Publications by authors named "Athina Bakopoulou"

48 Publications

A finite element model of an osteoblast to quantify the transduction of exogenous forces to cellular components.

Med Eng Phys 2021 Aug 3;94:61-69. Epub 2021 Jul 3.

School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki GR-54124, Thessaloniki, Greece; Division of Postgraduate Prosthodontics, Tufts University School of Dental Medicine, Boston, MA, 02111, USA. Electronic address:

Encouraged by recent advances of biophysical and biochemical assays we introduce a 3D finite element model of an osteoblast, seeking an analogue between exogenous forces and intracellularly activated sensory mechanisms. The cell was reverse engineered and the dimensions of the internal cellular structures were based on literature data. The model was verified and validated against atomic force microscopy experiments and four loading scenarios were considered. The stress distributions developing on the main cellular components were calculated along with their corresponding strain values. The nucleus and mitochondria exhibited similar loading trends, with the mitochondria being stressed by an order of magnitude higher than the nucleus (e.g. 1.4 vs. 0.16 MPa). Equivalent stiffness was determined to increase by almost 50%, from the apex to the cell's periphery, as was the cell's elasticity, which was lowest when the load was exerted directly above the nucleus. The assessment of how extrinsic loads are propagated to a cell's internal structures is inherently a problem of high complexity. The findings presented in this study can provide important insight into biophysical and biochemical responses elicited in cells through mechanical stimulus. This was evident in both the nuclear and mitochondrial loading and would stipulate the important contribution of even more accurate models in the interpretation of cellular events. One Sentence Summary: The results of this numerical biomechanical study demonstrated that even minor extrinsic loads irrespective of the application site, are transduced by a fraction of the cytoskeleton to its internal structure (primarily to its mitochondria and secondary to the cell's nucleus), indicating mechanical stimulus as the dominant pathway to cell expression.
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http://dx.doi.org/10.1016/j.medengphy.2021.06.010DOI Listing
August 2021

Biophysical interactions between components of the tumor microenvironment promote metastasis.

Biophys Rev 2021 Jun 4;13(3):339-357. Epub 2021 Jun 4.

Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece.

During metastasis, tumor cells need to adapt to their dynamic microenvironment and modify their mechanical properties in response to both chemical and mechanical stimulation. Physical interactions occur between cancer cells and the surrounding matrix including cell movements and cell shape alterations through the process of mechanotransduction. The latter describes the translation of external mechanical cues into intracellular biochemical signaling. Reorganization of both the cytoskeleton and the extracellular matrix (ECM) plays a critical role in these spreading steps. Migrating tumor cells show increased motility in order to cross the tumor microenvironment, migrate through ECM and reach the bloodstream to the metastatic site. There are specific factors affecting these processes, as well as the survival of circulating tumor cells (CTC) in the blood flow until they finally invade the secondary tissue to form metastasis. This review aims to study the mechanisms of metastasis from a biomechanical perspective and investigate cell migration, with a focus on the alterations in the cytoskeleton through this journey and the effect of biologic fluids on metastasis. Understanding of the biophysical mechanisms that promote tumor metastasis may contribute successful therapeutic approaches in the fight against cancer.
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http://dx.doi.org/10.1007/s12551-021-00811-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214652PMC
June 2021

A tissue-engineered biocomplex for periodontal reconstruction. A proof-of-principle randomized clinical study.

J Clin Periodontol 2021 Aug 14;48(8):1111-1125. Epub 2021 May 14.

Department of Preventive Dentistry, Periodontology and Implant Biology, School of Dentistry, Faculty of Health Sciences (FHS), Aristotle University of Thessaloniki (AUTh), Thessaloniki, Greece.

Aim: To assess the safety/efficacy of a tissue-engineered biocomplex in periodontal reconstruction.

Methods: Twenty-seven intrabony defects were block-randomized across three treatment groups: Group-A (N  = 9) received autologous clinical-grade alveolar bone marrow mesenchymal stem cells (a-BMMSCs), seeded into collagen scaffolds, enriched with autologous fibrin/platelet lysate (aFPL). In Group-B (N  = 10), the collagen scaffold/aFPL devoid of a-BMMSCs filled the osseous defect. Group-C (N  = 8) received Minimal Access Flap surgery retaining the soft tissue wall of defects identically with Groups-A/-B. Subjects were clinically/radiographically assessed before anaesthesia (baseline) and repeatedly over 12 months.

Results: Quality controls were satisfied before biocomplex transplantation. There were no adverse healing events. All approaches led to significant clinical improvements (p < .001) with no inter-group differences. At 12 months, the estimated marginal means for all groups were as follows: 3.0 (95% CI: 1.9-4.1) mm for attachment gain; 3.7 (2.7-4.8) mm for probing pocket depth reduction; 0.7 (0.2-1.3) mm increase in recession. An overall greater mean reduction in the radiographic Cemento-Enamel Junction to Bottom Defect (CEJ-BD) distance was found for Groups-A/-C over Group-B (p < .023).

