Tchilalo Boukpessi

Elvire Le Norcy

Céline Gaucher

Marjolaine Gosset

Gaël Rochefort

Caroline Gorin

Dominique Septier

Dominique Le-Denmat

Brigitte Baroukh

Annie Llorens

Jeremy Sadoine

Sandy Ribes

Elisabeth Jimenez

Maheva Garcia

Tania Selbonne

Alexandra Benoist

EA S Opsahl-Vital

F Mangione

M Ezeldeen

C Bardet

J Lesieur

M Bonneau

F Decup

B Salmon

R Jacobs

C Chaussain

Implanted Dental Pulp Cells Fail to Induce Regeneration in Partial Pulpotomies

Keywords: stem/progenitor cell therapy, tissue engineering, reparative dentin, miniature pig, microCT, tissue engineering

Cell-based partial pulp regeneration is one of the promising approaches to obtain newly formed functional dentin-pulp complex. It relies on the preservation of the healthy tissue while regenerating the damaged pulp. The aim of this study was to investigate whether this regenerative process could be achieved by implanting porcine dental pulp cells (pDPCs) in pulp defects in the minipig. By split-mouth model, self-assembling injectable nanopeptide hydrogel, with and without pDPCs, was implanted after cameral pulpotomy in premolars and molars. At day 21 after surgery, 3-dimensional morphometric characterization, Masson's trichrome staining, and immunolabeling for DSP and BSP (dentin sialoprotein and bone sialoprotein) were performed on treated teeth. This study demonstrated no pulp regeneration but systematic reparative dentinogenesis. In fact, regardless of the presence of pDPCs in the scaffold, an osteodentin bridge-the microarchitecture of which significantly differed from the native dentin-was systematically obtained. Furthermore, the presence of pDPCs significantly affected the microstructure of the dentin bridges. In the radicular area of each treated tooth, hyperemia in the remaining pulp and external root resorptions were observed. Under the conditions tested in this work, pulp regeneration was not achieved, which highlights the need of further investigations to develop favorable regenerative microenvironment.

At the conclusion of my PhD, I would like to sincerely thank all the persons who directly or indirectly contributed to this important experience of my professional as well as personal life.

First of all my gratitude goes to Prof. Sibylle Vital who directed this work and patiently guided my evolution as a researcher.

I am very grateful to Prof. Catherine Chaussain who accepted me in her laboratory 4 years ago and constantly gave support to Sibylle and me during decisive moments of this experience.

My most sincere thank you is addressed to Prof. Galler and Prof. Lambrichts for accepting to be the referees and part of the committee of my thesis.

For sure I am grateful to Prof. Jacobs, Prof. Simon, Prof. Vital and Prof. Chaussain for being part of the committee: I am very honoured of your presence.

Then I would like to address a special thanks to Prof. Jacobs for her very kind welcome in her laboratory in Leuven during 3 months: it has been a honour for me and this experience has been amazing.

So I cannot forget to sincerely thank all the team of Oral and Maxillo-facial Surgery -Imaging & Pathology (OMFS-IMPATH) of the University of Leuven (KU Leuven), and especially my friends Mostafa, Laura, Jeroen and Yan: you all teached me a lot and you gave me a lot of support.

I am very thankful to the laboratory Cr2i of INRA (Jouy-en-Josas) in particular to Michel Bonneau and Chantal Kang for they very precious collaboration and constant help.

I am grateful to University Paris Descartes, National French Agency for Research (Grant PulpCell ANR-14-CE16-0006-01) and Fondation des Maladies Rares (Grant large animal experiment LAM-RD-U201140104) that supported this work with their grants.

For sure I want to thank all the members of the laboratory EA2496: Anne Polliard, Bernard Pellat, Jean-Louis Saffar, Marie-Laure Colombier, Philippe Bouchard, Claire Bardet, Benjamin Salmon,

Abstract

The dental pulp is a connective tissue, which is highly innervated and vascularized, encapsulated in a mineralized inextensible structure formed by enamel, dentin and cementum, ensuring the homeostasis and sensibility of the tooth. The pulp is often damaged by caries and trauma, resulting in infection or necrosis. In such situations, the routine clinical treatment is a root canal therapy, which consists in the elimination of the affected tissue and filling of the endodontic canal system with bioinert synthetic materials. In spite of satisfactory clinical outcomes, none of the original functions is restored and the lack of sensitivity as well as natural defence may lead to tooth fracture and reinfection.

Cell-based pulp regeneration could provide a valid alternative to traditional endodontic treatment of damaged teeth. This strategy focuses, in fact, on the preservation of the healthy pulp tissue and the regeneration of the damaged one, by combining stem cells, scaffolds and growth factors.

In case of trauma or carious lesion, as the pulp inflammatory reaction is compartmentalized in first instance, such conservative approach could be indicated. Regarding non-rodent animal model, to our knowledge, only Iohara et al. (2009) reported the achievement of partial pulp regeneration in canine tooth by implantation of subfractions of autologous pulp cells; however, in the perspective of a transfer to the human clinic, larger animal models should be developed to test the feasibility and the success of the therapy mimicking the clinical conditions of pulpotomy.

Due to dental anatomical and physiological similarities with human, the minipig constitutes a model of choice for preclinical pulp engineering studies.

The aim of this study was to develop a preclinical model of partial dental pulp regeneration in minipig, by implanting a pulp construct, made by self-assembling nano-peptide injectable hydrogel and porcine minipig dental pulp cells (pDPCs), in artificially created pulp defects.

Secondarily, in the context of this preclinical model, two different techniques of analysis of the regeneration process have been developed. In particular, an in vivo 3D subtraction angiography has been set for the visualization of dental pulp vascular network. Indeed, further developments of this modality open promising perspectives of its application for the morphometric characterization of angiogenesis process in newly formed dental tissues and bone defects.

Moreover, using specific morphometric parameters, initially developed to characterize bone, a micro-CT morphometric analysis of the mineralized reparative tissues, obtained by the partial pulp regeneration protocol, has been elaborated. By split mouth model, pulp constructs made with self-assembling injectable nano-peptide hydrogel with and without porcine dental pulp cells (pDPCs) were implanted, after pulp chamber amputation in premolars and molars. At day 21 after surgery, three-dimensional morphometric characterization, Masson's trichrome and immunolabeled for DSP and BSP were performed on treated teeth. 3D subtraction angiographies have been performed before and after partial pulp regeneration procedure.

Regardless of the presence of pDPCs, the implantation of pulp construct induces the formation of an osteodentin bridge, whose microarchitecture sensibly differs from the native dentin. Furthermore, the presence of pDPCs in the construct slightly impairs this reparative process. The latter was led the remaining pulp cells, instead of the pDPCs in the scaffold. Angiographies could show entire vascularization of jaws and continuously growing teeth but blood supply of treated mature permanent teeth could not be displayed.

The failure of partial pulp regeneration cell based strategy, in these near-real clinical conditions, highlights the importance of preclinical models, to identify the factors promoting a favourable regenerative environment, in the perspective of a transfer to the human clinics.

L'objectif principal de cette étude est de tester la faisabilité de la régénération pulpaire partielle, en implantant des cellules pulpaires porcines (pDPCs) contenues des hydrogels injectables dans des défauts pulpaires artificiellement créés chez le miniporc.

Au cours ce travail, différentes techniques d'imagerie d'évaluation du processus de régénération ont été développées. En particulier, un protocole d'angiographie tridimensionnelle in-pour la visualisation du réseau vasculaire pulpaire a été mise au point.

Par ailleurs, en utilisant des paramètres morphométriques spécifiques, initialement développés pour caractériser l'os, une analyse tridimentionnelle par micro-CT des tissus minéralisés de réparation a été élaborée.

En appliquant un "split mouth model", les hydrogels injectables ensemencés ou non par des pDPCs ont été implantés dans des molaires et des prémolaires, après amputation de la pulpe camérale. À 21 jours après la chirurgie, les analyses d'imagerie, d'histologie et d'immunologie ont mis en évidence, qu'indépendamment de la présence des pDPCs, l'implantation des hydrogels a induit la formation d'un pont d'ostéodentine. La caractérisation morphométrique tridimensionnelle a montré que la microarchitecture de ce pont différait largement de la dentine native. De plus, en présence des pDPCs, le processus de réparation était modifié, avec une moins bonne étanchéité du pont.

Au cours de ce travail, une technique de suivi non invasive de la régénération a tenté d'être mise au point. Une angiographie tridimensionnelle par soustraction a été réalisée avant et après la procédure de régénération pulpaire partielle. Si les angiographies ont révélé l'entière vascularisation des mâchoires et des dents à croissance continue, l'apport vasculaire des dents matures traitées, du fait de son faible flux, n'a pas pu être mis en évidence.

L'absence de régénération partielle de la pulpe dans les conditions testées souligne l'importance des modèles précliniques pour identifier les facteurs promouvant un environnement favorable à la régénération, dans la perspective d'un transfert vers la clinique humaine.

TABLE OF ABBREVIATIONS

TABLE OF FIGURES

INTRODUCTION

The dental pulp is the connective tissue enclosed into the mineralized inextensible structure of dental walls. It ensures the homeostasis and sensibility of the tooth. When this tissue is damaged, consecutively to caries or trauma, the routine clinical treatment is a root canal therapy. This procedure consists in the elimination of the affected tissue and filling of the endodontic canal system with bioinert synthetic materials. In spite of satisfactory clinical outcomes, this treatment does not restore the original sensitivity, neither the natural defence of the pulp.

Thus, there is an increasing desire to provide alternative options for endodontic therapy and one of the most significant challenges in modern dentistry is the regeneration of a vital dental pulp.

Cell-based pulp regeneration could provide a valid alternative to traditional endodontic treatment of damaged teeth. This strategy focuses, in fact, on the preservation of the healthy pulp tissue and the regeneration of the damaged one, by combining stem cells, scaffolds and growth factors.

In the laboratory EA2496 directed by Prof. Catherine Chaussain, research is conducted on pulp regeneration. In particular Gorin et al. have largely investigated the growth factors implicated in the angiogenic capacity of SHED seeded into hydrogels and in vivo subcutaneous implantations (Gorin et al., 2016). Souron et al., studied partial pulp regeneration approach in a rat model, showing positive outcomes after implantation of a pulp construct made by collagen I and DPCs in artificially cretaed pulp defects (Souron et al., 2013). 20 Thus, in the perspective of a preclinical application of these results and thanks to the opportunity to work with the laboratory CR2i of INRA (Jouy-en-Josas) directed by Prof.

Laurent the present project has been carried on minipigs.

AIM

The aim of this study was to develop a preclinical model of partial dental pulp regeneration in minipig, by implanting a pulp construct, made by self-assembling nano-peptide injectable hydrogel and porcine minipig dental pulp cells (pDPCs), in artificially created pulp defects.

In parallel, an interventional imaging method was explored to follow up the tissue engineering process, by interventional CBCT angiography.

RESEARCH HYPOTHESIS

This work relies on the hypothesis that autologous transplanted pDPCs could regenerate in vivo a functional parenchyma in pulpotomized permanent teeth and that this functionality could be detected by advanced imaging techniques

OBJECTIVES

DENTAL PULP AND DENTAL PULP STEM CELLS (DPSCs)

The dental pulp is a unique and complex connective tissue serving to support the dentin. It is highly innervated and vascularized, encapsulated in a mineralized inextensible structure formed by enamel, dentin and cementum, assuring the homeostasis and sensibility of the tooth organ [START_REF] Piette | La dent normale et pathologique[END_REF][START_REF] Goldberg | Pulp healing and regeneration: more questions than answers[END_REF][START_REF] Rosa | Pluripotency of stem cells from human exfoliated deciduous teeth for tissue engineering[END_REF].

The pulp tissue is composed of collagen type I and type III along with a variety of noncollagenous proteins, including a large proteoglycan component [START_REF] Colombo | Scaffolds to control inflammation and facilitate dental pulp regeneration[END_REF].

The pulp contains a variety of cell types, including immune cells, fibroblasts, mesenchymal progenitor cells, vascular cells, and nerve cells [START_REF] Jontell | Immune defense mechanisms of the dental pulp[END_REF].

The fibroblasts are the most numerous cells in the dental pulp, they located in the central part of the parenchyma. Their main role is synthesis of collagen I and III fibers, proteoglycans and glycoproteins as well as cytokines [START_REF] Mathieu | Role of injured endothelial cells in the recruitment of human pulp cells[END_REF]. The odontoblasts are disposed with a palisade pattern in contact with dentin and they show the Tomes' process, which is a cytoplasmic extension located into dentin tubules, associated with a system of fluid-filled tubules actively involved in dentin mineralization. These cells secrete the primary dentin, then the secondary and tertiary dentin [START_REF] Magloire | Odontoblast primary cilia: facts and hypotheses[END_REF] and they also contribute to proprioception and nociception [START_REF] Magloire | Odontoblast: a mechano-sensory cell[END_REF][START_REF] Magloire | Topical review. Dental pain and odontoblasts: facts and hypotheses[END_REF][START_REF] Maurin | Odontoblast: a key cellinvolved in the perception of dentinal pain[END_REF].

The population of immune cells, in particular tissue macrophages, hold themselves ready to respond to microbial incursion [START_REF] Colombo | Scaffolds to control inflammation and facilitate dental pulp regeneration[END_REF].

A network of blood vessels runs throughout the pulp, perfusing the tissue and networks of nerve fibers provide enervation to the tissue, linking it with the central nervous system and providing sensory output.

Finally, as in many other dental tissues such as periodontal ligament, apical papilla and dental follicle, a resident population of mesenchymal progenitor cells provides a reservoir of pluripotent cells capable of differentiation into a variety of cells as required for tissue maintenance and repair.

In addition to cellular elements, pulp presents an extracellular matrix (ECM), mainly composed by collagen, glycosaminoglycans, glycoproteins and metallo-proteases (Bogovic et al., 2011). Such a composition of the ECM allows the maintenance of hydration by water molecules storage. They manage the transit of metabolites, nutrients and cells debris among blood vessels and pulp cells [START_REF] Goldberg | Dentin extracellular matrix molecules implanted into exposed pulps generate reparative dentin: a novel strategy in regenerative dentistry[END_REF]. Indeed, in the perspective of a conservative approach of the pathologies affecting the pulp, regenerative strategies are increasingly investigated. However, the peculiar heterogeneity of the tissue makes pulp regeneration challenging.

DENTAL PULP STEM CELLS

Gronthos' team has highlighted the presence of stem cells in the pulp of permanent teeth, in 2000. These cells, named Dental Pulp Stem Cells (DPSCs), showed their attitude to form the dentin-pulp complex in vitro (Gronthos et al., 2000).

Few years later, the same researchers have showed that stem cells were also present within pulp of temporary teeth. This population, named Stem Cells from Human Exfoliated Deciduous teeth (SHED) [START_REF] Batouli | Comparison of stemcell-mediated osteogenesis and dentinogenesis[END_REF], thanks to the physiological exfoliation of temporary teeth, represents a unique and natural source of stem cells [START_REF] Batouli | Comparison of stemcell-mediated osteogenesis and dentinogenesis[END_REF][START_REF] Kerkis | Stem cells in dental pulp of deciduous teeth[END_REF]. Some surface markers in common with BMSC have been identified (Gronthos et al., 2000) by (Gronthos et al., 2000). Endogenous alkaline phosphatase (L) (Gronthos et al., 2000).

Phenotypically, DPSCs are similar to BMSCs [START_REF] Eleuterio | Proteome of human stem cells from periodontal ligament and dental pulp[END_REF]. Surface markers and proteins distribution are also comparably found (Gronthos et al., 2000;[START_REF] Gronthos | Stem cell properties of human dental pulp stem cells[END_REF][START_REF] Eleuterio | Proteome of human stem cells from periodontal ligament and dental pulp[END_REF][START_REF] Cao | Adenovirus-mediated transfer of hepatocyte growth factor gene to human dental pulp stem cells under good manufacturing practice improves their potential for periodontal regeneration in swine[END_REF].

Moreover, DPSCs seem to present a higher proliferation rate than BMSCs, without loosing their multipotency (Gronthos et al., 2000;[START_REF] Seong | Stem cells in bone tissue engineering[END_REF]Gong et al., 2016).

Isolation

DPSCs can be isolated either by the enzymatic digestion or outgrowth methods [START_REF] Hilkens | Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells[END_REF]. Since they have a high renewal property, Gronthos et al. in 2000 proposed to individualize cells, to seed them and to select clones having the highest proliferation rate.

This property is maintained beyond 25 passages (Rodriguez-Lozano et al., 2012).

Several markers are proposed for human dental pulp stem cells characterization [START_REF] Kawashima | Characterisation of dental pulp stem cells: A new horizon for tissue regeneration?[END_REF]Gong et al., 2016): the most of them, like OCT3/4, CD90, STRO1, are in common with BMSCs. So stem cells markers can be used to sort them by FACS or by antibodies linked to spheres [START_REF] Kawashima | Characterisation of dental pulp stem cells: A new horizon for tissue regeneration?[END_REF]. Nevertheless, no specific DPSCs markers have been identified yet.

Differentiation

In vitro, DPSCs can differentiate into several germ cell lines [START_REF] Kawashima | Characterisation of dental pulp stem cells: A new horizon for tissue regeneration?[END_REF]. In fact, many protocols allow their differentiation into mesodermic line cells such as myocites, osteoblasts, chondrocytes, adipocytes and cardiomyocytes (Jo et al., 2007;Koyama et al., 2009;[START_REF] Gronthos | A method to isolate and culture expand human dental pulp stem cells[END_REF][START_REF] D' Alimonte | Vascular endothelial growth factor enhances in vitro proliferation and osteogenic differentiation of human dental pulp stem cells[END_REF]Dhillon et al., 2015). Endodermic line cells (hepatocytes) differentiation can also be obtained (Iohara et al., 2006;Rodriguez-Lozano et al., 2012;Dhillon et al., 2015). The differentiation into neural cells has been demonstrated [START_REF] Iohara | Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1[END_REF][START_REF] Mao | Regenerative endodontics: barriers and strategies for clinical translation[END_REF][START_REF] Fang | Intraventricular injection of human dental pulp stem cells improves hypoxic-ischemic brain damage in neonatal rats[END_REF] but the maturation into neurons is still controversial [START_REF] Gervois | Neurogenic maturation of human dental pulp stem cells following neurosphere generation induces morphological and electrophysiological characteristics of functional neurons[END_REF][START_REF] Iohara | Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1[END_REF].

