570 Biowissenschaften, Biologie
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Different types of raw cotton were investigated by a commercial ultraviolet-visible/near infrared (UV-Vis/NIR) spectrometer (210–2200 nm) as well as on a home-built setup for NIR hyperspectral imaging (NIR-HSI) in the range 1100–2200 nm. UV-Vis/NIR reflection spectroscopy reveals the dominant role proteins, hydrocarbons and hydroxyl groups play in the structure of cotton. NIR-HSI shows a similar result. Experimentally obtained data in combination with principal component analysis (PCA) provides a general differentiation of different cotton types. For UV-Vis/NIR spectroscopy, the first two principal components (PC) represent 82 % and 78 % of the total data variance for the UV-Vis and NIR regions, respectively. Whereas, for NIR-HSI, due to the large amount of data acquired, two methodologies for data processing were applied in low and high lateral resolution. In the first method, the average of the spectra from one sample was calculated and in the second method the spectra of each pixel were used. Both methods are able to explain ≥90 % of total variance by the first two PCs. The results show that it is possible to distinguish between different cotton types based on a few selected wavelength ranges. The combination of HSI and multivariate data analysis has a strong potential in industrial applications due to its short acquisition time and low-cost development. This study opens a novel possibility for a further development of this technique towards real large-scale processes.
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.
Thermoplastic polycarbonate urethane elastomers (TPCU) are potential implant materials for treating degenerative joint diseases thanks to their adjustable rubber-like properties, their toughness, and their durability. We developed a water-containing high-molecular-weight sulfated hyaluronic acid-coating to improve the interaction of TPCU with the synovial fluid. It is suggested that trapped synovial fluid can act as a lubricant that reduces the friction forces and thus provides an enhanced abrasion resistance of TPCU implants. Aims of this work were (i) the development of a coating method for novel soft TPCU with high-molecular sulfated hyaluronic acid to increase the biocompatibility and (ii) the in vitro validation of the functionalized TPCUs in cell culture experiments.
Polyurethane-bases block copolymers (TPCUs) are block-copolymers with systematically varied soft and hard segments. They have been suggested to serve as material for chondral implants in joint regeneration. Such applications may require the adhesion of chondrocytes to the implant surface, facilitating cell growth while keeping their phenotype. Thus, aims of this work were (1) to modify the surface of soft biostable polyurethane-based model implants (TPCU and TSiPCU) with high-molecular weight hyaluronic acid (HA) using an optimized multistep strategy of immobilization, and (2) to evaluate bioactivity of the modified TPCUs in vitro. Our results show no cytotoxic potential of the TPCUs. HAbioactive molecules (Mw =700kDa) were immobilized onto the polyurethane surface via polyethylenimine (PEI) spacers, and modifications were confirmed by several characterization methods. Tests with porcine chondrocytes indicated the potential of the TPCU-HA for inducing enhanced cell proliferation.
The early detection of head and neck cancer is a prolonged challenging task. It requires a precise and accurate identification of tissue alterations as well as a distinct discrimination of cancerous from healthy tissue areas. A novel approach for this purpose uses microspectroscopic techniques with special focus on hyperspectral imaging (HSI) methods. Our proof-of-principle study presents the implementation and application of darkfield elastic light scattering spectroscopy (DF ELSS) as a non-destructive, high-resolution, and fast imaging modality to distinguish lingual healthy from altered tissue regions in a mouse model. The main aspect of our study deals with the comparison of two varying HSI detection principles, which are a point-by-point and line scanning imaging, and whether one might be more appropriate in differentiating several tissue types. Statistical models are formed by deploying a principal component analysis (PCA) with the Bayesian discriminant analysis (DA) on the elastic light scattering (ELS) spectra. Overall accuracy, sensitivity, and precision values of 98% are achieved for both models whereas the overall specificity results in 99%. An additional classification of model-unknown ELS spectra is performed. The predictions are verified with histopathological evaluations of identical HE-stained tissue areas to prove the model’s capability of tissue distinction. In the context of our proof-of-principle study, we assess the Pushbroom PCA-DA model to be more suitable for tissue type differentiations and thus tissue classification. In addition to the HE-examination in head and neck cancer diagnosis, the usage of HSI-based statistical models might be conceivable in a daily clinical routine.
Pre-clinical evaluation of advanced nerve guide conduits using a novel 3D in vitro testing model
(2018)
Autografts are the current gold standard for large peripheral nerve defects in clinics despite the frequently occurring side effects like donor site morbidity. Hollow nerve guidance conduits (NGC) are proposed alternatives to autografts, but failed to bridge gaps exceeding 3 cm in humans. Internal NGC guidance cues like microfibres are believed to enhance hollow NGCs by giving additional physical support for directed regeneration of Schwann cells and axons. In this study, we report a new 3D in vitro model that allows the evaluation of different intraluminal fibre scaffolds inside a complete NGC. The performance of electrospun polycaprolactone (PCL) microfibres inside 5 mm long polyethylene glycol (PEG) conduits were investigated in neuronal cell and dorsal root ganglion (DRG) cultures in vitro. Z-stack confocal microscopy revealed the aligned orientation of neuronal cells along the fibres throughout the whole NGC length and depth. The number of living cells in the centre of the scaffold was not significantly different to the tissue culture plastic (TCP) control. For ex vivo analysis, DRGs were placed on top of fibre-filled NGCs to simulate the proximal nerve stump. In 21 days of culture, Schwann cells and axons infiltrated the conduits along the microfibres with 2.2 ± 0.37 mm and 2.1 ± 0.33 mm, respectively. We conclude that this in vitro model can help define internal NGC scaffolds in the future by comparing different fibre materials, composites and dimensions in one setup prior to animal testing.
