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1.
Carbohydr Polym ; 254: 117438, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33357911

RESUMO

Layer-by-layer self-assembly (LBL) is an effective method to prepare potential biomaterial with multilayer coatings, and few reports have focused on the variation of oriented microstructure during LBL process. In this study, polycaprolactone (PCL) and type І collagen (COL) were electrospun to oriented nanofibrous mats, and chitosan (CS) and COL molecules were then deposited on the mats by LBL technique. Zeta potential, FT-IR analysis and XPS measurement indicated the successful fabrication and modification. Changes in surface morphology and increase in surface roughness were observed in LBL process. Additionally, LBL-structured mats exhibited improved mechanical properties with the maximal tensile strength of 35.1 ± 7.0 MPa and the best elongation of 106.0 ± 11.5 %. CCK-8 and live/dead assays illustrated that the cell viability of the mats increased more than 20 % after LBL modification. More importantly, cells seeded onto the mats showed oriented adhesion and growth along the direction of nanofiber arrangement in LBL modified mats, which provided an effective strategy for realizing the controlled growth of cells.


Assuntos
Materiais Biocompatíveis/química , Quitosana/química , Colágeno Tipo I/química , Nanofibras/química , Poliésteres/química , Animais , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quitosana/farmacologia , Colágeno Tipo I/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos , Nanofibras/ultraestrutura , Resistência à Tração , Tecidos Suporte
2.
Food Funct ; 11(1): 328-338, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31799535

RESUMO

Skeletal muscle regeneration is a complicated process, requiring the proliferation, migration and differentiation of myoblasts whose processes are highly regulated by the extracellular matrix (ECM) surrounding the muscle tissues in vivo. However, the effects of respective ECM components on the regulation of myoblast behaviors are unknown. In this study, we report on the effect of collagen I, a major ECM component in muscle tissue and a popular food supplement, on mouse C2C12 myoblast proliferation, migration and differentiation as well as the underlying mechanisms. Collagen I (col 1) enhances the migration and myogenic differentiation of C2C12 cells, but has no effect on cell proliferation. Col I significantly promotes the production and release of interleukin-6 via nuclear translocation of nuclear factor κB (NF-κB) p65. The release of IL-6 plays a critical role in the col I-enhanced migration and differentiation of C2C12 cells. Furthermore, col I increases phosphorylation of focal adhesion kinase (FAK) that is involved in the nuclear translocation of NF-κB p65. Collectively, col I enhances the migration and differentiation of C2C12 cells through IL-6 release induced by FAK/NF-κB p65 activation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Quinase 1 de Adesão Focal/metabolismo , Interleucina-6/metabolismo , Mioblastos/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Animais , Linhagem Celular , Camundongos , Desenvolvimento Muscular , Mioblastos/citologia
3.
Biomolecules ; 9(12)2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31779287

RESUMO

BACKGROUND: Tumor cell binding to the microenvironment is regarded as the onset of therapeutic resistance, referred to as cell adhesion mediated drug resistance (CAM-DR). Here we elucidate whether CAM-DR occurs in ovarian cancer cells and contributes to still-existing cisplatin resistance. METHODS: Cultivation of W1 and cisplatin-resistant W1CR human ovarian cancer cells on collagen-type I (COL1) was followed by whole genome arrays, MTT assays focusing cisplatin cytotoxicity, and AAS detection of intracellular platinum levels. Expression of cisplatin transporters Ctr1 and MRP2 was analyzed. Mechanistic insight was provided by lentiviral ß1-integrin (ITGB1) knockdown, or inhibition of integrin-linked kinase (ILK). RESULTS: EC50 values of cisplatin cytotoxicity increased twofold when W1 and W1CR cells were cultivated on COL1, associated with significantly diminished intracellular platinum levels. Transporter deregulation could not be detected at mRNA levels but appears partially responsible at protein levels. The ITGB1 knockdown confirms that CAM-DR follows a COL1/ITGB1 signaling axis in W1 cells; thus, a blockade of ILK re-sensitized W1 cells on COL1 for cisplatin. In contrast, CAM-DR adds to cisplatin resistance in W1CR cells independent of ITGB1. CONCLUSIONS: CAM-DR appears relevant for ovarian cancer cells, adding to existing genetic resistance and thus emerges as a target for sensitization strategies.


