Renata Vecchiatini

Role of Slug transcription factor in human mesenchymal stem cells

The pathways that control mesenchymal stem cells (MSCs) differentiation are not well understood, and although some of the involved transcription factors (TFs) have been characterized, the role of others remains unclear. We used human MSCs from tibial plateau (TP) trabecular bone, iliac crest (IC) bone marrow and Wharton’s jelly (WJ) umbilical cord demonstrating a variability in their mineral matrix deposition, and in the expression levels of TFs including Runx2, Sox9, Sox5, Sox6, STAT1 and Slug, all involved in the control of osteochondroprogenitors differentiation program. Because we reasoned that the basal expression level of some TFs with crucial role in the control of MSC fate may be correlated with osteogenic potential, we considered the possibility to affect the hMSCs behaviour by using gene silencing approach without exposing cells to induction media. In this study we found that Slug‐silenced cells changed in morphology, decreased in their migration ability, increased Sox9 and Sox5 and decreased Sox6 and STAT1 expression. On the contrary, the effect of Slug depletion on Runx2 was influenced by cell type. Interestingly, we demonstrated a direct in vivo regulatory action of Slug by chromatin immunoprecipitation, showing a specific recruitment of this TF in the promoter of Runx2 and Sox9 genes. As a whole, our findings have important potential implication on bone tissue engineering applications, reinforcing the concept that manipulation of specific TF expression levels may elucidate MSC biology and the molecular mechanisms, which promote osteogenic differentiation.

Production of polymeric micelles by microfluidic technology for combined drug delivery: Application to osteogenic differentiation of human periodontal ligament mesenchymal stem cells (hPDLSCs)

The current paper reports the production of polymeric micelles (PMs), based on pluronic block-copolymers, as drug carriers, precisely controlling the cellular delivery of drugs with various physico-chemical characteristics. PMs were produced with a microfluidic platform to exploit further control on the size characteristic of the PMs. PMs were designed for the co-delivery of dexamethasone (Dex) and ascorbyl-palmitate (AP) to in vitro cultured human periodontal ligament mesenchymal stem cells (hPDLSCs) for the combined induction of osteogenic differentiation. Mixtures of block-copolymers and drugs in organic, water miscible solvent, were conveniently converted in PMs within microfluidic channel leveraging the fast mixing at the microscale. Our results demonstrated that the drugs can be efficiently co-encapsulated in PMs and that different production parameters can be adjusted in order to modulate the PM characteristics. The comparative analysis of PM produced by microfluidic and conventional procedures confirmed that the use of microfluidics platforms allowed the production of PMs in a robust manner with improved controllability, reproducibility, smaller size and polydispersity. Finally, the analysis of the effect of PMs, containing Dex and AP, on the osteogenic differentiation of hPDLSCs is reported. The data demonstrated the effectiveness and safety of PM treatment on hPDLSC. In conclusion, this report indicates that microfluidic approach represents an innovative and useful method for PM controlled preparation, warrant further evaluation as general methodology for the production of colloidal systems for the simultaneous drug delivery.

Human mesenchymal stem cells seeded on extracellular matrix-scaffold: Viability and osteogenic potential

The development and the optimization of novel culture systems of mesenchymal osteoprogenitors are some of the most important challenges in the field of bone tissue engineering (TE). A new combination between cells and extracellular matrix (ECM)‐scaffold, containing ECM has here been analyzed. As source for osteoprogenitors, mesenchymal stem cells obtained from human umbilical cord Whartons Jelly (hWJMSCs), were used. As ECM‐scaffold, a powder form of isolated and purified porcine urinary bladder matrix (pUBM), was employed. The goals of the current work were: (1) the characterization of the in vitro hWJMSCs behavior, in terms of viability, proliferation, and adhesion to ECM‐scaffold; (2) the effectiveness of ECM‐scaffold to induce/modulate the osteoblastic differentiation; and (3) the proposal for a possible application of cells/ECM‐scaffold construct to the field of cell/TE. In this respect, the properties of the pUBM‐scaffold in promoting and guiding the in vitro adhesion, proliferation, and three‐dimensional colonization of hWJMSCs, without altering viability and morphological characteristics of the cells, are here described. Finally, we have also demonstrated that pUBM‐scaffolds positively affect the expression of typical osteoblastic markers in hWJMSCs. J. Cell. Physiol. 227: 857–866, 2012.

