Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model.

Periodontitis remains an unsolved oral disease, prevalent worldwide and resulting in tooth loss due to dysfunction of the periodontal ligament (PDL), a tissue connecting the tooth root with the alveolar bone. A scaffold-free three-dimensional (3D) organoid model for in vitro tenogenesis/ligamentogeneis has already been described. As PDL tissue naturally arises from the dental follicle, the aim of this study was to investigate the ligamentogenic differentiation potential of dental follicle cells (DFCs) in vitro by employing this 3D model. Human primary DFCs were compared, in both two- and three-dimensions, to a previously published PDL- hTERT cell line. The 3D organoids were evaluated by haematoxylin and eosin, 4',6-diamidino-2-phenylindole and F-actin staining combined with detailed histomorphometric analyses of cell-row structure, angular deviation and cell density. Furthermore, the expression of 48 tendon/ligament- and multilineage-related genes was evaluated using quantitative polymerase chain reaction, followed by immunofluorescent analyses of collagen 1 and 3. The results showed that both cell types were successful in the formation of scaffold-free 3D organoids. DFC organoids were comparable to PDL-hTERT in terms of cell density; however, DFCs exhibited superior organoid morphology, cell-row organisation (p < 0.0001) and angular deviation (p < 0.0001). Interestingly, in 2 dimensions as well as in 3D, DFCs showed significantly higher levels of several ligament- related genes compared to the PDL-hTERT cell line. In conclusion, DFCs exhibited great potential to form PDL-like 3D organoids in vitro suggesting that this strategy can be further developed for functional PDL engineering.

Correction to: Induced surface proteins of Staphylococcus epidermidis adhering to titanium implant substrata.

There is a mistake in the original published version of this article. The word 'Streptococcus' in the article title should have been 'Staphylococcus'.

Induced surface proteins of Staphylococcus [corrected] epidermidis adhering to titanium implant substrata.

OBJECTIVE: Staphylococcus epidermidis, as a primary colonizer, is strongly associated with infections of (dental) implants (i.e., peri-implantitis), but little is known about the surface proteome of this bacterium. For the identification of bacterial adhesins, this study investigated the surface proteome of S. epidermidis adhering directly to titanium implant substrata. MATERIALS AND METHODS: S. epidermidis strain ATTC 35984 was cultured either planktonically or on titanium implant specimens. The surface proteomes were isolated by mutanolysin digestion, and proteins were separated by 2D gel electrophoreses to reveal highly expressed proteins only. Protein spots were visualized by silver staining and proteins were identified by mass spectrometry. RESULTS: Surface proteome analyses of S. epidermidis on titanium identified six expressed proteins. Three proteins were highly expressed on the titanium implants including accumulation-associated protein Q8CQD9. These specific proteins could be potential pathogenicity factors of bacteria in peri-implant biofilms. CONCLUSION: For the first time, our study identified S. epidermidis surface proteins, which are expressed after adhesion to titanium implant materials. CLINICAL RELEVANCE: Our study reveals possible candidates for a newly protein-based vaccine against peri-implantitis.

WNT3A and the induction of the osteogenic differentiation in adipose tissue derived mesenchymal stem cells.

Adipose tissue derived stem cells (ASCs) can easily be isolated, but the osteogenic differentiation potential is limited. To improve this differentiation potential, more investigations are required about signaling proteins for the induction of the osteogenic differentiation. This study focused on the WNT3A protein, because little is known about the canonical WNT signaling pathway and the osteogenic differentiation of ASCs. The alkaline phosphatase (ALP) activity was measured for the evaluation of the osteogenic differentiation. WNT3A and Dickkopf-related protein 1 (DKK1) were used for the activation and the inhibition of the canonical WNT signaling pathway, respectively. For control we manipulated the bone morphogenetic protein (BMP) pathway in ASCs with BMP2 and NOGGIN (BMP pathway inhibitor). WNT3A stimulated significantly the ALP activity in ASCs, while BMP2, DKK1 and NOGGIN did not induce highly the ALP activity in ASCs. Moreover, an osteogenic differentiation medium with dexamethasone and WNT3A increased the ALP activity, but the gene expression of osteoblast markers and the biomineralization after long-term cultures were not increased. In contrast, ASCs differentiated into adipocyte-like cells in all tested differentiation media. WNT3A did not repress the expression of the adipogenic transcription factor Peroxisome Proliferator-Activated Receptor Gamma (PPARG). In conclusion, WNT3A supports early stages such as the ALP activity, but it does neither improve later stages of the osteogenic differentiation nor it inhibits the genuine adipogenic differentiation of ASCs.

