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Tibiletti M.,Istituto Ortopedico Galeazzi | Tibiletti M.,University of Ulm | Kregar Velikonja N.,Educell Ltd. | Urban J.P.G.,University of Oxford | Fairbank J.C.T.,University of Oxford
European Spine Journal | Year: 2014

Background: Disc cell therapies, in which cells are injected into the degenerate disc in order to regenerate the matrix and restore function, appear to be an attractive, minimally invasive method of treatment. Interest in this area has stimulated research into disc cell biology in particular. However, other important issues, some of which are discussed here, need to be considered if cell-based therapies are to be brought to the clinic. Purpose: Firstly, a question which is barely addressed in the literature, is how to identify patients with 'degenerative disc disease' who would benefit from cell therapy. Pain not disc degeneration is the symptom which drives patients to the clinic. Even though there are associations between back pain and disc degeneration, many people with even severely degenerate discs, with herniated discs or with spinal stenosis, are pain-free. It is not possible using currently available techniques to identify whether disc repair or regeneration would remove symptoms or prevent symptoms from occurring in future. Moreover, the repair process in human discs is very slow (years) because of the low cell density which can be supported nutritionally even in healthy human discs. If repair is necessary for relief of symptoms, questions regarding quality of life and rehabilitation during this long process need consideration. Also, some serious technical issues remain. Finding appropriate cell sources and scaffolds have received most attention, but these are not the only issues determining the feasibility of the procedure. There are questions regarding the safety of implanting cells by injection through the annulus whether the nutrient supply to the disc is sufficient to support implanted cells and whether, if cells are able to survive, conditions in a degenerate human disc will allow them to repair the damaged tissue. Conclusions: If cell therapy for treatment of disc-related disorders is to enter the clinic as a routine treatment, investigations must examine the questions related to patient selection and the feasibility of achieving the desired repair in an acceptable time frame. Few diagnostic tests that examine whether cell therapies are likely to succeed are available at present, but definite exclusion criteria would be evidence of major disc fissures, or disturbance of nutrient pathways as measured by post-contrast MRI. © 2014 Springer-Verlag.

Gorgieva S.,University of Maribor | Modic M.,Jozef Stefan Institute | Dovgan B.,Educell Ltd. | Kaisersberger-Vincek M.,University of Maribor | Kokol V.,University of Maribor
Plasma Processes and Polymers | Year: 2015

Novel, polypropylene mesh (PPmesh) - gelatin scaffold (GELscaffold) composites are prepared from O2-plasma activated PPmesh, coated with GEL solution being in-situ structured in a 3D scaffold with by-preparation tuned, gradual porosity. The fabricated biomimetic-like composites demonstrate frequency-dependent rheological behaviour and high (up to 100%) intrinsic and/or by Ïμ -poly-L-lysine evoked antibacterial activity against S. aureus and E. coli, latter demonstrating preparation-dependant releasing profile, without triggering in-vitro cytotoxicity. Finally, long-term (up to 2 months) biodegradation resistance highlight the composite potential as active, biomimetic barrier in hernia treatment. Potential of novel, PPmesh-GELscaffold composites for active hernia treatment is demonstrated by means of extracellular matrix-like gradual μ-porosity, high elasticity, excellent intrinsic/ by μ-poly-L-lysine-evoked antibacterial activity against S. aureus and E. coli, for 60% diminished fibrinogen adsorption, up to 2 months biodegradation resistance, and non cytotoxicity towards human ASC cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Grayson W.L.,Columbia University | Frohlich M.,Columbia University | Frohlich M.,Educell Ltd. | Yeager K.,Columbia University | And 7 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010

The ability to engineer anatomically correct pieces of viable and functional human bone would have tremendous potential for bone reconstructions after congenital defects, cancer resections, and trauma.We report that clinically sized, anatomically shaped, viable human bone grafts can be engineered by using human mesenchymal stem cells (hMSCs) and a "biomimetic" scaffold-bioreactor system. We selected the temporomandibular joint (TMJ) condylar bone as our tissue model, because of its clinical importance and the challenges associated with its complex shape. Anatomically shaped scaffolds were generated from fully decellularized trabecular bone by using digitized clinical images, seeded with hMSCs, and cultured with interstitial flow of culture medium. A bioreactor with a chamber in the exact shape of a human TMJ was designed for controllable perfusion throughout the engineered construct. By 5 weeks of cultivation, tissue growth was evidenced by the formation of confluent layers of lamellar bone (by scanning electron microscopy), markedly increased volume of mineralized matrix (by quantitative microcomputer tomography), and the formation of osteoids (histologically). Within bone grafts of this size and complexity cells were fully viable at a physiologic density, likely an important factor of graft function. Moreo-ver, the density and architecture of bone matrix correlated with the intensity and pattern of the interstitial flow, as determined in experimental and modeling studies. This approach has potential to overcome a critical hurdle - in vitro cultivation of viable bone grafts of complex geometries - to provide patient-specific bone grafts for craniofacial and orthopedic reconstructions.

Dovgan B.,Educell Ltd. | Barlic A.,Educell Ltd. | Knezevic M.,Educell Ltd. | Miklavcic D.,University of Ljubljana
Journal of Membrane Biology | Year: 2016

New cryopreservation approaches for medically applicable cells are of great importance in clinical medicine. Current protocols employ the use of dimethyl sulfoxide (DMSO), which is toxic to cells and causes undesirable side effects in patients, such as cardiac arrhythmias, neurological events, and others. Trehalose, a nontoxic disaccharide, has been already studied as a cryoprotectant. However, an efficient approach for loading this impermeable sugar into mammalian cells is missing. In our study, we assessed the efficiency of combining reversible electroporation and trehalose for cryopreservation of human adipose-derived stem cells. First, we determined reversible electroporation threshold by loading of propidium iodide into cells. The highest permeabilization while maintaining high cell viability was reached at 1.5 kV/cm, at 8 pulses, 100 µs, and 1 Hz. Second, cells were incubated in 250 or 400 mM trehalose and electroporated before cryopreservation. After thawing, 83.8 ± 1.8 % (mean ± SE) cell recovery was obtained at 250 mM trehalose. By using a standard freezing protocol (10 % DMSO in 90 % fetal bovine serum), cell survival after thawing was about 91.5 ± 1.6 %. We also evaluated possible effects of electroporation on cells’ functionality before and after thawing. Successful cell growth and efficient adipogenic and osteogenic differentiation were achieved. In conclusion, electroporation seems to be an efficient method for loading nonpermeable trehalose into human adipose-derived stem cells, allowing long-term cryopreservation in DMSO-free and xeno-free conditions. © 2016 Springer Science+Business Media New York

Bas T.,Worcester Polytechnic Institute | Veber M.,Educell Ltd. | Kosir A.,University of Ljubljana | Dominko T.,Worcester Polytechnic Institute | Page R.,Worcester Polytechnic Institute
Histochemistry and Cell Biology | Year: 2013

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets. © 2012 Springer-Verlag Berlin Heidelberg.

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