Moreover, many authors have demonstrated that DPSCs can differentiate into endothelial cells in vitro and in vivo (Gorin et al., 2016[START_REF] Iohara | Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1[END_REF][START_REF] Hilkens | Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells[END_REF][START_REF] Iohara | Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1[END_REF][START_REF] Ratajczak | The neurovascular properties of dental stem cells and their importance in dental tissue engineering[END_REF]. This capacity is particularly suitable for tissue engineering.

.

TISSUE ENGINEERING

SCAFFOLDS

In the tissue-engineering triade, the scaffold plays a very important role. In fact, it represents a three-dimensional substrate mimicking the ECM (Ma, 2008) for cell adhesion, proliferation and differentiation. At the same time the scaffold has to be biocompatible and biodegradable and it has to be mechanically, biologically and chemically adaptable for specific tissues regeneration (Galler et al., 2010;Galler et al., 2011;Yuan et al., 2011).

Scaffold can be made by natural or synthetic biodegradable polymers, inorganic materials and composites. Moreover, they can be shaped as porous scaffolds, nanofibrous materials, microparticles, and hydrogels.

The choice of an appropriate scaffold relies on the specific application and it is strongly influenced by the availability and the ease of manipulation, as well as by the balance cost/ effectiveness (Galler et al., 2010;[START_REF] Sun | Biological approaches toward dental pulp regeneration by tissue engineering[END_REF]Yuan et al., 2011).

Natural polymers are mechanically resistant, bioactive and biodegradable but they are not easy to be processed and they can induce immune reaction.

Synthetic polymers show good processing, manipulation and mechanical properties but they have low bioactivity and they induce high inflammatory reaction (Yuan et al., 2011).

Regarding pulp regeneration, collagen type 1, PLGA and PLA have been reported as the scaffolds providing the best outcomes [START_REF] Albuquerque | Tissue-engineering-based strategies for regenerative endodontics[END_REF].

A valid alternative is represented by hydrogels, which are characterized by high water content, viscoelastic properties matching with those of soft tissues and high capacity of cell encapsulation and delivery (Nicodemus et al., 2008;Galler et al., 2010).

Even if collagen hydrogels have been widely used for pulp-dentin complex regeneration, they are difficult to be purified and not stable. Thus, synthetic biomimetic hydrogels are being increasingly used.

Galler et al. showed that self-assembling multidomain peptides (MDP) supported pulp regeneration (Galler et al., 2008;Galler et al., 2010;Galler et al., 2012).

GROWTH FACTORS

The third factor of tissue engineering is represented by growth factors. They determine cellular fate by a signalling action within the regenerative environment [START_REF] Bansal | Regenerative endodontics: a state of the art[END_REF]. These factors can derive from degraded dentin matrix, blood clot or be synthesized in vitro [START_REF] Bessho | Human dentin-matrix-derived bone morphogenetic protein[END_REF].

Nakashima showed that recombinant human BMP2 and BMP4 were directly involved in dentin and osteodentin deposition in dog pulp defects capped with inactivated dentinmatrix (Nakashima, 1994). The same factors have been reported by Casagrande et al.

as inducing SHEDs differentiation into odontoblasts when seeded in tooth slices treated with EDTA (Casagrande et al., 2010). VEGF is a proangiogenic factor. Its activity has been demonstrated on SHED (Sakai et al., 2010) and in co-cultures of SCAPs and HUVECs (Yuan et al., 2015). bFGF is involved in odontogenesis and in fibroblasts' reaction to injuries (Shimabukuro et al., 2009). It has been reported that this factor inhibits differentiation and increases survivability of DPSCs (He et al., 2008;[START_REF] Yang | Autophagy in SDF-1αmediated DPSC migration and pulp regeneration[END_REF].

However, the use of growth factors is complicated because their nature, their dosage, and the delivery timing may induce controversial biological effects on cellular behaviours (Gong et al., 2016).

CELL-BASED PULP REGENERATION

DPSCs have been employed in the regeneration of periodontal bone defects and dentin-pulp complex (Zheng et al., 2012, Hilkens et al., 2015).

More specifically, DPSCs showed their capacity of differentiation into several types of pulp cells. Very important aspect is their capacity to differentiate into odontoblasts and synthetize dentin [START_REF] Yu | Differentiation of dental pulp stem cells into regular-shaped dentin-pulp complex induced by tooth germ cell conditioned medium[END_REF][START_REF] Onyekwelu | Tooth development: 2. Regenerating teeth in the laboratory[END_REF], Hilkens et al., 2015).

In immunocompromised mice, DPSC transplanted with hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold formed a pulp tissue characterized by blood vessels and human odontoblastlike cells (Gronthos et al., 2000;[START_REF] Gronthos | Stem cell properties of human dental pulp stem cells[END_REF][START_REF] Batouli | Comparison of stemcell-mediated osteogenesis and dentinogenesis[END_REF]. The same result has been achieved in more standardized model using human tooth slices and DPSC or SHED implanted in immunodeficient mice (Cordeiro et al., 2008;Prescott et al., 2008;Gorin et al., 2016).

Vascularized pulp / dentin-like tissue has been obtained by Huang et al. (2010) by implanting subcutaneously in immunodeficient mice an emptied human root (with an apical foramen of 2.5 mm) seeded with a combination of DPSCs and poly(lactic-co-glycolic acid) (PLGA) scaffold. The same results has been demonstrated in ectopic root transplantation using SHEDs associated with self-assembling peptide hydrogel (Rosa et al., 2013).

In presence of a narrower apical foramen (1 mm) Dissanayaka et al. could achieve pulp regeneration after transplantation of DPSCs or DPSCs-HUVECs co-cultures (Dissanayaka et al., 2015). This result is consistent with the study of Yang et al. based on the transplantation of DPSCs in a silk fibroin scaffold [START_REF] Yang | Autophagy in SDF-1αmediated DPSC migration and pulp regeneration[END_REF].

Regarding in situ transplantation, Iohara et al. obtained reparative dentin by implanting autologous BMP2-treated DPSC pellets into canine pulp defects [START_REF] Iohara | Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2[END_REF]Iohara et al., 2006).

The same authors achieved pulp regeneration in canine pulpectomy models [START_REF] Iohara | Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1[END_REF][START_REF] Ishizaka | Regeneration of dental pulp following pulpectomy by fractionated stem/progenitor cells from bone marrow and adipose tissue[END_REF][START_REF] Wang | Preliminary study on dental pulp stem cell-mediated pulp regeneration in canine immature permanent teeth[END_REF] and more recently they showed pulp-dentin complex regeneration by using G-CSF mobilized DPSCs in canine mature teeth (apical foramen 0.6 mm diameter) [START_REF] Iohara | A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration[END_REF][START_REF] Nakashima | Mobilized dental pulp stem cells for pulp regeneration: initiation of clinical trial[END_REF].

OTHER DENTAL TISSUES STEM CELLS

In addition to DPSCs, many other types of stem cells from dental tissues have been then isolated and characterized: Periodontal Ligament Stem Cells (PDLSCs), Stem Cells From Apical Papilla (SCAPs) and Dental Follicle Stem Cells (DFSCs).

Stem cells from the apical papilla

This population of stem cells isolated from the apical papilla which is a connective tissue located at the apex of developing permanent teeth is increasing the interest in pulp regeneration field [START_REF] Sonoyama | Mesenchymal stem cell-mediated functional tooth regeneration in swine[END_REF]Sonoyama et al., 2008).

In fact, SCAPs show a higher capacity of proliferation and migration in comparison with DPSCs. Already in 2006, Sonayama et al. achieved dentin pulp complex regeneration by transplanting SCAPs with HA/TCP scaffold in immunocompromised mice [START_REF] Sonoyama | Mesenchymal stem cell-mediated functional tooth regeneration in swine[END_REF].

Regarding ectopic root transplantation, better results have been showed by Huang et al.

with SCAPs-PLGA than in the model with DPSCs-PLGA construct (Huang et al., 2010).

Moreover, well documented is the role of SCAPs in differentiating into odontoblasts and promoting root development in immature teeth affected by periodontitis or apical abscesses [START_REF] Hilkens | Dental Stem Cells in Pulp Regeneration: Near Future or Long Road Ahead?[END_REF].

Dental follicle precursor cells

This population of stem cells is found in the dental follicle, the connective tissue surrounding the developing tooth and generating the periodontium.

Indeed, most of studies have investigated the capacity of DFSCs in the regeneration of periodontal tissues (Yaguu et al., 2010;Guo et al., 2012;Guo et al., 2013).

Regarding pulp regeneration, Guo et al. showed that FSCs seeded in treated dentin matrix (TDM) were able to give rise to odontoblast and dentin in the omental pouch of adult rats (Guo et al., 2009). Cementum-like, periodontal ligament and dentin-pulp complex were obtained by the same group of researchers transplanting DFSCs-TDM construct in rat alveolar sockets (Guo et al., 2012).

The ability of these cells to regenerate such a variety of dental tissues was further confirmed by implanting subcutaneously in immunocompromised mice the same construct made with human DFSCs [START_REF] Yang | Fibrin glue mixed with platelet-rich fibrin as a scaffold seeded with dental bud cells for tooth regeneration[END_REF]Jiao et al., 2014).

Periodontal ligament stem cells

PDLSCs can differentiate into osteoblasts and regenerate bone (Seo et al., 2004;Gay et al., 2007;Lindroos et al., 2008;Xu et al., 2009). However, they form mineralized tissues only when cultured in tricalcium phosphate/hydroxyapatite (TCP/HA) or when a medium from apical papilla tooth germ is used (Shi et al., 2005;Yang et al., 2009).

Regarding pulp regeneration, the role of these cells is controversial because they express dentin markers only when co-cultured with DPSCs (Suh et al., 2014).

BANKING

Since the extraction of third molars, premolars and deciduous teeth is a common practice, dental stem cells are considered easily available. Moreover, it was demonstrated that their properties are maintained when cryopreserved [START_REF] Ding | Allogeneic periodontal ligament stem cell therapy for periodontitis in swine[END_REF]Lee et al., 2012). Even thought DSCs present low immunogenicity and immunomodulatory activity [START_REF] Kim | Regeneration of dental-pulplike tissue by chemotaxis-induced cell homing[END_REF]Tomic et al., 2011;Liu et al., 2012), at present allogeneic transplantation is not possible and only autologous DSCs could be used for pulp-dentin complex regeneration.

However, the small amount of cells isolated from the dental pulp needs to be processed before to be used for cell-based procedures.

DSCs banks exist, allowing a rapid access to stem cells in case of necessity of cell based therapies (Egusa et al., 2012;Huang et al., 2013).

Nowadays, several private societies propose the cryopreservation of dental pulp stem cells (Genecell International ® , USA) from extracted teeth. For the moment, these societies do not operate in France and in the most of European countries.

Unfortunately none of the private tooth banks provide clinical-grade MSCs because of the absence of good manufacturing practice allowing the standardization of cell banking taking into account the origin and donor-associated differences among stem cells (Gong et al., 2016). In fact, many studies have investigated the effect of epigenetic and genetic modifications induced by cell culture on the regenerative potential [START_REF] Duncan | Epigenetic modulation of dental pulp stem cells: implications for regenerative endodontics[END_REF][START_REF] Gopinathan | Epigenetic marks define the lineage and differentiation potential of two distinct neural crest-derived intermediate odontogenic progenitor populations[END_REF]. Thus cell banking should apply standardized expansion of stem cells in order to provide a genetically stable population, with specific tissue regeneration ability and lack of tumorigenicity [START_REF] Shahdadfar | In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability[END_REF]. Moreover these cells should not be contaminated by microorganisms: for this reason studies are conducted to develop culture media the without animal serum able to favour cell proliferation and preservation of their stemness properties [START_REF] Sotiropoulou | Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells[END_REF][START_REF] Mannello | Concise review: no breakthroughs for human mesenchymal and embryonic stem cell culture: conditioned medium, feeder layer, or feeder-free; medium with fetal calf serum, human serum, or enriched plasma; serum-free, serum replacement nonconditioned medium, or ad hoc formula? All that glitters is not gold![END_REF].

Finally standardized procedures should be developed for all the following steps regarding cell purification from the media, harvesting without porcine trypsin, as well as use of performing carriers [START_REF] Bakopoulou | Stem cells of dental origin: current research trends and key milestones towards clinical application[END_REF].

REGENERATIVE ENDODONTICS

It is widely established that the pulp has an innate capacity for self-repair and contains all the necessary components to regenerate both the mineralized dentin and the soft tissues of the pulpal matrix.

Indeed, considering the outcomes reported in literature, pulp regeneration became a clinical reality. In fact, if techniques such as revascularization and induction of reparative dentinogenesis are already widely applied on patients, on the other hand experimental procedures including cell homing, partial and total pulp regeneration still require further development.

REVASCULARIZATION

The revascularization is an approach applicable in necrotic/infected permanent immature teeth with an apical foramen of more than 1 mm. It relies on the intentional laceration of periapical area's tissues to obtain the formation of a blood clot in a decontaminated root canal system. The clot, which acts like a scaffold, is colonized by SCAPs and growth factors and promotes root development and elongation (Diogenes et al., 2013;[START_REF] Albuquerque | Tissue-engineering-based strategies for regenerative endodontics[END_REF].

However, this procedure seems promising only in very immature teeth, because the nature of the regenerated parenchyma and the possibility of root elongation are still debated [START_REF] Neha | Management of immature teeth by dentin-pulp regeneration: a recent approach[END_REF][START_REF] Nosrat | Regenerative endodontic treatment (revascularization) for necrotic immature permanent molars: a review and report of two cases with a new biomaterial[END_REF][START_REF] Andreasen | Pulp regeneration after non-infected and infected necrosis, what type of tissue do we want? A review[END_REF].

The regenerated tissue is constituted by cementum, bone and fibrous tissue (Wang et al., 2010;Cao et al., 2013;Martin et al. 2013). Thus, all these outcomes show that revascularization rather induces resolution of apical periodontitis by tissue repair [START_REF] Cao | Adenovirus-mediated transfer of hepatocyte growth factor gene to human dental pulp stem cells under good manufacturing practice improves their potential for periodontal regeneration in swine[END_REF].

REPARATIVE DENTINOGENESIS

As mentioned above, the presence of a population of progenitor cells within pulp tissue is demonstrated. In case of pulp injury, these cells can differentiate into odontoblasts-like and replace damaged odontoblasts [START_REF] Nakashima | The application of tissue engineering to regeneration of pulp and dentin in endodontics[END_REF][START_REF] Huang | A paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration[END_REF]Huang, 2009;[START_REF] Huang | Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine[END_REF][START_REF] Scheller | Tissue engineering: state of the art in oral rehabilitation[END_REF][START_REF] Sun | Biological approaches toward dental pulp regeneration by tissue engineering[END_REF].

Therapeutic approaches aiming pulp vitality preservation by formation of a reparative dentin bridge, after reversible pulp injury, rely on this principle. Nevertheless, the prognosis of a direct pulp capping in case of deep caries or traumas is still uncertain and this procedure needs to be applied only on few selected cases [START_REF] Bashutski | Periodontal and endodontic regeneration[END_REF].

In fact, the outcomes of such therapy are strictly dependent to pulp exposure type, inflammation degree, tooth age, materials choice and restoration sealing (Mjor, 2002;Murray et al., 2002;[START_REF] Ward | Vital pulp therapy in cariously exposed permanent teeth and its limitations[END_REF][START_REF] Murray | Odontoblast morphology and dental repair[END_REF][START_REF] Tziafas | The future role of a molecular approach to pulp-dentinal regeneration[END_REF].

Furthermore, the clinical diagnosis of inflammation degree is hard to be established and at present there are no guidelines allowing the clinicians to identify the cases suitable for direct capping treatment [START_REF] Huang | A paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration[END_REF][START_REF] Sun | Biological approaches toward dental pulp regeneration by tissue engineering[END_REF]Zanini et al., 2017).

As a matter of fact, it is frequent that so treated teeth subsequently undergo endodontic therapy.

The progress into the comprehension of molecular composition and cellular mechanisms regulating dentinogenesis favours new regeneration strategies in which the damaged pulp is partially or totally eliminated and replaced by a regenerated healthy functional tissue.

Thus, two different approaches are distinguished: in situ partial pulp and de novo pulp regeneration [START_REF] Nakashima | The application of tissue engineering to regeneration of pulp and dentin in endodontics[END_REF][START_REF] Huang | Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine[END_REF][START_REF] Sun | Biological approaches toward dental pulp regeneration by tissue engineering[END_REF].

CELL HOMING

The cell homing approach is a cell-free method to regenerate pulp-dentin complex. It represents an alternative to the revascularization and it relies on the stimulation and the migration of the endogenous MSCs on the area of the tissue to be regenerated [START_REF] Mao | Regenerative endodontics: barriers and strategies for clinical translation[END_REF]. In fact, many studies have reported regeneration of a functional pulp tissue into empty endodontic spaces by combining cytokines and growth factors promoting cellular chemotaxis and cell homing.

Kim et al. showed positives outcomes in vivo using associations of bFGF, PDGF or VEGF and NGF and BMP-7 in endodontically treated teeth [START_REF] Kim | Regeneration of dental-pulplike tissue by chemotaxis-induced cell homing[END_REF].

Suzuki demonstrated that the combination of bFGF and SDF1 recruits DPSCs and that BMP-7 promotes their differentiation (Suzuki et al., 2011). Moreover, Yang et al., obtained complete functional pulp regeneration using silk-fibroin scaffolds and SDF1 in mature canine teeth [START_REF] Yang | Autophagy in SDF-1αmediated DPSC migration and pulp regeneration[END_REF].

