RESUMO
Gold nanoparticles decorated with analyte recognition units can form the basis of colorimetric (bio)sensors. The presentation of those recognition units may play a critical role in determining sensor sensitivity. Herein, we use a model system to investigate the effect of the architecture of a polymeric linker that connects gold nanoparticles with the recognition units. Our results show that the number of the latter that can be adsorbed during the assembly of the colorimetric sensors depends on the linker topology. We also show that this may lead to substantial differences in colorimetric sensor performance, particularly in situations in which the interactions with the analyte are comparably weak. Finally, we discuss design principles for efficient colorimetric sensor materials based on our findings.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Técnicas Biossensoriais/métodos , Colorimetria/métodos , Ouro , PolímerosRESUMO
Glycosaminoglycan (GAG)-based biohybrid hydrogels of varied GAG content and GAG sulfation pattern were prepared and applied to sequester cytokines. The binding of strongly acidic and basic cytokines correlated with the integral space charge density of the hydrogel, while the binding of weakly charged cytokines was governed by the GAG sulfation pattern.
Assuntos
Citocinas/química , Glicosaminoglicanos/química , Hidrogéis/química , Animais , Química Click , Heparina/química , Humanos , Polietilenoglicóis/química , Sulfatos/química , Compostos de Sulfidrila/químicaRESUMO
Glycosaminoglycan (GAG)-based hydrogels gain increasing interest in regenerative therapies. To support specific applications, the biomolecular functionality of gel matrices needs to be customized via conjugation of peptide sequences that mediate cell adhesion, expansion and differentiation. Herein, we present an orthogonal strategy for the formation and chemoselective functionalization of starPEG-GAG hydrogels, utilizing the uniform and specific conjugation of peptides and GAGs for customizing the resulting materials. The introduced approach was applied for the incorporation of three different types of RGD peptides to analyze the influence of peptide sequence and conformation on adhesion and morphogenesis of endothelial cells (ECs) grown on the peptide-containing starPEG-GAG hydrogels. The strongest cellular response was observed for hydrogels functionalized with cycloRGD followed by linear forms of RGDSP and RGD, showing that morphogenesis and growth rate of ECs is controlled by both type and quantity of the conjugated peptides.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Glicosaminoglicanos/química , Hidrogéis/química , Oligopeptídeos/química , Polietilenoglicóis/química , Sequência de Aminoácidos , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Heparina/química , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Maleimidas/química , Modelos Moleculares , Conformação Molecular , Especificidade por SubstratoRESUMO
Sulfation patterns of glycosaminoglycans (GAG) govern the electrostatic complexation of biomolecules and thus allow for modulating the release profiles of growth factors from GAG-based hydrogels. To explore options related to this, selectively desulfated heparin derivatives were prepared, thoroughly characterized, and covalently converted with star-shaped poly(ethylene glycol) into binary polymer networks. The impact of the GAG sulfation pattern on the network characteristics of the obtained hydrogels was theoretically evaluated by mean field methods and experimentally analyzed by rheometry and swelling measurements. Sulfation-dependent differences of reactivity and miscibility of the heparin derivatives were shown to determine network formation. A theory-based design concept for customizing growth factor affinity and physical characteristics was introduced and validated by quantifying the release of fibroblast growth factor 2 from a set of biohybrid gels. The resulting new class of cell-instructive polymer matrices with tunable GAG sulfation will be instrumental for multiple applications in biotechnology and medicine.
