A Study of Bone Formation around Titanium Implants Using Frozen Sections

Titanium is most widely used for implants. Almost all the histological studies of the implants have been carried out with polished specimens and the use of frozen sections are challenging. This study focused to define the formation process of bone around titanium with frozen sections made from tissue implanted with a commercially pure titanium foil (Tifoil). We inserted the Ti-foil with a 30-μm thickness into rat femurs and extracted it after 1, 3, 5, 7, 10, 15, and 30 days. After freezing, they were sliced into 3-μm thick serial frozen sections and the sections were used for hematoxylin and eosin (H&E) staining, Masson trichrome staining, Alizarin Red S staining, enzymatic staining (ALPase and TRAPase), immunostaining (osteopontin, osteocalcin, and collagen type I), and elemental analysis. At 1 day after the implantation, the area around the Ti-foil was filled with blood and there are no collagen fibers and no proliferated cells in the area. At 3 days, the new collagen fibers were observed on the marrow side of the blood clot. The proliferated cells were observed around the fibers and they were positive for immunostaining to osteopontin, osteocalcin, and collagen type I. After 5 days, calcium precipitation was observed on the newly formed collagen fibers. After 7 days, the calcification progressed and started to reach the Ti-foil surface. After 10 days, calcification progressed to the area around the Ti-foil. At 15 days, a remarkable bone resorption appears on the marrow side. At 30 days, further resorption was observed and bony tissue was seen on the Ti-foil surface only. These results clearly showed that the bone around the Ti-foil is formed after undergoing the blood clotting process around the Ti-foil, osteoblast and fibroblast proliferation, calcium precipitation on the collagen fibers, bone formation around the Ti-foil, and bone resorption by osteoclasts.