Sample preparation of bone tissue for MALDI-MSI for forensic and (pre)clinical applications.

In the past decades, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been applied to a broad range of biological samples, e.g., forensics and preclinical samples. The use of MALDI-MSI for the analysis of bone tissue has been limited due to the insulating properties of the material but more importantly the absence of a proper sample preparation protocol for undecalcified bone tissue. Undecalcified sections are preferred to retain sample integrity as much as possible or to study the tissue-bone bio interface in particular. Here, we optimized the sample preparation protocol of undecalcified bone samples, aimed at both targeted and untargeted applications for forensic and preclinical applications, respectively. Different concentrations of gelatin and carboxymethyl cellulose (CMC) were tested as embedding materials. The composition of 20% gelatin and 7.5% CMC showed to support the tissue best while sectioning. Bone tissue has to be sectioned with a tungsten carbide knife in a longitudinal fashion, while the sections need to be supported with double-sided tapes to maintain the morphology of the tissue. The developed sectioning method was shown to be applicable on rat and mouse as well as human bone samples. Targeted (methadone and EDDP) as well as untargeted (unknown lipids) detection was demonstrated. DHB proved to be the most suitable matrix for the detection of methadone and EDDP in positive ion mode. The limit of detection (LOD) is estimated to approximately 50 pg/spot on bone tissue. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle. The best matrices for the untargeted detection of unknown lipids in mouse hind legs in positive ion mode were CHCA and DHB based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations.

Optimization of MALDI-TOF mass spectrometry imaging for the visualization and comparison of peptide distributions in dry-cured ham muscle fibers.

In this article, a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI)-based study was designed in order to selectively map and compare the peptides present in slices of Biceps femoris, Istrian dry-cured ham muscles, from the same production batch. A systematic sample preparation process was optimized, which comprises embedding samples of Biceps femoris, cryo-sectioning, glass slide mounting, a nine-step washing protocol, MALDI matrix sublimation and recrystallization. This process efficiently preserved sample morphology and removed the high salt and lipid content, which was present in the samples as a result of the dry-curing production process. We show that MALDI MSI, in combination with principal component analysis, can be used to monitor subtle changes in proteolysis outcome within the same dry-cured ham muscle type, resulting from differentially resolved spatial data. The peptides identified in Istrian dry-cured ham may therefore be studied further, as putative biomarkers for this specific product.

Multimodal imaging of undecalcified tissue sections by MALDI MS and µXRF.

Matrix-assisted laser desorption/ionisation mass spectrometric imaging (MALDI MSI) is a technique that provides localized information on intact molecules in a sample. Micro X-ray fluorescence (µXRF) imaging allows the examination of the spatial distribution of elements in a sample without any morphological changes. These methods have already been applied separately to different tissues, organs, plants and bacterial films, but, to the best of our knowledge, they have yet to be coupled in a multimodal analysis. In this proof-of-principle study, we established and tested sample preparation strategies, allowing the multimodal analysis of lipids (sphingomyelin and phosphatidylcholines) and elements relevant to bone structures as calcium, phosphorous and sulphur in the very same sample section of a chicken phalanx without tissue decalcification. The results of the investigation of such parameters as adhesive tapes supporting tissue sections, and the sequence of the imaging experiments are presented. We show specific lipid distributions in skin, cartilage, muscle, nail, and the intact morphology of bone by calcium and phosphorus imaging. A combination of molecular and elemental imaging was achieved, thus, providing now for the first time the possibility of gathering MALDI MSI and µXRF information from the very same sample without any washing steps omitting therefore the analytical artifacts that inevitably occur in approaches using consecutive tissue sections. The proposed combination can benefit in research studies regarding bone diseases, osteoporosis, osteoarthritis, cartilage failure, bone/tendon distinguishing, where elemental and lipid interaction play an essential role.

Vinyl Sulfonate Esters: Efficient Chain Transfer Agents for the 3D Printing of Tough Photopolymers without Retardation.

The formation of networks through light-initiated radical polymerization allows little freedom for tailored network design. The resulting inhomogeneous network architectures and brittle material behavior of such glassy-type networks limit the commercial application of photopolymers in 3D printing, biomedicine, and microelectronics. An ester-activated vinyl sulfonate ester (EVS) is presented for the rapid formation of tailored methacrylate-based networks. The chain transfer step induced by EVS reduces the kinetic chain length of the photopolymer, thus shifting the gel point to higher conversion, which results in reduced shrinkage stress and higher overall conversion. The resulting, more homogeneous network is responsible for the high toughness of the material. The unique property of EVS to promote nearly retardation-free polymerization can be attributed to the fact that after the transfer step no polymerizable double bond is formed, as is usually seen in classical chain transfer agents. Laser flash photolysis, theoretical calculations, and photoreactor studies were used to elucidate the fast chain transfer reaction and exceptional regulating ability of EVS. Final photopolymer networks exhibit improved mechanical performance making EVS an outstanding candidate for the 3D printing of tough photopolymers.

Green and highly efficient synthesis of perylene and naphthalene bisimides in nothing but water.

High-purity, symmetrically substituted perylene and naphthalene bisimides were obtained by hydrothermal condensation of monoamines with the corresponding bisanhydride. The hydrothermal imidization proceeds quantitatively, without the need for organic solvents, catalysts or excess of the amines.

Mass spectrometric imaging of in vivo protein and lipid adsorption on biodegradable vascular replacement systems.

Cardiovascular diseases present amongst the highest mortality risks in Western civilization and are frequently caused by arteriosclerotic vessel failure. Coronary artery and peripheral vessel reconstruction necessitates the use of small diameter systems that are mechanically stress-resistant and biocompatible. Expanded polytetrafluorethylene (ePTFE) is amongst the materials used most frequently for non-degradable and bio-degradable vessel reconstruction procedures, with thermoplastic polyurethanes (TPU) representing a promising substitute. The present study describes and compares the biological adsorption and diffusion occurring with both materials following implantation in rat models. Gel electrophoresis and thin-layer chromatography, combined with mass spectrometry and mass spectrometry imaging, were utilized to identify the adsorbed lipids and proteins. The results were compared with the analytes present in native aorta tissue. It was revealed that both polymers were severely affected by biological adsorption after 10 min in vivo. Proteins associated with cell growth and migration were identified, especially on the luminal graft surface, while lipids were found to be located on both the luminal and abluminal surfaces. Lipid adsorption and cholesterol diffusion were found to be correlated with the polymer modifications identified on degradable thermoplastic urethane graft samples, with the latter revealing extensive cholesterol adsorption. The present study demonstrates an interaction between biological matter and both graft materials, and provides insights into polymer changes, in particular, those observed with thermoplastic urethanes already after 10 min in vivo exposure. ePTFE demonstrated minor polymer modifications, whereas several different polymer signals were observed for TPU, all were co-localized with biological signals.