Overall, cell homing seems to be a promising economical approach for pulp regeneration and endodotic space revascularization.

IN SITU PARTIAL PULP REGENERATION

Biotechnologies focusing on partial pulp regeneration are based on the observation that in the first instance pulp inflammation is compartmentalized before that whole tissue is affected [START_REF] Huang | A paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration[END_REF]Huang, 2009;[START_REF] Huang | Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine[END_REF]. Current data suggest that the healthy part of the pulp could not only be conservable but also have the potential to regenerate the lost tissue (Huang, 2009). In order to favour such regeneration, pulp constructs constituted by DPSCs and scaffolds can be implanted in the endodontic space inducing pulp-dentin complex reformation (Huang, 2009;[START_REF] Sun | Biological approaches toward dental pulp regeneration by tissue engineering[END_REF].

In vivo, in large animal models, two different approaches of partial pulp regeneration have been proposed. The first consists in the implantation of a pellet of autologous DPSC in tooth underwent to pulp amputation. The second relies on the use of different kinds of scaffolds containing sorted DPSCs (expressing for example CD31-, CD146-antigens).

At present, only Iohara et al. have regenerated pulp tissue in dogs by implantation of autologous DPSC pellets (Iohara et al., 2009).

DE NOVO PULP SYNTHESIS

When pulp parenchyma is completely destroyed, de novo synthesis has to be done to regenerate the tissue. The volume of mature pulp is very small (about 10-100 μl), so the regeneration of pulp should be easier than big organs or tissues. Nevertheless, this tissue is hard to be regenerated because of the following characteristics: 1) it is placed within rigid calcified walls; 2) it has reduced blood supply due to the narrow apical foramen and 3) it is an heterogeneous tissue whose cells are distributed in several innervation layers.

Recently, many teams of researchers demonstrated the feasibility of the implantation and the survival of cells within the tooth in vivo. En 2009, Huang (Huang, 2009) showed the formation of a mineralized tissue by stem periodontal cells within pulp chamber in dog. In the same animal model, Iohara et al. [START_REF] Iohara | A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration[END_REF] obtained a functional vascularized and innervated pulp tissue by using different stem cells.

In 2017, Nakashima et al. published the first clinical trial outcomes of de novo pulp synthesis procedure and they demonstrated by MRI and CBCT functional dentin-pulp complex regeneration (Nakashima et al., 2017).

MONITORING METHODS OF ANGIOGENESIS IN TISSUE ENGINEERING

Vascularization has been studied for years using several in vitro and in vivo models.

Different mechanisms are distinguished: angiogenesis, arteriogenesis and vasculogenesis.

Angiogenesis relies on growth of capillaries from pre-existing vessels and it is activated by tissue ischemia. Arteriogenesis is process mediated by monocytes and macrophages, which consists on pre-existing vessels dilatation and remodelling as response to augmented physiological demands. Finally, vasculogenesis is a de novo blood vessel development by bone marrow-derived endothelial progenitor cells [START_REF] Duvall | Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury[END_REF].

Angiogenesis and vasculogenesis are deemed important for regenerative strategies of all soft and calcified tissues. In fact, a key factor in tissue engineering is the possibility to obtain a functional microvascular network within constructs providing oxygen and nutrients for engraftment, differentiation, and tissue functionality. Thus, the distance of the stem cells constructs from vascular supply is critical for integration to the receiving tissue [START_REF] Bolland | Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs[END_REF][START_REF] Duvall | Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury[END_REF].

Dental pulp is a highly vascularized tissue. Blood vessels penetrate within pulp by the apical and secondary foramina. They proceed to the root canal centre in direction of the pulp chamber in which they progressively brunch out to form in the periphery a thin subodontoblastic capillaries net of about 5 to 10 μm diameter [START_REF] Trubiani | Human dental pulp vasculogenesis evaluated by CD34 antigen expression and morphological arrangement[END_REF].

Blood vessels penetrate inside the pulp and go out from it by apical and secondary foramina in the form of one or sometimes two arterioles (35-45μm) passing through the centre of the root canal. Venous return is also made by the apical foramen and it's ensured by post-capillary veinules gathering to form collector veinules in the central part of the root canal.

The presence of artero-venous anastomosis contributes to the regulation of blood flux as well as intra-pulp pressure and they allow blood flux drift and loop isolation in case of injury.

Regarding pulp regeneration, blood supply ensures the survival of pulp constructs and their engraftment. Nevertheless, several factors like dental anatomy, narrow diameter of root apical foramen, presence of a terminal vascular net and characteristics of calcified tissues embedding the pulp, complicate these procedures. So, the preservation of a part of pulp parenchyma allows to rely on the root remaining vascularization and to put the construct in direct contact with blood vessels, to obtain its engraftment [START_REF] Bolland | Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs[END_REF] However, this therapeutic option is strictly dependent to the knowledge and the managing of the local blood supply, which must be precisely and reproductively quantified and 4a) (Blery et al., 2015). Among direct imaging techniques microscopy, angiography, computed tomography (CT)

and micro-CT both in association or not with a synchrotron system are the most used (Blery et al., 2015)(fig 4d).

In vivo angiogenesis of tissue-engineered constructs is mainly detected by two-dimensional evaluation of microvessel concentration within histological sections [START_REF] Pabst | Imaging angiogenesis: perspectives and opportunities in tumour research -a method display[END_REF].

The preclinical high-resolution imaging technology of micro-CT is customized to visualize mineralized tissue. However, more recently, this technique allowed a three-dimensional characterization of organs' vascular networks [START_REF] Lu | Detection of microvasculature in rat hind limb using synchrotron radiation[END_REF]. Besides, a little has been published about its use for the assessment of the vascularization in implanted tissue engineering scaffold.

In combination with radiological techniques, many contrast agents are implied: iodine, liquid fatty oil, resin, gold nanoparticles, Microfil and barium sulphate [START_REF] Pabst | Imaging angiogenesis: perspectives and opportunities in tumour research -a method display[END_REF].

Recently, some authors have assessed vascular microarchitecture parameters, deduced from bone microarchitecture analysis [START_REF] Bolland | Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs[END_REF][START_REF] Jia | Imaging and Quantitative Assessment of Long Bone Vascularization in the Adult Rat Using Microcomputed Tomography[END_REF][START_REF] Langer | Simultaneous 3D imaging of bone and vessel microstructure in a rat model[END_REF][START_REF] Prisby | Intermittent PTH(1-84) is osteoanabolic but not osteoangiogenic and relocates bone marrow blood vessels closer to bone-forming sites[END_REF][START_REF] Roche | Structure and quantification of microvascularisation within mouse long bones:what and how should we measure?[END_REF] using micro-CT and radio-opaque contrast agents. For this morphometric analysis, the vascular network should be entirely visualized (fig 5). By injecting barium sulphate the bone blood supply is not completely permeated, due to the heterogeneity of the network. Microfil, which is a silicon with chromate, perfuses entirely soft tissues, but it has the same gray level of mineralized structures. Thus, decalcification is required when using this contrast medium [START_REF] Bentley | The use of microcomputed tomography to study microvasculature in small rodents[END_REF][START_REF] Duvall | Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury[END_REF][START_REF] Marxen | MicroCT scanner performance and considerations for vascular specimen imaging[END_REF][START_REF] Bolland | Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs[END_REF][START_REF] Young | Microcomputed tomography characterization of neovascularization in bone tissue engineering applications[END_REF]Mondy et al., 2009a[START_REF] Nyangoga | Three-dimensional characterization of the vascular bed in bone metastasis of the rat by microcomputed tomography (MicroCT)[END_REF][START_REF] Roche | Structure and quantification of microvascularisation within mouse long bones:what and how should we measure?[END_REF].

In fact, thresholding is crucial for blood supply assessment. Clearly separating the vessels from the bone is important for a good estimation of the vascular and bone microarchitecture parameters. Synchrotron-CT is considered as the gold standard for bone microarchitecture and vascularization morphometric analysis [START_REF] Eppel | Contrast angiography of the rat renal microcirculation in vivo using synchrotron radiation[END_REF]Langer et al., 2009;[START_REF] Chien | Synchrotron microangiography studies of angiogenesis in mice with microemulsions and gold nanoparticles[END_REF][START_REF] Jia | Imaging and Quantitative Assessment of Long Bone Vascularization in the Adult Rat Using Microcomputed Tomography[END_REF][START_REF] Lu | Detection of microvasculature in rat hind limb using synchrotron radiation[END_REF][START_REF] Peyrin | Local plate/rod descriptors of 3D trabecular bone micro-CT images from medial axis topologic analysis[END_REF][START_REF] Roche | Structure and quantification of microvascularisation within mouse long bones:what and how should we measure?[END_REF][START_REF] Matsumoto | Subtraction micro-computed tomography of angiogenesis and osteogenesis during bone repair using synchrotron radiation with a novel contrast agent[END_REF]Holme et al., 2014;[START_REF] Neldam | Synchrotron μCT imaging of bone, titanium implants and bone substitutes-a systematic review of the literature[END_REF].

In fact, the collimated monochromatic beam of Synchrotron radiation (SR) provides micrometric spatial resolution for the identification of different structures and it allowed the visualization of vascular canals in human and rats cortical bone (Bousson et al., 2004; focused on the study of osteoporotic bone and fractures [START_REF] Ito | Assessment of bone quality using micro-computed tomography (micro-CT) and synchrotron micro-CT[END_REF][START_REF] Kazakia | Assessment of bone tissue mineralization by conventional X-ray microcomputed tomography: comparison with synchrotron radiation microcomputed tomography and ash measurements[END_REF]Cooper et al., 2011a), microcracks [START_REF] Voide | Timelapsed assessment of microcrack initiation and propagation in murine cortical bone at submicrometer resolution[END_REF][START_REF] Larrue | Synchrotron radiation micro-CT at the micrometer scale for the analysis of the threedimensional morphology of microcracks in human trabecular bone[END_REF], and alveolar bone modifications caused by orthodontic treatments [START_REF] Dalstra | Synchrotron radiation-based microtomography of alveolar support tissues[END_REF].

In vivo microangiography in rat hind limb has been reported to not allow visualization of capillaries (10 μm diameters) [START_REF] Lu | Detection of microvasculature in rat hind limb using synchrotron radiation[END_REF]. The Doppler flowmetry is a simple modality but it is not completely reliable and the probe can reach a very limited depth. Colour doppler ultrasound gives information about both arteries and veins, but it requires high competence in performance and interpretation of the exam. Moreover it does not allow three-dimensional (3D) visualization of anatomy [START_REF] Roche | Structure and quantification of microvascularisation within mouse long bones:what and how should we measure?[END_REF].

- ✓ ✓ - - +⁄- Casting/ Perfusion - ✓ - ✓ - +⁄- CT-Angiography ✓ - ✓ ✓ ✓ + SPECT ✓ - ✓ ✓ ✓ +/- Doppler ✓ - ✓ - ✓ +⁄- MRI ✓ - ✓ ✓ ✓ +/-
MRI for vascular supply visualization requires the use of a contrast agent gadoliniumchelated that is injected into a large vein. However, this technique has a limited spatial resolution (130microm).

SPECT is performed by injection in the bloodstream of a radioisotope (isotope of gallium) associated to a ligand to create a tissue specific radioligand whose concentration is registered by a gamma camera This modality presents low spatial resolution (1cm), soft tissue attenuation and exposition to ionising radiations (Johns et al., 2016;[START_REF] Yabuuchi | Anterior mediastinal solid tumours in adults: characterisation using dynamic contrast-enhanced MRI, diffusion-weighted MRI, and FDG-PET/CT[END_REF]. Indeed, the translation of regenerative endodontics to human clinics and the development of imaging techniques for the quali-quantitative analysis of regenerated tissues require in vivo studies in large animal models.

LARGE ANIMAL PRE-CLINICAL MODELS IN ORAL AND DENTAL TISSUES ENGINEERING

The development of regenerative medicine, based on stem cells requires animal models. For oral and dental tissue engineering, literature mainly reports in vivo studies in rodent models. However, rodents display limitations, due to their size and physiology [START_REF] Lee | Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency[END_REF]. The development of larger animal models is crucial in perspective to human application. The selection of the most pertinent animal species for a specific study relies on the evaluation of the several elements. First, the animal should be anatomically, physiologically as well as pathologically similar with humans and its involvement should allow the observation of a high number of subjects easily accessible, in a reasonable time period. The animal should be resistant to infections, disease and surgery and all biological information about the species should be available. In addition, one should establish in details the budget required for purchasing and caring the animals, also considering their acceptance to captivity and all housing characteristics. Finally, the use of the animal should be culturally acceptable (Pearce et al., 2007).

However, according to the purpose, in a specific field of research, several animal models can be pertinent and, in general, the use of multiple models is necessary for an extensive knowledge [START_REF] Hazzard | Selection of an appropriate animal model to study aging processes with special emphasis on the use of rat strains[END_REF].

Indeed, sheep, dog and pig are the most used large animal models for oro-facial tissue engineering. Obviously, each model presents specific limits: pigs represent a good alternative in terms of size and oro-dental anatomy and physiology, but they can reach big dimensions and be very unfriendly and uncooperative (Pearce et al., 2007;[START_REF] Pautke | Bisphosphonate related osteonecrosis of the jaw: A minipig large animal model[END_REF].

SHEEP

The use of sheep in oro-facial research increased over the last decade, because of the ethical issues of employing companion animals like dogs for in vivo studies [START_REF] Martini | Sheep model in orthopedic research: a literature review[END_REF]Pearce et al., 2007). Sheep display the advantage to be extensively available, easy to manage and not to require expensive housing. In terms of size, sheep represent a more advantageous model than rodent models or small dog breeds, but they have a ruminant digestion, which conducts to some anatomical differences and a more basic pH of saliva in comparison with humans.

Moreover, important dissimilarities in jaw bone tissue have been reported, regarding histological properties (i.e. higher density and mechanical resistance), as well as age related changes in structure and bone remodelling (i.e. the Haversian remodelling is predominant with age) [START_REF] Liebschner | Biomechanical considerations of animal models used in tissue engineering of bone[END_REF]. These characteristics can complicate, in case of age differences, the comparison between studies, or clearly exclude this animal as model for a specific field as, for example, the research about bysphosphonates-related osteonecrosis of the jaw (BRONJ) [START_REF] Pautke | Bisphosphonate related osteonecrosis of the jaw: A minipig large animal model[END_REF].

With regards to dental anatomy, sheep are diphyodont and in permanent denture their dental formula is composed for each quadrant of 3 premolars and molars. Incisors are present only on the lower jaw and their features are comparable with human teeth (fig 7) (Altaii et al., 2016).

Sheep models in cell-based regenerative dentistry

Sheep have been mostly implied in bone and periodontal regeneration studies. In 2006, Gronthos and collaborators showed ovine periodontal ligament stem cells' (oPDLSC) regeneration potential, when seeded in HA/TCP scaffold in immunocompromised mice [START_REF] Gronthos | Ovine periodontal ligament stem cells: isolation, characterization, and differentiation potential[END_REF]. [START_REF] Mrozik | Regeneration of periodontal tissues using allogeneic periodontal ligament stem cells in an ovine model[END_REF] and they found that two-teeth stage provided the most pertinent radicular anatomical criteria with humans for endodontic regeneration (Altaii et al., 2016). Then they tested revitalization in immature infected teeth and found positive outcomes for pulp regeneration and root elongation. Subsequently they tested the regenerative potential of ovine dental pulp cells (oDPCs) seeded onto PRP scaffold. oDPCs proliferation and migration seemed to be enhanced by PRP [START_REF] Altaii | Endodontic regeneration and tooth revitalization in immature infected sheep teeth[END_REF].

-DOG

Dog is one of the most used large animal models in oral and dental regeneration, because they are easy to manipulate [START_REF] Wang | Preliminary study on dental pulp stem cell-mediated pulp regeneration in canine immature permanent teeth[END_REF] and they present growth, physiology and pathology similar to humans. However, ethical issues are increasing, because they are considered companion animals and their use in medical research is negatively perceived by the society (Pearce et al., 2007).

A lot of literature concerning oro-facial regeneration compares canines and humans. In fact, instead of an obvious difference in size and shape of the oral cavity, dogs seem to show some important characteristics in common with human musculoskeletal system. It has been reported that dogs' bone turnover, composition and mechanical properties are the most similar to humans among large animal models; even though jaws show a denser and more resistant bone, they have been widely involved in implantology studies [START_REF] Wang | An interspecies comparison of bone fracture properties[END_REF].

Regarding dental anatomy, dogs are dyphiodont. They display 3 incisors, 1 canine, 4

premolars and 2 molars per quadrant in the upper jaw and 3 molars in the mandible (fig 8) [START_REF] Kim | In Vivo Experiments with Dental Pulp Stem Cells for Pulp-Dentin Complex Regeneration[END_REF]. The periodontal regeneration with dPDLSCs has been examined in several studies about class III furcation defects and peri-implant defects healing and tooth reimplantation [START_REF] Suaid | Autologous periodontal ligament cells in the treatment of Class III furcation defects: a study in dogs[END_REF][START_REF] Park | Ex vivo bone morphogenetic protein 2 gene delivery using periodontal ligament stem cells for enhanced reosseointegration in the regenerative treatment of peri-implantitis[END_REF]Lee, 2015).

Another source of stem cells used in canine periodontal regeneration are adipose tissue derived stem cells (ASCs). This kind of stem cells has shown high regeneration potential of periodontal structures, also in association with PRP, as it has been reported by Aziz [START_REF] Aly | Influence of Autologus Adipose Derived Stem Cells and PRP on Regeneration of Dehiscence-Type Defects in Alveolar Bone: A Comparative Histochemical and Histomorphometric Study in Dogs[END_REF], [START_REF] Tobita | Periodontal tissue regeneration by combined implantation of adipose tissue-derived stem cells and platelet-rich plasma in a canine model[END_REF] and [START_REF] Sawada | Trophic factors from adipose tissue-derived multi-lineage progenitor cells promote cytodifferentiation of periodontal ligament cells[END_REF].

