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Single
Immobilization

The antibodies against TGF-b3 and IGF-I were
immobilized at the surface of activated and functionalized NFM, in a wide range
of concentrations (0-10  mg mL-1),
to determine the maximum immobilization capacity for each antibody by using an
indirect quantification method (Figure 1ab).

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The maximum concentration of immobilized primary antibody was achieved at 4 ?g mL-1,
for both TGF-?3 and IGF-I antibodies assessed, representing the saturation
point of the nanofibrous substrate.

The spatial distribution of the TGF-?3 and IGF-I antibodies immobilized
at the surface of the nanofibrous substrate at the 4 ?g mL-1
concentration and in a single way, is shown in Figure
1 d) and e), respectively.  The
immobilized antibodies cover uniformly the nanofibers surface, resembling the
typical morphology of electrospun NFMs.

The binding capacity of the biofunctionalized nanofibrous substrate,
with immobilized single primary antibodies at 4 ?g mL-1, was
assessed by quantification the total amount of recombinant protein that can be
bound by the biofunctionalized nanofibrous substrate, by means the indirect of
sandwich method (i.e. FLISA) and by the quantification of unbound protein with
commercially available ELISAs. For FLISA (Figure
2), an increase on the total amount of recombinant protein leads to a
decrease in the fluorescence signal of the secondary antibody, meaning that
less secondary antibody is unbound. On the other hand, in ELISAs, an increase
on the amount of bound recombinant protein occurs due to an increase in the total
amount of recombinant protein used. The recombinant human GF binding capacity
of the biofunctionalized nanofibrous substrate reaches its maximum at a
concentration from which no statistically significant differences were observed.

Figure 2 shows that the recombinant human GF
binding capacity of the biofunctionalized nanofibrous substrates was reached a
maximum at 4 ?g mL-1, both for rTGF-b3 and for rIGF-I. At this concentration, the binding efficiency of the
biofunctionalized nanofibrous surface is 37 ± 11 % for rTGF-b3 and 42
± 21 % for
rIGF-I.

Mixed
Immobilization

Mixed immobilization of these two different, but complementary,
chondrogenic GF at the surface of the same nanofibrous substrate was performed.

To successfully implement this strategy, the single antibody concentration
previous optimized (i.e. 4 ?g mL-1) was considerate, and the
antibodies were mixed at the 1:10 proportion and incubated over the same
nanofibrous substrate. A single and a mixed immobilization strategies were
applied in order to assess the competition for the NH2 groups, available
at the surface of an activated and functionalized NFM (Figure 3ab). No statistically significant differences were observed
between the single and mixed immobilization strategy, being the immobilization
efficiency similar. The results show that no competition occurs between these
two antibodies (anti-TGF-?3 and anti-IGF-I) for the same amount of NH2
groups available at nanofibrous substrate once no statistically
significant differences were observed.

A uniform spatial distribution of both antibodies immobilized over the
same nanofibrous substrate in a mixed fashion (Figure
3 d) and e) was confirmed. The yellow color of the merged image (Figure 3 f) demonstrates the colocalization of both
antibodies, in a mixed fashion, over the same nanofibrous substrate.

Following the immobilization of the antibodies (i.e. anti-TGF-b3 and anti-IGF-I) in a mixed
fashion at the nanofibrous substrate, the corresponding recombinant proteins
solutions (single or mixed) were incubated at concentration previously
optimized (i.e. 4 ?g mL-1) for single substrates. The results were
analyzed by FLISA (Figure 4 a) and
b) and further confirmed with commercially available ELISAs (Figure 4 c). No
competition between the TGF-b3 and IGF-I
recombinant human proteins was observed, since the bioactivity of the immobilized
mixed antibodies, using single or mixed recombinant GFs solution, was similar.

When single recombinant protein solutions were used, the binding efficiency was
49 ± 5 %  for rTGF-b3 and 62 ± 15 % for rIGF-I. A
binding efficiency of 49 ± 10 %  for rTGF-b3 and 65 ± 15 % for rIGF-I was
achieved when a mixed recombinant protein solution was used. This result shows
the specificity of the antibody-antigen bond.

The amount of each autologous GF derived from PL of three independent
donors was quantified by ELISA, as well as its binding efficiency to the
biofunctionalized nanofibrous substrates (Table 1). A wide range of
concentrations was obtained from the different donors. However, the binding efficiency
of GFs derived from PL samples is similar for the different PL samples, showing
the consistency of the immobilization strategy in capturing the GFs. A PL-pool
of the three donors, containing 0.27 ± 0.03 ng mL-1 of TGF-b3 and 6.86 ± 1.09 ng mL-1
of IGF-I, and achieving a binding efficiency of 99.3 ± 0.4 % for TGF-b3 and 68.9 ± 4.9 %
for IGF-I, was used as autologous source of GF in the subsequent biological
assays.

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