570 Biowissenschaften, Biologie
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Artificial adipose tissue (AT) constructs are urgently needed to treat severe wounds, to replace removed tissue, or for the use as in vitro model to screen for potential drugs or study metabolic pathways. The clinical translation of products is mostly prevented by the absence of a vascular component that would allow a sustainable maintenance and an extension of the construct to a relevant size. With this study, we aimed to evaluate the suitability of a novel material based on bacterial cellulose (CBM) on the defined adipogenic differentiation of human adipose-derived stem cells (ASCs) and the maintenance of the received adipocytes (diffASCs) and human microvascular endothelial cells (mvECs) in mono- and coculture. A slight acceleration of adipogenic differentiation over regular tissue culture polystyrene (TCPS) was seen on CBM under defined conditions, whereas on the maintenance of the generated adipocytes, comparable effects were detected for both materials. CBM facilitated the formation of vascular like structures in monoculture of mvECs, which was not observed on TCPS. By contrast, vascular-like structures were detected in CBM and TCPS in coculture by the presence of diffASCs. Concluding, CBM represents a promising material in vascularized AT engineering with the potential to speed up and simplify the in vitro setup of engineered products.
Introduction
Despite its high accuracy, polysomnography (PSG) has several drawbacks for diagnosing obstructive sleep apnea (OSA). Consequently, multiple portable monitors (PMs) have been proposed.
Objective
This systematic review aims to investigate the current literature to analyze the sets of physiological parameters captured by a PM to select the minimum number of such physiological signals while maintaining accurate results in OSA detection.
Methods
Inclusion and exclusion criteria for the selection of publications were established prior to the search. The evaluation of the publications was made based on one central question and several specific questions.
Results
The abilities to detect hypopneas, sleep time, or awakenings were some of the features studied to investigate the full functionality of the PMs to select the most relevant set of physiological signals. Based on the physiological parameters collected (one to six), the PMs were classified into sets according to the level of evidence. The advantages and the disadvantages of each possible set of signals were explained by answering the research questions proposed in the methods.
Conclusions
The minimum number of physiological signals detected by PMs for the detection of OSA depends mainly on the purpose and context of the sleep study. The set of three physiological signals showed the best results in the detection of OSA.
Continuous manufacturing is becoming more important in the biopharmaceutical industry. This processing strategy is favorable, as it is more efficient, flexible, and has the potential to produce higher and more consistent product quality. At the same time, it faces some challenges, especially in cell culture. As a steady state has to be maintained over a prolonged time, it is unavoidable to implement advanced process analytical technologies to control the relevant process parameters in a fast and precise manner. One such analytical technology is Raman spectroscopy, which has proven its advantages for process monitoring and control mostly in (fed-) batch cultivations. In this study, an in-line flow cell for Raman spectroscopy is included in the cell-free harvest stream of a perfusion process. Quantitative models for glucose and lactate were generated based on five cultivations originating from varying bioreactor scales. After successfully validating the glucose model (Root Mean Square Error of Prediction (RMSEP) of ∼0.2 g/L), it was employed for control of an external glucose feed in cultivation with a glucose-free perfusion medium. The generated model was successfully applied to perform process control at 4 g/L and 1.5 g/L glucose over several days, respectively, with variability of ±0.4 g/L. The results demonstrate the high potential of Raman spectroscopy for advanced process monitoring and control of a perfusion process with a bioreactor and scale-independent measurement method.
Critical size bone defects and non-union fractions are still challenging to treat. Cell-loaded bone substitutes have shown improved bone ingrowth and bone formation. However, a lack of methods for homogenously colonizing scaffolds limits the maximum volume of bone grafts. Additionally, therapy robustness is impaired by heterogeneous cell populations after graft generation. Our aim was to establish a technology for generating grafts with a size of 10.5 mm in diameter and 25 mm of height, and thus for grafts suited for treatment of critical size bone defects. Therefore, a novel tailor-made bioreactor system was developed, allowing standardized flow conditions in a porous poly(L-lactide co-caprolactone) material. Scaffolds were seeded with primary human mesenchymal stem cells derived from four different donors. In contrast to static experimental conditions, homogenous cell distributions were accomplished under dynamic culture. Additionally, culture in the bioreactor system allowed the induction of osteogenic lineage commitment after one week of culture without addition of soluble factors. This was demonstrated by quantitative analysis of calcification and gene expression markers related to osteogenic lineage. In conclusion, the novel bioreactor technology allows efficient and standardized conditions for generating bone substitutes that are suitable for the treatment of critical size defects in humans.
