regeneration. It is now evident that biophysical and biomechanical stimuli are as crucial for regulating stem cell behavior as biochemical stimuli. Despite this, the influence of the biophysical and biomechanical environment presented by biomaterials is less widely accounted for in stem cell-based tissue regeneration studies. This Review focuses on key studies in the field of stem cell mechanobiology, which have uncovered how matrix properties of biomaterial substrates and 3D scaffolds regulate stem cell migration, self-renewal, proliferation and differentiation, and activation of specific biological responses. First, we provide a primer of stem cell biology and mechanobiology in isolation. This is followed by a critical review of key experimental and computational studies, which have unveiled critical information regarding the importance of the biophysical and biomechanical cues for stem cell biology. This review aims to provide an informed understanding of the intrinsic role that physical and mechanical stimulation play in regulating stem cell behavior so that researchers may design strategies that recapitulate the critical cues and develop effective regenerative medicine approaches.

Scl-Ab reverts pro-osteoclastogenic signalling and resorption in estrogen deficient osteocytes.

BACKGROUND: Neutralising antibodies to sclerostin (Scl-Ab) have shown significant potential to induce bone formation and decrease bone resorption, increase strength and substantially reduce fracture risk in animal studies and clinical trials. Mechanical loading negatively regulates sclerostin expression, and sclerostin has been shown to induce RANKL synthesis in osteocytes. However, how Scl-Ab governs osteocyte regulation of osteoclast differentiation and function is not fully understood. We have recently discovered that osteoblasts and osteocytes alter osteoclastogenic signalling (RANKL/OPG) during estrogen-deficiency, and that osteoblast-induced osteoclastogenesis and resorption are exacerbated. However, it is not known whether estrogen deficient osteocytes exacerbate osteoclastogenesis. The aims of this study were to (1) establish whether osteocytes induce osteoclastogenesis and bone resorption during estrogen deficiency in vitro (2) investigate whether the sclerostin antibody can revert osteocyte-mediated osteoclastogenesis and resorption by attenuating RANKL/OPG expression. RESULTS: Using conditioned media and co-culture experiments we found increased osteocyte-induced osteoclastogenesis and bone resorption in estrogen deficient conditions. This is the first study to report that administration of Scl-Ab has the ability to revert osteocyte-mediated osteoclastogenesis and resorption by decreasing RANKL/OPG ratio expression and increasing WISP1 expression in estrogen deficient osteocytes. CONCLUSIONS: This study provides an enhanced understanding of the biological changes underpinning decreases in bone resorption following Scl-Ab treatment observed in vivo by revealing that Scl-Ab can reduce pro-osteoclastogenic cell signalling between osteocytes and osteoclasts.

Secondary alterations in bone mineralisation and trabecular thickening occur after long-term estrogen deficiency in ovariectomised rat tibiae, which do not coincide with initial rapid bone loss.

This study delineates the time sequence of changes in bone tissue mineralisation in ovariectomised rats. We report that changes in bone mineral distribution arise secondary to the initial rapid bone loss but coincide with trabecular thickening. We propose that these changes compensate for elevated stresses in remaining trabeculae after bone resorption. INTRODUCTION: Recent studies have shown that osteoporosis is not simply a disease of bone loss and microarchitectural degradation but that important changes in tissue composition also occur. Such changes may be a secondary response to early bone loss, but the time sequence of changes in bone mineral distribution is not fully understood. The objective of this study was to quantify the temporal effects of estrogen deficiency on trabecular mineral distribution in the tibia of ovariectomised (OVX) rats. METHODS: Weekly in vivo micro-CT scans and morphometric and bone mineral density distribution analyses of the proximal tibia were conducted for the first 4 weeks of estrogen deficiency and then at 8, 14 and 34 weeks. RESULTS: Here we report that although trabecular bone volume and architecture are significantly deteriorated within the first 4 weeks of estrogen deficiency, there is no change in the distribution of bone mineral within trabeculae during this initial period. The rate of bone loss in OVX animals dramatically reduced between week 4 and week 14, which coincided with the initiation of increases in trabecular thickness and mineralisation in the OVX group. CONCLUSIONS: Together this study reveals for the first time that alterations in bone mineralisation and trabecular thickening arise secondary to the initial rapid bone loss. We propose that these secondary mineralisation changes act to reinforce the trabecular network in an attempt to compensate for the increased loading that ensues after severe bone loss. This study provides an insight into temporal changes in bone mineral distribution in estrogen deficiency.

