Dae Gyun Woo

Co-delivery of SOX9 genes and anti-Cbfa-1 siRNA coated onto PLGA nanoparticles for chondrogenesis of human MSCs.

Some genes expressed in stem cells interrupt and/or enhance differentiation. Therefore, the aim of this study was to inhibit the expression of unnecessary genes and enhance the expression of specific genes involved in stem cell differentiation by using small interfering RNA (siRNA) and plasmid DNA (pDNA) incorporated into cationic polymers as co-delivery factors. To achieve co-delivery of siRNA and pDNA to human mesenchymal stem cells (hMSCs), two different genes were complexed with poly(ethyleneimine) (PEI) and then coated onto poly(lactide-co-glycolic acid) (PLGA) nanoparticles (NP). To evaluate co-delivery of siRNA and pDNA into hMSCs, cells were transfected with green fluorescence protein (GFP) pDNA (GFP pDNA) and GFP siRNA (GFP siRNA). The percentage of GFP-expressing hMSCs decreased from 25.35 to 3.7% after transfection with GFP-DNA/PLGA NP (NPs) or GFP siRNA/PLGA NPs, whereas GFP-DNA/PLGA NPs and scramble siRNA (MOCK)/PLGA NPs had no effect on GFP expression. hMSCs cotransfected with coSOX9-pDNA/NPs and Cbfa-1-siRNA/NPs were tested both in vitro and in vivo using gel retardation, dynamic light scattering (DLS), and scanning electron microscope (SEM). The expression of genes and proteins associated with chondrogenesis was evaluated by FACS, RT-PCR, real time-qPCR, Western blotting, immunohistochemistry, and immunofluorescence imaging.

The use of biodegradable PLGA nanoparticles to mediate SOX9 gene delivery in human mesenchymal stem cells (hMSCs) and induce chondrogenesis

In stem cell therapy, transfection of specific genes into stem cells is an important technique to induce cell differentiation. To perform gene transfection in human mesenchymal stem cells (hMSCs), we designed and fabricated a non-viral vector system for specific stem cell differentiation. Several kinds of gene carriers were evaluated with regard to their transfection efficiency and their ability to enhance hMSCs differentiation. Of these delivery vehicles, biodegradable poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles yielded the best results, as they complexed with high levels of plasmid DNA (pDNA), allowed robust gene expression in hMSCs, and induced chondrogenesis. Polyplexing with polyethylenimine (PEI) enhanced the cellular uptake of SOX9 DNA complexed with PLGA nanoparticles both in vitro and in vivo. The expression of enhanced green fluorescent protein (EGFP) and SOX9 increased up to 75% in hMSCs transfected with PEI/SOX9 complexed PLGA nanoparticles 2 days after transfection. SOX9 gene expression was evaluated by RT-PCR, real time-qPCR, glycosaminoglycan (GAG)/DNA levels, immunoblotting, histology, and immunofluorescence.

Chondrogenesis of human mesenchymal stem cells mediated by the combination of SOX trio SOX5, 6, and 9 genes complexed with PEI-modified PLGA nanoparticles

Target gene transfection for desired cell differentiation has recently become a major issue in stem cell therapy. For the safe and stable delivery of genes into human mesenchymal stem cells (hMSCs), we employed a non-viral gene carrier system such as polycataionic polymer, poly(ethyleneimine) (PEI), polyplexed with a combination of SOX5, 6, and 9 fused to green fluorescence protein (GFP), yellow fluorescence protein (YFP), or red fluorescence protein (RFP) coated onto PLGA nanoparticles. The transfection efficiency of PEI-modified PLGA nanoparticle gene carriers was then evaluated to examine the potential for chondrogenic differentiation by carrying the exogenous SOX trio (SOX5, 6, and 9) in hMSCs. Additionally, use of PEI-modified PLGA nanoparticle gene carriers was evaluated to investigate the potential for transfection efficiency to increase the potential ability of chondrogenesis when the trio genes (SOX5, 6, and 9) polyplexed with PEI were delivered into hMSCs. SOX trio complexed with PEI-modified PLGA nanoparticles led to a dramatic increase in the chondrogenesis of hMSCs in in vitro culture systems. For the PEI/GFP and PEI/SOX5, 6, and 9 genes complexed with PLGA nanoparticles, the expressions of GFP as reporter genes and SOX9 genes with PLGA nanoparticles showed 80% and 83% of gene transfection ratios into hMSCs two days after transfection, respectively.

