SaOS2 Osteosarcoma Cells as an In Vitro Model for Studying the Transition of Human Osteoblasts to Osteocytes (original) (raw)
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Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties
Cancer Research, 1987
This study examines the osteoblastic properties of the established human osteosarcoma cell line Saos-2. Saos-2 cells inoculated into diffu sion chambers, which were implanted i.p. into nude mice, produced mineralized matrix in 4 of 6 chambers at 8 weeks. In 5 of 6 chambers there was a strong positive alkaline phosphatase reaction. In culture the alkaline phosphatase levels increased with time and cell density, reaching very high levels at confluence: 4-7 /imol/mg protein/min. The cells show a sensitive adenylate cyclase response to parathyroid hormone, 50% effective dose = 2.8 UM,which increases with cell density and is further raised by dexamethasone treatment. They also exhibit typical binding of 1-25-dihydroxyvitamin I), to 3.2S receptor protein with an apparent K¿ of 0.21 IIM;the numbers of sites per cell were 3,300 at 50,000 cells/cm2 and 1,800 at 280,000 cells/cm2. The presence of osteonectin was visual ized with a monoclonal antibody which revealed a reticular pattern on the cell surface. Osteonectin was also detected in the medium by Western blots, migrating at around M, 40,000 in nonreduced gels and M, 44,000 in reduced gels. The Saos-2 cells thus possess several osteoblastic features and could be useful as a permanent line of human osteoblastlike cells and as a source of bone-related molecules.
Journal of Bone and Mineral Research, 1998
Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1␣,25-dihydroxyvitamin D 3 (1,25(OH 2)D 3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10 ؊8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10 ؊8 M 1,25(OH 2)D 3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH 2)D 3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and -glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH 2)D 3 , or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans. (
Experimental Cell Research, 2010
Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiationa period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation. Published by Elsevier Inc. a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m w w w . e l s e v i e r . c o m / l o c a t e / y e x c r
Journal of Biomedical Materials Research, 2004
Differential expression of multiple osteogenic factors may be responsible for the different osteoinductivity of osteosarcoma cell lines. We compared in vivo osteoinductivity of human osteosarcoma cell lines (Saos-2 vs. U-2 OS) in nude mice, and their in vitro expression of various osteogenic factors of protein level by quantitative immunocytochemistry and mRNA level by RT-PCR and/or in situ hybridization. Saos-2 cells, but not U-2 OS, were osteoinductive in vivo. Significantly higher expression (independent t-test, all p Ͻ 0.005) of osteogenic factors were observed in Saos-2 cells compared with U-2 OS, which included bone morphogenetic proteins (particularly BMPs-2, 3, 4, and 7), transforming growth factor-beta (TGF-), BMP receptor (BMPR)-1A, receptor-regulated Smads (R-Smads), Smads 1, 2, and 5, and common-mediator Smad (Co-Smad), Smad 4. In contrast, U-2 OS cells expressed higher levels of inhibitory Smad 6 (I-Smad) protein than Saos-2 cells (p Ͻ 0.001). These results suggest that a combination of osteogenic factors (BMPs, TGF-, BMPRs, and R/Co-Smads) against I-Smad may play important roles in the Saos-2 cell osteoinductivity. This may have a clinical implication in selecting key osteogenic factors for combined therapy for bone defect diseases. The characterized cell lines can be used as positive and negative controls for the assessments of both in vitro and in vivo bone formation capabilities of designed tissues or biomaterials.
Journal of Cellular Biochemistry, 2011
Osteosarcoma is usually associated with a disturbed bone metabolism. The aim of this work was to characterize the reciprocal interactions between MG63 osteosarcoma cells and osteoclasts, in a coculture system. Co-cultures were characterized throughout 21 days for the osteoclastogenic response and the expression of osteoblastic markers. Monocultures of MG63 cells and PBMC and co-cultures of PBMC + human bone marrow cells (hBMC) were also performed. Compared to PBMC cultures, co-cultures yielded significantly increased gene expression of osteoclast-related markers, TRAP activity, TRAP-positive multinucleated cells, cells with actin rings and vitronectin and calcitonin receptors and calcium phosphate resorbing ability. Results showed that the development of functional osteoclasts required a very low number of MG63 cells, suggesting a high osteoclastogenic-triggering capacity of this cell line. Subjacent mechanisms involved the pathways MEK and NFkB, although with a lower relevance than that observed on PBMC monocultures or cocultures of hBMC + PBMC; PGE2 production also had a contribution. Compared to MG63 cell monocultures, the co-culture expressed lower levels of COL1 and ALP and higher levels of BMP-2, suggesting that PBMC also modulated the osteoblastic behaviour. While M-CSF appeared to be involved in the osteoclastogenic response on the MG63+PBMC co-cultures, RANKL does not seem to be a key player in the process. On the other hand, sphingosine-1-phosphate production might contribute to the modulation of the osteoblastic behaviour. Results suggest that the reciprocal modulation between osteosarcoma and osteoclastic cells might contribute to the disturbed bone metabolism associated with bone tumours.
