Copyright © 2009 by ASME 1
The annulus fibrosus (AF) of the intervertebral
disc is a multi$lamellar fibrocartilage that, together with the nucleus
pulposus, confers mechanical support and flexibility to the spine.
Function of the AF is predicated on a high degree of structural
organization over multiple length scales: aligned collagen fibers reside
within each lamella, and the direction of alignment alternates between
adjacent lamellae from +30
o
to $30
o
with respect to the transverse axis
of the spine. Electrospinning permits fabrication of scaffolds
consisting of aligned arrays of nanofibers, and has proven effective for
directing the alignment of both cells and extracellular matrix (ECM)
deposition [1$3]. We recently employed electrospinning to engineer
the primary functional unit of the AF, a single lamella [4]. However, it
remains a challenge to engineer a multi$lamellar tissue that replicates
the cross$ply fiber architecture of the native AF. Moreover, relatively
few studies have considered functional properties of engineered AF,
and, when measured, tensile properties of these constructs have been
inferior to native AF [4]. In this study, mesenchymal stem cells
(MSCs) were seeded onto aligned nanofibrous scaffolds organized into
bi$lamellar constructs with opposing or parallel fiber orientations, and
their functional maturation was evaluated with time. Additionally, we
determined a novel role for lamellar ECM in reinforcing the
tensile response of bilayers, and confirmed this mechanism by testing
acellular bilayers with controllable interface properties.
Poly(ε$caprolactone) (PCL) was electrospun
as described [3] to generate an aligned nanofibrous sheet 250 8m
thick. Scaffolds (5 x 30 mm
2
) were excised with the long axis rotated
30
o
from the fiber direction (Fig. 1A).
Scaffolds were coated in fibronectin, seeded with bovine
bone marrow derived MSCs and cultured for 2 weeks in a chemically
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defined growth media containing 10 ng/ml TGF$β3 [1]. Samples were
then assembled into bilayers with either parallel (+30
o
/+30
o
) or
opposing (±30
o
) fiber orientations and cultured between pieces of
porous polypropylene (PP) wrapped with foil (F) (Fig. 1B) [5]. After 2
weeks, PP and F were removed. . ’ At 2, 4, 6
and 10 weeks samples tested in uniaxial tension (n=5) followed by
biochemical analyses to determine glycosaminoglycan (GAG),
collagen, and DNA contents [1]. Modulus was computed from the
slope of the linear portion of the stress$strain curve. Additional
samples (n=3) were cryotomed to 8 Dm across the fiber plane, and
stained with Picrosirius Red (PR). Polarized light microscopy was
used to visualize and quantify collagen alignment [6]. (/
’ An acellular study was carried out to relate the tensile
behavior of nanofibrous bilayers to the properties of the interlamellar
matrix. Aligned PCL scaffolds ~1 mm thick were fabricated (as above)
and formed into bilayers by applying molten agarose to the
interlamellar space and allowing it to set. Agarose concentration was
varied to alter material properties of the interface [7,8]. Parallel and
opposing bilayers were tested in uniaxial tension as above, for agarose
concentrations of 2, 4, 5, and 6 % in PBS. ’’ Statistical
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