RESUMO
While most of the in vivo extracellular matrices are 3D, most of the in vitro cultures are 2D--where only ventral adhesion is permitted--thus modifying cell behavior as a way to self-adaptation to this unnatural environment. We hypothesize that the excitation of dorsal receptors in cells already attached on a 2D surface (sandwich culture) could cover the gap between 2D and 3D cell-material interactions and result in a more physiological cell behavior. In this study we investigate the role of dorsal stimulation on myoblast differentiation within different poly(L-lactic acid) (PLLA) sandwich-like microenvironments, including plain material and aligned fibers. Enhanced cell differentiation levels were found for cells cultured with dorsal fibronectin-coated films. Seeking to understand the underlying mechanisms, experiments were carried out with (i) different types of dorsal stimuli (FN, albumin, FN after blocking the RGD integrin-binding site and activating dorsal cell integrin receptors), (ii) in the presence of an inhibitor of cell contractility, and (iii) increasing the frequency of culture medium changes to assess the effect of paracrine factors. Furthermore, FAK and integrin expressions, determined by Western blotting, revealed differences between cell sandwiches and 2D controls. Results show a stimuli-dependent response to dorsal excitation, proving that integrin outside-in signaling is involved in the enhanced cell differentiation. Due to their easiness and versatility, these sandwich-like systems are excellent candidates to get deeper insights into the study of 3D cell behavior and to direct cell fate within multilayer constructs.
Assuntos
Diferenciação Celular , Mioblastos/fisiologia , Transdução de Sinais , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Meios de Cultura/química , CamundongosRESUMO
The aim of this work is to compare the effect of hydroxyapatite (HAp) or bioglass (BG) nanoparticles in a polycaprolactone composite scaffold aimed to bone regeneration. To allow a comparison of the influence of both types of fillers, scaffolds made of PCL or composites containing up to 20 % by weight HAp or BG were obtained. Scaffolds showed acceptable mechanical properties for its use and high interconnected porosity apt for cellular colonization. To study the effect of the different materials on pre-osteoblast cells differentiation, samples with 5 % mineral reinforcement, were cultured for up to 28 days in osteogenic medium. Cells proliferated in all scaffolds. Nevertheless, differentiation levels for the selected markers were higher in pure PCL scaffolds than in the composites; inclusion of bioactive particles showed no positive effects on cell differentiation. In osteogenic culture conditions, the presence of bioactive particles is thus not necessary in order to observe good differentiation.
Assuntos
Osso e Ossos , Cerâmica/química , Durapatita/química , Poliésteres/química , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Animais , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiologia , Células Cultivadas , Cerâmica/síntese química , Cerâmica/farmacologia , Força Compressiva , Durapatita/farmacologia , Módulo de Elasticidade , Teste de Materiais , Camundongos , Nanocompostos/química , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Poliésteres/farmacologia , PorosidadeRESUMO
Fibronectin (FN) assembles into fibrillar networks by cells through an integrin-dependent mechanism. We have recently shown that simple FN adsorption onto poly(ethyl acrylate) surfaces (PEA), but not control polymer (poly(methyl acrylate), PMA), also triggered FN organization into a physiological fibrillar network. FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules. In the present study, we investigate the influence of topological cues on the material-driven FN assembly and the myogenic differentiation process. Aligned and random electrospun fibers were prepared. While FN fibrils assembled on the PEA fibers as they do on the smooth surface, the characteristic distribution of globular FN molecules observed on flat PMA transformed into non-connected FN fibrils on electrospun PMA, which significantly enhanced cell differentiation. The direct relationship between the fibrillar organization of FN at the material interface and the myogenic process was further assessed by preparing FN gradients on smooth PEA and PMA films. Isolated FN molecules observed at one edge of the substrate gradually interconnected with each other, eventually forming a fully developed network of FN fibrils on PEA. In contrast, FN adopted a globular-like conformation along the entire length of the PMA surface, and the FN gradient consisted only of increased density of adsorbed FN. Correspondingly, the percentage of differentiated cells increased monotonically along the FN gradient on PEA but not on PMA. This work demonstrates an interplay between material chemistry and topology in modulating material-driven FN fibrillogenesis and cell differentiation.
