ACVR1R206H FOP mutation alters mechanosensing and tissue stiffness during heterotopic ossification.

An activating bone morphogenetic proteins (BMP) type I receptor ACVR1 (ACVR1R206H) mutation enhances BMP pathway signaling and causes the rare genetic disorder of heterotopic (extraskeletal) bone formation fibrodysplasia ossificans progressiva. Heterotopic ossification frequently occurs following injury as cells aberrantly differentiate during tissue repair. Biomechanical signals from the tissue microenvironment and cellular responses to these physical cues, such as stiffness and rigidity, are important determinants of cell differentiation and are modulated by BMP signaling. We used an Acvr1R206H/+ mouse model of injury-induced heterotopic ossification to examine the fibroproliferative tissue preceding heterotopic bone and identified pathologic stiffening at this stage of repair. In response to microenvironment stiffness, in vitro assays showed that Acvr1R206H/+ cells inappropriately sense their environment, responding to soft substrates with a spread morphology similar to wild-type cells on stiff substrates and to cells undergoing osteoblastogenesis. Increased activation of RhoA and its downstream effectors demonstrated increased mechanosignaling. Nuclear localization of the pro-osteoblastic factor RUNX2 on soft and stiff substrates suggests a predisposition to this cell fate. Our data support that increased BMP signaling in Acvr1R206H/+ cells alters the tissue microenvironment and results in misinterpretation of the tissue microenvironment through altered sensitivity to mechanical stimuli that lowers the threshold for commitment to chondro/osteogenic lineages.

High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation.

Extracellular matrix dynamics are key to tissue morphogenesis, homeostasis, injury, and repair. The spatiotemporal organization of this matrix has profound biological implications, but is challenging to monitor using standard techniques. Here, we address these challenges by using noncanonical amino acid tagging to fluorescently label extracellular matrix synthesized in the presence of bio-orthogonal methionine analogs. This strategy labels matrix proteins with high resolution, without compromising their distribution or mechanical function. We demonstrate that the organization and temporal dynamics of the proteinaceous matrix depend on the biophysical features of the microenvironment, including the biomaterial scaffold and the niche constructed by cells themselves. Pulse labeling experiments reveal that, in immature constructs, nascent matrix is highly fibrous and interdigitates with pre-existing matrix, while in more developed constructs, nascent matrix lacks fibrous organization and is retained in the immediate pericellular space. Inhibition of collagen crosslinking increases matrix synthesis, but compromises matrix organization. Finally, these data demonstrate marked cell-to-cell heterogeneity amongst both chondrocytes and mesenchymal stem cells undergoing chondrogenesis. Collectively, these results introduce fluorescent noncanonical amino acid tagging as a strategy to investigate spatiotemporal matrix organization, and demonstrate its ability to identify differences in phenotype, microenvironment, and matrix assembly at the single cell level.

Single-cell differences in matrix gene expression do not predict matrix deposition.

Mesenchymal stem cells (MSCs) display substantial cell-to-cell heterogeneity, complicating their use in regenerative medicine. However, conventional bulk assays mask this variability. Here we show that both chondrocytes and chondrogenically induced MSCs exhibit substantial mRNA expression heterogeneity. Single-molecule RNA FISH to measure mRNA expression of differentiation markers in single cells reveals that sister cell pairs have high levels of mRNA variability, suggesting that marker expression is not heritable. Surprisingly, this variability does not correlate with cell-to-cell differences in cartilage-like matrix production. Transcriptome-wide analysis suggests that no combination of markers can predict functional potential. De-differentiating chondrocytes also show a disconnect between mRNA expression of the cartilage marker aggrecan and cartilage-like matrix accumulation. Altogether, these quantitative analyses suggest that sorting subpopulations based on these markers would only marginally enrich the progenitor population for 'superior' MSCs. Our results suggest that instantaneous mRNA abundance of canonical markers is tenuously linked to the chondrogenic phenotype at the single-cell level.

Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage.

Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.

Pericellular conditions regulate extent of cell-mediated compaction of collagen gels.

Cell-mediated compaction of the extracellular matrix (ECM) plays a critical role in tissue engineering, wound healing, embryonic development, and many disease states. The ECM is compacted as a result of cellular traction forces. We hypothesize that a cell mechanically remodels the nearby ECM until some target conditions are obtained, and then the cell stops compacting. A key feature of this hypothesis is that ECM compaction primarily occurs in the pericellular region and the properties of the ECM in the pericellular region govern cellular force generation. We developed a mathematical model to describe the amount of macroscopic compaction of cell-populated collagen gels in terms of the initial cell and collagen densities, as well as the final conditions of the pericellular environment (defined as the pericellular volume where the collagen is compacted (V(*)) and the mass of collagen within this volume (m(*))). This model qualitatively predicts the effects of varying initial cell and collagen concentrations on the extent of gel compaction, and by fitting V(*) and m(*), provides reasonable quantitative agreement with the extent of gel compaction observed in experiments with endothelial cells and fibroblasts. Microscopic analysis of compacted gels supports the assumption that collagen compaction occurs primarily in the pericellular environment.