Conclusion: Radiographic evidence of bone fill was less pronounced in Group-B, although clinical improvements were similar across groups. All approaches aimed to trigger the innate healing potential of tissues. Cell-based therapy is justified for periodontal reconstruction and remains promising in selected cases.
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http://dx.doi.org/10.1111/jcpe.13474DOI Listing
August 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

Biocompatibility assessment of resin-based cements on vascularized dentin/pulp tissue-engineered analogues.

Dent Mater 2021 05 7;37(5):914-927. Epub 2021 Mar 7.

Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (AUTh), Thessaloniki, GR-54124, Greece. Electronic address:

Objectives: A three-dimensional (3D) dentin/pulp tissue analogue, resembling the human natural tissue has been engineered in an in vitro setup, aiming to assess the cytocompatibility of resin-based dental restorative cements.

Methods: Stem Cells from Apical Papilla (SCAP) and Human Umbilical Vein Endothelial Cells (HUVEC) were embedded in Collagen-I/Fibrin hydrogels at 1:3 ratio within 24-well plates. Hanging culture inserts were placed over the hydrogels, housing an odontoblast-like cell layer and a human treated-dentin barrier. Shear modulus of the hydrogels at 3.5 and 5 mg/ml was evaluated by dynamic mechanical analysis. Eluates of two resin-based cements, a dual-cure- (Breeze™, Pentron: Cement-1/C1), and a self-adhesive cement (SpeedCEMplus™, Ivoclar-Vivadent: Cement-2/C2) were applied into the dentin/pulp tissue analogue after pre-stimulation with LPS. Cytocompatibility was assessed by MTT assay, live/dead staining and real-time PCR analysis.

Results: Both hydrogel concentrations showed similar shear moduli to the natural pulp until day (D) 7, while the 5 mg/ml-hydrogel substantially increased stiffness by D14. Both cements caused no significant toxicity to the dentin/pulp tissue analogue. C1 induced stimulation (p < 0.01) of cell viability (158 ± 3%, 72 h), while pre-stimulation with LPS attenuated this effect. C2 (±LPS) caused minor reduction of viability (15-20%, 24 h) that recovered at 72 h for the LPS+ group. Both cements caused upregulation of VEGF, ANGP-1, and downregulation of the respective receptors VEGFR-2 and Tie-1.

Significance: Both resin-based cements showed good cytocompatibility and triggered angiogenic response within the dentin/pulp tissue analogue, indicating initiation of pulp repair responses to the released xenobiotics.
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http://dx.doi.org/10.1016/j.dental.2021.02.019DOI Listing
May 2021

Evaluation of stemness properties of cells derived from granulation tissue of peri-implantitis lesions.

Clin Exp Dent Res 2021 Feb 18. Epub 2021 Feb 18.

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

Objectives: Peri-implantitis (PI) is an inflammatory disease associated with peri-implant bone loss and impaired healing potential. There is limited evidence about the presence of mesenchymal stromal cells (MSCs) and their regenerative properties within the granulation tissue (GT) of infrabony peri-implantitis defects. The aim of the present study was to characterize the cells derived from the GT of infrabony PI lesions (peri-implantitis derived mesenchymal stromal cells-PIMSCs).

Material And Methods: PIMSC cultures were established from GT harvested from PI lesions with a pocket probing depth ≥6 mm, bleeding on probing/suppuration, and radiographic evidence of an infrabony component from four systemically healthy individuals. Cultures were analyzed for embryonic (SSEA4, NANOG, SOX2, OCT4A), mesenchymal (CD90, CD73, CD105, CD146, STRO1) and hematopoietic (CD34, CD45) stem cell markers using flow cytometry. PIMSC cultures were induced for neurogenic, angiogenic and osteogenic differentiation by respective media. Cultures were analyzed for morphological changes and mineralization potential (Alizarin Red S method). Gene expression of neurogenic (NEFL, NCAM1, TUBB3, ENO2), angiogenic (VEGFR1, VEGFR2, PECAM1) and osteogenic (ALPL, BGLAP, BMP2, RUNX2) markers was determined by quantitative RT-PCR.

Results: PIMSC cultures demonstrated high expression of embryonic and mesenchymal stem cell markers with inter-individual variability. After exposure to neurogenic, angiogenic and osteogenic conditions, PIMSCs showed pronounced tri-lineage differentiation potential, as evidenced by their morphology and expression of respective markers. High mineralization potential was observed.

Conclusions: This study provides evidence that MSC-like populations reside within the GT of PI lesions and exhibit a multilineage differentiation potential. Further studies are needed to specify the biological role of these cells in the healing processes of inflamed PI tissues and to provide indications for their potential use in regenerative therapies.
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http://dx.doi.org/10.1002/cre2.406DOI Listing
February 2021

Influence of 2-hydroxyethyl methacrylate (HEMA) exposure on angiogenic differentiation of dental pulp stem cells (DPSCs).

Dent Mater 2021 03 10;37(3):534-546. Epub 2021 Feb 10.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, D-30625 Hannover, Germany. Electronic address:

Objective: The angiogenic differentiation of dental pulp stem cells (DPSCs) is important for tissue homeostasis and wound healing. In this study the influence of 2-hydroxyethyl methacrylate (HEMA) on angiogenic differentiation was investigated.

