Biomaterial use is a promising approach to facilitate wound healing of the bone tissue. Biomaterials induce the formation of membrane capsules and the recruitment of different types of macrophages. Macrophages are immune cells that produce diverse combinations of cytokines playing an important role in bone healing and regeneration, but the exact mechanism remains to be studied. Our work aimed to identify in vivo macrophages in the Masquelet induced membrane in a rat model. Most of the macrophages in the damaged area were M2-like, with smaller numbers of M1-like macrophages. In addition, high expression of IL-1β and IL-6 cytokines were detected in the membrane region by RT-qPCR. Using an innovative combination of two hybridization techniques (in situ hybridization and in situ hybridization chain reaction (in situ HCR)), M2b-like macrophages were identified for the first time in cryosections of non-decalcified bone. Our work has also demonstrated that microspectroscopical analysis is essential for macrophage characterization, as it allows the discrimination of fluorescence and autofluorescence. Finally, this work has revealed the limitations of immunolabelling and the potential of in situ HCR to provide valuable information for in vivo characterization of macrophages.
Flower morphologies shape the accessibility to nectar and pollen, two major traits that determine plant–pollinator interactions and reproductive success. Melon is an economically important crop whose reproduction is completely pollinator-dependent and, as such, is a valuable model for studying crop-ecological functions. High-resolution imaging techniques, such as micro-computed tomography (micro-CT), have recently become popular for phenotyping in plant science. Here, we implemented micro-CT to study floral morphology and honey bees in the context of nectar-related traits without a sample preparation to improve the phenotyping precision and quality. We generated high-quality 3D models of melon male and female flowers and compared the geometric measures. Micro-CT allowed for a relatively easy and rapid generation of 3D volumetric data on nectar, nectary, flower, and honey bee body sizes. A comparative analysis of male and female flowers showed a strong positive correlation between the nectar gland volume and the volume of the secreted nectar. We modeled the nectar level inside the flower and reconstructed a 3D model of the accessibility by honey bees. By combining data on flower morphology, the honey bee size and nectar volume, this protocol can be used to assess the flower accessibility to pollinators in a high resolution, and can readily carry out genotypes comparative analysis to identify nectar-pollination-related traits.
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.
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