Evaluation and Optimization of a Three-Dimensional Construct Model for Equine Superficial Digital Flexor Tendon

Musculoskeletal injuries in equine athletes represent a leading cause for sport-induced and career-ending injuries because of slow or incomplete tendon repair creating clinical challenges in functional repair. Three-dimensional (3D) tendon constructs provide an in vitro model highly representative of in vivo tendon and provide an ideal strategy for testing therapies to improve tendon repair outcomes. Exploring different 3D construct protocols found in literature led us to consider the need for protocol optimization for horse superficial digital flexor tendon (SDFT) cells. The objective of this study was to assess both cell seeding density and seeding technique for equine SDFT 3D constructs by analyzing functionality and ultrastructure via biochemical, morphological, and mechanical characterization of engineered tendons under varying conditions of cell seeding density and seeding technique. Engineered tendons produced with varying cell seeding densities were assessed for their biochemical, collagen content, and fibril ultrastructure organization analysis. Although not statistically significant, using 300,000 cells per construct produced a more functionally and structurally reproducible construct as compared with 100,000 and 500,000 cells. A localized seeding method produced significantly stronger engineered tendons than those produced using a spread method as assessed by their biomechanical properties and collagen content. Collectively, our findings show evidence that although cell seeding number did not ultimately affect the construct, localized seeding method improved collagen content and mechanical properties for the model tendon. The tendon construct strategy described in this study is now being used as an initial “in vitro” step in the analysis of therapeutic strategies for equine tendon repair.