Stem cell plasticity, osteogenic differentiation and the third dimension

Different cues present in the cellular environment control basic biological processes. A previously established 3D microwell array was used to study dimensionality-related effects on osteogenic differentiation and plasticity of marrow stromal cells. To enable long-term culture of single cells in the array a novel surface functionalization technique was developed, using the principle of subtractive micro contact printing of fibronectin and surface passivation with a triblock-copolymer. Immunohistochemical stainings showed that when cultivated in 3D microenvironments, marrow stromal cells can be maintained in the wells for up to 7days and be induced to commit to the osteogenic lineage. In conclusion, this work shows the modification of a 3D microwell array allowing the long term study of single stem cell plasticity and fate in controlled microenvironment

Nylon-6/chitosan core/shell antimicrobial nanofibers for the prevention of mesh-associated surgical site infection

The state-of-the-art hernia meshes, used in hospitals for hernia repair, are predominantly polymeric textile-based constructs that present high mechanical strength, but lack antimicrobial properties. Consequently, preventing bacterial colonization of implanted prosthetic meshes is of major clinical relevance for patients undergoing hernia repair. In this study, the co-axial electrospinning technique was investigated for the development of a novel mechanically stable structure incorporating dual drug release antimicrobial action. Core/shell structured nanofibers were developed, consisting of Nylon-6 in the core, to provide the appropriate mechanical stability, and Chitosan/Polyethylene oxide in the shell to provide bacteriostatic action. The core/shell structure consisted of a binary antimicrobial system incorporating 5-chloro-8-quinolinol in the chitosan shell, with the sustained release of Poly(hexanide) from the Nylon-6 core of the fibers. Homogeneous nanofibers with a "beads-in-fiber" architecture were observed by TEM, and validated by FTIR and XPS. The composite nanofibrous meshes significantly advance the stress-strain responses in comparison to the counterpart single-polymer electrospun meshes. The antimicrobial effectiveness was evaluated in vitro against two of the most commonly occurring pathogenic bacteria; S. aureus and P. aeruginosa, in surgical site infections. This study illustrates how the tailoring of core/shell nanofibers can be of interest for the development of active antimicrobial surfaces

Plasma-deposited AgOx-doped TiOx coatings enable rapid antibacterial activity based on ROS generation

Abstract To enable a rapid-acting antibacterial mechanism without the release of biocidal substances, TiO2 catalysts have been considered based on the generation of reactive oxygen species (ROS). Doping with dissimilar metals generates electron-hole pairs with narrow band gaps promoting the production of ROS. Here, plasma technology is investigated to deposit Ag nano islets on defective TiOx films, stabilized by plasma postoxidation suppressing Ag ion release. Importantly, ROS generation is maintained upon storage in the dark yet with diminishing efficacy; however, it can be restored by exposure to visible light. The rapid-acting antibacterial properties are found to strongly correlate with ROS generation, which can even be maintained by functionalization with hydrophobic plasma polymer films. The cytocompatible coatings offer promising applications for implants and other medical devices

Ribosomal protein l13a as a reference gene for human bone marrow-derived mesenchymal stromal cells during expansion, adipo-, chondro-, and osteogenesis

In the field of human mesenchymal stromal cell (MSC) research, quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the method of choice to study changes in gene expression patterns upon differentiation, application of stimuli, or of factors such as inhibitors or siRNAs. To reliably detect small changes, the use of a reference gene (RG) that is stably expressed under all conditions is essential. The large number of different RGs used in the field and the lack of validation of their suitability make the comparison between studies impossible. Therefore, this work aims to establish one single RG for mesodermal differentiation studies that use MSCs. Seven commonly used RGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ribosomal protein L13a [RPL13a], beta-actin [ACTB], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta-polypeptide [YWHAZ], eukaryotic translational elongation factor 1 alpha [EF1α], β2-microglobulin [B2M], and 18S ribosomal RNA [18S]) were investigated concerning their mRNA expression stability during expansion of bone marrow-derived MSCs up to four passages as well as during their adipo-, chondro-, and osteogenenic differentiation on days 9, 16, and 22 after induction. RPL13a was validated for qPCR studies of MSCs (bone marrow- and placenta-derived) and, additionally, for primary human bone cells (HBCs) and the osteosarcoma cell line MG-63. GAPDH and ACTB, the two most frequently used RGs, showed the highest expression variance. The superior performance of RPL13a should make it the RG of choice for all MSC studies addressing mesodermal differentiation

Uncoupling bacterial attachment on and detachment from polydimethylsiloxane surfaces through empirical and simulation studies

