Bone Lining Cells Research Articles

Heterozygous inactivation of Gnas in adipose-derived mesenchymal progenitor cells enhances osteoblast differentiation and promotes heterotopic ossification

Human genetic disorders sharing the common feature of subcutaneous heterotopic ossification (HO) are caused by heterozygous inactivating mutations in GNAS, a gene encoding multiple transcripts including two stimulatory G proteins, the α subunit of the stimulatory G protein (G(s)α) of adenylyl cyclase, and the extralong form of G(s)α, XLαs. In one such disorder, progressive osseous heteroplasia (POH), bone formation initiates within subcutaneous fat before progressing to deeper tissues, suggesting that osteogenesis may involve abnormal differentiation of mesenchymal precursors that are present in adipose tissues. We determined by immunohistochemical analysis that GNAS protein expression is limited to G(s)α in bone-lining cells and to G(s)α and XLαs in osteocytes. By contrast, the GNAS proteins G(s)α, XLαs, and NESP55 are detected in adipocytes and in adipose stroma. Although Gnas transcripts, as assessed by quantitative RT-PCR, show no significant changes on osteoblast differentiation of bone-derived precursor cells, the abundance of these transcripts is enhanced by osteoblast differentiation of adipose-derived mesenchymal progenitors. Using a mouse knockout model, we determined that heterozygous inactivation of Gnas (by disruption of the G(s)α-specific exon 1) abrogates upregulation of multiple Gnas transcripts that normally occurs with osteoblast differentiation in wild-type adipose stromal cells. These transcriptional changes in Gnas(+/-) mice are accompanied by accelerated osteoblast differentiation of adipose stromal cells in vitro. In vivo, altered osteoblast differentiation in Gnas(+/-) mice manifests as subcutaneous HO by an intramembranous process. Taken together, these data suggest that Gnas is a key regulator of fate decisions in adipose-derived mesenchymal progenitor cells, specifically those which are involved in bone formation.

Monosodium urate monohydrate crystals inhibit osteoblast viability and function: implications for development of bone erosion in gout

BackgroundBone erosion is a common manifestation of chronic tophaceous gout.ObjectivesTo investigate the effects of monosodium urate monohydrate (MSU) crystals on osteoblast viability and function.MethodsThe MTT assay and flow cytometry were...

Sustained swimming increases the mineral content and osteocyte density of salmon vertebral bone

This study addresses the effects of increased mechanical load on the vertebral bone of post-smolt Atlantic salmon by forcing them to swim at controlled speeds. The fish swam continuously in four circular tanks for 9 weeks, two groups at 0.47 body lengths (bl) × s(-1) (non-exercised group) and two groups at 2 bl × s(-1) (exercised group), which is just below the limit for maximum sustained swimming speed in this species. Qualitative data concerning the vertebral structure were obtained from histology and electron microscopy, and quantitative data were based on histomorphometry, high-resolution X-ray micro-computed tomography images and analysis of bone mineral content, while the mechanical properties were tested by compression. Our key findings are that the bone matrix secreted during sustained swimming had significantly higher mineral content and mechanical strength, while no effect was detected on bone in vivo architecture. mRNA levels for two mineralization-related genes bgp and alp were significantly upregulated in the exercised fish, indicating promotion of mineralization. The osteocyte density of the lamellar bone of the amphicoel was also significantly higher in the exercised than non-exercised fish, while the osteocyte density in the cancellous bone was similar in the two groups. The vertebral osteocytes did not form a functional syncytium, which shows that salmon vertebral bone responds to mechanical loading in the absence of an extensive connecting syncytial network of osteocytic cell processes as found in mammals, indicating the existence of a different mechanosensing mechanism. The adaptive response to increased load is thus probably mediated by osteoblasts or bone lining cells, a system in which signal detection and response may be co-located. This study offers new insight into the teleost bone biology, and may have implications for maintaining acceptable welfare for farmed salmon.

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: A cryo-electron microscopy study

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.

