by Keyword: Osteoclast

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Diez-Escudero, A., Torreggiani, E., Di Pompo, G., Espanol, M., Persson, C., Ciapetti, G., Baldini, N., Ginebra, M. P., (2019). Effect of calcium phosphate heparinization on the in vitro inflammatory response and osteoclastogenesis of human blood precursor cells Journal of Tissue Engineering and Regenerative Medicine 13, (7), 1217-1229

The immobilization of natural molecules on synthetic bone grafts stands as a strategy to enhance their biological interactions. During the early stages of healing, immune cells and osteoclasts (OC) modulate the inflammatory response and resorb the biomaterial, respectively. In this study, heparin, a naturally occurring molecule in the bone extracellular matrix, was covalently immobilized on biomimetic calcium‐deficient hydroxyapatite (CDHA). The effect of heparin‐functionalized CDHA on inflammation and osteoclastogenesis was investigated using primary human cells and compared with pristine CDHA and beta-tricalcium phosphate (β-TCP). Biomimetic substrates led to lower oxidative stresses by neutrophils and monocytes than sintered β-TCP, even though no further reduction was induced by the presence of heparin. In contrast, heparinized CDHA fostered osteoclastogenesis. Optical images of stained TRAP positive cells showed an earlier and higher presence of multinucleated cells, compatible with OC at 14 days, while pristine CDHA and β-TCP present OC at 21–28 days. Although no statistically significant differences were found in the OC activity, microscopy images evidenced early stages of degradation on heparinized CDHA, compatible with osteoclastic resorption. Overall, the results suggest that the functionalization with heparin fostered the formation and activity of OC, thus offering a promising strategy to integrate biomaterials in the bone remodelling cycle by increasing their OC-mediated resorption.

Keywords: Biomaterial, Heparin, Hydroxyapatite, Inflammation, Osteoclastogenesis

Sadowska, Joanna M., Wei, Fei, Guo, Jia, Guillem-Marti, Jordi, Ginebra, Maria-Pau, Xiao, Yin, (2018). Effect of nano-structural properties of biomimetic hydroxyapatite on osteoimmunomodulation Biomaterials 181, 318-332

Immune cells are sensitive to the microstructural and textural properties of materials. Tuning the structural features of synthetic bone grafts could be a valuable strategy to regulate the specific response of the immune system, which in turn modulates the activity of bone cells. The aim of this study was to analyse the effect of the structural characteristics of biomimetic calcium deficient hydroxyapatite (CDHA) on the innate immune response of macrophages and the subsequent impact on osteogenesis and osteoclastogenesis. Murine RAW 264.7 cells were cultured, under standard and inflammatory conditions, on chemically identical CDHA substrates that varied in microstructure and porosity. The impact on osteogenesis was evaluated by incubating osteoblastic cells (SaOS-2) with RAW-CDHA conditioned extracts. The results showed that macrophages were sensitive to different textural and structural properties of CDHA. Under standard conditions, the impact of inflammatory cytokine production by RAW cells cultured on CDHA played a significant role in the degradation of substrates, suggesting the impact of resorptive behaviour of RAW cells on biomimetic surfaces. Osteoblast differentiation was stimulated by the conditioned media collected from RAW cells cultured on needle-like nanostructured CDHA. The results demonstrated that needle-like nanostructured CDHA was able to generate a favourable osteoimmune environment to regulate osteoblast differentiation and osteogenesis. Under inflammatory conditions, the incubation of RAW cells with less porous CDHA resulted in a decreased gene expression and release of pro-inflammatory cytokines.

Keywords: Calcium phosphates, Biomimetic hydroxyapatite, Osteoimmunomodulation, Inflammation, Osteogenesis, Osteoclastogesis

Ciapetti, G., Di Pompo, G., Avnet, S., Martini, D., Diez-Escudero, A., Montufar, E. B., Ginebra, M. P., Baldini, N., (2017). Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates Acta Biomaterialia 50, 102-113

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium. Statement of Significance: The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors’ differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.

Keywords: Bone resorption, Differentiation, Hydroxyapatite, Ionic exchange, Osteoclasts, Topography

Diez-Escudero, A., Espanol, M., Montufar, E. B., Di Pompo, G., Ciapetti, G., Baldini, N., Ginebra, M. P., (2017). Focus ion beam/scanning electron microscopy characterization of osteoclastic resorption of calcium phosphate substrates Tissue Engineering Part C: Methods , 23, (2), 118-124

This article presents the application of dual focused ion beam/scanning electron microscopy (FIB-SEM) imaging for preclinical testing of calcium phosphates with osteoclast precursor cells and how this high-resolution imaging technique is able to reveal microstructural changes at a level of detail previously not possible. Calcium phosphate substrates, having similar compositions but different microstructures, were produced using low-and high-Temperature processes (biomimetic calcium-deficient hydroxyapatite [CDHA] and stoichiometric sintered hydroxyapatite, respectively). Human osteoclast precursor cells were cultured for 21 days before evaluating their resorptive potential on varying microstructural features. Alternative to classical morphological evaluation of osteoclasts (OC), FIB-SEM was used to observe the subjacent microstructure by transversally sectioning cells and observing both the cells and the substrates. Resorption pits, indicating OC activity, were visible on the smoother surface of high-Temperature sintered hydroxyapatite. FIB-SEM analysis revealed signs of acidic degradation on the grain surface under the cells, as well as intergranular dissolution. No resorption pits were evident on the surface of the rough CDHA substrates. However, whereas no degradation was detected by FIB sections in the material underlying some of the cells, early stages of OC-mediated acidic degradation were observed under cells with more spread morphology. Collectively, these results highlight the potential of FIB to evaluate the resorptive activity of OC, even in rough, irregular, or coarse surfaces where degradation pits are otherwise difficult to visualize.

Keywords: Bone Regeneration, Calcium Phosphate, Focus Ion Beam, Osteoclast, Resorption, Scanning Electron Microscopy