Ultrasound Assisted Extraction of Saponins from Hedera helix L. and an In Vitro Biocompatibility Evaluation of the Extracts.

The aim of this study was to establish the best ultrasound assisted extraction (UAE) conditions of saponins from Hedera helix L. leaves and to evaluate the in vitro biocompatibility of the extracts richest in saponins. Different parameters, such as extraction time, temperature, ultrasound power, solvent to plant material ratio, and solvent concentration, were investigated. The most efficient extraction conditions were a temperature of 50 °C, an ultrasound amplitude of 40%, an extraction time of 60 min, a plant material to solvent ratio of 1:20 (w:v), and 80% ethanol as solvent. In vitro cytotoxicity of the extracts richest in saponins and their influence on the DNA content of L929 (NCTC) fibroblasts were tested. Until 200 µg/mL, the studied extracts were cytocompatible with L929 fibroblast cell lines at 48 h of treatment. These in vitro cell culture results provide useful information for further applications of Hedera helix extracts in a pharmaceutical field.

New Pyrazolo-Benzimidazole Mannich Bases with Antimicrobial and Antibiofilm Activities.

A new series of pyrazolo-benzimidazole hybrid Mannich bases were synthesized, characterized by 1H-NMR, 13C-NMR, IR, UV-Vis, MS, and elemental analysis. In vitro cytotoxicity of the new compounds studied on fibroblast cells showed that the newly synthesized pyrazolo-benzimidazole hybrid derivatives were noncytotoxic until the concentration of 1 µM and two compounds presented a high degree of biocompatibility. The antibacterial and antibiofilm activity of the newly synthesized compounds was assayed on Gram-positive Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC29212, and Gram-negative Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922 strains. All synthesized compounds 5a-g are more active against all three tested bacterial strains Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC29212, and Escherichia coli ATCC25922 than reference drugs (Metronidazole, Nitrofurantoin), with the exception of compounds 5d and 5g, which are less active compared to Nitrofurantoin, and all synthesized compounds 5a-g are more active against Pseudomonas aeruginosa ATCC27853 compared to reference drugs (Metronidazole, Nitrofurantoin). Compound 5f showed the best activity against Staphylococcus aureus ATCC 25923, with a MIC of 150 µg/mL and has also inhibited the biofilm formed by all the bacterial strains, having an MBIC of 310 µg/mL compared to the reference drugs (Metronidazole, Nitrofurantoin).

Marine and Agro-Industrial By-Products Valorization Intended for Topical Formulations in Wound Healing Applications.

Over the past years, research attention has been focusing more on waste-derived, naturally derived, and renewable materials, in the view of a more sustainable economy. In this work, different topical formulations were obtained from the valorization of marine and agro-industrial by-products and the use of Carbopol 940 as gelling agent. In particular, the combination of extracts obtained from the marine snail, Rapanosa venosa, with Cladophora vagabunda and grape pomace extracts, was investigated for wound healing purposes. Rapana venosa has demonstrated wound healing properties and antioxidant activity. Similarly, grape pomace extracts have been shown to accelerate the healing process. However, their synergic use has not been explored yet. To this aim, four different formulations were produced. Three formulations differed for the presence of a different extract of Rapana venosa: marine collagen, marine gelatin, and collagen hydrolysate, while another formulation used mammalian gelatin as further control. Physico-chemical properties of the extracts as well as of the formulations were analyzed. Furthermore, thermal stability was evaluated by thermogravimetric analysis. Antioxidant capacity and biological behavior, in terms of cytocompatibility, wound healing, and antimicrobial potential, were assessed. The results highlighted for all the formulations (i) a good conservation and thermal stability in time, (ii) a neutralizing activity against free radicals, (iii) and high degree of cytocompatibility and tissue regeneration potential. In particular, collagen, gelatin, and collagen hydrolysate obtained from the Rapana venosa marine snail represent an important, valuable alternative to mammalian products.

Fluorescent Flavin/PVP-Coated Silver Nanoparticles: Design and Biological Performance.

