Modulated mesenchymal stromal cells improve skin wound healing.

Mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are considered to be promising candidates for cell-based therapy of numerous skin disorders and wound healing. It is believed that MSCs differentiation into epithelial cells fill the area of the wound, while secretion of paracrine factors regulates cell homeostasis and adaptation. MSCs have been found to accelerate wound healing and recovery of skin appendages, however, it has been stated that the majority of exogenously applied MSCs may not survive nor were detectable incorporated in the target area. These ambivalent data of localization and persistence of MSCs after their administration evoke some doubts about the effectiveness of MSCs. To elucidate these ambiguities and overcome the problem, different methods of improving the survival and homing capacity of MSCs have been developed. This article will review research data and strategies that may increase MSC's therapeutic efficacy in aging and damaged skin.

Detection of intracellular biomarkers in viable cells using millisecond pulsed electric fields.

Reversible electroporation is a temporary permeabilization of cell membrane through the formation of transient pores created by short high voltage electric pulses. This method has numerous applications in biology and biotechnology and has become an important technique in molecular medicine. Reversible electroporation is usually used to transfer macromolecules into the cells. However, the delivery of large molecules such as proteins into cells without loss of cell viability remains a challenge. In our study, we investigated whether electroporation can be used for this purpose. The study was performed with the primary mouse splenocytes and Jurkat cell line. The electroporation efficacy was evaluated by flow cytometry. We used the reversible electroporation for intracellular marker detection investigating antibody and fluorescein-conjugated dextran transfer efficiency, cell viability and metabolic activity. We have found that reversible electroporation parameters can be optimized for efficient transfer of large molecules such as antibodies/proteins into live cells without a significant loss of cell viability. We conclude that a well-established and relatively easy method of reversible electroporation can be adjusted to detect intracellular biomarkers in viable cells. This is a new approach on how electroporation could be utilised in medicine and biological research to detect rare subpopulations of cells that produce specific markers and to keep cells viable. This would allow the use of these rare subpopulations of isolated cells for further research and personalized medicine.

Regenerative potential of partially differentiated mesenchymal stromal cells in a mouse model of a full-thickness skin wound.

Mesenchymal stromal cells (MSCs, known as mesenchymal stem cells) are considered to be a promising therapeutic tool for many diseases. But it is still unclear which cells are more efficient and safe for wound healing and tissue regeneration for clinical applications: undifferentiated, partially differentiated stem cells or differentiated cells. In this study, we modified MSCs with keratinocyte-conditioned medium (KCM) and examined MSCs, partially differentiated MSCs (PMSCs) and differentiated cell migration, accumulation in the wounded area as well as cell regenerative efficiency in a full-thickness skin wound model. In addition to that, the impact of intradermal and intravenous cell delivery methods of wound healing was evaluated. C57BL/6J mouse compact bone MSCs were treated with a KCM for 14 days. Flow cytometry analysis showed the appearance of keratinocyte surface markers which were absent in MSCs, whereas the specific markers for MSCs were lost. Cells were injected either intravenously or intradermally in C57BL/6J mice. Wound closure, cell migration and accumulation in the wounded area were further analysed. Wound healing was assessed by the rate of wound closure and by histological evaluation. Cells were monitored using optical imaging. We demonstrated that PMSCs showed morphology similar to keratinocyte cells, had enhanced migration and increased survival at the site of injury. PMSCs had a beneficial effect on wound healing and tissue regeneration. This effect was reinforced when these cells were injected intravenously. Due to their partial differentiation status, we assume that PMSCs can differentiate more rapidly into epidermal cell lineages thus causing faster and qualitatively improved wound healing.

Bone marrow-derived lineage-negative cells accelerate skin regeneration in vivo.

