Tauroursodeoxycholic Acid Enhances Osteogenic Differentiation through EGFR/p-Akt/CREB1 Pathway in Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are pluripotent stromal cells that are among the most appealing candidates for regenerative medicine and may aid in the repair and regeneration of skeletal disorders through multiple mechanisms, including angiogenesis, differentiation, and response to inflammatory conditions. Tauroursodeoxycholic acid (TUDCA) has recently been used in various cell types as one of these drugs. The mechanism of osteogenic differentiation by TUDCA in hMSCs remains unknown. METHODS: Cell proliferation was performed by the WST-1 method, and alkaline phosphatase activity and alizarin red-sulfate staining were used to confirm the osteogenic differentiation indicator. Expression of genes related to bone differentiation and specific genes related to signaling pathways was confirmed by quantitative real-time polymerase chain reaction. RESULTS: We found that cell proliferation was higher as the concentration increased, and showed that the induction of osteogenic differentiation was significantly enhanced. We also show that osteogenic differentiation genes were upregulated, with the expression of the epidermal growth factor receptor (EGFR) and cAMP responsive element binding protein 1 (CREB1) being specifically high. To confirm the participation of the EGFR signaling pathway, the osteogenic differentiation index and expression of osteogenic differentiation genes were determined after using an EGFR inhibitor. As a result, EGFR expression was remarkably low, and that of CREB1, cyclin D1, and cyclin E1 was also significantly low. CONCLUSIONS: Therefore, we suggest that TUDCA-induced osteogenic differentiation of human MSCs is enhanced through the EGFR/p-Akt/CREB1 pathway.

Tauroursodeoxycholic acid induces angiogenic activity in endothelial cells and accelerates bone regeneration.

Angiogenesis is a crucial process during bone tissue regeneration. The aim of this study was to investigate the angiogenic activity and the potentiation of bone regeneration via angiogenesis using tauroursodeoxycholic acid (TUDCA) in vitro and in vivo. We investigated the effect of TUDCA on proliferation and angiogenic differentiation in human umbilical vein endothelial cells (HUVECs) and the associated signaling pathway. Proliferation was determined using crystal violet assay. Angiogenic effects were evaluated based on cell migration and tube formation. In order to explore TUDCA-signaling pathways, phosphorylation of mitogen activated protein kinase, protein kinase B (AKT), and endothelial nitric oxide synthase (eNOS) was determined using western blot. Furthermore, in vivo bone formation and angiogenesis were determined using a New Zealand outbred albino rabbit calvarial defect model, while angiogenesis and bone formation were evaluated using micro-CT and histological analysis. Our results show that TUDCA significantly increased cell proliferation. Moreover, TUDCA enhanced cell migration and tube formation in HUVECs. TUDCA increased the phosphorylation of AKT, ERK1/2, c-Jun N-terminal kinase, and eNOS. Specific inhibitors of ERK1/2 (PD98059), JNK (SP600125), and AKT (AKT1/2) inhibited the TUDCA-induced migration and tube formation, while the p38 inhibitor (SB203580) did not. The in vivo study used TUDCA to accelerate new blood vessel formation and promoted bone formation in rabbit calvarial defect model. These results indicate that TUDCA plays a critical role in enhancing the angiogenesis of endothelial cells and in vivo new bone regeneration. The use of TUDCA may contribute to the regeneration of bone tissue by improving angiogenesis.

Incorporation of BMP-2 nanoparticles on the surface of a 3D-printed hydroxyapatite scaffold using an ε-polycaprolactone polymer emulsion coating method for bone tissue engineering.

