Osteocyte Markers Research Articles

CD99 Drives Terminal Differentiation of Osteosarcoma Cells by Acting as a Spatial Regulator of ERK 1/2

Differentiation therapy is an attractive treatment for osteosarcoma (OS). CD99 is a cell surface molecule expressed in mesenchymal stem cells and osteoblasts that is maintained during osteoblast differentiation while lost in OS. Herein, we show that whenever OS cells regain CD99, they become prone to reactivate the terminal differentiation program. In differentiating conditions, CD99-transfected OS cells express osteocyte markers, halt proliferation, and largely die by apoptosis, resembling the fate of mature osteoblasts. CD99 induces ERK activation, increasing its membrane-bound/cytoplasmic form rather than affecting its nuclear localization. Through cytoplasmic ERK, CD99 promotes activity of the main osteogenic transcriptional factors AP1 and RUNX2, which in turn enhance osteocalcin and p21WAF1/CIP1, leading to G0/G1 arrest. These data underscore the alternative positions of active ERK into distinct subcellular compartments as key events for determining OS fate. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

DNA Aneuploidy in Porcine Bone Marrow–Derived Mesenchymal Stem Cells Undergoing Osteogenic and AdipogenicIn VitroDifferentiation

In this study, we estimated the distribution of DNA diploidy and aneuploidy in porcine mesenchymal stem cells (pMSCs) that were subjected to osteoblast/osteocyte and adipocyte differentiation to determine the impact of long-term in vitro culture and differentiation on the cell cycle distribution and nuclear DNA profile. This determination could be helpful to confirm or exclude the suitability of physico-chemical culture conditions for the purposes of both the maintenance of an undifferentiated state and to promote differentiation in pMSCs. Flow cytometry was applied to analyze the cell cycle and occurrence of aneuploidy/diploidy, and real-time PCR was used to quantify aP2 and osteocalcin, markers of adipocytes and osteocytes, respectively. The chi-squared test was used to compare the total rates of G0/G1-, S-, and G2/M-phase cell fractions with diploid and aneuploid DNA and the DNA index ratios between three experimental groups of pMSCs. Five weeks of in vitro culture under differentiating conditions resulted in a considerable reduction of DNA stability and a remarkable increase in the rate of cells exhibiting an aneuploid DNA stem line; however, a similar dependence was not found in the nondifferentiated MSCs. Furthermore, the cell fraction rates in each phase of the mitotic cycle and the DNA index (DI) were calculated. The results of real-time PCR for aP2 and osteocalcin proved positive MSC differentiation toward adipocytes and osteocytes. In terms of the possible use of differentiated MSC lines in tissue engineering and regenerative medicine, we propose cytokinetic diagnostics using flow cytometry as an objective and useful method for screening the tumor-forming capacity and malignancy potential of both in vitro long-term cultured MSCs and MSCs subjected to ectopic differentiation.

Calcium induces pro-anabolic effects on human primary osteoblasts associated with acquisition of mature osteocyte markers

Calcium, in combination with vitamin D, is an effective treatment for osteoporosis. Since bone mineralisation occurs concurrently with osteoblast to osteocyte transition, we hypothesised that calcium would stimulate this process. The effect of calcium (1.8–11.8mM) was tested on human primary osteoblast (NHBC) differentiation in vitro. Cultures were assayed for cell-associated mineral and gene expression associated with osteoblast differentiation and mineralisation. Treatment with calcium resulted in a striking dose- and time-dependent increase in cell-associated mineralisation. Calcium appeared to promote osteoblast to osteocyte differentiation, as indicated by increased expression of osteocalcin (OCN), E11, dentin matrix protein 1 (DMP1) and SOST mRNA. The expression of the osteoclast inhibitor, osteoprotegerin, was dramatically enhanced by calcium. Calcium also increased the ratio of PHEX mRNA expression relative to that of MEPE, suggesting a mechanism for the pro-anabolic effect. Consistent with this, calcium-dependent mineralisation was reversed in the presence of MEPE–ASARM peptides. This study suggests that calcium promotes osteoblast to osteocyte transition and concurrent matrix mineralisation, at least in part through the PHEX–MEPE axis.

