Figure 6.
Figure 6. Cultivation and osteogenic differentiation of DFCs on PA after modification with collagen I. (Left) Relative cell number and (Right) normalized ALP activity.
Figure 5. resentative results are shown for dNC-PCs.
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.
Figure 4. dishes for control.
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.
Figure 3. (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.
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), apop...
Figure 2. spheroid cell clusters on silicone (representative pictures for DFCs); Silicone (24 and 48 h).
Figure 2. C ell 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).
Figure 1. ative pictures of DFCs.
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.
Gosau, M., Viale-Bouroncle, S., Eickhoff, H. et al. Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions.
Int J Implant Dent 1, 2 (2015). https://doi.org/10.1186/s40729-014-0002-y
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Received: 17 September 2014
Accepted: 20 November 2014
Published: 12 February 2015
DOI: https://doi.org/10.1186/s40729-014-0002-y
DFCs and dNC-PCs expressed typical markers for dental stem cells.
Martin Gosau, Sandra Viale-Bouroncle, Hannah Eickhoff, Esthera Prateeptongkum, Anja Reck, W Götz, Christoph Klingelhöffer, Steffen Müller and Christian Morsczeck declare that they have no competing interests.
SVB, HE, and EP carried out all cell biology experiments, performed real-time PCRs, Western blots, and the statistical analysis and made figures for the manuscript. AR carried out and ana...
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Correspondence to
Christian Morsczeck.
Department of Cranio- and Maxillofacial Surgery, Hospital of the University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany
Martin Gosau, Sandra Viale-Bouroncle, Hannah Eickhoff, Esthera Prateeptongkum, Anja Reck, Christoph Klingelhöffer, Steffen Müller & Christian Morsczeck
Department of Oral and Maxillofacial Surgery, Paracelsus Medical University Nuernberg, B...
This study was supported by a grant from the Deutschen Gesellschaft für Implantologie (DGI) e.V.
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In conclusion, our work supports our hypothesis that soft implant materials are not suitable for dental tissue engineering. Moreover, our study also supports the results of our previous studies with DFCs and TCP that induction of apoptosis did not impair the proliferation and the differentiation in dental stem cells.
In a previous study, we showed that TCP induced the programmed cell death (apoptosis) in DFCs [11]. Our new study investigated therefore the induction of apoptosis in dental cells. While SB and soft materials did not induce apoptosis or cell death, AP induced obviously cell death and apoptosis in dental cells. Here, the results for dNC-PCs and DFCs were almost the same. Interestingly, neither sili...
Scaffolds play an important role in tissue engineering. However, little is known about the proliferation and differentiation of DFCs and dNC-PCs on different types of materials. As we have learned from previous studies mechanical properties such as surface stiffness are decisive for a successful osteogenic differentiation of dental stem cells [13,14]. Moreover, we showed that bone substitute mater...
The soft material PA was also treated with the extracellular matrix protein collagen to improve cell adherence. We tested representatively DFCs with collagen I modified PA. DFCs adhered and proliferated on modified PA, but, however, the specific ALP activity was reduced in comparison to that of DFCs on standard cell culture dishes (Figure 6). This reduction of the specific ALP activity was simila...
Dental cells were cultivated in standard cell culture media until passage 6. Cell adherence and cell proliferation/growth were measured for the estimation of cell viability on tested rigid and soft materials. In Figure 1, the cell adherence of dNC-PCs on bone substitute materials was better than that of DFCs. However, both dental cells types adhered very well on silicone. Unluckily, dental cells ...
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 ...
Numbers of vital cells were evaluated after days 1, 2, 3, and 6. For cell counting, cell cultures were incubated with the cell counting kit 8 (CCK8) ready to use solution according to manufactures instructions (Dojindo, Rockville, MD, USA). The optical density (O.D.) was measured at a wavelength of 450 nm. For the evaluation of the cell adherence (normalized to standard cell culture dishes), cell...
The bone substitutes Maxgraft® (AP) and Maxresorb® (SB) were obtained from the company Botiss (botiss dental GmbH, Berlin, Germany). Maxgraft® is a sterile, high-safety allograft product (AP), derived from human donor bone. It is processed by an audited and certified bone bank (Cells+ Tissue Bank Austria, Berlin, Germany). In contrast, Maxresorb® is a fully synthetic bone graft substitute (SB)...
The isolation and characterization of DFCs and dNC-PCs were described in previous studies [4,7,12]. DFCs were routinely cultivated in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/ml penicillin/streptomycin (standard cell culture medium). dNC-PCs were cultivated in DMEM (Sigma-Aldrich) supplemented with 15% fetal ...
