**1. Introduction**

In the face of large bone defects, surgery is required to restore the shape and function of the bone. The induced membrane technique (IMT), also known as the Masquelet technique, is a widely used two-stage surgical procedure. This technique is unique in preparing the bed graft by molding a polymethylmethacrylate (PMMA) spacer to fill the bone defect [1]. The implantation of this spacer leads to the formation of an induced membrane (IM)—granulation tissue surrounding the spacer. In the second step, the spacer

**Citation:** Durand, M.; Oger, M.; Nikovics, K.; Venant, J.; Guillope, A.-C.; Jouve, E.; Barbier, L.; Bégot, L.; Poirier, F.; Rousseau, C.; et al. Influence of the Immune Microenvironment Provided by Implanted Biomaterials on the Biological Properties of Masquelet-Induced Membranes in Rats: Metakaolin as an Alternative Spacer. *Biomedicines* **2022**, *10*, 3017. https://doi.org/10.3390/ biomedicines10123017

Academic Editor: Matthias Wiens

Received: 29 September 2022 Accepted: 17 November 2022 Published: 23 November 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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 France is removed while preserving the integrity of the IM. A standard autologous bone graft is then implanted into the IM cavity to repair the bone.

The IM, which acts as a biologically privileged membrane at the site of the defect, is the key element in this procedure. Preclinical and clinical studies have highlighted the various roles of the IM. It prevents graft resorption and muscle invasion of the defective bone by acting as a barrier membrane. It also creates an osteogenic and osteoinductive environment by secreting many growth factors and cytokines. These factors include bone morphogenic protein-2 (BMP-2), interleukin-6 (IL-6), transforming growth factor-β (TGF-β), vascular endothelial growth factor A (VEGF-A), von Willebrand factor (vWF) and metalloproteinase-9 (MMP-9) [2–6]. The IM has also been shown to be highly vascularized and to serve as a source of bone progenitor cells.

Biologically, the IM results from a foreign-body reaction (FBR). Upon implantation, all biomaterials elicit a FBR, a natural immunoinflammatory process that isolates the implant from the rest of the body in a collagenous capsule [7]. Macrophages are plastic cells that play a key role in the FBR by orchestrating the inflammatory environment around the implanted biomaterial. Indeed, macrophages can adopt diverse functional phenotypes upon activation, ranging from M1 (pro-inflammatory) to the M2 (pro-healing) profiles. Interestingly, macrophage activation depends on the shape and surface properties of the biomaterial (chemistry, porosity, wettability, roughness and stiffness) [8].

We hypothesized that changing the chemical composition of the spacer generating the IM in the Masquelet technique would modify the immune microenvironment in which the FBR occurred, thereby altering the osteogenic properties of the IM and potentially enhancing bone regeneration. We tested this hypothesis by replacing the PMMA of standard spacers with an alternative biomaterial, a metakaolin-based geopolymer (Davidovits) [9]. This polymer is synthesized by an alkaline activator solution's reaction (geopolymerization) with metakaolin particles. Chemically, metakaolin is a dehydroxylated form of the clay mineral kaolinite, an aluminosilicate material. Aluminosilicates and their derivatives are known to have immunostimulatory effects due to induction of macrophage activation [10]. Metakaolin was, therefore, chosen for this study based on its immunogenicity, and its innocuity relative to other clay minerals, such as bentonite [11]. Wiemann et al. [12] recently showed that the intratracheal instillation of kaolin in rats induced transient macrophagebased hypercellularity in rat lungs, with no signs of inflammation or structural change in the lung parenchyma, whereas bentonite instillation leads to a very intense lung inflammation with changes to the structure of the lung epithelium. Metakaolin is listed in the US Pharmacopeia, suggesting that its transfer into clinical practice might be facilitated in terms of the requirements for medical device regulation. Kaolinite and its chemical derivatives have been widely used in the pharmaceutical domain for decades as wellcharacterized pharmaceutical excipients: diluents, binders, disintegrants, pelleting agents, granulating agents, amorphizing agents, film-coating additives or even drug carriers [13,14]. They are also used as active pharmaceutical ingredients in hemostatic wound dressings, dermatological protectors, gastrointestinal protectors and antidiarrheal agents.

We used a validated Masquelet model in rats to assess the osteogenic properties of metakaolin-generated IM with histological and immunohistochemical methods. We first analyzed the distribution of M1-like and M2-like macrophage populations within the IM. Finally, we determined the impact of metakaolin spacers as an alternative to PMMA on bone-healing outcomes.

### **2. Materials and Methods**
