Osteocytes as a record of bone formation dynamics: A mathematical model of osteocyte generation in bone matrix

The formation of new bone involves both the deposition of bone matrix, and the formation of a network of cells embedded within the bone matrix, called osteocytes. Osteocytes derive from bone-synthesising cells (osteoblasts) that become buried in bone matrix during bone deposition. The generation of osteocytes is a complex process that remains incompletely understood. Whilst osteoblast burial determines the density of osteocytes, the expanding network of osteocytes regulates in turn osteoblast activity and osteoblast burial. In this paper, a spatiotemporal continuous model is proposed to investigate the osteoblast-to-osteocyte transition. The aims of the model are (i) to link dynamic properties of osteocyte generation with properties of the osteocyte network imprinted in bone, and (ii) to investigate Marotti's hypothesis that osteocytes prompt the burial of osteoblasts when they become covered with sufficient bone matrix. Osteocyte density is assumed in the model to be generated at the moving bone surface by a combination of osteoblast density, matrix secretory rate, rate of entrapment, and curvature of the bone substrate, but is found to be determined solely by the ratio of the instantaneous burial rate and matrix secretory rate. Osteocyte density does not explicitly depend on osteoblast density nor curvature. Osteocyte apoptosis is also included to distinguish between the density of osteocyte lacuna and the density of live osteocytes. Experimental measurements of osteocyte lacuna densities are used to estimate the rate of burial of osteoblasts in bone matrix. These results suggest that: (i) burial rate decreases during osteonal infilling, and (ii) the control of osteoblast burial by osteocytes is likely to emanate as a collective signal from a large group of osteocytes, rather than from the osteocytes closest to the bone deposition front.Comment: 11 pages, 6 figures. V2: substantially augmented version. Addition of Section 4 (osteocyte apoptosis

Tumor-associated Endo180 requires stromal-derived LOX to promote metastatic prostate cancer cell migration on human ECM surfaces

The diverse composition and structure of extracellular matrix (ECM) interfaces encountered by tumor cells at secondary tissue sites can influence metastatic progression. Extensive in vitro and in vivo data has confirmed that metastasizing tumor cells can adopt different migratory modes in response to their microenvironment. Here we present a model that uses human stromal cell-derived matrices to demonstrate that plasticity in tumor cell movement is controlled by the tumor-associated collagen receptor Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) and the crosslinking of collagen fibers by stromal-derived lysyl oxidase (LOX). Human osteoblast-derived and fibroblast-derived ECM supported a rounded ‘amoeboid-like’ mode of cell migration and enhanced Endo180 expression in three prostate cancer cell lines (PC3, VCaP, DU145). Genetic silencing of Endo180 reverted PC3 cells from their rounded mode of migration towards a bipolar ‘mesenchymal-like’ mode of migration and blocked their translocation on human fibroblast-derived and osteoblast-derived matrices. The concomitant decrease in PC3 cell migration and increase in Endo180 expression induced by stromal LOX inhibition indicates that the Endo180-dependent rounded mode of prostate cancer cell migration requires ECM crosslinking. In conclusion, this study introduces a realistic in vitro model for the study of metastatic prostate cancer cell plasticity and pinpoints the cooperation between tumor-associated Endo180 and the stiff microenvironment imposed by stromal-derived LOX as a potential target for limiting metastatic progression in prostate cancer

Role of galectin-3 in bone cell differentiation, bone pathophysiology and vascular osteogenesis

Galectin-3 is expressed in various tissues, including the bone, where it is considered a marker of chondrogenic and osteogenic cell lineages. Galectin-3 protein was found to be increased in the differentiated chondrocytes of the metaphyseal plate cartilage, where it favors chondrocyte survival and cartilage matrix mineralization. It was also shown to be highly expressed in differentiating osteoblasts and osteoclasts, in concomitance with expression of osteogenic markers and Runt-related transcription factor 2 and with the appearance of a mature phenotype. Galectin-3 is expressed also by osteocytes, though its function in these cells has not been fully elucidated. The effects of galectin-3 on bone cells were also investigated in galectin-3 null mice, further supporting its role in all stages of bone biology, from development to remodeling. Galectin-3 was also shown to act as a receptor for advanced glycation endproducts, which have been implicated in age-dependent and diabetes-associated bone fragility. Moreover, its regulatory role in inflammatory bone and joint disorders entitles galectin-3 as a possible therapeutic target. Finally, galectin-3 capacity to commit mesenchymal stem cells to the osteoblastic lineage and to favor transdifferentiation of vascular smooth muscle cells into an osteoblast-like phenotype open a new area of interest in bone and vascular pathologies

