CHONDROGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS

IN CHITOSAN-BASED FIBER MESHES USING A FLOW PERFUSION BIOREACTOR

University of Minho
School of Engineering
MARTA L. ALVES DA SILVA*
3B´S Research Group Supervisors: Nuno M. Neves and Rui L. Reis
* msilva@dep.uminho.pt

Introduction Results and discussion Results and discussion

Bioreactor Static
Human Bone Marrow Stromal Cells (hBMSCs) are multipotent cells Proliferation (DNA quantification) SEM analysis
present in several mesenchymal tissues. hMSCs can be differentiated
into several cell lineages, including chondrogenic. Native articular
cartilage is subjected to synovial fluid flow during normal joint ØBioreactor samples
function. Thus, it is believed that the morphogenesis of articular In terms of DNA quantification, observation shows that cells
7D 7D

cartilage may be positively regulated by the application of similar no significant difference

stimulation in vitro. We studied the effect of the stimulation provided acquired a round-shaped
between static and bioreactor morphology at early stages of
by a flow perfusion bioreactor in the chondrogenic differentiation of
human MSCs when seeded onto fiber meshes scaffolds, consisting of cultures was found at 14 days culture (i.e. 7 and 14 days)
a blend of 50/50 chitosan and Poly(Butylene Terephtalate Adipate)– (p=0.18), but static cultures 14D 14D

CPBTA. We intended to ascertain if shear stress was capable of displayed a significantly higher ØCells cultured in static
augmenting the differentiation process. DNA quantification than the conditions are stretched and
bioreactor ones at 21 and 28 flat until at least 21 days of
days of culture (p=0.002). culture 21D 21D
Figure 1 - DNA quantification in static
and bioreactor cultures. Data analyzed
Materials and Methods
by nonparametric way of a Mann–
hBMSCs Isolation and Characterization Whitney U-test (* p<0.01, vs Static).
28D 28D

Cartilage-related genes
ECM localization
Spin DMEM
Bioreactor Static ØAggrecan, Collagen type II
Flow cytometry analysis and Sox9 were expressed in
Characterization of hBMSCs Stemness: both cultures
Ø

H&E
ØPhenotypic analysis: CD43-; CD45- ØhBMSCs cultured ØStatic cultures expressed a
D29+;CD44+;CD73+;CD90+;CD105+;CD106+ under perfusion were significant difference in the
ØDifferentiation potential for several lineages, 50μm
able to produce expression of Collagen Type I
proteoglycans Figure 4 – Bar plots of chondrogenic markers
including chondrogenesis Toluidine blue staining of
ECM
Safranin O characteristic of fibrotic normalized for the reference gene, GAPDH.
Ø
cartilage Data were analyzed with a Mann–Whitney U
test (* p<0.01, vs static culture conditions).
Scaffold production
ØhBMSCs cultured in
ØCPBTA fibers were obtained using a single screw micro-extruder static conditions also Conclusions
coupled to a capillary die. Fiber bonded meshes were cut with a produced We found that shear stress has a beneficial effect on the
Toluidine Blue

circular die. proteoglycans, but in chondrogenic differentiation of hMSCs. Thus, shear stress generated
less expressive in the bioreactor had a positive influence in the chondrogenic
Seeding amounts than the ones differentiation of hBM-MSCs cultured in chitosan-based scaffolds.
cultured in the
ØhBMSCs were seeded statically onto the bioreactor
Figure 3 – Histological sections at the
Acknowledgements
CPBTA fiber meshes during 24 hours
end of the experiment. Portuguese Foundation for Science and Technology (PhD Grant to M.
Alves da Silva, SFRH/BD/28708/2006). European NoE
ØConstructs were cultured in a bioreactor, using chondrogenic EXPERTISSUES (NMP3-CT-2004-500283). European FP7 Project
differentiation medium, for 28 days Find and Bind (NMP4-SL-2009-229292).

Engenharia para a Qualidade de Vida: SAÚDE, LAZER E AMBIENTE– Semana da Escola de Engenharia -11 a 16 de Outubro de 2010