Influence of milling time on the structural refinement and in vitro biological performance of porcine-derived hydroxyapatite via the drilling method

Animal-derived hydroxyapatite (HA) is considered a promising alternative for bone applications. In this study, its production was focused on via a drilling–calcination–milling route applied to porcine femurs, aiming to identify the critical milling threshold required to transition from a polydisperse, cytotoxic material to a biocompatible nanostructure. The powders were characterized by X-ray diffraction (XRD) coupled with Rietveld refinement, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and transmission electron microscopy (TEM). Concurrently, the in vitro performance was evaluated in the L929 cell line using cytotoxicity and proliferation assays at 24 and 48 h. The primary particle and crystallite sizes were reduced by extended milling, shifting the material toward the nanometric regime and increasing morphological homogeneity, as was observed via SEM and TEM. A chemical composition compatible with biological HA, along with the presence of trace elements characteristic of the animal origin, was qualitatively confirmed through EDS screening. The biological assays revealed that cytotoxic HA was produced by short milling times (2 h) due to size-associated polydispersity. Conversely, non-cytotoxic nano-HA was successfully achieved through prolonged milling (6 h), corresponding to sample D-S45-M6H. A significant increase in cell proliferation was exhibited by the latter at 48 h (34.81% above the control), thereby surpassing the standard biogenic responses reported in recent literature. A sustainable and high-precision alternative to synthetic precursors is represented by this approach, as livestock waste is effectively transformed into a high-value biomaterial for advanced bone regeneration.