RESUMO
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.
Assuntos
Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Sobrevivência Celular , Células Cultivadas , Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Humanos , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Receptores de Superfície Celular/metabolismo , Especificidade por Substrato , Inibidores Teciduais de Metaloproteinases/genética , Inibidores Teciduais de Metaloproteinases/metabolismoRESUMO
RATIONALE: If carbohydrates are investigated by nuclear magnetic resonance (NMR) spectroscopy, they are normally dissolved in deuterated solvents, such as D2 O. The incorporation of deuterium leads to a high complexity of subsequently recorded mass spectra and reduced sensitivity because different deuterated ions become detectable. Here, we demonstrate that the applied matrix-assisted laser desorption/ionization (MALDI) matrix solution has a considerable impact on the observed isotopic distribution. METHODS: Unsaturated disaccharides of chondroitin (CS) and dermatan sulfate (DS) were prepared by enzymatic digestion of the polysaccharides in D2 O and analyzed by MALDI time-of-flight mass spectrometry (TOF MS) using 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA) according to previously published protocols. RESULTS: The extent of deuteration of a given compound can be easily determined by using the mass shift between the non-deuterated and deuterated ions. However, such a determination is more difficult when considering sugars due to their high content of exchangeable groups. Therefore, both the solvent and the matrix have a considerable impact on the MS patterns. Additionally, there are significant differences if the spectra are recorded at different laser fluences. CONCLUSIONS: Great caution should be taken when the deuterium content of disaccharides is determined by MALDI MS. Aside from the use of non-protic solvents, DHB is the matrix of choice, whereas 9-AA shows a considerable influence on the observed isotope pattern in dependence on the applied laser fluence. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Dissacarídeos/química , Glicosaminoglicanos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Deutério/química , Gentisatos/química , Espectroscopia de Ressonância Magnética , Peso MolecularRESUMO
PURPOSE: The influence of the pore size of biodegradable poly(lactic-co-glycolic acid) scaffolds on bone regeneration was investigated. METHODS: Cylindrical poly(lactic-co-glycolic acid) scaffolds were implanted into a defect in the tibial head of rats. Pore sizes of 100-300, 300-500, and 500-710 µm were tested and compared to untreated defects as control. Two and four weeks after implantation, the specimens were explanted and defect regeneration and de novo extracellular matrix generation were investigated by MRI, quantitative solid-state NMR, and mass spectrometry. RESULTS: The pore size of the scaffolds had a pronounced influence on the quantity of the extracellular matrix synthesized in the graft; most collagen was synthesized within the first 2 weeks of implantation, while the amount of hydroxyapatite increased in the second 2 weeks. After 4 weeks, the scaffolds contained large quantities of newly formed lamellar bone while the control defects were filled by inhomogenous woven bone. Best results were obtained for scaffolds of a pore size of 300-500 µm. CONCLUSION: Our analysis showed that the structure and dynamics of the regenerated extracellular matrix was very similar to that of the native bone, suggesting that biomineralization was significantly enhanced by the choice of the most appropriate implant material.
Assuntos
Implantes Absorvíveis , Regeneração Óssea/fisiologia , Transplante Ósseo/instrumentação , Regeneração Tecidual Guiada/instrumentação , Ácido Láctico/química , Ácido Poliglicólico/química , Fraturas da Tíbia/fisiopatologia , Fraturas da Tíbia/cirurgia , Animais , Calcificação Fisiológica , Análise de Falha de Equipamento , Feminino , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética , Teste de Materiais , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Porosidade , Desenho de Prótese , Ratos , Ratos Wistar , Fraturas da Tíbia/patologia , Resultado do TratamentoRESUMO
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.
