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Local inflammation plays a pivotal role in the process of secondary damage after spinal cord injury. We recently reported that acute intravenous application of extracellular vesicles (EVs) secreted by human umbilical cord mesenchymal stromal cells dampens the induction of inflammatory processes following traumatic spinal cord injury. However, systemic application of EVs is associated with delayed delivery to the site of injury and the necessity for high doses to reach therapeutic levels locally. To resolve these two constraints, we injected EVs directly at the lesion site acutely after spinal cord injury. We report here that intralesional application of EVs resulted in a more robust improvement of motor recovery, assessed with the BBB score and sub-score, as compared to the intravenous delivery. Moreover, the intralesional application was more potent in reducing inflammation and scarring after spinal cord injury than intravenous administration. Hence, the development of EV-based therapy for spinal cord injury should aim at an early application of vesicles close to the lesion.
Bone grafts can be engineered by differentiating human mesenchymal stromal cells (MSCs) via the endochondral and intramembranous ossification pathways. We evaluated the effects of each pathway on the properties of engineered bone grafts and their capacity to drive bone regeneration. Bone-marrow-derived MSCs were differentiated on silk scaffolds into either hypertrophic chondrocytes (hyper) or osteoblasts (osteo) over 5 weeks of in vitro cultivation, and were implanted subcutaneously for 12 weeks. The pathways' constructs were evaluated over time with respect to gene expression, composition, histomorphology, microstructure, vascularization and biomechanics. Hypertrophic chondrocytes expressed higher levels of osteogenic genes and deposited significantly more bone mineral and proteins than the osteoblasts. Before implantation, the mineral in the hyper group was less mature than that in the osteo group. Following 12 weeks of implantation, the hyper group had increased mineral density but a similar overall mineral composition compared with the osteo group. The hyper group also displayed significantly more blood vessel infiltration than the osteo group. Both groups contained M2 macrophages, indicating bone regeneration. These data suggest that, similar to the body's repair processes, endochondral pathway might be more advantageous when regenerating large defects, whereas intramembranous ossification could be utilized to guide the tissue formation pattern with a scaffold architecture.
Regeneration of bone defects is often limited due to compromised bone tissue physiology. Previous studies suggest that engineered extracellular matrices enhance the regenerative capacity of mesenchymal stromal cells. In this study, we used human-induced pluripotent stem cells, a scalable source of young mesenchymal progenitors (hiPSC-MPs), to generate extracellular matrix (iECM) and test its effects on the osteogenic capacity of human bone-marrow mesenchymal stromal cells (BMSCs). iECM was deposited as a layer on cell culture dishes and into three-dimensional (3D) silk-based spongy scaffolds. After decellularization, iECM maintained inherent structural proteins including collagens, fibronectin and laminin, and contained minimal residual DNA. Young adult and aged BMSCs cultured on the iECM layer in osteogenic medium exhibited a significant increase in proliferation, osteogenic marker expression, and mineralization as compared to tissue culture plastic. With BMSCs from aged donors, matrix mineralization was only detected when cultured on iECM, but not on tissue culture plastic. When cultured in 3D iECM/silk scaffolds, BMSCs exhibited significantly increased osteogenic gene expression levels and bone matrix deposition. iECM layer showed a similar enhancement of aged BMSC proliferation, osteogenic gene expression, and mineralization compared with extracellular matrix layers derived from young adult or aged BMSCs. However, iECM increased osteogenic differentiation and decreased adipocyte formation compared with single protein substrates including collagen and fibronectin. Together, our data suggest that the microenvironment comprised of iECM can enhance the osteogenic activity of BMSCs, providing a bioactive and scalable biomaterial strategy for enhancing bone regeneration in patients with delayed or failed bone healing.
A major challenge for breath research is the lack of standardization in sampling and analysis. To address this, a test that utilizes a standardized intervention and a defined study protocol has been proposed to explore disparities in breath research across different analytical platforms and to provide benchmark values for comparison. Specifically, the Peppermint Experiment involves the targeted analysis in exhaled breath of volatile constituents of peppermint oil after ingestion of the encapsulated oil. Data from the Peppermint Experiment performed by proton transfer reaction mass spectrometry (PTR-MS) and selected ion flow tube mass spectrometry (SIFT-MS) are presented and discussed herein, including the product ions associated with the key peppermint volatiles, namely limonene, α- and β-pinene, 1,8-cineole, menthol, menthone and menthofuran. The breath washout profiles of these compounds from 65 individuals were collected, comprising datasets from five PTR-MS and two SIFT-MS instruments. The washout profiles of these volatiles were evaluated by comparing the log-fold change over time of the product ion intensities associated with each volatile. Benchmark values were calculated from the lower 95% confidence interval of the linear time-to-washout regression analysis for all datasets combined. Benchmark washout values from PTR-MS analysis were 353 min for the sum of monoterpenes and 1,8-cineole (identical product ions), 173 min for menthol, 330 min for menthofuran, and 218 min for menthone; from SIFT-MS analysis values were 228 min for the sum of monoterpenes, 281 min for the sum of monoterpenes and 1,8-cineole, and 370 min for menthone plus 1,8-cineole. Large inter- and intra-dataset variations were observed, whereby the latter suggests that biological variability plays a key role in how the compounds are absorbed, metabolized and excreted from the body via breath. This variability seems large compared to the influence of sampling and analytical procedures, but further investigations are recommended to clarify the effects of these factors.
