Biological Biomarkers of Response and Resistance to Immune Checkpoint Inhibitors in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) represents around 2% of cancer-related deaths worldwide per year. RCC is an immunogenic malignancy, and treatment of metastatic RCC (mRCC) has greatly improved since the advent of the new immunotherapy agents, including immune checkpoint inhibitors (ICIs). However, it should be stressed that a large proportion of patients does not respond to these therapies. There is thus an urgent need to identify predictive biomarkers of efficacy or resistance associated with ICIs or ICI/Tyrosine kinase inhibitor (TKI) combinations; this is a major challenge to achieve precision medicine for mRCC in routine practice. To identify potential biomarkers, it is necessary to improve our knowledge on the biology of immune checkpoints. A lot of efforts have been made over the last decade in the field of immuno-oncology. We summarize here the main data obtained in this field when considering mRCC. As for clinical biomarkers, clinician and scientific experts of the domain are facing difficulties in identifying such molecular entities, probably due to the complexity of immuno-oncology and the constant adaptation of tumor cells to their changing environment.

Complete Response in Metastatic Clear Cell Renal Cell Carcinoma Patients Treated with Immune-Checkpoint Inhibitors: Remission or Healing? How to Improve Patients' Outcomes?

Renal-cell carcinoma (RCC) accounts for 2% of cancer diagnoses and deaths worldwide. Clear-cell RCCs represent the vast majority (85%) of kidney cancers and are considered morphologically and genetically as immunogenic tumors. Indeed, the RCC tumoral microenvironment comprises T cells and myeloid cells in an immunosuppressive state, providing an opportunity to restore their activity through immunotherapy. Standard first-line systemic treatment for metastatic patients includes immune-checkpoint inhibitors (ICIs) targeting PD1, in combination with either another ICI or with antiangiogenic targeted therapy. During the past few years, several combinations have been approved with an overall survival benefit and overall response rate that depend on the combination. Interestingly, some patients achieve prolonged complete responses, raising the question of whether these metastatic RCC patients can be cured. This review will focus on recent therapeutic advances in RCC and the clinical and biological aspects underpinning the potential for healing.

Primary failure of eruption and tooth resorption / Défaut primaire d'éruption et résorption radiculaire externe

Introduction: The number of adult patients who seek an orthodontic treatment is increasing. These Primary failure of eruption (PFE) is defined as the partial or complete failure of eruption of at least one posterior tooth, without any mechanical obstacle. A better understanding of the biological mechanisms involved in PFE would enable to refine the diagnostic and prognostic criteria. This rare disease is currently related to PTHR1 gene variants. This gene codes for a transmembrane receptor involved in bone metabolism. However, there is few evidence associating PFE and bone remodeling abnormalities such as external root resorption. External root resorption is the loss of cementum and dentin tissues, resulting from the activation of clastic cells. Materials and Methods: Human teeth affected by PFE were extracted and histological sections were made after fixation of the tissues in 4% PFA. The observations were correlated with three-dimensional imaging by cone beam computed tomography (CBCT) carried out in the preoperative phase. Results: Histological and radiographic analysis confirm the presence of ankylosis area in patients with no history of orthodontic treatment. Large areas of resorption of external root replacement were detected. Discussion: The results call the causal link between the appearance of ankylosis areas and the establishment of orthodontic traction in patients with PFE into question. The installation of an orthodontic force in this context could be only an aggravating factor, accelerating the processes of ankylosis or triggering them more prematurely. Conclusion: With or without orthodontic treatment, teeth with PFE are likely to progress to ankylosis and resorption of replacement external root.

Introduction: Les défauts primaires d'éruption (DPE) se caractérisent par l'échec total ou partiel de l'éruption d'une ou plusieurs dents postérieures, sans obstacle mécanique. Une meilleure compréhension des mécanismes biologiques impliqués dans les DPE permettrait d'affiner les critères diagnostiques et pronostiques. Cette pathologie rare est actuellement imputée à des variants du gène PTHR1. Ce gène code pour un récepteur transmembranaire impliqué dans le métabolisme osseux. Cependant, on trouve peu de données associant DPE et anomalies du remodelage osseux de type résorption radiculaire externe. La résorption radiculaire externe correspond à la perte de tissus cémentaire et dentinaire résultant de l'activation de cellules clastiques. Matériels et méthodes: Des dents d'origine humaine atteintes de DPE ont été avulsées et des coupes histologiques réalisées après fixation des tissus. Les observations ont été corrélées avec l'imagerie tridimensionnelle par tomographie volumique à faisceau conique (TVFC ou encore CBCT). Résultats: Les analyses histologiques et radiographiques montrent la présence de plage d'ankylose chez des patients sans antécédent de prise en charge orthodontique. De larges zones de résorptions radiculaires externes de remplacement ont été détectées. Discussion: Les résultats remettent en cause le lien de causalité entre l'apparition d'ankylose et la mise en place de traction orthodontique chez les patients atteints de DPE. La mise en place d'une force orthodontique dans ce contexte pourrait n'être qu'un facteur aggravant, accélérant les processus d'ankylose ou les déclenchant plus prématurément. Conclusion: Avec ou sans traitement orthodontique, les dents atteintes de DPE sont susceptibles d'évoluer vers l'ankylose et la résorption radiculaire externe de remplacement.

