Expression of Osteocalcin and Transglutaminase C during Fracture Healing and Distraction Osteogenesis in Rat's Tibia

Choi, In Ho; Seong, Sang Cheol; Lee, Myung Chul; Song, Gye Yong; Park, Sang Chul

Abstract

Incorporation of bromodeoxyuridine(BrdU) and expression of osteocalcin and transglutaminase C(TGase C) during fracture healing and distraction osteogenesis were investigated in the rat with immunohistochemical studies. Transverse osteotomy was made at the proximal tibia. Bilateral dynamic mini-fixator was applied to immobilize the fracture and also to lengthen the leg. Distraction was started, at the rate of 0.25 mm twice daily, from the 4th operative day and continued for 7 days. Animals were killed for immunohistochemical studies on the 1st, 3rd, 5th, 7th, 14th, 28th, 42nd, 56th, and 84th day after osteotomy or distraction. Longitudinal histologic sections of the healing bone were stained with monoclonal antibodies against BrdU, osteocalcin, and TGase C. Radiologically, complete fracture healing was achieved in 6 weeks after osteotomy, while neo-osteogenesis was successfully achieved in the distracted gap in 7 weeks after the completion of distraction, During active healing stage of the fracture and distraction osteogenesis, BrdU was mainly expressed in the perisoteal and endosteal osteoprogenitor cells while osteocalcin was expressed in the proliferating osteoprogenitor cells, osteoblast, osteocyte, osteoid matrix, and chondrocyte. The expression of BrdU and osteocalcin in the mesenchymal cells from the surrounding soft tissues around the osteotomy site was negligible. At the site of enchondral bone formation, TGase C was expressed in the cytomplasm of more centrally located and matured chondrocytes, while oseocalcin was mainly expressed in the cytoplasm of peripherally located chondrocyte. These findings may suggest that osteocalcin participates in early phase of enchondral bone formation, while TGase C in the late phase, suggesting the role of TGase C in matrix stabilization. At the site of intramern-branous bone formation, the expression of TGase C was weakly positive in both osteoprogenitor cell and osteoblast. The reason of the difference in the expression of TGase C between the enchondral bone formation and intrarnembranous bone formation should be further investigated. Fracture healing and distraction osteogenesis was predominantly induced by intramembranous ossification rather than enchondral ossification. Periosteal osteoprogenitor cells appeared to initiate and to lead bone formation after osteotomy and distraction. Active proliferation and differentiation of osteoprogenitor cell ocurred during entire periods of distraction. Also, active osteoid matrix formation and mineralization was started from the 5th day of distraction and continued thereafter for further 4 weeks after completion of the lengthening. These findings indicate that preservation of the periosteum is essential to achieve successful fracture healing and distraction osteogenesis.