Osteogenic differentiation of human dental pulp stem cells in decellularised adipose tissue solid foams.

3D cell culture systems based on biological scaffold materials obtainable from both animal and human tissues constitute very interesting tools for cell therapy and personalised medicine applications. The white adipose tissue (AT) extracellular matrix (ECM) is a very promising biomaterial for tissue engineering due to its easy accessibility, malleability and proven biological activity. In the present study, human dental pulp stem cells (hDPSCs) were combined in vitro with ECM scaffolds from porcine and human decellularised adipose tissues (pDAT, hDAT) processed as 3D solid foams, to investigate their effects on the osteogenic differentiation capacity and bone matrix production of hDPSCs, compared to single-protein-based 3D solid foams of collagen type I and conventional 2D tissue-culture-treated polystyrene plates. pDAT solid foams supported the osteogenic differentiation of hDPSCs to similar levels to collagen type I, as assessed by alkaline phosphatase and alizarin red stainings, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and osteocalcin/bone gamma-carboxyglutamate protein (BGLAP) immunostaining. Interestingly, hDAT solid foams showed a markedly lower capacity to sustain hDPSC osteogenic differentiation and matrix calcification and a higher capacity to support adipogenesis, as assessed by RT-qPCR and oil red O staining. White ATs from both human and porcine origins are relatively abundant and available sources of raw material to obtain high quality ECM-derived biomedical products. These biomaterials could have promising applications in tissue engineering and personalised clinical therapy for the healing and regeneration of lesions involving not only a loss of calcified bone but also its associated soft non-calcified tissues.

Adhesion, integration and osteogenesis of human dental pulp stem cells on biomimetic implant surfaces combined with plasma derived products.

Dental implants are the usual therapy of choice in the dental clinic to replace a loss of natural teeth. Over recent decades there has been an important progress in the design and manufacturing of titanium implant surfaces with the goal of improving their osteointegration. In the present work, the aim was to evaluate the usefulness of hDPSCs (human dental pulp stem cells), in combination with autologous plasma components, for in vitro bone generation on biomimetic titanium dental implant materials. In this context, the combination of hDPSCs stimulated by PRGF or PRF and cultured on standard Ti6A14V and biomimetic BAS™ (Avinent Implant System) titanium surfaces were studied in order to evaluate possible enhancements in the osteoblastic differentiation process out of human mesenchymal cells, as well as bone matrix secretion on the implant surface. The results obtained in this in vitro model of osteogenesis suggested a combination of biomimetic rough titanium surfaces, such as BAS™, with autologous plasma-derived fibrin-clot membranes such as PRF and/or insoluble PRGF formulations, but not with an addition of water-soluble supplements of plasma-derived growth factors, to maximise osteoblastic cell differentiation, bone generation, anchorage and osteointegration of titanium-made dental implants.

Notch/Wnt cross-signalling regulates stemness of dental pulp stem cells through expression of neural crest and core pluripotency factors.

Dental pulp stem cells (DPSCs) from adult teeth express neural crest (NC) markers together with core transcriptional factors associated with stem cell pluripotency, such as Oct4a, Sox2, c-Myc, Rex1, Stella/Dppa3, Ssea1/Fut4, Lin28 and Nanog. The possibility to boost the natural stemness features of DPSCs by mild methods, that do not involve gene and/or chromatin modification or gene transfection, is highly desirable for cell therapy. Canonical Wnt and Notch are two highly conserved developmental signalling pathways that are involved in NC emergence and stem cell self-renewal. We determined that both pathways coordinate to regulate the expression of core pluripotency and NC factors in DPSCs. Pharmacological inhibition of the Notch pathway for 48 h, by the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), abolished the expression of NC and core factors. In addition, it induced a silencing of the canonical Wnt signalling and a clear reduction in the stemness potential of DPSCs, as shown by a reduced ability to generate mature, fully differentiated osteoblasts and adipocytes. Conversely, pharmacological activation of the Wnt pathway for 48 h, by either the glycogen synthase kinase 3 beta (GSK3-ß) inhibitor 6-bromoindirubin-3´-oxime (BIO) or the human recombinant protein Wnt-3a, not only largely increased the expression of NC and core factors, but also increased the efficiency of DPSCs to differentiate into mature osteoblasts and adipocytes. These results showed that a short preconditioning activation of Wnt/Notch signalling by small molecules and/or recombinant proteins enhanced the stemness and potency of DPSCs in culture, which could be useful for optimising the therapeutic use of these and other tissue-specific stem cells.

