Dental pulp stem cells-derived cannabidiol-pretreated micro-spheroids showed robust osteogenic potential via upregulation of WNT6

Abstract

Abstract Dental pulp stem cells (DPSC)-derived 3D-aggregates/spheroids have shown robust soft/hard tissue regeneration potential. However, difficulties to control the cell number, size, and shape of DPSC-aggregates/spheroids cause cell necrosis and difficulties in homogenous seeding of aggregates/spheroids in the 3D-printed microporous bone grafts. Moreover, inducing the osteogenic potential of these aggregates/spheroids is still a challenge. This study developed cannabidiol (CBD)-pretreated, self-assembled, and injectable DPSC-derived osteogenic micro-spheroids (70 μm) that robustly promoted in situ bone regeneration. We developed micro-spheroids by seeding 250 cells/microwell in agarose gel microwells of 200 µm diameter developed using prefabricated mold and cultured with CBD for 14 days to induce osteogenic potential. In vitro study results showed that CBD did not affect the viability of DPSC but promoted osteogenic differentiation during 2D culture. In micro-spheroids, 3D cytoskeleton visualization showed better integrity and robustly higher expression of osteogenic markers and promoted in situ bone regeneration compared with DPSC. CBD-pretreated micro-spheroids showed robustly higher bone-regenerative capacity via upregulation of WNT6. Taken together, our approach of developing organoid-like injectable osteogenic micro-spheroids can be used as the effective carrier of the effect of in vitro drug treatment during in situ bone tissue engineering which eliminates the direct in vivo drug application-related adverse effects.