3.4.2. TNF-α Capturing during 15 Days

To confirm the efficiency of the primary antibody immobilized at the NFMs' surface to clear TNF-α from the conditioned medium over time, as well as to evaluate the action time of the immobilized antibody, an extended assay (15 days) was performed. Experimental results show that the sAb only has a biological effect for the first 3 days, since from the fourth time point on, it does not present significant differences when compared to the UV-O activated NFM (Figure 4). The NFM + Ab has a significantly higher antigen-neutralizing activity ranging from the 2nd day until the 11th day when compared to the sAb condition. Moreover, the biofunctionalized NFM + Ab does not have a significant biological effect from the 11th day on, as no significant differences were found when compared to the control NFM.

**Figure 4.** TNF-α captured (median and interquartile range) over 15 days. Data were analyzed by nonparametric way of a Kruskal-Wallis test, followed by Tukey's HSD test: (**a**) denotes significant differences compared to immobilized anti-TNF at the surface of electrospun NFM (condition NFM + Ab); and (**b**) denotes significant differences compared to soluble anti-TNF (condition sAb).

These results confirm the initial hypothesis that the immobilization of the primary antibody at the NFMs' surface had a longer effect than the soluble antibody. This longer effect might be supported by the increased stability that the antibody immobilization provides [53]. Moreover, the physical attachment prevents the antibody clearance from the system by biological means. As each NFM has a high specific surface area, it allows the immobilization of high concentrations of primary antibody, requiring a small amount of material for the development of an effective cytokine capture system. Moreover, as the NFMs are made of polycaprolactone (PCL), a well-known biodegradable polymer, these membranes will be completely degraded within a maximum period of 2 years. Thus, the degradation time ensures that the captured TNF-α, as well as the attached antibody, may be slowly degraded/metabolized by biological processes and that there is no abrupt release of these molecules to the exterior of the synovial cavity.
