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Cell migration is essential in numerous living processes, including embryonic development, wound healing, immune responses, and cancer metastasis. From individual cells to collectively migrating epithelial sheets, the locomotion of cells is tightly regulated by multiple structural, chemical, and biological factors. However, the high complexity of this process limits the understanding of the influence of each factor. Recent advances in materials science, tissue engineering, and microtechnology have expanded the toolbox and allowed the development of biomimetic in vitro assays to investigate the mechanisms of cell migration. Particularly, three-dimensional (3D) hydrogels have demonstrated a superior ability to mimic the extracellular environment. They are therefore well suited to studying cell migration in a physiologically relevant and more straightforward manner than in vivo approaches. A myriad of synthetic and naturally derived hydrogels with heterogeneous characteristics and functional properties have been reported. The extensive portfolio of available hydrogels with different mechanical and biological properties can trigger distinct biological responses in cells affecting their locomotion dynamics in 3D. Herein, we describe the most relevant hydrogels and their associated physico-chemical characteristics typically employed to study cell migration, including established cell migration assays and tracking methods. We aim to give the reader insight into existing literature and practical details necessary for performing cell migration studies in 3D environments.
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Autologous fat grafting is hampered by unpredictable outcomes due to high tissue resorption. Hydrogels based on enzymatically pretreated tunicate nanocellulose (ETC) and alginate (ALG) are biocompatible, safe, and present physiochemical properties capable of promoting cell survival. Here, we compared in situ and ex situ crosslinking of ETC/ALG hydrogels combined with lipoaspirate human adipose tissue (LAT) to generate an injectable formulation capable of retaining dimensional stability in vivo. We performed in situ crosslinking using two different approaches; inducing Ca2+ release from CaCO3 microparticles (CMPs) and physiologically available Ca2+ in vivo. Additionally, we generated ex situ-crosslinked, 3D-bioprinted hydrogel-fat grafts. We found that in vitro optimization generated a CMP-crosslinking system with comparable stiffness to ex situ-crosslinked gels. Comparison of outcomes following in vivo injection of each respective crosslinked hydrogel revealed that after 30 days, in situ crosslinking generated fat grafts with less shape retention than 3D-bioprinted constructs that had undergone ex situ crosslinking. However, CMP addition improved fat-cell distribution and cell survival relative to grafts dependent on physiological Ca2+ alone. These findings suggested that in situ crosslinking using CMP might promote the dimensional stability of injectable fat-hydrogel grafts, although 3D bioprinting with ex situ crosslinking more effectively ensured proper shape stability in vivo.
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BACKGROUND: The main hurdles to the widespread use of islet transplantation for the treatment of type 1 diabetes continue to be the insufficient number of appropriate donors and the need for immunosuppression. Microencapsulation has been proposed as a means to protect transplanted islets from the host's immune system. METHODS: This study investigated the function of human pancreatic islets encapsulated in Ca(2+) /Ba(2+) -alginate microbeads intraperitoneally transplanted in diabetic Balb/c mice. RESULTS: All mice transplanted with encapsulated human islets (n = 29), at a quantity of 3000 islet equivalent (IEQ), achieved normoglycemia 1 day after transplantation and retained normoglycemia for extended periods of time (mean graft survival 134 ± 17 days). In comparison, diabetic Balb/c mice transplanted with an equal amount of non-encapsulated human islets rejected the islets within 2 to 7 days after transplantation (n = 5). Microbeads retrieved after 232 days (n = 3) were found with little to no fibrotic overgrowth and contained viable insulin-positive islets. Immunofluorescent staining on the retrieved microbeads showed F4/80-positive macrophages and alpha smooth muscle actin-positive fibroblasts but no CD3-positive T lymphocytes. CONCLUSIONS: The Ca(2+) /Ba(2+) -alginate microbeads can protect human islets from xenogeneic rejection in immunocompetent mice without immunosuppression. However, grafts ultimately failed likely secondary to a macrophage-mediated foreign body reaction.