Methods: To evaluate HEMA effects on angiogenic differentiation, DPSCs were cultivated in angiogenic differentiation medium (ADM) in the presence or absence of non-toxic HEMA concentrations (0.1 mM and 0.5 mM). Subsequently, angiogenic differentiation was analyzed on the molecular level by qRT-PCR and protein profiler analyzes of angiogenic markers and flow cytometry of PECAM1. The influence of HEMA on angiogenic phenotypes was analyzed by cell migration and sprouting assays.

Results: Treatment with 0.5 mM HEMA during differentiation can lead to a slight reduction of angiogenic markers on mRNA level. HEMA also seems to slightly reduce the quantity of angiogenic cytokines (not significant). However, these HEMA concentrations have no detectable influence on cell migration, the abundance of PECAM1 and the formation of capillaries. Higher concentrations caused primary cytotoxic effects in angiogenic differentiation experiments conducted for longer periods than 72 h.

Significance: Non-cytotoxic HEMA concentrations seem to have a minor impact on the expression of angiogenic markers, essentially on the mRNA level, without affecting the angiogenic differentiation process itself on a detectable level.
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http://dx.doi.org/10.1016/j.dental.2020.12.008DOI 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

Advanced in Vitro Experimental Models for Tissue Engineering-based Reconstruction of a 3D Dentin/pulp Complex: a Literature Review.

Stem Cell Rev Rep 2021 Jun 3;17(3):785-802. Epub 2020 Nov 3.

Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), GR-54124, Thessaloniki, Greece.

Objective: Experimental procedures have been used to monitor cellular responses at the dentin/pulp interface. Aiming to divert from in vivo studies and oversimplified two-dimensional assays, three-dimensional (3D) models have been developed. This review provides an overview of existing literature, regarding 3D in vitro dentin/pulp reconstruction.

Material & Methods: PubMed, Scopus, Cochrane Library and Web of Science- were systematically searched for attributes between 1998 and 2020. The search focused on articles on the development of three-dimensional tools for the reconstruction of a dentin/pulp complex under in vitro conditions, which were then screened and qualitatively assessed. Article grouping according to mode of implementation, resulted in five categories: the customised cell perfusion chamber (CPC) (n = 8), the tooth bud model (TBM) (n = 3), the 3D dentin/pulp complex manufactured by tissue engineering (DPC) (n = 6), the entire tooth culture (ETC) (n = 4) and the tooth slice culture model (TSC) (n = 5).

Results: A total of 26 publications, applying nine and eight substances for pulp and dentin representation respectively, were included. Natural materials and dentin components were the most widely utilized. The most diverse category was the DPC, while the CPC group was the test with the highest longevity. The most consistent categories were the ETC and TSC models, while the TBM presented as the most complete de novo approach.

Conclusions: All studies presented with experimental protocols with potential upgrades. Solving the limitations of each category will provide a complete in vitro testing and monitoring tool of dental responses to exogenous inputs.

Clinical Relevance: The 3D dentin/pulp complexes are valid supplementary tools for in vivo studies and clinical testing. Graphical Abstract.
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http://dx.doi.org/10.1007/s12015-020-10069-8DOI Listing
June 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

Prospects of Advanced Therapy Medicinal Products-Based Therapies in Regenerative Dentistry: Current Status, Comparison with Global Trends in Medicine, and Future Perspectives.

J Endod 2020 Sep;46(9S):S175-S188

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

Introduction: Regenerative medicine offers innovative approaches to restore damaged tissues on the basis of tissue engineering (TE). Although research on advanced therapy medicinal products (ATMPs) has been very active in recent years, the number of licensed products remains surprisingly low and restricted to the treatment of severe, incurable diseases.

Methods: This paper provides a critical review of current literature on the regulatory, clinical, and commercial status of ATMP-based therapies in the EU and worldwide and the hurdles to overcome for their broader application in Regenerative Dentistry.

Results: Competent authorities have focused on developing regulatory pathways to address unmet patient needs. Oncology represents the dominating field, followed by cardiovascular, musculoskeletal, neurodegenerative, immunologic, and inherited diseases. Yet, the status remains in early development, and scientific, regulatory, and cost-effectiveness issues impose considerable hurdles toward marketing authorization, technology adoption, and patient accessibility. In this context, although regenerative dentistry has achieved breakthrough innovations in TE of several dental/oral tissues in preclinical models, it has hardly harnessed research progress to integrate innovative regenerative treatments into clinical practice.

Conclusion: Global demographic changes, which demonstrate a steady increase of the aging population, highlight the societal need for the application of ATMP-based therapies in the treatment of noncommunicable diseases (NCDs). Although oral diseases, as an integral part of NCDs, are not life-threatening and largely preventable, they sustain high prevalence, with severe burden on economy and quality of life. In this perspective, the urgent request to ultimately translate draining research in dental TE conducted during the last decades into innovative treatments brought safely and cost-effectively into society at large still holds the stage. This review provides an overview of the regulatory, clinical, and commercial status of ATMP-based therapies in the European Union and worldwide and the hurdles to overcome for their broader application in regenerative dentistry.
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http://dx.doi.org/10.1016/j.joen.2020.06.026DOI Listing
September 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

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

Salivary Gland Stem Cells and Tissue Regeneration: An Update on Possible Therapeutic Application.

