Goldner s trichrome

After euthanasia, the subcutaneous implants were fixed with 10% formalin. Following fixation, the implants were washed and placed into a processor that dehydrated the samples in 70% alcohol, followed by 95%, 100%, and xylene. The samples were then embedded in paraffin and cut into slices of 5 microns using a microtome (Accu-Cut SRM 200 Rotary Microtomoe, Sakura Finetek USA, CA). Slides were stained with hematoxylin and eosin (H&E) and Goldner’s trichrome (Sigma-Aldrich). Images were obtained with Lionheart LX (Biotek Instruments Inc, Winooski, VT) at 4× and captured using Gen 5 software.

After euthanasia, the femora were collected and fixed in 4% paraformaldehyde solution. For non-decalcified sections, the samples were dehydrated with gradient ethanol and embedded with methyl-methacrylate (MMA, Technovit 9100 New, Heraeus Kulzer, Hanau, Germany) after using xylene as a transition. Then, the embedded samples were cut into slices with a thickness of 200 µm and micro-ground to a thickness of 50–70 µm. The selected sections were stained with Goldner’s trichrome (Sigma-Aldrich) staining. For decalcified sections, the samples were decalcified with 12.5% ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich) for 6 weeks. The specimens were then dehydrated in ethanol, embedded in paraffin, and cut into 5 µm-thick sections using a rotary microtome (RM215, Leica Microsystems, Germany). Haematoxylin and eosin (H&E) staining and TRAP staining (Sigma-Aldrich) was performed on selected sections from each sample following the manufacturer’s instructions. Images were captured using polarizing microscopy (Nikon Eclipse VL100POL, Nikon, Tokyo, Japan). The quantification of osteoblasts and osteoclasts in the grafted defect area was done on ×10 histological images using ImageJ software (NIH, USA).

Histomorphometric parameters were measured on femurs from mice in the 5TGM.1 cohort. Isolated femurs were dehydrated in ascending alcohol concentrations, defatted in xylene and embedded in methylmethacrylate. All histomorphometric parameters were recorded as recommended by the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee [76 (link)] and measured using the Bonolab software package designed for bone histomorphometry (Microvision, Evry, France). Osteoid surface and osteoblast surface were measured on sections stained with toluidine blue, aniline blue and Goldner's trichrome (Sigma-Aldrich). Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining (Sigma-Aldrich). Briefly, sections were stained for acid phosphatase using naphthol ASTR phosphate as substrate in the presence of 50 mM tartrate with hexazotised pararosaline, and counterstained with methyl green. TRAP-positive cells were counted in the whole epiphysis (×25), and expressed as the number of osteoclasts per bone volume. In addition, osteoclast surface was determined.