Mechanistic Illustration: How Newly-Formed Blood Vessels Stopped by the Mineral Blocks of Bone Substitutes Can Be Avoided by Using Innovative Combined Therapeutics.

One major limitation for the vascularization of bone substitutes used for filling is the presence of mineral blocks. The newly-formed blood vessels are stopped or have to circumvent the mineral blocks, resulting in inefficient delivery of oxygen and nutrients to the implant. This leads to necrosis within the implant and to poor engraftment of the bone substitute. The aim of the present study is to provide a bone substitute currently used in the clinic with suitably guided vascularization properties. This therapeutic hybrid bone filling, containing a mineral and a polymeric component, is fortified with pro-angiogenic smart nano-therapeutics that allow the release of angiogenic molecules. Our data showed that the improved vasculature within the implant promoted new bone formation and that the newly-formed bone swapped the mineral blocks of the bone substitutes much more efficiently than in non-functionalized bone substitutes. Therefore, we demonstrated that our therapeutic bone substitute is an advanced therapeutical medicinal product, with great potential to recuperate and guide vascularization that is stopped by mineral blocks, and can improve the regeneration of critical-sized bone defects. We have also elucidated the mechanism to understand how the newly-formed vessels can no longer encounter mineral blocks and pursue their course of vasculature, giving our advanced therapeutical bone filling great potential to be used in many applications, by combining filling and nano-regenerative medicine that currently fall short because of problems related to the lack of oxygen and nutrients.

Eruption of Bioengineered Teeth: A New Approach Based on a Polycaprolactone Biomembrane.

Obtaining a functional tooth is the ultimate goal of tooth engineering. However, the implantation of bioengineered teeth in the jawbone of adult animals never allows for spontaneous eruption due mainly to ankylosis within the bone crypt. The objective of this study was to develop an innovative approach allowing eruption of implanted bioengineered teeth through the isolation of the germ from the bone crypt using a polycaprolactone membrane (PCL). The germs of the first lower molars were harvested on the 14th day of embryonic development, cultured in vitro, and then implanted in the recipient site drilled in the maxillary bone of adult mice. To prevent the ankylosis of the dental germ, a PCL membrane synthesized by electrospinning was placed between the germ and the bone. After 10 weeks of follow-up, microtomography, and histology of the implantation site were performed. In control mice where germs were directly placed in contact with the bone, a spontaneous eruption of bioengineered teeth was only observed in 3.3% of the cases versus 19.2% in the test group where PCL biomembrane was used as a barrier (p < 0.1). This preliminary study is the first to describe an innovative method allowing the eruption of bioengineered tooth implanted directly in the jawbone of mice. This new approach is a hope for the field of tooth regeneration, especially in children with oligodontia in whom titanium implants are not an optimal solution.

Influence of parathyroid hormone on periodontal healing in animal models: A systematic review.

OBJECTIVES: The purpose of this systematic review was to determine the potential interest of parathyroid hormone (PTH) as an adjunct to periodontal treatment based on studies performed in rodents. MATERIALS & METHODS: Electronic databases (MEDLINE, Web of Science) were searched up to December 2019. Studies assessing the impact of PTH administration in experimental periodontitis in rodents have been identified. RESULTS: Amongst the 247 identified articles, 10 met the inclusion criteria and were included in this systematic review. Experimental periodontitis was mainly induced by ligature placement or surgically with a dental bur. All studies considered bone healing after PTH administration at different frequencies as primary outcome. Results showed that an intermittent administration of PTH promoted bone healing and neovascularization. Nevertheless, a decrease of soft tissue inflammation was also observed. CONCLUSION: Intermittent administration of PTH appears to enhance significantly periodontal healing and to promote alveolar bone regeneration. However, due to the risk of side effects, the development of scaffolds allowing its local and time-controlled delivery is of importance.

