The Effect of NaHCO3 on the Growth of Tissue Engineered Cartilage in a Continuous Flow Bioreactor
1,2
Khan, A A; +1,2,Waldman, S D
+1Department of Chemical Engineering, Queen's University, Canada, 2Human Mobility Research Centre, Kingston General Hospital, Canada
[email protected]
INTRODUCTION
The use of bioreactors for cartilage tissue engineering has become
increasing important as traditional batch-fed (static) culture is not
optimal for tissue growth in vitro. We have previously reported on the
use of a continuous flow bioreactor that improves tissue growth by
supplying the cells with a near infinite supply of media [1]. As the
continuous flow of fresh media also helped to maintain extracellular pH
in the reactor, it was postulated that this effect could be maximized with
the use of additional buffering agents (e.g. NaHCO3) to media. NaHCO3
was chosen as it has been shown to be an effective means to maintain
media pH and stimulate ECM synthesis by chondrocytes [2, 3] in static
culture,
METHODS
Cell Isolation and High Density 3D Culture: Cartilaginous tissue
constructs were generated from isolated chondrocytes harvested from
the metacarpal joints of 12-18 month old calves, under sterile conditions
and by enzymatic digestion. The cells were seeded on the surface of type
II collagen-coated Millicell™ filters in high-density 3D culture (2 x 106
cells/filter). Cultures were maintained in Ham’s F12 media
supplemented with 25 mM HEPES, 20% FBS and 100 µg/mL ascorbate.
After two weeks of culture, the constructs were cultivated in a
continuous flow bioreactor [1] under a media flow rate of 10 µL/min
with or without 14 mM NaHCO3 added to the media for a period of 5
weeks. Control cultures were maintained in static, no-flow culture with
or without additional NaHCO3 for the same period of time. All cultures
were housed in an incubator maintained at 37°C and 95% relative
humidity supplemented with 5% CO2. At the end of the culture period,
the pH of the conditioned media of each of the culture conditions was
determined.
Determination of ECM Formation and Thickness: After the 5 weeks
culture period, tissue constructs were harvested and the thickness of the
developed tissue was measured using a needle probe method [4]. Tissues
were then digested with papain for 48 hours at 65ºC and stored at -20°C
until analysis. Proteoglycan content was estimated by quantifying the
amount of sulphated glycosaminoglycans using the dimethylmethylene
blue dye binding assay [5]. To determine the collagen content, aliquots
of the papain digest were hydrolyzed in 6 N HCl at 110ºC for 18 hours.
Hydroxyproline content of the hydrolyzate was determined using
chloramine-T/Ehrlich’s reagent assay [6]. DNA content was determined
using the Hoechst dye 33258 assay [7].
Histological and Immunohistochemical Assessment: Upon harvest,
representative tissue constructs were fixed overnight in 4%
paraformaldehyde and embedded in paraffin. Sections were cut (5 µm)
and stained with hemotoxylin-eosin (H&E) or, toluidine blue (TB).
Immunohistochemistry with antibodies against type II was performed
according to a standard ABC protocol (Vector Laboratories Inc.) with
diaminobenzidine for colour development [8].
Statistical Analyses: All data is presented as mean ± standard error
(SEM). Data was analyzed using a one-way ANOVA and the Fisher’s
LSD post-hoc test, A sample size of n = 6 was used for each condition.
RESULTS
Effect on Buffering on ECM Accumulation, Cellularity and Tissue
Thickness: Media supplemented with NaHCO3 significantly affected the
growth of cartilaginous tissue in both traditional batch-fed (static) and
bioreactor culture (Table 1). This effect was maximized when NaHCO3
containing media was used in the continuous flow bioreactor. Tissue
cultivated under these conditions contained the highest amount of
collagen and proteoglycans (5-fold and 18-fold increase over static
control, respectively). As well, these cultures were substantially thicker
(static: 340 ± 17; static + NaHCO3: 480 ± 33; bioreactor: 777 ± 200;
bioreactor + NaHCO3: 1175 ± 330 µm). Similarly, the presence of
NaHCO3 appeared to induce a proliferative effect which was also
maximized under the continuous flow of media (3.7-fold increase in
DNA content compared to static control).
