Publications by authors named "Aristeidis Kritis"

20 Publications

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Electrospun wound dressings containing bioactive natural products: physico-chemical characterization and biological assessment.

Biomater Res 2021 Jul 16;25(1):23. Epub 2021 Jul 16.

Laboratory of Organic Chemistry, School of Chemical Engineering, Aristotle University of Thessaloniki (AUTh), 54124, Thessaloniki, Greece.

Background: Current research on skin tissue engineering has been focusing on novel therapies for the effective management of chronic wounds. A critical aspect is to develop matrices that promote growth and uniform distribution of cells across the wound area, and at the same time offer protection, as well as deliver drugs that help wound healing and tissue regeneration. In this context, we aimed at developing electrospun scaffolds that could serve as carriers for the bioactive natural products alkannin and shikonin (A/S).

Methods: A series of polymeric nanofibers composed of cellulose acetate (CA) or poly(ε-caprolactone) (PCL) and varying ratios of a mixture of A/S derivatives, has been successfully fabricated and their physico-chemical and biological properties have been explored.

Results: Scanning electron microscopy revealed a uniform and bead-free morphology for CA scaffolds, while for PCL beads along the fibers were observed. The average diameters for all nanofibers ranged between 361 ± 47 and 487 ± 88 nm. During the assessment of physicochemical characteristics, CA fiber mats exhibited a more favored profile, while the assessment of the biological properties of the scaffolds showed that CA samples containing A/S mixture up to 1 wt.% achieved to facilitate attachment, survival and migration of Hs27 fibroblasts. With respect to the antimicrobial properties of the scaffolds, higher drug-loaded (1 and 5 wt.%) samples succeeded in inhibiting the growth of Staphylococcus epidermidis and S. aureus around the edges of the fiber mats. Finally, carrying out a structure-activity relationship study regarding the biological activities (fibroblast toxicity/proliferation and antibacterial activity) of pure A/S compounds - present in the A/S mixture - we concluded that A/S ester derivatives and the dimeric A/S augmented cell proliferation after 3 days, whereas shikonin proved to be toxic at 500 nM and 1 μM and alkannin only at 1 μM. Additionally, alkannin, shikonin and acetyl-shikonin showed more pronounced antibacterial properties than the other esters, the dimeric derivative and the A/S mixture itself.

Conclusions: Taken together, these findings indicate that embedding A/S derivatives into CA nanofibers might be an advantageous drug delivery system that could also serve as a potential candidate for biomedical applications in the field of skin tissue engineering.
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http://dx.doi.org/10.1186/s40824-021-00223-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284004PMC
July 2021

Novel electrospun poly-hydroxybutyrate scaffolds as carriers for the wound healing agents alkannins and shikonins.

Regen Biomater 2021 Jun 10;8(3):rbab011. Epub 2021 Jun 10.

Laboratory of Organic Chemistry, School of Chemical Engineering, Aristotle University of Thessaloniki (AUTh), Thessaloniki 54124, Greece.

The aim of this study was to investigate the potential of novel electrospun fiber mats loaded with alkannin and shikonin (A/S) derivatives, using as carrier a highly biocompatible, bio-derived, eco-friendly polymer such as poly[(R)-3-hydroxybutyric acid] (PHB). PHB fibers containing a mixture of A/S derivatives at different ratios were successfully fabricated via electrospinning. Αs evidenced by scanning electron microscopy, the fibers formed a bead-free mesh with average diameters from 1.25 to 1.47 μm. Spectroscopic measurements suggest that electrospinning marginally increases the amorphous content of the predominantly crystalline PHB in the fibers, while a significant drug amount lies near the fiber surface for samples of high total A/S content. All scaffolds displayed satisfactory characteristics, with the lower concentrations of A/S mixture-loaded PHB fiber mats achieving higher porosity, water uptake ratios, and entrapment efficiencies. The dissolution studies revealed that all samples released more than 70% of the encapsulated drug after 72 h. All PHB scaffolds tested by cell viability assay were proven non-toxic for Hs27 fibroblasts, with the 0.15 wt.% sample favoring cell attachment, spreading onto the scaffold surface, as well as cell proliferation. Finally, the antimicrobial activity of PHB meshes loaded with A/S mixture was documented for and .
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http://dx.doi.org/10.1093/rb/rbab011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240617PMC
June 2021

The Marine Polysaccharide Ulvan Confers Potent Osteoinductive Capacity to PCL-Based Scaffolds for Bone Tissue Engineering Applications.

