Despite advances in bone regenerative medicine, the relationship between stress-induced premature senescence (SIPS) in cells and bone regeneration remains largely unknown. Herein, we demonstrated that the implantation of a lipopolysaccharide (LPS) sustained-release gelatin sponge (LS-G) increases the number of SIPS cells and that the elimination of these cells promotes bone formation in critical-sized bone defects in the rat calvaria. Histological (hematoxylin–eosin and SA-β-gal) and immunohistological (p16 and p21 for analyzing cellular senescence and 4-HNE for oxidation) staining was used to identify SIPS cells and elucidate the underlying mechanism. Bone formation in defects were analyzed using microcomputed tomography, one and four weeks after surgery. Parallel to LS-G implantation, local epigallocatechin gallate (EGCG) administration, and systemic senolytic (dasatinib and quercetin: D+Q) administration were used to eliminate SIPS cells. After LS-G implantation, SA-β-gal-, p16-, and p21-positive cells (SIPS cells) accumulated in the defects. However, treatment with LS-G+EGCG and LS-G+D+Q resulted in lower numbers of SIPS cells than that with LS-G in the defects, resulting in an augmentation of newly formed bone. We demonstrated that SIPS cells induced by sustained stimulation by LPS may play a deleterious role in bone formation. Controlling these cell numbers is a promising strategy to increase bone regeneration.

Freeze-drying, also known as lyophilization, is widely used in the preparation of porous biomaterials. Nevertheless, limited information is known regarding the effect of gas permeability on molds to obtain porous materials. We demonstrated that the different levels of gas permeability of molds remarkably altered the pore distribution of prepared gelatin sponges and distinct bone formation at critical-sized bone defects of the rat calvaria. Three types of molds were prepared: silicon tube (ST), which has high gas permeability; ST covered with polyvinylidene chloride (PVDC) film, which has low gas permeability, at the lateral side (STPL); and ST covered with PVDC at both the lateral and bottom sides (STPLB). The cross sections or curved surfaces of the sponges were evaluated using scanning electron microscopy and quantitative image analysis. The gelatin sponge prepared using ST mold demonstrated wider pore size and spatial distribution and larger average pore diameter (149.2 µm) compared with that prepared using STPL and STPLB. The sponges using ST demonstrated significantly poor bone formation and bone mineral density after 3 weeks. The results suggest that the gas permeability of molds critically alters the pore size and spatial pore distribution of prepared sponges during the freeze-drying process, which probably causes distinct bone formation.