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1.
Acta Physiologica Sinica ; (6): 381-391, 2022.
Article in Chinese | WPRIM | ID: wpr-939573

ABSTRACT

Extracellular matrix (ECM) stiffness is closely related to the physiological and pathological states of breast tissue. The current study was aimed to investigate the effect of silk fibroin/collagen composite hydrogels with adjustable matrix stiffness on the growth and phenotype of normal breast epithelial cells. In this study, the enzymatic reaction of horseradish peroxidase (HRP) with hydrogen peroxide (H2O2) was used to change the degree of cross-linking of the silk fibroin solution. The rotational rheometer was used to characterize the composite hydrogel's biomechanical properties. Human normal mammary epithelial cell line MCF-10A were inoculated into composite hydrogels with various stiffness (19.10-4 932.36 Pa) to construct a three dimensional (3D) culture system of mammary epithelial cells. The CCK-8 assay was applied to detect the cell proliferation rate and active states in each group. Hematoxylin-Eosin (HE) staining and whole-mount magenta staining were used for histological evaluation of cell morphology and distribution. The results showed that with the increase of matrix stiffness, MCF-10A cells exhibited inhibited proliferation rate, decreased formation of acinus structures and increased branching structures. Meanwhile, with the increase of matrix stiffness, the polarity of MCF-10A cells was impeded. And the increase of matrix stiffness up-regulated the expression levels of mmp-2, mmp-3, and mmp-9 in MCF-10A cells. Among the genes related to epithelial-mesenchymal transition (EMT), the expression level of the epithelial marker gene E-cadherin was significantly down-regulated, while the interstitial cell marker gene Vimentin was up-regulated, and the expression levels of Snail, Wnt5b and Integrin β1 in the Wnt pathway were up-regulated. These results suggest that the silk fibroin/collagen composite hydrogels with adjustable matrix stiffness regulates the proliferation and the phenotype of MCF-10A cells. The effects of increased matrix stiffness may be closely related to the changes of the polar structures and function of MCF-10A cells, as well as the occurrence of ECM-remodeling and EMT.


Subject(s)
Humans , Collagen/metabolism , Epithelial Cells/metabolism , Fibroins/pharmacology , Hydrogels/metabolism , Hydrogen Peroxide , Phenotype
2.
Article in Chinese | WPRIM | ID: wpr-936344

ABSTRACT

OBJECTIVE@#To assess the efficacy of GelMA hydrogel loaded with bone marrow stem cell-derived exosomes for repairing injured rat knee articular cartilage.@*METHODS@#The supernatant of cultured bone marrow stem cells was subjected to ultracentrifugation separate and extract the exosomes, which were characterized by transmission electron microscopy, particle size analysis and Western blotting of the surface markers. The changes in rheology and electron microscopic features of GelMA hydrogel were examined after loading the exosomes. We assessed exosome release from the hydrogel was detected by BCA protein detection method, and labeled the exosomes with PKH26 red fluorescent dye to observe their phagocytosis by RAW264.7 cells. The effects of the exosomes alone, unloaded hydrogel, and exosome-loaded hydrogel on the polarization of RAW264.7 cells were detected by q-PCR and immunofluorescence assay. We further tested the effect of the exosome-loaded hydrogel on cartilage repair in a Transwell co-culture cell model of RAW264.7 cells and chondrocytes in a rat model of knee cartilage injury using q-PCR and immunofluorescence assay and HE and Masson staining.@*RESULTS@#GelMA hydrogel loaded with exosomes significantly promoted M2-type polarization of RAW264.7 cells (P < 0.05). In the Transwell co-culture model, the exosome-loaded GelMA hydrogel significantly promoted the repair of injured chondrocytes by regulating RAW264.7 cell transformation from M1 to M2 (P < 0.05). HE and Masson staining showed that the exosome-loaded hydrogel obviously promoted cartilage repair in the rat models damage.@*CONCLUSION@#GelMA hydrogel loaded with bone marrow stem cell-derived exosomes can significantly promote the repair of cartilage damage in rats by improving the immune microenvironment.


Subject(s)
Animals , Rats , Bone Marrow Cells , Cartilage , Chondrocytes , Exosomes , Hydrogels/metabolism
3.
An. acad. bras. ciênc ; 89(1,supl): 745-755, May. 2017. tab, graf
Article in English | LILACS | ID: biblio-886671

ABSTRACT

ABSTRACT Enzymatically crossliked gelatin hydrogel was submitted to two different drying methods: air drying and freeze drying. The resulting polymeric tridimensional arrangement (compact or porous, respectively) led to different thermal and swelling properties. Significant differences (p < 0.05) on thermal and mechanical characteristics as well as swelling in non-enzymatic gastric and intestinal simulated fluids (37 ºC) were detected. Water absorption data in such media was modelled according to Higuchi, Korsmeyer-Peppas, and Peppas-Sahlin equations. Freeze dried hydrogel showed Fickian diffusion behavior while air dried hydrogels presented poor adjustment to Higuchi model suggesting the importance of the relaxation mechanism at the beginning of swelling process. It was possible to conclude that the same gelatin hydrogel may be suitable to different applications depending on the drying process used.


