Engineering Fibrous Tissues and Their Interfaces with Bone - Structural Interfaces and Attachments in Biology - page 327

Cells can respond differently when cultured in a two-dimensional (2D) or a
three-dimensional (3D) system [ 126 ]. 2D culture allows for confluent and uniform
cell seeding, but does not create an environment similar to the native tissue
environment, while 3D cultures can more closely mimic natural tissue architec-
ture. In a bioreactor, a 3D environment allows mechanical stimulation to be
uniform across and through the scaffold. Mechanical stimulation in the form of
cyclic tensile stress has been recently identified as a critical component in
the tissue engineering approach for tendon and ligament regeneration [ 127 ].
ThewidelyusedTissueTrain ® culture system is a method for culture that
provides tensile loading of cells in a 3D matrix or hydrogel [ 128 ]. The system
consists of a loading well plate to first seed the matrix or gel and cells on a flexible
membrane. The loading well plate is removed and a loading post is attached.
When a vacuum is applied to the loading post, the membrane deforms the sides of
the flexible membrane and, thus, the construct undergoes uniaxial strain [ 129 ]. In
one study using this system for tendon/ligament applications, avian tendon
fibroblasts were suspended in a collagen type I gel in each well of the culture
plates. The loading regimen consisted of uniaxial cyclic strain of 1% at 1 Hz for
1 hour/day up to 11 days. Histological staining revealed cells aligned in the
direction of the axial load, similar towhatisfoundinnativetendontissue.
Mechanical testing also revealed that the elastic modulus increased over time
with strain and the ultimate tensile strength was significantly higher than that of
the non-loaded controls [ 129 ].
In other experiments using bioreactors for tensile stimulation, human patellar
tendon fibroblasts were cultured and seeded on micro-grooved silicone surfaces and
subject to uniaxial stretch at 0, 4, and 8% at 0.5 Hz for 4 h. After stretching, cells
were incubated statically for 4 hours and then analysis was performed. RT-PCR
results revealed that stretching induced increased gene expression of collagen type I
and collagen type III. Enzyme-linked immunosorbent assays (ELISA) of proteins
released into the media also indicated increases in collagen type I in the media for
stretched cells [ 122 ]. The results of this study confirm that strain is necessary for
fibroblast ECM protein expression. The stimulation of cyclic loading is also
necessary for fibroblasts to adopt an elongated spindle shape that is seen in native
fibroblasts [ 123 , 130 ].
MSCs under mechanical strain have also been shown to increase fibrous tissue
ECM gene expression and protein production [ 27 , 115 , 127 , 131 , 132 ]. In one study
that applied a 10% translational followed by a 25% rotational strain on collagen
gels seeded with bone marrow-derived cells for 14 days, real time RT-PCR results
indicated collagen type I, collagen type III, and tenascin-C were upregulated
in strained samples when compared to the baseline measurements of the control
group and immunostaining images detected the presence of collagen type I and III.
In addition, mechanically stimulated constructs did not show an upregulation
of typical markers for bone and cartilage (i.e., bone sialoprotein and collagen
type II) [ 132 ].