The Effect of Growth-Mimicking Continuous Strain on the Early Stages of Skeletal Development in Micromass Culture

Embryonic skeletogenesis involves proliferation, condensation and subsequent chondrogenic differentiation of mesenchymal precursor cells, and the strains and stresses inherent to these processes have been hypothesized to influence skeletal development. The aim of this study was to determine the effect of growth-mimicking strain on the process of early skeletal development in vitro. To this end, we applied continuous uniaxial strain to embryonic skeletal precursor cells in micromass culture. Strain was applied at different times of culture to specifically address the effect of mechanical loading on the sequential stages of cellular proliferation, condensation and differentiation. We found that growth-mimicking strain at all three times did not affect proliferation or chondrogenic differentiation under the tested conditions. However, the timing of the applied strain did play a role in the density of mesenchymal condensations. This finding suggests that a mechanically dynamic environment, and specifically strain, can influence skeletal patterning. The growth-mimicking micromass model presented here may be a useful tool for further studies into the role of mechanical loading in early skeletal development

From Skeletal Development to Tissue Engineering: Lessons from the Micromass Assay

Damage and degeneration of the skeletal elements due to disease, trauma, and aging lead to a significant health and economical burden. To reduce this burden, skeletal tissue engineering strategies aim to regenerate functional bone and cartilage in the adult body. However, challenges still exist. Such challenges involve the identification of the external cues that determine differentiation, how to control chondrocyte hypertrophy, and how to achieve specific tissue patterns and boundaries. To address these issues, it could be insightful to look at skeletal development, a robust morphogenetic process that takes place during embryonic development and is commonly modeled in vitro by the micromass assay. In this review, we investigate what the tissue engineering field can learn from this assay. By comparing embryonic skeletal precursor cells from different anatomic locations and developmental stages in micromass, the external cues that guide lineage commitment can be identified. The signaling pathways regulating chondrocyte hypertrophy, and the cues required for tissue patterning, can be elucidated by combining the micromass assay with genetic, molecular, and engineering tools. The lessons from the micromass assay are limited by two major differences between developmental and regenerative skeletogenesis: cell type and scale. We highlight an important difference between embryonic and adult skeletal progenitor cells, in that adult progenitors are not able to form mesenchymal condensations spontaneously. Also, the mechanisms of tissue patterning need to be adjusted to the larger tissue engineering constructs. In conclusion, mechanistic insights of skeletal tissue generation gained from the micromass model could lead to improved tissue engineering strategies and construct

Cell mediated contraction in 3D cell-matrix constructs leads to spatially regulated osteogenic differentiation

During embryonic development, morphogenetic processes give rise to a variety of shapes and patterns that lead to functional tissues and organs. While the impact of chemical signals on these processes is widely studied, the role of physical cues is less understood. The aim of this study was to test the hypothesis that the interplay of cell mediated contraction and mechanical boundary conditions alone can result in spatially regulated differentiation in simple 3D constructs. An experimental model consisting of a 3D cell-gel construct and a finite element (FE) model were used to study the effect of cellular traction exerted by mesenchymal stem cells (MSCs) on an initially homogeneous matrix under inhomogeneous boundary conditions. A robust shape change is observed due to contraction under time-varying mechanical boundary conditions, which is explained by the finite element model. Furthermore, distinct local differences in osteogenic differentiation are observed, with a spatial pattern independent of osteogenic factors in the culture medium. Regions that are predicted to have experienced relatively high shear stress at any time during contraction correlate with the regions of distinct osteogenesis. Taken together, these results support the underlying hypothesis that cellular contractility and mechanical boundary conditions alone can result in spatially regulated differentiation. These results will have important implications for tissue engineering and regeneratio

Linear patterning of mesenchymal condensations is modulated by geometric constraints

The development of the vertebral column starts with the formation of a linear array of mesenchymal condensations, forming the blueprint for the eventual alternating pattern of bone and cartilage. Despite growing insight into the molecular mechanisms of morphogenesis, the impact of the physical aspects of the environment is not well understood. We hypothesized that geometric boundary conditions may play a pivotal role in the linear patterning of condensations, as neighbouring tissues provide physical constraints to the cell population. To study the process of condensation and the patterning thereof under tightly controlled geometric constraints, we developed a novel in vitro model that combines micropatterning with the established micromass assay. The spacing and alignment of condensations changed with the width of the cell adhesive patterns, a phenomenon that could not be explained by cell availability alone. Moreover, the extent of chondrogenic commitment was increased on substrates with tighter geometric constraints. When the in vivo pattern of condensations was investigated in the developing vertebral column of chicken embryos, the measurements closely fit into the quantitative relation between geometric constraints and inter-condensation distance found in vitro. Together, these findings suggest a potential role of geometric constraints in skeletal patterning in a cellular process of self-organizatio

Experimental model.

<p><b>(A)</b> Bird view of a PDMS well containing five mini-wells. <b>(B)</b> Fluorescent micrographs of a representative well cultured for 60 hours in the absence of strain, stained with DAPI to indicated nuclei (blue, left) and peanut agglutinin lectin (PNA) to indicate condensations (green, right). Scale bar is 500μm. <b>(C)</b> Fluorescent micrographs of representative cultures after 60 hours of culture in the absence of strain. Cells are stained with DAPI (blue), peanut agglutinin lectin (PNA, green), and an anti-avian fibronectin (FN) antibody to indicate deposited FN (red). Lower magnification (top row): scale bar is 100μm. Higher magnification (bottom row): scale bar is 50μm. <b>(D)</b> Schematic representation of the time line of the experiments. Cells are freshly isolated and directly plated onto the PDMS wells. Continuous strain is subsequently initiated at 0hr, 20hr, or 40hr, for a total of 25% strain over 20hours. Cultures are terminated at 60 hours.</p

Proliferation.

<p><b>(A)</b> Total DNA content is measured after 60 hrs. Data are normalized to the non-strained condition and represent means ± standard deviations, n≥8. <b>(B)</b> Percentages of proliferating cells, indicated by the incorporation of EdU, were measured at 48 hour of culture. Data are normalized to the non-strained condition and represent means ± standard deviations, n≥5. <b>(C)</b> Fluorescent micrographs of 48-hour cultures stained for nuclei (DAPI, blue) and EdU (red), after incubation with EdU for 4 hours. Scale bar is 100μm.</p

Mesenchymal condensation.

<p><b>(A)</b> Fluorescent micrographs of samples cultured for 60 hours. Cultures are stained with DAPI (blue, top row) and peanut agglutinin lectin (PNA, green, bottom row). Scale bar is 200μm. <b>(B)</b> The number of condensations per squared mm is quantified at 60 hours, using images of PNA staining. Data represent means ± standard deviations, n≥7. *, p < 0.05. <b>(C)</b> The number of condensations per squared mm for the three strained conditions is corrected for the applied strain, thus multiplied by a factor of 1.25. The graph displays the number of condensations per squared mm of original surface area. Data represent means ± standard deviations, n≥7. *, p < 0.05.</p

Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer