The growth factors are stimulatory
molecules which help in regulating cartilage development and maintenance of the
chondrocyte phenotype of the stem cells.(5, 21, 22) The
TGF-b3 and IGF-I have significant and
complementary activities to induce, accelerate, and/or enhance cartilage tissue
formation, being commonly used to supplement the chondrogenic inducing culture
medium at concentrations of 10 ng mL-1 and 100 ng mL-1,
respectively.(17-20, 23, 24, 33-36) This
proportion (1:10) was taken into consideration in our biofunctionalized
nanofibrous substrate when both antibodies were immobilized in a mixed fashion.
The maximum amount of recombinant growth factors bound at the biofunctionalized
nanofibrous substrate is approximately 2 ?g mL-1 of TGF-b3 and 2.5 ?g mL-1 of IGF-I (Figure 4). Accordingly, our
biofunctional nanofibrous substrate enables the immobilization of high
concentrations of growth factors (on the mg mL-1 order), whereas common approaches report values
that are on the ng mL-1 order, reflecting the positive effect of the
high specific surface area of electrospun nanofibers to maximize the potential
to immobilize GFs.
The use of platelet-rich plasma (PRP) to
stimulate tissue regeneration is growing at the research and clinical levels
for being employed in various fields of surgery, namely orthopedics.(27, 32, 37-41)
Different bioactive factors are released from platelet activation, including
TGF-b3 and IGF-I. The amount of these two
growth factors, in the three-independent human PL` samples, varies between 0.10
– 0.27 ng mL-1 of TGF-b3 and
1.66 – 11.31 ng mL-1 of IGF-I. The differences among the quantified
GFs and their variability are related to intrinsic differences between the
platelets obtained from the different donors. Comparing this data to values
reported in the literature, i.e. TGF-b3 (no
data reporting), TGF-b1 (79.7
ng mL-1) and IGF-I (69.5 ng mL-1), (42) we obtained lower amounts then these reported in the literature.
The amount of bound autologous GFs varies according to their concentration
found in the PL sample. For PL-derived TGF-?3, the binding efficiency (between
95?99%) was high as in the case of the recombinant protein, which makes sense,
since the concentration present in PL is very low (on the rg mL-1 order). For IGF-I, only around 52?78% of
PL-derived GF was bound to the biofunctionalized nanofibrous substrate.
Furthermore, the binding efficiency of the GFs are in the same range for the
three independent donors, showing the reproducibility of this approach (Table 1).
Despite the differences in the concentration of GFs present in the PL (ranging
from rg mL-1 for TGF-?3 to ng mL-1
for IGF-I), these concentrations are much lower than the maximum binding
capacity of the biofunctionalized nanofibrous substrate, where recombinant
proteins were used at micrograms per milliliter concentration. Herein, a chondrogenesis-inductive nanofibrous substrate was
developed, by the immobilization of defined antibodies at the surface of
electrospun nanofiber, enabling to bound TGF-?3 (0.27 ± 0.03 ng mL-1) and/or IGF-I
(4.7 ± 1.1 ng mL-1) from a PL´ pool or from a recombinant-origin.