In-vitro biodegradability of the pure chitosan and chitosan/BNNT-OH scaffolds was examined by incubating both types of materials in an enzymatic solution that contained a concentration of lysozyme that was similar to that present during cellular digestion [27]. Particularly the solution contained 2 mL of DMEM and 1 mg of lysozyme and the weight of the scaffolds was measured after 1, 3 and 7 days. Afterwards they were washed in distilled water and dried. The degradation ratio after 7 days of incubation was similar in both cases, as it was 5.2% for the chitosan/ BNNT-OH and 6.2% for the pure chitosan. However, for the latter the biodegradation process was irregular, and at a higher rate, and therefore could not be controlled.

Although the degradation rates do not differ significantly between the two scaffolds it is clear that the addition of BNNT-OH does result in a slower and more controlled biodegradability. A low biodegradation rate is preferred since it allows the accommodation of the degradation products by the body, minimizing hence possible toxic effects they may have on cells, and allowing for ingrown tissue within the scaffold. In order to verify that the BNNT-OH reinforced materials did not have cytotoxic effects, cell cultures with human dermal fibroblasts (HDF) were performed using a WST-1 cell viability assay. Prior to the toxicity tests, the HDF cells was seeded and incubated for 24 h. Then the scaffolds were incubated for 1, 3 and 7 days (at 37 °C) in a solution containing 2 mL of DMEM. After each incubation time the solution which contained the scaffold degradation products was used to replace the cell culture medium and incubate the cells for 24 h. Then their viability was tested.

It was shown that the cell viability decreased by 80% on the pure chitosan scaffolds, while it increased to 115% for the ones that included BNNT-OH. This highly enhanced cell viability of HDF cells is not only due to the lower degradation rate, but also to the ability of chitosan/BNNT-OH to retain a higher strength during immersion in lysozyme. Particularly after allowing the scaffolds to degrade in lysozyme the chitosan/BNNT-OH scaffolds required 35 kPa to reach a strain of 70% while the pure chitosan scaffold needed 18 kPa [27].

In addition to viability tests, it is important to examine how the scaffold type affects the morphology of the adhered cells. Fluorescent and scanning electron microscopy was therefore performed 1, 3 and 7 days after incubation. After the first day the HDF cells in both cases had a round shape without their natural extensions. Such extensions were visible at the end of the third day for both scaffolds and they had an expanded/relaxed morphology which is their natural state. After the seventh day, however, this preferred state was lost for the cells on the chitosan scaffold (due to degradation of the chitosan and loss of adhesion with the cells), but retained for the chitosan/BNNT-OH, which degraded at lower rates. Representative SEM and fluorescent microscopy images from the 1st and 7th culture days are depicted in Fig. 4.4a-h [27].

Fluorescence and scanning electron microscopy images of cell cultures on the pure chi- tosan and chitosan/BNNT-OH scaffolds

Fig. 4.4 Fluorescence and scanning electron microscopy images of cell cultures on the pure chi- tosan and chitosan/BNNT-OH scaffolds. (a) & (c) Correspond to the first day of culture on pure chitosan; (b) & (d) correspond to the first day of culture on chitosan/BNNT-OH scaffolds; (e) & (g) correspond to the seventh day of culture on pure chitosan; (f) & (h) correspond to the seventh day of culture on chitosan/BNNT-OH scaffolds (Reproduced with permission from Ref. [27])

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