Hydroxyapatite (HAp)-based three-dimensional (3D) scaffolding is an excellent method for the fabrication of complex-shaped scaffolds to reconstruct bone defects. This study aimed at improving the osteoinductivity and compressive strength of the HAp-based 3D scaffold for bone regeneration. Bone morphogenetic protein-2-loaded nanoparticles (BMP-2/NPs) were prepared by a double emulsion-solvent evaporation method and incorporated onto the surface of 3D scaffolds using ε-polycaprolactone (PCL) and NPs emulsion solution. The surface morphology of the scaffold was characterized using scanning electron microscopy and its biocompatibility and osteogenic effects evaluated in vitro using human mesenchymal stem cells. The in vivo bone regeneration efficiency was determined using a rabbit calvarial bone defect model. We obtained 3D HAp scaffolds with NPs using PCL coating process. BMP-2/NPs were uniformly distributed on the scaffold surface and BMP-2 was gradually released. Furthermore, PCL coating improved the compressive strength of the scaffold. The cell proliferation, adhesion, and osteogenic differentiation properties were improved with PCL_BMP-2/NPs coated scaffold. In vivo experiments showed that the formation of new bone was significantly higher in the PCL_BMP-2/NPs group than in the uncoated scaffold-implanted group. The coating method using PCL and NPs emulsion solutions was useful not only to incorporate BMP-2/NPs onto the surface of the scaffold, but also to improve the compressive strength, which enhanced bone regeneration.

Zoledronate suppresses VEGF­induced capillary tube formation and inhibits expression of interferon­induced transmembrane protein­1 in human umbilical vein endothelial cells.

Interferon­induced transmembrane protein 1 (IFITM1) is a member of the interferon­induced transmembrane protein family and has recently been identified as a novel protein participant in angiogenesis. Zoledronate (ZON), a nitrogen­containing bisphosphonate, is widely used in the treatment of osteoporosis and to prevent bone metastases of certain cancer types. However, the association between ZON and IFITM1 has remained elusive. The present study investigated the effect of ZON on the expression of IFITM1 during vascular endothelial growth factor (VEGF)­induced capillary tube formation in human umbilical vein endothelial cells. It was observed that cell proliferation and VEGF­induced tube formation were significantly inhibited by treatment with 10 µM ZON. The expression of IFITM1 increased during VEGF­induced tube formation. However, the VEGF­induced increase in IFITM1 expression exhibited a dose­ and time­dependent decrease with ZON treatment at the mRNA and protein level. Furthermore, matrix metalloproteinase­9 activation was markedly decreased by ZON treatment. These results suggest that induction of IFITM1 expression may be involved in the anti­angiogenic activity of ZON.

Fucoidan-induced osteogenic differentiation promotes angiogenesis by inducing vascular endothelial growth factor secretion and accelerates bone repair.

Osteogenesis and angiogenesis, including cell-cell communication between blood vessel cells and bone cells, are essential for bone repair. Fucoidan is a chemical compound that has a variety of biological activities. It stimulates osteoblast differentiation in human mesenchymal stem cells (MSCs), which in turn induces angiogenesis. However, the mechanism by which this communication between osteoblasts and endothelial cells is mediated remains unclear. Thus, the aim of this study was to clarify the relationship between fucoidan-induced osteoblastic differentiation in MSCs and angiogenesis in endothelial cells. First, the effect was confirmed of fucoidan on osteoblast differentiation in MSCs and obtained conditioned media from these cells (Fucoidan-MSC-CM). Next, the angiogenic activity of Fucoidan-MSC-CM was investigated and it was found that it stimulated angiogenesis, demonstrated by proliferation, tube formation, migration and sprout capillary formation in human umbilical vein endothelial cells. Messenger ribonucleic acid expression and protein secretion of vascular endothelial growth factor (VEGF) were dramatically increased during fucoidan-induced osteoblast differentiation and that its angiogenic activities were reduced by a VEGF/VEGF receptor-specific binding inhibitor. Furthermore, Fucoidan-MSC-CM increased the phosphorylation of mitogen-activated protein kinase and PI3K/AKT/eNOS signalling pathway, and that its angiogenic effects were markedly suppressed by SB203580 and AKT 1/2 inhibitor. Finally, an in vivo study was conducted and it was found that fucoidan accelerated new blood vessel formation and partially promoted bone formation in a rabbit model of a calvarial bone defect. This is the first study to investigate the angiogenic effect of fucoidan-induced osteoblastic differentiation through VEGF secretion, suggesting the therapeutic potential of fucoidan for enhancing bone repair.

Improvement of mechanical strength and osteogenic potential of calcium sulfate-based hydroxyapatite 3-dimensional printed scaffolds by ε-polycarbonate coating.