The hedgehog-signaling pathway is repressed during the osteogenic differentiation of dental follicle cells.

Dental follicle stem cells (DFCs) are precursor cells of alveolar osteoblasts, and previous studies have shown that the growth factor bone morphogenetic protein (BMP)2 induces the osteogenic differentiation of DFCs. However, the molecular mechanism down-stream of the induction of the osteogenic differentiation by BMP2 remains elusive. We investigated therefore the phosphoproteome of DFCs after the induction of the osteogenic differentiation with BMP2. In this study, phosphoproteins of the hedgehog "off" state were differentially expressed. Further analyses revealed that BMP2 induced the expression of repressors of the hedgehog-signaling pathway such as Patched 1 (PTCH1), Suppressor of Fused (SUFU), and Parathyroid Hormone-Related Peptide (PTHrP). Previous studies suggested that hedgehog proteins induce the osteogenic differentiation of mesenchymal stem cells via a paracrine pathway. Indian hedgehog (IHH) induced the expression of the osteogenic transcription factor RUNX2. However, a supplementation of the BMP2-based osteogenic differentiation medium with IHH did not induce the expression of RUNX2. Moreover, IHH inhibited slightly the ALP activity and the mineralization of osteogenic-differentiated DFCs. In conclusion, our results suggest that BMP2 inhibits the hedgehog signaling after the induction of the osteogenic differentiation in DFCs.

The extracellular concentration of osteocalcin decreased in dental follicle cell cultures during biomineralization.

The secretion of osteocalcin (OCN) is an excellent differentiation marker for the osteogenic differentiation. This study investigated the secretion of OCN during the osteogenic differentiation of DFCs. During the differentiation of DFCs the extracellular concentrations of OCN were higher in standard cell culture medium than in osteogenic differentiation medium. However, after 4 weeks in the osteogenic differentiation medium the extracellular OCN concentration decreased strongly, whereas the concentration remains high in the control medium. At this point in time DFCs formed connective tissue like structures with mineralized clusters and OCN. Real-time RT-PCR analyses and western-blot analyses proved that OCN was expressed in both cell culture media. However, the expression of the mRNA was inhibited in the osteogenic differentiation medium. These results suggest that DFCs secrete constitutively OCN into the cell culture medium and that the osteogenic differentiation medium suppresses the gene expression of OCN. Moreover, OCN imbeds into the extracellular matrix after the formation of connective tissue like structures, and the soluble OCN in the cell culture medium disappears. Hence, extracellular OCN in the cell culture medium is not a marker for the osteogenic differentiation of DFCs.

The parathyroid hormone-related protein is secreted during the osteogenic differentiation of human dental follicle cells and inhibits the alkaline phosphatase activity and the expression of DLX3.

The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5pg/ml±345.7SD. in osteogenic differentiation medium vs. 315.7pg/ml±156.2SD. in standard cell culture medium; Student's t Test: p<0.05 (n=3)). We showed that the supplementation of the osteogenic differentiation medium with PTHrP inhibited the alkaline phosphatase activity and the expression of the transcription factor DLX3, but the depletion of PTHrP did not support the differentiation of DFCs. Previous studies have shown that Indian Hedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs.

Characterization of progenitor cells and stem cells from the periodontal ligament tissue derived from a single person.

BACKGROUND: Periodontal ligament progenitor cells (PDLPs) and PDL stem cells (PDLSCs) are progenitor and stem cells that were isolated from PDL tissues using the outgrowth and single cell isolation methods respectively. The differences between PDLPs and PDLSCs characteristics could be observed from previous studies. However, these cells were obtained from different patients. This study was the first report to compare the characterization of PDLPs and PDLSCs from the same person. MATERIAL AND METHODS: The characterization of PDLPs and PDLSCs includes flow cytometry analysis, cell proliferation assay and the assessment of the colony-forming unit fibroblast. The osteogenic differentiation was evaluated by alkaline phosphatase activity, biomineralization (alizarin red staining) and gene expression of osteogenic markers. The adipogenic differentiation was examined by Oil Red O staining and adipocyte-related gene expression. RESULTS: Mesenchymal stem cell marker expression and colony-forming unit fibroblast analysis of PDLPs and PDLSCs were similar. However, PDLSCs grew faster than PDLPs on days 3 and 5 of the cell proliferation assay. Both PDLPs and PDLSCs could differentiate into osteoblast and adipocyte-like cells. However, the mineralization of PDLSCs was stronger than that of PDLPs. CONCLUSIONS: The characteristics of undifferentiated PDL cells in our study were not significantly impacted by the isolation method. We assumed that both PDLPs and PDLSCs are valuable cell sources for periodontal regeneration. However, PDLSCs have a possible advantage for the regeneration of alveolar bone.