Mesenchymal Stem Cells Derived from Rat Epicardial Versus Epididymal Adipose Tissue

Objective(s) Some investigation has indicated that adipose-derived stem cells possess different surface epitopes and differentiation potential according to the localization of fat pad from which the cells were derived. In the present study proliferation capacity and aging of such cells were explored. Materials and Methods Adherent cells were isolated from the collagenase digests of adipose tissues excised from rat epicardial and epididymal regions and propagated with several subcultures. The cells were then investigated whether or not they were able to differentiate into bone, cartilage and adipose cell lineages. Studied cells from two adipose tissues were also compared with respect to their in vitro proliferation capacity. The presence of senescent cells in the culture was determined and compared using senescence-associated (SA) s-galactosidase staining method. Results Successful differentiations of the cells were indicative of their mesenchymal stem cells (MSCs) identity. Epicardial adipose-derived cells tended to have a short population doubling time (45±9.6 hr) than the epididymal adipose-derived stem cells (69±16 hr, P< 0.05). Colonogenic activity and the growth curve characteristics were all better in the culture of stem cells derived from epicardial compared to epididymal adipose tissue. Comparatively more percentage of senescent cells was present at the cultures derived from epididymal adipose tissue (P< 0.05). Conclusion Our data emphasize on the differences existed between the stem cells derived from adipose depots of different anatomical sites in terms of their proliferative capacity and in vitro aging. Such data can help understand varying results reported by different laboratories involved in adipose stem cell investigations.

Effects of age and shear rate on the rheological properties of human yellow bone marrow

Bone marrow is the niche for stem cells and is within close proximity to bone lining cells. Forces experienced by these cells guide their differentiation and proliferation. As these forces are dependent on the viscosity of the medium, the knowledge about the viscosity of marrow is essential to modeling the mechanical environment of bone. This study sought to examine the effects of age on the rheological properties of human yellow bone marrow. Samples were harvested from the femurs of male donors ranging from 22 to 82 years of age (N=19) and subjected to stress and frequency sweeps to determine viscosity and dynamic moduli, respectively. The viscosity of bone marrow at physiologically plausible shear rates ranged from 44 to 142 mPa·s. The coefficients of variation ranged up to 0.40 within subjects and 0.14 between subjects. Regressions of viscosity values against age did not generate a strong level of significance; therefore, earlier reported changes in the composition of marrow with age did not translate into variation in viscosity of marrow. Since age does not seem to offer a governing effect, the observed variation within and between donors may stem from other factors (genetic, nutrition, etc.). The wide range of variation in the viscosity of marrow within subject, between subjects and with age implies that the fluid shear experienced by cells resident in marrow may also vary substantially.

Mechanosensing in Bone

The rationale for this issue of Clinical Reviews in Bone and Mineral Metabolism is rooted in the recognition of the revolution of the role of the osteocyte in bone biology. Since Wolff formulated his law of bone remodeling more than a century ago, the notion that mechanical function determines skeletal design and bone tissue organization has become widely accepted, and the mechanism whereby bone tissue detects mechanical loading is now becoming more and more clear. Basically, the cellular response mechanism to altered load starts with an adequate mechanical signal that is detected by the sensor cells, leading to the generation of an adequate signal that modulates bone formation and/or bone resorption. Bone formation and bone resorption result from the activity of osteoblasts and osteoclasts on the bone surface, while osteocytes are the cells within the tissue proper, and bone lining cells cover inactive bone surfaces. It is now known that osteocytes act as mechanosensors regulating mechanical adaptation and that load placed on an intact bone activates them. Our current knowledge of osteocytes still lags behind what we know of the properties and functions of both osteoblasts and osteoclasts, but the pace with which our knowledge of the osteocyte is advancing is impressive and illustrates an important role for osteocytes in bone biology. This issue of Clinical Reviews in Bone and Mineral Metabolism on ‘‘Mechanosensing in bone’’ is intended to review the concept and background information about the topic of mechanosensing in bone with the latest information on putative chemical/mechanical regulators along with speculation/impact on disease manifestations and is designed with a logic that might compel to read on. The first chapter, ‘‘Mechanisms of osteocyte mechanotransduction’’ by Bakker and Klein-Nulend, covers and provides a critical analysis of our current knowledge on how bone transduction takes place from the level of mechanical loads placed upon the bone as an organ to activation of a cell. The second chapter, ‘‘Application of bioimaging to osteocyte biology’’ by Kamioka and Adachi, is specifically devoted to the application of a new bioimaging technique to reveal the morphology of osteocytes as well as part of their biological nature. The application of this new technique has important relevance for understanding the osteocytes behavior in response to an extracellular stimulus. The third chapter, ‘‘Stress response by bone cells and implications on microgravity environment’’ by Bacabac and Van Loon, addresses the great advances that have been made in understanding the mechanisms involved in the adaptation of bone tissue to mechanical loading, leading to the notion that the mechanical properties of cells are a prerequisite to their tissue-level emergent mechanosensing behavior. A condition of disturbed mechanics due to extreme unloading, as in an environment under microgravity, may lead to impaired mechanosensing. The fourth chapter, ‘‘Gap junctions and biophysical regulation of bone cells’’ by Lloyd and Donahue, demonstrates the integral role of gap junctional intercellular communication in skeletal physiology and bone cell mechanosensing. This direct cell–cell communication via gap junctions is important for the communication between osteoblasts, osteoclasts, and osteocytes and is integral to J. Klein-Nulend (&) Department of Oral Cell Biology, Academic Centre for Dentistry, Amsterdam-University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands e-mail: j.kleinnulend@acta.nl