Assuntos
Materiais Biocompatíveis , Diferenciação Celular , Fibronectinas/metabolismo , Animais , Linhagem Celular , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de VarreduraRESUMO
Repair of central nervous system (CNS) lesions is difficulted by the lack of ability of central axons to regrow, and the blocking by the brain astrocytes to axonal entry. We hypothesized that by using bridges made of porous biomaterial and permissive olfactory ensheathing glia (OEG), we could provide a scaffold to permit restoration of white matter tracts. We implanted porous polycaprolactone (PCL) bridges between the substantia nigra and the striatum in rats, both with and without OEG. We compared the number of tyrosine-hydroxylase positive (TH+) fibers crossing the striatal-graft interface, and the astrocytic and microglial reaction around the grafts, between animals grafted with and without OEG. Although TH+ fibers were found inside the grafts made of PCL alone, there was a greater fiber density inside the graft and at the striatal-graft interface when OEG was cografted. Also, there was less astrocytic and microglial reaction in those animals. These results show that these PCL grafts are able to promote axonal growth along the nigrostriatal pathway, and that cografting of OEG markedly enhances axonal entry inside the grafts, growth within them, and re-entry of axons into the CNS. These results may have implications in the treatment of diseases such as Parkinson's and others associated with lesions of central white matter tracts. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 190-196, 2019.
Assuntos
Astrócitos/metabolismo , Axônios/metabolismo , Corpo Estriado/metabolismo , Microglia/metabolismo , Doença de Parkinson , Cimento de Policarboxilato , Animais , Astrócitos/patologia , Axônios/patologia , Corpo Estriado/patologia , Microglia/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Cimento de Policarboxilato/química , Cimento de Policarboxilato/farmacologia , Porosidade , Ratos , Ratos Transgênicos , Ratos Wistar , Substância BrancaRESUMO
Chondrogenesis of mesenchymal stem cells (MSCs) is known to be regulated by a number of environmental factors, including local oxygen levels. The hypothesis of this study is that the response of MSCs to hypoxia is dependent on the physical and chemical characteristics of the substrate used. The objective of this study was to explore how different modifications to polycaprolactone (PCL) scaffolds influenced the response of MSCs to hypoxia. PCL, PCL-hyaluronic acid (HA), and PCL-Bioglass® (BG) scaffolds were seeded with MSCs derived from bone marrow and cultured for 35 days under normoxic or low oxygen conditions, and the resulting biochemical properties of the MSC laden construct were assessed. Low oxygen tension has a positive effect over cell proliferation and macromolecules biosynthesis. Furthermore, hypoxia enhanced the distribution of collagen and glycosaminoglycans (GAGs) deposition through the scaffold. On the other hand, MSCs displayed certain material dependent responses to hypoxia. Low oxygen tension had a positive effect on cell proliferation in BG and HA scaffolds, but only a positive effect on GAGs synthesis in PCL and HA scaffolds. In conclusion, hypoxia increased cell viability and expression of chondrogenic markers but the cell response was modulated by the type of scaffold used. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1684-1691, 2017.
Assuntos
Condrogênese , Células-Tronco Mesenquimais/metabolismo , Oxigênio/metabolismo , Poliésteres/química , Alicerces Teciduais/química , Animais , Hipóxia Celular , Células Cultivadas , Cerâmica/química , Ácido Hialurônico/análogos & derivados , Células-Tronco Mesenquimais/citologia , Suínos , Engenharia TecidualRESUMO
BACKGROUND: Cancer research focuses increasingly on cancer stem cell study as those cells are thought to be the root of chemo and radioresistance of the most aggressive cancer types. Nevertheless, two-dimensional (2D) cell culture and even three-dimensional (3D) spheroid models, with their limited ability to reflect cell-extracellular matrix interactions, are not ideal for the study of cancer stem cells (CSCs). In this study, we establish a 3D in vitro cancer model using a synthetic and natural scaffold with tunable features and show that U87 cells cultured in this system acquire a stem-cell like phenotype. METHODS: U87 astrocytoma cells were grown on polycaprolactone (PCL)-2D flat substrates (2D) and PCL-3D scaffolds (3D) eventually containing hyaluronic acid (3D-HA). Cell viability, growth patterns, morphology, and cell surface marker expression (CD44, RHAMM and CD133) were studied to assess the effect of 3D culture and presence of HA. RESULTS: 3D scaffold, but most prominently presence of HA induced changes in cell morphology and marker expression; 3D-HA cultures showed features of aggregates; moreover, markedly increased expression of Nestin, CD44, RHAMM, and CD133 in 3D-HA scaffolds were found. CONCLUSIONS: the behavior of U87 in our 3D-HA model is more similar to tumor growth in vivo and a stem-like phenotype is promoted. Thus, the 3D-HA scaffold could provide a useful model for CSCs study and anti-cancer therapeutics research in vitro and may have preclinical application for the screening of drug candidates.
Assuntos
Astrocitoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Ácido Hialurônico/química , Proteínas de Neoplasias/biossíntese , Células-Tronco Neoplásicas/metabolismo , Astrocitoma/patologia , Células Imobilizadas/metabolismo , Células Imobilizadas/patologia , Humanos , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologiaRESUMO
Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior. This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.
