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- ItemGlycerylphytate as an ionic crosslinker for 3D printing of multi-layered scaffolds with improved shape fidelity and biological features(London : Royal Society of Chemistry, 2020) Mora-Boza, A.; Włodarczyk-Biegun, M.K.; Del Campo, A.; Vázquez-Lasa, B.; Román, J.S.The fabrication of intricate and long-term stable 3D polymeric scaffolds by a 3D printing technique is still a challenge. In the biomedical field, hydrogel materials are very frequently used because of their excellent biocompatibility and biodegradability, however the improvement of their processability and mechanical properties is still required. This paper reports the fabrication of dual crosslinked 3D scaffolds using a low concentrated (<10 wt%) ink of gelatin methacryloyl (GelMA)/chitosan and a novel crosslinking agent, glycerylphytate (G1Phy) to overcome the current limitations in the 3D printing field using hydrogels. The applied methodology consisted of a first ultraviolet light (UV) photopolymerization followed by a post-printing ionic crosslinking treatment with G1Phy. This crosslinker provides a robust framework and avoids the necessity of neutralization with strong bases. The blend ink showed shear-thinning behavior and excellent printability in the form of a straight and homogeneous filament. UV curing was undertaken simultaneously to 3D deposition, which enhanced precision and shape fidelity (resolution ≈150 μm), and prevented the collapse of the subsequent printed layers (up to 28 layers). In the second step, the novel G1Phy ionic crosslinker agent provided swelling and long term stability properties to the 3D scaffolds. The multi-layered printed scaffolds were mechanically stable under physiological conditions for at least one month. Preliminary in vitro assays using L929 fibroblasts showed very promising results in terms of adhesion, spreading, and proliferation in comparison to other phosphate-based traditional crosslinkers (i.e. TPP). We envision that the proposed combination of the blend ink and 3D printing approach can have widespread applications in the regeneration of soft tissues.
- ItemQuantifying ligand-cell interactions and determination of the surface concentrations of ligands on hydrogel films: The measurement challenge(Melville, NY : AIP Publishing, 2015) Beer, Meike V.; Hahn, Kathrin; Diederichs, Sylvia; Fabry, Marlies; Singh, Smriti; Spencer, Steve J.; Salber, Jochen; Möller, Martin; Shard, Alexander G.; Groll, JürgenHydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.
- ItemAntimicrobial Efficacy of Two Surface Barrier Discharges with Air Plasma against In Vitro Biofilms(San Francisco, CA : Public Library of Science, 2013) Matthes, R.; Bender, C.; Schlüter, R.; Koban, I.; Bussiahn, R.; Reuter, S.; Lademann, J.; Weltmann, K.-D.; Kramer, A.The treatment of infected wounds is one possible therapeutic aspect of plasma medicine. Chronic wounds are often associated with microbial biofilms which limit the efficacy of antiseptics. The present study investigates two different surface barrier discharges with air plasma to compare their efficacy against microbial biofilms with chlorhexidine digluconate solution (CHX) as representative of an important antibiofilm antiseptic. Pseudomonas aeruginosa SG81 and Staphylococcus epidermidis RP62A were cultivated on polycarbonate discs. The biofilms were treated for 30, 60, 150, 300 or 600 s with plasma or for 600 s with 0.1% CHX, respectively. After treatment, biofilms were dispensed by ultrasound and the antimicrobial effects were determined as difference in the number of the colony forming units by microbial culture. A high antimicrobial efficacy on biofilms of both plasma sources in comparison to CHX treatment was shown. The efficacy differs between the used strains and plasma sources. For illustration, the biofilms were examined under a scanning electron microscope before and after treatment. Additionally, cytotoxicity was determined by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with L929 mouse fibroblast cell line. The cell toxicity of the used plasma limits its applicability on human tissue to maximally 150 s. The emitted UV irradiance was measured to estimate whether UV could limit the application on human tissue at the given parameters. It was found that the UV emission is negligibly low. In conclusion, the results support the assumption that air plasma could be an option for therapy of chronic wounds.
- ItemGeometry-Driven Cell Organization Determines Tissue Growths in Scaffold Pores: Consequences for Fibronectin Organization(San Francisco, CA : Public Library of Science, 2013) Joly, P.; Duda, G.N.; Schöne, M.; Welzel, P.B.; Freudenberg, U.; Werner, C.; Petersen, A.To heal tissue defects, cells have to bridge gaps and generate new extracellular matrix (ECM). Macroporous scaffolds are frequently used to support the process of defect filling and thus foster tissue regeneration. Such biomaterials contain micro-voids (pores) that the cells fill with their own ECM over time. There is only limited knowledge on how pore geometry influences cell organization and matrix production, even though it is highly relevant for scaffold design. This study hypothesized that 1) a simple geometric description predicts cellular organization during pore filling at the cell level and that 2) pore closure results in a reorganization of ECM. Scaffolds with a broad distribution of pore sizes (macroporous starPEG-heparin cryogel) were used as a model system and seeded with primary fibroblasts. The strategies of cells to fill pores could be explained by a simple geometrical model considering cells as tensioned chords. The model matched qualitatively as well as quantitatively by means of cell number vs. open cross-sectional area for all pore sizes. The correlation between ECM location and cell position was higher when the pores were not filled with tissue (Pearson's coefficient ρ = 0.45±0.01) and reduced once the pores were closed (ρ = 0.26±0.04) indicating a reorganization of the cell/ECM network. Scaffold pore size directed the time required for pore closure and furthermore impacted the organization of the fibronectin matrix. Understanding how cells fill micro-voids will help to design biomaterial scaffolds that support the endogenous healing process and thus allow a fast filling of tissue defects.
