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Glucosamine (GlcN) functions as a building block of the cartilage matrix, and its multifaceted roles in promoting joint health have been extensively investigated. However, the role of GlcN in osteogenesis and bone tissue is poorly understood, mainly due to the lack of adequate experimental models. As a result, the benefit of GlcN application in bone disorders remains controversial. In order to further elucidate the pharmacological relevance and potential therapeutic/nutraceutic efficacy of GlcN, the effect of GlcN treatment was investigated in human primary osteoclasts (hOCs) and osteoblasts (hOBs) that were cultured with two‑dimensional (2D) traditional methods or co‑cultured in a 3D dynamic system more closely resembling the in vivo bone microenvironment. Under these conditions, osteoclastogenesis was supported by hOBs and sizeable self‑assembling aggregates were obtained. The differentiated hOCs were evaluated using tartrate‑resistant acid phosphatase assays and osteogenic differentiation was monitored by analyzing mineral matrix deposition via Alizarin Red staining, with expression of specific osteogenic markers determined via reverse transcription‑quantitative PCR. It was found that crystalline GlcN sulfate was effective in decreasing osteoclastic cell differentiation and function. hOCs isolated from patients with OA were more sensitive compared with those from healthy donors. Additionally, GlcN exhibited anabolic effects on hOCs both in 2D conventional cell culture and in hOC/hOB 3D dynamic co‑culture. The present study demonstrated for the first time the effectiveness of a 3D dynamic co‑culture system for characterizing the spectrum of action of GlcN on the bone microenvironment, which may pave the way for more fully determining the potential applications of a compound such as GlcN, which is positioned between pharmaceuticals and nutraceuticals. Based on the present findings, it is hypothesized that GlcN may have potential benefits in the treatment of osteopenic diseases such as osteoporosis, as well as in bone maintenance.

Conventional orthopedic implants are composed from titanium. To improve some characteristics (ie, volumetric porosity, modulus of elasticity, frictional modulus), a new porous tantalum biomaterial has been developed and its biocompatibility reported. By using DNA microarrays containing 20,000 genes, several genes whose expression were significantly up- or down-regulated were identified in an osteoblastlike cell line (MG63) cultured with tantalum powder (TP). The differentially expressed genes cover a broad range of functional activities: signaling transduction; transcription; cell cycle regulation, proliferation, and apoptosis; and cytoskeleton formation. To the authors' knowledge, the data reported represent the first genetic portrait of TP.

Polylactide, polyglycolide materials or devices have been utilized routinely during maxillofacial, craniofacial, and orthopaedic reconstructive surgical procedures.(1) These materials combine the benefits of rigid fixation with the advantages of biodegradation, avoiding the need for implant removal and minimizing the risk of other complications.(2) To study how polylactide, polyglycolide acids plates (PLPG plates) can induce osteoblast differentiation and proliferation in mesenchymal stem cells, the expression levels of bone related genes (RUNX2, SP7, ALPL, SPP1, COL1A1, COL3A1 and FOSL1) and mesenchymal stem cells marker (ENG) were measured in adipose derived stem cells (ADSCs) and normal osteoblast (NO) cultivated on PLPG plates after 15 and 30 days of treatment using real time Reverse Transcription-Polymerase Chain Reaction. Significantly differentially expressed genes among ADSCs and NO were SP7, ENG, FOSL1, RUNX, ALPL and SPP1 in the first 15 days of treatment and SP7, ENG FOSL1, COL3A1 COL1A1, SPP1 and ALPL after 30 days. The present study demonstrated that PLPG plates strongly influences the behavior of ADSCs in vitro by enhancing proliferation, differentiation and deposition of matrix.

Background: Although pulsed electromagnetic fields (PEMFs) are used to treat delayed unions and nonunions, their mechanisms of action are not completely clear. However, PEMFs are known to affect the expression of certain genes., Questions/purposes: We asked (1) whether PEMFs affect gene expression in human osteoblastlike cells (MG63) in vitro, and (2) whether and to what extent stimulation by PEMFs induce cell proliferation and differentiation in MG-63 cultures., Methods: We cultured two groups of MG63 cells. One group was treated with PEMFs for 18 hours whereas the second was maintained in the same culture condition without PEMFs (control). Gene expression was evaluated throughout cDNA microarray analysis containing 19,000 genes spanning a substantial fraction of the human genome., Results: PEMFs induced the upregulation of important genes related to bone formation (HOXA10, AKT1), genes at the transductional level (CALM1, P2RX7), genes for cytoskeletal components (FN1, VCL), and collagenous (COL1A2) and noncollagenous (SPARC) matrix components. However, PEMF induced downregulation of genes related to the degradation of extracellular matrix (MMP-11, DUSP4)., Conclusions and Clinical Relevance: PEMFs appear to induce cell proliferation and differentiation. Furthermore, PEMFs promote extracellular matrix production and mineralization while decreasing matrix degradation and absorption. Our data suggest specific mechanisms of the observed clinical effect of PEMFs, and thus specific approaches for use in regenerative medicine.