Osteogenesis of Heterotopically Transplanted Mesenchymal Stromal Cells in Rat Models of Chronic Kidney Disease

The current study is based on the hypothesis of mesenchymal stromal cells (MSCs) contributing to soft-tissue calcification and ectopic osteogenesis in chronic kidney disease (CKD). Rat MSCs were transplanted intraperitoneally in an established three-dimensional collagen-based model in healthy control animals and two rat models of CKD and vascular calcification: (1) 5/6 nephrectomy + high phosphorus diet; and (2) adenine nephropathy. As internal controls, collagen gels without MSCs were transplanted in the same animals. After 4 and 8 weeks, MSCs were still detectable and proliferating in the collagen gels (fluorescence-activated cell sorting [FACS] analysis and confocal microscopy after fluorescence labeling of the cells). Aortas and MSC-containing collagen gels in CKD animals showed distinct similarities in calcification (micro-computed tomography [µCT], energy-dispersive X-ray [EDX] analysis, calcium content), induction of osteogenic markers, (ie, bone morphogenic protein 2 [BMP-2], Runt related transcription factor 2 [Runx2], alkaline phosphatase [ALP]), upregulation of the osteocytic marker sclerostin and extracellular matrix remodeling with increased expression of osteopontin, collagen I/III/IV, fibronectin, and laminin. Calcification, osteogenesis, and matrix remodeling were never observed in healthy control animals and non-MSC-containing collagen gels in all groups. Paul Karl Horan 26 (PKH-26)-labeled, 3G5-positive MSCs expressed Runx2 and sclerostin in CKD animals whereas PKH-26-negative migrated cells did not express osteogenic markers. In conclusion, heterotopically implanted MSCs undergo osteogenic differentiation in rat models of CKD-induced vascular calcification, supporting our hypothesis of MSCs as possible players in heterotopic calcification processes of CKD patients.

Constitutive protein kinase A activity in osteocytes and late osteoblasts produces an anabolic effect on bone

Osteocytes have been implicated in the control of bone formation. However, the signal transduction pathways that regulate the biological function of osteocytes are poorly defined. Limited evidence suggests an important role for the Gs/cAMP pathway in osteocyte function. In the present study, we explored the hypothesis that cAMP-dependent kinase A (PKA) activation in osteocytes plays a key role in controlling skeletal homeostasis. To test this hypothesis, we mated mice harboring a Cre-conditional, mutated PKA catalytic subunit allele that encodes a constitutively active form of PKA (CαR) with mice expressing Cre under the control of the osteocyte-specific promoter, DMP1. This allowed us to direct the expression of CαR to osteocytes in double transgenic progeny. Examination of Cre expression indicated that CαR was also expressed in late osteoblasts. Cortical and trabecular bone parameters from 12-week old mice were determined by μCT. Expression of CαR in osteocytes and late osteoblasts altered the shape of cortical bone proximal to the tibia-fibular junction (TFJ) and produced a significant increase in its size. In trabecular bone of the distal femur, fractional bone volume, trabecular number, and trabecular thickness were increased. These increases were partially the results of increased bone formation rates (BFRs) on the endosteal surface of the cortical bone proximal to the TFJ as well as increased BFR on the trabecular bone surface of the distal femur. Mice expressing CαR displayed a marked increase in the expression of osteoblast markers such as osterix, runx2, collagen 1α1, and alkaline phosphatase (ALP). Interestingly, expression of osteocyte marker gene, DMP1, was significantly up-regulated but the osteocyte number per bone area was not altered. Expression of SOST, a presumed target for PKA signaling in osteocytes, was significantly down-regulated in females. Importantly, no changes in bone resorption were detected. In summary, constitutive PKA signaling in osteocytes and late osteoblasts led to a small expansion of the size of the cortical bone proximal to the TFJ and an increase in trabecular bone in female mice. This was associated with down-regulation of SOST and up-regulation of several osteoblast marker genes. Activation of the PKA pathway in osteocytes and late osteoblasts is sufficient for the initiation of an anabolic skeletal response.

Pax6 expressed in osteocytes inhibits canonical Wnt signaling.