A red-emitting fluorescent Riboflavin (RF)/Polyvinylpyrrolidone (PVP)-coated silver nanoparticles system, λem = 527 nm, Φ = 0.242, with a diameter of the metallic core of 27.33 nm and a zeta potential of - 25.05 mV was prepared and investigated regarding its biological activity. We found that PVP has a key role in RF adsorption around the SNPs surface leading to an enhancement of antioxidant properties (∼70%), low cytotoxicity (> 90% cell viability, at 50 µL/mL, after 48 h of incubation) as well as to an efficient process of its cellular uptake (∼ 60%, after 24 h of incubation) in L929 cells. The results are relevant concerning the involvement of RF and its coenzymes forms in SNPs - based systems, in cellular respiration as well as for future studies as antioxidant marker system on tumoral cells for viewing and monitoring them, by cellular imaging.

Enhanced Wound Healing Activity of Undenatured Type I Collagen Isolated from Discarded Skin of Black Sea Gilthead Bream (Sparus aurata) Conditioned as 3D Porous Dressing.

Acid-soluble, undenatured, type I collagen (BSC) isolated, for the first time, from gilthead bream skin and the novel fabricated 3D porous wound dressing were analyzed for physicochemical and biological properties, in order to offer a safe alternative to commercial bovine collagen (BC) products. SDS-polyacrylamide analysis confirmed the purity of BSC preparation. The hydroxyproline content and temperature of denaturation of BSC were lower than those of BC, in accordance with the structural data recorded by FT-IR spectroscopy. However, certain concentrations of BSC stimulated the cell metabolism of L929 fibroblasts in a higher proportion than BC. The 3D wound dressing presented high porosity and low surface hydrophobicity that could help cell attachment and growth. The rapid biodegradation of BSC wound dressing could explain the improved in vitro cell migration and wound closure rate. In conclusion, the skin of gilthead bream from the Black Sea coast represented a valuable source for the biomedical industry, providing biocompatible, biodegradable collagen and 3D porous wound dressing, as novel material with enhanced wound healing activity.

Spectroscopic, molecular dynamics simulation and biological studies of Flavin MonoNucleotide and Flavin Adenine Dinucleotide in biomimetic systems.

The present study describes a comprehensive investigation of the spectroscopic characteristics, stability and in vitro antioxidant and cytotoxic properties of the Flavin MonoNucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) in Dextran70 (Dx70) and Dx70/phospatidylcholine (PC) biomimetic systems by means of the UV-Vis absorption, fluorescence spectroscopy, chemiluminescence and Neutral Red assay. The affinity of FMN, FAD and the precursor riboflavin (RF) to an unsaturated phospholipid bilayer model as well as the location of the probes within the lipid bilayer were assessed from united-atom molecular dynamics simulations carried out on an unsaturated phospholipid bilayer model system, and the theoretical and experimental characterization of the two probes within biomembranes was complemented with the light microscopy survey of the cell morphology of L929 fibroblast cells cultivated in the presence of various dosage of FAD/FMN. In lipid bilayers, FMN/FAD resulted in a noticeable improvement of the antioxidant activity (the scavenging of reactive oxygen species up to 40%) and a significant effect on cellular viability in the L929 fibroblast cells. The results are important in the oxidative stress process concerning the redox reactions of flavins in humans as well as in further studies on different systems belonging to the category of flavoenzymes/flavoproteins, required for cellular respiration.

Physicochemical and Biological Properties of Gelatin Extracted from Marine Snail Rapana venosa.

In this study, we aimed to obtain gelatin from the marine snail Rapana venosa using acidic and enzymatic extraction methods and to characterize these natural products for cosmetic and pharmaceutical applications. Marine gelatins presented protein values and hydroxyproline content similar to those of commercial mammalian gelatin, but with higher melting temperatures. Their electrophoretic profile and Fourier transform infrared (FTIR) spectra revealed protein and absorption bands situated in the amide region, specific for gelatin molecule. Scanning electron microscopy (SEM) analysis showed significant differences in the structure of the lyophilized samples, depending on the type of gelatin. In vitro studies performed on human keratinocytes showed no cytotoxic effect of acid-extracted gelatin at all tested concentrations and moderate cytotoxicity of enzymatic extracted gelatin at concentrations higher than 0.5 mg/mL. Also, both marine gelatins favored keratinocyte cell adhesion. No irritant potential was recorded as the level of IL-1α and IL-6 proinflammatory cytokines released by HaCaT cells cultivated in the presence of marine gelatins was significantly reduced. Together, these data suggest that marine snails are an alternative source of gelatins with potential use in pharmaceutical and skincare products.