J Contemp Dent Pract 2019 Aug 1;20(8):978-986. Epub 2019 Aug 1.

Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece.

The aim of this review is to combine literature and experimental data concerning the impact of salivary gland (SG) stem cells (SCs) and their therapeutic prospects in tissue regeneration. So far, SCs were isolated from human and rodent major and minor SGs that enabled their regeneration. Several scaffolds were also combined with "SCs" and different "proteins" to achieve guided differentiation, although none have been proven as ideal. A new aspect of SC therapy aims to establish a relationship between SG and other ecto- or endodermal organs such as the pancreas, liver, kidneys, and thyroid. SC therapy could be a cheap and simple, non-traumatic, and individualized therapy for medically challenging cases like xerostomia and major organ failures. Functional improvement has been achieved in these organs, but till date, the whole organ regeneration was not achieved. Concerns about malignant formations and possible failures are yet to be resolved. In this review article, we highlight the basic embryology of SGs, existence of SG SCs with a detailed exploration of various cellular markers, scaffolds for tissue engineering, and, in the later part, cover potential therapeutic applications with a special focus on the pancreas and liver. Keywords: Salivary gland stem cells, Stem cell therapy, Tissue regeneration.
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August 2019

Three-dimensional tissue engineering-based Dentin/Pulp tissue analogue as advanced biocompatibility evaluation tool of dental restorative materials.

Dent Mater 2020 02 30;36(2):229-248. Epub 2019 Nov 30.

Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), GR-54124 Thessaloniki, Greece. Electronic address:

Objective: Two-dimensional (2D) in vitro models have been extensively utilized for cytotoxicity assessment of dental materials, but with certain limitations in terms of direct in vitro-in vivo extrapolation (IVIVE). Three-dimensional (3D) models seem more appropriate, recapitulating the structure of human tissues. This study established a 3D dentin/pulp analogue, as advanced cytotoxicity assessment tool of dental restorative materials (DentCytoTool).

Methods: DentCytoTool comprised two compartments: the upper, representing the dentin component, with a layer of odontoblast-like cells expanded on microporous membrane of a cell culture insert and covered by a treated dentin matrix; and the lower, representing a pulp analogue, incorporating HUVEC/SCAP co-cultures into collagen I/fibrin hydrogels. Representative resinous monomers (HEMA: 1-8mM; TEGDMA: 0.5-5mM) and bacterial components (LPS: 1μg/ml) were applied into the construct. Cytotoxicity was assessed by MTT and LDH assays, live/dead staining and real-time PCR for odontogenesis- and angiogenesis-related markers.

Results: DentCytoTool supported cell viability and promoted capillary-like network formation inside the pulp analogue. LPS induced expression of odontogenesis-related markers (RUNX2, ALP, DSPP) without compromising viability of the odontoblast-like cells, while co-treatment with LPS and resin monomers induced cytotoxic effects (live/dead staining, MTT and LDH assays) in cells of both upper and lower compartments and reduced expression angiogenesis-related markers (VEGF, VEGFR2, ANGPT-1, Tie-2, PECAM-1) in a concentration- and time- dependent manner. LPS treatment aggravated TEGDMA-induced and -in certain concentrations (2-4mM)- HEMA-induced cytotoxicity.

Significance: DentCytoTool represents a promising tissue-engineering-based cytotoxicity assessment tool, providing more insight into the mechanistic aspects of interactions of dental materials to the dentin/pulp complex.
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http://dx.doi.org/10.1016/j.dental.2019.11.013DOI Listing
February 2020

Variables affecting the fit of zirconia fixed partial dentures: A systematic review.

J Prosthet Dent 2020 May 5;123(5):686-692.e8. Epub 2019 Nov 5.

Professor and Chairman, Department of Prosthodontics, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.

Statement Of Problem: Different parameters affect the marginal and internal fit of zirconia fixed partial dentures (FPDs) on natural teeth. Determining a way to optimize these restorations is essential.

Purpose: The purpose of this systematic review was to determine the variables affecting the marginal fit and internal accuracy of zirconia FPDs on natural teeth.

Material And Methods: An electronic search was conducted by 2 independent reviewers by using the MEDLINE, Cochrane, Web of Science, and Scopus databases, as well as Google Scholar, for studies published up to July 2018, and a manual search was conducted from the reference lists of related articles. Eligibility criteria included articles in English published in peer-reviewed journals that assessed the marginal and/or internal adaptation of zirconia FPDs on teeth with 3 or more units, with at least 1 of the experimental groups being frameworks or FPDs fabricated from zirconia. Risk of bias was assessed with the aid of the Cochrane Handbook for Systematic Reviews of Interventions.

