Young Pil Yun

The effect of immobilization of heparin and bone morphogenic protein-2 (BMP-2) to titanium surfaces on inflammation and osteoblast function

The aim of this study was to investigate biologic function of bone morphorgenic protein-2 (rhBMP-2) immobilized on the heparin-grafted Ti surface. Ti surfaces were first modified by 3-aminopropyltriethoxysilane (ATPES), followed by grafting of heparin. BMP-2 was then immobilized on the heparin-grafted Ti surfaces. Pristine Ti and functionalized Ti surfaces were characterized by X-ray photoelectron spectroscopy (XPS), measurement of water contact angles, and protein adsorption. The biological activity of MG-63 cells on pristine and functionalized Ti surfaces was investigated by cell proliferation assays, measurement of alkaline phosphate (ALP) activity, and determination of calcium deposition. Anti-inflammatory effects were assessed by RT-PCR to measure the transcript levels of IL-6 and TNF-α. XPS revealed that heparin and BMP-2 were successfully grafted and immobilized on the Ti surfaces, respectively. In addition, Ti surfaces with BMP-2 immobilized were more hydrophilic than pristine Ti. Furthermore, BMP-2 immobilized Ti promoted significantly higher ALP activity and calcium deposition by MG-63 cells than pristine Ti. The inflammatory response was also decreased when cells were grown on heparin-grafted, BMP-2-immobilized Ti surfaces. The results of this study suggest that by grafting heparin and immobilizing BMP-2 on Ti surfaces, inflammation can be inhibited and osteoblast function promoted.

Simvastatin inhibits osteoclast differentiation by scavenging reactive oxygen species

Osteoclasts, together with osteoblasts, control the amount of bone tissue and regulate bone remodeling. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. Reactive oxygen species (ROS) acts as a signal mediator in osteoclast differentiation. Simvastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A, is a hypolipidemic drug which is known to affect bone metabolism and suppresses osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL). In this study, we analyzed whether simvastatin can inhibit RANKL-induced osteoclastogenesis through suppression of the subsequently formed ROS and investigated whether simvastatin can inhibit H2O2-induced signaling pathways in osteoclast differentiation. We found that simvastatin decreased expression of tartrate-resistant acid phosphatase (TRAP), a genetic marker of osteoclast differentiation, and inhibited intracellular ROS generation in RAW 264.7 cell lines. ROS generation activated NF-κB, protein kinases B (AKT), mitogen-activated protein kinases signaling pathways such as c-JUN N-terminal kinases, p38 MAP kinases as well as extracellular signal-regulated kinase. Simvastatin was found to suppress these H2O2-induced signaling pathways in osteoclastogenesis. Together, these results indicate that simvastatin acts as an osteoclastogenesis inhibitor through suppression of ROS-mediated signaling pathways. This indicates that simvastatin has potential usefulness for osteoporosis and pathological bone resorption.

Fabrication of a BMP-2-immobilized porous microsphere modified by heparin for bone tissue engineering.

The purpose of this study was to fabricate BMP-2-immobilized porous poly(lactide-co-glycolide) (PLGA) microspheres (PMS) modified with heparin for bone regeneration. A fluidic device was used to fabricate PMS and the fabricated PMS was modified with heparin-dopamine (Hep-DOPA). Bone morphogenic protein-2 (BMP-2) was immobilized on the heparinized PMS (Hep-PMS) via electrostatic interactions. Both PMS and modified PMS were characterized using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). MG-63 cell activity on PMS and modified PMS were assessed via alkaline phosphatase (ALP) activity, calcium deposition, and osteocalcin and osteopontin mRNA expression. Immobilized Hep-DOPA and BMP-2 on PMS were demonstrated by XPS analysis. BMP-2-immobilized Hep-PMS provided significantly higher ALP activity, calcium deposition, and osteocalcin and osteopontin mRNA expression compared to PMS alone. These results suggest that BMP-2-immobilized Hep-PMS effectively improves MG-63 cell activity. In conclusion, BMP-2-immobilized Hep-PMS can be used to effectively regenerate bone defects.