Morphology and ultrastructure of the somatic cells in Astacus leptodactylus ovary.

We defined the somatic environment in which female germinal cells develop, and performed ultrastructural analyses of various somatic cell types, with particular reference to muscle cells and follicle cells, that reside within the ovary at different stages of oogenesis. Our findings show that ovarian wall of the crayfish is composed of long muscle cells, blood cells, blood vessels and hemal sinuses. The follicle and germinal cells lie within a common compartment of ovarian follicles that is defined by a continuous basal matrix. The follicle cells form branching cords and migrate to surround the developing oocytes. A thick basal matrix separates the ovarian interstitium from ovarian follicles compartment. Transmission electron microscopy shows that inner layer of basal matrix invaginates deeply into the ovarian compartment. Our results suggest that before being surrounded by follicle cells to form follicles, oogonia and early previtellogenic oocytes reside within a niche surrounded by a basal matrix that separates them from ovarian interstitium. We found coated pits and coated vesicles in the cortical cytoplasm of previtellogenic and vitellogenic oocytes, suggesting the receptor mediated endocytosis for transfer of material from the outside of the oocytes, via follicle cells. The interstitial compartment between the inner muscular layer of the ovarian wall and the basal matrix of the ovarian follicle compartment contains muscle cells, hemal sinuses, blood vessels and blood cells. Granular hemocytes, within and outside the vessels, were the most abundant cell population in the ovarian interstitium of crayfish after spawning and in the immature ovary.

Comparative studies of mesenchymal stem cells derived from different cord tissue compartments - The influence of cryopreservation and growth media.

INTRODUCTION: We have identified some critical aspects concerning umbilical cord tissue mesenchymal stem cells: the lack of standards for cell isolation, expansion and cryopreservation, the lack of unanimous opinions upon their multilineage differentiation potential and the existence of very few results related to the functional characterization of the cells isolated from cryopreserved umbilical cord tissue. Umbilical cord tissue cryopreservation appears to be the optimal solution for umbilical cord tissue mesenchymal stem cells storage for future clinical use. Umbilical cord tissue cryopreservation allows mesenchymal stem cells isolation before expected use, according with the specific clinical applications, by different customized isolation and expansion protocols agreed by cell therapy institutions. METHODS: Using an optimized protocol for umbilical cord tissue cryopreservation in autologous cord blood plasma, isolation explant method and growth media supplemented with FBS or human serum, we performed comparative studies with respect to the characteristics of mesenchymal stem cells (MSC) isolated from different compartments of the same umbilical cord tissue such as Wharton's jelly, vein, arteries, before cryopreservation (pre freeze) and after cryopreservation (post thaw). RESULTS: Expression of histochemical and immunohistochemical markers as well as electron microscopy observations revealed similar adipogenic, chondrogenic and osteogenic differentiation capacity for cells isolated from pre freeze and corresponding post thaw tissue fragments of Wharton's jelly, vein or arteries of the same umbilical cord tissue, regardless growth media used for cells isolation and expansion. DISCUSSION: Our efficient umbilical cord tissue cryopreservation protocol is reliable for clinical applicability of mesenchymal stem cells that could next be isolated and expanded in compliance with future accepted standards.

Physicochemical Characterization and In Vitro Cytotoxic Effect of 3-Hydroxyflavone in a Silver Nanoparticles Complex.

The aim of this work was to characterize the physico-chemical properties of 3-hydroxyflavone (3-HF) in a silver nanoparticles complex (SNPs) using UV-vis and Fluorescence spectroscopy, Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM) analysis. One also evaluated its effect on the cell viability and morphology of L929 mouse fibroblast cells in vitro. The contribution of the carrier protein, Bovine Serum Albumin (BSA) to 3-HF properties has also been investigated. 3-HF in BSA/SNPs systems presented no cytotoxic effect in L929 mouse fibroblast cells at any of the tested concentrations. The results are discussed with relevance to the oxidative stress process.

