The and IGF-I bound from PL. Glycosaminoglycans (GAGs)

The biological activity of biofunctionalized NFM, in single or mixed
fashion, was assessed by seeding and culturing hBM-MSCs during 28 days. The hBM-MSCs
were cultured on eight different nanofibrous substrate conditions: (i) activated NFM without GFs (Basal
medium), (ii) activated NFM without
GFs (Chondro medium) (iii) NFM with immobilized
TGF-b3 from recombinant-origin
(TGF-b3_rGF), (iv) NFM with immobilized TGF-b3 from PL (TGF-b3_PL), (v) NFM with immobilized IGF-I from recombinant-origin (IGF-I_rGF),
(vi) NFM with immobilized IGF-I from
PL (IGF-I_PL), (vii) NFM with immobilized
TGF-b3 and IGF-I from recombinant-origin
(TGF-b3 & IGF-I_rGF), (viii) NFM with immobilized TGF-b3 and IGF-I from PL (TGF-b3 & IGF-I_PL). In condition i, the hBM-MSCs were cultured under
basal medium without chondrogenic factors as a negative control of the
chondrogenesis. and in condition ii,
the hBM-MSCs were cultured under standard chondrogenic differentiation medium,
aiming at achieving the differentiation of hBM-MSCs into the chondrogenic
lineage. In conditions iii?viii, the hBM-MSCs
were cultured in basal culture medium in order to evaluate the action of the
immobilized GFs, form recombinant or PL-origin.

Biological data (Figure 5) confirms
the bioactivity of bound TGF-b3 and IGF-I, since
the biofunctional nanofibrous substrates did not induce significant changes
over hBM-MSCs` viability and proliferation (Figure 5ab) during the different
culture times (7, 14 and 28 days), when compared to the controls (condition i-ii). Although no statistically
significant differences were observed for the nanofibrous substrates
functionalized with TGF-b3 and IGF-I, in a
single or mixed fashion, they seem to have higher viability and proliferation
compared to the positive control condition (chondro medium). Likewise, the
substrates where the GFs were bound from PL performs marginally better, although
not statistically different. Concerning the hBM-MSCs` total protein synthesis
(Figure 5c), no statistically significant differences were observed between the
biofunctional nanofibrous substrates and the controls. Even though, on the 28th
day, the controls tendentially displayed a lower protein concentration than the
nanofibrous substrates biofunctionalized with single or mixed TGF-b3 and IGF-I bound from PL.

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Glycosaminoglycans (GAGs) production (Figure 5d) was detected in all
testing conditions, excepted in the negative condition. In the
biofunctionalized nanofibrous substrates there was a trend to obtain increased
accumulation of GAGs along the time. Although no significant differences were
observed between the conditions, the single immobilized TGF-b3 and IGF-I (PL-derived)
tendentially display higher GAGs concentration, at time point 28, when compared
to the others conditions.

Further analysis of gene expression revealed that in all condition
hBM-MSCs expressed cartilage-related genes, indicating that the chondrogenic
differentiation was induced (Figure 6).

A similar expression pattern of Aggrecan
and Sox9 was observed in all
conditions, whose expression was maintained along the time course of the experiment.

Furthermore, Collagen II expression
in biofunctional nanofibrous substrates was significantly higher than in the
positive condition (chondro medium) for the 14th day of culture.

Interestingly, the Collagen Ia was expressed at low
levels and showed a decreasing trend of expression towards longer culturing
times. This result confirms that the hBM-MSCs are not differentiating into the
osteogenic linage.

SEM analysis of the hBM-MSCs cultured during 28 days on the seven
different conditions, shows that the cells began to acquire a round-shaped
morphology, being more evident in the biofunctionalized nanofibrous substrates
(Figure 7).

For the detection of ECM sulfated proteoglycans (Figure 7), all culture
conditions at 28 days were stained with alcian blue. In the conditions ii-viii, the cells positively stain to Alcian
blue demonstrating the presence of cartilaginous ECM components.

Immunolocalization of collagen type II confirms the deposition of a
cartilaginous ECM on the conditions ii-viii
as observed by the slight spots of collagen type II spread on the scaffolds
(Figure 7). These observations are consistent with the previously obtained
results for GAGs production, as well as by the expression of cartilage-related