Multilayered electrospun scaffolds for tendon tissue engineering

Abstract

Full-thickness rotator cuff tears are one of the most common causes of shoulder pain in people over the age of 65. High retear rates and poor functional outcomes are common after surgical repair, and currently available extracellular matrix scaffold patches have limited abilities to enhance new tendon formation. In this regard, tissue-engineered scaffolds may provide a means to improve repair of rotator cuff tears. Electrospinning provides a versatile method for creating nanofibrous scaffolds with controlled architectures, but several challenges remain in its application to tissue engineering, such as cell infiltration through the full thickness of the scaffold as well as control of cell growth and differentiation. Previous studies have shown that ligament-derived extracellular matrix may enhance differentiation toward a tendon or ligament phenotype by human adipose stem cells (hASCs). In this study, we investigated the use of tendon-derived extracellular matrix (TDM)-coated electrospun multilayered scaffolds compared to fibronectin (FN) or phosphate-buffered saline (PBS) coating for use in rotator cuff tendon tissue engineering. Multilayered poly(ɛ-caprolactone) scaffolds were prepared by sequentially collecting electrospun layers onto the surface of a grounded saline solution into a single scaffold. Scaffolds were then coated with TDM, FN, or PBS and seeded with hASCs. Scaffolds were maintained without exogenous growth factors for 28 days in culture and evaluated for protein content (by immunofluorescence and biochemical assay), markers of tendon differentiation, and tensile mechanical properties. The collagen content was greatest by day 28 in TDM-scaffolds. Gene expression of type I collagen, decorin, and tenascin C increased over time, with no effect of scaffold coating. Sulfated glycosaminoglycan and dsDNA contents increased over time in culture, but there was no effect of scaffold coating. The Young's modulus did not change over time, but yield strain increased with time in culture. Histology demonstrated cell infiltration through the full thickness of all scaffolds and immunofluorescence demonstrated greater expression of type I, but not type III collagen through the full thickness of the scaffold in TDM-scaffolds compared to other treatment groups. Together, these data suggest that nonaligned multilayered electrospun scaffolds permit tenogenic differentiation by hASCs and that TDM may promote some aspects of this differentiation.

FIG. 1.

Scanning electron micrographs of edge (A, B) and surface (C, D) of unseeded (A, C) scaffolds, and phosphate-buffered saline (PBS)-coated scaffolds seeded with human adipose stem cells and cultured for 28 days (B, D).

FIG. 2.

DNA (A), sulfated glycosaminoglycan (s-GAG) (B), and collagen (C) content (normalized to dry weight) of tendon-derived extracellular matrix (TDM)-, fibronectin (FN)-, and PBS-coated multilayered electrospun scaffolds at day 0 (unseeded), 1, 14, and 28, after seeding with human adipose-derived stem cells. Groups having different letters are significantly different from each other (p≤0.05, n=5).

FIG. 3.

Expression of type 1 collagen (COL1A1), type III collagen (COL3A1), decorin (DCN), and tenascin C (TNC) by human adipose-derived stem cells after seeding on TDM-, FN-, and PBS-coated multilayered electrospun scaffolds at day 4, 7, and 14 of culture, normalized to 18S expression and unseeded cell pellets at day 0. Groups having different letters are significantly different from each other (p≤0.05, n=5).

FIG. 4.

Immunofluorescence of human type I collagen of (−) unseeded and (+) human adipose-derived stem cell-seeded TDM-, FN-, and PBS-coated multilayered, electrospun scaffolds cultured for 28 days. Scale bar=50 μm. Color images available online at www.liebertpub.com/tea

FIG. 5.

Immunofluorescence of human type III collagen of (−) unseeded and (+) human adipose-derived stem cell-seeded TDM-, FN-, and PBS-coated multilayered, electrospun scaffolds cultured for 28 days. Scale bar=50 μm. Color images available online at www.liebertpub.com/tea

FIG. 6.

Safranin O/fast green (A, B) and hematoxylin/eosin (C, D) micrographs of unseeded (A, C) and human adipose-derived stem cell-seeded (B, D) TDM-, FN-, and PBS-coated multilayered, electrospun scaffolds cultured for 28 days. Scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 7.

The Young's modulus of linear region (A), yield strain (B), and yield stress (C) of human adipose-derived stem-cell seeded TDM-, FN-, and PBS-coated multilayered electrospun scaffolds at day 0 and after 28 days of culture. Groups having different letters are significantly different from each other (p≤0.05, n=5).