- ItemBreast Cancer Stem Cell–Derived Tumors Escape from γδ T-cell Immunosurveillance In Vivo by Modulating γδ T-cell Ligands(Philadelphia, Pa. : AACR, 2023) Raute, Katrin; Strietz, Juliane; Parigiani, Maria Alejandra; Andrieux, Geoffroy; Thomas, Oliver S.; Kistner, Klaus M.; Zintchenko, Marina; Aichele, Peter; Hofmann, Maike; Zhou, Houjiang; Weber, Wilfried; Boerries, Melanie; Swamy, Mahima; Maurer, Jochen; Minguet, SusanaThere are no targeted therapies for patients with triple-negative breast cancer (TNBC). TNBC is enriched in breast cancer stem cells (BCSC), which play a key role in metastasis, chemoresistance, relapse, and mortality. γδ T cells hold great potential in immunotherapy against cancer and might provide an approach to therapeutically target TNBC. γδ T cells are commonly observed to infiltrate solid tumors and have an extensive repertoire of tumor-sensing mechanisms, recognizing stress-induced molecules and phosphoantigens (pAgs) on transformed cells. Herein, we show that patient-derived triple-negative BCSCs are efficiently recognized and killed by ex vivo expanded γδ T cells from healthy donors. Orthotopically xenografted BCSCs, however, were refractory to γ δ T-cell immunotherapy. We unraveled concerted differentiation and immune escape mechanisms: xenografted BCSCs lost stemness, expression of γ δ T-cell ligands, adhesion molecules, and pAgs, thereby evading immune recognition by γ δ T cells. Indeed, neither promigratory engineered γ δ T cells, nor anti–PD-1 checkpoint blockade, significantly prolonged overall survival of tumor-bearing mice. BCSC immune escape was independent of the immune pressure exerted by the γ δ T cells and could be pharmacologically reverted by zoledronate or IFNα treatment. These results pave the way for novel combinatorial immunotherapies for TNBC.
- ItemBicyclic RGD peptides enhance nerve growth in synthetic PEG-based Anisogels(Cambridge : RSC, 2021) Vedaraman, Sitara; Bernhagen, Dominik; Haraszti, Tamas; Licht, Christopher; Castro Nava, Arturo; Omidinia Anarkoli, Abdolrahman; Timmerman, Peter; De Laporte, LauraNerve regeneration scaffolds often consist of soft hydrogels modified with extracellular matrix (ECM) proteins or fragments, as well as linear and cyclic peptides. One of the commonly used integrin-mediated cell adhesive peptide sequences is Arg-Gly-Asp (RGD). Despite its straightforward coupling mechanisms to artificial extracellular matrix (aECM) constructs, linear RGD peptides suffer from low stability towards degradation and lack integrin selectivity. Cyclization of RGD improves the affinity towards integrin subtypes but lacks selectivity. In this study, a new class of short bicyclic peptides with RGD in a cyclic loop and 'random screened' tri-amino acid peptide sequences in the second loop is investigated as a biochemical cue for cell growth inside three-dimensional (3D) synthetic poly(ethylene glycol) (PEG)-based Anisogels. These peptides impart high integrin affinity and selectivity towards either αvβ3 or α5β1 integrin subunits. Enzymatic conjugation of such bicyclic peptides to the PEG backbone enables the formulation of an aECM hydrogel that supports nerve growth. Furthermore, different proteolytic cleavable moieties are incorporated and compared to promote cell migration and proliferation, resulting in enhanced cell growth with different degradable peptide crosslinkers. Mouse fibroblasts and primary nerve cells from embryonic chick dorsal root ganglions (DRGs) show superior growth in bicyclic RGD peptide conjugated gels selective towards αvβ3 or α5β1, compared to monocyclic or linear RGD peptides, with a slight preference to αvβ3 selective bicyclic peptides in the case of nerve growth. Synthetic Anisogels, modified with bicyclic RGD peptides and containing short aligned, magneto-responsive fibers, show oriented DRG outgrowth parallel to the fibers. This report shows the potential of PEG hydrogels coupled with bicyclic RGD peptides as an aECM model and paves the way for a new class of integrin selective biomolecules for cell growth and nerve regeneration.