Titanium is the gold standard among materials used for prosthetic devices, because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide that is biocompatible and able to induce osseointegration. Three allotropic forms of titanium dioxide exist, that is brookite, rutile, and anatase. Anatase can be prepared as a colloidal suspension and then used to coat surfaces. Anatase coating (AC) can potentially have specific biological effects. Here we are testing the effect of AC on osteoblast-like cells (MG63) by using microarray techniques to identify genes that are differently regulated in osteoblasts exposed to AC. By using DNA microarrays containing 20,000 genes, we identified in osteoblast-like cell lines (MG-63) cultured on AC, several genes whose expression was significantly up- or downregulated. They cover a broad range of functional activities: signaling transduction, immunity, cell cycle regulation, lysosomes composition and vesicular transport, cell adhesion, cytoskeleton and extracellular matrix components, proliferation, and apoptosis. The data reported constitute, to our knowledge, the first genetic portrait of AC effects. They can be relevant to a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Titanium is the criterion standard among materials used for prosthetic devices because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide that is biocompatible and able to induce osseointegration. There are 3 allotropic forms of titanium dioxide: brookite, rutile, and anatase. Anatase can be prepared as a colloidal suspension and then used to coat surfaces. Anatase coating (AC) can potentially have specific biologic effects and specifically induce bone formation. To get more information as regards the osteogenic effect of AC, we used microRNA (miRNA) microarray techniques to investigate translation regulation in osteoblasts exposed to AC. Transduction, transcription, and translation are the 3 levels of regulation of cell activity. Recently, a new type of translation regulation has been identified: RNA interference. RNA interference is a process in which miRNA (i.e., noncoding RNAs of 19-23 nucleotides) can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes a few hundred miRNAs that can posttranscriptionally repress thousands of genes. miRNA oligonucleotide microarray provides a novel method of carrying out genome-wide miRNA profiling in human samples. By using miRNA microarrays containing 329 probes designed from human miRNA sequences, we identified in osteoblast-like cell line (MG-63) cultured with AC several miRNA whose expression had been significantly modified. The data reported constitute, to our knowledge, the first study on translation regulation in osteoblasts exposed to AC. They can be relevant to a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Zirconium is widely used as material for prosthetic devices because its good mechanical and chemical properties. When exposed to oxygen, zirconium becomes zirconium oxide (ZrO(2)), which is biocompatible. ZrO(2) can be also prepared as a colloidal suspension and then used to coat surfaces. Zirconium oxide coating (ZrO(2)C) can potentially have specific biologic effects, and among them is bone formation related to implant osseointegration. How this biomaterial alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to identify genes that are differently regulated in osteoblasts exposed to ZrO(2)C. By using DNA microarrays containing 20,000 genes, we identified in osteoblast-like cell lines (MG-63) cultured with ZrO(2)C several genes whose expression was significantly upregulated or downregulated. The differentially expressed genes cover a broad range of functional activities: (a) cell cycle regulation, (b) signal transduction, (c) immunity, and (d) cytoskeleton component. The data reported are, to our knowledge, the first genetic portrait of ZrO(2)C effects. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects., ((c) 2007 Wiley Periodicals, Inc.)

In this study, the methylation status of the distal promoter F of estrogen receptor alfa (ERalpha) gene in human osteoblastic cells was investigated. The activity of this promoter is responsible for the ERalpha gene transcription in bone tissue. The methylation status of promoter F was here evaluated, for the first time, by direct sequencing of bisulfite-treated genomic DNA, at 10 CpG specific sites localized in a region of about 800 bp. An heterogeneous methylation pattern was observed. The most notable difference was found at four particular CpGs, distant from the exon F transcription start site, showing a methylation status that correlates with the expression level, being ERalpha mRNA transcription reduced in a partially methylated cells but preserved in demethylated cells. The other CpG sites, localized around the transcription start site, were always demethylated except for MG-63 cells showing the lowest level of ERalpha expression. By quantitative RT-PCR analysis we demonstrated that ERalpha gene expression was higher in primary osteoblasts than in bone-derived cells (MG-63 and SaOS-2) and in all cases the ERalpha mRNA is represented by the isoform F. The same 10 CpG sites were investigated in non-osseous cell lines and were found fully methylated in ERalpha-negative breast cancer cells (MDA-MB-231) and completely demethylated in ERalpha-positive breast cancer cells (MCF7). The overall results suggest that methylation of the CpG sites inside ERalpha gene promoter F here analyzed may contribute to ERalpha transcriptional control, directly or indirectly, influencing the tissue specific expression of the gene.