Assuntos
Portadores de Fármacos/química , Fator 2 de Crescimento de Fibroblastos/química , Glicosaminoglicanos/química , Somatomedinas/química , Fenômenos Químicos , Heparina/química , Hidrogéis/química , Polietilenoglicóis/química , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade EstáticaRESUMO
For physical and chemical characterization of polymers, a wide range of analytical methods is available. Techniques like NMR and X-ray are often combined for a detailed characterization of polymers used in medical applications. Over the past few years, MALDI mass-spectrometry has been developed as a powerful tool for space-resolved analysis, not least because of its mass accuracy and high sensitivity. MALDI imaging techniques combine the potential of mass-spectrometric analysis with imaging as additional spatial information. MALDI imaging enables the visualization of localization and distribution of biomolecules, chemical compounds, and other molecules on different surfaces. In this study, surfaces of polymeric dialyzer membranes, consisting of polysulfone (PS) and polyvinylpyrrolidone (PVP), were investigated, regarding chemical structure and the compound's distribution. Flat membranes as well as hollow fiber membranes were analyzed by MALDI imaging. First, analysis parameters like laser intensity and laser raster step size (spatial resolution in resulting image) were established in accordance with polymer's characteristics. According to the manufacturing process, luminal and abluminal membrane surfaces are characterized by differences in chemical composition and physical characteristics. The MALDI imaging demonstrated that the abluminal membrane surface consists more of polysulfone than polyvinylpyrrolidone, and the luminal membrane surface displayed more PVP than PS. The addition of PVP as hydrophilic modifier to polysulfone-based membranes increases the biocompatibility of the dialysis membranes. The analysis of polymer distribution is a relevant feature for characterization of dialysis membranes. In conclusion, MALDI imaging is a powerful technique for polymer membrane analysis, regarding not only detection and identification of polymers but also localization and distribution in membrane surfaces.
Assuntos
Membranas Artificiais , Polímeros/química , Povidona/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sulfonas/química , Imagem Molecular , Propriedades de SuperfícieRESUMO
A model describing the binding of biological signaling proteins to highly charged polymer networks is presented. The networks are formed by polyelectrolyte chains for which the distance between two charges at the chain is smaller than the Bjerrum length. Counterion condensation on such highly charged chains immobilizes a part of the counterions. The Donnan-equilibrium between the polymer network and the aqueous solution with salt concentration c s b $c_s^b$ is used to calculate the salt concentration of the co- and counterions c s g $c_s^g$ entering the network. Two factors are decisive: i) The electrostatic interaction between the network and the protein is given by the Donnan-potential of the network and the net charge of the protein. In addition to this leading term, a second term describes the change in the Born-energy of the proteins when entering the network. ii) The interaction of the protein with the highly charged chains within the network is governed by counterion release: Patches of positive charge at the protein become multivalent counterions of the polyelectrolyte chains thus releasing a concomitant number of condensed counterions. The model compares favorably to experimental data obtained on a set of biohybrid polymer networks composed of crosslinked glycosaminoglycan chains that interact with a mixture of key signaling proteins.
Assuntos
Eletrólitos , Polímeros , Polieletrólitos , Citocinas , TermodinâmicaRESUMO
Adipose tissue-derived stem cells (ASCs) have been shown to assist regenerative processes after spinal cord injury (SCI) through their secretome, which promotes several regenerative mechanisms, such as inducing axonal growth, reducing inflammation, promoting cell survival, and vascular remodeling, thus ultimately leading to functional recovery. However, while systemic delivery (e.g., i.v. [intravenous]) may cause off-target effects in different organs, the local administration has low efficiency due to fast clearance by body fluids. Herein, a delivery system for human ASCs secretome based on a hydrogel formed of star-shaped poly(ethylene glycol) (starPEG) and the glycosaminoglycan heparin (Hep) that is suitable to continuously release pro-regenerative signaling mediators such as interleukin (IL)-4, IL-6, brain-derived neurotrophic factor, glial-cell neurotrophic factor, and beta-nerve growth factor over 10 days, is reported. The released secretome is shown to induce differentiation of human neural progenitor cells and neurite outgrowth in organotypic spinal cord slices. In a complete transection SCI rat model, the secretome-loaded hydrogel significantly improves motor function by reducing the percentage of ameboid microglia and systemically elevates levels of anti-inflammatory cytokines. Delivery of ASC-derived secretome from starPEG-Hep hydrogels may therefore offer unprecedented options for regenerative therapy of SCI.
Assuntos
Células-Tronco Neurais , Traumatismos da Medula Espinal , Ratos , Humanos , Animais , Glicosaminoglicanos , Preparações de Ação Retardada , Secretoma , Traumatismos da Medula Espinal/tratamento farmacológico , Heparina , Células-Tronco Neurais/metabolismo , Medula Espinal , Tecido Adiposo , Hidrogéis , Polietilenoglicóis/metabolismoRESUMO
Biohybrid hydrogels combining electrically neutral synthetic polymers and highly anionic glycosaminoglycans (GAGs) offer exciting options for regenerative therapies as they allow for the electrostatic conjugation of various growth factors. Unraveling details of ionization and structure within such networks defines an important analytical challenge that requires the extension of current methodologies. Here, we present a mean-field approach to quantify the density of ionizable groups, GAG concentration, and cross-linking degree of such hydrogels based on experimental data from microslit electrokinetics and ellipsometry. An exemplary poly(ethylene glycol)-heparin system was analyzed to demonstrate how electrostatic fingerprints of hydrogels obtained by the introduced strategy can sensitively display composition and structure of the polymer networks.
