H21 - Electrospraying-Induced Stem Cell Differentiation: Mechanistic Insights into Chondrogenesis via ATP Oscillations and P2X4 Activation

Introduction: : Cartilage tissue engineering seeks to create functional replacements for damaged cartilage, a tissue with limited regenerative capacity. One of the primary challenges is achieving chondrogenic differentiation of stem cells, such as human adipose-derived stem cells (hASCs), into chondrocytes. Traditionally, this process relies on the application of growth factors such as TGF-β and BMPs, which have shown promise but also come with significant drawbacks, including high costs, protein instability, and off-target differentiation. As a result, alternative strategies are urgently needed to promote chondrogenesis in a more cost-effective, scalable, and controlled manner. Recent advancements in biophysical stimuli, such as electrical and mechanical cues, offer promising avenues to achieve this. Specifically, electrospraying has emerged as a technique that can induce chondrogenesis in hASCs without the need for exogenous growth factors. Prior studies by our group demonstrated that electrospraying at specific voltages (10 kV and 15 kV) promoted spheroid formation and upregulated key chondrogenic markers such as Sox9 and Aggrecan. However, the underlying molecular mechanisms responsible for these observations remain unclear.

Materials and

Methods: : In this study, we aim to systematically investigate the mechanisms by which electrospraying induces chondrogenesis in hASCs. We will explore the role of ion channels (e.g., TRPC, TRPV, VGCCs) and downstream signaling pathways (e.g., cAMP/PKA, PKCα, ERK1/2) in the electrospraying process. Electrosprayed hASCs will be exposed to varying electric fields (10 kV, 15 kV) and their cellular response will be assessed through gene expression analysis, immunofluorescence staining, and calcium imaging to measure Ca²⁺ influx. We will also utilize specific inhibitors for ion channels and signaling molecules to identify the contribution of each pathway in promoting chondrogenesis.

Results, Conclusions, and Discussions:: Preliminary findings indicate that electrospraying significantly increases the expression of chondrogenic markers like Sox9 and Aggrecan in hASCs, particularly at 10 kV and 15 kV. These conditions also resulted in increased spheroid formation, suggesting that electrospraying influences cellular morphology and aggregation, key processes in chondrogenesis. Moreover, we observed a dose-dependent release of ATP following membrane depolarization, which corresponds with an increase in Ca²⁺ influx. Subsequent signaling pathway activation (e.g., PKCα, cAMP/PKA, ERK1/2) was detected, supporting the hypothesis that electrospraying-induced electrical signals initiate downstream signaling cascades that drive stem cell differentiation.This study demonstrates that electrospraying is a promising non-chemical approach for inducing chondrogenesis in human adipose-derived stem cells (hASCs). Our results highlight the critical role of ion channel activation, ATP release, and intracellular signaling pathways (e.g., PKCα, cAMP/PKA, ERK1/2) in driving the differentiation of stem cells into chondrocytes. By elucidating these mechanisms, we aim to provide a more comprehensive understanding of biophysical stimulation in cartilage tissue engineering, offering a potential strategy for regenerative therapies without relying on costly exogenous growth factors.

Acknowledgements and/or References (Optional):: "This research was funded by the National Science Foundation under Award No. 2337961 as part of the NSF CAREER Award."