Assuntos
Antineoplásicos/farmacologia , Adesão Celular/genética , Cisplatino/farmacologia , Integrina beta1/metabolismo , Neoplasias Ovarianas/metabolismo , Microambiente Tumoral/genética , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo I/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Ontologia Genética , Humanos , Integrina beta1/genética , Integrinas , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos
4.
Nat Commun ; 10(1): 4866, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653830

RESUMO

Despite the success of current therapies for acute myocardial infarction (MI), many patients still develop adverse cardiac remodeling and heart failure. With the growing prevalence of heart failure, a new therapy is needed that can prevent remodeling and support tissue repair. Herein, we report on injectable recombinant human collagen type I (rHCI) and type III (rHCIII) matrices for treating MI. Injecting rHCI or rHCIII matrices in mice during the late proliferative phase post-MI restores the myocardium's mechanical properties and reduces scar size, but only the rHCI matrix maintains remote wall thickness and prevents heart enlargement. rHCI treatment increases cardiomyocyte and capillary numbers in the border zone and the presence of pro-wound healing macrophages in the ischemic area, while reducing the overall recruitment of bone marrow monocytes. Our findings show functional recovery post-MI using rHCI by promoting a healing environment, cardiomyocyte survival, and less pathological remodeling of the myocardium.


Assuntos
Colágeno Tipo III/farmacologia , Colágeno Tipo I/farmacologia , Coração/efeitos dos fármacos , Infarto do Miocárdio/patologia , Proteínas Recombinantes/farmacologia , Função Ventricular/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Capilares/efeitos dos fármacos , Carbodi-Imidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cicatriz/patologia , Vasos Coronários/efeitos dos fármacos , Reagentes para Ligações Cruzadas/farmacologia , Dimetilaminas/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Monócitos/efeitos dos fármacos , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Succinimidas/farmacologia
5.
Lipids Health Dis ; 18(1): 166, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31470857

RESUMO

BACKGROUND: Atherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease. DESIGN AND METHODS: The aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen. RESULTS: Apo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites. CONCLUSIONS: These results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis.


Assuntos
Apolipoproteínas A/farmacologia , Colágeno Tipo I/farmacologia , Monócitos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Ácido Aminocaproico/farmacologia , Animais , Apolipoproteínas A/biossíntese , Apolipoproteínas A/química , Fibronectinas/farmacologia , Células HEK293 , Humanos , Metaloproteinase 9 da Matriz/biossíntese , Peso Molecular , Monócitos/citologia , Monócitos/metabolismo , Plasminogênio/farmacologia , Cultura Primária de Células , Ligação Proteica , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/farmacologia , Proteólise , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química
6.
J Mater Sci Mater Med ; 30(9): 107, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31512084

RESUMO

In the present study, collagen hydrogel containing naringin was fabricated, characterized and used as the scaffold for peripheral nerve damage treatment. The collagen was dissolved in acetic acid, naringin added to the collagen solution, and cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide powder (EDC; 0.10 mM) to form the hydrogel. The microstructure, swelling behavior, biodegradation, and cyto/hemocompatibility of the fabricated hydrogels were assessed. Finally, the healing efficacy of the prepared collagen hydrogel loaded with naringin on the sciatic nerve crush injury was assessed in the animal model. The characterization results showed that the fabricated hydrogels have a porous structure containing interconnected pores with the average pore size of 90 µm. The degradation results demonstrated that about 70% of the primary weight of the naringin loaded hydrogel had been lost after 4 weeks of storage in PBS. The in vitro study showed that the proliferation of Schwann cells on the collagen/naringin hydrogel was higher than the control group (tissue culture plate) at both 48 and 72 h after cell seeding and even significantly higher than pure collagen 72 h after cell seeding (*p < 0.005, **p < 0.001). The animal study implied that the sciatic functional index reached to -22.13 ± 3.00 at the end of 60th days post-implantation which was statistically significant (p < 0.05) compared with the negative control (injury without the treatment) (-82.60 ± 1.06), and the pure collagen hydrogel (-59.80 ± 3.20) groups. The hot plate latency test, the compound muscle action potential, and wet weight-loss of the gastrocnemius muscle evaluation confirmed the positive effect of the prepared hydrogels on the healing process of the induced nerve injury. In the final, the histopathologic examinations depicted that the collagen/naringin hydrogel group reduced all the histological changes induced from the nerve injury and showed more resemblance to the normal sciatic nerve, with well-arranged fibers and intact myelin sheath. The overall results implied that the prepared collagen/naringin hydrogel can be utilized as a sophisticated alternative to healing peripheral nerve damages.


