Multiply clustered gold-based nanoparticles complexed with exogenous pDNA achieve prolonged gene expression in stem cells.

Development of a stable and prolonged gene delivery system is a key goal in the gene therapy field. To this end, we designed and fabricated a gene delivery system based on multiply-clustered gold particles that could achieve prolonged gene delivery in stem cells, leading to improved induction of differentiation. Methods: Inorganic gold nanoparticles (AuNPs) underwent three rounds of complexation with catechol-functionalized polyethyleneimine (CPEI) and plasmid DNAs (pDNAs), in that order, with addition of heparin (HP) between rounds, yielding multiply-clustered gold-based nanoparticles (mCGNPs). Via metal-catechol group interactions, the AuNP surface was easily coordinated with positively charged CPEIs, which in turn allowed binding of pDNAs. Results: Negatively charged HP was encapsulated with the positive charge of CPEIs via electrostatic interactions, making the NPs more compact. Repeating the complexation process yielded mCGNPs with improved transfection efficiency in human mesenchymal stem cells (hMSCs); moreover, these particles exhibited lower cytotoxicity and longer expression of pDNAs than conventional NPs. This design was applied to induction of chondrogenesis in hMSCs using pDNA harboring SOX9, an important chondrogenic transcription factor. Prolonged expression of SOX9 induced by mCGNPs triggered expression of chondrocyte extracellular matrix (ECM) protein after 14 days, leading to more efficient chondrogenic differentiation in vitro and in vivo.

Aggrecan- and COMP-loaded poly-(lactic-co-glycolic acid) nanoparticles stimulate chondrogenic differentiation of human mesenchymal stem cells.

During embryogenesis, specific proteins expressed in cells have key roles in the formation of differentiated cells and tissues. Delivery of specific proteins into specific cells, both in vitro and in vivo, has proved to be exceedingly difficult. In this study, we developed a safe and efficient protein delivery system using encapsulation of proteins into biodegradable poly-(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). The PLGA NPs were used to deliver proteins into human mesenchymal stem cells (hMSCs). Fluorescent markers loaded into the PLGA NPs were used to verify the internalization of NPs into hMSCs using FACS analysis and confocal microscopy. With these methods, we demonstrated that the encapsulated model proteins are readily delivered into hMSCs, released from the NP vehicles, and, finally, moved into the cytosols. Using chondrogenesis-related proteins such as aggrecan and cartilage oligomeric matrix protein (COMP), chondrogenic differentiation of hMSCs treated with aggrecan and COMP encapsulated PLGA NPs was clearly observed and caused to differentiate into chondrocytes.

Poly(N-isopropylacrylamide-co-acrylic acid) nanogels for tracing and delivering genes to human mesenchymal stem cells.

Drugs, proteins, and cells can be macro- and micro-encapsulated by unique materials that respond to specific stimuli. The phases and hydrophobic interactions of these materials are reversibly altered by environmental stimuli such as pH and temperature. These changes can lead to self-assembly of the materials, which enables controlled drug release and safe gene delivery into cells and tissues. The fate of stem cells delivered by such methods is of great interest. The formation of transgenic tissues requires genes to be delivered safely into stem cells. A cell tracing vehicle and a gene delivery carrier were simultaneously introduced into human mesenchymal stem cells (hMSCs). A thermo-sensitive hydrogel, poly(N-isopropylacrylamide-co-acrylic acid) (p(NiPAAm-co-AAc)), was created to generate self-assembled nanoparticles with nanogel characteristics. Hydrophobic interactions mediated the binding of the carboxyl group on the outside of p(NiPAAm-co-AAc) with the amine group of iron oxide. Nanogels carrying iron oxide and a fluorescent dye were complexed with specific genes. These nanogels could be internalized by hMSCs, and the transplantation of these cells into mice was monitored by in vivo imaging. Self-assembled p(NiPAAm-co-dAAc) nanogels complexed with green fluorescent protein were highly expressed in hMSCs and are a potential material for gene delivery.

Multilineage differentiation of human-derived dermal fibroblasts transfected with genes coated on PLGA nanoparticles plus growth factors.

