Bovine alp

In order to biofunctionalize scaffolds, the ALP was grafted onto their structures. The scaffold cooled at 5.5 °C/h was selected for the assay as it exhibited improved mechanical strength. The ALP was chosen as the enzyme model because its activity plays an active role in bone formation and mineralization processes [20 (link),21 (link)]. To prepare ALP solution (250 µΜ), bovine ALP (from bovine intestinal mucosa, lyophilized powder, ≥10 DEA units/mg solid, Sigma–Aldrich) was dissolved in Tris (hydroxymethyl) aminomethane ((HOCH₂)₃CNH₂, 99.9+% ultrapure grade, Sigma–Aldrich) buffer (55 mΜ, pH 9) with ultrasonic stirring.
Three scaffolds were immersed in 1 mL of ALP solution for 24 h at 4 °C. When incubation ended, samples were rinsed in 5 mL of Tris buffer with magnetic stirring for 10 min and then dried at room temperature. Finally, they were stored hydrated in Tris buffer in a Petri dish at 4 °C. The functionalized samples’ effectiveness was investigated by running an enzymatic activity test as described below.

Cell-laden microfibers
after 7 days in culture were washed with 0.9% NaCl, soaked in 50 mM
EDTA and 0.2 mg/mL collagenase solution in 0.9% NaCl for 10 min at
room temperature and then incubated in trypsin solution for 10 min
to retrieve cells from the microfibers. The obtained cells were washed
in 0.9% NaCl, counted, and lysed for 30 min in a lysis buffer (0.1%
Triton X-100, 50 mM Tris-HCl) at 4 °C. The ALP activity was measured
with a pNPP Alkaline Phosphatase Assay Kit (Sigma) according to the
user’s manual. Bovine ALP (Sigma) was adopted to create a standard
curve. Absorbance determined at 405 nm was read by a fluorescent plate
reader (BioTek, USA) and normalized to cell counts.

To measure alkaline phosphatase (ALP) activity, cells were washed once with cold PBS, scraped into 1 mL PBS, pelleted by 5 minutes centrifugation at 2,000 RPM and resuspended in 100 µL ALP buffer (100 mM Tris-HCL pH 9.5, 100 mM NaCl, 5 mM MgCl, 1% Triton X-100). For analysis, ALP samples were diluted with ALP buffer (typically a 1:3 to 1:50 dilution). 50 µL of each sample was added to a flat bottom, clear plastic 96-well plate along with 50 µL of p-nitrophenyl phosphate (pNPP) chromogenic substrate (Sigma). A standard curve of purified bovine ALP (Sigma) was prepared in the range of 0–100 mU/mL using the batch information sheet to calculate units of enzymatic activity. Samples were incubated at 37 °C for 30 minutes and analysed by absorbance at 405 nm. All results were normalised to protein content as determined by BCA assay (Sigma).