Cell-cell and cell-extracellular matrix (ECM) adhesion regulates fundamental cellular functions and is crucial for cell-material contact. Adhesion is influenced by many factors like affinity and specificity of the receptor-ligand interaction or overall ligand concentration and density. To investigate molecular details of cell ECM and cadherins (cell-cell) interaction in vascular cells functional nanostructured surfaces were used Ligand-functionalized gold nanoparticles (AuNPs) with 6-8 nm diameter, are precisely immobilized on a surface and separated by non-adhesive regions so that individual integrins or cadherins can specifically interact with the ligands on the AuNPs. Using 40 nm and 90 nm distances between the AuNPs and functionalized either with peptide motifs of the extracellular matrix (RGD or REDV) or vascular endothelial cadherins (VEC), the influence of distance and ligand specificity on spreading and adhesion of endothelial cells (ECs) and smooth muscle cells (SMCs) was investigated. We demonstrate that RGD-dependent adhesion of vascular cells is similar to other cell types and that the distance dependence for integrin binding to ECM-peptides is also valid for the REDV motif. VEC-ligands decrease adhesion significantly on the tested ligand distances. These results may be helpful for future improvements in vascular tissue engineering and for development of implant surfaces.
Appropriate mechanical properties and fast endothelialization of synthetic grafts are key to ensure long-term functionality of implants. We used a newly developed biostable polyurethane elastomer (TPCU) to engineer electrospun vascular scaffolds with promising mechanical properties (E-modulus: 4.8 ± 0.6 MPa, burst pressure: 3326 ± 78 mmHg), which were biofunctionalized with fibronectin (FN) and decorin (DCN). Neither uncoated nor biofunctionalized TPCU scaffolds induced major adverse immune responses except for minor signs of polymorph nuclear cell activation. The in vivo endothelial progenitor cell homing potential of the biofunctionalized scaffolds was simulated in vitro by attracting endothelial colony-forming cells (ECFCs). Although DCN coating did attract ECFCs in combination with FN (FN + DCN), DCN-coated TPCU scaffolds showed a cell-repellent effect in the absence of FN. In a tissue-engineering approach, the electrospun and biofunctionalized tubular grafts were cultured with primary-isolated vascular endothelial cells in a custom-made bioreactor under dynamic conditions with the aim to engineer an advanced therapy medicinal product. Both FN and FN + DCN functionalization supported the formation of a confluent and functional endothelial layer.
Medical implants play a central role in modern medicine and both, naturally derived and synthetic materials have been explored as biomaterials for such devices. However, when implanted into living tissue, most materials initiate a host response. In addition, implants often cause bacterial infections leading to complications. Polyelectrolyte multilayer (PEM) coatings can be used for functionalization of medical implants improving the implant integration and reducing foreign body reactions. Some PEMs are also known to show antibacterial properties. We developed a PEM coating suggesting that it can decrease the risk of bacterial infections occurring after implantation while being highly biocompatible. We applied two different standard tests for evaluating the PEM’s antibacterial properties, the ISO norm (ISO 22196) and one ASTM norm (ASTM E2180) test. We found a reduction of bacterial growth on the PEM but to a different degree depending on the testing method. This result demonstrates the need for defining proper method to evaluate antibacterial properties of surface coatings.
The hard template method for the preparation of monodisperse mesoporous silica microspheres (MPSMs) has been established in recent years. In this process, in situ-generated silica nanoparticles (SNPs) enter the porous organic template and control the size and pore parameters of the final MPSMs. Here, the sizes of the deposited SNPs are determined by the hydrolysis and condensation rates of different alkoxysilanes in a base catalyzed sol–gel process. Thus, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), tetrapropyl orthosilicate (TPOS) and tetrabutyl orthosilicate (TBOS) were sol–gel processed in the presence of amino-functionalized poly (glycidyl methacrylate-co-ethylene glycol dimethacrylate) (p(GMA-co-EDMA)) templates. The size of the final MPSMs covers a broad range of 0.5–7.3 µm and a median pore size distribution from 4.0 to 24.9 nm. Moreover, the specific surface area can be adjusted between 271 and 637 m2 g−1. Also, the properties and morphology of the MPSMs differ according to the SNPs. Furthermore, the combination of different alkoxysilanes allows the individual design of the morphology and pore parameters of the silica particles. Selected MPSMs were packed into columns and successfully applied as stationary phases in high-performance liquid chromatography (HPLC) in the separation of various water-soluble vitamins.
