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Stress-induced premature senescent (SIPS) cells induced by various stresses deteriorate cell functions. Dasatinib and quercetin senolytics (DQ) can alleviate several diseases by eliminating senescent cells. α-tricalcium phosphate (α-TCP) is a widely used therapeutic approach for bone restoration but induces bone formation for a comparatively long time. Furthermore, bone infection exacerbates the detrimental prognosis of bone formation during material implant surgery due to oral cavity bacteria and unintentional contamination. It is essential to mitigate the inhibitory effects on bone formation during surgical procedures. Little is known that DQ improves bone formation in Lipopolysaccharide (LPS)-contaminated implants and its intrinsic mechanisms in the study of maxillofacial bone defects. This study aims to investigate whether the administration of DQ ameliorates the impairments on bone repair inflammation and contamination by eliminating SIPS cells. α-TCP and LPS-contaminated α-TCP were implanted into Sprague-Dawley rat calvaria bone defects. Simultaneously, bone formation in the bone defects was investigated with or without the oral administration of DQ. Micro-computed tomography and hematoxylin-eosin staining showed that senolytics significantly enhanced bone formation at the defect site. Histology and immunofluorescence staining revealed that the levels of p21- and p16-positive senescent cells, inflammation, macrophages, reactive oxygen species, and tartrate-resistant acid phosphatase-positive cells declined after administering DQ. DQ could partially alleviate the production of senescent markers and senescence-associated secretory phenotypes in vitro. This study indicates that LPS-contaminated α-TCP-based biomaterials can induce cellular senescence and hamper bone regeneration. Senolytics have significant therapeutic potential in reducing the adverse osteogenic effects of biomaterial-related infections and improving bone formation capacity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.) more...

Various stresses latently induce cellular senescence that occasionally deteriorates the functioning of surrounding tissues. Nevertheless, little is known about the appearance and function of senescent cells, caused by the implantation of beta-tricalcium phosphate (β-TCP)-used widely in dentistry and orthopedics for treating bone diseases. In this study, two varying sizes of β-TCP granules (<300 μm and 300-500 μm) were implanted, and using histological and immunofluorescent staining, appearances of senescent-like cells in critical-sized bone defects in the calvaria of Sprague Dawley rats were evaluated. Parallelly, bone formation in defects was investigated with or without the oral administration of senolytics (a cocktail of dasatinib and quercetin). A week after the implantation, the number of senescence-associated beta-galactosidase, p21-, p19-, and tartrate-resistant acid phosphatase-positive cells increased and then decreased upon administrating senolytics. This administration of senolytics also attenuated 4-hydroxy-2-nonenal staining, representing reactive oxygen species. Combining senolytic administration with β-TCP implantation significantly enhanced the bone formation in defects as revealed by micro-computed tomography analysis and hematoxylin-eosin staining. This study demonstrates that β-TCP granules latently induce senescent-like cells, and senolytic administration may improve the bone-forming ability of β-TCP by inhibiting senescence-associated mechanisms. more...

Heparin binds to and modulates various growth factors, potentially augmenting the bone-forming capability of biomaterials. Here, α-tricalcium phosphate (α-TCP) granules were modified with peptide containing the marine mussel-derived adhesive sequence, which reacts with α-TCP surface, and cationic sequence, which binds to heparin (α-Ph). α-Ph retained the α-TCP phase and intergranule spaces after the surface modification. The existence of heparin on α-Ph granules was confirmed using X-ray photoelectron spectroscopy. Granules of α-TCP and α-Ph were implanted into critical-size defects in rat calvaria for 4 weeks. Micro-computed tomography, histological evaluation, and Alcian blue staining revealed that α-Ph induced superior bone formation compared with α-TCP. Newly formed bone on α-Ph was preferentially in contact with the Alcian blue-stained surfaces of granules. These results suggested that heparinization enhanced the early osteogenic capacity of α-TCP, possibly by modulating the secretion of Alcian blue-stained extracellular matrixes. more...

