Osteocytes In Mice Research Articles

The induction of a catabolic phenotype in human primary osteoblasts and osteocytes by polyethylene particles

Polyethylene (PE) wear particles are associated with the osteolysis seen in aseptic loosening that leads to orthopaedic implant failure. While cells of the monocyte/macrophage lineage are implicated, evidence is now emerging that osteoblastic cells may also be affected by PE. In this study we investigated the effect of PE particles on osteoblasts, using a novel in vitro cell culture system that was developed to juxtapose cells and PE particles, replicating the 3-dimensional (3D) environment near implants. This system allowed normal human bone-derived cells (NHBC) to undergo differentiation into a mature osteocyte-like phenotype over a 21–28-day culture period. PE particles induced an increase in mRNA expression of the osteocyte markers E11, DMP-1 and SOST/sclerostin. NHBC responded to PE particles by increasing the mRNA expression of several genes associated with osteoclast formation and activity (RANKL, IL-8 and M-CSF) and decreased the expression of the osteoclast antagonist, OPG. PE also appeared to induce a switch in the RUNX2 control of gene expression from that of promoting matrix production (type I collagen) to inducing the expression of pro-osteoclastogenic genes. These results suggest that PE particles switch mature osteoblastic cells from an anabolic to a more catabolic phenotype. This concept was further supported by the finding that PE-induced expression of RANKL mRNA in the mouse osteocyte cell line, MLO-Y4. Overall, our results suggest that PE particles directly induce a change in the phenotype of mature osteoblasts and osteocytes, consistent with the net loss of bone near orthopaedic implants.

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

Collagen1α1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1alpha1 (Col1alpha1) promoter (Col1alpha1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1alpha1-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4(Co/Co)). Multiple tissue PCR of Col1alpha1-Cre;Smad4(Co/+)mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1alpha1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1alpha1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.

Distinct roles for intrinsic osteocyte abnormalities and systemic factors in regulation of FGF23 and bone mineralization in Hyp mice

X-linked hypophosphatemia (XLH) is characterized by hypophosphatemia and impaired mineralization caused by mutations of the PHEX endopeptidase (phosphate-regulating gene with homologies to endopeptidases on the X chromosome), which leads to the overproduction of the phosphaturic fibroblast growth factor 23 (FGF23) in osteocytes. The mechanism whereby PHEX mutations increase FGF23 expression and impair mineralization is uncertain. Either an intrinsic osteocyte abnormality or unidentified PHEX substrates could stimulate FGF23 in XLH. Similarly, impaired mineralization in XLH could result solely from hypophosphatemia or from a concomitant PHEX-dependent intrinsic osteocyte abnormality. To distinguish between these possibilities, we assessed FGF23 expression and mineralization after reciprocal bone cross-transplantations between wild-type (WT) mice and the Hyp mouse model of XLH. We found that increased FGF23 expression in Hyp bone results from a local effect of PHEX deficiency, since FGF23 was increased in Hyp osteocytes before and after explantation into WT mice but was not increased in WT osteocytes after explantation into Hyp mice. WT bone explanted into Hyp mice developed rickets and osteomalacia, but Hyp bone explanted into WT mice displayed persistent osteomalacia and abnormalities in the primary spongiosa, indicating that both phosphate and PHEX independently regulate extracellular matrix mineralization. Unexpectedly, we observed a paradoxical suppression of FGF23 in juvenile Hyp bone explanted into adult Hyp mice, indicating the presence of an age-dependent systemic inhibitor of FGF23. Thus PHEX functions in bone to coordinate bone mineralization and systemic phosphate homeostasis by directly regulating the mineralization process and producing FGF23. In addition, systemic counterregulatory factors that attenuate the upregulation of FGF23 expression in Hyp mouse osteocytes are present in older mice.

Evidence That the Amino-Terminal Composition of Non-(1–84) Parathyroid Hormone Fragments Starts before Position 19

