An Effect of Cyclosporin A in a Treatment of Temporal Bone Defect Using hBM-MSCs.

Background. The treatment of middle ear cholesteatoma requires surgical treatment and the reconstruction of the temporal bone, which represents an ongoing problem. Otologists have focused on the research of materials allowing an airy middle ear and the preservation of hearing function to reconstruct the temporal bone. Methods. This study evaluated the effect of cyclosporin A (CsA) and a combined biomaterial in the healing process of postoperative temporal bone defects in an animal model. Cultured human Bone Marrow Mesenchymal Stromal Cells (hBM-MSCs) were mixed with hydroxyapatite (Cem-Ostetic®), and subsequently applied as a bone substitute after middle ear surgery, showing that the therapeutic potential of hBM-MSCs associated with bone regeneration and replacement is directly influenced by CsA, confirming that it promotes the survival of MSCs in vivo. Results. The therapeutic efficacy of the combination of MSCs with CsA is greater than the sole application of MSCs in a hydroxyapatite carrier. Conclusion. The reconstruction of a temporal bone defect using hBM-MSCs requires an immunosuppressant to improve the results of treatment.

A Beginner's Introduction to Skin Stem Cells and Wound Healing.

The primary function of the skin is that of a physical barrier against the environment and diverse pathogens; therefore, its integrity is essential for survival. Skin regeneration depends on multiple stem cell compartments within the epidermis, which, despite their different transcriptional and proliferative capacity, as well as different anatomical location, fall under the general term of skin stem cells (SSCs). Skin wounds can normally heal without problem; however, some diseases or extensive damage may delay or prevent healing. Non-healing wounds represent a serious and life-threatening scenario that may require advanced therapeutic strategies. In this regard, increased focus has been directed at SSCs and their role in wound healing, although emerging therapeutical approaches are considering the use of other stem cells instead, such as mesenchymal stem cells (MSCs). Given its extensive and broad nature, this review supplies newcomers with an introduction to SSCs, wound healing, and therapeutic strategies for skin regeneration, thus familiarizing the reader with the subject in preparation for future in depth reading.

Simple Detection of Primary Cilia by Immunofluorescence.

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

The toxic effect of cytostatics on primary cilia frequency and multiciliation.

The primary cilium is considered as a key component of morphological cellular stability. However, cancer cells are notorious for lacking primary cilia in most cases, depending upon the tumour type. Previous reports have shown the effect of starvation and cytostatics on ciliogenesis in normal and cancer cells although with limited success, especially when concerning the latter. In this study, we evaluated the presence and frequency of primary cilia in breast fibroblasts and in triple-negative breast cancer cells after treatment with cytostatics finding that, in the case of breast fibroblasts, primary cilia were detected at their highest incidence 72 hours after treatment with 120 nM doxorubicin. Further, multiciliated cells were also detected after treatment with 80 nM doxorubicin. On the other hand, treatment with taxol increased the number of ciliated cells only at low concentrations (1.25 and 3.25 nM) and did not induce multiciliation. Interestingly, triple-negative breast cancer cells did not present primary cilia after treatment with either doxorubicin or taxol. This is the first study reporting the presence of multiple primary cilia in breast fibroblasts induced by doxorubicin. However, the null effect of these cytostatics on primary cilia incidence in the evaluated triple negative breast carcinomas cell lines requires further research.

Cell Stratification, Spheroid Formation and Bioscaffolds Used to Grow Cells in Three Dimensional Cultures.

The cell culture became an invaluable tool for studying cell behaviour, development, function, gene expression, toxicity of compounds and efficacy of novel drugs. Although most results were obtained from cell cultivation in two-dimensional (2D) systems, in which cells are grown in a monolayer, three-dimensional (3D) cultures are more promising as they correspond closely to the native arrangement of cells in living tissues. In our study, we focused on three types of 3D in vitro systems used for cultivation of one cell type. Cell morphology, their spatial distribution inside of resulting multicellular structures and changes in time were analysed with histological examination of samples harvested at different time periods. In multilayered cultures of WRL 68 hepatocytes grown on semipermeable membranes and non-passaged neurospheres generated by proliferation of neural progenitor cells, the cells were tightly apposed, showed features of cell differentiation but also cell death that was observable in short-term cultures. Biogenic scaffolds composed of extracellular matrix of the murine tibial anterior muscle were colonized with C2C12 myoblasts in vitro. The recellularized scaffolds did not reach high cell densities comparable with the former systems but supported well cell anchorage and migration without any signs of cell regression.

Extracorporeal Elimination of Circulating Pegylated Liposomal Doxorubicin (PLD) to Enhance the Benefit of Cytostatic Therapy in Platinum-Resistant Ovarian Cancer Patients.