Assuntos
Técnicas de Cultura de Células/métodos , Microambiente Celular , Técnicas de Cultura de Células/instrumentação , Humanos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Transdução de SinaisRESUMO
AIM: We introduced sandwich-like culture as a tool to engineer the cellular nanoenvironment by tuning protein presentation and activation of dorsal and ventral receptors. We aim at studying cell migration under more similar conditions to the 3D physiological one. MATERIALS & METHODS: We have investigated different nanoenvironments by changing the protein coating and using materials that adsorb proteins in different conformation, seeking to show their specific role in cell migration. RESULTS: Cell migration within sandwich cultures greatly differs from 2D cultures, shares some similarities with migration within 3D environments and is highly dependent on the protein nanoenvironment. Beyond differences in cell morphology and migration, dorsal stimulation promotes cell remodeling of the extracellular matrix over simple ventral receptor activation in traditional 2D cultures. CONCLUSION: Local(nano) stimulation of dorsal and ventral receptors within sandwich cultures alter cell migration in comparison to standard 2D environments.
Assuntos
Movimento Celular , Microambiente Celular , Animais , Adesão Celular , Técnicas de Cultura de Células/métodos , Engenharia Celular , Linhagem Celular , Movimento Celular/fisiologia , Microambiente Celular/fisiologia , Materiais Revestidos Biocompatíveis , Matriz Extracelular/metabolismo , Fibronectinas , Teste de Materiais , Camundongos , Nanomedicina , CicatrizaçãoRESUMO
Due to the attractive properties of poly(L-lactic acid) (PLLA) and chitosan (CHT) for tissue engineering applications, this study is aimed at analyzing the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (BM-MSCs) derived from osteoarthritic (OA) patients, in pure or CHT-coated PLLA, using different coating methodologies. Although PLLA scaffolds coated in one-step (PLLA-CHT1) yielded CHT smooth pellicles filling the PLLA macropores, a two-step strategy resulted in a CHT fiber-like thin coating covering PLLA pore walls (PLLA-CHT2). Both strategies led to the incorporation of similar content of CHT and a two-fold increase in the scaffold's water uptake capacity, providing elastic moduli values comparable to the ones found for human articular cartilage. After confirming OA-derived BM-MSCs, metabolic activity in the scaffolds, the chondrogenic potential was tested at 30 and 60 days, in a chondrogenic differentiation medium. PLLA scaffolds improved the chondrogenic differentiation of BM-MSCs, regarding cell pellet conventional culture and presented a typical cartilage zonal distribution, although was not able to revert a terminal differentiation. In PLLA-CHT1, on a short term, a rather heterogeneous tissue was formed, with confined areas of either slower cell infiltration or a faster maturation, with enhanced chondrogenic phenotype. In PLLA-CHT2, a similar tissue to PLLA was obtained, albeit on the long term, these scaffolds helped to maintain a hyaline-like phenotype and prevented the advance of the hypertrophic process. These results demonstrate the importance of the scaffolds microenvironment on the cellular events of chondrogenesis.
Assuntos
Quitosana/química , Condrócitos/patologia , Ácido Láctico/química , Células-Tronco Mesenquimais/patologia , Osteoartrite/patologia , Polímeros/química , Alicerces Teciduais , Adsorção , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Microambiente Celular , Condrogênese/fisiologia , Materiais Revestidos Biocompatíveis/síntese química , Humanos , PoliésteresRESUMO
Currently, cell culture systems that include nanoscale topography are widely used in order to provide cells additional cues closer to the in vivo environment, seeking to mimic the natural extracellular matrix. Electrospinning is one of the most common techniques to produce nanofiber mats. However, since many sensitive parameters play an important role in the process, a lack of reproducibility is a major drawback. Here we present a simple and robust methodology to prepare reproducible electrospun-like samples. It consists of a polydimethylsiloxane mold reproducing the fiber pattern to solvent-cast a polymer solution and obtain the final sample. To validate this methodology, poly(L-lactic) acid (PLLA) samples were obtained and, after characterisation, bioactivity and ability to direct cell response were assessed. C2C12 myoblasts developed focal adhesions on the electrospun-like fibers and, when cultured under myogenic differentiation conditions, similar differentiation levels to electrospun PLLA fibers were obtained.
