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1.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064719

RESUMO

Inflammation is a major cause of several chronic diseases and is reported to be recovered by the immuno-modulation of mesenchymal stem cells (MSCs). While most studies have focussed on the anti-inflammatory roles of MSCs in stem cell therapy, the impaired features of MSCs, such as the loss of homeostasis by systemic aging or pathologic conditions, remain incompletely understood. In this study, we investigated whether the altered phenotypes of human placenta-derived MSCs (hPD-MSCs) exposed to inflammatory cytokines, including TNF-α and IFN-γ, could be protected by MIT-001, a small anti-inflammatory and anti-necrotic molecule. MIT-001 promoted the spindle-like shape and cytoskeletal organization extending across the long cell axis, whereas hPD-MSCs exposed to TNF-α/IFN-γ exhibited increased morphological heterogeneity with an abnormal cell shape and cytoskeletal disorganization. Importantly, MIT-001 improved mitochondrial distribution across the cytoplasm. MIT-001 significantly reduced basal respiration, ATP production, and cellular ROS levels and augmented the spare respiratory capacity compared to TNF-α/IFN-γ-exposed hPD-MSCs, indicating enhanced mitochondrial quiescence and homeostasis. In conclusion, while TNF-α/IFN-γ-exposed MSCs lost homeostasis and mitochondrial quiescence by becoming over-activated in response to inflammatory cytokines, MIT-001 was able to rescue mitochondrial features and cellular phenotypes. Therefore, MIT-001 has therapeutic potential for clinical applications to treat mitochondrion-related inflammatory diseases.


Assuntos
Citoesqueleto/fisiologia , Células-Tronco Mesenquimais/fisiologia , Mitocôndrias/fisiologia , Compostos Orgânicos/farmacologia , Placenta/citologia , Citoesqueleto/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Espécies Reativas de Oxigênio/metabolismo
2.
Biomed Res Int ; 2021: 6695663, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937411

RESUMO

Background: When vascular endothelial cells are subjected to external stimuli, paracrine hormones and cytokines act on adjacent cells. The regulation of the biological behaviour of cells is closely related to the maintenance of organ function and the occurrence and development of disease. However, it is unclear whether vascular endothelial cells affect the biological behaviour of cells involved in wound repair through autocrine and paracrine mechanisms and ultimately play a role in wound healing. We aimed to verify the effect of the autocrine and paracrine functions of vascular endothelial cells on wound healing. Materials and Methods: ELISA was used to detect platelet-derived growth factor, basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor in human umbilical vascular endothelial cell-conditioned medium (HUVEC-CM). Different concentrations of HUVEC-CM were used to treat different stem cells. CCK-8 and scratch assays were used to detect the proliferation and migration ability of each cell. A full-thickness dorsal skin defect model was established in mice, and skin wound healing was observed after the local injection of HUVEC-CM, endothelial cell medium (ECM), or normal saline. H&E staining and immunofluorescence were used to observe the gross morphology of the wound tissue, the epithelial cell migration distance, and the expression of CD3 and CD31. Results: HUVEC-CM promotes the proliferation and migration of epidermal stem cells, skin fibroblasts, bone marrow mesenchymal stem cells, and HUVECs themselves. Furthermore, HUVEC-CM can promote angiogenesis in mouse skin wounds and granulation tissue formation and can accelerate wound surface epithelialization and collagen synthesis, thereby promoting wound healing. Conclusion: Our results clearly suggest that it is practicable and effective to promote wound healing with cytokines secreted by vascular endothelial cells in a mouse model.


Assuntos
Comunicação Autócrina , Células Endoteliais da Veia Umbilical Humana/metabolismo , Comunicação Parácrina , Pele/patologia , Cicatrização , Antígenos CD/metabolismo , Comunicação Autócrina/efeitos dos fármacos , Biomarcadores/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
3.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946412