Int J Mol Sci 2021 Mar 17;22(6). Epub 2021 Mar 17.

Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece.

Hybrid composites of synthetic and natural polymers represent materials of choice for bone tissue engineering. Ulvan, a biologically active marine sulfated polysaccharide, is attracting great interest in the development of novel biomedical scaffolds due to recent reports on its osteoinductive properties. Herein, a series of hybrid polycaprolactone scaffolds containing ulvan either alone or in blends with κ-carrageenan and chondroitin sulfate was prepared and characterized. The impact of the preparation methodology and the polysaccharide composition on their morphology, as well as on their mechanical, thermal, water uptake and porosity properties was determined, while their osteoinductive potential was investigated through the evaluation of cell adhesion, viability, and osteogenic differentiation of seeded human adipose-derived mesenchymal stem cells. The results verified the osteoinductive ability of ulvan, showing that its incorporation into the polycaprolactone matrix efficiently promoted cell attachment and viability, thus confirming its potential in the development of biomedical scaffolds for bone tissue regeneration applications.
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http://dx.doi.org/10.3390/ijms22063086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002638PMC
March 2021

Cartilage Regeneration with Cell-Seeded Natural Biomaterial Scaffold Implants: 15-Year Study.

Tissue Eng Part B Rev 2021 Mar 10. Epub 2021 Mar 10.

Department of Physiology and Pharmacology, Faculty of Health Sciences and cGMP Regenerative Medicine Facility, School of Medicine, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece.

Articular cartilage can be easily damaged from human's daily activities, leading to inflammation and to osteoarthritis, a situation that can diminish the patients' quality of life. For larger cartilage defects, scaffolds are employed to provide cells the appropriate three-dimensional environment to proliferate and differentiate into healthy cartilage tissue. Natural biomaterials used as scaffolds, attract researchers' interest because of their relative nontoxic nature, their abundance as natural products, their easy combination with other materials, and the relative easiness to establish Marketing Authorization. The last 15 years were chosen to review, document, and elucidate the developments on cell-seeded natural biomaterials for articular cartilage treatment . The parameters of the experimental designs and their results were all documented and presented. Considerations about the newly formed cartilage and the treatment of cartilage defects were discussed, along with difficulties arising when applying natural materials, research limitations, and tissue engineering approaches for hyaline cartilage regeneration.
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http://dx.doi.org/10.1089/ten.TEB.2020.0295DOI Listing
March 2021

Hybrid chitosan/gelatin/nanohydroxyapatite scaffolds promote odontogenic differentiation of dental pulp stem cells and in vitro biomineralization.

Dent Mater 2021 01 15;37(1):e23-e36. Epub 2020 Nov 15.

Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Greece. Electronic address:

Objective: Hybrid chitosan/gelatin/nanohydroxyapatite (CS/Gel/nHA) scaffolds have attracted considerable interest in tissue engineering (TE) of mineralized tissues. The present study aimed to investigate the potential of CS/Gel/nHA scaffolds loaded with dental pulp stem cells (DPSCs) to induce odontogenic differentiation and in vitro biomineralization.

Methods: CS/Gel/nHA scaffolds were synthesized by freeze-drying, seeded with DPSCs, and characterized with flow cytometry. Scanning Electron Microscopy (SEM), live/dead staining, and MTT assays were used to evaluate cell morphology and viability; real-time PCR for odontogenesis-related gene expression analysis; SEM-EDS (Energy Dispersive X-ray spectroscopy), and X-ray Diffraction analysis (XRD) for structural and chemical characterization of the mineralized constructs, respectively.

Results: CS/Gel/nHA scaffolds supported viability and proliferation of DPSCs over 14 days in culture. Gene expression patterns indicated pronounced odontogenic shift of DPSCs, evidenced by upregulation of DSPP, BMP-2, ALP, and the transcription factors RunX2 and Osterix. SEM-EDS showed the production of a nanocrystalline mineralized matrix inside the cell-based and - to a lesser extent - the cell-free constructs, with a time-dependent production of net-like nanocrystals (appr. 25-30nm in diameter). XRD analysis gave the crystallite size (D=50nm) but could not distinguish between the initially incorporated and the biologically produced nHA.

