Microscopic and Biological Characterization of Cryopreserved Equine Umbilical Tissue Allograft
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Published on: December 19, 2025
Britta S Leise1, Antonio P N Catunda1, Alicia L Bertone2
1Equine Health and Sports Performance Program and Department of Veterinary Clinical Sciences; Louisiana State University School of Veterinary Medicine, Baton Rouge, LA, USA.
Purpose:
Equicenta®CTM is a cryopreserved micronized equine umbilical-derived tissue allograft (eUTA) that is minimally processed and contains an abundance of fetal proteins in a connective tissue matrix. This study aimed to characterize this eUTA's microscopic structure, biodegradation, and potential to release biologically relevant proteins from a tissue scaffold reservoir, including cytokines, growth factors, and extracellular vesicles (EVs).
Methods:
Microscopic evaluation of eUTA was performed using stereoscopic culture and pelleted smears. In vitro enzymatic digestion with collagenase or trypsin (positive control) in tissue culture suspensions was used to assess morphological changes. A 24-hour digestion study quantified the total protein, collagen type 1 alpha chain 1 (Col1A1), and tissue inhibitor of metalloproteinase-2 (TIMP-2). Anabolic, anti-inflammatory, and anti-catabolic biomediators were measured using multiplex ELISA before and after detergent extraction. EVs were evaluated by nanoparticle tracking analysis and tetraspanin detection.
Results:
Microscopy revealed free-floating microparticle aggregates and fibrillar net-like extracellular matrix scaffolds in a proteinaceous ground substance. Collagenase caused gradual matrix breakdown, whereas trypsin resulted in rapid, complete degradation. The eUTA contained multiple cytokines and growth factors; selected anabolic and anti-catabolic proteins increased after scaffold digestion or extraction, including insulin-like growth factor (IGF-1), alpha-2 macroglobulin (A2M), and TIMP-2. (Col1A1) was released continuously over 24 hours during collagenase digestion. EV analysis confirmed abundant particles expressing CD9, CD63, and CD81.
Conclusion:
Microscopic evaluation of eUTA revealed an intricate filamentous microparticle matrix capable of serving as a scaffold for endogenous tissue repair in orthopedic injuries. This matrix can be degraded by naturally occurring proteolytic collagenases, thereby releasing matrix proteins and bioactive factors over time, which may support healing.