RESUMO

Despite the huge body of research on osteogenic differentiation and bone tissue engineering, the translation potential of in vitro results still does not match the effort employed. One reason might be that the protocols used for in vitro research have inherent pitfalls. The synthetic glucocorticoid dexamethasone is commonly used in protocols for trilineage differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). However, in the case of osteogenic commitment, dexamethasone has the main pitfall of inhibiting terminal osteoblast differentiation, and its pro-adipogenic effect is well known. In this work, we aimed to clarify the role of dexamethasone in the osteogenesis of hBMSCs, with a particular focus on off-target differentiation. The results showed that dexamethasone does induce osteogenic differentiation by inhibiting SOX9 expression, but not directly through RUNX2 upregulation as it is commonly thought. Rather, PPARG is concomitantly and strongly upregulated, leading to the formation of adipocyte-like cells within osteogenic cultures. Limiting the exposure to dexamethasone to the first week of differentiation did not affect the mineralization potential. Gene expression levels of RUNX2, SOX9, and PPARG were simulated using approximate Bayesian computation based on a simplified theoretical model, which was able to reproduce the observed experimental trends but with a different range of responses, indicating that other factors should be integrated to fully understand how dexamethasone influences cell fate. In summary, this work provides evidence that current in vitro differentiation protocols based on dexamethasone do not represent a good model, and further research is warranted in this field.


Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , PPAR gama/metabolismo , Fatores de Transcrição SOX9/metabolismo , Adulto , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , PPAR gama/genética , Fatores de Transcrição SOX9/genética
4.
Gene ; 788: 145662, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33887373

RESUMO

INTRODUCTION: Culture conditions and differentiation cocktails may facilitate cell maturation and extracellular matrix (ECM) secretion and support the production of engineered fibroblastic tissues with applications in ligament regeneration. The objective of this study is to investigate the potential of two connective tissue-related ligands (i.e., BMP6 and GDF5) to mediate collagenous ECM synthesis and tissue maturation in vitro under normoxic and hypoxic conditions based on the hypothesis that BMP6 and GDF5 are components of normal paracrine signalling events that support connective tissue homeostasis. METHODS: Human adipose-derived MSCs were seeded on 3D-printed medical-grade polycaprolactone (PCL) scaffolds using a bioreactor and incubated in media containing GDF5 and/or BMP6 for 21 days in either normoxic (5% oxygen) or hypoxic (2% oxygen) conditions. Constructs were harvested on Day 3 and 21 for cell viability analysis by live/dead staining, structural analysis by scanning electron microscopy, mRNA levels by RTqPCR analysis, and in situ deposition of proteins by immunofluorescence microscopy. RESULTS: Pro-fibroblastic gene expression is enhanced by hypoxic culture conditions compared to normoxic conditions. Hypoxia renders cells more responsive to treatment with BMP6 as reflected by increased expression of ECM mRNA levels on Day 3 with sustained expression until Day 21. GDF5 was not particularly effective either in the absence or presence of BMP6. CONCLUSIONS: Fibroblastic differentiation of MSCs is selectively enhanced by BMP6 and not GDF5. Environmental factors (i.e., hypoxia) also influenced the responsiveness of cells to this morphogen.


Assuntos
Proteína Morfogenética Óssea 6/farmacologia , Técnicas de Cultura de Células/métodos , Fibroblastos/citologia , Fator 5 de Diferenciação de Crescimento/farmacologia , Células-Tronco Mesenquimais/citologia , Reatores Biológicos , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibroblastos/química , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Tecidos Suporte
5.
Mar Drugs ; 19(4)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805470

RESUMO

Fucoidans, sulfated polysaccharides extracted from brown algae, are marine products with the potential to modulate bone formation and vascularization processes. The bioactivity and safety of fucoidans are highly associated with their chemical structure, which may vary with algae species and extraction method. Thus, in depth evaluation of fucoidan extracts in terms of endotoxin content, cytotoxicity, and their detailed molecular biological impact on the individual cell types in bone is needed. In this study, we characterized fucoidan extracts from three different Fucus species including Fucus vesiculosus (Fv), Fucus serratus (Fs), and Fucus distichus subsp. evanescens (Fe) for their chemical features, endotoxin content, cytotoxicity, and bioactive effects on human outgrowth endothelial cells (OEC) and human mesenchymal stem cells (MSC) as in vitro models for bone function and vascularization. Extracts contained mainly high molecular weight (HMW) fucoidans and were free of endotoxins that may cause inflammation or influence vascularization. OEC tolerated fucoidan concentrations up to 200 µg/mL, and no indication of cytotoxicity was observed. The inflammatory response, however, investigated by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) and endothelial barrier assessed by impedance measurement differed for the individual extracts. MSC in comparison with endothelial cells were more sensitive to fucoidans and showed partly reduced metabolic activity and proliferation at higher doses of fucoidans. Further results for MSC indicated impaired osteogenic functions in alkaline phosphatase and calcification assays. All tested extracts consistently lowered important molecular mediators involved in angiogenesis, such a VEGF (vascular endothelial growth factor), ANG-1 (angiopoietin 1), and ANG-2 (angiopoietin 2), as indicated by RT-PCR and ELISA. This was associated with antiangiogenic effects at the functional level using selected extracts in co-culture models to mimic bone vascularization processes during bone regeneration or osteosarcoma.


Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Fucus/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Polissacarídeos/farmacologia , Inibidores da Angiogênese/isolamento & purificação , Proteínas Angiogênicas/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Células-Tronco Mesenquimais/metabolismo , Peso Molecular , Polissacarídeos/isolamento & purificação , Transdução de Sinais
6.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924614

RESUMO

The physiological O2 microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O2, OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O2. At 21% O2, we measured significant increases in ultimate tensile strength (p < 0.0001) and Young's modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O2 hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties.


Assuntos
Materiais Biomiméticos/farmacologia , Medula Óssea/metabolismo , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Oxigênio/metabolismo , Tecidos Suporte/química , Animais , Biomarcadores/metabolismo , Bombyx , Medula Óssea/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Pressão Parcial , Ratos Sprague-Dawley , Resistência à Tração
7.
Int J Mol Sci ; 22(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33920046

RESUMO

SmartBone® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-ß.


Assuntos
Matriz Óssea/química , Regeneração Óssea/efeitos dos fármacos , Substitutos Ósseos/farmacologia , Transplante de Células-Tronco Mesenquimais , Animais , Substitutos Ósseos/química , Bovinos , Diferenciação Celular/efeitos dos fármacos , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Vesículas Extracelulares/química , Vesículas Extracelulares/genética , Humanos , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Procedimentos Cirúrgicos Reconstrutivos/métodos
8.
Molecules ; 26(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33801973

RESUMO

Obese subjects have an increased risk of developing triple-negative breast cancer (TNBC), in part associated with the chronic low-grade inflammation state. On the other hand, epidemiological data indicates that increased consumption of polyphenol-rich fruits and vegetables plays a key role in reducing incidence of some cancer types. Here, we tested whether green tea-derived epigallocatechin-3-gallate (EGCG) could alter adipose-derived mesenchymal stem cell differentiation into adipocytes, and how this impacts the secretome profile and paracrine regulation of the TNBC invasive phenotype. Here, cell differentiation was performed and conditioned media (CM) from preadipocytes and mature adipocytes harvested. Human TNBC-derived MDA-MB-231 real-time cell migration was performed using the exCELLigence system. Differential gene arrays and RT-qPCR were used to assess gene expression levels. Western blotting was used to assess protein expression and phosphorylation status levels. In vitro vasculogenic mimicry (VM) was assessed with Matrigel. EGCG was found to inhibit the induction of key adipogenic biomarkers, including lipoprotein lipase, adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. Increased TNBC-derived MDA-MB-231 cell chemotaxis and vasculogenic mimicry were observed in response to mature adipocytes secretome, and this was correlated with increased STAT3 phosphorylation status. This invasive phenotype was prevented by EGCG, the JAK/STAT inhibitors Tofacitinib and AG490, as well as upon STAT3 gene silencing. In conclusion, dietary catechin-mediated interventions could, in part through the inhibition of adipogenesis and modulation of adipocytes secretome profile, prevent the onset of an obesogenic environment that favors TNBC development.


Assuntos
Catequina/análogos & derivados , Células-Tronco Mesenquimais/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Adiponectina/metabolismo , Animais , Catequina/metabolismo , Catequina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados , Feminino , Humanos , Leptina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Comunicação Parácrina/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Chá/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo
9.
Int J Nanomedicine ; 16: 2789-2801, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33880024

RESUMO

Objective: Gold nanorods (AuNRs) show great potential for versatile biomedical applications, such as stem cell therapy and bone tissue engineering. However, as an indispensable shape-directing agent for the growth of AuNRs, cetyltrimethylammonium bromide (CTAB) is not optimal for biological studies because it forms a cytotoxic bilayer on the AuNR surface, which interferes with the interactions with biological cells. Methods: Citrate-stabilized AuNRs with various aspect-ratios (Cit-NRI, Cit-NRII, and Cit-NRIII) were prepared by the combination of end-selective etching and poly(sodium 4-styrenesulfonate)-assisted ligand exchange method. Their effects on osteogenic differentiation of the pre-osteoblastic cell line (MC3T3-E1), rat bone marrow mesenchymal stem cells (rBMSCs), and human periodontal ligament progenitor cells (PDLPs) have been investigated. Potential signaling pathway of citrate-stabilized AuNRs-induced osteogenic effects was also investigated. Results: The experimental results showed that citrate-stabilized AuNRs have superior biocompatibility and undergo aspect-ratio-dependent osteogenic differentiation via expression of osteogenic marker genes, alkaline phosphatase (ALP) activity and formation of mineralized nodule. Furthermore, Wnt/ß-catenin signaling pathway might provide a potential explanation for the citrate-stabilized AuNRs-mediated osteogenic differentiation. Conclusion: These findings revealed that citrate-stabilized AuNRs with great biocompatibility could regulate the osteogenic differentiation of multiple cell types through Wnt/ß-catenin signaling pathway, which promote innovative AuNRs in the field of tissue engineering and other biomedical applications.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Ácido Cítrico/farmacologia , Ouro/farmacologia , Nanotubos/química , Osteogênese/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Calcificação Fisiológica/efeitos dos fármacos , Células Cultivadas , Cetrimônio/farmacologia , Endocitose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Nanotubos/ultraestrutura , Osteogênese/genética , Ligamento Periodontal/citologia , Ratos , Tiazolidinas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos
10.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806897

RESUMO

Adipose tissue and more specifically micro-fragmented adipose tissue (MFAT) obtained from liposuction has recently been shown to possess interesting medicinal properties whereby its application supports pain reduction and may enhance tissue regeneration particularly in osteoarthritis. Here we have characterised samples of MFAT produced using the Lipogems® International Spa system from eight volunteer individuals in order to understand the critical biological mechanisms through which they act. A variation was found in the MFAT cluster size between individual samples and this translated into a similar variation in the ability of purified mesenchymal stem cells (MSCs) to form colony-forming units. Almost all of the isolated cells were CD105/CD90/CD45+ indicating stemness. An analysis of the secretions of cytokines from MFAT samples in a culture using targeted arrays and an enzyme-linked immunosorbent assay (ELISA) showed a long-term specific and significant expression of proteins associated with anti-inflammation (e.g., interleukin-1 receptor alpha (Il-1Rα) antagonist), pro-regeneration (e.g., hepatocyte growth factor), anti-scarring and pro-angiogenesis (e.g., transforming growth factor beta 1 and 2 (TGFß1/2) and anti-bacterial (e.g., chemokine C-X-C motif ligand-9 (CXCL-9). Angiogenesis and angiogenic signalling were notably increased in primary bovine aortic endothelial cells (BAEC) to a different extent in each individual sample of the conditioned medium whilst a direct capacity of the conditioned medium to block inflammation induced by lipopolysaccharides was shown. This work characterises the biological mechanisms through which a strong, long-lasting, and potentially beneficial effect can be observed regarding pain reduction, protection and regeneration in osteoarthritic joints treated with MFAT.


Assuntos
Tecido Adiposo/química , Indutores da Angiogênese/química , Indutores da Angiogênese/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Indutores da Angiogênese/isolamento & purificação , Animais , Anti-Inflamatórios/isolamento & purificação , Bovinos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Citocinas/biossíntese , Células Endoteliais , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925714