Reduced salivary gland size and increased presence of epithelial progenitor cells in DLK1-deficient mice.

DLK1 (PREF1, pG2, or FA1) is a transmembrane and secreted protein containing epidermal growth factor-like repeats. Dlk1 expression is abundant in many tissues during embryonic and fetal development and is believed to play an important role in the regulation of tissue differentiation and fetal growth. After birth, Dlk1 expression is abolished in most tissues but is possibly reactivated to regulate stem cell activation and responses to injury. We have recently reported that DLK1 regulates many aspects of salivary gland organogenesis. Here, we have extended our studies of the salivary gland phenotype of Dlk1 knock-out mice. We have observed that salivary glands are smaller and weigh significantly less in both Dlk1 knock-out males and females compared with gender and age-matched wild-type mice and regardless of the natural sexual dimorphism in rodent salivary glands. This reduced size correlates with a reduced capacity of Dlk1-deficient mice to secrete saliva after stimulation with pilocarpine. However, histological and ultrastructural analyses of both adult and developing salivary gland tissues have revealed no defects in Dlk1 ((-/-)) mice, indicating that genetic compensation accounts for the relatively mild salivary phenotype in these animals. Finally, despite their lack of severe anomalies, we have found that salivary glands from Dlk1-deficient mice present a higher amount of CK14-positive epithelial progenitors at various developmental stages, suggesting a role for DLK1 in the regulation of salivary epithelial stem cell balance.

Is There Such a Thing as a Genuine Cancer Stem Cell Marker? Perspectives from the Gut, the Brain and the Dental Pulp.

The conversion of healthy stem cells into cancer stem cells (CSCs) is believed to underlie tumor relapse after surgical removal and fuel tumor growth and invasiveness. CSCs often arise from the malignant transformation of resident multipotent stem cells, which are present in most human tissues. Some organs, such as the gut and the brain, can give rise to very aggressive types of cancers, contrary to the dental pulp, which is a tissue with a very remarkable resistance to oncogenesis. In this review, we focus on the similarities and differences between gut, brain and dental pulp stem cells and their related CSCs, placing a particular emphasis on both their shared and distinctive cell markers, including the expression of pluripotency core factors. We discuss some of their similarities and differences with regard to oncogenic signaling, telomerase activity and their intrinsic propensity to degenerate to CSCs. We also explore the characteristics of the events and mutations leading to malignant transformation in each case. Importantly, healthy dental pulp stem cells (DPSCs) share a great deal of features with many of the so far reported CSC phenotypes found in malignant neoplasms. However, there exist literally no reports about the contribution of DPSCs to malignant tumors. This raises the question about the particularities of the dental pulp and what specific barriers to malignancy might be present in the case of this tissue. These notable differences warrant further research to decipher the singular properties of DPSCs that make them resistant to transformation, and to unravel new therapeutic targets to treat deadly tumors.

Bone marrow cells can give rise to ameloblast-like cells.

