Biological in single or mixed fashion, was assessed

Biological Activity

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.

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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.

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 genes.