M. Grano

Alendronate Reduces Adhesion of Human Osteoclast-like Cells to Bone and Bone Protein-Coated Surfaces

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40–45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as αVβ3 integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration.

Soluble decoy receptor 3 modulates the survival and formation of osteoclasts from multiple myeloma bone disease patients

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor (TNF) receptor superfamily, is known to be involved in cell survival and osteoclast (OC) formation. In this study, we show that malignant plasma cells and T lymphocytes from multiple myeloma (MM) bone disease patients, as well as Karpas 909, a human myeloma cell line, directly produce DcR3. By interacting with FasL, this molecule could inhibit OC apoptosis. In fact, the use of a neutralizing anti-DcR3 antibody induces a reduction of cell viability with a consequent increase of apoptotic cell number, the activation of caspase-8 and -3, and DNA fragmentation. Furthermore, we show that DcR3 supports OC formation in samples from MM patients through the upregulation of RANKL and TNFα by T lymphocytes and only TNFα by CD14+ cells. In conclusion, our data provide the first evidence of the expression of DcR3 in MM, and the involvement of this molecule in supporting the survival and formation of OCs from MM bone disease patients. The production of DcR3 by T lymphocytes confers these cells a role in the pathogenesis of bone disease associated with MM.

Effects of Calcium-phosphate-based Materials on Proliferation and Alkaline Phosphatase Activity of Newborn Rat Periosteal Cells in vitro

The effects of dental materials, intended for bone substitution, on cell growth and alkaline phosphatase activity of newborn rat periosteal cells have been studied in vitro. Confluent periosteal cells were exposed to three apatite-based materials (400 μg/mL) with different physico-chemical properties. The materials were a β-tricalcium phosphate with a microporous granular structure obtained by sinterization (Synthograft, Johnson & Johnson, East Windsor, NY), a 40-60-mesh microporous durapatite ceramic (Periograf, Sterling Drug, Inc., Rensselaer, NY), and a 1-2-mm-diameter hydroxyapatite ceramic (Osprovit, Feldmuhle Aktiengeselschaft, Plochingen, Germany) with macropores larger than 100 μm. Cell proliferation and alkaline phosphatase activity were assessed by incorporation of 3H-thymidine into trichloroacetic-acid-precipitable material and by a fluorimetric method, respectively. Cell viability and compatibility with the materials were determined by morphology in phase-contrast microscopy. Periosteal cells showed increased proliferation following exposure to Synthograft, but were unaffected by Osprovit, whereas Periograf caused significantly reduced cell growth. Alkaline phosphatase activity was unaffected by Osprovit, but was decreased by both Synthograft and Periograf. The results indicated a differential response of periosteal cells to bone-substituting materials with heterogeneous physico-chemical characteristics.

Integrins and Cadherins could be important regulators of osteogenic differentiation and bone formation in dental stem cells

Regenerative medicine aims to translate the regeneration of tissues and organs into clinical application. Numerous studies have reported beneficial effects of Adult stem cells therapy in the treatment of disease and disabilities. New sources of mesenchymal stem cells (MSCs) are emerging in adult organisms, and dental tissues, that are easily accessible, have been identified as a source of postnatal MSCs capable of self-renewal and multipotency. Dental Follicle Stem Cells (DFSCs) isolated from tooth buds of healthy paediatric patients showed ≥95% expression of stemness makers (CD73, CD90, CD146, CD44, CD105, and HLA-I) [1]. Moreover DFSCs differentiated into osteoblastlike cells, produced mineralized matrix nodules and expressed typical osteoblastic markers. Cell interactions with extracellular matrix (ECM) and neighbor cells are critical for tissue morphogenesis and architecture, and are mediated by two classes of adhesion molecules, respectively Integrins and Cadherins, which also act intracellularly by modulating crucial pathways of proliferation and differentiation. Thus, in this study, DFSCs were characterized for the expression of adhesion molecules Cadherins and Integrins. In basal conditions DFSCs expressed higher levels of N-Cadherin and Cadherin-11 in comparison to E-Cadherin and P-Cadherin, which were low expressed. The examined Cadherins showed different behaviours during DFSCs osteogenic differentiation: N-cadherin expression was high during the first steps, while decreased at the later times; Cadherin-11 progressively increased; E-Cadherin and P-Cadherin did not change a Cadherin profile reflecting the osteoblastic commitment of the cells. DFSCs expressed the Integrin subunits alpha V, beta 3, alpha 5, and beta 1 in basal undifferentiated conditions but their expression increased time-dependently under osteogenic treatment. In addition we found that the subunits alpha V and beta 3 associated and formed the functional integrin, which localized at the focal adhesion in response to osteogenic trigger; similarly, alpha 5 and beta 1 subunits were found to associate and localize at the cell borders mostly in differentiated cells. Finally we found that osteogenic differentiation of DFSCs was prompted out by seeding the cells on ECM protein coated surfaces. Functional tissue engineering for bone regeneration requires the appropriate combination of MSCs with biocompatible scaffolds, thus acting on cell surface and ECM molecules could optimize osteogenic differentiation of MSCs and contribute to the successful regeneration of damaged bone tissue.