Enzyme-Mediated Crosslinking of Silk Fibroin/ N-Acetylcysteine-Modified Chitosan/ Hydroxyapatite Hydrogel Composites for Subchondral Bone Regeneration - additional experiments

The investigated materials were prepared using an enzymatic cross-linking approach and evaluated as composite hydrogel systems for potential application in subchondral bone regeneration. The data include the results of additional experiments conducted during the peer-review process and comprise: (1) direct biocompatibility assessment, including absorbance measurements and fluorescence microscopy observations; (2) alkaline phosphatase (ALP) activity measurements, together with total protein quantification at the corresponding time points; (3) Alizarin Red S mineralization assays, including absorbance measurements and microscopic observations; (4) ELISA measurements of osteocalcin (OCN) and RUNX2, normalized to total protein content determined using the BCA assay; (5) rheological measurements; (6) scanning electron microscopy (SEM) observations; and (7) assessment of superoxide anion (O₂•⁻) generation in THP-1 cells using the nitroblue tetrazolium (NBT) assay.

This research was funded by the National Science Centre, Poland, under the project “Hierarchical approaches to osteochondral tissue engineering” (grant no. UMO-2022/45/B/ST8/02557).