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Several factors are involved in angiogenesis. To form new blood vessels, we fabricated vehicles carrying an angiogenesis-related peptide (apelin) and gene (vascular endothelial growth factor (VEGF)165) that were internalized by human mesenchymal stem cells (hMSCs). These non-toxic poly-(DL)-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) easily entered hMSCs without cytotoxicity. The negatively charged outer surface of PLGA NPs can be easily complexed with highly positively charged polyethylenimine (PEI) to deliver genes into cells. PLGA NPs complexed with PEI could be coated with negatively charged VEGF plasmid DNA and loaded with apelin. The physical characteristics of these PLGA NPs were determined by size distribution, gel retardation, and morphological analyses. Transfection of VEGF-coated apelin-loaded PLGA NPs resulted in the differentiation of hMSCs into endothelial cells and vascular formation in Matrigel in vitro. Following injection of hMSCs transfected with these PLGA NPs into an ischemic hind limb mouse model, these cells differentiated into endothelial cells and accelerated neovascularization.

For electrical stimulation of hMSCs, gold nanoparticles were coated onto polyethyleneimine coated glass cover slips. The effects of pulsed or constant electrical stimulation upon cytotoxicity and differentiation of hMSCs were examined. The effects of co culturing hMSCs with neuronal cells were also tested. The neuronal differentiation of the stem cells was evaluated by determining the expression of neuron-specific genes and proteins using RT-PCR and Western blotting. Morphological changes were evaluated by scanning electron microscopy. The hMSCs co-cultured with mature neuronal cells and stimulated with electrical shock showed the greatest level of neurite outgrowth (>150 mm) and smaller cell body sizes.

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.

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.

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-β 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-β 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-β 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-β 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-β 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.

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.

In this study, to drive efficient adipogenic differentiation, the adipogenic transcription factors C/EBP-α and C/EBP-β 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-β, 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-β were confirmed by Western blotting and confocal laser microscopy. Overexpression of exogenous C/EBP-α and C/EBP-β 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-β 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.

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 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.

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.

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.

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.

This study examined the efficacy of poly(NiPAAm-co-AAc) as an injectable drug delivery vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. The hydrogel constructs, which consisted of embedded cells co-encapsulating dexamethasone (Dex) and TGF β-1 or unloaded Dex, were used as controls to determine the effects of Dex and TGF β-1 on chondrogenic differentiation. The level of Dex and TGF β-1 released was monitored using a bioimaging method. The amount of Dex released from hydrogel was faster than that of TGF β-1. TGF β-1 was present in hydrogel for more than 4 weeks after the injection. The level of the cartilage associated ECM proteins was examined by immunohistochemical staining for collagen type II as well as by Safranin-O and Alcian blue (GAG) staining. These results highlight the potential of a thermo-reversible hydrogel mixed with the chondrocytes and differentiation delivery material for applications in neocartilage formation. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res 2008

The aim of this study was to assess the efficacy of ectopic bone formation in a three-dimensional hybrid scaffold in combination with hydroxyapatite (HA) and poly(NiPAAm-co-AAc) as an injectable vehicle in the form of a supporting matrix for the osteogenic differentiation of rabbit mesenchymal stem cells (MSCs). Osteogenic differentiation of MSCs in the hybrid scaffold was greatly influenced by the addition of growth factors. When the osteoinduction activity of hybrid scaffold was studied following implantation into the back subcutis of nude mouse in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds containing growth factor (BMP-2: bone morphogenic protein-2). The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture group. We conclude that combination of MSC-seeded hybrid scaffold containing BMP-2 was a promising method by which to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.

Quantum dot (QDs) have been employed as bioimaging agents and delivery vehicles for gene therapeutics in several types of cells. In this study, we fabricated multiple QD bundled nanoparticles (NPs) to investigate the effect of QD size and poly(ethylenimine) (PEI) coating on the efficiency of gene delivery into human mesenchymal stem cells (hMSCs). Several types of QDs, which exhibit different ranges of particle size and fluorescence when employed, were coated with PEI to alter their negative charges and to enable them to be bundled into larger particles. Using specific wavelengths of QDs for bioimaging, gene-complexed QD bundled NPs were easily detected in the hMSCs using several different methods such as fluorescence-activated cell sorter, confocal laser scanning microscopy, and in vivo optical imaging. These PEI-coated, bundled QD NPs exhibited significantly higher gene transfection efficacy than single-type QDs. Particularly, the largest QD bundled NPs examined, QD655, had a much higher uptake capability and greater gene expression ability than the other QD NPs (QD525, QD565, and QD605). We believe that our findings help to enrich knowledge of design considerations that will aid in the engineering of QD NPs for stem cell application in the future.

