Min Soo Bae

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.

Photo-cured hyaluronic acid-based hydrogels containing simvastatin as a bone tissue regeneration scaffold

We describe in this study the positive influences on in vitro and in vivo osteogenesis of photo-cured hyaluronic acid (HA) hydrogels loaded with simvastatin (SIM). Prior to loading SIM, we first characterized the HA hydrogels for their mechanical properties and swelling ratios. The results from this testing indicated that these two factors improved as the substitution degree of 2-aminoethyl methacrylate (AEMA) increased. MTT and live/dead assays showed that the HA hydrogels have good biocompatibility for use as scaffolds for bone tissue regeneration. Moreover, another MTT assay showed that the photo-cured HA hydrogels III fabricated with 30% AEMA (300 mg) conjugated HA (HA-AEMA iii) loaded with between 0.1 and 1 mg of SIM had a similar cytotoxicity as compared to the HA hydrogel III itself. The sustained release of SIM was observed to occur in the HA hydrogel III loaded with 1 mg of SIM. In vitro and in vivo experiments showed that the HA hydrogel III loaded with 1 mg of SIM had a significant influence on osteogenesis.

Electrospun chitosan nanofibers with controlled levels of silver nanoparticles. Preparation, characterization and antibacterial activity

The ideal wound dressing would have properties that allow for absorption of exudates, and inhibition of microorganism for wound protection. In this study, we utilized an electrospinning (ELSP) technique to design a novel wound dressing. Chitosan (CTS) nanofibers containing various ratios of silver nanoparticles (AgNPs) were obtained. AgNPs were generated directly in the CTS solution by using a chemical reduction method. The formation and presence of AgNPs in the CTS/AgNPs composite was confirmed by x-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV) and thermogravimetric analysis (TGA). The electrospun CTS/AgNPs nanofibers were characterized morphologically by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). These nanofibers were subsequently tested to evaluate their antibacterial activity against gram-negative Pseudomonas aeruginosa (P. aeruginosa) and gram-positive Methicillin-resistant Staphylococcus aureus (MRSA). Results of this antibacterial testing suggest that CTS/AgNPs nanofibers may be effective in topical antibacterial treatment in wound care.

Enhanced bone regeneration with a gold nanoparticle–hydrogel complex

Gold nanoparticles (GNPs) are widely used in diagnostics, drug delivery, biomedical imaging, and photo-thermal therapy due to their surface plasmon resonance, fluorescence, and easy-surface functionalization. According to recent studies, GNPs display a positive effect on the osteogenic differentiation of mesenchymal stem cells (MSCs) and MC3T3-E1 osteoblast-like cells. The aim of this study was to develop a new approach for bone tissue regeneration based on the utilization of a biodegradable hydrogel loaded with GNPs. We have used photo-curable gelatin hydrogels (Gel) in order to provide a proof of principle of GNPs in regeneration strategies for bone tissue repair. We have investigated the effects of these Gel-GNP composite hydrogels both in vitro and in vivo. The in vitro results showed that the hydrogels loaded with GNPs promote proliferation, differentiation, and alkaline phosphate (ALP) activities of human adipose-derived stem cells (ADSCs) as they differentiate towards osteoblast cells in a dose-dependent manner. Moreover, the in vivo results showed that these hydrogels loaded with high concentrations of GNPs had a significant influence on new bone formation. Through these in vitro and vivo tests, we found that the Gel-GNP can be a useful material for bone tissue engineering.

Enhancement of ectopic bone formation by bone morphogenetic protein-2 delivery using heparin-conjugated PLGA nanoparticles with transplantation of bone marrow-derived mesenchymal stem cells.

This study was performed to determine if a combination of previously undifferentiated bone marrow-derived mesenchymal stem cells (BMMSCs) and exogenous bone morphogenetic protein-2 (BMP-2) delivered via heparin-conjugated PLGA nanoparticles (HCPNs) would extensively regenerate bone in vivo. In vitro testing found that the HCPNs were able to release BMP-2 over a 2-week period. Human BMMSCs cultured in medium containing BMP-2-loaded HCPNs for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) mRNA, while cells without BMP-2 expressed only ALP. In vivo testing found that undifferentiated BMMSCs with BMP-2-loaded HCPNs induce far more extensive bone formation than either implantation of BMP-2-loaded HCPNs or osteogenically differentiated BMMSCs. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of undifferentiated BMMSCs and BMP-2 delivery via HCPNs.

The effect of gold nanoparticle size on osteogenic differentiation of adipose-derived stem cells.

There have been many medical applications based on gold nanoparticles (GNPs) over the past several centuries. Recently, researchers have focused on bone tissue engineering applications utilizing GNPs. The effect of various sizes of gold nanoparticles on the differentiation of human adipose-derived stem cells (ADSCs) into osteoblasts was investigated. The concentration of gold nanoparticles was fixed at 1 μM and varying sizes of 15, 30, 50, 75 and 100 nm (spherical GNPs) were used. The lack of cytotoxicity was confirmed by establishing viability of ADSCs using cell counting kit-8 (CCK-8) and live/dead assays. The results showed that each size of GNPs had no significant toxicity on ADSCs during 1 week of incubation. Osteogenic differentiation of ADSCs was confirmed by alkaline phosphatase (ALP) staining, ALP activity, calcium deposition, and real time PCR experiments. It was found, through dark field assays and microscope cell images, that 30 nm and 50 nm GNPs were preferentially up taken into the ADSCs. As expected, all sizes of gold nanoparticles promoted the differentiation of ADSCs toward osteoblasts more than control. Among all sizes, 30 and 50 nm GNPs appeared to have the highest differentiation rates. The data consistently demonstrated that 30 and 50 nm GNPs are the most effective in promoting osteogenic differentiation of ADSCs.

