Single and by the quantification of unbound protein

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). 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.

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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.