Cell-based therapy is a promising strategy for promoting tissue regeneration when conventional treatments are not effective. ehT choice of the accessible source to obtain a sufficient cell amount and the use of suitable biomaterials to improve the cell delivery efficiency are the main tasks for safe, effective, and reliable application of stem cell therapy. In this study, we have compared the influence of bone marrow-derived Lin¯ cells on skin regeneration after local transplantation with or without type I collagen-based gel in a BALB/c mice full-thickness wound model. Lin¯ cells were isolated using magnetic-associated cell sorting and identified by flow cytometry. Cytokine gene expression was examined using real-time PCR. Our results show that the bone marrow-derived Lin¯ cell population demonstrates the properties to stimulate the skin tissue regeneration. Significant accelerated wound closure was revealed after cell transplantation (P < 0.05). Histological analysis indicated the earliest inhibition of inflammation, accelerated reepithelialization, and evenly distributed skin appendages in the neodermis after Lin¯ cell transplantation with type I collagen gel. eTh significant changes in mRNA levels of cytokines TNF-α, IL-10, TGF-ß, and VEGF after Lin¯ cell transplantation were confirmed by RT-PCR (P < 0.05). eTh ability to positively control the reactions taking place during the wound healing process gives the advantage to the bone marrow Lin¯ cell population to be used as a cell source for therapy.

Quantitative evaluation of the transplanted lin(-) hematopoietic cell migration kinetics.

Stem cells take part in organogenesis, cell maturation and injury repair. The migration is necessary for each of these functions to occur. The aim of this study was to investigate the kinetics of transplanted hematopoietic lin(-) cell population (which consists mainly of the stem and progenitor cells) in BALB/c mouse contact hypersensitivity model and quantify the migration to the site of inflammation in the affected foot and other healthy organs. Quantitative analysis was carried out with the real-time polymerase chain reaction method. Spleen, kidney, bone marrow, lung, liver, damaged and healthy foot tissue samples at different time points were collected for analysis. The quantitative data normalization was performed according to the comparative quantification method. The analysis of foot samples shows the significant migration of transplanted cells to the recipient mice affected foot. The quantity was more than 1000 times higher, as compared with that of the untreated foot. Due to the inflammation, the number of donor origin cells migrating to the lungs, liver, spleen and bone marrow was found to be decreased. Our data shows that transplanted cells selectively migrated into the inflammation areas of the foot edema. Also, the inflammation caused a secondary migration in ectopic spleen of hematopoietic stem cell niches and re-homing from the spleen to the bone marrow took place.

Exosomes from Human Dental Pulp Stem Cells Suppress Carrageenan-Induced Acute Inflammation in Mice.

The primary goal of this study was to examine the effects of human dental pulp stem cell-derived exosomes on the carrageenan-induced acute inflammation in mice. Exosomes were purified by differential ultracentrifugation from the supernatants of stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) cultivated in serum-free medium. At 1 h post-carrageenan injection, exosomes derived from supernatants of 2 × 10(6) SHEDs were administered by intraplantar injection to BALB/c mice; 30 mg/kg of prednisolone and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Edema was measured at 6, 24, and 48 h after carrageenan injection. For the in vivo imaging experiments, AngioSPARK750, Cat B 750 FAST, and MMPSense 750 FAST were administered into the mouse tail vein 2 h post-carrageenan injection. Fluorescence images were acquired at 6, 24, and 48 h after edema induction by IVIS Spectrum in vivo imaging system. Exosomes significantly reduced the carrageenan-induced edema at all the time points studied (by 39.5, 41.6, and 25.6% at 6, 24, and 48 h after injection, respectively), to similar levels seen with the positive control (prednisolone). In vivo imaging experiments revealed that, both exosomes and prednisolone suppress activities of cathepsin B and matrix metalloproteinases (MMPs) at the site of carrageenan-induced acute inflammation, showing more prominent effects of prednisolone at the early stages, while exosomes exerted their suppressive effects gradually and at later time points. Our study demonstrates for the first time that exosomes derived from human dental pulp stem cells suppress carrageenan-induced acute inflammation in mice.

Impact of differently modified nanocrystalline diamond on the growth of neuroblastoma cells.