Influence of obstetric factors on osteogenic potential of umbilical cord-derived mesenchymal stem cells

Whartons jelly from the umbilical cord is a noncontroversial source of mesenchymal stem cells (WJMSCs) with high plasticity, proliferation rate and ability to differentiate towards multiple lineages. WJMSCs from different donors have been characterized for their osteogenic potential. Although there is large evidence of WJMSCs plasticity, recently scientific debate has focused on MSCs selection, establishing predictable elements to discriminate the cells with most promising osteoprogenitor cell potential.In the present study a comparative study between the presence of osteoblastic markers and different parameters that pertain to both the newborn and the mother was performed. Umbilical cords were collected after all patients signed the informed consent and local ethical commettee approved the study. Obstetric parameters, including babys gender and birth weight, mothers age at delivery, gestational stage at parturition and mode of delivery were examined. After characterization and expansion, WJMSCs were analyzed for two osteoblastic markers, alkaline phosphatase (ALP) activity, and the expression level of RUNX-2 transcription factor, and for their ability to deposit mineralized matrix after osteogenic induction.We found that osteoblastic potential was not influenced by babys gender and mode of delivery. On the contrary, the highest degree of osteoblastic potential has been shown by WJMSCs with RUNX-2 high basal levels, selected from umbilical cords of the heaviest term babies.Even if further evaluation is required, our hypothesis is that our findings may help in selecting the optimal umbilical cord donors and in collecting high potential Whartons jelly-derived osteoprogenitors efficiently.

Effect of dynamic three‐dimensional culture on osteogenic potential of human periodontal ligament‐derived mesenchymal stem cells entrapped in alginate microbeads

BACKGROUND AND OBJECTIVE Bioreactors are devices that efficiently create an environment that enables cell cultures to grow in a three-dimensional (3D) context mimicking in vivo conditions. In this study, we investigate the effect of dynamic fluid flow on the osteogenic potential of human mesenchymal stem cells obtained from periodontal ligament and entrapped in alginate microbeads. MATERIAL AND METHODS After proper immunophenotyping, cells were encapsulated in barium alginate, cultured in 3D static or 3D dynamic conditions represented by a bioreactor system. Calcein-AM/propidium iodide staining was used to assess cellular viability. Quantitative real-time polymerase chain reaction was used to analyze the expression of osteogenic markers (Runx2 and COL1). Alizarin Red S staining and the Fourier transform infrared spectroscopy were used to assess mineral matrix deposition. RESULTS Optimal encapsulation procedure, in terms of polymer pumping rate, distance from droplet generator to the gelling bath and atomizing airflow was assessed. Cell viability was not affected by encapsulation in alginate microbeads. Bioreactor cell exposure was effective in anticipating osteogenic differentiation and improving mineral matrix deposition. CONCLUSION For the first time human mesenchymal stem cells obtained from periodontal ligaments encapsulated in alginate microbeads were cultured in a bioreactor system. This combination could represent a promising strategy to create a cell-based smart system with enhanced osteogenic potential useful for many different dental applications.

Explaining pain after lower third molar extraction by preoperative pain assessment.

PURPOSE To evaluate whether preoperative pain sensitivity testing and emotional perception of pain could explain the level of postoperative pain after lower third molar extraction. PATIENTS AND METHODS Twenty-three patients (16 women, 7 men) scheduled for lower third molar extraction were enrolled in the study. Patients preoperatively were submitted to a nociceptive stimulus by a cold pressor test (immersion of the hand into ice water). Preoperative pain tolerance (seconds), algosity and unpleasantness (visual analog scale), and dental anxiety (Modified Dental Anxiety Scale) were assessed. The duration of surgery was recorded (minutes). Postoperative pain ratings were taken by self-reported registrations on a 100-mm visual analog scale during the 6 days after surgery. Separate stepwise regression analyses were performed to evaluate the usefulness of preoperative scores in explaining the overall maximum postoperative pain level and postoperative pain rates at different intervals. RESULTS Preoperative unpleasantness related to the nociceptive stimulus was found to be the best predictor of maximum postoperative pain (adjusted R(2) = 0.39, P = .001). Demographic information (age) and preoperative (dental anxiety, pain tolerance, algosity) and intraoperative (duration of surgery) factors were not correlated with postoperative pain. CONCLUSIONS These results show that a simple preoperative test is useful to identify patients at risk of developing greater pain after third molar surgery. They are characterized by a higher level of reported pain or unpleasantness after exposure to a nociceptive stimulus. This test may be tailored to specific patient needs for postoperative treatment.