Results: The search provided 418 records, with 41 fulfilling the inclusion criteria. The selected studies showed considerable heterogeneity regarding materials, state of sintering, manufacturer and computer-aided design and computer-aided manufacturing (CAD-CAM) system, experimental methodology, sample size, and span length. Of the included articles, 36 were in vitro studies and 5 were clinical studies, most of which exhibited high-performance and detection biases. CAD-CAM systems provided more precise marginal and internal fit than CAM systems. An increase of framework span length to 6 or more units decreased both marginal and internal fit. The reported marginal gap tended to increase after the veneering process. The introduction of a conventional impression into an otherwise digital workflow seems to have a negative effect on the marginal fit.

Conclusions: The accuracy of zirconia FPDs or frameworks is considerably influenced by the processing procedure used and the choice of CAD-CAM system.
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http://dx.doi.org/10.1016/j.prosdent.2019.06.019DOI Listing
May 2020

Multiphoton 3D Printing of Biopolymer-Based Hydrogels.

ACS Biomater Sci Eng 2019 Nov 25;5(11):6161-6170. Epub 2019 Oct 25.

Institute of Electronic Structure and Laser, Foundation for Research and Technology-Hellas, 70013 Heraklion, Crete, Greece.

Multiphoton lithography, based on multiphoton polymerization, is a powerful technique for the fabrication of complex three-dimensional (3D) structures. Herein, we report on the photostructuring of novel biopolymer-based hybrid hydrogels, comprising gelatin methacrylamide and a water-soluble chitosan derivative, via multiphoton polymerization. The nontoxic, Food and Drug Administration-approved, biocompatible photosensitizer eosin Y was exploited as the sole photoinitiator, without the coinitiators and/or comonomer that are commonly used, allowing for further expansion of the available wavelengths up to 800 nm. Importantly, the obtained hybrid material exhibits excellent biocompatibility, evidenced by the increased proliferation of dental pulp stem cells, compared with the individual components and the polystyrene control, after 7 days in culture. Additionally, the 3D hybrid scaffolds promote the matrix mineralization, following their functionalization with bone morphogenetic protein 2. These tailor-made synthetic, biocompatible materials pave the way for further opportunities in 3D scaffold fabrication, including in situ and in vivo biofabrication.
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http://dx.doi.org/10.1021/acsbiomaterials.9b01300DOI Listing
November 2019

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

Assessment of cytotoxicity and antibacterial effects of silver nanoparticle-doped titanium alloy surfaces.

Dent Mater 2019 09 10;35(9):e220-e233. Epub 2019 Jul 10.

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

Objectives: This study aimed to develop silver nanoparticle (AgNP)-doped TiAlV alloy surfaces and investigate their antibacterial properties against representative periopathogens and potential cytotoxicity on osteoblastic cells.

Methods: AgNPs of different size distributions (5 and 30nm) were incorporated onto the TiAlV surfaces by electrochemical deposition, using colloid silver dispersions with increasing AgNP concentrations (100, 200 and 300ppm). The time-course silver release from the specimen surfaces to cell culture media was assessed by Atomic Absorption Spectroscopy (AAS). Cell attachment, viability and proliferation were investigated by SEM, live/dead staining MTT and BrdU assays. The antibacterial effects were assessed against P. gingivalis and P. intermedia by serial dilution spotting assays.

Results: A time- and concentration-dependent silver release from the experimental surfaces was observed. Overall, cell viability and attachment on the AgNP-doped surfaces, suggested adequate cytocompatibility at all concentrations. A transient cytotoxic effect was detected at 24h for the 5nm-sized groups that fully recovered at later time-points, while no cytotoxicity was observed for the 30nm-sized groups. A statistically significant, concentration-dependent decrease in cell proliferation rates was induced at 48h in all AgNP groups, followed by recovery at 72h in the groups coated with 5nm-sized AgNPs. A statistically significant, concentration-dependent antibacterial effect up to 30% was confirmed against both periopathogens.

Significance: This study sheds light to the optimal size-related concentrations of AgNP-doped TiAlV surfaces to achieve antibacterial effects, without subsequent cytotoxicity. These results significantly contribute to the development of antibacterial surfaces for application in oral implantology.
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http://dx.doi.org/10.1016/j.dental.2019.06.003DOI Listing
September 2019

Biological effects of low-level laser irradiation (LLLI) on stem cells from human exfoliated deciduous teeth (SHED).

Clin Oral Investig 2020 Jan 8;24(1):167-180. Epub 2019 May 8.

Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), 541 24, Thessaloniki, Greece.

Objectives: To investigate the effects of low-level laser irradiation (LLLI) on viability/proliferation, migration, osteo/odontogenic differentiation, and in vitro biomineralization of stem cells from human exfoliated deciduous teeth (SHED).

