PTHrP Induces Autocrine/Paracrine Proliferation of Bone Tumor Cells through Inhibition of Apoptosis

Giant Cell Tumor of Bone (GCT) is an aggressive skeletal tumor characterized by local bone destruction, high recurrence rates and metastatic potential. Previous work in our lab has shown that the neoplastic cell of GCT is a proliferating pre-osteoblastic stromal cell in which the transcription factor Runx2 plays a role in regulating protein expression. One of the proteins expressed by these cells is parathryroid hormone-related protein (PTHrP). The objectives of this study were to determine the role played by PTHrP in GCT of bone with a focus on cell proliferation and apoptosis. Primary stromal cell cultures from 5 patients with GCT of bone and one lung metastsis were used for cell-based experiments. Control cell lines included a renal cell carcinoma (RCC) cell line and a human fetal osteoblast cell line. Cells were exposed to optimized concentrations of a PTHrP neutralizing antibody and were analyzed with the use of cell proliferation and apoptosis assays including mitochondrial dehydrogenase assays, crystal violet assays, APO-1 ELISAs, caspase activity assays, flow cytometry and immunofluorescent immunohistochemistry. Neutralization of PTHrP in the cell environment inhibited cell proliferation in a consistent manner and induced apoptosis in the GCT stromal cells, with the exception of those obtained from a lung metastasis. Cell cycle progression was not significantly affected by PTHrP neutralization. These findings indicate that PTHrP plays an autocrine/paracrine neoplastic role in GCT by allowing the proliferating stromal cells to evade apoptosis, possibly through non-traditional caspase-independent pathways. Thus PTHrP neutralizing immunotherapy is an intriguing potential therapeutic strategy for this tumor

Development of three-dimensional tissue engineered bone-oral mucosal composite models

Tissue engineering of bone and oral mucosa have been extensively studied independently. The aim of this study was to develop and investigate a novel combination of bone and oral mucosa in a single 3D in vitro composite tissue mimicking the natural structure of alveolar bone with an overlying oral mucosa. Rat osteosarcoma (ROS) cells were seeded into a hydroxyapatite/tri-calcium phosphate scaffold and bone constructs were cultured in a spinner bioreactor for 3 months. An engineered oral mucosa was fabricated by air/liquid interface culture of immortalized OKF6/TERET-2 oral keratinocytes on collagen gel-embedded fibroblasts. EOM was incorporated into the engineered bone using a tissue adhesive and further cultured prior to qualitative and quantitative assessments. Presto Blue assay revealed that ROS cells remained vital throughout the experiment. The histological and scanning electron microscope examinations showed that the cells proliferated and densely populated the scaffold construct. Micro computed tomography (micro-CT) scanning revealed an increase in closed porosity and a decrease in open and total porosity at the end of the culture period. Histological examination of bone-oral mucosa model showed a relatively differentiated parakeratinized epithelium, evenly distributed fibroblasts in the connective tissue layer and widely spread ROS cells within the bone scaffold. The feasibility of fabricating a novel bone-oral mucosa model using cell lines is demonstrated. Generating human ‘normal’ cell-based models with further characterization is required to optimize the model for in vitro and in vivo applications

Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6-18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. beta-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner

Development of a 3D Collagen Model for the In Vitro Evaluation of Magnetic-assisted Osteogenesis

Abstract Magnetic stimulation has been applied to bone regeneration, however, the cellular and molecular mechanisms of repair still require a better understanding. A three-dimensional (3D) collagen model was developed using plastic compression, which produces dense, cellular, mechanically strong native collagen structures. Osteoblast cells (MG-63) and magnetic iron oxide nanoparticles (IONPs) were incorporated into collagen gels to produce a range of cell-laden models. A magnetic bio-reactor to support cell growth under static magnetic fields (SMFs) was designed and fabricated by 3D printing. The influences of SMFs on cell proliferation, differentiation, extracellular matrix production, mineralisation and gene expression were evaluated. Polymerase chain reaction (PCR) further determined the effects of SMFs on the expression of runt-related transcription factor 2 (Runx2), osteonectin (ON), and bone morphogenic proteins 2 and 4 (BMP-2 and BMP-4). Results demonstrate that SMFs, IONPs and the collagen matrix can stimulate the proliferation, alkaline phosphatase production and mineralisation of MG-63 cells, by influencing matrix/cell interactions and encouraging the expression of Runx2, ON, BMP-2 and BMP-4. Therefore, the collagen model developed here not only offers a novel 3D bone model to better understand the effect of magnetic stimulation on osteogenesis, but also paves the way for further applications in tissue engineering and regenerative medicine

Activated T lymphocytes support osteoclast formation in vitro.

Osteoblastic stromal cells are capable of supporting osteoclast formation from hematopoietic precursors in the presence of osteotropic factors such as 1alpha,25(OH)(2)D(3), PTH, and IL-11. Osteoblastic stromal cells produce receptor activator of NF-kappaB ligand (RANKL), a type II membrane protein of the TNF ligand family, in response to these agents. Activated T lymphocytes also produce RANKL; however, the ability of this cell type to support osteoclast formation in vitro is unknown. Human PBMC-derived T cells, extracted using alphaCD3-coated magnetic beads, were cocultured with adherent murine spleen cells in the presence of Con A and a panel of cytokines. In the presence of Con A, bona fide osteoclasts were formed in vitro with activated T cells: IL-1alpha and TGFbeta further enhanced osteoclast numbers. PBMC-derived lymphocytes showed an increase in the mRNA expression of RANKL within 24 h of treatment with the same agents that were used to induce osteoclast formation. In synovial tissue sections with lymphoid infiltrates from RA patients, the expression of RANKL was demonstrated in CD3(+) T cells. The ability of activated T lymphocytes to support osteoclast formation may provide a mechanism for the potentiation of osteoclast formation and bone resorption in disease states such as rheumatoid arthritis

Localization of RANKL (receptor activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues.

RANKL (receptor activator of NFkappaB ligand) is a membrane-associated osteoblastic molecule, and along with macrophage-colony-stimulating factor, is crucial for osteoclast formation. RANKL is known to be strongly expressed in osteoblasts and lymphoid tissues. We have sought to determine the skeletal and extraskeletal sites of production of RANKL mRNA and protein using the techniques of in situ hybridization and immunohistochemistry. Expression of RANKL mRNA and protein were determined in the developmental progression of endochondral bone formation in mouse, intramembranous bone formation in a rabbit model (mRNA only), in human giant cell tumors of bone, and at extraskeletal sites in the mouse. RANKL mRNA was expressed in prehypertrophic and hypertrophic chondrocytes at day E15 embryonic mouse long bone, and its expression was maintained at these sites throughout development. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar localization pattern of RANKL protein at the sites described. In the intramembranous bone formation model, RANKL mRNA was expressed in mesenchymal cells and in actively synthesizing osteoblasts, but not in flattened lining osteoblasts or late osteocytes. Expression of RANKL mRNA and protein in osteoclasts was variable with those within resorption lacunae showing the strongest signal/staining. Likewise, expression varied in osteoclasts from giant cell tumor of bone with a minority of tartrate-resistant acid phosphatase-positive multinucleated cells having no detectable RANKL mRNA or protein. In extraskeletal tissues, RANKL mRNA and protein were detected in the brain, heart, kidney, skeletal muscle, and skin throughout mouse development, suggesting the possibility of several other functions of the molecule. RANKL was also developmentally regulated, as evidenced by its expression in the intestine, liver, and lung at E15 and newborn mouse but not in the adult