Osteoblast Differentiation and Bone Matrix Formation In Vivo and In Vitro
Tissue engineering. Part B, Reviews, 2016
We review the characteristics of osteoblast differentiation and bone matrix synthesis. Bone in air breathing vertebrates is a specialized tissue that developmentally replaces simpler solid tissues, usually cartilage. Bone is a living organ bounded by a layer of osteoblasts that, because of transport and compartmentalization requirements, produce bone matrix exclusively as an organized tight epithelium. With matrix growth, osteoblasts are reorganized and incorporated into the matrix as living cells, osteocytes, which communicate with each other and surface epithelium by cell processes within canaliculi in the matrix. The osteoblasts secrete the organic matrix, which are dense collagen layers that alternate parallel and orthogonal to the axis of stress loading. Into this matrix is deposited extremely dense hydroxyapatite-based mineral driven by both active and passive transport and pH control. As the matrix matures, hydroxyapatite microcrystals are organized into a sophisticated compo...
BioResearch Open Access, 2015
Natural bone healing following fractures is initiated by osteoblasts (OBs) and mesenchymal stem cells (MSCs), a cell combination with possible potential in tissue engineering techniques for bony defects. The aim of the study was to investigate MSC/OB-crosstalk, in order to determine optimal cell culture conditions for osteogenic differentiation. Human OBs and MSCs interactions were investigated in an in vitro trans-well co-culture study over a time period of 28 days. Calcification was determined by optical density (OD) at 450 nm and Alizarin red staining. Messenger RNA expression was assessed by quantitative PCR. Osteogenic medium containing 1% fetal bovine serum resulted in superior levels of calcification in MSCs in co-culture with OBs compared to 2% or 5% fetal bovine serum ( p < 0.05). Comparing MSCs and OBs alone with the MSC/OB co-culture, calcification, as measured by OD 450 nm, increased over time in all groups. The highest values were recorded in the co-culture ( p < 0.05). Osteogenic differentiation potential showed significant interindividual differences. In order to predict differentiation potential, OD 450 nm measurements and mRNA expression of alkaline phosphatase were correlated with the population doubling rate during the expansion period. For OBs and MSCs, statistically significant associations of proliferation and differentiation potential were found (p < 0.001). The addition of transforming growth factor beta resulted in up-regulation of collagen type I and Sp7 mRNA, and down-regulation of alkaline phosphatase mRNA. The results suggest the idea of soluble paracrine factors being secreted by OBs to induce osteogenic differentiation of MSCs.
Journal of Cellular Biochemistry, 1994
Establishing regulatory mechanisms that mediate proliferation of osteoblasts while restricting expression of genes associated with mature bone cell phenotypic properties to post-proliferative cells is fundamental to understanding skeletal development. To gain insight into relationships between growth control and the developmental expression of genes during osteoblast differentiation, we have examined expression of three classes of genes during the cell cycle of normal diploid rat calvarial-derived osteoblasts and rat osteosarcoma cells (ROS 17/23): cell cycle and growth-related genes (e.g., histone), genes that encode major structural proteins (e.g., actin and vimentin), and genes related to the biosynthesis, organization, and mineralization of the bone extracellular matrix (e.g., alkaline phosphatase, collagen I, osteocalcin, and osteopontin). In normal diploid osteoblasts as well as in osteosarcoma cells we found that histone genes, required for cell progression, are selectively expressed during S phase. All other genes studied were constitutively expressed both at the transcriptional and posttranscriptional levels. Alkaline phosphatase, an integral membrane protein in both osteoblasts and osteosarcoma cells, exhibited only minimal changes in activity during the osteoblast and osteosarcoma cell cycles. Our findings clearly indicate that despite the loss of normal proliferationdifferentiation interrelationships in osteosarcoma cells, cell cycle regulation or constitutive expression of growth and phenotypic genes is maintained.