Impact of Electrospun Piezoelectric Core–Shell PVDFhfp/PDMS Mesh on Tenogenic and Inflammatory Gene Expression in Human Adipose-Derived Stem Cells: Comparison of Static Cultivation with Uniaxial Cyclic Tensile Stretching

Specific microenvironments can trigger stem cell tenogenic differentiation, such as specific substrates or dynamic cell cultivation. Electrospun meshes composed by core–shell fibers (random or aligned; PDMS core; piezoelectric PVDFhfp shell) were fabricated by coaxial electrospinning. Elastic modulus and residual strain were assessed. Human ASCs were seeded on such scaffolds either under static conditions for 1 week or with subsequent 10% dynamic stretching for 10,800 cycles (1 Hz, 3 h), assessing load elongation curves in a Bose® bioreactor system. Gene expression for tenogenic expression, extracellular matrix, remodeling, pro-fibrotic and inflammatory marker genes were assessed (PCR). For cell-seeded meshes, the E modulus increased from 14 ± 3.8 MPa to 31 ± 17 MPa within 3 h, which was not observed for cell-free meshes. Random fibers resulted in higher tenogenic commitment than aligned fibers. Dynamic cultivation significantly enhanced pro-inflammatory markers. Compared to ASCs in culture flasks, ASCs on random meshes under static cultivation showed a significant upregulation of Mohawk, Tenascin-C and Tenomodulin. The tenogenic commitment expressed by human ASCs in contact with random PVDFhfp/PDMS paves the way for using this novel highly elastic material as an implant to be wrapped around a lacerated tendon, envisioned as a functional anti-adhesion membrane.

Optimization of exopolysaccharide synthesys by medicinal fungus Trametes versicolor in submerged culture

The species Trametes versicolor refers to medicinal mushroom well known in traditional Asian medicine for over 2000 years. Due to the long time required for basidiocarp formation, attention has recently been given to the submerged cultivation method for the production of mycelial biomass and various bioactive components. Exopolysaccharides (EPS) produced by Trametes versicolor are essential components possessing numerous functionalities and exhibiting potential medicinal applications. The subject of this investigation is higher fungus Trametes versicolor isolated from Bulgaria. Four well-defined culture media were studied to select the medium that maximizes production of EPS in submerged cultivation. The M3 was shown to provide the highest yields of EPS and was further investigated to optimize EPS production conditions. The initial glucose concentration was found to be the most important factor in both EPS production and cell growth. The maximum biopolymer quantity of 1.067 g/L was obtained at 40 g/L glucose. For examination and evaluation of the correlation between the carbon source and the complex influence of the nitrogen sources over the mycelial growth and the EPS synthesis and the optimization of the media orthogonal central composition design 23 with star arm ±α=1.454671 was applied. The experimental design was based on 21 combinations. Dynamic cultivation was carried out after the optimization of the media for determination of the effect of the duration of the cultivation process over the Trametes versicolor growth and EPS gain. Maximum EPS yield was observed after 216 hours.

Critical parameters in cultivation of experimental biofilms using the example of Pseudomonas fluorescens

Formation and treatment of biofilms present a great challenge for health care and industry. About 80% of human infections are associated with biofilms including biomaterial centered infections, like infections of prosthetic heart valves, central venous catheters, or urinary catheters. Additionally, biofilms can cause food and drinking water contamination. Biofilm research focusses on application of experimental biofilm models to study initial adherence processes, to optimize physico-chemical properties of medical materials for reducing interactions between materials and bacteria, and to investigate biofilm treatment under controlled conditions. Exploring new antimicrobial strategies plays a key role in a variety of scientific disciplines, like medical material research, anti-infectious research, plant engineering, or wastewater treatment. Although a variety of biofilm models exist, there is a lack of standardization for experimental protocols, and designing experimental setups remains a challenge. In this study, a number of experimental parameters critical for material research have been tested that influence formation and stability of an experimental biofilm using the non-pathogenic model strain of Pseudomonas fluorescens. These parameters include experimental time frame, nutrient supply, inoculum concentration, static and dynamic cultivation conditions, material properties, and sample treatment during staining for visualization of the biofilm. It was shown, that all tested parameters critically influence the experimental biofilm formation process. The results obtained in this study shall support material researchers in designing experimental biofilm setups.

Crosstalk of Diabetic Conditions with Static Versus Dynamic Flow Environment—Impact on Aortic Valve Remodeling

Type 2 diabetes mellitus (T2D) is one of the prominent risk factors for the development and progression of calcific aortic valve disease. Nevertheless, little is known about molecular mechanisms of how T2D affects aortic valve (AV) remodeling. In this study, the influence of hyperinsulinemia and hyperglycemia on degenerative processes in valvular tissue is analyzed in intact AV exposed to an either static or dynamic 3D environment, respectively. The complex native dynamic environment of AV is simulated using a software-governed bioreactor system with controlled pulsatile flow. Dynamic cultivation resulted in significantly stronger fibrosis in AV tissue compared to static cultivation, while hyperinsulinemia and hyperglycemia had no impact on fibrosis. The expression of key differentiation markers and proteoglycans were altered by diabetic conditions in an environment-dependent manner. Furthermore, hyperinsulinemia and hyperglycemia affect insulin-signaling pathways. Western blot analysis showed increased phosphorylation level of protein kinase B (AKT) after acute insulin stimulation, which was lost in AV under hyperinsulinemia, indicating acquired insulin resistance of the AV tissue in response to elevated insulin levels. These data underline a complex interplay of diabetic conditions on one hand and biomechanical 3D environment on the other hand that possesses an impact on AV tissue remodeling.