The aim of this study was to assess the impact of nanocrystalline diamond (NCD) thin coatings on neural cell adhesion and proliferation. NCD was fabricated on fused silica substrates by microwave plasma chemical vapor deposition (MPCVD) method. Different surface terminations were performed through exposure to reactive hydrogen and by UV induced oxidation during ozone treatment. Boron doped NCD coatings were also prepared and investigated. NCD surface wettability was determined by contact angle measurement. To assess biocompatibility of the NCD coatings, the neuroblastoma SH-SY5Y cell line was used. Cells were plated directly onto diamond surfaces and cultured in medium with or without fetal bovine serum (FBS), in order to evaluate the ability of cells to adhere and to proliferate. The obtained results showed that these cells adhered and proliferated better on NCD surfaces than on the bare fused silica. The cell proliferation on NCD in medium with and without FBS after 48h from plating was on average, respectively, 20 and 58% higher than that on fused silica, irrespective of NCD surface modification. Our results showed that the hydrogenated, oxygenated and boron-doped NCD coatings can be used for biomedical purposes, especially where good optical transparency is required.

Impact of diamond nanoparticles on neural cells.

Diamond nanoparticles (DNPs) are very attractive for biomedical applications, particularly for bioimaging. The aim of this study was to evaluate the impact of DNPs on neural cancer cells and thus to assess the possible application of DNPs for these cells imaging. For this purpose, the neuroblastoma SH-SY5Y cell line was chosen. Cells were cultured in medium with different concentrations (15, 50, 100 and 150 µg/ml) of DNPs. After 48 h of incubation, cell metabolic activity was evaluated by the XTT assay. For assessment of cellular metabolic activity, cells were also cultured on differently terminated nanocrystalline diamond (NCD) coatings in medium with 150 µg/ml of DNPs. Cell adhesion and morphology were evaluated by brightfield microscopy. Diamond nanoparticle internalization was determined by confocal microscopy. The obtained results showed that low concentrations (15, 50 and 100 µg/ml) of nanoparticles did not significantly affect the SH-SY5Y cell metabolic activity. However, a higher concentration (150 µg/ml) of DNPs statistically significantly reduced SH-SY5Y cell metabolic activity. After 48 h incubation with 150 µg/ml DNPs, cell metabolic activity was 23% lower than in medium without DNPs on standard tissue culture polystyrene.

Evaluation of cytotoxicity of polypyrrole nanoparticles synthesized by oxidative polymerization.

Polypyrrole (Ppy) is known as biocompatible material, which is used in some diverse biomedical applications and seeming to be a very promising for advanced biotechnological applications. In order to increase our understanding about biocompatibility of Ppy, in this study pure Ppy nanoparticles (Ppy-NPs) of fixed size and morphology were prepared by one-step oxidative polymerization and their cyto-compatibility was evaluated. The impact of different concentration of Ppy nanoparticles on primary mouse embryonic fibroblasts (MEF), mouse hepatoma cell line (MH-22A), and human T lymphocyte Jurkat cell line was investigated. Cell morphology, viability/proliferation after the treatment by Ppy nanoparticles was evaluated. Obtained results showed that Ppy nanoparticles at low concentrations are biocompatible, while at high concentrations they became cytotoxic for Jurkat, MEF and MH-22A cells, and it was found that cytotoxic effect is dose-dependent.

Skin regeneration with bone marrow-derived cell populations.

Bone marrow-derived cells of distinct differentiation level could differently influence the process of skin regeneration. The results of our study revealed that hematopoietic stem cells (HSC) population influenced the repair of injured tissue slower in comparison with lineage negative (lin⁻) cell population containing not only HSC but also cell progenitors of different differentiation levels. Wound healing process was faster in lin⁻) cell suspension treated group, the stage of proliferation was more intensive and increased number of skin appendages occurred. The adaptation of purified HSC at the site of injury was longer and the stages of wound healing took place later. The results obtained show that in further experiments the complex procedure of HSC isolation and purification could be shortened and heavy skin injuries could be successfully treated with the help of lin⁻ cell population.

An anti-inflammatory effect of murine fetal liver cells in BALB/c mouse contact hypersensitivity model.