Non-(1-84) parathyroid hormone (PTH) fragments are large C-terminal fragments of PTH with a partially preserved N-terminal structure. They differ from other C-terminal PTH fragments, which do not have an N-terminal structure and do not react in intact PTH assays. We aimed to identify the minimal N-terminal structure common to all non-(1-84) PTH fragments. Sera obtained from six healthy individuals and six patients with primary hyperparathyroidism, and six serum pools from dialysis patients with different PTH concentrations were fractionated by HPLC and analyzed by four different PTH assays. Each assay was characterized by saturation analysis of its detection antibody and capacity to react with different PTH fragments. Human PTH(1-84) [hPTH(1-84)] calibrators were normalized to an in-house hPTH(1-84) calibrator. The cyclase-activating PTH (CA-PTH) assay had an early (1, 2,) epitope and reacted only with hPTH(1-84). The other assays had epitopes in region (13-34). Total and intact PTH assays had epitopes proximal to position 18 and reacted equally well with hPTH(1-84) and hPTH(7-84), and the Elecsys PTH assay had an epitope distal to position 19, being saturable by hPTH(18-48) and also reacting with [Tyr(34)]hPTH(19-84). The HPLC profiles obtained with these assays showed that non-(1-84) PTH fragments did not react in the CA-PTH assay, as expected. The amount of non-(1-84) PTH detected by the other three assays was similar when the assay results were normalized to a common calibrator. The results suggest that the amount of non-(1-84) PTH detected by epitopes proximal or distal to position 19 of the PTH structure is identical, indicating a common minimum structure starting before position 19. This in turn points to a probable high-affinity interaction with the C-PTH receptor, as observed previously with [Tyr(34)]hPTH(19-84) in various cell lines and in mouse osteocytes with PTH/PTHrP type I receptor ablation.

Hormonally-Regulated Expression of Voltage-Operated Ca2+ Channels in Osteocytic (MLO-Y4) Cells

Voltage operated calcium channels (VOCCs) are important in stimulus-response coupling in osteoblasts. We have investigated the expression of VOCCs in the mouse osteocyte cell line, MLO-Y4. Using the whole-cell patch clamp technique we were unable to detect any VOCC currents (n = 436) even in the presence of the L-type VOCC agonist Bay K 8644 (n = 350). Reverse transcription polymerase chain reaction (RT-PCR), using primers to detect α1C, α1D, and α1G VOCC subunits (all of which are expressed in primary osteoblasts), did not generate detectable products with mRNA from MLO-Y4 cells. However, after treatment with physiological levels of hormones, VOCC α1 subunit mRNAs were detected in MLO-Y4 cells. PTH, 17β-estradiol, and dexamethasone-treatment induced expression of L-type (α1C, α1D) subunit transcripts. ATP-treatment induced expression of T-type (α1G) transcripts. Using whole-cell patch clamp we detected VOCC currents in 5–10% of cells after treatment. Current characteristics (L- or T-type) were consistent with the transcript expressed.

Osteomalacia in hyp mice is associated with abnormal phex expression and with altered bone matrix protein expression and deposition.

To explore how the loss of Phex function contributes to the pathogenesis of osteomalacia, we examined the abnormalities of mineralization, Phex, and bone matrix protein expression occurring in Hyp mice in vivo and in ex vivo bone marrow cell cultures. The results in vivo show that mineralization was decreased significantly in Hyp mouse bone. Phex protein was identifiable in osteoblasts and osteocytes in wild-type mice, but not in Hyp mice. In Hyp mice, osteocalcin, bone sialoprotein, and vitronectin expression were down-regulated, whereas biglycan and fibrillin-1 expression were up-regulated in osteocytes and bone matrix relative to those in their wild-type counterparts. Parallel studies ex vivo demonstrated that cells derived from 18-day Hyp mouse bone marrow cell cultures had a 3'-Phex deletion, no Phex protein expression, decreased alkaline phosphatase activity, collagen deposition, and calcium accumulation, and reduced osteocalcin, bone sialoprotein, and vitronectin at both the protein and messenger RNA levels. Furthermore conditioned medium from Hyp mouse bone marrow cultures could induce analogous defects in bone marrow cell cultures of wild-type cells. These novel findings indicate that there is an intrinsic osteogenic cell differentiation defect in addition to the known hypomineralization of bone in Hyp mice, which may be inducible by an autocrine/paracrine secreted factor. These results suggest that alterations in the Phex gene may control bone matrix mineralization indirectly by regulating the synthesis and deposition of bone matrix proteins.

Mode of C-type virus production by bone cells of C3H/Fg mice

Electron microscopy of bone from the distal ends of femurs of six 3-month-old male C3H/Fg mice revealed varying modes and quantities of C-type virus production by cells in the preosteoblast to osteocyte developmental gradient. Production occurred by budding from the cell surface, either from very short pedicles or from longer microvillous-like processes. Although apparently absent in preosteoblasts, limited numbers of budding viruses (90-110 nM) were seen in 6 of 53 osteoblasts examined. Examination of 200 osteocytes revealed evidence of virus production in all but eight. In these cells, budding occurred from the cell body and its processes with release of viruses into the pericellular lacunar space. Budding from the longer microvillous-like processes sometimes resulted in binding together of viruses in bead-like fashion by thin strands of limiting membrane. In a few cases budding was also seen on the cell processes within the canaliculi. The quantities of viruses present within the lacunae usually increased with increasing age of the osteocytes, indicating continual virus production. Most of the viruses observed, however, were pre C-types along with lesser numbers of mature C particles. Thus, factors contributing to the production of mature C particles may be diminished in osteocytes of mice at this age.