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Composição de Medicamentos/métodos , Sobrevivência de Enxerto/fisiologia , Ilhotas Pancreáticas/citologia , Microesferas , Alginatos/metabolismo , Animais , Bário/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Tipo 1/terapia , Ácido Glucurônico/metabolismo , Sobrevivência de Enxerto/imunologia , Ácidos Hexurônicos/metabolismo , Humanos , Terapia de Imunossupressão/métodos , Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Alginate hydrogels have applications in biomedicine, ranging from delivery of cells and growth factors to wound management aids. However, they are mechanically soft and have shown little potential for the use in bone tissue engineering. Here, the viscoelastic properties of alginate hydrogel beads mineralized with calcium phosphate, both by a counter-diffusion (CD) and an enzymatic approach, are characterized by a micro-manipulation technique and mathematical modeling. Fabricated hydrogel materials have low mineral content (below 3 % of the total hydrogel mass, which corresponds to mineral content of up to 60 % of the dry mass) and low dry mass content (<5 %). For all samples compression and hold (relaxation after compression) data was collected and analyzed. The apparent Young's modulus of the mineralized beads was estimated by the Hertz model (compression data) and was shown to increase up to threefold upon mineralization. The enzymatically mineralized beads showed higher apparent Young's modulus compared to the ones mineralized by CD, even though the mineral content of the former was lower. Full compression-relaxation force-time profiles were analyzed using viscoelastic model. From this analysis, infinite and instantaneous Young's moduli were determined. Similarly, enzymatic mineralized beads, showed higher instantaneous and infinite Young's modulus, even if the degree of mineralization is lower then that achieved for CD method. This leads to the conclusion that both the degree of mineralization and the spatial distribution of mineral are important for the mechanical performance of the composite beads, which is in analogy to highly structured mineralized tissues found in many organisms.
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Alginatos/química , Elasticidade , Hidrogéis , Viscosidade , DifusãoRESUMO
This work explores the largely unknown surface microstructure and elastic modulus of soft calcium-alginate hydrogels (E = 100-4500 Pa) in their hydrated state by atomic force microscopy (AFM) in quantitative imaging mode. Alginate concentration influenced the surface topography with surface roughness measured to be 101 ± 6 nm and 57 ± 1 nm for 0.5 and 2.0% (w/v) alginate, respectively. The calculated range of pore sizes increased with decreasing alginate concentration, with radii smaller than 360 nm, 570 nm and 1230 nm for 2.0%, 1.0% and 0.5% alginate, respectively. Small changes in calcium concentration (from 20 to 25 mM, 1.5% alginate) did not induce changes in surface microstructure, although it increased the elastic modulus mean values and distribution. Introducing oxidized or peptide-grafted alginate in the gels resulted in rougher surfaces, larger pore sizes and lower elasticity than the respective hydrogels with no modified alginate.
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Engineered block polysaccharides is a relatively new class of biomacromolecules consisting of chemical assembly of separate block structures at the chain termini. In contrast to conventional, laterally substituted polysaccharide derivatives, the block arrangement allows for much higher preservation of inherent chain properties such as biodegradability and stimuli-responsive self-assembly, while at the same time inducing new macromolecular properties. Abundant, carbon neutral, and even recalcitrant biomass is an excellent source of blocks, opening for numerous new uses of biomass for a wide range of novel biomaterials. Among a limited range of methodologies available for block conjugation, bifunctional linkers allowing for oxyamine and hydrazide 'click' reactions have recently proven useful additions to the repertoire. This article focuses the chemistry and kinetics of these reactions. It also presents some new data with the aim to provide useful protocols and methods for general use towards new block polysaccharides.