Abnormally High Levels of E- and N-Cadherin Expression Add to Altered Regulation of b-Catenin In Multiple Myeloma

AbstractAbstract 2982Gene expression profiling (GEP) of normal and malignant plasma cells and B-cells, revealed that MM is uniquely characterized by elevated expression of E- or N-cadherin. Classical cadherins are integral plasma membrane proteins, that together with a- and b-catenin form calcium-dependent adherent junctions. Homotypic interaction of N-cadherin+ hematopoietic stem cells and N-cadherin+ bone lining cells in the endosteal niche regulates HSC function. Adherent junctions contribute to the regulation of Wnt/b-catenin signaling by modulating the balance between membrane-bound and free cytosolic b-catenin, the latter of which is required for TCF transcriptional activity. Overexpression of DKK1 and suppression of Wnt/b-catenin osteoblasts causes a loss in self-renewal of HSC and only stromal cells with active nuclear b-catenin can support hematopoiesis in-vitro. On the other hand, disruption of adherent junctions and release of b-catenin contributes to epithelial-to-mesenchymal transition and solid tumor metastases. We, and others, have demonstrated that Wnt/β-catenin signaling is active in MM. However, emerging evidence suggests that loss of Wnt/b-catenin activity, rather than its activation, is central to MM pathogenesis. Nearly 90% of primary MM cells express and secrete DKK1 and/or SFRP3 or SFRP2, potent inhibitors of Wnt/b-catenin signaling. Moreover, loss-of-function mutations of APC or axin genes or gain-of-function mutations in the β-catenin gene common in colon cancer have not been found in MM. We therefore hypothesized that elevated expression of N/E-cadherin in MM cells contributes to the abnormally increase of b-catenin in MM. We first assessed the steady-state levels of β-catenin protein in MMCL with immunoblotting analysis. β-Catenin protein was expressed in all tested MMCL, with variable levels in individual lines. Interestingly, relative levels of β-catenin protein were comparable to N-cadherin expression in all eight tested myeloma cell lines. CD138-enriched plasma cells from the BM of 72 patients newly diagnosed MM revealed β-catenin protein levels are highly variable. After normalization of β-catenin with β-tubulin levels we segregated cases into three groups: 39% had low, 23% moderate, and 38% high levels of β-catenin. Analysis of correlation of b-catenin protein levels with U133Plus microarray data revealed there are striking positive correlations between N- or E-cadherin mRNA levels with levels of b-catenin protein. Importantly, b-catenin levels were not correlated with known Wnt/b-catenin target genes. To evaluate the role of N-cadherin in regulating β-catenin signaling in MM, we used a lentiviral expression system to express wild-type N-cadherin (NCadW/MMS1) or empty vector (EV/MMS1) in MMS1 cells. Significant increases in total and free b-catenin correlated with N-cadherin protein expression. These results indicate that N-cadherin protein modulates b-catenin levels MM cells. Results of experiments to determine whether N-cadherin-mediated regulation of b-catenin translates into altered TCF/b-catenin transcriptional activity in MM cells will be reported. Disclosures:Shaughnessy:Myeloma Health LLC: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Genzyme: Patents & Royalties; Millennium: Honoraria; Celgene: Honoraria; OrthoBiotech: Honoraria; Array BioPharma: Honoraria.

Sclerostin: A gem from the genome leads to bone-building antibodies

Sclerostin: A gem from the genome leads to bone-building antibodies

Cellular communications in bone homeostasis and repair.