Materials And Methods: SHED cultures were established by enzymatic dissociation from pulps of deciduous teeth. SHED were irradiated with a diode laser (InGaAsP; 940 nm; 0.2 W, continuous mode) at energy fluences 4, 8, and 16 J/cm in the dark, while non-irradiated SHED served as control. Cell viability/proliferation was evaluated by MTT assay and cell mobilization by Transwell™ migration assay. Expression of osteo/odontogenesis-related genes (ALP, BMP-2, BGLAP, DSPP, MSX2, RUNX2) was assessed by real-time PCR, while in vitro biomineralization by Alizarin Red staining. Statistical analysis was performed by two-way ANOVA and Tukey's post hoc tests (*p < 0.05, **p < 0.01).

Results: Statistically significant stimulation of cell viability/proliferation was observed at all energy fluences, reaching the highest effect for the 4 and 16 J/cm. Although the 8 J/cm fluence showed the lowest stimulatory effect on cell viability/proliferation, it was the most effective in inducing SHED migration, upregulation of odontogenesis-related genes (DSPP, ALP, BMP-2) at specific time-points, and the in vitro biomineralization potential of SHED compared to the other two energy fluences.

Conclusions: LLLI proved beneficial in promoting SHED biological processes critical for pulp repair in deciduous teeth. Overall, the 8 J/cm energy fluence showed the most beneficiary effects.

Clinical Relevance: These results provide insights on a narrow "therapeutic window" of LLLI application in vital pulp therapies of deciduous teeth, paving the way for the establishment of effective clinical protocols.
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http://dx.doi.org/10.1007/s00784-019-02874-4DOI Listing
January 2020

Evaluation of the biological behaviour of various dental implant abutment materials on attachment and viability of human gingival fibroblasts.

Dent Mater 2019 07 3;35(7):1053-1063. Epub 2019 May 3.

Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), GR-54124, Thessaloniki, Greece. Electronic address:

Objective: This study aimed to investigate the biological effects of yttria-stabilized zirconia (Y-TZP) compared to other dental implant abutment materials, i.e. lithium disilicate (LS2) and titanium alloy (Ti), as well as the effects of aging of Y-TZP on viability/proliferation and attachment properties of Human Gingival Fibroblasts (HGFs).

Methods: Cylindrical specimens of each material were prepared as per manufacturer's instructions. Y-TZP specimens were divided into three groups: 1. no aging (Zr0), 2. aging for 5 h, 134 °C, 2 bars, 100% humidity (Zr5), 3. aging for 10 h under the same conditions (Zr10). Surface roughness was evaluated by optical profilometry; cell metabolic activity/viability by MTT assay, morphological changes by Scanning Electron Microscopy (SEM) and ratio of live/dead cells by confocal microscopy.

Results: Results showed statistically significant reduction of HGF metabolic activity/viability in contact with Y-TZP after aging. Nevertheless, non-aged zirconia showed no significant differences compared with LS2, Ti and control cultures. In contrast, significant stimulation of cell metabolic activity/viability was observed in HGFs exposed to LS2 eluates. Differential morphological patterns were observed for HGF in contact with different materials/treatments, with obviously increased number of dead cells and sparser distribution of HGFs cultured on Zr10 specimens. These effects were not correlated with surface topography, since Y-TZP aging did not alter surface micro-roughness.

Significance: These findings indicate that Y-TZP shows comparable biological properties to Ti and LS2 as implant abutment material. Nevertheless, Y-TZP aging might influence gingival cell attachment and proliferation properties, providing an alert to a potentially negative effect on the long-term maintenance of gingival architecture.
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http://dx.doi.org/10.1016/j.dental.2019.04.010DOI Listing
July 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

Dental pulp stem cells in chitosan/gelatin scaffolds for enhanced orofacial bone regeneration.

Dent Mater 2019 02 7;35(2):310-327. Epub 2018 Dec 7.

Department of Materials Science and Technology, University of Crete, Heraklion, Greece; Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), Heraklion, Greece. Electronic address:

Objective: Biomimetic chitosan/gelatin (CS/Gel) scaffolds have attracted great interest in tissue engineering of several tissues. However, limited information exists regarding the potential of combining CS/Gel scaffolds with oral cells, such as dental pulp stem cells (DPSCs), to produce customized constructs targeting alveolar/orofacial bone reconstruction, which has been the aim of the present study.

Methods: Two scaffold types, designated as CS/Gel-0.1 and CS/Gel-1, were fabricated using 0.1 and 1% (v/v) respectively of the crosslinker glutaraldehyde (GTA). Scaffolds (n=240) were seeded with DPSCs with/without pre-exposure to recombinant human BMP-2. In vitro assessment included DPSCs characterization (flow cytometry), evaluation of viability/proliferation (live/dead staining, metabolic-based tests), osteo/odontogenic gene expression analysis (qRT-PCR) and structural/chemical characterization (scanning electron microscopy, SEM; energy dispersive X-ray spectroscopy, EDX; X-ray powder diffraction, XRD; thermogravimetry, TG). In vivo assessment included implantation of DPSC-seeded scaffolds in immunocompromised mice, followed by histology and SEM-EDX. Statistical analysis employed one/two-way ANOVA and Tukey's post-hoc tests (significance for p<0.05).