Post-eruptive loss of ameloblasts requires identification of alternative sources for these cells to realize tooth-tissue-engineering strategies. Recent reports showed that bone-marrow-derived cells can give rise to different types of epithelial cells, suggesting their potential to serve as a source for ameloblasts. To investigate this potential, we mixed c-Kit(+)-enriched bone marrow cells with embryonic dental epithelial cells and cultured them in re-association with dental mesenchyme. Non-dividing, polarized, and secretory ameloblast-like cells were achieved without cell fusion. Before basement membrane reconstitution, some bone marrow cells migrated to the mesenchyme, where they exhibited morphological, molecular, and functional characteristics of odontoblasts. These results show, for the first time, that bone-marrow-derived cells can be reprogrammed to give rise to ameloblast-like cells, offering novel possibilities for tooth-tissue engineering and the study of the simultaneous differentiation of one bone marrow cell subpopulation into cells of two different embryonic lineages.

Dynamic assembly of tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin during mouse tooth development.

Tight junctions might play a role during tissue morphogenesis and cell differentiation. In order to address these questions, we have studied the distribution pattern of the tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin in the developing mouse tooth as a model. A specific temporal and spatial distribution of tight junction-associated proteins during tooth development was observed. ZO-1 appeared discontinuously in the cell membrane of enamel organ and dental mesenchyme cells. However, endothelial cells of the dental mesenchyme capillaries displayed a continuous fluorescence at the cell membrane. Inner dental epithelium first showed an evident signal for ZO-1 at the basal pole of the cells at bud/cap stage, but ZO-1 was accumulated at the basal and apical pole of preameloblast/ameloblasts at late bell stage. Surprisingly, in the incisor ZO-1 decreased as the inner dental epithelium differentiated, and was re-expressed in secretory and mature ameloblasts. On the contrary, ZO-2 was confined to continuous cell-cell contacts of the enamel organ in both molars and incisors. The lateral cell membrane of inner dental epithelial cells was specifically ZO-2 labeled. However, ZO-3 was expressed in oral epithelium whereas dental embryo tissues were negative. In addition, occludin was hardly detected in dental tissues at the early stage of tooth development, but was distributed continuously at the cell membrane of endothelial cells of ED19.5 dental mesenchyme. In incisors, occludin was detected at the cell membrane of the secretory pole of ameloblasts. The occurrence and relation during tooth development of tight junction proteins ZO-1, ZO-2 and occludin, but not ZO-3, suggests a combinatory assembly in tooth morphogenesis and cell differentiation.

Antimetastatic effects of liposome entrapped indomethacin.

The "in vivo"administration of sized liposomes encapsulating indomethacin to mice bearing 3LL tumor, significantly reduced the incidence and/or number of superficial lung metastases. Also liposomes encapsulating indomethacin had significant inhibitory effects on the experimentally induced lung metastases. We conclude: i) indomethacin encapsulated in liposomes is more efficient than the free drug in mediating the antimetastatic effects and ii) liposomes are an valuable vehicle in evading the side metastatic effects of this drug during indomethacin treatment of tumor bearing mice.

Monolignol ferulate transferase introduces chemically labile linkages into the lignin backbone.

Redesigning lignin, the aromatic polymer fortifying plant cell walls, to be more amenable to chemical depolymerization can lower the energy required for industrial processing. We have engineered poplar trees to introduce ester linkages into the lignin polymer backbone by augmenting the monomer pool with monolignol ferulate conjugates. Herein, we describe the isolation of a transferase gene capable of forming these conjugates and its xylem-specific introduction into poplar. Enzyme kinetics, in planta expression, lignin structural analysis, and improved cell wall digestibility after mild alkaline pretreatment demonstrate that these trees produce the monolignol ferulate conjugates, export them to the wall, and use them during lignification. Tailoring plants to use such conjugates during cell wall biosynthesis is a promising way to produce plants that are designed for deconstruction.

In vivo delivery of human alpha 1-antitrypsin gene to mouse hepatocytes by liposomes.