Cellular communication between the bone component cells osteoblasts, osteocytes and (pre-)osteoclasts is essential for bone remodeling which maintains bone integrity. As in the remodeling of other organs, cell death is a trigger for remodeling of bone. During the systematic process of bone remodeling, direct or indirect cell-cell communication is indispensable. Thus, osteoblasts induce migration and differentiation of preosteoclasts, which is followed by bone resorption (by mature multinuclear osteoclasts). After completion of bone resorption, apoptosis of mature osteoclasts and differentiation of osteoblasts are initiated. At this time, the osteoblasts do not support osteoclast differentiation but do support bone formation. Finally, osteoblasts differentiate to osteocytes in bone or to bone lining cells on bone surfaces. In this way, old bone areas are regenerated as new bone. In this review the role of cell-cell communication in bone remodeling is discussed.

Col3.6-HSD2 transgenic mice: A glucocorticoid loss-of-function model spanning early and late osteoblast differentiation

The goal of this study was to characterize the bone phenotype and molecular alterations in Col3.6-HSD2 mice in which a 3.6-kb Col1a1 promoter fragment drives 11ß-HSD2 expression broadly in the osteoblast lineage to reduce glucocorticoid signaling. Serum corticosterone was unchanged in transgenic females exluding a systemic effect of the transgene. Adult transgenic mice showed reduced vertebral trabecular bone volume and reduced femoral and tibial sub-periosteal and sub-endosteal areas as assessed by microCT. In adult female transgenic mice, histomorphometry showed that vertebral bone mass and trabecular number were reduced but that osteoblast and osteoclast numbers and the mineral apposition and bone formation rates were not changed, suggesting a possible developmental defect in the formation of trabeculae. In a small sample of male mice, osteoblast number and percent osteoid surface were increased but the mineral apposition bone formation rates were not changed, indicating subtle sex-specific phenotypic differences in Col3.6-HSD2 bone. Serum from transgenic mice had decreased levels of the C-terminal telopeptide of α1(I) collagen but increased levels of osteocalcin. Transgenic calvarial osteoblast and bone marrow stromal cultures showed decreased alkaline phosphatase and mineral staining, reduced levels of Col1a1, bone sialoprotein and osteocalcin mRNA expression, and decreased cell growth and proliferation. Transgenic bone marrow cultures treated with RANKL and M-CSF showed greater osteoclast formation; however, osteoclast activity as assessed by resorption of a calcium phosphate substrate was decreased in transgenic cultures. Gene profiling of cultured calvarial osteoblasts enriched in the Col3.6-HSD2 transgene showed modest but significant changes in gene expression, particularly in cell cycle and integrin genes. In summary, Col3.6-HSD2 mice showed a low bone mass phenotype, with decreased ex vivo osteogenesis. These data further strengthen the concept that endogenous glucocorticoid signaling is required for optimal bone mass acquisition and highlight the complexities of glucocorticoid signaling in bone cell lineages.

The effect of osteocyte apoptosis on the signalling in the osteocyte network and at the bone lining cell surface: A computer simulation

The effect of osteocyte apoptosis on the signalling in the osteocyte network and at the bone lining cell surface: A computer simulation

Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

BackgroundHuman multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.ResultsAfter transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.ConclusionPhysiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

Immature and mature megakaryocytes enhance osteoblast proliferation and inhibit osteoclast formation

Recent data suggest that megakaryocytes (MKs) play a role in skeletal homeostasis. In vitro and in vivo data show that MKs stimulate osteoblast (OB) proliferation and inhibit osteoclast (OC) formation, thus favoring net bone deposition. There are several mouse models with dysregulated megakaryopoiesis and resultant high bone mass phenotypes. One such model that our group has extensively studied is GATA-1 deficient mice. GATA-1 is a transcription factor required for normal megakaryopoiesis, and mice deficient in GATA-1 have increases in immature MK number and a striking increase in bone mass. While the increased bone mass could simply be a result of increased MK number, here we take a more in depth look at the MKs of these mice to see if there is a unique factor inherent to GATA-1 deficient MKs that favors increased bone deposition. We show that increased MK number does correspond with increased OB proliferation and decreased OC formation that stage of maturation does not alter the effect of MKs on bone cell lineages beyond the megakaryoblast stage, and that GATA-1 deficient MKs survive longer than wild-type controls. So while increased MK number in GATA-1 deficient mice likely contributes to the high bone mass phenotype, we propose that the increased longevity of this lineage also plays a role. Since GATA-1 deficient MKs live longer they are able to exert both more proliferative influence on OBs and more inhibitory influence on OCs.

