After the μCT scan bone histology was performed to investigate the micro-architecture of the callus.
For calcified histology the bones were fixed in 10 % neutral buffered formalin for 14 days at room temperature, dehydrated with ascending concentrations of ethanol and embedded in methylmethacrylate for 5 days at 4 °C.
Afterwards the tissue was soaked in methyl methacrylat monomer, nonpylphenyl-polyethyleneglycol acetate and azoisobutyronitrile (all from Sigma-Aldrich, St. Louis, USA). The blocks were released from the glass vials and sections of 40 μm were sawed and grinded using an EXAKT diamond saw system (Exakt, Norderstedt, Germany). The samples were lubricated with 0.1 % formic acid for 2 minutes and washed with water afterwards. The sections were submerged in 20 % methanol for one hour and stained with toluidine blue or haematoxylin and eosin.
For calcified histology the bones were fixed in 10 % neutral buffered formalin for 14 days at room temperature, dehydrated with ascending concentrations of ethanol and embedded in methylmethacrylate for 5 days at 4 °C.
Afterwards the tissue was soaked in methyl methacrylat monomer, nonpylphenyl-polyethyleneglycol acetate and azoisobutyronitrile (all from Sigma-Aldrich, St. Louis, USA). The blocks were released from the glass vials and sections of 40 μm were sawed and grinded using an EXAKT diamond saw system (Exakt, Norderstedt, Germany). The samples were lubricated with 0.1 % formic acid for 2 minutes and washed with water afterwards. The sections were submerged in 20 % methanol for one hour and stained with toluidine blue or haematoxylin and eosin.