Bioactive cations, including calcium, copper and magnesium, have shown the potential to become the alternative to protein growth factor-based therapeutics for bone healing. Ion substitutions are less costly, more stable, and more effective at low concentrations. Although they have been shown to be effective in providing bone grafts with more biological functions, the precise control of ion release kinetics is still a challenge. Moreover, the synergistic effect of three or more metal ions on bone regeneration has rarely been studied. In this study, vaterite-calcite CaCO3 particles were loaded with copper (Cu2+) and magnesium (Mg2+). The polyelectrolyte multilayer (PEM) was deposited on CaCuMg-CO3 particles via layer-by-layer technique to further improve the stability and biocompatibility of the particles and to enable controlled release of multiple metal ions. The PEM coated microcapsules were successfully combined with collagen at the outmost layer, providing a further stimulating microenvironment for bone regeneration. The in vitro release studies showed remarkably stable release of Cu2+ in 2 months without initial burst release. Mg2+ was released in relatively low concentration in the first 7 days. Cell culture studies showed that CaCuMg-PEM-Col microcapsules stimulated cell proliferation, extracellular maturation and mineralization more effectively than blank control and other microcapsules without collagen adsorption (Ca-PEM, CaCu-PEM, CaMg-PEM, CaCuMg-PEM). In addition, the CaCuMg-PEM-Col microcapsules showed positive effects on osteogenesis and angiogenesis in gene expression studies. The results indicate that such a functional and controllable delivery system of multiple bioactive ions might be a safer, simpler and more efficient alternative of protein growth factor-based therapeutics for bone regeneration. It also provides an effective method for functionalizing bone grafts for bone tissue engineering.
Surface topographies are often discussed as an important parameter influencing basic cell behavior. Whereas most in vitro studies deal with microstructures with sharp edges, smooth, curved microscale topographies might be more relevant concerning in-vivo situations. Addressing the lack of highly defined surfaces with varying curvature, we present a topography chip system with 3D curved features of varying spacing, curvature radii as well as varying overall dimensions of curved surfaces. The CurvChip is produced by low-cost photolithography with thermal reflow, subsequent (repetitive) PDMS molding and hot embossing. The platform facilitates the systematic in-vitro investigation of the impact of substrate curvature on cell types like epithelial, endothelial, smooth muscle cells, or stem cells. Such investigations will not only help to further understand the mechanism of curvature sensation but may also contribute to optimize cell-material interactions in the field of regenerative medicine.
Continuous manufacturing is becoming more important in the biopharmaceutical industry. This processing strategy is favorable, as it is more efficient, flexible, and has the potential to produce higher and more consistent product quality. At the same time, it faces some challenges, especially in cell culture. As a steady state has to be maintained over a prolonged time, it is unavoidable to implement advanced process analytical technologies to control the relevant process parameters in a fast and precise manner. One such analytical technology is Raman spectroscopy, which has proven its advantages for process monitoring and control mostly in (fed-) batch cultivations. In this study, an in-line flow cell for Raman spectroscopy is included in the cell-free harvest stream of a perfusion process. Quantitative models for glucose and lactate were generated based on five cultivations originating from varying bioreactor scales. After successfully validating the glucose model (Root Mean Square Error of Prediction (RMSEP) of ∼0.2 g/L), it was employed for control of an external glucose feed in cultivation with a glucose-free perfusion medium. The generated model was successfully applied to perform process control at 4 g/L and 1.5 g/L glucose over several days, respectively, with variability of ±0.4 g/L. The results demonstrate the high potential of Raman spectroscopy for advanced process monitoring and control of a perfusion process with a bioreactor and scale-independent measurement method.
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.
Digital light microscopy techniques are among the most widely used methods in cell biology and medical research. Despite that, the automated classification of objects such as cells or specific parts of tissues in images is difficult. We present an approach to classify confluent cell layers in microscopy images by learned deep correlation features using deep neural networks. These deep correlation features are generated through the use of gram-based correlation features and are input to a neural network for learning the correlation between them. In this work we wanted to prove if a representation of cell data based on this is suitable for its classification as has been done for artworks with respect to their artistic period. The method generates images that contain recognizable characteristics of a specific cell type, for example, the average size and the ordered pattern.
The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used.
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.
The establishment of adipose tissue test systems is still a major challenge in the investigation of cellular and molecular interactions responsible for the pathogenesis of inflammatory diseases involving adipose tissue. Mature adipocytes are mainly involved in these pathologies, but rarely used in vitro, due to the lack of an appropriate culture medium which inhibits dedifferentiation and maintains adipocyte functionality. In our study, we showed that Dulbecco's Modified Eagle's Medium/Ham's F-12 with 10% fetal calf serum (FCS) reported for the culture of mature adipocytes favors dedifferentiation, which was accompanied by a high glycerol release, a decreasing release of leptin, and a low expression of the adipocyte marker perilipin A, but high expression of CD73 after 21 days. Optimized media containing FCS, biotin, pantothenate, insulin, and dexamethasone decelerated the dedifferentiation process. These cells showed a lower lipolysis rate, a high level of leptin release, as well as a high expression of perilipin A. CD73-positive dedifferentiated fat cells were only found in low quantity. In this work, we showed that mature adipocytes when cultured under optimized conditions could be highly valuable for adipose tissue engineering in vitro.
