RESUMO
Mesenchymal stem cells (MSCs), pivotal for tissue repair, utilize collagen to restore structural integrity in damaged tissue, preserving its organization through concomitant remodeling. The non-enzymatic glycation of collagen potentially compromises MSC communication, particularly upon advancing the process, underlying various pathologies such as late-stage diabetic complications and aging. However, an understanding of the impact of early-stage collagen glycation on MSC interaction is lacking. This study examines the fate of in vitro glycated rat tail collagen (RTC) upon exposure to glucose for 1 or 5 days in contact with MSCs. Utilizing human adipose tissue-derived MSCs (ADMSCs), we demonstrate their significantly altered interaction with glycated collagen, characterized morphologically by reduced cell spreading, diminished focal adhesions formation, and attenuated development of the actin cytoskeleton. The morphological findings were confirmed by ImageJ 1.54g morphometric analysis with the most significant drop in the cell spreading area (CSA), from 246.8 µm2 for the native collagen to 216.8 µm2 and 163.7 µm2 for glycated ones, for 1 day and 5 days, respectively, and a similar trend was observed for cell perimeter 112.9 µm vs. 95.1 µm and 86.2 µm, respectively. These data suggest impaired recognition of early glycated collagen by integrin receptors. Moreover, they coincide with the reduced fibril-like reorganization of adsorbed FITC-collagen (indicating impaired remodeling) and a presumed decreased sensitivity to proteases. Indeed, confirmatory assays reveal diminished FITC-collagen degradation for glycated samples at 1 day and 5 days by attached cells (22.8 and 30.4%) and reduced proteolysis upon exogenous collagenase addition (24.5 and 40.4%) in a cell-free system, respectively. The mechanisms behind these effects remain uncertain, although differential scanning calorimetry confirms subtle structural/thermodynamic changes in glycated collagen.
Assuntos
Colágeno , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Colágeno/metabolismo , Glicosilação , Animais , Ratos , Comunicação Celular , Células Cultivadas , Glucose/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/citologia , Adesões Focais/metabolismo , Adesões Focais/efeitos dos fármacosRESUMO
Graphene and graphene oxide (GO), due to their unique chemical and physical properties, possess biochemical characteristics that can trigger intercellular signals promoting tissue regeneration. Clinical applications of thin GO-derived sheets have inspired the development of various tissue regeneration and repair approaches. In this study, we demonstrate that ultrathin sheets of plasma-functionalized and reduced GO, with the oxygen content ranging from 3.2 % to 22 % and the nitrogen content from 0 % to 8.3 %, retain their essential mechanical and molecular integrity, and exhibit robust potential for regenerating bone tissue and blood vessels across multiple cellular and animal models. Initially, we observed the growth of blood vessels and bone tissue in vitro using these functionalized GO sheets on human adipose-derived mesenchymal stem cells and umbilical vein endothelial cells. Remarkably, our study indicates a 2.5-fold increase in mineralization and two-fold increase in tubule formation even in media lacking osteogenic and angiogenic supplements. Subsequently, we observed the initiation, conduction, and formation of bone and blood vessels in a rat tibial osteotomy model, evident from a marked 4-fold increase in the volume of low radio-opacity bone tissue and a significant elevation in connectivity density, all without the use of stem cells or growth factors. Finally, we validated these findings in a mouse critical-size calvarial defect model (33 % higher healing rate) and a rat skin lesion model (up to 2.5-fold increase in the number of blood vessels, and 35 % increase in blood vessels diameter). This study elucidates the pro-osteogenic and pro-angiogenic properties of both pristine and plasma-treated GO ultrathin films. These properties suggest their significant potential for clinical applications, and as valuable biomaterials for investigating fundamental aspects of bone and blood vessel regeneration.
