Histology Staining Protocol Adaptation and Blood Smear Analysis

Once a bone substitute, previously seeded with human bone progenitor cells, is implanted in a femoral condyle of a rat, it’s of importance to determine the intensity of a cell-triggered immune response of the host. Different techniques can be used, such as the analysis of blood, the analysis of RNA expression of immune/inflammation-related genes or immunohistology stainings between groups of rats implanted either with cell-seeded or cell-free scaffolds. In this study, we were first interested in developing an accurate histology staining protocol for bone embedded samples in MMA resin. The three-targeted stainings are the Berliner blue staining, which targets the iron phagocyted by macrophages, the Trichrom of Goldner, which stains osteoblasts and osteoids, and finally the Tratrate resistant acid phosphatase (TRAP) stainings, which is highly present in osteoclasts and active macrophages. The procedures were adapted and improved accordingly. In a second step of this study, we analyzed blood smears taken from different experimental groups of rat implanted with three types of scaffolds. The blood was taken before implantation, 3, 7 and 14 days after the implantation. The implanted scaffolds were either seeded with human bone progenitor cells, osteogenic induced human bone progenitor cells or without any cells. We observed significant differences between the different scaffold conditions after 7 and 14 days of implantation for lymphocytes, monocytes and, non-segmented and juvenile neutrophils. Based on other publications, we found that they were within the normal range for rats.