About 3D printability of thermoplastic collagen for biomedical applications

With more than 1.5 million total knee and hip implants placed each year, there is an urgent need for a drug delivery system that can effectively support the repair of bone infections. Scaffolds made of natural biopolymers are widely used for this purpose due to their biocompatibility, biodegradability, and suitable mechanical properties. However, the poor processability is a bottleneck, as highly customizable scaffolds are desired. The aim of the present research is to develop a scaffold made of thermoplastic collagen (TC) using 3D printing technology. The viscosity of the material was measured using a rheometer. A 3D bioplotter was used to fabricate the scaffolds out of TC. The mechanical properties of the TC scaffolds were performed using tension/compression testing on a Zwick/Roell universal testing machine. TC shows better compressibility with increasing temperature and a decrease in dynamic viscosity (η), storage modulus (G'), and loss modulus (G″). The compressive strength of the TC scaffolds was between 3-10 MPa, depending on the geometry (cylinder or cuboid, with different infills). We have demonstrated for the first time that TC can be used to fabricate porous scaffolds by 3D printing in various geometries.The article processing charge was funded by the Baden-Wuerttemberg Ministry of Science, Research and Art and the University of Freiburg in the funding program Open Access Publishing

Biliverdin-copper complex at the physiological pH

Biliverdin (BV) is a degradation product of heme catabolism, which is rapidly converted to bilirubin (BR) by BV reductase 1. Biliverdin and unconjugated BR, commonly named bile pigments, have important function in biochemical processes. The presence of copper and other biological and toxic transitional metals at significant concentrations in bile implies the possibility that metal complexes with bile pigments can be formed 2. Consequently, our interest was to study the complex of BV with copper in physiological conditions – phosphate buffer with pH 7.4. UV-Vis spectrophotometry was applied to investigate formation/degradation of complex of BV with copper ions and to check stoichiometry by titration, showing that BV interacted with Cu2+ in 1:1 stoichiometry. Mass spectroscopy analysis confirmed this – ion at m/z 643.36 was detected. The results of Raman spectroscopy of BV were in good agreement with previous reports 3. Comparing spectra of BV and BV-Cu complex, the following differences were observed: a new band at low wave number is emerged for the complex may be attributed to Cu-N bond vibration; the band which was shifted to lower energies implicates increased stability of BV in the complex; intensity changes imply a more planar structure of BV in the complex, while stronger bands in complex imply higher delocalization of π-electrons and consequently a higher stability of the BV structure. Pertinent to this, it has been proposed that complexes of BV model compounds with Cu2+ may show unusual electronic structures that exhibit a significant ligand radical character. 1H NMR spectrum of BV in phosphate buffer had a poor resolution of signals, which may originate from aggregation, but this was of little relevance here, since the addition of copper ions led to very strong effect - the complete loss of almost all lines. The loss of signals represents the result of strong paramagnetic effects that may come from an unpaired e- that is delocalized in pπ orbitalss of the ring/ligand influencing all protons in the complex. The EPR spectrum of Cu2+ (S = 1/2; I = 3/2) in phosphate buffer shows that Cu2+ is weakly coordinated in an axial symmetry with one gr line and four lines coming from hyperfine coupling along gs. The addition of BV in equimolar concentration led to the loss of Cu2+ signal. The remaining signal in the [BV]/[Cu2+] = 1 system was broad, and did not show hyperfine structure. The g-value of the isotropic signal of BV-Cu complex was significantly lower than the average g-value of Cu2+ in the phosphate buffer indicating delocalization of the spin away from the metal nucleus. Similar EPR signals have been reported previously 4. Parallel-mode EPR showed no signal. Furthermore, the spectra were run over a wide field range and no half field lines were observed, either in parallel or in perpendicular mode. These results are consistent with S = 0 for the copper center. Further, redox properties of the complex were examined. BV showed a well-defined anodic peak. The [BV]/[Cu2+] = 2 system showed two additional oxidation peaks at much lower potentials than BV. The former potential corresponds to the oxidation of Cu1+, as we have shown previously 5. There was a slight consumption of O2 in [BV]/[Cu2+] = 1 system, which may be explained by traces of ‘free’ copper. However, in the presence of an excess of copper ([BV]/[Cu2+] = 0.5), the consumption of O2 was significant. This implies that ‘free’ Cu2+ reacts with the complex and ‘shuttles’ an e- to O2. The complex was susceptible to oxidizing agents but not to reducing agents. Considering the results obtained we conclude that, at physiological pH, BV builds a complex with copper ions in 1:1 stoichiometry. The formation of complex involves the rearrangement of electronic structure which provides increased energetic stability and strong paramagnetic effects. We believe that a complex with a highly delocalized unpaired e- and the formal BV•+-Cu1+ character best suites the outlined properties, but other structures of the complex cannot be completely ruled out. The presented results may shed new light on long-standing issues of BV chemistry and catalysis in biological systems

