The human dental apical papilla promotes spinal cord repair through a paracrine mechanism.

Traumatic spinal cord injury is an overwhelming condition that strongly and suddenly impacts the patient's life and her/his entourage. There are currently no predictable treatments to repair the spinal cord, while many strategies are proposed and evaluated by researchers throughout the world. One of the most promising avenues is the transplantation of stem cells, although its therapeutic efficiency is limited by several factors, among which cell survival at the lesion site. In our previous study, we showed that the implantation of a human dental apical papilla, residence of stem cells of the apical papilla (SCAP), supported functional recovery in a rat model of spinal cord hemisection. In this study, we employed protein multiplex, immunohistochemistry, cytokine arrays, RT- qPCR, and RNAseq technology to decipher the mechanism by which the dental papilla promotes repair of the injured spinal cord. We found that the apical papilla reduced inflammation at the lesion site, had a neuroprotective effect on motoneurons, and increased the apoptosis of activated macrophages/ microglia. This therapeutic effect is likely driven by the secretome of the implanted papilla since it is known to secrete an entourage of immunomodulatory or pro-angiogenic factors. Therefore, we hypothesize that the secreted molecules were mainly produced by SCAP, and that by anchoring and protecting them, the human papilla provides a protective niche ensuring that SCAP could exert their therapeutic actions. Therapeutic abilities of the papilla were demonstrated in the scope of spinal cord injury but could very well be beneficial to other types of tissue.

The combined administration of LNC-encapsulated retinoic acid and calcitriol stimulates oligodendrocyte progenitor cell differentiation in vitro and in vivo after intranasal administration.

Intranasal administration is an efficient strategy for bypassing the BBB, favoring drug accumulation in the brain, and improving its efficiency. Lipid nanocapsules (LNC) are suitable nanocarriers for the delivery of lipophilic drugs via this route and can be used to encapsulate lipophilic molecules such as retinoic acid (RA) and calcitriol (Cal). As the hallmarks of multiple sclerosis (MS) are neuroinflammation and oligodendrocyte loss, our hypothesis was that by combining two molecules known for their pro-differentiating properties, encapsulated in LNC, and delivered by intranasal administration, we would stimulate oligodendrocyte progenitor cells (OPC) differentiation into oligodendrocytes and provide a new pro-remyelinating therapy. LNC loaded with RA (LNC-RA) and Cal (LNC-Cal) were stable for at least 8 weeks. The combination of RA and Cal was more efficient than the molecules alone, encapsulated or not, on OPC differentiation in vitro and decreased microglia cell activation in a dose-dependent manner. After the combined intranasal administration of LNC-RA and LNC-Cal in a mouse cuprizone model of demyelination, increased MBP staining was observed in the corpus callosum. In conclusion, intranasal delivery of lipophilic drugs encapsulated in LNC is a promising strategy for myelinating therapies.

Quality of Cure in Depth of Commercially Available Bulk-fill Composites: A Layer-by-layer Mechanical and Biological Evaluation.

Despite their popularity, the use of bulk-fill composites remains controversial, both in terms of their properties and their in-depth development. The objectives of the present work were (1) to provide a more comprehensive evaluation of the quality of cure in depth of commercially available bulk-fill composites by combining various key mechanical and biological characterization methods, (2) to evaluate the inter-material differences when optimally cured, and (3) to evaluate the efficiency of an antioxidant-N-acetyl-cysteine (NAC)-to restrain the adverse effects of the leached components on cell viability. Nine bulk-fill composites (including flowable and high-viscosity materials) were investigated and compared to two conventional resin-based composites, one flowable and one high-viscosity restorative material. The materials were injected or packed into Teflon molds of various configurations, up to 6 mm material thickness. They were then light-cured from the top for 20 seconds with Bluephase G2 (Ivoclar Vivadent, irradiance = 1050 mW/cm2). The following physico-mechanical properties were measured for the upper (0-2 mm), intermediate (2-4 mm), and lower (4-6 mm) layers: degree of conversion using Raman Spectrometry (DC, in %), microhardness using a Vickers micro-indenter before (VHN dry) and after 24 hours of storage in ethanol (VHN EtOH), and flexural strength (in MPa) and flexural modulus (in GPa) using a three-point bend test. Each composite layer and an uncured layer were also stored for one week in a standard cell growth medium to generate conditioned media. Human dental pulp cells were then cultured for 24 hours with the latter and cell viability was measured using an MTS assay. A similar experiment was repeated with conditioned media produced in contact with uncured composites, with and without the addition of 4 mM NAC. The data were subjected to a Shapiro-Wilk test, then one-way ANOVA or Kruskal-Wallis test, followed either by Tukey's test (inter-material comparison) or by Dunnett's or Dunn's test (comparison between layers relative to the upper one). The level of statistical significance was set at 0.05. Some materials (EverX, X-traF, VenusBF, X-traB) did not show any significant differences (p>0.05) for any of the properties considered between the intermediate layers compared to the upper one (considered as reference). Others displayed significant differences, at least for some properties, highlighting the value of combining various key mechanical and biological characterization methods when investigating the quality of cure in depth. Significant inter-material differences (p<0.05) were observed when comparing the properties of their upper layer, considered as "optimally" polymerized. Hence, one needs to consider the absolute property values, not only their relative evolution concerning layer thickness. Finally, the use of NAC appeared as beneficial to reduce the risk of harmful effects to dental pulp cells, especially in case of excessive thickness use, and may therefore be of potential interest as an additive to composites in the future.