Purpose: Interferon (IFN)-γ is a major cytokine produced by immune cells that plays diverse roles in modulating both the immune system and bone metabolism, but its role in autogenous bone grafting remains unknown. Here, we present that local IFN-γ administration improved the efficacy of autogenous bone graft treatment in an experimental rat model., Methods: An autogenous bone graft model was prepared with critically sized rat calvariae defects. Four weeks (w) after bone graft implantation, rats were treated locally with IFN-γ or were not treated. The effect of IFN-γ on bone formation was evaluated for up to 8w with micro-computed tomography, quantitative histomorphometry, and Von Kossa staining. Osteoclastogenesis was assessed by tartrate-resistant acid phosphatase staining. Immunohistochemistry staining or quantitative polymerase chain reactions were used to estimate the expression of osteoclast differentiation factor and inflammatory cytokines including tumor necrosis factor (TNF)-α, a well-known stimulant of osteoclastogenesis and an inhibitor of osteoblast activity, in defects., Results: Newly formed bone gradually replaced the autogenous bone grafts within 4w, although severe bone resorption with osteoclastogenesis and TNF-α expression occurred after 6w in the absence of IFN-γ administration. IFN-γ administration markedly attenuated bone loss, osteoclastogenesis, and TNF-α expression, while it enhanced bone formation at 8w., Conclusion: Local IFN-γ administration promoted bone formation in autogenous bone grafts possibly via regulating osteoclastogenesis and TNF-α expression. The data provide insights into the potential roles of IFN-γ in autogenous bone grafting., (Copyright © 2016 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.) more...

Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT) cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG), the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25-10 μM) in osteogenic medium (OM) with or without 100 nM dexamethasone (Dex) for 12 days (hereafter two osteogenic media were designated as OM(Dex) and OM). Supplementation of 1.25 μM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1) and runt-related transcription factor 2 (RUNX2) and also increased proliferation and mineralization. Compared to OM(Dex) with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex) with 10 μM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex) with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy. more...

Catechins are extensively used in health care treatments. Nevertheless, there is scarce information about the feasibility of local administration with polyphenols for bone regeneration therapy, possibly due to lack of effective delivery systems. Here we demonstrated that the epigallocatechin-3-gallate-conjugated gelatin (EGCG/Gel) prepared by an aqueous chemical synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholinium chloride (DMT-MM) gradually disintegrated with time and facilitated bone formation in a critical size defect of a mouse calvaria. Conjugation of EGCG with the Gel generated cross-linking between the two molecules, thereby leading to a retardation of the degradation of the EGCG/Gel and to a delayed release of EGCG. The prepared EGCG/Gels represented significant osteogenic capability compared with that of the uncross-linked Gel and the cross-linked Gel with uncombined-EGCG. In vitro experiments disclosed that the EGCG/Gel induced osteoblastogenesis of a mouse mesenchymal stem cell line (D1 cells) within 14 days. Using fluorescently-labeled EGCG/Gel, we found that the fraction of EGCG/Gel adsorbed onto the cell membrane of the D1 cells possibly via a Gel-cell interaction. The interaction might confer the long-term effects of EGCG on the cells, resulting in a potent osteogenic capability of the EGCG/Gel in vivo. These results should provide insight into local controlled release of polyphenols for bone therapy. more...

Stem cell-based disease modeling presents unique opportunities for mechanistic elucidation and therapeutic targeting. The stable induction of fate-specific differentiation is an essential prerequisite for stem cell-based strategy. Bone morphogenetic protein 2 (BMP-2) initiates receptor-regulated Smad phosphorylation, leading to the osteogenic differentiation of mesenchymal stromal/stem cells (MSC) in vitro; however, it requires supra-physiological concentrations, presenting a bottleneck problem for large-scale drug screening. Here, we report the use of a double-objective feedback system control (FSC) with a differential evolution (DE) algorithm to identify osteogenic cocktails of extrinsic factors. Cocktails containing significantly reduced doses of BMP-2 in combination with physiologically relevant doses of dexamethasone, ascorbic acid, beta-glycerophosphate, heparin, retinoic acid and vitamin D achieved accelerated in vitro mineralization of mouse and human MSC. These results provide insight into constructive approaches of FSC to determine the applicable functional and physiological environment for MSC in disease modeling, drug screening and tissue engineering. more...

Although the efficacy of the in vivo osteogenic capabilities of synthetic octacalcium phosphate (OCP) crystal implantation can be explained through its stimulatory capacity for the differentiation of the host osteoblastic cell lineage, direct evidence that OCP supports bone regeneration by osteogenic cells in vivo has not been shown. Mesenchymal stem cells (MSCs) isolated from 4-week-old male Wistar rat long bones were pre-incubated in osteogenic or maintenance medium in the presence or absence of basic fibroblast growth factor (bFGF). OCP/Collagen (OCP/Col) or collagen disks were seeded with MSCs that had been pre-incubated in osteogenic medium containing bFGF, which exhibited the highest differentiation induction, and then incubated for an additional day. The disks were implanted in critical-sized calvaria defects of 12-week-old male Wistar rats and the specimens were analysed radiographically, histologically, histomorphometrically, and by micro-computed tomography (CT) imaging at 4 and 8 weeks after the implantation. The OCP/Col·MSCs group rapidly induced more bone regeneration, even within 4 weeks, compared to the OCP/Col group without MSCs. The bone mineral density of the OCP/Col·MSCs group was also greater than the OCP/Col group. The Col·MSCs group did not exhibit prominent osteogenicity. These results indicate that OCP crystals in a collagen matrix efficiently promote exogenously introduced osteogenic cells to initiate bone regeneration if the cells are pre-treated in a suitable differentiation condition. more...