Assuntos
Regeneração Óssea , Grafite , Células Endoteliais da Veia Umbilical Humana , Células-Tronco Mesenquimais , Animais , Grafite/química , Humanos , Ratos , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/fisiologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Camundongos , Vasos Sanguíneos , Ratos Sprague-Dawley , Osso e Ossos/irrigação sanguínea , Osso e Ossos/efeitos dos fármacos , Gases em Plasma/farmacologia , Gases em Plasma/química , Tíbia/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Engenharia Tecidual/métodosRESUMO
Extracellular matrix (ECM) provides various mechanical cues that are able to affect the self-renewal and differentiation of mesenchymal stem cells (MSC). Little is known, however, how these cues work in a pathological environment, such as acute oxidative stress. To better understand the behavior of human adipose tissue-derived MSC (ADMSC) in such conditions, we provide morphological and quantitative evidence for significantly altered early steps of mechanotransduction when adhering to oxidized collagen (Col-Oxi). These affect both focal adhesion (FA) formation and YAP/TAZ signaling events. Representative morphological images show that ADMSCs spread better within 2 h of adhesion on native collagen (Col), while they tended to round up on Col-Oxi. It also correlates with the lesser development of the actin cytoskeleton and FA formation, confirmed quantitatively by morphometric analysis using ImageJ. As shown by immunofluorescence analysis, oxidation also affected the ratio of cytosolic-to-nuclear YAP/TAZ activity, concentrating in the nucleus for Col while remaining in the cytosol for Col-Oxi, suggesting abrogated signal transduction. Comparative Atomic Force Microscopy (AFM) studies show that native collagen forms relatively coarse aggregates, much thinner with Col-Oxi, possibly reflecting its altered ability to aggregate. On the other hand, the corresponding Young's moduli were only slightly changed, so viscoelastic properties cannot explain the observed biological differences. However, the roughness of the protein layer decreased dramatically, from RRMS equal to 27.95 ± 5.1 nm for Col to 5.51 ± 0.8 nm for Col-Oxi (p < 0.05), which dictates our conclusion that it is the most altered parameter in oxidation. Thus, it appears to be a predominantly topographic response that affects the mechanotransduction of ADMSCs by oxidized collagen.
Assuntos
Colágeno , Mecanotransdução Celular , Células-Tronco Mesenquimais , Humanos , Colágeno/química , Colágeno/farmacologia , Matriz Extracelular/metabolismo , Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Transdução de SinaisRESUMO
Mesenchymal stem cells (MSCs) are involved in the process of extracellular matrix (ECM) remodeling where collagens play a pivotal role. We recently demonstrated that the remodeling of adsorbed collagen type I might be disordered upon oxidation following its fate in the presence of human adipose-derived MSC (ADMSCs). With the present study we intended to learn more about the effect of polyphenolic antioxidant Epigallocatechin-3-gallate (EGCG), attempting to mimic the conditions of oxidative stress in vivo and its putative prevention by antioxidants. Collagen Type I was isolated from mouse tail tendon (MTC) and labelled with FITC before being oxidized according to Fe2+/H2O2 protocol. FITC-collagen remodeling by ADMSC was assessed morphologically before and after EGCG pretreatment and confirmed via detailed morphometric analysis measuring the anisotropy index (AI) and fluorescence intensity (FI) in selected regions of interest (ROI), namely: outside the cells, over the cells, and central (nuclear/perinuclear) region, whereas the pericellular proteolytic activity was measured by de-quenching fluorescent collagen probes (FRET effect). Here we provide morphological evidence that MTC undergoes significant reorganization by the adhering ADMSC and is accompanied by a substantial activation of pericellular proteolysis, and further confirm that both processes are suppressed upon collagen oxidation. An important observation was that this abrogated remodeling cannot be prevented by the EGCG pretreatment. Conversely, the detailed morphometric analysis showed that oxidized FITC-collagen tends to accumulate beneath cells and around cell nuclei, suggesting the activation of alternative routes for its removal, such as internalization and/or transcytosis. Morphometric analysis also revealed that both processes are supported by EGCG pretreatment.
RESUMO
This study describes the effect of collagen type I (Col I) oxidation on its physiological remodeling by adipose tissue-derived mesenchymal stem cells (ADMSCs), both mechanical and proteolytic, as an in vitro model for the acute oxidative stress that may occur in vivo upon distinct environmental changes. Morphologically, remodeling was interpreted as the mechanical rearrangement of adsorbed FITC-labelled Col I into a fibril-like pattern. This process was strongly abrogated in cells cultured on oxidized Col I albeit without visible changes in cell morphology. Proteolytic activity was quantified utilizing fluorescence de-quenching (FRET effect). The presence of ADMSCs caused a significant increase in native FITC-Col I fluorescence, which was almost absent in the oxidized samples. Parallel studies in a cell-free system confirmed the enzymatic de-quenching of native FITC-Col I by Clostridial collagenase with statistically significant inhibition occurring in the oxidized samples. Structural changes to the oxidized Col I were further studied by differential scanning calorimetry. In the oxidized samples, an additional endotherm with sustained enthalpy (∆H) was observed at 33.6 °C along with Col I's typical one at 40.5 °C. Collectively, these data support that the remodeling of Col I by ADMSCs is altered upon oxidation due to intrinsic changes to the protein's structure, which represents a novel mechanism for the control of stem cell behavior.
