Expression of Osteocalcin and Transglutaminase and Labelling of Bromodeoxyuridine during Fracture Healing in the Rat Tibia

Abstract

The expression of osteocalcin and transglutaminase C(TGase C) during fracture healing was inwstigated with immunohistochemical studies. A transverse osteotomy was made at the proximal tibia in Sprague-Dawley male rats and immobilized with a small external skeletal fixator. The animals lU!l'e sacrificed serially I, 3, 5, 7, 14, 42 days respectively after fracture. Longitudinal sections of the healing bone were stained with pohclonal antibody against osteocalcin and TGase C, and monoclonal antibody against bromodeoxyuridine. During the intramembranous bone formation at the periosteum around the fracture site, osteocalcin was strongly expressed in the proliferating osteoprogenitor cells from the 1st day of fracture, and then, in osteoblasts, osteoid matrix and osteocytes. The expression of TGase C was weakly positive in both osteoprogenitor cells and osteoblasts. Ai the site of endochondral bone formation, which was first reoealed 5 days after fracture, cell proliferation occurred at the periphery of cartilaginous callus where the number of cells stained with BrdU was highest During the maturation of callus, those cells uere entrapped in the chondroid matrix and became larger and larger. Osteocalcin was demonstrated in the cytoplasm of chondrocytes, while chondroid matrix was negatiwly stained. TGase C was found in the cytoplasm of more centrally located and matured chondrocytes as compared with osteocalcin. Osteoid matrix was stained with osteocalcin but not with TGase C. These finding may suggest that osteocalcin participates in the early phase of endochondral bone formation, while TGase C participates in the late phase, suggesting the role of TGase C in matrix stabilization. But the reason for the difference in the expression of TGase C between the endochondral bone formation and intramembranous bone formation should be further inwstigated. Healing of IAA2Il immobilized fracture in this study was predominantly induced by intramembranous ossification rather than endochondral ossification. Periosteal osteoprogenitor cells appeared to initiate and to lead bone formation after osteotomy. These findings indicate that preservation of the periosteum is essential to achieve successful fracture healing.