Production and CharacterizationElectrospinning of nanofibrous substrate The production of polycaprolactone (PCL) NFMswere performed as describedpreviously.(30) Functionalization of the nanofibrous substratesurfaceThe surface activation andfunctionalization of the PCL NFMs were performed as described elsewhere.(31) Antibody immobilizationThe antibody immobilization at the surfaceof activated and functionalized nanofibrous substrate was performed asdescribed previously.(32)Single Antibody Immobilization: The maximum immobilization capacity of a single antibody (Mouseanti-human TGF-?3 monoclonal antibody (Clone 44922), goat anti-human IGF-Ipolyclonal antibody Bio-techne/R&D SystemsTM) on thenanofibrous substrate was determined by using a wide range of concentrations(from 0 to 10 mg/mL).
After primary antibodyimmobilization at the surface of the nanofibrous substrate, a blocking step wasperformed by a 3 % bovine serum albumin (BSA; Sigma-Aldrich) incubation step (1h at RT), followed by the secondary antibody (1:200 in PBS) incubation (1 h atRT). Alexa Fluor 594® donkey anti-mouse IgG (H+L) and Alexa Fluor®488 rabbit anti-goat IgG (H+L) (Life Technologies) were used as secondaryantibodies. The unbound secondary antibody fluorescence was measured by amicroplate reader (Synergy HT, Bio-TEK), as an indirect method to determine theprimary antibody immobilization efficiency. The nanofibrous substrate withoutprimary antibody was used as a negative control to evaluate nonspecificimmobilization.Mixed Antibodies Immobilization: The TGF-b3 andIGF-I antibodies were mixed in a PBS solution at the proportion 1:10 ofanti-TGF-b3 and anti-IGF-I. Mixed antibodiesimmobilization was performed as previously described for single antibodyimmobilization.
In order to determine the degree of immobilization, thefluorescence of unbound secondary antibody was measured. For that, nanofibroussubstrate with mixed antibodies immobilization was firstly incubated with AlexaFluor® 594 (1 h at RT) followed by Alexa Fluor® 488incubation (1 h at RT). A washing step was performed between the secondaryantibodies incubation.
The samples were further analyzed by laser scanningconfocal (LSCM; Leica TCS SP8; Leica Microsystems CMS GmbH) microscopy todetect their distribution at the surface.Platelet lysatesPlatelet concentrates were obtained fromthree O Pos female donors at the Imuno-hemotherapy Service of the Hospital deSão João in Porto, under an established cooperation protocol, which wasestablished in accordance to the ethical and legal regulations. The number ofplatelets was counted and the sample volume adjusted to 106platelets/mL using the own plasma. Platelet lysate(PL) was prepared as a pool of platelet concentrates according as previouslyreported.(32) Theamount of each growth factor of interest (ie.TGF-b3 and IGF-I) was quantified by Enzyme-LinkedImmunosorbent Assay (ELISA). Assays (human TGF-beta3 and IGF-I DuoSet®development ELISA R&D Systems, Inc.
) were performed according to themanufacturer`s instructions.Growth factors bindingThe growth factors binding capacity of the biofunctionalizednanofibrous substrate was performed as described elsewhere,(32) by using a mixtureof TGF-b3 and IGF-I fromrecombinant-origin (TGF-?3 PeproTech Inc. and IGF-I Bio-techne/R&DSystemsTM) or from the PL. The unbound protein solutions (fromrecombinant or PL-origin) were collected and stored at -20 °C, until furtherquantification by ELISA. For the quantification of bound growth factors, a fluorescence-linked immunosorbent assay(FLISA) was performed as an indirect method. The biofunctionalized nanofibroussubstrate with bound growth factors were incubated overnight at 4 °C with thecorresponding primary antibody, followed by a PBS washing and BSA blockingsteps. The corresponding secondary antibodies were incubated and the unbound secondaryantibodies fluorescence were measured as previous described.