Assuntos
Colágeno Tipo I , Células-Tronco Mesenquimais , Colágeno/química , Colágeno Tipo I/química , Fluoresceína-5-Isotiocianato/farmacologia , Células-TroncoRESUMO
In this paper, we created a dynamic adhesive environment (DAE) for adipose tissue-derived mesenchymal stem cells (ADMSCs) cultured on smart thermo-responsive substrates, i.e., poly (N-isopropyl acrylamide) (PNIPAM), via introducing periodic changes in the culture temperature. We further explored the particular role of adsorbed fibronectin (FN), an important cell adhesive protein that was recently attributed to the recruitment of stem cells in the niche. The engineered FN/PNIPAM DAE system significantly increased the symmetric renewal of ADMSCs, particularly between passages 7 and 9 (p7-p9), before it dropped down to the level of the control (FN-coated TC polystyrene). This decline in the growth curve was consistent with the increased number of senescent cells, the augmented average cell size and the suppressed FN matrix secretion at late passages (p10-p12), all of them characteristic for stem cells ageing, which equivocally tended to slow down at our DAE system. FN supported also the osteogenic response of ADMSCs (apart from the previous observations with plain PNIPAM substrata) indicated by the significant increase of alkaline phosphatase (ALP) activity at days 7 and 14. The minimal changes in the Ca deposition, however, suggest a restricted effect of DAE on the early osteogenic response of ADMSCs only. Thus, the engineering of niche-like DAE involving FN uncovers a new tissue engineering strategy for gaining larger amounts of functionally active stem cells for clinical application.
Assuntos
Fibronectinas/química , Células-Tronco Mesenquimais/citologia , Polímeros/química , Engenharia Tecidual/métodos , Alicerces Teciduais , Resinas Acrílicas/química , Tecido Adiposo/citologia , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Adesão Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Senescência Celular , Meios de Cultura , Humanos , Técnicas In Vitro , Teste de Materiais , Osteogênese , Células-Tronco/metabolismo , TemperaturaRESUMO
Mimicking the complex organization of the extracellular matrix (ECM), especially its structure and dimensionality, is necessary to produce living tissues from stem cells. In compliance with a previously established role of nanofiber organization for the osteogenic differentiation of stem cells, here we used hybrid fibrinogen/poly(l-lactide-ε-caprolactone) (FBG/PLCL) nanofibers arranged in aligned and honeycomb configurations, to recapitulate the highly oriented ECM of the cortical bone and the sponge-like (i.e., honeycomb) environment of the cancellous one, respectively. Using special bilayered constructs, we demonstrate that the dimensionality (i.e., 2D vs. 3D) of the nanofibers as well as their architecture (i.e., honeycomb vs. aligned) affects differently the overall morphology and the expression of multiple osteogenic genes of human adipose-derived mesenchymal stem cells (ADMSCs). The cells had elongated shape with markedly increased cell mobility when seeded on aligned nanofibers. Conversely, on honeycomb-shaped nanofibers, ADMSCs initially concentrated inside the honeycomb curvatures adopting rounded morphology, but late, they formed network-like structures overlaying the honeycomb curvatures. By employing quantitative polymerase chain reaction (qPCR), we further show that a 3D environment generally supports the multiple osteogenic response of ADMSCs, but honeycomb and aligned architectures promote rather different differentiation pathways.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Osteogênese , Alicerces Teciduais/química , Diferenciação Celular/genética , Forma Celular , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Nanofibras/ultraestrutura , Osteogênese/genéticaRESUMO