RESUMO

A pericyte-like differentiation of human adipose-derived mesenchymal stem cells (ASCs) was tested in in vitro experiments for possible therapeutic applications in cases of diabetic retinopathy (DR) to replace irreversibly lost pericytes. For this purpose, pericyte-like ASCs were obtained after their growth in a specific pericyte medium. They were then cultured in high glucose conditions to mimic the altered microenvironment of a diabetic eye. Several parameters were monitored, especially those particularly affected by disease progression: cell proliferation, viability and migration ability; reactive oxygen species (ROS) production; inflammation-related cytokines and angiogenic factors. Overall, encouraging results were obtained. In fact, even after glucose addition, ASCs pre-cultured in the pericyte medium (pmASCs) showed high proliferation rate, viability and migration ability. A considerable increase in mRNA expression levels of the anti-inflammatory cytokines transforming growth factor-ß1 (TGF-ß1) and interleukin-10 (IL-10) was observed, associated with reduction in ROS production, and mRNA expression of pro-inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), and angiogenic factors. Finally, a pmASC-induced better organization of tube-like formation by retinal endothelial cells was observed in three-dimensional co-culture. The pericyte-like ASCs obtained in these experiments represent a valuable tool for the treatment of retinal damages occurring in diabetic patients.


Assuntos
Glucose/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Pericitos/metabolismo , Tecido Adiposo/metabolismo , Adulto , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Retinopatia Diabética/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Itália , Células-Tronco Mesenquimais/metabolismo , Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo
12.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924332

RESUMO

Mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs), have the most impressive ability to reduce inflammation through paracrine growth factors and cytokines that participate in inflammation. Tumor necrosis factor (TNF)-α bioactivity is a prerequisite in several inflammatory and autoimmune disease models. This study investigated the effects of TNF-α stimulate on ADSCs in the tumor microenvironment. The RNAseq analysis and cytokines assay demonstrated that TNF-α stimulated ADSCs proliferation and pro-inflammatory genes that correlated to leukocytes differentiation were upregulated. We found that upregulation of TLR2 or PTGS2 toward to IRF7 gene-associated with immunomodulatory and antitumor pathway under TNF-α treatment. In TNF-α-treated ADSCs cultured with the bladder cancer (BC) cell medium, the results showed that apoptosis ratio and OCT-4 and TLR2 genes which maintained the self-renewal ability of stem cells were decreased. Furthermore, the cell survival regulation genes including TRAF1, NF-kB, and IRF7 were upregulated in TNF-α-treated ADSCs. Additionally, these genes have not been upregulated in BC cell medium. A parallel study showed that tumor progressing genes were downregulated in TNF-α-treated ADSCs. Hence, the study suggests that TNF-α enhances the immunomodulatory potential of ADSCs during tumorigenesis and provides insight into highly efficacious MSC-based therapeutic options for BC.


Assuntos
Inflamação/patologia , Células-Tronco Mesenquimais/patologia , Microambiente Tumoral , Fator de Necrose Tumoral alfa/farmacologia , Neoplasias da Bexiga Urinária/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Imunomodulação/efeitos dos fármacos , Imunossupressão , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/imunologia
13.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802984

RESUMO

Hybrid composites of synthetic and natural polymers represent materials of choice for bone tissue engineering. Ulvan, a biologically active marine sulfated polysaccharide, is attracting great interest in the development of novel biomedical scaffolds due to recent reports on its osteoinductive properties. Herein, a series of hybrid polycaprolactone scaffolds containing ulvan either alone or in blends with κ-carrageenan and chondroitin sulfate was prepared and characterized. The impact of the preparation methodology and the polysaccharide composition on their morphology, as well as on their mechanical, thermal, water uptake and porosity properties was determined, while their osteoinductive potential was investigated through the evaluation of cell adhesion, viability, and osteogenic differentiation of seeded human adipose-derived mesenchymal stem cells. The results verified the osteoinductive ability of ulvan, showing that its incorporation into the polycaprolactone matrix efficiently promoted cell attachment and viability, thus confirming its potential in the development of biomedical scaffolds for bone tissue regeneration applications.


Assuntos
Organismos Aquáticos/química , Osso e Ossos/fisiologia , Osteogênese/efeitos dos fármacos , Poliésteres/química , Polissacarídeos/farmacologia , Engenharia Tecidual , Tecidos Suporte/química , Osso e Ossos/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Elasticidade , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Polissacarídeos/ultraestrutura , Espectroscopia de Infravermelho com Transformada de Fourier , Termogravimetria , Água/química
14.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809794