Assuntos
Plasmalogênios/análise , Plasmalogênios/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espermatozoides/química , Espectrometria de Massas em Tandem/métodos , Animais , Gatos , Bovinos , Cervos , Masculino , Especificidade da Espécie , Sus scrofaRESUMO
Hypochlorous acid (HOCl) is a strong non-radical oxidant, which is generated during inflammatory processes under the catalysis of the enzyme myeloperoxidase (MPO). HOCl reacts particularly with sulfhydryl and amino acid residues but affects also many other biomolecules. For instance, the glycosaminoglycans of articular cartilage and synovial fluids (such as hyaluronan) undergo degradation in the presence of HOCl at which the native polysaccharide is fragmented into oligosaccharides in a complex reaction. This is an initial mass spectrometry (MS)-based investigation dealing with the HOCl-induced degradation of glycosaminoglycans and the conversion of the related monosaccharides into chlorinated products. In particular, it will be shown that the reaction between HOCl and hyaluronan is slower than originally assumed and results in the generation of different products (particularly the hyaluronan monosaccharides) by the cleavage of the ß-1,3/1,4-glycosidic linkages. The MS detection of chlorinated products is, however, only possible in the case of the monosaccharides. Potential reasons will be discussed.
RESUMO
The self-healing capacity of skin is limited, and medical intervention is often unavoidable. Skin may be generated ex vivo from cultured fibroblasts. Because the molecular composition of de novo formed skin (mostly collagen and glycosaminoglycans [GAGs]) is crucial, analytical methods are required for the quality control of tissue-engineered products. Here, we show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of fibroblast cultures subsequent to digestion with chondroitinase ABC is a reliable and fast method to monitor the GAG content of native and bioengineered skin. Furthermore, the supplementation of the fibroblast medium with ¹³C-labeled glucose provides insights into the biosynthesis of GAGs.
Assuntos
Condroitina ABC Liase/análise , Matriz Extracelular/metabolismo , Fibroblastos/enzimologia , Glicosaminoglicanos/biossíntese , Pele/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Células Cultivadas , Meios de Cultura , Glucose/química , HumanosRESUMO
The determination of the collagen and glycosaminoglycan (GAG) contents of native and particularly bioengineered tissues is of considerable interest because the collagen-to-GAG ratio determines the water content of the tissue, which is crucial regarding its mechanical properties. (1)H NMR spectroscopy subsequent to the hydrolysis of the sample by aqueous 6 M DCl at 353 K is used to determine the GAG and collagen contents simultaneously. Under these strongly acidic conditions the biopolymers of the extracellular matrix, collagen, and GAG are fragmented into their individual monomers, that is, free amino acids from collagen and monosaccharides from the polymer repeat units of GAGs. The amino acid amount can be easily determined in the presence of an internal standard by (1)H NMR spectroscopy because amino acids proved to be stable under acidic conditions. The carbohydrates are subject to charring in the presence of concentrated DCl, but glucosamine and galactosamine were found to be sufficiently stable for quantification under the chosen conditions.
Assuntos
Colágeno/química , Glicosaminoglicanos/química , Ácido Clorídrico/química , Acetilgalactosamina/química , Aminoácidos/química , Animais , Cartilagem/química , Bovinos , Sulfatos de Condroitina/química , Deutério , Matriz Extracelular/química , Glucosamina/química , Ácido Hialurônico/química , Hidrólise , Espectroscopia de Ressonância Magnética , Tendões/químicaRESUMO
Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) is an indispensable tool in modern lipid research since it is fast, sensitive, tolerates sample impurities and provides spectra without major analyte fragmentation. We will discuss some methodological aspects, the related ion-forming processes and the MALDI MS characteristics of the different lipid classes (with the focus on glycerophospholipids) and the progress, which was achieved during the last ten years. Particular attention will be given to quantitative aspects of MALDI MS since this is widely considered as the most serious drawback of the method. Although the detailed role of the matrix is not yet completely understood, it will be explicitly shown that the careful choice of the matrix is crucial (besides the careful evaluation of the positive and negative ion mass spectra) in order to be able to detect all lipid classes of interest. Two developments will be highlighted: spatially resolved Imaging MS is nowadays well established and the distribution of lipids in tissues merits increasing interest because lipids are readily detectable and represent ubiquitous compounds. It will also be shown that a combination of MALDI MS with thin-layer chromatography (TLC) enables a fast spatially resolved screening of an entire TLC plate which makes the method competitive with LC/MS.
Assuntos
Lipídeos , Cromatografia em Camada Fina/métodos , Lipídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
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.
Assuntos
Colágeno/química , Colágeno/metabolismo , Colagenases/metabolismo , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Espectroscopia de Ressonância Magnética , Peptídeos/química , Desnaturação ProteicaRESUMO
Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.