Tissue engineering demands the development of scaffolds that mimic natural extracellular matrices (ECM). Despite the success in obtaining synthetic interstitial ECM, the production of an artificial basement membrane (BM), the specialized thin sheet of ECM that is pivotal for the functional organization of most tissues and internal organs, is still not achieved. With the long-term aim of developing a flat BM-like structure here we investigated the behavior of acid-soluble Col IV during simultaneous assembly with laminin (LM) in acidic conditions. The underlying rationale was the previously observed phenomenon of acid-triggered LM polymerization, giving rise to biomimetic polylaminin (polyLM) that can be adsorbed on the substrate. Unexpectedly, we found that Col IV (that does not polymerize in acidic conditions) readily incorporated into the polyLM layer, forming a network that mimics to a great extent the characteristic polygonal morphology of single polyLM observable at micrometric scale. Scanning calorimetry and light scattering measurements supported the notion that polyLM and Col IV could directly interact. The biological properties of the proposed artificial BM-like structure were characterized using human keratinocytes (HACAT) and umbilical vein endothelial cells (HUVEC). HACAT formed stratified cell layers on the hybrid polyLM/Col IV layer, but not on Matrigel, nor on LM or Col IV alone, while HUVEC improved cortical F-actin and tight juctions organization on polyLM/Col IV. Thus, the proposed artificial BM reproduces not only morphological but also some functional properties of the natural BM. STATEMENT OF SIGNIFICANCE: Basement membranes (BMs) are flat biological matrices separating tissue compartments in the body. Their peculiar sheet-like structure is thought to result from the association of two independent protein networks of laminin and collagen IV. While pursuing the development of an artificial BM, we found that, when mixed with acid-induced polymerized laminin, collagen IV immediately conformed to the laminin shape. This implies that the protein networks may not be independently assembled as believed so far, but instead that laminin may command the assembly of collagen IV. Our hybrid matrix was structurally more stable than the commercial BM extract Matrigel and, unlike the latter, supported in vitro formation of a stratified layer of keratinocytes that approximated the organization of the natural epidermis.
Assuntos
Colágeno Tipo IV , Células Endoteliais , Membrana Basal , Matriz Extracelular , Humanos , Laminina , Engenharia TecidualRESUMO
PIKfyve phosphoinositide kinase produces PtdIns(3,5)P2 and PtdIns5P and governs a myriad of cellular processes including cytoskeleton rearrangements and cell proliferation. The latter entails rigorous investigation since the cytotoxicity of PIKfyve inhibition is a potential therapeutic modality for cancer. Here we report the effects of two PIKfyve-specific inhibitors on the attachment/spreading and viability of mouse embryonic fibroblasts (MEFs) and C2C12 myoblasts. Importantly, 18-h treatment of adherent cells with YM201636 (800â¯nM) and apilimod (20â¯nM) in serum-containing culture media did not affect cell viability despite the presence of multiple cytoplasmic vacuoles, a hallmark of PIKfyve inhibition. Strikingly, at the same dose and duration the inhibitors caused excessive cytoplasmic vacuolation, initial suppression of cell attachment/spreading and subsequent marked detachment/death in serum-deprived cells. The remaining adherent cells under serum-deprived conditions had smaller surface area, lacked vinculin/actin-positive focal adhesions and displayed vacuoles occupying the entire cytoplasm. Serum or growth factors protected against PIKfyve inhibitor cytotoxicity. This protection required Akt activation evidenced by the abrogated beneficial effect of serum upon treatment with the clinically-relevant Akt inhibitor MK-2206. Moreover, Akt inhibition triggered cell detachment/death even in serum-fed adherent MEFs treated with apilimod. Intriguingly, BafilomycinA1 (H+-vacuolar ATPase inhibitor), which prevents the cytoplasmic vacuolation under PIKfyve perturbations, rescued all defects in attaching/spreading as well as in adherent cells under serum-starved or serum-fed conditions, respectively. Together, the results indicate that the cytotoxicity of PIKfyve inhibitors in MEFs and C2C12 myoblasts requires Akt suppression and excessive cytoplasmic vacuolation.
Assuntos
Antineoplásicos/farmacologia , Citoplasma/efeitos dos fármacos , Proteína Oncogênica v-akt/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Vacúolos/efeitos dos fármacos , Aminopiridinas/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Contagem de Células , Morte Celular/efeitos dos fármacos , Citoplasma/ultraestrutura , Inibidores Enzimáticos/farmacologia , Fibroblastos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Macrolídeos/farmacologia , Camundongos , Mioblastos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases , Vacúolos/ultraestruturaRESUMO
Extracellular matrix (ECM) proteins are key mediators of cell/material interactions. The surface density and conformation of these proteins adsorbed on the material surface influence cell adhesion and the cellular response. We have previously shown that subtle variations in surface chemistry lead to drastic changes in the conformation of adsorbed fibronectin (FN). On poly(ethyl acrylate) (PEA), FN unfolds and displays domains for cell adhesion and FN-FN interaction, whereas on poly(methyl acrylate) (PMA) - with only one methyl group less - FN remains globular as it is in solution. The effect of the strength of the protein/material interaction in cell response, and its relation to protein density and conformation, has received limited attention so far. In this work, we used FN-functionalized AFM cantilevers to evaluate, via force spectroscopy, the strength of interaction between fibronectin and the underlying polymer which controls FN conformation (PEA and PMA). We found that the strength of FN/PEA interaction is significantly higher than FN/PMA, which limits the mobility of FN layer on PEA, reduces the ability of cells to mechanically reorganize FN and then leads to enhanced proteolysis and degradation of the surrounding matrix with compromised cell viability. By contrast, both PEA and PMA support cell adhesion when FN density is increased and also in the presence of serum or other serum proteins, including vitronectin (VN) and bovine serum albumin (BSA), which provide a higher degree of mobility to the matrix. STATEMENT OF SIGNIFICANCE: The identification of parameters influencing cell response is of paramount importance for the design of biomaterials that will act as synthetic scaffolds for cells to anchor, grow and, eventually, become specialised tissues. Cells interact with materials through an intermediate layer of proteins adsorbed on the material surface. It is known that the density and conformation of these proteins determine cell behaviour. Here we show that the strength of protein/material interactions, which has received very limited attention so far, is key to understand the cellular response to biomaterials. Very strong protein/material interactions reduce the ability of cells to mechanically reorganize proteins at the material interface which results in enhanced matrix degradation, leading ultimately to compromised cell viability.
Assuntos
Resinas Acrílicas/química , Linhagem da Célula , Matriz Extracelular/metabolismo , Fibronectinas/química , Células 3T3 , Adsorção , Animais , Materiais Biocompatíveis/química , Adesão Celular , Diferenciação Celular , Sobrevivência Celular , Humanos , Camundongos , Microscopia de Força Atômica , Microscopia de Fluorescência , Soroalbumina Bovina/química , Propriedades de Superfície , Vitronectina/químicaRESUMO
AIM: To develop a nanofiber (NF)-based biomimetic coating on titanium (Ti) that mimics the complex spatiotemporal organization of the extracellular matrix (ECM). MATERIALS & METHODS: Recombinant cell attachment site (CAS) of fibronectin type III8-10 domain was co-electrospun with polylactic acid (PLA) and covalently bound on polished Ti discs. Osteoblast-like SaOS-2 cells were used to evaluate their complex bioactivity. RESULTS: A significant increase of cell spreading was found on CAS/PLA hybrid NFs, followed by control pure PLA NFs and bare Ti discs. Cell proliferation showed similar trend being about twice higher on CAS/PLA NFs. The significantly increased ALP activity at day 21 indicated an enhanced differentiation of SaOS-2 cells. CONCLUSION: Coating of Ti implants with hybrid CAS/PLA NFs may improve significantly their osseointegration potential.
Assuntos
Fibronectinas/farmacologia , Nanofibras/química , Osteoblastos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fibronectinas/química , Humanos , Osseointegração/efeitos dos fármacos , Osteoblastos/citologia , Poliésteres/química , Poliésteres/farmacologia , Próteses e Implantes , Proteínas Recombinantes/química , Propriedades de Superfície , Titânio/química , Titânio/farmacologiaRESUMO
Stem cells therapy offers a viable alternative for treatment of bone disorders to the conventional bone grafting. However clinical therapies are still hindered by the insufficient knowledge on the conditions that maximize stem cells differentiation. Hereby, we introduce a novel 3D honeycomb architecture scaffold that strongly support osteogenic differentiation of human adipose derived mesenchymal stem cells (ADMSCs). The scaffold is based on electrospun hybrid nanofibers consisting of poly (L-lactide ε-caprolactone) and fibrinogen (PLCL/FBG). Classical fibers orientations, random or aligned were also produced and studied for comparison. The overall morphology of ADMSC's generally followed the nanofibers orientation and dimensionality developing regular focal adhesions and direction-dependent actin cytoskeleton bundles. However, there was an initial tendency for cells rounding on honeycomb scaffolds before ADMSCs formed a distinct bridging network. This specific cells organization appeared to have significant impact on the differentiation potential of ADMSCs towards osteogenic lineage, as indicated by the alkaline phosphatase production, calcium deposition and specific genes expression. Collectively, it was observed synergistic effect of nanofibers with honeycomb architecture on the behavior of ADMSCs entering osteogenic path of differentiation which outlines the potential benefits from insertion of such bioinspired geometrical cues within scaffolds for bone tissue engineering.
