Bioreactors - Articular Cartilage Tissue Engineering - page 68

68 CHAPTER 4. BIOREACTORS
systems was a reduction in shear force, since high or even moderate levels of shear are undesirable
in the formation of hyaline cartilage. A rotating fluid environment was found to be the best way to
produce a low-shear, high-mass transfer bioreactor [ 460 , 549 - 552 ].
The rotating bioreactor is capable of adjusting shear levels associated with fluid/construct
interaction because of its unique design. Shear forces can induce either positive or negative responses
from cultured cells, and a threshold level of 0.092 Pa seems to demarcate that point for rotating
bioreactor systems [ 542 ]. Constructs cultured in this environment remain suspended in the media
by two forces: gravity and fluid flow. Samples 'fall' through the media while rotating fluid flow acts in
the upward direction, keeping the samples suspended but also exerting a slight shear force. Altering
the rotation rate of the vessel can effect different flow lines and shear environments around the
sample. Low-shear environments are typically produced by slowly rotating both the inner and outer
cylinders at nearly the same rate. Initial experiments used cell-seeded microcarriers to investigate
the effect of “microgravity” on cell growth and development [ 542 , 552 , 553 ]. Over time in culture,
the microcarriers slowly aggregated to form larger cell-matrix constructs [ 543 , 554 ]. Subsequent
experiments with larger constructs showed that the samples tended to remain near the ends of the
media chamber, not distributing as widely as smaller particles. Because of this, newer versions of the
rotating bioreactor (Synthecon, Inc.) have altered the aspect ratio of the media chamber, producing
an environment more conducive to culturing large constructs [ 554 ]. The culture environment is not
ideal, though, since the flow patterns inside the bioreactor tend to slowly tumble large constructs
through the media; an action that introduces higher shear levels caused by turbulent fluid motion
across the construct surface [ 553 ]. Small-amplitude, long-period oscillations in the fluid-wake at
this interface may be the source of mechanical stimuli felt by the cells [ 549 ]. Additionally, the
magnitude and direction of shear on the construct constantly changes, which might be good (dynamic
mechanical stimulus) or bad (un-definable forces). The stress exerted on a construct in a bioreactor
rotating at 19 rpm was calculated to be 0.15 Pa (1.5 dyne/cm 2 )[ 549 ], which is significantly higher ∼

0.0005 Pa (0.005 dyne/cm 2 ) measured for microcarrier beads in the same
environment [ 552 ]. However, this shear stress is still significantly lower than many other fluid flow
bioreactors.
Published results show wide use of rotating bioreactors for cartilage tissue engineering. Past
findings have shown that rotating bioreactors produce higher fractions of glycosaminoglycans and
collagen than mixed flasks or static culture [ 353 , 551 ]. Constructs cultured for six weeks produced tis-
sue that had glycosaminoglycan and total collagen compositions that were 68% and 33%, respectively,
of native cartilage levels [ 353 ]. Similar results were obtained in a subsequent study, with engineered
constructs accumulating 75% of native glycosaminoglycan and 39% of native type II collagen compo-
sitions [ 551 ]. Additionally, extending culture to seven months increased glycosaminoglycan content
beyond physiological levels although collagen remained at 39%. The accumulation of matrix also
affected the mechanical properties, with equilibrium moduli (950 kPa) and hydraulic permeability
(5

than the shear level of ∼

10 − 15 m 4 /N-s) reaching values comparable to healthy cartilage. ×