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In recent years, the development and application of decellularized extracellular matrices (ECMs) for use as biomaterials have grown rapidly. These cell-derived matrices (CDMs) represent highly bioactive and biocompatible materials consisting of a complex assembly of biomolecules. Even though CDMs mimic the natural microenvironment of cells in vivo very closely, they still lack specifically addressable functional groups, which are often required to tailor a biomaterial functionality by bioconjugation. To overcome this limitation, metabolic glycoengineering has emerged as a powerful tool to equip CDMs with chemical groups such as azides. These small chemical handles are known for their ability to undergo bioorthogonal click reactions, which represent a desirable reaction type for bioconjugation. However, ECM insolubility makes its processing very challenging. In this contribution, we isolated both the unmodified ECM and azide-modified clickECM by osmotic lysis. In a first step, these matrices were concentrated to remove excessive water from the decellularization step. Next, the hydrogel-like ECM and clickECM films were mechanically fragmentized, resulting in easy to pipette suspensions with fragment sizes ranging from 7.62 to 31.29 μm (as indicated by the mean d90 and d10 values). The biomolecular composition was not impaired as proven by immunohistochemistry. The suspensions were used for the reproducible generation of surface coatings, which proved to be homogeneous in terms of ECM fragment sizes and coating thicknesses (the mean coating thickness was found to be 33.2 ± 7.3 μm). Furthermore, they were stable against fluid-mechanical abrasion in a laminar flow cell. When primary human fibroblasts were cultured on the coated substrates, an increased bioactivity was observed. By conjugating the azides within the clickECM coatings with alkyne-coupled biotin molecules, a bioconjugation platform was obtained, where the biotin–streptavidin interaction could be used. Its applicability was demonstrated by equipping the bioactive clickECM coatings with horseradish peroxidase as a model enzyme.
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.
Bone tissue is highly vascularized. The crosstalk of vascular and osteogenic cells is not only responsible for the formation of the strongly divergent tissue types but also for their physiological maintenance and repair. Extrusion-based bioprinting presents a promising fabrication method for bone replacement. It allows for the production of large-volume constructs, which can be tailored to individual tissue defect geometries. In this study, we used the all-gelatin-based toolbox of methacryl-modified gelatin (GM), non-modified gelatin (G) and acetylated GM (GMA) to tailor both the properties of the bioink towards improved printability, and the properties of the crosslinked hydrogel towards enhanced support of vascular network formation by simple blending. The vasculogenic behavior of human dermal microvascular endothelial cells (HDMECs) and human adipose-derived stem cells (ASCs) was evaluated in the different hydrogel formulations for 14 days. Co-culture constructs including a vascular component and an osteogenic component (i.e. a bone bioink based on GM, hydroxyapatite and ASCs) were fabricated via extrusion-based bioprinting. Bioprinted co-culture constructs exhibited functional tissue-specific cells whose interplay positively affected the formation and maintenance of vascular-like structures. The setup further enabled the deposition of bone matrix associated proteins like collagen type I, fibronectin and alkaline phosphatase within the 30-day culture.
Improvement of a three-layered in vitro skin model for topical application of irritating substances
(2020)
In the field of skin tissue engineering, the development of physiologically relevant in vitro skin models comprising all skin layers, namely epidermis, dermis, and subcutis, is a great challenge. Increasing regulatory requirements and the ban on animal experiments for substance testing demand the development of reliable and in vivo-like test systems, which enable high-throughput screening of substances. However, the reproducibility and applicability of in vitro testing has so far been insufficient due to fibroblast-mediated contraction. To overcome this pitfall, an advanced 3-layered skin model was developed. While the epidermis of standard skin models showed an 80% contraction, the initial epidermal area of our advanced skin models was maintained. The improved barrier function of the advanced models was quantified by an indirect barrier function test and a permeability assay. Histochemical and immunofluorescence staining of the advanced model showed well-defined epidermal layers, a dermal part with distributed human dermal fibroblasts and a subcutis with round-shaped adipocytes. The successful response of these advanced 3-layered models for skin irritation testing demonstrated the suitability as an in vitro model for these clinical tests: only the advanced model classified irritative and non-irritative substances correctly. These results indicate that the advanced set up of the 3-layered in vitro skin model maintains skin barrier function and therefore makes them more suitable for irritation testing.
