*Article*

### **Influence of the Immune Microenvironment Provided by Implanted Biomaterials on the Biological Properties of Masquelet-Induced Membranes in Rats: Metakaolin as an Alternative Spacer**

**Marjorie Durand 1,\*, Myriam Oger 2, Krisztina Nikovics 2, Julien Venant 1,3, Anne-Cecile Guillope 1, Eugénie Jouve 1, Laure Barbier 4, Laurent <sup>B</sup>égo<sup>t</sup> 2, Florence Poirier 3, Catherine Rousseau 4, Olivier Pitois 5, Laurent Mathieu 1,6, Anne-Laure Favier 2, Didier Lutomski 3 and Jean-Marc Collombet 1**

	- Laboratoire Navier, Gustave Eiffel University, Ecole des Ponts ParisTech, CNRS, 77447 Marne-la-Vallée,
	- Department of Surgery, Ecole du Val-de-Grace, French Military Health Service Academy, 1 Place Alphonse Laveran, 75005 Paris, France

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> **Abstract:** Macrophages play a key role in the inflammatory phase of wound repair and foreign body reactions—two important processes in the Masquelet-induced membrane technique for extremity reconstruction. The macrophage response depends largely on the nature of the biomaterials implanted. However, little is known about the influence of the macrophage microenvironment on the osteogenic properties of the induced membrane or subsequent bone regeneration. We used metakaolin, an immunogenic material, as an alternative spacer to standard polymethylmethacrylate (PMMA) in a Masquelet model in rats. Four weeks after implantation, the PMMA- and metakaolin-induced membranes were harvested, and their osteogenic properties and macrophage microenvironments were investigated by histology, immunohistochemistry, mass spectroscopy and gene expression analysis. The metakaolin spacer induced membranes with higher levels of two potent pro-osteogenic factors, transforming growth factor-β (TGF-β) and bone morphogenic protein-2 (BMP-2). These alternative membranes thus had greater osteogenic activity, which was accompanied by a significant expansion of the total macrophage population, including both the M1-like and M2-like subtypes. Microcomputed tomographic analysis showed that metakaolin-induced membranes supported bone regeneration more effectively than PMMA-induced membranes through better callus properties (+58%), although this difference was not significant. This study provides the first evidence of the influence of the immune microenvironment on the osteogenic properties of the induced membranes.

**Keywords:** Masquelet-induced membrane; macrophages; PMMA; metakaolin