Dental Apical Papilla as Therapy for Spinal Cord Injury.

Stem cells of the apical papilla (SCAP) represent great promise regarding treatment of neural tissue damage, such as spinal cord injury (SCI). They derive from the neural crest, express numerous neurogenic markers, and mediate neurite outgrowth and axonal targeting. The goal of the present work was to investigate for the first time their potential to promote motor recovery after SCI in a rat hemisection model when delivered in their original stem cell niche-that is, by transplantation of the human apical papilla tissue itself into the lesion. Control groups consisted of animals subjected to laminectomy only (shams) and to lesion either untreated or injected with a fibrin hydrogel with or without human SCAP. Basso-Beattie-Bresnahan locomotor scores at 1 and 3 d postsurgery confirmed early functional decline in all SCI groups. This significant impairment was reversed, as seen in CatWalk analyses, after transplantation of apical papilla into the injured spinal cord wound, whereas the other groups demonstrated persistent functional impairment. Moreover, tactile allodynia did not develop as an unwanted side effect in any of the groups, even though the SCAP hydrogel group showed higher expression of the microglial marker Iba-1, which has been frequently associated with allodynia. Notably, the apical papilla transplant group presented with reduced Iba-1 expression level. Masson trichrome and human mitochondria staining showed the preservation of the apical papilla integrity and the presence of numerous human cells, while human cells could no longer be detected in the SCAP hydrogel group at the 6-wk postsurgery time point. Altogether, our data suggest that the transplantation of a human apical papilla at the lesion site improves gait in spinally injured rats and reduces glial reactivity. It also underlines the potential interest for the application of delivering SCAP in their original niche, as compared with use of a fibrin hydrogel.

In vitro identification of targeting ligands of human M cells by phage display.

To improve transport of vaccine-loaded nanoparticles, the phage display technology was used to identify novel lead peptides targeting human M cells. Using an in vitro model of the human follicle-associated epithelium (FAE) which contains both Caco-2 and M cells, a T7 phage display library was screened for its ability either to bind the apical cell surface of or to undergo transcytosis across Caco-2 cells or FAE. The selection for transcytosis across both enterocytes and FAE identified three different peptide sequences (CTGKSC, PAVLG and LRVG) with high frequency. CTGKSC and LRVG sequences enhanced phage transport across M-like cells. When polymeric nanoparticles were grafted with the sequences CTGKSC and LRVG, their transport by FAE was significantly enhanced. These peptides could therefore be used to enhance the transport of vaccine-loaded nanoparticles across the intestinal mucosal barrier.

Transport mechanisms of mmePEG750P(CL-co-TMC) polymeric micelles across the intestinal barrier.

Monomethylether poly(ethyleneglycol)(750)-poly(caprolactone-co-trimethylene carbonate) (mmePEG750)P(CL-co-TMC)) which spontaneously form micelles, can cross lipid bilayers via passive diffusion and demonstrate an oral bioavailability of 40% in rats. The aim of the current work was to study the transport mechanism(s) of drug-loaded mmePEG750P(CL-co-TMC) micelles across the intestinal barrier. The transport of radiolabelled polymer across Caco-2 cell monolayer was investigated by disrupting tight junctions and by inhibiting endocytosis. The polymer and drugs loaded in micelles independently crossed Caco-2 cell monolayers and did not use either the paracellular route or M-cells. The polymer did not affect P-gp pumps. This mechanistic study suggests that whereas drug-loaded micelles were absorbed by fluid-phase endocytosis, polymeric unimers diffused passively across the membrane concomitantly with micellar endocytosis.