Significance: This is the first study validating the potential of CS/Gel/nHA scaffolds to support viability and proliferation of DPSCs, and to provide a biomimetic microenvironment favoring odontogenic differentiation and in vitro biomineralization without the addition of any inductive factors, including dexamethasone and/or growth/morphogenetic factors. These results reveal a promising strategy towards TE of mineralized dental tissues.
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http://dx.doi.org/10.1016/j.dental.2020.09.021DOI Listing
January 2021

An effective device and method for enhanced cell growth in 3D scaffolds: Investigation of cell seeding and proliferation under static and dynamic conditions.

Mater Sci Eng C Mater Biol Appl 2020 Sep 11;114:111060. Epub 2020 May 11.

Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece; cGMP Regenerative Medicine Facility, Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece.

Cell adhesion on 3D-scaffolds is a challenging task to succeed high cell densities and even cell distribution. We aimed to design a 3D-cell Culture Device (3D-CD) for static seeding and cultivation, to be used with any kind of scaffold, limiting cell loss and facilitating nutrient supply. 3D printing technology was used for both scaffold and device fabrication. Apart from testing the device, the purpose of this study was to assess and compare static and dynamic seeding and cultivation methods, of wet and dry scaffolds, under normoxic and hypoxic conditions and their effects on parameters such as cell seeding efficiency, cell distribution and cell proliferation. Human adipose tissue was harvested and cultured in 3D-printed poly(epsilon-caprolactone) scaffolds. Micro-CT scans were performed and projection images were reconstructed into cross section images. We created 3D images to visualize cell distribution and orientation inside the scaffolds. The group of prewetted scaffolds was the most favorable to cell attachment. The 3D-cell Culture Device (3D-CD) enhanced cell seeding efficiency with almost no cell loss. We suggest that the most favorable outcome can be produced with static seeding in the device for 24 h, followed either by static cultivation in the same device or by dynamic cultivation.
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http://dx.doi.org/10.1016/j.msec.2020.111060DOI Listing
September 2020

Mechanical properties of human glioma.

Neurol Res 2020 Dec 24;42(12):1018-1026. Epub 2020 Jul 24.

First Department of Neurosurgery, AHEPA University Hospital, Aristotle University of Thessaloniki , Thessaloniki, Greece.

Brain gliomas represent some of the most aggressive tumors encountered by modern medicine and, despite major efforts to optimize early diagnosis and treatment, the prognosis remains poor. Due to the complex structure of the brain and the unique mechanical properties of the extracellular matrix, gliomas invade and expand into the brain parenchyma, along white matter tracts and within perivascular spaces, usually sparing normal vessels. Different methods have been developed to study the mechanical properties of gliomas in a wide range of scales, from cells and the microscale to tissues and the macroscale. In this review, the current view on glioma mechanics is presented and the methods used to determine glioma mechanical properties are outlined. Their principles and current state of affairs are discussed.
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http://dx.doi.org/10.1080/01616412.2020.1796381DOI Listing
December 2020

The Efficacy of Stem Cells Secretome Application in Osteoarthritis: A Systematic Review of In Vivo Studies.

Stem Cell Rev Rep 2020 12;16(6):1222-1241

Department of Prosthodontics, Faculty of Dentistry, Aristotle University of Thessaloniki, University Campus, Dentistry Building, GR-54124, Thessaloniki, Greece.

Purpose: Mesenchymal stem cells (MSCs) have appeared as a promising regenerative cell-based therapeutic, for degenerative conditions, such as OA, while the beneficial results from the application of MSCs have been attributed to the MSCs-derived secretome, which is the sum of cytoprotective factors produced by the MSCs. Aim of this study was to systematically review the literature in order to assess whether stem cell secretome (conditioned medium-CM, exosome-Exos or microvesicles-MV)(CM/Exos/MVs) treatment reduces inflammation and enhances cartilage regeneration in preclinical studies of experimental arthritis.

Materials And Methods: An extensive electronic search was conducted by 2 independent reviewers by using the PubMed, Cochrane Library, Web of Science, and Scopus database, as well as Google Scholar, in order to identify the studies that met our inclusion criteria until August 2019. Included studies were assessed for quality and Risk of Bias (RoB) using the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines and a modification of Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) RoB tool for animal studies, respectively.

