Production and Characterization

Electrospinning of nanofibrous substrate

The production of polycaprolactone (PCL) NFMs
were performed as described

Functionalization of the nanofibrous substrate

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The surface activation and
functionalization of the PCL NFMs were performed as described elsewhere.(31)

Antibody immobilization

The antibody immobilization at the surface
of activated and functionalized nanofibrous substrate was performed as
described previously.(32)

Single Antibody Immobilization: The maximum immobilization capacity of a single antibody (Mouse
anti-human TGF-?3 monoclonal antibody (Clone 44922), goat anti-human IGF-I
polyclonal antibody Bio-techne/R&D SystemsTM) on the
nanofibrous substrate was determined by using a wide range of concentrations
(from 0 to 10 mg/mL). After primary antibody
immobilization at the surface of the nanofibrous substrate, a blocking step was
performed by a 3 % bovine serum albumin (BSA; Sigma-Aldrich) incubation step (1
h at RT), followed by the secondary antibody (1:200 in PBS) incubation (1 h at
RT). Alexa Fluor 594® donkey anti-mouse IgG (H+L) and Alexa Fluor®
488 rabbit anti-goat IgG (H+L) (Life Technologies) were used as secondary
antibodies. The unbound secondary antibody fluorescence was measured by a
microplate reader (Synergy HT, Bio-TEK), as an indirect method to determine the
primary antibody immobilization efficiency. The nanofibrous substrate without
primary antibody was used as a negative control to evaluate nonspecific

Mixed Antibodies Immobilization: The TGF-b3 and
IGF-I antibodies were mixed in a PBS solution at the proportion 1:10 of
anti-TGF-b3 and anti-IGF-I. Mixed antibodies
immobilization was performed as previously described for single antibody
immobilization. In order to determine the degree of immobilization, the
fluorescence of unbound secondary antibody was measured. For that, nanofibrous
substrate with mixed antibodies immobilization was firstly incubated with Alexa
Fluor® 594 (1 h at RT) followed by Alexa Fluor® 488
incubation (1 h at RT). A washing step was performed between the secondary
antibodies incubation. The samples were further analyzed by laser scanning
confocal (LSCM; Leica TCS SP8; Leica Microsystems CMS GmbH) microscopy to
detect their distribution at the surface.

Platelet lysates

Platelet concentrates were obtained from
three O Pos female donors at the Imuno-hemotherapy Service of the Hospital de
São João in Porto, under an established cooperation protocol, which was
established in accordance to the ethical and legal regulations. The number of
platelets was counted and the sample volume adjusted to 106
platelets/mL using the own plasma. Platelet lysate
(PL) was prepared as a pool of platelet concentrates according as previously
reported.(32) The
amount of each growth factor of interest (ie.TGF-b3 and IGF-I) was quantified by Enzyme-Linked
Immunosorbent Assay (ELISA). Assays (human TGF-beta3 and IGF-I DuoSet®
development ELISA R&D Systems, Inc.) were performed according to the
manufacturer`s instructions.

Growth factors binding

The growth factors binding capacity of the biofunctionalized
nanofibrous substrate was performed as described elsewhere,(32) by using a mixture
of TGF-b3 and IGF-I from
recombinant-origin (TGF-?3 PeproTech Inc. and IGF-I Bio-techne/R&D
SystemsTM) or from the PL. The unbound protein solutions (from
recombinant or PL-origin) were collected and stored at -20 °C, until further
quantification by ELISA. For the quantification of bound growth factors, a fluorescence-linked immunosorbent assay
(FLISA) was performed as an indirect method. The biofunctionalized nanofibrous
substrate with bound growth factors were incubated overnight at 4 °C with the
corresponding primary antibody, followed by a PBS washing and BSA blocking
steps. The corresponding secondary antibodies were incubated and the unbound secondary
antibodies fluorescence were measured as previous described.


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