The 10 ng mL-1 and 100 ng mL-1,

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. 

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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.

TGF-?3 is the essential
growth factor for promoting chondrogenesis, both in vivo and in vitro. (14, 16, 21, 33, 43, 44) Attisano and Wrana (45) reported that TGF-? signal was
transmitted into the nucleus via smad pathway. This pathway is involved in the
activation of Sox 9, a transcription
factor inductor of other cartilage-specific gene expression, including Collagen type II and Aggrecan.(46) We also found that bound TGF-?3 induced Sox 9 expression by culturing hBM-MSCs,
with concomitant expression of cartilaginous extracellular matrix genes, namely
Collagen type II and Aggrecan, as shown by the real-time PCR
results. Meanwhile, IGF-I is involved in cartilage
repair and is considered an essential mediator of cartilage homeostasis and
metabolism, mainly due to its capability to promote the chondrocytes survival
and proliferation, induce chondrogenic differentiation and to stimulate
proteoglycan synthesis.(47-50)
Messai et al. (51)
reported that IGF-I is involved in chondrogenesis differentiation by regulating
the synthesis of Aggrecan and Collagen type II at the transcription
level in rat articular chondrocytes. Our real-time
PCR data also shows that hBM-MSCs cultured on biofunctionalized
nanofibrous substrate with bound IGF-I expressed cartilaginous genes, namely Sox 9, Aggrecan and Collagen type II.

Many in
vitro studies have used TGF-? and IGF-I in combination, showing enhanced
chondrogenesis.(18, 34-36, 50)
Indrawattana et al. (17)
reported that MSCs cultured in the presence of 
TGF-?3 and IGF-I, combined and in cycling patterns, showed strong
expression of Sox 9 and cartilage
extracellular matrix genes. By its side, Matsuda et al. (28)
reported that the combination of TGF-?3, dexamethasone and IGF-I was the most
effective cocktail to stimulate differentiation of MSCs into chondrocytes.

Conversely, our results showed that hBM-MSCs cultured in the simultaneous
presence of TGF-?3 and IGF-I did not have a higher expression of cartilaginous
markers, showing a chondrogenic gene expression
profile similar to BM-MSCs cultured on biofunctionalized nanofibrous substrates
with individually bound TGF-?3 or IGF-I. We also found that both
autologous and recombinant growth factors bound at the surface of biofunctionalized
nanofibrous substrates are able to regulate the
BM-MSCs chondrogenesis. Our results showed that the bound IGF-I, similarly to the bound TGF-?3, induces
chondrogenic differentiation of BM-MSCs, stimulating proliferation, protein
synthesis, GAGs production and expression of chondrogenic markers. Furthermore,
we showed that the combined action of the two GFs induces the differentiation
of BM-MSCs into chondrocytes, presenting a chondrogenic gene expression profile
similar to BM-MSCs cultured in standard chondrogenic differentiation medium.

Interestingly, the results show a trend to have stronger chondrogenic induction
by the autologous PL-derived GFs, as compared to the recombinant-GFs. Based on
these results, the advantage of this novel biofunctional nanofibrous
substrate consists in the maximization of the spatial delivery of defined GF
signals, by providing direct contact between cultured hBM-MSCs and immobilized
bioactive GFs.

This study shows the successful development
of a unique chondrogenesis-inductive nanofibrous substrate, able to spatially
present autologous GFs immobilized at the surface of a biomaterial scaffold. Cell
culture assays with hBM-MSCs demonstrates that the developed biofunctional
nanofibrous substrates are able to promote chondrogenesis, as effective as the
standard differentiation condition. The present manuscript describes a new
autologous regeneration strategy, where both the cells and the bioactive agents,
namely TGF-b3 and/or IGF-I, can be obtained from
the same patient, providing a personalize therapy for cartilage regeneration.