Mercury porosimetery revealed comparable (F = 0.057, P < 0.92) average pore diameter (85 ± 24 μm) for all the prepared scaffolds. Smallest pore diameter was 34 ± 2 μm and the largest pore diameter was 720 ± 13 μm. After filling the pores with hydroxyapatite, there was a significant (F = 16.1, P < 0.01) reduction in total porosity percent from 83 to 44 wt.% indicating that the nano-particles filled almost half of the available pores. There was also a significant reduction in average pore diameter from 85 ± 24 to 46 ± 29 μm. Amount of measured porosity was directly related to the measured bulk density of the scaffold structure. Agglomerates of hydroxyapatite particles were observed filling the porous structure (Fig. 1a, b).

EDX analysis of the enriched scaffolds revealed that Ca/P ratio was 1.67 indicating the presence of pure hydroxyapatite in the enriched scaffolds. XRD pattern revealed the characteristic peaks specific for the hexagonal HA crystal phase represented by (211), (112), and (300) peaks. Peaks of tetragonal yttrium zirconium oxide were detected for all specimens.

Histomorphometric analysis revealed that hydroxyapatite-enriched scaffolds had significantly (F = 14, P < 0.02) higher amount of new bone formation (33% ± 14) compared to the controls (21% ± 11). Amount of new bone formation was calculated as a percent of the total pore volume measured on each image. New bone growth started by lining pore cavity and propagated to gradually fill the entire pore volume (Fig. 2a). Bone ingrowth proceeded from the periphery of the scaffold and propagated towards its center (Fig. 2b). The surface under the guided tissue membrane was filled with unmineralized connective tissue. Regional areas of entrapped hydroxyapatite were observed inside the pore cavity of the enriched scaffolds (Fig. 2b). Entrapped islands of hydroxyapatite were surrounded by mineralized tissue. Lower amount of mineralized bone was observed for uncoated scaffolds (Fig. 3a, b).

Radiographic examination revealed clear margins separating newly inserted scaffolds from surrounding bone defect (Fig. 4a). After completion of healing time, the margins between the scaffold and bone defect became less demarcated due to deposition and ingrowth of new bone (Fig. 4b).