Sleep quality and in general, behavior in bed can be detected using a sleep state analysis. These results can help a subject to regulate sleep and recognize different sleeping disorders. In this work, a sensor grid for pressure and movement detection supporting sleep phase analysis is proposed. In comparison to the leading standard measuring system, which is Polysomnography (PSG), the system proposed in this project is a non invasive sleep monitoring device. For continuous analysis or home use, the PSG or wearable actigraphy devices tends to be uncomfortable. Besides this fact, they are also very expensive. The system represented in this work classifies respiration and body movement with only one type of sensor and also in a non invasive way. The sensor used is a pressure sensor. This sensor is low cost and can be used for commercial proposes. The system was tested by carrying out an experiment that recorded the sleep process of a subject. These recordings showed the potential for classification of breathing rate and body movements. Although previous researches show the use of pressure sensors in recognizing posture and breathing, they have been mostly used by positioning the sensors between the mattress and bedsheet. This project however, shows an innovative way to position the sensors under the mattress.
The spreading area of cells has been shown to play a central role in the determination of cell fate and tissue morphogenesis; however, a clear understanding of how spread cell area is determined is still lacking. The observation that cell area and force generally increase with substrate rigidity suggests that cell area is dictated mechanically, by means of a force-balance between the cell and the substrate. A simple mechanical model, corroborated by experimental measurements of cell area and force is presented to analyze the temporal force balance between the cell and the substrate during spreading. The cell is modeled as a thin elastic disc that is actively pulled by lamellipodia protrusions at the cell front. The essential molecular mechanisms of the motor activity at the cell front, including, actin polymerization, adhesion kinetics, and the actin retrograde flow, are accounted for and used to predict the dynamics of cell spreading on elastic substrates; simple, closed-form expressions for the evolution of cell size and force are derived. Time-resolved, traction force microscopy, combined with measurements of cell area are performed to investigate the simultaneous variations of cell size and force. We find that cell area and force increase simultaneously during spreading but the force develops with an apparent delay relative to the increase in cell area. We demonstrate that this may reflect the strain-stiffening property of the cytoskeleton. We further demonstrate that the radial cell force is a concave function of spreading speed and that this may reflect the strengthening of cell–substrate adhesions during spreading.
The extracellular matrix (ECM) is the non-cellular part of tissues and represents the natural environment of the cells. Next to structural stability, it provides various physical, chemical, and mechanical cues that strongly regulate and influence cellular behavior and are required for tissue morphogenesis, differentiation, and homeostasis. Due to its promising characteristics, ECM is used in a wide range of tissue engineering and regenerative medicine approaches as a biomaterial for coatings and scaffolds. To date, there are two sources for ECM material. First, native ECM is generated by the removal of the residing cells of a tissue or organ (decellularized ECM; dECM). Secondly, cell-derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. Although both types of ECM were intensively used for tissue engineering and regenerative medicine approaches, studies directly characterizing and comparing them are rare. Hence, in the first part of this thesis, dECM from adipose tissue and cdECM from stem cells and adipogenic differentiated stem cells from adipose tissue (ASCs) were characterized towards their macromolecular composition, structural features, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans compared to cdECMs. Structural characteristics revealed an immature state of collagen fibers in cdECM samples. The obtained results revealed differences between the two ECMs that can relevantly impact cellular behavior and subsequently experimental outcome and should therefore be considered when choosing a biomaterial for a specific application. The establishment of a functional vascular system in tissue constructs to realize an adequate nutrient supply remains challenging. In the second part, the supporting effect of cdECM on the self‐assembled formation of prevascular‐like structures by microvascular endothelial cells (mvECs) was investigated. It could be observed that cdECM, especially adipogenic differentiated cdECM, enhanced the formation of prevascular-like structures. An increased concentration of proangiogenic factors was found in cdECM substrates. The demonstration of cdECMs capability to induce the spontaneous formation of prevascular‐like structures by mvECs highlights cdECM as a promising biomaterial for adipose tissue engineering. Depending on the purpose of the ECM material chemical modification might be necessary. In the third and last part, the chemical functionalization of cdECM with dienophiles (terminal alkenes, cyclopropene) by metabolic glycoengineering (MGE) was demonstrated. MGE allows the chemical functionalization of cdECM via the natural metabolism of the cells and without affecting the chemical integrity of the cdECM. The incorporated dienophile chemical groups can be specifically addressed via catalysts-free, cell-friendly inverse electron-demand Diels‐Alder reaction. Using this system, the successful modification of cdECM from ASCs with an active enzyme could be shown. The possibility to modify cdECM via a cell-friendly chemical reaction opens up a wide range of possibilities to improve cdECM depending on the purpose of the material. Altogether, this thesis highlighted the differences between adipose dECM and cdECM from ASCs and demonstrated cdECM as a promising alternative to native dECM for application in tissue engineering and regenerative medicine approaches.