Structural heterogenity of intraluminal content of the prostate: a histochemical and ultrastructural study.

Intraluminal contents of benign and malignant prostatic tissue are associated with varying forms of acellular structures. These include corpora amylacea, prostatic calculi, and prostatic crystalloids. There are relatively few microscopy studies about the characterization of intraluminal structures from benign and malignant prostatic glands and little is known about their chemical composition. In the present study, we used a combination of special histochemical methods, immunohistochemistry, and transmission electron microscopy to characterize intraluminal contents of benign and malignant prostate glands. The study was done on 33 radical prostatectomy and four transurethral resections of prostate specimens. Histochemical methods such as von Kossa, autometallography (AMG), as well as PSA immunohistochemistry and transmission electron microscopy were performed to characterize intraluminal contents of benign and malignant prostate glands. Von Kossa staining was observed in acellular structures, corpora amylacea, prostatic calculi, and calcified blood vessels. AMG staining was observed in the lumen of small glands, in the epithelium lining prostate glands, and corpora amylacea. PSA staining showed prostatic glands with both positive and negative corpora amylacea and epithelial cells. Ultrastructural observation revealed the presence of a variety of highly heterogeneous aggregates composed of fibrillar elements that were similar to those of amyloid.

Autologous mesenchymal stem cells applied on the pressure ulcers had produced a surprising outcome in a severe case of neuromyelitis optica.

Recent studies provided evidence that mesenchymal stem cells (MSCs) have regenerative potential in cutaneous repair and profound immunomodulatory properties making them a candidate for therapy of neuroimmunologic diseases. Neuromyelitis optica (NMO) is an autoimmune, demyelinating central nervous system disorder characterized by a longitudinally extensive spinal cord lesion. A 46-year-old male diagnosed with NMO had relapses with paraplegia despite treatment and developed two stage IV pressure ulcers (PUs) on his legs. The patient consented for local application of autologous MSCs on PUs. MSCs isolated from the patient's bone marrow aspirate were multiplied in vitro during three passages and embedded in a tridimensional collagen-rich matrix which was applied on the PUs. Eight days after MSCs application the patient showed a progressive healing of PUs and improvement of disability. Two months later the patient was able to walk 20 m with bilateral assistance and one year later he started to walk without assistance. For 76 months the patient had no relapse and no adverse event was reported. The original method of local application of autologous BM-MSCs contributed to healing of PUs. For 6 years the patient was free of relapses and showed an improvement of disability. The association of cutaneous repair, sustained remission of NMO and improvement of disability might be explained by a promotion/optimization of recovery mechanisms in the central nervous system even if alternative hypothesis should be considered. Further studies are needed to assess the safety and efficacy of mesenchymal stem cells in NMO treatment.

Evaluation of pristine and Eu 2O3-added MgB 2 ceramics for medical applications: hardness, corrosion resistance, cytotoxicity and antibacterial activity.

Nano- or micropowders of Eu2O3 were added to MgB2, resulting in a composition of (MgB2)0.975(EuO1.5)0.025. Pristine and doped samples were prepared using spark plasma sintering and tested for (i) Vickers hardness, (ii) pH evolution in phosphate-buffered saline solution, (iii) corrosion resistance (Tafel polarization curves), (iv) cytotoxicity (in vitro tests), and (v) antibacterial activity. Eu2O3 addition influenced the investigated properties. Solutions of MgB2-based samples show a relatively high saturation pH of 8.5. This value is lower than that of solutions incubated with Mg or other Mg-based biodegradable alloys reported in the literature. MgB2-based samples have lower electro-corrosion rates than Mg. Their Vickers hardness is 6.8-10.2GPa, and these values are higher than those of biodegradable Mg-based alloys. MgB2 has low in vitro biocompatibility, good antibacterial activity against Escherichia coli, and mild activity against Staphylococcus aureus. Our results suggest that MgB2-based materials deserve attention in biomedical applications, such as implants or sterile medical instruments.