Inhibition of osteoclastogenesis by mechanically stimulated osteoblasts is attenuated during estrogen deficiency.

Osteoporotic bone loss and fracture have long been regarded to arise upon depletion of circulating estrogen, which increases osteoclastogenesis and bone resorption. Osteoblasts from human osteoporotic patients also display deficient osteogenic responses to mechanical loading. However, while osteoblasts play an important role in regulating osteoclast differentiation, how this relationship is affected by estrogen deficiency is unknown. This study seeks to determine how mechanically stimulated osteoblasts regulate osteoclast differentiation and matrix degradation under estrogen deficiency. Here, we report that osteoblast-induced osteoclast differentiation (indicated by tartrate-resistant acid phosphatase, cathepsin K, and nuclear factor of activated T cells, cytoplasmic 1) and matrix degradation were inhibited by estrogen treatment and mechanical loading. However, estrogen-deficient osteoblasts exacerbated osteoclast formation and matrix degradation in conditioned medium and coculture experiments. This was accompanied by higher expression of cyclooxygenase-2 and macrophage colony-stimulating factor, but not osteoprotegerin, by osteoblasts under estrogen deficiency. Interestingly, this response was exacerbated under conditions that block the Rho-Rho-associated protein kinase signaling pathway. This study provides an important, but previously unrecognized, insight into bone loss in postmenopausal osteoporosis, whereby estrogen-deficient osteoblasts fail to produce inhibitory osteoprotegerin after mechanical stimulation but upregulate macrophage colony-stimulating factor and cyclooxygenase-2 expression and, thus, leave osteoclast activity unconstrained.

The role of adhesion junctions in the biomechanical behaviour and osteogenic differentiation of 3D mesenchymal stem cell spheroids.

Osteogenesis of mesenchymal stem cells (MSC) can be regulated by the mechanical environment. MSCs grown in 3D spheroids (mesenspheres) have preserved multi-lineage potential, improved differentiation efficiency, and exhibit enhanced osteogenic gene expression and matrix composition in comparison to MSCs grown in 2D culture. Within 3D mesenspheres, mechanical cues are primarily in the form of cell-cell contraction, mediated by adhesion junctions, and as such adhesion junctions are likely to play an important role in the osteogenic differentiation of mesenspheres. However the precise role of N- and OB-cadherin on the biomechanical behaviour of mesenspheres remains unknown. Here we have mechanically tested mesenspheres cultured in suspension using parallel plate compression to assess the influence of N-cadherin and OB-cadherin adhesion junctions on the viscoelastic properties of the mesenspheres during osteogenesis. Our results demonstrate that N-cadherin and OB-cadherin have different effects on mesensphere viscoelastic behaviour and osteogenesis. When OB-cadherin was silenced, the viscosity, initial and long term Young's moduli and actin stress fibre formation of the mesenspheres increased in comparison to N-cadherin silenced mesenspheres and mesenspheres treated with a scrambled siRNA (Scram) at day 2. Additionally, the increased viscoelastic material properties correlate with evidence of calcification at an earlier time point (day 7) of OB-cadherin silenced mesenspheres but not Scram. Interestingly, both N-cadherin and OB-cadherin silenced mesenspheres had higher BSP2 expression than Scram at day 14. Taken together, these results indicate that N-cadherin and OB-cadherin both influence mesensphere biomechanics and osteogenesis, but play different roles.