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Aminas/farmacologia , Hidrazonas/farmacologia , Polissacarídeos/antagonistas & inibidores , Aminas/química , Configuração de Carboidratos , Química Click , Hidrazonas/químicaRESUMO
Cell encapsulation in alginate microbeads is a promising approach to provide immune isolation in cell therapy without immunosuppression. However, the efficacy is hampered by pericapsular fibrotic overgrowth (PFO), causing encapsulated cells to lose function. Stability of the microbeads is important to maintain immune isolation in the long-term. Here, we report alginate microbeads with minimal PFO in immunocompetent C57BL/6JRj mice. Microbead formulations included either alginate with an intermediate (47 %) guluronate (G) content (IntG) or sulfated alginate (SA), gelled in Ca2+/Ba2+ or Sr2+. A screening panel of eleven microbead formulations were evaluated for PFO, yielding multiple promising microbeads. Two candidate formulations were evaluated for 112 days in vivo, exhibiting maintained stability and minimal PFO. Microbeads investigated in a human whole blood assay revealed low cytokine and complement responses, while SA microbeads activated coagulation. Protein deposition on microbeads explanted from mice investigated by confocal laser scanning microscopy (CLSM) showed minimal deposition of complement C3. Fibrinogen was positively associated with PFO, with a high deposition on microbeads of high G (68 %) alginate compared to IntG and SA microbeads. Overall, stable microbeads containing IntG or SA may serve in long-term therapeutic applications of cell encapsulation. STATEMENT OF SIGNIFICANCE: Alginate-based hydrogels in the format of micrometer size beads is a promising approach for the immunoisolation of cells in cell therapy. Clinical trials in type 1 diabetes have so far had limited success due to fibrotic responses that hinder the diffusion of nutrients and oxygen to the encapsulated cells, resulting in graft failure. In this study, minimal fibrotic response towards micrometer size alginate beads was achieved by chemical modification of alginate with sulfate groups. Also, the use of alginate with intermediate guluronic acid content resulted in minimally fibrotic microbeads. Fibrinogen deposition was revealed to be a good indicator of fibrosis. This study points to both new microsphere developments and novel insight in the mechanisms behind the fibrotic responses.
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Alginatos , Sulfatos , Alginatos/farmacologia , Animais , Fibrose , Ácido Glucurônico , Ácidos Hexurônicos , Camundongos , Camundongos Endogâmicos C57BL , MicroesferasRESUMO
Protein adsorption to biomaterial surfaces is considered a determining factor for the host response. Here we detail the protein adsorption profiles of alginate hydrogel microspheres relevant for cell therapy using mass spectrometry (MS)-based proteomics. The investigated microspheres include sulfated alginate (SA), high G alginate (HiG), and poly-l-lysine coated alginate (AP), which previously have been shown to exhibit different inflammatory and fibrotic responses. The biological significance was assessed in lepirudin-anticoagulated human whole blood (hWB) by functional analysis of the acute-phase responses (complement and coagulation). Proteomic profiling revealed distinct signatures for the microspheres, wherein Ingenuity Pathway Analysis identified complement and coagulation as the top enriched canonical pathways. The levels of complement and coagulation activators and inhibitors were distinctly different, which was reflected in the functional hWB analyses: SA was highly enriched with inhibitory factors of complement and coagulation (e.g. C1 inhibitor, factor H, antithrombin-III, heparin cofactor 2), other heparin-binding proteins and factors promoting fibrinolysis (factor XII, plasma kallikrein), conforming to an anti-inflammatory and anti-fibrotic profile. HiG enriched moderate levels of complement inhibitors, conforming to a low-inflammatory and pro-fibrotic profile. AP showed the most prominent enrichment of complement activators (e.g. C3, properdin, C-reactive protein) and low levels of inhibitors, conforming to a pro-inflammatory and highly pro-fibrotic profile. In conclusion, the extensive enrichment of inhibitory acute-phase proteins on SA could be a determining factor for its reduced host response. The interactions between the plasma proteins and hydrogel surfaces shown herein point to proteomics as an important supplement to existing in vitro and in vivo methods for designing biocompatible alginate-based hydrogels.
