PIERRE-YVES COLLART DUTILLEUL (GROUP LEADER)

• Valérie ORTI (Professor, University of Paris)
• Stéphane BARTHELEMI (Professor of Medicine)
• Ivan PANAYOTOV (MCU PH)
• Jean Claude EGEA (Associate Professor)
• Habib BELAID (Associate Professor)
• Jerome PRADOS (Assistant Professor)
• Marc ZINDEL (Technician)

TASKS

• Porous silicon for dental pulp stem cell proliferation and differentiation
• PEEK functionalization for cell adhesion and mineralized tissue formation
• Bifunctional peptide for epithelial adhesion on titanium
• Bone and cartilage formation followed by Raman spectroscopy
• DPSC for anticancer drug delivery
• DPSC Conditioned Medium for cell and tissue regeneration

AZEL ZINE (PU, GROUP LEADER)

GROUP MEMBERS

• Azel ZINE (PU, Group Leader)
• Veronique MONTERO (MCU)
• Olivier ROMIEU (MCU, PH)
• Bayan AASAR (Ph.D. student)
• Meryam ZAROUKI (Research Technician)
• Maëlis LUCAS (Project Manager)
• Damien VERET (Postdoctoral Researcher)

Stem Cells, Organoids, and Otic Neurosensory Regeneration

Sensorineural deafness is often caused by the degeneration of spiral ganglion neurons (SGN) in the inner ear that relay auditory signals to the brain. These SGN do not regenerate after degeneration. Dental pulp stem cells (DPSCs) are neural crest-derived ecto-mesenchymal stem cells as the cranial neural crest cells (NCC). These NCC partially contribute to the SGN during development of the inner ear. DPSCs are promising candidates for cell therapy approaches because they have migratory properties, enabling migration after transplantation, can differentiate into sensory neurons and glial cells, and can be harvested in relatively high numbers. We hypothesize that human DPSCs can be used for cell-based regeneration and repair of the SGN.

Project 1: Production of human SGN from DPSC and iPSC using 2D and 3D cultures                           

The main goal of this task is to differentiate and characterize human otic neurosensory progenitors and SGN using a cellular platform for the in vitro production of differentiated cell types from either iPSCs or DPSCs. The stepwise guided differentiation of stem cells into SGN is not straightforward, since there is no single marker which can be applied to distinguish this specific cell type. However, by using RNA-seq high throughput gene expression, it will be possible to more thoroughly determine whether cohorts of known otic neurosensory progenitor and SGN markers over a sequential time course of in vitro differentiation. We will compare a global trajectory towards human otic neurosensory lineage in both DPSCs and iPSCs-derived otic neuronal progenitors and SGN. The development of identity/purity control experiments of otic neuroprogenitors and their neurosensory trajectories will be performed using confocal Raman Spectroscopy in collaboration with Team 3 and Edmos Platform.

Project 2: Potential of sugar-based angiogenic agents on organoid vascularization during iPSC and DPSC toward SGN neurosensory differentiation

One of the unique aspects of organoid models is the ability to resemble, to some extent, the in vivo organ or tissue from which they were derived, and which allow for relevant cell-cell and cell-matrix interactions. One of the key limitations in using organoid approaches to generate tissues is that upon reaching a certain size, organoids cease to proliferate and develop a necrotic core. In order to maintain the complexity of organoids, it is necessary to prevent the appearance of the necrotic inner core leading to the premature differentiation in the outer layers of organoids. This phenomenon, in turn, was largely ascribed to a lack of organoid vascularization. We will explore a panel of angiogenic effectors derived from Mannose-6-phosphate (M6P) on the neurodifferentiation of either DPSC or iPSCs, and in the development of organoid vascularization.

Project 3: Effects of transplantation of neurosensory progenitors in models of SGN degeneration To test the regeneration and synaptogenesis abilities of DPSC/iPSC-derived otic neurosensory progenitors following engraftment, we will first usean in vitro co-culturemodel with denervated cochlear organotypic explants. We have previously demonstrated that hiPSCs-derived otic progenitors are able to engraft and differentiate in the damaged cochlea (Lopez et al., 2019).

