Muriel Voisin

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 e12¼24,000 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 cell–cell 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 (maximume12¼48,000 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 e12¼41,000 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.

Osteoblasts response to microstructured and nanostructured polyimide film, processed by the use of silica bead microlenses

UNLABELLED The surface of polyimide films was modified by the use of silica microspheres as microlenses to focus radiation emitted by an excimer laser. The resultant surface had both microstructures and nanostructures. Physical and chemical characterization was performed by atomic force and Fourier transform-infrared microscopy. Laser processing resulted in surfaces that had similar roughness but different component frequencies. Chemical changes were not observed with the techniques used. The response of osteoblasts to the surface was assayed by measuring their metabolic activity and the enzyme alkaline phosphatase activity, after 24 hours of growth. Cytoskeleton and expression were both investigated. Metabolic activity was similar on treated and untreated samples. Total cell number and size were increased on microstructured polymer, where specific structures were observed (protrusions). Adhesion was noted, and the actin cytoskeleton showed normal morphology. Cells on nanostructured samples had a diffuse actin network and less mature adhesions as compared with the control. FROM THE CLINICAL EDITOR Polyimide films with microstructure and nanostructure surface elements were studied from the standpoint of osteoblast response. Total cell number and size were increased on microstructured polymer and protrusions were also observed. Adhesion was noted and the actin cytoskeleton exhibited normal morphology. Cells on nanostructured samples had a diffuse actin network and less mature adhesions.

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.

Thermally induced osteocyte damage initiates a remodelling signaling cascade

Thermal elevations experienced by bone during orthopaedic procedures, such as cutting and drilling, exothermal reactions from bone cement, and thermal therapies such as tumor ablation, can result in thermal damage leading to death of native bone cells (osteocytes, osteoblasts, osteoclasts and mesenchymal stem cells). Osteocytes are believed to be the orchestrators of bone remodeling, which recruit nearby osteoclast and osteoblasts to control resorption and bone growth in response to mechanical stimuli and physical damage. However, whether heat-induced osteocyte damage can directly elicit bone remodelling has yet to be determined. This study establishes the link between osteocyte thermal damage and the remodeling cascade. We show that osteocytes directly exposed to thermal elevations (47°C for 1 minute) become significantly apoptotic and alter the expression of osteogenic genes (Opg and Cox2). The Rankl/Opg ratio is consistently down-regulated, at days 1, 3 and 7 in MLO-Y4s heat-treated to 47°C for 1 minute. Additionally, the pro-osteoblastogenic signaling marker Cox2 is significantly up-regulated in heat-treated MLO-Y4s by day 7. Furthermore, secreted factors from heat-treated MLO-Y4s administered to MSCs using a novel co-culture system are shown to activate pre-osteoblastic MSCs to increase production of the pro-osteoblastic differentiation marker, alkaline phosphatase (day 7, 14), and calcium deposition (day 21). Most interestingly, an initial pro-osteoclastogenic signaling response (increase Rankl and Rankl/Opg ratio at day 1) followed by later stage pro-osteoblastogenic signaling (down-regulation in Rankl and the Rankl/Opg ratio and an up-regulation in Opg and Cox2 by day 7) was observed in non-heat-treated MLO-Y4s in co-culture when these were exposed to the biochemicals produced by heat-treated MLO-Y4s. Taken together, these results elucidate the vital role of osteocytes in detecting and responding to thermal damage by means of thermally induced apoptosis followed by a cascade of remodelling responses.

Wash-free highly sensitive detection of C-reactive protein using gold derivatised triangular silver nanoplates

A rapid, wash-free highly sensitive detection method for C-reactive protein (hs-CRP) is reported with both triangular silver nanoplate (TSNP) and Au-edge coated TSNP biosensor sols. The latter shows higher assay sensitivity as well as excellent stability under assay conditions including centrifugation and salinity. A series of Au derived TSNP sols including Au-edge-coated TSNP, AuAg nanomesh and nanobox derived sols are examined as statistically representative saline-stable enhanced ensemble local surface plasmon resonance (LSPR) refractive index sensitive sensors. Samples with plasmon bands spanning the biological spectral window are prepared showing enhancement compared with the original TSNP sols. Refractive index sensitivities as high as 1816 nm RIU−1 are exhibited by Au-edge-coated TSNP sols. The priority of ensemble refractive index sensitivity values over figures of merit in characterising these sols is examined using discrete dipole approximation calculations and single nanostructure dark field microscopy measurements. We anticipate that the high ensemble LSPR refractive index sensitivities, saline stability and ultra-low detection limit capacity of the Au derived TSNP sols, in particular Au-edge coated TSNP sols, present them as excellent candidates for sensing within biological environments.