Wounded tissues and cells may be treated with growth factors and specific genes for the purpose of tissue repair and regeneration. To deliver specific genes into tissues and cells, this study presents the use of fabricated poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) complexed with the cationic polymer poly (ethleneimine) (PEI). Through complexation with PEI, several types of genes (SOX9, Cbfa1, and C/EBP-α) were coated into PLGA NPs, which enhanced gene uptake into normal human-derived dermal fibroblast cells (NFDHCs) in vitro and in vivo. Several cell types (293T, HeLa, and fibroblast cells) were transfected with fluorescence-tagged PEI/SOX9, PEI/Cbfa1, and PEI/C/EBP-α gene-complexed PLGA NPs. The gene and protein expression levels in the cells were evaluated by RT-PCR, real-time quantitative PCR, Western blotting, and confocal laser microscopy. Fibroblast cells encapsulated in fibrin gels were transfected with the gene-complexed NPs plus specific growth factors (TGF-ß3, BMP-2, or IGF/bFGF), which induced chondrogenesis, osteogenesis, or adipogenesis both in vitro and after transplantation into nude mouse.

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.

Transfection of VEGF(165) 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.

SOX9 gene plus heparinized TGF-ß 3 coated dexamethasone loaded PLGA microspheres for inducement of chondrogenesis of hMSCs.

Microparticulated types of scaffolds have been widely applied in stem cell therapy and the tissue engineering field for the regeneration of wound tissues. During application of simple genes or growth factors and cell delivery vehicles, we designed a method that employs dexamethsone loaded PLGA microspheres consisting of polyplexed SOX9 genes plus heparinized TGF-ß 3 on the surface of polymeric microspheres prepared using a layer-by-layer (LbL) method. The fabrication of the polyplexed SOX9 genes plus heparinized TGF-ß 3 and their subsequent coating onto dexamethsone loaded PLGA microspheres represents a method for functionalization of the polymeric matrix. The use of SOX9 gene plus heparinized TGF-ß 3 coated dexamethsone loaded PLGA microspheres was evaluated to determine their potential as both gene carriers and cell delivery vehicle. By adhesion of hMSCs onto SOX9 gene plus heparinized TGF-ß 3 coated dexamethsone loaded PLGA microspheres, the chondrogenesis-related specific genes of collagen type II were increased 30 times comparing to control. Also, the specific extracellular matrix of glycosaminoglycan (GAG) production of hMSCs adhered onto SOX9 gene plus heparinized TGF-ß 3 coated dexamethasone loaded PLGA microspheres increased more 2.5 times than control group. Not only in vitro culture but in vivo results, the specific genes of COMP, aggrecan, collagen type II, and SOX9 showed much more gene expressions such as 20, 15, 10, 8 times.

Exogenous Nurr1 gene expression in electrically-stimulated human MSCs and the induction of neurogenesis.

In this study, synergistic effects of electrical stimulation and exogenous Nurr1 gene expression were examined to induce the differentiation of human mesenchymal stem cells (hMSCs) into nerve cells in in vitro culture system. A two-step procedure was designed to evaluate the effects of electrical stimulus and exogenous gene delivery for inducing neurogenesis. First, an electrical stimulation device was designed using gold nanoparticles adsorbed to the surface of a cover glass. Gold nanoparticles, as an electrical conductor for stem cells, are well-defined particles adsorbed to a polyethyleneimine (PEI)-coated cover glass. The nanoparticle morphology was examined by scanning electron microscope (SEM). Second, a plasmid carrying Nurr1 cDNA was complexed with biodegradable poly-(DL)-lactic-co-glycolic acid (PLGA) nanoparticles to support neurogenesis. To evaluate the neuronal differentiation of stem cells mediated by the treatment with either electrical stimulation and exogenous Nurr1 gene delivery, or both, the expression of neuron-specific genes and proteins was examined by RT-PCR and Western blotting. Cells transfected with exogenous Nurr1 genes plus electrical stimulation (250 mV for 1000 s) showed the greatest level of neurite outgrowth with a mean neurite length of 150 µm. Neurite length in cells treated with only one stimulus was not significant, approximately 10-20 µm. These results indicate that electrical stimulation and exogenous Nurr1 gene expression together may be adequate to induce nerve regeneration using stem cells.