Human adipose-derived stem cells (hASCs) have become an important cell source for the use in tissue engineering and other medical applications. Not every biomaterial is suitable for human cell culture and requires surface modifications to enable cell adhesion and proliferation. Our hypothesis is that chemical surface modifications introduced by low-discharge plasma enhance the adhesion and proliferation of hASCs. Polystyrene (PS) surfaces were modified either by ammonia (NH3), carbon dioxide (CO2) or acrylic acid (AAc) plasma. The results show that the initial cell adhesion is significantly higher on all modified surfaces than on unmodified material as evaluated by bright field microscopy, live/dead staining, total DNA amount and scanning electron microscopy. The formation of focal adhesions was well pronounced on the Tissue Culture PS, NH3-, and CO2 plasma modified samples. The number of matured fibrillar adhesions was significantly higher on NH3 plasmamodified surfaces than on all other surfaces. Our study validates the suitability of chemical plasma activation and represents a method to enhance hASCs adhesion and improved cell expansion. All chemical modification promoted hASCs adhesion and can therefore be used for the modification of different scaffold materials whereby NH3-plasma modified surfaces resulted in the best outcome concerning hASCs adhesion and proliferation.
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.
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.
New approaches to respiratory assist: bioengineering an ambulatory, miniaturized bioartificial lung
(2019)
Although state-of-the-art treatments of respiratory failure clearly have made some progress in terms of survival in patients suffering from severe respiratory system disorders, such as acute respiratory distress syndrome (ARDS), they failed to significantly improve the quality of life in patients with acute or chronic lung failure, including severe acute exacerbations of chronic obstructive pulmonary disease or ARDS as well. Limitations of standard treatment modalities, which largely rely on conventional mechanical ventilation, emphasize the urgent, unmet clinical need for developing novel(bio)artificial respiratory assist devices that provide extracorporeal gas exchange with a focus on direct extracorporeal CO2 removal from the blood. In this review, we discuss some of the novel concepts and critical prerequisites for such respiratory lung assist devices that can be used with an adequate safety profile, in the intensive care setting, as well as for long-term domiciliary therapy in patients with chronic ventilatory failure. Specifically, we describe some of the pivotal steps, such as device miniaturization, passivation of the blood-contacting surfaces by chemical surface modifications, or endothelial cell seeding, all of which are required for converting current lung assist devices into ambulatory lung assist device for long-term use in critically ill patients. Finally, we also discuss some of the risks and challenges for the long-term use of ambulatory miniaturized bioartificial lungs.
Size and function of bioartificial tissue models are still limited due to the lack of blood vessels and dynamic perfusion for nutrient supply. In this study, we evaluated the use of cytocompatible methacryl-modified gelatin for the fabrication of a hydrogel-based tube by dip-coating and subsequent photo-initiated cross-linking. The wall thickness of the tubes and the diameter were tuned by the degree of gelatin methacryl-modification and the number of dipping cycles. The dipping temperature of the gelatin solution was adjusted to achieve low viscous fluids of approximately 0.1 Pa s and was different for gelatin derivatives with different modification degrees. A versatile perfusion bioreactor for the supply of surrounding tissue models was developed, which can be adaped to several geometries and sizes of blood-vessel mimicking tubes. The manufactured bendable gelatin tubes were permeable for water and dissolved substances, like Nile Blue and serum albumin. As a proof of concept, human fibroblasts in a three-dimensional collagen tissue model were sucessfully supplied with nutrients via the central gelatin tube under dynamic conditions for 2 days. Moreover, the tubes could be used as scaffolds to build-up a functional and viable endothelial layer. Hence, the presented tools can contribute to solving current challenges in tissue engineering.
Natural extracellular matrix (ECM) represents an ideal biomaterial for tissue engineering and regenerative medicine approaches. For further functionalization, there is a need for specific addressable functional groups within this biomaterial. Metabolic glycoengineering (MGE) provides a technique to incorporate modified monosaccharide derivatives into the ECM during their assembly, which was shown by us earlier for the production of a modified fibroblast-derived dermal ECM.