Anna Theocharidou

Sol-Gel Derived Mg-Based Ceramic Scaffolds Doped with Zinc or Copper Ions: Preliminary Results on Their Synthesis, Characterization, and Biocompatibility

Glass-ceramic scaffolds containing Mg have shown recently the potential to enhance the proliferation, differentiation, and biomineralization of stem cells in vitro, property that makes them promising candidates for dental tissue regeneration. An additional property of a scaffold aimed at dental tissue regeneration is to protect the regeneration process against oral bacteria penetration. In this respect, novel bioactive scaffolds containing Mg2+ and Cu2+ or Zn2+, ions known for their antimicrobial properties, were synthesized by the foam replica technique and tested regarding their bioactive response in SBF, mechanical properties, degradation, and porosity. Finally their ability to support the attachment and long-term proliferation of Dental Pulp Stem Cells (DPSCs) was also evaluated. The results showed that conversely to their bioactive response in SBF solution, Zn-doped scaffolds proved to respond adequately regarding their mechanical strength and to be efficient regarding their biological response, in comparison to Cu-doped scaffolds, which makes them promising candidates for targeted dental stem cell odontogenic differentiation and calcified dental tissue engineering.

Human treated dentin matrices combined with Zn-doped, Mg-based bioceramic scaffolds and human dental pulp stem cells towards targeted dentin regeneration.

OBJECTIVE This study aimed to investigate the potential of Mg-based bioceramic scaffolds combined with human treated-dentin matrices (hTDMs) and dentinogenesis-related morphogens to promote odontogenic differentiation and dentin-like tissue formation by Dental Pulp Stem Cells-DPSCs. METHODS DPSC cultures were established and characterized by flow cytometry. Experimental cavities were prepared inside crowns of extracted teeth and demineralized by EDTA (hTDMs). Zn-doped, Mg-based bioceramic scaffolds, synthesized by the sol-gel technique, were hosted inside the hTDMs. DPSCs were spotted inside the hTDMs/scaffold constructs with/without additional exposure to DMP-1 or BMP-2 (100ng/ml, 24h). Scanning Electron Microscopy-SEM, live/dead fluorescence staining and MTT assay were used to evaluate cell attachment and viability; Real time PCR for expression of osteo/odontogenic markers; Inductively Coupled Plasma-Atomic Emission Spectrometry-ICP/AES for scaffold elemental release analysis; ELISA for hTDM growth factor release analysis; SEM and X-ray Diffraction-XRD for structural/chemical characterization of the regenerated tissues. RESULTS Scaffolds constantly released low concentrations of Mg(2+), Ca(2+), Zn(2+) and Si(4+), while hTDMs growth factors, like DMP-1, BMP-2 and TGFβ-1. hTDMs/scaffold constructs supported DPSC viability, inducing their rapid odontogenic shift, indicated by upregulation of DSPP, BMP-2, osteocalcin and osterix expression. Newly-formed Ca-P tissue overspread the scaffolds partially transforming into bioapatite. Exposure to DMP-1 or BMP-2 pronouncedly enhanced odontogenic differentiation phenomena. SIGNIFICANCE This is the first study to validate that combining the bioactivity and ion releasing properties of bioceramic materials with growth factor release by treated natural dentin further supported by exogenous addition of key dentinogenesis-related morphogens (DMP-1, BMP-2) can be a promising strategy for targeted dentin regeneration.

Effective Cell Growth Potential of Mg-Based Bioceramic Scaffolds towards Targeted Dentin Regeneration

SUMMARY New emerging approaches in tissue engineering include incorporation of metal ions involved in various metabolic processes, such as Cu, Zn, Si into bioceramic scaffolds for enhanced cell growth and differentiation of specific cell types. The aim of the present work was to investigate the attachment, morphology, growth and mineralized tissue formation potential of Dental Pulp Stem Cells (DPSCs) seeded into Mg-based glassceramic scaffolds with incorporated Zn and Cu ions. Bioceramic scaffolds containing Si 60%, Ca 30%, Mg 7.5% and either Zn or Cu 2.5%, sintered at different temperatures were synthesized by the foam replica technique and seeded with DPSCs for up to 21 days. Scanning Electron Microscopy with associated Energy Dispersive Spectroscopy (SEM-EDS) was used to evaluate their ability to support the DPSCs’s attachment and proliferation, while the structure of the seeded scaffolds was investigated by X-Ray Diffraction Analysis (XRD). Zn-doped bioceramic scaffolds promoted the attachment and growth of human DPSCs, while identically fabricated scaffolds doped with Cu showed a cytotoxic behaviour, irrespective of the sintering temperature. A mineralized tissue with apatite-like structure was formed on both Cu-doped scaffolds and only on those Zn-doped scaffolds heat-treated at lower temperatures. Sol-gel derived Zn-doped scaffolds sintered at 890oC support DPSC growth and apatite-like tissue formation, which renders them as promising candidates towards dental tissue regeneration.

