Particle stability and structure on the peritoneal surface in pressurized intra-peritoneal aerosol chemotherapy (PIPAC) analysed by electron microscopy: First evidence of a new physical concept for PIPAC.

Pressurized intra-peritoneal aerosol chemotherapy (PIPAC) has been introduced to the clinical setting as a novel approach for the treatment of peritoneal metastasis. The local interaction of chemoaerosol droplets with the peritoneal surface as well as their distribution pattern is considered the main advantage over conventional liquid intraperitoneal chemotherapy. The aim of the present study was to investigate the behavior of these aerosol particles during PIPAC application via electron microscopy. Solutions of doxycycline, liposomal doxorubicin and macrophage cells were aerosolized using an established ex-vivo model. PIPAC was performed on peritoneum samples via microcatheter (MC) at a pressure of 12 mmHg C02 at 27°C. Following PIPAC the surface structure of applied particles was measured via electron microscopy. The aerosol particle contact of doxycyclin created a nanofilm of ~200 nm height on the peritoneal surface, and this height was revealed to be independent of the size of the initial particle hitting. These nanofilm blocks of 'cylinders' are of different diameters depending on the initial aerosol particle hitting that spot. Diameters of these 'cylinders' are far wider than the original diameter of the initial aerosol particle. However, coated particles such as liposomal doxorubicin and macrophages remained intact following contact with the peritoneal surface. Based on this and other data, the concept that aerosol particles exhibit a gas-like behavior in the abdomen creating a therapeutic capnoperitoneum should be revised. Fluid aerosol particles collide with the peritoneum creating a nanofilm. The interaction of pressurized intraperitoneal aerosol on the peritoneum is therefore closer to the distribution of a liquid film than to that of a gas. Further studies are required to further analyze the interaction of this nanofilm on the peritoneum.

Effect of Liposomal Doxorubicin in Pressurized Intra-Peritoneal Aerosol Chemotherapy (PIPAC).

Background: This ex-vivo study was performed to compare the impact of doxorubicin vs. liposomal doxorubicin on penetration depth in peritoneal tissue during Pressurized Intra-Peritoneal Aerosol Chemotherapy (PIPAC) via microcatheter (MC). Methods: Fresh post mortem swine peritoneum was cut into proportional sections. One group of samples was treated with PIPAC with Doxorubicin (D), and the other was treated with PIPAC with liposomal doxorubicin (LD). Tissue specimens were placed as follows: at the bottom of the plastic box (1), at the side wall (2), at the top cover (3) and the side of the box covered by a plastic tunnel (4). In-tissue doxorubicin penetration was measured using fluorescence microscopy on frozen thin sections. Results: Medium penetration levels with D were 325 µm (1), 152 µm (2), 84 µm (3) and 71 µm (4), respectively. Medium penetration levels with LD were significantly lower with 10 µm (1), 2 µm (2), 0 µm (3) and 0 µm (4), respectively. In most samples that were treated with LD no doxorubicin could be detected at all. Conclusion: Our data indicate that liposomal coating of doxorubicin and possibly other chemotherapeutical drugs might inhibit their interaction with the peritoneal surface. This inhibition appears to be relatively strong, since doxorubicin is partially undetectable due to liposomal coating. Further studies are warranted to investigate this interaction and its potential benefit in peritoneal applications.

Antiviral Activity of Tannic Acid Modified Silver Nanoparticles: Potential to Activate Immune Response in Herpes Genitalis.

(1) Background: Tannic acid is a plant-derived polyphenol showing antiviral activity mainly because of an interference with the viral adsorption. In this work, we tested whether the modification of silver nanoparticles with tannic acid (TA-AgNPs) can provide a microbicide with additional adjuvant properties to treat genital herpes infection. (2) Methods: The mouse model of the vaginal herpes simplex virus 2 (HSV-2) infection was used to test immune responses after treatment of the primary infection with TA-AgNPs, and later, after a re-challenge with the virus. (3) Results: The mice treated intravaginally with TA-AgNPs showed better clinical scores and lower virus titers in the vaginal tissues soon after treatment. Following a re-challenge, the vaginal tissues treated with TA-AgNPs showed a significant increase in the percentages of IFN-gamma+ CD8+ T-cells, activated B cells, and plasma cells, while the spleens contained significantly higher percentages of IFN-gamma+ NK cells and effector-memory CD8+ T cells in comparison to NaCl-treated group. TA-AgNPs-treated animals also showed significantly better titers of anti-HSV-2 neutralization antibodies in sera; and (4) Conclusions: Our findings suggest that TA-AgNPs sized 33 nm can be an effective anti-viral microbicide to be applied upon the mucosal tissues with additional adjuvant properties enhancing an anti-HSV-2 immune response following secondary challenge.

