Histological and Immunohistochemical Analysis of Engineered Constructs

Following the in vitro and in vivo studies, the constructs were fixed in 4% paraformaldehyde for 24 h. The in vivo constructs were decalcified using EDTA for up to 3 weeks. The constructs were then embedded in paraffin and sectioned at 6 mm thickness. After rehydration, in vitro sections were stained with safranin O/Fast Green, Alizarin Red, and Von Kossa; the in vivo sections were stained with hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP). For immunohistochemical analysis, in vivo sections were treated with 3% hydrogen peroxide to quench endogenous peroxidase activity. For antigen retrieval, the sections were incubated with 5 mg/ml hyaluronidase for 30 min at 37°C, followed by digestion with 1 mg/mL pepsin in 0.5 M acetic acid for 30 min at 37°C, after which the sections were incubated in blocking solution (5% Bloc-Ace, Dainippon Sumitomo Pharma, Osaka, Japan) for 30 min at RT. The sections were then incubated overnight with the following primary antibodies: rabbit polyclonal type I (1:400, Proteintech), type II (1:100, Santa Cruz Biotechnology), type X (1:400, Cloud-Clone Corp.) collagens, osteocalcin (1:400, Proteintech), CD31 (1:100, Abcam), SOX-9 (1:100, Santa Cruz Biotechnology), mouse monoclonal type X collagen (1:100, eBioscience), MMP-13 (1:100, Santa Cruz Biotechnology) or normal rabbit IgG (1:100, Santa Cruz Biotechnology). Peroxidase-conjugated Histofine Simple Stain MAX PO (MULTI) or MAX-PO (R, Nichirei, Tokyo, Japan) was used for secondary antibodies. The signal was detected using the ImmPACT NovaRED Substrate kit (Vector). The circularity of cells was quantified by measuring the number of cells in five different areas (200 μm × 200 μm) each, for the periphery and center of constructs.