(E-183) Matrix Mechanics Regulates Temporal Changes in Mesenchymal Stroma

Introduction::
Cellular infiltration into matrices and successive proliferation and differentiation of stem cells is a well-coordinated sequence involving mechanical and biochemical cues, including but not limited to matrix density, access, and attachment. While native musculoskeletal extracellular matrix (ECM) is primarily organized Collagen I (Col), tissue damage results in replacement by a transient fibrin (Fib) matrix within a blood clot and disorganized Col. Cell attachment to this ECM is integrin mediated, and the density of the matrix affects both physical migration and proliferation of cells, as well as cytoskeletal stress in the cells which affects cell state. Our objective is to observe temporal changes of cellular behavior that occur via matrix mechanics and to determine at each stage, if differences in mesenchymal stroma is predominately driven by matrix mechanics, cellular response, or a combination of the two. For this purpose, we use mechanically entangled fibrin and collagen gels at varying densities and observe cell shape, proliferation and the changes in the mechanics of the matrix over 14 days. The results of this study will inform the varying temporal support available for rudimentary vascular networks to form within the regenerating stroma.




Materials and Methods::
Rat bone marrow derived mesenchymal stem cells (MSCs) were seeded within fibrin, collagen:fibrin (col:fib) and collagen hydrogels at the following concentrations and combinations: fibrin at fibrinogen density of 10 mg/mL (F10), collagen gels at a collagen concentration of 2, 4, and 6, mg/mL (C2, C4, C6), col:fib gels at a ratio of 25 Col:75 Fib at each of the previously listed concentrations (C2F10, C4F10, and C6F10) (representative image of a blended hydrogel Figure 1). The study was carried out over 14 days with timepoints at days 0, 3, 7, and 14. Following fixation, samples were stained with Actin (yellow) and DAPI (blue) and imaged with an Operetta CLS (Perkin Elmer) at a 40x water magnification with a z slice thickness of 10 mm. Samples were then treated with sucrose treatments, flash frozen, cryo-sectioned at a slice thickness of 20 mm, and sample stiffness was characterized using a Piuma Nanoindenter (Optics11) with a probe stiffness of 4.38 N/m and tip radius of 28.5 mm via a 5x5 matrix scan. Statistical analysis was performed using one-way ANOVA across material composition for n=3/group/time point and Kruskal-Wallis’ post hoc test for significance determined at p< 0.05 (GraphPad Prism).




Results, Conclusions, and Discussions::
Matrix density clearly impacted cell morphology and proliferation early and throughout the 14 day study. Collagen, especially at increased concentration prevented infiltration of MSCs and cells had a rounded morphology, unlike with the addition of fibrin or in fibrin-only gels, where cells formed tracks and spindle like morphology in 3D prior to highly spread-out morphologies at later times (Figure 2). At day 3, matrix stiffness of F10 groups was significantly higher than all others except C2F10 (Figure 3). Due to a self-secreted matrix, cells show more migratory and proliferative behavior, as matrix signaling allows cells to secrete less enzymes to infiltrate the matrix. The stiffness of C6F10 samples due to high collagen concentration resulted in significantly different morphologies from all other hydrogels, with MSCs forming clumps by day 3 and concentrated spindle tracks by day 7 (Figure 2). Matrix stiffness of F10 samples was significantly different from both C2F10 and C4F10 samples, even though similar proliferative and migratory behaviors were observed in C2F10 and F10 samples. The matrix stiffnesses measured are a combination (within the distribution of stiffnesses within the sample) of initial hydrogel proteins, degraded hydrogel matrix, cellular contributions and cell secreted matrix, all of which could potentially be within the 10 kPa to 1 MPa range measured (Figure 3). While the C2F10 and C4F10 samples exhibit similar matrix stiffnesses, they signaled different cell states, the similar stiffnesses can be attributed to the ECM produced by the MSCs. By day 14, there were no significant differences within matrix stiffness (not shown), indicating that either MSCs produced enough of their own matrix in groups that were less stiff or enzymatic activity resulted in matrix degradation to an appropriate level of stiffness in groups that were initially more stiff.




In conclusion, we find that the fibrin that constitutes the temporary blood clot matrix that forms at wound sites is crucial from a mechanical perspective in allowing cellular infiltration and proliferation and resultant matrix remodeling over time. It is anticipated that temporal changes in matrix stiffness and extent of stromal proliferation will synergistically support which hydrogels are most conducive for concurrent vascularization.




Acknowledgements (Optional): : This work is supported in part by NSF CBET#1847103 and the Jacobson, Lutcher Brown, and USAA Foundation endowments at the University of Texas at San Antonio. MLP is supported by UTSA’s ESTEEMED Program (NIH R25EB027605).




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