Bacterial cellulose yield increased over 500% by supplementation of medium with vegetable oil.

Bacterial cellulose (BC), produced by Komagataeibacter xylinus, has numerous applications to medicine and industry. A major limitation of BC use is relatively low production rates and high culturing media costs. By supplementing culture media with 1% vegetable oil, we achieved BC yield exceeding 500% over the yield obtained in standard media. BC properties were similar to cellulose cultured in standard methods with regard to cytotoxicity but displayed significantly higher water swelling capacity and mechanical strength. As we demonstrated herein, this significantly increased BC yield is the result of microscopic and macroscopic physiochemical processes reflecting a complex interaction between K. xylinus biophysiology, chemical processes of BC synthesis, and physiochemical forces between BC membranes, oil and culturing vessel walls. Our findings have significant translational implications to biomedical and clinical settings and can be transformative for the cellulose biopolymer industry.

The effect of the bioactive sphingolipids S1P and C1P on multipotent stromal cells--new opportunities in regenerative medicine.

Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) belong to a family of bioactive sphingolipids that act as important extracellular signaling molecules and chemoattractants. This study investigated the influence of S1P and C1P on the morphology, proliferation activity and osteogenic properties of rat multipotent stromal cells derived from bone marrow (BMSCs) and subcutaneous adipose tissue (ASCs). We show that S1P and C1P can influence mesenchymal stem cells (MSCs), each in a different manner. S1P stimulation promoted the formation of cellular aggregates of BMSCs and ASCs, while C1P had an effect on the regular growth pattern and expanded intercellular connections, thereby increasing the proliferative activity. Although osteogenic differentiation of MSCs was enhanced by the addition of S1P, the effectiveness of osteoblast differentiation was more evident in BMSCs, particularly when biochemical and molecular marker levels were considered. The results of the functional osteogenic differentiation assay, which includes an evaluation of the efficiency of extracellular matrix mineralization (SEM-EDX), revealed the formation of numerous mineral aggregates in BMSC cultures stimulated with S1P. Our data demonstrated that in an appropriate combination, the bioactive sphingolipids S1P and C1P may find wide application in regenerative medicine, particularly in bone regeneration with the use of MSCs.

Polyurethane/polylactide-based biomaterials combined with rat olfactory bulb-derived glial cells and adipose-derived mesenchymal stromal cells for neural regenerative medicine applications.

Research concerning the elaboration and application of biomaterial which may support the nerve tissue regeneration is currently one of the most promising directions. Biocompatible polymer devices are noteworthy group among the numerous types of potentially attractive biomaterials for regenerative medicine application. Polylactides and polyurethanes may be utilized for developing devices for supporting the nerve regeneration, like nerve guide conduits or bridges connecting the endings of broken nerve tracts. Moreover, the combination of these biomaterial devices with regenerative cell populations, like stem or precursor cells should significantly improve the final therapeutic effect. Therefore, the composition and structure of final device should support the proper adhesion and growth of cells destined for clinical application. In current research, the three polymer mats elaborated for connecting the broken nerve tracts, made from polylactide, polyurethane and their blend were evaluated both for physical properties and in vitro, using the olfactory-bulb glial cells and mesenchymal stem cells. The evaluation of Young's modulus, wettability and roughness of obtained materials showed the differences between analyzed samples. The analysis of cell adhesion, proliferation and morphology showed that the polyurethane-polylactide blend was the most neutral for cells in culture, while in the pure polymer samples there were significant alterations observed. Our results indicated that polyurethane-polylactide blend is an optimal composition for culturing and delivery of glial and mesenchymal stem cells.

The osteogenic properties of multipotent mesenchymal stromal cells in cultures on TiO2 sol-gel-derived biomaterial.

The biocompatibility of the bone implants is a crucial factor determining the successful tissue regeneration. The aim of this work was to compare cellular behavior and osteogenic properties of rat adipose-derived multipotent stromal cells (ASCs) and bone marrow multipotent stromal cells (BMSCs) cultured on metallic substrate covered with TiO2 sol-gel-derived nanolayer. The morphology, proliferation rate, and osteogenic differentiation potential of both ASCs and BMSCs propagated on the biomaterials were examined. The potential for osteogenic differentiation of ASCs and BMSCs was determined based on the presence of specific markers of osteogenesis, that is, alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCL). Additionally, the concentration of calcium and phosphorus in extracellular matrix was determined using energy-dispersive X-ray spectroscopy (SEM-EDX). Obtained results showed that TiO2 layer influenced proliferation activity of ASCs, which manifested by shortening of population doubling time and increase of OPN secretion. However, characteristic features of cells morphology and growth pattern of cultures prompted us to conclude that ultrathin TiO2 layer might also enhance osteodifferentiation of BMSCs. Therefore in our opinion, both populations of MSCs should be used for biological evaluation of biomaterials compatibility, such results may enhance the area of investigations related to regenerative medicine.