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BACKGROUND: The anatomical spatial distribution of microencapsulated islets transplanted into the peritoneal cavity of large animals remains a relatively unexplored area of study. In this study, we developed a new implantation approach using laparoscopy in order to avoid microcapsule amalgamation. This approach constitutes a clinically relevant method, which can be used to evaluate the distribution and in vivo biocompatibility of various types of transplanted microcapsules in the future. MATERIALS AND METHODS: Two healthy baboons were implanted intraperitoneally with microencapsulated islets through mini-laparotomy and observed at 76 d after implantation. Nine baboons underwent laparoscopic implantation of approximately 80,000 empty microcapsules. Microcapsule distribution was observed by laparoscopic camera during and after implantation at 1, 2, and 4 wk. At each time point, microcapsules were retrieved and evaluated with brightfield microscopy and histologic analysis. RESULTS: Mini-laparotomic implantation resulted in microcapusle aggregation in both baboons. In contrast, laparoscopic implantation resulted in even distribution of microcapsules throughout the peritoneum without sedimentation to the Douglas space in all animals. In eight out of nine animals, retrieved microcapsules were evenly distributed in the peritoneal cavity and presented with no pericapsular overgrowth and easily washed out during laparoscopic procedure. The one exception was attributed to microcapsule contamination with blood from the abdominal wall following trocar insertion. CONCLUSIONS: Laparoscopic implantation of microcapsules in non-human primates can be successfully performed and prevents microcapsule aggregation. Given the current widespread clinical application of laparoscopy, we propose that this presented laparoscopy technique could be applied in future clinical trials of microencapsulated islet transplantation.
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Cápsulas , Transplante das Ilhotas Pancreáticas/métodos , Laparoscopia/métodos , Cavidade Peritoneal/cirurgia , Animais , Feminino , Masculino , Modelos Animais , Papio anubis , Fatores de Tempo , Resultado do TratamentoRESUMO
Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the mechanisms governing the developmental processes is limited, impeding the quality of differentiation protocols. Generating insulin-expressing cells in vitro is no exception, with the guided series of differentiation events producing heterogeneous cell populations that display mixed pancreatic islet phenotypes and immaturity. The achievement of terminal differentiation ultimately requires the in vivo transplantation of, usually, encapsulated cells. Here we show the impact of cell confinement on the pancreatic islet signature during the guided differentiation of alginate encapsulated human induced pluripotent stem cells (hiPSCs). Our results show that encapsulation improves differentiation by significantly reshaping the proteome landscape of the cells towards an islet-like signature. Pathway analysis is suggestive of integrins transducing the encapsulation effect into intracellular signalling cascades promoting differentiation. These analyses provide a molecular framework for understanding the confinement effects on hiPSCs differentiation while confirming its importance for this process.
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Alginatos/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Integrinas/metabolismo , Ilhotas Pancreáticas/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Fenótipo , Transdução de SinaisRESUMO
Controlling the rate of release of molecules from a hydrogel is of high interest for various drug delivery systems and medical devices. A strategy to alter the release profiles of soluble and poorly soluble active ingredients from hydrogels can be to combine the hydrogel forming ability of alginate with the inclusion forming ability of cyclodextrins (CyD). Here, ß-CyD was grafted to alginate in a three-step synthesis using periodate oxidation, reductive amination and copper(I)-catalyzed azide-alkyne cycloaddition. A grafting degree of 4.7% mol ß-CyD/mol sugar residues was obtained. The grafting degree was controlled by varying the reaction parameters where the amount of linker used in reductive amination was especially influential. Ca-alginate gel beads grafted with ß-CyD showed increased uptake of the model molecule methyl orange. Release experiments showed that the grafted material had a prolonged release of methyl orange and an increased total amount of released methyl orange. These results show that the ß-CyD grafted alginate is still able to form a hydrogel while the grafted cyclodextrins retain their ability to form inclusion complex with methyl orange. Further testing should be done with this system to investigate capability for drug delivery applications.
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Alginatos/química , Ciclodextrinas/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Hidrogéis/química , Compostos Azo/química , Química Click , Peso Molecular , SolubilidadeRESUMO
In this work, the mechanical properties and stability of alginate hydrogels containing functionalized alginates (peptide and ß-cyclodextrin) were studied. There is an increasing interest in the modification of alginates to add functions such as cell attachment and increased solubility of hydrophobic drugs, for better performance in tissue engineering and drug release, respectively. Functionalization was achieved in this study via periodate oxidation followed by reductive amination, previously shown to give a high and controllable degree of substitution. Young's modulus and the stress at rupture of the hydrogels were in general lowered when exchanging native alginate with the modified alginate. Still, the gel strength could be adjusted by the fraction of modified alginate in the mixed hydrogels as well as the degree of oxidation. No notable difference in deformation at rupture was observed while syneresis was influenced by the degree of oxidation and possibly by the nature and amount of the grafted molecules. The mixed hydrogels were less stable than hydrogels with only native alginate, and modified alginate was released from the hydrogels. Furthermore, the hydrogels in general rather disintegrated than swelled upon saline treatments.
