Nutrient deprivation of tendon-derived cells and its effect on collagen matrix integrity – mimicking graft remodelling after ACL reconstruction in vitro

This study investigated whether the nutrient-poor environment experienced by tendon grafts after ACL reconstruction (and the presence of dead/dying cells in auto-/allografts) directly compromises collagen matrix integrity via a cell-driven catabolic response. Rat tendon-derived cells were embedded in reconstituted collagen type I to create in vitro “tendon-mimics” using a constrained culture system, then exposed for 7 days to media conditions designed to mimic post-surgical nutrient restriction: complete medium (5 mM glucose, 2.5% serum), mild deprivation (0.5 mM glucose, no serum), extreme deprivation (0 mM glucose, no serum), and a frozen control (freeze–thaw cycles plus 0 mM glucose, no serum). Cell viability (PI/DAPI, LDH), mimic compaction, collagen fiber alignment (collagen probe + confocal; orientation analysis), mechanics, and secreted/activated MMP activity (collagen/gelatin zymography) were quantified. While extreme deprivation and freezing significantly reduced viability, tendon-mimic mechanical properties did not deteriorate under nutrient deprivation or freezing; instead, only the complete medium group showed increased stiffness and failure stress over time, consistent with cell-driven compaction/remodeling when nutrients are available. Zymography showed no nutrient-deprivation–induced increase in catabolic enzymes; rather, higher active MMP2 and MMP8 signals were most evident in the complete medium condition, supporting the interpretation that nutrient-deprived viable cells lack the energy to generate contractile forces or produce/activate MMPs at levels that alter bulk mechanics. Overall, the work suggests that nutrient deprivation alone does not cause tendon-derived cells to compromise collagen matrix integrity in this simplified in vitro model, and that early graft weakening in vivo likely involves additional factors beyond direct resident-cell catabolism under starvation.