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BACKGROUND: In the last decade, graphene surfaces have consistently supported osteoblast development of stem cells, holding promise as a therapeutic implant for degenerative bone diseases. However, until now no study has specifically examined the genetic changes when stem cells undergo osteogenic differentiation on graphene. RESULTS: In this study, we provide a detailed overview of gene expressions when human mesenchymal stem cells (MSCs) derived from either adipose tissue (AD-MSCs) or bone marrow (BM-MSCs), are cultured on graphene. Genetic expressions were measured using osteogenic RT2 profiler PCR arrays and compared either over time (7 or 21 days) or between each cell source at each time point. Genes were categorized as either transcriptional regulation, osteoblast-related, extracellular matrix, cellular adhesion, BMP and SMAD signaling, growth factors, or angiogenic factors. Results showed that both MSC sources cultured on low oxygen graphene surfaces achieved osteogenesis by 21 days and expressed specific osteoblast markers. However, each MSC source cultured on graphene did have genetically different responses. When compared between each other, we found that genes of BM-MSCs were robustly expressed, and more noticeable after 7 days of culturing, suggesting BM-MSCs initiate osteogenesis at an earlier time point than AD-MSCs on graphene. Additionally, we found upregulated angiogenic markers in both MSCs sources, suggesting graphene could simultaneously attract the ingrowth of blood vessels in vivo. Finally, we identified several novel targets, including distal-less homeobox 5 (DLX5) and phosphate-regulating endopeptidase homolog, X-linked (PHEX). CONCLUSIONS: Overall, this study shows that graphene genetically supports differentiation of both AD-MSCs and BM-MSCs but may involve different signaling mechanisms to achieve osteogenesis. Data further demonstrates the lack of aberrant signaling due to cell-graphene interaction, strengthening the application of specific form and concentration of graphene nanoparticles in bone tissue engineering.
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Medula Óssea , Diferenciação Celular , Grafite/metabolismo , Células-Tronco Mesenquimais , Osteogênese/fisiologia , Transdução de Sinais , Tecido Adiposo/citologia , Humanos , Células-Tronco Mesenquimais/citologia , OsteoblastosRESUMO
Non-viral transfection is a promising technique that could be used to increase the therapeutic potential of stem cells. The purpose of this study was to explore practical culture parameters of relevance in potential human mesenchymal stem cell (hMSC) clinical and tissue engineering applications, including type of polycationic transfection reagent, N/P ratio and dose of polycation/pDNA polyplexes, cell passage number, cell density and cell proliferation. The non-viral transfection efficiency was significantly influenced by N/P ratio, polyplex dose, cell density and cell passage number. hMSC culture conditions that inhibited cell division also decreased transfection efficiency, suggesting that strategies to promote hMSC proliferation may be useful to enhance transfection efficiency in future tissue engineering studies. Non-viral transfection treatments influenced hMSC phenotype, including the expression level of the hMSC marker CD105 and the ability of hMSCs to differentiate down the osteogenic and adipogenic lineages. The parameters found here to promote hMSC transfection efficiency, minimize toxicity and influence hMSC phenotype may be instructive in future non-viral transfection studies and tissue engineering applications.