Cyclic Tensile Stress Induces Skeletal Muscle Hypertrophy and Myonuclear Accretion in a 3D Model
(2023)
Skeletal muscle is highly adaptive to mechanical stress due to its resident stem cells and the pronounced level of myotube plasticity. Herein, we study the adaptation to mechanical stress and its underlying molecular mechanisms in a tissue-engineered skeletal muscle model. We subjected differentiated 3D skeletal muscle-like constructs to cyclic tensile stress using a custom-made bioreactor system, which resulted in immediate activation of stress-related signal transducers (Erk1/2, p38). Cell cycle re-entry, increased proliferation, and onset of myogenesis indicated subsequent myoblast activation. Furthermore, elevated focal adhesion kinase and β-catenin activity in mechanically stressed constructs suggested increased cell adhesion and migration. After 3 days of mechanical stress, gene expression of the fusogenic markers MyoMaker and MyoMixer, myotube diameter, myonuclear accretion, as well as S6 activation, were significantly increased. Our results highlight that we established a promising tool to study sustained adaptation to mechanical stress in healthy, hypertrophic, or regenerating skeletal muscle.
The rat median nerve model is a well-established and frequently used model for peripheral nerve injury and repair. The grasping test is the gold-standard to evaluate functional recovery in this model. However, no comprehensive review exists to summarize the course of functional recovery in regard to the lesion type. According to PRISMA-guidelines, research was performed, including the databases PubMed and Web of Science. Groups were: (1) crush injury, (2) transection with end-to-end or with (3) end-to-side coaptation and (4) isogenic or acellular allogenic grafting. Total and respective number, as well as rat strain, type of nerve defect, length of isogenic or acellular allogenic allografts, time at first signs of motor recovery (FSR) and maximal recovery grasping strength (MRGS), were evaluated. In total, 47 articles met the inclusion criteria. Group I showed earliest signs of motor recovery. Slow recovery was observable in group III and in graft length above 25 mm. Isografts recovered faster compared to other grafts. The onset and course of recovery is heavily dependent from the type of nerve injury. The grasping test should be used complementary in addition to other volitional and non-volitional tests. Repetitive examinations should be planned carefully to optimize assessment of valid and reliable data.
Extracorporeal shockwave therapy (ESWT) can stimulate processes to promote regeneration, including cell proliferation and modulation of inflammation. Specific miRNA expression panels have been established to define correlations with regulatory targets within these pathways. This study aims to investigate the influence of low-energy ESWT-applied within the subacute and chronic phase of SCI (spinal cord injury) on recovery in a rat spinal cord contusion model. Outcomes were evaluated by gait analysis, µCT and histological analysis of spinal cords. A panel of serum-derived miRNAs after SCI and after ESWT was investigated to identify injury-, regeneration- and treatment-associated expression patterns. Rats receiving ESWT showed significant improvement in motor function in both a subacute and a chronic experimental setting. This effect was not reflected in changes in morphology, µCT-parameters or histological markers after ESWT. Expression analysis of various miRNAs, however, revealed changes after SCI and ESWT, with increased miR-375, indicating a neuroprotective effect, and decreased miR-382-5p potentially improving neuroplasticity via its regulatory involvement with BDNF. We were able to demonstrate a functional improvement of ESWT-treated animals after SCI in a subacute and chronic setting. Furthermore, the identification of miR-375 and miR-382-5p could potentially provide new targets for therapeutic intervention in future studies.
Investigations reporting positive effects of extracorporeal shockwave therapy (ESWT) on nerve regeneration are limited to the rat sciatic nerve model. The effects of ESWT on muscle-in-vein conduits (MVCs) have also not been investigated yet. This study aimed to evaluate the effects of ESWT after repair of the rat median nerve with either autografts (ANGs) or MVCs. In male Lewis rats, a 7 mm segment of the right median nerve was reconstructed either with an ANG or an MVC. For each reconstructive technique, one group of animals received one application of ESWT while the other rats served as controls. The animals were observed for 12 weeks, and nerve regeneration was assessed using computerized gait analysis, the grasping test, electrophysiological evaluations and histological quantification of axons, blood vessels and lymphatic vasculature. Here, we provide for the first time a comprehensive analysis of ESWT effects on nerve regeneration in a rat model of median nerve injury. Furthermore, this study is among the first reporting the quantification of lymphatic vessels following peripheral nerve injury and reconstruction in vivo. While we found no significant direct positive effects of ESWT on peripheral nerve regeneration, results following nerve repair with MVCs were significantly inferior to those after ANG repair.
