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Prolyl-Hydroxyproline Promotes Structural Matrix Organization in 3D Equine Tendon Proper and Peritenon Post-Injury Model.
Abstract: 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.
© 2026 The Author(s). Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.
Publication Date: 2026-09-21 PubMed ID: 42765573PubMed Central: PMC13591663DOI: 10.1002/jor.70284Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This study investigates the effect of the collagen-derived dipeptide prolyl hydroxyproline (H-Pro-Hyp-OH) on tendon cells from different regions of equine tendons, specifically the tendon proper (TP) and peritenon (PERI), under normal and simulated post-injury conditions.
- The research aims to understand how H-Pro-Hyp-OH could improve structural matrix organization and potentially aid tendon healing after injury in horses.
Background and Purpose
- Tendinopathy, particularly of the superficial digital flexor tendon in horses, causes significant health and performance issues due to slow healing and high rates of re-injury.
- The healing process often leads to the formation of scar tissue that is mechanically weaker than healthy tendon tissue.
- Type I collagen is a major component of tendons and critical for their mechanical strength.
- Previous research in mouse models suggests that the dipeptide prolyl hydroxyproline (H-Pro-Hyp-OH) can enhance collagen regeneration and reduce scar formation post-injury.
- This study aims to explore whether H-Pro-Hyp-OH has similar benefits on horse tendon cells, focusing on two distinct niches: the tendon proper (TP) and the peritenon (PERI), under both normal and simulated injury (inflammatory or glucocorticoid) conditions.
Methods
- Three-dimensional fibrin-based constructs mimicking the tendon proper (TP) and peritenon (PERI) environments were created to cultivate equine tenocytes.
- The constructs were treated under various conditions:
- Control (normal conditions)
- H-Pro-Hyp-OH supplementation alone
- H-Pro-Hyp-OH combined with inflammatory stimulus (Interleukin-1 beta, IL-1β)
- H-Pro-Hyp-OH combined with glucocorticoid stimulus (dexamethasone, DEX)
- Several experimental techniques were employed to analyze outcomes:
- RT-qPCR to assess gene expression changes, indicating cellular responses at the transcriptional level.
- Hydroxyproline assay to quantify collagen content in the tendon constructs.
- Second harmonic generation imaging to evaluate collagen fibril structure and alignment—this noninvasive imaging technique highlights collagen’s ultrastructural organization.
Key Findings
- Under normal control conditions:
- H-Pro-Hyp-OH improved collagen fibril alignment in the tendon proper (TP) constructs, indicating enhanced matrix organization.
- Under simulated post-injury conditions (with IL-1β inflammatory stimulus or DEX glucocorticoid stress):
- H-Pro-Hyp-OH did not reverse gene expression changes or restore collagen content reduced by stress, showing limited effects on bulk biochemical collagen levels or transcriptional shifts.
- Despite this, H-Pro-Hyp-OH improved collagen fibril organization and alignment, particularly in the peritenon (PERI) constructs, demonstrating a rescue effect on tendon ultrastructure.
- The dissociation between biochemical collagen quantity and improved collagen microstructure suggests that H-Pro-Hyp-OH helps organize existing collagen rather than increasing its production during stress.
- The peritenon (extrinsic) cells were more responsive to the treatment than the tendon proper cells, highlighting the role of the extrinsic niche in promoting functional tendon repair.
Implications and Significance
- This study highlights the potential therapeutic role of prolyl hydroxyproline in improving collagen matrix organization in equine tendons, especially after injury.
- Improved collagen alignment and ultrastructural organization could enhance the mechanical properties of the tendon matrix, potentially reducing scar formation and re-injury risks.
- Targeting the peritenon, often considered the extrinsic repair niche, may provide a novel strategy to promote better structural repair of tendons.
- Since H-Pro-Hyp-OH did not increase collagen quantity under stress, combining it with other treatments might be necessary to fully restore tendon health post-injury.
- Overall, this research adds to the growing evidence that matrix organization, not just collagen amount, is critical in tendon healing and function.
Cite This Article
APA
Wong AO, Mienaltowski MJ.
(2026).
Prolyl-Hydroxyproline Promotes Structural Matrix Organization in 3D Equine Tendon Proper and Peritenon Post-Injury Model.
J Orthop Res, 44(9), e70284.
https://doi.org/10.1002/jor.70284 Publication
Researcher Affiliations
- Department of Animal Science, College of Agricultural & Environmental Sciences, University of California Davis, Davis, California, USA.
- Department of Animal Science, College of Agricultural & Environmental Sciences, University of California Davis, Davis, California, USA.
MeSH Terms
- Animals
- Horses
- Tendon Injuries / drug therapy
- Tendons / drug effects
- Tendons / ultrastructure
- Dipeptides / pharmacology
- Dipeptides / therapeutic use
- Tenocytes / drug effects
Grant Funding
- 22A / UC Davis Center for Equine Health
- Division of Agriculture and Natural Resources, University of California
- College of Agricultural and Environmental Sciences, University of California, Davis
Conflict of Interest Statement
The authors declare no conflicts of interest.
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