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How mechanical and physicochemical material characteristics influence adipose-derived stem cell fate
(2023)
Adipose-derived stem cells (ASCs) are a subpopulation of mesenchymal stem cells. Compared to bone marrow-derived stem cells, they can be harvested with minimal invasiveness. ASCs can be easily expanded and were shown to be able to differentiate into several clinically relevant cell types. Therefore, this cell type represents a promising component in various tissue engineering and medical approaches (e.g., cell therapy). In vivo cells are surrounded by the extracellular matrix (ECM) that provides a wide range of tissue-specific physical and chemical cues, such as stiffness, topography, and chemical composition. Cells can sense the characteristics of their ECM and respond to them in a specific cellular behavior (e.g., proliferation or differentiation). Thus, in vitro biomaterial properties represent an important tool to control ASCs behavior. In this review, we give an overview of the current research in the mechanosensing of ASCs and current studies investigating the impact of material stiffens, topography, and chemical modification on ASC behavior. Additionally, we outline the use of natural ECM as a biomaterial and its interaction with ASCs regarding cellular behavior.
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 world population is growing and alternative ways of satisfying the increasing demand for meat are being explored, such as using animal cells for the fabrication of cultured meat. Edible biomaterials are required as supporting structures. Hence, we chose agarose, gellan and a xanthan-locust bean gum blend (XLB) as support materials with pea and soy protein additives and analyzed them regarding material properties and biocompatibility. We successfully built stable hydrogels containing up to 1% pea or soy protein. Higher amounts of protein resulted in poor handling properties and unstable gels. The gelation temperature range for agarose and gellan blends is between 23–30 °C, but for XLB blends it is above 55 °C. A change in viscosity and a decrease in the swelling behavior was observed in the polysaccharide-protein gels compared to the pure polysaccharide gels. None of the leachates of the investigated materials had cytotoxic effects on the myoblast cell line C2C12. All polysaccharide-protein blends evaluated turned out as potential candidates for cultured meat. For cell-laden gels, the gellan blends were the most suitable in terms of processing and uniform distribution of cells, followed by agarose blends, whereas no stable cell-laden gels could be formed with XLB blends.
Due to its wide-ranging endocrine functions, adipose tissue influences the whole body’s metabolism. Engineering long-term stable and functional human adipose tissue is still challenging due to the limited availability of suitable biomaterials and adequate cell maturation. We used gellan gum (GG) to create manual and bioprinted adipose tissue models because of its similarities to the native extracellular matrix and its easily tunable properties. Gellan gum itself was neither toxic nor monocyte activating. The resulting hydrogels exhibited suitable viscoelastic properties for soft tissues and were stable for 98 days in vitro. Encapsulated human primary adipose-derived stem cells (ASCs) were adipogenically differentiated for 14 days and matured for an additional 84 days. Live-dead staining showed that encapsulated cells stayed viable until day 98, while intracellular lipid staining showed an increase over time and a differentiation rate of 76% between days 28 and 56. After 4 weeks of culture, adipocytes had a univacuolar morphology, expressed perilipin A, and secreted up to 73% more leptin. After bioprinting establishment, we demonstrated that the cells in printed hydrogels had high cell viability and exhibited an adipogenic phenotype and function. In summary, GG-based adipose tissue models show long-term stability and allow ASCs maturation into functional, univacuolar adipocytes.
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.
An advanced ‘clickECM’ that can be modified by the inverse-electron demand Diels-Alder reaction
(2021)
The extracellular matrix (ECM) represents the natural environment of cells in tissue and therefore is a promising biomaterial in a variety of applications. Depending on the purpose, it is necessary to equip the ECM with specific addressable functional groups for further modification with bioactive molecules, for controllable cross-linking and/or covalent binding to surfaces. Metabolic glycoengineering (MGE) enables the specific modification of the ECM with such functional groups without affecting the native structure of the ECM. In a previous approach (S. M. Ruff, S. Keller, D. E. Wieland, V. Wittmann, G. E. M. Tovar, M. Bach, P. J. Kluger, Acta Biomater. 2017, 52, 159–170), we demonstrated the modification of an ECM with azido groups, which can be addressed by bioorthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC). Here, we demonstrate the modification of an ECM with dienophiles (terminal alkenes, cyclopropene), which can be addressed by an inverse-electron-demand Diels-Alder (IEDDA) reaction. This reaction is cell friendly as there are no cytotoxic catalysts needed. We show the equipment of the ECM with a bioactive molecule (enzyme) and prove that the functional groups do not influence cellular behavior. Thus, this new material has great potential for use as a biomaterial, which can be individually modified in a wide range of applications.
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.
Highly viscous bioinks offer great advantages for the three-dimensional fabrication of cell-laden constructs by microextrusion printing. However, no standardised method of mixing a high viscosity biomaterial ink and a cell suspension has been established so far, leading to non-reproducible printing results. A novel method for the homogeneous and reproducible mixing of the two components using a mixing unit connecting two syringes is developed and investigated. Several static mixing units, based on established mixing designs, were adapted and their functionality was determined by analysing specific features of the resulting bioink. As a model system, we selected a highly viscous ink consisting of fresh frozen human blood plasma, alginate, and methylcellulose, and a cell suspension containing immortalized human mesenchymal stem cells. This bioink is crosslinked after fabrication. A pre-crosslinked gellan gum-based bioink providing a different extrusion behaviour was introduced to validate the conclusions drawn from the model system. For characterisation, bioink from different zones within the mixing device was analysed by measurement of its viscosity, shape fidelity after printing and visual homogeneity. When taking all three parameters into account, a comprehensive and reliable comparison of the mixing quality was possible. In comparison to the established method of manual mixing inside a beaker using a spatula, a significantly higher proportion of viable cells was detected directly after mixing and plotting for both bioinks when the mixing unit was used. A screw-like mixing unit, termed “HighVisc”, was found to result in a homogenous bioink after a low number of mixing cycles while achieving high cell viability rates.
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.