*2.4. Stability Tests*

Sample preparation and stability tests were performed according to UNI EN ISO 10993-9 [34], ISO 10993-13 [35] and ISO 13871:1995 [36] guidelines. The test was performed in triplicate, and was designed over 5 study times (from a few seconds to 15 days). For each study time, DAC® hydrogel was uniformly distributed over the roughened surface of the titanium disk. The system was topped with a second titanium disk, obtaining a "sandwich". Each double disk system was immersed in PBS solution at pH 7.2, each container was closed and placed at 37 ◦C, without stirring, to simulate the physiological condition of the medullary cavity. At each study time, for each sample, the double disk system was separated from its buffered solution, and disk samples and solutions were freeze-dried separately. For the initial study time, sudden disk removal, no sample is recovered from the buffer solution. For the final study time, 15 days, no sample is recovered from the interface of the two disks. A total of 24 samples are therefore recovered for the High Performance Liquid Chromatography (HPLC) and NMR study (Table 1).

**Table 1.** The 24 samples of HA-PLA (Novagenit DAC®) recovered for HPLC and NMR study both from gel on disk and in buffer solution as indicated (see text).


Sterile titanium disks 8.0 cm diameter and 0.4 cm thick were purchase by Adler Ortho (Via dell'Innovazione, 9, 20032 Cormano, Italy). Titanium disks 6.5 cm diameter and 0.4 cm thick were purchased by Adler and sterilized by gamma irradiation.