Lipedema is a chronic, progressive disease of adipose tissue with unknown etiology. Based on the relevance of the stromal vascular fraction (SVF) cell population in lipedema, we performed a thorough characterization of subcutaneous adipose tissue, SVF isolated thereof and the sorted populations of endothelial cells (EC), pericytes and cultured adipose-derived stromal/stem cells (ASC) of early-stage lipedema patients. We employed histological and gene expression analysis and investigated the endothelial barrier by immunofluorescence and analysis of endothelial permeability in vitro. Although there were no significant differences in histological stainings, we found altered gene expression of factors relevant for local estrogen metabolism (aromatase), preadipocyte commitment (ZNF423) and immune cell infiltration (CD11c) in lipedema on the tissue level, as well as in distinct cellular subpopulations. Machine learning analysis of immunofluorescence images of CD31 and ZO-1 revealed a morphological difference in the cellular junctions of EC cultures derived from healthy and lipedema individuals. Furthermore, the secretome of lipedema-derived SVF cells was sufficient to significantly increase leakiness of healthy human primary EC, which was also reflected by decreased mRNA expression of VE-cadherin. Here, we showed for the first time that the secretome of SVF cells creates an environment that triggers endothelial barrier dysfunction in early-stage lipedema. Moreover, since alterations in gene expression were detected on the cellular and/or tissue level, the choice of sample material is of high importance in elucidating this complex disease.
Rheumatoid arthritis is characterised by a progressive, intermittent inflammation at the synovial membrane, which ultimately leads to the destruction of the synovial joint. The synovial membrane as the joint capsule's inner layer is lined with fibroblast-like synoviocytes that are the key player supporting persistent arthritis leading to bone erosion and cartilage destruction. While microfluidic models that model molecular aspects of bone erosion between bone-derived cells and synoviocytes have been established, RA's synovial-chondral axis has not yet been realised using a microfluidic 3D model based on human patient in vitro cultures. Consequently, we established a chip-based three-dimensional tissue coculture model that simulates the reciprocal cross talk between individual synovial and chondral organoids. When co-cultivated with synovial organoids, we could demonstrate that chondral organoids induce a higher degree of cartilage physiology and architecture and show differential cytokine response compared to their respective monocultures highlighting the importance of reciprocal tissue-level cross talk in the modelling of arthritic diseases.
Coculture systems employing adipose tissue-derived mesenchymal stromal/stem cells (ASC) and endothelial cells (EC) represent a widely used technique to model vascularization. Within this system, cell-cell communication is crucial for the achievement of functional vascular network formation. Extracellular vesicles (EVs) have recently emerged as key players in cell communication by transferring bioactive molecules between cells. In this study we aimed to address the role of EVs in ASC/EC cocultures by discriminating between cells, which have received functional EV cargo from cells that have not. Therefore, we employed the Cre-loxP system, which is based on donor cells expressing the Cre recombinase, whose mRNA was previously shown to be packaged into EVs and reporter cells containing a construct of floxed dsRed upstream of the eGFP coding sequence. The evaluation of Cre induced color switch in the reporter system via EVs indicated that there is no EV-mediated RNA transmission either between EC themselves or EC and ASC. However, since Cre mRNA was not found present in EVs, it remains unclear if Cre mRNA is generally not packaged into EVs or if EVs are not taken up by the utilized cell types. Our data indicate that this technique may not be applicable to evaluate EV-mediated cell-to-cell communication in an in vitro setting using EC and ASC. Further investigations will require a functional system showing efficient and specific loading of Cre mRNA or protein into EVs.
Extracellular vesicles (EVs), such as exosomes, microvesicles, and apoptotic bodies, are cell-derived, lipid bilayer-enclosed particles mediating intercellular communication and are therefore vital for transmitting a plethora of biological signals. The vascular endothelium substantially contributes to the circulating particulate secretome, targeting important signaling pathways that affect blood cells and regulate adaptation and plasticity of endothelial cells in a paracrine manner. Different molecular signatures and functional properties of endothelial cells reflect their heterogeneity among different vascular beds and drive current research to understand varying physiological and pathological effects of blood and lymphatic endothelial EVs. Endothelial EVs have been linked to the development and progression of various vascular diseases, thus having the potential to serve as biomarkers and clinical treatment targets. This review aims to provide a brief overview of the human vasculature, the biology of extracellular vesicles, and the current knowledge of endothelium-derived EVs, including their potential role as biomarkers in disease development.
Multi-photon lithography (MPL) has proven to be a suitable tool to precisely control the microenvironment of cells in terms of the biochemical and biophysical properties of the hydrogel matrix. In this work, we present a novel method, based on multi-photon photografting of 4,4′-diazido-2,2′-stilbenedisulfonic acid (DSSA), and its capabilities to induce cell alignment, directional cell migration and endothelial sprouting in a gelatin-based hydrogel matrix. DSSA-photografting allows for the fabrication of complex patterns at a high-resolution and is a biocompatible, universally applicable and straightforward process that is comparably fast. We have demonstrated the preferential orientation of human adipose-derived stem cells (hASCs) in response to a photografted pattern. Co-culture spheroids of hASCs and human umbilical vein endothelial cells (HUVECs) have been utilized to study the directional migration of hASCs into the modified regions. Subsequently, we have highlighted the dependence of endothelial sprouting on the presence of hASCs and demonstrated the potential of photografting to control the direction of the sprouts. MPL-induced DSSA-photografting has been established as a promising method to selectively alter the microenvironment of cells.
