Hironori Sugimoto1, Yusuke Inagaki2, Akira Furukawa1, Tsutomu Kira1, Sachiko Kawasaki1, Yoshinobu Uchihara1, Manabu Akahane3, Yasuhito Tanaka1. 1. Department of Orthopaedic Surgery, Nara Medical University, Shijocho 840, 634-8521, Kashihara, Nara, Japan. 2. Department of Orthopaedic Surgery, Nara Medical University, Shijocho 840, 634-8521, Kashihara, Nara, Japan. yinagaki@naramed-u.ac.jp. 3. Department of Health and Welfare Services, National Institute of Public Health, 2-3-6 Minami, 351-0197, Wako, Saitama, Japan.
Abstract
BACKGROUND: β-Tricalcium phosphate (β-TCP) is a popular synthetic bone graft substitute with excellent osteoconductive properties and bioabsorbability. However, its osteoinductive properties are inferior to those of autologous or allogeneic bone. Trace elements such as strontium (Sr), silica (Si), and zinc (Zn) have been reported to promote osteogenesis in materials. In this study, we aimed to determine whether a Si/Zn-substituted Sr apatite coating of β-TCP could enhance osteoinductive properties. METHODS: The apatite-coated β-TCP disks were prepared using nanoparticle suspensions of silicate-substituted Sr apatite (SrSiP) or silicate- and Zn-co-substituted Sr apatite (SrZnSiP). Bone marrow mesenchymal cells (BMSCs) from rat femur were cultured and subsequently seeded at a density of 1.0 × 106/cm2 onto apatite-coated and non-coated β-TCP disks. In vitro, the β-TCP disks were then placed in osteogenic medium, and lactate dehydrogenase (LDH) activity was measured from supernatants after culture for 2 days. Additionally, after culture for 14 days, the mRNA expression of genes encoding osteocalcin (OC), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was evaluated by qRT-PCR. In vivo, the β-TCP disks were transplanted subcutaneously into rats that were sacrificed after 4 weeks. Then, the harvested disks were evaluated biochemically (ALP activity, OC content, mRNA expression of OC, ALP, BMP-2, and VEGF measured by qRT-PCR), radiologically, and histologically. RESULTS: Significantly higher mRNA expression of almost all evaluated osteogenic and angiogenic genes was observed in the SrZnSiP and SrSiP groups than in the non-coated group, with no significant cytotoxicity elicited by the apatite coating in vitro. Moreover, in vivo, the SrZnSiP and SrSiP groups showed significantly higher osteogenic and angiogenic gene expression and higher ALP activity and OC content than the non-coated group (P < 0.05). Radiological and histopathological findings revealed abundant bone formation in the apatite-coated group. CONCLUSIONS: Our findings indicate that apatite coating of β-TCP improves osteoinductive properties without inducing significant cytotoxicity.
BACKGROUND: β-Tricalcium phosphate (β-TCP) is a popular synthetic bone graft substitute with excellent osteoconductive properties and bioabsorbability. However, its osteoinductive properties are inferior to those of autologous or allogeneic bone. Trace elements such as strontium (Sr), silica (Si), and zinc (Zn) have been reported to promote osteogenesis in materials. In this study, we aimed to determine whether a Si/Zn-substituted Sr apatite coating of β-TCP could enhance osteoinductive properties. METHODS: The apatite-coated β-TCP disks were prepared using nanoparticle suspensions of silicate-substituted Sr apatite (SrSiP) or silicate- and Zn-co-substituted Sr apatite (SrZnSiP). Bone marrow mesenchymal cells (BMSCs) from rat femur were cultured and subsequently seeded at a density of 1.0 × 106/cm2 onto apatite-coated and non-coated β-TCP disks. In vitro, the β-TCP disks were then placed in osteogenic medium, and lactate dehydrogenase (LDH) activity was measured from supernatants after culture for 2 days. Additionally, after culture for 14 days, the mRNA expression of genes encoding osteocalcin (OC), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was evaluated by qRT-PCR. In vivo, the β-TCP disks were transplanted subcutaneously into rats that were sacrificed after 4 weeks. Then, the harvested disks were evaluated biochemically (ALP activity, OC content, mRNA expression of OC, ALP, BMP-2, and VEGF measured by qRT-PCR), radiologically, and histologically. RESULTS: Significantly higher mRNA expression of almost all evaluated osteogenic and angiogenic genes was observed in the SrZnSiP and SrSiP groups than in the non-coated group, with no significant cytotoxicity elicited by the apatite coating in vitro. Moreover, in vivo, the SrZnSiP and SrSiP groups showed significantly higher osteogenic and angiogenic gene expression and higher ALP activity and OC content than the non-coated group (P < 0.05). Radiological and histopathological findings revealed abundant bone formation in the apatite-coated group. CONCLUSIONS: Our findings indicate that apatite coating of β-TCP improves osteoinductive properties without inducing significant cytotoxicity.