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.

Chondrogenic potential of stem cells derived from amniotic fluid, adipose tissue, or bone marrow encapsulated in fibrin gels containing TGF-ß3.

In this study, several types of hMSCs, derived from bone marrow, adipose tissue, or amniotic fluid, were encapsulated in a fibrin hydrogel mixed with TGF-ß3 and then evaluated for their capacity for differentiation in vitro and in vivo. For determination of stem cell differentiation, RT-PCR, real time quantitative PCR (qPCR), histology, and immunohistochemical assays were used for analysis of chondrogenesis. Using these analysis methods, several of the cultured hMSCS were found to highly express genes and proteins specific to cartilage forming tissues. Additionally, similar trends in expression were found in tissue recovered from nude mice transplanted with several types of hMSCs encapsulated in a fibrin hydrogel containing TGF-ß3. The results of both in vitro and in vivo analyses showed that cultured or transplanted hMSCs mixed with TGF-ß3 in a fibrin hydrogel differentiated into chondrocytes, suggesting that these cells would be suitable for reconstruction of hyaline articular cartilage.

Chondrogenesis of mesenchymal stem cells and dedifferentiated chondrocytes by transfection with SOX Trio genes.

In this study, bone marrow-derived mesenchymal stem cells (MSCs), adipose-derived mesenchymal stem cells (ASCs) and dedifferentiated chondrocytes were transfected with SOX5, 6, and 9 genes (SOX Trio) and grown under pellet culture conditions (encapsulated in a fibrin hydrogel) to evaluate the chondrogenic potential in vitro and in vivo. RT-PCR, real-time quantitative PCR (qPCR), histology, and immunohistochemical assays were performed to determine the chondrogenic potential of the stem cells and dedifferentiated chondrocytes. Chondrogenic genes and proteins were more highly expressed in SOX Trio-expressing cells than in untransfected cells. In addition, not only specific genes and proteins, but cartilage-forming tissues were observed in nude mice transplanted with fibrin hydrogel encapsulated SOX Trio-expressing MSCs, ASCs, and dedifferentiated chondrocytes. Both in vitro and in vivo analyses revealed that fibrin hydrogel encapsulated cultured or transplanted cells transfected with the SOX Trio successfully differentiated into mature chondrocytes and could be used for the reconstruction of hyaline articular cartilage.

C/EBP-α and C/EBP-ß-mediated adipogenesis of human mesenchymal stem cells (hMSCs) using PLGA nanoparticles complexed with poly(ethyleneimmine).

In this study, to drive efficient adipogenic differentiation, the adipogenic transcription factors C/EBP-α and C/EBP-ß fused to green fluorescent protein (GFP) or red fluorescent protein (RFP) were complexed with poly-ethyleneimine (PEI) coupled with biodegradable PLGA nanospheres and delivered to human mesenchymal stem cell (hMSC). FACS analysis revealed that the transfection efficiency of C/EBP-α, C/EBP-ß, or both genes complexed with PEI-coated PLGA nanospheres was 12.59%, 21.74%, and 28.96% of hMSCs. Expression and localization of C/EBP-α and C/EBP-ß were confirmed by Western blotting and confocal laser microscopy. Overexpression of exogenous C/EBP-α and C/EBP-ß significantly elevated adipogenic differentiation processes as indicated by RT-PCR, real-time PCR, Western blotting, histology, and immunofluorescence microscopy. During adipogenesis, PEI-coupled PLGA nanospheres complexed with C/EBP-α and C/EBP-ß greatly increased the adipogenic capability of in vitro cultured cells, as well of in vivo transplanted cells. The expression of genes and proteins specific to adipogenic differentiation in hMSCs was significantly elevated compared to the controls.

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.

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.

Multi-lineage differentiation of hMSCs encapsulated in thermo-reversible hydrogel using a co-culture system with differentiated cells.