Ultrastructural features of human adipose-derived multipotent mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MMSCs) are plastic-adherent cells with a well-established phenotype. Equine, but not human, adipose MMSCs have been characterized ultrastructurally. The purpose of our study was to evaluate ultrastructurally the adipose-derived human MMSCs. Cell cultures were prepared from human lipoaspirate. The flow cytometry evaluation of surface markers of cultured cells confirmed the expected profile of MMSCs, that were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. We examined these human adipose-derived MMSCs in transmission electron microscopy (TEM) by Epon en-face embedding the fixed MMSCs. The main ultrastructural features of MMSCs were the extremely rich content of endosomal/vesicular elements, long mitochondria, dilated RER (rough endoplasmic reticulum) cisternae, and abundant intermediate filaments and microtubules. We found two types of MMSCS prolongations: (a) thick processes, with opposite, vesicular and filaments-rich, sides and (b) slender processes (pseudopodes and filopodes), with occasional proximal dilated segments housing mitochondria, vesicles and secretory granules. These TEM features of MMSCs characterized an in vitro cell population and could use to distinguish between different cell types in culture.

Liposomal formulation of chondroitin sulfate enhances its antioxidant and anti-inflammatory potential in L929 fibroblast cell line.

Liposomes have the capacity to be used as efficient, biodegradable and nontoxic carriers of bioactive molecules and are able to better control their delivery at the site of interest. The objective of this study was to obtain and characterize an appropriate liposomal formulation of the bioactive molecule chondroitin sulfate (CS) for its use in the local treatment of inflammatory and degenerative disorders, specifically osteoarthritis (OA). Empty liposomes (L) and CS-entrapping liposomes (L-CS) were prepared by thin film hydration method followed by sonication and extrusion. They were characterized in terms of size, polydispersity index and ζ-potential by dynamic light scattering (DLS) and morphology by transmission electron microscopy. The effect of L-CS formulation on viability and morphology of mouse fibroblast cells and its biologic activity in hydrogen peroxide-stimulated cells were compared to those of L, non-encapsulated CS and a mixture of L and CS (L + CS). Our results demonstrated a high biocompatibility of L-CS and a more efficient cell protection against oxidative damage using L-CS treatment than CS or L + CS treatment. Also, L-CS exhibited a higher anti-inflammatory activity than CS in stimulated cells by reducing the level of IL-8 and TNF-α proinflammatory cytokines. The overall results suggest that the delivery of CS in liposomal formulation could improve its therapeutic potential in intra-articular treatment of OA.

Inhibition of caudal fin regeneration in Corydoras aeneus by lithium chloride.

In the present study we examined the effects of lithium chloride on the Corydoras aeneus caudal fin regeneration. After caudal fin amputation, the fish were exposed 3h daily to 35 mM lithium chloride for 9 days. The effects of lithium chloride treatment were evaluated by analyzing the caudal fin structure at 3, 6 and 9 days after amputation. Comparison of normal and LiCl treated fish clearly shows that regeneration of amputated caudal fins was inhibited or delayed after lithium treatment. By the third day after amputation (dpa) either no epidermal cap or blastema ever formed or the epidermal cap had an abnormal morphology in lithium treated fish. By the 3 and 6 dpa no lepidotrichial matrix deposition was observed in the lithium treated fish compared to control fish. Unlike the control fish that completely regenerate their caudal fins after 9 dpa and have fully mineralized lepidotrichia, lithium treated fish have small blastema. In some treated fish, small amounts of new lepidotrichial matrix were observed at this time, in some fin rays. Ultrastructural observations have shown differences between control and lithium treated fish. Thus, in the lithium treated fish we observed expanded intercellular spaces between epidermal cells and many apoptotic cells. Results of this study suggest the use of this model in elucidating the molecular mechanisms that are responsible for regeneration of complex structures such as fish fins.

Evaluation of antioxidant and cytoprotective activities of Arnica montana L. and Artemisia absinthium L. ethanolic extracts.

