European Cells & Materials (Jun 2025)
Alendronate nanoparticles promote fracture healing by inhibiting LRP4 to activate the Wnt/β-catenin pathway
Abstract
Background: Fracture is a common skeletal disorder, and fracture healing is a complex biological process. The Wingless-Int-Integrated/beta-catenin (Wnt/β-catenin) signaling pathway plays an important role in bone formation and fracture healing. Low-density lipoprotein receptor-related protein 4 (LRP4) has been reported to negatively regulate the Wnt/β-catenin pathway. This study investigated whether alendronate nanoparticles (alendronate-NPs) can activate the Wnt/β-catenin pathway by inhibiting LRP4, thereby promoting fracture healing. Methods: We initially synthesized alendronate-NPs. Then, the impact of alendronate, alendronate-NPs, and LRP4 on the osteogenic differentiation of rat osteoblasts was investigated. The proliferation capacity of osteoblasts was evaluated using cell-counting kit-8. The effects on the messenger ribonucleic acid (mRNA) and protein levels of proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), osteopontin (OPN), LRP4, Wingless-type MMTV integration site family, member 4 (Wnt4), glycogen synthase kinase 3 beta (GSK-3β), p-GSK-3β, and β-catenin were analyzed by quantitative reverse-transcription polymerase chain reaction and Western blot. The impact on osteoblast apoptosis and calcium deposition was assessed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and alizarin red staining assays. A rat fracture model was then established, followed by treatments with either alendronate or alendronate-NPs. Bone formation during fracture healing was evaluated using alkaline phosphatase. The effects of alendronate and alendronate-NPs on fracture healing were assessed through hematoxylin-eosin (HE) staining and immunohistochemistry staining of collagen I. Results: Alendronate and alendronate-NPs significantly downregulated the expression of LRP4 during osteoblast differentiation and upregulated the expression of downstream targets of the Wnt/β-catenin pathway (p < 0.01). The co-treatment of osteoblasts with alendronate-NPs and LRP4 overexpression partially offset the bone-formation-promoting effects of alendronate-NPs. LRP4 overexpression reversed the proliferative and osteogenic effects of alendronate-NPs (p < 0.01). Compared with alendronate-NP treatment alone, the combined treatment of alendronate-NPs and LRP4 overexpression downregulated the expression of downstream targets of the Wnt/β-catenin pathway in osteoblasts (p < 0.01). Alendronate and alendronate-NPs significantly accelerated fracture healing in rats. They also promoted the proliferation and bone-formation capacity of osteoblasts, with alendronate-NPs showing a more pronounced therapeutic effect on fracture healing than alendronate. Conclusions: Alendronate-NPs may promote fracture healing by inhibiting LRP4 to activate the Wnt/β-catenin pathway. This study provides experimental evidence for a new mechanism of alendronate-NPs in promoting fracture healing.
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