Local delivery of alendronate eluting chitosan scaffold can effectively increase osteoblast functions and inhibit osteoclast differentiation

The aim of this study was to investigate the effect of alendronate released from chitosan scaffolds on enhancement of osteoblast functions and inhibition of osteoclast differentiation in vitro. The surface and cell morphologies of chitosan scaffolds and alendronate-loaded chitosan scaffolds were characterized by variable pressure field emission scanning electron microscope (VP-FE-SEM). Alendronate was released in a sustained manner. For evaluating osteoblast functions in MG-63 cells, we investigated cell proliferation, alkaline phosphatase (ALP) activity, and calcium deposition. Furthermore, for evaluating inhibition of osteoclast differentiation in RAW 264.7 cells, we investigated tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and gene expressions. The in vitro studies revealed that osteoblasts grown on alendronate-loaded chitosan scaffold showed a significant increment in cell proliferation, ALP activity, and calcium deposition as compared to those grown on chitosan scaffolds. In addition, the in vitro study showed that osteoclast differentiation in RAW 264.7 cells cultured on alendronate-loaded chitosan scaffolds was greatly inhibited as compared to those cultured on chitosan scaffolds by the results of TRAP activity, TRAP staining, and gene expressions. Taken together, alendronate-loaded chitosan scaffolds could achieve the dual functions of improvement in osteoblast functions and inhibition of osteoclast differentiation. Thus, alendronate-eluting chitosan substrates are promising materials for enhancing osteoblast functions and inhibiting osteoclast differentiation in orthopedic and dental fields.

The effect of titanium with heparin/BMP-2 complex for improving osteoblast activity.

The objective of this study was to investigate the enhanced osteoblast activity of MG-63 cells cultured on titanium (Ti) with a heparin/BMP-2 (Hep/BMP-2) complex. The Ti substrates were initially modified by chemical grafting poly-L-lysine (PLL) using condensing agent, followed by immobilizing the heparin/BMP-2 complex to the PLL-grafted Ti substrate via electrostatic interactions. The surface modification of Ti substrates with PLL and/or Hep/BMP-2 complex were confirmed with scanning electron microscopy, contact angle measurements, and X-ray photoelectron spectroscopy. Immobilized BMP-2 was released from the Hep/BMP-2/Ti substrate in a sustained manner. In vitro studies revealed that osteoblasts grown on Hep/BMP-2/Ti substrate increased ALP activity, calcium deposition, ALP and osteocalcin levels as compared to those grown on pristine Ti or PLL-Ti. These results indicated that heparin/BMP-2 complex immobilized Ti substrate can be useful to effectively improve osteoblast activity.

Effect of lactoferrin-impregnated porous poly(lactide-co-glycolide) (PLGA) microspheres on osteogenic differentiation of rabbit adipose-derived stem cells (rADSCs).

The aim of this study was to develop lactoferrin (LF)-impregnated porous poly(lactide-co-glycolide) (PLGA) microspheres (PMs) to induce osteogenic differentiation of rabbit adipose-derived stem cells (rADSCs). Porous PLGA PMs were fabricated by a fluidic device and their surfaces were modified with heparin-dopamine (Hep-DOPA). Then, LF (100μg, 500μg, and 1000μg) was impregnated on the surface of heparinized PMs (Hep-PMs) via electrostatic interactions to yield LF-impregnated PMs. PMs and modified PMs were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Osteogenic differentiation of rADSCs on PMs and modified PMs was demonstrated by alkaline phosphatase (ALP) activity, calcium deposition, and mRNA expression of osteocalcin and osteopontin. Successful immobilization of Hep-DOPA and LF on the surface of PMs was confirmed by XPS analysis. LF-impregnated PMs generated significantly greater ALP activity, calcium deposition, and mRNA expression of osteocalcin and osteopontin compared with PMs. These results suggested that LF-impregnated PMs effectively induced osteogenic differentiation of rADSCs.

In vitro and in vivo evaluation of bone formation using solid freeform fabrication-based bone morphogenic protein-2 releasing PCL/PLGA scaffolds

The aim of this study was to develop novel polycaprolactone/poly(lactic-co-glycolic acid) (PCL/PLGA) scaffolds with a heparin-dopamine (Hep-DOPA) conjugate for controlled release of bone morphogenic protein-2 (BMP-2) to enhance osteoblast activity in vitro and also bone formation in vivo. PCL/PLGA scaffolds were prepared by a solid freeform fabrication method. The PCL/PLGA scaffolds were functionalized with Hep-DOPA and then BMP-2 was sequentially coated onto the Hep-DOPA/PCL/PLGA scaffolds. The characterization and surface elemental composition of all scaffolds were evaluated by scanning electron microscope and x-ray photoelectron spectroscopy. The osteoblast activities on all scaffolds were assessed by cell proliferation, alkaline phosphatase (ALP) activity and calcium deposition in vitro. To demonstrate bone formation in vivo, plain radiograph, micro-computed tomography (micro-CT) evaluation and histological studies were performed after the implantation of all scaffolds on a rat femur defect. Hep-DOPA/PCL/PLGA had more controlled release of BMP-2, which was quantified by enzyme-linked immunosorbent assay, compared with Hep/PCL/PLGA. The in vitro results showed that osteoblast-like cells (MG-63 cells) grown on BMP-2/Hep-DOPA/PCL/PLGA had significantly enhanced ALP activity and calcium deposition compared with those on BMP-2/Hep/PCL/PLGA and PCL/PLGA. In addition, the plain radiograph, micro-CT evaluation and histological studies demonstrated that the implanted BMP-2/Hep-DOPA/PCL/PLGA on rat femur had more bone formation than BMP-2/Hep/PCL/PLGA and PCL/PLGA in vivo.

