We have developed and validated a method for fabricating ring-shaped tissue constructs entirely from aggregated SMCs that are strong enough to withstand handling and mechanical testing within 8 days of cell seeding. To our knowledge, this is the first study to report biomechanical evaluation of tissue constructs generated in a one-step process from aggregated cells and cell-derived ECM in static culture in this time frame. Our results demonstrate that facilitated cell aggregation can be used to create strong three-dimensional tissue constructs within the diameter range of clinically useful vascular grafts (2–6 mm). Although the focus of the current study was to characterize the strength and structure of ring-shaped constructs as a function of ring size and culture duration, we believe that this system will be useful for screening the effects of cell source and culture conditions on material properties of cell-derived tissues. Finally, we have provided evidence that these ring constructs can be used as building blocks to generate cell-derived tissue tubes, suggesting that information gained from functional ring studies may be directly translated to the design and construction of tubular structures such as vascular grafts.
Overall, cell-derived tissue rings were stronger than ring segments from engineered vascular tissue equivalents cultured for similar time periods. For example, the average UTS (100–500 kPa) far exceeded that reported for engineered tissues made with SMCs cultured statically within collagen (16 kPa at 8 days) [Seliktar et al., 2000
] and collagen/fibrin mixtures (28 kPa at 7 days) [Rowe and Stegemann, 2006
]. Without growth factor supplementation, the strength of our rings at 8 days approached that reported for SMC-populated fibrin gels cultured for 3 weeks with TGF-β1 and insulin (476 kPa) [Grassl et al., 2003
]. The MTM (0.5–2 MPa) of the cell-derived tissue rings also compared favorably to other engineered tissue rings (0.07–5.35 MPa) [Seliktar et al., 2000
; Gildner et al., 2004
] and tissue ring toughness values (12–71 kJ/m3
) were also high relative to what has been observed for collagen gel-based model vessels (0.5 kJ/m3
) [Gildner et al., 2004
]. However, all of these values are low compared to native arteries (for example, porcine carotid artery UTS approx. 6.6 MPa) [Dahl et al., 2007
]. In future studies, optimization of culture conditions to increase ECM synthesis and tissue strength may be performed, such as treatment with soluble factors (for example, sodium ascorbate [Ahlfors and Billiar, 2007
], TGF-β1 [Long and Tranquillo, 2003
] and insulin [Long and Tranquillo, 2003
]) or mechanical conditioning [Niklason et al., 1999
; Isenberg and Tranquillo, 2003
] to further strengthen cell-derived tissue rings.
Previous studies in which the strength and composition of planar cell-derived tissues were compared to constructs comprised of an equal number of cells seeded within fibrin or collagen gels demonstrated that cell-derived constructs exhibit greater tensile strength and ECM synthesis [Ahlfors and Billiar, 2007
]. Similarly, robust synthesis of ECM, comprised primarily of glycosaminoglycans and collagen, may be the basis for the observed strength and stiffness of the cell-derived tissue rings. Quantitative biochemical analysis of ECM composition, organization and cross-linking will be performed in future studies to evaluate the molecular basis of tissue ring structure and material properties.
Ring size had a significant effect on tissue mechanical properties, with lower force at failure, UTS and MTM recorded for the smallest (2-mm) rings at all time points. Ring wall thickness was consistent across samples of different dimensions (cultured for the same duration; fig. ), therefore the length-to-cross-sectional-area ratio of the constructs at the initial gauge length differed as a function of ring inside diameter. We attempted to account for the effects of ring inside diameter by defining and reporting the functional stiffness (data shown in fig. ). As stated in the Methods section, this calculation was performed to normalize samples with different inner diameters (lg~½πdi). As a result of the high thickness-to-inside diameter ratio in smaller (2-mm) compared to larger (6-mm) rings, there may be greater bending stiffness associated with the smaller rings, therefore a greater load would be applied to the smaller rings to straighten them prior to pre-cycling. Consequently, the smaller rings may be subjected to higher stresses prior to the pull-to-failure test, which could result in lower recorded UTS and force at failure values in the 2-mm rings. However, regardless of the lower strength and modulus compared to larger rings, the 2-mm rings in this study were mechanically robust compared to those reported in other studies of engineered vascular tissue, as detailed above, and tissue ring fusion studies demonstrated that 2-mm rings cultured for 7 days were strong enough to withstand transfer and manipulation on silicone tubes. Histologically, the tissue rings were indistinguishable on the basis of size at a given time point (data not shown), and thickness and failure strain values were not statistically different.
