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

Abstract

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.

Keywords: MSC; Masquelet's induced membrane technique; bone graft; coculture; macrophage.

Figure 1

In vivo microCT images obtained the day before sacrifice and dissection. Induced membrane development was allowed to proceed for 4 weeks (left; N = 7) or 8 weeks (right; N = 7) after MIMT surgery.

Figure 2

Masson's trichrome histology of IM at the anterior‐lateral facing side of the spacer. After removing IM tissue from the medial face of the spacer, bones were processed for histology (N = 4 per IM development interval). At both 4 (A) and 8 (B) weeks, the IM was composed of a variably thick fibrous layer (denoted with with bars and quantified in [C]) adjacent to the spacer plus a second layer containing immune cell infiltrate and blood vessels. *PMMA spacer.

Figure 3

IM attributes after clodronate pretreatment and coculture with MSC. (A–D): Wet weight and DNA content of 4‐week and 8‐week IM were determined after pretreatment with PBS (A, C) or clodronate (B, D), followed by 72 h coculture with MSC. (E and F) Expression of the macrophage surface marker CD11b (integrin alpha M) was reduced in 4‐week (Ei) and 8‐week (Fi) IM treated with clodronate. Raw Ct values (Eii, Eiii, Fii, Fiii) verify that the housekeeping gene was minimally affected by clodronate treatment. Each data point represents IM tissue harvested from a single animal. Data are from 5 to 7 animals per outcome measure.

Figure 4

Paracrine effects of IM on primary MSC. After 72 h of indirect coculture with 4‐ or 8‐week IM pretreated with PBS or clodronate, the MSC were photographed (A) and expression of osteoblastic genes were determined in MSC. Gene expression levels were normalized to MSC cultured alone without IM (B–G). Open circles: MSC only. Closed circles MSC cocultured with IM pretreated with PBS. Closed squares: MSC cocultured with IM pretreated with clodronate. *p < 0.05, **p < 0.01 versus MSC or between IM ages as indicated by lines between groups. IM tissue from N = 7 rats per IM development interval was subject to coculture. Each data point represents a single culture well.

Figure 5

Secretomes of 4‐ and 8‐week IM have similar composition. (A) Volcano plot demonstrating time associated increases in 7 of 150 proteins secreted by IM (red dots). (B) GO Biological Process Terms enrichment analysis of the whole IM secretome. (C) Cell type enrichment analysis of the whole IM secretome (https://maayanlab.cloud/Enrichr/) IM conditioned medium from 2 to 3 different animals was combined to achieve 3 biological replicates per IM development interval for proteomics.