Assuntos
Colágeno Tipo I/química , Flavanonas/farmacologia , Regeneração Tecidual Guiada/métodos , Hidrogéis/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Nervo Isquiático/lesões , Nervo Isquiático/fisiologia , Animais , Células Cultivadas , Colágeno Tipo I/farmacologia , Flavanonas/química , Humanos , Hidrogéis/química , Masculino , Teste de Materiais , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/terapia , Ratos , Ratos Wistar , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/fisiologia , Nervo Isquiático/efeitos dos fármacos , Tecidos Suporte/química , Cicatrização/efeitos dos fármacos
7.
J Am Soc Nephrol ; 30(9): 1605-1624, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31383731

RESUMO

BACKGROUND: The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus via a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with transcription factors. METHODS: To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation. RESULTS: We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and ß-actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. CONCLUSIONS: These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.


Assuntos
Colágeno Tipo IV/genética , Colágeno Tipo I/metabolismo , Receptor com Domínio Discoidina 1/metabolismo , Túbulos Renais Proximais/metabolismo , Actinas/metabolismo , Lesão Renal Aguda/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Núcleo Celular , Cromatina/metabolismo , Colágeno Tipo I/farmacologia , Colágeno Tipo IV/metabolismo , Receptor com Domínio Discoidina 1/genética , Humanos , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Sinais de Localização Nuclear , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Canais de Translocação SEC/metabolismo , Transcrição Genética
8.
Adv Healthc Mater ; 8(17): e1900595, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31328896

RESUMO

This report addresses the issue of optimizing extracellular matrix protein density required to support osteogenic lineage differentiation of mesenchymal stem cells (MSCs) by culturing MSCs on surface-bound density gradients of immobilized collagen type I (COL1) and osteopontin (OPN). A chemical surface gradient is prepared by tailoring the surface chemical composition from high hydroxyl groups to aldehyde groups using a diffusion-controlled plasma polymerization technique. Osteogenesis on the gradient surface is determined by immunofluorescence staining against Runx2 as an early marker and by staining of calcium phosphate deposits as a late stage differentiation marker. The Runx2 intensity and calcified area increase with increasing COL1 density up to a critical value corresponding to 124.2 ng cm-2 , above which cell attachment and differentiation do not rise further, while this critical value for OPN is 19.0 ng cm-2 . This gradient approach may facilitate the screening of an optimal biomolecule surface density on tissue-engineered scaffolds, implants, or tissue culture ware to obtain the desired cell response, and may generate opportunities for more cost-effective regenerative medicine.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Células-Tronco Mesenquimais/citologia , Osteogênese/efeitos dos fármacos , Aldeídos/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Etanol/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteopontina/farmacologia , Ratos Wistar
9.
Sci Adv ; 5(6): eaaw4991, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206025

RESUMO

The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration.