Unfortunately, an additional study showed that TCP induced apoptosis in DFCs [11]. However, the induction of apoptosis exposed a risk for cellular therapies. We decided therefore to evaluate additional implant materials for the identification of a suitable scaffold for dental stem cells. Soft materials such as silicone are successfully used in regenerative medicine, and they are suitable for tissu...
While bone substitute materials are routinely used, especially vertical bone, augmentation of the jaws is still a problematic step. Dental stem cells in combination with bone substitute materials may accelerate the augmentation of alveolar bone and perhaps, stem cell-based therapies can become an alternative to autologous, allogenic, or synthetic bone transplants and substitutes [1,2]. However, sc...
Dental stem cells in combination with implant materials may become an alternative to autologous bone transplants. For tissue engineering different types of soft and rigid implant materials are available, but little is known about the viability and the osteogenic differentiation of dental stem cells on these different types of materials. According to previous studies we proposed that rigid bone sub...
Figure 6. Cultivation and osteogenic differentiation of DFCs on PA after modification with collagen I. (Left) Relative cell number and (Right) normalized ALP activity.
Figure 6. Cultivation and osteogenic differentiation of DFCs on PA after modification with collagen I. (Left) Relative cell number and (Right) normalized ALP activity.
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.
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 result...
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.
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 standar...
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.
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).
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.
In a previous study, we showed that TCP induced the programmed cell death (apoptosis) in DFCs. Our new study investigated therefore the induction of apoptosis in dental cells. While SB and soft materials did not induce apoptosis or cell death, AP induced obviously cell death and apoptosis in dental cells. Here, the results for dNC-PCs and DFCs were almost the same. Interestingly, neither sil...
Discussion
Scaffolds play an important role in tissue engineering. However, little is known about the proliferation and differentiation of DFCs and dNC-PCs on different types of materials. As we have learned from previous studies mechanical properties such as surface stiffness are decisive for a successful osteogenic differentiation of dental stem cells. Moreover, we showed that bone substi...
Results
Cell viability
Dental cells were cultivated in standard cell culture media until passage 6. Cell adherence and cell proliferation/growth were measured for the estimation of cell viability on tested rigid and soft materials. In Figure 1, the cell adherence of dNC-PCs on bone substitute materials was better than that of DFCs. However, both dental cells types adhered very well on silicone...
Cells positive for Caspase3/7 Green Detection Reagent were identified as apoptotic cells, while dead cells were positive for SYTOX® AADvanced dead cell stain. However, vital cells were negatively stained for both staining solutions.
Western blotting
For protein isolation, cells were treated with lysis buffer (250 μl phosphatase, 100 mM Na3VO4, 137 mM NaCl, 200 mM Tris, 480 mM NaF, 1% NP-4...
This incubation step with the implant material was repeated twice with fresh cell culture media. Three eluates were pooled for cell culture experiments. DFCs were seeded onto cell culture plates and cultivated in standard cell culture media. After cell seeding (12 to 24 h), cell culture media were changed, and cells were cultivated in cell culture media with material eluates. After 24 h of cultiva...
After washing with PBS, the gels were stored in PBS at 4°C. Before platting the cells, the gel was exposed to UV for 15 min for the sterilization and replace PBS with complete culture medium for 1 h at 37°C.
Implant materials
The bone substitutes Maxgraft® (AP) and Maxresorb® (SB) were obtained from the company Botiss (botiss dental GmbH, Berlin, Germany). Maxgraft® is a sterile, high-saf...
Methods
Cell culture
The isolation and characterization of DFCs and dNC-PCs were described in previous studies. DFCs were routinely cultivated in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/ml penicillin/streptomycin (standard cell culture medium). dNC-PCs were cultivated in DMEM (Sigma-Aldrich) supplemented ...
Background
While bone substitute materials are routinely used, especially vertical bone, augmentation of the jaws is still a problematic step. Dental stem cells in combination with bone substitute materials may accelerate the augmentation of alveolar bone and perhaps, stem cell-based therapies can become an alternative to autologous, allogenic, or synthetic bone transplants and substitutes. How...
Abstract
Background
Dental stem cells in combination with implant materials may become an alternative to autologous bone transplants. For tissue engineering different types of soft and rigid implant materials are available, but little is known about the viability and the osteogenic differentiation of dental stem cells on these different types of materials. According to previous s...