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

Abstract

Introduction

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.

Graphical Abstract

Keywords:

• bioactive microdroplets
• PLA scaffold
• MSC-Exo
• biocompatibility
• osteoclast formation
• hBMSCs
• osteogenesis

Acknowledgments

Peng Luo and Yi Zhang are co-first authors for this study.

Disclosure

The authors declare that they have no competing interests in this work.

Additional information

Funding

This research received financial support from the National Natural Science Foundation of China (Grant no. 82060620 and 31960209), Outstanding Youth Scientific Fund of Guizhou Province (Grant no. Qian Ke He Platform Talents YQK[2023] 039), Guizhou Science and Technology Program Project (Grant no. Qiankehe Foundation - ZK[2023] General 502), Scientific Research Program of Guizhou Provincial Department of Education (Grant no. QJJ [2023] 019), Science and Technology Foundation of Guizhou Provincial Department of Education (Grant no. QJJ[2023]020), Zunyi Science and Technology Fund Project (Grant no. Zunyi Kehe HZ Zi [2021]40), and Future Eminent Clinician Plan of Zunyi Medical University (Grant no. 2022-02).