Other dog models of periodontal defects healing have been developed to assess the regeneration of cementum, periodontal fibers and bone, employing gingiva-derived mesenchymal stromal cells (GMSCs) [START_REF] Yu | Comparison of tissue-engineered bone from different stem cell sources for maxillary sinus floor augmentation: a study in a canine model[END_REF], dental pulp stem cells (DPSCs) [START_REF] Khorsand | Autologous dental pulp stem cells in regeneration of defect created in canine periodontal tissue[END_REF] and stem cells from extraction socket-derived early healing tissue (ESEHT) [START_REF] Luo | The In Vitro and In Vivo Osteogenic Capability of the Extraction Socket-Derived Early Healing Tissue[END_REF].

Regarding bone regeneration, sinus lift procedures using bovine hydroxy-apataite Bio-Oss and BMSCs (Wang, 2016;[START_REF] Liu | Maxillary sinus floor augmentation and dental implant placement using dentin matrix protein-1 gene-modified bone marrow stromal cells mixed with deproteinized boving bone: A comparative study in beagles[END_REF], or Bio-Oss and PDLSCs [START_REF] Yu | Comparison of tissue-engineered bone from different stem cell sources for maxillary sinus floor augmentation: a study in a canine model[END_REF] have been evaluated in canine models and high rate of bone regeneration for implants placing has been reported. Many studies focused on critical size bone defects healing. Yao et al.

(2011) described bone regeneration with trans-sutural distraction osteogenesis (TSDO).

ADCs or BMScs and HS/TCP scaffolds have been reported improving bone regeneration within marginal bone defects or cleft defects in dogs [START_REF] Mylonas | Investigation of a thermoplastic polymeric carrier for bone tissue engineering using allogeneic mesenchymal stem cells in granular scaffolds[END_REF][START_REF] Bressan | Adipose-Derived Stem Cells as a Tool for Dental Implant Osseointegration: an Experimental Study in the Dog[END_REF]Pourebrahim et al., 2012). Three-walls critical-size alveolar ridge defects have been about DPSC characterization and implantations in dog's teeth (Iohara et al., 2009[START_REF] Iohara | Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1[END_REF][START_REF] Iohara | A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration[END_REF].

In reported that these cells differentiation was enhanced by conditioned medium (CM) from murine pre-ameloblasts.

Many scaffolds have been validated for DPSCs implantation in regenerative endodontics such as Gelfaom [START_REF] Wang | Preliminary study on dental pulp stem cell-mediated pulp regeneration in canine immature permanent teeth[END_REF] treated dentine matrix (TDM) (Bakhtiar et al., 2016), PRP [START_REF] Zhu | Transplantation of dental pulp stem cells and platelet-rich plasma for pulp regeneration[END_REF] and PRP associated with intracanal dressing of modified triantibiotics paste (mTAP) [START_REF] Stambolsky | Histologic characterization of regenerated tissues after pulp revascularization of immature dog teeth with apical periodontitis using tri-antibiotic paste and platelet-rich plasma[END_REF]. In contrast with these outcomes, Zhu et al.

(2014), found that PRP in regenerative endodontics provided manly periodontal tissue formation instead of pulp. Moradi et al., in 2016 demonstrated that PRP could not increase the formation of functional tissues in root canals of immature, non-vital teeth.

Finally, many studies have focused on DPSCs activity enhancement by involving different agents such as calcium hydroxide [START_REF] Ji | Dental stem cell therapy with calcium hydroxide in dental pulp capping[END_REF], vimentin [START_REF] Murakami | Identification of novel function of vimentin for quality standard for regenerated pulp tissue[END_REF], simvastatine [START_REF] Jia | Simvastatin Promotes Dental Pulp Stem Cellinduced Coronal Pulp Regeneration in Pulpotomized Teeth[END_REF] and recombinant DNA-produced amelogenin protein [START_REF] Mounir | Recombinant Amelogenin Protein Induces Apical Closure and Pulp Regeneration in Open-apex, Nonvital Permanent Canine Teeth[END_REF].

PIG

The pig is the animal model of choice in many biomedical research fields because it is very similar to humans in terms of size of body, characteristics of the teguments, cardiovascular and urinary systems (Stembirek et al., 2012). Also gastrointestinal and immune systems are functionally comparable to human ones [START_REF] Lee | Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency[END_REF]. Thus, the pig provides several disease models for arteriosclerosis, metabolic syndromes, gastric ulcer and wound healing.

Moreover, the similarities of haematological and cardiovascular system allow the use of pig in toxicology (Stembirek et al., 2012;[START_REF] Swindle | Swine as models in biomedical research and toxicology testing[END_REF].

Also in orofacial tissue engineering, porcine models are widely used, because of their similitudes with humans in terms of anatomy, physiology and pathology as well as for economic advantages and ethical reasons (Pearce et al., 2007;[START_REF] Swindle | Swine as models in biomedical research and toxicology testing[END_REF].

Nevertheless, pigs are more difficult than sheep or dogs to be managed, because they can be noisy and aggressive (Pearce et al., 2007, Stembirek et al., 2012).

With regards to the oral cavity, the mucosa of the pig is a valid model for scarless wound healing and the tongue is used to assess surgical procedures of volumetric reduction for apnoea syndrome therapy. The same salivary glands and saliva rate are found in pig and humans (Stembirek et al., 2012). Porcine jaws bone is very resembling to humans in terms of anatomy, morphology, healing, remodelling and mechanical properties. In fact, the lamellar bone is similar to humans but the trabecular system is denser. Also, mineral density and concentration are very similar in pig and humans (Pearce et al., 2007;Stembirek et al., 2012;[START_REF] Yeh | Molecular and structural assessment of alveolar bone during tooth eruption and function in the miniature pig, sus scrofa[END_REF]. Moreover, the maxillary and mandible blood supply distribution is analogous to humans [START_REF] Saka | Experimental and comparative study of the blood supply to the mandibular cortex in Göttingen minipigs and in man[END_REF].

For these reasons, pigs are preferentially selected as models for research in implantology (Stembirek et al., 2012), Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ) [START_REF] Pautke | Bisphosphonate related osteonecrosis of the jaw: A minipig large animal model[END_REF][START_REF] Sharma | Animal models for bisphosphonate-related osteonecrosis of the jaws an appraisal[END_REF], Wang et al., 2007), jaws distraction osteogenesis [START_REF] Wan | Craniofacial bone tissue engineering[END_REF][START_REF] Papadaki | Minipig model of maxillary distraction osteogenesis: immunohistochemical and histomorphometric analysis of the sequence of osteogenesis[END_REF]Stembirek et al., 2012[START_REF] Hansen | BMP4 localization and PCNA expression during distraction osteogenesis of the porcine mandible[END_REF] as well as critical size defects healing [START_REF] Henkel | Repair of bone defects by applying biomatrices with and without autologous osteoblasts[END_REF][START_REF] Sun | Establishing a critical-size mandibular defect model in growing pigs: characterization of spontaneous healing[END_REF].

Regarding the masticatory system, pig temporo-mandibular joint (TMJ) is reported to have the same components, morphology and structure of human one. These elements make the pig an optimal non-primate model for TMJ disorders [START_REF] Herring | The dynamics of mastication in pigs[END_REF][START_REF] Ström | Gross anatomy of the mandibular joint and masticatory muscles in the domestic pig (Sus scrofa)[END_REF]Stembirek et al., 2012).

With regard to dental anatomy and physiology, pigs are dyphiodont and 3 incisors, 1 canine, 61 dentition (fig 9). The canine is a continuously growing tooth. The teeth present the same eruption time and sequence than humans (Stembirek et al., 2012;Wang et al., 2007).

Porcine models in cell-based regenerative dentistry

Porcine models in cell-based regenerative dentistry have been developed for periodontal and bone regeneration as well as for endodontic regeneration.

With regard to periodontal tissue engineering, Ibi et al., in 2007, isolated Literature reports many studies about regenerative endodontics in pigs. [START_REF] Nakamura | Enamel matrix derivative promotes reparative processes in the dental pulp[END_REF][START_REF] Nakamura | The induction of reparative dentine by enamel proteins[END_REF][START_REF] Nakamura | Immunohistochemical characterization of rapid dentin formation induced by enamel matrix derivative[END_REF] showed a role of enamel matrix derivative (EMD) in the induction of reparative dentinogenesis in amputated porcine dental parenchyma. In 2012, Zheng et al.

found that autologous porcine deciduous pulp stem/progenitor cells (PDPSCs) seeded onto β-TCP scaffold significantly regenerated the dentin-like structures of pulp chamber roof, at 16 weeks after direct capping of pulp defects in minipig. Finally, treated dentin matrix (TDM) associated with minipig dental follicle cells (DFCs) has been reported to be a performing scaffold for dentin and tooth root regeneration (Chen et al., 2015;[START_REF] Luo | CAD based design sensitivity analysis and shape optimization of scaffolds for bio-root regeneration in swine[END_REF][START_REF] Chen | Treated dentin matrix paste as a novel pulp capping agent for dentin regeneration[END_REF].

AIMS

This work aimed to investigate the possibility to achieve partial pulp regeneration by implanting autologous porcine dental pulp cells seeded in an injectable hydrogel scaffold in mature premolars and molars in a minipig model.

In parallel, in order to monitor the success of the grafting, we aimed to follow-up neoangiogenesis by a dynamic imaging technique in mini-pig tooth. In vivo 3D subtraction angiography has been set for the visualization of dental pulp vascular network.

Moreover, an innovating image analysis techniques for the quali-quantification of newly

MATERIALS AND METHODS

ANIMALS

This study was designed conformingly to the ARRIVE guidelines and approved by the Ethics Committee for animal experiments of COMITHEA 45/INRA (number 03302.02).

Three healthy minipigs, aged 18 to 20 months, from Bretoncelles breeding farm (France)

were used. They were housed at stable conditions (22 ± 2 °C) with a 12 h dark/light cycle and with ad libitum access to water and pellet food, in the animal facility of the National Institute of Agronomic Research (INRA, Jouy-en-Josas, France).

For each procedure, animals were anesthetized with intramuscular ketamine (15mg/kg), intubated, and then maintained over time with a mix of isoflurane (1.5%) and oxygen (98.5%). Animals received intra muscular injection of 1ml/10kg oxytetracycline (Terralon LA 20%, Virbac, France) before and 72 hours after surgery. Intramuscular flunixine based anti-inflammatory therapy (Finadyne, MSD Animal Health, France) was administrated 2mg/kg per day before the procedure and during the following 3 days.

CELLS CULTURE

Multicolony-derived minipig dental pulp cells (pDPC) were obtained using a protocol

CELL PROLIFERATION IN PURAMATRIX™

Three-dimensional self-assembling peptide (RADA-16I) BD PuraMatrix™ (BD Biosciences, Bedford, MA, USA) was employed. PuraMatrix™ stock solution viscosity (1% w/v) was reduced by vortexing, and air bubbles were removed by centrifuging. According to the manufacturer's guidelines, the 0,2% PuraMatrix™ was prepared by diluting the stock with sterile 20% sucrose to generate a 2X concentration of PuraMatrix™ in 10% sucrose.

pDPCs were washed in 10% sucrose solution to remove salts. Cells were suspended in 0.2%

PuraMatrix™ and added to the wells of a 96-well plate, then gelation was induced by careful addition of an equal volume of culture medium to give final concentration of 2x10 7 /ml.

Cell proliferation was assayed at day 1, 3 and 7, using the Vybrant MTT cell proliferation assay kit (invitrogen). For each time point a triplicate of pDPCs/scaffold was analyzed.

According to the manufacturer recommendations, 100 μL of medium was removed from the well and replaced with 100 μL of fresh medium along with 10 μL of the 12mM MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) stock solution.

The cells were incubated at 37°C for 2 hours. 85 μL of medium were removed and 50 μL of DMSO were added, followed by a 10 min incubation at 37°C. The samples were transferred to a microplate, and the absorbance at 540 nm was determined.

PARTIAL PULPOTOMY PROTOCOL

Forty-eight teeth (second, third and fourth premolar and first molar for each minipig) of both jaws were selected for experimentation. Ultrasonic scaling was performed. Dam isolation was placed from the second molar to the canine for each quadrant and the pulpotomy was executed on selected teeth. An occlusal cavity was drilled to the pulp chamber with high-speed rotary 0.6-mm-diameter round bur (Maillefer Dentsply). The cameral pulp parenchyma was eliminated with a rotary instrument. Hemostasis was obtained by sterile cotton pellets compression.

Simultaneously, pDPCs/scaffolds were prepared as described above. For no-pDPCs/scaffolds, only sucrose was added to 0,2% hydrogel. As soon as gelation was achieved, the hydrogels were aspirated in 1ml syringes and injected in each selected tooth by 27G needle. A volume of 100 μL pDPCs/scaffold was implanted within each empty pulp chamber of the left maxillary and mandibular teeth, while 100 μL no-pDPCs/scaffold were implanted in the right side teeth, according to a split mouth model. The occlusal cavities were sealed with a calcium silicate based restorative cement (Biodentine; Septodont) and covered with a light-cured composite (FloRestore; Denmat) (fig 11).

Teeth were extracted under general anesthesia at day 21 after surgery and fixed in a 10% formalin solution (Antigenfix, DiaPath).

Teeth were extracted under general anesthesia at day 21 after surgery and fixed in a 10% formalin solution (Antigenfix, DiaPath).

MINIPIG ANGIOGRAPHY

Via femoral arterial approach external carotid arteriography with a 5F guide catheter (Envoy; Johnson & Johnson, Tokyo, Japan) was performed. Then, a 2.2F microcatheter (Masters, Asahi-Intecc, Aichi, Japan) was inserted into the common branche of internal maxillary artery. Systemic heparin administration was performed before external carotid arteriography by injecting 5000 heparin units/body.

A dose of 8 ml of iodine contrast medium (Hexabrit 2,5%) was injected with a speed of 4 ml/seconds and pressure of 300 kPa. The CBCT X-rays (FOV 20,kV,mA) were taken before and after contrast medium injection. Acquired volumes have been analysed in multi planar the imaging analysis tool applies semi-interactive livewire boundary extraction (Barrett and Mortensen, 1997) to create a set of orthogonal contours, followed by a variational interpolation algorithm that reconstructs the surface of an object using energy-minimizing, smooth and implicit functions (fig 12) [START_REF] Heckel | Interactive 3D medical image segmentation with energy-minimizing implicit functions[END_REF]. For each dentin bridge, a geometrically equivalent region was extracted from the native dentine, by super-imposing the contours created during the segmentation of the newly formed tissue, to be used as a control (fig 13).

The nature of the dentine bridges was characterized using specific bone-derived morphometric parameters commonly used for mineralized tissues analysis (Parfitt et al., 1983), using dedicated protocols in CTAn (Antwerp, Belgium) and Amira (FEI Visualization Sciences Group, Hillsboro, OR, USA) software (fig 14-21).

For mineralized areas of the segmented volumes, the following parameters were calculated:

Mineralized

HISTOLOGY AND IMMUNOHISTOCHEMISTRY

Teeth were decalcified in 10% nitric acid during 1 month. After tissue embedding in Paraplast, serial sections (7 μm) were stained for Masson's trichrome.

For immunohistochemical analysis, the sections were incubated with primary antibodies, against BSP (LF-84, kindly donated by Larry Fisher) diluted at 1/200, and against the DSP (LF-153, kindly donated by Larry Fisher) at 1/200. Tissue sections were then incubated with anti-rabbit immunoglobulin (Dakocytomation) diluted at 1/100. Color reaction was developed by using 3,3'-diaminodenzidine tetrahydrochloride. A negative control was included by using an equal concentration of rabbit pre-immune serum as primary antibody.

STATISTICAL ANALYSIS

The dentin bridge was used as statistical unit. Comparisons between the two groups newly formed mineralized tissue (bridge-with/without cells) and dentin control (controlwith/without cells) were performed using the Wilcoxon test (StatSoft STATISTICA 8.0).

Comparison between the two groups with cells and without cells was performed by Tstudent test (StatSoft STATISTICA 8.0). The level of significance was set at p < 0.05.

RESULTS

PURAMATRIX™ SUPPORTED THE PROLIFERATION OF PDPCs

To assess pDPCs proliferation in 0.2% PuraMatrix™, cells were seeded in the scaffold at a density of 2x10 7 /ml. Cells proliferated at an average rate of 1.4 from day 1 to 7 in vitro (Fig 26). Therefore, these conditions were used for in vivo implantations.

IMPLANTATION OF PDPCS/SCAFFOLD INDUCED REPARATIVE DENTINOGENESIS AND FAILED TO INDUCE PARTIAL PULP REGENERATION

To investigate whether cell-based partial pulp regeneration could be achieved, pDPCs/scaffolds and no-pDPCs/scaffolds were implanted after cameral pulpotomy in premolars and molars.

At day 21 after implantation, microCT analysis of the treated teeth revealed the formation of a reparative mineralized bridge in the residual pulp, regardless of the presence of cells in the hydrogel (fig 27A).

Three-dimensional volume rendering of the entire tooth showed that the dentin bridge was located at each root canal entrance, corresponding to the apical limit of the pulp amputation

THE DENTIN BRIDGE MICROSTRUCTURE DIFFERED FROM THE NATIVE DENTIN CONTROL AND IT WAS AFFECTED BY THE PRESENCE OF PDPCS IN THE SCAFFOLD.

Using specific bone-derived morphometric parameters, the nature of newly formed dentin has been characterized (fig 28). First of all, each bridge (with and without cells) was compared to a corresponding volume of native dentin control from the same tooth.

The mineralized part of each volume was first analyzed. The newly formed reparative dentin showed a lower mineralized volume fraction and a higher mineralized surface density, meaning that its mineralized volume was lower but denser in comparison with native dentin. Moreover, the analysis of the specific mineralized surface revealed higher values in the newly formed mineralized dentin, indicating that the reparative dentin was more compact than the native one. Besides, a significantly higher thickness of mineralized areas has been measured in the dentin bridges.

