Mineral coated microparticles doped with fluoride and complexed with mRNA prolong transfection in fracture healing

Anna Laura Nelson; Gianluca Fontana; Laura Chubb; Josh Choe; Katherine Williams; Dan Regan; Johnny Huard; William Murphy; Nicole Ehrhart; Chelsea Bahney

doi:10.3389/fbioe.2023.1295313

Mineral coated microparticles doped with fluoride and complexed with mRNA prolong transfection in fracture healing

Front Bioeng Biotechnol. 2024 Jan 9:11:1295313. doi: 10.3389/fbioe.2023.1295313. eCollection 2023.

Authors

Anna Laura Nelson^{1

2}, Gianluca Fontana³, Laura Chubb⁴, Josh Choe³, Katherine Williams^{4

5}, Dan Regan⁵, Johnny Huard^{1

4}, William Murphy^{3

6}, Nicole Ehrhart⁴, Chelsea Bahney^{1

4

7}

Affiliations

¹ Center for Regenerative and Personalized Medicine, Steadman Philippon Research Institute (SPRI), Vail, CO, United States.
² School of Biomedical Engineering, Colorado State University, Fort Collins, CO, United States.
³ Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI, United States.
⁴ Department of Clinical Sciences, Colorado State University, Fort Collins, CO, United States.
⁵ Department of Microbiology, Colorado State University, Fort Collins, CO, United States.
⁶ Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, United States.
⁷ Orthopaedic Trauma Institute, University of California, San Francisco, CA, United States.

Abstract

Introduction: Impaired fracture healing, specifically non-union, has been found to occur up to 14% in tibial shaft fractures. The current standard of care to treat non-union often requires additional surgeries which can result in long recovery times. Injectable-based therapies to accelerate fracture healing have the potential to mitigate the need for additional surgeries. Gene therapies have recently undergone significant advancements due to developments in nanotechnology, which improve mRNA stability while reducing immunogenicity. Methods: In this study, we tested the efficacy of mineral coated microparticles (MCM) and fluoride-doped MCM (FMCM) to effectively deliver firefly luciferase (FLuc) mRNA lipoplexes (LPX) to the fracture site. Here, adult mice underwent a tibia fracture and stabilization method and all treatments were locally injected into the fracture. Level of osteogenesis and amount of bone formation were assessed using gene expression and histomorphometry respectively. Localized and systemic inflammation were measured through gene expression, histopathology scoring and measuring C-reactive protein (CRP) in the serum. Lastly, daily IVIS images were taken to track and measure transfection over time. Results: MCM-LPX-FLuc and FMCM-LPX-FLuc were not found to cause any cytotoxic effects when tested in vitro. When measuring the osteogenic potential of each mineral composition, FMCM-LPX-FLuc trended higher in osteogenic markers through qRT-PCR than the other groups tested in a murine fracture and stabilization model. Despite FMCM-LPX-FLuc showing slightly elevated il-1β and il-4 levels in the fracture callus, inflammation scoring of the fracture callus did not result in any differences. Additionally, an acute systemic inflammatory response was not observed in any of the samples tested. The concentration of MCM-LPX-FLuc and FMCM-LPX-FLuc that was used in the murine fracture model did not stimulate bone when analyzed through stereological principles. Transfection efficacy and kinetics of delivery platforms revealed that FMCM-LPX-FLuc prolongs the luciferase signal both in vitro and in vivo. Discussion: These data together reveal that FMCM-LPX-FLuc could serve as a promising mRNA delivery platform for fracture healing applications.

Keywords: biomimetic; bone regeneration; fluoride; mRNA delivery; transfection.

Abstract

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