General outlook of otic neurosensory differentiation in vitro and engraftment in SGN degeneration models

COLLABORATIONS:

• Albert Edge (Harvard Medical School)
• John Devos (IRMB, University Hospital, Montpellier)
• Christian Chabbert (CNRS, AMU Marseille)
• Pierre Gaudriault (Cherry Biotech, Rennes)
• Annelies Schrott-Fischer (Medical University of Innsbruck, Austria)

AMEL SLIMANI (GROUP LEADER)

• Frédéric CUISINIER (Associate Professor)
• Jean Valcarcel (University Professor-Hospital Physician)
• Michel Fages (Professor and Hospital Physician)
• Ivan Panayotov (MCU-PH)
• Jean Cédric Durand (Associate Professor)
• Alban Desoutter (technical assistant, LBN)
• Elias Estephan (Associate Professor)

Our scientific objective is to identify new photonic biomarkers for oral diseases, mainly dental caries, periodontal diseases, and oral cancer. These pathologies will be studied using different spectroscopic methods and devices. We organize this project according to the methods, even if the methods can be combined to increase the specificity and sensitivity of biomarkers.

1. Intraoral 3D scanners (IOS):

Oral cancer is a significant health problem often identified at a later stage. Oral cancers are characterized by a change in tissue color as well as thickening of the oral mucosa. Concerning tissue thickening, obtaining a 3D mesh of the oral cavity is an interesting tool for creating standardized biomarkers of cancerous lesions. Autofluorescence, color, and 3D meshes could be combined to detect oral cancer. We will focus our work on exploring the use of the two IOS in order to optimize cancer detection. For this purpose, 3D meshes from malignant lesions and healthy tissue will be collected. Segmentation and color spectral analysis will lead to the identification of biomarkers through multivariate statistics. This can be extended to periodontal disease.

2. BCARS to detect oral cancer

The diagnosis of malignant and cancerous lesions in the oral cavity is the main focus in the development of new imaging techniques. Malignant tissues undergo biochemical changes called tumor markers, which induce variations in the vibrational spectra. Raman spectra from diseased and healthy tissue are compared for diagnosis. Raman biomarkers complement biopsies and provide real-time diagnosis. The first part of the task is to compare the existing standard biopsy analysis with our label-free B-CARS signal. The relevant data analysis could detect changes in tissue with the potential to be used in operating rooms.

3. Early caries lesion detection using Brillouin/Raman biomarkers

The initial caries lesions are challenging to detect for a dentist and even more so in teledentistry. In our laboratory, we have developed aWSLin vitromodeland we used confocal Raman spectroscopy and multiphoton microscopy images to evaluate structural changes in the white spot lesion. The goal of this task is to adapt a new non-invasive biomedical imaging technology based on Brillouin light scattering to measure the biomechanical properties of hard tissues and biomaterials, which is the main aim of this project. Molecular analysis of dental tissues using Raman spectroscopy has been used to characterize the mineral phase in synthetic compounds and natural tissues. Our project is to use confocal Raman microscopy coupled with a home-built Brillouin spectrometer. The first step will be to demonstrate the possibility of simultaneous biomechanical/biochemical imaging to obtain photonic biomarkers for early caries and WSL diagnosis by studying in vitro incipient caries lesions and animal-induced periodontal disease. Comparison with healthy samples will allow statistical analysis to find a biomarker to detect caries at an early stage, which is the final step.

Partnerships:

1- L2C- UMR5221 CNRS: we will continue our strong collaborations through our involvement in the financing and building of the BCARS (Nanobiophonic team) and the building of the Brillouin Spectrometer (B. Rufflé, D. Felbacq, glassy state physics team). We will develop a partnership for colorimetry in teledentistry (F. Geniet, complex systems and nonlinear phenomena team).

2- LIRMM: all metrology work for IOS is discussed and carried out with LIRMM (G. Subsol, ICAR team).

3- LNE: concerning the metrology of IOS, characterization of objects and samples tested, we have a collaboration with a team from LNE (Laboratoire National de Métrologie et d’Essai, Nimes).