Assuntos
Reabsorção Óssea/genética , Calcificação Fisiológica/genética , Osteogênese/genética , Osteoprotegerina/genética , Tecidos Suporte , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/genética , Reabsorção Óssea/prevenção & controle , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Técnicas de Cocultura , Colágeno Tipo I/química , Colágeno Tipo I/farmacologia , Reagentes para Ligações Cruzadas/química , Expressão Gênica , Glicosaminoglicanos/química , Glicosaminoglicanos/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Cultura Primária de Células , Engenharia Tecidual , Transgenes
10.
Biomacromolecules ; 20(6): 2360-2371, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31050892

RESUMO

Structurally and functionally well-defined recombinant proteins are an interesting class of sequence-controlled macromolecules to which different crosslinking chemistries can be applied to tune their biological properties. Herein, we take advantage of a 571-residue recombinant peptide based on human collagen type I (RCPhC1), which we functionalized with supramolecular 4-fold hydrogen bonding ureido-pyrimidinone (UPy) moieties. By grafting supramolecular UPy moieties onto the backbone of RCPhC1 (UPy-RCPhC1), increased control over the polymer structure, assembly, gelation, and mechanical properties was achieved. In addition, by increasing the degree of UPy functionalization on RCPhC1, cardiomyocyte progenitor cells were cultured on "soft" (∼26 kPa) versus "stiff" (∼68-190 kPa) UPy-RCPhC1 hydrogels. Interestingly, increased stress fiber formation, focal adhesions, and proliferation were observed on stiffer compared to softer substrates, owing to the formation of stronger cell-material interactions. In conclusion, a bioinspired hydrogel material was designed by a combination of two well-known natural components, i.e., a protein as sequence-controlled polymer and UPy units inspired on nucleobases.


Assuntos
Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I , Miócitos Cardíacos/metabolismo , Células-Tronco/metabolismo , Animais , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular Transformada , Colágeno Tipo I/química , Colágeno Tipo I/farmacologia , Humanos , Camundongos , Miócitos Cardíacos/citologia , Células-Tronco/citologia
11.
J Cell Biochem ; 120(9): 15572-15584, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31038807

RESUMO

The major resolution of the study was to develop a dynamic form of natural biopolymer material to improve the wound healing by inhibition of biofilm formation on the surface. The extraction of collagen was effectively prepared from Scomberomorus lineolatus fish skin. Lyophilized collagen sheet was liquefied in 0.5M acetic acid to form acidic solubilized collagen (ASC) for further analysis. Physicochemical characterization of ASC was performed by various techniques using a standard protocol. The yield of ASC form S.lineolatus is higher (21.5%) than the previous reported studies. The effect of collagen solubility is gradually decreases with increasing concentration of NaCl and collagen is mostly soluble in acidic pH conditions. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of ASC contains α chain composition of α1 and α2 subunits and was characterized as type I collagen. Ultraviolet absorption was regulated as the appropriate wavelength to optimize the collagen. Fourier-transform infrared spectroscopy and X-ray diffraction confirmed that the isolated collagen is a triple-helical structure. The biofilm formation of Pseudomonas aeruginosa was significantly reduced by collagen incorporated with isolated 3,5,7-trihydroxyflavone (collagen-TF) sheet up to 70%. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay executed on fibroblast cell lines (L929) shows that the collagen-TF sheet was 100% compatible to enrich the cell adhesion and proliferation. The current study was the first report to extract, purify, and characterize ASC from S. lineolatus fish skin and characterize as type I collagen. Based on the result, we design the natural biodegradable collagen loaded with TF compound (collagen-TF) for antibiofilm properties. Compared with different sources of polymer, fish skin collagen is more effective and can be used as a biopolymer sheet for wound healing, food, drug delivery, tissue engineering, and pharmaceutical application.


