Z16 - Effects of bone-derived nanoparticles on mesenchymal stem cell dynamics in collagen

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

GM

Gabriel Medina

Research Assistant
University of New Haven
Wallingford, Connecticut, United States

Co-Author(s)

Xiangyu Gong, PhD

Research Scientist
Stony Brook University, United States
MM

Michael Mak

Associate Professor
Stony Brook University, United States

Primary Investigator(s)

SW

Shue Wang

Associate Professor
University of New Haven
West Haven, Connecticut, United States

Introduction: : Bone degenerative disorders, including osteopetrosis, osteopenia, and osteoporosis, arise from dysregulated bone remodeling due to impaired osteogenesis and/or bone resorption. Current therapeutic approaches exhibit adverse side effects and are limited in capability and resources. Bone marrow-derived mesenchymal stem cells (MSCs), are multipotent and immunomodulatory, making them key candidates for regenerative strategies aimed at enhancing osteogenic repair. Further, collagen type I is the predominant extracellular matrix (ECM) protein in native bone microenvironments that facilitate adhesion, integrin signaling, and osteogenic differentiation. Previously, we have shown that bone-derived nano particle (BNP) treatment can enhance osteogenesis on 2D rigid substrates that do not accurately recapitulate tissue mechanical properties. However, the exact mechanisms behind how BNPs regulate cell fate in a physiologically relevant environment remain unexplored.
Here, we conducted high resolution timelapse imaging to capture cell dynamics and uptake events upon the addition of BNPs. Furthermore, we develop a new collagen hydrogel platform that enables generation of real-time, high-resolution imaging of cell–ECM interactions under physiologically relevant conditions for quantitative analysis. This platform allows further investigation to understand MSC cytoskeleton dynamics and the role of collagen in osteogenic processes under the BNP treatment. This study aims to gain more insight into the mechanistic role of BNPs in osteogenesis, and how that process is impacted by a biomimetic ECM substrate.

Materials and

Methods: : To adhere collagen cushions, glass-bottom well plates were coated with 3-hydroxytyramine hydrochloride (1.8 mg/mL in 10 mM Tris-HCl), sterile-filtered (0.4 µm), and incubated at room temperature (RT) for 1.5 h. Wells were washed with pure water, dried in a warm oven, and cooled to RT before use.
Collagen hydrogel cushions were prepared by neutralizing acidified TeloCol-6 (95% type I, 5% type III collagen, 6.2 mg/mL) to 2 mg/mL with a pre-prepared basic buffer. Droplets of 35 μL collagen were dispensed into wells and overlaid with 10 mm coverslips. After gelling at 37 °C, coverslips were removed. Collagen cushions were fluorescently labeled with Alexa Fluor 647 NHS Ester.
GFP-labelled bone-derived nanoparticles (BNPs) were sonicated and diluted in Opti-MEM to 20 µg/mL. Mouse mesenchymal stem cells (mMSCs) tagged with LifeAct-RFP were cultured to 50% confluency, treated overnight with BNP media, then seeded onto collagen cushions (5,000 cells/well). Cells were cultured for 1–3 days.
Confocal microscopy (Leica) was performed at 10x magnification, maintaining constant exposure, laser intensity, and acquisition settings across conditions. For each medium condition, image sets were collected from five regions with cells per cushion.
For 2D timelapse imaging, cells were cultured to 25% confluency. Media was replaced with BNP media and cells were immediately placed in confocal for live imaging. Images were captured every 30 seconds for 1 hour.
Quantitative image analysis was performed using ImageJ. Actin cytoskeleton morphology and fluorescence intensity were assessed to evaluate cell response. Collagen fiber densification and fluorescence changes were measured to assess ECM remodeling.

Results, Conclusions, and Discussions:: Collagen cushions were successfully fabricated with uniform thickness within working distance of the high resolution confocal microscopy system. The flat collagen hydrogel cushions supported robust cell adhesion and proliferation, enabling high-resolution imaging of cytoskeletal and matrix architecture remodelling. 2D timelapse imaging demonstrated that BNP uptake events can be observed within 10 minutes. Under this brief BNP treatment, cells quickly exhibit less morphological changes and displacement. After overnight BNP treatment changes in cell dynamics can also be observed on the fibrous collagen cushions. In specific, cells treated with BNPs exhibit an increase in cell spreading area, reduced circularity, and a decrease in collagen plastic remodelling. Our data revealed that early stage physical contact with BNPs is sufficient to regulate cell dynamics. These observations have revealed novel cell-ECM interactions during osteogenic processes under BNP treatment. This effect is sustained and can be further reflective of a physiologically relevant environment.
Overall, our collagen system provides cells with a biomimetic environment while maintaining accessibility for quality imaging and analysis. Future investigation will be focused on how this unique cell-ECM interaction may influence long-term osteogenic differentiation. Our collagen cushions can be further modulated to mimic diverse physiological tissue environments. The tunability of these cushions can be used to further investigate how stiffness and ECM structure mechanistically affects osteogenesis in MSCs under BNP treatment.

Acknowledgements and/or References (Optional): :