The micro-environment is an important factor in the differentiation of cultured stem cells for the purpose of site specific transplantation. In an attempt to optimize differentiation conditions, co-culture systems composed of both stem cells and primary cells or cell lines were used in hydrogel with in vitro and in vivo systems. Stem cells encapsulated in hydrogel, under certain conditions, can undergo increased differentiation both in vitro and in vivo; therefore, reconstruction of transplanted stem cells in a hydrogel co-culture system is important for tissue regeneration. In order to construct such a co-culture system, we attempted to create a hydrogel scaffold which could induce neo-tissue growth from the recipient bed into the material. This material would enable encapsulation of stem cells in vitro after which they could be transferred to an in vivo system utilizing nude mice. In this case, the hydrogel was implanted in the subfascial space of nude mice and excised 4 weeks later. Cross-sections of the excised samples were stained with von Kossa or safranin-O and tubular formations into the gel were observed with and tested by doppler imaging. The data showed that the hydrogel markedly induced growth of osteogenic, chondrogenic, and vascular-rich tissue into the hydrogel by 4 weeks, which surpassed that after transplantation in a co-culture system. Further, a co-culture system with differentiated cells and stem cells potentially enhanced chondrogenesis, osteogenesis, and vascularization. These findings suggest that a co-culture system with hydrogel as scaffold material for neo-tissue formation is a useful tools for multi-lineage stem cell differentiation.

Chondrogenic differentiation of mesenchymal stem cells embedded in a scaffold by long-term release of TGF-beta 3 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.

Chondrogenesis of human mesenchymal stem cells encapsulated in a hydrogel construct: neocartilage formation in animal models as both mice and rabbits.

In this study, in vivo studies, both nude mouse and rabbit cartilage defect, were tested for chondrogenesis using stem cells (SCs) using growth factor. Specifically, human mesenchymal stem cells (hMSCs) were embedded in a hydrogel scaffold, which was coencapsulated with transforming growth factor-beta3 (TGF-beta3). The specific extracellular matrices (ECMs) released from hMSCs transplanted into the animal were assessed via glycosaminoglycan (GAG)/DNA content, RT-PCR, real time-QPCR, immunohistochemical (IHC), and Safranin-O staining and were observed up to 7 weeks after injection. By detection of ECMs the GAG content per cell remained constant for all formulations, indicating that the dramatic increase in cell number for samples with TGF-beta3 was accompanied by the maintenance of the cell phenotypes. The histological and IHC staining of the newly repaired tissues observed after treatment with TGF-beta3 mixed with hMSCs evidenced hyaline cartilage-like characteristics. Moreover, the results observed with the animal model (rabbit) treated with hMSCs embedded in the growth factor-containing hydrogel indicate that the implantation of mixed cells with TGF-beta3 may constitute a clinically efficient method for the regeneration of hyaline articular cartilage.

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.

Transforming growth factor beta-3 bound with sulfate polysaccharide in synthetic extracellular matrix enhanced the biological activities for neocartilage formation in vivo.

The thermoreversible hydrogel of poly(N-isopropylacrylamide-co-acrylic acid) [p(NiPAAm-co-AAc)] was utilized in an injectable protein (growth factor) delivery system for use in tissue regeneration protocols. The transforming growth factor beta (TGF beta) proteins, which have been shown to bind to heparin, have been well established to induce chondrogenic differentiation in chondrocytes. In this study, we assessed the effects of heparin on the TGF beta-3 activities in rabbit chondrocytes embedded in thermoreversible hydrogel. The hydrogels embedded with chondrocytes and heparin-bound TGF beta-3 were injected subcutaneously into nude mice, which were monitored for up to 8 weeks after injection. P(NiPAAm-co-AAc) hydrogels with TGF beta-3 were compared with the hydrogels with heparin bound to TGF beta-3 for the assessment of the effects of heparin binding on chondrogenic differentiation. To monitor the release of growth factor via in vivo analysis, we assessed the Cy5.5-NHS-conjugated growth factors via the bioimaging method. In terms of the release of growth factors, the release of heparin-binding growth factor was slower than that of the growth factor itself. Additionally, the proliferation rate and cartilage-specific ECM production were both found to be significantly higher in the presence of heparin binding than was observed with the controls. The quantity of cartilage-associated ECM proteins was assessed via immunohistochemical staining (collagen type II) and safranin-O staining. These data indicate the potential use of heparin-binding TGF beta-3 for the reconstruction of neocartilage formation.

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.