Anti-inflammatory effects of murine fetal liver (FL) cells were studied using BALB/c mouse contact hypersensitivity (paw edema) model. Paw weight differences, lymphatic organ weights, hematological and histological indices as well as proinflammatory (TNF-alpha) and anti-inflammatory (IL-10) cytokine levels in sera were evaluated. Immunophenotyping revealed that both murine FL homogenate cells (HC) and FL hematopoietic stem cells (HSC) express CD117 and CD38 surface markers. Single doses of 1x10(6) cells/mouse and 2x10(6) cells/mouse of FL HC as well as of FL HSC, when used separately, all statistically significantly (p<0.05) inhibited paw edema, but the lower dose was more effective and giving results similar to that of prednisolone. Either dose of FL HC or FL HSC studied had no significant influence on lymphatic organ weights; no significant changes were also observed in blood indices. The data of cytokine studies showed that TNF-alpha concentration in sera of mice treated with either FL HC or FL HSC at a dose of 1x10(6) cells/mouse was statistically significantly (p<0.001) lower than that of the control mice. A concentration of IL-10 was statistically significantly higher (p<0.01) in mice treated with a dose of 1x10(6) cells/mouse of FL HC but not with the same dose of FL HSC as compared to the control group. Histological examination revealed better effects of a dose of 1x10(6) cells/mouse of FL HC when compared with the same dose of FL HSC as in regard to reduction of edema thickness and cell infiltration.

Oral tryptic casein hydrolysate enhances phagocytosis by mouse peritoneal and blood phagocytic cells but fails to prevent induced inflammation.

Mouse experiments were conducted in order to find whether oral application of tryptic casein hydrolysate (TCH) results in enhancement of phagocytosing capacity of murine phagocytic cells as well as whether such application might be of use for prevention of inflammatory processes. Phagocytosing capacity of phagocytic cells of mice that received oral TCH once daily in a dose of 1.0 mg/g body weight dissolved in 0.5 ml of distilled water for five successive days was significantly higher (p < 0.05) than that of mice given equivalent volumes of distilled water, with a phagocytosing capacity enhancement index being 1.39 and 1.34 regarding peritoneal macrophages and blood phagocytic cells, respectively. Taken on the other hand, the immunostimulatory effects of oral TCH were found to be not enough to prevent mice from inflammation that was induced experimentally using acute (paw edema) and contact hypersensitivity models. A possibility for development of food protein enzymatic hydrolysates as antimicrobial immunostimulants acting through improvement of phagocytic cell functioning is discussed.

Non-specific immunity-enhancing effects of tryptic casein hydrolysate versus Fermosorb for treatment/prophylaxis of newborn calf colibacillosis.

The effects of treatment/prophylaxis of newborn calf colibacillosis with tryptic casein hydrolysate (TCH), recently shown to be a novel type of antimicrobial acting through stimulation of the microbial autolytic system, versus an authorized veterinary drug, Fermosorb, were evaluated. Both products showed similar high therapeutic and prophylactic efficacies, but hematological indices and daily weight gain of cured/protected animals were better with TCH. The differences in hemoglobin and hematocrit levels, total protein, gamma-globulin and sulfhydryl group quantities, bactericidal and lysozyme activities as well as daily weight gain at the end of treatment/prophylaxis were statistically significant (P<0.05-0.000005). Statistically significant differences (P<0.05-0.0005) in favor of TCH were also observed when bactericidal activity, total protein quantity of serum as well as daily weight gain of the animals were compared on the 90th day after birth. We conclude that TCH acts not only as an antimicrobial, but also as an immunostimulant (and growth promoter). The immunostimulatory activity of TCH most probably derives from a synergistic action of bioactive peptides encrypted in the preparation itself and the cell wall fragments resulting from microbial autolysis induction.

Quantitative evaluation of macrophage phagocytosing capacity by a fluorometric assay.

This paper reviews sensitive and simple quantitative evaluation of macrophage phagocytosing capacity by applying fluoresecin-labeled Sacharomyces cerevisiae cells. Yeast cells were conjugated with fluoresceinisothiocyanate (FITC) and used as fluorescent particles. A time course analysis within this method showed that phagocytosis of yeast cells was temperature dependent and that the number of that ones ingested by macrophages increased rapidly during the initial 60 min of incubation at 37 degrees C. Free fluorescent cells can be effectively removed by aspiration from the well. Furthermore, yeast cells required preopsonization with serum to achieve optimal uptake of the cells. The uptake of nonopsonized yeast cells by macrophages was significantly lower than that of opsonized cells (P < 0.05). We propose that about 50% of mouse macrophages can carry functionally active FcR responsible for phagocytosis.