Biliverdin-copper complex at the physiological pH

Biliverdin (BV) is a degradation product of heme catabolism, which is rapidly converted to bilirubin (BR) by BV reductase 1. Biliverdin and unconjugated BR, commonly named bile pigments, have important function in biochemical processes. The presence of copper and other biological and toxic transitional metals at significant concentrations in bile implies the possibility that metal complexes with bile pigments can be formed 2. Consequently, our interest was to study the complex of BV with copper in physiological conditions – phosphate buffer with pH 7.4. UV-Vis spectrophotometry was applied to investigate formation/degradation of complex of BV with copper ions and to check stoichiometry by titration, showing that BV interacted with Cu2+ in 1:1 stoichiometry. Mass spectroscopy analysis confirmed this – ion at m/z 643.36 was detected. The results of Raman spectroscopy of BV were in good agreement with previous reports 3. Comparing spectra of BV and BV-Cu complex, the following differences were observed: a new band at low wave number is emerged for the complex may be attributed to Cu-N bond vibration; the band which was shifted to lower energies implicates increased stability of BV in the complex; intensity changes imply a more planar structure of BV in the complex, while stronger bands in complex imply higher delocalization of π-electrons and consequently a higher stability of the BV structure. Pertinent to this, it has been proposed that complexes of BV model compounds with Cu2+ may show unusual electronic structures that exhibit a significant ligand radical character. 1H NMR spectrum of BV in phosphate buffer had a poor resolution of signals, which may originate from aggregation, but this was of little relevance here, since the addition of copper ions led to very strong effect - the complete loss of almost all lines. The loss of signals represents the result of strong paramagnetic effects that may come from an unpaired e- that is delocalized in pπ orbitalss of the ring/ligand influencing all protons in the complex. The EPR spectrum of Cu2+ (S = 1/2; I = 3/2) in phosphate buffer shows that Cu2+ is weakly coordinated in an axial symmetry with one gr line and four lines coming from hyperfine coupling along gs. The addition of BV in equimolar concentration led to the loss of Cu2+ signal. The remaining signal in the [BV]/[Cu2+] = 1 system was broad, and did not show hyperfine structure. The g-value of the isotropic signal of BV-Cu complex was significantly lower than the average g-value of Cu2+ in the phosphate buffer indicating delocalization of the spin away from the metal nucleus. Similar EPR signals have been reported previously 4. Parallel-mode EPR showed no signal. Furthermore, the spectra were run over a wide field range and no half field lines were observed, either in parallel or in perpendicular mode. These results are consistent with S = 0 for the copper center. Further, redox properties of the complex were examined. BV showed a well-defined anodic peak. The [BV]/[Cu2+] = 2 system showed two additional oxidation peaks at much lower potentials than BV. The former potential corresponds to the oxidation of Cu1+, as we have shown previously 5. There was a slight consumption of O2 in [BV]/[Cu2+] = 1 system, which may be explained by traces of ‘free’ copper. However, in the presence of an excess of copper ([BV]/[Cu2+] = 0.5), the consumption of O2 was significant. This implies that ‘free’ Cu2+ reacts with the complex and ‘shuttles’ an e- to O2. The complex was susceptible to oxidizing agents but not to reducing agents. Considering the results obtained we conclude that, at physiological pH, BV builds a complex with copper ions in 1:1 stoichiometry. The formation of complex involves the rearrangement of electronic structure which provides increased energetic stability and strong paramagnetic effects. We believe that a complex with a highly delocalized unpaired e- and the formal BV•+-Cu1+ character best suites the outlined properties, but other structures of the complex cannot be completely ruled out. The presented results may shed new light on long-standing issues of BV chemistry and catalysis in biological systems

Supplementary data for article: Dimitrijević, M. S.; Bogdanović Pristov, J.; Žižić, M.; Stanković, D. M.; Bajuk-Bogdanović, D.; Stanić, M.; Spasić, S.; Hagen, W.; Spasojević, I. Biliverdin-Copper Complex at Physiological PH. Dalton Transactions 2019, 48 (18), 6061–6070. https://doi.org/10.1039/c8dt04724c