Results: Both scaffolds supported cell viability/proliferation over 14 days in culture, showing extensive formation of a hydroxyapatite-rich nanocrystalline calcium phosphate phase. Differential expression patterns indicated GTA concentration to significantly affect the expression of osteo/odontogenic genes, with CS/Gel-0.1 scaffolds being more effective in upregulating DSPP, IBSP and Osterix. In vivo analysis demonstrated time-dependent production of a nanocrystalline, mineralized matrix at 6, 8 and 10 weeks, being more prominent in constructs bearing rhBMP-2 pre-treated cells. The latter showed higher amounts of osteoid and fully mineralized bone, as well as empty space reduction.

Significance: These results reveal a promising strategy for orofacial bone tissue engineering.
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http://dx.doi.org/10.1016/j.dental.2018.11.025DOI Listing
February 2019

Impaired angiogenic differentiation of dental pulp stem cells during exposure to the resinous monomer triethylene glycol dimethacrylate.

Dent Mater 2019 01 28;35(1):144-155. Epub 2018 Nov 28.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, D-30625 Hannover, Germany. Electronic address:

Objective: Dental pulp stem cells (DPSCs) can differentiate into tissue specific lineages to support dental pulp regeneration after injuries. Triethylene glycol dimethacrylate (TEGDMA) is a widely used co-monomer in restorative dentistry with adverse effects on cellular metabolism. Aim of this study was to analyze the impact of TEGDMA on the angiogenic differentiation potential of DPSCs.

Methods: DPSCs were characterized by flow cytometry. Short-term (max. 72h) cytotoxicity of TEGDMA was assessed by MTT assay. To evaluate TEGDMA effects on angiogenic differentiation, DPSCs were cultivated in angiogenic differentiation medium (ADM) in the presence or absence of short-term non-toxic TEGDMA concentrations (0.1mM and 0.25mM). Subsequently, angiogenic differentiation was analyzed by qRT-PCR analysis of mRNA markers and in vitro spheroid sprouting assays.

Results: DPSCs treated with 0.25mM TEGDMA revealed downregulation of angiogenesis-related marker genes PECAM1 (max. 3.8-fold), VEGF-A (max. 2.4-fold) and FLT1 (max. 2.9-fold) compared to respective untreated control. In addition, a reduction of the sprouting potential of DPSCs cultured in the presence of 0.25mM TEGDMA was detectable. Larger spheroidal structures were detectable in the untreated control in comparison to cells treated with 0.25mM TEGDMA. In contrast, TEGDMA at 0.1mM was not affecting angiogenic potential in the investigated time period (up to 28 days).

Significance: The results of the present study show that TEGDMA concentration dependently impair the angiogenic differentiation potential of DPSCs and may affect wound healing and the formation of granulation tissue.
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http://dx.doi.org/10.1016/j.dental.2018.11.006DOI Listing
January 2019

Biological interactions of a calcium silicate based cement (Biodentine™) with Stem Cells from Human Exfoliated Deciduous teeth.

Dent Mater 2018 12 11;34(12):1797-1813. Epub 2018 Oct 11.

Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, GR-54124, Greece. Electronic address:

Objective: To investigate the biological interactions of a calcium silicate based cement (Biodentine™) with Stem Cells from Human Exfoliated Deciduous teeth (SHED), focusing on viability/proliferation, odontogenic differentiation, biomineralization and elemental release/exchange.

Methods: Biodentine™ specimens were used directly or for eluate preparation at serial dilutions (1:1-1:64). SHED cultures were established from deciduous teeth of healthy children. Viability/proliferation and morphological characteristics were evaluated by live/dead fluorescent staining, MTT assay and Scanning Electron Microscopy. Odontogenic differentiation by qRT-PCR, biomineralization by Alizarin red S staining, while ion elution by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES).

Results: SHED effectively attached within the crystalline surface of Biodentine™ specimens acquiring a spindle-shaped phenotype. Statistically significant stimulation of cell proliferation was induced at day 3 by eluates in dilutions from 1:16 to 1:64. Differential, concentration- and time-dependent expression patterns of odontogenic genes were observed under non-inductive and inductive (osteogenic) conditions, with significant up-regulation of DSPP and Runx2 at higher dilutions and a peak in expression of BMP-2, BGLAP and MSX-2 at 1:8 dilution on day 7. Progressive increase in mineralized tissue formation was observed with increasing dilutions of Biodentine™ eluates. ICP-OES indicated that Biodentine™ absorbed Ca, Mg and P ions from culture medium, while releasing Si and Sr ions from its backbone.

Significance: Biodentine™ interacts through elemental release/uptake with the cellular microenvironment, triggering odontogenic differentiation and biomineralization in a concentration-dependent manner. These results reveal a promising strategy for application of the calcium silicate based cement (Biodentine™) for vital pulp therapies of deciduous teeth in Paediatric Dentistry.
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http://dx.doi.org/10.1016/j.dental.2018.09.014DOI Listing
December 2018

Erythropoietin (rhEPOa) promotes endothelial transdifferentiation of stem cells of the apical papilla (SCAP).

Arch Oral Biol 2018 Dec 3;96:96-103. Epub 2018 Sep 3.