Subject(s)
Water , Hydrogels/metabolism , Freeze Drying , Gelatin/metabolism , Time Factors , Calorimetry, Differential Scanning , Microscopy, Electron, Scanning , Hydrogels/chemistry , Mechanical Phenomena , Gelatin/ultrastructure , Gelatin/chemistry
4.
São Paulo; s.n; s.n; 2017. 191 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-848096

ABSTRACT

A esterilidade é um requisito indispensável para a maioria dos biomateriais. Devido à sensibilidade dos hidrogéis, a sua esterilização apresenta-se como um desafio, sobretudo quando estão em causa nanoestruturas ou a presença de fármacos. O objetivo deste trabalho foi estudar os principais efeitos de diversos métodos de esterilização nas propriedades de diferentes tipos de sistemas à base de hidrogel. Além dos métodos convencionais (calor húmido e radiação gama) foi aferida a aplicabilidade da esterilização por ozono, um processo que embora se revele recente e ainda em fase de desenvolvimento, não deixa, no entanto, de ser bastante promissor. Estudou-se uma formulação de hidrogel natural nanoparticulado à base de quitosano (nanogel), com um largo espectro de possíveis aplicações, e duas formulações de hidrogéis sintéticos para aplicações oftálmicas (modelos de lentes de contato convencional e da nova geração de silicone-hidrogel), com e sem fármacos incorporados. Avaliaram-se alterações nas principais propriedades vitais ao bom desempenho e integridade dos materiais. No caso do nanogel: tamanho médio de partícula, potencial zeta, índice de polidispersão, absorvância, morfologia, estrutura química e citotoxicidade. No caso dos hidrogéis sintéticos: intumescimento, propriedades ópticas, permeabilidade iónica, molhabilidade, morfologia e topografia, estrutura química, propriedades mecânicas, citotoxicidade e perfil de libertação dos fármacos. Para garantir a melhor eficácia dos métodos de esterilização, realizaram-se testes de esterilidade após a contaminação intencional das amostras com diferentes cargas dos respetivos indicadores biológicos. A esterilização por calor húmido revelou-se o método mais agressivo, no caso do nanogel, e o menos agressivo para os hidrogéis sintéticos, quer na presença quer na ausência de fármacos. Relativamente à irradiação gama, para o nanogel, verificou-se que a sua resistência à irradiação aumenta consideravelmente na presença de açucares protetores. Já os hidrogéis sintéticos mostram-se resistentes à técnica, apresentando sinais de degradação evidente apenas para a dose mais elevada (25 kGy). Na presença de fármacos, a irradiação gama mostrou-se inadequada devido à sensibilidade dos mesmos. Os resultados obtidos são promissores no que diz respeito à aplicabilidade da esterilização por ozono, tanto para o nanogel como para os hidrogéis sintéticos, sem fármacos incorporados. Os fármacos sofrem, na generalidade, degradação quando expostos a este agente oxidante. Em suma, face à evidente complexidade dos fatores envolvidos (e.g. natureza, composição e propriedades dos materiais, estabilidade dos fármacos, condições e parâmetros dos processos esterilização), torna-se difícil generalizar os efeitos e prever o resultado dos métodos de esterilização. A escolha do processo mais adequado deve, portanto, ser feita caso a caso.


Sterility is mandatory requirement for most biomaterials. Because of their known sensitivity, hydrogel sterilization poses as a challenge, particularly when it comes to nanostructures or when drugs are incorporated. The main goal of this work was to study the key effects of different sterilization methods on the properties of different types of hydrogel-based systems. In addition to the conventional methods (steam heat and gamma radiation) the applicability of a promising new ozone method was assessed. Two formulations of synthetic hydrogels for ophthalmic applications (representing conventional contact lenses and new generation silicone-hydrogel contact lens), unloaded and loaded with ophthalmic drugs, were studied; and a natural chitosan-based nanostructured hydrogel (nanogel), with a broad spectrum of possible applications. Changes in the essential properties were evaluated. For synthetic hydrogels: swelling, optical properties, ionic permeability, wettability, morphology and topography, chemical structure, mechanical properties, cytotoxicity and drug release profile. For the nanogel: average particle size, zeta potential, polydispersity index, absorbance, morphology, chemical structure and cytotoxicity. In order to ensure the effectiveness of the sterilization methods, sterility tests were carried out after purposely contaminating the samples with different loads of biological indicators. Seam heat sterilization proved to be the most aggressive method for the nanogel, while being the least aggressive for the synthetic hydrogels in the presence and absence of drugs. Regarding gamma irradiation, the nanogel resistance to irradiation increased considerably in the presence of protective sugars. The synthetic hydrogels were resistant to this technique, showing signs of severe degradation only at the highest dose (25 kGy). In the presence of drugs, gamma irradiation proved to be inadequate due to drug degradation. The obtained results were encouraging with regard to the applicability of ozone sterilization for both the nanogel and the unloaded synthetic hydrogels. The drugs generally undergo degradation when exposed to this oxidizing agent. In view of the evident complexity of the factors involved (e.g. nature, composition and properties of materials, drug stability, conditions and parameters of sterilization processes), it is difficult to generalize the effects and predict the outcome of the sterilization methods. The selection of the most suitable procedure must be made on a case-by-case basis.


Subject(s)
Biocompatible Materials , Hydrogels/metabolism , Sterilization/methods , Gamma Rays , Ozone/analysis , Radiation
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