Assuntos
Fibrinogênio/química , Fibrinogênio/farmacologia , Células-Tronco Mesenquimais/citologia , Nanofibras/química , Osteogênese/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Tecido Adiposo/citologia , Fosfatase Alcalina/metabolismo , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Nanofibras/ultraestrutura , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
Engineering dynamic stem cell niche-like environment offers opportunity to obtain better control of the fate of stem cells. We identified, for the first time, that periodic changes in the adhesive environment of human adipose derived mesenchymal stem cells (ADSCs) alters dramatically their asymmetric division but not their ability for symmetric renewal. Hereby, we used smart thermo-responsive polymer (PNIPAM) to create a dynamic adhesive environment for ADSCs by applying periodic temperature cycles to perturb adsorbed adhesive proteins to substratum interaction. Cumulative population doubling time (CPDT) curves showed insignificant decline in the symmetric cell growth studied for up to 13th passages accompanied with small changes in the overall cell morphology and moderately declined fibronectin (FN) matrix deposition probably as a functional consequence of ADSCs ageing. However, a substantial alteration in the differentiation potential of ADSCs from both early and late passages (3rd and 14th, respectively) was found when the cells were switched to osteogenic differentiation conditions. This behavior was evidenced by the significantly altered alkaline phosphatase activity and Ca deposition (Alizarin red) assayed at 3, 14 and 21day in comparison to the control samples of regular TC polystyrene processed under same temperature settings.
Assuntos
Células-Tronco Mesenquimais , Adesivos , Tecido Adiposo , Diferenciação Celular , Células Cultivadas , Humanos , OsteogêneseRESUMO
Novel, hybrid fibrinogen/polylactic acid (FBG/PLA) nanofibers with different configuration (random vs aligned) and dimensionality (2-D vs 3-D environment) were used to control the overall behavior and the osteogenic differentiation of human adipose-derived mesenchymal stem cells (ADMSCs). Aligned nanofibers in both the 2-D and 3-D configurations are proved to be favored for osteodifferentiation. Morphologically, we found that on randomly configured nanofibers, the cells developed a stellate-like morphology with multiple projections; however, time-lapse analysis showed significantly diminished cell movements. Conversely, an elongated cell shape with advanced cell spreading and extended actin cytoskeleton accompanied with significantly increased cell mobility were observed when cells attached on aligned nanofibers. Moreover, a clear tendency for higher alkaline phosphatase activity was also found on aligned fibers when ADMSCs were switched to osteogenic induction medium. The strongest accumulation of Alizarin red (AR) and von Kossa stain at 21 days of culture in osteogenic medium were found on 3-D aligned constructs while the rest showed lower and rather undistinguishable activity. Quantitative reverse transcription-polymerase chain reaction analysis for Osteopontin (OSP) and RUNX 2 generally confirmed this trend showing favorable expression of osteogenic genes activity in 3-D environment particularly in aligned configuration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2065-2074, 2017.
Assuntos
Diferenciação Celular , Fibrinogênio/química , Células-Tronco Mesenquimais/metabolismo , Nanofibras/química , Osteogênese , Poliésteres/química , Humanos , Células-Tronco Mesenquimais/citologiaRESUMO
Mimicking the complex intricacies of the extra cellular matrix including 3D configurations and aligned fibrous structures were traditionally perused for producing cartilage tissue from stem cells. This study shows that human adipose derived mesenchymal stem cells (hADMSCs) establishes significant chondrogenic differentiation and may generate quality cartilage when cultured on 2D and randomly oriented fibrinogen/poly-lactic acid nanofibers compared to 3D sandwich-like environments. The adhering cells show well-developed focal adhesion complexes and actin cytoskeleton arrangements confirming the proper cellular interaction with either random or aligned nanofibers. However, quantitative reverse transcription-polymerase chain reaction analysis for Collagen 2 and Collagen 10 genes expression confirms favorable chondrogenic response of hADMSCs on random nanofibers and shows substantially higher efficacy of their differentiation in 2D configuration versus 3D constructs. These findings introduce a new direction for cartilage tissue engineering through providing a simple platform for the routine generation of transplantable stem cells derived articular cartilage replacement that might improve joint function.