Assuntos
Colágeno Tipo I/farmacologia , Proteínas de Peixes/farmacologia , Pele/química , Cicatrização/efeitos dos fármacos , Animais , Bandagens/microbiologia , Plásticos Biodegradáveis/química , Plásticos Biodegradáveis/farmacologia , Biofilmes/efeitos dos fármacos , Linhagem Celular , Colágeno Tipo I/química , Fibroblastos/efeitos dos fármacos , Proteínas de Peixes/química , Peixes/metabolismo , Humanos , Pele/metabolismo , Solubilidade
12.
Colloids Surf B Biointerfaces ; 180: 334-343, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31075687

RESUMO

Breast cancer cell lines lose the inherent gene expression profiles of their source tumor and when cultured as monolayers (two-dimensional) are unable to represent patient tumors. Thus, we engineered a biochemico- and mechano-mimetic three-dimensional (3D) culture platform for primary breast cancer cells by decellularizing cancer-associated fibroblasts (CAFs) cultured on 3D macroporous polymer scaffolds to recapitulate tumor behavior and drug response more realistically. The presence of the CAF-derived extracellular matrix deposited on the polycaprolactone scaffold promoted cell attachment and viability, which is ascribed to higher levels of phosphorylated Focal Adhesion Kinase that mediates cell attachment via integrins. Single cells from primary breast cancers self-organized into tumoroids on prolonged culture. Response of the tumoroids to two chemotherapeutic drugs, doxorubicin and mitoxanthrone, varied significantly across patient samples. This model could be used as an ex vivo platform to culture primary cells toward developing effective and personalized chemotherapy regimens.


Assuntos
Materiais Biomiméticos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Organoides/patologia , Medicina de Precisão , Técnicas de Cultura de Tecidos , Tecidos Suporte/química , Fibroblastos Associados a Câncer/patologia , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Junções Célula-Matriz/efeitos dos fármacos , Junções Célula-Matriz/metabolismo , Colágeno Tipo I/farmacologia , Matriz Extracelular/metabolismo , Feminino , Fibronectinas/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Pessoa de Meia-Idade , Poliésteres , Porosidade , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Células Tumorais Cultivadas
13.
Biomed Res Int ; 2019: 7343957, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31111065

RESUMO

Biocompatible scaffolding materials play an important role in bone tissue engineering. This study sought to develop and characterize a nano-hydroxyapatite (nHA)/collagen I (ColI)/multi-walled carbon nanotube (MWCNT) composite scaffold loaded with recombinant bone morphogenetic protein-9 (BMP-9) for bone tissue engineering by in vitro and in vivo experiments. The composite nHA/ColI/MWCNT scaffolds were fabricated at various concentrations of MWCNTs (0.5, 1, and 1.5% wt) by blending and freeze drying. The porosity, swelling rate, water absorption rate, mechanical properties, and biocompatibility of scaffolds were measured. After loading with BMP-9, bone marrow mesenchymal stem cells (BMMSCs) were seeded to evaluate their characteristics in vitro and in a critical sized defect in Sprague-Dawley rats in vivo. It was shown that the 1% MWCNT group was the most suitable for bone tissue engineering. Our results demonstrated that scaffolds loaded with BMP-9 promoted differentiation of BMMSCs into osteoblasts in vitro and induced more bone formation in vivo. To conclude, nHA/ColI/MWCNT scaffolds loaded with BMP-9 possess high biocompatibility and osteogenesis and are a good candidate for use in bone tissue engineering.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Osso e Ossos , Colágeno Tipo I/farmacologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Fator 2 de Diferenciação de Crescimento/farmacologia , Engenharia Tecidual , Animais , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Durapatita/farmacologia , Liofilização , Células-Tronco Mesenquimais , Nanoestruturas , Osteoblastos , Osteogênese/efeitos dos fármacos , Porosidade , Ratos , Ratos Sprague-Dawley , Tecidos Suporte
14.
Cells ; 8(4)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939867

