Extracellular Matrix Synthesis and Remodeling by Mesenchymal Stromal Cells Is Context-Sensitive.

Matrix remodeling could be an important mode of action of multipotent mesenchymal stromal cells (MSC) in extracellular matrix (ECM) disease, but knowledge is limited in this respect. As MSC are well-known to adapt their behavior to their environment, we aimed to investigate if their mode of action would change in response to healthy versus pathologically altered ECM. Human MSC-derived ECM was produced under different culture conditions, including standard culture, culture on Matrigel-coated dishes, and stimulation with the pro-fibrotic transforming growth factor-ß1 (TGFß1). The MSC-ECM was decellularized, characterized by histochemistry, and used as MSC culture substrate reflecting different ECM conditions. MSC were cultured on the different ECM substrates or in control conditions for 2 days. Culture on ECM increased the presence of surface molecules with ECM receptor function in the MSC, demonstrating an interaction between MSC and ECM. In MSC cultured on Matrigel-ECM and TGFß1-ECM, which displayed a fibrosis-like morphology, gene expression of collagens and decorin, as well as total matrix metalloproteinase (MMP) activity in the supernatant were decreased as compared with control conditions. These results demonstrated that MSC adapt to their ECM environment, which may include pathological adaptations that could compromise therapeutic efficacy.

A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry.

Plasmalogens (alkenylacyl glycerophospholipids) are important lipid constituents of many tissues and cells (e.g., selected spermatozoa). Since the molecular weights of plasmalogens overlap with that of diacyl- or alkyl acyl lipids, sophisticated mass spectrometry (MS; including MS/MS) analysis is normally used for the unequivocal identification of plasmalogens. We will show here that a simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (without MS/MS capability) in combination with acidic hydrolysis and subsequent derivatization with 2,4-dinitrophenylhydrazine (DNPH) and/or digestion with phospholipase A2 (PLA2) is sufficient to determine the contributions of ether lipids in spermatozoa extracts. As neither diacyl nor alkylacyl lipids are sensitive to acids and do not react with DNPH, the comparison of the mass spectra before and after treatment with acids and/or DNPH addition readily provides unequivocal information about the plasmalogen content. Additionally, the released aldehydes are readily converted into the 2,4-dinitrophenylhydrazones and can be easily identified in the corresponding negative ion mass spectra. Finally, PLA2 digestion is very useful in confirming the presence of plasmalogens. The suggested method was validated by analyzing roe deer, bovine, boar, and domestic cat spermatozoa extracts and comparing the results with isolated phospholipids.

Quantitative analysis of denatured collagen by collagenase digestion and subsequent MALDI-TOF mass spectrometry.

Collagens are the most abundant proteins in vertebrate tissues and constitute significant moieties of the extracellular matrix (ECM). The determination of the collagen content is of relevance not only in the field of native tissue research, but also regarding the quality assessment of bioengineered tissues. Here, we describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen with clostridial collagenase (clostridiopeptidase A) in order to obtain characteristic oligopeptides. Among the resulting peptides, Gly-Pro-Hyp, which is highly indicative of collagen, has been used to assess the amount of collagen by comparing the Gly-Pro-Hyp peak intensities with the intensities of a spiked tripeptide (Arg-Gly-Asp). The approach presented herein is both simple and convenient and allows the determination of collagen in microgram quantities. In tissue samples such as cartilage, the actual collagen content has additionally been determined for comparative purposes by nuclear magnetic resonance spectroscopy subsequent to acidic hydrolysis. Both methods give consistent data within an experimental error of ±10%. Although the differentiation of the different collagen types cannot be achieved by this approach, the overall collagen contents of tissues can be easily determined.