RESUMO

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5ß1, αvß3, and αIIbß3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30-40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fibronectinas/química , Células-Tronco Mesenquimais/citologia , Peptídeos/farmacologia , Alginatos , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células HeLa , Humanos , Integrinas/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Modelos Moleculares , Osteogênese/efeitos dos fármacos , Domínios Proteicos , Ratos , Receptores de Superfície Celular/metabolismo
15.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810359

RESUMO

Despite the strong evidence for the immunomodulatory activity of mesenchymal stromal cells (MSCs), clinical trials have so far failed to clearly show benefit, likely reflecting methodological shortcomings and lack of standardization. MSC-mediated tissue repair is commonly believed to occur in a paracrine manner, and it has been stated that extracellular vesicles (EVs) secreted by MSCs (EVMSC) are able to recapitulate the immunosuppressive properties of parental cells. As a next step, clinical trials to corroborate preclinical studies should be performed. However, effective dose in large mammals, including humans, is quite high and EVs industrial production is hindered by the proliferative senescence that affects MSCs during massive cell expansion. We generated a genetically modified MSC cell line overexpressing hypoxia-inducible factor 1-alpha and telomerase to increase the therapeutic potency of EVMSC and facilitate their large-scale production. We also developed a cytokine-based preconditioning culture medium to prime the immunomodulatory response of secreted EVs (EVMSC-T-HIFc). We tested the efficacy of this system in vitro and in a delayed-type hypersensitivity mouse model. MSC-T with an HIF-1α-GFP lentiviral vector (MSC-T-HIF) can be effectively expanded to obtain large amounts of EVs without major changes in cell phenotype and EVs composition. EVMSC-T-HIFc suppressed the proliferation of activated T-cells more effectively than did EVs from unmodified MSC in vitro, and significantly blunted the ear-swelling response in vivo by inhibiting cell infiltration and improving tissue integrity. We have developed a long-lived EV source that secretes high quantities of immunosuppressive EVs, facilitating a more standard and cost-effective therapeutic product.


Assuntos
Vesículas Extracelulares/transplante , Hipersensibilidade Tardia/terapia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imunomodulação , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Linfócitos T/imunologia , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Citocinas/farmacologia , Polpa Dentária/citologia , Vesículas Extracelulares/imunologia , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lentivirus/genética , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfócitos T/fisiologia , Telomerase/genética , Telomerase/metabolismo , Adulto Jovem
16.
Mol Med Rep ; 23(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33760123

RESUMO

Lipopolysaccharide (LPS) from oral pathogenic bacteria is an important factor leading to alveolar bone absorption and the implant failure. The present study aimed to evaluate the modulation of berberine hydrochloride (BBR) on the LPS-mediated osteogenesis and adipogenesis imbalance in rat bone marrow-derived mesenchymal stem cells (BMSCs). Cell viability, osteoblastic and adipogenic differentiation levels were measured using the Cell Counting Kit-8 assay, alkaline phosphatase (ALP) staining and content assay, and oil red O staining, respectively. Reverse transcription-quantitative PCR and immunoblotting were used to detect the related gene and protein expression levels. In undifferentiated cells, BBR increased the mRNA expression levels of the osteoblastic genes (Alp, RUNX family transcription factor 2, osteocalcin and secreted phosphoprotein 1) but not the adipogenic genes (fatty acid binding protein 4, Adipsin and peroxisome proliferator-activated receptorγ). LPS-induced osteoblastic gene downregulation, adipogenic gene enhancement and NF-κB activation were reversed by BBR treatment. In osteoblastic differentiated cells, decreased ALP production by LPS treatment was recovered with BBR co-incubation. In adipogenic differentiated cells, LPS-mediated lipid accumulation was decreased by BBR administration. The mRNA expression levels of the pro-inflammatory factors (MCP-1, TNF-α, IL-6 and IL-1ß) were increased by LPS under both adipogenic and osteoblastic conditions, which were effectively ameliorated by BBR. The actions of BBR were attenuated by compound C, suggesting that the role of BBR may be partly due to AMP-activated protein kinase activation. The results demonstrated notable pro-osteogenic and anti-adipogenic actions of BBR in a LPS-stimulated inflammatory environment. This indicated a potential role of BBR for bacterial infected-related peri-implantitis medication.