Assuntos
Mioblastos/citologia , Polímeros/química , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Animais , Adesão Celular , Diferenciação Celular , Proliferação de Células , Técnicas Eletroquímicas , Camundongos , Polímeros/síntese químicaRESUMO
Scaffolds for cartilage tissue engineering should promote both adequate biomechanical environment and chondrogenic stimulation. Hyaluronic acid (HA) has been used in cartilage engineering for its chondrogenic and chondroprotective properties, nevertheless its mechanical properties are limited. Influence of HA microstructure in chondrocyte response has not been addressed yet. In this work, polycaprolactone (PCL) scaffolds were modified using HA following two coating strategies: coating in one step (PCL-HA1s) yields a gel-like phase within the scaffold, whereas a two-step reaction (PCL-HA2s) yields a thin HA layer coating internal surfaces of PCL structure. Chondrocytes were seeded in the scaffolds and cultured in dedifferentiating conditions up to 3 weeks and analyzed using a total DNA assay and sulfated glycosaminoglycan (sGAG) determination assay; cell morphology and extracellular matrix secretion were assessed by electron microscopy as well as immunofluorescent imaging (collagen I, collagen II, aggrecan, CD44). Cells proliferate in all samples and no cytotoxicity is observed. PCL-HA1s shows higher sGAG production per cell than PCL and PCL-HA2s at all times. Presence of hyaluronic acid promotes qualitative expression of CD44 surface markers and aggrecan (more visible in PCL-HA1s than PCL-HA2s), whereas in dedifferentiating conditions, expression of CD44 and aggrecan can hardly be detected in pure PCL scaffolds. Collagen type II seems more prominent in PCL-HA2s; although PCL-HA2s shows markers for COL II, aggrecan and CD44, quantitative ECM production is not improved with respect to PCL. It is thus likely that CD44 activation is not sufficient for explaining the better response in PCL-HA1s.
Assuntos
Cartilagem Articular/citologia , Condrócitos/citologia , Materiais Revestidos Biocompatíveis/farmacologia , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Poliésteres/farmacologia , Alicerces Teciduais/química , Idoso , Células Cultivadas , Condrócitos/efeitos dos fármacos , Força Compressiva/efeitos dos fármacos , DNA/metabolismo , Módulo de Elasticidade/efeitos dos fármacos , Imunofluorescência , Glicosaminoglicanos/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Masculino , Microscopia Eletrônica de Varredura , Água/químicaRESUMO
Cells behave differently between bidimensional (2D) and tridimensional (3D) environments. While most of the in vitro cultures are 2D, most of the in vivo extracellular matrices are 3D, which encourages the development of more relevant culture conditions, seeking to provide more physiological models for biomedicine (e.g., cancer, drug discovery and tissue engineering) and further insights into any dimension-dependent biological mechanism. In this study, cells were cultured between two protein coated surfaces (sandwich-like culture). Cells used both dorsal and ventral receptors to adhere and spread, undergoing morphological changes with respect to the 2D control. Combinations of fibronectin and bovine serum albumin on the dorsal and ventral sides led to different cell morphologies, which were quantified from bright field images by calculating the spreading area and circularity. Although the mechanism underlying these differences remains to be clarified, excitation of dorsal receptors by anchorage to extracellular proteins plays a key role on cell behavior. This approach--sandwich-like culture--becomes therefore a versatile method to study cell adhesion in well-defined conditions in a quasi 3D environment.
Assuntos
Fibroblastos/citologia , Propriedades de Superfície , Animais , Bovinos , Adesão Celular , Técnicas de Cultura de Células , Linhagem Celular , Forma Celular , Fibroblastos/fisiologia , Fibronectinas/metabolismo , Humanos , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Soroalbumina Bovina/metabolismoRESUMO
BACKGROUND: Cartilage tissue engineering using synthetic scaffolds allows maintaining mechanical integrity and withstanding stress loads in the body, as well as providing a temporary substrate to which transplanted cells can adhere. PURPOSE: This study evaluates the use of polycaprolactone (PCL) scaffolds for the regeneration of articular cartilage in a rabbit model. STUDY DESIGN: Controlled laboratory study. METHODS: Five conditions were tested to attempt cartilage repair. To compare spontaneous healing (from subchondral plate bleeding) and healing due to tissue engineering, the experiment considered the use of osteochondral defects (to allow blood flow into the defect site) alone or filled with bare PCL scaffold and the use of PCL-chondrocytes constructs in chondral defects. For the latter condition, 1 series of PCL scaffolds was seeded in vitro with rabbit chondrocytes for 7 days and the cell/scaffold constructs were transplanted into rabbits' articular defects, avoiding compromising the subchondral bone. Cell pellets and bare scaffolds were implanted as controls in a chondral defect. RESULTS: After 3 months with PCL scaffolds or cells/PCL constructs, defects were filled with white cartilaginous tissue; integration into the surrounding native cartilage was much better than control (cell pellet). The engineered constructs showed histologically good integration to the subchondral bone and surrounding cartilage with accumulation of extracellular matrix including type II collagen and glycosaminoglycan. The elastic modulus measured in the zone of the defect with the PCL/cells constructs was very similar to that of native cartilage, while that of the pellet-repaired cartilage was much smaller than native cartilage. CONCLUSION: The results are quite promising with respect to the use of PCL scaffolds as aids for the regeneration of articular cartilage using tissue engineering techniques.