Hindlimb unloading reversibly attenuates osteogenic potential of rat skeletal stem and progenitor cells ex vivo.

Abstract

Introduction: Prolonged space flights negatively affect skeleton. Stromal cells of mesenchymal origin play a crucial role in maintaining homeostasis and in regulating the physiological remodeling of various tissues, and this has particular significance for bone.

Methods: Hindlimb unloading (HU) of rats as a ground-based model for simulation of microgravity was implemented. The functional activity of skeletal stem and progenitor cells (SSPCs) from rat femoral bones was assessed in vitro after 2 weeks of HU and after 2 weeks of subsequent recovery of load support (HU+R). To characterize the growth of the SSPCs, the number of population doublings (PD) was calculated. Histochemical detection of the activity of alkaline phosphatase (AP) - an early marker of osteo-differentiation - on day 7, and of extracellular matrix (ECM) mineralization - as a sign of late osteo-differentiation - on day 21, were carried out. Quantitative real-time PCR was performed to detect the expression of the genes encoding proteins associated with the functional activity of osteoprogenitor cells (Pparg, Runx2, Alpl, Cxcl12) and bone tissue homeostasis (Mmp9, Spp1, RANKL, OPG, Ibsp, BMP10, Sost).

Results: After HU, a decrease in AP activity and a significant attenuation of extracellular matrix mineralization were detected. There was also significant downregulation of the genes those for bone matrix proteins (RANKL, OPG, Ibsp), and of the master-genes controlling osteo- and adipo-differentiation (Runx2, Alpl), as well as of Mmp9, encoding a regulatory molecule of bone matrix remodeling. By contrast, sclerostin (Sost) was upregulated. After HU+R, the PD, an AP activity and the level of extracellular matrix mineralization were restored.

Conclusions: HU leads to inhibition of the osteoplastic function of SSPCs. The presented data are significant for the elucidation of microgravity-induced mechanisms of bone impairment and for the development of countermeasures for astronauts as well as for osteo-deficient patients after prolonged immobilization.