Gene Expression during Fracture Healing in Normal Mouse: In Situ Hybridization on Hard Tissues for Investigation of Regenerative Mechanisms.

Abstract

Fracture repair is a sequence of events involving formation of hematoma, recruitment of mesenchymal cells, induction of cartilaginous and bony callus, and remodeling of woven bone tissues. Decalcified tissues of mouse fracture model were subjected to in situ hybridization with PCR-derived single-stranded DNA probes. Bone morphogenetic protein (BMP)-2 transcripts were detected primarily in mesenchymal cells at the fracture site. Strong signals for BMP-2 and -3 and moderate signals for BMP-4 and -6 were detected in chondrocytes of the cartilaginous callus (days 7-10) and in osteoblasts of the bony callus (days 10-14), where transcripts of Runx2/Cbfa1 (an osteoblastic transcription factor downstream of BMP) were detected in chondrocytes and osteoblasts. BMP signals finally decreased 21 days after the fracture. Since immature cells expressing BMPs differentiated into chondrocytes or osteoblasts, BMPs might promote fracture healing through paracrine and autocrine mechanisms. During fracture healing, on the other hand, a number of osteoclasts were involved in the resorption of cartilaginous and bony callus; receptor activator of NF-kB ligand (RANKL), recently identified as a requisite to osteoclastogenesis, was expressed on mesenchymal cells (day 4) as well as on osteoblasts (days 10-21). Since Runx2/Cbfa1 binding sites are found on the mouse RANKL gene promoter, RANKL expression and osteoclastogenesis could also be promoted through Runx2/Cbfa1 at the phase of accelerated bone formation during fracture healing.