Time-sequential fibroblast-to-myofibroblast transition in elastin-variable 3D hydrogel environments by collagen...

Area of Science:

• Biomaterials Science
• Cell Biology
• Extracellular Matrix Research

Background:

• Fibrosis involves fibroblast differentiation into myofibroblasts, crucial in physiological and pathological processes.
• Fibroblast activation is mediated by extracellular matrix (ECM) interactions.
• Regulation of fibroblast-to-myofibroblast transition (FMT) by 3D microenvironment elastin concentration is unexplored.

Purpose of the Study:

• To investigate the effect of varying elastin concentrations in a 3D hydrogel on fibroblast-to-myofibroblast transition (FMT).
• To elucidate the molecular mechanisms regulating FMT in response to the extracellular matrix (ECM) microenvironment.
• To develop a 3D elastin-gradient hydrogel system for studying fibroblast activation.

Main Methods:

• Fabrication of insoluble elastin-gradient 3D hydrogels with varying elastin concentrations (0-80%) in collagen.
• Long-term cell culture of embedded fibroblasts within the hydrogel system.
• Analysis of molecular mechanisms using nanoflow LC-MS/MS, real-time PCR, and immunofluorescence staining.

Main Results:

• Low elastin concentration (20%) significantly enhanced fibroblast proliferation and induced FMT.
• Mass spectrometry revealed a two-stage differentiation process involving calcium-dependent proteins (e.g., calmodulin, S100A4) and myofibroblast markers (e.g., α-SMA).
• Low elastin induced cancer-associated fibroblast (CAF) markers (PDGFR-β) and fibrosis markers (THY-1).

Conclusions:

• A 3D elastin-gradient hydrogel system effectively demonstrated that low elastin concentrations induce FMT and increase CAF levels via calcium signaling.
• The study analyzed protein expression changes throughout the FMT process across multiple cell passages.
• The developed system provides a platform for studying fibroblast-ECM interactions, highlighting the ECM's pivotal role.