Non-mineralized areas characteristics were further examined within the dentin. In comparison with the native dentin, the reparative one showed significantly higher values for the total porosity percentage, mineralized areas separation and mean volume of pores.

These results confirmed that the reparative dentin presented higher percentage of non mineralized areas, whose volume was also higher than in native dentin. In addition, a significantly higher connectivity percentage was found in the reparative dentin, meaning that within the dentin bridge non mineralized areas were more extensively inter-connected, in comparison with those of native dentin.

Secondly, the effect of pDPCs on the reparative dentin was determined by comparing the morphometric features of bridges with and without cells. The results showed a significantly lower mineralized volume fraction with pDPCs implantation than without, meaning that the mineralized component in the entire volume of the dentin bridge was less represented when cells were seeded in the scaffold. Moreover, in presence of cells, the reparative dentin was less dense, as demonstrated by the mineralized surface density values that were significantly lower than in samples without cells. Similar compactness and thickness of the mineralized area were detected in both conditions, since no significant differences were found in terms of specific mineralized tissue surface and thickness of mineralized areas. The total porosity percentage, the connectivity and the separation of mineralized areas were significantly higher in teeth implanted with cells than in those without cells. These results

show that in the presence of cells the reparative bridge presented more numerous and connected non mineralized areas. Furthermore, the latter did not differ in both conditions in terms of volume, since no significant differences were found for the parameter mean volume of pores.

All these data suggest that the implantation of pDPCs/scaffold or no-pDPCs/scaffold after cameral pulpotomy induced the formation of a mineralized dentin bridge, whose microarchitecture sensibly differs from the native dentin. Furthermore, the presence of pDPCs significantly affected the microarchitecture of the dentin bridge.

PULP AMPUTATION AND SCAFFOLD IMPLANTATION IMPACTED RADICULAR AREA.

Since the reparative dentinogenesis constitutes a defense mechanism of the pulp, we further characterized the dentin bridge by histological analysis.

Masson's trichrome staining (fig 29 b, c, e and f) confirmed that the reparative bridge closed the root canal entrance, independently of the presence of the pDPCs in the scaffold. This indicates that the reparative dentinogenesis was not driven by implanted pDPCs.

Moreover, the presence of cells in the scaffold led to a mineralized tissue with more numerous cell inclusions, compared to those obtained without cells, consistent with our microCT morphometric characterization. The remaining root pulp presented signs of vasodilatation of blood vessels (hyperaemia), alluding to the presence of an inflammatory process.

The cells associated with the reparative dentin bridge displayed processes within the dentin and they showed a palisade organization. Moreover, they expressed both DSP and BSP, evocating odontoblast-like cells (Golderg et al., 2008;Sagomonyants et al., 2017) (fig 29 g-n). In addition, immunolabeled cells for these markers were also detected within the cell inclusions of the reparative bridge. These data indicate that the mineralization tissue constituted osteodentin. At day 21, a dentin bridge was detected at the root canal entries apically to the pulpotomy limit as showed in the microCT sections (a and d). Masson's trichrome confirmed the presence of a large reparative dentin bridge in both groups (b and e), in contact with dentin walls. Hyperaemia was observed in the radicular pulp. At higher magnification (c and f), cell inclusions appeared within the bridge in both conditions, with or without cells in the scaffold. Characterization of the reparative dentin bridge was performed by DSP and BSP immunostaining. BSP and DSP labeling were observed associated to the cells adjacent to the reparative bridge as well as in cell inclusions, in both conditions (g, i, k and m). At higher magnification of specific areas (squares), BSP and DSP labeled cells presented a polarized morphology (arrows) and were organized as a palisade at the dentin bridge -pulp interface as well as inside the inclusions, in both conditions (h, j, l and n). N = 48 teeth. Scale bar: 50 μm. DB= dentin bridge P= pulp.

Furthermore, tridimensional radiographic analysis of implanted teeth highlighted radiolucent areas, located at several radicular levels, suggesting external root resorptions.

In fact, independently of the presence of pDPCs, these radiological images were identified in every sample (fig 30).

MAXILLARY AND MANDIBULAR BLOOD SUPPLY DISPLAY

In order to display and analyze dental and maxillary blood supply, iodine contrast medium has been selectively injected in the internal maxillary artery or directly into the inferior alveolar artery.

For the validation of the protocol of selective injection the procedure was first simulated using Patent blue V stain. The vital staining was detected in the pulp of all teeth (fig 31). Mini-pigs' scans performed after injection in the internal maxillary artery have showed the entire blood supply of its district. In particular sinus, infraorbitary, superior alveolar, inferior alveolar and soft tissues branches have been clearly displayed.

Scans obtained by more selective injection into inferior alveolar artery allowed the visualization of the entire trajectory of this vessel from the Spyx spine to the mental and incisor foramina.

In both conditions the vascular network was characterized by a constant and homogeneous radiological signal. The radiopacity of the contrast medium was not distinguishable from that one of teeth and cortical bone (fig 32). In addition, independently of the way of injection, no signal was detected in the pulp of the treated teeth. In fact, at the apex of premolars and molars, contrast medium collection has been observed (fig 33). The morphometrical analysis provided quali-quantitative characterization of the vessels surrounding the hemimandible in terms of non-connected vessels trees, nodes, branches, segments and average vessels' diameter distribution (tab 3, fig 37).

PARAMETER VALUE

Non-Connected Vessels Trees 90

Nodes 900

Branches 855

Segments 5770

DISCUSSION

In the present study, we investigated whether partial pulp regeneration could be achieved by implanting pDPCs/scaffold, after cameral pulp amputation. Our results showed systematic reparative dentinogenesis instead of pulp regeneration. Indeed, a dentin bridge, that completely closed each root canal entrance, was observed at 3 weeks after implantation. This finding is important for cell-based regenerative endodontic strategies and their further clinical implications, notably regarding the benefit to implant pulp cells. In fact, the reparative dentin bridge was obtained independently of the presence of cells in the scaffold.

CHARACTERIZATION OF THE REPARATIVE DENTINE

By dedicated morphometric characterization derived from bone microstructure analysis (Parfitt et al., 1983;Bouxsein et al., 2010), we showed that the sealing of the bridge was impaired in teeth implanted with pDPCs. Since the reparative process was detected in teeth with no-pDPCs/scaffold, the synthesis of the osteodentin was not driven by the implanted pDPCs. Considering the positive immunostaining for DSP and BSP, it is likely that the dentin bridge was formed by host odonto/osteoblasts-like cells located in the remaining pulp or brought to the injury site by the blood supply (Tran et al., 2012;Saito et al., 2016;Sagomonyants et al., 2017, Vidovic et al., 2017). Moreover, we applied an innovative microCT analysis of this reparative osteodentin. This non-destructive and non-invasive technique allowed quali-quantitative tridimensional characterization of the microarchitecture of the newly formed tissue. In particular we could study in details the morphometric features of mineralized and non-mineralized areas into dentin bridges and understand the relation between each component. Such an evaluation provided important information regarding the shape, the internal organization and the mechanical properties of the osteodentin bridges. Thus, one could assume that, with the future improvement of highresolution cone beam computed tomography, this standardized morphometric analysis could find application in clinical trials focusing on dentin bridge formation and root walls elongation.

SCAFFOLD

An ideal scaffold should be easily manipulable by clinicians and it should provide, at the same time, a temporary 3 dimensional framework in which cells can be seeded, adhere and proliferate, as well as a bioactive microenvironment for cells metabolism and communication [START_REF] Amrollahi | Recent advancements in regenerative dentistry: A review[END_REF].

Collagen is a natural scaffold extremely biocompatibile and it has been one of the material of choice for dental tissue engineering. Numerous studies performed in different animal models have reported the formation of a functional dentin-pulp complex by using collagen and DPSCs (Prescott et al., 2008;Rosa et al., 2013;[START_REF] Kodonas | Experimental formation of dentinlike structure in the root canal implant model using cryopreserved swine dental pulp progenitor cells[END_REF]. In previous works of the lab, collagen scaffolds were used for pulp regeneration after pulpotomy (Souron et al., 2013) or in a tooth-slide model (Gorin et al., 2016).

However, this scaffold shows an important contraction when cells are seeded and this characteristic impairs their activity and so the regeneration [START_REF] Chan | Effects of TGF-beta s on the growth, collagen synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro[END_REF][START_REF] Huang | In vitro characterization of human dental pulp cells: various isolation methods and culturing environments[END_REF]. Moreover, the allogenic nature of collagen can induce inflammatory and immune reactions (Galler et al., 2011;[START_REF] Sharma | Biomaterials in tooth tissue engineering: a review[END_REF].

The group of Galler et al., reported in 2010 the use of a customized self assembling peptide hydrogel for dentin-pulp complex regeneration. Self-assembling peptides are biocompatible and highly biodegradable composed by natural amino acids organized in a network.

In our model, pDPCs were implanted in an injectable self-assembling nanopeptide hydrogel (PuraMatrix TM ), that has been reported in literature as an optimal tridimensional matrix inducing DPSCs differentiation (Cavalcanti et al., 2013;Dissanayaka et al., 2015).

Characteristically, this hydrogel can be easily adapted according to the necessities of the regenerative model and it provides a biodegradable, biocompatible, fast assembling matrix made by 16-mer peptide in 99% water solution that polymerizes as soon as placed in physiological conditions (Cavalcanti et al., 2013). We tested the ability of the pDPCs to proliferate in this PuraMatrix TM in vitro.

CELL SORTING

So far, there is no distinct marker characterising DPSCs, which are a heterogeneous and mixed population. DPSCs are isolated by their high proliferative potential. They are positive for classical mesenchymal stem cells markers, including CD29, CD44, CD73, CD90, CD105, CD166, CD271, Stro1, and CD146, but negative for CD34, CD45, and CD14 in vitro [START_REF] Zhao | Stem Cells in Teeth and Craniofacial Bones[END_REF]. DPSCs also exhibit hematopoietic (CD34 and CD117) and stemness (OCT-3/4

and NANOG) markers. Several markers in common with human DPSCs are expressed by pDPSCs (Iohara et al., 2006;[START_REF] Ishizaka | Stimulation of angiogenesis, neurogenesis and regeneration by side population cells from dental pulp[END_REF]. It is important to note that DPSCs do not necessarily express all of these surface markers, with each DPSC expressing some of these markers at different ratios.

Recently, techniques for dental pulp cells sorting have been reported. In particular, Iohara et al., showed the regeneration of a vascularized and innervated pulp tissue into canine pulp amputation model. In this study a subpopulation of cells expressing CD31-/CD146-were transplanted with a collagen I and II scaffold (Iohara et al., 2009). The same group of researcher also demonstrated functional pulp and dentin regeneration in a dog pulpectomy model by using CD31-/CD146-SP cells and CD105+ cells with stromal cell-derived factor-1 (SDF-1) [START_REF] Iohara | Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1[END_REF][START_REF] Iohara | Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1[END_REF]. However in this study, dentinpulp complex regeneration was achieved also in the teeth treated with unfractioned cells.

Moreover, the most of the studies in literature use unfractioned dental pulp cells. At present, there is no evidence that cell sorting is a real advantage in pulp regeneration.

In our model, we decided to use unfractioned pDPCs. This decision was support by our previous results on rat (Souron et al., 2013) and by the fact that we tested the minipig model for the first time. The large volume of pulp tissue to regenerate required a high amount of cells rapidly available, making difficult the cell sorting.

At the time we designed the experiment, we hypothesized an engraftment of the implanted pDPCs with the remaining host pulp, promoted by the paracrine mediation of angiogenesis by secretion of regulatory proteins by pDPCs [START_REF] Bakopoulou | Stem cells of dental origin: current research trends and key milestones towards clinical application[END_REF][START_REF] Ratajczak | The neurovascular properties of dental stem cells and their importance in dental tissue engineering[END_REF]Rombouts et al., 2016).

REGENERATIVE ENVIRONMENT

Although dentin-pulp complex regeneration has been achieved in vitro and in small animal models in vivo, a more translational approach requires the use of larger animals. In fact, only these models allow to investigate the influence of the environment on the regenerative potential of DPSCs implanted in situ in teeth [START_REF] Hilkens | Dental Stem Cells in Pulp Regeneration: Near Future or Long Road Ahead?[END_REF].

In our model, we tested the cell-based pulp regeneration in a specific environment, represented by mature pulpotomized pluriradicular teeth. We selected these conditions because they represented the most frequent situation in clinics: partial inflammation of the pulp due to caries. Our results might be explained by the limitation of the blood supply, due to reduced root canal volume and apical closure, which might be insufficient to ensure the engraftment of implanted cells (Zheng et al., 2012).

It might be interesting to test the cell-based pulp regeneration in immature teeth in future experiments.

Moreover, inflammation might play a critical role in our results. Hyperaemia and root resorptions, which are indirect signs of pulp inflammation [START_REF] Kassa | Histological comparison of pulpal inflammation in primary teeth with occlusal or proximal caries[END_REF][START_REF] Patel | Internal root resorption: a review[END_REF], were observed in all the treated teeth. We were not able to put in evidence by immunolabelling the markers of inflammation such as CD45, because it is likely that the aggressive demineralization process, made by 10% nitric acid, damaged the antigenic sites.

The inflammatory process has been reported to influence negatively the neoangiogenesis (Boyle et al., 2014;Vidovic et al., 2017;Sagomonyants et al., 2017) and to promote, when limited, the synthesis of a reparative bridge (Goldberg et al., 2015). The origin of the inflammation and the respective implication of i) the severe injury of the pulp amputation and ii) the implantation of the hydrogels, have to be determined in our model to expect a successful pulp regeneration therapy.

Hence, to clarify this point, further investigations in a "pulpotomy-only" model are still required.

Finally, another parameter might have largely influenced the formation of the dentine bridge: the use of Biodentine. Indeed, Biodentine a tricalcium silicate-based cement presents bioactive properties and induces biological effect on pulp cells. In addition this cupping material displays short setting time high mechanical characteristics as well as sealing ability (Tran et al., 2012;Zanini et al., 2012).

Even though, this cement appeared as the material of choice to be applied in contact with cells, due to its biocompatibility and its ease of use.

Once again, other experiments would be necessary to assess the role of Biodentine itself in our results.

The failure of partial pulp regeneration in this study raises many questions regarding local factors influencing the regenerative environment and highlighted the necessity to develop preclinical models.

MINI-PIG MODEL AND NEAR-REAL CLINICAL CONDITIONS

Indeed, literature mainly reports in vitro studies and murine models of cell-based partial pulp regeneration, but the clinical feasibility of this strategy in preclinical models is still missing (Rosa et al., 2017). The large animal models recognized and developed in translational dentistry are the dog (Iohara et al., 2009), the minipig (Zheng et al., 2012) and more recently the sheep (Altaii et al., 2016). In the present work, we selected the minipig, because it has anatomical and physiological characteristics similar to humans and it is the only omnivore out of the three species (Stembirek et al., 2012). As mentioned above, our choice was also to reproduce as close as possible the clinical conditions. Thus, the pulpotomies were performed on posterior mature permanent teeth, which are more frequently affected by caries. Due to the size and the anatomy of minipig's teeth, the conventional clinical operative conditions were established (i.e. isolation by dam, high speed rotational instruments under copious sterile irrigation, calcium silicate based restorative cement and composite used in clinics).

However, this model implies elevated cost, time-consuming procedures and technical limits (Pearce et al., 2007;[START_REF] Yamamoto-Fukud | Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling[END_REF]. Indeed, one of the most important limits of the minipig model is the lack of specific antibodies for the immunolabelling and the necessity to use an aggressive demineralization process (10% nitric acid) to perform the histological analysis. These aspects did not allow the complete characterization of the outcomes of the regenerative procedure.

TRIDIMENSIONAL SUBTRACTION ANGIOGRAPHY

In order to analyze the regenerative process in our model, we performed an in vivo 3D CT subtraction angiography.

In fact, the knowledge of vascularization and neo-angiogenesis is crucial for precise diagnosis and follow-up as well as for the development of ever more performing regeneration strategies. However, no accurate non destructive methods of vessels' qualiquantitative analysis from radiological data are still available for this anatomic area.

Vascular anatomy of the dental and oro-facial regions seems to be especially hard to explore and to provides high quality images, because of the heterogeneity of the involved tissues, the complexity of the muscle-skeletal associated system and the terminal nature of its blood supply. Moreover, the employ of an iodine contrast medium can induce hypersensitivity reactions, thyrotoxicosis and a nephrotoxic effect on kidney function.

This risk complicates the estimation of the volume of contrast medium to be injected and, as consequence, the choice of the most performing acquisition parameters.

Thus, the setting of the scan is supposed to minimize any kind of artefact, responsible of non-diagnostic examinations or false-positive diagnoses, like patient breathing and motion, streak artefacts from non-diluted contrast media, vascular bifurcations and the presence of other pathologies. Angio-CT made by intraarterial injection of a reduced volume of diluted contrast agent may be a useful and safer alternative. In fact, the procedure can be easily performed and the time required for the exam reduced [START_REF] Garcarek | Intraarterial CT Angiography Using Ultra Low Volume of Iodine Contrast -Own Experiences[END_REF] In this study we set two protocols of arteriography with the perspective of qualiquantitative morphometric analysis of dental and maxillary blood supply.

Regarding internal maxillary artery arteriography, all the main branches could be clearly displayed. In fact infraorbital artery, superior alveolar artery, sinus and nasal branches, inferior alveolar artery and mental brunches were shown. When the selective protocol was performed, inferior alveolar artery and its terminal ramus could be visualized.

In both procedures, dental vascularization could be observed only in the canines that are continuously growing teeth so having root open apexes. For all mature premolars and molars collection of the contrast medium was detected in the apical region and pulp vascularization could not be highlighted. These results further confirm the presence of a reduced apical foramina diameter and a lower blood supply of pulp parenchyma in mature permanent teeth. In addition, it is likely that the contrast agent could not pass through the apical constriction because of its high viscosity. In fact, when the protocol was simulated by In both models the follow-up was not possible. A similar radiological intensity of contrast medium and mineralized tissues did not allow the segmentation of the vascular network and its morphometric analysis.