Bacterial infections related to medical devices can cause severe problems, whose solution requires in-depth understanding of the interactions between bacteria and surfaces. This work investigates the influence of surface physicochemistry on bacterial attachment and detachment under flow through both empirical and simulation studies. We employed polydimethylsiloxane (PDMS) substrates having different degrees of crosslinking as the model material and the extended Derjaguin - Landau - Verwey - Overbeek model as the simulation method. Experimentally, the different PDMS materials led to similar numbers of attached bacteria, which can be rationalized by the identical energy barriers simulated between bacteria and the different materials. However, different numbers of residual bacteria after detachment were observed, which was suggested by simulation that the detachment process is determined by the interfacial physicochemistry rather than the mechanical property of a material. This finding is further supported by analyzing the bacteria detachment from PDMS substrates from which non-crosslinked polymer chains had been removed: similar numbers of residual bacteria were found on the extracted PDMS substrates. The knowledge gained in this work can facilitate the projection of bacterial colonization on a given surface

A new mechanism for mtDNA pathogenesis: impairment of post-transcriptional maturation leads to severe depletion of mitochondrial tRNA(Ser(UCN)) caused by T7512C and G7497A point mutations

We have studied the consequences of two homoplasmic, pathogenic point mutations (T7512C and G7497A) in the tRNA(Ser(UCN)) gene of mitochondrial (mt) DNA using osteosarcoma cybrids. We identified a severe reduction of tRNA(Ser(UCN)) to levels below 10% of controls for both mutations, resulting in a 40% reduction in mitochondrial protein synthesis rate and in a respiratory chain deficiency resembling that in the patients muscle. Aminoacylation was apparently unaffected. On non-denaturating northern blots we detected an altered electrophoretic mobility for G7497A containing tRNA molecules suggesting a structural impact of this mutation, which was confirmed by structural probing. By comparing in vitro transcribed molecules with native RNA in such gels, we also identified tRNA(Ser(UCN)) being present in two isoforms in vivo, probably corresponding to the nascent, unmodified transcripts co-migrating with the in vitro transcripts and a second, faster moving isoform corresponding to the mature tRNA. In cybrids containing either mutations the unmodified isoforms were severely reduced. We hypothesize that both mutations lead to an impairment of post-transcriptional modification processes, ultimately leading to a preponderance of degradation by nucleases over maturation by modifying enzymes, resulting in severely reduced tRNA(Ser(UCN)) steady state levels. We infer that an increased degradation rate, caused by disturbance of tRNA maturation and, in the case of the G7497A mutant, alteration of tRNA structure, is a new pathogenic mechanism of mt tRNA point mutations

Extraction of Biofilms From Ureteral Stents for Quantification and Cultivation-Dependent and -Independent Analyses

Ureteral stenting is a common surgical procedure, which is associated with a high morbidity and economic burden, but the knowledge on the link between biofilms on these stents, morbidity, and the impact of the involved microbiota is still limited. This is partially due to a lack of methods that allow for a controlled extraction of the biofilms from stents. Development of an appropriate in vitro model to assess prevention of biofilm formation by antimicrobial coatings and biomaterials requires a profound understanding of the biofilm composition, including the involved microbiota. This work describes an analytical pipeline for the extraction of native biofilms from ureteral stents for both cultivation-dependent and -independent analysis, involving a novel mechanical abrasion method of passing stent samples through a tapered pinhole. The efficiency of this novel method was evaluated by quantifying the removed biofilm mass, numbers of cultivable bacteria, calcium content, and microscopic stent analysis after biofilm removal using 30 clinical stent samples. Furthermore, the extraction of in vitro formed Escherichia coli biofilms was evaluated by universal 16S quantitative PCR, a cultivation-independent method to demonstrate efficient biofilm removal by the new approach. The novel method enables effective contamination-free extraction of the biofilms formed on ureteral stents and their subsequent quantification, and it represents a useful tool for comprehensive examinations of biofilms on ureteral stents

Multifunctional nano‐biointerfaces: cytocompatible antimicrobial nanocarriers from stabilizer‐free cubosomes

The rational design of alternative antimicrobial materials with reduced toxicity toward mammalian cells is highly desired due to the growing occurrence of bacteria resistant to conventional antibiotics. A promising approach is the design of lipid‐based antimicrobial nanocarriers. However, most of the commonly used polymer‐stabilized nanocarriers are cytotoxic. Herein, the design of a novel, stabilizer‐free nanocarrier for the human cathelicidin derived antimicrobial peptide LL‐37 that is cytocompatible and promotes cell proliferation for improved wound healing is reported. The nanocarrier is formed through the spontaneous integration of LL‐37 into novel, stabilizer‐free glycerol mono‐oleate (GMO)‐based cubosomes. Transformations in the internal structure of the cubosomes from Pn3m to Im3m‐type and eventually their transition into small vesicles and spherical micelles are demonstrated upon the encapsulation of LL‐37 into their internal bicontinuous cubic structure using small angle X‐ray scattering, cryogenic transmission electron microscopy, and light scattering techniques. Additional in vitro biological assays show the antimicrobial activity of the stabilizer‐free nano‐objects on a variety of bacteria strains, their cytocompatibility, and cell‐ proliferation enhancing effect. The results outline a promising strategy for the comprehensive design of antimicrobial, cytocompatible lipid nanocarriers for the protection and delivery of bioactive molecules with potential for application as advanced wound healing materials