Radiation induces primary osteocyte senescence phenotype and affects osteoclastogenesis in vitro

Abstract

Irradiation-induced bone remodeling imbalances arise as a consequence of the dysregulation of bone formation and resorption. due to the abundance of osteocytes, their long life and their dual-regulatory effects on both osteoblast and osteoclast function, they serve as critical coordinators of bone remolding. In the present study, femur and tibia-derived primary osteocytes were cultured and irradiated to observe the functional changes and the cellular senescence phenotype in vitro. Irradiation directly reduced cell viability, affected the crucial dendritic morphology and altered the expression of functional proteins, including upregulation of receptor activator of nuclear factor-κB ligand and sclerostin, and downregulation of osteoprotegerin. Irradiated osteocytes were shown to exhibit notable dNA damage, which resulted in the initiation of a typical cellular senescence phenotype. Furthermore, it was found that irradiation-induced prematurely senescent osteocytes stimulate molecular secretion, referred to as senescence-associated secretory phenotype (SASP), which may be involved in modulation of the bone microenvironment, including the promotion of osteoclastogenesis. Taken together, the results showed that irradiation triggered osteocyte senescence and the acquisition of an associated secretory phenotype. This further resulted in an imbalance of bone remodeling through senescent influence on proliferation, morphology and marker protein production, but also indirectly via a paracrine pathway through SASP secretion. The results of the present study may highlight the potential of SASP-targeted interventions for the management of radiation-induced bone loss.

Total body proton and heavy-ion irradiation causes cellular senescence and promotes pro-osteoclastogenic activity in mouse bone marrow

“…Moreover, radiation quality-dependent accelerated-senescence in BM cells, likely involve the acquisition of the senescence associated secretory phenotype (SASP) and pro-osteoclastogenic secretion from these cells plausibly results in persistent osteoclastogenesis ( Wang et al., 2021 ; Gorissen et al., 2018 ), as observed in aged mice ( Cao et al., 2003 , 2005 ; Shahnazari et al., 2012 ). The role of SASP-associated overexpression of RANKL in osteoclastogenesis is well established and emerging reports on IR-induced premature aging have also demonstrated the role of RANKL in osteoclastogenesis ( Pignolo et al., 2021 ).…”

Free Transplantation of a Tissue Engineered Bone Graft into an Irradiated, Critical-Size Femoral Defect in Rats

“…However, regulation of the housekeeping genes cannot be fully ruled out. A recent study showed an increase in osteocyte senescence after irradiation [ 17 ]. Although PPIA has been identified as a very stable and GAPDH as fairly stable reference genes during in vitro senescence in mesenchymal stem cells [ 18 ], it is noteworthy that GAPDH expression decreases during in vivo aging in fibroblasts [ 19 ].…”

Radiation-Induced Osteocyte Senescence Alters Bone Marrow Mesenchymal Stem Cell Differentiation Potential via Paracrine Signaling

“…Immunofluorescence detection for γ-H2AX foci: We used an immunofluorescence assay to detect γ-H2AX foci, a marker of DNA damage. The γH2AX foci were tested according to the previous description [50], and then the γ-H2AX foci number was quantified with a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany).…”

“…Mixed supernatants were centrifuged and resuspended in α-MEM medium containing 5% fetal bovine serum, 5% calf serum, and 1% penicillin/streptomycin, which were inoculated together with the final bone fragments in dishes coated with rat tail collagen type I (Solarbio, Beijing, China) and incubated in a humidified incubator at 37 • C and 5% CO 2 . A more detailed protocol for primary osteoblast extraction can be found in our previous publication [50]. The production of dendritic osteocytes was microscopically visible after 3-7 days.…”