Primary cilia expression in bone marrow in response to mechanical stimulation in explant bioreactor culture.

Bone marrow contains a multitude of mechanically sensitive cells that may participate in mechanotransduction. Primary cilia are sensory organelles expressed on mesenchymal stem cells (MSCs), osteoblasts, osteocytes, and other cell types that sense fluid flow in monolayer culture. In marrow, cilia could similarly facilitate the sensation of relative motion between adjacent cells or interstitial fluid. The goal of this study was to determine the response of cilia to mechanical stimulation of the marrow. Bioreactors were used to supply trabecular bone explants with low magnitude mechanical stimulation (LMMS) of 0.3 ×g at 30 Hz for 1 h/d, 5 d/week, inducing shear stresses in the marrow. Four groups were studied: unstimulated (UNSTIM), stimulated (LMMS), and with and without chloral hydrate (UNSTIM+CH and LMMS+CH, respectively), which was used to disrupt cilia. After 19 days of culture, immunohistochemistry for acetylated α-tubulin revealed that more cells expressed cilia in culture compared to in vivo controls. Stimulation decreased the number of cells expressing cilia in untreated explants, but not in CH-treated explants. MSCs represented a greater fraction of marrow cells in the untreated explants than CH-treated explants. MSCs harvested from the stimulated groups were more proliferative than in the unstimulated explants, but this effect was absent from CH treated explants. In contrast to the marrow, neither LMMS nor CH treatment affected bone formation as measured by mineralising surface. Computational models indicated that LMMS does not induce bone strain, and the reported effects were thus attributed to shear stress in the marrow. From a clinical perspective, genetic or pharmaceutical alterations of cilia expression may affect marrow health and function.

Osteocyte differentiation and the formation of an interconnected cellular network in vitro.

Extracellular matrix (ECM) stiffness and cell density can regulate osteoblast differentiation in two dimensional environments. However, it is not yet known how osteoblast-osteocyte differentiation is regulated within a 3D ECM environment, akin to that existing in vivo. In this study we test the hypothesis that osteocyte differentiation is regulated by a 3D cell environment, ECM stiffness and cell density. We encapsulated MC3T3-E1 pre-osteoblastic cells at varied cell densities (0.25, 1 and 2 × 106 cells/mL) within microbial transglutaminase (mtgase) gelatin hydrogels of low (0.58 kPa) and high (1.47 kPa) matrix stiffnesses. Cellular morphology was characterised from phalloidin-FITC and 4',6-diamidino-2-phenylindole (DAPI) dilactate staining. In particular, the expression of cell dendrites, which are phenotypic of osteocyte differentiation, were identified. Immunofluorescent staining for the osteocytes specific protein DMP-1 was conducted. Biochemical analyses were performed to determine cell number, alkaline phosphatase activity and mineralisation at 2.5 hours, 3, 21 and 56 days. We found that osteocyte differentiation and the formation of an interconnected network between dendritic cells was significantly increased within low stiffness 3D matrices, compared to cells within high stiffness matrices, at high cell densities. Moreover we saw that this network was interconnected, expressed DMP-1 and also connected with osteoblast-like cells at the matrix surface. This study shows for the first time the role of the 3D physical nature of the ECM and cell density for regulating osteocyte differentiation and the formation of the osteocyte network in vitro. Future studies could apply this method to develop 3D tissue engineered constructs with an osteocyte network in place.

Investigation of the optimal timing for chondrogenic priming of MSCs to enhance osteogenic differentiation in vitro as a bone tissue engineering strategy.

