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

https://doi.org/10.1016/j.jevs.2018.10.011Get rights and content

Highlights

  • Tendon constructs were made by seeding cells in fibrinogen/thrombin matrix.

  • Three-dimensional constructs were anchored by phosphate-rich brushite anchors.

  • Effective cell seeding densities included 100,000–500,000 cells per construct.

  • Consideration of seeding technique was essential to tendon construct generation.

  • Tendon constructs can be used as in vitro models for testing repair strategies.

Abstract

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.

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

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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.

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