The pTG7101 plasmid containing the full length human alpha 1-Antitrypsin was encapsulated in large (142 +/- 15 nm of diameter) and small (54 +/- 11 nm of diameter) liposomes and administered i.v. to mice (80 ng/mouse). Control animals were treated with empty (small and large) liposomes plus free DNA and with the liposome solvent buffer. The immunohistochemical results on liver cryosections and cytophotometric analysis of hepatocyte chromophore absorbance, after peroxidase reaction, indicated that significant presence of immunoreactive human alpha 1-antitrypsin was present 7 days after mice treatment with encapsulated DNA in small liposomes but not when large liposomes were used. This effect was observed in a great number of liver parenchymal cells. These results agree with the observation that only small liposomes have easy access to hepatocytes and support the idea that small liposomes are appropriate vehicles for in vivo delivery of specific genetic material to liver parenchymal cells, with high efficiency.

Effects of aFGF, bFGF, TGFbeta1 and IGF-I on odontoblast differentiation in vitro.

In this work, we investigated the effects of aFGF and bFGF alone or combined with TGFbeta1 or IGF-I on odontoblast differentiation. Trypsin-isolated dental papillae from day 17 mandibular first molar were cultured in semisolid-agar medium for 6 d. Our results demonstrated that aFGF, bFGF or combinations of these promoted cell polarization at the periphery of the dental papillae. Moreover, simultaneous addition of aFGF and TGFbeta1 to dental papillae cultures induced both polarization and functional differentiation of odontoblast-like cells, as well as extracellular matrix deposition. Combination of aFGF or bFGF with IGF-I caused cell polarization at the surface of dental papillae, but matrix secretion was restricted to a few explants. In the presence of bFGF and TGFbeta1, the explants had pronounced cell elongations but no matrix deposition. These results indicate that aFGF or bFGF is not able to induce odontoblast differentiation alone. However, both aFGF and bFGF can act synergistically with TGFbeta1 and IGF-I to strengthen their inductive effects and promote gradients of cytological and functional changes in odontoblast-like cells.

FGFs-1 and -2, and TGF beta 1 as inductive signals modulating in vitro odontoblast differentiation.

We have studied the expression of FGF1 and FGF2 during mouse odontogenesis by immunohistochemistry. FGF1 was detected in differentiated odontoblasts and at the secretory pole of ameloblasts. Localization of FGF2 was mainly observed within the basement membrane interposed between dental epithelium and dental mesenchyme. These findings indicate that FGF1 and FGF2 may participate in the control of odontoblast and ameloblast differentiation. Thereafter, we studied the ability of FGF1 and FGF2, alone or in combination with TGF beta 1, to induce polarization and/or functional differentiation of preodontoblasts. Dental papillae (DP) obtained from first lower molars of 17-day-old mouse embryo were cultured in the presence or the absence of growth factors. DP cultured with FGF1 + TGF beta 1 showed gradients of odontoblast-like cell differentiation, which displayed alkaline phosphatase reactivity. DP treated with FGF2 + TGF beta 1 exhibited pre-odontoblast cell polarization, and the cell bodies displayed long cytoplasm processes. However, following this treatment we did not observe extracellular matrix secretion, and alkaline phosphatase activity was completely inhibited. In summary, our results show that exogenous addition of FGF1 to pre-odontoblasts induces their terminal differentiation, by synergistically acting with TGF beta 1. In contrast, FGF2 may regulate the effect of TGF beta 1, permitting cell polarization but restraining pre-odontoblast functions.

Recombinant cDNA encapsulation in small liposomes with hepatocyte access ability.

Liposomal encapsulation efficiency of a recombinant cDNA was studied by several procedures. We observed that supernatant fraction of ultracentrifuged liposomes prepared by extrusion through polycarbonate filters of 400 nm pore size yielded a very homogeneous suspension of small (50 nm diameter) unilamellar liposomes with highest DNA/lipid ratio and great ability to access to hepatocytes.

Dissection of the odontoblast differentiation process in vitro by a combination of FGF1, FGF2, and TGFbeta1.

