570 Biowissenschaften, Biologie
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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.
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.
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.
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.
The coculture of osteogenic and angiogenic cells and the resulting paracrine signaling via soluble factors are supposed to be crucial for successfully engineering vascularized bone tissue equivalents. In this study, a coculture system combining primary human adiposederived stem cells (hASCs) and primary human dermal microvascular endothelial cells (HDMECs) within two types of hydrogels based on methacryloyl‐modified gelatin (GM) as three‐dimensional scaffolds was examined for its support of tissue specific cell functions. HDMECs, together with hASCs as supporting cells, were encapsulated in soft GM gels and were indirectly cocultured with hASCs encapsulated in stiffer GM hydrogels additionally containing methacrylate‐modified hyaluronic acid and hydroxyapatite particles. After 14 days, the hASC in the stiffer gels (constituting the “bone gels”) expressed matrix proteins like collagen type I and fibronectin, as well as bone‐specific proteins osteopontin and alkaline phosphatase. After 14 days of coculture with HDMEC‐laden hydrogels, the viscoelastic properties of the bone gels were significantly higher compared with the gels in monoculture. Within the soft vascularization gels, the formed capillary‐like networks were significantly longer after 14 days of coculture than the structures in the control gels. In addition, the stability as well as the complexity of the vascular networks was significantly increased by coculture. We discussed and concluded that osteogenic and angiogenic signals from the culture media as well as from cocultured cell types, and tissue‐specific hydrogel composition all contribute to stimulate the interplay between osteogenesis and angiogenesis in vitro and are a basis for engineering vascularized bone.
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 this article, liposome-based coatings aiming to control drug release from therapeutic soft contact lenses (SCLs) materials are analyzed. A PHEMA based hydrogel material loaded with levofloxacin is used as model system for this research. The coatings are formed by polyelectrolyte layers containing liposomes of 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) and DMPC1cholesterol (DMPC1 CHOL). The effect of friction and temperature on the drug release is investigated. The aim of the friction tests is to simulate the blinking of the eyelid in order to verify if the SCLs materials coated with liposomes are able to keep their properties, in particular the drug release ability. It was observed that under the study conditions, friction did not affect significantly the drug release from the liposome coated PHEMA material. In contrast, increasing the temperature of release leads to an increase of the drug diffusion rate through the hydrogel. This phenomenon is recorded both in the control and in the coated samples.
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.
Positively charged metallic oxides prevent blood coagulation whereas negatively charged metallic oxides are thrombogenic. This study was performed to examine whether this effect extends to metallic oxide nanoparticles. Oscillation shear rheometry was used to study the effect of zinc oxide and silicon dioxide nanoparticles on thrombus formation in human whole blood. Our data show that oscillation shear rheometry is a sensitive and robust technique to analyze thrombogenicity induced by nanoparticles. Blood without previous contact with nanoparticles had a clotting time (CT) of 16.7 ± 1.0 min reaching a maximal clot strength (CS) of 16 ± 14 Pa (G') after 30 min. ZnO nanoparticles (diameter 70 nm, +37 mV zeta-potential) at a concentration of 1 mg/mL prolonged CT to 20.8 ± 3.6 min and provoked a weak clot (CS 1.5 ± 1.0 Pa). However, at a lower concentration of 100 µg/mL the ZnO particles dramatically reduced CT to 6.0 ± 0.5 min and increased CS to 171 ± 63 Pa. This procoagulant effect decreased at lower concentrations reaching the detection limit at 10 ng/mL. SiO2 nanoparticles (diameter 232 nm, −28 mV zeta-potential) at high concentrations (1 mg/mL) reduced CT (2.1 ± 0.2 min) and stimulated CS (249 ± 59 Pa). Similar to ZnO particles, this procoagulant effect reached a detection limit at 10 ng/mL. Nanoparticles in high concentrations reproduce the surface charge effects on blood coagulation previously observed with large particles or solid metal oxides. However, nanoparticles with different surface charges equally well stimulate coagulation at lower concentrations. This stimulation may be an effect which is not directly related to the surface charge.
Poly(dimethylsiloxane) can be covalently coated with ultrathin NCO-sP(EO-stat-PO) hydrogel layers which permit covalent binding of cell adhesive moieties, while minimizing unspecific cell adhesion on non-functionalized areas. We applied long term uniaxial cyclic tensile strain (CTS) and revealed (a) the preservation of protein and cell-repellent properties of the NCO-sP(EO-stat-PO) coating and (b) the stability and bioactivity of a covalently bound fibronectin (FN) line pattern. We studied the adhesion of human dermal fibroblast (HDFs) on non-modified NCO-sP(EO-stat-PO) coatings and on the FN. HDFs adhered to FN and oriented their cell bodies and actin fibers along the FN lines independently of the direction of CTS. This mechanical long term stability of the bioactive, patterned surface allows unraveling biomechanical stimuli for cellular signaling and behavior to understand physiological and pathological cell phenomenon. Additionally, it allows for the application in wound healing assays, tissue engineering, and implant development demanding spatial control over specific cell adhesion.