Assuntos
Hidrogéis/química , Eletricidade Estática , Glicosaminoglicanos/química , Heparina/química , Cinética , Polietilenoglicóis/químicaRESUMO
Macroporous scaffolds with adaptable mechanical and biomolecular properties can be instrumental in enabling cell-based therapies. To meet these requirements, a cryostructuration method was adapted to prepare spongy hydrogels based on chemically cross-linked star-shaped poly(ethylene glycol) (starPEG) and heparin. Subzero temperature treatment of the gel forming reaction mixtures and subsequent lyophilization of the incompletely frozen gels resulted in macroporous biohybrid cryogels showing rapid swelling, porosity of up to 92% with interconnected large pores (30-180 µm), low bulk stiffness, and high mechanical stability upon compression. The applicability of the cryogel scaffolds was investigated using human umbilical vein endothelial cells. Cell attachment and three-dimensional spreading resulted in evenly distributed viable cells within the macroporous starPEG-heparin materials, demonstrating the significant translational potential of the developed three-dimensional cell carriers.
Assuntos
Criogéis/síntese química , Heparina/química , Polietilenoglicóis/química , Alicerces Teciduais/química , Adsorção , Algoritmos , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Força Compressiva , Reagentes de Ligações Cruzadas/química , Criogéis/química , Módulo de Elasticidade , Etildimetilaminopropil Carbodi-Imida/química , Células Endoteliais da Veia Umbilical Humana/fisiologia , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Conformação Molecular , Permeabilidade , Porosidade , Água/químicaRESUMO
Excessive inflammation often impedes the healing of chronic wounds. Scavenging of chemokines by multiarmed poly(ethylene glycol)-glycosaminoglycan (starPEG-GAG) hydrogels has recently been shown to support regeneration in a diabetic mouse chronic skin wound model. Herein, a textile-starPEG-GAG composite wound contact layer (WCL) capable of selectively sequestering pro-inflammatory chemokines is reported. Systematic variation of the local and integral charge densities of the starPEG-GAG hydrogel component allows for tailoring its affinity profile for biomolecular signals of the wound milieu. The composite WCL is subsequently tested in a large animal (porcine) model of human wound healing disorders. Dampening excessive inflammatory signals without affecting the levels of pro-regenerative growth factors, the starPEG-GAG hydrogel-based WCL treatment induced healing with increased granulation tissue formation, angiogenesis, and deposition of connective tissue (collagen fibers). Thus, this biomaterials technology expands the scope of a new anti-inflammatory therapy toward clinical use.
Assuntos
Materiais Biocompatíveis/farmacologia , Quimiocinas/metabolismo , Tecido de Granulação/metabolismo , Hidrogéis/farmacologia , Inflamação/prevenção & controle , Cicatrização , Animais , Modelos Animais de Doenças , Humanos , Camundongos , SuínosRESUMO
Poly(ethylene glycol) (PEG)-glycosaminoglycan (GAG) hydrogel networks are established as very versatile biomaterials. Herein, the synthetic gel component of the biohybrid materials is systematically varied by combining different poly(2-alkyl-2-oxazolines) (POx) with heparin applying a Michael-type addition crosslinking scheme: POx of gradated hydrophilicity and temperature-responsiveness provides polymer networks of distinctly different stiffness and swelling. Adjusting the mechanical properties and the GAG concentration of the gels to similar values allows for modulating the release of GAG-binding growth factors (VEGF165 and PDGF-BB) by the choice of the POx and its temperature-dependent conformation. Adsorption of fibronectin, growth of fibroblasts, and bacterial adhesion scale with the hydrophobicity of the gel-incorporated POx. In vitro hemocompatibility tests with freshly drawn human whole blood show advantages of POx-based gels compared to the PEG-based reference materials. Biohybrid POx hydrogels can therefore enable biomedical technologies requiring GAG-based materials with customized and switchable physicochemical characteristics.
