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Ravnihar K.,Orthopedic Hospital Valdoltra | Barlic A.,Educell Ltd | Drobnic M.,University of Ljubljana
Arthroscopy - Journal of Arthroscopic and Related Surgery | Year: 2014

Purpose To evaluate the hypothetical toxic effect of local anesthetics on the articular cartilage using patient data from autologous chondrocyte cultivation with different anesthesia types used for arthroscopic cartilage biopsy specimen procurement. Methods A retrospective analysis of patient data from the national autologous chondrocyte implantation registry and the corresponding hospital records was approved by the National Medical Ethics Committee. Articular cartilage biopsy specimens from the knees of 49 consecutive patients assigned for autologous chondrocyte implantation (aged 14 to 44 years) were procured from the non-weight-bearing articular surface during arthroscopy under general anesthesia (12 patients), spinal anesthesia (18 patients), or local anesthesia (intra-articular injection of 15 to 20 mL of 2% lidocaine hydrochloride) (19 patients). All the biopsy specimens were further manipulated following the same chondrocyte cultivation protocol. General patient data and surgery-related parameters, together with chondrocyte viability, population doublings, and chondrocyte morphology in biopsy specimens and primary cell cultures, were analyzed and compared across different types of anesthesia. Results Patients in the general, spinal, and local anesthesia groups showed no statistical differences in age (27 years, 29 years, and 32 years, respectively), duration of surgery (36 minutes, 37 minutes, and 39 minutes, respectively), weight of biopsy specimens (110 mg, 178 mg, and 130 mg, respectively), cell viability in cartilage biopsy specimens (67%, 69%, and 78%, respectively) or primary cultures (95%, 95%, and 95%, respectively), and population doublings (5.2, 5.2, and 5.2, respectively). Similar chondrocyte morphology in primary cell cultures was observed among the 3 groups. Conclusions This retrospective study showed that a single intra-articular injection of lidocaine hydrochloride used for knee arthroscopy did not influence the viability, morphology, and cultivation potential of chondrocytes in articular cartilage biopsy specimens assigned for autologous chondrocyte implantation. Level of Evidence Level IV, retrospective comparative study. © 2014 by the Arthroscopy Association of North America.

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.

PubMed | Slovenian National Institute of Biology and Educell Ltd.
Type: Clinical Trial | Journal: Biologicals : journal of the International Association of Biological Standardization | Year: 2015

Tumor necrosis factor-alpha (TNF) antagonists are efficacious in the treatment of various immune-mediated inflammatory diseases. Because of rapidly growing demand for developing new or biosimilar versions of these biologicals, the need to create invitro testing models that best represent physiological conditions is increasing. Primary human chondrocytes were used for potency evaluation and comparison between the molecular effects of anti-TNF biologicals. Infliximab and etanercept were chosen to assess the suitability of chondrocyte cell culture for determination of anti-TNF neutralization efficacy employing quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technology. Use of both anti-TNF biologics resulted in decrease of TNF-stimulated expression of various matrix metalloproteinases, interleukins and other inflammation-related genes in our cell model. Significant differences in inhibition efficacy of etanercept and infliximab were observed, which were confirmed also on protein level. To evaluate the potency of anti-TNF biologicals, a selection of TNF-responsive target genes was made from the gene array data. The selected genes were employed in development of statistical model, which enables comparability of anti-TNF biologicals. The presented analytical approach is suitable for assessment of the neutralization efficacy of various anti-TNF biologicals. As such, it can be used for additional comprehensive characterization and comparability of TNF antagonists in preclinical drug testing.