Supplementary material for: [https://pubs.rsc.org/en/content/articlelanding/2019/DT/C8DT04724C#!divAbstract]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3066]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3068

Experimental and numerical investigation on soot formation and evolution of particle size distribution in laminar counterflow ethylene flames

A detailed investigation of the process of soot formation in ethylene-fueled laminar counterflow diffusion flames is conducted using dedicated experiments and numerical simulations. Two different strategies based on the Discrete Sectional Method (DSM) and the Split-based Quadrature Method of Moments (S-EQMOM) are considered to model the evolution of soot particle size distributions, and their comparative assessment is carried out for soot formation prediction and particle growth. A consistent chemical reaction mechanism describing the oxidation of hydrocarbon fuels and the prediction of soot precursors with the growth of polycyclic aromatic hydrocarbons (PAHs) up to pyrene ( ) is examined. Experiments for various strain rates and fuel compositions are performed to assess the sensitivity of soot production to these two parameters. The results show that both modeling strategies captured well the qualitative trends of soot volume fraction under variations in strain rate and mixture composition, with slight over-prediction of the peak values. For both soot models, a higher sensitivity of soot formation is noticed by changes in mixture composition compared to those of strain rate variation. Additionally, the soot models demonstrated promising performance in capturing the experimentally observed evolution of the soot particle size distribution (PSD)

Experimental and numerical investigation on soot formation and evolution of particle size distribution in laminar counterflow ethylene flames

A detailed investigation of the process of soot formation in ethylene-fueled laminar counterflow diffusion flames is conducted using dedicated experiments and numerical simulations. Two different strategies based on the Discrete Sectional Method (DSM) and the Split-based Quadrature Method of Moments (S-EQMOM) are considered to model the evolution of soot particle size distributions, and their comparative assessment is carried out for soot formation prediction and particle growth. A consistent chemical reaction mechanism describing the oxidation of hydrocarbon fuels and the prediction of soot precursors with the growth of polycyclic aromatic hydrocarbons (PAHs) up to pyrene ( ) is examined. Experiments for various strain rates and fuel compositions are performed to assess the sensitivity of soot production to these two parameters. The results show that both modeling strategies captured well the qualitative trends of soot volume fraction under variations in strain rate and mixture composition, with slight over-prediction of the peak values. For both soot models, a higher sensitivity of soot formation is noticed by changes in mixture composition compared to those of strain rate variation. Additionally, the soot models demonstrated promising performance in capturing the experimentally observed evolution of the soot particle size distribution (PSD)

Supplementary data for article: Dimitrijević, M. S.; Bogdanović Pristov, J.; Žižić, M.; Stanković, D. M.; Bajuk-Bogdanović, D.; Stanić, M.; Spasić, S.; Hagen, W.; Spasojević, I. Biliverdin-Copper Complex at Physiological PH. Dalton Transactions 2019, 48 (18), 6061–6070. https://doi.org/10.1039/c8dt04724c

Supplementary material for: [https://pubs.rsc.org/en/content/articlelanding/2019/DT/C8DT04724C#!divAbstract]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3066]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3068

Methylation Markers of Early-Stage Non-Small Cell Lung Cancer

Despite of intense research in early cancer detection, there is a lack of biomarkers for the reliable detection of malignant tumors, including non-small cell lung cancer (NSCLC). DNA methylation changes are common and relatively stable in various types of cancers, and may be used as diagnostic or prognostic biomarkers.We performed DNA methylation profiling of samples from 48 patients with stage I NSCLC and 18 matching cancer-free lung samples using microarrays that cover the promoter regions of more than 14,500 genes. We correlated DNA methylation changes with gene expression levels and performed survival analysis.We observed hypermethylation of 496 CpGs in 379 genes and hypomethylation of 373 CpGs in 335 genes in NSCLC. Compared to adenocarcinoma samples, squamous cell carcinoma samples had 263 CpGs in 223 hypermethylated genes and 513 CpGs in 436 hypomethylated genes. 378 of 869 (43.5%) CpG sites discriminating the NSCLC and control samples showed an inverse correlation between CpG site methylation and gene expression levels. As a result of a survival analysis, we found 10 CpGs in 10 genes, in which the methylation level differs in different survival groups.We have identified a set of genes with altered methylation in NSCLC and found that a minority of them showed an inverse correlation with gene expression levels. We also found a set of genes that associated with the survival of the patients. These newly-identified marker candidates for the molecular screening of NSCLC will need further analysis in order to determine their clinical utility