Microdroplets Encapsulated with NFATc1-siRNA and Exosomes-Derived from MSCs Onto 3D Porous PLA Scaffold for Regulating Osteoclastogenesis and Promoting Osteogenesis

Abstract

Peng Luo,1,* Yi Zhang,2,3,* Maodi Huang,1 Guochen Luo,1 Yaping Ma,1,4 Xin Wang1,4 1Department of Orthopaedic Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou, 563003, People’s Republic of China; 2Department of Hygiene Toxicology, School of Public Health, Zunyi Medical University, Zunyi, Guizhou, 563000, People’s Republic of China; 3Key Laboratory of Maternal & Child Health and Exposure Science of Guizhou Higher Education Institutes, Zunyi Medical University, Zunyi, Guizhou, 563000, People’s Republic of China; 4Guizhou Provincial Key Laboratory of Medicinal Biotechnology in Colleges and Universities, Zunyi Medical University, Zunyi, Guizhou, 563000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xin Wang, Department of Orthopaedic Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou, 563003, People’s Republic of China, Tel [phone], Fax [phone], Email [email protected]: Osteoporotic-related fractures remains a significant public health concern, thus imposing substantial burdens on our society. Excessive activation of osteoclastic activity is one of the main contributing factors for osteoporosis-related fractures. While polylactic acid (PLA) is frequently employed as a biodegradable scaffold in tissue engineering, it lacks sufficient biological activity. Microdroplets (MDs) have been explored as an ultrasound-responsive drug delivery method, and mesenchymal stem cell (MSC)-derived exosomes have shown therapeutic effects in diverse preclinical investigations. Thus, this study aimed to develop a novel bioactive hybrid PLA scaffold by integrating MDs-NFATc1-silencing siRNA to target osteoclast formation and MSCs-exosomes (MSC-Exo) to influence osteogenic differentiation (MDs-NFATc1/PLA-Exo).Methods: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) were used for exosome isolation. Transmission electron microscopy (TEM) and confocal laser scanning microscopy were used for exosome and MDs morphological characterization, respectively. The MDs-NFATc1/PLA-Exo scaffold was fabricated through poly(dopamine) and fibrin gel coating. Biocompatibility was assessed using RAW 264.7 macrophages and hBMSCs. Osteoclast formations were examined via TRAP staining. Osteogenic differentiation of hBMSCs and cytokine expression modulation were also investigated.Results: MSC-Exo exhibited a cup-shaped structure and effective internalization into cells, while MDs displayed a spherical morphology with a well-defined core-shell structure. Following ultrasound stimulation, the internalization study demonstrated efficient delivery of bioactive MDs into recipient cells. Biocompatibility studies indicated no cytotoxicity of MDs-NFATc1/PLA-Exo scaffolds in RAW 264.7 macrophages and hBMSCs. Both MDs-NFATc1/PLA and MDs-NFATc1/PLA-Exo treatments significantly reduced osteoclast differentiation and formation. In addition, our results further indicated MDs-NFATc1/PLA-Exo scaffold significantly enhanced osteogenic differentiation of hBMSCs and modulated cytokine expression.Discussion: These findings suggest that the bioactive MDs-NFATc1/PLA-Exo scaffold holds promise as an innovative structure for bone tissue regeneration. By specifically targeting osteoclast formation and promoting osteogenic differentiation, this hybrid scaffold may address key challenges in osteoporosis-related fractures. Keywords: bioactive microdroplets, PLA scaffold, MSC-Exo, biocompatibility, osteoclast formation, hBMSCs, osteogenesis

Keywords

• bioactive microdroplets
• pla scaffold
• msc-exo
• biocompatibility
• osteoclast formation
• hbmscs
• osteogenesis.