Assuntos
Cartilagem Articular/citologia , Diferenciação Celular/efeitos dos fármacos , Fibrinogênio/farmacologia , Células-Tronco Mesenquimais/citologia , Nanofibras/química , Tecido Adiposo/citologia , Animais , Bovinos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Colágeno/genética , Colágeno/metabolismo , Humanos , Imageamento Tridimensional , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Nanofibras/ultraestrutura , Poliésteres/química , Reação em Cadeia da Polimerase em Tempo RealRESUMO
UNLABELLED: The effect of molecular composition of multilayers, by pairing type I collagen (Col I) with either hyaluronic acid (HA) or chondroitin sulfate (CS) was studied regarding the osteogenic differentiation of adhering human adipose-derived stem cells (hADSCs). Polyelectrolyte multilayer (PEM) formation was based primarily on ion pairing and on additional intrinsic cross-linking through imine bond formation with Col I replacing native by oxidized HA (oHA) or CS (oCS). Significant amounts of Col I fibrils were found on both native and oxidized CS-based PEMs, resulting in higher water contact angles and surface potential under physiological condition, while much less organized Col I was detected in either HA-based multilayers, which were more hydrophilic and negatively charged. An important finding was that hADSCs remodeled Col I at the terminal layers of PEMs by mechanical reorganization and pericellular proteolytic degradation, being more pronounced on CS-based PEMs. This was in accordance with the higher quantity of Col I deposition in this system, accompanied by more cell spreading, focal adhesions (FA) formation and significant α2ß1 integrin recruitment compared to HA-based PEMs. Both CS-based PEMs caused also an increased fibronectin (FN) secretion and cell growth. Furthermore, significant calcium phosphate deposition, enhanced ALP, Col I and Runx2 expression were observed in hADSCs on CS-based PEMs, particularly on oCS-containing one. Overall, multilayer composition can be used to direct cell-matrix interactions, and hence stem cell fates showing for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal protein layers, which seems to enable cells to form a more adequate extracellular matrix-like environment. STATEMENT OF SIGNIFICANCE: Natural polymer derived polyelectrolyte multilayers (PEMs) have been recently applied to adjust biomaterials to meet specific tissue demands. However, the effect of molecular composition of multilayers on both surface properties and cellular response, especially the fate of human adipose derived stem cells (hADSCs) upon osteogenic differentiation has not been studied extensively, yet. In addition, no studies exist that investigate a potential cell-dependent remodeling of PEMs made of extracellular matrix (ECM) components like collagens and glycosaminoglycans (GAGs). Furthermore, there is no knowledge whether the ability of cells to remodel PEM components may provide an added value regarding cell growth and differentiation. Finally, it has not been explored yet, how intrinsic cross-linking of ECM derived polyelectrolytes that improve the stability of PEMs will affect the differentiation potential of hADSCs. The current work aims to address these questions and found that the type of GAG has a strong effect on properties of multilayers and osteogenic differentiation of hADSCs. Additionally, we also show for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal layers as completely new finding, which allows cells to form an ECM-like environment supporting differentiation upon osteogenic lineage. The finding of this work may open new avenues of application of PEM systems made by layer by layer (LbL) technique in tissue engineering and regenerative medicine.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Colágeno Tipo I/química , Glicosaminoglicanos/química , Osteogênese , Células-Tronco/citologia , Fosfatase Alcalina/metabolismo , Biomarcadores/metabolismo , Adesão Celular , Proliferação de Células , Células Cultivadas , Eletrólitos/química , Humanos , Integrina alfa2beta1/metabolismo , Proteólise , Propriedades de SuperfícieRESUMO
Considering that vitronectin (VN) can promote both cell adhesion and matrix degradation, it is likely to play a dual role at the cell-biomaterial interface. In this paper we therefore describe details of the dynamic interplay between matrix adhesion and pericellular proteolysis in endothelial cells adhered to glass model substratum. Initially we show that coating concentration determines protein organization at the surface. When the protein coating density approached saturation (63 ng cm-2), VN spontaneously organized itself in multimeric aggregates at the surface (30-50 nm in diameter). At subsaturation protein density (17 ng cm-2) VN molecules were present predominantly as single entities, indicating that a minimum coating density was required for VN multimerization. By fluorescent visualization of surface-associated VN in different ways, we provide the first evidence of significant proteolytic remodelling of VN by endothelial cells (HUVECs) at the sites of αv integrin clusters. The degree of proteolysis was estimated using a novel approach relying on dequenching of FITC-labeled VN upon proteolytic activity, showing that about one-third of the surface-associated VN was proteolytically altered by adhering HUVECs. In addition, we demonstrate that HUVECs can internalize surface-associated VN and deposit it in a linear pattern along longitudinal actin filaments. Deposited VN was partly colocalized with urokinase receptors. Taken altogether, we elucidate the complex and dynamic behavior of VN during initial cell-biomaterials interactions, the equilibrium if which could have a significant impact on the biocompatibility of any blood contacting implants.
