Differences in macrophage expression in induced membranes by fixation method - Masquelet technique using a mouse's femur critical-sized bone defect model

Masquelet's induced membrane technique (MIMT) is an emerging method for reconstructing critical-sized bone defects. However, an incomplete understanding of the underlying biological and physical processes hinders further optimization. This study investigated the effect of different bone-defect fixation methods on macrophage expression in an induced membrane using a novel mouse plate-fixed Masquelet model.

Mice were divided into Plate-fixed Masquelet (P-M), Intramedullary-fixed Masquelet (IM-M), Plate-fixed Control (P-C), and Back subfascial (B) groups. In the P-M and IM-M groups, a polymethylmethacrylate (PMMA) spacer was implanted into a 3 mm bone defect, while the defect in the P-C group remained unfilled. In group B, a spacer was inserted under the back fascia to examine membrane formation caused by a simple foreign body reaction. Tissues were collected at 1, 2, and 4 weeks postoperatively. Hematoxylin and eosin (H&E) staining and immunohistochemistry (CD68 and CD163: macrophage markers) were performed to assess macrophage expression within the membrane. qPCR was performed to measure the expression of CD68, CD163, and fibroblast growth factor 2 (FGF2).

Four weeks post-operation, the P-M group presented with minimal callus growth, whereas the IM-M group exhibited vigorous growth. The P-M and IM-M groups displayed a tri-layered membrane structure, which is consistent with the results of previous studies. The IM-M group had significantly thicker membranes, whereas the P-M group exhibited higher expression levels of CD68, CD163, and FGF2. Group P-C showed no osteogenesis, whereas group B maintained a thin, cell-dense membrane structure. The P-M group consistently showed higher gene expression levels than the P-C and P-B groups.

This study introduced a mouse plate fixation model for MIMT. The induced membranes could be adequately evaluated in this model. Induced membranes are formed by foreign body reactions to PMMA spacers; however, their properties are clearly different from those of simple foreign body reaction capsules and granulation tissues that infiltrate bone defects, suggesting that they are more complex tissues. The characteristics and expression of macrophages within these induced membranes varied according to the bone defect fixation method.