Fibrin hydrogels have proven highly suitable scaffold materials for skeletal muscle tissue engineering in the past. Certain parameters of those types of scaffolds, however, greatly affect cellular mechanobiology and therefore the myogenic outcome. The aim of this study was to identify the influence of apparent elastic properties of fibrin scaffolds in 2D and 3D on myoblasts and evaluate if those effects differ between murine and human cells. Therefore, myoblasts were cultured on fibrin-coated multiwell plates (“2D”) or embedded in fibrin hydrogels (“3D”) with different elastic moduli. Firstly, we established an almost linear correlation between hydrogels’ fibrinogen concentrations and apparent elastic moduli in the range of 7.5 mg/ml to 30 mg/ml fibrinogen (corresponds to a range of 7.7–30.9 kPa). The effects of fibrin hydrogel elastic modulus on myoblast proliferation changed depending on culture type (2D vs 3D) with an inhibitory effect at higher fibrinogen concentrations in 3D gels and vice versa in 2D. The opposite effect was evident in differentiating myoblasts as shown by gene expression analysis of myogenesis marker genes and altered myotube morphology. Furthermore, culture in a 3D environment slowed down proliferation compared to 2D, with a significantly more pronounced effect on human myoblasts. Differentiation potential was also substantially impaired upon incorporation into 3D gels in human, but not in murine, myoblasts. With this study, we gained further insight in the influence of apparent elastic modulus and culture type on cellular behavior and myogenic outcome of skeletal muscle tissue engineering approaches. Furthermore, the results highlight the need to adapt parameters of 3D culture setups established for murine cells when applied to human cells.
Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types' migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC-LEC interaction.
Temporary scaffolds that mimic the extracellular matrix's structure and provide a stable substratum for the natural growth of cells are an innovative trend in the field of tissue engineering. The aim of this study is to obtain and design porous 2D fibroin-based cell matrices by femtosecond laser-induced microstructuring for future applications in muscle tissue engineering. Ultra-fast laser treatment is a non-contact method, which generates controlled porosity-the creation of micro/nanostructures on the surface of the biopolymer that can strongly affect cell behavior, while the control over its surface characteristics has the potential of directing the growth of future muscle tissue in the desired direction. The laser structured 2D thin film matrices from silk were characterized by means of SEM, EDX, AFM, FTIR, Micro-Raman, XRD, and 3D-roughness analyses. A WCA evaluation and initial experiments with murine C2C12 myoblasts cells were also performed. The results show that by varying the laser parameters, a different structuring degree can be achieved through the initial lifting and ejection of the material around the area of laser interaction to generate porous channels with varying widths and depths. The proper optimization of the applied laser parameters can significantly improve the bioactive properties of the investigated 2D model of a muscle cell matrix.
Keywords: biopolymers; femtosecond laser processing; muscle cell matrix 2D model; muscle tissue engineering; silk fibroin.
Tele-rehabilitation at home is one of the promising approaches in increasing rehabilitative success and simultaneously decreasing the
financial burden on the healthcare system. Objectives: Novel and mostly mobile devices are already in use, but shall be used in the future to a higher extent for allowing at home rehabilitation processes at a high quality level. The combination of exercises, assessments and available equipment is the basic objective of the
presented database. Methods: The database has been structured in order to allow easy-to-use and fast access for the three main user groups. Therapists – looking for exercise and equipment combinations – patients – rechecking their tasks for home exercises – and manufacturers – entering their equipment for specific use cases.
Results: The database has been evaluated by a proof of concept study and shows a high degree of applicability for the field of rehabilitative medicine. Currently it contains 110 exercises/assessments and 111 equipment/systems. Conclusion: Foundations of presented database are already established in the rehabilitative field of application, but can and will be enhanced in its functionality to be usable for a higher variety of medical fields and specificatios.