Large, deep full-thickness skin wounds from high-graded burns or trauma are not able to reepithelialize sufficiently, resulting in scar formation, mobility limitations, and cosmetic deformities. In this study, in vitro-constructed tissue replacements are needed. Furthermore, such full-skin equivalents would be helpful as in vivo-like test systems for toxicity, cosmetic, and pharmaceutical testing. Up to date, no skin equivalent is available containing the underlying subcutaneous fatty tissue. In this study, we composed a full-skin equivalent and evaluated three different media for the coculture of mature adipocytes, fibroblasts, and keratinocytes. Therefore, adipocyte medium was supplemented with ascorbyl-2-phosphate and calcium chloride, which are important for successful epidermal stratification (Air medium). This medium was further supplemented with two commercially available factor combinations often used for the in vitro culture of keratinocytes (Air-HKGS and Air- KGM medium). We showed that in all media, keratinocytes differentiated successfully to build a stratified epidermal layer and expressed cytokeratin 10 and 14. Perilipin A-positive adipocytes could be found in all tissue models for up to 14 days, whereas adipocytes in the Air-HKGS and Air-KGM medium seemed to be smaller. Adipocytes in all tissue models were able to release adipocyte-specific factors, whereas the supplementation of keratinocyte-specific factors had a slightly negative effect on adipocyte functionality. The permeability of the epidermis of all models was comparable since they were able to withstand a deep penetration of cytotoxic Triton X in the same manner. Taken together, we were able to compose functional three-layered fullskin equivalents by using the Air medium.
Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146 cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146 cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146 cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146⁺ perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries.
Engineering of large vascularized adipose tissue constructs is still a challenge for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Communication between mature adipocytes and endothelial cells is important for homeostasis and the maintenance of adipose tissue mass but, to date, is mainly neglected in tissue engineering strategies. Thus, new coculture strategies are needed to integrate adipocytes and endothelial cells successfully into a functional construct. This review focuses on the cross-talk of mature adipocytes and endothelial cells and considers their influence on fatty acid metabolism and vascular tone. In addition, the properties and challenges with regard to these two cell types for vascularized tissue engineering are highlighted.
Human adipose-derived mesenchymal stem/stromal cells (Ad-MSCs) have great potential for bone tissue engineering. Cryogels, mimicking the three-dimensional structure of spongy bone, represent ideal carriers for these cells. We developed poly(2-hydroxyethyl methacrylate) cryogels, containing hydroxyapatite to mimic inorganic bone matrix. Cryogels were additionally supplemented with different types of proteins, namely collagen (Coll), platelet rich plasma (PRP), immune cells-conditioned medium (CM), and RGD peptides (RGD). The different protein components did not affect scaffolds’ porosity or water-uptake capacity, but altered pore size and stiffness. Stiffness was highest in scaffolds with PRP (82.3 kPa), followed by Coll (55.3 kPa), CM (45.6 kPa), and RGD (32.8 kPa). Scaffolds with PRP, CM, and Coll had the largest pore diameters (~60 µm). Ad MSCs were osteogenically differentiated on these scafffolds for 14 days. Cell attachment and survival rates were comparable for all four scaffolds. Runx2 and osteocalcin levels only increased in Ad-MSCs on Coll, PRP and CM cryogels. Osterix levels increased slightly in Ad-MSCs differentiated on Coll and PRP cryogels. With differentiation alkaline phosphatase activity decreased under all four conditions. In summary, besides Coll cryogel our PRP cryogel constitutes as an especially suitable carrier for bone tissue engineering. This is of special interest, as this scaffold can be generated with patients’ PRP.
Collagen-based barrier membranes are an essential component in Guided Bone Regeneration (GBR) procedures. They act as cell-occlusive devices that should maintain a micromilieu where bone tissue can grow, which in turn provides a stable bed for prosthetic implantation. However, the standing time of collagen membranes has been a challenging area, as native membranes are often prematurely resorbed. Therefore, consolidation techniques, such as chemical cross-linking, have been used to enhance the structural integrity of the membranes, and by consequence, their standing time. However, these techniques have cytotoxic tendencies and can cause exaggerated inflammation and in turn, premature resorption, and material failures. However, tissues from different extraction sites and animals are variably cross-linked. For the present in vivo study, a new collagen membrane based on bovine dermis was extracted and compared to a commercially available porcine-sourced collagen membrane extracted from the pericardium. The membranes were implanted in Wistar rats for up to 60 days. The analyses included well-established histopathological and histomorphometrical methods, including histochemical and immunohistochemical staining procedures, to detect M1- and M2-macrophages as well as blood vessels. Initially, the results showed that both membranes remained intact up to day 30, while the bovine membrane was fragmented at day 60 with granulation tissue infiltrating the implantation beds. In contrast, the porcine membrane remained stable without signs of material-dependent inflammatory processes. Therefore, the bovine membrane showed a special integration pattern as the fragments were found to be overlapping, providing secondary porosity in combination with a transmembraneous vascularization. Altogether, the bovine membrane showed comparable results to the porcine control group in terms of biocompatibility and standing time. Moreover, blood vessels were found within the bovine membranes, which can potentially serve as an additional functionality of barrier membranes that conventional barrier membranes do not provide.
