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The crustacean processing industry produces large quantities of waste by-products (up to 70%). Such wastes could be used as raw materials for producing chitosan, a polysaccharide with a unique set of biochemical properties. However, the preparation methods and the long-term stability of chitosan-based products limit their application in biomedicine. In this study, different scale structures, such as aggregates, photo-crosslinked films, and 3D scaffolds based on mechanochemically-modified chitosan derivatives, were successfully formed. Dynamic light scattering revealed that aggregation of chitosan derivatives becomes more pronounced with an increase in the number of hydrophobic substituents. Although the results of the mechanical testing revealed that the plasticity of photo-crosslinked films was 5â»8% higher than that for the initial chitosan films, their tensile strength remained unchanged. Different types of polymer scaffolds, such as flexible and porous ones, were developed by laser stereolithography. In vivo studies of the formed structures showed no dystrophic and necrobiotic changes, which proves their biocompatibility. Moreover, the wavelet analysis was used to show that the areas of chitosan film degradation were periodic. Comparing the results of the wavelet analysis and X-ray diffraction data, we have concluded that degradation occurs within less ordered amorphous regions in the polymer bulk.
Assuntos
Materiais Biocompatíveis , Quitosana/química , Engenharia Tecidual , Animais , Configuração de Carboidratos , Quitosana/análogos & derivados , Teste de Materiais , Porosidade , Ratos , Ratos Wistar , Resistência à Tração , Alicerces TeciduaisRESUMO
This communication outlines the advances made in the development of thermoresponsive substrates for human mesenchymal stem cell (hMSC) expansion and subsequent controlled specific and multilineage differentiation from a previous study performed by this group. Previously, the development of an inexpensive and technically accessible method for hMSC expansion and harvesting was reported, using the solvent casting deposition method and thermoresponsive poly(N-isopropylacrylamide). Here, the logical continuation of this work is reported with the multipassage expansion of hMSCs with phenotypic maintenance followed by induced adipogenic, osteogenic, and chondrogenic differentiation. Interestingly, 1 µm thick solvent cast films are not only capable of hosting an expanding population of phenotypically preserved hMSCs similar to tissue culture plastic controls, but also the cells detached via temperature control better maintain their ability to differentiate compared to conventionally trypsinized cells. This approach to hMSC expansion and differentiation can be highly attractive to stem cell researchers where clinical therapies have seen a collective deviation away from the employment of animal derived products such as proteolytic trypsin.
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Human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest in tissue engineering. We obtained hWJ-MSCs from four patients, and then we stimulated their chondrogenic phenotype formation in vitro by adding resveratrol (during cell expansion) and a canonical Wnt pathway activator, LiCl, as well as a Rho-associated protein kinase inhibitor, Y27632 (during differentiation). The effects of the added reagents on the formation of hWJ-MSC sheets destined to repair osteochondral injuries were investigated. Three-dimensional hWJ-MSC sheets grown on P(NIPAM-co-NtBA)-based matrices were characterized in vitro and in vivo. The combination of resveratrol and LiCl showed effects on hWJ-MSC sheets similar to those of the basal chondrogenic medium. Adding Y27632 decreased both the proportion of hypertrophied cells and the expression of the hyaline cartilage markers. In vitro, DMSO was observed to impede the effects of the chondrogenic factors. The mouse knee defect model experiment revealed that hWJ-MSC sheets grown with the addition of resveratrol and Y27632 were well integrated with the surrounding tissues; however, after 3 months, the restored tissue was identical to that of the naturally healed cartilage injury. Thus, the combination of chondrogenic supplements may not always have additive effects on the progress of cell culture and could be neutralized by the microenvironment after transplantation.
Assuntos
Condrogênese , Células-Tronco Mesenquimais , Geleia de Wharton , Animais , Humanos , Camundongos , Células Cultivadas , Indicadores e Reagentes , Resveratrol/farmacologia , Geleia de Wharton/citologiaRESUMO
Coil to globule transition in poly(N-isopropylacrylamide) aqueous solutions was studied using spin probe continuous-wave electronic paramagnetic resonance (CW EPR) spectroscopy with an amphiphilic TEMPO radical as a guest molecule. Using Cu(II) ions as the "quencher" for fast-moving radicals in the liquid phase allowed obtaining the individual spectra of TEMPO radicals in polymer globule and observing inhomogeneities in solutions before globule collapsing. EPR spectra simulations confirm the formation of molten globules at the first step with further collapsing and water molecules coming out of the globule, making it denser.