Thus the prosecution of this study is still required to establish a protocol for dental and orofacial blood supply visualization and characterization. In future, this technique could be applied in minipig models with the possibility of follow-up of neoangiogenesis process in mature and immature teeth and in bone defects, in order to transfer new knowledge at the service of human tissues engineering.

10-CONCLUSION AND PERSPECTIVES

This work represents an important step for preclinical application of cell-based regenerative dentistry. The use of a large animal model anatomically and physiologically similar to humans such as the minipig allowed us to assess the feasibility of partial pulp regeneration by implanting autologous pDPCs in near-real conditions with a follow-up of 21 days.

At the same time, this model offered the opportunity to set new imaging techniques for the characterization of newly formed mineralized tissues and to visualize maxillary and dental blood supply.

Our results showed that the implantation of pDPCs in mature permanent teeth after pulp chamber parenchyma amputation failed to regenerate a functional tissue and that the cells of remaining pulp synthetized a reparative dentin morphometrically and histologically different from native dentin. The position of the reparative bridge at the entrance of the root canal space, created two separated areas within the endodontic system, completely isolating the chamber and impeding the creeping of new blood vessels and the engraftment of the matrix. The reduced blood supply and the narrow diameter of apical foramina in the treated teeth have been also highlighted by 3D angiography.

At present, preclinical models are still required for a better understanding of DPCs behaviour and the development of an environment supporting the regeneration. In fact, in spite of the elevated cost and time-consuming procedures, the use of large animal models represents the most relevant strategy to recreate clinical conditions and to take a step towards human application.

Introduction

Dental pulp is a highly innervated and vascularized connective tissue, which is encapsulated in a mineralized inextensible structure formed by enamel, dentin, and cementum. It ensures the homeostasis and sensibility of the tooth [START_REF] Rosa | Pluripotency of stem cells from human exfoliated deciduous teeth for tissue engineering[END_REF]).

The pulp is often damaged by caries and trauma, resulting in infection or necrosis. In such situations, the routine clinical treatment is a root canal therapy, which consists of eliminating the affected tissue and filling the endodontic canal system with bioinert synthetic materials. In spite of satisfactory clinical outcomes, none of the original functions is restored, and the lack of sensitivity, as well as natural defense, may lead to tooth fracture and reinfection (Gong et al. 2016).

In 2000, Gronthos et al. identified, for the first time, adult mesenchymal stem cells populations (dental pulp stem cells [DPSCs]) from dental pulp and demonstrated their abilities to regenerate a tissue. Subsequently, researchers have examined several ways to transfer this capacity to the clinical practice. Indeed, cell-based tissue regeneration is a promising strategy to obtain newly formed functional dentin-pulp complex by combining stem cells, scaffolds, and growth factors. Depending on the clinical situation, 2 cell-based pulp-engineering strategies can be considered (Huang 2009). The first is the de novo synthesis, which consists in the regeneration of the whole pulp tissue in the canal space. Positive outcomes have been reported for this approach, mostly with ectopic implantation of DPSCs in murine animal models (Cordeiro et al. 2008;Gorin et al. 2016;[START_REF] Khayat | GelMA-encapsulated hDPSCs and HUVECs for dental pulp regeneration[END_REF]. Furthermore, a very recent pilot study, conducted on 5 patients, showed complete pulp regeneration after DPSC transplantation following pulpectomy (Nakashima et al. 2017).

The second is the cell-based partial pulp regeneration, which is indicated in case of trauma or carious lesion, when the pulp inflammatory reaction is still compartmentalized (Bjørndal 2008). This conservative strategy relies on the preservation of the healthy tissue while regenerating the damaged pulp (Huang 2009). Cell-based partial pulp regeneration has been achieved in rat molars (Souron et al. 2014) and monoradicular teeth in dog (Iohara et al. 2009). However, to test the feasibility and the success of such a regenerative therapy mimicking the clinical conditions, more studies should be performed in large animal models (Luiz de Oliveira da Rosa et al. 2017). The minipig is widely used in oral-facial research, thanks to its similarities with humans, in terms of oral anatomy and physiology, as well as its omnivorous diet (Wang et al. 2007;[START_REF] Štembirek | The pig as an experimental model for clinical craniofacial research[END_REF]. Moreover, the regenerative potential of porcine DPSCs has been largely demonstrated (Iohara et al. 2006;Iohara et al. 2008).

The aim of this study was to investigate whether partial pulp regeneration could be achieved by implanting porcine dental pulp cells (pDPCs) in artificially created pulp defects in a minipig animal model.

Materials and Methods

Animals

This study was designed in conformity to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Ethics Committee for animal experiments of COMITHEA 45/INRA (03302.02).

Three healthy minipigs, aged 18 to 20 mo, from Bretoncelles breeding farm (France) were used. They were housed at stable conditions (22 ± 2 °C) with a 12-h dark/light cycle and with ad libitum access to water and pellet food in the animal facility of the National Institute of Agronomic Research (INRA).

For each procedure, the animals were anesthetized with intramuscular ketamine (15 mg/kg), intubated, and then maintained over time with a mix of isoflurane (1.5%) and oxygen (98.5%). They received intramuscular injection of oxytetracycline (1 mL/10 kg, Terralon LA 20%; Virbac) before and 72 h after surgery. Intramuscular flunixin-based anti-inflammatory therapy (Finadyne; MSD Animal Health) was administrated before the procedure (2 mg/kg/d) and during the following 3 d.

Cell Culture

Multicolony-derived minipig dental pulp cells were obtained from lower second lateral incisors extracted under sterile and atraumatic conditions, as previously described (Souron et al. 2014;Appendix). The isolated cells were cultured in α-MEM (Gibco) supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen) at 37 °C under 5% CO 2 atmosphere. The required cell number was reached after 3 passages.

Cell Proliferation in PuraMatrix

Three-dimensional self-assembling peptide (RADA-16I) BD PuraMatrix (BD Biosciences) was employed. PuraMatrix stock solution viscosity (1% w/v) was reduced by vortexing, and air bubbles were removed by centrifuging. According to the manufacturer's guidelines, the 0.2% PuraMatrix was prepared by diluting the stock with sterile 20% sucrose to generate a 2× concentration of PuraMatrix in 10% sucrose.

pDPCs were washed in 10% sucrose solution to remove salts. Cells were suspended in 0.2% PuraMatrix and added to the wells of a 96-well plate; then, gelation was induced by careful addition of an equal volume of culture medium to give a final concentration of 2 × 10 7 /mL.

Cell proliferation was assayed at days 1, 3, and 7 with the Vybrant MTT cell proliferation assay kit (Invitrogen). For each time point, a triplicate of pDPCs/scaffold was analyzed. According to the manufacturer recommendations, 100 μL of medium was removed from the well and replaced with 100 μL of fresh medium with 10 μL of the 12mM MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) stock solution.

The cells were incubated at 37 °C for 2 h; 85 μL of medium was removed, and 50 μL of dimethyl sulfoxide (DMSO) was added, followed by a 10-min incubation at 37 °C. The samples were transferred to a microplate, and the absorbance at 540 nm was determined.

Partial Pulpotomy Protocol

Forty-eight teeth (second, third, and fourth premolars and first molar for each minipig) of both jaws were selected for experimentation. Ultrasonic scaling was performed. Dam isolation was placed from the second molar to the canine for each quadrant, and the pulpotomy was executed on selected teeth. An occlusal cavity was drilled to the pulp chamber with a round high-speed rotary bur (0.6-mm diameter; Maillefer Dentsply). The cameral pulp parenchyma was eliminated with a rotary instrument. Hemostasis was obtained by sterile cotton pellet compression.

Simultaneously, pDPCs/scaffolds were prepared as described. For no-pDPCs/scaffold, only sucrose was added to 0.2% hydrogel. As soon as gelation was achieved, the hydrogels were aspirated in 1-mL syringes and injected into each selected tooth by 27G needle. A volume of 100 μL of pDPCs/scaffold was implanted within each empty pulp chamber of the left maxillary and mandibular premolars and molars, while 100 μL of no-pDPCs/ scaffold was implanted in premolars and molars of the right side, according to a split-mouth model. The occlusal cavities were sealed with a calcium silicate-based restorative cement (Biodentine; Septodont) and covered with a light-cured composite (FloRestore, Denmat; Appendix Fig. 1).

Teeth were extracted under general anesthesia at day 21 after surgery and fixed in a 10% formalin solution (Antigenfix; DiaPath).

Micro-computed tomography Imaging and Analysis

Image Acquisition and Export. All harvested teeth were scanned ex vivo at day 21 after surgery with an x-ray micro-computed tomography (microCT) device (Quantum FX Caliper; Life

Statistical Analysis

The dentin bridge was used as a statistical unit. Comparisons between the 2 groups' newly formed mineralized tissue (bridge: with/without cells) and dentin control (control: with/ without cells) were performed via the Wilcoxon test (StatSoft STATISTICA 8.0). Comparison between the 2 groups with and without cells was performed by Student's t test (StatSoft STATISTICA 8.0). The level of significance was set at P < 0.05.

Results

PuraMatrix Supported the Proliferation of pDPCs

To assess pDPC proliferation in 0.2% PuraMatrix, cells were seeded in the scaffold at a density of 2 × 10 7 /mL. Cells proliferated at an average rate of 1.4 from day 1 to 7 in vitro (Fig. 1). Therefore, these conditions were used for in vivo implantations.

Implantation of pDPCs/Scaffold Induced Reparative Dentinogenesis and Failed to Induce Partial Pulp Regeneration

To investigate whether cell-based partial pulp regeneration could be achieved, pDPCs/scaffolds and no-pDPCs/scaffolds were implanted after cameral pulpotomy in premolars and molars.

At day 21 after implantation, microCT analysis of the treated teeth revealed the formation of a reparative mineralized bridge in the residual pulp, regardless of the presence of cells in the hydrogel (Fig. 2A). Three-dimensional volume rendering of the entire tooth showed that the dentin bridge was located at each root canal entrance, corresponding to the apical limit of the pulp amputation (Fig. 2B). This reparative dentin was in contact with the lateral dentin walls and completely closed the entrance of the root canal space. Furthermore, the observation of the segmented volume of each bridge revealed a thick defined mineralized structure perfectly matching the anatomy of the endodontic space at its coronal part (Fig. 2C).

Dentin Bridge Microstructure Differed from the Native Dentin Control and Was Affected by the Presence of pDPCs in the Scaffold

With specific bone-derived morphometric parameters, the nature of newly formed dentin was characterized (Fig. 3).

First of all, each bridge (with and without cells) was compared with a corresponding volume of native dentin control from the same tooth.

The mineralized part of each volume was first analyzed. The newly formed reparative dentin showed a lower mineralized volume fraction (Fig. 3a) and a higher mineralized surface density (Fig. 3b), meaning that its mineralized volume was lower but denser in comparison with native dentin. Moreover, the analysis of the specific mineralized surface (Fig. 3c) revealed higher values in the newly formed mineralized dentin, indicating that the reparative dentin was more compact than the native one. Besides, a significantly higher thickness of mineralized areas was measured in the dentin bridges (Fig. 3f).

Nonmineralized areas characteristics were further examined within the dentin. In comparison with the native dentin, the reparative one showed significantly higher values for the total porosity percentage (Fig. 3d), mineralized areas separation (Fig. 3e), and mean volume of pores (Fig. 3g). These results confirmed that the reparative dentin presented a higher percentage of nonmineralized areas, whose volume was also higher than in native dentin. In addition, a significantly higher connectivity percentage (Fig. 3h) was found in the reparative dentin, meaning that within the dentin bridge, nonmineralized areas were more extensively interconnected in comparison with those of native dentin.

Second, the effect of pDPCs on the reparative dentin was determined by comparing the morphometric features of bridges with and without cells. The results showed a significantly lower mineralized volume fraction with pDPC implantation than without (Fig. 3a), meaning that the mineralized component in the entire volume of the dentin bridge was less represented when cells were seeded in the scaffold. Moreover, in presence of cells, the reparative dentin was less dense, as demonstrated by the mineralized surface density values that were significantly lower than in samples without cells (Fig. 3b). Similar compactness and thickness of the mineralized areas were detected in both conditions, since no significant differences were found in terms of specific mineralized tissue surface (Fig. 3c) and thickness of mineralized areas (Fig. 3f). The total porosity percentage (Fig. 3d), the connectivity (Fig. 3h), and the separation of mineralized areas (Fig. 3e) were significantly higher in teeth implanted with cells than in those without cells. These results show that in the presence of cells, the reparative bridge presented more numerous and no cells vs. control (P = 0.0020); mineralized surface density (BS/TV), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0016); specific mineralized surface (BS/BV), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0008). The dentin bridge presented more nonmineralized areas (d and e) separated by thicker mineralized tissue (f) than in dentin control: total porosity percentage (porosity), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0020); mineralized areas thickness (Tb.Th.), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0016); mineralized areas separation (Tb.Sp.), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0010). Nonmineralized areas of dentin bridge, corresponding to cellular inclusions, showed a higher volume than the dentin control, and these areas were highly interconnected (g and h): mean volume of pores, cells vs. control (P = 0.0098) and no cells vs. control (P = 0.0342); connectivity of pores, cells vs. control (P = 0.0195) and no cells vs. control (P = 0.010). When the influence of cells on the microstructure of the bridges was analyzed, it was found that the mineralized component in the entire volume of dentin bridge was reduced when cells were seeded in the hydrogel (a). Moreover, in the presence of cells, the reparative dentin was less dense (b) but similar in thickness (f) and compactness (c): mineralized volume fraction (BV/TV), no cells vs. cells (P = 0.0061); mineralized surface density (BS/TV), no cells vs. cells (P < 0.0017); mineralized areas thickness (Tb.Th.), no cells vs. cells (P = 0.2541); specific mineralized surface (BS/BV), no cells vs. cells (P = 0.3751). The dentin bridge, in the presence of cells, presented more numerous and interconnected nonmineralized areas (d, e, h) whose volume, however, did not differ from reparative dentin of samples treated without cells (g): total porosity percentage, no cells vs. cells (P < 0.0001); mineralized areas separation (Tb.Sp.), no cells vs. cells (P < 0.0001); mean volume of pores, no cells vs. cells (P = 0.8110); connectivity of pores, no cells vs. cells (P < 0.0001). *P < 0.05. N = 144 dentin bridges. BS/BV, specific mineralized surface; BS/TV, mineralized surface density; BV/TV, mineralized volume fraction; Tb.Sp. mineralized areas separation, ; Tb.Th. mineralized areas thickness,.

and connected nonmineralized areas. Furthermore, the latter did not differ in both conditions in terms of volume, since no significant differences were found for the parameter mean volume of pores (Fig. 3g).

All these data suggest that the implantation of pDPCs/scaffold or no-pDPCs/scaffold after cameral pulpotomy induced the formation of a mineralized dentin bridge, whose microarchitecture sensibly differs from the native dentin. Furthermore, the presence of pDPCs significantly affected the microarchitecture of the dentin bridge.

Pulp Amputation and Scaffold Implantation Affected the Radicular Area

Since the reparative dentinogenesis constitutes a defense mechanism of the pulp, we further characterized the dentin bridge by histologic analysis.

Masson's trichrome staining (Fig. 4b, c, e, f) confirmed that the reparative bridge closed the root canal entrance, independent of the presence of the pDPCs in the scaffold. This indicates that the reparative dentinogenesis was not driven by implanted pDPCs.

Moreover, the presence of cells in the scaffold led to a mineralized tissue with more numerous cell inclusions when compared with those obtained without cells, consistent with our microCT morphometric characterization. The remaining root pulp presented signs of vasodilatation of blood vessels (hyperemia), alluding to the presence of an inflammatory process.

The cells associated with the reparative dentin bridge displayed processes within the dentin and showed a palisade organization. Moreover, they expressed DSP and BSP, evocating odontoblast-like cells (Golderg et al. 2008;Sagomonyants et al. 2017;Fig. 4g-n). In addition, immunolabeled cells for these markers were detected within the cell inclusions of the reparative bridge. These data indicate that the mineralization tissue constituted osteodentin.

Furthermore, tridimensional radiographic analysis of implanted teeth highlighted radiolucent areas, located at several radicular Hyperemia was observed in the radicular pulp. At higher magnification (c and f), cell inclusions appeared within the bridge in both conditions, with or without cells in the scaffold. Characterization of the reparative dentin bridge was performed by DSP and BSP immunostaining. BSP and DSP labeling were observed associated to the cells adjacent to the reparative bridge as well as in cell inclusions, in both conditions (g, i, k, and m). At higher magnification of specific areas (squares), BSP-and DSP-labeled cells presented a polarized morphology (arrows) and were organized as a palisade at the dentin bridge-pulp interface as well as inside the inclusions, in both conditions (h, j, l, and n). N = 48 teeth. Scale bar: 50 μm. DB, dentin bridge; P, pulp. levels, suggesting external root resorptions. In fact, independent of the presence of pDPCs, these radiologic images were identified in every sample (Appendix Fig. 3).

Discussion

In the present study, we investigated whether partial pulp regeneration could be achieved by implanting pDPCs/scaffold after cameral pulp amputation. Our results showed systematic reparative dentinogenesis instead of pulp regeneration. Indeed, a dentin bridge that completely closed each root canal entrance was observed at 3 wk after implantation. This finding is important for cell-based regenerative endodontic strategies and their further clinical implications, notably regarding the benefit to implant pulp cells. In fact, the reparative dentin bridge was obtained independent of the presence of cells in the scaffold. Even more, by dedicated morphometric characterization derived from bone microstructure analysis (Parfitt et al. 1983;Bouxsein et al. 2010), we showed that the sealing of the bridge was impaired in teeth implanted with pDPCs. Since the reparative process was detected in teeth with no-pDPCs/scaffold, the synthesis of the osteodentin was not driven by the implanted pDPCs. Considering the positive immunostaining for DSP and BSP, it is likely that the dentin bridge was formed by host odonto-/osteoblast-like cells located in the remaining pulp or brought to the injury site by the blood supply (Tran et al. 2012;Saito et al. 2016;Sagomonyants et al. 2017;Vidovic et al. 2017). Moreover, we applied a microCT analysis of this reparative osteodentin. This nondestructive and noninvasive technic allowed quali-/quantitative tridimensional characterization of the microarchitecture. One could assume that with the future improvement of high-resolution cone beam computed tomography, this standardized morphometric analysis could find application in clinical trials focusing on dentin bridge formation and root wall elongation.