RESUMO
Developing appropriate cell culturing techniques to populate scaffolds has become a great challenge in tissue engineering. This work describes the use of spinner flask dynamic cell cultures to populate hydroxyapatite microcarriers for bone tissue engineering. The microcarriers were obtained through the emulsion of a self-setting aqueous α-tricalcium phosphate slurry in oil. After setting, hydroxyapatite microcarriers were obtained. The incorporation of gelatin in the liquid phase of the α-tricalcium phosphate slurry allowed obtaining hybrid gelatin/hydroxyapatite-microcarriers. Initial cell attachment on the microcarriers was strongly influenced by the speed of the dynamic culture, achieving higher attachment at low speed (40 r/min) as compared to high speed (80 r/min). Under moderate culture speeds (40 r/min), the number of cells present in the culture as well as the number of microcarrier-containing cells considerably increased after 3 days, particularly in the gelatin-containing microcarriers. At longer culture times in dynamic culture, hydroxyapatite-containing microcarriers formed aggregates containing viable and extracellular matrix proteins, with a significantly higher number of cells compared to static cultures.
RESUMO
Protein remodeling at the cell-material interface is an important phenomenon that should be incorporated into the design of advanced biomaterials for tissue engineering. In this work, we address the relationship between fibronectin (FN) activity at the material interface and remodeling, including proteolytic cascades. To do so, we studied FN adsorption on two chemically similar substrates, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA), which resulted in different distribution and conformation of the protein at the material interface: FN organized spontaneously upon adsorption on PEA into physiological-like fibrils, through a process called material-driven FN fibrillogenesis. The amount of adsorbed FN and its conformation were investigated in two different coating concentrations (2 and 20 µg/mL). Since FN activity at the material interface determines the initial cellular response, we followed the formation of focal adhesions (vinculin) and subsequent cell signaling by focal adhesion kinase (FAK) expression and its phosphorylation (pFAK). More detailed studies were performed to get further insights into integrin binding by crosslinking and extraction followed by immunofluorescence, as well as protein and gene expression for α5 and αv. To correlate cell adhesion with matrix degradation, gene expression and activity (zymography) of matrix metalloproteinases (MMPs) were measured. Overall, we demonstrated that the material-driven FN fibrillogenesis triggers proteolytic activity: MMP activity was higher on the material-driven FN fibrils, as a compensatory mechanism to the inability of cells to reorganize this FN network.
RESUMO
Cells assemble fibronectin (FN) into fibrils in a process mediated by integrins. For this process to occur, it is known that the presence of other serum proteins is necessary. However, the individual effect of these proteins on FN fibrillogenesis has not been addressed so far. In this study, the effect of vitronectin (VN), an ECM adhesion protein, on material-driven FN fibrillogenesis and cell-mediated FN reorganization is investigated. Poly(ethyl acrylate), PEA, which has previously shown the ability to induce the organization of FN into well-developed physiological-like networks upon adsorption, was employed as a material substrate. FN adsorption, cell adhesion and cellular FN reorganization in the presence or absence of VN were studied. Both FN surface density, quantified via western blot, and its distribution on PEA surfaces, determined via atomic force microscopy, were altered when FN was adsorbed competitively with VN at certain compositions. Moreover, the presence of VN on the material surfaces enhanced cell-mediated FN reorganization and secretion, in comparison with the process which took place in the presence of serum proteins.