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.
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.
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.
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.
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.