Patients with chronic kidney disease (CKD) experience multiple comorbidities, among which mineral/bone disorders (MBD) contribute to high mortality due to increased facture risk . CKD affects the quality of bones which become weaker and easily break. Recently, endogenous metabolites, such indoxyl sulfate (IS), were reported to have an active role in the development of uremic bone as they accumulate in blood due to poor kidney function . However, the effects of these metabolites on the bone matrix is not well understood, due to lack of appropriate models. For this, we established an in vitro 3D model of human bone to investigate the activity of osteocytes, master regulators of bone remodeling, under exposure to IS. We investigate the osteocyte phenotypic signature (gene and secretion of remodeling molecules) accompanied by analysis of the quality and quantity of the deposited bone matrix.
Materials and Methods
We encapsulated human adipose-derived stem cells (hASCs) into fibrin hydrogels and differentiated them into osteoblasts- and osteocytes-like cells. The osteogenic differentiation was performed either under standard conditions or under treatment with IS. To assess the effect of IS treatment on the newly formed bone-like matrix, we examined the differential expression of the major osteoblastic (Opg, Col1a1, Opn, Osteonectin, Tnfsf11) and osteocytic genes (Fgf23, Col1a1, Mepe, Sclerostin, Opg, Osteonectin), and evaluated the secretion of soluble key regulators of bone remodeling (OPG, FGF23, SOST). The composition and mineralization of the bone matrix was also analyzed by staining for calcium (Alizarin Red) and collagen (Sirius Red and immunοstainings), accompanied by mechanical testing of the bone-like construct.
HASCs successfully differentiated into osteoblasts and osteocytes-like cells in standard osteogenic differentiation conditions, as evidenced by a highly mineralized and collagen enriched matrix. IS treatment was shown to significantly downregulate the expression of bone remodeling genes. Osteocytes-secreted bone remodeling mediators were also significantly lower (p<0.05) when compared to healthy cultures. Even more, the quality and the quantity of the deposited bone were impaired. The amount of collagen, as well as the degree of mineralization were reduced by 43.9%, p<0.001 and by 29.7%, p<0.05, respectively. Via X-ray imaging, we could identify a poor calcium-containing matrix while under exposure to IS. These changes were reflected in the decrease (p<0.05) of the mechanical properties of the uremic bone-like construct.
Our results suggest that IS impairs the osteogenic differentiation of hASCs and induces alterations of the bone extracellular matrix, in terms of collagen deposition, amount of inorganic matter and mechanical properties. Furthermore, exposure to IS during the transition of osteoblasts to osteocytes affects the acquisition of key regulatory features of bone remodeling.
This project received funding from the 3Rs stimulus Funds of the Utrecht University.
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