Directing the controlled differentiation and tracking of stem cells is essential to achieve successful stem cell therapy. In this work, we describe a multi-modal (MR/optical) transfection agent (MTA) for efficient gene delivery and cell tracking of human mesenchymal stem cells (hMSCs). The MTA was synthesized through a facile two-step approach with 1) ligand exchange of a catechol-functionalized polypeptide (CFP) and 2) chemical immobilization of fluorescence labelled cationic polymer via aminolysis reaction. Cationic polymer-immobilized MTAs with size of ∼40 nm exhibit greatly enhanced colloidal stability in aqueous solution. In addition, the MTAs were capable of binding DNA molecules for transfection. The MTA/pDNA complex showed relatively good transfection efficiency in hMSCs (compared to the commercial transfection agent, Lipofectamine) and good biocompatibility. MTA-treated hMSCs were successfully visualized after transplantation via MR and optical imaging system over 14 days. These studies highlight the challenges associated with the potential advantages of designing multi-modal nanostructured materials as tools for genetic materials delivery and cell-tracking in stem cell therapy.

Specific genes and growth factors are involved in stem cell differentiation. In this study, we fabricated a delivery carrier for both protein and gene delivery that was introduced into human endothelial progenitor cells (EPCs). The highly negative charge carried by the heparin-modified pluronic nanogels allowed for binding to growth factors and localization in the core of nanogels. The residues of negatively charged heparin can complex with positively charged cationic materials, making it suitable for gene delivery. Supramolecular nanogels can be easily encapsulated the hydrophilic drugs and highly positive surfaces can be complexed with negative charge carrying plasmid DNA (pDNA). The size distribution, gel retardation, and denaturation of encapsulated growth factors and supramolecular nanogels modified with heparin were evaluated. The supramolecular nanogels containing basic fibroblast growth factors and complexing VEGF165 pDNA internalized into EPCs have been well formed vascular formation in matrigel gels. Proteins and genes introduced into EPCs using nanogels promoted neovascularization in an animal model of limb ischemia. EPCs that differentiated into endothelial cells both in vitro and in vivo were tested.

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-β3, BMP-2, or IGF/bFGF), which induced chondrogenesis, osteogenesis, or adipogenesis both in vitro and after transplantation into nude mouse.

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.

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.

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.

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-β3 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-β3. The results of both in vitro and in vivo analyses showed that cultured or transplanted hMSCs mixed with TGF-β3 in a fibrin hydrogel differentiated into chondrocytes, suggesting that these cells would be suitable for reconstruction of hyaline articular cartilage.

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.

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-β3 (TGF-β3). 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-β3 was accompanied by the maintenance of the cell phenotypes. The histological and IHC staining of the newly repaired tissues observed after treatment with TGF-β3 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-β3 may constitute a clinically efficient method for the regeneration of hyaline articular cartilage. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010

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.

The aim of this study was to assess the efficacy of poly(NiPAAm-co-AAc) blended with hyaluronic acid (HA) as an injectable cell vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. Specially, rabbit chondrocytes were embedded in blended hydrogels co-encapsulation with dexamethasone (Dex) and growth factors for enhancing the chondrogenic differentiation. Blended hydrogel constructs consisting of embedded cells co-encapsulating Dex and TGF β-3 or unloaded Dex and sTGF β-3 served as controls to assess the effects of Dex on chondrogenic differentiation. Hydrogel constructs consisting of embedded cells co-encapsulating Dex and TGF β-3 on chondrogenic differentiation. The hydrogel constructs were injected subcutaneously into the nude mice and monitored for 1, 4, and 8 weeks after the injection. The level of the cartilage-associated ECM proteins was determined by immunohistochemical (collagen type II; specific marker for chondrogenic differentiation), Safranin-O, and Alcian blue (GAG) staining. Over the same time period, the glycosamingoglycan content per cell remained constant for all formulations, indicating that the dramatic increase in cell number for samples with Dex and TGF β-3 loaded in hydrogel constructs was accompanied by maintenance of the cell phenotypes. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007