Mechanistic Study of Phytoestrogenic Icaritin and Its Osteopromotive Effects after Incorporation into a Composite Scaffold for Enhancing Bone Defect Repair in Steroid Associated Osteonecrosis (SAON)

Part I: to study the effect and molecular mechanism of Icaritin on the differentiation of human bone marrow-derived MSCs. Human MSC was identified first by flow cytometery and result showed our cultured human MSC expressed standard surface markers of MSCs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation ability of MSCs was not affected by Icaritin. Differentiation assay showed that without oseteogenic supplements (OS), Icaritin had no effect on osteogenic differentiation of MSCs. With presence of OS, Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs. Real-time polymerase chain reaction (RT-PCR) showed that Icaritin up-regulated osteoblastic marker genes expression during osteogenic differentiation of MSCs and inhibited adipogenic gene expression. Further studies showed that Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis. All these findings indicated Icaritin possessed osteopromotive but not osteoinductive potentials during the differentiation of MSCs. Icaritin regulated osteogenic differentiation of MSCs in BMP2 pathway dependent manner.;Part II: to evaluate the differentiation potential of MSCs derived from rabbit with SAON and the effect of Icaritin on the altered differentiation of MSCs. The results showed that Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs derived from normal rabbit. Osteogenic differentiation potential of mesenchymal stem cells derived from rabbit with SAON declined and Icaritin partly rescued the declined osteogenic differentiation potential in dose-dependent manner. Adipogenic differentiation potential of MSCs derived from rabbit with SAON enhanced while the enhanced adipogenesis could be depressed by Icaritin. The proliferation ability of MSCs derived from rabbit with SAON declined while could not be rescued by Icaritin. VEGF expression decreased in MSCs derived from rabbit with SAON but its expression could not be influenced by Icaritin. These findings showed that the differentiation potential of MSCs destroyed during SAON development and this potential could be partially restored by Icaritin.;Part III: to evaluate the in vitro angiogenic effect of Icaritin. The proliferation, migration and tube formation ability of human umbilical vein cells (HUVECs) were detected. The results showed that Icaritin did not affect HUVECs proliferation, migration and tube-like structure formation of HUVECs. Real time PCR showed that VEGF, HIF1a, FGF2 and TGF-beta expression in HUVECs was not changed when HUVECs were treated by Icaritin. These data indicated Icaritin did not directly impact angiogenesis in vitro. Combined with in vivo findings, we supposed Icaritin promoted angiogenesis through its enhanced osteogenesis during bone defect repair.;Part IV: to study Icaritin and scaffold impact on stem cell homing in vitro and in vivo. It was found Icaritin promoted the migration of rabbit MSCs and increased vascular cell adhesion molecule 1 (VCAM1) expression. Composite scaffolds PLGA/TCP and PLGA/TCP/Icaritin could recruit rabbit MSCs under in vitro culture condition. When labeled with SPIO SiO2-NH2, the differentiation potential of rabbit MSCs retained while proliferation and migration ability of rabbit MSCs declined. Two weeks after SAON establishment, PLGA/TCP and PLGA/TCP/Icaritin scaffolds were implanted into the bone tunnel after core-decompression in initial necrotic bone defect in rabbits with SAON, immediately with SPIO SiO2-NH2 labeled MSCs injected into bone marrow cavity locally. The results showed that without scaffold implantation, the tunnel was filled with fat cells and fibrotic tissues and there was no label MSC in the tunnel while there were more labeled cells appeared in bone marrow near the tunnel than far away the tunnel, with both PLGA/TCP and PLGA/TCP/Icaritin implantation, the labeled MSCs migrated into scaffold after its implantation into the bone tunnel while there was no labeled cell next to the tunnel but some were shown away from the tunnel. No significant difference was found in SPIO positive MSCs in bone tunnel between PLGA/TCP and PLGA/TCP/Icaritin group. The findings indicated that at least PLGA/TCP scaffold itself promoted MSCs homing in vitro and in vivo where the released icaritin could execute its osteopromotive effects. (Abstract shortened by UMI.).