Results: The initial search provided 525 records, with 28 fulfilling the inclusion criteria. The included studies presented great heterogeneity regarding the stem cells used, the preparation of therapeutic agent as well as the animal models used for testing. In addition, most studies presented with an unclear or high risk bias.

Conclusion: In summary, the positive results of CM/Exos/MVs application in preclinical models of experimentally induced OA in terms of resolution of inflammation and cartilage regeneration are highlighted in this review, presenting a promising therapeutic solution for OA.
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http://dx.doi.org/10.1007/s12015-020-09980-xDOI Listing
December 2020

Hybrid Sponge-Like Scaffolds Based on Ulvan and Gelatin: Design, Characterization and Evaluation of Their Potential Use in Bone Tissue Engineering.

Materials (Basel) 2020 Apr 9;13(7). Epub 2020 Apr 9.

Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece.

Ulvan, a bioactive natural sulfated polysaccharide, and gelatin, a collagen-derived biopolymer, have attracted interest for the preparation of biomaterials for different biomedical applications, due to their demonstrated compatibility for cell attachment and proliferation. Both ulvan and gelatin have exhibited osteoinductive potential, either alone or in combination with other materials. In the current work, a series of novel hybrid scaffolds based on crosslinked ulvan and gelatin was designed, prepared and characterized. Their mechanical performance, thermal stability, porosity, water-uptake and degradation ability were assessed, while their morphology was analyzed through scanning electron microscopy. The prepared hybrid ulvan/gelatin scaffolds were characterized by a highly porous and interconnected structure. Human adipose-derived mesenchymal stem cells (hADMSCs) were seeded in selected ulvan/gelatin hybrid scaffolds and their adhesion, survival, proliferation, and osteogenic differentiation efficiency was evaluated. Overall, it was found that the prepared hybrid sponge-like scaffolds could efficiently support mesenchymal stem cells' adhesion and proliferation, suggesting that such scaffolds could have potential uses in bone tissue engineering.
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http://dx.doi.org/10.3390/ma13071763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178717PMC
April 2020

Therapeutic Potential of Mesenchymal Stromal Stem Cells in Rheumatoid Arthritis: a Systematic Review of In Vivo Studies.

Stem Cell Rev Rep 2020 04;16(2):276-287

Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece.

Standard treatment options for rheumatoid arthritis (RA) often fail to deliver a long-term therapeutic outcome and in many cases cause intractable adverse events leading to treatment discontinuation or readjustment. Treatment with mesenchymal stem cells (MSCs) has been recently studied in RA due to its immunomodulatory and anti-inflammatory capacities. Thus, this study aims at systematically search and review the literature for randomized or non-randomized clinical trials comparing interventions of MSCs with placebo in RA patients. Electronic searches were conducted on PubMed, SCOPUS, Cochrane-CENTRAL, registries of clinical trials and grey literature. Selected studies were estimated for risk of bias with the Cochrane RoB tool 2 or the ROBINS-I tool. Four trials met the eligibility criteria and entered the review process. Identified MSCs treatments varied from allogeneic to autologous or umbilical cord-derived cells. Enrolled patients had an active RA and had poor responses to previous standard medications. In general, the safety evaluation revealed that treatment with MSCs was safe and well tolerated. Regarding the efficacy measurements, modest improvements were found in RA symptoms and RA-related indices. Significant decreases were found in inflammatory molecules such as C-reactive protein, tumor necrosis factor alpha and interleukin 6. However, clinical response criteria related to RA were achieved by a low-to-moderate percentage of patients. In conclusion, treatment of RA with MSCs appears to have a short-term therapeutic effect. Better-designed randomized trials with sufficient follow-up periods are needed so that the long-term safety and efficacy interventions with MSCs would be elucidated.
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http://dx.doi.org/10.1007/s12015-020-09954-zDOI Listing
April 2020

Assessment of cartilage regeneration on 3D collagen-polycaprolactone scaffolds: Evaluation of growth media in static and in perfusion bioreactor dynamic culture.

Colloids Surf B Biointerfaces 2019 Nov 29;183:110403. Epub 2019 Jul 29.

Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece; cGMP Regenerative Medicine Facility, Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece. Electronic address:

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-β based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-β medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.
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http://dx.doi.org/10.1016/j.colsurfb.2019.110403DOI Listing
November 2019

Hyaline cartilage next generation implants from adipose-tissue-derived mesenchymal stem cells: Comparative study on 3D-printed polycaprolactone scaffold patterns.