The influence of the donor on dental apical papilla stem cell properties.

Stem cells from the human dental apical papilla (SCAP) can be obtained from almost all extracted wisdom teeth with an immature tooth root. Although different stem cell lines are used for studies, it remains elusive whether specific characteristics of the dental stem cell cultures such as proliferation rates or the cell differentiation potential are related to the cell source, e.g. the donor tissue of the dental apical papilla. To answer this question, we compared two independent SCAP cell lines from the same donor and compared them with a third cell line from another donor. We investigated the expression of stem cell markers, the efficiency of colony forming units, cell proliferation and the differentiation potential. Results showed particular differences for typical stem cell attributes such as stem cell marker expression, cell proliferation and the adipogenic differentiation. These differences were regardless of the donor of the cell lines. In conclusion, we suppose that stem cell characteristics of SCAP cell cultures are independent from the donor.

The AKT signaling pathway sustains the osteogenic differentiation in human dental follicle cells.

Signaling transduction pathways are established by interactions between growth factors, protein kinases, and transcription factors, and they play a crucial role in tooth development. Precursor cells of the dental follicle (DFCs) are used for in vitro studies about molecular mechanisms during periodontal development. Previous studies have already shown that the growth factor BMP2 and the transcription factor EGR1 are involved in the osteogenic differentiation in DFCs while interactions with protein kinase-based pathways remain elusive. In this current study, we investigated the role of the AKT kinase signaling pathway for the osteogenic differentiation in DFCs. The AKT signaling pathway was activated in DFCs after the induction of the osteogenic differentiation by BMP2. The inhibition of AKT in DFCs repressed the differentiation and the expression of the transcription factor EGR1. Interestingly, EGR1 bound to the phosphorylated form of SMAD1/5 (pSMAD). The binding of pSMAD to EGR1 was increased after the induction with BMP2. Moreover, the overexpression EGR1 increased the osteogenic differentiation of DFCs. Our results suggest that the AKT signaling pathway submits the BMP2-dependent osteogenic differentiation in DFCs via the expression of the transcription factor EGR1.

The WNT inhibitor APCDD1 sustains the expression of ß-catenin during the osteogenic differentiation of human dental follicle cells.

In hair follicle cells APCDD1 inhibits the canonical WNT/ß-Catenin pathway and its inactivation is associated with an autosomal dominant form of hair loss. We analyzed the role of APCDD1 for the osteogenic differentiation in dental follicle cells (DFCs) and identified a new and surprising function. Contrarily to hair follicle cells APCDD1 was crucial for the expression of ß-Catenin and for the activity of the TCF/LEF reporter assay in DFCs. In addition, a depletion of APCDD1 inhibits the expression of osteogenic markers such as RUNX2 and decreased the matrix mineralization. However, similar to hair follicle cells in previous studies a control cell culture with oral squamous carcinoma cells showed that APCDD1 inhibits the expression of ß-Catenin and of typical target genes of the canonical WNT/ß-Catenin pathway. In conclusion, our data disclosed an unusual role of APCDD1 in DFCs during the osteogenic differentiation. APCDD1 sustains the expression and activation of ß-Catenin.

A protein kinase A (PKA)/ß-catenin pathway sustains the BMP2/DLX3-induced osteogenic differentiation in dental follicle cells (DFCs).