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Meios de Cultura/farmacologia , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Transfecção/métodos , Animais , Antígenos CD/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Endoglina , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células NIH 3T3 , Plasmídeos/metabolismo , Polietilenoimina/química , Receptores de Superfície Celular/metabolismo , Vírus/efeitos dos fármacos , Vírus/metabolismoRESUMO
Autologous protein solution (APS) has been used anecdotally for intralesional treatment of tendon and ligament injuries, however, its use in these injuries has never been studied in vivo. Our objective was to evaluate the effect of APS on tendon healing in an equine superficial digital flexor (SDF) tendonitis model. We hypothesized intralesional injection of APS would result in superior structural and biomechanical healing. SDF tendonitis was induced in both forelimbs of eight horses using collagenase injection. One forelimb was randomly assigned to receive an intralesional injection of APS, while the other was injected with saline. Ultrasonographic examinations were performed at weeks -1, 0, 2, 4, 8, and 12 following treatment. At 12 weeks, horses were euthanized and SDF samples harvested. Histologic evaluation, biomechanical testing, gene expression analysis, total glycosaminoglycan (GAG) and total DNA quantification were performed. Collagen type III (COL3A1) expression was significantly higher (p = 0.028) in saline treated tendon than in normal tendon. Otherwise, there were no significant differences in gene expression. There were no significant differences in histologic or ultrasonographic scores between groups. Mean total DNA content was significantly higher (p = 0.024) in saline treated tendons than normal tendons, whereas total DNA content was not significantly different between APS treated tendon and normal tendon. Elastic modulus was higher in APS treated than saline treated tendon, but the difference was not significant. Reduced expression of COL3A1 in APS treated tendon may indicate superior healing. Increased total DNA content in saline treated tendon may indicate ongoing healing processes, vs. APS treated tendons which may be in the later stages of healing. Limitations include a relatively short study period and inconsistency in size and severity of induced lesions. Intralesional injection of APS resulted in some improvements in healing characteristics.
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PURPOSE: The extracellular matrix (ECM) labyrinthine network secreted by mesenchymal stem cells (MSCs) provides a microenvironment that enhances cell adherence, proliferation, viability, and differentiation. The potential of graphene-based nanomaterials to mimic a tissue-specific ECM has been recognized in designing bone tissue engineering scaffolds. In this study, we investigated the expression of specific ECM proteins when human fat-derived adult MSCs adhered and underwent osteogenic differentiation in the presence of functionalized graphene nanoparticles. METHODS: Graphene nanoparticles with 6-10% oxygen content were prepared and characterized by XPS, FTIR, AFM and Raman spectroscopy. Calcein-am and crystal violet staining were performed to evaluate viability and proliferation of human fat-derived MSCs on graphene nanoparticles. Alizarin red staining and quantitation were used to determine the effect of graphene nanoparticles on osteogenic differentiation. Finally, immunofluorescence assays were used to investigate the expression of ECM proteins during cell adhesion and osteogenic differentiation. RESULTS: Our data show that in the presence of graphene, MSCs express specific integrin heterodimers and exhibit a distinct pattern of the corresponding bone-specific ECM proteins, primarily fibronectin, collagen I and vitronectin. Furthermore, MSCs undergo osteogenic differentiation spontaneously without any chemical induction, suggesting that the physicochemical properties of graphene nanoparticles might trigger the expression of bone-specific ECM. CONCLUSION: Understanding the cell-graphene interactions resulting in an osteogenic niche for MSCs will significantly improve the application of graphene nanoparticles in bone repair and regeneration.