Powder-based three-dimensional (3D) printing is an excellent method to fabricate complex-shaped scaffolds for tissue engineering. However, their lower mechanical strength restricts their application in bone tissue engineering. Here, we created a 3D-printed scaffold coated with a ε-polycaprolactone (PCL) polymer solution (5 and 10 w/v %) to improve the mechanical strength of the scaffold. The 3D scaffold was fabricated from calcium sulfate hemihydrate powder (CaSO4-1/2 H2O), transformed into hydroxyapatite (HAp) by treatment with a hydrothermal reaction in an NH4H2PO4 solution. The surface properties and composition of the scaffold were evaluated using scanning electron microscopy and X-ray diffraction analysis. We demonstrated that the 3D scaffold coated with PCL had an improved mechanical modulus. Coating with 5 and 10% PCL increased the compressive strength significantly, by about 2-fold and 4-fold, respectively, compared with that of uncoated scaffolds. However, the porosity was reduced significantly by coating with 10% PCL. In vitro biological evaluation demonstrated that MG-63 cells adhered well and proliferated on the 3D scaffold coated with PCL, and the scaffold was not cytotoxic. In addition, alkaline phosphatase activity and real time polymerase chain reaction demonstrated that osteoblast differentiation also improved in the PCL-coated 3D scaffolds. These results indicated that PCL polymer coating could improve the compressive strength and biocompatibility of 3D HAp scaffolds for bone tissue engineering applications.

Precoating of biphasic calcium phosphate bone substitute with atelocollagen enhances bone regeneration through stimulation of osteoclast activation and angiogenesis.

Type I collagen (Col) is a naturally polymerizing protein and important extracellular matrix bone component. The aim of this study was to improve bone regeneration capacity by precoating the surface of biphasic calcium phosphate (BCP) granules with AT-Col, and evaluating its biological effects. BCP granules were precoated with AT-Col using adsorption and lyophilization method. Morphology of AT-Col precoated surfaces was observed using scanning electron microscopy (SEM). Biocompatibility and osteogenic activity of AT-Col were determined in vitro with human mesenchymal stem cell (hMSCs). In vivo bone healing efficiency and related biological effects were determined using a rabbit calvarial defect model. SEM results revealed numerous irregularly distributed AT-Col polymer clusters on BCP granule surface. Biocompatibility experiments demonstrated that AT-Col was non-cytotoxic, and that cell proliferation, adhesion, and osteogenic activity were improved by AT-Col precoating. After in vivo surgical implantation into bone defects, new bone formation was improved by AT-Col granule precoating. Specifically, 8 weeks post-surgery, percentage bone volume was significantly higher in AT-Col/BCP animals (35.02 ± 1.89%) compared with BCP-treated animals (8.94 ± 1.47%) (p < 0.05). Furthermore, tartrate-resistant acid phosphatase staining and CD31 immunohistochemical staining revealed that osteoclast activation and new blood vessel formation in vivo were also induced by AT-Col precoating. Collectively, these data indicate that AT-Col/BCP may be potentially used as a bone substitute to enable effective bone regeneration through enhanced new blood vessel formation and osteoclast activation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1446-1456, 2017.

Enhanced bone regeneration by silicon-substituted hydroxyapatite derived from cuttlefish bone.

OBJECTIVE: There is growing interest in the use of cuttlefish bone (CB) as a bone graft material. Silicon (Si) plays an important role in bone formation and calcification. This study aimed to prepare Si-substituted CB-derived hydroxyapatite (Si-CB-HAp) using a natural CB to improve the bioactivity for bone formation. MATERIALS AND METHODS: We prepared Si-HAp from CB (Si-CB-HAp) using a hydrothermal and solvothermal method. The microstructure and chemical composition were characterized by scanning electron microscope (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectrometer (EDS). The bioactivity of the Si-CB-HAp was evaluated using human mesenchymal stem cells. Furthermore, the in vivo bone regeneration efficiency was evaluated using a rabbit calvarial defect model. RESULTS: Our results show that the Si content was 0.77 wt% in Si-CB-HAp, and its original microstructure was conserved. The presence of Si was shown to enhance cell proliferation and early cellular attachment of human mesenchymal stem cells. Additionally, results of alkaline phosphatase activity and real-time PCR for osteoblast marker genes show that Si substitution into CB-HAp enhanced osteoblast differentiation. In addition, in vivo bone defect healing experiments show that the formation of bone with Si-CB-HAp is higher than that with CB-HAp. CONCLUSION: These results indicate that Si-CB-HAp may potentially be used as a bone graft material to enhance bone healing.

