Human Dental Follicle Precursor Cells of Wisdom Teeth: Isolation and Differentiation towards Osteocytes for Implants and Scaffolds
Skroch1, M., Rehhahn1, A., Haddouti1, E.-M., Birova1, B., Langenbach1, F., Zippel>1, N., Müller1, C.,
Roitzheim1, B., Pansky1, A., Kleinfeld;1, C., Winter2, M., Tobiasch1, E.
1 Fachhochschule Bonn-Rhein-Sieg, von-Liebig-Str. 20, 53359 Rheinbach, Germany;
2 Oralchirurgische Praxis, Martinstr. 26-28, 53359 Rheinbach, Germany
Abstract
Introduction: The human dental follicle derived from ectomesenchyme is a developmental precursor for essential periodontal tissues such as periodontal ligament and root development. Precursor cells out of dental follicle can be differentiated into osteocytes [1,3]. Thus, they could be a source for tissue regeneration with or without growing on scaffold biomaterials in applications such as autologous transplantation for reconstruction of large bone defects and incorporation of teeth implants [6,7]. Therefore, the aim of this study was to develop a fast and efficient method to isolate stem cells out of the dental follicle of wisdom teeth and to investigate their differentiation potential. Furthermore, a microbial approach was performed, to examine the presence of pathogenic microorganisms, thus the quality of the cell material.
Materials and Methods: Singularisation of dental follicle cells (DFCs) derived from wisdom teeth tissues was achieved through mincing, collagenase digestion and cell adhesion to plastic [2]. To confirm the presence of precursor cells, specific markers were used during RT-PCR. Additionally, their differentiation potential towards the osteogenic and adipogenic lineages was defined by Alizarin Red S and Oil Red O staining, respectively. For the determination of the potential microbial contamination dilutions were inoculated onto CBA and FAA agar plates. Incubations were done under aerobic and anaerobic conditions. The determination of the cell number was done in total and per species. With respect to morphological differences single colonies were picked and characterised by the means of gram staining, catalase- and oxidase-tests.
Results: The lineage specific differentiation potential of dental follicle derived mesenchymal stem cells was proven by specific gene expression. Typical markers confirmed a stem cell character of the isolated cells. After their selective induction the lineage specific differentiation potential has been verified by special stainings as well. The microbial investigations showed that all samples contained microbial species, even after pre-treatment of patients with antibiotics. The majority of organisms were found to be members of the group of gram-positive cocci being either catalase-positive and oxidase-negative or catalase- and oxidase-negative. Most microorganisms belonged to the families of Streptococcaceae and Staphylococcaceae. The results of the microbiological investigation showed that the pre-treatment of patients with antibiotics will reduce the number of microorganisms greatly. The contamination with microorganisms could be easily suppressed by usage of standard cell culture condition with penicillin and streptomycin during cultivation.
Discussion: Precursor cells from wisdom teeth follicles are very interesting. The higher lineage commitment of dental follicle precursor cells towards osteocytes might imply a decreased tumor potential if compared to mesenchymal stem cells. Human DFCs can be obtained in a high yields from young and healthy donors without additional invasive intervention and ethical issues. The combination of antibiotic treatment of patients and cell growth with antibiotic containing media does not interfere with the quality of DFCs and therefore their usage in future transplantation application. Large amounts of autologes osteocytes as can be obtained via this method could substantiate treatment of large bone defects with biomaterial scaffolds and therefore contribute in this field of regenerative medicine.
INTRODUCTION
The dental follicle is an ectomesenchymal derived portion of the outer layer of the tooth bud, the primordial tooth forming structure (Fig. 1). The cardinal function of the dental follicle is the generation of tooth supportive structures like the periodontal ligament, the cementum and alveolar bone of the tooth socket. These structures are thought to be descended from a heterogenous cell population composed of precursor cells or stem cells of unclear differentiation potential [1].
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| Figure 1. Wisdom tooth germ with surrounding follicle. |
Figure 2. DFCs of passage 0; 200 x. |
The main problem in dental regenerative medicine is bone reconstruction and the osseointegration of implants. Materials used are mainly artificial (hydroxyapatite), bovine derived xenotransplants, autologous material from intraoral areas or for extensive reconstructions from the hip. Disadvantages of these materials can be fibrosis or rejection of synthetic, surgical trauma for autologous and potential risk for prion infection by bovine material. Therefore DFCs could, after negation of potential problems occuring from microbial pathogens [5], serve as a superior alternative in the future.
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RT-PCR detection of stem cell marker CD73 in DFCs and ADSCs. |
Figure 4. follicel tissue. Main frame: Cocci; insert: rods; 100 x. |
RESULTS
Dental follicle tissue was enzymatically digested and DFCs were isolated using their adherence to
plastic (Fig. 2). Typical stem cell markers, such as CD73, were tested positive in most samples (Fig. 3). In order to investigate the multi-lineage differentiation potential of human DFCs in comparison to ADSCs, both cell types were induced towards the osteogenic as well as the adipogenic lineage. After induction with osteogenic differentiation medium (Fig. 5) the typical calcium mineralisation was observed in both cell types, but Alizarin Red S staining was more intense in DFCs than in ADSCs (Fig. 5A and Fig. 5C). In contrast, adipogenic differentiation of both cell types, detected with Oil Red O, showed lipid formation only in ADSCs (Fig. 5E and 5G).
The probability of DFCs usage in dental regenerative medicine precipitates the need for protective measures against pathogenious microbial organisms originating from the donor's oral cavity. So far only gram+bacteria were observed in our samples, most of wich belong to the family of cocci (Fig. 4). Only very few microbial organisms from the family of rods could be detected (Fig. 4 insert).
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Figure 5. Comparison of osteogenic induced DFCs and ADSCs. (B) DFCs,negative control; (C) ADSCs, osteogenic differentiation; (D) ADSCs, negative control. Magnification 380 x. |
| DFCs and ADSCs were grown in DMEM with 10% FCS supplemented with Dexamethasone, β-Glycerophosphate and Ascorbic-acid-2-phosphate [2]. After 28 days the cells were stained with Alizarin Red S. The corresponding controls were incubated in DMEM with 10% FCS only. | ||
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Comparison of
adipogenic induced DFCs and ADSCs.
(F) DFCs,negative control; (G) ADSCs, adipogenic differentiation; (H) ADSCs, negative control. Magnifications F, H, G 380 x; G 760 x. |
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DFCs and ADSCs were grown in DMEM with 10% FCS supplemented with Dexamethasone, Insulin and Indomethacin [2]. After 21 days the cells were stained with Oil Red O. The corresponding controls were incubated in DMEM with 10% FCS only.
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DISCUSSION