Assuntos
Heparina , Hidrogéis , Materiais Biocompatíveis , Glicosaminoglicanos , Humanos , PolietilenoglicóisRESUMO
Precision surface engineering is key to advanced biomaterials. A new platform of PEGylated styrene-maleic acid copolymers for adsorptive surface biofunctionalization is reported. Balanced amphiphilicity renders the copolymers water-soluble but strongly affine for surfaces. Fine-tuning of their molecular architecture provides control over adsorptive anchorage onto specific materials-which is why they are referred to as "anchor polymers" (APs)-and over structural characteristics of the adsorbed layers. Conjugatable with an array of bioactives-including cytokine-complexing glycosaminoglycans, cell-adhesion-mediating peptides and antimicrobials-APs can be applied to customize materials for demanding biotechnologies in uniquely versatile, simple, and robust ways. Moreover, homo- and heterodisplacement of adsorbed APs provide unprecedented means of in situ alteration and renewal of the functionalized surfaces. The related options are exemplified with proof-of-concept experiments of controlled bacterial adhesion, human umbilical vein endothelial cell, and induced pluripotent cell growth on AP-functionalized surfaces.
Assuntos
Materiais Biocompatíveis/química , Polímeros/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Citocinas/química , Glicosaminoglicanos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Maleatos/química , Oligopeptídeos/química , Polietilenoglicóis/química , Polímeros/farmacologia , Estireno/química , Propriedades de SuperfícieRESUMO
Glycosaminoglycan (GAG)-based, biohybrid hydrogels offering far-reaching control over their physical and biomolecular signaling properties have been successfully used in various cell and tissue culture applications. To explore the suitability of the materials for in vivo use, we herein studied the host reaction to in situ-assembling star(PEG)-GAG hydrogel variants upon subcutaneous implantation in immunocompetent C57BL/6J mice for up to 28 days. Specifically, we investigated the immune reaction and the angiogenic response to hydrogels with systematically varied cytokine functionalizations, physical network (and mechanical) properties, cell adhesiveness, and enzymatic degradability. The GAG-based hydrogel elicited only minor foreign body reaction with low immune cell infiltration and collagen deposition compared to similarly implanted medical grade silicone. Adjusting of the physical properties, biofunctionalization, and degradability allowed to program the host response from nearly no degradation and infiltration to fast integration of the gel scaffolds into the tissue within days. The results demonstrate that foreign body reactions and starPEG-GAG hydrogel tissue integration can be effectively controlled by defined adjustments of the hydrogel system, suggesting the in situ-assembling materials as safe and effective for in vivo tissue engineering applications.
Assuntos
Glicosaminoglicanos , Hidrogéis , Animais , Colágeno , Camundongos , Camundongos Endogâmicos C57BL , Polietilenoglicóis , Engenharia TecidualRESUMO
Materials capable of directing cell fate by providing spatially-graded mechanical and biomolecular cues are critically important in the reconstitution of living matter. Herein, we report a multi-component inkjet bioprinting method that allows for spatially varying composition and network properties in cell-instructive glycosaminoglycan (GAG)-based biohybrid and pure poly(ethylene glycol) hydrogels with unprecedented (50 µm) resolution. The principle relies on the covalent crosslinking of different polymeric precursors through a very rapid bio-orthogonal Michael type addition scheme adjusted in ways to occur during the fusion of bio-ink droplets prior to and upon contact with the target. Exemplary data show that chemotactic molecular gradients produced by this approach within printed GAG-gels of defined zonal architecture can effectively direct migratory activity and morphogenesis of embedded human bone-marrow derived mesenchymal stem cells. The introduced methodology is expected to enable a new, holistic level of control over reductionistic tissue and organoid models.