The transcription factor Pax6, which belongs to the paired box-containing gene family, regulates developmental processes, especially in the eyes, central nervous tissues and craniofacial structures. However, the role of Pax6 in bone has never been studied exclusively. Here we report that Pax6 is expressed at both the mRNA and protein level in the calvaria and long bones of adult mice as well as osteocyte-like MLOY4 cells and suppresses the canonical Wnt signaling pathway. Moreover, the expression levels of Pax6 were much higher in the calvaria than the long bones, and Pax6 was also expressed at E16 to E18 in both the calvaria and long bones. Knockdown of Pax6 in MLOY4 cells did not affect cell proliferation or survival; however, the expression of Sost, an osteocyte marker gene, was significantly decreased. In addition, the overexpression of Pax6 suppressed the canonical Wnt signaling pathway by enhancing the expression of Sost. Furthermore, we also demonstrated that Pax6 binds to the Sost promoter and that stimulation of Sost transcription by Pax6 was dependent on a specific Pax6-binding sequence within the promoter. In conclusion, the results of the present study suggest that Pax6 is expressed in bone and may play an important role in osteocyte differentiation by controlling canonical Wnt signaling.

Characteristics of alveolar bone associated with physiological movement of molar in mice: a histological and histochemical study

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar.

Acute Effects of Glucocorticoids on Serum Markers of Osteoclasts, Osteoblasts, and Osteocytes

The aim of this study was to investigate the acute effects of oral glucocorticoids in doses used in clinical practice on biochemical indices of the function of osteoclasts, osteoblasts, and osteocytes. In 17 adult patients suffering from various medical pathologies requiring systemic steroid therapy that were never before treated with glucocorticoids, glucocorticoid treatment was initiated (mean prednisolone equivalent dose of 23.1 ± 12.7 mg/day, range 10-50). Fasting morning serum concentrations of osteocalcin (OC), amino-terminal propeptide of type I procollagen (PINP), type 1 collagen cross-linked C-telopeptide (βCTX), soluble receptor activator of nuclear factor kappaB ligand (sRANKL), osteoprotegerin (OPG), sclerostin, Dickkopf-1 (Dkk-1), and high-sensitivity C-reactive protein (hsCRP) were measured at baseline and on three consecutive days. Significant reductions in serum OC, PINP, OPG, sclerostin, and hsCRP were observed during 96 h of glucocorticoid administration, while serum βCTX showed a significant percentual increase. A significant positive correlation was found between serum concentrations of Dkk-1 and βCTX after 96 h of treatment with glucocorticoids. A significant drop in serum sclerostin, OPG, and OC observed in this study may reflect the rapid glucocorticoid-induced apoptosis of osteocytes.

Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice.

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38. The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease.

Extracellular Matrix Mineralization Promotes E11/gp38 Glycoprotein Expression and Drives Osteocytic Differentiation

Osteocytes are terminally differentiated osteoblasts which reside in a mineralized extracellular matrix (ECM). The factors that regulate this differentiation process are unknown. We have investigated whether ECM mineralization could promote osteocyte formation. To do this we have utilised MLO-A5 pre-osteocyte-like cells and western blotting and comparative RT-PCR to examine whether the expression of osteocyte-selective markers is elevated concurrently with the onset of ECM mineralization. Secondly, if mineralization of the ECM is indeed a driver of osteocyte formation, we reasoned that impairment of ECM mineralization would result in a reversible inhibition of osteocyte formation. Supplementation of MLO-A5 cell cultures with ascorbic acid and phosphate promoted progressive ECM mineralization as well as temporally associated increases in expression of the osteocyte-selective markers, E11/gp38 glycoprotein and sclerostin. Consistent with a primary role for ECM mineralization in osteocyte formation, we also found that inhibition of ECM mineralization, by omitting phosphate or adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, resulted in a 15-fold decrease in mineral deposition that was closely accompanied by lower expression of E11 and other osteocyte markers such as Dmp1, Cd44 and Sost whilst expression of osteoblast markers Ocn and Col1a increased. To rule out the possibility that such restriction of ECM mineralization may produce an irreversible modification in osteoblast behaviour to limit E11 expression and osteocytogenesis, we also measured the capacity of MLO-A5 cells to re-enter the osteocyte differentiation programme. We found that the mineralisation process was re-initiated and closely allied to increased expression of E11 protein after re-administration of phosphate or omission of sodium pyrophosphate, indicating an ECM mineralization-induced restoration in osteocyte formation. These results emphasise the importance of cell-ECM interactions in regulating osteoblast behaviour and, more importantly, suggest that ECM mineralization exerts pivotal control during terminal osteoblast differentiation and acquisition of the osteocyte phenotype.