RESUMO

A hallmark of ageing is the redistribution of body fat. Particularly, subcutaneous fat decreases paralleled by a decrease of skin collagen I are typical for age-related skin atrophy. In this paper, we hypothesize that collagen I may be a relevant molecule stimulating the differentiation of adipose-derived stem cells (ASCs) into adipocytes augmenting subcutaneous fat. In this context lipogenesis, adiponectin, and collagen I receptor expression were determined. Freshly isolated ASCs were characterized by stemness-associated surface markers by FACS analysis and then transdifferentiated into adipocytes by specific medium supplements. Lipogenesis was evaluated using Nile Red staining and documented by fluorescence microscopy or quantitatively measured by using a multiwell spectrofluorometer. Expression of adiponectin was measured by real-time RT-PCR and in cell-free supernatants by ELISA, and expression of collagen I receptors was observed by western blot analysis. It was found that supports coated with collagen I promote cell adhesion and lipogenesis of ASCs. Interestingly, a reverse correlation to adiponectin expression was observed. Moreover, we found upregulation of the collagen receptor, discoidin domain-containing receptor 2; receptors of the integrin family were absent or downregulated. These findings indicate that collagen I is able to modulate lipogenesis and adiponectin expression and therefore may contribute to metabolic dysfunctions associated with ageing.


Assuntos
Adipócitos/citologia , Tecido Adiposo/citologia , Colágeno Tipo I/farmacologia , Células-Tronco/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Integrina alfa2beta1/metabolismo , Lipogênese/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Colágeno/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos
15.
Tissue Eng Part A ; 25(21-22): 1550-1563, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30896295

RESUMO

Nowadays autologous fibroblast application for skin repair presents an important clinical interest. In most cases, in vitro skin cell culture is mandatory. However, cell expansion using xenogeneic or allogenic culture media presents some disadvantages, such as the risk of infection transmission or slow cell expansion. In this study, we investigated an autologous culture system to expand human skin fibroblast cells in vitro with the patient's own platelet-rich plasma (PRP). Human dermal fibroblasts were isolated from patients undergoing abdominoplasty, and blood was collected to prepare nonactivated PRP using the CuteCell™ PRP medical device. Cultures were followed up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Fibroblasts cultured in medium supplemented with PRP showed dose-dependently significantly higher proliferation rates (up to 7.7 times with 20% of PRP) and initiated a faster migration in the in vitro wound healing assay compared with FBS, while chromosomal stability was maintained. At high concentrations, PRP changed fibroblast morphology, inducing cytoskeleton rearrangement and an increase of alpha-smooth muscle actin and vimentin expression. Our findings show that autologous PRP is an efficient and cost-effective supplement for fibroblast culture, and should be considered as a safe alternative to xenogeneic/allogenic blood derivatives for in vitro cell expansion. Impact Statement Autologous dermal fibroblast graft is an important therapy in skin defect repair, but in vitro skin cell culture is mandatory in most cases. However, cell expansion using xenogeneic/allogenic culture media presents some disadvantages, such as the risk of infection transmission. We demonstrated that an autologous culture system with the patient's own platelet-rich plasma is an efficient, cost-effective, and safe supplement for fibroblast culture. As it respects the good manufacturing practices and regulatory agencies standards, it should be considered as a potent alternative and substitute to xenogeneic or allogenic blood derivatives for the validation of future clinical protocols using in vitro cell expansion.


Assuntos
Fibroblastos/citologia , Plasma Rico em Plaquetas/metabolismo , Actinas/metabolismo , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Derme/citologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Humanos , Laminina/farmacologia , Contagem de Plaquetas , Vimentina/metabolismo
16.
Mater Sci Eng C Mater Biol Appl ; 99: 905-918, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30889765

