Final Report Summary - 3D MULTICELL GROWTH (How mechanical forces regulate tissue growth in defined 3D geometries) 1) Project objectivesThe formation and regeneration of complex biological structures and tissues requires the coordinated action of a large number of cells. How individual cell behavior is integrated to give rise to this emergent phenomenon is a key topic in developmental biology as well as in regenerative medicine and tissue engineering. This project aims at defining the role of intercellular forces and cellular mechanosensing for regulating tissue growth in 3D scaffolds. A better understanding of the interplay of mechanics and biological growth will lead to new design strategies in bioengineering and thereby greatly facilitate the translation of basic knowledge into clinical applications.Specifically, the objective of this project was to develop an improved quantitative in vitro tissue growth setup, and to integrate it with leading-edge bioengineering techniques to measure matrix strains, force fluctuations and cellular mechanotransduction. Using this multidisciplinary approach, the hypothesis was tested that mechanical forces regulate tissue growth by translating macroscopic 3D substrate geometry into microscopic cell behavior.2) Work performed and main results2.1 Development of improved model system for in vitro microtissue growthIn the first work package, a new model system for quantitative in vitro imaging of 3D multicellular growth was developed and characterized. Transparent silicone scaffolds containing millimeter-sized pores of controlled shape and size were microfabricated with a novel modified projection lithography process, and subsequently functionalized covalently with the extracellular matrix protein Fibronectin. Primary human dermal fibroblast cells were then cultured on these substrates during several weeks. During this time, the cells proliferate and deposit extracellular matrix in the concave regions of the pores, resulting in the formation of large multicellular in vitro microtissues. These microtissues were then imaged under a confocal microscope, either while still alive or after being fixed and stained for different intracellular or extracellular proteins. To improve imaging of fixed microtissues, a recently developed tissue clearing method was successfully adapted to this setup. This system was shown to provide an excellent platform to study ECM deposition and tissue formation both with live imaging as well as after fixation.2.2 Measurement of matrix strain and force fluctuationsTo clarify the role of mechanical forces for the geometrically guided coordination of growing tissue the mechanical stretch of the extracellular matrix was determined by measuring the relative molecular extension of Fibronectin using FRET. New image segmentation and analysis software was developed to quantify the spatial distribution of extracellular matrix extension. We found that the matrix is significantly more extended on the surface of the tissue compared to the bulk, and that this gradient in matrix extension disappears if cell contractility is inhibited biochemically. In contrast, upregulation of cell contractility using TGF-beta resulted in an increase of matrix extension. While it was previously known that cells mechanically extend Fibronectin during ECM assembly, we show here for the first time that during 3D tissue growth, this process takes place along the surface of the growing tissue, but not in the interior.Complementary to these experiments, force fluctuations in the growing tissue were measured using fluorescent marker particles. Both the direction of the spontantous force-induced motion as well as their spatial correlation were found to be mainly along the surface of the microtissues. Together, these results highlight a novel mechanism for tissue deposition: cells along the growth surface collectively pull on each other and stretch the ECM, akin to an effective surface tension, which together with the curvature of the tissue surface leads to subsequent addition of new ECM.2.3 Characterization of cell behavior and mechanotransductionThe role of cell behavior during tissue growth was studied by quantifying the spatial distribution of proliferation in growing microtissues. For this purpose, the incorporation of the proliferation marker EdU during a 16-hour incubation period was imaged and spatiotemporally quantified using custom segmentation software. Results show that cells proliferate predominantly in the first cell layer along the tissue surface. Morphological analysis revealed that higher proliferation rates correlate with a more spread and flattened appearance of nuclei and cells. Treatment with TGF-beta resulted in an increase of cell proliferation, resulting in a significantly increased tissue volume after three weeks of growth. The role of downstream mechanobiological signaling was assessed by quantifying expression of alpha smooth muscle actin (aSMA) and the nuclear translocation of the transcription factor YAP/TAZ. The final data analysis on these experiments is still being carried out at the end of the project.2.4 Summary of significant resultsTaken together, we developed and implemented an improved tissue growth imaging setup, which enabled us to find that 1) the proliferation rate of fibroblasts is significantly increased near the surface of the growing microtissues, 2) the Fibronectin within newly assembled ECM is significantly more stretched by cells along the surface compared to the bulk, and that 3) upregulation of cell contractility increases Fibronectin extension and the amount of tissue produced. These results show that cell-generated forces, extracellular matrix properties, and cell signaling and response are highly coordinated during the geometry-dependent de-novo growth of microtissues in millimeter-sized scaffold pores.3) Impact and implicationsBy revealing a link between cell contractility, extracellular matrix properties, and biological growth during de novo tissue formation in an in vitro growth setup, this study offers novel insights not only into fibroblast tissue deposition, but into many other physiological scenarios in development and disease where collective cell behavior in geometrically defined environments is mechanically coordinated. The results highlight an important regulatory feedback mechanism on a collective multicellular scale that acts independently of specific signaling factors and is based on physical principles. Furthermore, by bridging the technological gap between single-cell studies and in vivo experiments, this project sets a new standard for realistic, quantitative investigations of multicellular organization, thereby facilitating the translation of basic knowledge on cellular mechanotransduction into clinical applications in tissue engineering and regenerative medicine. The results could serve as inspiration for new types of scaffold designs based on this multicellular mechanobiological control mechanism, and the tissue growth and matrix deposition assay developed in this project can easily be adapted for other cell types and to answer different questions, and thereby provides an innovative and versatile platform for future studies on cell-cell and cell-matrix interactions in a realistic in vitro tissue-like environment.