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Research Papers

Static and Cyclic Mechanical Loading of Mesenchymal Stem Cells on Elastomeric, Electrospun Polyurethane Meshes

[+] Author and Article Information
Robyn D. Cardwell, Patrick S. Thayer

Department of Biomedical Engineering,
Virginia Tech,
Blacksburg, VA 24061

Jonathan A. Kluge, David L. Kaplan

Department of Biomedical Engineering,
Tufts University,
Medford, MA 02155

Scott A. Guelcher

Chemical and Biomolecular Engineering,
Vanderbilt University,
Nashville, TN 37235

Linda A. Dahlgren

Large Animal Clinical Sciences,
Virginia-Maryland
Regional College of Veterinary Medicine,
Blacksburg, VA 24061

Aaron S. Goldstein

Department of Biomedical Engineering,
Virginia Tech,
Blacksburg, VA 24061
Department of Chemical Engineering,
Virginia Tech,
Blacksburg, VA 24061
e-mail: goldst@vt.edu

1Corresponding author.

Manuscript received May 30, 2014; final manuscript received April 14, 2015; published online June 3, 2015. Assoc. Editor: Carlijn V. C. Bouten.

J Biomech Eng 137(7), 071010 (Jul 01, 2015) (8 pages) Paper No: BIO-14-1238; doi: 10.1115/1.4030404 History: Received May 30, 2014; Revised April 14, 2015; Online June 03, 2015

Biomaterial substrates composed of semi-aligned electrospun fibers are attractive supports for the regeneration of connective tissues because the fibers are durable under cyclic tensile loads and can guide cell adhesion, orientation, and gene expression. Previous studies on supported electrospun substrates have shown that both fiber diameter and mechanical deformation can independently influence cell morphology and gene expression. However, no studies have examined the effect of mechanical deformation and fiber diameter on unsupported meshes. Semi-aligned large (1.75 μm) and small (0.60 μm) diameter fiber meshes were prepared from degradable elastomeric poly(esterurethane urea) (PEUUR) meshes and characterized by tensile testing and scanning electron microscopy (SEM). Next, unsupported meshes were aligned between custom grips (with the stretch axis oriented parallel to axis of fiber alignment), seeded with C3H10T1/2 cells, and subjected to a static load (50 mN, adjusted daily), a cyclic load (4% strain at 0.25 Hz for 30 min, followed by a static tensile loading of 50 mN, daily), or no load. After 3 days of mechanical stimulation, confocal imaging was used to characterize cell shape, while measurements of deoxyribonucleic acid (DNA) content and messenger ribonucleic acid (mRNA) expression were used to characterize cell retention on unsupported meshes and expression of the connective tissue phenotype. Mechanical testing confirmed that these materials deform elastically to at least 10%. Cells adhered to unsupported meshes under all conditions and aligned with the direction of fiber orientation. Application of static and cyclic loads increased cell alignment. Cell density and mRNA expression of connective tissue proteins were not statistically different between experimental groups. However, on large diameter fiber meshes, static loading slightly elevated tenomodulin expression relative to the no load group, and tenascin-C and tenomodulin expression relative to the cyclic load group. These results demonstrate the feasibility of maintaining cell adhesion and alignment on semi-aligned fibrous elastomeric substrates under different mechanical conditions. The study confirms that cell morphology is sensitive to the mechanical environment and suggests that expression of select connective tissue genes may be enhanced on large diameter fiber meshes under static tensile loads.

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Figures

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Fig. 1

Bioreactor components and operations. (a) Individual mesh between a pair of custom-designed polyetherimide grips and laid over a cotton pad. (b) Troughs to seed multiple meshes and maintain tension. (c) Diagram of the assembled bioreactor [25]. (d) Diagram of the mechanical stimulation time-course. For the no load group (1), meshes were hung slack. For the static load group (2), meshes were strained to 50 mN each day. For the cyclic load group (3), meshes were strained to 50 mN each day and then cyclically strained to an additional 4% at 0.25 Hz for 30 min.

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Fig. 2

Electron micrographs of electrospun segmented polyurethane meshes. Meshes had mean fiber diameters of: (a) 0.60 μm and (b) 1.74 μm. Scale bars correspond to 10 μm.

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Fig. 3

Representative stress–strain curves for PEUUR electrospun meshes tested wet at room temperature. (a) Strain to failure testing of meshes with mean fiber diameters of 0.60 μm (small, gray trace) and 1.74 μm (large, black trace). Cyclic loading of meshes with: (b) a mean fiber diameter of 1.74 μm (large) and (c) a mean fiber diameter of 0.60 μm (small). Meshes were preloaded to 50 mN at a strain rate of 0.1% s−1 and then cyclically stretched between 0% and 4% elongation at 0.25 Hz for 30 min (450 cycles). Traces for the first (black) and last cycles (gray) are shown.

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Fig. 4

Cell density on electrospun PEUUR meshes after 3 days of mechanical treatment. Bars correspond to the mean ± standard deviation for n = 2 samples, except for small diameter fiber, static load, where n = 1.

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Fig. 5

Confocal images of C3H10T1/2 cells following 3 days of: (a) and (d) no load, (b) and (e) static load, or (c) and (f) cyclic load on meshes with either a small (0.60 μm) ((a)–(c)) or large (1.74 μm) ((d)–(f)) mean fiber diameter. Scale bars correspond to 300 μm.

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Fig. 6

Histogram of the distribution of cell orientations relative to the mean for: (a)–(c) small and (d)–(f) large diameter fiber meshes. Orientations were determined after the third daily application of mechanical stimulation and panels correspond to: (a) and (d) no load, (b) and (e) static load, and (c) and (f) cyclic load. Curve corresponds to the best fit of a wrapped normal distribution based on angular standard deviations of: (a) 18.8 deg, (b) 14.8 deg, (c) 13.9 deg, (d) 22.9 deg, (e) 13.9 deg, and (f) 11.3 deg. (These numbers are plotted in Fig. S2(c) (Supplemental figures are available under the “Supplemental Data” tab for this paper on the ASME Digital Collection)).

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Fig. 7

Relative gene expression of tendon/ligament primary collagens ((a) and (b)), tendon/ligament matrix accessory genes ((c) and (d)), and tendon/ligament selective genes ((e) and (f)) after 3 days of no load, static load, or cyclic load culture on electrospun polyurethane meshes with mean fiber diameter size of 0.60 μm (small) or 1.74 μm (large). Bars correspond to the mean ± SEM for n = 4 for all groups except for small fiber diameter meshes group, static load, where n = 2.

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Fig. 8

SEM images of C3H10T1/2 cells on: (a)–(c) small diameter and (d)–(f) large diameter electrospun polyurethane meshes after 3 days of: (a) and (d) no load, (b) and (e) static load, or (c) and (f) cyclic load. Scale bars correspond to 20 μm.

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