Cell-specific response of NSIP- and IPF-derived fibroblasts to the modification of the elasticity, biological properties, and 3D architecture of the substrate

The fibrotic fibroblasts derived from idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP) are surrounded by specific environments, characterized by increased stiffness, aberrant extracellular matrix (ECM) composition, and altered lung architecture. The presented research was aimed at investigating the effect of biological, physical, and topographical modification of the substrate on the properties of IPF- and NSIP-derived fibroblasts, and searching for the parameters enabling their identification. Soft and stiff polydimethylsiloxane (PDMS) was chosen for the basic substrates, the properties of which were subsequently tuned. To obtain the biological modification of the substrates, they were covered with ECM proteins, laminin, fibronectin, and collagen. The substrates that mimicked the 3D structure of the lungs were prepared using two approaches, resulting in porous structures that resemble natural lung architecture and honeycomb patterns, typical of IPF tissue. The growth of cells on soft and stiff PDMS covered with proteins, traced using fluorescence microscopy, confirmed an altered behavior of healthy and IPF- and NSIP-derived fibroblasts in response to the modified substrate properties, enabling their identification. In turn, differences in the mechanical properties of healthy and fibrotic fibroblasts, determined using atomic force microscopy working in force spectroscopy mode, as well as their growth on 3D-patterned substrates were not sufficient to discriminate between cell lines

Thermoresponsive smart copolymer coatings based on P(NIPAM co-HEMA) and P(OEGMA-co-HEMA) brushes for regenerative medicine

The fabrication of multifunctional, thermoresponsive platforms for regenerative medicine based on polymers that can be easily functionalized is one of the most important challenges in modern biomaterials science. In this study, we utilized atom transfer radical polymerization (ATRP) to produce two series of novel smart copolymer brush coatings. These coatings were based on copolymerizing 2-hydroxyethyl methacrylate (HEMA) with either oligo(ethylene glycol) methyl ether methacrylate (OEGMA) or N-isopropylacrylamide (NIPAM). The chemical compositions of the resulting brush coatings, namely, poly(oligo(ethylene glycol) methyl ether methacrylate-co-2-hydroxyethyl methacrylate) (P(OEGMA-co-HEMA)) and poly(N-isopropylacrylamide-co-2- hydroxyethyl methacrylate) (P(NIPAM-co-HEMA)), were predicted using reactive ratios of the monomers. These predictions were then verified using time-of-flight-secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). The thermoresponsiveness of the coatings was examined through water contact angle (CA) measurements at different temperatures, revealing a transition driven by lower critical solution temperature (LCST) or upper critical solution temperature (UCST) or a vanishing transition. The type of transition observed depended on the chemical composition of the coatings. Furthermore, it was demonstrated that the transition temperature of the coatings could be easily adjusted by modifying their composition. The topography of the coatings was characterized using atomic force microscopy (AFM). To assess the biocompatibility of the coatings, dermal fibroblast cultures were employed, and the results indicated that none of the coatings exhibited cytotoxicity. However, the shape and arrangement of the cells were significantly influenced by the chemical structure of the coating. Additionally, the viability of the cells was correlated with the wettability and roughness of the coatings, which determined the initial adhesion of the cells. Lastly, the temperature-induced changes in the properties of the fabricated copolymer coatings effectively controlled cell morphology, adhesion, and spontaneous detachment in a noninvasive, enzyme-free manner that was confirmed using optical microscopy

The effect of butein on TGF-β1-induced phenotypic transition of asthmatic human bronchial fibroblasts to myofibroblasts