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A new bioactive scaffold was prepared from a binary polysaccharide mixture composed of a polyanion (alginate) and a polycation (a lactose-modified chitosan, chitlac). Its potential use for articular chondrocytes encapsulation and cartilage reconstructive surgery applications has been studied. The hydrogel combines the ability of alginate to act as a 3D supporting structure with the capability of the second component (chitlac) to provide interactions with porcine articular chondrocytes. Physico-chemical characterization of the scaffold was accomplished by gel kinetics and compression measurements and demonstrated that alginate-chitlac mixture (AC-mixture) hydrogels exhibit better mechanical properties when compared with sole alginate hydrogels. Furthermore, biochemical and biological studies showed that these 3D scaffolds are able to maintain chondrocyte phenotype and particularly to significantly stimulate and promote chondrocyte growth and proliferation. In conclusion, the present study can be considered as a first step towards an engineered, biologically active scaffold for chondrocyte in vitro cultivation, expansion, and cell delivery.
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Alginatos/química , Materiais Biocompatíveis/química , Quitosana/química , Condrócitos/efeitos dos fármacos , Hidrogéis/química , Lactose/química , Animais , Biomarcadores/metabolismo , Cálcio/química , Cartilagem Articular/citologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/biossíntese , Colágeno/genética , Glicosaminoglicanos/biossíntese , Glicosaminoglicanos/genética , Cinética , Laminaria/química , Espectroscopia de Ressonância Magnética , Microscopia Confocal , RNA/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Reologia , SuínosRESUMO
BACKGROUND: A recent study from our laboratory documented significant improvements in post-transplant viability in an experimental model of intestinal transplantation when a novel, nutrient-rich preservation solution was used during cold storage. The current study investigated the relationship between energetic/oxidative stress responses and fundamental kinase signaling events during the period of organ storage. This relationship may be a key factor contributing to improved graft viability after storage in a nutrient-rich preservation solution. METHODS: Rat small intestine was harvested and flushed intraluminally with University of Wisconsin (UW) solution or an amino acid-rich (AA) solution as follows: Group 1, no luminal flush (clinical control); Group 2, luminal UW solution; Group 3, luminal AA solution. Energetics (ATP, total adenylates), oxidative stress (malondialdehyde), histology, and MAPK (P38, JNK, ERK)/AMPK/Caspase-3 were assessed throughout 12-hour cold storage. RESULTS: P38 and JNK were upregulated strongly in Group 2 after 1- and 12-hour storage. Group 3 exhibited a delayed activation and subsequent downregulation of these pre-apoptotic signals. Between 6 to 12 hours, a strong upregulation of ERK was observed in Group 3. AMPK downregulation correlated with a reduction in AMP/ATP ratio, ERK upregulation, and P38/JNK downregulation in Group 3. After 12-hour storage, histology indicated superior preservation of mucosal architecture in Group 3 tissues. CONCLUSIONS: A nutrient-rich preservation solution abrogates pre-apoptotic signaling (JNK and P38) and upregulates cytoprotective signals (ERK). Our data support the concept of a concerted effort facilitating cellular protection in response to ischemic stress.