Angiogenin-loaded fibrin/bone powder composite scaffold for vascularized bone regeneration.

BACKGROUND: Angiogenin (ANG) is a potent stimulator of angiogenesis. The aim of this study was to fabricate an ANG-loaded scaffold and to evaluate its angiogenic and osteogenic effects. In this study, we fabricated an ANG-loaded scaffold using bovine bone powder and fibrin glue. We then evaluated the structural, morphological, and mechanical properties of the scaffold and the in vitro release profile of ANG. Cell proliferation, viability, and adhesion were evaluated using endothelial cells in vitro, and angiogenesis and new bone formation were evaluated using a rabbit calvarial defect model in vivo. RESULTS: Micro-computed tomography imaging showed that the bone powder was uniformly distributed in the scaffold, and scanning electron microscopy showed that the bone powder was bridged by polymerized fibrin. The porosity and compressive strength of the scaffolds were ~60 % and ~0.9 MPa, respectively, and were not significantly altered by ANG loading. In vitro, at 7 days, approximately 0.4 µg and 1.3 µg of the ANG were released from the FB/ANG 0.5 and FB/ANG 2.0, respectively and sustained slow release was observed until 25 days. The released ANG stimulated cell proliferation and adherence and was not cytotoxic. Furthermore, in vivo implantation resulted in enhanced angiogenesis, and new bone formation depended on the amount of loaded ANG. CONCLUSIONS: These studies demonstrate that a fibrin and bone powder scaffold loaded with ANG might be useful to promote bone regeneration by enhanced angiogenesis.

Comparison of in vitro and in vivo bioactivity: cuttlefish-bone-derived hydroxyapatite and synthetic hydroxyapatite granules as a bone graft substitute.

Bone reconstruction in clinical settings often requires bone substitutes. Hydroxyapatite (HAp) is a widely used bone substitute due to its osteoconductive properties and bone bonding ability. The aim of this study was to evaluate HAp granules derived from cuttlefish bone (CB-HAp) as a substitute biomaterial for bone grafts. In this study, HAp granules were prepared from raw CB by using a hydrothermal reaction. The formation of HAp from CB was confirmed by scanning electron microscopy and x-ray diffraction analysis. The bioactivity of the CB-HAp granules was evaluated both in vitro and in vivo. Our results show that CB-HAp is non-toxic and that CB-HAp granules supported improved cell adhesion, proliferation and differentiation compared to stoichiometric synthetic HAp granules. Furthermore, in vivo bone defect healing experiments show that the formation of bone with CB-HAp is higher than that with pure HAp. These results show that CB-HAp granules have excellent potential for use as a bone graft material.

A polycaprolactone/cuttlefish bone-derived hydroxyapatite composite porous scaffold for bone tissue engineering.

Cuttlefish bone (CB) is an attractive natural biomaterial source to obtain hydroxyapatite (HAp). In this study, a porous polycaprolactone (PCL) scaffold incorporating CB-derived HAp (CB-HAp) powder was fabricated using the solvent casting and particulate leaching method. The presence of CB-HAp in PCL/CB-HAp scaffold was confirmed by X-ray diffraction (XRD). Scanning electron microscopy (SEM) and porosity analysis showed that the average pore dimension of the fabricated scaffold was approximately 200-300 µm, with ∼85% porosity, and that the compressive modulus increased after addition of CB-HAp powders. In vitro tests such as cell proliferation assay, cytotoxicity analysis, cell attachment observations, and alkaline phosphatase activity assays showed that the PCL/CB-HAp scaffold could improve the proliferation, viability, adherence, and osteoblast differentiation rate of MG-63 cells. When surgically implanted into rabbit calvarial bone defects, consistent with the in vitro results, PCL/CB-HAp scaffold implantation resulted in significantly higher new bone formation than did implantation of PCL alone. These findings suggest that addition of CB-HAp powder to the PCL scaffold can improve cellular response and that the PCL/CB-HAp composite scaffold has great potential for use in bone tissue engineering.