Matrix metalloproteinase–13 is required for osteocytic perilacunar remodeling and maintains bone fracture resistance

Like bone mass, bone quality is specified in development, actively maintained postnatally, and disrupted by disease. The roles of osteoblasts, osteoclasts, and osteocytes in the regulation of bone mass are increasingly well defined. However, the cellular and molecular mechanisms by which bone quality is regulated remain unclear. Proteins that remodel bone extracellular matrix, such as the collagen-degrading matrix metalloproteinase (MMP)-13, are likely candidates to regulate bone quality. Using MMP-13-deficient mice, we examined the role of MMP-13 in the remodeling and maintenance of bone matrix and subsequent fracture resistance. Throughout the diaphysis of MMP-13-deficient tibiae, we observed elevated nonenzymatic cross-linking and concentric regions of hypermineralization, collagen disorganization, and canalicular malformation. These defects localize to the same mid-cortical bone regions where osteocyte lacunae and canaliculi exhibit MMP-13 and tartrate-resistant acid phosphatase (TRAP) expression, as well as the osteocyte marker sclerostin. Despite otherwise normal measures of osteoclast and osteoblast function, dynamic histomorphometry revealed that remodeling of osteocyte lacunae is impaired in MMP-13(-/-) bone. Analysis of MMP-13(-/-) mice and their wild-type littermates in normal and lactating conditions showed that MMP-13 is not only required for lactation-induced osteocyte perilacunar remodeling, but also for the maintenance of bone quality. The loss of MMP-13, and the resulting defects in perilacunar remodeling and matrix organization, compromise MMP-13(-/-) bone fracture toughness and postyield behavior. Taken together, these findings demonstrate that osteocyte perilacunar remodeling of mid-cortical bone matrix requires MMP-13 and is essential for the maintenance of bone quality.

Inhibitory effects of beta-tricalciumphosphate wear particles on osteocytes via apoptotic response and Akt inactivation

Wear debris-induced osteolysis, a major contributing factor of orthopedic implant aseptic loosening, affects long-term survival of orthopedic prostheses following joint replacement and revision surgery. Pathogenic effects of wear debris on various cell types including macrophages/monocytes, osteoblasts, and osteoclasts have been well studied. However, the interactions between wear debris particles and osteocytes, which make up over 90% of all bone cells, have not been clearly illustrated. Here, we explored the biological effects of endotoxin-free beta-tricalciumphosphate (β-TCP) wear particles with the average diameter of 1.997μm (range 1.3–3.2μm) on osteocytes in vitro. Our results showed that 24h or 48h incubation of β-TCP particles dose-dependently inhibited cell viability of osteocytes MLO-Y4. Alternatively, β-TCP particles treatment for 24h significantly increased the osteocytic marker SOST/sclerostin mRNA expression and the release of inflammatory cytokines including TNF-α and IL-1β into the culture media, but decreased the mRNA expression of another osteocytic marker dentin matrix protein-1 (DMP-1). Furthermore, these osteocytes dysfunctions were accompanied by F-actin disassembly, cell apoptosis, sustained enhancement of intracellular reactive oxygen species (ROS) and mitochondrial injury upon β-TCP particles stimulation. In addition, β-TCP particles also caused Akt inactivation at Ser473 resides with a dose- and time-dependent pattern. Taken together, β-TCP wear particles could cause osteocytes dysfunctions, which may be mediated by apoptotic death and Akt inactivation in MLO-Y4 cells. These findings strongly suggest that osteocytes may play an important role in the β-TCP wear particles-induced osteolysis, and provide valuable insights for understanding the molecular mechanisms of osteocytes death involved in tissue damage during bone cement and intolerance of cemented prostheses.