Dry powder inhalers are used by a large number of patients worldwide to treat respiratory diseases. The objective of this work is to experimentally investigate changes in aerosol particle diameter and particle number concentration of pharmaceutical aerosols generated by four dry powder inhalers under realistic inhalation and exhalation conditions. To simulate patients undergoing inhalation therapy, the active respiratory system model (xPULM™) was used. A mechanical upper airway model was developed, manufactured, and introduced as a part of the xPULM™ to represent the human upper respiratory tract with high fidelity. Integration of optical aerosol spectrometry technique into the setup allowed for evaluation of pharmaceutical aerosols. The results show that there is a significant difference (p < 0.05) in mean particle diameter between inhaled and exhaled particles with the majority of the particles depositing in the lung, while particles with the size of (>0.5 μm) are least influenced by deposition mechanisms. The fraction of exhaled particles ranges from 2.13% (HandiHaler®) over 2.94% (BreezHaler®), and 6.22% (Turbohaler®) to 10.24% (Ellipta®). These values are comparable to previously published studies. Furthermore, the mechanical upper airway model increases the resistance of the overall system and acts as a filter for larger particles (>3 μm). In conclusion, the xPULM™ active respiratory system model is a viable option for studying interactions of pharmaceutical aerosols and the respiratory tract regarding applicable deposition mechanisms. The model strives to support the reduction of animal experimentation in aerosol research and provides an alternative to experiments with human subjects.
Ziel dieser Studie war das überlagerte Elektromyogramm (ueEMG) und das decompositionierte (dEMG) auf deren Reliabilität und Aussagekraft zu überprüfen. Hierfür vollzogen zehn gesunde männliche Probanden zu drei Testzeitpunkten isometrische Ermüdungsmessungen. Hintergrund Bei der Messung des ueEMG werden die Motor Units Aktionspotentiale (MUAPS) elektrisch überlagert. Physiologische Muskelermüdung zeigt sich hier unter anderem in der Veränderung des Frequenzspektrums und der Signalamplitude. Letzteres steigt bei Ermüdung an, wohingegen die mediane Frequenz (MF) sinkt. Eine weitere Möglichkeit die Muskelermüdung mittels Oberflächen-EMG zu ermitteln ist die Anwendung des dEMGs. Dieses ermöglicht mithilfe einer Mustererkennungssoftware einzelne MUAPS zu erkennen. Studien zeigten, dass aktive Motor Units (MU) bei wiederholten oder anhaltenden submaximalen Kontraktionen als Muskelermüdung ihre Feuerungsrate erhöhen und neue MU rekrutiert werden. Bisher wurden diese Methoden jeweils einzeln betrachtet. Um sicherzustellen welcher Ansatz für klinische Daten geeigneter ist, wurden Datensätze zu beiden Methoden gleichzeitig generiert und hinsichtlich ihrer Aussagekraft und Wiederholbarkeit mit statistischen Mitteln verglichen.
Comparison of breathing patterns for aerosol inhalation using an electro-mechanical lung simulator
(2018)
Hybrid courses with a focus on practice-orientated education and self-guided learning phases are on the rise on the higher education sector. Disciplines in Life Sciences implicate a high degree of practical laboratory expertise. The University of Applied Sciences (UAS) in Vienna, Austria, has thus been endeavoured offering students a high qualitative education integrating hybrid courses based on PBL principles, which consist of on-site (including the transmission of necessary background and practical laboratory training) and off-site (including self-study phases) sessions. As practical laboratory units are central in those courses, the restrictive measures, including the transition to a complete online teaching format due to the first Covid-19-pandemic lock-down, had severe effects on the implementation and the quality of the curriculum. According to surveys made specifically to address this problematic situation, it can be concluded that on-site practical units are fundamental for certain disciplines such as Life Sciences.
The repair of large bone defects remains challenging and often requires graft material due to limited availability of autologous bone. In clinical settings, collagen sponges loaded with excessive amounts of bone morphogenetic protein 2 (rhBMP-2) are occasionally used for the treatment of bone non-unions, increasing the risk of adverse events. Therefore, strategies to reduce rhBMP-2 dosage are desirable. Silk scaffolds show great promise due to their favorable biocompatibility and their utility for various biofabrication methods. For this study, we generated silk scaffolds with axially aligned pores, which were subsequently treated with 10× simulated body fluid (SBF) to generate an apatitic calcium phosphate coating. Using a rat femoral critical sized defect model (CSD) we evaluated if the resulting scaffold allows the reduction of BMP-2 dosage to promote efficient bone repair by providing appropriate guidance cues. Highly porous, anisotropic silk scaffolds were produced, demonstrating good cytocompatibility in vitro and treatment with 10× SBF resulted in efficient surface coating. In vivo, the coated silk scaffolds loaded with a low dose of rhBMP-2 demonstrated significantly improved bone regeneration when compared to the unmineralized scaffold. Overall, our findings show that this simple and cost-efficient technique yields scaffolds that enhance rhBMP-2 mediated bone healing.