Wyciąg z roślin zawierających ekstrakt buteiny od tysiąca lat wykorzystywany był w medycynie Dalekiego Wschodu do leczenia różnych dolegliwości. Buteina jest naturalnym flawonoidem, należącym do grupy chalkonów i ma potwierdzone właściwości antyoksydacyjne, przeciwzapalne, przeciwnowotworowe i przeciwzwłóknieniowe.Astma to heterogenna choroba, charakteryzująca się przewlekłym zapaleniem dróg oddechowych. Jest globalnym problemem zdrowotnym dotyczącym wszystkich grup wiekowych. Obecne terapie sprowadzają się właściwie tylko do kontroli i łagodzenia bieżących objawów, ale nie są w stanie przewrócić zmian zwłóknieniowych oskrzeli i doprowadzić do wyzdrowienia. Wiadomo, że TGF-β odgrywa istotną rolę w większości procesów zachodzących podczas przebudowy oskrzeli, m.in. odpowiada za indukcję fenotypowego przejścia fibroblastów w miofibroblasty (FMT). Wzrost liczby miofibroblastów w tkance łącznej prowadzi do nadmiernej akumulacji białek macierzy zewnątrzkomórkowej, a dzięki ekspresji alfa aktyny mięśni gładkich (α-SMA) miofibroblasty mogą generować siłę kurczliwą.Celem pracy było zbadanie w modelu in vitro wpływu buteiny na indukowany TGF-β1 proces FMT fibroblastów oskrzelowych pochodzących od astmatyków. Proces FMT indukowany TGF-β1 jest znaczniej nasilony w populacjach fibroblastów pochodzących od pacjentów z astmą w porównaniu od fibroblastów izolowanych od osób zdrowych. Wykonano badania na pierwotnych populacjach komórek, które lepiej odzwierciedlają zachodzące procesy i uwzględniają różnorodność odpowiedzi pacjentów. Zastosowano nowoczesne metody biotechnologii i biologii komórki dla oceny żywotności, poziomu proliferacji komórek, aktywności potencjału FMT.W niniejszej pracy magisterskiej po raz pierwszy wykazano, że buteina w hodowli in vitro, w niecytotoksycznym i niecytostatycznym stężeniu, hamuje zajście procesu FMT stymulowanego TGF-β1, obniżając znacząco liczbę miofibroblastów w populacjach fibroblastów oskrzelowych pochodzących od pacjentów z astmą. Otrzymane wyniki analiz in-cell ELISA i Western Blot wykazują obniżenie poziomu białek profibrotycznych (α-SMA, fibronektyny, kolagenu I, Cx43) pod wpływem buteiny, co dodatkowo wspiera hipotezę o hamowaniu przez buteinę procesu FMT. Jednocześnie potwierdzono barwieniem immunofluorescencyjnym białek p-Smad po aktywacji komórek TGF-β1, że buteina ogranicza translokację dojądrową białka p-Smad2, co może być ważnym mechanizmem przyhamowania procesu FMT. Ponieważ obserwowane przyhamowanie kanonicznej ścieżki TGF-β/Smad2/3 obserwuje się na dość niskim poziomie można przypuszczać, że buteina wpływa i na aktywność innych niekanonicznych szlaków sygnalizacyjnych od TGF-β. Otrzymane wyniki wydają się bardzo obiecujące pod względem aktywności przeciwzwłóknieniowych buteiny i mogą stanowić podstawę dalszych badań dla opracowania nowych terapii astmy.Plants containing butein extract have been used for thousands of years in Far Eastern medicine to treat a variety of illnesses. Butein is a natural flavonoid that belongs to the chalcone group and has proven antioxidant, anti-inflammatory, anti-tumor, and anti-fibrotic properties.Asthma is a heterogeneous disease characterized by chronic inflammation of the airways. It is a global health problem affecting all age groups. Current therapies are limited to control and relief of current symptoms but are unable to reverse fibrotic changes in the bronchial wall or lead to recovery. TGF-β is known to play an important role in most of the processes occurring during bronchial remodeling, including the induction of transition of the fibroblasts to myofibroblasts (FMT). The increase in the number of myofibroblasts in connective tissue leads to an excessive accumulation of extracellular matrix proteins, and through the expression of alpha-smooth muscle actin (α-SMA), myofibroblasts can generate contractile force.This paper is aimed to investigate the effect of butein on the TGF-β1-induced FMT process of bronchial fibroblasts of asthma patients in an in vitro model. The FMT process is significantly enhanced in fibroblast populations derived from patients with asthma compared to fibroblasts isolated from healthy individuals. Studies have been performed on primary cell populations that better reflect the processes involved and take into account the diversity of patient responses. Modern biotechnology and cell biology methods were applied to assess viability, cell proliferation levels, FMT potential activity.In this thesis, it is shown for the first time that butein in culture in vitro, at non-cytotoxic and non-cytostatic concentrations, inhibits the TGF-β1-stimulated FMT by significantly reducing the number of myofibroblasts in bronchial fibroblast populations from patients with asthma. The obtained results of in-cell ELISA and Western Blot analyses show a decrease in the level of profibrotic proteins (α-SMA, fibronectin, collagen I, Cx43) under the influence of butein, which further supports the hypothesis that butein inhibits the FMT process. At the same time, immunofluorescence staining of p-Smad proteins after TGF-β1 cell activation confirmed that butein reduces the nuclear translocation of p-Smad2 protein, which may be an important mechanism of FMT inhibition. Since the observed inhibition of the canonical TGF-β/Smad2/3 pathway is observed at a rather low level, it can be speculated that butein also affects the activity of other non-canonical TGF-β signaling pathways. The results obtained seem very promising in terms of the anti-fibrotic activity of butein and may provide a basis for further studies to develop new therapies for asthma