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Proteínas da Matriz Extracelular/metabolismo , Grafite/farmacologia , Células-Tronco Mesenquimais/metabolismo , Nanopartículas/química , Osteogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Integrinas/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Oxigênio/química , Espectroscopia Fotoeletrônica , Multimerização ProteicaRESUMO
Cartilage injury occurs commonly in equine athletes, often precipitating posttraumatic osteoarthritis (PTOA). Orthobiologics such as autologous conditioned serum (ACS) and autologous protein solution (APS) may be useful in decreasing posttraumatic inflammation, thereby preventing PTOA. The objective of this study was to quantify cytokine concentrations in ACS and APS and evaluate the protective effects of ACS and APS on inflamed chondrocytes cultured in vitro. We hypothesized that the combination of platelet-derived growth factors (PDGF) and anti-inflammatory cytokines present in APS would be superior in decreasing the inflammatory and catabolic cascade in inflamed chondrocytes when compared to ACS in which platelets are excluded from the preparation. Chondrocytes were isolated from the cartilage of femoral trochlear ridges of 6 horses and cultured in 12-well transwell plates. Treatment groups included: (1) control, (2) APS (Pro-Stride; Owl Manor), and (3) ACS (IRAP II; Arthrex). Each group was unstimulated or stimulated with IL-1ß and TNF-α for 48 h. The concentration of IL-1ß, IL-6, TNF-α, MMP-3, MMP-13, and IL-10 was quantified using a fluorescent bead-based multiplex assay. IL-1Ra concentration was quantified using ELISA. APS and ACS both had significantly increased concentrations of IL-1Ra without a concurrent increase in IL-1ß concentration. After 48 h of culture, media from chondrocytes treated with APS contained significantly increased concentrations of IL-1Ra and IL-10. APS-treated cultures had increased concentrations of IL-6. Overall, APS effectively concentrated IL-1Ra without an incubation period and media from APS-treated chondrocytes had increased concentrations of chondroprotective (IL-1Ra and IL-10) and modulatory (IL-6) cytokines, which may be beneficial in the treatment of inflammatory conditions such as PTOA.
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Hydrogels have been commonly used as model systems for 3-dimensional (3-D) cell biology, as they have material properties that resemble natural extracellular matrices (ECMs), and their cell-interactive properties can be readily adapted in order to address a particular hypothesis. Natural and synthetic hydrogels have been used to gain fundamental insights into virtually all aspects of cell behavior, including cell adhesion, migration, and differentiated function. However, cell responses to complex 3-D environments are difficult to adequately explore due to the large number of variables that must be controlled simultaneously. Here we describe an adaptable, automated approach for 3-D cell culture within hydrogel arrays. Our initial results demonstrate that the hydrogel network chemistry (both natural and synthetic), cell type, cell density, cell adhesion ligand density, and degradability within each array spot can be systematically varied to screen for environments that promote cell viability in a 3-D context. In a test-bed application we then demonstrate that a hydrogel array format can be used to identify environments that promote viability of HL-1 cardiomyocytes, a cell line that has not been cultured previously in 3-D hydrogel matrices. Results demonstrate that the fibronectin-derived cell adhesion ligand RGDSP improves HL-1 viability in a dose-dependent manner, and that the effect of RGDSP is particularly pronounced in degrading hydrogel arrays. Importantly, in the presence of 70mum RGDSP, HL-1 cardiomyocyte viability does not decrease even after 7 days of culture in PEG hydrogels. Taken together, our results indicate that the adaptable, array-based format developed in this study may be useful as an enhanced throughput platform for 3-D culture of a variety of cell types.
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Materiais Biocompatíveis , Técnicas de Cultura de Células/métodos , Hidrogéis , Animais , Materiais Biocompatíveis/química , Adesão Celular , Contagem de Células , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Células Cultivadas , Colágeno Tipo I/química , Meios de Cultura/química , Células Endoteliais/citologia , Humanos , Hidrogéis/química , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Camundongos , Miócitos Cardíacos/citologia , Células NIH 3T3 , Polietilenoglicóis/químicaRESUMO
Critical limb ischemia (CLI) is a terminal disease with high morbidity and healthcare costs due to limb loss. There are no effective medical therapies for patients with CLI to prevent amputation. Cell-based therapies are currently being investigated to address this unmet clinical need and have shown promising preliminary results. The purpose of this study was to characterize the output of a point-of-care cell separator (MarrowStim P.A.D. Kit), currently under investigation for the treatment of CLI, and compare its output with Ficoll-based separation. The outputs of the MarrowStim P.A.D. Kit and Ficoll separation were characterized using an automated hematology analyzer, colony-forming unit (CFU) assays, and tubulogenesis assays. Hematology analysis indicated that the MarrowStim P.A.D. Kit concentrated the total nucleated cells, mononuclear cells, and granulocytes compared with baseline bone marrow aspirate. Cells collected were positive for VEGFR-2, CD3, CD14, CD34, CD45, CD56, CD105, CD117, CD133, and Stro-1 antigen. CFU assays demonstrated that the MarrowStim P.A.D. Kit output a significantly greater number of mesenchymal stem cells and hematopoietic stem cells compared with cells output by Ficoll separation. There was no significant difference in the number of endothelial progenitor cells output by the two separation techniques. Isolated cells from both techniques formed interconnected nodes and microtubules in a three-dimensional cell culture assay. This information, along with data currently being collected in large-scale clinical trials, will help instruct how different cellular fractions may affect the outcomes for CLI patients.