Osteoblasts have been shown to express both isoforms of estrogen receptor (ER alpha and ER beta). As a tool for the study of endogenous regulation of these genes the decoy strategy was employed. Human MG-63 osteoblast-like cells were transfected with a DNA decoy molecule containing a putative negative cis-element (DNA-102) located in the C distal promoter of ER alpha gene. Using real-time quantitative RT-PCR, we found that the DNA-102, but not scrambled DNA, produced a 36-fold increase in the level of total ER alpha mRNA and a 12-fold increase in the level of mRNA for the F isoform that is transcribed from the upstream F promoter, which is predominantly used in osteoblasts. This effect appears to be controlled by estrogen since 17-beta-estradiol downregulated the mRNA increase. Notably, the same decoy was able to induce a 6-fold increase in ER beta mRNA transcription, indicating the coregulation of the ER alpha and ER beta expression. An increase in OPN but not in BMP4 expression was also observed. In addition, in decoy-treated cells, the cell growth decreased together with an increase in alkaline phosphatase activity. These findings indicated that DNA-102 decoy was able to induce a more differentiated osteoblastic phenotype. The augmentation of ER alpha and ER beta expression by the decoy approach may offer a further possibility for patient response to estrogenic therapy in the treatment of diseases related to estrogen deficiency.

We have exposed cultured bone cells to a pulsed electromagnetic field (PEMF) for different times to find the minimal exposure time necessary to stimulate an increase of DNA synthesis. We used two different human osteosarcoma cell lines, TE-85 and MG-63, and human normal osteoblast cell (NHOC) obtained from surgical bone specimens. The cells were placed in multiwell plates and set in a tissue culture incubator between a pair of Helmoltz coils powered by a pulse generator (1.3-ms pulse, repeated at 75 Hz) for different periods of time. [3H]Thymidine incorporation was used to evaluate cell proliferation. The two osteosarcoma cell lines increase their thymidine incorporation when exposed to a PEMF for at least 30 min, both in a medium containing 10% fetal calf serum and in a serum-free medium. NHOC are known to increase their cell proliferation when exposed to PEMF but only if cultured in the presence of 10% fetal calf serum. In this experimental condition, three of the four cell lineages studied required at least 9 h of PEMF exposure to increase their DNA synthesis, whereas one cell lineage increased its cell proliferation after 6 h of PEMF exposure. Our observations confirm the hypothesis that the proliferative responses of NHOC and human osteosarcoma cell lines to PEMF exposure are quite different. Moreover, NHOC required minimal exposure times to PEMF to increase their cell proliferation, similar to that needed to stimulate bone formation in vivo.

We have studied the effects of low-energy, low-frequency pulsed electromagnetic fields (PEMF) on cell proliferation, in both human osteoblast-like cells obtained from bone specimens and in human MG-63 osteosarcoma cell line. Assessment of osteoblastic phenotype was performed both by immunolabeling with antiosteonectin antibody and by verifying the presence of parathyroid hormone receptors. The cells were placed in multiwell plates and set in a tissue culture incubator between a pair of Helmholtz coils powered by a pulse generator (1.3 ms, 75 Hz) for different periods of time. [3H]Thymidine incorporation was used to evaluate cell proliferation. Since it had previously been observed that the osteoblast proliferative response to PEMF exposure may also be conditioned by the presence of serum in the medium, experiments were carried out at different serum concentrations. [3H]Thymidine incorporation increases in osteoblast-like cells, when they are exposed to PEMF in the presence of 10% fetal calf serum (FCS). The greatest effect is observed after 24 hours of PEMF exposure. No effects on cell proliferation are observed when osteoblast-like cells are exposed to PEMF in the presence of 0.5% FCS or in a serum-free medium. On the other hand, PEMF-exposed MG-63 cells show increased cell proliferation either at 10% FCS, 0.5% FCS and in serum-free medium. Nevertheless, the maximum effect of PEMF exposure on MG-63 cell proliferation depends on the percentage of FCS in the medium. The higher the FCS concentration, the faster the proliferative response to PEMF exposure. Our results show that, although MG-63 cells display some similarity with human bone cells, their responses to PEMF's exposure are quite different from that observed in normal human bone cells.