Original ResearchEvaluation and Optimization of a Three-Dimensional Construct Model for Equine Superficial Digital Flexor Tendon
Introduction
Musculoskeletal injuries contribute significantly to morbidity and mortality rates in equine athletes. Catastrophic musculoskeletal injuries in racehorses account for 67%–88% of racetrack-related fatalities [1], [2], [3], [4]. Most of these injuries affect the forelimb, more specifically the superficial digital flexor tendon (SDFT), as compared with the deep digital flexor tendon or common digital extensor tendon [4], [5], [6], [7]. The SDFT is particularly susceptible to injury because the energy storage and transmission of elastic forces sustained create vulnerabilities within the structure, such as microtrauma, from the maximum load and straining power during competition and high-speed work [8], [9]. The SDFT is required to undergo constant recoil and elongation along with inhibiting the hyperextension of the metacarpophalangeal joint [10]. Tendon healing after injury is slow and incomplete thereby leading to high rates of reinjury and the inability to compete, meaning that these musculoskeletal injuries are often career-ending [11], [12].
To test therapeutic interventions ahead of implementation, it is beneficial to use model systems, such as three-dimensional (3D) tendon constructs that provide an in vitro model representative of tendon [13]. Seeding tenocytes in a 3D fibrin gel providing cell-induced unidirectional tension between two anchor points results in structural and collagen fibril organization that closely resembles the developmentally immature in vivo state [14], [15]. For some model species, protocols for cell seeding have been established, yet this is not the case for horse cells [14], [16]. Moreover, two methods of seeding the fibrin gel matrix—either spreading the initial gel volume over an entire plate (spread method) or localizing the gel between the anchors—could affect structural or material properties because of differences in progression of fibrinogen processing, collagen production, and alignment. Based on previous work in other species and cell types, we hypothesized that 300,000 cells seeded per 1-cm long construct would result in functionally and structurally sound 3D tendon constructs [14], [17]. To test this hypothesis, we assessed the mechanical properties of engineered tendons produced with different initial cell densities. Furthermore, we evaluated collagen content and fibril ultrastructure to understanding how seeding density affected structural properties. We also hypothesized that concentrating the gel within the core of the plate would produce a more robust model of tendon. To test this hypothesis, we measured mechanical properties, assessed bulk collagen content, and measured fibril diameter distributions in relation to plating area to discern which method produced more functionally and structurally representative model of native tissue.
Section snippets
Tendon Harvest and Cell Isolation
Equine SDFTs were harvested from horses that were euthanized for reasons unrelated to this study with approval from the University of California Davis's Institutional Animal Care and Use Committee. Five mature nonbreed specific horses aged between 6 and 17 years of age were used for the cell seeding and cell density studies. Each horse was considered a biological replicate. From each horse, an inch of healthy SDFT was harvested using sterile technique and transported in Dulbecco's phosphate
Cell Density
We observed that some constructs narrowed to the point of failure before appropriate tests could be conducted and as such construct survivability was recorded. Construct survival at day 14 and average days to failure were 81.25% and 10.0 days for 100k cells, 65.15% and 10.5 days for 300k, and 68.91% and 8.5 days for 500k, respectively. Average cross-sectional area was calculated at 0.30 mm, 0.33 mm, and 0.29 mm for 100k, 300k, and 500k, respectively.
Five biological replicates with a minimum of
Discussion
We were able to optimize the original construct protocols for use with horse cells. Original protocols relied on chicken, mouse, and human cells [14], [15], [16], [17], [18], [19], [20]. Although protocols for other species used silk suture [14], [18], [19], [20], we used the brushite anchors [16], [17] for horse tenocytes. From our findings, we were able to characterize several aspects of the 3D equine tendon construct protocol. For varying cell density, construct survivability to 14 days was
Acknowledgments
The authors thank their funding support and the University of California—Davis TEAM Biomedical Engineering Facility for assistance with design and printing of model molds. Sources of funding were Henry A. Jastro Graduate Research Award, College of Agriculture and Environmental Sciences, Animal Biology Graduate Group, University of California—Davis; University of California—Davis, Center for Equine Health; UC Davis Agricultural Experiment Station.
Financial disclosure: We have no financial
References (31)
Injuries of the flexor tendons: focus on the superficial digital flexor tendon
Clin Tech Equine Pract
(2007)- et al.
The pathobiology and repair tendon and ligament injury
Vet Clin North Am Equine Pract
(1994) - et al.
Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3
Matrix Biol
(2010) - et al.
Tension is required for fibripositor formation
Matrix Biol
(2008) The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid
Arch Biochem Biophys
(1961)- et al.
Modified assay for determination of hydroxyproline in a tissue hydrolyzate
Clin Chim Acta
(1980) - et al.
Glycosaminoglycans in tendon physiology, pathophysiology, and therapy
Bioconjug Chem
(2015) - et al.
Fatal musculoskeletal injuries of quarter horse racehorses: 314 cases (1990-2007)
J Am Vet Med Assoc
(2012) - et al.
Causes of death in racehorses over a 2 year period
Equine Vet J
(1994) - et al.
A pilot study on factors influencing the career of Dutch sport horses
Equine Vet J Suppl
(2010)
Diseases and problems of tendons, ligaments, and tendon sheaths
Superficial digital flexor tendonitis in the horse
Equine Vet J
A review of tendon injury: why is the equine superficial digital flexor tendon most at risk?
Equine Vet J
Extracellular matrix remodeling: the role of matrix metalloproteinases
J Pathol
Mechanical properties of the equine superficial digital flexor tendon relate to specific collagen cross-link levels
Equine Vet J Suppl
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Animal welfare/ethical statement: Samples were collected from euthanized horses that were euthanized for reasons other than this study; thus IACUC exempted the tissue collection protocol.
Conflict of interest statement: The authors have no competing interests.