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PURPOSE: To examine the role of the CD28-CD80-CD86 pathway of T-lymphocyte costimulation in corneal allograft rejection and the effect of blockade of that pathway on graft survival. METHODS: Kinetics of CD80 and CD86 expression in the cornea and draining lymph nodes were examined by RT-PCR and immunohistochemistry in untreated allograft recipients in a high-responder rat model. The effect of blockade of CD28-mediated costimulation was first examined by ex vivo incubation of excised Brown Norway rat donor cornea with the inhibitory protein CTLA4-Ig or an adenovirus vector (AdCTLA) expressing CTLA4-Ig, before grafting into Lewis rat recipients. A second group of graft recipients received systemic posttransplantation treatment with either CTLA4-Ig or AdCTLA. RESULTS: Expression of CD80 mRNA was increased in both donor and recipient cornea 16 hours after transplantation, whereas CD86 was detected constitutively, with no significant early increase. Immunohistochemistry on day 5 after transplantation demonstrated major histocompatibility complex (MHC) class II expression, no CD80, and only a trace of CD86 in corneal allografts. In lymph nodes strong MHC class II, weak CD80, and moderate CD86 expression was noted. Both donor cornea and recipient treatment with CTLA4-Ig resulted in prolonged allograft survival. AdCTLA was found to induce sustained secretion of bioactive CTLA4-Ig from corneas infected ex vivo. Survival of corneal allografts incubated with AdCTLA was marginally prolonged, and systemic treatment with AdCTLA significantly prolonged survival. CONCLUSIONS: Protein- or gene-based administration of CTLA4-Ig prolongs allograft survival by treatment of either the recipient or the donor tissue ex vivo before grafting.
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Antígenos de Diferenciação/administração & dosagem , Antígenos de Diferenciação/genética , Córnea/metabolismo , Transplante de Córnea , DNA Complementar/administração & dosagem , Sobrevivência de Enxerto/efeitos dos fármacos , Imunoconjugados , Imunossupressores/administração & dosagem , Abatacepte , Adenoviridae/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2 , Antígenos CD28/genética , Antígenos CD28/metabolismo , Antígeno CTLA-4 , Feminino , Terapia Genética , Vetores Genéticos , Técnicas Imunoenzimáticas , Fragmentos Fc das Imunoglobulinas , Linfonodos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Proteínas Recombinantes de Fusão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HomólogoRESUMO
Fipronil is a highly effective, broad-spectrum insecticide with potential value for the control of a wide range of crop, public hygiene, amenity, and veterinary pests. It can generally be applied at low to very low dose rates to achieve effective pest control. Application rates vary between 0.6 and 200 g a.i./ha, depending on the target pest and formulation. It belongs to the phenyl pyrazole or fiprole group of chemicals and is a potent disrupter of the insect central nervous system via interference with the gamma-aminobutyric acid (GABA-) regulated chloride channel. Fipronil degrades slowly on vegetation and relatively slowly in soil and in water, with a half-life ranging between 36 hr and 7.3 mon depending on substrate and conditions. It is relatively immobile in soil and has low potential to leach into groundwater. One of its main degradation products, fipronil desulfinyl, is generally more toxic than the parent compound and is very persistent. There is evidence that fipronil and some of its degradates may bioaccumulate, particularly in fish. Further investigation on bioaccumulation is warranted, especially for the desulfinyl degradate. The suitability of fipronil