Debatin, T. | Search bioRxiv

bioRxiv2026

Mechanophenotype of synovial fibroblasts correlates with patients' pathotypes and treatment response in Rheumatoid Arthritis

The mechanical features of synovial fibroblasts may encode clinically relevant information for the management of rheumatoid arthritis. Indeed, it remains poorly understood the links between cellular and microenvironment mechanics with patient stratification and therapeutic response. Here we show that combining organotypic synovial cultures with multimodal mechanophenotyping and transcriptomics identifies distinct ''soft'' and ''stiff'' fibroblast states. Healthy and rheumatoid arthritis fibroblasts were cultured in two or three dimensions (organotypic cell cultures), alone or with endothelial cells, and characterized by high-throughput nanoindentation, picobalance measurements, digital holographic microscopy, Brillouin microscopy and RNA sequencing. Three-dimensional culture reduced single-cell Young's modulus and total mass while increasing dry-mass surface density; endothelial co-culture increased fibroblast stiffness and the longitudinal modulus of intact organotypic cultures. Stiffness-based grouping revealed transcriptional programmes related to extracellular-matrix remodelling, cytoskeletal contractility, and TGF-beta, WNT, and Hippo signalling. Mapping these signatures to publicly available rheumatoid arthritis clinical transcriptomic datasets associated the ''stiff'' state with fibrotic and myeloid-fibrotic pathotypes and treatment-refractory programmes, whereas the ''soft'' state aligned with lymphoid pathotypes and rituximab- or tocilizumab-responsive programmes. These findings support mechanophenotype-associated transcriptional signatures as potential biomarkers for rheumatoid arthritis stratification.

biophysics↗

bioRxiv2023

2-photon laser printing to mechanically stimulate multicellular systems in 3D

Most biological activities take place in 3D environments, where cells communicate with each other in various directions and are located in a defined, often microstructured, space. To investigate the effect of defined cyclic mechanical forces on a multicellular system, we develop a sub-millimeter sized stretching device for mechanical stimulation of a structurally restricted, soft multicellular microenvironment. For the stretching device, a multimaterial 3D microstructure made of PDMS and gelatine-based hydrogel is printed via 2-photon polymerization (2PP) method. The printed structures are first characterized microscopically and mechanically to study the effect of different printing parameters. With 2PP, organotypic cell cultures are then directly printed into the hydrogel structures to achieve true 3D cell culture systems. These are mechanically stimulated with a cantilever by indenting the stretching device at a defined point. As a most important result, the cells in the 3D organotypic cell culture change morphology and actin orientation when exposed to cyclic mechanical stretch, even within short timescales of just 30 minutes. As a proof of concept, we encapsulated a Medaka retinal organoid in the same structure to demonstrate that even preformed organoids can be stimulated by our method. The results demonstrate the power of 2PP to manufacturing multifunctional soft devices for mechanically controlling multicellular systems at micrometer resolution and thus mimicking mechanical stress situations, as they occur in vivo.

biophysics↗