Tissue constructs of physiologically relevant scale require a vascular system to maintain cell viability. However, in vitro vascularization of engineered tissues is still a major challenge. Successful approaches are based on a feeder layer (FL) to support vascularization. Here, we investigated whether the supporting effect on the self‐assembled formation of prevascular‐like structures by microvascular endothelial cells (mvECs) originates from the FL itself or from its extracellular matrix (ECM). Therefore, we compared the influence of ECM, either derived from adipose‐derived stem cells (ASCs) or adipogenically differentiated ASCs, with the classical cell‐based FL. All cell‐derived ECM (cdECM) substrates enabled mvEC growth with high viability. Prevascular‐like structures were visualized by immunofluorescence staining of endothelial surface protein CD31 and could be observed on all cdECM and FL substrates but not on control substrate collagen I. On adipogenically differentiated ECM, longer and higher branched structures could be found compared with stem cell cdECM. An increased concentration of proangiogenic factors was found in cdECM substrates and FL approaches compared with controls. Finally, the expression of proteins associated with tube formation (E‐selectin and thrombomodulin) was confirmed. These results highlight cdECM as promising biomaterial for adipose tissue engineering by inducing the spontaneous formation of prevascular‐like structures by mvECs.
The development of in vitro adipose tissue constructs is highly desired to cope with the increased demand for substitutes to replace damaged soft tissue after high graded burns, deformities or tumor removal. To achieve clinically relevant dimensions, vascularization of soft tissue constructs becomes inevitable but still poses a challenge. Adipose-derived stem cells (ASCs) represent a promising cell source for the setup of vascularized fatty tissue constructs as they can be differentiated into adipocytes and endothelial cells in vitro and are thereby available in sufficiently high cell numbers.
This review summarizes the currently known characteristics of ASCs and achievements in adipogenic and endothelial differentiation in vitro. Further, the interdependency of adipogenesis and angiogenesis based on the crosstalk of endothelial cells, stem cells and adipocytes is addressed at the molecular level. Finally, achievements and limitations of current co-culture conditions for the construction of vascularized adipose tissue are evaluated.
Age-dependent migratory behavior of human endothelial cells revealed by substrate microtopography
(2019)
Cell migration is part of many important in vivo biological processes and is influenced by chemical and physical factors such as substrate topography. Although the migratory behavior of different cell types on structured substrates has already been investigated, up to date it is largely unknown if specimen's age affects cell migration on structures. In this work, we investigated age-dependent migratory behavior of human endothelial cells from young (≤ 31 years old) and old (≥ 60 years old) donors on poly(dimethylsiloxane) microstructured substrates consisting of well-defined parallel grooves. We observed a decrease in cell migration velocity in all substrate conditions and in persistence length perpendicular to the grooves in cells from old donors. Nevertheless, in comparison to young cells, old cells exhibited a higher cell directionality along grooves of certain depths and a higher persistence time. We also found a systematic decrease of donor age dependent responses of cell protrusions in orientation, velocity and length, all of them decreased in old cells. These observations lead us to hypothesize a possible impairment of actin cytoskeleton network and affected actin polymerization and steering systems, caused by aging.
Gelatin is one of the most prominent biopolymers in biomedical material research and development. It is frequently used in hybrid hydrogels, which combine the advantageous properties of bio‐based and synthetic polymers. To prevent the biological component from leaching out of the hydrogel, the biomolecules can be equipped with azides. Those groups can be used to immobilize gelatin covalently in hydrogels by the highly selective and specific azide–alkyne cycloaddition. In this contribution, we functionalized gelatin with azides at its lysine residues by diazo transfer, which offers the great advantage of only minimal side‐chain extension. Approximately 84–90% of the amino groups are modified as shown by 1H‐NMR spectroscopy, 2,4,6‐trinitrobenzenesulfonic acid assay as well as Fourier‐transform infrared spectroscopy, rheology, and the determination of the isoelectric point. Furthermore, the azido‐functional gelatin is incorporated into hydrogels based on poly(ethylene glycol) diacrylate (PEG‐DA) at different concentrations (0.6, 3.0, and 5.5%). All hydrogels were classified as noncyctotoxic with significantly enhanced cell adhesion of human fibroblasts on their surfaces compared to pure PEG‐DA hydrogels. Thus, the new gelatin derivative is found to be a very promising building block for tailoring the bioactivity of materials.
