Paracrine Influence of Masquelet's Induced Membrane on Marrow Derived Mesenchymal Stem Cells

Masquelet's induced membrane technique (MIMT) is a staged surgical procedure that leverages the foreign body induced membrane (IM) that forms around a cement spacer placed into a segmental bone defect to support subsequent bone grafting. The mechanisms by which the IM supports bone consolidation are not fully understood. We present an indirect coculture system for studying IM-MSC interactions using a rat model of MIMT. Compared to control cells, MSC Tnap (alkaline phosphatase) was induced by 4- but not 8-week IM. MSC Spp1 (Osteopontin) was attenuated by both 4- and 8-week IM. Although Tnfrsf11b (osteoprotegrin) in MSC exposed to IM was not different from control cells, it was induced by 8-week IM compared to 4-week IM. MSC Tnfsf11 (RANKL) was reduced by 4-week and 8-week IM. MSC Thbs2 (Tsp2) was induced by 8-week but not 4-week IM. Ablation of macrophages in IM blocked the induction of Thbs2 by 8-week IM. MSC Col1a1 expression was not affected under any condition tested. TMT proteomics analysis of IM-conditioned medium revealed 150 unique secreted proteins, 7 of which were differentially abundant (fold change ≥ 2 and FDR corrected p ≤ 0.05) in 8-week versus 4-week IM secretomes. All differentially abundant proteins were elevated in medium conditioned by 8-week IM. Our data suggest that factor(s) secreted by IM resident cells affect MSC gene expression, and that duration of IM development influences the potency and nature of this paracrine effect. Patient-specific factors including age and interval between MIMT surgeries may affect IM biological potency and graft to bone consolidation.