Tissue ring wall thicknesses were greater (up to 0.94 mm after 14 days in culture) than those reported for other cell-derived tissue constructs. This may be partially explained by the high density of cell seeding used to form rings and greater proliferation rate of the rat cell line used in this study compared to primary human cells. By comparison, cell-derived tissue sheets generated from human dermal fibroblasts seeded at 10,000 cells/cm2
and cultured for 6 weeks were 43 μm thick (more than 20-fold thinner), which increased by 5 μm per week thereafter (up to 15 weeks [L'Heureux et al., 2006
]). Thicker constructs (125–395 μm) were created from human dermal fibroblasts within 3 weeks by seeding at a higher density (2 million cells seeded in a 4.5-cm2
well) and culturing in chemically defined medium [Ahlfors and Billiar, 2007
]. In our tissues, high thicknesses may have contributed to necrosis observed at the tissue centers. This necrosis may have contributed to a reduction in structural integrity due to a loss of cells, which may partially explain the observed decrease in UTS despite an increase in ring thickness between days 8 and 14. The polarized light microscopy data suggested that collagen synthesis and remodeling increased in the tissue rings between days 8 and 14, although this did not coincide with an increase in tissue strength or stiffness. Interestingly, preliminary studies suggest that culturing tissue rings in culture medium supplemented with sodium ascorbate and amino caproic acid, conditions that have been shown to increase collagen synthesis and cross-linking, also improved tissue ring strength and stiffness (data not shown). It may be possible to optimize culture conditions (by decreasing or eliminating serum, and adding growth factors or mechanical stimulation, as described above) to make tissue rings stronger without increasing thickness.
Given the large number of cells needed to generate 4 batches of tissue rings in 3 different sizes to establish the basic parameters (for example, initial cell seeding number per well, culture duration and mechanical testing protocol) for creating and analyzing cell-derived tissue rings, we chose to use the WKY 3M-22 rat SMC line for the experiments reported in this study. However, we recently applied the same techniques to successfully assemble primary human coronary artery SMCs into cell-derived tissue rings, which were then cultured for 14 days. Despite their slower doubling time, in preliminary experiments the human SMC rings exhibited greater mechanical strength than the rat SMCs reported here (data not shown), thereby demonstrating that this cell aggregation system can be applied to create tissue rings from primary cells. Ongoing studies are focused on histological and biochemical analysis of the human SMC tissue constructs.
An important difference between the tissue ring constructs and vascular ring segments from native arteries is the lack of an endothelium or adventitia. Like many in vitro reports of TEBV construction, our study focused on a single cell type, SMCs, to mimic the vascular media. Recent studies have shown that cell sheet-based vascular grafts comprised of both SMCs and fibroblasts exhibit greater ECM synthesis and higher burst pressures compared to constructs made from SMCs alone [Gauvin et al., 2010
]. Furthermore, microtissue aggregation studies have shown that endothelial cells can co-aggregate with fibroblasts to form spheroids [Napolitano et al., 2007a
; Kelm et al., 2010
]. It may therefore be possible to add fibroblasts and endothelial cells to SMCs to increase strength and more closely mimic blood vessel structure and function in cell-derived tissue rings.
Upon successful fabrication and handling of cell-based ring constructs, it became evident that cell-derived tissue rings could be used as building blocks to form tissue tubes. Here, we report proof of concept that tissue rings cultured in close proximity fuse to form a cohesive tissue tube within 14 days (7 days for ring fabrication and 7 days for fusion). Culturing the tubes for an extended period may result in further fusion and elimination of ring boundaries. A recent study by Livoti and Morgan 
showed that toroid microtissues (600 μm inner diameter) self-assembled from H35 hepatocytes cultured for 48 h could be stacked and cultured, with fusion of adjacent toroids within 72 h. The ease with which 2-mm SMC rings could be handled after 7 days in our study suggests that it may be possible to harvest our rings even earlier to accelerate the process of graft fabrication. Finally, histological evaluation demonstrated that individual rings had fused to form a contiguous tissue mass within 7 days. However, burst pressure analysis will be a critical benchmark to determine the feasibility of transplanting vascular grafts created with this method.
In conclusion, we have shown that tissue constructs that are suitable for manipulation and functional testing can be created from aggregated SMCs within a few days. Although these rings are not as strong as ring segments of native blood vessels or TEBV generated from cultured cell sheets for 2–3 months, their strength compares favorably to other engineered tissue constructs reported to date. Given the short time frame and simplicity of this system (which relies on commercially available materials and methods), it may enable systematic assessment of a variety of parameters on tissue structure and function (for example, cell source, culture medium composition and dynamic culture regimens). The ring-shaped geometry of these constructs is useful for mechanical testing, and based on the ease with which they could be mounted onto wire grips, may also be used in a myograph system to measure tissue responses to pharmacologic agents. This system has potential as a new three-dimensional in vitro model of vascular tissue function, and a versatile tool to advance development of cell-derived vascular grafts.