*3.5. Biologic Assays*

After the successful assembly of the TNF-capturing system, we then tested it in contact with arthritic joint cells. Therefore, the toxicity of the biofunctionalized NFMs was assessed by culturing them with human articular chondrocytes (hACs). These cells were isolated from diseased knee arthroplasties, thus presenting a phenotype that is associated with RA and osteoarthritis pathologies, where inflammation was present. Different biological assays were conducted to assess the hACs morphology, proliferation, total protein, and glycosaminoglycans (GAGs) synthesis. hACs were cultured at the surface of three different substrates: (1) anti-TNF-α antibody immobilized at the surface of NFMs (NFM + Ab), (2) UV-O activated NFM (NFM) and (3) tissue culture polystyrene (TCPs) as positive control. The influence of the anti-TNF-α immobilization at the surface of NFMs over hACs morphology was assessed by SEM. Micrographs (Figure 5) showed that hACs maintained their round-shape morphology, typical of these chondrocytic cells.

**Figure 5.** SEM micrographs of hACs cultures on tissue culture polystyrene (TCPs), UV-O treated electrospun PCL NFM (NFMs) and anti-TNF-α immobilized at the NFMs' surface (NFM + Ab) for 14, 21 and 28 days.

Chondrocytes proliferation was also evaluated by quantifying the dsDNA concentration. Experimental results showed that the TCP's condition displayed a significantly higher DNA concentration, for 14 and 28 days of culture, when compared to all other culture conditions (*p* < 0.001) (Figure 6A). Furthermore, on the 1st and 21st days of hACs culture, the control condition (TCPs) present similar proliferation to the NFM + Ab testing condition. Finally, the NFM condition presented a significantly lower cell proliferation at 21 and 28 days of culture, when compared to the NFM + AB condition (*p* < 0.001). Taken together, these results indicate that the immobilized anti-TNF-α may inhibit chondrocytes growth by mimicking an inflammatory environment, where cells would respond by limiting their growth and proliferation. This effect is not negative, since our primary goal was not to promote an extensive cell proliferation, but merely to assess the device's potential cell toxicity. Nevertheless, hACs keep proliferating, even if at a lower rate when compared to the controls. Indeed, IL1-β and TNF-α have been shown to inhibit the migratory potential of chondrogenic progenitor cells in osteoarthritic cartilage [54]. Furthermore, OA-derived hACs cultures have been reported to excrete around 3000 pm/μL of TNF-α [55], thus these cells have mechanisms to respond to high concentrations of this cytokine in the surrounding environment.

**Figure 6.** Box plot of human articular chondrocytes proliferation (**A**) and protein synthesis (**B**) when cultured on tissue culture polystyrene (TCPs), UV-O treated electrospun PCL NFM (NFM) and anti-TNF-α immobilized at the NFMs' surface (NFM + Ab). Data were analysed by nonparametric way of a Kruskal-Wallis test, followed by Tukey's HSD test: (**a**) denotes significant differences compared to NFM + Ab and (**b**) denotes significant differences compared to NFM.

Quantification of total protein synthesis is presented in Figure 6B. Experimental data showed that TCP's control condition displayed significantly higher concentration values than all the other testing conditions for the 1st and 21st days of culture (*p* < 0.01). On the other hand, the NFM + Ab testing condition displayed a significantly lower protein concentration when compared to all other culture conditions for 28 days of culture (*p* < 0.01). At 14 days of hACs culture, no significant differences between conditions were found (*p* = 0.148). The protein synthesis in the NFM + Ab condition was lower for longer culture periods, which is possibly due to the extracellular accumulation of proteins by hACs, rather than the intracellular synthesizing rate.

Furthermore, the impact of the anti-TNF-α antibody immobilization at the surface of NFMs on GAG production and accumulation (Figure 7A) by hACs was also assessed. No significant differences were found between all culture conditions for the 1st (*p* = 0.186) and 14th days (*p* = 0.088) of the experiment. On the 21st day, the NFM + Ab testing condition displayed significantly lower GAG concentrations than control condition TCPs (*p* < 0.01). Moreover, on the 28th day, TCPs presented significant higher GAGs concentration than all other culture conditions (*p* < 0.01). Concerning the GAGs production by hACs cultured onto the NFM + Ab, the obtained results confirm the evidences found in the intracellular protein synthesis. There is a decrease in the total protein concentration values for longer time points, with a concomitant increase of GAGs production for these same periods of time, indicating that protein synthesis was being directed by the GAGs production. This increase of GAGs

concentration for late time points of the experiment are in accordance to our hypothesis, as they will protect chondrocytes against the inflammatory environment.

**Figure 7.** Blox plot of human articular chondrocytes GAGs accumulation when cultured on tissue culture polystyrene (TCPs), UV-O treated electrospun PCL NFM (NFM) and anti-TNF-α immobilized at the NFMs' surface (NFM + Ab) (**A**). Data were analysed by nonparametric way of a Kruskal-Wallis test, followed by Tukey's HSD test: (**a**) denotes significant differences compared to NFM + Ab and (**b**) denotes significant differences compared to NFM. Alcian blue staining evidencing sulphated proteoglycans deposition in samples from TCPs (**B**), NFMs (**C**), and NFM + Ab (**D**) on the 28th day of the experiment.

As it can be observed in Figure 7B–D, sulphated proteoglycans, an important component of articular cartilage ECM, were detected on all culture conditions by alcian blue staining. Furthermore, these observations are reassured by the quantification of GAGs presented in the box plot of Figure 7A. Altogether, these results on intra- and extra-cellular protein synthesis confirmed that hACs are not only able to proliferate and maintain their typical round morphology, but are also capable of performing their metabolic functions properly, in the presence of the NFM + Ab testing system. With this study, we validated the use of this TNF-α capturing system nearby cartilaginous structures. There is, indeed, an influence on these parameters when cells are cultured onto NFM + Ab nanofibers, but it is not enough to restrain human articular chondrocytes functions. In fact, it has been described that the blockade of TNF-α by Infliximab treatment, during fracture healing, leads to an increased callus size. This increase was accompanied by an increase in cartilage tissue [56].
