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The dynamic metabolism and the numerous roles of bone tissue necessitate a suitable in vitro model to represent them. In order to investigate the interaction among the several cell types composing bone microenvironment, we studied a tri-culture model including human osteoblasts (OBs), osteoclasts (OCs), and endothelial cells (HUVEC). While OBs are essential for bone deposition and OCs for bone resorption, the vasculature is necessary to provide growth factors, nutrients, and oxygen in the mature tissue. The results of this study showed a strong mutual influence between OBs, OCs, and HUVEC in term of proliferation, viability, and activity (release of ALP, Coll I, OPG, RANKL, VEGF, CTSK, TGFβ, and IL-6). The behavior of the single cultures demonstrated to be different compared to the bi- or tri-cultures and depending on the cell types involved: the coexistence of OBs and OCs stimulated the synthetic activity of both cell types, while the presence of HUVEC induced a stimulating role for OBs but mainly an inhibitory effect for OC. In addition, evidence of the effects of OBs and OCs on HUVEC is highlighted by their morphology: regular and able to "sketch" little vessels in presence of OBs, more disorganized and heterogeneous in presence of OCs. Taken together, these observations well characterize an advanced cellular model to be used as starting point for mimicking bone microenvironment in vivo, thus reducing the use of animals in the preclinical phase and offering a more reliable tool to test new and innovative biomaterials., (© 2017 Wiley Periodicals, Inc.)

To reduce the burden of bone metastases, the pathophysiology of the metastatic niche should be elucidated and targeted. The aim of the present study was to assess the effect of tumor cells on osteoclast (OC) recruitment and activity in the presence of altered bone remodelling. Peripheral blood mononuclear cells (PBMC) were isolated from healthy and ovariectomized (OVX) rats and co-cultured with MRMT-1 rat breast carcinoma cells or with their conditioned medium for 1 and 2 weeks. Alamar Blue viability test, synthesis of cathepsin K, transforming growth factor-beta 1 (TGF-β1), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), metalloproteinase (MMP)-7, MMP-9, FITC-conjugate phalloidin staining and tartrate-resistant acid phosphatase (TRAP) staining were evaluated. The results indicate that breast carcinoma cells induced different responses in PBMC derived from rats affected by estrogen deficiency osteoporosis (OP) in comparison with healthy ones, with a significant increase in proliferation rate, OC differentiation, synthesis of TNF-α, MMP-7 and MMP-9. The data support the "proof of concept" that OP due to estrogen deficiency might offer a receptive site for cancer cells to form bone metastases.

Bone loss occurs insidiously and initially asymptomatically; therefore, osteoporosis is frequently diagnosed only after the first clinical fracture. The aim of this study was to test the hypothesis is that by simply observing the behavior of cultured peripheral monocytes, it might be possible to diagnose altered bone remodeling and, therefore, limit the complications associated with osteoporosis, especially fractures. Monocytes isolated as mononuclear precursors from healthy and ovariectomized rats were cultured both in basal and differentiation medium for up to 3 weeks. Viability and differentiation capability towards the osteoclastic phenotype was checked by light microscopy at early times, whereas differentiation state and synthetic activity (tartrate-resistant acid phosphatase (TRAP) staining; phalloidin, fluorescin isothiocynate (FITC) staining, cathepsin K, metalloproteinase 7 and 9, MMP-7 and MMP-9) were measured at 1, 2, and 3 weeks. Compared to their controls, monocytes isolated from ovariectomized rats proliferate and lean toward the osteoclastic phenotype in the absence of differentiating factors. In both culture conditions, osteoclasts from ovariectomized rats showed significantly higher productions of cathepsin K, MMP-7, and MMP-9 than those of cells isolated from healthy rats, steadily over time. These results obtained in an animal osteoporotic model, if confirmed by clinical studies, open up the possibility to assess the presence of an alteration in bone remodeling with a simple in vitro diagnostic test requiring a small blood sample and less than 48 h. This might allow to early select patients with a spontaneous viability and differentiation of monocytes to osteoclasts for further diagnostic techniques.

In this study, we investigate the molecular mechanisms by which human osteoblasts (HOB) challenged with wear debris promote the differentiation of osteoclast precursors. HOB were obtained from trabecular bone and exposed to alumina (Al(2)O(3)) or 'ultra-high molecular weight polyethylene' (UHMWPE) particles for 24h. The supernatant (HOB-CM) was used for the immunoenzymatic detection of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG), as well as for inducing the osteoclast differentiation from peripheral blood mononuclear cells (PBMC). The OPG-to-RANKL ratio was significantly decreased in the conditioned medium of UHMWPE-challenged HOB. Morphological and cytochemical analysis showed that HOB-CM induced by itself the osteoclast formation, but a large amount of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive giant cells were obtained when PBMCs were cultured with 1 microg/mL UHMWPE HOB-CM. The expression of genes involved in osteoclast differentiation and activation was evaluated, i.e. c-fms, RANK, c-src, c-fos, cathepsin-K (CATK), TRAP, and calcitonin R (CTR). The UHMWPE HOB-CM increases c-src expression, suggesting that polyethylene debris favour the paracrine activity of HOB in inducing the pathway involved in osteoclast polarization and adhesion. On the contrary, Al(2)O(3) HOB-CM downregulates c-fos expression, suggesting that the passage from macrophages into the osteoclast lineage is deviated. These results show that Al(2)O(3) wear debris is less active than UHMWPE in inducing osteoclast differentiation. Moreover, they provide new insight into the molecular basis of particle-induced osteoclastogenesis, that is the starting point for planning mode-specific targeting of periprosthetic osteolysis.

The integration of an implant material with bone tissue depends on the chemistry and physics of the implant surface. In this study we applied matrix assisted pulsed laser evaporation (MAPLE) in order to synthesize calcium alendronate monohydrate (a bisphosphonate obtained by calcium sequestration from octacalcium phosphate by alendronate) and calcium alendronate monohydrate/octacalcium phosphate composite thin films on titanium substrates. Octacalcium phosphate coatings were prepared as reference material. The powders, which were synthesized in aqueous medium, were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets for MAPLE experiments. The transfer was conducted with a KrF* excimer laser source ( λ = 248 nm, τ FWHM ≤ 25 ns) in mild conditions of temperature and pressure. XRD, FTIR and SEM analyses confirmed that the coatings contain the same crystalline phases as the as-prepared powder samples. Osteoblast derived from stem cells and osteoclast derived from monocytes of osteoporotic subjects were co-cultured on the coatings up to 14 days. Osteoclast displayed significantly reduced proliferation and differentiation in the presence of calcium alendronate monohydrate, pointing to a clear role of the coatings containing this bisphosphonate on inhibiting excessive bone resorption. At variance, osteoblast production of alkaline phosphatase and type I pro-collagen were promoted by the presence of bisphosphonate, which also decreased the production of interleukin 6. The positive influence towards osteoblast differentiation was even more enhanced in the composite coatings, thanks to the presence of octacalcium phosphate. [ABSTRACT FROM AUTHOR]