A New Polycaprolactone-Based Biomembrane Functionalized with BMP-2 and Stem Cells Improves Maxillary Bone Regeneration.

Oral diseases have an impact on the general condition and quality of life of patients. After a dento-alveolar trauma, a tooth extraction, or, in the case of some genetic skeletal diseases, a maxillary bone defect, can be observed, leading to the impossibility of placing a dental implant for the restoration of masticatory function. Recently, bone neoformation was demonstrated after in vivo implantation of polycaprolactone (PCL) biomembranes functionalized with bone morphogenic protein 2 (BMP-2) and ibuprofen in a mouse maxillary bone lesion. In the present study, human bone marrow derived mesenchymal stem cells (hBM-MSCs) were added on BMP-2 functionalized PCL biomembranes and implanted in a maxillary bone lesion. Viability of hBM-MSCs on the biomembranes has been observed using the "LIVE/DEAD" viability test and scanning electron microscopy (SEM). Maxillary bone regeneration was observed for periods ranging from 90 to 150 days after implantation. Various imaging methods (histology, micro-CT) have demonstrated bone remodeling and filling of the lesion by neoformed bone tissue. The presence of mesenchymal stem cells and BMP-2 allows the acceleration of the bone remodeling process. These results are encouraging for the effectiveness and the clinical use of this new technology combining growth factors and mesenchymal stem cells derived from bone marrow in a bioresorbable membrane.

Potential Implantable Nanofibrous Biomaterials Combined with Stem Cells for Subchondral Bone Regeneration.

The treatment of osteochondral defects remains a challenge. Four scaffolds were produced using Food and Drug Administration (FDA)-approved polymers to investigate their therapeutic potential for the regeneration of the osteochondral unit. Polycaprolactone (PCL) and poly(vinyl-pyrrolidone) (PVP) scaffolds were made by electrohydrodynamic techniques. Hydroxyapatite (HAp) and/or sodium hyaluronate (HA) can be then loaded to PCL nanofibers and/or PVP particles. The purpose of adding hydroxyapatite and sodium hyaluronate into PCL/PVP scaffolds is to increase the regenerative ability for subchondral bone and joint cartilage, respectively. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were seeded on these biomaterials. The biocompatibility of these biomaterials in vitro and in vivo, as well as their potential to support MSC differentiation under specific chondrogenic or osteogenic conditions, were evaluated. We show here that hBM-MSCs could proliferate and differentiate both in vitro and in vivo on these biomaterials. In addition, the PCL-HAp could effectively increase the mineralization and induce the differentiation of MSCs into osteoblasts in an osteogenic condition. These results indicate that PCL-HAp biomaterials combined with MSCs could be a beneficial candidate for subchondral bone regeneration.

Osteochondral repair combining therapeutics implant with mesenchymal stem cells spheroids.

Functional articular cartilage regeneration remains challenging, and it is essential to restore focal osteochondral defects and prevent secondary osteoarthritis. Combining autologous stem cells with therapeutic medical device, we developed a bi-compartmented implant that could promote both articular cartilage and subchondral bone regeneration. The first compartment based on therapeutic collagen associated with bone morphogenetic protein 2, provides structural support and promotes subchondral bone regeneration. The second compartment contains bone marrow-derived mesenchymal stem cell spheroids to support the regeneration of the articular cartilage. Six-month post-implantation, the regenerated articular cartilage surface was 3 times larger than that of untreated animals, and the regeneration of the osteochondral tissue occurred during the formation of hyaline-like cartilage. Our results demonstrate the positive impact of this combined advanced therapy medicinal product, meeting the needs of promising osteochondral regeneration in critical size articular defects in a large animal model combining not only therapeutic implant but also stem cells.

Temporomandibular joint damage in K/BxN arthritic mice.

