Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure

Abstract

Low-dose radiotherapy (LD-RT) is a local treatment option for patients with chronic degenerative and inflammatory diseases, in particular musculoskeletal diseases. Despite reported analgesic and anti-inflammatory effects, cellular and molecular mechanisms related to osteoimmunological effects are still elusive. Here we test the hypothesis that X-irradiation inhibits the differentiation of precursor osteoclasts into mature osteoclasts (mOC) and their bone resorbing activity. Circulating monocytes from healthy donors were isolated and irradiated after attachment with single or fractionated X-ray doses, comparable to an LD-RT treatment scheme. Then monocytes underwent ex vivo differentiation into OC during cultivation up to 21 days, under conditions mimicking the physiological microenvironment of OC on bone. After irradiation, apoptotic frequencies were low, but the total number of OC precursors and mOC decreased up to the end of the cultivation period. On top, we observed an impairment of terminal differentiation, i.e. a smaller fraction of mOC, reduced resorbing activity on bone, and release of collagen fragments. We further analyzed the effect of X-irradiation on multinucleation, resulting from the fusion of precursor OC, which occurs late during OC differentiation. At 21 days after exposure, the observation of smaller cellular areas and a reduced number of nuclei per mOC suggest an impaired fusion of OC precursors to form mOC. Before, at 14 days, the nuclear translocation of Nuclear Factor Of Activated T Cells 1 (NFATc1), a master regulator of osteoclast differentiation and fusion, was decreased. In first results, obtained in the frame of a longitudinal LD-RT study, we previously reported a pain-relieving effect in patients. However, in a subgroup of patients suffering from Calcaneodynia or Achillodynia, we did not observe a consistent decrease of established blood markers for resorption and formation of bone, or modified T cell subtypes involved in regulating these processes. To assess the relevance of changes in bone metabolism for other diseases treated with LD-RT will be subject of further studies. Taken together, we observed that in vitro X-irradiation of monocytes results in an inhibition of the differentiation into bone-resorbing OC and a concomitant reduction of resorbing activity. The detected reduced NFATc1 signaling could be one underlying mechanism.

Osteo-immunological impact of radon spa treatment: due to radon or spa alone? Results from the prospective, thermal bath placebo-controlled RAD-ON02 trial

“…In addition, the fraction of Treg and Th17 cells were determined in the PBMCs and related to lymphocytes, according to Eckert et al. (see 2.5) ( 38 ). Furthermore, the concentration of markers related to bone metabolism and inflammatory key players, such as adipokines, were investigated in serum or plasma of the patients before and at different time points after treatment.…”

“…For the isolation of human PBMCs, blood from patients was collected in vacutainer tubes (BD vacutainer ® blood collection tubes, BD Biosciences, Heidelberg, Germany at the state-certified health resort in Bad Steben, and was shipped to GSI Helmholtzzentrum für Schwerionenforschung (Darmstadt, Germany) as described by Eckert et al. ( 38 ). Briefly, the next day, vacutainer tubes were centrifuged at 1500 xg for 20 min at room temperature (RT).…”

“…Fluorescent triple staining of OC precursors and mOCs was performed as previously described ( 38 ). Briefly, cells were fixed with 3.7% PFA (Merck KGaA, Darmstadt, Germany) (15 min at RT).…”

“…The bone resorbing activity was visualized by staining with toluidine blue as described elsewhere ( 38 ). For this, cells were removed from the bone slices by incubation with ammonium hydroxide solution (0.25 M ammonium hydroxide solution, Sigma Aldrich Chemie GmbH, diluted with dist.…”

Radiation induced therapeutic effects in cancerous and tumor cells: A review

“…IR is used at high doses and has detrimental effects on bone. 105 Bone marrow dysfunction has historically been one of the main causes of morbidity and mortality after ionizing radiation (Table 3). 106 Effects of radiation on the eye: High doses of ultraviolet (UV) radiation can be harmful to your eyes.…”