Vladimir J. Cvetković

Antioxidant, Antimicrobial and Antiproliferative Activities of Five Lichen Species

The antioxidative, antimicrobial and antiproliferative potentials of the methanol extracts of the lichen species Parmelia sulcata, Flavoparmelia caperata, Evernia prunastri, Hypogymnia physodes and Cladonia foliacea were evaluated. The total phenolic content of the tested extracts varied from 78.12 to 141.59 mg of gallic acid equivalent (GA)/g of extract and the total flavonoid content from 20.14 to 44.43 mg of rutin equivalent (Ru)/g of extract. The antioxidant capacities of the lichen extracts were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging. Hypogymnia physodes with the highest phenolic content showed the strongest DPPH radical scavenging effect. Further, the antimicrobial potential of the lichen extracts was determined by a microdilution method on 29 microorganisms, including 15 strains of bacteria, 10 species of filamentous fungi and 4 yeast species. A high antimicrobial activity of all the tested extracts was observed with more potent inhibitory effects on the growth of Gram (+) bacteria. The highest antimicrobial activity among lichens was demonstrated by Hypogymnia physodes and Cladonia foliacea. Finally, the antiproliferative activity of the lichen extracts was explored on the colon cancer adenocarcinoma cell line HCT-116 by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) viability assay and acridine orange/ethidium bromide staining. The methanol extracts of Hypogymnia physodes and Cladonia foliacea showed a better cytotoxic activity than the other extracts. All lichen species showed the ability to induce apoptosis of HCT-116 cells.

Platismatia glaucia and Pseudevernia furfuracea lichens as sources of antioxidant, antimicrobial and antibiofilm agents

The antioxidative, antimicrobial and antibiofilm potentials of acetone, ethyl acetate and methanol extracts of lichen species Platismatia glauca and Pseudevernia furfuracea were evaluated. The phytochemical analysis by GC, GC/MS and NMR revealed caperatic acid, atraric acid, atranorin and chloroatranorin as the predominant compounds in Platismatia glauca. Atraric acid, olivetoric acid, atranorin and chloroatranorin were the major constituents in Pseudevernia furfuracea. The strong antioxidant capacities of the Platismatia glauca and Pseudevernia furfuracea extracts were assessed by their total phenolic and flavonoid contents and DPPH scavenging activities. The methanol extracts of both species exhibited the strongest antioxidant activities with the highest IC50 value for Pseudevernia furfuracea (95.33 µg/mL). The lichen extracts demonstrated important antibacterial activities against 11 bacterial strains with detectable MIC values from 0.08 mg/mL to 2.5 mg/mL for Platismatia glauca and from 0.005 mg/mL to 2.5 mg/mL for Pseudevernia furfuracea. While the antibacterial activities of Pseudevernia furfuracea were solvent–independent, the acetone and ethyl acetate extracts of Platismatia glauca showed higher antibacterial activities compared to its methanol extract. The methanol extracts of both species demonstrated significant antifungal activities against 9 fungal strains with detectable MIC values from 0.04 mg/mL to 2.5 mg/mL. The best antifungal activities were determined against Candida species in Pseudevernia furfuracea extracts with remarkable MIC values which were lower than the MIC values of the positive contol fluconazole. The acetone and ethyl acetate extracts of Platismatia glauca showed better antibiofilm activities on Staphylococcus aureus and Proteus mirabilis with BIC value at 0.63 mg/mL then its methanol extract. On the other hand, the methanol extract of Pseudevernia furfuracea was more potent with BIC value at 1.25 mg/mL on Staphylococcus aureus and 0.63 mg/mL on Proteus mirabilis compared to other types of extracts. Our study indicates a possible use of lichens Platismatia glauca and Pseudevernia furfuracea as natural antioxidants and preservatives in food, pharmaceutical and cosmetic industry.

Effects of human food grade titanium dioxide nanoparticle dietary exposure on Drosophila melanogaster survival, fecundity, pupation and expression of antioxidant genes

The fruitfly, Drosophila melanogaster was exposed to the human food grade of E171 titanium dioxide (TiO2). This is a special grade of TiO2 which is frequently omitted in nanotoxicology studies dealing with TiO2, yet it is the most relevant grade regarding oral exposure of humans. D. melanogaster larvae were exposed to 0.002 mg mL(-1), 0.02 mg mL(-1), 0.2 mg mL(-1), and 2 mg mL(-1) of TiO2 in feeding medium, and the survival, fecundity, pupation time, and expression of genes involved in oxidative stress response were monitored. TiO2 did not affect survival but significantly increased time to pupation (p < 0.001). Fecundity of D. melanogaster was unaffected by the treatment. Expression of the gene for catalase was markedly downregulated by the treatment, while the effect on the downregulation of superoxide dismutase 2 was less pronounced. After four days of dietary exposure TiO2 was present in a significant amount in larvae, but was not transferred to adults during metamorphosis. Two individuals with aberrant phenotype similar to previously described gold nanoparticles induced mutant phenotypes were detected in the group exposed to TiO2. In general, TiO2 showed little toxicity toward D. melanogaster at concentrations relevant to oral exposure of humans.