Assuntos
Bioimpressão/métodos , Hidrogéis/química , Becaplermina/farmacologia , Movimento Celular , Módulo de Elasticidade , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Polietilenoglicóis/química , Impressão Tridimensional , Alicerces Teciduais/químicaRESUMO
Multiphasic in vitro models with cross-scale heterogeneity in matrix properties and/or cellular composition can reflect the structural and compositional complexity of living tissues more faithfully, thereby creating new options for pathobiology and drug development studies. Herein, a new class of tunable microgel-in-gel materials is reported that build on a versatile platform of multifunctional poly(ethylene glycol)-heparin gel types and integrates monodisperse, cell-laden microgels within cell-laden bulk hydrogel matrices. A novel microfluidic approach was developed to enable the high-throughput fabrication of microgels of in situ adjustable diameters, stiffness, degradability and biomolecular functionalization. By choosing structure and composition of the microgel and the bulk gel compartments independently, our microgel-in-gel arrangements provide cross-scale control over tissue-mimetic features and pave the way for culture systems with designed mesoenvironmental characteristics. The potentialities of the introduced approach are exemplarily shown by creating a reductionistic in vitro model of vascularized prostate cancer tissue.
Assuntos
Microgéis/química , Neoplasias da Próstata/patologia , Engenharia Tecidual/métodos , Humanos , Hidrogéis , Masculino , Técnicas Analíticas Microfluídicas/instrumentação , Modelos BiológicosRESUMO
The mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using 2D substrates, much less is known about it in a physiologically more relevant 3D context. Here it is demonstrated that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa build up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy reveals that tumor spheroid stiffness increases with hydrogel stiffness. Also, constituent cell stiffness increases in a Rho associated kinase (ROCK)- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase, and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition not only reverses cell stiffening upon culture in stiff hydrogels but also increases tumor spheroid growth. Taken together, a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment is revealed here. Thus, the findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica , Hidrogéis/farmacologia , Mecanotransdução Celular/genética , Esferoides Celulares/efeitos dos fármacos , Quinases Associadas a rho/genética , Resinas Acrílicas/química , Resinas Acrílicas/farmacologia , Actinas/genética , Actinas/metabolismo , Fenômenos Biomecânicos , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Heparina/química , Heparina/farmacologia , Humanos , Hidrogéis/síntese química , Células MCF-7 , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Análise de Célula Única/métodos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Quinases Associadas a rho/metabolismoRESUMO
Repaired cartilage tissue lacks the typical zonal structure of healthy native cartilage needed for appropriate function. Current grafts for treatment of full thickness cartilage defects focus primarily on a nonzonal design and this may be a reason why inferior nonzonal regeneration tissue developed in vivo. No biomaterial-based solutions have been developed so far to induce a proper zonal architecture into a non-mineralized and a calcified cartilage layer. The objective was to grow bizonal cartilage with a calcified cartilage bottom zone wherein main tissue development is occurring in vivo. We hypothesized that starPEG/heparin-hydrogel owing to the glycosaminoglycan heparin contained as a building-block would prevent mineralization of the upper cartilage zone and be beneficial in inhibiting long-term progression of calcified cartilage into bone. MSCs were pre-cultured as self-assembling non-mineralized cell discs before a chondrocyte-seeded fibrin- or starPEG/heparin-hydrogel layer was cast on top directly before ectopic implantation. Bizonal cartilage with a calcified bottom-layer developed in vivo showing stronger mineralization compared to in vitro samples, but the hydrogel strongly determined outcome. Zonal fibrin-constructs lost volume and allowed non-organized expansion of collagen type X, ALP-activity and mineralization from the bottom-layer into upper regions, whereas zonal starPEG/heparin-constructs were of stable architecture. While non-zonal MSCs-derived discs formed bone over 12 weeks, the starPEG/heparin-chondrocyte layer prevented further progression of calcified cartilage into bone tissue. Conclusively, starPEG/heparin-hydrogel-controlled and cell-type mediated spatiotemporal regulation allowed in vivo growth of bizonal cartilage with a stable calcified cartilage layer. Altogether our work is an important milestone encouraging direct in vivo growth of organized cartilage after biofabrication.