Magnesium Based Sol-Gel Derived Bioactive Glass Ceramics for Dental Tissue Regeneration

Scaffold-based tooth engineering is currently the most popular approach towards replacing dental tissues or even engineering a bio-tooth. Although, various scaffold materials have been employed in tooth regeneration, the scaffold-based tooth design has, until now, achieved only limited success. Recently, bioactive Mg-based ceramics have attracted interest as Mg plays an important role on skeletal metabolism and affects the quality and structure of hard dental tissues. Mg has been reported to improve the mechanical properties of calcium phosphate ceramics, control biodegradation rate and stabilize the cell-material interface improving cell attachment and growth. The aim of this study was the development of an experimental Mg-based ceramic material, with enhanced bioactivity and adequate mechanical properties, in order to be potentially used in dental tissue regeneration. The Mg-based ceramic was prepared by the sol-gel method, while the stabilization was performed at 1300, 1400 and 1450oC in order a fully crystalline material to be obtained. The characterization of the materials -before and after immersion is Simulated Body Fluid (SBF)- was performed by Fourier Tranform Infrared Spectroscopy (FTIR), X-Ray Diffractometry (XRD) and Scanning Electron Microscopy associated with an EDS analyzer (SEM-EDS), while the flexural strength of uniaxially pressed pellets was measured using a universal testing machine for 3- point bending tests (Instron 3344). FTIR spectra and XRD patterns of all powder samples before immersion in SBF solution confirmed the presence of three crystalline phases; akermanite, merwinite and diopside. The onset of apatite formation on the surface of all powders was observed even after three days of immersion, while the apatite formation on the surface of the sintered pellets was slightly delayed. Flexural strength values were in the range of 30Mpa. In conclusion, Mg-based glass-ceramics attain adequate mechanical integrity and high rate of bioactivity and could be potentially used in the construction of ceramic scaffolds for dental tissue regeneration.

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

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.

SEM Observation of Composite Ceramic Scaffolds’ Surface during Incubation in Culture Medium with or without Human PDL Fibroblasts

Chitin is a polysaccharide abundant in nature. Its’ deacetylation product-chitosan- in combination with gelatin (collagen product) is commonly used asbiopolymer scaffold for tissue engineering. The aim of this study was to investigate diffrerences in surface characteristics of chitin (CHN CCS) and chitosan –gelatin (CHS-G CCS) composite ceramic scaffolds (CCS), during their incubation in culture medium (DMEM) with or without human periodontal ligament fibroblasts (HPDLF). CHN CCS and CHS- G CCS, with pore size 70-200μm, were fabricated on the surface of ceramic disks, being coated with a mixture of bioactive glass – ceramic (1:1 wt). Three CCSs of each type were constructed. Each CCS was incubated at 37 °C up to 10 days, either only in DMEM supplemented with 10% FCS or in DMEM with the presence of 105 HPDLF. SEM microphotographs and EDS analysis, before and after incubation, were used to investigate CCSs’ surface alterations. Before incubation, all type of CCSs appeared to be macro porous with high interconnectivity. Exposed to incubation, CHN CCSs’ surface porosity seemed to be rapidly reduced and a rough surface without pores was observed with or without HPDLF. Attached HPDLF were rarely detected. CHS-G CCSs appeared to retain surface porosity in DMEM without cells. In HPDLF culture an almost uniform surface with organic aggregates and attached cells was observed. Until day 10, HPDLF could only be detected at CHS-G CCS’s surface. Conclusion: SEM microphotographs observations indicate that CHN CCSs’ incubation in DMEM led in early and rapid coalescence of surface pores, thus inhibiting HPDLF attachment. HPDLF attachment on CHS-G CCSs confirm the beneficial role of gelatin, while differences in CHS-G CCSs’ surface with and without HPDLF culture indicate that not only sedimentation of mediums ingredients, but cell attachment and function could decrease surface’s porosity, affecting consequently HPDLF proliferation.