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Cell aggregates, including sheets and spheroids, represent a simple yet powerful model system to study both biochemical and biophysical intercellular interactions. However, it is becoming evident that, although the mechanical properties and behavior of multicellular structures share some similarities with individual cells, yet distinct differences are observed in some principal aspects. The description of mechanical phenomena at the level of multicellular model systems is a necessary step for understanding tissue mechanics and its fundamental principles in health and disease. Both cell sheets and spheroids are used in tissue engineering, and the modulation of mechanical properties of cell constructs is a promising tool for regenerative medicine. Here, we review the data on mechanical characterization of cell sheets and spheroids, focusing both on advances in the measurement techniques and current understanding of the subject. The reviewed material suggest that interplay between the ECM, intercellular junctions, and cellular contractility determines the behavior and mechanical properties of the cell aggregates.
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Cell sheet technology has remained quite popular among tissue engineering techniques over the last several years. Meanwhile, there is an apparent trend in modern scientific research towards combining different approaches and strategies. Accordingly, a large body of work has arisen where cell sheets are used not as separate structures, but in combination with scaffolds as supporting constructions. The aim of this review is to analyze the intersection of these two vast areas of tissue engineering described in the literature mainly within the last five years. Some practical and technical details are emphasized to provide information that can be useful in research design and planning. The first part of the paper describes the general issues concerning the use of combined technology, its advantages and limitations in comparison with those of other tissue engineering approaches. Next, the detailed literature analysis of in vivo studies aimed at the regeneration of different tissues is performed. A significant part of this section concerns bone regeneration. In addition to that, other connective tissue structures, including articular cartilage and fibrocartilage, ligaments and tendons, and some soft tissues are discussed. STATEMENT OF SIGNIFICANCE: This paper describes the intersection of two technologies used in designing of tissue-engineered constructions for regenerative medicine: cell sheets as extracellular matrix-rich structures and supporting scaffolds as essentials in tissue engineering. A large number of reviews are devoted to each of these scientific problems. However, the solution of complex problems of tissue engineering requires an integrated approach that includes both three-dimensional scaffolds and cell sheets. This manuscript serves as a description of advantages and limitations of this method, its use in regeneration of bones, connective tissues and soft tissues and some other details.
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Engenharia Tecidual , Alicerces Teciduais , Osso e Ossos , Medicina Regenerativa , TecnologiaRESUMO
SIGNIFICANCE: Currently, various scaffolds with immobilized cells are widely used in tissue engineering and regenerative medicine. However, the physiological activity and cell viability in such constructs might be impaired due to a lack of oxygen and nutrients. Photobiomodulation (PBM) is a promising method of preconditioning cells to increase their metabolic activity and to activate proliferation or differentiation. AIM: Investigation of the potential of PBM for stimulation of cell activities in hydrogels. APPROACH: Mesenchymal stromal cells (MSCs) isolated from human gingival mucosa were encapsulated in modified fibrin hydrogels with different thicknesses and concentrations. Constructs with cells were subjected to a single-time exposure to red (630 nm) and near-infrared (IR) (840 nm) low-intensity irradiation. After 3 days of cultivation, the viability and physiological activity of the cells were analyzed using confocal microscopy and a set of classical tests for cytotoxicity. RESULTS: The cell viability in fibrin hydrogels depended both on the thickness of the hydrogels and the concentration of gel-forming proteins. The PBM was able to improve cell viability in hydrogels. The most pronounced effect was achieved with near-IR irradiation at the 840-nm wavelength. CONCLUSIONS: PBM using near-IR light can be applied for stimulation of MSCs metabolism and proliferation in hydrogel-based constructs with thicknesses up to 3 mm.
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Células-Tronco Mesenquimais , Engenharia Tecidual , Diferenciação Celular , Sobrevivência Celular , Humanos , HidrogéisRESUMO
The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.