The directed expression of osteogenic transcription factors via a balanced activation of signaling pathways is an important prerequisite for the development of mineralized tissues. A positive-feedback loop of the BMP2-dependent SMAD signaling pathway and the DLX3 transcription factor (BMP2/DLX3 pathway) directs the osteogenic differentiation of periodontal precursor cells from the dental follicle (DFCs). However, little is known how this BMP2/DLX3 pathway interacts with other crucial signaling pathways such as the WNT/ß-catenin signaling pathway. This study investigated the interaction between the BMP2/DLX3 pathway and the WNT pathway during the osteogenic differentiation of DFCs. BMP2 induced the WNT/ß-catenin pathway in DFCs and phosphorylates ß-catenin via protein kinase A (PKA). Moreover, only BMP2 facilitated the binding of LEF1/SMAD4/ß-catenin complex to the DLX3 promoter, while an inducer of the canonical WNT pathway, WNT3A, act as an inhibitor. Although WNT3A inhibits the osteogenic differentiation of DFCs the expression of ß-catenin was crucial for both the expression of DLX3 and for the osteogenic differentiation. In conclusion, while the activation of the canonical WNT pathway inhibits the osteogenic differentiation of DFCs, ß-catenin sustains the BMP2/DLX3-mediated osteogenic differentiation via the activation of PKA.

EGR1 supports the osteogenic differentiation of dental stem cells.

AIM: To evaluate whether and how the transcription factor early growth response gene 1 (EGR1) affects the osteogenic differentiation of dental stem cells. METHODOLOGY: Dental stem cells from apical papilla (SCAPs) and from the dental follicle (DFCs) were transfected with EGR1-specific siRNA or EGR-1 expression plasmid. Gene regulation was verified at protein level by Western blotting. The expression of the transcription factors distal-less homeobox 3 (DLX3), alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP2), which are all regulators and markers of the osteogenic differentiation in dental stem cells, was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). To investigate mineralization, SCAP long-term cultures were stained with alizarin red after EGR1 over-expression. RESULTS: EGR1 was induced in SCAPs during osteogenic differentiation. DLX3 and bone morphogenetic protein 2 (BMP2) were up-regulated after EGR1 over-expression and down-regulated after EGR1 depletion. The expression of ALP was also down-regulated after EGR1 depletion. The over-expression of EGR1 in SCAPs promoted mineralization after osteogenic differentiation. CONCLUSIONS: EGR1 supported the osteogenic differentiation of dental stem cells by potentially regulating the expression of DLX3 and BMP2.

Migration of human dental follicle cells in vitro.

BACKGROUND AND OBJECTIVES: The objective of this study was to elucidate the effects of different growth factors on the migration of dental follicle cells (DFCs). DFCs are ectomesenchymally derived easily accessible multipotent stem cells. Cell migration is a crucial step in many biological processes but also for tissue engineering. Growth factors such as epidermal growth factor (EGF), bone morphogenetic protein-2 (BMP2) or transforming growth factor ß1 (TGF-ß1) can be used to modify the behavior of cells. MATERIAL AND METHODS: We used different migration assays (gel spot assay, scratch assay, transwell assay) to evaluate the influence of EGF, BMP2 and TGF-ß1 on the migration of DFCs. We investigated the expression of migration-related genes after growth factor stimulation using the PCR array human cell motility. RESULTS: DFCs treated with BMP2 or TGF-ß1 migrated faster than DFCs treated with EGF. Additionally, more migration-related genes are regulated after treatment with BMP2 or TGF-ß1 than with EGF. TGF-ß1 additionally functions as a chemoattractant for DFCs. Osteogenic differentiation markers were regulated after BMP2 treatment only. CONCLUSION: Whereas the strong migration induced by BMP2 was accompanied by beginning osteogenic differentiation the strong migration induced by TGF-ß1 was directional. EGF exhibited not only the weakest migration stimulation but also the weakest induction of differentiation into mineralizing cells.

ß-Tricalcium phosphate induces apoptosis on dental follicle cells.

Dental stem cells represent a good treatment option in regenerative dentistry. Regeneration of large bone defects can be achieved by a cell-based therapy consisting of osteogenic progenitor cells, such as dental follicle precursor cells (DFCs), in combination with bone substitute material used as a scaffold. A previous trial had shown that ß-tricalcium phosphate (TCP) improves the osteogenic differentiation of DFCs. In the present trial, we investigated the attachment, survival, and proliferation of DFCs on TCP in more detail. A high initial cell number was required for the adhesion, attachment, and sufficient proliferation of DFCs on a TCP scaffold. The TCP scaffold released fine soluble particles enriched in TCP eluates that induced cell death and showed typical characteristics of programmed cell death (apoptosis) in DFCs. During cultivation on the TCP scaffold, DFCs showed a highly upregulated expression of antiapoptotic genes but a downregulated expression of proapoptotic markers. In conclusion, TCP supports osteogenic differentiation in DFCs but also induces programmed cell death. Our data suggest that surviving DFCs avoid programmed cell death by inducing antiapoptotic genes.

