Icariin-Enhanced Osteoclast-Derived Exosomes Promote Repair of Infected Bone Defects by Regulating Osteoclast and Osteoblast Communication

Yang Zhang,1 Minjie Zhang,2 Mengying Li,2 Maomao Miao,2 Dan Shou,2 Peijian Tong1 1Institute of Orthopaedics and Traumatology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese Medicine), Hangzhou, 310053, People’s Republic of China; 2School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of ChinaCorrespondence: Dan Shou; Peijian Tong, Email shoudanok@163.com; peijiantong@zcmu.edu.cnBackground: Infected bone defects pose a challenging clinical issue due to an imbalance of osteoclasts (OC) and osteoblasts (OB). Exosomes are crucial for intercellular signaling of OC and OB in bone repair. Icariin, has been shown to regulate the balance between OC and OB. However, the specific mechanisms by which icariin influences exosomes derived from osteoclasts, and subsequently impacts osteoblast activity, remain unclear. This study aims to investigate the effects of icariin-treated osteoclast-derived exosomes (ICA-OC-Exo) on osteoblast function and bone repair in cases of infected bone defects.Methods: We investigated the exosome profile and localization of multivesicular bodies (MVB) and quantification of intraluminal vesicles (ILVs) in osteoclasts by using transmission electron microscopy. Additionally, the expressions of Rab27A and MITF, which are associated with exosome release, were determined through immunofluorescence staining and Western blot. The profiling of exosomal miRNA expression was conducted via miRNA-sequencing. The effects of ICA-OC-Exo on osteoblast differentiation were determined using RT-qPCR, Western blot, alkaline phosphatase staining. Additionally, ICA-OC-Exo was administered into the localized bone defect of the infected bone rat models, and bone formation was assessed using Micro-CT.Results: Icariin increased the presence of MVBs in the cytoplasm through modulation of the MITF/Rab27A signaling pathway, resulting in higher number of ICA-OC-Exo compared to OC-Exo. Additionally, miR-331-3p expression in ICA-OC-Exo was found to be elevated compared to OC-Exo. ICA-OC-Exo was observed to stimulate osteoblast function by targeting FGF23, reducing DKK1, and subsequently upregulating ALP. In the in vivo study, ICA-OC-Exo exhibited the capacity to enhance bone healing at the site of a local bone defect following anti-infection treatment.Conclusion: Icariin enhanced the quantification of OC-Exo and the expression of miRNA-331-3p in OC-Exo, leading to the regulation of osteoblast function via activation of the miRNA-331-3p/FGF23/DKK1 pathway. ICA-OC-Exo demonstrated potential clinical applicability in bone repair of infected bone defects. Keywords: infected bone defects, icariin, osteoclast, exosomes, osteoblast