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Apoptose , Citoproteção , Metabolismo Energético , Intestino Delgado , Estresse Oxidativo , Fosfotransferases/metabolismo , Preservação Biológica , Transdução de Sinais , Animais , Caspase 3/metabolismo , Isquemia Fria , Intestino Delgado/patologia , Masculino , Soluções para Preservação de Órgãos/farmacologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Transplantation of pancreatic islets in immune protective capsules holds the promise as a functional cure for type 1 diabetes, also about 40 years after the first proof of principal study. The concept is simple in using semipermeable capsules that allow the ingress of oxygen and nutrients, but limit the access of the immune system. Encapsulated human islets have been evaluated in four small clinical trials where the procedure has been evaluated as safe, but lacking long-term efficacy. Host reactions toward the biomaterials used in the capsules may be one parameter limiting the long-term function of the graft in humans. The present article briefly discusses important capsule properties such as stability, permeability and biocompatibility, as well as possible strategies to overcome current challenges. Also, recent progress in capsule development as well as the production of insulin-producing cells from human stem cells that gives promising perspectives for the transplantation of encapsulated insulin-producing tissue is briefly discussed. Stem Cells Translational Medicine 2017;6:1053-1058.
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Alginatos/química , Ilhotas Pancreáticas/citologia , Células-Tronco/citologia , Animais , Cápsulas , HumanosRESUMO
UNLABELLED: The modification of soft hydrogels with hard inorganic components is a method used to form composite materials with application in non-load-bearing bone tissue engineering. The inclusion of an inorganic component may provide mechanical enhancement, introduce osteoconductive or osteoinductive properties, or change other aspects of interactions between native or implanted cells and the material. A thorough understanding of the interactions between such components is needed to improve the rational design of such biomaterials. To achieve this goal, model systems which could allow study of the formation and transformation of mineral phases within a hydrogel network with a range of experimental methods and high spatial and time resolution are needed. Here, we report a detailed investigation of the formation and transformation process of calcium phosphate mineral within an alginate hydrogel matrix. A combination of optical microscopy, confocal Raman microspectroscopy and electron microscopy was used to investigate the spatial distribution, morphology and crystal phase of the calcium phosphate mineral, as well as to study transformation of the mineral phases during the hydrogel mineralization process and upon incubation in a simulated body fluid. It was found, that under the conditions used in this work, mineral initially formed as a metastable amorphous calcium phosphate phase (ACP). The ACP particles had a distinctive spherical morphology and transformed within minutes into brushite in the presence of brushite seed crystals or into octacalcium phosphate, when no seeds were present in the hydrogel matrix. Incubation of brushite-alginate composites in simulated body fluid resulted in formation of hydroxyapatite. The characterization strategy presented here allows for non-destructive, in situ observation of mineralization processes in optically transparent hydrogels with little to no sample preparation. STATEMENT OF SIGNIFICANCE: The precipitation and transformations of calcium phosphates (CaP) is a complex process, where both formation kinetics and the stability of different mineral phases control the outcome. This situation is even more complex if CaP is precipitated in a hydrogel matrix, where one can expect the organic matrix to modulate crystallization by introducing supersaturation gradients or changing the nucleation and growth kinetics of crystals. In this study we apply a range of characterization techniques to study the mineral formation and transformations of CaP within an alginate matrix with spatiotemporal resolution. It demonstrates how a detailed investigation of the mineral precipitation and transformations can aid in the future rational design of hydrogel-based materials for bone tissue engineering and studies of biomineralization processes.
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Alginatos/química , Fosfatos de Cálcio/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Análise Espaço-Temporal , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Concentração de Íons de Hidrogênio , Microscopia de Contraste de Fase , Imagem Óptica , Análise Espectral RamanRESUMO
Peptide coupled alginates obtained by chemical functionalization of alginates are commonly used as scaffold materials for cells in regenerative medicine and tissue engineering. We here present an alternative to the commonly used carbodiimide chemistry, using partial periodate oxidation followed by reductive amination. High and precise degrees of substitution were obtained with high reproducibility, and without formation of by-products. A protocol was established using l-Tyrosine methyl ester as a model compound and the non-toxic pic-BH3 as the reducing agent. DOSY was used to indirectly verify covalent binding and the structure of the product was further elucidated using NMR spectroscopy. The coupling efficiency was to some extent dependent on alginate composition, being most efficient on mannuronan. Three different bioactive peptide sequences (GRGDYP, GRGDSP and KHIFSDDSSE) were coupled to 8% periodate oxidized alginate resulting in degrees of substitution between 3.9 and 6.9%. Cell adhesion studies of mouse myoblasts (C2C12) and human dental stem cells (RP89) to gels containing various amounts of GRGDSP coupled alginate demonstrated the bioactivity of the material where RP89 cells needed higher peptide concentrations to adhere.