Assuntos
Adipogenia/efeitos dos fármacos , Berberina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Adipogenia/genética , Animais , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/genética , Ratos
17.
Am J Physiol Endocrinol Metab ; 320(4): E760-E771, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33645251

RESUMO

Apigenin (API), a natural plant flavone, is abundantly found in common fruits and vegetables. As a bioactive flavonoid, API exhibits several activities including antiproliferation and anti-inflammation. A recent study showed that API could retard osteoporosis progress, indicating its role in the skeletal system. However, the detailed function and mechanism remain obscure. In the present study, API was found to promote osteogenic differentiation of mesenchymal stem cells (MSCs). And further investigation showed that API could enhance the expression of the critical transcription factor ß-catenin and several downstream target genes of Wnt signaling, thus activated Wnt/ß-catenin signaling. Using a rat femoral fracture model, API was found to improve new bone formation and accelerate fracture healing in vivo. In conclusion, our data demonstrated that API could promote osteogenesis in vitro and facilitate the fracture healing in vivo via activating Wnt/ß-catenin signaling, indicating that API may be a promising therapeutic candidate for bone fracture repair.NEW & NOTEWORTHY 1) API promoted osteogenic differentiation of human MSCs in vitro; 2) API facilitated bone formation and accelerated fracture healing in vivo; 3) API stimulated Wnt/ß-catenin signaling during osteogenesis of human MSCs.


Assuntos
Apigenina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Adulto , Animais , Apigenina/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Fraturas Ósseas/tratamento farmacológico , Fraturas Ósseas/fisiopatologia , Humanos , Masculino , Células-Tronco Mesenquimais/fisiologia , Ratos , Ratos Sprague-Dawley , Via de Sinalização Wnt/efeitos dos fármacos
18.
Carbohydr Polym ; 261: 117878, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33766365

RESUMO

Hydrogels have gained great attentions as wound dressing. Binding to the tissue and preventing wound infection were the basic requirements for an "ideal dressing". We employed l-DOPA and ε-Poly-l-lysine to modify thermo-sensitive hydroxybutyl chitosan (HBC) to obtain (l-DOPA) - (ε-Poly-l-lysine)-HBC hydrogels (eLHBC). The eLHBC exhibited an almost 1.5 fold (P < 0.01) increase in wet adhesion strength compared to HBC. Upon the introduction of ε-Poly-l-lysine, eLHBC presented inherent antimicrobial property and prevented wound infection and inflammation response. Bone marrow mesenchymal stem cells (BMSCs) encapsulated in the eLHBC (BMSCs ⊂ eLHBC) could secret cytokins and growth factors via paracrine and promote the migration of fibroblast cells. BMSCs ⊂ eLHBC enhanced the complete skin-thickness wound healing via promoting collagen deposition and inhibiting infection and inflammation in vivo with wound closure rate being above 99 % after 15 days. The bioinspired, tissue-adhesive eLHBC could serve as advanced wound dressings for facilitating tissue repair and regeneration.


Assuntos
Adesivos , Curativos Hidrocoloides , Quitosana/análogos & derivados , Células-Tronco Mesenquimais/efeitos dos fármacos , Tecidos Suporte/química , Adesivos/síntese química , Adesivos/química , Adesivos/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Bioengenharia/métodos , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Bivalves/química , Bivalves/metabolismo , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Quitosana/síntese química , Quitosana/química , Quitosana/farmacologia , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Hidrogéis/síntese química , Hidrogéis/química , Hidrogéis/farmacologia , Masculino , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Testes de Sensibilidade Microbiana , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Coelhos , Ratos , Ratos Sprague-Dawley , Temperatura , Cicatrização/efeitos dos fármacos
19.
Phytomedicine ; 85: 153485, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33743412

