Utility of Thermal Cross-Linking in Stabilizing Hydrogels with Beta-Tricalcium Phosphate and/or Epigallocatechin Gallate for Use in Bone Regeneration Therapy.

ß-tricalcium phosphate (ß-TCP) granules are commonly used materials in dentistry or orthopedic surgery. However, further improvements are required to raise the operability and bone-forming ability of ß-TCP granules in a clinical setting. Recently, we developed epigallocatechin gallate (EGCG)-modified gelatin sponges as a novel biomaterial for bone regeneration. However, there is no study on using the above material for preparing hydrogel incorporating ß-TCP granules. Here, we demonstrate that vacuum heating treatment induced thermal cross-linking in gelatin sponges modified with EGCG and incorporating ß-TCP granules (vhEc-GS-ß) so that the hydrogels prepared from vhEc-GS-ß showed high stability, ß-TCP granule retention, operability, and cytocompatibility. Additionally, microcomputed tomography morphometry revealed that the hydrogels from vhEc-GS-ß had significantly higher bone-forming ability than ß-TCP alone. Tartrate-resistant acid phosphatase staining demonstrated that the number of osteoclasts increased at three weeks in defects treated with the hydrogels from vhEc-GS-ß compared with that around ß-TCP alone. The overall results indicate that thermal cross-linking treatment for the preparation of sponges (precursor of hydrogels) can be a promising process to enhance the bone-forming ability. This insight should provide a basis for the development of novel materials with good operativity and bone-forming ability for bone regenerative medicine.

Augmentation of Bone Regeneration by Depletion of Stress-Induced Senescent Cells Using Catechin and Senolytics.

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-ß-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-ß-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.