Characterization of bioactive substances MHGF-68 on tumour cell lines with LiveFlow In Vitro Technology

As part of experimental research, growth factor-like substances associated with MHV-68, named MHGF-68, were discovered in our laboratory. MHGF-68 activity was manifested by the ability to alter cell morphology, that is, normal phenotype to transformed, resp. suppresses the transformed phenotype of tumour cells. The aim of the experiments was to monitor the effect of MHGF-68 on the change of the cell actin cytoskeleton in the tumour cell line Hepa1c1c7, as well as the normal cell line NIH3T3, and compare conventional stationary cultivation and dynamic cultivation conditions using a LiveFlow system (In Vitro Technologies). LiveFlow is an advanced system to test the impact of different compounds on the cell cultures, which allows simulation of in vivo conditions thanks to continuous flow of cultivation medium. MHGF-68 was prepared with the infection of BHK-21 cells with MHV-68 virus under non-permissive conditions (41°C). After dynamic cultivation with MHGF-68, we observed changes in morphology on Hepa1c1c7 cells. In cells cultured in a dynamic environment, we observed more pronounced changes in cell morphology in comparison with cells cultured statically. We observed no changes in the cytoskeletal structures in the NIH 3T3 cell line affected by MHGF-68 in both types of cultivation. The advantage of LiveFlow in comparison to in vivo testing is that the experiments performed in this system are less time and money consuming. Dynamic cultivation in the LiveFlow system is suitable for optimizing experiments before testing substances in vivo.

Bioreactor Processed Stromal Cell Seeding and Cultivation on Decellularized Pericardium Patches for Cardiovascular Use

(1) Background: Decellularized xenogeneic tissues are promising matrices for developing tissue-engineered cardiovascular grafts. In vitro recellularization of these tissues with stromal cells can provide a better in vivo remodelling and a lower thrombogenicity of the graft. The process of recellularization can be accelerated using a cultivation bioreactor simulating physiological conditions and stimuli. (2) Methods: Porcine pericardium was decellularized using a custom-built decellularization system with an optimized protocol. Autologous porcine adipose-derived stromal cells (PrASCs), isolated from the subcutaneous fat tissue, were used for recellularizing the decellularized pericardium. A custom cultivation bioreactor allowing the fixing of the decellularized tissue into a special cultivation chamber was created. The bioreactor maintained micro-perfusion and pulsatile pressure stimulation in order to promote the ingrowth of PrASCs inside the tissue and their differentiation. (3) Results: The dynamic cultivation promoted the ingrowth of cells into the decellularized tissue. Under static conditions, the cells penetrated only to the depth of 50 µm, whereas under dynamic conditions, the tissue was colonized up to 250 µm. The dynamic cultivation also supported the cell differentiation towards smooth muscle cells (SMCs). In order to ensure homogeneous cell colonization of the decellularized matrices, the bioreactor was designed to allow seeding of the cells from both sides of the tissue prior to the stimulation. In this case, the decellularized tissue was recolonized with cells within 5 days of dynamic cultivation. (4) Conclusions: Our newly designed dynamic bioreactor markedly accelerated the colonization of decellularized pericardium with ASCs and cell differentiation towards the SMC phenotype.

Cultivation and characterization of human midbrain organoids in sensor integrated microfluidic chips

1.ABSTRACTWith its ability to emulate microarchitectures and functional characteristics of native organs in vitro, induced pluripotent stem cell (iPSC) technology has enabled the generation of a plethora of organotypic constructs, including that of the human midbrain. However, reproducibly engineering and differentiating such human midbrain organoids (hMOs) under a biomimetic environment favorable for brain development still remains challenging. This study sets out to address this problem by combining the potential of iPSC technology with the advantages of microfluidics, namely its precise control over fluid flow combined with sensor integration. Here, we present a novel sensor-integrated platform for the long-term cultivation and non-invasive monitoring of hMOs under an interstitial flow regime. Our results show that dynamic cultivation of iPSC-derived hMOs maintains high cellular viabilities and dopaminergic neuron differentiation over prolonged cultivation periods of up to 50 days.

Yield Data Provide New Insight into the Dynamic Evaluation of Maize’s Climate Suitability: A Case Study in Jilin Province, China

Examining the effects of climate change on spring maize, and its suitability under dynamic cultivation patterns, will aid strategic decision-making for future agricultural adaptation. This paper investigates the climate suitability of spring maize, based on daily data from 50 meteorological stations, and statistics on maize yield and area at the county level in Jilin Province, China, between 1986 and 2015. Based on a significant correlation between the cultivation patterns indicator ≥10 °C accumulated temperature (AAT10) and the average yield (R2 = 0.503), the yield data are used to determine suitable thresholds for meteorological factors under the dynamic cultivation pattern, and a fuzzy fitness approach is used to evaluate the climate suitability. The results showed a good agreement between suitability estimates and scaled observed yields (average d = 0.705). Moreover, good consistency between cultivation patterns, climate suitability and yield show that the late-maturing varieties of maize have gradually moved northward and eastward, and the areas of high suitability and high yield have gradually expanded eastward. In addition, drought and chilling hazard factors limit the suitability of climate resources, especially in the eastern and western regions.