Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM–ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM–ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.

Formation of a barrier capable of protecting tissue from external damage, chemical factors, and pathogens is one of the main functions of the epidermis. Furthermore, upon development and during aging, mechanoprotective epidermal functions change dramatically. However, comparative studies between embryonic and adult skin in comparison to skin equivalents are still scarce which is especially due to the lack of appropriate measurement systems with sufficient accuracy and long-term tissue compatibility. Our studies fill this gap by developing a combined bioreactor and tensile testing machine for biomechanical analysis of living epithelia. Based on this tissue stretcher, our data clearly show that viscoelastic and plastic deformation behavior of embryonic and adult skin differ significantly. Tissue responses to static strain compared to cyclic strain also show a clear dependence on differentiation stage. Multilayered unkeratinized epidermis equivalents, on the other hand, respond very similar to mechanical stretch as adult tissue. This mechanical similarity is even more evident after a single cycle of mechanical preconditioning. Our studies therefore suggest that skin equivalents are well suited model systems to analyze cellular interactions of epidermal cells in natural tissues.

Tissue‐engineered constructs have great potential in many intervention strategies. In order for these constructs to function optimally, they should ideally mimic the cellular alignment and orientation found in the tissues to be treated. Here we present a simple and reproducible method for the production of cell‐laden pure fibrin micro‐fibers with longitudinal topography. The micro‐fibers were produced using a molding technique and longitudinal topography was induced by a single initial stretch. Using this method, fibers up to 1 m in length and with diameters of 0.2–3 mm could be produced. The micro‐fibers were generated with embedded endothelial cells, smooth muscle cell/fibroblasts or Schwann cells. Polarized light and scanning electron microscopy imaging showed that the initial stretch was sufficient to induce longitudinal topography in the fibrin gel. Cells in the unstretched control micro‐fibers elongated randomly in both the floating and encapsulated environments, whereas the cells in the stretched micro‐fibers responded to the introduced topography by adopting a similar orientation. Proof of concept bottom‐up tissue engineering (TE) constructs are shown, all displaying various anisotropic organization of cells within. This simple, economical, versatile and scalable approach for the production of highly orientated and cell‐laden micro‐fibers is easily transferrable to any TE laboratory.

A correction to this paper has been published: https://doi.org/10.1007/s00441-021-03472-1

Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell–cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.

Basically all mammalian tissues are constantly exposed to a variety of environmental mechanical signals. Depending on the signal strength, mechanics intervenes in a multitude of cellular processes and is thus capable to induce simple cellular adaptations but also complex differentiation processes and even apoptosis. The underlying recognition typically depends on mechanosensitive proteins, which most often sense the mechanical signal for the induction of a cellular signaling cascade by changing their protein conformation. However, the fate of mechanosensors after mechanical stress application is still poorly understood and it remains unclear whether protein degradation pathways affect the mechanosensitivity of cells. Here, we show that cyclic stretch induces autophagosome formation in a time-dependent manner. Formation depends on the cochaperone BAG3 and thus likely involves BAG3-mediated chaperone-assisted selective autophagy. Furthermore, we demonstrate that strain-induced cell reorientation is clearly delayed upon inhibition of autophagy, suggesting a bidirectional crosstalk between mechanotransduction and autophagic degradation. The strength of the observed delay depends on stable adhesion structures and stress fiber formation in a RhoA-dependent manner.

No abstract