Phenotypic Characterization of Osteoarthritic Osteocytes from the Sclerotic Zones: A Possible Pathological Role in Subchondral Bone Sclerosis

Subchondral bone sclerosis is a well-recognised manifestation of osteoarthritis (OA). The osteocyte cell network is now considered to be central to the regulation of bone homeostasis; however, it is not known whether the integrity of the osteocyte cell network is altered in OA patients. The aim of this study was to investigate OA osteocyte phenotypic changes and its potential role in OA subchondral bone pathogenesis. The morphological and phenotypic changes of osteocytes in OA samples were investigated by micro-CT, SEM, histology, immunohistochemistry, TRAP staining, apoptosis assay and real-time PCR studies. We demonstrated that in OA subchondral bone, the osteocyte morphology was altered showing rough and rounded cell body with fewer and disorganized dendrites compared with the osteocytes in control samples. OA osteocyte also showed dysregulated expression of osteocyte markers, apoptosis, and degradative enzymes, indicating that the phenotypical changes in OA osteocytes were accompanied with OA subchondral bone remodelling (increased osteoblast and osteoclast activity) and increased bone volume with altered mineral content. Significant alteration of osteocytes identified in OA samples indicates a potential regulatory role of osteocytes in subchondral bone remodelling and mineral metabolism during OA pathogenesis.

The Appearance and Modulation of Osteocyte Marker Expression during Calcification of Vascular Smooth Muscle Cells

BackgroundVascular calcification is an indicator of elevated cardiovascular risk. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in medial vascular calcification, can undergo phenotypic transition to both osteoblastic and chondrocytic cells within a calcifying environment.Methodology/Principal FindingsIn the present study, using in vitro VSMC calcification studies in conjunction with ex vivo analyses of a mouse model of medial calcification, we show that vascular calcification is also associated with the expression of osteocyte phenotype markers. As controls, the terminal differentiation of murine calvarial osteoblasts into osteocytes was induced in vitro in the presence of calcifying medium (containing ß-glycerophosphate and ascorbic acid), as determined by increased expression of the osteocyte markers DMP-1, E11 and sclerostin. Culture of murine aortic VSMCs under identical conditions confirmed that the calcification of these cells can also be induced in similar calcifying medium. Calcified VSMCs had increased alkaline phosphatase activity and PiT-1 expression, which are recognized markers of vascular calcification. Expression of DMP-1, E11 and sclerostin was up-regulated during VSMC calcification in vitro. Increased protein expression of E11, an early osteocyte marker, and sclerostin, expressed by more mature osteocytes was also observed in the calcified media of Enpp1−/− mouse aortic tissue.Conclusions/SignificanceThis study has demonstrated the up-regulation of key osteocytic molecules during the vascular calcification process. A fuller understanding of the functional role of osteocyte formation and specifically sclerostin and E11 expression in the vascular calcification process may identify novel potential therapeutic strategies for clinical intervention.

Podoplanin expression in adamantinoma of long bones and osteofibrous dysplasia

Adamantinoma of long bones (ALB) and osteofibrous dysplasia (OFD) are rare osteolytic bone tumours that principally arise in the tibia. Both ALB and OFD contain epithelial and stromal elements, as well as areas of fibro-osseous proliferation. We assessed expression of podoplanin, a glycoprotein found in osteocytes, in OFD and ALB as well as in fibrous dysplasia and metastatic cancer. Forty-two cases of ALB and OFD, 20 cases of fibrous dysplasia and 20 cases of metastatic carcinoma to bone were stained by immunohistochemistry for expression of podoplanin, epithelial (cytokeratin, epithelial membrane antigen) and vascular (CD34, LYVE-1) markers. Podoplanin was expressed in epithelial cells and tumour glands in ALB as well as in scattered intertrabecular stromal cells in both ALB and OFD. Podoplanin was not expressed by intertrabecular stromal cells in fibrous dysplasia or in metastatic adenocarcinoma. Podoplanin was expressed by osteocytes but not osteoblasts of woven and lamellar bone trabeculae in ALB, OFD, fibrous dysplasia and skeletal metastases. The finding of a common osteocyte marker in OFD/ALB stromal cells is in keeping with a close histogenetic relationship between OFD and ALB; this may reflect the prominence of fibro-osseous proliferation in these tumours. The expression of podoplanin in an osteolytic tumour of the tibia may be useful as a diagnostic discriminant in distinguishing OFD from fibrous dysplasia and ALB from metastatic adenocarcinoma.