RESUMO

BACKGROUND: Due to unmet need for bone augmentation, our aim was to promote osteogenic differentiation of human adipose stem cells (hASCs) encapsulated in gellan gum (GG) or collagen type I (COL) hydrogels with bioactive glass (experimental glass 2-06 of composition [wt-%]: Na2O 12.1, K2O 14.0, CaO 19.8, P2O5 2.5, B2O3 1.6, SiO2 50.0) extract based osteogenic medium (BaG OM) for bone construct development. GG hydrogels were crosslinked with spermidine (GG-SPD) or BaG extract (GG-BaG). METHODS: Mechanical properties of cell-free GG-SPD, GG-BaG, and COL hydrogels were tested in osteogenic medium (OM) or BaG OM at 0, 14, and 21 d. Hydrogel embedded hASCs were cultured in OM or BaG OM for 3, 14, and 21 d, and analyzed for viability, cell number, osteogenic gene expression, osteocalcin production, and mineralization. Hydroxyapatite-stained GG-SPD samples were imaged with Optical Projection Tomography (OPT) and Selective Plane Illumination Microscopy (SPIM) in OM and BaG OM at 21 d. Furthermore, Raman spectroscopy was used to study the calcium phosphate (CaP) content of hASC-secreted ECM in GG-SPD, GG-BaG, and COL at 21 d in BaG OM. RESULTS: The results showed viable rounded cells in GG whereas hASCs were elongated in COL. Importantly, BaG OM induced significantly higher cell number and higher osteogenic gene expression in COL. In both hydrogels, BaG OM induced strong mineralization confirmed as CaP by Raman spectroscopy and significantly improved mechanical properties. GG-BaG hydrogels rescued hASC mineralization in OM. OPT and SPIM showed homogeneous 3D cell distribution with strong mineralization in BaG OM. Also, strong osteocalcin production was visible in COL. CONCLUSIONS: Overall, we showed efficacious osteogenesis of hASCs in 3D hydrogels with BaG OM with potential for bone-like grafts.


Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Colágeno Tipo I/farmacologia , Vidro/química , Osteogênese , Polissacarídeos Bacterianos/farmacologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Força Compressiva , Reagentes para Ligações Cruzadas/química , Durapatita/química , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Íons , Pessoa de Meia-Idade , Minerais/química , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ratos , Soro/metabolismo , Análise Espectral Raman , Células-Tronco/efeitos dos fármacos , Tecidos Suporte/química
17.
Connect Tissue Res ; 60(5): 463-476, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30871385

RESUMO

Extracellular matrix (ECM) molecules have multiple functions; prevention of cytotoxicity, provision of mechanical support, cell adhesive substrates and structural integrity in addition to mediation of cellular signaling. In this study, we report that the proliferation of INS-1 cells cultured on collagen I-coated dishes is enhanced, but it is inhibited on collagen V-coated dishes. Inhibitory proliferation on collagen V-coated is not due to apoptosis induction. Silibinin decreases hepatic glucose production and protects pancreatic ß-cells, as a potential medicine for type II diabetes. Silibinin up-regulates the proliferation of cells cultured on both collagen I- and V-coated dishes. Collagen-coating regulates gene expression of collagen in a collagen type-related manner. Silibinin increases mRNA expression of collagen I in the cells on collagen I- and V-coated dishes; however, silibinin decreases collagen V mRNA expression on collagen I- and V-coated dishes. Collagen I-coating significantly enhances nuclear translocation of ß-catenin, while collagen V-coating reduces it. Differential effects of silibinin on collagen I mRNA and collagen V mRNA can be accounted for by the finding that silibinin enhances nuclear translocation of ß-catenin on both collagen I- and V-coated dishes, since phenomenologically nuclear translocation of ß-catenin enhances collagen I mRNA but represses collagen V mRNA. These results demonstrate that nuclear translocation of ß-catenin up-regulates proliferation and collagen I gene expression, whereas it down-regulates collagen V gene expression of INS-1 cells. Differential gene expressions of collagen I and V by nuclear ß-catenin could be important for understanding fibrosis where collagen I and V may have differential effects.


Assuntos
Núcleo Celular/metabolismo , Colágeno Tipo I/farmacologia , Colágeno Tipo V/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Silibina/farmacologia , beta Catenina/metabolismo , Animais , Bovinos , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos
18.
J Tissue Eng Regen Med ; 13(3): 482-494, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30746894

