(B-77) Stromal Cell Identity Affects Perivascular ECM Stiffness and Regulates Capillary Morphogenesis in a 3D Fibrin Model

Introduction: Vascularization of tissues with functionally normal blood vessels remains a significant challenge. We have shown that robust microvasculature can be formed by co-delivery of endothelial cells (ECs) and supportive stromal cells (SCs), and that SC identity critically regulates the rate of neovascularization and vessel functionality both in vitro and in vivo. For instance, multipotent bone marrow stem cells (BMSCs) induce more stable, less permeable vasculature with a slower growth rate as compared to fibroblasts from various tissues. We posit a mechanobiological mechanism that links stromal cell identity, extracellular matrix (ECM) mechanical properties, rate of vascularization, and the functional qualities of the developed microvasculature. Here we hypothesize that fibrin hydrogels containing EC-fibroblast co-cultures dynamically stiffen the ECM locally and globally, which in turn accelerates vascular morphogenesis in comparison to EC-BMSC co-cultures.

Materials and Methods: A 3D model of capillary morphogenesis was established by co-embedding human umbilical vein endothelial cells (HUVECs) and SCs of different origins at a 1:1 ratio within 2.5 mg/mL fibrin gels to achieve a total cell density of 100k/mL. Normal human dermal fibroblasts (DFs), normal human lung fibroblasts (LFs), and BMSCs were evaluated as the SC populations in this study. Cultures were monitored for 14 days. Vascular morphogenesis was assessed via confocal microscopy and total vessel network lengths were quantified at each time point using the Angiogenesis Tube Formation module in Metamorph. Bulk mechanical properties of the fibrin-based gels were measured via parallel plate shear rheology. Perivascular mechanical properties were measured via active microrheology (AMR) using a dual-laser optical tweezers system. AMR measurements were taken at locations as far as 125 µm from growing capillaries

Results, Conclusions, and Discussions:
By the end of 14 days in culture, interconnected vascular networks had developed (Fig. 1A). As previously observed, the rates of capillary morphogenesis were higher in EC-DF and EC-LF co-cultures as compared to EC-BMSC co-cultures (Fig. 1B). To our surprise, bulk rheological data suggested a softening at day 7 when accelerated network growth was first observed, although significance was not detected in all cases (ANOVA; p< 0.05) (Fig. 1C). In contrast, AMR, which we used to interrogate the perivascular volume, revealed statistically significant local softening within EC-DF (Fig. 1D) but not EC-BMSC (Fig. 1E) cultures at day 7. Further, as we hypothesized, BMSCs did not alter perivascular stiffness when comparing all time points (Kruskal- Wallis; p < 0.05). This stasis in perivascular stiffness was commensurate with a relatively modest rate of vascular network growth (Fig. 1A). Taken together, these results and our previous work suggest the rate and quality of capillary formation may be dependent on SC-mediated time-varying local stiffness through poorly understood biophysical mechanisms, currently under investigation by our group.

Here we report correlations between perivascular stiffness dynamics and the rate of capillary morphogenesis within fibrin hydrogels. Such dynamics depend on stromal cell type, where BMSCs maintain static pericellular stiffness values that correlate with a slower-growing vascular network as compared to those induced by fibroblasts. Our findings are of importance to the development of vascularized implants and to furthering our understanding of microvascular mechanobiology.






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