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.
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.
Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (αVβ3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts.
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.
In the current study the in vitro outcome of a degradable magnesium alloy (AZ91D) and standard titanium modified by nanostructured-hydroxyapatite (n-HA) coatings concerning cell adhesion and osteogenic differentiation was investigated by direct cell culture. The n-HA modification was prepared via radio-frequency magnetron sputtering deposition and proven by field emission scanning electron microscopy and X-ray powder diffraction patterns revealing a homogenous surface coating. Human mesenchymal stem cell (hMSCs) adhesion was examined after one and 14 days displaying an enhanced initial cell adhesion on the n-HA modified samples. The osteogenic lineage commitment of the cells was determined by alkaline phosphatase (ALP) quantification. On day one n-HA coated AZ91D exhibited a comparable ALP expression to standard tissue culture polystyrene samples. However, after 14 days solely little DNA and ALP amounts were measurable on n-HA coated AZ91D due to the lack of adherent cells. Titanium displayed excellent cell adhesion properties and ALP was detectable after 14 days. An increased pH of the culture was measured for AZ91D as well as for n-HA coated AZ91D. We conclude that n-HA modification improves initial cell attachment on AZ91D within the first 24 h. However, the effect does not ersist for 14 days in in vitro conditions.
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.
Polyelectrolyte multi-layer (PEM) coatings are prepared by alternative deposition of single polyelectrolyte monolayers on charged surfaces using the Layer-by-Layer (LbL) dip coating procedure. These are nanometre scaled coatings which allow fulfilling of different technical or biological requirements. The build-up process is based on selfassembly and self organization of polycations and polyanions on different substrates including complex geometrical structures and even closed volumes, forming homogeneous layer without defects. Depending on the proper selection of the applied polyelectrolytes, coatings with different stabilities can be prepared. Some of the coatings are stable and cannot be removed from the surface. Others are degradable and can be used as systems for controlled local drug delivery. Here we summarise the results of our experience in preparation of PEM coatings with different functionalities. PEM coatings can be used as controllable delivery system for siRNA polyplexes. They can be used to control the adhesion of different cell types on the surfaces and support e.g. the endothelialisation process on cardio-vascular medical devices as e.g. stents or reduce the immunological response of the tissue after implantation. We summarise results from physical characterisation of the coatings (e.g. film thickness, roughness, electrical charge and hydrophilicity) combined with in-vitro biological studies on adhesion of HUVEC cells.
A series of novel biomedical TPCUs with different percentages of hard segment and a silicone component in the soft segment were synthesized in a multi stage one-pot method. The kinetic profiles of the urethane formation in TPCU-based copolymer systems were monitored by rheological, in line FTIR spectroscopic (React IR) and real-time calorimetric (RC1) methods. This process-analytically monitored multi step synthesis was successfully used to optimize the production of medical-grade TPCU elastomers on preparative scale (in lots of several kg) with controlled molecular structure and mechanical properties. Various surface and bulk analytical methods as well as systematic studies of the mechanic response of the elastomer end-products towards compression and tensile loading were used to estimate the bio-stability of the prepared TPCUs in vitro after 3 months. The tests suggested that high bio-stability of all polyurethane formulations using accelerating in vitro test can be attributed to the synthetic design as well as to the specific techniques used for specimen preparation, namely: (1) the annealing for reducing residual polymer surface stress and preventing IES, (2) stabilization of the morphology by long time storage of the specimens after processing before being immersed in the test liquids, (3) purification by extraction to remove the shot chain oligomers which are the most susceptible to degradation. All mechanical tests were performed on cylindrical and circular disc specimens for modelling the thickness of the meniscus implants under application-relevant stress conditions.
Vitamin E (VitE) additives are important in treating osteoarthritis inclusive cartilage regeneration due to their antioxidant and anti-inflammatory properties. The present research study focuses on the ability of biological antioxidant VitE (alpha-tocopherol isoform) to reduce or minimize oxidative degradation of soft implantable polyurethane (PU) elastomers after extended periods of time (5 months) in vitro. The effect of the oxidation storage media on the morphology of the segmented PUs was evaluated by mechanical softening, crystallization and melting behavior of both soft and hard segments (SS, HS) using dynamic mechanical analysis (DMA). Bulk mechanical properties of the potential implant materials during ageing were predicted from comprehensive mechanical testing of the biomaterials under tension and compression cyclic loads. 5-months in vitro data suggest that the prepared siloxane-poly(carbonate urethane) formulations have sufficient resistance against degradation to be suitable materials for chondral long term bio-stable implants. Most importantly, the positive effect of incorporating VitE (0.5 or 1.0% w/w) as bio-antioxidant and lubricant on the bio-stability was observed for all PU types. VitE-additives protected the surface layer from erosion and cracking during chemical oxidation in vitro as well as from thermal oxidation during extrusion re-processing.
