Although IL-6 mRNA expression in rat is restricted to the first day post-fracture, the inflammatory phase, the protein has been observed later in the healing process, indicating additional roles. The importance of IL-6 was demonstrated by delayed healing in knockout mice through diminished osteoclast numbers, formation thereof being stimulated by IL-6. The aim of our study was to investigate with which cells this cytokine is associated and when during fracture healing.

A closed fracture of the lower right limb was created in rats. The tibia was obtained from six animals at each of 1, 3, 7, 14 and 28 days post-fracture, decalcified and prepared for standard immunohistochemistry with an IL-6-specific polyclonal antibody. The number and types of cells positively stained for IL-6 along the whole length of the periosteal callus on one surface and in the fracture was evaluated.

Mostly inflammatory cells were initially stained, becoming virtually absent by day 7 when this phase has normally ended. Within the immediate vicinity of the fracture where endochondrial ossification occurred, staining of chondrocytes was significant (69%) by day 7 when this cell was laying down cartilaginous tissue that was also calcified. Distally to the fracture where direct bone formation occurred through intra-membranous ossification by osteoblasts, staining of these cells was observed, peaking at day 14 (56%). As this bone started to take on the appearance of cortex and surviving embedded osteoblasts differentiated to osteocytes, the latter cells were stained, suggesting a role in remodelling. At the fracture as bone replaced the cartilaginous tissue and union occurred, staining of chondrocytes decreased, whereas local osteoblasts were positive.

IL-6 appears to play a role throughout fracture healing, in endochondrial and intra-membranous ossification. The level of staining of each cell type reflected the degree of their activity with respect to production of related tissue.