In the last decade, the development of organoids has generated real enthusiasm, allowing the study of new treatments and the implementation of a personalized medicine approach while reducing experiments on animals. The generation of bone organoids from human mesenchymal stem cells capable of mimicking certain bone functionalities requires exposing the cells to physiologically relevant conditions. The proposed project aims to develop a smart bone bioreactor capable, for the first time, of measuring and adapting, in a dynamic and controlled manner, the mechanical stress, oxygen tension and nutrient supply to the cells throughout the entire genesis of the bone organoid. This project will rely on the consortium's leading-edge expertise in the field of bone and in particular on the development of a pre-existing bioreactor. The ultimate validation phase will be an efficient treatment against bone fragility on the organoid generated from the Smart Bone Bioreactor.

Any inner and outer body surface is covered by epithelia. Epithelia are shaped by a layer of cells that, at the same time, form a barrier against the loss of salts and water, and transport salts and water in a coordinated manner. Transport across epithelial layers uses two pathways: the transcellular and the paracellular route. So far, mainly the transcellular pathway has been investigated. Recently, it became evident that this paracellular pathway is highly specific, tightly regulated and causally involved in human diseases. Tight junctions (TJ) are the backbone of the paracellular pathway of epithelial ion transport. Most important for the properties of TJ are members of the claudin (CLDN) family. The set of CLDN expressed at a TJ is extremely tissue-specific; it is responsible for the permeability and selectivity properties of every TJ. CLDN10, -16 and -19 are co-expressed at TJ in the thick ascending limb of the loop of Henle and in ameloblasts. Both tissues express the calcium-sensing receptor CaSR that decreases the paracellular permeability to divalent ions. The aforementioned CLDNs are causally linked to Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (CLDN16 and 19) that associates renal and dental defects, and to HELIX syndrome (CLDN10) that associates dental, renal, cutaneous, and salivary defects. The bases for the functional properties of the TJ and for the control of those properties are poorly understood. The consequences of the alteration in claudin composition of a TJ to the surrounding tissues are clinically obvious but their pathophysiology remains elusive. In addition, an efficient treatment of TJ diseases is currently not available. Based on those pending questions, on preliminary results recently obtained by the 3 partners of the consortium, and on available animal and cell models and techniques, we aim at i) identifying how claudins determine the permeability and selectivity of the paracellular pathway, and how claudin properties are controlled ii) understanding how mutations in claudins are responsible for the development of abnormal mineralization in the close environment of the epithelium (nephrocalcinosis in the kidney (abnormal parenchymal mineralization) and amelogenesis imperfecta (enamel defect) in tooth), and iii) developing therapeutic strategies of the diseases caused by defects in the paracellular pathway properties; the strategies are based on use of drugs that affect the signalling pathways studied in i). The proposed study is highly multidisciplinary as it requires a mixed biological and structural approach and will integrate renal physiologists, odontologists, and cell and molecular biologists. The studies conducted in this proposal will provide key insights regarding the roles of claudins as determinants of permeability, selectivity and structure of tight junctions. They will also help to decipher the mechanisms involved in pathologic consequences of TJ impairment, such as nephrocalcinosis and amelogenesis imperfecta. Finally, they will pave new pathways towards treatment of TJ disorders.

