Prolyl-Hydroxyproline Promotes Structural Matrix Organization in 3D Equine Tendon Proper and Peritenon Post-Injury Model.

Tendinopathy of the superficial digital flexor tendon affects horse health and performance, with slow healing and high re-injury rates due to the formation of mechanically weaker scar tissue. Tendons are rich in Type I collagen and may benefit from supplementation with collagen-derived dipeptide prolyl hydroxyproline (H-Pro-Hyp-OH) which improves collagen regeneration and reduces scarring in murine injury models. However, its effects on equine tenocytes in the tendon proper (TP) and peritenon (PERI) under stress are unknown. Three-dimensional fibrin-based TP and PERI constructs were used to evaluate the effects of H-Pro-Hyp-OH within TP and PERI niches under control conditions and simulated post-injury environments. Constructs were treated with H-Pro-Hyp-OH alone or in combination with inflammatory (Interleukin-1 beta, IL-1β) or glucocorticoid (dexamethasone, DEX) stimuli. Gene expression was assessed by RT-qPCR, collagen content was quantified using a hydroxyproline assay, and tendon ultrastructure was evaluated using second harmonic generation imaging. Under control conditions, H-Pro-Hyp-OH enhanced alignment in TP. Under inflammatory and DEX stress, H-Pro-Hyp-OH did not reverse stress-induced transcriptional shifts or restore collagen content. However, H-Pro-Hyp-OH treatment improved collagen fibril organization and alignment, with structural rescue seen in PERI constructs. In stressful conditions induced by IL-1β or DEX, structural improvements did not always correspond with biochemical measurements of collagen, suggesting that H-Pro-Hyp-OH promotes matrix organization rather than bulk collagen accumulation in adverse post-injury environments. The responsiveness of PERI cells highlights the importance of targeting the extrinsic repair niche to promote functional tendon repair and limit fibrotic scarring.