Abstract 484: Persistent activation of mesenchymal stromal cells (MSCs) and tissue repair in the etiology of desmoid tumors (DT)

Abstract Mesenchymal stromal cells (MSCs) are multipotent cells that can be isolated from various adult tissues and are implicated in a growing list of sarcomas. Desmoid tumors (DTs) have a clonal origin, are linked with wound healing, and mostly arise in the abdomen of patients with familial adenomatous polyposis (FAP) or sporadically. MSCs home to sites of tissue damage and the persistent activation of these cells in a wound healing context combined with deregulated Wnt signaling may lead to neoplasia. To test this idea, we performed immunohistochemistry on 17 sporadic and FAP-associated DTs from different individuals for the expression of multiple MSC markers (CD73, CD90, and Bmi1). While present in differing numbers, all specimens showed positive cells for all markers, and immunofluorescence (IF) showed co-localization of these markers and CD105 in cells within tumors. Matching adjacent normal fascia from a subset was negative for these antigens. A FAP DT-derived cell line was then made that also co-expressed these MSC markers but not CD34, a negative marker. Fluorescence-activated cell sorting analysis of surface antigen profiles confirmed that >90% of the cell population bore MSC positive, but not negative, markers. Although not genotyped, IF for the C- vs. N-termini of APC confirmed that only truncated APC product was expressed in our DT-derived MSC line. Upon growth for 5-6 weeks in differentiation medium, we confirmed by co-expression of CTGF and Sox9 (obligate chondrocyte markers) plus Alcian blue staining, PPARγ and c/EBPα (obligate adipocyte markers) plus Oil Red staining, and OPN and Runx2 (obligate osteocyte markers) plus von Kossa staining that the DT-derived MSCs were tripotent. In conclusion, this study supports the view that DT etiology involves the persistence of MSCs with deregulated Wnt signaling. The differentiation potential of these cells combined with expression of Bmi1, a transcriptional repressor downstream of Hedgehog and Notch signaling, suggest that inhibitors targeting these pathways may provide a rational systemic therapy to prevent primary or recurrent DT in high-risk patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 484. doi:10.1158/1538-7445.AM2011-484

Bone in vitro 3D osteoblast-osteocyte co-culture model

Event Abstract Back to Event Bone in vitro 3D osteoblast-osteocyte co-culture model M Vazquez1*, BAJ Evans2, J Ralphs3, D Riccardi3 and DJ Mason3 1 Cardiff University, Arthritis Research UK Biomechanics and Bioengineering Centre, School of Biosciences, United Kingdom 2 Cardiff Universit, Department of Child Health, School of Medicine, United Kingdom 3 Cardiff University, Division of Pathophysiology and Repair, School of Biosciences, United Kingdom Background: Osteoporosis occurs when the activities of bone cells become unbalanced leading to increased bone remodelling and weakening of bones. Anabolic therapies are limited, necessitating further investigation of the mechanisms that regulate bone formation. Normal mechanical loading potently induces bone formation via effects on osteocytes. Current investigations of mechanical loading of bone do not reflect the interactions of the cells within it, with most focusing on mechanical loading of osteoblasts in monolayers. Of the 3D models that do exist, none elucidate the osteoblast-osteocyte interactions that regulate mechanically-induced bone formation. We are developing a novel in vitro 3D co-culture model of bone to investigate osteoblast-osteocyte interactions (1) and the role of glutamate, calcium and adenosine signalling in bone mechanotransduction.{BR}Methods: MLO-Y4 cells (1.5x10^6 cells/ml) were incorporated into acid-soluble rat tail tendon type I collagen (2 mg/ml in alpha MEM, pH7.4) gels and MC3T3-E1(1.0x10^5 cells/well) layered on top and cultured at 37C in SMEM (1 mM CaCl2, 2 mM Glutamax, 50 microgram/ml ascorbic acid, 5 % dialysed FBS) for 1 week. Co-cultures were fixed with 1 % paraformaldehyde, infiltrated with OCT, cryosectioned and labelled with phalloidin and DAPI to assess cell morphology, ethidium homodimer and DAPI to assess cell viability or immunostained using anti-connexin 43 antibody to assess cell connectivity. Osteoblast and osteocyte phenotype were determined by RT-PCR of RNA extracted (Trizol) separately from surface osteoblasts and encased osteocytes.{BR}Results: Preliminary data show co-cultures survive, for at least one week, with osteocyte cell death within gels (18 +/- 2.16 %, n=3) comparable to monolayer cultures (2). MLO-Y4 and MC3T3-E1 cells maintain their morphology, form a network through connexin 43, and express osteocyte and osteoblast phenotypic markers respectively, and components of the glutamate, calcium and adenosine signalling pathways.{BR}Conclusion: We have established a mouse osteoblast-osteocyte 3D co-culture system which can be used to investigate co-ordination of mechanical signalling through glutamate, calcium and adenosine delineating their roles in mechanically-induced bone formation.{BR}