Osteogenic potential of in vitro osteo-induced adipose-derived mesenchymal stem cells combined with platelet-rich plasma in an ectopic model

PurposeThe osteogenic potential of in vitro osteo-induced adipose-derived mesenchymal stem cells (ADSCs) combined with platelet-rich plasma (PRP) and implanted on bone mineral matrix (BMM) carrier was examined in a subcutaneous model in Balb/c mice.MethodsIn vitro osteogenic differentiation of ADSCs was monitored by relative bone-related gene expression and osteocalcin expression at the third, seventh and 15th day. Test implants consisting of in vitro osteo-induced ADSCs, PRP and BMM (OPC implants) and control implants consisting of PRP and BMM (PC implants) were examined. The relative expression of the bone-related genes encoding osterix, osteocalcin, collagen type I α1 and alkaline phosphatase was examined in implants extracted at one, two, four and eight weeks. Histochemical, immunohistochemical and histomorphometric analyses of implants extracted at two and eight weeks were performed.ResultsThe highest relative expression of bone-related genes and osteocalcin expression was found at the 15th day of in vitro osteogenic induction of the ADSCs. Permanent and continuous increased expression of bone-related genes was noticed in OPC implants at eight weeks. Expression peaks of bone-related genes in PC implants were at two and four weeks, but they significantly decreased at eight weeks. The signs of resorption, formation of callus-like tissue positive for osteocalcin and increased presence of bone cells were found in OPC implants compared with PC implants. A higher percentage of infiltrated tissue and vascularisation was found in OPC than in PC implants.ConclusionsThe combination of in vitro osteo-induced ADSCs and PRP on BMM carrier represents a promising approach for bone regeneration.

Effects of bone tissue engineering triad components on vascularization process: comparative gene expression and histological evaluation in an ectopic bone-forming model

ABSTRACT Vascular development has a great significance in the osteogenic process and in bone tissue engineering (BTE). BTE is based on various combinations of three principal types of components: biomaterials as scaffolds, regulatory signals and cells. The aim of our study was to evaluate, at gene expression and histological level, the effect of BTE triad components on the vascularization process in an ectopic bone-forming model in mice. Bone mineral matrix (BMM) was used as a scaffold and a carrier, platelet-rich plasma (PRP) as a source of regulatory signals and adipose stem cells (ASCs) as a source of cells for endothelial differentiation, in order to show how and to what extent the biological enrichment of BMM influences the outcome of the osteogenic process and its key precondition, vascularization. Implants composed of BMM, PRP and ASCs in vitro induced into endothelial cells (EPB implants) and implants composed of BMM and PRP (PB implants) were compared with implants composed of BMM only (B implants). More pronounced endothelial-related gene expression and stronger VCAM-1 (vascular cell adhesion molecule-1) immunoexpression were observed in EPB implants in comparison with PB and B ones at later time points of the in vivo experimental period. Osteopontin gene expression and immunoexpression of osteopontin were significantly higher in EPB compared to PB and B implants. Therefore, addition of ASCs combined with PRP to BMM improved the vascularization process in the ectopic bone-forming model, which makes this BTE composition the most favourable among the examined types of implants for application in BTE.

Genotoxicity and Mutagenicity Testing of Biomaterials

Rapid progress in biomedical fields and development of new technologies in bioengineering and tissue engineering, such as 3D bioprinting technologies, lead us to extensive and rapid designing of new biomaterials and medical devices (MDs). The main feature that newly engineered biomaterials must possess, in order to be applied in clinical practice, is to be biocompatible. Besides toxicity testing, genotoxicity and mutagenicity assays are very important in assessment of biocompatibility. Genotoxicity/mutagenicity is a feature that can completely disable the use of some biomaterial and MDs in biomedical and clinical applications especially if material is intended for long term use. Mutagens are agents that can cause heritable changes in DNA and their capacity to cause mutations is defined as mutagenicity. Genotoxicity is a wider term which, besides mutagenicity, refers to the property of an agent to cause various damages to DNA and other disturbances which can affect genes function. In this chapter, we gave an overview of in vitro and in vivo assays that are mostly used in biocompatibility assessment with a focus on the most promising assays for evaluations of genotoxicity and mutagenicity of biomaterials.

Osteogenic capacity of diluted platelet-rich plasma in ectopic bone-forming model: Benefits for bone regeneration

Platelet-rich plasma (PRP) with normal and below-normal physiological concentrations of platelets is designated as diluted PRP (dPRP). The aims of this study are to evaluate whether bone mineral matrix in combination with dPRP possesses osteogenic capacity; and whether the differences in dynamics and osteogenic process pattern depend on different platelet concentrations, to what extent, and also what could be benefits for bone regeneration in clinical practice. Three types of implants were made: BMM-bone mineral matrix alone; dPRP/10-bone mineral matrix mixed with dPRP (concentration of platelets 10 times lower than physiological level) and dPRP/3-bone mineral matrix mixed with dPRP (concentration of platelets 3 times lower than physiological level). A subcutaneous implantation model in Balb/c mice was used. The implants were analyzed using expression analysis of bone-related genes, histochemical, immunohistochemical and histomorphometrical analysis. All types of implants induced creation of necessary preconditions for supporting osteogenic processes, but did not induce visible young bone growth. Implant types dPRP/10 and dPRP/3 showed very similar and significantly better stimulatory effects on osteogenic processes than bone matrix alone. In this study, significant ectopic osteogenic potential of concentration of platelets in PRP that are lower than physiological level in blood plasma in combination with bone mineral matrix was demonstrated. Diluted platelet-rich plasma could be a promising and useful adjuvant therapeutic agent in bone regeneration.