<b>Immunolocalization of osteocyte-derived molecules during bone fracture healing of mouse </b><b>ribs </b>

Abstract

We employed a well-standardized murine rib fracture model to assess the distribution, in the cortical bone, of three important osteocyte-derived molecules-dentine matrix protein 1 (DMP1), sclerostin and fibroblast growth factor 23 (FGF 23). Two days after the fracture, the periosteum thickened, and up to the seventh day post-fracture, the cortical surfaces were promoting neoformation of two tissue types depending on the distance from the fracture site: chondrogenesis was taking place near the fracture, and osteogenesis distant from it. The cortical bones supporting chondrogenesis featured several empty lacunae, while in the ones underlying newly-formed woven bone, empty lacunae were hardly seen. DMP1-immunopositive osteocytic lacunae and canaliculi were seen both close and away from the fracture. In contrast, the region close to the fracture had only few sclerostin-and FGF23-immunoreactive osteocytes, whereas the distant region revealed many osteocytes immunopositive for these markers. Mature cortical bone encompassing the native cortical bone was observed at two-, three-and four-weeks post-fracture, and the distribution of DMP1, sclerostin and FGF23 appeared to have returned to normal. In summary, early stages of fracture healing seem to be important for triggering chondrogenesis and osteogenesis that may be regulated by osteocytes via their secretory molecules.Fracture healing is a sequence of biological processes that include new formation of cartilage and bone. The tissue composed of newly-formed cartilage and bone, referred to as "callus", is the product of a coordinated physiological cascade that involves proliferation and differentiation of various lineages of inflammatory cells, angioblasts, fibroblasts, chondroblasts, and osteoblasts (24, 32). The initial cellular event is supposed to be cell replication of periosteal mesenchymal cells in the tissues surrounding the fracture site. After that, chondroblastic and osteoblastic differentiation ensues to promote the growth of a scaffold of cartilaginous and bony tissue, which is needed for initial bridging of the fracture gap. Through endochondral ossification, bone tissue gradually replaces the cartilaginous callus that was essential for primary stabilization of the fractured bone. There are reports showing that periosteal mesenchymal cells respond to locally increased levels of growth factors and cytokines, and may differentiate into chondrocytes or osteoblasts (41), so that, the periosteal mesenchymal environment appears to influence such cell fate (4). In spite of the local ef-

Rib histology after unsuccessful cardiopulmonary resuscitation in infants suffering sudden death from natural causes; implications for injury mechanism and timing

“…Verborgt et al ., using DNA fragmentation as a marker of apoptosis in rat microfractures, showed a process progressing over many days, with empty lacunae not appearing above baseline incidence until 3 days and not peaking until 7 days 19 . Similar findings have been reported by others using different biomarkers of cell death 20–22 . These cell death processes, involving loss of nuclear and cell wall integrity, DNA fragmentation, and cytoplasmic vesiculation, correlate with the stages of osteocyte death shown by light microscopy, including pyknosis and cell pallor without a visible nucleus, before phagocytosis leads to an empty lacuna 16,17 .…”

“…19 Similar findings have been reported by others using different biomarkers of cell death. [20][21][22] These cell death processes, involving loss of nuclear and cell wall integrity, DNA fragmentation, and cytoplasmic vesiculation, correlate with the stages of osteocyte death shown by light microscopy, including pyknosis and cell pallor without a visible nucleus, before phagocytosis leads to an empty lacuna. 16,17 In conclusion, there is evidence from animal experiments using differing methodologies and from human observational studies that osteocyte cell death processes-whether necrotic or apoptotic-take time, and that empty lacunae become visible in days rather than hours, in keeping with the timescales given in standard texts.…”

Osteocytes

“…Zuletzt konnte eine schnellere knöcherne Konsolidierung in einem cre/loxP-Knockout-Mausmodell für IGF-1 in Osteozyten nachgewiesen werden [68]. Außerdem konnte zu Beginn der Frakturheilung bei frakturnah lokalisierten Osteozyten ein Ausbleiben der Sclerostin-und FGF23-Expression gezeigt werden, welche sich im Rahmen der Frakturheilung wieder normalisiert, was wiederum auf eine Steuerung der Frakturheilung durch Osteozyten schließen lässt [69].…”