At the beginning of 2022, Frontiers in Bioengineering and Biotechnology - Biomaterials Section has published a Research Topic on “Functional Surfaces and Biomaterials.” The aim of this Research Topic is to summarize the current state of research and development in the field of functional surfaces and biomaterials with a particular focus on biotechnological and medical applications.
The guest editorial team would like to thank all colleagues from around the world who submitted their reviews and research articles for the Research Topic. By the end of August 2022, we have successfully collected 20 articles by 138 participating authors following the peer review process. We also tried to select manuscripts from different research areas to cover the most relevant Research Topic of interest, from drug delivery systems to bone tissue engineering to biosensors and general aspects in biomedicine. By the end of December, the 20 articles had been viewed for more than 21000 times with downloads more than 4,000 times, and 11 articles have reached more than 1,000 views.
Soft lithography, a tool widely applied in biology and life sciences with numerous applications, uses the soft molding of photolithography-generated master structures by polymers. The central part of a photolithography set-up is a mask-aligner mostly based on a high-pressure mercury lamp as an ultraviolet (UV) light source. This type of light source requires a high level of maintenance and shows a decreasing intensity over its lifetime, influencing the lithography outcome. In this paper, we present a low-cost, bench-top photolithography tool based on ninety-eight 375 nm light-emitting diodes (LEDs). With approx. 10 W, our presented lithography set-up requires only a fraction of the energy of a conventional lamp, the LEDs have a guaranteed lifetime of 1000 h, which becomes noticeable by at least 2.5 to 15 times more exposure cycles compared to a standard light source and with costs less than 850 C it is very affordable. Such a set-up is not only attractive to small academic and industrial fabrication facilities who want to enable work with the technology of photolithography and cannot afford a conventional set-up, but also microfluidic teaching laboratories and microfluidic research and development laboratories, in general, could benefit from this cost-effective alternative. With our self-built photolithography system, we were able to produce structures from 6 μm to 50 μm in height and 10 μm to 200 μm in width. As an optional feature, we present a scaled-down laminar flow hood to enable a dust-free working environment for the photolithography process.
Endogenous electrical fields play an important role in various physiological and pathological events. Yet the effects of electrical cues on processes such as wound healing, tumor development or metastasis are still rarely investigated, though it is known that direct current electrical fields can alter cell migration or proliferation in vitro. Several 2D experimental models for studying cell responses to direct current electrical fields have been presented and characterized but suitable experimental models for electrotaxis studies in 3D are rare. Here we present a novel, easy-to-produce, multi-well-based galvanotactic-chamber for the use in 2D and 3D cell experiments for investigations on the influence of electrical fields on tumor cell migration and tumor spheroid growth. Our presented system allows the simultaneous application of electrical field to cells in four chambers, either cultured on the bottom of the culture-plate (2D) or embedded in hydrogel filled channels(3D). The set-up is also suitable for, live-cell-imaging. Validation tests show stable electrical fields and high cell viabilities inside the channel. Tumor spheroids of various diameters can be exposed to direct current electrical fields up to one week.
The extracellular matrix (ECM) is the non-cellular part of tissues and represents the natural environment of the cells. Next to structural stability, it provides various physical, chemical, and mechanical cues that strongly regulate and influence cellular behavior and are required for tissue morphogenesis, differentiation, and homeostasis. Due to its promising characteristics, ECM is used in a wide range of tissue engineering and regenerative medicine approaches as a biomaterial for coatings and scaffolds. To date, there are two sources for ECM material. First, native ECM is generated by the removal of the residing cells of a tissue or organ (decellularized ECM; dECM). Secondly, cell-derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. Although both types of ECM were intensively used for tissue engineering and regenerative medicine approaches, studies directly characterizing and comparing them are rare. Hence, in the first part of this thesis, dECM from adipose tissue and cdECM from stem cells and adipogenic differentiated stem cells from adipose tissue (ASCs) were characterized towards their macromolecular composition, structural features, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans compared to cdECMs. Structural characteristics revealed an immature state of collagen fibers in cdECM samples. The obtained results revealed differences between the two ECMs that can relevantly impact cellular behavior and subsequently experimental outcome and should therefore be considered when choosing a biomaterial for a specific application. The establishment of a functional vascular system in tissue constructs to realize an adequate nutrient supply remains challenging. In the second part, the supporting effect of cdECM on the self‐assembled formation of prevascular‐like structures by microvascular endothelial cells (mvECs) was investigated. It could be observed that cdECM, especially adipogenic differentiated cdECM, enhanced the formation of prevascular-like structures. An increased concentration of proangiogenic factors was found in cdECM substrates. The demonstration of cdECMs capability to induce the spontaneous formation of prevascular‐like structures by mvECs highlights cdECM as a promising biomaterial for adipose tissue engineering. Depending on the purpose of the ECM material chemical modification might be necessary. In the third and last part, the chemical functionalization of cdECM with dienophiles (terminal alkenes, cyclopropene) by metabolic glycoengineering (MGE) was demonstrated. MGE allows the chemical functionalization of cdECM via the natural metabolism of the cells and without affecting the chemical integrity of the cdECM. The incorporated dienophile chemical groups can be specifically addressed via catalysts-free, cell-friendly inverse electron-demand Diels‐Alder reaction. Using this system, the successful modification of cdECM from ASCs with an active enzyme could be shown. The possibility to modify cdECM via a cell-friendly chemical reaction opens up a wide range of possibilities to improve cdECM depending on the purpose of the material. Altogether, this thesis highlighted the differences between adipose dECM and cdECM from ASCs and demonstrated cdECM as a promising alternative to native dECM for application in tissue engineering and regenerative medicine approaches.