Bone morphogenetic proteins (BMPs) possess osteoinductive activities and are useful for clinical treatments, including bone regeneration. We found that transforming growth factor (TGF)-β1 strongly enhances the osteoinductive activity of BMP-2. Collagen sponges containing 5 μg of BMP-2 were implanted into mouse muscle tissues, after which lump-like masses appeared and grew until day 7. Subsequently, calcification occurred in the lump-like masses by day 14. Addition of 50 ng of TGF-β1 to the BMP-2-containing sponges markedly accelerated the growth of the lump-like masses and resulted in a fivefold increase in total bone volume as compared with BMP-2 alone. The number of osteoblasts in ectopic bone tissues at 14 days after implantation induced by BMP-2+TGF-β1 was twofold greater than that with BMP-2 alone, whereas the number of osteoclasts was decreased by half. On the other hand, TGF-β1 accelerated the differentiation of both osteoblasts and osteoclasts in the early stage (2-7 days after implantation) of ectopic bone formation. We also implanted collagen sponges into bone defects surgically created in mouse calvaria. Sponges containing 2.5 μg of BMP-2 and 25 ng of TGF-β1 caused complete filling of the defects with orthotopic bone, whereas those containing 2.5 μg of BMP-2 alone caused only partial filling. These results suggest that TGF-β1 enhances BMP-2-induced ectopic bone formation by accelerating the growth of lump-like masses, and regulates osteoblast and osteoclast generation. Our findings may contribute to the development of a new treatment method for skeletal disorders. more...

The present study was designed to investigate whether octacalcium phosphate (OCP)-precipitated alginate (Alg) promotes osteoblastic cell proliferation and bone regeneration in comparison with Alg itself. Alg, known to lack mammal cell attachment capability, was used as the matrix to test the distribution effect of OCP in a three-dimensional environment. A series of Alg/OCP scaffolds with different pore sizes was prepared by centrifuging Alg gels precipitated by OCP crystals. The scaffolds had a bimodal distribution of pores (ultrafine pores: approximately 100 nm; relatively large pores: from 6.0 to 51.7 microm) and over 86% porosity. The osteoconductive capability of Alg/OCP was determined by examining mouse bone marrow stromal ST-2 cell proliferation after 3 days in vitro and bone regeneration in mouse calvaria critical-sized defect after 21 days. The analyses showed that ST-2 cell proliferation and bone regeneration increased with an increase in the pore size and reached the highest level in the 51.7 microm pore scaffold. The results suggest that OCP-precipitated Alg provides a better scaffold for osteoblasts to attach and proliferate in a three-dimensional environment and promotes bone regeneration, indicating that OCP is a candidate material to modify the surface of non-cell-interactive polymeric scaffolds, such as Alg, into an osteogenic condition. more...

Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP--apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage. more...

The present study was designed to investigate whether hydrolysis of synthetic octacalcium phosphate (OCP) into hydroxyapatite affects bone formation. Mouse bone marrow stromal ST-2 cells and primary calvarial osteoblastic cells were cultured on the dishes pre-coated with OCP or its hydrolyzed Ca-deficient hydroxyapatite (OCP hydrolyzate; HL). The capacity of proliferation and differentiation was determined up to day 20. Granules of OCP and HL were implanted into critical-size rat calvaria defects for 4 and 12 weeks, and then bone formation was measured by histomorphometry. Structural changes of incubated and implanted OCP were determined by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The proliferation of both ST-2 and primary osteoblasts cultured on OCP or HL was initially inhibited, whereas their differentiation to osteoblasts was promoted at last. Implantation of OCP in bone defect more significantly enhanced bone formation than that of HL until 12 weeks. OCP tended to convert to apatite in vitro and in vivo. The conversion of the implanted OCP was ascertained to advance gradually with implantation periods. Taken together, these results suggest that OCP supports appositional bone formation and OCP-apatite conversion may be involved in this stimulatory capacity of OCP. more...