A cross-talk between bone homeostasis and the kidney has been revealed by the association of chronic kidney disease (CKD) and mineral bone disorder (MBD). The abnormalities observed in CKD-MBD also contribute to vascular calcification, a major cause of mortality in patients undergoing dialysis. This underlines the need to identify sensitive markers in order to i) rapidly determine the origin of the kidney pathology and ii) select for each patient the therapeutic with the best efficacy. Vasorin (VASN), a type I membrane protein involved in TGF-beta signaling, was identified in the urine of patients with nephropathies. Initially studied in the context of the development, Vasn was shown to be strongly expressed in several adult tissues, including large arterial vessels, bone, teeth, and kidney. In this later, it was shown to be expressed both in the collecting ducts and in the glomeruli. The investigation of Vasn knock-out (KO) mice by our Consortium has provided little information regarding Vasn function(s) as mice die around 21 days. When compared to Wild type (WT) or heterozygous (HET) mice, post-natal (P) day 21 Vasn KO mice display a very severe phenotype with short stature, severe nephrotic syndrome (NS) from P15 onwards and abnormal bone and tooth mineralization. In the kidney, major abnormalities were observed in glomeruli, and podocytes were the most affected cells are characterized by loss of their foot processes and absence of slit diaphragm, which is essential for proper selective urine filtration. In addition, the media of the large vessels was strongly altered, manifested by disorganization of the elastic fibers and hypertrophic vascular smooth muscle cells (VSMCs) with some areas devoid of cells. Thus, having access to conditional Vasn KO mice, our Consortium will aim to further understand the pathophysiological functions of Vasn, specifically in 1) bone and tooth formation, and bone homeostasis, 2) large vessel organization and the development of ectopic calcifications, 3) renal tubule function and the maintenance of mineral homeostasis, 4) glomerulus and podocyte homeostasis. In that purpose, we will develop Cre-lox models including deletion of Vasn specifically in either bones and teeth, large vessels, tubules or nephrons and analyze the phenotype of these mice by sharing our expertise as a role for soluble Vasn (sVasn) that may mediate interorgan crosstalk. We will perform functional tests in conditional KO mice such as tubule perfusion, vascular hemodynamic, models of bone, tooth, or vascular injury. In parallel, in vitro experiments will also be carried out by Partners on primary cell cultures thanks to cells sampled in tamoxifen inducible Vasn KO for the different tissues (primary culture of podocytes, osteoblasts or VSMCs). In both in vivo and in vitro experiments, we will investigate the molecular pathways associated to Vasn by conducting several transcriptomic studies and determine whether the interaction with TGF-beta signaling is restricted to large vessels or common with the other organs. Finally, we will investigate human tissue collections and databases of patients with NS and CKD and/or vascular and bone diseases to analyze more in depth Vasn role(s) in these disorders. In a clinical perspective, VASN variant(s) may have a central role in rare or acquired nephrotic syndromes, bone disorders or more generally in cardiovascular diseases, and may constitute a therapeutic target.

The rehabilitation of large bone defects, such as mandibulectomies, is a major challenge in maxillofacial surgery. As an alternative to autografts, a device reproducing the mechanical loading properties of bone while containing progenitor cells and growth factors, promoting both osteogenesis and angiogenesis, is highly sought after. This project aims to develop a 3D printed “custom-made” insert, mechanically-robust but porous acting as a support for a hydrogel containing mesenchymal stem cells derived from the dental pulp. An in vitro approach will be conducted to optimize the porous structure of the insert to obtain suitable mechanical properties while allowing a dialogue between the enclosed cells and the surrounding tissues, in particular neo-angiogenesis. A rodent preclinical mandible model adapted to load-bearing defects will be optimized and then used to evaluate the bone regeneration capacity of our devices.

Given the unique properties of dental tissues, teeth can be considered as fossil records in which the history of an individual's early environmental exposures is permanently imprinted. Characterization of resulting developmental dental defects (DDDs) may be used as early biomarker of exposure to specific stressors during the perinatal period corresponding to the time of tooth development. Based on encouraging preliminary data in mice and rats, the hypothesis being tested in the DStress project is that chronic psychological stress early in life can cause DDDs by involving stress hormones. The DStress project aims to characterize DDDs in three experimental models resulting from maternal separation stress in pups, chronic social stress in adults and cortisone treatment (WP1), in order to demonstrate the involvement of the corticosteroid signaling pathway in dental development and DDDs (WP2). The use of modified experimental models for the mineralocorticoid pathway (MR and NGAL) combined with pharmacological treatments with a specific MR inhibitor, and chronic stresses will demonstrate the in vivo functional role of corticosteroïd signaling pathways in DDD (WP2). The experimental data expected from the DStress project will be immediately transposed to humans (WP3), thanks to the extension of the consortium including public health researchers and dentists coordinating ELFE and EPIPAGE 2, two national cohorts including children. DStress is a project that brings together researchers from different fields - dentists, neurobiologists, physiologists, molecular biologists and mineral engineers - forming a consortium with complementary expertise, who recently published papers in high-level journals. Altogether, these strengths increase the likelihood of the project's success, as well as make it possible to propose innovative and risky tasks. If, in the future, DDDs could be used as markers of psychiatric and neuronal (or even cardiovascular) pathologies, this would enable early diagnosis of DDD patients exposed to chronic psychological stress during the perinatal period, and appropriate follow-up as early as possible to increase the effectiveness of proposed therapies.