Adipose tissue is related to the development and manifestation of multiple diseases, demonstrating the importance of suitable in vitro models for research purposes. In this study, adipose tissue lobuli were explanted, cultured, and used as an adipose tissue control to evaluate in vitro generated adipose tissue models. During culture, lobule exhibited a stable weight, lactate dehydrogenase, and glycerol release over 15 days. For building up in vitro adipose tissue models, we adapted the biomaterial gelatin methacryloyl (GelMA) composition and handling to homogeneously mix and bioprint human primary mature adipocytes (MA) and adipose-derived stem cells (ASCs), respectively. Accelerated cooling of the bioink turned out to be essential for the homogeneous distribution of lipid-filled MAs in the hydrogel. Last, we compared manual and bioprinted GelMA hydrogels with MA or ASCs and the explanted lobules to evaluate the impact of the printing process and rate the models concerning the physiological reference. The viability analyses demonstrated no significant difference between the groups due to additive manufacturing. The staining of intracellular lipids and perilipin A suggest that GelMA is well suited for ASCs and MA. Therefore, we successfully constructed physiological in vitro models by bioprinting MA-containing GelMA bioinks.
Blood vessel reconstruction is still an elusive goal for the development of in vitro models as well as artificial vascular grafts. In this study, we used a novel photo curable cytocompatible polyacrylate material (PA) for freeform generation of synthetic vessels. We applied stereolithography for the fabrication of arbitrary 3D tubular structures with total dimensions in the centimeter range, 300 µm wall thickness, inner diameters of 1 to 2 mm and defined pores with a constant diameter of approximately 100 µm or 200 µm. We established a rinsing protocol to remove remaining cytotoxic substances from the photo-cured PA and applied thio-modified heparin and RGDC-peptides to functionalize the PA surface for enhanced endothelial cell adhesion. A rotating seeding procedure was introduced to ensure homogenous endothelial monolayer formation at the inner luminal tube wall. We showed that endothelial cells stayed viable and adherent and aligned along the medium flow under fluid-flow conditions comparable to native capillaries. The combined technology approach comprising of freeform additive manufacturing (AM), biomimetic design, cytocompatible materials which are applicable to AM, and biofunctionalization of AM constructs has been introduced as BioRap® technology by the authors.
Though bioprinting is a forward-looking approach in bone tissue engineering, the development of bioinks which are on the one hand processable with the chosen printing technique, and on the other hand possess the relevant mechanical as well as osteoconductive features remains a challenge. In the present study, polymer solutions based on methacrylated gelatin and methacrylated hyaluronic acid modified with hydroxyapatite (HAp) particles (5 wt%) were prepared. Encapsulation of primary human adipose derived stem cells in the HAp-containing gels and culture for 28 d resulted in a storage moduli significantly increased to 126% ± 9.6% compared to the value on day 1 by the sole influence of the HAp. Additional use of osteogenic media components resulted in an increase of storage module up to 199% ± 27.8%. Similarly, the loss moduli was increased to 370% ± 122.1% under the influence of osteogenic media components and HAp. Those changes in rheological material characteristics indicate a distinct change in elastic and viscous hydrogel properties, and are attributed to extensive matrix production in the hydrogels by the encapsulated cells, what could also be proven by staining of bone matrix components like collagen I, fibronectin, alkaline phosphatase and osteopontin. When using the cell-laden polymer solutions as bioinks to build up relevant geometries, the ink showed excellent printability and the printed grid structure's integrity remained intact over a culture time of 28 d. Again, an intense matrix formation as well as upregulation of osteogenic markers by the encapsulated cells could be shown. In conclusion, we demonstrated that our HAp-containing bioinks and hydrogels on basis of methacrylated gelatin and hyaluronic acid are on the one hand highly suitable for the build up of relevant three-dimensional geometries with microextrusion bioprinting, and on the other hand exhibit a significant positive effect on bone matrix development and remodeling in the hydrogels, as indicated by rheological measurements and staining of bone components. This makes the developed composite hydrogels an excellent material for bone bioprinting approaches.