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.
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.
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 spreading area of cells has been shown to play a central role in the determination of cell fate and tissue morphogenesis; however, a clear understanding of how spread cell area is determined is still lacking. The observation that cell area and force generally increase with substrate rigidity suggests that cell area is dictated mechanically, by means of a force-balance between the cell and the substrate. A simple mechanical model, corroborated by experimental measurements of cell area and force is presented to analyze the temporal force balance between the cell and the substrate during spreading. The cell is modeled as a thin elastic disc that is actively pulled by lamellipodia protrusions at the cell front. The essential molecular mechanisms of the motor activity at the cell front, including, actin polymerization, adhesion kinetics, and the actin retrograde flow, are accounted for and used to predict the dynamics of cell spreading on elastic substrates; simple, closed-form expressions for the evolution of cell size and force are derived. Time-resolved, traction force microscopy, combined with measurements of cell area are performed to investigate the simultaneous variations of cell size and force. We find that cell area and force increase simultaneously during spreading but the force develops with an apparent delay relative to the increase in cell area. We demonstrate that this may reflect the strain-stiffening property of the cytoskeleton. We further demonstrate that the radial cell force is a concave function of spreading speed and that this may reflect the strengthening of cell–substrate adhesions during spreading.
Focal adhesion clusters (FAC) are dynamic and complex structures that help cells to sense physicochemical properties of their environment. Research in biomaterials, cell adhesion or cell migration often involves the visualization of FAC by fluorescence staining and microscopy, which necessitates quantitative analysis of FAC and other cell features in microscopy images using image processing. Fluorescence microscopy images of human umbilical vein endothelial cells (HUVEC) obtained at 63x magnification were quantitatively analysed using ImageJ software. A generalised algorithm for selective segmentation and morphological analysis of FAC, nucleus and cell morphology is implemented. Further, a method for discrimination of FACnear the nucleus and around the periphery is implemented using masks. Our algorithm is able to effectively quantify different morphological characteristics of cell components and shows a high sensitivity and specificity while providing a modular software implementation.
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.
Cytocompatibility analyses of new implant materials or biomaterials are not only prescribed by the Medical Device Regulation (MDR), as defined in the DIN ISO Norm 10993-5 and -12, but are also increasingly replacing animal testing. In this context, jellyfish collagen has already been established as an alternative to mammalian collagen in different cell culture conditions, but a lack of knowledge exists about its applicability for cytocompatibility analyses of biomaterials. Thus, the present study was conducted to compare well plates coated with collagen type 0 derived from Rhizostoma pulmo with plates coated with bovine and porcine collagen. The coated well plates were analysed in vitro for their cytocompatibility, according to EN ISO 10993-5/−12, using both L929 fibroblasts and MC3T3 pre-osteoblasts. Thereby, the coated well plates were compared, using established materials as positive controls and a cytotoxic material, RM-A, as a negative control. L929 cells exhibited a significantly higher viability (#### p < 0.0001), proliferation (## p < 0.01), and a lower cytotoxicity (## p < 0.01 and # p < 0.05)) in the Jellagen® group compared to the bovine and porcine collagen groups. MC3T3 cells showed similar viability and acceptable proliferation and cytotoxicity in all collagen groups. The results of the present study revealed that the coating of well plates with collagen Type 0 derived from R. pulmo leads to comparable results to the case of well plates coated with mammalian collagens. Therefore, it is fully suitable for the in vitro analyses of the cytocompatibility of biomaterials or medical devices.
This article contains data on the synthesis and mechanical characterization of polysiloxane-based urea-elastomers (PSUs) and is related to the research article entitled “Influence of PDMS molecular weight on transparency and mechanical properties of soft polysiloxane-urea-elastomers for intraocular lens application” (Riehle et al., 2018) [1]. These elastomers were prepared by a two-step polyaddition using the aliphatic diisocyanate 4,4′-Methylenbis(cyclohexylisocyanate) (H12MDI), a siloxane-based chain extender 1,3-Bis(3-aminopropyl)-1,1,3,3-tetramethyldisiloxane (APTMDS) and amino-terminated polydimethylsiloxanes (PDMS) or polydimethyl-methyl-phenyl-siloxane-copolymers (PDMS-Me,Ph), respectively. (More details about the synthesis procedure and the reaction scheme can be found in the related research article (Riehle et al., 2018) [1]).