for use in IPM must be evaluated on a case-by-case basis. In certain situations, fipronil may disrupt natural enemy populations, depending on the groups and species involved and the timing of application. The indications are that fipronil may be incompatible with locust IPM; hence, this possibility requires further urgent investigation. It is very highly toxic to termites and has severe and long-lasting negative impacts on termite populations. It thus presents a long-term risk to nutrient cycling and soil fertility where termites are "beneficial" key species in these ecological processes. Its toxicity to termites also increases the risk to the ecology of habitats in which termites are a dominant group, due to their importance as a food source to many higher animals. This risk has been demonstrated in Madagascar, where two endemic species of lizard and an endemic mammal decline in abundance because of their food chain link to termites. Fipronil is highly toxic to bees (LD50 = 0.004 microgram/bee), lizards [LD50 for Acanthodactylus dumerili (Lacertidae) is 30 micrograms a.i./g bw], and gallinaceous birds (LD50 = 11.3 mg/kg for Northern bobwhite quail), but shows low toxicity to waterfowl (LD50 > 2150 mg/kg for mallard duck). It is moderately toxic to laboratory mammals by oral exposure (LD50 = 97 mg/kg for rats; LD50 = 91 mg/kg for mice). Technical fipronil is in toxicity categories II and III, depending on route of administration, and is classed as a nonsensitizer. There are indications of carcinogenic action in rats at 300 ppm, but it is not carcinogenic to female mice at doses of 30 ppm. The acute toxicity of fipronil varies widely even in animals within the same taxonomic groups. Thus, toxicological findings from results on standard test animals are not necessarily applicable to animals in the wild. Testing on local species seems particularly important in determining the suitability of fipronil-based products for registration in different countries or habitats and the potential associated risk to nontarget wildlife. Risk assessment predictions have shown that some fipronil formulations present a risk to endangered bird, fish, and aquatic and marine invertebrates. Great care should thus be taken in using these formulations where they may impact any of these endangered wildlife groups. Work in Madagascar has highlighted field evidence of this risk. The dose levels at which fipronil produces thyroid cancer in rats are very high and are unlikely to occur under normal conditions of use. There is also dispute as to whether this is relevant to human health risk. However, as fipronil is a relatively new insecticide that has not been in use for long enough to evaluate the risk it may pose to human health, from data on human exposure to the product, a precautionary approach may be warranted. The use of some fipronil-based products on domestic animals is not recommended where handlers spend significant amounts of time grooming or handling treated animals. In general, it would appear unwise to use fipronil-based insecticides without accompanying environmental and human health monitoring, in situations, regions, or countries where it has not been used before, and where its use may lead to its introduction into the wider environment or bring it into contact with people. Further work is needed on the impacts of fipronil on nontarget vertebrate fauna (amphibians, reptiles, birds, and mammals) in the field before the risk to wildlife from this insecticide can be adequately validated. Further field study of the effects of fipronil on the nutrient cycling and soil water-infiltration activities of beneficial termites is required to assess the ecological impacts of the known toxicity of fipronil to these insects.