Biological effects of sol-gel derived ZrO2 and SiO2/ZrO2 coatings on stainless steel surface--In vitro model using mesenchymal stem cells.

The objective of this study was to determine biocompatibility of zirconia-based coatings obtained by the sol-gel method. Two matrices, ZrO2 and SiO2/ZrO2, were created and applied on stainless steel type 316L with dip-coating technique. The morphology and topography of biomaterials' surface were characterized using energy-dispersive X-ray spectroscopy and atomic force microscopy, while chemical composition was analyzed by Raman spectroscopy. Additionally, wettability and surface free energy were characterized. Biocompatibility of obtained biomaterials was evaluated using an in vitro model employing mesenchymal stem cells (MSCs) of adipose and bone marrow origin. Biological analysis included determination of proliferation activity and morphology of MSCs in cultures on synthesized biomaterials. Osteoinductive properties of biomaterials were determined both in non-osteogenic, as well as osteogenic conditions. The results showed that investigated biomaterials exerted different impact on MSCs. Biomaterial with ZrO2 layer was more biocompatible for adipose-derived MSCs, while SiO2/ZrO2 layer promoted proliferation of bone marrow derived MSCs. Moreover, hybrid coating exhibited greater osteoinductive properties than ZrO2 coating, both on cultures with adipose-derived stromal (stem) cells and bone marrow stromal cells. Observed biological effects may result not only from different chemical composition, but also from diverse wettability. The ZrO2 coating was characterized as hydrophobic layer, while SiO2/ZrO2 exhibited hydrophilic properties. The results obtained suggest that behavior of MSCs in response to the biomaterial may vary depending on their origin, therefore we postulate, that screening analysis of implants' biocompatibility, should incorporate model applying both adipose- and bone marrow derived MSCs.

Influence of modified alginate hydrogels on mesenchymal stem cells and olfactory bulb-derived glial cells cultures.

BACKGROUND: Great potential of cellular therapies has generated extensive research in the field of cells harvesting and culturing. Transplantation of cell cultures has been used in a variety of therapeutic programs but in many cases it appeared that biomaterial scaffold or sheath would enhance cells regenerative potential. OBJECTIVE: Hydrogels composed of different proportions sodium and calcium alginates, were undertaken to evaluate their influence on mesenchymal stem cells and olfactory bulb-derived glial cells cultures. Additionally, these biomaterials were also enriched with fibrin protein. METHODS: The structure of materials was visualized by means of scanning electron microscopy. After seeding with cells - hydrogels were observed with inverted and fluorescence microscope. Cell's morphology, behavior and phenotype were analyzed in investigated materials by means of light, fluorescence and scanning electron microscopes. Also, viability assay was performed with Alamar Blue cytotoxic test. RESULTS: Our observations showed that basic alginate hydrogels had significant influence on both cell types. Materials maintained cells alive, which is desired attribute, however none of them kept cells in normal, flat form. Alginates with significant calcium component kept cells alive for longer period of culture. CONCLUSIONS: Addition of fibrin protein resulted in material's biocompatibility properties improvement, by creation of adhesion surface, which helps cells to keep proper morphology and behavior. Our findings suggest that addition of fibrin protein to alginate hydrogels improves them as cell carriers for regenerative medicine applications.

Effects of steroids on the morphology and proliferation of canine and equine mesenchymal stem cells of adipose origin - in vitro research.