RESUMO
Musculoskeletal diseases are extremely widespread and a significant burden on the health systems of the industrialized countries. The use of mesenchymal stromal cells is a promising approach to cure cartilage and tendon injuries, which often also occur in younger people as consequences of sport accidents. Although particular interest is on the collagen and the glycosaminoglycan composition of the tendon and potential alterations compared to healthy tissue, there is nowadays also increasing evidence that some selected phospholipids (PL) are potential mediators of tissue regeneration. Therefore, PL (and potential changes thereof) attract increasing interest in this field. We have used positive and negative ion matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to elucidate the lipid compositions of human mesenchymal stromal cells in dependence on the composition of the cell culture medium and the cultivation time. The de novo biosynthesis of PL was monitored by adding 13C labeled glucose or deuterated palmitic acid (d31-PA) to the cells and the incorporation of 13C or 2H into the different PL classes was investigated by electrospray ionization (ESI) mass spectrometry (MS). It is remarkable that all PL classes (for instance, phosphatidylcholine and -inositol) exhibited 13C incorporation - but not the sphingomyelin (SM) which is the most abundant sphingolipid in the majority of human tissues and body fluids. Using suitable internal standards it could be shown, that only 12C-containing SM is de novo generated while no 13C-labeled SM could be monitored - independent of the cultivation time, which was varied between 7 and 28 days. SM impurities stemming from the cell culture medium and the used MALDI matrix compounds (2,5-dihydroxybenzoic acid (DHB) or 9-aminoacridine (9-AA)) could be ruled out. However, incorporation of deuterated palmitic acid (d31-PA) could be observed for multiple PL, including SM. Therefore, it is suggested that there must exist another, so far unknown SM biosynthesis pathway. This pathway does not make use of glucose but relies on the use of other molecules as energy sources. Potential pathways to explain the experimental observations are discussed.
Assuntos
Fosfolipídeos/biossíntese , Esfingomielinas/biossíntese , Humanos , Cinética , Ácido Palmítico/química , Ácido Palmítico/metabolismo , Fosfolipídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esfingomielinas/química , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a soft ionization MS technique providing only minor fragmentation of the analyte. Therefore, the method is basically suitable for mixture analysis, although the ion yields strongly depend on the basicity/acidity of the analyte in relation to the applied matrix. Accordingly, less sensitively detectable compounds may be suppressed by more sensitively detectable compounds. Thus, separation of the mixture into the individual compounds is normally indispensable. This paper demonstrates the capabilities and limitations of a direct, simple, and inexpensive MALDI-high-performance thin-layer chromatography (HPTLC) coupling for the analysis of a crude lipid extract from porcine brain. Brain lipids were chosen because they represent a rather complex mixture and are of currently significant research interest. It was found that normal-phase HPTLC-separated lipids can be easily characterized by direct MALDI-TOF-MS analysis with sufficient resolution to allow the assignment of virtually all lipid classes, even rather minor species such as phosphorylated phosphoinositides or complex glycolipids as gangliosides. Advantages and disadvantages of this approach are discussed.
Assuntos
Química Encefálica , Lipídeos/análise , Animais , Cromatografia em Camada Fina , Densitometria , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta , SuínosAssuntos
Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Proteínas de Insetos/química , Fragmentos de Peptídeos/química , Vertebrados , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Proteínas de Insetos/farmacologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The human spermatozoa membrane is characterized by a unique fatty acyl composition with significant amounts of highly unsaturated fatty acids, particularly docosahexaenoic acid (22:6), whereby phosphatidylcholine (PtdCho) (16:0/22:6) is the most abundant glycerophospholipid. The large amount of highly unsaturated fatty acyl residues is crucial for the fluidity of the membrane and, therefore, the successful fertilization process. Consequently, however, the spermatozoa are very sensitive to reactive oxygen species (ROS) that are generated under conditions of "oxidative stress" and key players in many pathological conditions. Lipid oxidation of the sperm membrane is accompanied by the loss of the oxidatively modified unsaturated residue (normally in the sn-2 position) and the generation of saturated lysophosphatidylcholine (LysoPtdCho). Although other lysolipids are also generated, LysoPtdCho is the "marker" lipid of choice due to the high abundance of PtdCho. In particular, obesity (body mass index >30 kg/m(2)) is characterized by increased ROS generation and negatively affects the reproductive potential. We will show here that the LysoPtdCho/PtdCho ratio can be easily determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The data found do correlate with clinical markers of sperm quality. A very interesting aspect is that the LysoPtdCho/PtdCho ratios determined in the spermatozoa extracts correlate with the LysoPtdCho/PtdCho values determined in the organic extracts of erythrocytes. Thus, there is no absolute need for a sperm investigation, but an estimation of the fertilizing ability of the corresponding male could be also made directly from the blood which is more readily available than the spermatozoa.