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Monitoramento Ambiental/métodos , Inseticidas , Pirazóis , Animais , Animais Selvagens , Biodegradação Ambiental , Humanos , Inseticidas/química , Inseticidas/metabolismo , Inseticidas/toxicidade , Plantas/efeitos dos fármacos , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/toxicidade , Poluentes do Solo/análiseRESUMO
Human mesenchymal stem cells (hMSCs) have been extensively explored for drug delivery applications due to their safety, immunomodulatory properties, and ability to differentiate into new tissues. The experiments presented in this study were designed to determine peptide-based mechanisms to increase the adenoviral transduction of hMSCs for the purpose of improving their capacity as drug delivery vehicles. Specifically, we demonstrated that cyclic- RGD peptides increased the internalization of adenoviruses into MSCs. MSCs treated with cyclic-RGD peptides had a transduction efficiency of 76.6%±4%, which was significantly greater than the 23.5%±12.2% transduction efficiency of untreated stem cells (P<0.05). Blocking endocytosis with inhibitors of dynamin or actin polymerization decreased the cyclic-RGD-mediated increase in transduction efficiency. MSCs treated with cyclic-RGD and adenoviruses carrying the gene for bone morphogenetic protein-2 produced significantly greater concentrations of this growth factor compared to stem cells treated with only adenoviruses or adenoviruses cocultured with cyclic-RAD peptides. Furthermore, this stem cell-produced bone morphogenetic protein induced alkaline phosphatase expression in C2C12 cells indicating growth factor bioactivity. Taken together, these studies suggest that cyclic-RGD peptides could be used to increase the adenoviral transduction of hMSCs and increase their therapeutic potential.
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Adenoviridae , Antineoplásicos/farmacologia , Células-Tronco Mesenquimais , Oligopeptídeos/farmacologia , Transdução Genética/métodos , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismoRESUMO
Biomaterial scaffolds have been extensively used to deliver growth factors to induce new bone formation. The pharmacokinetics of growth factor delivery has been a critical regulator of their clinical success. This review will focus on the surface interactions that control the non-covalent incorporation of growth factors into scaffolds and the mechanisms that control growth factor release from clinically relevant biomaterials. We will focus on the delivery of recombinant human bone morphogenetic protein-2 from materials currently used in the clinical practice, but also suggest how general mechanisms that control growth factor incorporation and release delineated with this growth factor could extend to other systems. A better understanding of the changing mechanisms that control growth factor release during the different stages of preclinical development could instruct the development of future scaffolds for currently untreatable injuries and diseases.
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Proteína Morfogenética Óssea 2/administração & dosagem , Sistemas de Liberação de Medicamentos , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/química , Proteína Morfogenética Óssea 2/farmacocinética , Osso e Ossos/metabolismo , Portadores de Fármacos/química , Desenho de Fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/farmacocinéticaRESUMO
pH-induced protein aggregation facilitated the formation of nucleation centers for the chain growth polymerization of hydrogel microspheres.
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Hidrogel de Polietilenoglicol-Dimetacrilato/síntese química , Microesferas , Proteínas/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Concentração de Íons de Hidrogênio , Estrutura Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Dynamic hydrogels have emerged as an important class of biomaterials for temporal control over growth factor delivery. In this study we formed dynamic hydrogel microspheres from protein-polymer conjugates using an aqueous two-phase suspension polymerization process. This polymerization process enabled rapid microsphere formation without the use of an organic phase, surfactants, mechanical strain or toxic radical initiators. The microspheres' size distribution was modulated by varying the protein-polymer conformation in the pre-polymer solution. Notably, the protein's ligand-induced, nanometer-scale conformational change translated to maximum hydrogel volume changes of 76±10%. The magnitude of the microspheres' volume change was tuned by varying the crosslinking time and ligand identity. After characterizing the microspheres' dynamic properties, we encapsulated two important therapeutic proteins, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2), in the hydrogel microspheres and characterized how the microspheres' dynamic properties controlled their release. Significantly, the aqueous two-phase suspension polymerization process enabled high encapsulation efficiencies (65.8±4.8% and 79.5±3.0% for VEGF and BMP-2, respectively). Also, the microspheres' ligand-induced volume change triggered VEGF and BMP-2 release at specific, predetermined times. There are hundreds of proteins that undergo well-characterized conformational changes that could be processed into hydrogel microspheres via aqueous two-phase suspension polymerizations. Therefore, this approach could be used to form dynamic, growth-factor-releasing hydrogel microspheres that respond to a broad range of specific biochemical ligands.