PubMed | Bishop Moore College, Lulea University of Technology, University of Stockholm and Educell ltd.
Type: Journal Article | Journal: Biomacromolecules | Year: 2016

Double cross-linked interpenetrating polymer network (IPN) hydrogels of sodium alginate and gelatin (SA/G) reinforced with 50 wt % cellulose nanocrystals (CNC) have been prepared via the freeze-drying process. The IPNs were designed to incorporate CNC with carboxyl surface groups as a part of the network contribute to the structural integrity and mechanical stability of the hydrogel. Structural morphology studies of the hydrogels showed a three-dimensional (3D) network of interconnected pores with diameters in the range of 10-192 m and hierarchical pores with a nanostructured pore wall roughness, which has potential benefits for cell adhesion. Significant improvements in the tensile strength and strain were achieved in 98% RH at 37 C for CNC cross-linked IPNs. The high porosity of the scaffolds (>93%), high phosphate buffered saline (PBS) uptake, and cytocompatibility toward mesenchymal stem cells (MSCs) are confirmed and considered beneficial for use as a substitute for cartilage.

Frohlich M.,Columbia University | Frohlich M.,Educell Ltd | Grayson W.L.,Columbia University | Marolt D.,Columbia University | And 3 more authors.
Tissue Engineering - Part A | Year: 2010

We report engineering of half-centimeter-sized bone constructs created in vitro using human adipose-derived stem cells (hASCs), decellularized bone scaffolds, and perfusion bioreactors. The hASCs are easily accessible, can be used in an autologous fashion, are rapidly expanded in culture, and are capable of osteogenic differentiation. hASCs from four donors were characterized for their osteogenic capacity, and one representative cell population was used for tissue engineering experiments. Culture-expanded hASCs were seeded on fully decellularized native bone scaffolds (4mm diameter×4mm thick), providing the necessary structural and mechanical environment for osteogenic differentiation, and cultured in bioreactors with medium perfusion. The interstitial flow velocity was set to a level necessary to maintain cell viability and function throughout the construct volume (400μm/s), via enhanced mass transport. After 5 weeks of cultivation, the addition of osteogenic supplements (dexamethasone, sodium-β-glycerophosphate, and ascorbic acid-2-phosphate) to culture medium significantly increased the construct cellularity and the amounts of bone matrix components (collagen, bone sialoprotein, and bone osteopontin). Medium perfusion markedly improved the distribution of cells and bone matrix in engineered constructs. In summary, a combination of hASCs, decellularized bone scaffold, perfusion culture, and osteogenic supplements resulted in the formation of compact and viable bone tissue constructs. © Copyright 2009, Mary Ann Liebert, Inc. 2009.

PubMed | Hemoteq Inc., Catholic University of Leuven, Biotech International, SP Food and Bioscience and Educell Ltd
Type: Journal Article | Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society | Year: 2016

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2191-2198, 2016.

Kregar Velikonja N.,Educell Ltd. | Kregar Velikonja N.,Faculty for Health science Novo Mesto | Urban J.,University of Oxford | Frohlich M.,Educell Ltd. | And 5 more authors.
European Spine Journal | Year: 2014

Purpose: There is increasing interest in the development of cell therapy as a possible approach for the treatment of degenerative disc disease. To regenerate nucleus pulposus tissue, the cells must produce an appropriate proteoglycan-rich matrix, as this is essential for the functioning of the intervertebral disc. The natural environment within the disc is very challenging to implanted cells, particularly if they have been subcultured in normal laboratory conditions. The purpose of this work is to discuss parameters relevant to translating different proposed cell therapies of IVD into clinical use. Results: Several sources of cells have been proposed, including nucleus pulposus cells, chondrocytes and mesenchymal stem cells derived from bone marrow or adipose tissue. There are some clinical trials and reports of attempts to regenerate nucleus pulposus utilising either autologous or allogenic cells. While the published results of clinical applications of these cell therapies do not indicate any safety issues, additional evidence will be needed to prove their long-term efficacy. Conclusion: This article discusses parameters relevant for successful translation of research on different cell sources into clinically applicable cell therapies: the influence of the intervertebral disc microenvironment on the cell phenotype, issues associated with cell culture and technical preparation of cell products, as well as discussing current regulatory requirements. There are advantages and disadvantages of each proposed cell type, but no strong evidence to favour any one particular cell source at the moment. © 2013 Springer-Verlag.

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.

Tibiletti M.,IRCCS Instituto 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.

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