In Vitro and In Vivo Chondrogenesis of Rabbit Bone Marrow–Derived Stromal Cells in Fibrin Matrix Mixed with Growth Factor Loaded in Nanoparticles

The effects of growth factor loaded in nanoparticles mixed in fibrin constructs on chondrogenic differentiation were investigated by evaluating the specific cartilage extracellular matrix components in vitro and in vivo using a special cell source of bone marrow-derived stromal cells (BMSCs). The proliferation of cultured and transplanted BMSCs was found to be greater in fibrin constructs that contained TGF-beta3-loaded nanoparticles and TGF-beta3 alone than in constructs that contained unloaded nanoparticles or in fibrin hydrogel alone. Further, reverse transcriptase-polymerase chain reaction revealed that BMSCs cultured in the presence of TGF-beta3 in vitro and in vivo expressed high levels of aggrecan, cartilage oligomer matrix protein, SOX9, and type II collagen. However, a decrease in type I collagen expression was observed from 1 to 4 weeks in the presence of TGF-beta3. Moreover, histological and immunohistochemical assays revealed that large amounts of type II and proteoglycan were released from BMSCs embedded in fibrin constructs, while decreased levels of collagen type I were observed in BMSCs cultured in constructs that contained nanoparticles that were loaded with TGF-beta both in vitro and in vivo. These findings indicate that use of fibrin constructs that contained BMSCs and were provided with sustained levels of growth factors for a long period of time enabled the formation of hyaline cartilage tissue in vitro and in vivo. Overall, these results indicate that the system evaluated here may be useful for minimally invasive transplantation, BMSC differentiation, and engineering of composite tissue structures with multiple cellular phenotypes.

Chondrogenic differentiation of mesenchymal stem cells embedded in a scaffold by long-term release of TGF-β3 complexed with chondroitin sulfate

In this study, mesenchymal stem cells (MSCs) embedded in biodegradable and water-swollen, elastic block copolymer scaffolds were assessed for MSC chondrogenesis. To determine the optimal conditions for chondrogenesis of the embedded rMSCs, transforming growth factor-beta 3 (TGF-beta 3) was physically conjugated with chondroitin sulfate (CS) and mixed into scaffolds, which were subsequently evaluated for the differentiation of transplanted rMSCs. In determination of CS-bound growth factors for chondrogenesis, scaffold mixed with rMSCs and TGF-beta 3 was then tested by growth factor release profiles, confocal laser microscopy, RT-PCR analysis, real time-QPCR, and histology. The results of several different analyses of the transplanted rMSCs embedded in the scaffolds showed that rMSCs coupled with a CS-bound TGF-beta 3 encapsulated scaffold evidenced superior cartilage tissue formation as measured by an assay of specific gene and protein expression. Moreover, the scaffold exhibited more rapid and more distinct morphology of differentiated rMSCs than was observed with other scaffolds, as determined by histology and immunochemical histology analysis. These results indicate that the elastic block copolymer scaffolds combined with a CS-bound TGF-beta 3 should prove very suitable matrix for cell-based cartilage tissue engineering.

PLGA Microsphere Construct Coated with TGF-β 3 Loaded Nanoparticles for Neocartilage Formation

Polymeric microsphere system has been widely used in tissue-regeneration matrix and drug delivery systems. To apply these biomaterials as novel cell supporting matrix for stem cell delivery, we have devised a novel method for the fabrication of nanostructured 3D scaffolds that growth factor loaded heparin/poly(L-lysine) nanoparticles were physically attached on the positively charged surface of PLGA microspheres precoated with low molecular weight of poly(ethyleneimmine) (PEI) via a layer-by-layer (LbL) system. Based on a previous study, we have prepared poly(lactide-co-glycolide) (PLGA) microspheres harboring heparin/poly(L-lysine) loaded with growth factors. Growth factor loaded heparin/poly(L-lysine) nanoparticles, which were simply produced as polyion complex micelles (PICM) with diameters of 50-150 nm, were fabricated in the first step. Microsphere matrix (size, 20 approximately 80 nm) containing TGF-beta 3 showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study in vitro culture of mesenchymal stem cells. Thus, growth factor delivery of PLGA microsphere can be used to engineer synthetic extracellular matrix. This PLGA microsphere matrix containing TGF-beta 3 showed promise as coatings for implantable biomedical devices to improve biocompatibility and ensure in vivo performance.

Electrical Pulsed Stimulation of Surfaces Homogeneously Coated with Gold Nanoparticles to Induce Neurite Outgrowth of PC12 Cells

In this study, the potential of gold nanoparticles (20 nm) to deliver electrical stimulation to nerve cell cultures in vitro to induce nerve regeneration was evaluated. In order to use these biomaterials to deliver an electrical stimulus, we devised a novel method for the fabrication of a nanostructured 2D substrate comprising gold nanoparticles attached to the surface of a cover glass via an adsorption system. In this strategy, gold nanoparticles are created and then coated onto a positively charged cover glass that has been pretreated with polyethyleneimine (PEI). Scanning electron microscopy (SEM) revealed that the PC 12 cells extended neurites well on the gold nanoparticles in the presence of electrical stimulation. In addition, the neurite outgrowth of PC12 cells in response to pulsed and constant electrical stimulation was evaluated by live/dead cell determination, by reverse transcriptase polymerase chain reaction (RT-PCR) analysis, as well as by beta-tubulin and NF-200 expression. By electrical stimulation (250 mV for 1 h), PC12 cells with their neurite outgrowth length were highly increased, with a mean diameter of 98.5 microm. However, the neurite outgrowth length without electrical stimulation was approximately 10 approximately 20 microm. Moreover, the alternating current stimulation also showed good viability (<90%), while a high amount of cell death (more than 30%) was observed with constant current stimulation. Thus, the gold nanoparticles with pulsed current stimulation may provide suitable tools for the nerve regeneration using neuronal cells.