Recent in vitro tissue engineering approaches have shown that chondrogenic priming of human bone marrow mesenchymal stem cells (MSCs) can have a positive effect on osteogenesis in vivo. However, whether chondrogenic priming is an effective in vitro bone regeneration strategy is not yet known. In particular, the appropriate timing for chondrogenic priming in vitro is unknown albeit that in vivo cartilage formation persists for a specific period before bone formation. The objective of this study is to determine the optimum time for chondrogenic priming of MSCs to enhance osteogenic differentiation by MSCs in vitro. Pellets derived from murine and human MSCs were cultured in six different media groups: two control groups (chondrogenic and osteogenic) and four chondrogenic priming groups (10, 14, 21 and 28 days priming). Biochemical analyses (Hoechst, sulfate glycosaminoglycan (sGAG), Alkaline Phosphate (ALP), calcium), histology (Alcian Blue, Alizarin Red) and immunohistochemistry (collagen types I, II and X) were performed on the samples at specific times. Our results show that after 49 days the highest amount of sGAG production occurred in MSCs chondrogenically primed for 21 days and 28 days. Moreover we found that chondrogenic priming of MSCs in vitro for specific amounts of time (14 days, 21 days) can have optimum influence on their mineralization capacity and can produce a construct that is mineralized throughout the core. Determining the optimum time for chondrogenic priming to enhance osteogenic differentiation in vitro provides information that might lead to a novel regenerative treatment for large bone defects, as well as addressing the major limitation of core degradation and construct failure.

An Experimental and Computational Investigation of Bone Formation in Mechanically Loaded Trabecular Bone Explants.

Understanding how bone marrow multipotent stromal cells (MSCs) contribute to new bone formation and remodeling in vivo is of principal importance for informing the development of effective bone tissue engineering strategies in vitro. However, the precise in situ stimuli that MSCs experience have not been fully established. The shear stress generated within the bone marrow of physiologically loaded samples has never been determined, but could be playing an important role in the generation of sufficient stimulus for MSCs to undergo osteogenic differentiation. In this study fluid structure interaction (FSI) computational models were used in conjunction with a bioreactor which physiologically compresses explanted trabecular bone samples to determine whether MSCs can be directly stimulated by mechanical cues within the bone marrow. Experimentally loaded samples were found to have greater osteogenic activity, as verified by bone histomorphometry, compared to control static samples. FSI models demonstrated a linear relationship between increasing shear stress and decreasing bone volume. The FSI models demonstrated that bone strain, not marrow shear stress, was likely the overall driving mechanical signal for new bone formation during compression. However, the shear stress generated in the models is within the range of values which has been shown previously to generate an osteogenic response in MSCs.

Mechanical stimulation of bone marrow in situ induces bone formation in trabecular explants.

Low magnitude high frequency (LMHF) loading has been shown to have an anabolic effect on trabecular bone in vivo. However, the precise mechanical signal imposed on the bone marrow cells by LMHF loading, which induces a cellular response, remains unclear. This study investigates the influence of LMHF loading, applied using a custom designed bioreactor, on bone adaptation in an explanted trabecular bone model, which isolated the bone and marrow. Bone adaptation was investigated by performing micro CT scans pre and post experimental LMHF loading, using image registration techniques. Computational fluids dynamic models were generated using the pre-experiment scans to characterise the mechanical stimuli imposed by the loading regime prior to adaptation. Results here demonstrate a significant increase in bone formation in the LMHF loaded group compared to static controls and media flow groups. The calculated shear stress in the marrow was between 0.575 and 0.7 Pa, which is within the range of stimuli known to induce osteogenesis by bone marrow mesenchymal stem cells in vitro. Interestingly, a correlation was found between the bone formation balance (bone formation/resorption), trabecular number, trabecular spacing, mineral resorption rate, bone resorption rate and mean shear stresses. The results of this study suggest that the magnitude of the shear stresses generated due to LMHF loading in the explanted bone cores has a contributory role in the formation of trabecular bone and improvement in bone architecture parameters.

Multiscale modeling of trabecular bone marrow: understanding the micromechanical environment of mesenchymal stem cells during osteoporosis.