Disorders of the locomotive system, especially those occurring due to degenerative changes of the joints, are serious problems in daily veterinary medical practice. Steroid injections are the main way of treating these disorders. However, this approach brings usually only temporary effects of pain relief, and may cause many side effects. Alternative therapies focus on regeneration of damaged tissue using adult mesenchymal stem cells (MSCs). Since 2002, the great plasticity and immunomodulatory properties of MSCs isolated from adipose tissue (AdMSCs) have been used successfully in the treatment of degenerative joint diseases (DJD) of both dogs and horses. Possible simultaneous application of steroid therapy and stem cell transplantation could improve the commonly used clinical procedure. In this paper, the influence of the two steroid drugs (betamethasone and methylprednisolone) on AdMSCs was evaluated on the basis of morphology and proliferation rate. Both steroids positively influenced the viability and proliferation state of cells in a concentration of 0.01 mg/ml and 0.1 mg/ml, respectively. However, the concentration of 1 mg/ml had a cytotoxic effect. Moreover, the lower dosage of steroid drugs used in the experiment did not affect the morphology of cells and significantly increased cellular activity. In conclusion, our data demonstrate the stimulating effect of steroid drugs on cell morphology, proliferation rate and cytophysiological activity. These findings may influence the use of stem cells and steroids in applied regenerative veterinary medical practice in the future.

Application of bone marrow and adipose-derived mesenchymal stem cells for testing the biocompatibility of metal-based biomaterials functionalized with ascorbic acid.

In this study, metal-based biomaterials were functionalized with ascorbic acid (LAA). Two types of substrates were used: austenitic steel 316L and titanium Ti6Al4V. Coatings were prepared with the sol-gel method and applied on metal surfaces using the dip-coating technique. Ascorbic acid was delivered with SiO2-coating at concentrations of 0.1 and 0.4 M. The morphology of the surfaces and coatings was determined using scanning electron microscope (SEM), whereas their elemental composition by SEM-EDX. Immobilization of ascorbic acid in the coatings was confirmed with Raman spectroscopy. The biocompatibility of the materials obtained was tested in vitro using both bone marrow- and adipose-derived mesenchymal stem cells (BMMSC and ADMSC, respectively). Proliferation rate and morphology of cells cultured in the presence of designed biomaterials were monitored after 24, 48, 120 and 168 h of propagation. The results obtained indicated that silica coatings doped with 0.4 M LAA had a positive effect on the proliferation rate of investigated cells, and in some cases on the growth pattern of culture.

Morphological characterization of gecko's (eublepharis macularius) glial cells in culture / Caracterización morfológica de las células gliales del gecko (eublepharis macularius) en cultivo

Central nervous system of reptiles has the ability to grow and regenerate during adult life of the animal. Therefore, cells creating CNS of this animal class should compound substances or molecules enabling neuroregeneration. Cells directly involved in this process have not been clearly characterized, especially in cell culture environment. Morphology of reptilian glial adherent cells should be known better to find any differences from mammalian CNS cells. We isolated glial cells from olfactory bulb and cerebrum from gecko (Eublepharis macularius) and cultured separately. We have observed populations of cells with proliferative capacity in both types of cultures. Also, we have detected lipid molecules deposits within their cytoplasm, which localization was correlated with mitochondria position. This information can be helpful in searching new bioactive substances involved in regeneration of central nervous system.

El sistema nervioso central de los reptiles tiene la capacidad de crecer y regenerarse durante la vida adulta del animal. Por lo tanto, las células de SNC creadas de esta clase de animales deberían componerse de sustancias o moléculas que permiten la neuroregeneración. Las células que participan directamente en este proceso no han sido claramente caracterizadas, especialmente en el entorno de cultivo celular. La morfología de las células adherentes gliales de reptiles deben ser reconocidas y diferenciarse respecto a las células del SNC de mamíferos. Se aislaron células gliales del bulbo olfatorio y el cerebro del Gecko (Eublepharis macularius) y se cultivaron por separado. Se observaron poblaciones de células con capacidad proliferativa en ambos tipos de cultivos. Además, se detectaron moléculas de depósitos lipídicos dentro de su citoplasma, y su localización se correlacionó con la posición de las mitocondrias. Esta información puede ser útil en la búsqueda de nuevas sustancias bioactivas que participan en la regeneración del sistema nervioso central.

Comparison of behavior, morphology and morphometry of equine and canine adipose derived mesenchymal stem cells in culture / Comparación del comportamiento, morfología y morfometría del tejido adiposo equino y canino derivado de células madre mesenquimales en cultivo

Recent studies revealed multipotent properties of fat tissue isolated mesenchymal stem cells. These cells are successfully used as therapeutic factor for many locomotive disorders, being even more effective than stem cells from bone marrow. Isolated and cultured, AD-MSCs were observed, photographed and measured to compare cells from two different species.

Estudios recientes han revelado propiedades pluripotentes del tejido graso aislado de células madre mesenquimales. Estas células se utilizan con éxito como factor terapéutico para muchos trastornos locomotores, siendo aún más eficaz que las células madre de médula ósea. Aisladas y cultivadas, AD-MSC se observaron, fotografiaron y midieron comparar células de dos especies diferentes.