Sabine Kuchler-Bopp

Smart Hybrid Materials Equipped by Nanoreservoirs of Therapeutics

Nanobiotechnology enables the emergence of entirely new classes of bioactive devices intended for targeted intracellular delivery for more efficacies and less toxicities. Among organic and inorganic approaches currently developed, controlled release from polymer matrices promises utmost clinical impact. Here, a unique nanotechnology strategy is used to entrap, protect, and stabilize therapeutic agents into polymer coatings acting as nanoreservoirs enrobing nanofibers of implantable membranes. Upon contact with cells, therapeutic agents become available through enzymatic degradation of the nanoreservoirs. As cells grow, divide, and infiltrate deeper into the porous membrane, they trigger slow and progressive release of therapeutic agents that, in turn, stimulate further cell proliferation. This constitutes the first instance of a smart living nanostructured hybrid membrane for regenerative medicine. The cell contact-dependent bioerodable nanoreservoirs described here will permit sustained release of drugs, genes, growth factors, etc., opening a general route to the design of sophisticated cell-therapy implants capable of robust and durable regeneration of a broad variety of tissues.

Tooth Engineering: Searching for Dental Mesenchymal Cells Sources

The implantation of cultured re-associations between embryonic dental mesenchymal cells and epithelial cells from mouse molars at embryonic day 14 (ED14) allowed making full teeth with crown, root, periodontal ligament fibers, and bone. Although representing valuable tools to set up methodologies embryonic cells are not easily available. This work thus aimed to replace the embryonic cells by dental mesenchymal cell lines or cultured expanded embryonic cells, and to test their ability to mediate tooth development in vitro when re-associated with a competent dental epithelium. Histology, immunostaining and RT-PCR allowed getting complementary sets of results. Two different immortalized cell lines from ED18 dental mesenchyme failed in mediating tooth formation. The potentialities of embryonic dental mesenchymal cells decreased from ED14 to ED16 and were lost at ED18. This is likely related to a change in the mesenchymal cell phenotype and/or populations during development. Attempts to cultivate ED14 or ED16 embryonic dental mesenchymal cells prior to re-association led to the loss of their ability to support tooth development. This was accompanied by a down-regulation of Fgf3 transcription. Supplementation of the culture medium with FGF2 allowed restoring Fgf3 expression, but not the ability of mesenchymal cells to engage in tooth formation. Altogether, these observations suggest that a competent cell population exists in the dental mesenchyme at ED14, progressively decreases during development, and cannot as such be maintained in vitro. This study evidenced the need for specific conditions to maintain the ability of dental mesenchymal cells to initiate whole tooth formation, when re-associated with an odontogenic epithelium. Efforts to improve the culture conditions will have to be combined with attempts to characterize the competent cells within the dental mesenchyme.

Nanofibers Implant Functionalized by Neural Growth Factor as a Strategy to Innervate a Bioengineered Tooth

Current strategies for jaw reconstruction require multiple procedures, to repair the bone defect, to offer sufficient support, and to place the tooth implant. The entire procedure can be painful and time-consuming, and the desired functional repair can be achieved only when both steps are successful. The ability to engineer combined tooth and bone constructs, which would grow in a coordinated fashion with the surrounding tissues, could potentially improve the clinical outcomes and also reduce patient suffering. A unique nanofibrous and active implant for bone-tooth unit regeneration and also the innervation of this bioengineered tooth are demonstrated. A nanofibrous polycaprolactone membrane is functionalized with neural growth factor, along with dental germ, and tooth innervation follows. Such innervation allows complete functionality and tissue homeostasis of the tooth, such as dentinal sensitivity, odontoblast function, masticatory forces, and blood flow.

