Calcific aortic stenosis (CAS) is more prevalent, occurs earlier, progresses faster and has worse outcomes in patients with chronic kidney disease (CKD). The uremic toxin indoxyl sulfate (IS) is powerful predictor of cardiovascular mortality in these patients and a strong promoter of ectopic calcification whose role in CAS remains poorly studied. The objective of this study was to evaluate whether IS influences the mineralization of primary human valvular interstitial cells (hVICs) from the aortic valve. Primary hVICs were exposed to increasing concentrations of IS in osteogenic medium (OM). The hVICs' osteogenic transition was monitored by qRT-PCRs for BMP2 and RUNX2 mRNA. Cell mineralization was assayed using the o-cresolphthalein complexone method. Inflammation was assessed by monitoring NF-κB activation using Western blots as well as IL-1β, IL-6 and TNF-α secretion by ELISAs. Small interfering RNA (siRNA) approaches enabled us to determine which signaling pathways were involved. Indoxyl-sulfate increased OM-induced hVICs osteogenic transition and calcification in a concentration-dependent manner. This effect was blocked by silencing the receptor for IS (the aryl hydrocarbon receptor, AhR). Exposure to IS promoted p65 phosphorylation, the blockade of which inhibited IS-induced mineralization. Exposure to IS promoted IL-6 secretion by hVICs, a phenomenon blocked by silencing AhR or p65. Incubation with an anti-IL-6 antibody neutralized IS's pro-calcific effects. IS promotes hVIC mineralization through AhR-dependent activation of the NF-κB pathway and the subsequent release of IL-6. Further research should seek to determine whether targeting inflammatory pathways can reduce the onset and progression of CKD-related CAS.

Bone fracture healing is a complex biological process involving four phases coordinated over time: hematoma formation, granulation tissue formation, bony callus formation, and bone remodelling. Bone fractures represent a significant health problem, particularly among the elderly population and patients with comorbidities. Therapeutic strategies proposed to treat such fractures include the use of autografts, allografts, and tissue engineering strategies. It has been shown that bone morphogenetic protein 2 (BMP-2) has a therapeutic potential to enhance fracture healing. Despite the clinical efficacy of BMP-2 in osteoinduction and bone repair, adverse side effects and complications have been reported. Therefore, in this in vitro study, we propose the use of a disaccharide compound (DP2) to improve the mineralisation process. We first evaluated the effect of DP2 on primary human osteoblasts (HOb), and then investigated the mechanisms involved. Our findings showed that (i) DP2 improved osteoblast differentiation by inducing alkaline phosphatase activity, osteopontin, and osteocalcin expression; (ii) DP2 induced earlier in vitro mineralisation in HOb cells compared to BMP-2 mainly by earlier activation of Runx2; and (iii) DP2 is internalized in HOb cells and activates the protein kinase C signalling pathway. Consequently, DP2 is a potential therapeutical candidate molecule for bone fracture repair.

Background/Objectives: Delays in bone healing and complications of remodeling constitute a major medical problem—particularly in older adults and patients with comorbidities. Current therapeutic approaches are based on strategies that promote bone regeneration. We recently identified a disaccharide compound (DP2) that enhances in vitro mineralization in human osteoblast cells via the early activation of Runx2 and the induction of osteoblast differentiation. Methods: First, a calcium quantification assay was performed to assess mineralization in MC3T3-E1 cells. Next, microcomputed tomography and histological analyses were used to examine in vivo bone repair in a rat 5 mm cranial defect model following the implantation of DP2 coupled to a micro/macroporous biphasic CaP ceramic (MBCP⁺) or collagen scaffold. Results: Here, we demonstrated that DP2 induced osteogenic differentiation and significantly elevated calcium matrix deposition in the murine preosteoblast cell line MC3T3-E1. We found that treatment with DP2 coupled to MBCP⁺ repaired the calvarial defect on post-implantation day 91. It significantly increased bone mineral density starting on day 29 post-treatment. In addition, DP2 did not induce ectopic bone formation. Conclusions: Taken as a whole, these results show that DP2 is a promising candidate treatment for delayed bone healing.