Assuntos
Cartilagem Articular/crescimento & desenvolvimento , Condrócitos/citologia , Heparina/química , Hidrogéis/química , Polietilenoglicóis/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Calcificação Fisiológica , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Proliferação de Células , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo X/metabolismo , Glicosaminoglicanos/química , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Suínos , Porco Miniatura , Engenharia Tecidual/instrumentaçãoRESUMO
Cell-based tissue engineering is a promising approach for treating cartilage lesions, but available strategies still provide a distinct composition of the extracellular matrix and an inferior mechanical property compared to native cartilage. To achieve fully functional tissue replacement more rationally designed biomaterials may be needed, introducing bioactive molecules which modulate cell behavior and guide tissue regeneration. This study aimed at exploring the impact of cell-instructive, adhesion-binding (GCWGGRGDSP called RGD) and collagen-binding (CKLER/CWYRGRL) peptides, incorporated in a tunable, matrixmetalloprotease (MMP)-responsive multi-arm poly(ethylene glycol) (starPEG)/heparin hydrogel on cartilage regeneration parameters in vitro and in vivo. MMP-responsive-starPEG-conjugates with cysteine termini and heparin-maleimide, optionally pre-functionalized with RGD, CKLER, CWYRGRL or control peptides, were cross-linked by Michael type addition to embed and grow mesenchymal stromal cells (MSC) or chondrocytes. While starPEG/heparin-hydrogel strongly supported chondrogenesis of MSC according to COL2A1, BGN and ACAN induction, MMP-degradability enhanced cell viability and proliferation. RGD-modification of the gels promoted cell spreading with intense cell network formation without negative effects on chondrogenesis. However, CKLER and CWYRGRL were unable to enhance the collagen content of constructs. RGD-modification allowed more even collagen type II distribution by chondrocytes throughout the MMP-responsive constructs, especially in vivo. Collectively, peptide-instruction via heparin-enriched MMP-degradable starPEG allowed adjustment of self-renewal, cell morphology and cartilage matrix distribution in order to guide MSC and chondrocyte-based cartilage regeneration towards an improved outcome. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Cartilagem Articular/metabolismo , Forma Celular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Heparina/farmacologia , Hidrogéis/farmacologia , Mitógenos/farmacologia , Peptídeos/farmacologia , Polietilenoglicóis/farmacologia , Animais , Cartilagem Articular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Humanos , Camundongos SCID , Oligopeptídeos/farmacologia , Reologia , SuínosRESUMO
Distinct micro-environmental properties have been reported to be essential for maintenance of neural precursor cells (NPCs) within the adult brain. Due to high complexity and technical limitations, the natural niche can barely be studied systematically in vivo. By reconstituting selected environmental properties (adhesiveness, proteolytic degradability, and elasticity) in geldrop cultures, we show that NPCs can be maintained stably at high density over an extended period of time (up to 8 days). In both conventional systems, neurospheres and monolayer cultures, they would expand and (in the case of neurospheres) differentiate rapidly. Further, we report a critical dualism between matrix adhesiveness and degradability. Only if both features are functional NPCs stay proliferative. Lastly, Rho-associated protein kinase was identified as part of a pivotal intracellular signaling cascade controlling cell morphology in response to environmental cues inside geldrop cultures. Our findings demonstrate that simple manipulations of the microenvironment in vitro result in an important preservation of stemness features in the cultured precursor cells.
Assuntos
Técnicas de Cultura de Células/métodos , Hidrogéis/farmacologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Glicosaminoglicanos/química , Hidrogéis/química , Camundongos , Células-Tronco Neurais/metabolismo , Polietilenoglicóis/química , Quinases Associadas a rho/metabolismoRESUMO
Charging and swelling of cellulose in aqueous environments are of highest interest with respect to the performance of cellulose based products and applications. To unravel the interplay between ionization and structural features of the biopolymer hydrogel we compared non-crosslinked and crosslinked cellulose thin films based on a determination of the Donnan potential [S.S. Dukhin, R. Zimmermann, C. Werner, J. Colloid Interface Sci. 274 (2004) 309] from microslit electrokinetic (streaming potential/streaming current) experiments and layer thicknesses from ellipsometry in aqueous electrolyte solutions. The pH dependence of the Donnan potential, reflecting the ionization of carboxylic acid groups within the cellulose films, was found to be significantly different from the related trend of the streaming current which reflects the characteristics of the topmost surface of the layers: While carboxylic acid groups on the surface of the films dissociate as isolated functionalities, the electrostatic interactions of ionized groups within the cellulose layers cause an incomplete dissociation (pK shift) of the carboxylic acid and a layer expansion (swelling) in the alkaline pH range. The system was found to restrict its volume charge density even after structural restrictions (crosslinking) of the layer and at lower ionic strength of the solutions through a further decrease of the degree of dissociation of the carboxylic acid functions. These findings were attributed to the local accumulation of the carboxylic acid groups caused by preferential oxidation of the amorphous regions of the cellulose and to the ordered water structure within the layer.