Fig. 4. Temporal and spatial progression of peri-implant osteogenesis triggered by L-WNT3A.

(A,A’) Strain map obtained from FE analyses, displaying a continuum of increasing strains, from Zone 1 (light blue, at the implant surface, between the threads) to Zone 3 (bright red, at the thread tips). (B) Illustration of L-WNT3A treatment at time of surgery where liposomes are evenly dispersed throughout the peri-implant blood clot. (C) GFP immunostaining for WNT responsive cells in Axin2^CreERT2/+;R26R^mTmG/+ mice; the majority of Wnt-responding cells are localized in the lower strain, Zone 1 region. These data are summarized in (D) an illustration depicting the distribution of WNT-responsive cells (green) and undifferentiated cells (light blue) 3 days after L-WNT3A treatment. (E) Pentachrome staining at PID7 assessed the state of ossification in Zone 1. (F) Illustration of cell activity at PID7 where osteoblasts (yellow squares) are confined to Zone 1 while the outer gap region remains fibrous (blue cells). (G) ALP activity in the peri-implant gap at PID14. (H) Illustration of tissue layers comprising the gap region at PID14. (I) Osterix IHC showing the strong osteogenic activity in the gap region at PID14. (J) Lateral stiffness testing of L-PBS and L-WNT3A treated groups at PID14. Scale bars = 50μm (B, H). Abbreviations: b, bone; IM, implant. Asterisk indicates p<.05.