Scaffold-free tenogenic macro-tissues engineered by multi-layered bioassembly of rat tendon fibroblast spheroids

Abstract Tissue-engineered tendon constructs that replicate the structural and functional properties of natural tendons are crucial in regenerative medicine to improve treatment outcomes after tendon injury. This study aimed to engineer 3-dimensional biomimetic tendon macro-tissues through bioassembly of cell spheroids. Rat tendon fibroblasts seeded at different cell numbers (1 × 10 ⁴ , 5 × 10 ⁴ , 1 × 10 ⁵ , 2 × 10 ⁵ , 3 × 10 ⁵ ) were analysed for spheroid formation and development for 28 d. The spheroid diameters decreased over time with a reduction in cell density while synthesising their own collagen fibres. Spheroids were then bioassembled to form fused macro-tissue constructs using pillar array temporary supports. With the presence of ascorbic acid in growth media, the spheroids fused within 6 d after bioassembly, during which the supports were removed, leaving the constructs scaffold-free. The fused spheroids reorganised over time with increased fibrillar collagen content, showing elongated cell morphology in peripheral regions, which were parallelly aligned with collagen fibres resembling natural tendon micro-anatomy. The presence of scleraxis and tenomodulin gene markers also supports the tenogenic nature of tissue constructs. These biomimetic scaffold-free tenogenic macro-tissues have promising applications in tendon repair and regeneration, as in vitro models and tendon graft substitutes.