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Alginatos/química , Materiais Biocompatíveis/química , Oligopeptídeos/química , Células-Tronco/citologia , Aminação , Animais , Adesão Celular , Linhagem Celular , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Camundongos , Mioblastos/citologia , Oxirredução , Ácido Periódico/química , Tirosina/análogos & derivados , Tirosina/químicaRESUMO
UNLABELLED: Due to their large water content and structural similarities to the extracellular matrix, hydrogels are an attractive class of material in the tissue engineering field. Polymers capable of ionotropic gelation are of special interest due to their ability to form gels at mild conditions. In this study we have developed an experimental toolbox to measure the gelling kinetics of alginate upon crosslinking with calcium ions. A reaction-diffusion model for gelation has been used to describe the diffusion of calcium within the hydrogel and was shown to match experimental observations well. In particular, a single set of parameters was able to predict gelation kinetics over a wide range of gelling ion concentrations. The developed model was used to predict the gelling time for a number of geometries, including microspheres typically used for cell encapsulation. We also demonstrate that this toolbox can be used to spatiotemporally investigate the formation and evolution of mineral within the hydrogel network via correlative Raman microspectroscopy, confocal laser scanning microscopy and electron microscopy. STATEMENT OF SIGNIFICANCE: Hydrogels show great promise in cell-based tissue engineering, however new fabrication and modification methods are needed to realize the full potential of hydrogel based materials. The inclusion of an inorganic phase is one such approach and is known to affect both cell-material interactions and mechanical properties. This article describes the development of a correlative experimental approach where gel formation and mineralization has been investigated with spatial and temporal resolution by applying Raman microspectroscopy, optical and electron microscopy and a reaction-diffusion modeling scheme. Modeling allows us to predict gelling kinetics for other geometries and sizes than those investigated experimentally. Our experimental system enables non-destructive study of composite hydrogel systems relevant for, but not limited to, applications within bone tissue engineering.
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Alginatos/química , Hidrogéis/química , Análise Espaço-Temporal , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Concentração de Íons de Hidrogênio , Cinética , Microscopia , Minerais/química , Modelos Teóricos , Análise Espectral Raman , Fatores de TempoRESUMO
The effect of alginate-based scaffolds with added basement membrane proteins on the in vitro development of hESC-derived pancreatic progenitors was investigated. Cell clusters were encapsulated in scaffolds containing the basement membrane proteins collagen IV, laminin, fibronectin, or extracellular matrix-derived peptides, and maintained in culture for up to 46 days. The cells remained viable throughout the experiment with no signs of central necrosis. Whereas nonencapsulated cells aggregated into larger clusters, some of which showed signs of morphological changes and tissue organization, the alginate matrix stabilized the cluster size and displayed more homogeneous cell morphologies, allowing culture for long periods of time. For all conditions tested, a stable or declining expression of insulin and PDX1 and an increase in glucagon and somatostatin over time indicated a progressive reduction in beta cell-related gene expression. Alginate scaffolds can provide a chemically defined, xeno-free and easily scalable alternative for culture of pancreatic progenitors. Although no increase in insulin and PDX1 gene expression after alginate-immobilized cell culture was seen in this study, further optimization of the matrix physicochemical and biological properties and of the medium composition may still be a relevant strategy to promote the stabilization or maturation of stem cell-derived beta cells.
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Alginatos/química , Células-Tronco Embrionárias Humanas/citologia , Células Secretoras de Insulina/citologia , Células-Tronco/citologia , Alicerces Teciduais/química , Técnicas de Cultura de Células , Linhagem Celular , Expressão Gênica , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Proteínas de Homeodomínio/genética , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Células-Tronco/metabolismo , Transativadores/genéticaRESUMO
Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.