observations 11.31 ng mL-1 of IGF-I. The differences

observations are consistent with thepreviously obtained results for GAGs production, as well as by the expressionof cartilage-related genes. DiscussionThe growth factors are stimulatorymolecules which help in regulating cartilage development and maintenance of thechondrocyte phenotype of the stem cells.(5, 21, 22) TheTGF-b3 and IGF-I have significant andcomplementary activities to induce, accelerate, and/or enhance cartilage tissueformation, being commonly used to supplement the chondrogenic inducing culturemedium at concentrations of 10 ng mL-1 and 100 ng mL-1,respectively.(17-20, 23, 24, 33-36) Thisproportion (1:10) was taken into consideration in our biofunctionalizednanofibrous substrate when both antibodies were immobilized in a mixed fashion.

The maximum amount of recombinant growth factors bound at the biofunctionalizednanofibrous substrate is approximately 2 ?g mL-1 of TGF-b3 and 2.5 ?g mL-1 of IGF-I (Figure 4). Accordingly, ourbiofunctional nanofibrous substrate enables the immobilization of highconcentrations of growth factors (on the mg mL-1 order), whereas common approaches report valuesthat are on the ng mL-1 order, reflecting the positive effect of thehigh specific surface area of electrospun nanofibers to maximize the potentialto immobilize GFs.  The use of platelet-rich plasma (PRP) tostimulate tissue regeneration is growing at the research and clinical levelsfor being employed in various fields of surgery, namely orthopedics.(27, 32, 37-41)Different bioactive factors are released from platelet activation, includingTGF-b3 and IGF-I. The amount of these twogrowth factors, in the three-independent human PL` samples, varies between 0.10- 0.

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27 ng mL-1 of TGF-b3 and1.66 – 11.31 ng mL-1 of IGF-I. The differences among the quantifiedGFs and their variability are related to intrinsic differences between theplatelets obtained from the different donors.

Comparing this data to valuesreported in the literature, i.e. TGF-b3 (nodata reporting), TGF-b1 (79.7ng 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 concentrationfound in the PL sample. For PL-derived TGF-?3, the binding efficiency (between95?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% ofPL-derived GF was bound to the biofunctionalized nanofibrous substrate.Furthermore, the binding efficiency of the GFs are in the same range for thethree independent donors, showing the reproducibility of this approach (Table 1).Despite the differences in the concentration of GFs present in the PL (rangingfrom rg mL-1 for TGF-?3 to ng mL-1for IGF-I), these concentrations are much lower than the maximum bindingcapacity of the biofunctionalized nanofibrous substrate, where recombinantproteins were used at micrograms per milliliter concentration. Herein, a chondrogenesis-inductive nanofibrous substrate wasdeveloped, by the immobilization of defined antibodies at the surface ofelectrospun 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 essentialgrowth factor for promoting chondrogenesis, both in vivo and in vitro. (14, 16, 21, 33, 43, 44) Attisano and Wrana (45) reported that TGF-? signal wastransmitted into the nucleus via smad pathway.

This pathway is involved in theactivation of Sox 9, a transcriptionfactor 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, namelyCollagen type II and Aggrecan, as shown by the real-time PCRresults. Meanwhile, IGF-I is involved in cartilagerepair and is considered an essential mediator of cartilage homeostasis andmetabolism, mainly due to its capability to promote the chondrocytes survivaland proliferation, induce chondrogenic differentiation and to stimulateproteoglycan synthesis.(47-50)Messai et al. (51)reported that IGF-I is involved in chondrogenesis differentiation by regulatingthe synthesis of Aggrecan and Collagen type II at the transcriptionlevel in rat articular chondrocytes.

Our real-timePCR data also shows that hBM-MSCs cultured on biofunctionalizednanofibrous substrate with bound IGF-I expressed cartilaginous genes, namely Sox 9, Aggrecan and Collagen type II.Many invitro studies have used TGF-? and IGF-I in combination, showing enhancedchondrogenesis.(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 strongexpression of Sox 9 and cartilageextracellular matrix genes. By its side, Matsuda et al. (28)reported that the combination of TGF-?3, dexamethasone and IGF-I was the mosteffective cocktail to stimulate differentiation of MSCs into chondrocytes.Conversely, our results showed that hBM-MSCs cultured in the simultaneouspresence of TGF-?3 and IGF-I did not have a higher expression of cartilaginousmarkers, showing a chondrogenic gene expressionprofile similar to BM-MSCs cultured on biofunctionalized nanofibrous substrateswith individually bound TGF-?3 or IGF-I. We also found that bothautologous and recombinant growth factors bound at the surface of biofunctionalizednanofibrous substrates are able to regulate theBM-MSCs chondrogenesis.

Our results showed that the bound IGF-I, similarly to the bound TGF-?3, induceschondrogenic differentiation of BM-MSCs, stimulating proliferation, proteinsynthesis, GAGs production and expression of chondrogenic markers. Furthermore,we showed that the combined action of the two GFs induces the differentiationof BM-MSCs into chondrocytes, presenting a chondrogenic gene expression profilesimilar to BM-MSCs cultured in standard chondrogenic differentiation medium.Interestingly, the results show a trend to have stronger chondrogenic inductionby the autologous PL-derived GFs, as compared to the recombinant-GFs.

Based onthese results, the advantage of this novel biofunctional nanofibroussubstrate consists in the maximization of the spatial delivery of defined GFsignals, by providing direct contact between cultured hBM-MSCs and immobilizedbioactive GFs.This study shows the successful developmentof a unique chondrogenesis-inductive nanofibrous substrate, able to spatiallypresent autologous GFs immobilized at the surface of a biomaterial scaffold. Cellculture assays with hBM-MSCs demonstrates that the developed biofunctionalnanofibrous substrates are able to promote chondrogenesis, as effective as thestandard differentiation condition. The present manuscript describes a newautologous regeneration strategy, where both the cells and the bioactive agents,namely TGF-b3 and/or IGF-I, can be obtained fromthe same patient, providing a personalize therapy for cartilage regeneration.


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