RESUMO

BACKGROUND: Icariin (ICA) is a bioactive compound isolated from epimedium-derived flavonoids that modulates bone mesenchymal stem cell osteogenesis and adipogenesis. However, its precise mechanism in this process is unknown. PURPOSE: The purpose of this study was to elucidate the role of ICA on human bone mesenchymal stem cell (hBMSC) osteogenesis and adipogenesis by focusing on miR-23a mediated activation of the Wnt/ß-catenin signaling pathway. METHODS: After ICA treatment, hBMSC osteogenesis and adipogenesis were evaluated using alkaline phosphatase staining, an alkaline phosphatase activity assay, Oil Red O staining, and cellular triglyceride levels. Moreover, the mRNA and protein expression levels of osteogenic and adipogenic markers as well as key factors of the Wnt/ß-catenin signaling pathway were measured using quantitative reverse transcription polymerase chain reaction and western blotting. Lithium chloride, an activator of the Wnt/ß-catenin signaling pathway, was used as a positive control. Finally, to investigate the role of miR-23a in ICA-induced activation of the Wnt/ß-catenin signaling pathway, hBMSCs were transfected with miR-23a mimics or a miR-23a inhibitor. RESULTS: ICA significantly promoted hBMSC osteogenic differentiation by upregulating alkaline phosphatase activity and the expression of bone sialoprotein II (BSPII) and runt-related transcription factor-2 (Runx-2). In contrast, ICA inhibited hBMSC adipogenic differentiation by reducing lipid droplet formation and cellular triglyceride levels as well as by downregulating the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT enhancer-binding protein-α (C/EBP-α). ICA mediated its effects on hBMSCs by activating the Wnt/ß-catenin signaling pathway. It did so by upregulating ß-catenin, low density lipoprotein receptor-related protein 5 (LRP5), and T cell factor 1 (TCF1). Notably, the up-regulation of these proteins was blocked by Dickkopf-related protein 1 (DKK1). Critically, the effects of ICA on hBMSCs were similar to that of the positive control, lithium chloride. Notably, ICA-induced activation of the Wnt/ß-catenin signaling pathway was significantly attenuated following miR-23a up-regulation. Conversely, miR-23a downregulation affected hBMSCs in the same manner as ICA; i.e., it activated the Wnt/ß-catenin signaling pathway. CONCLUSION: ICA promotes and inhibits, respectively, hBMSC osteogenesis and adipogenesis via miR-23a-mediated activation of the Wnt/ß-catenin signaling pathway.


Assuntos
Adipogenia/efeitos dos fármacos , Flavonoides/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , MicroRNAs/genética , Osteogênese/efeitos dos fármacos , Via de Sinalização Wnt , Osso e Ossos/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Epimedium/química , Humanos , Sialoproteína de Ligação à Integrina/metabolismo , Células-Tronco Mesenquimais/citologia , beta Catenina/metabolismo
20.
Molecules ; 26(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669598

RESUMO

Synthetic arylamines and dietary phytophenolics could inhibit ferroptosis, a recently discovered regulated cell death process. However, no study indicates whether their inhibitory mechanisms are inherently different. Herein, the ferroptosis-inhibitory mechanisms of selected ferrostatin-1 (Fer-1) and two dietary stilbenes (piceatannol and astringin) were compared. Cellular assays suggested that the ferroptosis-inhibitory and electron-transfer potential levels decreased as follows: Fer-1 >> piceatannol > astringin; however, the hydrogen-donating potential had an order different from that observed by the antioxidant experiments and quantum chemistry calculations. Quantum calculations suggested that Fer-1 has a much lower ionization potential than the two stilbenes, and the aromatic N-atoms were surrounded by the largest electron clouds. By comparison, the C4'O-H groups in the two stilbenes exhibited the lowest bond disassociation enthalpies. Finally, the three were found to produce corresponding dimer peaks through ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry analysis. In conclusion, Fer-1 mainly depends on the electron transfer of aromatic N-atoms to construct a redox recycle. However, piceatannol and astringin preferentially donate hydrogen atoms at the 4'-OH position to mediate the conventional antioxidant mechanism that inhibits ferroptosis, and to ultimately form dimers. These results suggest that dietary phytophenols may be safer ferroptosis inhibitors for balancing normal and ferroptotic cells than arylamines with high electron-transfer potential.


Assuntos
Cicloexilaminas/farmacologia , Dieta , Ferroptose/efeitos dos fármacos , Glucosídeos/farmacologia , Fenilenodiaminas/farmacologia , Estilbenos/farmacologia , Animais , Antioxidantes/análise , Óxidos N-Cíclicos/química , Cicloexilaminas/química , Glucosídeos/química , Imidazóis/química , Concentração Inibidora 50 , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Modelos Moleculares , Fenilenodiaminas/química , Piperazinas/farmacologia , Ratos Sprague-Dawley , Eletricidade Estática , Estilbenos/química
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