Rheumatoid arthritis (RA) is an autoimmune disease affecting 1% of the world population and is characterized by chronic inflammation of the joints sometimes accompanied by extra-articular manifestations. K/BxN mice, originally described in 1996 as a model of polyarthritis, exhibit knee joint alterations. The aim of this study was to describe temporomandibular joint (TMJ) inflammation and damage in these mice. We used relevant imaging modalities, such as micro-magnetic resonance imaging (µMRI) and micro-computed tomography (µCT), as well as histology and immunofluorescence techniques to detect TMJ alterations in this mouse model. Histology and immunofluorescence for Col-I, Col-II, and aggrecan showed cartilage damage in the TMJ of K/BxN animals, which was also evidenced by µCT but was less pronounced than that seen in the knee joints. µMRI observations suggested an increased volume of the upper articular cavity, an indicator of an inflammatory process. Fibroblast-like synoviocytes (FLSs) isolated from the TMJ of K/BxN mice secreted inflammatory cytokines (IL-6 and IL-1ß) and expressed degradative mediators such as matrix metalloproteinases (MMPs). K/BxN mice represent an attractive model for describing and investigating spontaneous damage to the TMJ, a painful disorder in humans with an etiology that is still poorly understood.

Polymer-Based Instructive Scaffolds for Endodontic Regeneration.

The challenge of endodontic regeneration is modulated by clinical conditions which determine five kinds of tissue requirements: pulp connective-tissue formation, dentin formation, revascularization, reinnervation and radicular edification. Polymer scaffolds constitute keystone of the different endodontic regenerative strategies. Indeed, scaffolds are crucial for carrying active molecules and competent cells which optimize the regeneration. Hydrogels are very beneficial for controlling viscosity and porosity of endodontic scaffolds. The nanofibrous and microporous scaffolds mimicking extracellular matrix are also of great interest for promoting dentin-pulp formation. Two main types of polymer scaffolds are highlighted: collagen and fibrin. Collagen scaffolds which are similar to native pulp tissue, are adequate for pulp connective tissue formation. Functionnalization by active biomolecules as BMP, SDF-1, G-CSF enhances their properties. Fibrin or PRF scaffolds present the advantage of promoting stem cell differentiation and concomitant revascularisation. The choice of the type of polymers (polypeptide, PCL, chitosan) can depend on its ability to deliver the active biomolecule or to build as suitable hydrogel as possible. Since 2010s, proposals to associate different types of polymers in a same scaffold have emerged for adding advantages or for offsetting a disadvantage of a polymer. Further works would study the synergetic effects of different innovative polymers composition.

Porphyromonas gingivalis bypasses epithelial barrier and modulates fibroblastic inflammatory response in an in vitro 3D spheroid model.

Porphyromonas gingivalis-induced inflammatory effects are mostly investigated in monolayer cultured cells. The aim of this study was to develop a 3D spheroid model of gingiva to take into account epithelio-fibroblastic interactions. Human gingival epithelial cells (ECs) and human oral fibroblasts (FBs) were cultured by hanging drop method to generate 3D microtissue (MT) whose structure was analyzed on histological sections and the cell-to-cell interactions were observed by scanning and transmission electron microscopy (SEM and TEM). MTs were infected by P. gingivalis and the impact on cell death (Apaf-1, caspase-3), inflammatory markers (TNF-α, IL-6, IL-8) and extracellular matrix components (Col-IV, E-cadherin, integrin ß1) was evaluated by immunohistochemistry and RT-qPCR. Results were compared to those observed in situ in experimental periodontitis and in human gingival biopsies. MTs exhibited a well-defined spatial organization where ECs were organized in an external cellular multilayer, while, FBs constituted the core. The infection of MT demonstrated the ability of P. gingivalis to bypass the epithelial barrier in order to reach the fibroblastic core and induce disorganization of the spheroid structure. An increased cell death was observed in fibroblastic core. The development of such 3D model may be useful to define the role of EC-FB interactions on periodontal host-immune response and to assess the efficacy of new therapeutics.

Periodontal Tissues, Maxillary Jaw Bone, and Tooth Regeneration Approaches: From Animal Models Analyses to Clinical Applications.

This review encompasses different pre-clinical bioengineering approaches for periodontal tissues, maxillary jaw bone, and the entire tooth. Moreover, it sheds light on their potential clinical therapeutic applications in the field of regenerative medicine. Herein, the electrospinning method for the synthesis of polycaprolactone (PCL) membranes, that are capable of mimicking the extracellular matrix (ECM), has been described. Furthermore, their functionalization with cyclosporine A (CsA), bone morphogenetic protein-2 (BMP-2), or anti-inflammatory drugs' nanoreservoirs has been demonstrated to induce a localized and targeted action of these molecules after implantation in the maxillary jaw bone. Firstly, periodontal wound healing has been studied in an induced periodontal lesion in mice using an ibuprofen-functionalized PCL membrane. Thereafter, the kinetics of maxillary bone regeneration in a pre-clinical mouse model of surgical bone lesion treated with BMP-2 or BMP-2/Ibuprofen functionalized PCL membranes have been analyzed by histology, immunology, and micro-computed tomography (micro-CT). Furthermore, the achievement of innervation in bioengineered teeth has also been demonstrated after the co-implantation of cultured dental cell reassociations with a trigeminal ganglia (TG) and the cyclosporine A (CsA)-loaded poly(lactic-co-glycolic acid) (PLGA) scaffold in the jaw bone. The prospective clinical applications of these different tissue engineering approaches could be instrumental in the treatment of various periodontal diseases, congenital dental or cranio-facial bone anomalies, and post-surgical complications.