The natural extracellular matrix (ECM) provides the optimal environment for cells. Many enzymatic or non-enzymatic based strategies to extract ECM proteins from tissues were published over the past years. However, every single isolation strategy reported so far is associated with specific bottlenecks. In this study, frequently used strategies to isolate ECM from human placenta or adipose tissue using Tris-, serum-, or pepsin-based buffers were compared. The resulting ECM proteins were biochemically characterized by analysis of cellular remnants using Hoechst DNA staining, glycosaminoglycan (GAG) content by dimethylmethylene blue, visualization of protein bands using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis combined with amino acid quantification, and assessment of the proangiogenic profile using an angiogenesis array. Tris-NaCl-extracted ECM proteins showed a high heterogenic degree of extracted proteins, bioactive growth factors, and GAGs, but no collagen-I. Active serum-extracted ECM showed significant lower DNA remnants when compared with the Tris-NaCl isolation strategy. Pepsin-extracted ECM was rich in collagen-I and low amounts of remaining bioactive growth factors. This strategy was most effective to reduce DNA amounts when compared with the other isolation strategies. Pepsin-extracted ECM from both tissues easily gelled at 37°C, whereas the other extracted ECM strategies did not gel at 37°C (Tris-NaCl: liquid; serum: sponge). All relevant characteristics (DNA residues, ECM diversity and bioactivity, shape) of the extracted ECM proteins highly depend on its isolation strategy and could still be optimized. Impact statement The natural human extracellular matrix (ECM) is the ideal cell niche. Various strategies were reported to isolate human ECM components from various sources. In this article, we compared frequently used methods and compared their characteristics (DNA remnants, glycosaminoglycan content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, amino acid quantification, angiogenesis array, and gel formation). We conclude that more research is still necessary to optimize current isolation approaches for in vitro or in vivo applications of human ECM.
The additive manufacturing of low elastic modulus alloys that have a certain level of porosity for biomedical needs is a growing area of research. Here, we show the results of manufacturing of porous and dense samples by a laser powder bed fusion (LPBF) of Ti-Nb alloy, using two distinctive fusion strategies. The nanostructured Ti-Nb alloy powders were produced by mechanical alloying and have a nanostructured state with nanosized grains up to 90 nm. The manufactured porous samples have pronounced open porosity and advanced roughness, contrary to dense samples with a relatively smooth surface profile. The structure of both types of samples after LPBF is formed by uniaxial grains having micro- and nanosized features. The inner structure of the porous samples is comprised of an open interconnected system of pores. The volume fraction of isolated porosity is 2 vol. % and the total porosity is 20 vol. %. Cell viability was assessed in vitro for 3 and 7 days using the MG63 cell line. With longer culture periods, cells showed an increased cell density over the entire surface of a porous Ti-Nb sample. Both types of samples are not cytotoxic and could be used for further in vivo studies.
Due to their strong biomimetic potential, silk fibroin (SF) hydrogels are impressive candidates for tissue engineering, due to their tunable mechanical properties, biocompatibility, low immunotoxicity, controllable biodegradability, and a remarkable capacity for biomaterial modification and the realization of a specific molecular structure. The fundamental chemical and physical structure of SF allows its structure to be altered using various crosslinking strategies. The established crosslinking methods enable the formation of three-dimensional (3D) networks under physiological conditions. There are different chemical and physical crosslinking mechanisms available for the generation of SF hydrogels (SFHs). These methods, either chemical or physical, change the structure of SF and improve its mechanical stability, although each method has its advantages and disadvantages. While chemical crosslinking agents guarantee the mechanical strength of SFH through the generation of covalent bonds, they could cause some toxicity, and their usage is not compatible with a cell-friendly technology. On the other hand, physical crosslinking approaches have been implemented in the absence of chemical solvents by the induction of β-sheet conformation in the SF structure. Unfortunately, it is not easy to control the shape and properties of SFHs when using this method. The current review discusses the different crosslinking mechanisms of SFH in detail, in order to support the development of engineered SFHs for biomedical applications.
Injury of articular cartilage leads to an imbalance in tissue homeostasis, and due to the poor self-healing capacity of cartilage the affected tissue often exhibits osteoarthritic changes. In recent years, injectable and highly tunable composite hydrogels for cartilage tissue engineering and drug delivery have been introduced as a desirable alternative to invasive treatments. In this study, we aimed to formulate injectable hydrogels for drug delivery and cartilage tissue engineering by combining different concentrations of hyaluronic acid-tyramine (HA-Tyr) with regenerated silk-fibroin (SF) solutions. Upon enzymatic crosslinking, the gelation and mechanical properties were characterized over time. To evaluate the effect of the hydrogel compositions and properties on extracellular matrix (ECM) deposition, bovine chondrocytes were embedded in enzymatically crosslinked HA-Tyr/SF composites (in further work abbreviated as HA/SF) or HA-Tyr hydrogels. We demonstrated that all hydrogel formulations were cytocompatible and could promote the expression of cartilage matrix proteins allowing chondrocytes to produce ECM, while the most prominent chondrogenic effects were observed in hydrogels with HA20/SF80 polymeric ratios. Unconfined mechanical testing showed that the compressive modulus for HA20/SF80 chondrocyte-laden constructs was increased almost 10-fold over 28 days of culture in chondrogenic medium which confirmed the superior production of ECM in this hydrogel compared to other hydrogels in this study. Furthermore, in hydrogels loaded with anabolic and anti-inflammatory drugs, HA20/SF80 hydrogel showed the longest and the most sustained release profile over time which is desirable for the long treatment duration typically necessary for osteoarthritic joints. In conclusion, HA20/SF80 hydrogel was successfully established as a suitable injectable biomaterial for cartilage tissue engineering and drug delivery applications.