Transcription factor decoy against NFATc1 in human primary osteoblasts

The present study describes, for the first time, the removal of the nuclear factor of activated T cells cytoplasmic 1 (NFATc1) by a decoy approach in human primary osteoblasts (hOBs). hOBs with different NFATc1 expression levels were used. The functionality of endogenous NFAT proteins in our experimental model was analyzed by monitoring the transcriptional activity on a luciferase reporter construct driven by three copies of an NFAT response element (pNFAT-TA-luc). Cell treatment with the decoy against NFATc1 resulted in a significant increase in the expression of osteoblastic markers, including ERα and ColXV. On the contrary, the expression of Runx2, which is known to not be transcriptionally regulated by NFATc1, was not altered, indicating the specificity of the decoy effect. To our knowledge, this is the first time that transcription factor decoy has been successful in hOBs to allow the investigation of the role of NFATc1 in an experimental model that, compared to the use of cell lines, more closely resembles an in vivo model. In addition, by using chromatin immunoprecipitation we found that in vivo NFATc1 is recruited on the ColXV gene promoter. The specific role of NFATc1 in osteoblast differentiation is not well understood, however, our findings reinforce the action of NFATc1 in the transcriptional program of osteoblasts, also supporting the therapeutic potential for the proper manipulation of NFATc1-mediated events in different bone disorders. At the same time, our data add important information on the regulation of the expression of ColXV, which only recently has been proposed as an osteoblastic marker.

Milled bar-supported implant overdenture after mandibular resection: a case report.

After surgical treatment for oral cancer, patients often are affected by disfigurements, thwarted function, and psychological and social problems. Prosthodontic rehabilitation has the aim of restoring function and esthetics. Implant-supported prosthodontic rehabilitation is useful for patients with compromised residual ridge anatomy, such as patients with oral cancer following treatment. This clinical report describes the rehabilitation of a patient after mandibular resection with a milled bar-supported implant overdenture. Overdenture achieves best hygienic maintenance, easy soft tissue follow-up, and low realization cost. This rehabilitation increased prosthesis retention and stability and improved oral conditions and the patients quality of life.

Microencapsulation Procedures for the Immunoisolation of Wharton’s Jelly Mesenchymal Stem Cells: A Review

The entrapment of cells is one of the most promising and usefulness tool in tissue transplantation and regenerative medicine. Cell encapsulation procedures allow the physical isolation of cells from the surrounding environment, after their transplantation and the maintenance of the normal cellular physiology. In this paper, different microencapsulation procedures for Wharton’s Jelly Mesenchymal Stem Cells (WJMSCs) are reported, including coaxial bead generator, vibrating-nozzle procedure and microfluidic based approach. The produced microcapsules were characterized by excellent morphological characteristics and a very narrow size distribution. The experiments demonstrated that the microencapsulation procedure did not alter the morphology, viability and osteogenic differentiation of the enveloped WJMSCs. In conclusion, the encapsulation technologies, here presented, represent a promising strategy for the possible in vivo applications of WJMSCs in tissue engineering and biomedicine.

Osteogenic potential of cells derived from nasal septum

BACKGROUND The research addressed to detect new molecular targets in the development of therapeutic strategies aimed to repair bone tissues. The AIM OF THIS STUDY was to determine the potential osteogenic activity of bone cells from the nasal septum and their use to perform accurate molecular analysis from a single sample. METHODOLOGY The cells, after nasal septum surgery, were subjected to gene silencing, Reverse Transcriptase - Polymerase Chain reactions, immunocytochemistry and chromatin immunoprecipitation. RESULTS Cells from the nasal septum can give rise to mature osteoblasts that express osteogenic markers (ALP, Runx2, Slug) and are able to mineralize. We demonstrated that Runx2, a transcription factor critical in early osteospecific differentiation, interacts in vivo with the promoter of the SLUG gene, a marker of osteoblast maturation. CONCLUSIONS We demonstrated that nasal septum-derived osteoblasts represent an interesting alternative source for bone forming cells, and a promising material to be utilized in bone cellular therapy.