In our model, pDPCs were implanted in an injectable selfassembling nanopeptide hydrogel (PuraMatrix) that has been reported in literature as an optimal tridimensional matrix inducing DPSC differentiation (Cavalcanti et al. 2013;Dissanayaka et al. 2015) and pulp regeneration in vivo (Rosa et al. 2013). Although the pDPCs were able to proliferate in this scaffold in vitro, a functional mesenchyme was not obtained in the teeth. This issue highlights the importance of the other specific parameters influencing the regenerative process in vivo. Indeed, we tested the cell-based pulp regeneration in a specific environment, represented by mature pulpotomized pluriradicular teeth. In mature teeth, blood supply is limited due to reduced root canal volume and apical closure, which might be insufficient to ensure the engraftment of implanted cells (Zheng et al. 2012). In addition, hyperemia and root resorptions, which are indirect signs of pulp inflammation [START_REF] Kassa | Histological comparison of pulpal inflammation in primary teeth with occlusal or proximal caries[END_REF][START_REF] Patel | Internal root resorption: a review[END_REF], were observed in all the treated teeth. The inflammatory process has been reported to negatively influence the neoangiogenesis (Boyle et al. 2014;Sagomonyants et al. 2017;Vidovic et al. 2017) and to promote, when limited, the synthesis of a reparative bridge (Goldberg et al. 2015). The origin of the inflammation and the respective implication of 1) the severe injury of the pulp amputation and 2) the implantation of the hydrogels have to be determined in our model to expect a successful pulp regeneration therapy. Moreover, the influence of Biodentin in the dentin bridge formation might not be neglected, since the material presents bioactive properties, even though this cement appeared as the material of choice to be applied in contact with cells, due to its biocompatibility and ease of use (Tran et al. 2012).

The failure of partial pulp regeneration in this study raises many questions regarding local factors influencing the regenerative environment and highlighted the necessity to develop preclinical models. Indeed, literature mainly reports in vitro studies and murine models of cell-based partial pulp regeneration, but the clinical feasibility of this strategy in preclinical models is still missing [START_REF] Rosa | Pluripotency of stem cells from human exfoliated deciduous teeth for tissue engineering[END_REF]. The large animal models recognized and developed in translational dentistry are the dog (Iohara et al. 2009), the minipig (Zheng et al. 2012), and, more recently, the sheep (Altaii et al. 2016). In the present work, we selected the minipig because it has anatomic and physiologic characteristics similar to humans and it is the only omnivore out of the 3 species (Štembirek et al. 2012). However, this model implies elevated cost, time-consuming procedures, and technical limits [START_REF] Yamamoto-Fukud | Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling[END_REF]Pearce et al. 2007).

Overall, this preclinical study in a large animal model showed that cell-based partial pulp regeneration could not be achieved in mature pluriradicular teeth. Further investigations are required to develop favorable microenvironmental conditions supporting the regeneration.

3D:

  three-dimensional ASCs: adipose tissue derived stem cells B-TCP: beta tricalcium phospahte BBM: bovine bone mineral bFGF: basic fibroblasts growth factor BMAC: bone marrow aspirate centrifugation BMP-7: bone morphogenetic protein 7 BMP2: bone morphogenetic protein 2 BMP4: bone morphogenetic protein 4 BMSCs: bone marrow stem cells BRONJ: bisphosphonate-related osteonecrosis of the Jaw BS/BV: specific mineralized surface BS/TV: mineralized surface density BSP: bone sialoprotein BV/TV: mineralized volume fraction CAD-CAM: computer-aided design and computer-aided manufacturing CBCT: cone beam computed tomography cBMMSC: canine bone marrow stem cells CD105: cluster of differentiation 105 CD105+: cluster of differentiation 105+ CD117: cluster of differentiation 117 CD14: cluster of differentiation 14 CD146: cluster of differentiation 146 CD166: cluster of differentiation 166 CD271: cluster of differentiation 271 CD29: cluster of differentiation 29 CD31: cluster of differentiation 31 CD34: cluster of differentiation 34 CD44: cluster of differentiation 44 CD45: cluster of differentiation 45 CD73: cluster of differentiation 73 CD90: cluster of differentiation 90 CM: conditioned medium cMSCs: canine mesenchymal stem cells DBCs: dental bud cells dBMSCs: dog bone marrow stem cells dDFCsd: dog dental follicles cells DFCs: dental follicle cells DFSCs: dental follicle stem cells DPCs: dental pulp cells dPDLSCs: dog periodontal ligament stem cells DSCs: dental stem cells DSP: dentin sialoprotein DTSCs: stem cells from deciduous teeth EDTA: ethylenediaminetetraacetic acid EMD: enamel matrix derivative EPCs: endothelial progenitor cells ESCs: embryonic stem cells ESEHT: extraction socket-derived early healing tissue FACS: fluorescence activated cell sorting FBS: fetal bovine serum FDMBB: freeze-dried mineral bone block FHA: fluorohydroxyapatite FOV: field of view G-CSF: granulocyte-colony stimulating factor GBP-L: gabapentin -lactam GCHT: gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer HA/TCP: hydroxyapatite/ tricalcium phosphate HIF-1a: hypoxia-inducible factor 1-alpha HUVECs: human umbilical vein endothelial cells LF-153: Larry Fisher 153 LF-84: Larry Fisher -84 MDP: multi domain peptides microCT: micro computed tomography MPR: multiplanar reconstruction MRI: magnentic resonance imaging MSCs: mesenchymal stem cells mTAP: modified tri-antibiotics paste NGF: nerve growth factor oAFMC: ovine amniotic fluid mesenchymal cells oBMSC: ovine bone marrow stem cells OCC: osteoblast cell culture OCT-3: octamer-binding transcription factor 3 oDPCs: ovine dental pulp cells oPDLSC: ovine periodontal ligament stem cells PBS: phosphate-buffered saline PCL-TCP: polycaprolactone-tricalcium phosphate PCs: periostal cells PDGF: platelet derived growth factor PDLSCs: periodontal ligament stem cells pDPC: porcine dental pulp cells pDPCs: porcine dental pulp cells PDPSCs: porcine deciduous pulp stem/progenitor cells pDPSCs: porcine dental pulp stem cells PLA: polylactic acid PLGA: polyglycolic acid PRF: platelet rich fibrin PRP: platelet rich plasma pSHED: porcine stem cells from from Exfoliated Deciduous teeth rh PDGF-BB: recombinant platelet-derived growth factor-BB SCAPs: stem cells from apical papilla SDF-1a: stromal cell-derived factor-1a SDF-1: stromal cell-derived factor-1 SHED: stem cells from human exfoliated deciduous teeth SP: side population SPECT: single photon emission computed tomography SRmCT: synchrotron radiation micro computed tomography TbSp: mineralized areas separation, TbTh: mineralized areas thickness TCP/HA: tricalcium phosphate/ hydroxyapatite TDM: treated dentine matrix TMJ: temporomandibular joint TSDO: trans-sutural distraction osteogenesis VEGF: vascular endothelial growth factor vWF: von Willebrand factor β-TCP: beta tricalcium phosphate αMEM: alpha minimum essential medium

Figure 1 -Figure 2 -

 12 Figure 1-Primary colture of Dental Pulp Cells. ......................................................................................... Figure 2-Immunophenotype of DPSCs. ......................................................................................................... Figure 3-Paradigm of tissue engineering ....................................................................................................Figure 4-Techniques of visualization of blood supply. ......................................................................... Figure 5-MicroCT visualization of mandibular blood supply in rat after barium sulphate injection ............................................................................................................................................................ Figure 6-Intra-arterial angiography in minipig. ...................................................................................... Figure 7-Sheep denture. ..................................................................................................................................... Figure 8-Dog's denture. ...................................................................................................................................... Figure 9-Pig's denture. ........................................................................................................................................ Figure 10-pDPCs in colture. .............................................................................................................................. Figure 11-Description of the partial pulp regeneration model. ........................................................ Figure 12-Dentin bridge segmenation (MeVisLab) ................................................................................ Figure 13-Native dentin control segmentation (MeVisLab) .............................................................. Figure 14-Thresholding of the dentin bridge (CTan) ............................................................................ Figure 15-Dentin bridge segmentation (CTan). ....................................................................................... Figure 16-Thresholding of the native dentin control (CTan) ............................................................ Figure 17-Native dentin control segmentation (CTan). ....................................................................... Figure 18-Segmentation of pores in the dentin bridge volume (Amira) ...................................... Figure 19-Segmentation of pores in the dentin bridge volume (Amira) ......................................Figure 20-Segmentation of pores in the native dentin control volume (Amira) ....................... Figure 21-Segmentation of pores in the native dentin control volume (Amira) ....................... Figure 22-Segmentation of the entire tooth volume (MeVisLab) .................................................... Figure 23-Volume rendering of the dentin bridge (3Matic) .............................................................. Figure 24-Volume rendering of the entire tooth (3Matic) .................................................................. Figure 25-Volume rendering of the tooth showing the location of the dentin bridge within the endodontic space (3Matic) ............................................................................................................... Figure 26-pDPCs proliferation in 0.2% PuraMatrix™ in vitro. ............................................................. Figure 27-MicroCT analysis of reparative dentinogenesis in teeth implanted with pDPCs/scaffold and no-pDPCs/scaffold. ............................................................................................ Figure 28-Morphometric analysis of dentin bridge structure in comparison with native dentin control and influence of pDPCs on dentin bridge microstructure. ........................... Figure 29-Masson's trichrome staining, DSP and BSP labeling in the pulp of teeth, at day 21 after surgery. .................................................................................................................................................. Figure 30-External resorptions at 3 weeks post implantation. ........................................................ Figure 31-Selective injection of Patent blue V into the inferior alveolar artery. ....................... Figure 32-Maxillary and mandibular blood supply in minipig. ........................................................ Figure 33-Contrast agent collection at the apical area of mature teeth in minipig. ................ Figure 34-Selective injection of iodine contrast medium in minipig.. ........................................... Figure 35-Soft tissues surrounding mandible. Volume rendering of soft tissues surrounding mandible. .......................................................................................................................................................... Figure 36-Segmentation of mandible soft tissues blood supply. ...................................................... Figure 37-Example of morphometric analysis of mandibular soft tissue vascular network

Figure 4 -

 4 Figure 1-Primary colture of Dental Pulp Cells. ......................................................................................... Figure 2-Immunophenotype of DPSCs. ......................................................................................................... Figure 3-Paradigm of tissue engineering ....................................................................................................Figure 4-Techniques of visualization of blood supply. ......................................................................... Figure 5-MicroCT visualization of mandibular blood supply in rat after barium sulphate injection ............................................................................................................................................................ Figure 6-Intra-arterial angiography in minipig. ...................................................................................... Figure 7-Sheep denture. ..................................................................................................................................... Figure 8-Dog's denture. ...................................................................................................................................... Figure 9-Pig's denture. ........................................................................................................................................ Figure 10-pDPCs in colture. .............................................................................................................................. Figure 11-Description of the partial pulp regeneration model. ........................................................ Figure 12-Dentin bridge segmenation (MeVisLab) ................................................................................ Figure 13-Native dentin control segmentation (MeVisLab) .............................................................. Figure 14-Thresholding of the dentin bridge (CTan) ............................................................................ Figure 15-Dentin bridge segmentation (CTan). ....................................................................................... Figure 16-Thresholding of the native dentin control (CTan) ............................................................ Figure 17-Native dentin control segmentation (CTan). ....................................................................... Figure 18-Segmentation of pores in the dentin bridge volume (Amira) ...................................... Figure 19-Segmentation of pores in the dentin bridge volume (Amira) ......................................Figure 20-Segmentation of pores in the native dentin control volume (Amira) ....................... Figure 21-Segmentation of pores in the native dentin control volume (Amira) ....................... Figure 22-Segmentation of the entire tooth volume (MeVisLab) .................................................... Figure 23-Volume rendering of the dentin bridge (3Matic) .............................................................. Figure 24-Volume rendering of the entire tooth (3Matic) .................................................................. Figure 25-Volume rendering of the tooth showing the location of the dentin bridge within the endodontic space (3Matic) ............................................................................................................... Figure 26-pDPCs proliferation in 0.2% PuraMatrix™ in vitro. ............................................................. Figure 27-MicroCT analysis of reparative dentinogenesis in teeth implanted with pDPCs/scaffold and no-pDPCs/scaffold. ............................................................................................ Figure 28-Morphometric analysis of dentin bridge structure in comparison with native dentin control and influence of pDPCs on dentin bridge microstructure. ........................... Figure 29-Masson's trichrome staining, DSP and BSP labeling in the pulp of teeth, at day 21 after surgery. .................................................................................................................................................. Figure 30-External resorptions at 3 weeks post implantation. ........................................................ Figure 31-Selective injection of Patent blue V into the inferior alveolar artery. ....................... Figure 32-Maxillary and mandibular blood supply in minipig. ........................................................ Figure 33-Contrast agent collection at the apical area of mature teeth in minipig. ................ Figure 34-Selective injection of iodine contrast medium in minipig.. ........................................... Figure 35-Soft tissues surrounding mandible. Volume rendering of soft tissues surrounding mandible. .......................................................................................................................................................... Figure 36-Segmentation of mandible soft tissues blood supply. ...................................................... Figure 37-Example of morphometric analysis of mandibular soft tissue vascular network

  In the context of this preclinical model of pulp regeneration, three objectives were established: 1. To obtain pulp tissue regeneration after pDPCs implantation in partial pulpotomies in minipig 2. To follow-up the regenerative process by interventional CBCT angiography; 3. To characterize the newly formed tissues by non-invasive and non-destructive microCT imaging techniques. Thanks to the collaboration with the laboratory Cr2i of INRA, the interventional CBCT angiography procedure has been set and performed. Then, during 3 months of scholarship in the departement of Oral and Maxillo-facial Surgery -Imaging & Pathology (OMFS-IMPATH) of the University of Leuven (KU Leuven), directed by Prof. Reinhilde Jacobs, different techniques of analysis have been developed. In particular, combining the high level of professionalism and competences of Prof. Jacobs' team on CBCT and microCT image analysis and my previous experience in the same field, the data from the in vivo 3D subtraction angiography have been processed in order to analyze dental pulp vascular network. Indeed, further developments of this modality open promising perspectives of its application for the morphometric characterization of angiogenesis process in newly formed dental tissues and bone defects. Moreover, using specific morphometric parameters, initially developed to characterize bone, a micro-CT morphometric analysis of the mineralized reparative tissues, obtained by the partial pulp regeneration protocol, has been elaborated. This work has led to a publication accepted in the Journal of Dental Research (Mangione F, EzEldeen M, Bardet C, Lesieur J, Bonneau M, Decup F, Salmon B, Jacobs R, Chaussain C, Opsahl-Vital S. 2017. Implanted dental pulp cells fail to induce regeneration in partial pulpotomies) and several communications in international congresses.

1. 1 . 1

 11 Phenotype characteristicsDental pulp stem cells have a phenotype similar to Bone Marrow Stem Cells (BMSCs). In culture, they look like big, spindle with a big central nucleus and a voluminous cytoplasm.(fig1).

Figure 1 -

 1 Figure 1-Primary colture of Dental Pulp Cells. Scale bar 50 μm (EA 2496).

Figure 2 -

 2 Figure 2-Immunophenotype of DPSCs. Integrin β1 (A); CD44 (B); collagen type I (C); collagen type III (D); fibroblast growth factor-2 (E); osteonectin (F); osteocalcin (G); MUC-18 (CD146) (H); α-smooth muscle actin (I); osteopontin (J); and vascular cell adhesion molecule 1 (K). Endogenous alkaline phosphatase (L)(Gronthos et al., 2000).

  Tissue engineering relies on the simple as well as combined use of DPSCs, scaffolds and growth factors, to achieve tissue regeneration (fig 3) (Dhillon et al., 2015).

Figure 3 -

 3 Figure 3-Paradigm of tissue engineering (Malhotra and Mala, 2012).

  highlighting microvascular architecture can be employed in vivo and ex vivo, in 2-D or 3-D such as histology, immunohistochemistry, animal perfusion, casts and imaging (fig 4a, 4b, 4c)(Tab. 2). Histological staining (Hematoxylin-Eosin, Movat pentachrome) and immunohistochemistry (CD31 and vWF labelling) allow to identify vessels and their rapport with contiguous structures and tissues. Animal perfusions and the corrosion casts give a 3D high-quality representation of microvascular architecture (fig

Figure 4 -

 4 Figure 4-Techniques of visualization of blood supply. Casts of pig head vassels (a) ; haematoxylin and eosin staining of pig brain histological section showing blood vassels (scale bar 20 μm) (b) ; immunolabeling of vWF on pig skin section (scale bar 20 μm) (c) ; 3D angiography of minipig mandible (scale bar 1 cm) (d).

Figure 5 -

 5 Figure 5-MicroCT visualization of mandibular blood supply in rat after barium sulphate injection (Blery et al., 2016).

  allowing, at the same time, a selective action of surgical resection and injection of radionucleotides, as well as the evaluation of the amount of blood supply of the donor and receiving sites of reconstruction grafts (fig6)[START_REF] Sakakibara | Three-dimensional venous visualization with phase-lag computed tomography angiography for reconstructive microsurgery[END_REF].

Figure 6 -

 6 Figure 6-Intra-arterial angiography in minipig. Injection of Iodine contrast medium in the maxillary artery (a) ; volume rendering of the maxillary artery branches (b). Bar scale 1 cm. (EA 2496).