Ovarian cancer is the fifth most common malignancy in the world's female population and with the highest lethality index among gynecological tumors. The prognosis of metastatic disease is usually poor, especially in platinum-resistant cases. There are several options for the treatment of metastatic disease resistant to platinum derivates (e.g. paclitaxel, topotecan and pegylated liposomal doxorubicin), all of which are considered equipotent. Pegylated liposomal doxorubicin (PLD) is a liposomal form of the anthracycline antibiotic doxorubicin. It is characterized by more convenient pharmacokinetics and a different toxicity profile. Cardiotoxicity, the major adverse effect of conventional doxorubicin, is reduced in PLD as well as hematotoxicity, alopecia, nausea and vomiting. Skin toxicity and mucositis, however, emerge as serious issues since they represent dose and schedule-limiting toxicities. The pharmacokinetics of PLD (prolonged biological half-life and preferential distribution into tumor tissue) provide new possibilities to address these toxicity issues. The extracorporeal elimination of circulating liposomes after PLD saturation in the tumor tissue represents a novel and potent strategy to diminish drug toxicity. This article intends to review PLD characteristics and the importance of extracorporeal elimination to enhance treatment tolerance and benefits.

Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts.

Primary cilia act as physical-chemical sensors and their functions include the perception of the extracellular milieu, regulation of organogenesis, and cell polarity. In general, these cells are monociliated and the single cilium possesses diverse receptors and channels which are involved in morphogenesis and growth signaling, and are, therefore, important for cell proliferation and differentiation. In this study, we used an in vitro model of C2C12 myoblasts to evaluate the effect of DNA damage induced by gamma ionizing radiation on primary cilia incidence. A significantly higher number of ciliated cells were observed after 1 day post-irradiation with 2-20 Gy when compared with non-irradiated cells. After 3 days post-irradiation, the cilia incidence in cells had decreased slightly when treated with 2, 6, and 10 Gy, although an increase in incidence rate was observed in cells treated with 20 Gy. Multi-ciliated cells were also detected in myoblasts irradiated with 10 and 20 Gy but not in non-irradiated cells or after low irradiation (2-6 Gy). Irradiation also caused a dose-dependent decrease in cell viability and proliferation and corresponding cell cycle arrest. Furthermore, an activation of caspases 3/7, 8, and 9 was observed after higher radiation (10 and 20 Gy) with increased apoptosis. Together, our results show that irradiation by gamma rays promotes myoblast ciliogenesis, with pronounced effects observed after 3 days post-irradiation. We conclude that irradiation doses of 10 and 20 Gy are sufficient to induce cell death and are responsible for the formation of multiple cilia originating from multiple basal bodies.

Breast cancer and cancer stem cells: a mini-review.

Breast cancer is the most common type of malignant disease in women worldwide. In developing countries the past few years have sustained an increasing incidence of this type of cancer. Currently, breast cancer is the second leading cause of death due to cancer in women. In 2008 alone it was diagnosed in more than 1 million patients and each year the number of breast cancer-related deaths is estimated to be ~450,000. The mortality rate in breast cancer patients has been decreasing over the years thanks to the development of early diagnostic methods and more effective treatments. But despite the new advances in cancer diagnosis and treatment, the risk of recurrence and metastasis is ever present. It has been theorized that cancer stem cells are involved in the process of tumor growth and metastases. Due to their self-renewing and differentiation capabilities, they are now considered the underlying factor in tumor recurrence and the main reason for therapy resistance. Therefore, the characterization of cancer stem cells may contribute to the development of more effective treatment strategies that should make it possible to eliminate cancer stem cells in order to prevent tumor relapse and metastasis in diagnosed patients.

The role of mesenchymal stem cells in bone repair and regeneration.

Despite the undisputed modern development of synthetic biomaterials that range from bioactive unresorbable to restorable materials, clinically applied osteoconduction bone substitutes still have limitations in the treatment of bone defects. These are the result of the physical and chemical properties of the utilized materials and the biological interactions associated with both local and general reactions of the organism. Mesenchymal stem cells constitute a promising treatment alternative in orthopedics. Preclinical studies regarding the use of mesenchymal stem cells have shown good therapeutic results. However, it is still necessary to advance further in this area and enable the treatment of patients with critically large bone defects. The aim of this review is to describe the role of mesenchymal stem cells in bone repair and regeneration, describe the techniques used in the clinical application of mesenchymal stem cells and outline future research endeavors in this area.

Osteointegration of an uncemented modular revision stem implanted during revision hip surgery.

BACKGROUND: Though mid-term survival rates of over 95% in several series have been published, there is still a paucity of related literature regarding the role of vertical stem instability in the osteointegration of fluted tapered stems. This paper presents a comprehensive and prospective assessment on short-term experiences with uncemented modular femoral stem in the treatment of defective femur during revision surgery of total hip replacement. MATERIALS AND METHODS: Clinical and radiological monitoring of 20 consecutive patients with implanted tapered fluted revision stem (Lima Corporate, Udine, Italy) was of 27 months in average (20-35 months). The average pre-operative Merle d'Aubigné and Postel method score was 6.3 points (3-10 points). The frequency of femur defects, classified according to Paprosky, was IIIA = 9 and IIIB = 11. RESULTS: During last follow-up, the Merle d'Aubigné and Postel hip score was on average 11.7 (6-16 points). Compared to post-operation radiograph, stem migration of 1.9 mm (0-11 mm) on average was found. This vertical stem migration was observed only when comparing hip radiographs immediately after surgery, and at 6 weeks post-surgery. The Paprosky IIIA defects group, presented a subsided stem by an average of 1.5 mm. In the group of Paprosky IIIB defects, the stem subsidence was on average 2.3 mm. All 20 patients in the study showed excellent osteointegration of the uncemented revision modular stem. CONCLUSIONS: This study found and excellent osteointegration of the Lima uncemented tapered fluted revision modular stem in defective femur with a cortical bone segment present in the diaphyseal isthmus area. The initial vertical instability leading to stem migrating during the first six weeks following surgery did not, however, affect its osteointegration.

NANOTECHNOLOGY - NEW TRENDS IN THE TREATMENT OF BRAIN TUMOURS.

High grade gliomas are some of the deadliest human tumours. Conventional treatments such as surgery, radiotherapy and chemotherapy have only a limited effect. Nowadays, resection is the common treatment of choice and although new approaches, such as perioperative magnetic resonance imaging or fluorescent microscopy have been developed, the survival rate of diagnosed patients is still very low. The inefficacy of conventional methods has led to the development of new strategies and the significant progress of nanotechnology in recent years. These platforms can be used either as novel imaging tools or to improve anticancer drug delivery into tumours while minimizing its distribution and toxicity in healthy tissues. Amongst the new nanotechnology platforms used for delivery into the brain tissue are: polymeric nanoparticles, liposomes, dendrimers, nanoshells, carbon nanotubes, superparamagnetic nanoparticles and nucleic acid based nanoparticles (DNA, RNA interference [RNAi] and antisense oligonucleotides [ASO]). These nanoparticles have been applied in the delivery of small molecular weight drugs as well as macromolecules - proteins, peptides and genes. The unique properties of these nanoparticles, such as surface charge, particle size, composition and ability to modify their surface with tissue recognition ligands and antibodies, improve their biodistribution and pharmacokinetics. All of the above mentioned characteristics make of nanoplatforms a very suitable tool for its use in targeted, personalized medicine, where they could possibly carry large doses of therapeutic agents specifically into malignant cells while avoiding healthy cells. This review poses new possibilities in the large field of nanotechnology with special interest in the treatment of high grade brain tumours.

Tumor necrosis factor-α -308G/A polymorphism is associated with active vitiligo vulgaris in a northeastern Mexican population.

Vitiligo is a skin disease characterized by depigmentation. Its etiopathogenesis is unclear, but it has been associated with autoimmune processes. Gene polymorphisms in the tumor necrosis factor-α (TNF-α) have been associated with several imflammatory diseases. In particular, the -308G/A polymorphism in the gene promoter region has been reported to be associated with increased plasma levels of TNF-α and with an increased risk to develop autoimmune diseases. To date, this polymorphism has not been associated with vitiligo. To assess a possible association between the TNF-α -308G/A and vitiligo vulgaris (VV), 198 vitiligo patients and 395 control subjects were recruited for the study. A complete demographic and clinical profile of each case was registered to analyze the possible risk factors of vitiligo. Genomic DNA isolated from peri pheral blood was subjected to PCR-RFLP for genotyping of the TNF-α -308G/A polymorphism. Causal associations were determined by χ(2) test and their respective OR was assessed in a 2×2 contingency table. When population variables of type of vitiligo, gender, age of disease onset, and active disease status were considered, an association between active VV and the TNF-α GA genotype was found (P=0.0295, OR=2.0; 95% CI 1.01-3.93). All other variables were irrelevant to vitiligo. Our data suggest a possible association between the TNF-α -308 GA genotype and the active form of VV in a Mexican population.

The Fsr quorum-sensing system of Enterococcus faecalis modulates surface display of the collagen-binding MSCRAMM Ace through regulation of gelE.

Ace, a known virulence factor and the first identified microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Enterococcus faecalisis associated with host cell adherence and endocarditis. The Fsr quorum-sensing system of E. faecalis, a two-component signal transduction system, has also been repeatedly linked to virulence in E. faecalis, due in part to the transcriptional induction of an extracellular metalloprotease, gelatinase (GelE). In this study, we discovered that disruption of the Fsr pathway significantly increased the levels of Ace on the cell surface in the latter phases of growth. Furthermore, we observed that, in addition to fsrB mutants, other strains identified as deficient in GelE activity also demonstrated a similar phenotype. Additional experiments demonstrated the GelE-dependent cleavage of Ace from the surface of E. faecalis, confirming that GelE specifically reduces Ace cell surface display. In addition, disruption of the Fsr system or GelE expression significantly improved the ability of E. faecalis to adhere to collagen, which is consistent with higher levels of Ace on the E. faecalis surface. These results demonstrate that the display of Ace is mediated by quorum sensing through the action of GelE, providing insight into the complicated world of Gram-positive pathogen adhesion and colonization.