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

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 (ß-galactosidase (SA-ß-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.

Assessment of oxygen saturation in dental pulp of permanent teeth with periodontal disease.

INTRODUCTION: In individuals with periodontal disease, dental pulp status should be determined before a treatment plan is made. Pulse oximeters are promising diagnostic tools to evaluate pulp vascularization. This study used pulse oximetry to determine the level of oxygen saturation in dental pulp of intact permanent teeth with periodontal attachment loss (PAL) and gingival recession (GR) and to evaluate the correlation between periodontal disease and level of oxygen saturation in the pulp. METHODS: This study included 67 anterior teeth of 35 patients; all teeth showed intact crowns, PAL, a periodontal pocket (PP), and GR. The teeth underwent periodontal examination, cold and electric pulp testing, and pulse oximetry measurements. The Pearson correlation coefficient and a linear regression coefficient were calculated to evaluate the degree of correlation between periodontal disease markers (PAL, PP, and GR) and the level of oxygen saturation in dental pulp. These tests also evaluated possible associations between oxygen saturation and cold and electric pulp testing. RESULTS: PAL, PP, and GR had negative correlations with oxygen saturation in dental pulp. Conversely, no statistically significant association was found between oxygen saturation in dental pulp and the response to electric sensibility testing. CONCLUSIONS: Oxygen saturation was lower in the pulp of permanent teeth with PAL, PP, and GR, indicating that periodontal disease correlates with the level of oxygen saturation in the pulp.

Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.

Human deciduous teeth have been proposed as a promising source of mesenchymal stem cells for application in bone and dental tissue engineering. We established cultures of mesenchymal stem cells from the pulp of human deciduous teeth (deciduous teeth stem cells, DTSCs) and analyzed their morphologic, growth, immunophenotypic, and osteo/odontogenic differentiation characteristics using different isolation methods and culturing environments. We compared the biologic behavior of DTSCs isolated either by enzymatic dissociation (DTSCs-ED) or by direct outgrowth from pulp tissue explants (DTSCs-OG). We found that different isolation methods give rise to different populations/lineages of cells with respect to their phenotypic and differentiation characteristics. DTSCs-ED cultures comprised heterogeneous cell populations, whereas DTSCs-OG comprised more homogenous spindle-shaped cells. We have characterized DTSCs as STRO-1(+)/CD146(+)/CD34(+)/CD45(-) cells. However, the percentage of STRO-1(+) and CD34(+) cells was higher in DTSCs-ED (STRO-1, 17.01 ± 5.04%; CD34, 19.79 ± 4.66%) compared to DTSCs-OG cultures (STRO-1, 5.18 ± 2.39%; CD34, 9.94 ± 3.41%), probably as a result of a higher release of stem/progenitor cells from the perivascular niche during enzymatic dissociation. DTSCs isolated using either method displayed an active potential for cellular migration and biomineralization, giving rise to 3D mineralized structures when challenged with dexamethasone, monopotassium phosphate, and ß-glycerophosphate. These cellular aggregates progressively expressed differentiation markers of functional odontoblasts, including dentin sialophosphoprotein, bone sialoprotein, osteocalcin, and alkaline phosphatase, having the characteristics of osteodentin. However, in DTSCs-ED, the mineralization rate and the amount of mineralized matrix produced was higher compared to DTSCs-OG cultures. Therefore, DTSCs-ED cells display enhanced biomineralization potential, which might be of advantage for application in clinical therapy.

Tooth slice organ culture and established cell line culture models for cytotoxicity assessment of dental materials.

The aim was to compare the use of different cell-material contact test methods with two different biological systems (cell line and tooth slice cultures) for cytotoxicity assessment of dental materials. Cytotoxicity of composites polymerized with two halogen-based and two light-emitting diode (LED) light-curing units (LCUs) served as the basis for comparison. Disk shaped specimens (7 x 2 mm) were fabricated using the four light sources. Composites were tested using L-929 cell line using direct/indirect/extract tests in accordance to standard protocols. Cytotoxicity was assessed using neutral red uptake. Tooth slice organ cultures were also employed to test the dental materials using direct/indirect test methods. Histomorphometric cell counting of intact odontoblasts and pulp fibroblasts and the use of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were applied for cytotoxicity evaluation. Discrepancy in result presentation was observed in the different tests used with L-929. Sensitivity levels of the L-929 tests ranked as follows: extract test < direct contact test < indirect contact test. Tooth slice tests confirmed that L-929 direct contact test proved to be the most reliable test among the three. In conclusion, this study highlights the risk involved when relying on a single test method for cytotoxicity assessment. It would be advisable to test different culture models and then proceed using more clinically relevant biological system that stimulate the in vivo situation for confirmation.