Mechanical loading directs the differentiation of mesenchymal stem cells (MSCs) in vitro and it has been hypothesized that the mechanical environment plays a role in directing the cellular fate of MSCs in vivo. However, the complex multicellular composition of trabecular bone marrow means that the precise nature of mechanical stimulation that MSCs experience in their native environment is not fully understood. In this study, we developed a multiscale model that discretely represents the cellular constituents of trabecular bone marrow and applied this model to characterize mechanical stimulation of MCSs in vivo.We predicted that cell-level strains in certain locations of the trabecular marrow microenvironment were greater in magnitude (maximum e12»24,000 le) than levels that have been found to result in osteogenic differentiation of MSCs in vitro (>8000 le),which may indicate that the native mechanical environment of MSCs could direct cellular fate in vivo. The results also showed that cell­cell adhesions could play an important role in mediating mechanical stimulation within the MSC population in vivo. The model was applied to investigate how changes that occur during osteoporosis affected mechanical stimulation in the cellular microenvironment of trabecular bone marrow. Specifically,a reduced bone volume (BV) resulted in an overall increase in bone deformation, leading to greater cell-level mechanical stimulation in trabecular bone marrow (maximume12»48,000 le). An increased marrow adipocyte content resulted in slightly lower levels of stimulation within the adjacent cell population due to a shielding effect caused by the more compliant behavior of adipocytes (maximum e12»41,000 le). Despite this reduction, stimulation levels in trabecular bone marrow during osteoporosis remained much higher than those predicted to occur under healthy conditions. It was found that compensatory mechanobiological responses that occur during osteoporosis, such as increased trabecular stiffness and axial alignment of trabeculae, would be effective in returning MSC stimulation in trabecular marrow to normal levels. These results have provided novel insight into the mechanical stimulation of the trabecular marrow MSC population in both healthy and osteoporotic bone, and could inform the design three dimensional(3D) in vitro bioreactor strategies techniques, which seek to emulate physiological conditions.

Bone cell mechanosensation of fluid flow stimulation: a fluid-structure interaction model characterising the role integrin attachments and primary cilia.

Load-induced fluid flow acts as an important biophysical signal for bone cell mechanotransduction in vivo, where the mechanical environment is thought to be monitored by integrin and primary cilia mechanoreceptors on the cell body. However, precisely how integrin- and primary cilia-based mechanosensors interact with the surrounding fluid flow stimulus and ultimately contribute to the biochemical response of bone cells within either the in vitro or in vivo environment remains poorly understood. In this study, we developed fluid-structure interaction models to characterise the deformation of integrin- and primary cilia-based mechanosensors in bone cells under fluid flow stimulation. Under in vitro fluid flow stimulation, these models predicted that integrin attachments on the cell-substrate interface were highly stimulated ε(eq) > 200,000 µÎµ, while the presence of a primary cilium on the cell also resulted in significant strain amplifications, arising at the ciliary base. As such, these mechanosensors likely play a role in mediating bone mechanotransduction in vitro. Under in vivo fluid flow stimulation, integrin attachments along the canalicular wall were highly stimulated and likely play a role in mediating cellular responses in vivo. The role of the primary cilium as a flow sensor in vivo depended upon its configuration within the lacunar cavity. Specifically, our results showed that a short free-standing primary cilium could not effectively fulfil a flow sensing role in vivo. However, a primary cilium that discretely attaches the lacunar wall can be highly stimulated, due to hydrodynamic pressure in the lacunocanalicular system and, as such, could play a role in mediating bone mechanotransduction in vivo.

Cell morphology and focal adhesion location alters internal cell stress.

Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues.

Estrogen withdrawal from osteoblasts and osteocytes causes increased mineralization and apoptosis.

Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested two separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated with or without estrogen (17ß-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis.

Effects of ageing, prolonged estrogen deficiency and zoledronate on bone tissue mineral distribution.

