Comparative Matrix Compaction and Cell Survival Analysis of Linear 3D Bioartificial Tissues
McGee CE, Amegashie A, Brown F, Grant D, Banes A, Banes AJ. Regenerative Medicine Symposium, Research Triangle Park, NC, October 15, 2013.
Introduction: The Tissue Train® culture systems facilitate the in vitro growth of cultured primary and continuous cells in a three-dimensional (3D) collagen matrix thereby subjecting them to more native growth conditions with the potential for applying static or cyclic mechanical loading. Bioartificial tissues (BATs) prepared in this way undergo significant morphological changes including integration into the anchoring material, alignment with the axis of attachment/strain, and compaction of the matrix. Furthermore, cell survivorship (time to failure or breaking of the construct), is related to cell type, cell density, and composition/architecture of the anchoring material. Methods: The relative gross morphology, compaction kinetics and survivorship of various cell types including tenocytes (isolated from primary human and equine explants) and osteoblasts (human bone MG63 cells) were evaluated and compared. Briefly, BATs were cast as linear collagen hydrogels (Collagel® kit, Flexcell Intl. Corp.) in Tissue Train®culture plates using 200µL volume and linear trough loaders beneath silicone elastomer membranes in the Tissue Train® wells that were subjected to static vacuum tension (FX-5000™ tension system). BATs developed into bioartificial tendons as the matrix was compacted by cell-driven collagen fiber and cell alignment. BATs were imaged every hour for the first 24 hours and every 6 hours thereafter using the ScanFlex™ system and serial kinetic images analyzed (XyFlex™ software) to determine change in surface area over time and/or time to failure when the BAT broke in vitro. Results: Matrix compaction rates varied in accordance with tissue origin, cells derived from fibrous connective tissue demonstrating the greatest compaction but decreased survivorship as compared to cells derived from mineralized connective tissue (bone). Furthermore, differences in the architecture/composition of the material used to facilitate anchoring of the 3D linear construct were observed to have an impact on time to failure with BATs comprised of primary tenocytes anchored to a 3D open cell foam exhibiting significantly increased survivorship as compared to identical BATs adhered to linear nonwoven mesh anchor tabs despite comparable compaction kinetics. Conclusions: Taken together, these data suggest that although various cell types are capable of matrix re-organization and compaction to form linear 3D BATs, the gross biological properties of a given BAT are largely dependent upon both cell type and architecture/composition of the anchoring material.