2Hollow hydroxyapatite (HA) microspheres showed the ability to facilitate bone regeneration 3 in rats with non-healing calvarial defects. However, new bone formation in the rat calvarial defect 4 implanted with the closed HA microspheres was limited. The objective of this work is to evaluate 5 size-, time, and structure-dependent bone regeneration between open and closed HA microspheres in 6 an osseous model. Open HA microspheres were obtained by sectioning closed HA microspheres. 7 The open HA microsphere had dense convex surface and rough and porous concave surface. For 8 both size ranges (ϕ106-150 μm vs. ϕ212-250 μm), the open HA microsphere were more effective in 9 facilitating bone regeneration than the closed HA microspheres in rat calvarial defects. Bone 10 regeneration in the open HA microspheres (49 ± 7% for ϕ106-150 μm; 40 ± 8% for ϕ212-250 μm)11 were higher than the closed HA microsphere (26 ± 8% for ϕ106-150 μm; 30 ± 9% for ϕ212-250 μm)12 at 12 weeks. Furthermore, the open HA microspheres of smaller size showed a significant increase in 13 bone regeneration than the open HA microspheres of larger size at both 6 weeks and 12 weeks. The 14 difference in bone regeneration between these microspheres could be due to their differences in 15 microstructures, namely curvature, concavity, porosity, surface roughness, and total surface area 16 available for cells to attached to.