Magnetic resonance angiography for free fibula harvest: anatomy and perforator mapping.

The purpose of this study was to outline lower leg vessel anatomy and to investigate reliability and limitations of magnetic resonance angiography (MRA) in patients proposed for microvascular fibula transplantation (free fibula flap (FFF)). We retrospectively investigated MRAs of 99 patients considered for FFF. Frontal MRA planes and maximal intensity projections (MIPs) were evaluated for fibula lengths, anatomical branching pattern, arterial stenoses and fibular perforator positions in both legs (n=198). Normal branching patterns were observed in 168 (85.3%) legs. Twenty-nine (14.7%) legs presented abnormal branching patterns. Once (0.5%) the anterior, 19 times (9.6%) the posterior tibial artery were absent or hypoplastic. Nine (4.6%) lower legs presented an arteria peronea magna. Average length of the tibiofibular trunk (TFT) was 3.3±0.15cm. A total of 492 perforators were found with an average of 2.5 (±0.82±0.99) perforators per leg. A mapping of perforator run-offs was illustrated true to scale. Lower limb stenoses were distributed in the anterior tibial artery (14.1%), in the posterior tibial artery (11.1%) and in the fibular artery (8.1%). Smoking (P=0.828), diabetes (P=0.727) and peripheral arterial occlusive disease (P=0.172) did not correlate with presence of stenoses. Preoperative lower limb angiography avoids postoperative complications. MRA reliably and non-invasively identifies anatomical variants and arterial stenoses without radiation. Illustration of perforator run-offs enhances incision planning for fibula harvest.

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.