FGF-2 treatment of high passage human mesenchymal stem cells enhances chondrogenesis in pellet culture through upregulation of Sox9 and integrin alpha 10, key markers of chondrocyte differentiation. However, exposing MSCs to FGF-2 does not improve their ability to regenerate cartilage defects in explant culture. Similarly, FGF-2 increases integrin alpha 10 in bovine chondrocytes but does not enhance repair of cartilage lesions. Pretreating MSCs with FGF-18 does not affect chondrogenesis in pellet culture or expression of chondrogenic markers.

1. (A) Semi-quantitative RT-PCR analysis of high passage monolayer culture hMSCs. Among the
chondrogenic markers, the expression levels of Itga10 and Sox9 were upregulated, while the
expression of Agc1 was downregulated after FGF-2 treatment. The mRNA levels of the
fibroblastic markers Itga11 and Col1a1 were reduced, whereas the expression of Fn1 was not
changed compared to non-FGF-2 treated cultures. Col2a1 mRNA was not detected in the
monolayer cultures. (B) GAPDH-normalized expression of selected genes.
FGF-2 enhances chondrogenesis of p8 hMSCs in pellet culture
FGF-2 pretreated hMSCs have no increased regenerative potential
FGF-2 pretreatment elevates Itga10 expression in monolayer
cultured bovine chondrocytes but it has no impact on regeneration
FGF-18 pretreatment of p8 hMSCs has no effect on chondrogenesis
FGF-2 treatment of high passage human mesenchymal stem cells
enhances chondrogenesis in pellet culture
Introduction
The potential of human mesenchymal stem cells (hMSCs) to differentiate into chondrocytes
declines with increased passaging. Recent studies have demonstrated that hMSCs expanded
in medium under hypoxia or supplemented with FGF-2 have enhanced chondrogenic capacity.
FGF-2, in addition to being a potent mitogen, elevates the expression of the chondrogenic
master transcription factor Sox9 and the cartilage-specific collagen-binding integrin alpha 10
(Itga10) in low passage hMSC monolayer cultures. In this work, we have investigated the
effect of FGF-2 and FGF-18 1) on the chondrogenic potential of high passage hMSCs in pellet
culture and 2) on the regenerative capacity of bovine chondrocytes in explant culture.
Methods
Passage 8 hMSCs (Lonza) from four different donors were expanded for 14 days with or
without FGF-2 supplementation (10 ng/ml) followed by hypoxic (2% O2) preconditioning for 5
days. Expression levels of integrins a10 and a11, Sox9, aggrecan (Agc1), collagens I and II,
and fibronectin were monitored by RT-PCR. The chondrogenic potential of hMSCs was
assessed in in-vitro pellet culture using serum-free chondrogenic differentiation medium with
or without addition of TGF-ß1 and TGF-ß1/BMP-2. Pellets were grown under hypoxia for 22-
28 days. Chondrogenesis in the pellets was characterized by toluidine blue histochemistry and
immunohistochemistry for aggrecan, collagen II and collagen X. Primary bovine chondrocytes
were isolated from the femoral condyle and cultured with or without FGF-2. FGFs-treated and
non-treated hMSCs and passage 1 chondrocytes were applied into defined chondral lesions of
bovine articular cartilage explants and cultured under normoxic or hypoxic conditions.
Itga10 and Sox9 is upregulated in p8 hMSCs upon FGF-2 treatment
Summary and Conclusions
Pretreatment of high passage hMSC cultures with FGF-2 but not FGF-18 enhances
chondrogenesis in pellet culture likely through the upregulation of Sox9 and the chondrocyte-
specific integrin alpha 10. However, exposure of MSCs to FGF-2 did not improve their
regenerative potential in cartilage defect explant culture. FGF-2 treatment also upregulates
Itga10 expression in bovine chondrocytes but does not enhance regeneration of chondral
lesions.
(A) Preparation of bovine articular cartilage explants with chondral defects. (B) Typical
regenerative tissues using high passage hMSCs show the formation of fibrous and amorphous
matrices after 4 weeks of explant culture. (C) Quantification of the repair tissues suggests that
FGF-2 pretreatment of hMSCs does not improve regeneration in our system.
(A) RT-PCR shows that the
expression levels of Itga10 and
Col2a1 were reduced, while the
expression of the fibroblastic
marker Col2a1 was increased by
one passaging of primary
bovine chondrocytes. FGF-2
pretreat-ment of p0
chondrocytes significantly
increased the Itga10/Itga11 ratio
but it reduced the expression of
Col2a1.
(B) Quantification of repair
tissue after 4 weeks of normoxic
or hypoxic culture with or
without growth factor
supplementation. Note that
FGF-2 has no positive effect on
chondrogenic regeneration.
Boris Schmalz*, Michael Schmutzer*, Zsuzsanna Farkas, Matthias Schieker and Attila Aszodi
Experimentelle Chirurgie und Regenerative Medizin, Klinik für Allgemeine, Unfall-, Hand- und Plastische Chirurgie, München. * Equal contribution
(A) Representative micrographs of p8 hMSC pellets at days 2 and 22. Pellets were cultured
under hypoxia in chondrogenic differentiation medium (control, C) with or without
supplementation of Tgf-ß1 (T) and Tgf-ß1/BMP2 (T+B). (B) Toluidine blue staining shows
increased deposition of sulfated proteoglycans in growth factor supplemented pellets. (C)
Diagram of the relative growth (difference between days 2 and 22) demonstrates that FGF-2
pretreatment increases pellet size during chondrogenic differentiation of all four donor-
specific hMSCs.
(A) Representative RT-PCR of p8 hMSCs indicates no obvious difference in the expression of
chondrogenic/fibroblastic marker genes between FGF-18-treated and non-treated monolayer
cultures. (B) Normalization for GAPDH reveals a moderate increase of Itga10 expression in
donor 2 and 4 hMSCs upon FGF-18 pretreatment. Representative micrographs (C) and
diagram (D) showing the relative pellet growth of FGF-18-treated and non-treated hMSCs. (E)
TB staining and IHC on pellet sections show no enhanced chondrogenic potential of FGF-18
pretreated hMSCs.