Heparin-Bound Transforming Growth Factor-β3 Enhances Neocartilage Formation by Rabbit Mesenchymal Stem Cells

Background. Heparin binds growth factors to form a stable complex that maintains the biological activity and can retard the release pattern. To differentiate the embedded the stem cells, heparin-bind transforming growth factor (TGF)-&bgr;3 was mixed with rabbit mesenchymal stem cells encapsulated with thermo-reversible hydrogel. It is suggested that the heparin-bound TGF-&bgr;3 would help to increase the chondrogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel. Methods. To determine the optimal condition for neocartilage formation, we characterized hydrogel constructs with growth factor release profiles, confocal laser microscopy, reverse transcription-polymerase chain reaction analysis, and histology. Results. Reverse transcription-polymerase chain reaction analysis of the resultant cartilage tissue revealed that a thermo-reversible hydrogels with a heparin-bound TGF-&bgr;3 was optimal for cartilage tissue formation as measured by production of collagen Type II, aggrecan, and SOX9, and cartilage oligomeric matrix protein gene expression. Additionally, the proliferation rate and cartilage specific ECM production were both significantly greater in the presence of heparin-bound TGF-&bgr;3 than in the control. The amount of cartilage-associated ECM proteins was examined by immunohistochemical staining (collagen type II), Safranin-O staining, and Alcian blue staining. Conclusions. These data indicate the potential use of heparin-binding TGF&bgr;-3 for reconstruction of neocartilage formation.

Transfection of VEGF165 genes into endothelial progenitor cells and in vivo imaging using quantum dots in an ischemia hind limb model

Endothelial progenitor cells (EPCs) were transfected with fluorescently labeled quantum dot nanoparticles (QD NPs) with or without VEGF(165) plasmid DNA (pDNA) to probe the EPCs after in vivo transplantation and to test whether they presented as differentiated endothelial cells (ECs). Bare QD NPs and QD NPs coated with PEI or PEI + VEGF(165) genes were characterized by dynamic light scattering, scanning electron microscopy, and atomic force microscopy. Transfection of EPCs with VEGF(165) led to the expression of specific genes and proteins for mature ECs. A hind limb ischemia model was generated in nude mice, and VEGF(165) gene-transfected EPCs were transplanted intramuscularly into the ischemic limbs. At 28 days after transplantation, the VEGF(165) gene-transfected EPCs significantly increased the number of differentiated ECs compared with the injection of medium or bare EPCs without VEGF(165) genes. Laser Doppler imaging revealed that blood perfusion levels were increased significantly by VEGF(165) gene-transfected EPCs compared to EPCs without VEGF(165). Moreover, the transplantation of VEGF(165) gene-transfected EPCs increased the specific gene and protein expression levels of mature EC markers and angiogenic factors in the animal model.

The use of anti-COX2 siRNA coated onto PLGA nanoparticles loading dexamethasone in the treatment of rheumatoid arthritis.

In drug delivery systems, some genes have the potential to interrupt unnecessary gene expression in specific target cells. In this study, two types of drug, glucocorticoids and siRNA, were co-delivered into conditioned cells to inhibit the expression of unnecessary genes and proteins involved in arthritis. To deliver the two factors into a human chondrocyte cell line (C28/I2), dexamethasone was first loaded into PLGA nanoparticles, and then drug-loaded PLGA nanoparticles were complexed with poly(ethyleneimine) (PEI)/siRNA. To test the co-delivery of siRNA and dexamethasone into chondrocytes, cells were transfected with green fluorescence protein siRNA (GFP siRNA) and drugs. After transfection with GFP siRNA, 70% reduction of C28/I2 cells demonstrated GFP expression, whereas MOCK carrying PLGA nanoparticles and PLGA nanoparticles without siRNA showed no differences of GFP expressions. COX-2 and iNOS productions in C28/I2 cells were examined after TNF-α pre-treatment to induce expression of arthritis-related molecules in vitro. The reduction of gene and protein expression associated with arthritis by transfection with dexamethasone-loaded and COX-2 siRNA-complexed PLGA nanoparticles was evaluated by RT-PCR, real time-qPCR, immunoblotting, immunohistochemistry, and immunofluorescence imaging.