Effect of Buffering on Extracellular pH: The presence of NaHCO3
helped to regulate the pH of the media (static: 6.6 – 6.7; static +
NaHCO3: 6.7 – 6.9; bioreactor: 6.7 – 6.8; bioreactor + NaHCO3: 6.9 –
7.0). This effect was also greatest in the bioreactor and had extracellular
pH values closest to neutral pH.
Bioreactor
+ NaHCO3
– NaHCO3
Dry weight
DNA
Collagen
Proteoglycans
3.2* ± 0.3
3.7* ± 0.4
5.4* ± 1.0
18.1* ± 3.2
2.2* ± 0.1
1.9 ± 0.3
3.5* ± 0.2
7.1* ± 0.5
Static
+ NaHCO3
– NaHCO3
1.4 ± 0.2
1.9 ± 0.4
2.3* ± 0.4
4.2* ± 1.0
1.0 ± 0.08
1.0 ± 0.11
1.0 ± 0.06
1.0 ± 0.16
* significantly different from all other groups (p<0.01)
Table 1: Effect of NaHCO3 and Culture Type on Tissue Formation (all
values normalized to static control without NaHCO3)
Histology and Immunohistochemisty Assessment: Although all of the
developed tissues stained positive for sulfated proteoglycans (Figure 1 e,
f,g,h), tissues cultured in the presence of NaHCO3 appeared to display
depth-dependent staining that was increasingly more intense at the
bottom of the culture (Figure 1 e,g). This was in contrast to the control,
which displayed more uniform proteoglycan staining (Figure 1 h).
Tissues cultivated in the bioreactor stained positive for type II collagen
throughout the entire ECM (Figure 1 i) whereas the static cultures
appeared to stained more intensively at the tissue surface (Figure 1 j).
Figure 1: Histology and Immunohistochemical Assessment
DISCUSSION
The presence of NaHCO3 in the culture media has been shown to have
the ability to maintain extracellular pH and stimulate cartilaginous ECM
production in static culture [2,3]. As our previously described
continuous flow bioreactor also improves tissue growth by helping to
maintain extracellular pH [1], we postulated whether the combination of
bioreactor culture and media containing NaHCO3 would further
stimulate cartilaginous tissue growth in vitro. Not only did these cultures
accumulate substantially more ECM (5- to 18-fold increase), they also
were similar in thickness of native bovine cartilage [9]. Even though the
presence of NaHCO3 helped to maintain media pH in both static and
bioreactor culture, the effect of NaHCO3 on media pH was minimal
(within 0.1 – 0.2 pH). While only relatively small changes in media pH
are required to affect matrix synthesis [2,3], the improved tissue growth
observed may also be a result of changes in intracellular pH. Previous
studies have demonstrated that NaHCO3 can also regulate the
intracellular pH of chondrocytes as NaHCO3 can be transported into the
cells by electroneutral channels [10]. Future studies will be directed at
determining chondrocyte intracellular pH under these different culturing
conditions.
REFERENCES
[1] Khan et al., Biotechnol Progess, 2009, 25: 508-515.
[2] Waldman et al., Tissue Eng, 2004, 10: 1633-1640.
[3] Xu et al., Osteoarthritis Cartilage, 2007, 15(4) : 396-402.
[4] Hoch et al., JOR, 1993, 1: 4-12.
[5] Farndale et al., BBA, 1986, 863: 173-177.
[6] Woesner et al., Arch Biochem Biophys, 1961, 93: 440-447.
[7] Kim et al., JBC, 1972, 251: 6210-6217.
[8] Poole et al., J Histochem Cytochem, 1991, 39: 1175-1187.
[9] Waldman et al., JBMR, 2003, 62: 323-330.
[10] Sterling et al., Biochem J, 1999. 344: 221-229.
Poster No. 1317 • 56th Annual Meeting of the Orthopaedic Research Society