Mesenchymal dental stem cells in regenerative dentistry

In the last decade, tissue engineering is a field that has been suffering an enormous expansion in the regenerative medicine and dentistry. The use of cells as mesenchymal dental stem cells of easy access for dentist and oral surgeon, immunosuppressive properties, high proliferation and capacity to differentiate into odontoblasts, cementoblasts, osteoblasts and other cells implicated in the teeth, suppose a good perspective of future in the clinical dentistry. However, is necessary advance in the known of growth factors and signalling molecules implicated in tooth development and regeneration of different structures of teeth. Furthermore, these cells need a fabulous scaffold that facility their integration, differentiation, matrix synthesis and promote multiple specific interactions between cells. In this review, we give a brief description of tooth development and anatomy, definition and classification of stem cells, with special attention of mesenchymal stem cells, commonly used in the cellular therapy for their trasdifferentiation ability, non ethical problems and acceptable results in preliminary clinical trials. In terms of tissue engineering, we provide an overview of different types of mesenchymal stem cells that have been isolated from teeth, including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells (PDLSCs), dental follicle progenitor stem cells (DFPCs), and stem cells from apical papilla (SCAPs), growth factors implicated in regeneration teeth and types of scaffolds for dental tissue regeneration

Amniotic Membrane Modifies the Genetic Program Induced by TGFß, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds

<div><p>Background</p><p>Post-traumatic large-surface or deep wounds often cannot progress to reepithelialisation because they become irresponsive in the inflammatory stage, so intervention is necessary to provide the final sealing epidermis. Previously we have shown that Amniotic Membrane (AM) induced a robust epithelialisation in deep traumatic wounds.</p><p>Methods and Findings</p><p>To better understand this phenomenon, we used keratinocytes to investigate the effect of AM on chronic wounds. Using keratinocytes, we saw that AM treatment is able to exert an attenuating effect upon Smad2 and Smad3 TGFß-induced phosphorylation while triggering the activation of several MAPK signalling pathways, including ERK and JNK1, 2. This also has a consequence for TGFß-induced regulation on cell cycle control key players CDK1A (p21) and CDK2B (p15). The study of a wider set of TGFß regulated genes showed that the effect of AM was not wide but very concrete for some genes. TGFß exerted a powerful cell cycle arrest; the presence of AM however prevented TGFß-induced cell cycle arrest. Moreover, AM induced a powerful cell migration response that correlates well with the expression of c-Jun protein at the border of the healing assay. Consistently, the treatment with AM of human chronic wounds induced a robust expression of c-Jun at the wound border.</p><p>Conclusions</p><p>The effect of AM on the modulation of TGFß responses in keratinocytes that favours proliferation together with AM-induced keratinocyte migration is the perfect match that allows chronic wounds to move on from their non-healing state and progress into epithelialization. Our results may explain why the application of AM on chronic wounds is able to promote epithelialisation.</p></div

AM modified the genetic response induced by TGFß in HaCaT cells.

<p>Several TGFß inducible genes were measured in HaCaT cells stimulated with TGFß or TGFß and AM. (A), isolated RNA from HaCaT stimulated with AM, TGFß, or both was analyzed by qPCR, represented as a ratio to <i>GAPDH</i> and represented as fold change of the untreated control sample. (B), isolated RNA from HaCaT stimulated for 24 h with AM and TGFß for the indicated times, or only with TGFß was analysed by qPCR, represented as a ratio to <i>GAPDH</i> and represented as fold change of the untreated control sample. The asterisks denote statistically significant differences between the treatments according Student’s <i>t</i>-test. *p<0.05, **p<0.005 and ***p<0.001, ****p<0.0001.</p

In HaCaT cells, AM induced motility and the expression of c-Jun at the migratory front.

<p>Wound healing scratch assay was performed in HaCaT cells in the presence of AM, EGF or combinations of AM with different inhibitors. (A), cells forming a confluent epithelium were wounded and immediately treated as indicated for 21 h. Representative pictures were taken at the beginning of the treatment and 21 h later. (B), treatment of HaCaT cells with AM caused the cells to express c-Jun at the migratory front. Wound healing scratch assay was treated with AM, EGF or combinations of AM with different inhibitors. Cells were wounded and treated for 24 h, afterwards cells were fixed and immunostained for c-Jun. Images of c-Jun fluorescence were converted into pseudo-colour to show the intensity of c-Jun staining. Colour rainbow scale represents fluorescence intensity for c-Jun. Co-staining with phalloidin and Hoechst-33258 was used to show cells structure and nuclei, respectively. Images were taken by confocal microscopy using a Zeiss 510 LSM confocal microscope. This experiment was repeated at least three times. A representative result is shown. (C), HaCaT cells forming a confluent epithelium were treated with Mitomycin C, wounded and immediately treated for 21 h as indicated. Results were compared to non-treated cells. Representative pictures were taken at the beginning of the treatment and 21 h later. Scale Bars 100 μm.</p

AM modified the genetic response induced by TGFß in human primary keratinocytes.

<p>Several TGFß inducible genes were measured in human primary keratinocytes in response to TGFß or in response to the combined treatment of TGFß and AM. Isolated RNA from primary keratinocytes stimulated for 24 h with AM and TGFß for the indicated times, or only with TGFß was analysed by qPCR, represented as a ratio to <i>GAPDH</i> and represented as a fold change of the untreated control sample. The asterisks denote statistically significant differences between the treatments according Student’s <i>t</i>-test. *p<0.05, **p<0.005 and ***p<0.001, ****p<0.0001.</p

AM attenuated cell cycle proliferation arrest of TGFß on HaCaT cells and induced the expression of c-Jun protein in HaCaT and in human primary keratinocytes.

<p>Treatment of HaCaT cells with AM attenuates TGFß-induced cell cycle arrest in G1. (A), Cell cycle analysis of HaCaT cells in different conditions, treatment with AM, combined with serum starvation (SS) or TGFß is indicated. The histogram shows cells at G0/G1, S or G2/M stage respectively. AM induced the expression of c-Jun in HaCaT and human primary keratinocytes in clear synergy with TGFß. (B), HaCaT cells or, (D), human primary keratinocytes were stimulated with AM, TGFß or both simultaneously for the indicated times. Additionally, (C), HaCaT cells, or, (E), human primary keratinocytes, were stimulated for 24 h with AM and then treated with TGFß for the indicated times, as a control non treated cells were used. Indicated proteins were detected by Western blot. Grb2 or Zo-1 were used as loading controls where indicated. This experiment was performed at least three times. A representative result is shown.</p

Expression of c-Jun at the epidermal leading edge.

<p>Histopathological study of AM induced epithelialisation from a patient´s wound that had been treated with AM. (A), microscopic section from wound border before AM treatment. (B), and (C), microscopic section of the wound border 5 and 15 days after AM treatment, respectively. Insets in Fig A to C show roughly the area where confocal microscopy images have been taken. (D), to (F), microscopic sections were also immune-stained against c-Jun (green) and F-Actin (red). Cell nuclei were revealed by Hoechst-33258 staining. (D), same as in A; (E), same as in (B) and (F), same as in (C). Arrows in (E) and (F) point to the epidermal leading edge. Several patients were analysed in this experiment, single patient data is shown for illustrative purposes. Images were taken by confocal microscopy using a Zeiss 510 LSM confocal microscope.</p