Effect of heparin and alendronate coating on titanium surfaces on inhibition of osteoclast and enhancement of osteoblast function

The failure of orthopedic and dental implants has been attributed mainly to loosening of the implant from host bone, which may be due to weak bonding of the implant material to bone tissue. Titanium (Ti) is used in the field of orthopedic and dental implants because of its excellent biocompatibility and outstanding mechanical properties. Therefore, in the field of materials science and tissue engineering, there has been extensive research to immobilize bioactive molecules on the surface of implant materials in order to provide the implants with improved adhesion to the host bone tissue. In this study, chemically active functional groups were introduced on the surface of Ti by a grafting reaction with heparin and then the Ti was functionalized by immobilizing alendronate onto the heparin-grafted surface. In the MC3T3-E1 cell osteogenic differentiation study, the alendronate-immobilized Ti substrates significantly enhanced alkaline phosphatase activity (ALP) and calcium content. Additionally, nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was inhibited with the alendronate-immobilized Ti as confirmed by TRAP analysis. Real time PCR analysis showed that mRNA expressions of osteocalcin and osteopontin, which are markers for osteogenesis, were upregulated in MC3T3-E1 cells cultured on alendronate-immobilized Ti. The mRNA expressions of TRAP and Cathepsin K, markers for osteoclastogenesis, in RAW264.7 cells cultured on alendronate-immobilized Ti were down-regulated. Our study suggests that alendronate-immobilized Ti may be a bioactive implant with dual functions to enhance osteoblast differentiation and to inhibit osteoclast differentiation simultaneously.

Photo-cured hyaluronic acid-based hydrogels containing growth and differentiation factor 5 (GDF-5) for bone tissue regeneration

In this study we describe the generation and influences on in vitro and in vivo osteogenesis of photo-cured hyaluronic acid (HA) hydrogels loaded with growth and differentiation factor 5 (GDF-5). Prior to loading GDF-5, we characterized the release profiles from these hydrogels and tested their respective cell viability, differentiation and in vivo bone regeneration. The results from this testing indicated that GDF-5 was observed to release in a sustained manner from the HA hydrogels I-III. MTT and Live/Dead assays showed that the HA hydrogels I-III have good biocompatibility for use as scaffolds for bone tissue regeneration. In vitro cell tests showed a higher level of MC3T3-E1 cell proliferation and differentiation on HA hydrogels I-III than on HA hydrogel 0. Moreover, in vivo animal tests showed that the HA hydrogels I and III had a significant improvement on osteogenesis. Overall, our results suggest that the HA-based hydrogel is a good biomaterial to deliver osteogenic differentiation factors such as GDF-5, and GDF-5 can be useful as an effective alternative to aid new bone formation.

Inhibition of Osteoclast Differentiation by Gold Nanoparticles Functionalized with Cyclodextrin Curcumin Complexes

Gold nanoparticles (GNPs) have been previously reported to inhibit osteoclast (OC) formation. However, previous research only confirmed the osteoclastogenesis inhibitory effect under in vitro conditions. The aim of this study was to develop a therapeutic agent for osteoporosis based on the utilization of GNPs and confirm their effect both in vitro and in vivo. We prepared β-cyclodextrin (CD) conjugated GNPs (CGNPs), which can form inclusion complexes with curcumin (CUR-CGNPs), and used these to investigate their inhibitory effects on receptor activator of nuclear factor-κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). The CUR-CGNPs significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells in BMMs without inducing cytotoxicity. The mRNA expressions of genetic markers of OC differentiation including c-Fos, nuclear factor of activated T cells 1 (NFATc1), TRAP, and osteoclast associated receptor (OSCAR) were significantly decreased in the presence of CUR-CGNPs. In addition, the CUR-CGNPs inhibited OC differentiation of BMMs through suppression of the RANKL-induced signaling pathway. Additionally, CUR-CGNPs caused a decrease in RANKL-induced actin ring formation, which is an essential morphological characteristic of OC formation allowing them to carry out bone resorption activity. Furthermore, the in vivo results of an ovariectomy (OVX)-induced osteoporosis model showed that CUR-CGNPs significantly improved bone density and prevented bone loss. Therefore, CUR-CGNPs may prove to be useful as therapeutic agents for preventing and treating osteoporosis.

Poly(l-Lactic Acid)/Gelatin Fibrous Scaffold Loaded with Simvastatin/Beta-Cyclodextrin-Modified Hydroxyapatite Inclusion Complex for Bone Tissue Regeneration

Recently, the application of nanostructured materials in the field of tissue engineering has garnered attention to mediate treatment and regeneration of bone defects. In this study, poly(l-lactic acid) (PLLA)/gelatin (PG) fibrous scaffolds are fabricated and β-cyclodextrin (βCD) grafted nano-hydroxyapatite (HAp) is coated onto the fibrous scaffold surface via an interaction between βCD and adamantane. Simvastatin (SIM), which is known to promote osteoblast viability and differentiation, is loaded into the remaining βCD. The specimen morphologies are characterized by scanning electron microscopy. The release profile of SIM from the drug loaded scaffold is also evaluated. In vitro proliferation and osteogenic differentiation of human adipose derived stem cells on SIM/HAp coated PG composite scaffolds is characterized by alkaline phosphatase (ALP) activity, mineralization (Alizarin Red S staining), and real time Polymerase chain reaction (PCR). The scaffolds are then implanted into rabbit calvarial defects and analyzed by microcomputed tomography for bone formation after four and eight weeks. These results demonstrate that SIM loaded PLLA/gelatin/HAp-(βCD) scaffolds promote significantly higher ALP activity, mineralization, osteogenic gene expression, and bone regeneration than control scaffolds. This suggests the potential application of this material toward bone tissue engineering.