Background: In spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage.
Methods: Human articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold.
Findings: Crossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix.
Interpretation: Due to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment.
Funding: Austrian Research Promotion Agency FFG ("CartiScaff" #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08).
Keywords: Cartilage regeneration; Decellularisation; Ectopic animal model; Laser engraving; Mechanical testing; Repopulation.
Vascular grafts with a diameter of less than 6 mm are made from a variety of materials and techniques to provide alternatives to autologous vascular grafts. Decellularized materials have been proposed as a possible approach to create extracellular matrix (ECM) vascular prostheses as they are naturally derived and inherently support various cell functions. However, these desirable graft characteristics may be limited by alterations of the ECM during the decellularization process leading to decreased biomechanical properties and hemocompatibility. In this study, arteries from the human placenta chorion were decellularized using two distinct detergents (Triton X-100 or SDS), which differently affect ECM ultrastructure. To overcome biomechanical strength loss and collagen fiber exposure after decellularization, riboflavin-mediated UV (RUV) crosslinking was used to uniformly crosslink the collagenous ECM of the grafts. Graft characteristics and biocompatibility with and without RUV crosslinking were studied in vitro and in vivo. RUV-crosslinked ECM grafts showed significantly improved mechanical strength and smoothening of the luminal graft surfaces. Cell seeding using human endothelial cells revealed no cytotoxic effects of the RUV treatment. Short-term aortic implants in rats showed cell migration and differentiation of host cells. Functional graft remodeling was evident in all grafts. Thus, RUV crosslinking is a preferable tool to improve graft characteristics of decellularized matrix conduits.
Erosion of the epigenetic DNA methylation landscape is a widely recognized hallmark of aging. Emerging advances in high throughput sequencing techniques, in particular DNA methylation data analysis, have resulted in the establishment of precise human and murine age prediction tools. In vertebrates, methylation of cytosine at the C5 position of CpG dinucleotides is executed by DNA methyltransferases (DNMTs) whereas the process of enzymatic demethylation is highly dependent on the activity of the ten-eleven translocation methylcytosine dioxygenase (TET) family of enzymes. Here, we report the identification of the key players constituting the DNA methylation machinery in the short-lived teleost aging model Nothobranchius furzeri. We present a comprehensive spatio-temporal expression profile of the methylation-associated enzymes from embryogenesis into late adulthood, thereby covering the complete killifish life cycle. Data mining of the N. furzeri genome produced five dnmt gene family orthologues corresponding to the mammalian DNMTs (DNMT1, 2, 3A, and 3B). Comparable to other teleost species, N. furzeri harbors multiple genomic copies of the de novo DNA methylation subfamily. A related search for the DNMT1 recruitment factor UHRF1 and TET family members resulted in the identification of N. furzeri uhrf1, tet1, tet2, and tet3. Phylogenetic analysis revealed high cross-species similarity on the amino acid level of all individual dnmts, tets, and uhrf1, emphasizing a high degree of functional conservation. During early killifish development all analyzed dnmts and tets showed a similar expression profile characterized by a strong increase in transcript levels after fertilization, peaking either at embryonic day 6 or at the black eye stage of embryonic development. In adult N. furzeri, DNA methylation regulating enzymes showed a ubiquitous tissue distribution. Specifically, we observed an age-dependent downregulation of dnmts, and to some extent uhrf1, which correlated with a significant decrease in global DNA methylation levels in the aging killifish liver and muscle. The age-dependent DNA methylation profile and spatio-temporal expression characteristics of its enzymatic machinery reported here may serve as an essential platform for the identification of an epigenetic aging clock in the new vertebrate model system N. furzeri.
There is critical unmet need for new vascularized tissues to support or replace injured tissues and organs. Various synthetic and natural materials were already established for use of two-dimensional (2D) and three-dimensional (3D) in vitro neovascularization assays, however, they still cannot mimic the complex functions of the sum of the extracellular matrix (ECM) in native intact tissue. Currently, this issue is only addressed by artificial products such as Matrigel™, which comprises a complex mixture of ECM proteins, extracted from animal tumor tissue. Despite its outstanding bioactivity, the isolation from tumor tissue hinders its translation into clinical applications. Since nonhuman ECM proteins may cause immune reactions, as are frequently observed in clinical trials, human ECM proteins represent the best option when aiming for clinical applications. Here, we describe an effective method of isolating a human placenta substrate (hpS) that induces the spontaneous formation of an interconnected network of green fluorescence-labeled human umbilical vein endothelial cells (gfpHUVECs) in vitro. The substrate was biochemically characterized by using a combination of bicinchoninic acid (BCA) assay, DNA, and glycosaminoglycan (GAG) content assays, sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot, angiogenesis arrays, chromatographic thrombin detection, high performance liquid chromatography (HPLC)-based amino acid quantification analysis, and assessment of antimicrobial properties. 2D in vitro cell culture experiments have been performed to determine the vasculogenic potential of hpS, which demonstrated that cell networks developed on hpS show a significantly higher degree of complexity (number of tubules/junctions; total/mean tube length) when compared with Matrigel. As 3D cell culture techniques represent a more accurate representation of the in vivo condition, the substrate was 3D solidified using various natural polymers. 3D in vitro vasculogenesis assays have been performed by seeding gfpHUVECs in an hpS-fibrinogen clot. In conclusion, hpS provides a potent human/material-based alternative to xenogenic-material-based biomaterials for vascularization strategies in tissue engineering.
