ABSTRACT
Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.
As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].
Subject(s)
Animals , Rats , Spectrum Analysis, Raman/methods , Mesenchymal Stem Cells , Biochemical PhenomenaABSTRACT
Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.
Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.
ABSTRACT
Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.
Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.
Subject(s)
Animals , Rats , Mesenchymal Stem Cells , Osteogenesis , Polyesters , Spectrum Analysis, Raman , Culture Media, Conditioned , Cell Proliferation , Tissue ScaffoldsABSTRACT
BACKGROUND: Kidney diseases are a global health problem. Currently, over 2 million people require dialysis or transplant which are associated with high morbidity and mortality; therefore, new researches focused on regenerative medicine have been developed, including the use of stem cells. RESULTS: In this research, we generate differentiated kidney cells (DKCs) from mouse pluripotent stem cells (mPSCs) analyzing their morphological, genetic, phenotypic, and spectroscopic characteristics along differentiation, highlighting that there are no reports of the use of Fourier transform infrared (FTIR) spectroscopy to characterize the directed differentiation of mPSCs to DKCs. The genetic and protein experiments proved the obtention of DKCs that passed through the chronological stages of embryonic kidney development. Regarding vibrational spectroscopy analysis by FTIR, bands related with biomolecules were shown on mPSCs and DKCs spectra, observing distinct differences between cell lineages and maturation stages. The second derivative of DKCs spectra showed changes in the protein bands compared to mPSCs. Finally, the principal components analysis obtained from FTIR spectra allowed to characterize chemical and structurally mPSCs and their differentiation process to DKCs in a rapid and non-invasive way. CONCLUSION: Our results indicated that we obtained DKCs from mPSCs, which passed through the chronological stages of embryonic kidney development. Moreover, FTIR spectroscopy resulted in a non-invasive, rapid and precise technic that together with principal component analysis allows to characterize chemical and structurally both kind of cells and also discriminate and determine different stages along the cell differentiation process.
Subject(s)
Animals , Mice , Cell Differentiation/physiology , Spectroscopy, Fourier Transform Infrared/methods , Pluripotent Stem Cells/physiology , Kidney/cytology , Immunohistochemistry , Gene Expression , Cells, Cultured , Fluorescent Antibody Technique , Principal Component Analysis , Pluripotent Stem Cells/cytology , Real-Time Polymerase Chain ReactionABSTRACT
The aim of this work was to verify the existence of correlation between Raman spectroscopy readings of phosphate apatite (∼960 cm−1), fluoridated apatite (∼575 cm−1) and organic matrix (∼1450 cm−1) levels and Diagnodent® readings at different stages of dental caries in extracted human teeth. The mean peak value of fluorescence in the carious area was recorded and teeth were divided in enamel caries, dentin caries and sound dental structure. After fluorescence readings, Raman spectroscopy was carried out on the same sites. The results showed significant difference (ANOVA, p<0.05) between the fluorescence readings for enamel (16.4 ± 2.3) and dentin (57.6 ± 23.7) on carious teeth. Raman peaks of enamel and dentin revealed that ∼575 and ∼960 cm−1 peaks were more intense in enamel caries. There was significant negative correlation (p<0.05) between the ∼575 and ∼960 cm−1 peaks and dentin caries. It may be concluded that the higher the fluorescence detected by Diagnodent the lower the peaks of phosphate apatite and fluoridated apatite. As the early diagnosis of caries is directly related to the identification of changes in the inorganic tooth components, Raman spectroscopy was more sensitive to variations of these components than Diagnodent.
O objetivo desse estudo foi verificar por meio da espectroscopia Raman, a existência de correlação entre os níveis de apatita fosfatada (∼960 cm−1), apatita fluoretada (∼575 cm−1) e matriz orgânica (∼1450 cm−1) e as leituras do Diagnodent® em diferentes estágios de cárie dental em dentes humanos extraídos. O valor médio do pico de fluorescência na área da cárie foi anotado e os dentes divididos em cárie de esmalte, dentina e dente hígido. Após as leituras de fluorescência, foi realizada a espectroscopia Raman nos mesmos sítios. Os resultados mostraram diferença significante (ANOVA p<0,05) entre as leituras de fluorescência para esmalte (16,4 ± 2,3) e dentina (57,6 ± 23,7) nos dentes cariados. Os picos Raman para esmalte e dentina evidenciaram que os picos ∼575 e ∼960 cm−1 foram mais intensos em cárie de esmalte. Houve correlação negativa e significante (p<0,05) entre os picos ∼575 e ∼960 cm−1 e cárie de dentina. Pode-se concluir que quanto maior a fluorescência detectada pelo Diagnodent menor o pico da apatita fosfatada e fluoretada. O diagnóstico precoce da cárie está diretamente relacionado com a identificação de mudanças nos componentes inorgânicos do dente, assim a espectroscopia Raman foi mais sensível para variações desses componentes quando comparada ao Diagnodent.
Subject(s)
Humans , Dental Caries/diagnosis , Dental Enamel/chemistry , Dentin/chemistry , Durapatite/analysis , Lasers, Semiconductor , Spectrum Analysis, Raman , Analysis of Variance , Apatites/analysis , Fluorescence , Fluorometry , Organic Chemicals/analysis , Statistics, Nonparametric , VibrationABSTRACT
Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have a far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand-binding domain and for subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction to the insight gained from X-ray crystallography and nuclear magnetic resonance investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer to study the behavior of the isolated ligand-binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation.