Hugo Murua Escobar

Poly-ε-caprolactone Coated and Functionalized Porous Titanium and Magnesium Implants for Enhancing Angiogenesis in Critically Sized Bone Defects.

For healing of critically sized bone defects, biocompatible and angiogenesis supporting implants are favorable. Murine osteoblasts showed equal proliferation behavior on the polymers poly-ε-caprolactone (PCL) and poly-(3-hydroxybutyrate)/poly-(4-hydroxybutyrate) (P(3HB)/P(4HB)). As vitality was significantly better for PCL, it was chosen as a suitable coating material for further experiments. Titanium implants with 600 µm pore size were evaluated and found to be a good implant material for bone, as primary osteoblasts showed a vitality and proliferation onto the implants comparable to well bottom (WB). Pure porous titanium implants and PCL coated porous titanium implants were compared using Live Cell Imaging (LCI) with Green fluorescent protein (GFP)-osteoblasts. Cell count and cell covered area did not differ between the implants after seven days. To improve ingrowth of blood vessels into porous implants, proangiogenic factors like Vascular Endothelial Growth Factor (VEGF) and High Mobility Group Box 1 (HMGB1) were incorporated into PCL coated, porous titanium and magnesium implants. An angiogenesis assay was performed to establish an in vitro method for evaluating the impact of metallic implants on angiogenesis to reduce and refine animal experiments in future. Incorporated concentrations of proangiogenic factors were probably too low, as they did not lead to any effect. Magnesium implants did not yield evaluable results, as they led to pH increase and subsequent cell death.

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.

Identification of a gene rearranged by 2p21 aberrations in thyroid adenomas.

Thyroid adenomas belong to the cytogenetically best investigated human epithelial tumors. Cytogenetic studies of about 450 benign lesions allow one to distinguish between different cytogenetic subgroups. Two chromosomal regions, that is, 19q13 and 2p21, are frequently rearranged in these tumors. Although 2p21 aberrations only account for about 10% of the benign thyroid tumors with clonal cytogenetic deviations, 2p21 rearrangements belong to the most common cytogenetic rearrangements in epithelial tumors due to the high frequency of these benign lesions. The 2p21 breakpoint region recently has been delineated to a region of 450 kbp, but the gene affected by the cytogenetic rearrangements still has escaped detection. Positional cloning and 3′ RACE–PCR allowed us to clone that gene which we will refer to as thyroid adenoma associated (THADA) gene. In cells from two thyroid adenomas characterized by translocations t(2;20;3) (p21;q11.2;p25) and t(2;7)(p21;p15), respectively, we performed 3′-RACE–PCRs and found two fusions of THADA with a sequence derived from chromosome band 3p25 or with a sequence derived from chromosome band 7p15. The THADA gene spans roughly 365 kbp and, based on preliminary results, encodes a death receptor-interacting protein.

Cloning, characterisation, and comparative quantitative expression analyses of receptor for advanced glycation end products (RAGE) transcript forms

RAGE is a member of the immunoglobulin superfamily of cell surface molecules playing key roles in pathophysiological processes, e.g. immune/inflammatory disorders, Alzheimers disease, diabetic arteriosclerosis and tumourigenesis. In humans 19 naturally occurring RAGE splicing variants resulting in either N-terminally or C-terminally truncated proteins were identified and are lately discussed as mechanisms for receptor regulation. Accordingly, deregulation of sRAGE levels has been associated with several diseases e.g. Alzheimers disease, Type 1 diabetes, and rheumatoid arthritis. Administration of recombinant sRAGE to animal models of cancer blocked tumour growth successfully. In spite of its obvious relationship to cancer and metastasis data focusing sRAGE deregulation and tumours is rare. In this study we screened a set of tumours, healthy tissues and various cancer cell lines for RAGE splicing variants and analysed their structure. Additionally, we analysed the ratio of the mainly found transcript variants using quantitative Real-Time PCR. In total we characterised 24 previously not described canine and 4 human RAGE splicing variants, analysed their structure, classified their characteristics, and derived their respective protein forms. Interestingly, the healthy and the neoplastic tissue samples showed in majority RAGE transcripts coding for the complete receptor and transcripts showing insertions of intron 1.

Role of miRNA let-7 and its major targets in prostate cancer.

Prostate cancer is worldwide the sixth leading cause of cancer related death in men thus early detection and successful treatment are still of major interest. The commonly performed screening of the prostate-specific antigen (PSA) is controversially discussed, as in many patients the prostate-specific antigen levels are chronically elevated in the absence of cancer. Due to the unsatisfying efficiency of available prostate cancer screening markers and the current treatment outcome of the aggressive hormone refractory prostate cancer, the evaluation of novel molecular markers and targets is considered an issue of high importance. MicroRNAs are relatively stable in body fluids orchestrating simultaneously the expression of many genes. These molecules are currently discussed to bear a greater diagnostic potential than protein-coding genes, being additionally promising therapeutic drugs and/or targets. Herein we review the potential impact of the microRNA let-7 family on prostate cancer and show how deregulation of several of its target genes could influence the cellular equilibrium in the prostate gland, promoting cancer development as they do in a variety of other human malignant neoplasias.

Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine

BackgroundIn molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells.ResultsThe parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization.ConclusionThis methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

TNF-α induced secretion of HMGB1 from non-immune canine mammary epithelial cells (MTH53A)

BACKGROUND Mammary neoplasias are one of the most frequent and spontaneously occurring malignancies in dogs and humans. Due to the similar anatomy of the mammary gland in both species, the dog has become an important animal model for this cancer entity. In human breast carcinomas, the overexpression of a protein named high-mobility group box 1 (HMGB1) was reported. Cells of the immune system were described to release HMGB1 actively exerting cytokine function. Thereby it is involved in the immune system activation, tissue repair, and cell migration. Passive release of HMGB1 by necrotic cells at sites of tissue damage or in necrotic hypoxic regions of tumors induces cellular responses e.g. release of proinflammatory cytokines leading to elevated inflammatory response and neo-vascularization of necrotic tumor areas. Herein we investigated if a time-dependent stimulation with the separately applied proinflammatory cytokines TNF-α and IFN-γ can cause secretion of HMGB1 in a non-immune related HMGB1-non-secreting epithelial canine mammary cell line (MTH53A) derived from non-neoplastic tissue. METHODS The canine cell line was transfected with recombinant HMGB1 bicistronic expression vectors and stimulated after transfection with the respective cytokine independently for 6, 24 and 48 h. HMGB1 protein detection was performed by Western blot analysis and quantified a by enzyme-linked immunosorbent assay. Live cell laser scanning multiphoton microscopy of MTH53A cells expressing a HMGB1-GFP fusion protein was performed in order to examine, if secretion of HMGB1 under cytokine stimulating conditions is also visible by fluorescence imaging. RESULTS The observed HMGB1 release kinetics showed a clearly time-dependent manner with a peak release 24h after TNF-α stimulation, while stimulation with IFN-γ had only small effects on the HMGB1 release. Multiphoton HMGB1 live cell microscopy showed diffuse cell membrane structure changes 29 h after cytokine-stimulation but no clear secretion of HMGB1-GFP after TNF-α stimulation was visible. CONCLUSION Our results demonstrate that non-immune HMGB1-non-secreting cells of epithelial origin derived from mammary non-neoplastic tissue can be induced to release HMGB1 by single cytokine application. This indicates that tumor and surrounding tissue can be stimulated by tumor present inflammatory and necrotic cytokines to release HMGB1 acting as neo-vascularizing factor thus promoting tumor growth.

Biophysical effects in off-resonant gold nanoparticle mediated (GNOME) laser transfection of cell lines, primary- and stem cells using fs laser pulses

Gold nanoparticle mediated (GNOME) laser transfection is a powerful technique to deliver small biologically relevant molecules into cells. However, the transfection of larger and especially negatively charged DNA remains challenging. The efficiency for pDNA was 0.57% using parameter that does not influence the endo- and exogenous DNA. In order to gain a deeper understanding of the actual molecule uptake process, the uptake efficiency was determined using molecules of different sizes. It was evaluated that uncharged dextran molecules (2000 kDa) were delivered with an efficiency of 68%. The intracellular distribution of injected molecules was visualized and larger molecules were primary found in the cytoplasm. Patch clamp measurements suggested a permeabilization time up to 15 minutes. The uptake efficiency depended on the size and charge of the molecule to deliver as well as the cell size. A minor role for transfection plays the cell type since primary stem cells were successfully transfected. The perforation efficiency of semi-adherent and suspension cells is influenced by the cell and molecule size.

Plasmonic laser treatment for Morpholino oligomer delivery in antisense applications.

Several cell transfection techniques have been developed in the last decades for specific applications and for various types of molecules. In this context, laser based approaches are of great interest due to their minimal invasiveness and spatial selectivity. In particular, laser induced plasmon based delivery of exogenous molecules into cells can have great impact on future applications. This approach allows high-throughput laser transfection by excitation of plasmon resonances at gold nanoparticles non-specifically attached to the cell membrane. In this study, we demonstrate specific gene-knockdown by transfection of Morpholino oligos using this technique with optimized particle size. Furthermore, we evaluated the cytotoxicity of plasmonic laser treatment by various assays, including LDH activity and ROS formation. In summary, this study gives important insights into this new approach and clearly demonstrates its relevance for possible biological applications.

HMGA1 and HMGA2 expression and comparative analyses of HMGA2, Lin28 and let-7 miRNAs in oral squamous cell carcinoma

BackgroundHumans and dogs are affected by squamous cell carcinomas of the oral cavity (OSCC) in a considerably high frequency. The high mobility group A2 (HMGA2) protein was found to be highly expressed in human OSCC and its expression was suggested to act as a useful predictive and prognostic tool in clinical management of oral carcinomas. Herein the expression of HMGA2 and its sister gene HMGA1 were analysed within human and canine OSCC samples. Additionally, the HMGA negatively regulating miRNAs of the let-7 family as well as the let-7 regulating gene Lin28 were also comparatively analysed. Deregulations of either one of these members could affect the progression of human and canine OSCC.MethodsExpression levels of HMGA1, HMGA2, Lin28, let-7a and mir-98 were analysed via relative qPCR in primary human and canine OSCC, thereof derived cell lines and non-neoplastic samples. Additionally, comparative HMGA2 protein expression was analysed by immunohistochemistry.ResultsIn both species, a significant up-regulation of the HMGA2 gene was found within the neoplastic samples while HMGA1 expression did not show significant deregulations. Comparative analyses showed down-regulation of mir-98 in human samples and up-regulation of let-7a and mir-98 in canine neoplastic samples. HMGA2 immunostainings showed higher intensities within the invasive front of the tumours than in the centre of the tumour in both species.ConclusionsHMGA2 could potentially serve as tumour marker in both species while HMGA1 might play a minor role in OSCC progression. Comparative studies indicate an inverse correlation of HMGA2 and mir-98 expression in human samples whereas in dogs no such characteristic could be found.