Novel geometries for tissue-engineered tendonous collagen constructs

A promising approach to addressing the performance limitations of currently available mechanical and bioprosthetic heart valves lies in tissue engineering. Tissue-engineered valves should incorporate the complex microstructure of the native valves to mimic their unique mechanics. This would include a layered topology, mesh networks, and branched collagen fiber bundles. Our approach to heart valve tissue engineering is to develop the functional components of the aortic valve cusps separately in vitro and, once they are mature, integrate them into a composite valve structure. Here we report on our efforts to create more complex collagenous structures, suitable for heart valve tissue engineering. Collagen fiber bundles were fabricated using the principle of directed collagen gel contraction, using neonatal rat aortic smooth muscle cells and acid-soluble type I rat-tail tendon collagen. The collagen gels were cast into rectangular or branched wells with porous end holders that constrained the gels longitudinally but allowed contraction to occur transverse to the long axis. Pairs of such constructs were placed in direct contact with each other and cultured further to determine whether they integrated to form continuous tissue. After 6-8 weeks of culture, highly compacted and aligned collagen fiber bundles formed. Mechanical testing revealed that linear constructs (2 free ends) with an 8:1 aspect ratio were significantly stronger than similar constructs with an aspect ratio of 2:1 (mean +/- SD, 298 +/- 90 kPa vs. 152 +/- 49 kPa; p < .001). Branching reduced mechanical strength considerably. Constructs fabricated with 4 free ends were significantly weaker than constructs with 3 ends (31 +/- 32 kPa vs. 116 +/- 66 kPa; p < .003). Histologic images demonstrated the integration of the crossed collagen bundles, with a bonding strength of 2.1 +/- 1.1 g (0.02 N). We found that the geometry of the molds into which the collagen constructs are cast can greatly affect their mechanical strength: multibranched constructs were the weakest, and long, linear constructs were the strongest. We also found that integration of collagen constructs occurs in vitro and that the fabrication of a composite structure in vitro is probably feasible.