Rest Periods during Bioreactor Culture Restore Mechanosensitivity and Enhance Osteogenesis
+1,2 Partap, S; 1,2 Plunkett, N A; 2 Kelly, D J; 1,2O’Brien, F J
+1Department of Anatomy, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland, 2Trinity Centre for Bioengineering,
Department of Mechanical Engineering, Trinity College, Dublin, Dublin 2, Ireland
[email protected]
INTRODUCTION
Bioreactors can be used to influence biological processes by the
application of a mechanical stimulus. In particular, osteoblasts have been
shown to respond to mechanical stimulation in the form of flow
perfusion. However, after extended periods of stimulation they lose their
ability to respond and become densensitized to the stimulus. In a
previous study in our laboratory, the application of different dynamic
flow profiles using a flow perfusion bioreactor was shown to have a
positive stimulatory effect on osteoblasts seeded on collagenglycosaminoglycan (CG) scaffolds following up to 49 hrs stimulation
[1,2]. Mechanosensitivity has been shown to be restored by the
introduction of rest periods between bouts of loading [3]; therefore, the
aim of this study was to analyse the effect of short and long term restinsertion on the response of osteoblasts seeded on CG scaffolds in the
same flow perfusion system over culture periods of up to 14 days.
METHODS
Scaffold samples were seeded with 2x106 MC3T3-E1 pre-osteoblast
cells. Constructs were pre-cultured in static conditions (37°C, 5% CO2)
for 6 days after which point the constructs were cultured in either the
flow perfusion bioreactor or static culture [1]. The constructs were
stimulated with a flow pattern incorporating both short- and long-term
rest periods. Short-term periods of no flow were incorporated into 1 hr
bouts of stimulation. They were of duration: 0 (steady flow group) or 5,
10 and 15 seconds (rest-inserted groups) and were inserted between
bouts of 10 seconds of 1 mL/min flow. This hr of stimulation was
followed by a 7 hr long-term rest period. This 8 hr cycle was repeated
for the duration of the culture period.
Cellular spatial distribution, cell number and expression of a number
of genes including Cyclooxygenase-2 (COX-2), collagen I (COL-1),
alkaline phosphatase (ALP) and osteopontin (OPN) were then assessed
for each construct at each time point using Haematoxylin & Eosin
staining, Hoechst DNA assay and Real Time Polymerase Chain
Reaction (RT-PCR) respectively. In addition, a PGE2 enzyme assay was
used to look at PGE2 concentration. Results are expressed as mean ±
standard deviations (SD). A general linear model ANOVA with the
Holm-Sidak post-hoc multiple comparison test was used. Statistical
significance was taken at p<0.05.
RESULTS
Significant changes in the expression of genes associated with bone
formation were observed in response to the different stimulation patterns
used. COL-1 expression and ALP expression decreased over time (from
1 hr to 14 days) for all groups. However, OPN expression increased due
to bioreactor culture for the 10 second rest-inserted group over the 14
day time period when compared to the static control and steady flow
group (figure 1).
(a)
(b)
(c)
Figure 2 (a) Cell number up to 14 days, * represents p≤0.025.
Transverse H&E images of constructs cultured for 14 days in (b) the
flow perfusion bioreactor using rest-inserted flow and (c) static
conditions. Note the formation of an external capsule on the static group
as highlighted by the arrow.
DISCUSSION
The higher levels of OPN expression which were observed on the 10
second rest-inserted group compared to the static control group
(p≤0.017) and steady flow group (p≤0.025) is a trend that has also been
observed using rest-inserted flow in 2-D culture [4]. The flow groups
had a significantly lower expression of COL-1 compared to the static
control group (p≤0.025), whereas there was no significant difference in
ALP expression between any of the groups. COL-1 is expressed during
proliferation in 2-D culture, is downregulated post-proliferatively but is
continuously expressed at low levels throughout osteoblast
differentiation and maturation. Taken together, increased OPN
expression coupled with decreased COL-1 and ALP expression may
indicate that bioreactor culture has enhanced expression of post
proliferative genes at the expense of those found during proliferation on
CG constructs.
Additionally, reduced cell numbers were seen on constructs cultured
in the bioreactor, albeit with a more homogenous cell distribution
compared to constructs cultured statically, which was attributed to flow
induced cellular loss that occurred preferentially from the edges and
surfaces of constructs. Thus, bioreactor culture may also overcome
problems associated with static culture conditions where cells tend to
concentrate on the construct periphery (‘encapsulation effect’) which
leads to poor nutrient and waste exchange, limited cell viability and core
degradation that effects the resulting mechanical properties of the
engineered tissue [5]. Therefore, the decrease in cell number seen under
bioreactor culture may potentially be of benefit to tissue development.
These results indicate that the insertion of short term rest periods
during flow enhances cellular distribution and osteogenic responses on
CG constructs cultured in a flow perfusion bioreactor which is
promising for the development of homogeneous bone graft substitutes.
REFERENCES
Figure 1 Gene expression of Osteopontin (OPN), * represents p≤0.017
and ** represents p≤0.025
Higher cell numbers were observed on statically cultured constructs
(ca. 2 x 106) compared to the flow groups (ca. 1 x 106) (figure 2a);
however, cell distribution was improved on all flow groups while static
culture controls exhibited peripheral encapsulation (figure 2b and c).
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ACKNOWLEDGEMENTS
Science Foundation Ireland (President of Ireland Young Researcher Award,
04/Yl1/B531), Research Frontiers Programme (07/RFP/ENMF142) and the Irish
Research Council for Science, Engineering and Technology (RS/2005/173).
Paper No. 377 • 56th Annual Meeting of the Orthopaedic Research Society