Assuntos
Biomarcadores/metabolismo , Eritrócitos/metabolismo , Fertilidade , Lisofosfatidilcolinas/metabolismo , Fosfatidilcolinas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espermatozoides/metabolismo , Humanos , Masculino , Estresse OxidativoRESUMO
The lipids of human spermatozoa are characterized by unique fatty acyl compositions, i.e. considerable amounts of highly unsaturated, in particular docosahexaenoyl (22:6) fatty acyl residues. This makes spermatozoa very sensitive to oxidation. It has already been shown that the most abundant lipid of sperm, phosphatidylcholine (PC), is converted into lysophosphatidylcholine (LPC) under conditions of oxidative stress, and, thus, the PC/LPC ratio may be used as a measure of sperm quality. However, direct correlations between the PC/LPC ratios and certain pathologies are so far missing. We will show here for the first time (by using matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)) that sperm from obese donors (BMI>35kg/m(2)) are characterized by (a) a significantly increased LPC as well as (b) an increased sphingomyelin content. This does exclusively hold true for the lipid extracts of the spermatozoa but is not valid for the related seminal fluids.
Assuntos
Lisofosfatidilcolinas/análise , Obesidade/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espermatozoides/química , Esfingomielinas/análise , Adulto , Índice de Massa Corporal , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/patologia , Motilidade dos Espermatozoides , Espermatozoides/fisiologiaRESUMO
The important roles of lipids particularly certain phospholipids in signal transduction processes and as important disease markers are becoming increasingly evident. Unfortunately, however, sensitive methods of lipid analysis are established to a much lesser extent than, e.g., methods of protein analysis. Mass spectrometry (MS) is an increasingly used technique of lipid analysis and electrospray ionization (ESI) MS is the so far most established ionization method. Although matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was so far primarily used for protein analysis, however, this method has itself proven to be very useful in the field of lipid analysis, too. This chapter gives an overview of methodological aspects of MALDI-TOF MS in lipid research and summarizes the specific advantages and drawbacks of this soft-ionization method. In particular, suppression effects of some lipid classes, especially those with quaternary ammonia groups such as phosphatidylcholine, will be highlighted and possible ways to overcome this problem (use of different matrices, separation of the relevant lipid mixture prior to analysis) will be discussed on the example of an organic liver extract.
Assuntos
Fosfolipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia em Camada FinaRESUMO
Chondroitin sulfate (CS) is an important glycosaminoglycan of the extracellular matrix and its quantitative detection is of interest in different pathologies. Although there are already methods of quantitative CS determination, many of them are laborious, require time-consuming sample workup and/or suffer from low sensitivity. It will be shown here that the CS content of biological samples can be easily assessed in the picomole range subsequent to enzymatic digestion. MALDI-TOF MS (matrix-assisted laser desorption and ionization time-of-flight mass spectrometry) was used to determine the concentrations of the unsaturated disaccharide of CS obtained by enzymatic digestion of native CS with chondroitin ABC lyase. The signal-to-noise (S/N) ratio can be used as a quantitative measure: amounts of CS (measured as the disaccharide) down to at least 500fmol could be detected and there is a direct correlation between the S/N ratio and the amount of CS between about 2 and 200pmol although the curve per se is sigmoidal. The influence of different parameters such as the used matrix, the applied laser intensity and different methods of data analysis were also tested. Advantages and drawbacks of this approach are critically discussed. Finally, the method was validated by the determination of the CS content in samples of known concentration as well as in enzymatically digested bovine nasal cartilage and compared with two further established methods of CS determination (Carbazole and Alcian Blue method).