The appearance and modulation of osteocyte marker expression during calcification of vascular smooth muscle cells

The appearance and modulation of osteocyte marker expression during calcification of vascular smooth muscle cells

Induction of an osteocyte-like phenotype by fibroblast growth factor-2

The purpose of this study was to characterize the molecular phenotype that occurs during the profound morphological shift of cultured osteogenic cells upon treatment with fibroblast growth factor-2 (FGF2). A time course of treatment with FGF2 was performed on an osteoblast cell line, primary bone marrow stromal cells and an osteocyte-like cell line. Morphologic changes were recorded, and gene profiling was carried out by real time PCR. By 8 h of FGF2 treatment, there is a striking morphological shift of osteoblast and stromal cells to an elongated dendritic-like morphology that is remindful of osteocytes. In osteoblasts treated with FGF2, this morphologic shift is preceded by an induction of several osteocyte markers, including dentin matrix protein 1 (>20-fold) and E11 (>5-fold). There is a transient increase in the gene expression of sclerostin (3.5-fold) and PHEX (2.5-fold). Sclerostin regulation by FGF2 is complex, as gene expression becomes markedly inhibited by FGF2 at times points after 8 h of treatment before rebounding at day 12. Analogous modulation of osteocyte markers is seen in bone marrow stromal cells and MLO-Y4 osteocyte-like cells. In conclusion, this study shows that FGF2 can regulate the transition of osteogenic cells towards the osteocyte lineage, as well as, regulate the expression of critical genes in osteocytes.

Effect of bone graft substitute on marrow stromal cell proliferation and differentiation

Marrow stromal cells (MSCs) are ideally suited for tissue engineered bone grafts since they have the potential to regenerate bone, but may also maintain the homeostasis of the repaired tissue through their ability for self-renewal. An ideal bone graft substitute should support MSC self-renewal as well as differentiation to ensure complete bone defect regeneration and maintenance. The purpose of this investigation was to determine the effect of different substrate materials on MSC expansion and differentiation. Calcium polyphosphate (CPP), bone and hydroxyapatite/tricalcium phosphate (HA/TCP) were seeded with rat MSCs and maintained in culture conditions that promote cell expansion. At 0, 3, 7, 14, and 21 days cell numbers were determined by measuring their metabolic activity using a MTT assay and the frequency of cycling cells by 24 hr BrdU incorporation. Osteogenic, chondrogenic, and adipogenic marker expression in these cultures was measured by qRT-PCR. An initial drop in cell numbers was observed on all substrates. CPP and bone, but not HA/TCP supported an increase in proliferating cells at day 14 and 21. In addition, no upregulation of mature bone markers was observed in cells cultured on CPP and bone, which suggests that these substrates support the expansion of undifferentiated MSCs. In contrast, cell numbers on HA/TCP decreased with time and only rare BrdU positive cells were observed. This decrease in proliferation correlated with the down regulation of osteogenic progenitor markers and the substantial increase in mature osteocyte markers, indicating that HA/TCP favors MSC differentiation and maturation along the osteogenic lineage.