Maxillary Bone Regeneration Based on Nanoreservoirs Functionalized ε-Polycaprolactone Biomembranes in a Mouse Model of Jaw Bone Lesion.

Current approaches of regenerative therapies constitute strategies for bone tissue reparation and engineering, especially in the context of genetical diseases with skeletal defects. Bone regeneration using electrospun nanofibers' implant has the following objectives: bone neoformation induction with rapid healing, reduced postoperative complications, and improvement of bone tissue quality. In vivo implantation of polycaprolactone (PCL) biomembrane functionalized with BMP-2/Ibuprofen in mouse maxillary defects was followed by bone neoformation kinetics evaluation using microcomputed tomography. Wild-Type (WT) and Tabby (Ta) mice were used to compare effects on a normal phenotype and on a mutant model of ectodermal dysplasia (ED). After 21 days, no effect on bone neoformation was observed in Ta treated lesion (4% neoformation compared to 13% in the control lesion). Between the 21st and the 30th days, the use of biomembrane functionalized with BMP-2/Ibuprofen in maxillary bone lesions allowed a significant increase in bone neoformation peaks (resp., +8% in mutant Ta and +13% in WT). Histological analyses revealed a neoformed bone with regular trabecular structure, areas of mineralized bone inside the membrane, and an improved neovascularization in the treated lesion with bifunctionalized membrane. In conclusion, PCL functionalized biomembrane promoted bone neoformation, this effect being modulated by the Ta bone phenotype responsible for an alteration of bone response.

Temporomandibular Joint Regenerative Medicine.

The temporomandibular joint (TMJ) is an articulation formed between the temporal bone and the mandibular condyle which is commonly affected. These affections are often so painful during fundamental oral activities that patients have lower quality of life. Limitations of therapeutics for severe TMJ diseases have led to increased interest in regenerative strategies combining stem cells, implantable scaffolds and well-targeting bioactive molecules. To succeed in functional and structural regeneration of TMJ is very challenging. Innovative strategies and biomaterials are absolutely crucial because TMJ can be considered as one of the most difficult tissues to regenerate due to its limited healing capacity, its unique histological and structural properties and the necessity for long-term prevention of its ossified or fibrous adhesions. The ideal approach for TMJ regeneration is a unique scaffold functionalized with an osteochondral molecular gradient containing a single stem cell population able to undergo osteogenic and chondrogenic differentiation such as BMSCs, ADSCs or DPSCs. The key for this complex regeneration is the functionalization with active molecules such as IGF-1, TGF-ß1 or bFGF. This regeneration can be optimized by nano/micro-assisted functionalization and by spatiotemporal drug delivery systems orchestrating the 3D formation of TMJ tissues.

Semaphorin 3A receptor inhibitor as a novel therapeutic to promote innervation of bioengineered teeth.

The sensory innervation of the dental pulp is essential for tooth function and protection. It is mediated by axons originating from the trigeminal ganglia and is spatio-temporally regulated. We have previously shown that the innervation of bioengineered teeth can be achieved only under immunosuppressive conditions. The aim of this study was to develop a model to determine the role of Semaphorin 3A (Sema3A) in the innervation of bioengineered teeth. We first analysed innervation of the dental pulp of mandibular first molars in newborn (postnatal day 0: PN0) mice deficient for Sema3A (Sema3A-/- ), a strong inhibitor of axon growth. While at PN0, axons detected by immunostaining for peripherin and NF200 were restricted to the peridental mesenchyme in Sema3A+/+ mice, they entered the dental pulp in Sema3A-/- mice. Then, we have implanted cultured teeth obtained from embryonic day-14 (E14) molar germs of Sema3A-/- mice together with trigeminal ganglia. The dental pulps of E14 cultured and implanted Sema3A-/- teeth were innervated, whereas the axons did not enter the pulp of E14 Sema3A+/+ cultured and implanted teeth. A "Membrane Targeting Peptide NRP1," suppressing the inhibitory effect of Sema3A, has been previously identified. The injection of this peptide at the site of implantation allowed the innervation of the dental pulp of bioengineered teeth obtained from E14 dental dissociated mesenchymal and epithelial cells reassociations of ICR mice. In conclusion, these data show that inhibition of only one axon repellent molecule, Sema3A, allows for pulp innervation of bioengineered teeth.