Restoring physiological cell heterogeneity in the mesenchyme during tooth engineering

Tooth development is controlled by reciprocal epithelial-mesenchymal interactions. Complete teeth can form when culturing and implanting re-associations between single embryonic dental epithelial and mesenchymal cells. Although epithelial histogenesis is clear, very little is known about cell diversity and patterning in the mesenchyme. The aim of this work was to compare the situation in engineered and developing teeth at similar developmental stages. To this end, the expression of cell surface markers in the mesenchyme was investigated by immunostaining in: 1) embryonic mouse molars at embryonic day 14, as the initial cell source for re-associations, 2) cultured cell re-associations just before their implantation and 3) cultured cell re-associations implanted for two weeks. Surface markers allowed visualization of the complex patterning of different cell types and the differential timing in their appearance. The phenotype of mesenchymal cells rapidly changed when they were grown as a monolayer, even without passage. This might explain the rapid loss of their potential to sustain tooth formation after re-association. Except for markers associated with vascularization, which is not maintained in vitro, the staining pattern in the mesenchyme of cultured re-associations was similar to that observed in situ. After implantation, vascularization and the cellular heterogeneity in the mesenchyme were similar to what was observed in developing molars. Besides tissue oxygenation and its role in mineralization of dental matrices, vascularization is involved in the progressive increase in mesenchymal cell heterogeneity, by allowing external cells to enter the mesenchyme.

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.

Tooth Organ Engineering: Biological Constraints Specifying Experimental Approaches

Basically, two types of approaches are currently being developed for tooth engineering. The first one consists in the engineering of dental constituents, such as the periodontium, the pulp/dentin, or the enamel/dentin complexes (Duan et al., 2011; Honda et al., 2009; Huang, 2009; Park et al., 2010). In parallel with these experiments of tissue engineering, attempts are also made to reconstruct a whole tooth (Arany et al., 2009; Honda et al., 2008; Hu et al., 2006a; Komine et al., 2007; Nakao et al., 2007; Ohazama et al., 2004). Most of this chapter will consider the second goal, and only use data from tooth tissue engineering, when they bring information about the cellular or molecular mechanisms that are involved and/or about the specific constraints, which they may illustrate. Few groups are interested in a biomimetic approach to engineer a whole tooth, including crown, roots, and periodontium by using cultured cell-cell re-associations and trying to recapitulate the successive steps of tooth development (Arany et al., 2009; Honda et al., 2008; Hu et al., 2006a; Nakao et al., 2007; Ohazama et al., 2004). Specific questions arising from this research concern the experimental design, and the search for easily available cell sources. The panel of approaches is progressively restricted by two types of biological constraints: those specifically related to tooth functionality and those related to more general biological aspects such as the maintenance of the cell heterogeneity in the dental mesenchyme and in the periodontium, keeping the gradients of cell differentiation and their 3D-geometry, or the maintenance of the cell kinetic parameters to ensure the differential cusp timing and growth. These complementary points will be discussed in light of parallel approaches being developed by different groups.

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

Abstract 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. Graphical abstract Figure. No caption available. HighlightsCombining 2D endothelial cells/3D tumor cells leads to a well‐organized network.This network consists of tubule‐like structures able to infiltrate 3D tumor.These vessels present a lumen and express specific marker as CD31 and Collagen IV.Combined 2D/3D strategy allows to an increased expression of angiogenic factors.

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-β1 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.

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.

Tooth Organ Engineering

Abstract As part of regenerative medicine, tooth organ engineering raises a series of distinct biological challenges to be solved before any clinical development can be considered. These include the finding of easily available cell sources, the maintenance of odontogenetic properties of cultured cells, and the synchronization of very distinct biological processes. Summarizing the main achievements concerning the engineering of a complete tooth and attachment tissues (crown and root formation, functional differentiation of odontoblasts, ameloblasts and cementoblasts, periodontium attached to newly formed bone), this review aims to point out the main questions that still need to be addressed. Taking into account the specificities of tooth development, the search for competent cells to be used for engineering will need to determine the simplest minimal requirements that have to be preserved. The loss of odontogenic properties in vitro raises the question of possible cell selection versus the relationship between microenvironment and cell phenotype. Addressing these questions will require a better understanding of cell homeostasis and heterogeneity during tooth development and engineering. Although methodological adjustments will be necessary when changing the cell sources, a lot remains to be done, still working with embryonic mouse dental cells. With this system, it will be necessary to search for the molecular mechanisms sustaining the “instructive potential” of the epithelium, as required for the specification of mesenchymal cells, the initiation of odontogenesis and later, the specification of a dental epithelial-mesenchymal junction.