RESUMO

HepaRG is a bipotent stem cell line that can be differentiated towards hepatocyte-like and biliary-like cells. The entire cultivation process requires 1 month and relies on the addition of 2% dimethyl sulfoxide (DMSO) to the culture. Our motivation in this research is to differentiate HepaRG cells (progenitor cells and undifferentiated cells) towards hepatocyte-like cells by minimizing the cultivation time and without using DMSO treatment by instead using a microfluidic device combined with the following strategies: (a) comparison of extracellular matrices (matrigel and collagen I), (b) types of flow (one or both sides), and (c) effects of DMSO. Our results demonstrate that matrigel promotes the differentiation of progenitor cells towards hepatocytes and biliary-like cells. Moreover, the frequent formation of HepaRG cell clusters was observed by a supply of both sides of flow, and the cell viability and liver specific functions were influenced by DMSO. Finally, differentiated HepaRG progenitor cells cultured in a microfluidic device for 14 days without DMSO treatment yielded 70% of hepatocyte-like cells with a highly polarized organization that reacted to stimulation with IL-6 to produce C-reactive protein (CRP). This culture model has high potential for investigating cell differentiation and liver pathophysiology research.


Assuntos
Diferenciação Celular , Fígado/citologia , Microfluídica , Células-Tronco/citologia , Proteína C-Reativa/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Polaridade Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno/farmacologia , Colágeno Tipo I/farmacologia , Dimetil Sulfóxido/farmacologia , Combinação de Medicamentos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Interleucina-6/farmacologia , Laminina/farmacologia , Proteoglicanas/farmacologia , Reologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
19.
J Tissue Eng Regen Med ; 13(5): 874-891, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30811090

RESUMO

The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.


Assuntos
Colágeno Tipo I , Fibroblastos , Queratinócitos , Pele/metabolismo , Traumatismos dos Tendões , Tendões , Engenharia Tecidual , Tecidos Suporte/química , Animais , Células Imobilizadas/metabolismo , Células Imobilizadas/patologia , Células Imobilizadas/transplante , Colágeno Tipo I/química , Colágeno Tipo I/farmacologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/transplante , Xenoenxertos , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/transplante , Ovinos , Pele/patologia , Traumatismos dos Tendões/metabolismo , Traumatismos dos Tendões/patologia , Traumatismos dos Tendões/terapia , Tendões/metabolismo , Tendões/patologia
20.
Biofabrication ; 11(3): 035002, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30769331

RESUMO

Bioprinting is rapidly developing into a powerful tool in tissue engineering, for both organ printing and the development of in vitro models that can be used in drug discovery, toxicology and in vitro bioreactors. Nevertheless, the ability to create complex 3D culture systems with different types of cells and extracellular matrices integrated with perfusable channels has been a challenge. Here we develop an approach that combines the xurography of a scaffold material (cellulose) with extrusion printing of bioinks onto it, followed by assembly in a layer-by-layer fashion to create complex 3D culture systems that could be used as in vitro models of biological processes. This new method, termed ExCeL, can recapitulate the complexities of natural tissues with a proper 3D distribution of cells, extracellular matrices, and different molecules, while providing the whole structure with mechanical stability, and direct and easy access to the cells, even the ones that are positioned deep in the bulk of the structure, without the need to fix or section the samples. Briefly, the bioprinting of predefined patterns with a feature size of ∼1 mm has been made possible by treating paper with the hydrogel's crosslinker and printing cell-embedded hydrogel that will solidify immediately upon contact with the paper. These impregnated layers can be used as single layers or in a layer-by-layer approach by stacking them (here up to four layers) for applications such as cell migration and proliferation in 3D structures composed of collagen or alginate. Cells are generally sensitive to the amount of FBS in their culture media and we have shown how FBS amount will effect cell migration. By cutting the paper in certain patterns, printing hydrogel on the remaining parts of it, and stacking the paper in layers, features like embedded channels are formed that will provide cells will better mass transfer in thick structures. This technique provides biologists with a unique tool to perform sophisticated in vitro assays.


Assuntos
Bioimpressão/métodos , Celulose/química , Modelos Biológicos , Tecidos Suporte/química , Células 3T3 , Animais , Cálcio/análise , Movimento Celular , Sobrevivência Celular , Colágeno Tipo I/farmacologia , Fibroblastos/citologia , Fluorescência , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Impressão Tridimensional
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