BACKGROUND: Arnica montana L. and Artemisia absinthium L. (Asteraceae) are medicinal plants native to temperate regions of Europe, including Romania, traditionally used for treatment of skin wounds, bruises and contusions. In the present study, A. montana and A. absinthium ethanolic extracts were evaluated for their chemical composition, antioxidant activity and protective effect against H2O2-induced oxidative stress in a mouse fibroblast-like NCTC cell line. RESULTS: A. absinthium extract showed a higher antioxidant capacity than A. montana extract as Trolox equivalent antioxidant capacity, Oxygen radical absorbance capacity and 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging activity, in correlation with its flavonoids and phenolic acids content. Both plant extracts had significant effects on the growth of NCTC cells in the range of 10-100 mg/L A. montana and 10-500 mg/L A. absinthium. They also protected fibroblast cells against hydrogen peroxide-induced oxidative damage, at the same doses. The best protection was observed in cell pre-treatment with 10 mg/L A. montana and 10-300 mg/L A. absinthium, respectively, as determined by Neutral red and lactate dehydrogenase assays. In addition, cell pre-treatment with plant extracts, at these concentrations, prevented morphological changes induced by hydrogen peroxide. Flow-cytometry analysis showed that pre-treatment with A. montana and A. absinthium extracts restored the proportion of cells in each phase of the cell cycle. CONCLUSIONS: A. montana and A. absinthium extracts, rich in flavonoids and phenolic acids, showed a good antioxidant activity and cytoprotective effect against oxidative damage in fibroblast-like cells. These results provide scientific support for the traditional use of A. montana and A. absinthium in treatment of skin disorders.

Collagen-chondroitin sulphate-hydroxyapatite porous composites: a histochemical and electron microscopy approach.

In this study the structure of collagen-chondroitin sulphate-hydroxyapatite porous composites is investigated by histochemical (Von Kossa staining), immunohistochemical, and transmission electron microscopy. Examination of composites on picrosirius red stained sections showed that polarization colors of collagen were generally in the range of orange-red. Immunofluorescence data indicate that chondroitin sulphate was either chemically incorporated into the bulk structure of collagen scaffolds or attached on surfaces of collagen bundles. Depending on the ratio between collagen:chondroitin sulphate:hydroxyapatite, von Kossa histochemical staining showed a progressive loading of collagen-chondroitin sulphate bundles with hydroxyapatite. Transmission electron microscopy studies have shown that composites contain mostly collagen fibrils aggregated with random orientation with very few collagen fibers showing the 67-nm banding pattern. Hydroxyapatite deposits of various sizes occurred among the collagen fibrils.

Transplantation of mesenchymal stem cells cultured on biomatrix support induces repairing of digestive tract defects, in animal model.

Transplanted mesenchymal stem cells (MSCs) appear to play a significant role in adult tissue repair. The aim of this research was to obtain MSCs enriched, three dimensional (3D) patches for transplant, and to test their ability to induce repair of iatrogenic digestive tract defects in rats. MSCs were obtained from human and rat bone marrow, cultured in vitro, and seeded in a collagen-agarose scaffold, where they showed enhanced viability and proliferation. The phenotype of the cultured cells was representative for MSCs (CD105+, CD90+, and CD34-, CD45-, CD3-, CD14-). The 3D patch was obtained by laying the MSCs enriched collagen-agarose scaffold on a human or swine aortic fragment. After excision of small portions of the rat digestive tract, the 3D patches were sutured at the edge of the defect using micro-surgical techniques. The rats were sacrificed at time-points and the regeneration of the digestive wall was investigated by immunofluorescence, light and electron microscopy. The MSCs enriched 3D patches were biocompatible, biodegradable, and prompted the regeneration of the four layers of the stomach and intestine wall in rats. Human cells were identified in the rat regenerated digestive wall as a hallmark of the transplanted MSCs. For the first time we constructed 3D patches made of cultured bone marrow MSCs, embedded into a collagen-rich biomatrix, on vascular bio-material support, and transplanted them in order to repair iatrogenic digestive tract defects. The result was a complete repair with preservation of the four layered structure of the digestive wall.