Three-dimensional printing of antibiotics-loaded poly-ε-caprolactone/poly(lactic-co-glycolic acid) scaffolds for treatment of chronic osteomyelitis

Osteomyelitis, an infection and inflammation of bone marrow, often progresses to chronic stage because of delay in diagnosis and treatment. Once it becomes chronic, intravenous antibiotics therapy is no longer effective as swollen surrounding tissue interrupts blood flow into the infected tissue. In severe cases, debridement of the necrotic tissue becomes necessary to prevent further infection. In this study, for the first time, we produced three-dimensional (3D) printed antibiotics-loaded biodegradable poly-e-caprolactone/poly(lactic-co-glycolic acid) scaffold for treatment of chronic osteomyelitis. Subsequent bone regeneration in debrided site was also observed with the customized scaffolds fabricated using 3D printing. Tobramycin, one of the most widely used antibiotics in orthopedic surgery, was chosen due to its thermostable nature compliant to the heat-based fabrication conditions. In in vitro tests, antibacterial and anti-inflammatory effects and release profile of tobramycin from the scaffold were evaluated to verify the potential of our scaffold as a drug delivery system. In addition, in vivo efficacy of the developed drug loaded scaffolds for treatment of chronic osteomyelitis was also examined in a rat model.

Bone formation in a rat tibial defect model using carboxymethyl cellulose/BioC/bone morphogenic protein-2 hybrid materials.

The objective of this study was to assess whether carboxymethyl cellulose- (CMC-) based hydrogel containing BioC (biphasic calcium phosphate (BCP); tricalcium phosphate (TCP) : hydroxyapatite (Hap) = 70 : 30) and bone morphogenic protein-2 (BMP-2) led to greater bone formation than CMC-based hydrogel containing BioC without BMP-2. In order to demonstrate bone formation at 4 and 8 weeks, plain radiographs, microcomputed tomography (micro-CT) evaluation, and histological studies were performed after implantation of all hybrid materials on an 8 mm defect of the right tibia in rats. The plain radiographs and micro-CT analyses revealed that CMC/BioC/BMP-2 (0.5 mg) led to much greater mineralization at 4 and 8 weeks than did CMC/BioC or CMC/Bio/BMP-2 (0.1 mg). Likewise, bone formation and bone remodeling studies revealed that CMC/BioC/BMP-2 (0.5 mg) led to a significantly greater amount of bone formation and bone remodeling at 4 and 8 weeks than did CMC/BioC or CMC/BioC/BMP-2 (0.1 mg). Histological studies revealed that mineralized bone tissue was present around the whole circumference of the defect site with CMC/BioC/BMP-2 (0.5 mg) but not with CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) at 4 and 8 weeks. These results suggest that CMC/BioC/BMP-2 hybrid materials induced greater bone formation than CMC/BioC hybrid materials. Thus, CMC/BioC/BMP-2 hybrid materials may be used as an injectable substrate to regenerate bone defects.

Improvement of osteoblast functions by sustained release of bone morphogenetic protein-2 (BMP-2) from heparin-coated chitosan scaffold

The aim of this study was to investigate the improvement in osteoblast functions by using bone morphogenetic protein-2 (BMP-2) immobilized heparin-coated chitosan scaffolds and comparing it with that using chitosan scaffold or BMP-2/chitosan scaffold in vitro. BMP-2 was released from the heparin-coated chitosan scaffold in a sustained manner compared to that released from the chitosan scaffold. The osteoblast functions of MG-63 cells grown on the chitosan scaffold, the BMP-2/chitosan scaffold, the BMP-2/Hep/chitosan scaffold were investigated by assessing cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and gene expression. The results of the in vitro studies demonstrated that MG-63 cells grown on the BMP-2/Hep/chitosan scaffold showed a significant increment in ALP activity, and calcium deposition as compared to those grown on the chitosan scaffold by sustained release of BMP-2 due to the influence of heparin. Therefore, BMP-2 immobilized heparin-coated chitosan scaffolds are promising materials for improving osteoblast functions through sustained release of BMP-2.