From Stem Cells to Bone: Phenotype Acquisition, Stabilization, and Tissue Engineering in Animal Models

The regeneration of bone tissue depends on the concerted actions of a plethora of signals that recruit mesenchymal stem cells for lineage-specific differentiation, with cellular phenotypes serving various functions throughout their life span. The signals are conveyed in hormones, growth factors, and mechanical forces, all of which ensure proper modeling and remodeling. Both processes are secured by indigenous and programmed metabolism in osteoblasts/osteocytes as well as in other stem cell (SC)-derived cell types (e.g., osteoclasts, bone lining cells) involved in the remodeling of the subject tissue. The focus of this review is the concerted action of these signals as well as the regulatory and/or stabilizing control circuits exhibited by a class of small RNAs, designated microRNAs. We discuss an in vitro approach for ensuring proper phenotype acquisition as well as the choice of scaffolds and animal models for in vivo tissue repair. This approach includes selection of SC niches to optimize bone formation in vivo, transcription factors important for osteoblastogenesis, the Wnt and Notch pathways of signaling, selection of delivery systems for gene therapy, use of appropriate matrices and scaffolds, in vivo mechanostimulation, choice of lesions to be repaired, and type of animal to use. We also discuss Wnt-related and SC-based treatment of osteoporosis. Throughout, we offer considerations for the selection of model systems and parameters to assess the entire procedure from initial SC selection to final bone repair, and conclude with a table summarizing our recommendations.

Claudin expression in the bone-lining cells of female rats exposed to long-standing acidemia

Besides enhancing osteoclast-mediated bone resorption, chronic metabolic acidosis (CMA) induces mineral efflux across the epithelial-like bone membrane formed by bone-lining cells (inactive osteoblasts), possibly via the paracellular pathway. However, there was a compensatory mechanism that restricted bone loss in the late phase of CMA, and changes in the expression of claudins, which are tight junction proteins known to regulate epithelial barrier function, were therefore anticipated in bone-lining cells. Herein, primary rat osteoblasts were found to express several transcripts of claudins, i.e., claudin-5, -11, -14, -15 and -16. Their protein expressions in bone-lining cells were demonstrated by immunohistochemistry in decalcified tibial sections. After exposure to CMA induced by oral administration of 1.5% NH4Cl for 21 days, expression of claudin-14, which normally seals the paracellular space and restricts ion movement, was increased, whereas that of claudin-15 and -16 which form pores for ion transport were decreased. Expressions of claudin-5 and -11 were not changed by CMA. In conclusion, the bone-lining cells of rats exposed to CMA for 21 days upregulated an ion-restrictive claudin (i.e., claudin-14), while downregulating ion-permeable claudins (i.e., claudin-15 and -16). These cellular responses might be parts of a compensatory mechanism accounting for deceleration of bone loss in late CMA.

Niche Regulation of Hematopoietic Stem Cells in the Endosteum

During postnatal life, the bone marrow (BM) supports both the self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized niches. The interaction of HSCs with their niches also regulates the quiescence of HSCs. HSC quiescence is critical to ensure lifelong hematopoiesis and to protect the HSC pool from myelotoxic insult and premature exhaustion under conditions of hematopoietic stress. Here we identified long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, Mpl, as a quiescent population in adult BM. THPO was produced by bone-lining cells in the endosteum. Inhibition and stimulation of the THPO/Mpl pathway produced opposite effects on the quiescence of LT-HSC. Exogenous THPO transiently increased the quiescent LT-HSC population, such as side-population and pyronin Y-negative cells. In contrast, administration of an anti-Mpl neutralizing antibody, AMM2, suppressed the quiescence of LT-HSCs and enabled HSC engraftment without irradiation, indicating that inhibition of THPO/Mpl signaling reduces HSC-niche interactions. Moreover, it suggests that inhibiting the HSC-niche interaction could represent a novel technique for bone marrow transplantation without irradiation. Altogether, these data suggest that the THPO/Mpl signaling pathway is a novel niche component in the endosteum, and in the steady-state condition, this signaling pathway plays a critical role in the regulation of LT-HSCs in the osteoblastic niche.

FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling

This study aimed to evaluate whether the immunolocalization of fibroblast growth factor (FGF) 23 and dentin matrix protein 1 (DMP1) is associated with the spatial regularity of the osteocyte lacunar canalicular system(s) (OLCS). Femora of 12-weeks-old male ICR mice were fixed with 4% paraformaldehyde, decalcified with a 10% EDTA solution and then embedded in paraffin. We have devised a triple staining procedure that combines silver impregnation, alkaline phosphatase (ALPase) immunohistochemistry and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry on a single paraffin section. This procedure permitted the visualization of ALPase-positive plump osteoblasts and several TRAPase-positive osteoclasts on those bone matrices featuring irregularly arranged OLCS, and of ALPase-positive bone lining cells on the bone matrix displaying the well-arranged OLCS. As observations proceeded from the metaphysis toward the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling, accompanied by increased regularity of the OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of the OLCS. While DMP1 was evenly localized in all regions of the cortical bones, FGF23 was more abundantly localized in osteocytes of cortical bones with regularly arranged OLCS. In cortical bones, the endosteal area featuring regular OLCS exhibited more intense FGF23 immunoreaction when compared to the periosteal region, which tended to display irregular OLCS. In summary, FGF23 appears to be synthesized principally by osteocytes in the regularly distributed OLCS that have been established after bone remodeling.

Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures

BackgroundThe possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs).MethodsLineages of hFTs were expanded, had their karyotype analyzed, were characterized by flow cytometry and underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation.ResultsHere we show for the first time that hFTs, which are discarded after some gynecological procedures, are a rich additional source of MSCs, which we designated as human tube MSCs (htMSCs).ConclusionHuman tube MSCs can be easily isolated, expanded in vitro, present a mesenchymal profile and are able to differentiate into muscle, fat, cartilage and bone in vitro.

The role of osteocytes in bone mechanotransduction

IntroductionBones are subjected to a variety of mechanical loads duringdaily activities. In the nineteenth century, Julius Wolffproposed that bones adapt their mass and 3D structure tothe loading conditions in order to optimize their load-bearing capacity, and that this process is driven bymechanical stress [1]. For the past centuries, an increasingnumber of theoretical and experimental results reveal thatosteocytes are the pivotal cells orchestrating this bio-mechanical regulation of bone mass and structure, whichis accomplished by the process of bone remodeling [2–5]Osteocytes are terminally differentiated cells of theosteogenic lineage that are derived from mesenchymalprecursor cells. A number of molecules have been iden-tified as important markers of osteocytes, such as matrixextracellular phosphoglycoprotein [6] sclerostin [7], dentinmatrix protein-1 [8], and phex protein [8]. The osteocytesare the most abundant cells in adult bone and are constantlyspaced throughout the mineralized matrix. Mature osteo-cytes have a characteristic dendritic cell shape, with pro-cesses radiating from the cell body through the canaliculi indifferent directions. These processes form an intercellularnetwork through gap and adherent junctions with surround-ing osteocytes, the cells lining the bone surface and bonemarrow. Through this unique 3D network, osteocytes areanatomically placed in a prime position not only to sensedeformations driven by stresses placed upon bone, butalso to respond with passage of signals to the neighboringcells [9].For more than a decade now, it is known that theosteocytes are very sensitive to stress applied to intact bonetissue [10–16]. Computer simulation models have shownthat mechanosensors lying at the surface of bone, asosteoblasts and bone lining cells do, would be less sensitiveto changes in the loading pattern than the osteocytes, lyingwithin the calcified matrix [3]. Interestingly, targetedablation of osteocytes in mice disturbs the adaptation ofbone to mechanical loading [16].Osteocytes as key players in the process of bonemechanotransductionIt is currently believed that when bones are loaded, theresulting deformation will drive the thin layer of interstitialfluid surrounding the network of osteocytes to flow fromregions under high pressure to regions under low pressure[17, 18]. This flow of fluid is sensed by the osteocyteswhich in turn produce signaling molecules that can regulatebone resorption through the osteoclasts, and bone formationthrough the osteoblasts, leading to adequate bone remodel-ing [17, 18]. This concept is known as the fluid flowhypothesis. Evidence has been increasing for the flow ofcanalicular interstitial fluid as the likely factor that informsthe osteocytes about the level of bone loading [2, 5, 17, 18].Nevertheless, Vatsa and colleagues [19, 20] proposed thatif osteocytes could sense matrix strains directly, the cellshape, cytoskeletal alignment and distribution of adhesionsites in osteocytes in situ would bear alignment to themechanical loading patterns. Indeed, it was shown that the