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Proteína Morfogenética Óssea 2/administração & dosagem , Hidrogéis , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Microesferas , PolímerosRESUMO
In this study, three-dimensional (3-D) poly(ethylene glycol) (PEG) hydrogel arrays were used to screen for the effects of fibroblast growth factor-2 (FGF2), combined with multiple hydrogel matrix parameters, on human mesenchymal stem cell (hMSC) viability and spreading. In particular, we examined the effects of FGF2 while co-varying hydrogel matrix degradability, cell adhesion ligand type, and cell adhesion ligand density. FGF2 significantly improved viability of hMSCs in a dose-dependent manner in both nondegrading and degrading PEG hydrogels in the absence of extracellular matrix-derived cell adhesion ligands. The presence of a small molecule that inhibits autophosphorylation of the FGF2 receptor blocked the effects of FGF2 on hMSC viability in PEG hydrogels, both in the presence and absence of the Arg-Gly-Asp-Ser-Pro (RGDSP) ligand. FGF2 effects on hMSC viability were less pronounced when FGF2 was presented in combination with the RGDSP cell adhesion ligand or the IKVAV cell adhesion ligand in nondegrading PEG hydrogels. Importantly, spread hMSC morphologies were observed and quantified in a select subset of hydrogel networks, which were degradable and included both FGF2 and RGDSP. These results indicate that the hydrogel arrays described here can be used to efficiently study the influence of soluble and insoluble hydrogel matrix parameters on stem cell behavior, and to identify synthetic, 3-D environments that promote specific hMSC behaviors.
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Movimento Celular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Difusão/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Humanos , Ligantes , Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Peso Molecular , Peptídeos/farmacologia , Solubilidade/efeitos dos fármacosRESUMO
In this study we formed and characterized dynamic hydrogel microspheres in which a protein conformational change was used to control microsphere volume changes and the release of an encapsulated drug. In particular, a specific biochemical ligand, trifluoperazine, induced calmodulin's nanometer scale conformation change, which translated to a 48.7% microsphere volume decrease. This specific, ligand-induced volume change triggered the release of a model drug, vascular endothelial growth factor (VEGF), at pre-determined times. After release from the microspheres, 85.6 +/- 10.5% of VEGF was in its native conformation. Taken together, these results suggest that protein conformational change could serve as a useful mechanism to control drug release from dynamic hydrogels.
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Preparações de Ação Retardada/química , Hidrogéis/uso terapêutico , Microesferas , Proteínas/química , Humanos , Hidrogéis/química , Conformação Proteica , Proteínas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
In this study we generated 3D poly(ethylene glycol) (PEG) hydrogel arrays to screen for the individual and combinatorial effects of extracellular matrix (ECM) degradability, cell adhesion ligand type, and cell adhesion ligand density on human mesenchymal stem cell (hMSC) viability. In particular, we explored the influence of two well-characterized ECM-derived cell adhesion ligands: the fibronectin-derived Arg-Gly-Asp-Ser-Pro (RGDSP) sequence, and the laminin-derived Ile-Lys-Val-Ala-Val (IKVAV) sequence. PEG network degradation, the RGDSP ligand, and the IKVAV ligand each individually increased hMSC viability in a dose-dependent manner. The RGDSP ligand also improved hMSC viability in a dose-dependent manner in degradable PEG hydrogels, while the effect of IKVAV was less pronounced in degradable hydrogels. Combinations of RGDSP and IKVAV promoted high viability of hMSCs in nondegradable PEG networks, while the combined effects of the ligands were not significant in degradable PEG hydrogels. Although hMSC spreading was not commonly observed within PEG hydrogels, we qualitatively observed hMSC spreading after 5 days only in degradable PEG hydrogels prepared with 2.5 mM of both RGDSP and IKVAV. These results suggest that the enhanced throughput approach described herein can be used to rapidly study the influence of a broad range of ECM parameters, as well as their combinations, on stem cell behavior.