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Gelatina/química , Gelatina/metabolismo , Transglutaminases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Gelatina/farmacologia , Glutaral/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Camundongos , Células NIH 3T3 , Suínos , Temperatura , Fatores de Tempo , Engenharia Tecidual/métodosRESUMO
Induced pluripotent stem cells (iPSCs) have attracted considerable attention from the public, clinicians, and scientists since their discovery in 2006, and raised huge expectations for regenerative medicine. One of the distinctive features of iPSCs is their propensity to differentiate into the cells of three germ lines in vitro and in vivo. The human iPSCs can be used to study the mechanisms underlying a disease and to monitor the disease progression, for testing drugs in vitro, and for cell therapy, avoiding many ethical and immunologic concerns. This technology offers the potential to take an individual approach to each patient and allows a more accurate diagnosis and specific treatment. However, there are several obstacles that impede the use of iPSCs. The derivation of fully reprogrammed iPSCs is expensive, time-consuming, and demands meticulous attention to many details. The use of biomaterials could increase the efficacy and safety while decreasing the cost of tissue engineering. The choice of a substrate utilized for iPSC culture is also important because cell-substrate contacts influence cellular behavior such as self-renewal, expansion, and differentiation. This Progress Report aims to summarize the advantages and drawbacks of natural and synthetic biomaterials, and to evaluate their role for maintenance and differentiation of iPSCs.
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Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Microambiente Celular/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Engenharia TecidualRESUMO
The facile regeneration of undifferentiated human mesenchymal stem cells (hMSCs) from thermoresponsive surfaces facilitates the collection of stem cells avoiding the use of animal derived cell detachment agents commonly used in cell culture. This communication proposes a procedure to fabricate coatings from commercially available pNIPAm which is both affordable and a significant simplification on alternative approaches used elsewhere. Solvent casting was used to produce films in the micrometer range and successful cell adhesion and proliferation was highly dependent on the thickness of the coating produced with 1 µm thick coatings supporting cells to confluence. 3T3 cell sheets and hMSCs were successfully detached from the cast coatings upon temperature reduction. Furthermore, results indicate that the hMSCs remained undifferentiated as the surface receptor profile of hMSCs was not altered when cells were detached in this manner.
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Resinas Acrílicas/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Resinas Acrílicas/química , Animais , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/química , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Células NIH 3T3 , TemperaturaRESUMO
A series of poly-(N-isopropyl acrylamide)-based copolymers were developed with a view to biomedical applications, specifically cell cultivation and recovery. Ethylpyrrolidone methacrylate (EPM), the monomer of poly-(ethylpyrrolidone methacrylate) (pEPM), which is itself thermoresponsive, was copolymerized with N-isopropylacrylamide in varying ratios to create this novel thermoresponsive copolymer series. Characterization indicated a moderate increase of copolymer lower critical solution temperature with increasing EPM content. Films of the copolymers successfully hosted cells to monolayer. Cells detached from the copolymers upon temperature reduction with cell to cell junctions maintained, avoiding the damage which can be caused using conventional detachment techniques. These results indicate that these copolymers are highly cell compatible and may be useful for a range of biomedical applications.
Assuntos
Resinas Acrílicas/síntese química , Resinas Acrílicas/farmacologia , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Temperatura , Células 3T3 , Resinas Acrílicas/química , Animais , Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Técnicas de Química Sintética , Interações Hidrofóbicas e Hidrofílicas , Metacrilatos/química , CamundongosRESUMO
The use of thermoresponsive surfaces as platforms for cell culture and cell regeneration has been explored over the last couple of decades. Poly-N-isopropylacrylamide (pNIPAm) is a well characterized thermoresponsive polymer which has an aqueous lower critical solution temperature (LCST) in a physiologically useful range, which allows it to reversibly attract (T < 32 °C) and repel water (T > 32 °C). It is this phenomenon that is exploited in temperature-controlled cell harvesting. pNIPAm coatings are generally poorly cell compatible and a number of complex or expensive techniques have been developed in order to overcome this issue. This study seeks to design a simple one-step system whereby commercially sourced pNIPAm is used to achieve similar results. Films were deposited using the operationally simple but rheologically complex spin coating technique. Reversible temperature modulated cell adhesion was achieved using a variety of different cell lines. This system offers a simplistic and cheaper alternative to methods used elsewhere.