J Tissue Eng Regen Med 2019 02;13(2):342-355

Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece.

We used additive manufacturing to fabricate 3D-printed polycaprolactone scaffolds of different geometry topologies and porosities. We present a comparative analysis of hyaline cartilage development from adipose-tissue-derived mesenchymal stem cells (ADMSCs) on three different, newly designed scaffold geometry patterns. The first scaffold design (MESO) was based on a rectilinear layer pattern. For the second pattern (RO45), we employed a 45° rotational layer loop. The design for the third scaffold (3DHC) was a three-dimensional honeycomb-like pattern with a hexagonal cellular distribution and small square shapes. We examined cell proliferation, colonization, and differentiation, in relation to the scaffold's structure, as well as to the mechanical properties of the final constructs. We gave emphasis on the scaffolds, both microarchitecture and macroarchitecture, for optimal and enhanced chondrogenic differentiation, as an important parameter, not well studied in the literature. Among the three patterns tested, RO45 was the most favourable for chondrogenic differentiation, whereas 3DHC better supported cell proliferation and scaffold penetration, exhibiting also the highest rate of increase onto the mechanical properties of the final construct. We conclude that by choosing the optimal scaffold architecture, the resulting properties of our cartilaginous constructs can better approximate those of the physiological cartilage.
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http://dx.doi.org/10.1002/term.2798DOI Listing
February 2019

Crocus sativus L. Causes a Non Apoptotic Calpain Dependent Death in C6 Rat Glioma Cells, Exhibiting a Synergistic Effect with Temozolomide.

Nutr Cancer 2019 1;71(3):491-507. Epub 2018 Oct 1.

c Laboratory of Physiology, Faculty of Medicine, School of Health Sciences , Aristotle University of Thessaloniki , Greece , Thessaloniki , Greece.

Crocus sativus L., a dietary herb, has been used for various diseases including cancer. This is an in vitro study investigating the antineoplastic effect of the extract of the plant against C6 glioma rat cell line. The mechanism of cellular death and the synergistic effect of the extract with the alkylating agent temozolomide (TMZ) were investigated. Cellular viability was examined in various concentrations of the extract alone or in combination with TMZ. Apoptosis was determined with flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and autophagy by western blotting of the light chain 3 (LC3)-II. Cellular viability was reduced after exposure to the extract with half maximal inhibition concentration at 3 mg/ml. Flow cytometry and TUNEL assay suggested that the extract does not induce apoptosis. Moreover, their combination increased the ratio dead/apoptotic cells 10-fold (P < 0.001). LC3-II protein levels reduced after Crocus extract while this effect was reversed when the calpain inhibitor MDL28170 was added, suggesting a calpain-dependent death possibly through autophagy. We concluded that the extract of Crocus increases dead cell number after 48 h of exposure. Our results suggest that the cell undergoes calpain-dependent programmed cell death while co-exposure to Crocus extract and TMZ enhances the antineoplastic effect of the latter.
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http://dx.doi.org/10.1080/01635581.2018.1506493DOI Listing
May 2020

Fentanyl and naloxone effects on glutamate and GABA release rates from anterior hypothalamus in freely moving rats.

Eur J Pharmacol 2018 Sep 19;834:169-175. Epub 2018 Jul 19.

Department of Clinical Pharmacology, Faculty of Medicine, School of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.

Fentanyl, a μ-opioid receptor agonist, has been studied for its neuro/psycho-pharmacological effects since its first clinical use; however, its effect on the release rate of the Central Nervous System (CNS) neurotransmitters has not been yet elucidated. In the present study the influence of fentanyl on the release rates of glutamate and GABA is investigated. Specifically, we examined the effects of intravenous (10 μg/kg) as well as intrahypothalamic (0.1nmol/min) fentanyl administration on the release rates of GABA and glutamate in the superfusate of anterior hypothalamus, under tail pinch manipulation. The release rate of the neurotransmitters was monitored by the push-pull superfusion technique. To investigate the role of fentanyl the opioid antagonist, naloxone 0.1 mg/kg was administered intravenously, or 50nmol/min intrahypothalamicaly. The amino acids were determined by High Performance Liquid Chromatography (HPLC) and fluorimetric detection after NBD-Cl derivatisation. After intravenous fentanyl administration a significant decrease of glutamate and increase of GABA release rates were observed. However during the pain manipulations, the release rate of glutamate was increased. Intravenous naloxone did not affect significantly the release rates of both amino acids, while intrahypothalamic antagonist administration reversed the alterations in both neurotransmitters release rates. Our results demonstrate that there is an opioid-glutamatergic transmission pathway, located in hypothalamus and that opioids can activate NMDA receptors, thus reducing the nociceptive threshold and the opioid analgesic effect.
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http://dx.doi.org/10.1016/j.ejphar.2018.07.029DOI Listing
September 2018

