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.

Study of the interaction of 1,4-dihydropyridine derivatives with glucocorticoid hormone receptors from the rat liver.

Seventeen derivatives of 1,4-dihydropyridine (DHP) series were tested in vitro for their ability to inhibit [1,2,4-(3)H]-dexamethasone binding to glucocorticoid receptor from the rat liver cytosol. Depending on structural features and inhibiting activities, the compounds can be divided into three groups. The first group (nifedipine, foridone, J-6-163, OSI-4164 and OSI-7724) had the highest activity: they inhibited specific ligand-receptor binding by 70-80% at concentrations of 10(-5) M and 10(-4) M, with apparent IC(50)values of 1.5-6.0 muM. The second group (cerebrocrast, diethone, OSI-1211 and OSI-7265) was active at concentration of 10(-4) M, and their IC(50) values were 23-45 muM; compound OSI-5003 was almost inactive. Both groups are compounds with scarce water solubility, more or less lipophilic. The third group of compounds comprises ionogenic compounds (organic cations or anions with corresponding inorganic counterions): most of them are water-soluble (glutapyrone, carbatone, gammapyrone, OSI-2780, OSI-1580, OSI-2140) or liposome-forming (A-74). They lack the above-mentioned activity. Among the first two groups, compounds possessing more bulky substituents in positions 3 and 5 are less active. The aromatic ring in the position 4 is essential for the optimal activity. It seems that there is a bell-shaped dependence of activity upon lipophilicity. In general, the compounds of the first group are strong Ca-antagonists, while the second group includes moderate Ca-antagonists, but each group comprises also compounds which lack Ca antagonistic activity. All compounds of the third group lack Ca antagonistic properties.