Amino-terminated polydimethylsiloxanes with varying molecular weights and PDMS-Me,Ph-copolymers were prepared prior by a base-catalyzed ring-chain equilibration of a cyclic siloxane and the endblocker APTMDS. This DiB article contains a procedure for the synthesis of the base catalyst tetramethylammonium-3-aminopropyl-dimethylsilanolate and a generic synthesis procedure for the preparation of a PDMS having a targeted number average molecular weight of 3000 g mol−1. Molecular weights and the amount of methyl-phenyl-siloxane within the polysiloxane-copolymers were determined by 1H NMR and 29Si NMR spectroscopy. The corresponding NMR spectra and data are described in this article.
Additionally, this DiB article contains processed data on in line and off line FTIR-ATR spectroscopy, which was used to follow the reaction progress of the polyaddition by showing the conversion of the diisocyanate. All relevant IR band assignments of a polydimethylsiloxane-urea spectrum are described in this article.
Finally, data on the tensile properties and the mechanical hysteresis-behaviour at 100% elongation of PDMS-based polyurea-elastomers are shown in dependence to the PDMS molecular weight.
Polyelectrolyte multilayer coatings (PEM) are prepared by alternative layer-by-layer deposition of cationic and anionic polyelectrolyte monolayers on charged surfaces. The thickness of the coatings ranges from nm to few μm. Their properties such as roughness, stiffness, surface charge and surface energy can be precisely tuned to fulfil different technical or biological requirements. The coating process is based on self-assembly of polyelectrolytes. Advantages of these coatings are their easy handling, no harsh chemistry and the possibility for coatings on complex geometries. The PEM coatings can be prepared from a variety of suitable polyelectrolytes. Their stability varies from very durable PEM coatings that are only soluble in strong solvents to quickly degradable, which may be applied as drug release system. One example of such a degradable PEM system is the one based on the polyelectrolyte pair Hyaluronan (HA) and Chitosan (CHI). These biopolymers originate from natural sources and show low toxicity towards human cells. However, HA/CHI multilayers show only weak adhesiveness for human umbilical vein endothelial cells (HUVEC). In this article, we summarize our approaches to enhance the HA/CHI multilayer by incorporation of a non-polymer substance –graphene oxide– to improve the cell adhesion and keep such properties as low cytotoxicity and biodegradability. Different approaches for incorporation of graphene oxide were performed and the cellular adhesion was tested by metabolic assay.
Drug-induced liver toxicity is one of the most common reasons for the failure of drugs in clinical trials and frequent withdrawal from the market. Reasons for such failures include the low predictive power of in vivo studies, that is mainly caused by metabolic differences between humans and animals, and intraspecific variances. In addition to factors such as age and genetic background, changes in drug metabolism can also be caused by disease-related changes in the liver. Such metabolic changes have also been observed in clinical settings, for example, in association with a change in liver stiffness, a major characteristic of an altered fibrotic liver. For mimicking these changes in an in vitro model, this study aimed to develop scaffolds that represent the rigidity of healthy and fibrotic liver tissue. We observed that liver cells plated on scaffolds representing the stiffness of healthy livers showed a higher metabolic activity compared to cells plated on stiffer scaffolds. Additionally, we detected a positive effect of a scaffold pre-coated with fetal calf serum (FCS)-containing media. This pre-incubation resulted in increased cell adherence during cell seeding onto the scaffolds. In summary, we developed a scaffold-based 3D model that mimics liver stiffness-dependent changes in drug metabolism that may more easily predict drug interaction in diseased livers.
Age-dependent migratory behavior of human endothelial cells revealed by substrate microtopography
(2019)
Cell migration is part of many important in vivo biological processes and is influenced by chemical and physical factors such as substrate topography. Although the migratory behavior of different cell types on structured substrates has already been investigated, up to date it is largely unknown if specimen's age affects cell migration on structures. In this work, we investigated age-dependent migratory behavior of human endothelial cells from young (≤ 31 years old) and old (≥ 60 years old) donors on poly(dimethylsiloxane) microstructured substrates consisting of well-defined parallel grooves. We observed a decrease in cell migration velocity in all substrate conditions and in persistence length perpendicular to the grooves in cells from old donors. Nevertheless, in comparison to young cells, old cells exhibited a higher cell directionality along grooves of certain depths and a higher persistence time. We also found a systematic decrease of donor age dependent responses of cell protrusions in orientation, velocity and length, all of them decreased in old cells. These observations lead us to hypothesize a possible impairment of actin cytoskeleton network and affected actin polymerization and steering systems, caused by aging.
Increasing number of studies are focused on how adherent cells respond, in vitro, to different properties of a material. Typical properties are the surface chemistry, topographical cues (at the nano- and micro-scale) of the surface, and the substrate stiffness. Cell Response studies are of importance for designing new biomaterials with applications in cell culture technologies, regenerative medicine, or for medical implants. However, only very few studies take the cell age factor, respectively the donor age, into account. In this work, we tested two types of human vascular cells (smooth muscle and endothelial cells) from old and young donors on (a) micro-structured surfaces made of pol (dimethylsiloxane) or on (b) flat polyacrylamide hydrogels with varying stiffnesses. These experiments reveal age-dependent and cell typedependent differences in the cell response to the topography and stiffness, and may establish the Basis for further studies focusing on cell age-dependent responses.