On PC12 oxygen glucose deprivation and cell death.

Biomed Pharmacother 2018 08 20;104:849-850. Epub 2018 Mar 20.

Laboratory of Physiology, Department of Physiology and Pharmacology, Faculty of Medicine, School of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), University Campus, 54124 Thessaloniki, Greece. Electronic address:

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http://dx.doi.org/10.1016/j.biopha.2018.03.035DOI Listing
August 2018

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

Stem Cell Res Ther 2017 Nov 2;8(1):247. Epub 2017 Nov 2.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School (MHH), Hannover, Germany.

Background: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion.

Methods: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR.

Results: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions.

Conclusions: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.
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http://dx.doi.org/10.1186/s13287-017-0705-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667471PMC
November 2017

Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells.

Cell Physiol Biochem 2015 13;37(5):1750-8. Epub 2015 Nov 13.

Background/aims: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis.

Methods: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level.

Results: Administration of glutamate in PC12 cells in doses as low as 10 μM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3.

Conclusion: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.
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http://dx.doi.org/10.1159/000430250DOI Listing
August 2016

Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model.

Cell Mol Neurobiol 2016 Jul 4;36(5):701-12. Epub 2015 Aug 4.

Laboratory of Physiology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece.

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen-glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.
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http://dx.doi.org/10.1007/s10571-015-0250-2DOI Listing
July 2016

Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments.

Stem Cells Dev 2015 Nov 2;24(21):2496-512. Epub 2015 Sep 2.

5 Laboratory of Pharmacology, School of Pharmaceutical Sciences, Aristotle University of Thessaloniki (A.U.TH.) , Thessaloniki, Greece .

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro- and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro- (angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.
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http://dx.doi.org/10.1089/scd.2015.0197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620528PMC
November 2015

Researching glutamate - induced cytotoxicity in different cell lines: a comparative/collective analysis/study.

Front Cell Neurosci 2015 17;9:91. Epub 2015 Mar 17.

Laboratory of Physiology, Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki Greece.

Although glutamate is one of the most important excitatory neurotransmitters of the central nervous system, its excessive extracellular concentration leads to uncontrolled continuous depolarization of neurons, a toxic process called, excitotoxicity. In excitotoxicity glutamate triggers the rise of intracellular Ca(2+) levels, followed by up regulation of nNOS, dysfunction of mitochondria, ROS production, ER stress, and release of lysosomal enzymes. Excessive calcium concentration is the key mediator of glutamate toxicity through over activation of ionotropic and metabotropic receptors. In addition, glutamate accumulation can also inhibit cystine (CySS) uptake by reversing the action of the CySS/glutamate antiporter. Reversal of the antiporter action reinforces the aforementioned events by depleting neurons of cysteine and eventually glutathione's reducing potential. Various cell lines have been employed in the pursuit to understand the mechanism(s) by which excitotoxicity affects the cells leading them ultimately to their demise. In some cell lines glutamate toxicity is exerted mainly through over activation of NMDA, AMPA, or kainate receptors whereas in other cell lines lacking such receptors, the toxicity is due to glutamate induced oxidative stress. However, in the greatest majority of the cell lines ionotropic glutamate receptors are present, co-existing to CySS/glutamate antiporters and metabotropic glutamate receptors, supporting the assumption that excitotoxicity effect in these cells is accumulative. Different cell lines differ in their responses when exposed to glutamate. In this review article the responses of PC12, SH-SY5Y, HT-22, NT-2, OLCs, C6, primary rat cortical neurons, RGC-5, and SCN2.2 cell systems are systematically collected and analyzed.
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http://dx.doi.org/10.3389/fncel.2015.00091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362409PMC
April 2015
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