Laboratory of Pharmacology, School of Pharmaceutical Sciences, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, GR-54124, Greece. Electronic address:

Objective: Mesenchymal stem cells (MSCs) have attracted worldwide attention for their capacity to repair damaged tissue, immunosuppression, ability to differentiate into several cell types and their secretome. Earlier studies have demonstrated their angiogenic potential in vitro and in vivo. However, little is known regarding pro-angiogenic inducers of stable endothelial transdifferentiation of MSCs. Here, we employed human MSCs from the Apical Papilla (SCAP) and investigated whether recombinant human erythropoietin-alpha (rhEPOa) could act as such inducer.

Design: Cultured SCAP cells were exposed to rhEPOa and assessed for cell growth kinetics, viability and morphology, as well as their capacity to form capillary tubule structures in selected microenvironments. RT-PCR was used to monitor endothelial markers and activation of EPO/EPOR pathway signaling components; while gelatin zymographies to assess activation of MMP-2.

Results: rhEPOa treatment initially (48 h) accelerated cell proliferation and allowed SCAP to sprout micro-tubular structures. Morphological and biochemical differentiation was accompanied by activation of MMP-2 and upregulation of PECAM-1, VEGFR2, vWF and VE-cadherin/CDH5. SCAP expressed the cognate EPO-R, while rhEPOa-treated SCAP exhibited higher expression of molecules involved in EPO/EPOR pathway (EPOR and JAK2).

Conclusion: rhEPOa is capable of promoting endothelial transdifferentiation of SCAP which may be of clinical value in treating of ischemic disorders.
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http://dx.doi.org/10.1016/j.archoralbio.2018.09.001DOI Listing
December 2018

Fibro/chondrogenic differentiation of dental stem cells into chitosan/alginate scaffolds towards temporomandibular joint disc regeneration.

J Mater Sci Mater Med 2018 Jun 26;29(7):97. Epub 2018 Jun 26.

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

Tissue engineering (TE) may provide effective alternative treatment for challenging temporomandibular joint (TMJ) pathologies associated with disc malpositioning or degeneration and leading to severe masticatory dysfunction. Aim of this study was to evaluate the potential of chitosan/alginate (Ch/Alg) scaffolds to promote fibro/chondrogenic differentiation of dental pulp stem cells (DPSCs) and production of fibrocartilage tissue, serving as a replacement of the natural TMJ disc. Ch/Alg scaffolds were fabricated by crosslinking with CaCl2 combined or not with glutaraldehyde, resulting in two scaffold types that were physicochemically characterized, seeded with DPSCs or human nucleus pulposus cells (hNPCs) used as control and evaluated for cell attachment, viability, and proliferation. The DPSCs/scaffold constructs were incubated for up to 8 weeks and assessed for extracellular matrix production by means of histology, immunofluorescence, and thermomechanical analysis. Both Ch/Alg scaffold types with a mass ratio of 1:1 presented a gel-like structure with interconnected pores. Scaffolds supported cell adhesion and long-term viability/proliferation of DPSCs and hNPCs. DPSCs cultured into Ch/Alg scaffolds demonstrated a significant increase of gene expression of fibrocartilaginous markers (COLI, COL X, SOX9, COM, ACAN) after up to 3 weeks in culture. Dynamic thermomechanical analysis revealed that scaffolds loaded with DPSCs significantly increased storage modulus and elastic response compared to cell-free scaffolds, obtaining values similar to those of native TMJ disc. Histological data and immunochemical staining for aggrecan after 4 to 8 weeks indicated that the scaffolds support abundant fibrocartilaginous tissue formation, thus providing a promising strategy for TMJ disc TE-based replacement.
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http://dx.doi.org/10.1007/s10856-018-6109-6DOI Listing
June 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

Oculo-Dento-Digital Dysplasia (ODDD) Due to a GJA1 Mutation: Report of a Case with Emphasis on Dental Manifestations.

Int J Prosthodont 2017 May/June;30(3):280–285. Epub 2017 Mar 20.

Oculo-dento-digital dysplasia (ODDD) is a congenital disorder manifesting with multiple phenotypic abnormalities involving the face, eyes, teeth, and limbs in addition to neurologic symptomatology. This report aims to present a female patient with ODDD who was referred due to extensive oral restorative needs. The presence of hypoplastic enamel triggered further evaluation. Characteristic facies with hypoplastic alae nasi and syndactyly offered greater insight into the phenotype of the syndrome. Clinical suspicion was confirmed by genetic sequencing revealing heterozygous mutation in GJA1. It is important to be aware of genetic disorders associated with characteristic dental malformations to offer appropriate counseling and treatment.
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http://dx.doi.org/10.11607/ijp.5130DOI Listing
July 2017

Advances and New Technologies towards Clinical Application of Oral Stem Cells and Their Secretome.

Stem Cells Int 2017 24;2017:6367375. Epub 2017 Jan 24.

Aix-Marseille University, CNRS, Institute of Movement Science (ISM), Marseille, France.

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http://dx.doi.org/10.1155/2017/6367375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294372PMC
January 2017
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