Figure 4

Illustration of the method used for the analysis of nerve profiles association to bone surfaces in human bone. (a–b) Adjacent sections were double-immunostained with either TH or PGP9.5 in combination with CD34, a marker of endothelial cells (a) or Masson’s trichrome stained (b). The images illustrate two adjacent sections. (b) In the Masson’s trichrome stained section, eroded and quiescent surfaces were collectively marked as remodeling surfaces (Rm.S), and these markings were transferred to the adjacent immunostained section showing CD34+ vascular structures (v, black) above the remodeling surface, which is associated with multiple PGP9.5+ (red) nerve profiles. (a) For the analysis of nerve density, the bone marrow was separated into different zones: the bone marrow below or above 100 µm (hatched line) of the bone surface, as well as the bone marrow above quiescent surfaces (QS) or remodeling surfaces (Rm.S) below 100 µm. A point-grid (yellow crosses) was used to estimate the area of the zones, in which the nerve density was estimated. Scale bars 50 µm.