Nonalcoholic fatty liver disease affects 30% of the United States population and its progression can lead to nonalcoholic steatohepatitis (NASH), and increased risks for cirrhosis and hepatocellular ...carcinoma. NASH is characterized by a highly heterogeneous liver microenvironment created by the fibrotic activity of hepatic stellate cells (HSCs). While HSCs have been widely studied in 2D, further advancements in physiologically relevant 3D culture platforms for the in vitro modeling of these heterogeneous environments are needed. In this study, the use of stiffness‐variable, extracellular matrix (ECM) protein‐conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation is demonstrated. These microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions are further employed. The 6 kPa fibronectin microgels are shown to significantly increase HSC matrix remodeling and metabolic activities in single or multiple‐component microenvironments. Overall, heterogeneous microenvironments consisting of multiple distinct ECM microgels promoted a decrease in HSC matrix remodeling and metabolic activities compared to homogeneous microenvironments. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.
The development and utility of stiffness‐variable, ECM protein‐conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate human hepatic stellate cell activation is demonstrated. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.
Skin tissue engineering is a developing technology to heal severe wounds. Combining polyvinyl alcohol (PVA) and silk fibroin (SF) nanofibers is a promising method of developing a skin scaffold ...because the resulting structure mimics collagen fibers. The aim of this research was to study the growth of human dermal fibroblasts (HDF) on a polyvinyl alcohol-silk fibroin (PVA-SF) nanofiber scaffold that was produced by electrospinning. Morphological characterization and chemical analysis of the scaffold were performed by scanning electron microscopy (SEM), Fourier transform infrared spectrophotometry (FTIR), and contact angle measurement. The biocompatibility of the scaffold was tested by MTT cytotoxicity assay, SEM analysis, adherence ratio calculation, and analysis of the HDF growth curve for 9 days. The FTIR results confirmed the presence of SF and PVA. The average fiber diameter and pore size of the PVA scaffold were greater than those of the PVA-SF scaffold. Both scaffolds had hydrophilic properties and were not cytotoxic. Thus, HDF can attach and grow on both types of scaffold better than HDF seeded on a polystyrene plate. In conclusion, the addition of SF to the PVA nanofibers caused bead formation, which affected the substrate topography, decreased hydrophilicity and also decreased the fiber diameter and pore size in the nanofiber scaffold compared to the PVA nanofiber scaffold without SF addition. SF addition increases cell attachment to the nanofiber scaffold and has potential to facilitate HDF cell growth.
Hydrogels have been used to design synthetic matrices that capture salient features of matrix microenvironments to study and control cellular functions. Recent advances in understanding of both ...extracellular matrix biology and biomaterial design have shown that biophysical cues are powerful mediators of cell biology, especially that of mesenchymal stromal cells (MSCs). MSCs have been tested in many clinical trials because of their ability to modulate immune cells in different pathological conditions. While roles of biophysical cues in MSC biology have been studied in the context of multilineage differentiation, their significance in regulating immunomodulatory functions of MSCs is just beginning to be elucidated. This review first describes design principles behind how biophysical cues in native microenvironments influence the ability of MSCs to regulate immune cell production and functions. We will then discuss how biophysical cues can be leveraged to optimize cell isolation, priming, and delivery, which can help improve the success of MSC therapy for immunomodulation. Finally, a perspective is presented on how implementing biophysical cues in MSC potency assay can be important in predicting clinical outcomes.
Stromal cells of mesenchymal origin are known to direct immune cell functions in vivo by secreting paracrine mediators. This property has been leveraged in developing mesenchymal stromal cell (MSC)-based therapeutics by adoptive transfer to treat immunological rejection and tissue injuries, which have been tested in over one thousand clinical trials to date, but with mixed success. Advances in biomaterial design have enabled precise control of biophysical cues based on how stromal cells interact with the extracellular matrix in microenvironments in situ. Investigators have begun to use this approach to understand how different matrix biophysical parameters, such as fiber orientation, porosity, dimensionality, and viscoelasticity impact stromal cell-mediated immunomodulation. The insights gained from this effort can potentially be used to precisely define the microenvironmental cues for isolation, priming, and delivery of MSCs, which can be tailored based on different disease indications for optimal therapeutic outcomes.
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One of the major drawbacks associated with autologous fat grafting is unpredictable graft retention. Various efforts to improve the survivability of these cells have been explored, but these methods ...are time‐consuming, complex, and demand significant technical skill. In our study, we examine the use of cryopreserved amniotic membrane as a source of exogenous growth factors to improve adipocyte survivability under normal and hypoxic conditions. Human primary preadipocytes were cultured in a gelatin‐ferulic acid (Gtn‐FA) hydrogel with variable oxygen concentration and treated with amniotic membrane‐derived condition medium (CM) for 7 days. This hydrogel provides a hypoxic environment and also creates a 3D cell culture to better mimic recipient site conditions. The O2 concentration in the hydrogel was measured by electron paramagnetic resonance oxygen imaging (EPROI). The conjugation of FA was confirmed by FTIR and NMR spectroscopy. The cell viability and adipocyte differentiation were analyzed by alamarBlue™ assay, Oil Red O staining, and RT‐qPCR. The expression of genes: Pref‐1, C/EBP β, C/EBP α, PPAR‐ƴ, SLC2A4, and VEGF‐A were quantified. The cell viability results show that the 50% CM showed significantly higher cell pre‐adipocyte cell viability. In addition, compared to normal conditions, hypoxia/CM provided higher PPAR‐ƴ (p < .05), SLC2A4, and VEGF‐A (p < .05) (early and terminal differentiating markers) mRNA expression. This finding demonstrates the efficacy of amniotic CM supplementation as a novel way to promote adipocyte survival and retention via the expression of key gene markers for differentiation and angiogenesis.
Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell ...population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia
. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD
and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD
without proton pumping
was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor
or NAD
precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD
regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD
regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.
The necessity of chondrocytes can be obtained by inducing Mesenchymal Stem Cells (MSCs) using internal and external stimuli. In this research, MSC was directed to be differentiated on modified ...substrate and L-ascorbic acid addition. The objective of this research is to direct human MSCs (hMSCs) to become chondrocytes on modified substrates by qualitative and quantitative evaluation using several spesific markers. Mesenchymal stem cells were obtained from adipose tissue (ADMSC). Cell stemness was evaluated using flow cytometry and multipotency analysis. Micropatterned substrate was developed using Parafilm ™ by doing mechanical method and pith size was measured after getting Scanning Electron Microscope (SEM) image and analyzedusing imageJ. The cells were seeded in 2.4× 104 cells/mL and incubated for 12 days. The expression of Collagen II was observed by immunocytochemistry method and sulphated-GAG using Alcian Blue Staining on day 6 and day 12. Cell morphology was observed using SEM imaging. ADMSCs were successfully obtained from adipose tissue and complied the mesenchymal stem cellscriteria. The micropatterned substrate was well developed using Parafilm ™ and could be used to modify the substrate because ADMSCs were only attached on pith (P) that was not covered by Parafilm ™ . ADMSCs expressed Collagen II and GAG on day 6 and 12 of observation.The cells morphology also confirmed that chondrogenicdifferentiation was affected by seeding density because ADMSCs were found in more rounded shape and also expressed more Collagen II in narrow pith than in widepith.
• Many ecologically important forest trees from dry areas have been insufficiently investigated for their ability to adapt to the challenges posed by climate change, which hampers the implementation ...of mitigation policies. We analyzed 14 common-garden experiments across the Mediterranean which studied the widespread thermophilic conifer Pinus halepensis and involved 157 populations categorized into five ecotypes.
• Ecotype-specific tree height responses to climate were applied to projected climate change (2071–2100 AD), to project potential growth patterns both locally and across the species’ range.
• We found contrasting ecotypic sensitivities to annual precipitation but comparatively uniform responses to mean temperature, while evidence of local adaptation for tree height was limited to mesic ecotypes. We projected intriguing patterns of response range-wide, implying either height inhibition or stimulation of up to 75%, and deduced that the ecotype currently experiencing more favorable (wetter) conditions will show the largest inhibition. Extensive height reductions can be expected for coastal areas of France, Greece, Spain and northern Africa.
• Our findings underline the fact that intraspecific variations in sensitivity to precipitation must be considered when projecting tree height responses of dry forests to future climate. The ecotype-specific projected performances call for management activities to ensure forest resilience in the Mediterranean through, for example, tailored deployment strategies.
Free radicals and oxidants are now implicated in physiological responses and in several diseases. Given the wide range of expertise of free radical researchers, application of the greater ...understanding of chemistry has not been uniformly applied to biological studies. We suggest that some widely used methodologies and terminologies hamper progress and need to be addressed. We make the case for abandonment and judicious use of several methods and terms and suggest practical and viable alternatives. These changes are suggested in four areas: use of fluorescent dyes to identify and quantify reactive species, methods for measurement of lipid peroxidation in complex biological systems, claims of antioxidants as radical scavengers, and use of the terms for reactive species.
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•Dye oxidation alone is insufficient evidence for detection of reactive species in biology.•Nonenzymatic lipid peroxidation assessment requires product analysis by mass spectroscopy.•Antioxidant claims require quantitative dose–response lowering of oxidative stress.•“ROS” and “RNS” should be used only when defined and the actual species are unknown.
Although clofazimine is used to treat multidrug-resistant tuberculosis (MDR-TB), there is scant information on its effectiveness and safety. The aim of this retrospective, observational study was to ...evaluate these factors as well as the tolerability of clofazimine in populations in Brazil, where it was administered at a daily dose of 100 mg·day
(body weight ≥45 kg) as part of a standardised MDR-TB treatment regimen until 2006 (thereafter pyrazinamide was used).All MDR-TB patients included in the Sistema de Informação de Tratamentos Especiais da Tuberculose (SITETB) individual electronic register were analysed. The effectiveness of clofazimine was assessed by comparing the treatment outcomes of patients undergoing clofazimine-containing regimens against those undergoing clofazimine-free regimens and its safety by describing clofazimine-attributed adverse events. A total of 1446 patients were treated with clofazimine-containing regimens and 1096 with pyrazinamide-containing regimens.Although success rates were similar in patients treated with clofazimine
those treated with pyrazinamide (880 out of 1446, 60.9%,
708 out of 1096, 64.6%; p=0.054), clofazimine-treated cases exhibited higher death rates due to tuberculosis than pyrazinamide-treated ones (314 out of 1446, 21.7%,
120 out of 1096, 10.9%) but fewer failures (78 out of 1446, 5.4%,
95 out of 1096, 8.7%) and less loss to follow-up (144 out of 1446, 10.0%,
151 out of 1096, 13.8%). No relevant differences were detected when comparing adverse events in patients treated with clofazimine-containing regimens to those treated with clofazimine-free regimens. However, the incidence of side-effects was less than previously reported (gastro-intestinal complaints: 10.5%; hyper-pigmentation: 50.2%; neurological disturbances: 9-13%).
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