Methods : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [4]
DFCs were cultivated until sub-confluence (>80%) in standard cell culture medium before the differentiation starts with the osteogenic differentiation medium (ODM) comprised DMEM (PAA) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 μmol/L ascorbic acid 2-phosphate, 10 mmol/L KH2PO4, 1 × 10−8 mol/L dexamethasone sodium phosphate (Sigma-Aldrich, St. Louis, MO, USA), HEPES (20 mmol/L) and 100 μg/ml penicillin/streptomycin. The differentiation was evaluated by qRT-PCR and ALP activity detection.
Cells were washed with PBS buffer and lysed by shock freezing (−80°C). Diluted 1:1 in 1 × PBS, 100 mM p-nitrophenyl phosphate (Sigma) was added to each sample. After incubation at 37°C for 60 min, the reaction was stopped by adding 300 μL of 0.3 M NaOH and the liberated p-nitrophenol was measured at 405 nm. ALP activity values were normalized to total DNA concentration, which were determined by the Quant-iT PicoGreen dsDNA Assay (Invitrogen).
For the evaluation of osteogenic marker expression, the Biorad PrimePCR array (Development - Hedgehog and PTH signaling pathways in bone and cartilage development) was used, which consists of the most important markers for the osteogenic differentiation. Total RNAs, which were isolated from differentiated dental cells at day 7, were reverse-transcripted with the iScript™ Advanced cDNA Synthesis Kit for RT-qPCR (Biorad) according to the manufacturers protocol. PCRs were made with SsoAdvanced™ Universal SYBR® Green Supermix (Biorad) on the StepOne real-time PCR machine (Life Technologies, Carlsbad, CA, USA). Results were analyzed with the PrimePCR™ Analysis Software (Biorad), and the output is presented as Clustergrams. While red tiles signify a high gene expression, black/gray and green tiles show a middle gene expression and a low gene expression, respectively. Black tiles with a cross designate no gene expression.
Combinations of SB with dNC-PCs and AP with dental cells yielded from cell cultures after 7 days of osteogenic differentiation were fixed in 4% formaldehyde/0.1 M PBS at 4°C for at least 24 h. Tissues were decalcified with EDTA and subsequently dehydrated in an ascending series of ethanol and embedded in paraffin. Serial sections of 5 μm were cut in different planes for orientation and stained with hematoxylin-eosin (HE).
Serial posts:
- Abstract : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions
- Background : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [1]
- Background : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [2]
- Methods : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [1]
- Methods : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [2]
- Methods : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [3]
- Methods : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [4]
- Results : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [1]
- Results : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [2]
- Discussion : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [1]
- Discussion : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [2]
- Conclusions : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions
- References : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [1]
- References : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [2]
- References : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [3]
- Acknowledgement : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions
- Author information : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [1]
- Author information : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions [2]
- Additional information : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions
- Additional file : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions
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- About this article : Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions
- Figure 1. Cell attachment on tested materials. (A) Relative cell adherence of DFCs and dNC-PCs; (B) dental cells did little adhere on PA; representative pictures of DFCs. : Evaluation of implant
- Figure 2. Cell proliferation of dNC-PCs and DFCs on tested materials. (A) and (B) Relative cell numbers; (C) spheroid cell clusters on silicone (representative pictures for DFCs); Silicone (24 and 48 h). : Evaluation of implant
- Figure 3. Evaluation of programmed cell death (apoptosis) in dental stem cells. (A) Flow cytometry analyses (for details materials and methods) show percentage of vital cells (black number), apoptotic cells (blue number), and dead cells (red number). (B) Western blot analyses show the expression of the pro-apoptotic marker BAX and the anti-apoptotic marker BCL2. : Evaluation of implant
- Figure 4. Osteogenic differentiation of dental stem cells. Normalized ALP activity of dNC-PCs and DFCs on AP and SB (A) and on silicone (B). Cells were differentiated on standard cell culture dishes for control. : Evaluation of implant
- Figure 5. Evaluation of osteogenic differentiation. (A) Clustergram of PCR-array results; (B-C) histology of differentiated dental cells on AP (B) and SB (C). Representative results are shown for dNC-PCs. : Evaluation of implant
- Figure 6. Cultivation and osteogenic differentiation of DFCs on PA after modification with collagen I. (Left) Relative cell number and (Right) normalized ALP activity. : Evaluation of implant