Negative pressure-induced fibroblast activation for wound healing through the Piezo1-Ca 2+-NFAT3 signaling pathway

Abstract Background Negative pressure wound therapy (NPWT) is widely used to promote wound healing, yet the mechanotransduction mechanisms underlying its efficacy remain unclear because of the absence of precise and controllable in vitro negative pressure loading devices for cellular-level studies. Methods We developed a high-precision negative pressure (NP) loading device compatible with six-well plates, facilitating independent and intelligent control over NP values, durations, and modes for each well. To validate the uniformity and stability of the NP environment, finite element analysis (FEA) was implemented. The mechanism was explored using proteomic profiling of NP-treated fibroblasts, complemented by molecular interrogation of Piezo1 expression and calcium signaling dynamics. The results of functional studies integrated genetic silencing and pharmacological modulation of the pathway, with in vivo confirmation through SD rat-based NPWT experiments. Results FEA confirmed a stable pressure distribution within the NP chamber. The cells experienced predominantly compressive stresses that scaled linearly with the applied pressure, reaching −2 kPa at −40 mmHg. We found that applying a continuous NP of −40 mmHg for 2 hours significantly activated dermal fibroblasts (DFs). TMT-based proteomic analysis revealed the upregulation of Piezo1 in NP-treated DFs, which was further confirmed by molecular and immunohistochemical analyses of both cellular and granulation tissue samples. Pharmacological inhibition or knockdown of Piezo1 attenuated NP-induced DF activation in vivo and in vitro. Mechanistically, we determined that Piezo1-mediated Ca2+ influx and the calcineurin/NFAT3 signaling pathway are critically enhanced during this process. Conclusion This study revealed that the Piezo1-Ca2+-NFAT3 mechanotransduction axis is the central pathway that mediates the therapeutic effects of NPWT. This work lays the foundational groundwork for elucidating the biomechanical mechanisms of negative pressure and reveals new approaches for research in tissue engineering and regenerative medicine.