A role for the p53 tumour suppressor in regulating the balance between homologous recombination and non-homologous end joining

Loss of p53, a transcription factor activated by cellular stress, is a frequent event in cancer. The role of p53 in tumour suppression is largely attributed to cell fate decisions. Here, we provide evidence supporting a novel role for p53 in the regulation of DNA double-strand break (DSB) repair pathway choice. 53BP1, another tumour suppressor, was initially identified as p53 Binding Protein 1, and has been shown to inhibit DNA end resection, thereby stimulating non-homologous end joining (NHEJ). Yet another tumour suppressor, BRCA1, reciprocally promotes end resection and homologous recombination (HR). Here, we show that in both human and mouse cells, the absence of p53 results in impaired 53BP1 focal recruitment to sites of DNA damage induced by ionizing radiation. This effect is largely independent of cell cycle phase and the extent of DNA damage. In p53-deficient cells, diminished localization of 53BP1 is accompanied by a reciprocal increase in BRCA1 recruitment to DSBs. Consistent with these findings, we demonstrate that DSB repair via NHEJ is abrogated, while repair via homology-directed repair (HDR) is stimulated. Overall, we propose that in addition to its role as an ‘effector’ protein in the DNA damage response, p53 plays a role in the regulation of DSB repair pathway choice.

Differential β3 and β1 Integrin Expression in Bone Marrow and Cortical Bone of Estrogen Deficient Rats

Integrin‐based (β3) attachments to the extracellular matrix (ECM) on osteocyte cell processes have recently been proposed to play an important role in facilitating osteocyte mechanosensation. However, it is not yet known whether integrin expression is altered in the mechanoregulatory osteocytes during osteoporosis. The objective of this study was to test the hypothesis that the expression of integrin‐based mechanosensory complexes (β1 and β3 integrins) is altered as a direct response to estrogen deficiency, in an estrogen deficient animal model of osteoporosis. Four weeks post‐operatively, immunohistochemistry was used to detect for β1 and β3 integrin subunits in bone tissue and marrow of ovariectomized (OVX; N = 4) and SHAM (N = 4) operated animals. A tartrate resistant acid phosphatase (TRAP) control stain was performed to quantify the presence of osteoclasts in the bone marrow and bone surfaces. Image analysis was performed to quantify expression patterns in different biological compartments, that is, bone marrow, endosteum, and cortical bone. Our results showed that β1 integrins were ubiquitously expressed throughout the bone and marrow, for both OVX and SHAM groups. β3 integrin subunit expression was lower in bone cells from osteoporotic animals compared to controls, whereas β3 expression in marrow cells did not differ significantly between groups. At the endosteum no difference was observed in β3 integrin subunit expression. As expected, the number of osteoclasts was higher in the OVX group validating an imbalance in bone remodeling. We propose that a reduction in β3 integrin expression in osteocytes might impair mechanosensation by bone cells during estrogen deficiency. Anat Rec, 298:1548–1559, 2015.

Primary Cilia Knockdown Reduces the Number of Stromal Cells in Three Dimensional Ex Vivo Culture

Mesenchymal stem cells (MSCs) are multipotent stromal cells that reside in the bone marrow and differentiate into connective cell lines, such as adipocytes and osteoblasts [1]. An appropriate balance of MSC differentiation toward adipocytes and osteoblasts is vital to bone homeostasis [6]. In vitro work demonstrates that differentiation of MSCs is influenced by mechanical stimuli [2, 3]. In a mouse model, the ratio of adipocytes to MSCs in the marrow was 19% lower compared to controls following treatment by low magnitude mechanical signals (LMMS) [4]. In mice, LMMS increased MSC number by 46% and the differentiation capacity of MSCs was biased towards osteoblastic compared to adipogenic differentiation [5]. Thus, mechanobiological stimuli may play an important role in maintaining balanced MSC differentiation.Copyright