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines.

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133(+) cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

Spontaneous immortalization of neural crest-derived corneal progenitor cells after chromosomal aberration.

OBJECTIVES: In a previous study, we have reported the existence of neural crest-derived stem cell-like cells originating from the corneal limbus of juvenile mice (termed murine corneal cells, MCCs). To yield a sufficient number of MCCs, for example, for cell-therapy approaches, here we have investigated MCCs' ability for extensive proliferation, and we have evaluated their stem cell qualities and genetic stability after large-scale culture. MATERIALS AND METHODS: MCCs were established from corneal limbal tissue of juvenile mice. To determine their cell proliferation and self-renewing potential, MTT tests and an estimation of colony forming unit efficiency were carried out. Multipotency of cell differentiation was examined by applying adipogenic and osteogenic differentiation protocols. Moreover, karyotyping was performed and expression of stem cell markers and cell cycle-associated genes was analysed. RESULTS: MCCs, as primary cells, could be cultured for more than 60 passages. We observed increased cell proliferation and high number of colony forming units (CFUs) after extensive culture. Interestingly, there were no changes in expression of MCC markers. Furthermore, cell differentiation potentials remained comparable with MCCs at early passages. However, karyotyping revealed numeric chromosomal aberrations at higher passages. Moreover, tumour suppressor genes such as p16 and p21 were found to be down-regulated after large-scale cell culture. CONCLUSIONS: MCCs immortalize spontaneously after extensive cell culture, but still demonstrate stem cell-like qualities.

Transcriptomes and proteomes of dental follicle cells.

Ectomesenchymal dental stem cells could be feasible tools for dental tissue engineering. Dental follicle cells are a promising example, since they are capable of differentiation into various dental tissue cells, such as osteoblasts or cementoblasts. However, cellular mechanisms of cell proliferation and differentiation are not understood in detail. Basic knowledge of these molecular processes may shorten the time before ectomesenchymal dental stem cells can be exploited for bone augmentation in regenerative medicine. Recent developments in proteomics and transcriptomics have made information about genome-wide expression profiles accessible, which can aid in clarifying molecular mechanisms of cells. This review describes the transcriptomes and proteomes of dental follicle cells before and after differentiation, and compares them with differentially expressed populations from dental tissue or bone marrow.

Streptococcus pneumoniae: proteomics of surface proteins for vaccine development.

Two formulations of pneumococcal vaccines are currently available to prevent invasive disease in adults and children. However, these vaccines will not protect against the majority of Streptococcus pneumoniae serotypes. The use of highly conserved cell-wall-associated proteins in vaccines may circumvent this problem. A proteomics approach was used to identify 270 S. pneumoniae cell-wall-associated proteins, which were then screened in a process that included in-silico, in-vitro and in-vivo validation criteria. Five potential candidates for inclusion in a vaccine were selected, expressed in Escherichia coli, and purified for use in immunisation experiments. These proteins were detected in at least 40 different serotypes of S. pneumoniae, and were expressed in S. pneumoniae isolates causing infection. Two of the five candidate proteins, the putative lipoate protein ligase (Lpl) and the ClpP protease, resulted in a reduced CFU titre and a trend towards reduced mortality in an animal sepsis model for investigating new S. pneumoniae protein vaccines.

Campylobacter jejuni proteomics for new travellers' diarrhoea vaccines.

Travellers' diarrhoea is defined as diarrhoea that develops while a person is abroad in or shortly after return from a developing country. Different pathogens cause diarrhoea in travellers. Campylobacter jejuni is one of the most prominent agents for this illness. Diarrhoea is defined as an abnormally increased frequency or decreased consistency of stools for less than one week. Antibiotics are effective in preventing travellers' diarrhoea, but routine prophylaxis with antibiotics, should be discouraged. Vaccination is promising but no vaccine against C. jejuni is available at the moment. This article presents the ACE BioSciences strategy for the discovery of protein based vaccine candidates using a cell surface proteomics approach of C. jejuni. New targets for C. jejuni protein vaccines were identified. As proof of concept, we could demonstrate decreased colonization of C. jejuni in mice after vaccination with some of these candidates. It is likely that the proteomics based ACE-Biosciences approach will result in reliable travellers' diarrhoea protein-vaccines in the future.