In autosomal recessive osteopetrosis due to mutations of the TNFSF11 gene, deficiency of the pro-osteoclastogenic cytokine RANKL prevents osteoclast formation. RANKL is a membrane-bound protein cleaved into active soluble (s)RANKL by various enzymes, including metalloproteinase 14 (MMP14). We created a bio-device that released sRANKL and induced osteoclastogenesis in tnfsf11−/− mice. We tested various RANKL cell sources, and used mouse primary calvarial osteoblasts, which are readily available, easy to handle and express RANKL in large amount and in a PTH-regulated fashion. Cells were cultured on 3D-hydroxyapatite scaffolds (3D-HASs) adsorbed with the catalytic domain of MMP14, creating a device enzymatically stable over time, that enhanced sRANKL release. These conditions allowed to achieve a free sRANKL concentration, calculated on the basis of 1:1 molar ratio with released OPG, three times higher than in standard cultures, seemly for osteoclastogenesis. These 3D-HASs were sealed in diffusion chambers (DCs) that isolate cells, preventing immune responses, but let molecular flow and release of soluble factors into the circulation. They were implanted in tnfsf11−/− mice of various ages (2140 days) and genetic background C57BL/6 and C57BL/6-CD1. Mice were sacrificed after 1 or 2 months from implants and received 1 or 2 DCs, once or twice. An increase of overall survival and body weight was observed in all implanted groups compared to non-implanted mice. Histological sections of tibias of non-implanted mice were negative for the osteoclast marker TRAcP, consistent with the lack of the osteoclast lineage. In contrast, tibias excided from implanted mice showed TRAcP-positive cells both in the bone marrow and on the bone surface, these latter morphologically similar to mature osteoclasts. Improved outcome was observed in longer treatments and double implants. We suggest that engineered DCs delivering sRANKL support the feasibility of an innovative experimental strategy to deliver the soluble cytokine and treat systemic deficiency.
17 - 20 May 2014
European Calcified Tissue Society