Using UV laser surface treatment to modify the wettability characteristics of polyamide 6,6 and its effects on osteoblast cell activity

Lasers can be used to modify the surface characteristics of a number of different materials for many applications. This paper details the way in which a KrF 248 nm excimer laser can be utilized to surface pattern and whole area irradiate nylon 6,6. 50 and 100 µm dimensioned trench and hatch patterns were induced in addition to the whole area irradiative processing which covered an area of 3.75 cm2 with fluencies ranging from 26 to 70 mJcm-2. The surface topography and roughness were determined with the use of a white light interferometer. From this it was found that the largest roughness, Sa, was 1.53 µm which arose from the 100 µm hatch excimer patterned sample. Wettability characteristics were obtained for each sample using a sessile drop device in which it was observed that the contact angle increased by up to 25° for the patterned samples and decreased by up to 15° for the large area processed samples. It is believed that the observed increase in contact angle can be attributed to the likely existence of a mixed-state wetting regime in which both Wenzel and Cassie-Baxter regimes are present over the liquid-solid interface. As a result of the small variation in surface roughness for the large area processed samples the observed decrease in contact angle can be explained by a modification of the surface chemistry and an increase in polar component (γp) and total surface energy (γT). Osteoblast cell activity was analyzed by carrying out cytotoxicity and alkaline leukocyte phosphatase (ALP) activity experiments, two major factors which are linked to sufficient cell growth and proliferation

Myocyte enhancer factor 2C: an osteoblast transcription factor identified by DMSO enhanced mineralization

Free to read on publisher website Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem-cell mediated therapies for fracture and other orthopaedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of simulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase (ALP) activity and extracellular matrix mineralization. Furthermore, similar DMSO mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1 we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased ALP activity and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. Flow on knockdown of bone specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts

Balancing porosity and mechanical properties of titanium samples to favor cellular growth against bacteria

Two main problems limit the success of titanium implants: bacterial infection, which restricts their osseointegration capacity; and the stiffness mismatch between the implant and the host cortical bone, which promotes bone resorption and risk of fracture. Porosity incorporation may reduce this difference in stiffness but compromise biomechanical behavior. In this work, the relationship between the microstructure (content, size, and shape of pores) and the antibacterial and cellular behavior of samples fabricated by the space-holder technique (50 vol % NH4HCO3 and three ranges of particle sizes) is established. Results are discussed in terms of the best biomechanical properties and biofunctional activity balance (cell biocompatibility and antibacterial behavior). All substrates achieved suitable cell biocompatibility of premioblast and osteoblast in adhesion and proliferation processes. It is worth to highlighting that samples fabricated with the 100–200 μm space-holder present better mechanical behavior—in terms of stiffness, microhardness, and yield strength—which make them a very suitable material to replace cortical bone tissues. Those results exposed the relationship between the surface properties and the race of bacteria and mammalian cells for the surface with the aim to promote cellular growth over bacteria.University of Seville (Spain) VI Plan Propio de Investigación y Transferencia—US 2018, I.3A

MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation

Mesenchymal stromal cells (hMSCs) display a pleiotropic function in bone regeneration. The signaling involved in osteoblast commitment is still not completely understood, and that determines the failure of current therapies being used. In our recent studies, we identified two miRNAs as regulators of hMSCs osteoblast differentiation driving hypoxia signaling and cytoskeletal reorganization. Other signalings involved in this process are epithelial to mesenchymal transition (EMT) and epidermal growth factor receptor (EGFR) signalings through the regulation of Yes-associated protein (YAP)/PDZ-binding motif (TAZ) expression. In the current study, we investigated the role of miR-33a family as a (i) modulator of YAP/TAZ expression and (ii) a regulator of EGFR signaling during osteoblast commitments. Starting from the observation on hMSCs and primary osteoblast cell lines (Nh-Ost) in which EMT genes and miR-33a displayed a specific expression, we performed a gain and loss of function study with miR-33a-5p and 3p on hMSCs cells and Nh-Ost. After 24 h of transfections, we evaluated the modulation of EMT and osteoblast genes expression by qRT-PCR, Western blot, and Osteoimage assays. Through bioinformatic analysis, we identified YAP as the putative target of miR-33a-3p. Its role was investigated by gain and loss of function studies with miR-33a-3p on hMSCs; qRT-PCR and Western blot analyses were also carried out. Finally, the possible role of EGFR signaling in YAP/TAZ modulation by miR-33a-3p expression was evaluated. Human MSCs were treated with EGF-2 and EGFR inhibitor for different time points, and qRT-PCR and Western blot analyses were performed. The above-mentioned methods revealed a balance between miR-33a-5p and miR-33a-3p expression during hMSCs osteoblast differentiation. The human MSCs phenotype was maintained by miR-33a-5p, while the maintenance of the osteoblast phenotype in the Nh-Ost cell model was permitted by miR-33a-3p expression, which regulated YAP/TAZ through the modulation of EGFR signaling. The inhibition of EGFR blocked the effects of miR-33a-3p on YAP/TAZ modulation, favoring the maintenance of hMSCs in a committed phenotype. A new possible personalized therapeutic approach to bone regeneration was discussed, which might be mediated by customizing delivery of miR-33a in simultaneously targeting EGFR and YAP signaling with combined use of drugs

Modulation of osteoblast cell response through laser surface processing of nylon 6,6

With an ageing population demand on medical facilities is growing, especially for bio-implants. Therefore, there is a need for cheaper, more efficient implants. This paper details how CO2 and KrF excimer lasers can be employed to modulate osteoblast cell growth on nylon 6,6 in relation to laser-modified wettability characteristics. Through patterning the contact angle, θ, increased by up to 19°, indicating the presence of a mixed state wetting regime; whereas θ decreased by up to 20° for the whole area irradiative processed samples. After 24 hours and 4 days incubation the cell cover density and cell count was somewhat modulated over the laser-modified samples compared to the as-received sample. A likely increase in surface toxicity gave rise to a hindered cell response for those samples with high energy densities and high incident pulse numbers. No strong correlations were determined for the laser-induced patterned samples which can be attributed to the likely mixed-state wetting regime. Correlative trends were found between the cell response, θ, polar component and surface oxygen content for the whole area irradiative processed samples. Thus, allowing one to identify the potential for this technology in regenerative medicine

Impaired osteoblast differentiation in annexin A2- and -A5-deficient cells.

Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts, inflammation, fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We employed shRNA to generate annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and determined whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to pSiren (Si) controls. We report that DNA content, a marker of proliferation, was significantly reduced in both AnxA2 and AnxA5 knockdown cells. Alkaline phosphatase expression and activity were also suppressed in AnxA2- or AnxA5-knockdown after 14 days of culture. The pattern of osteogenic gene expression was altered in knockdown cells, with Col1a1 expressed more rapidly in knock-down cells, compared to pSiren. In contrast, Runx2, Ibsp, and Bglap all revealed decreased expression after 14 days of culture. In both AnxA2- and AnxA5-knockdown, interleukin-induced STAT6 signaling was markedly attenuated compared to pSiren controls. These data suggest that AnxA2 and AnxA5 can influence bone formation via regulation of osteoprogenitor proliferation, differentiation, and responsiveness to cytokines in addition to their well-studied function in matrix vesicles

Bmp induces osteoblast differentiation through both Smad4 and mTORC1 signaling

The bone morphogenetic protein (Bmp) family of secreted molecules has been extensively studied in the context of osteoblast differentiation. However, the intracellular signaling cascades that mediate the osteoblastogenic function of Bmp have not been fully elucidated. By profiling mRNA expression in the bone marrow mesenchymal progenitor cell line ST2, we discover that BMP2 induces not only genes commonly associated with ossification and mineralization but also genes important for general protein synthesis. We define the two groups of genes as mineralization related versus protein anabolism signatures of osteoblasts. Although it induces the expression of several Wnt genes, BMP2 activates the osteogenic program largely independently of de novo Wnt secretion. Remarkably, although Smad4 is necessary for the activation of the mineralization-related genes, it is dispensable for BMP2 to induce the protein anabolism signature, which instead critically depends on the transcription factor Atf4. Upstream of Atf4, BMP2 activates mTORC1 to stimulate protein synthesis, resulting in an endoplasmic reticulum stress response mediated by Perk. Thus, Bmp signaling induces osteoblast differentiation through both Smad4- and mTORC1-dependent mechanisms