The responsibility of a lecturer is not only to share his or her knowledge with the students in an easy to understand manner, but also to help the students to embed new knowledge and to encourage the development of higher-order cognitive skills via applied exercises.
In order to meet the growing demand for blended learning approaches a new course concept was established in autumn 2018. To enhance comprehension and to provide opportunities for self-assessment, web-based training units were implemented by using the interactive learning software “Articulate Storyline”. Students had to prepare at home for the course units by completing interactive chapters. Their learning outcome was assessed by online quizzes at the end of each chapter. Online Training chapters allowed time to focus on selected topics and to repeat key messages in following presence units.
Additionally, guided group exercises were performed to promote analytic skills and abstract thinking. The students had to apply and combine their knowledge to solve problem-based challenges.
An optional revision course was offered to the students, which allowed for interactive repetition of the acquired knowledge with the focus on student-to-lecturer dialog.
An analysis based on a written evaluation of this course resulted in a positive feedback from the students, in particular regarding the guided exercises and the offered revision course. According to the students the group exercises allowed to process the learned subjects, promoted the group climate and were a convenient diversion from the frontal lecture format. Students who attended the revision course on a regular basis showed a better performance at the final exam and exceeded especially at interdisciplinary questions.
The first implementation of this master´s degree course indicated that the combination of web-based training elements with frontal lecture elements, guided exercises stimulating cognitive skills and an optional revision course can teach students the basics of biology in an understandable way. This course structure is especially applicable to teach basic subjects for groups of students with varying initial knowledge.
Financial support from the City of Vienna project PBL in Molecular Life Science (21-06) is gratefully acknowledged.
ELGA CDA Implementierungsleitfäden HL7 Implementation Guide for CDA® R2: Ambulanzbefund, ELGA GmbH
(2020)
Detection, quantification and monitoring of virus – host cell interactions are of great importance when evaluating the safety of pharmaceutical products. With the wide usage of viral based vector systems in combination with mammalian cell lines for the production of biopharmaceuticals, the presence of replication competent viral particles needs to be avoided and potential hazards carefully assessed. Consequently, regulatory agencies recommend viral clearance studies using plaque assays or TCID50 assays to evaluate the efficiency of the production process in removing viruses. While plaque assays provide reliable information on the presence of viral contaminations, they are still tedious to perform and can take up to two weeks to finish. To overcome some of these limitations, we have automated, miniaturized and integrated the dual cell culture bioassay into a common lab-on-a-chip platform containing embedded electrical sensor arrays to enrich and detect infectious viruses. Results of our microfluidic single step assay show that a significant reduction in assay time down to 3 to 4 days can be achieved using simultaneous cell-based viral amplification, release and detection of cytopathic effects in a target cell line. We further demonstrate the enhancing effect of continuous fluid flow on infection of PG-4 reporter cells by newly formed and highly active virions by M. dunni cells, thus pointing to the importance of physical relevant viral–cell interactions.
Biomechanical cues such as shear stress, stretching, compression, and matrix elasticity are vital in the establishment of next generation physiological in vitro tissue models. Matrix elasticity, for instance, is known to guide stem cell differentiation, influence healing processes and modulate extracellular matrix (ECM) deposition needed for tissue development and maintenance. To better understand the biomechanical effect of matrix elasticity on the formation of articular cartilage analogs in vitro, this study aims at assessing the redifferentiation capacity of primary human chondrocytes in three different hydrogel matrices of predefined matrix elasticities. The hydrogel elasticities were chosen to represent a broad spectrum of tissue stiffness ranging from very soft tissues with a Young's modulus of 1 kPa up to elasticities of 30 kPa, representative of the perichondral-space. In addition, the interplay of matrix elasticity and transforming growth factor beta-3 (TGF-β3) on the redifferentiation of primary human articular chondrocytes was studied by analyzing both qualitative (viability, morphology, histology) and quantitative (RT-qPCR, sGAG, DNA) parameters, crucial to the chondrotypic phenotype. Results show that fibrin hydrogels of 30 kPa Young's modulus best guide chondrocyte redifferentiation resulting in a native-like morphology as well as induces the synthesis of physiologic ECM constituents such as glycosaminoglycans (sGAG) and collagen type II. This comprehensive study sheds light onto the mechanobiological impact of matrix elasticity on formation and maintenance of articular cartilage and thus represents a major step toward meeting the need for advanced in vitro tissue models to study both re- and degeneration of articular cartilage.