Combining 2D angiogenesis and 3D osteosarcoma microtissues to improve vascularization.

Angiogenesis is now well known for being involved in tumor progression, aggressiveness, emergence of metastases, and also resistance to cancer therapies. In this study, to better mimic tumor angiogenesis encountered in vivo, we used 3D culture of osteosarcoma cells (MG-63) that we deposited on 2D endothelial cells (HUVEC) grown in monolayer. We report that endothelial cells combined with tumor cells were able to form a well-organized network, and that tubule-like structures corresponding to new vessels infiltrate tumor spheroids. These vessels presented a lumen and expressed specific markers as CD31 and collagen IV. The combination of 2D endothelial cells and 3D microtissues of tumor cells also increased expression of angiogenic factors as VEGF, CXCR4 and ICAM1. The cell environment is the key point to develop tumor vascularization in vitro and to be closer to tumor encountered in vivo.

A New Wnt1-CRE TomatoRosa Embryonic Stem Cell Line: A Tool for Studying Neural Crest Cell Integration Capacity.

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 RosaTomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.

Well-organized spheroids as a new platform to examine cell interaction and behaviour during organ development.

We present an experimental method allowing the production of three-dimensional organ-like structures, namely microtissues (MTs), in vitro without the need for exogenous extracellular matrix (ECM) or growth factors. Submandibular salivary glands (embryonic day ED14), kidneys (ED13) and lungs (ED13) were harvested from mouse embryos and dissociated into single cells by enzyme treatment. Single cells were seeded into special hanging drop culture plates (InSphero) and cultured for up to 14 days to obtain MTs. This strategy permitted full control of the quantity of seeded cells. The development of the MTs into organs was followed histologically and immunohistochemically. Well-organized epithelial structures surrounded by a basal lamina were formed, as confirmed by transmission electron microscopy. Expression of E-cadherin, vimentin, fibronectin and α-SMA was compared in organs and corresponding MTs by real-time quantitative polymerase chain reaction. Branching morphogenesis was induced in MTs (as shown by histology and immunostaining for fibronectin and perlecan) and was conserved even after 14 days of culture. MTs continued their development and their epithelial structures were comparable with those of the physiological organ at postnatal day 2 (PN2). Expression of aquaporins was investigated to obtain better support for the functional differentiation of epithelial cells. Histogenesis proceeded and led to the start of organogenesis. This experimental model might improve our knowledge of epithelial-mesenchymal histogenesis and can be employed to study development or cellular organization during the embryonic formation of organs.

Immunomodulation stimulates the innervation of engineered tooth organ.

The sensory innervation of the dental mesenchyme is essential for tooth function and protection. Sensory innervation of the dental pulp is mediated by axons originating from the trigeminal ganglia and is strictly regulated in time. Teeth can develop from cultured re-associations between dissociated dental epithelial and mesenchymal cells from Embryonic Day 14 mouse molars, after implantation under the skin of adult ICR mice. In these conditions however, the innervation of the dental mesenchyme did not occur spontaneously. In order to go further with this question, complementary experimental approaches were designed. Cultured cell re-associations were implanted together with trigeminal ganglia for one or two weeks. Although axonal growth was regularly observed extending from the trigeminal ganglia to all around the forming teeth, the presence of axons in the dental mesenchyme was detected in less than 2.5% of samples after two weeks, demonstrating a specific impairment of their entering the dental mesenchyme. In clinical context, immunosuppressive therapy using cyclosporin A was found to accelerate the innervation of transplanted tissues. Indeed, when cultured cell re-associations and trigeminal ganglia were co-implanted in cyclosporin A-treated ICR mice, nerve fibers were detected in the dental pulp, even reaching odontoblasts after one week. However, cyclosporin A shows multiple effects, including direct ones on nerve growth. To test whether there may be a direct functional relationship between immunomodulation and innervation, cell re-associations and trigeminal ganglia were co-implanted in immunocompromised Nude mice. In these conditions as well, the innervation of the dental mesenchyme was observed already after one week of implantation, but axons reached the odontoblast layer after two weeks only. This study demonstrated that immunodepression per se does stimulate the innervation of the dental mesenchyme.