Increasing number of studies are focused on how adherent cells respond, in vitro, to different properties of a material. Typical properties are the surface chemistry, topographical cues (at the nano- and micro-scale) of the surface, and the substrate stiffness. Cell Response studies are of importance for designing new biomaterials with applications in cell culture technologies, regenerative medicine, or for medical implants. However, only very few studies take the cell age factor, respectively the donor age, into account. In this work, we tested two types of human vascular cells (smooth muscle and endothelial cells) from old and young donors on (a) micro-structured surfaces made of pol (dimethylsiloxane) or on (b) flat polyacrylamide hydrogels with varying stiffnesses. These experiments reveal age-dependent and cell typedependent differences in the cell response to the topography and stiffness, and may establish the Basis for further studies focusing on cell age-dependent responses.
Due to its availability and minimal invasive harvesting human adipose tissue-derived extracellular matrix (dECM) is often used as a biomaterial in various tissue engineering and healthcare applications. Next to dECM, cell-derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. So far both types of ECM were investigated extensively toward their application as (bio)material in tissue engineering and healthcare. However, a systematic characterization and comparison of soft tissue dECM and cdECM is still missing. In this study, we characterized dECM from human adipose tissue, as well as cdECM from human adipose-derived stem cells, toward their molecular composition, structural characteristics, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans compared with cdECMs. Structural characteristics revealed an immature state of the fibrous part of cdECM samples. By the identified differences, we aim to support researchers in the selection of a suitable ECM-based biomaterial for their specific application and the interpretation of obtained results.
The extracellular matrix (ECM) naturally surrounds cells in humans, and therefore represents the ideal biomaterial for tissue engineering. ECM from different tissues exhibit different composition and physical characteristics. Thus, ECM provides not only physical support but also contains crucial biochemical signals that influence cell adhesion, morphology, proliferation and differentiation. Next to native ECM from mature tissue, ECM can also be obtained from the in vitro culture of cells. In this study, we aimed to highlight the supporting effect of cell-derived- ECM (cdECM) on adipogenic differentiation. ASCs were seeded on top of cdECM from ASCs (scdECM) or pre-adipocytes (acdECM). The impact of ECM on cellular activity was determined by LDH assay, WST I assay and BrdU assay. A supporting effect of cdECM substrates on adipogenic differentiation was determined by oil red O staining and subsequent quantification. Results revealed no effect of cdECM substrates on cellular activity. Regarding adipogenic differentiation a supporting effect of cdECM substrates was obtained compared to control. With these results, we confirm cdECM as a promising biomaterial for adipose tissue engineering.
The detection and characterisation of oxide layers on metallic copper samples plays an important role for power electronic modules in the automotive industry. However, since precise identification of oxide layers by visual inspection is difficult and time consuming due to inhomogeneous colour distribution, a reliable and efficient method for estimating their thickness is needed. In this study, hyperspectral imaging in the visible wavelength range (425–725 nm) is proposed as an in-line inspection method for analysing oxide layers in real-time during processing of copper components such as printed circuit boards in the automotive industry. For implementation in the production line a partial least square regression (PLSR) model was developed with a calibration set of n = 12 with about 13,000 spectra per sample to determine the oxide layer thickness on top of the technical copper surfaces. The model shows a good prediction performance in the range of 0–30 nm compared to Auger electron spectroscopy depth profiles as a reference method. The root mean square error (RMSE) is 1.75 nm for calibration and 2.70 nm for full cross validation. Applied to an external dataset of four new samples with about 13,000 spectra per sample the model provides an RMSE of 1.84 nm for prediction and demonstrates the robustness of the model during real-time processing. The results of this study prove the ability and usefulness of the proposed method to estimate the thickness of oxide layers on technical copper. Hence, the application of hyperspectral imaging for the industrial process control of electronic devices is very promising.
In vitro models of human adipose tissue may serve as beneficial alternatives to animal models to study basic biological processes, identify new drug targets, and as soft tissue implants. With this approach, we aimed to evaluate adipose-derived stem cells (ASC) and mature adipocytes (MA) comparatively for the application in the in vitro setup of adipose tissue constructs to imitate native adipose tissue physiology. We used human primary MAs and human ASCs, differentiated for 14 days, and encapsulated them in collagen type I hydrogels to build up a three-dimensional (3D) adipose tissue model. The maintenance of the models was analyzed after seven days based on a viability staining. Further, the expression of the adipocyte specific protein perilipin A and the release of leptin and glycerol were evaluated. Gene transcription profiles of models based on dASCs and MAs were analyzed with regard to native adipose tissue. Compared to MAs, dASCs showed an immature differentiation state. Further, gene transcription of MAs suggests a behavior closer to native tissue in terms of angiogenesis, which supports MAs as preferred cell type. In contrast to native adipose tissue, genes of de novo lipogenesis and tissue remodeling were upregulated in the in vitro attempts.