The quantity and distribution of bone tissue mineral are key determinants of bone strength. Recent research revealed altered mineral distribution within sheep femora following estrogen deficiency. Rapid increases in bone remodeling occur at the onset of estrogen deficiency and abate over time. Therefore, altered tissue mineralization might be a transient characteristic of osteoporosis. Bisphosphonates reduce fracture incidence by 40-60% but increases in bone mineral density are insufficient to explain such changes. In this study the hypotheses that bone tissue mineralization is altered over prolonged estrogen depletion and bisphosphonate treatment were tested. Quantitative backscattered imaging (qBEI) was used to quantify bone mineral density distribution (BMDD) parameters (mean, FWHM) in trabeculae from the proximal femora of an ovariectomized sheep model that underwent estrogen deficiency for 31 months, an ovariectomized group administered with Zoledronic acid and age-matched controls. To assess the effects of normal ageing and prolonged estrogen deficiency, data were compared to BMDD data from sheep that were estrogen deficient for 12 months and age-matched controls. This study reports that normal ageing increases mean mineralization and mineral heterogeneity at a trabecular level. In contrast, prolonged estrogen deficiency leads to significantly decreased mean mineralization and further exacerbates increases in mineral heterogeneity. Interestingly, ZOL treatment of OVX sheep significantly reduced tissue mineral variability, both at a trabecular level and between femoral regions. Together, these findings indicate that ZOL treatment acts to reverse the increased mineral heterogeneity occurring during estrogen deficiency, which may contribute to its capacity to reduce osteoporotic fractures.

Osteocyte differentiation is regulated by extracellular matrix stiffness and intercellular separation.

Osteocytes are terminally differentiated bone cells, derived from osteoblasts, which are vital for the regulation of bone formation and resorption. ECM stiffness and cell seeding density have been shown to regulate osteoblast differentiation, but the precise cues that initiate osteoblast-osteocyte differentiation are not yet understood. In this study, we cultured MC3T3-E1 cells on (A) substrates of different chemical compositions and stiffnesses, as well as, (B) substrates of identical chemical composition but different stiffnesses. The effect of cell separation was investigated by seeding cells at different densities on each substrate. Cells were evaluated for morphology, alkaline phosphatase (ALP), matrix mineralisation, osteoblast specific genes (Type 1 collagen, Osteoblast specific factor (OSF-2)), and osteocyte specific proteins (dentin matrix protein 1 (DMP-1), sclerostin (Sost)). We found that osteocyte differentiation (confirmed by dendritic morphology, mineralisation, reduced ALP, Col type 1 and OSF-2 and increased DMP-1 and Sost expression) was significantly increased on soft collagen based substrates, at low seeding densities compared to cells on stiffer substrates or those plated at high seeding density. We propose that the physical nature of the ECM and the necessity for cells to establish a communication network contribute substantially to a concerted shift toward an osteocyte-like phenotype by osteoblasts in vitro.

Computational modelling of the mechanics of trabecular bone and marrow using fluid structure interaction techniques.

Bone marrow found within the porous structure of trabecular bone provides a specialized environment for numerous cell types, including mesenchymal stem cells (MSCs). Studies have sought to characterize the mechanical environment imposed on MSCs, however, a particular challenge is that marrow displays the characteristics of a fluid, while surrounded by bone that is subject to deformation, and previous experimental and computational studies have been unable to fully capture the resulting complex mechanical environment. The objective of this study was to develop a fluid structure interaction (FSI) model of trabecular bone and marrow to predict the mechanical environment of MSCs in vivo and to examine how this environment changes during osteoporosis. An idealized repeating unit was used to compare FSI techniques to a computational fluid dynamics only approach. These techniques were used to determine the effect of lower bone mass and different marrow viscosities, representative of osteoporosis, on the shear stress generated within bone marrow. Results report that shear stresses generated within bone marrow under physiological loading conditions are within the range known to stimulate a mechanobiological response in MSCs in vitro. Additionally, lower bone mass leads to an increase in the shear stress generated within the marrow, while a decrease in bone marrow viscosity reduces this generated shear stress.