In-vivo imaging of the fracture healing in medaka revealed two types of osteoclasts before and after the callus formation by osteoblasts

Abstract

The fracture healing research, which has been performed in mammalian models not only for clinical application but also for bone metabolism, revealed that generally osteoblasts are induced to enter the fracture site before the induction of osteoclasts for bone remodeling. However, it remains unknown how and where osteoclasts and osteoblasts are induced, because it is difficult to observe osteoclasts and osteoblasts in a living animal. To answer these questions, we developed a new fracture healing model by using medaka. We fractured one side of lepidotrichia in a caudal fin ray without injuring the other soft tissues including blood vessels. Using the transgenic medaka in which osteoclasts and osteoblasts were visualized by GFP and DsRed, respectively, we found that two different types of functional osteoclasts were induced before and after osteoblast callus formation. The early-induced osteoclasts resorbed the bone fragments and the late-induced osteoclasts remodeled the callus. Both types of osteoclasts were induced near the surface on the blood vessels, while osteoblasts migrated from adjacent fin ray. Transmission electron microscopy revealed that no significant ruffled border and clear zone were observed in early-induced osteoclasts, whereas the late-induced osteoclasts had clear zones but did not have the typical ruffled border. In the remodeling of the callus, the expression of cox2 mRNA was up-regulated at the fracture site around vessels, and the inhibition of Cox2 impaired the induction of the late-induced osteoclasts, resulting in abnormal fracture healing. Finally, our developed medaka fracture healing model brings a new insight into the molecular mechanism for controlling cellular behaviors during the fracture healing.

Acute transcriptional up-regulation specific to osteoblasts/osteoclasts in medaka fish immediately after exposure to microgravity

“…Thus the fluorescent signals driven by osteoblast- and osteoclast- specific promoters were enhanced in the flight group. Both RANKL -GFP, which replicates the expression pattern of endogenous RANKL (data not shown), which is the key factor for osteoclast differentiation23, and cox2 -GFP24 (data not shown) lines showed no significant difference in the fluorescence intensity between the ground and flight groups (Supplementary Fig. S4).…”

“…We established 4 medaka double-transgenic lines: osterix -DsRed20/ TRAP -GFP1421, osteocalcin -DsRed12/ TRAP -GFP1421, RANKL -GFP/ MMP9 -DsRed, and cox2 -GFP24/ TRAP -DsRed24.…”

NPY and CGRP Inhibitor Influence on ERK Pathway and Macrophage Aggregation during Fracture Healing

“…Fracture healing is a complex process and generally divided into four stages: inflammation, soft callus (fibrocartilage) formation, hard callus formation and bone remodeling [21]. The inflammation process accounts for the broken tissue initially repairing while the fracture is bridged by both soft and hard calluses.…”

Osteoblast de- and redifferentiation is controlled by a dynamic response to retinoic acid during zebrafish fin regeneration

“…Resorption of bone matrix by osteoclasts is an obligatory process during bone growth, remodeling and fracture healing in mammals (Väänänen et al, 2000). Moreover, osteoclasts have been shown to be involved in healing of fin ray fractures in Medaka (Takeyama et al, 2014). Thus far, osteoclasts have not been implicated in fin regeneration.…”