Dental papillae (DP) isolated from first lower molars of 17-day-old mouse embryos were cultured in the presence of combinations of the following growth factors: FGF1, FGF2, and TGFbeta1. After 6 days in culture, only the DP treated with FGF1+TGFbeta1 contained differentiated odontoblast-like cells at the periphery of the explants, and these cells secreted extracellular matrix similar to predentin. Surprisingly, treatments with FGF2+TGFbeta1 induced cell polarization at the surface of the explants but no matrix secretion was observed. Electron microscopy and histochemical analysis of odontoblast markers showed that differentiated cells induced by FGF1+TGFbeta1 exhibited cytological features of functional odontoblasts with matrix vesicle secretion and mineral formation, positive alkaline-phosphatase activity, and type-I collagen production. DP cultured in the presence of FGF2+TGFbeta1 showed cell polarization and long and thin cell processes containing matrix vesicles; however, type-I collagen secretion was not detected and alkaline-phosphatase activity was completely inhibited. Our results indicate that, in our culture system, exogenous combinations of FGF1, FGF2, and TGFbeta1 interact with preodontoblasts and induce cell polarization or differentiation, which can be studied separately in vitro. Thus, FGF1 and TGFbeta1 do have a synergic effect to promote morphological and functional features of differentiated odontoblasts whereas FGF2 seems to modulate TGFbeta1 action, causing morphological polarization of preodontoblasts but limiting the functional activity of these cells in terms of type-I collagen secretion and alkaline-phosphatase activity.

Analysis of cDNAs from a mouse embryo tooth library: identification of novel genes during tooth development.

Signaling molecules, their receptors, and target genes from pathways and networks regulate the development of the tooth from initiation through cell differentiation. In order to identify genes involved in odontoblast and ameloblast differentiation, we constructed a cDNA library from E19.5 mouse molars. In this work, we report the partial cDNA sequences of 10 noncharacterized genes and we show cell expression of the transcripts on mouse embryo molars by in situ hybridization.

Efficacy of liposome-encapsulated indomethacin in response against metastatic 3LL and B16F1 tumor cells.

The ability of large liposomes to be taken up by tissue phagocytic cells, e.g., macrophages, has made it possible to increase the efficacy of several drugs as immunomodulating agents. In the present work, we have evaluated the effect of indomethacin, a prostaglandin synthesis inhibitor, both free and encapsulated in liposomes, on the spontaneous metastatic potential of 3LL and B16F1 tumor cells. Liposomes containing either carboxyfluorescein, indomethacin, or carboxyfluorescein plus indomethacin, were made in order to evaluate their in vitro plasma stability and in vivo clearance from the blood. The liposomes showed a high stability after 6 hours of plasma incubation and they were rapidly cleared in vivo. Liposomes encapsulating propidium iodide, a fluorescent DNA binding dye, were mainly taken up in vivo by hepatic and spleen macrophages 1 hour after intravenous injection, but not by lung macrophages. When C57BL/6 mice were intravenously inoculated with 10(5) 3LL or B16F1 tumor cells previously incubated with indomethacin (10(-7) M) for 48 hours, the number of experimental lung metastatic foci was increased with respect to their respective control groups. Also, in 3LL or B16F1 tumor-bearing mice, treatment with indomethacin (0.5 mg/kg weight/day) for 10 days enhanced the number of lung metastases, but not significantly. However, when mice received indomethacin encapsulated in liposomes, the number of metastases was significantly reduced. In addition, encapsulated indomethacin (0.5 mg/kg weight/day) inhibits prostaglandin E2 production by peritoneal and spleen macrophages, whereas no significant inhibitory effect was observed with control-liposomes or equivalent doses of free indomethacin. We conclude that intravenous administration of liposome-encapsulated indomethacin has an antimetastatic effect on tumor-bearing mice. Use of indomethacin in liposomes may avoid the stimulation of metastases observed when the drug is administered alone.