Bone Abstracts

Objective: In many traumatic or pathological conditions, bone turnover is low and osteoclast activity is reduced or abolished. We developed innovative hydroxyapatite (HA) scaffolds carrying RANKL expressing cells with the aim of supporting bone resorption when this is defective. Membrane-bound (m)RANKL is cleaved into soluble (s)RANKL by MMP14. We hypothesized that the osteoclastogenic potential of RANKL-producing cells could be improved if they were seeded on scaffolds engineered by immobilization of MMP14 to rise the shedding of sRANKL.

Methods: Primary osteoblasts (OBs) and stromal bone marrow cells from 10 days old WT CD1 mice were used. MC3T3 cells were stably transfected with sRANKL-vector. MMP14 was immobilized on 3D-HA scaffolds using procedures based on protein-to-substrate binding by glutaraldehyde (10% v/v). For in vivo studies, the scaffolds were entrapped in diffusion chambers (Millipore).

Results: We identified in OBs the best spontaneous sRANKL source, then on these cells we demonstrated a concentration-dependent RANKL shedding ability of the catalytic domain of MMP14. Next, we quantified in about 50% the enzymatic efficiency of MMP-14 functionalized scaffolds vs soluble MMP14, and tested the efficacy of MMP14-engineered 3D-HA scaffolds on OBs noting increased release of sRANKL vs scaffolds not subjected to MMP14 immobilization. Intact scaffolds are also seeded with MC3T3 cells stably overexpressing sRANKL. Moreover, we assembled devices with scaffolds embedded in diffusion chambers and proved the safety of their implants in WT mice. Finally, we tested the efficiency of devices harboring either OBS or sRANKL-MC3T3 implanted in RANKL KO mice. In tibial sections we noted the appearance of TRAcP positive cells in both groups of implanted animals in contrast with sham KO mice, which were TRAcP-negative.

Conclusion: Our results demonstrated the feasibility of a strategy based on engineered bio-device for supporting sRANKL release from osteoblast mRANKL.