Figure 7 -

 7 Figure 7-Sheep denture. Maxillary arcade presenting lack of incisors (a-b); mandibular arcade (c-d).

Figure 8 - 5 . 2 . 1

 8521 Figure 8-Dog's denture. Maxillary arcade.

  regenerated by ADSCs and TCP scaffold as described byAlvira-Gonzalez et al. (2016) and[START_REF] Sánchez-Garcés | Bone Regeneration Using Adipose-Derived Stem Cells with Fibronectin in Dehiscence-Type Defects Associated with Dental Implants: An Experimental Study in a Dog Model[END_REF].Implantation of cell sheets made by BMSCs and polylactic-co-glycolic acid (PLGA) was found to enhance bone formation in dog mandibular defects(Du et al., 2015).[START_REF] Khojasteh | The effect of PCL-TCP scaffold loaded with mesenchymal stem cells on vertical bone augmentation in dog mandible: a preliminary report[END_REF] studied bone critical size defects in dog's mandible by MSCs seeded onto polycaprolactone-tricalcium phosphate (PCL-TCP) scaffold and found early revascularization and bone regeneration. The same authors, in 2014, reported positive outcomes for bone defects healing using (BMSCs) loaded onto freeze-dried mineral bone block (FDMBB) in the presence or absence of recombinant platelet-derived growth factor-BB (rh PDGF-BB); in 2016 they also tested the effect of endothelial progenitor cells (EPCs) associated with b-TCP scaffolds coated with PLGA microspheres releasing and they didn't found better results than in previous studies.[START_REF] Zou | The bone-forming effects of HIF-1α-transduced BMSCs promote osseointegration with dental implant in canine mandible[END_REF] showed that cBMSCs in association with hypoxia-inducible factor-1a HIF-1a significantly promoted bone regeneration in mesi-implant defects in dogs.[START_REF] Yamada | A feasibility of useful cell-based therapy by bone regeneration with deciduous tooth stem cells, dental pulp stem cells, or bone-marrowderived mesenchymal stem cells for clinical study using tissue engineering technology[END_REF][START_REF] Yamada | Promising cell-based therapy for bone regeneration using stem cells from deciduous teeth, dental pulp, and bone marrow[END_REF][START_REF] Yamada | Injectable bone tissue engineering using expanded mesenchymal stem cells[END_REF] evaluated in a dog model and subsequently in a clinical study, the ability to induce bone healing of PRP seeded with different dog's stem cells, such as stem cells from deciduous teeth (DTSCs) and BMSCs. They showed positive outcomes mostly when DTSCs were implanted. These results are in line with those of Ito et al. (2011): they compared peri-implant bone regeneration obtained with DPSCs, BMSCs and Periostal Cells (PCs) and highlighted an improvement of osteintegration when DPSCs were used. Nakajima et al. (2014) showed bone regeneration of defects in dogs using a population of stem cells isolated from granulation tissue of dental sockets. Finally, Liu et al. (2014) demonstrated that BMSCs injected into long bone marrow in dogs present capacity of homing to appendicular and cranio-facial bone defects. Regarding regenerative endodontics, the most of the research about the orthotopic transplantation of DPSCs has used a canine model. Iohara et al. reported several studies

  2006, they demonstrated reparative dentinogenesis after pulp amputation and implantation of a side population of cells from dental pulp. In 2009, they described partial pulp regeneration after implantation of pellets of dental pulp cells expressing neoangiogenesis potential markers (CD31-/cd146-). The same team, in 2011, demonstrated complete pulp regeneration after pulpectomy in mature teeth, by transplantation of adipose CD105+ stem cells, expressing high potential of angiogenic/neurotrophic factors, with stromal cell-derived factor-1 (SDF-1). According to these results, in 2014, Yang et al. showed the active role of autophagy in stromal cell-derived factor-1a (SDF-1a)-mediated dental pulp stem cells (DPSC) migration and pulp regeneration, in pulpectomized canine teeth. In 2013, Iohara et al. achieved pulp regeneration in canine pulpectomy model, by using collagen scaffold and pulp cells mobilized with granulocyte-colony stimulating factor (G-CSF) (MDPSCs). These results were in line with those of Nakashima et al. (2014) and Murakami et al. (2015). Finally, in 2014, Iohara et al. reported an age-associated decrease of regeneration potential in MDPSCs.In 2016, the regenerative potential of the same population of cells was further evaluated, analyzing pulp function in canine pulpectomized teeth by MRI. A more intense signal was reported for teeth implanted with cells than controls implanted only with a collagen scaffold.[START_REF] Ishizaka | Regeneration of dental pulp following pulpectomy by fractionated stem/progenitor cells from bone marrow and adipose tissue[END_REF] demonstrated in a dog model, the pulp regeneration obtained after implantation of alternative sources of stem cells from bone marrow and adipose tissues. These outcomes are in line with those of[START_REF] Obeid | Mesenchymal stem cells promote hard-tissue repair after direct pulp capping[END_REF] demonstrating that autologous mesenchymal BMSCs were able to promote hard-tissue formation after direct pulp capping procedures. . Moreover,[START_REF] Yoo | Effect of conditioned medium from preameloblasts on regenerative cellular differentiation of the immature teeth with necrotic pulp and apical periodontitis[END_REF]

Figure 9 -

 9 Figure 9-Pig's denture. Maxillary arcade (a-b); mandibular arcade (c-d).

  Many authors have showed the possibility of tooth regeneration by implanting different kinds of scaffolds and stem cells from dental tissues in minipig jaws. In particular,[START_REF] Kodonas | Experimental formation of dentinlike structure in the root canal implant model using cryopreserved swine dental pulp progenitor cells[END_REF], demonstrated tooth-root regeneration seeding porcine dental follicle cells into collagen or PLGA scaffolds and transplanting these constructs in root canals of extracted teeth.[START_REF] Yang | Fibrin glue mixed with platelet-rich fibrin as a scaffold seeded with dental bud cells for tooth regeneration[END_REF] didn't achieve tooth regeneration using PRF and porcine dental follicle stem cells into mini-pig mandibular alveolar sockets. The same team, in 2016 investigated the possibility of whole-tooth formation by using pDPSCs (differentiated into odontoblasts and osteoblasts) and epithelial cells from gingiva. Cells were seeded into a bioactive scaffold and implanted in minipig alveolar sockets. At 13.5 months after surgery, they obtained eruption of teeth whose morphology was influenced by the site of implantation. Histological analysis confirmed the presence of enamel-like tissues, dentin, cementum, odontoblasts, and periodontal tissues[START_REF] Yang | Tooth Germ-Like Construct Transplantation for Whole-Tooth Regeneration: An In Vivo Study in the Miniature Pig[END_REF].[START_REF] Sonoyama | Mesenchymal stem cell-mediated functional tooth regeneration in swine[END_REF] demonstrated that HA/TCP root shaped scaffold containing porcine SCAP and coated with Gelfoam seeded with porcine PDLSCs into sockets in minipig provided tooth-root regeneration and allowed artificial crown support. The same scaffold associated with allogeneic mesenchymal stem cells was used by[START_REF] Wei | Functional tooth restoration by allogeneic mesenchymal stem cell-based bio-root regeneration in swine[END_REF] to obtain a functional bio-root formation in a minipig model. In 2011, Kuo et al. combined a gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer scaffold (GCHT scaffold) and dental bud cells DBCs conditioned with bone marrow fluid to regenerate a tooth in minipig alveolar sockets, 40 days after implantation.

  formed tissues and maxillary and dental blood supply have been developed, in collaboration with the department of Oral and Maxillo-facial Surgery -Imaging & Pathology (OMFS-IMPATH) of the University of Leuven (KU Leuven), directed by Prof. Reinhilde Jacobs.

  adapted from Gronthos et al. Briefly, under sterile and atraumatic conditions, lower second lateral incisors were harvested from each minipig. Teeth were cracked open and the dental pulp tissue was removed using forceps. The tissue was evenly sliced into 1-2-mm sections, and then incubated with phosphate-buffered saline (PBS) containing 3mg/mL collagenase type I (Worthington Biochem) and 2UI/mL dispase (Boehringer) in a shaking water bath at 37°C for 2h. The isolated cells were then centrifuged (1000 g) at 4°C for 5 min, resuspended, and plated in 25-cm2 flasks in the aMEM (Gibco) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen) at 37°C under 5% CO2 atmosphere. The cultures were daily monitored using light microscopy (fig10). Cell colony-forming units reached 80% confluence and the required cells number was reached after 3 passages.

Figure 10 -

 10 Figure 10-pDPCs in colture. Scale bar 50 μm.

Figure 11 -

 11 Figure 11-Description of the partial pulp regeneration model. After anesthesia, ultrasonic scaling to remove calculus accumulated on teeth (a). Isolation of the 2 nd , 3 rd , 4 th premolars and 1 st molar with a rubber dam to minimize the risk of bacterial contamination. Drilling of the occlusal cavity, elimination of the cameral pulp parenchyma by rotary instruments (b). Placement of the scaffold (with or without pDPCs) in the pulp chamber space (c). Sealing of the cavity with a calciumsilicate-based cement (Biodentine™, Septodont; France) and, thereafter, with a layer of dental composite (Filteck 250, 3M ESPE, Germany) (e). Post-operative radiography immediately after implantation showing the different layers (f), which are illustrated on the scheme (g). Scale bar 2 mm.

  volume fraction (BV/TV), Mineralized surface density (BS/TV), Specific mineralized surface (BS/BV), Mineralized areas thickness (TbTh). For non-mineralized areas, were calculated: Total porosity percentage (Porosity), Mineralized area separation (TbSp), Mean volume of pores and Connectivity of pores (Connectivity). After segmentation, the 3D triangle-based surface of the dentine bridges and the root canal were reconstructed and superimposed on the tooth structure of origin for spatial localization using 3matic (Materialise, Leuven, Belgium)(fig 22-25) .

Figure 12 -

 12 Figure 12-Dentin bridge segmenation (MeVisLab).

Figure 13 -

 13 Figure 13-Native dentin control segmentation (MeVisLab).

Figure 14 -

 14 Figure 14-Thresholding of the dentin bridge (CTan).

Figure 15 -

 15 Figure 15-Dentin bridge segmentation (CTan). Mineralized volume (white) and non mineralized volume (black) inside the total volume are segmented.

Figure 16 -

 16 Figure 16-Thresholding of the native dentin control (CTan).

Figure 17 -

 17 Figure 17-Native dentin control segmentation (CTan). Mineralized volume (white) and non mineralized volume (black) inside the total volume are segmented.

Figure 18 -

 18 Figure 18-Segmentation of pores in the dentin bridge volume (Amira).

Figure 19 -

 19 Figure 19-Segmentation of pores in the dentin bridge volume (Amira).

Figure 22 -

 22 Figure 22-Segmentation of the entire tooth volume (MeVisLab).

Figure 23 -

 23 Figure 23-Volume rendering of the dentin bridge (3Matic).

Figure 24 -

 24 Figure 24-Volume rendering of the entire tooth (3Matic).

Figure 25 -

 25 Figure 25-Volume rendering of the tooth showing the location of the dentin bridge within the endodontic space (3Matic).

Figure 26 -

 26 Figure26-pDPCs proliferation in 0.2% PuraMatrix™ in vitro. pDPCs proliferation has been tested in vitro. 2.10 6 cells were seeded in 100 μL 0.2% PuraMatrix hydrogel in a 96-well plate and cultured for 7 days. Cells proliferation was tested at day 1, 3 and 7. For each time point pDPCs/scaffolds have been made in triplicate. Cell proliferation was assessed at day 1, 3 and 7, using the Vybrant MTT cell proliferation assay kit (invitrogen). Absorbance measurements showed increasing cells proliferation from day 1 to 7.

(

  fig 27B). This reparative dentin was in contact with the lateral dentin walls and completely closed the entrance of the root canal space. Furthermore, the observation of the segmented volume of each bridge revealed a thick defined mineralized structure perfectly matching the anatomy of the endodontic space at its coronal part (fig27C).

Figure 27 -

 27 Figure 27-MicroCT analysis of reparative dentinogenesis in teeth implanted with pDPCs/scaffold and no-pDPCs/scaffold. A: MicroCT cross-sections focusing on the reparative dentin bridge, 3 weeks after implantation in 3rd premolars. A thick well-defined calcified tissue was present at the entrance of each root perfectly matching the endodontic anatomy and forming a canal obstruction, in both conditions (without or with cells in the scaffolds). Coronal views of tooth implanted without cells (i) and with cells (v). Sagittal views of tooth implanted without cells (ii and iii) and with cells (vi and vii). Axial views of tooth implanted without cells (iv) and with cells (viii). B: Teeth volume renderings, showing the precise location of the dentin bridges at each root entrance. (i-iv) The red arrows indicate mesio-lingual root, the yellow ones the mesio-vestibular root and the blue ones the disto-vestibular root. C: Volume renderings of segmented dentin bridges in the different roots. Dentin bridge of the mesio-vestibular root (i and iv); dentin bridge of mesio-lingual root (ii and v); dentin bridge of disto-vestibular root (iii and vi). Scale bar: 0.5 mm.

Figure 28 -

 28 Figure 28-Morphometric analysis of dentin bridge structure in comparison with native dentin control and influence of pDPCs on dentin bridge microstructure. The mineralized volume of the dentin bridges (ie. Mineralized Volume/Total Volume of the dentin bridge) (a) was lower but denser (b) and more compact (c) in comparison with control dentin [Mineralized volume fraction (BV/TV): cells vs. control p = 0.0020 and no cells vs. control p = 0.0020; Mineralized surface density (BS/TV): cells vs. control p = 0.0020 and no cells vs. control p = 0.0016; Specific mineralized surface (BS/BV): cells vs. control p = 0.0020 and no cells vs. control p = 0.0008]. The dentin bridge presented more non mineralized areas (d and e) separated by thicker mineralized tissue (f) than in dentin control [Total porosity percentage (Porosity): cells vs. control p = 0.0020 and no cells vs. control p = 0.0020; Mineralized areas thickness (Tb.Th.): cells vs. control p = 0.0020 and no cells vs. control p = 0.0016; Mineralized areas separation (Tb.Sp.): cells vs. control p = 0.0020 and no cells vs. control p = 0.0010]. Non mineralized areas of dentin bridge, corresponding to cellular inclusions, showed a higher volume than the dentin control and these areas were highly interconnected (g and h) [Mean volume of pores (Mean volume of pores): cells vs. control p = 0.0098 and no cells vs. control p = 0.0342; Connectivity of pores (Connectivity): cells vs. control p = 0.0195 and no cells vs. control p = 0.010]. When the influence of cells on the microstructure of the bridges was analyzed, it was found that the mineralized component in the entire volume of dentin bridge was reduced when cells were seeded in the hydrogel (a). Moreover, in the presence of cells, the reparative dentin was less dense (b) but similar in thickness (f) and compactness (c) [Mineralized volume fraction (BV/TV): no cells vs. cells p = 0.0061; Mineralized surface density (BS/TV): no cells vs. cells p < 0.0017; Mineralized areas thickness (Tb.Th.): no cells vs. cells p = 0.2541; Specific mineralized surface (BS/BV): no cells vs. cells p = 0.3751]. The dentin bridge, in the presence of cells, presented more numerous and interconnected non mineralized areas (d, e, h) whose volume, however, didn't differ from reparative dentin of samples treated without cells (g) ([Total porosity percentage (Porosity): no cells vs. cells p < 0.0001; Mineralized areas separation (Tb.Sp.): no cells vs. cells p < 0.0001; Mean volume of pores (Mean volume of pores): no cells vs. cells p = 0.8110; Connectivity of pores (Connectivity): no cells vs. cells p < 0,0001].* p < 0.05. N = 144 dentin bridges.

Figure 29 -

 29 Figure 29-Masson's trichrome staining, DSP and BSP labeling in the pulp of teeth, at day 21 after surgery.At day 21, a dentin bridge was detected at the root canal entries apically to the pulpotomy limit as showed in the microCT sections (a and d). Masson's trichrome confirmed the presence of a large reparative dentin bridge in both groups (b and e), in contact with dentin walls. Hyperaemia was observed in the radicular pulp. At higher magnification (c and f), cell inclusions appeared within the bridge in both conditions, with or without cells in the scaffold. Characterization of the reparative dentin bridge was performed by DSP and BSP immunostaining. BSP and DSP labeling were observed associated to the cells adjacent to the reparative bridge as well as in cell inclusions, in both conditions (g, i, k and m). At higher magnification of specific areas (squares), BSP and DSP labeled cells presented a polarized morphology (arrows) and were organized as a palisade at the dentin bridge -pulp interface as well as inside the inclusions, in both conditions (h, j, l and n). N = 48 teeth. Scale bar: 50 μm. DB= dentin bridge P= pulp.

Figure 30 -

 30 Figure 30-External resorptions at 3 weeks post implantation. Visualization by microCT scan of external resorptions. Several reconstructions in different orientations (axial, sagittal and coronal sections) were performed, showing external root resorptions, located on different root surfaces (a-g). Masson's Trichrome staining of sagittal section of distal root of the 3 rd premolar showing external resorption not associated with pulp infection (h). Scale bar 2mm.

Figure 31 -

 31 Figure 31-Selective injection of Patent blue V into the inferior alveolar artery.Presence of blue staining into the pulp of a treated tooth. Scale bar 1 cm.

Figure 32 -

 32 Figure 32-Maxillary and mandibular blood supply in minipig. Pre-visualization of internal maxillary artery (a); selective injection of iodine contrast medium into the internal maxillary artery (b and d) and into the inferior alveolar artery (c-e). Main branches were visualized in both injection protocols. Radiopacity of the contrast medium couldn't be distinguished from bone density. Bar scale 1 cm.

Figure 33 -

 33 Figure 33-Contrast agent collection at the apical area of mature teeth in minipig. The narrow diameter of the apical foramina of mature permanent teeth did not allow the contrast medium to diffuse into the pulp. Bar scale 1 cm.

Figure 34 -

 34 Figure 34-Selective injection of iodine contrast medium in minipig. Presence of radiological signal into canine pulp due to the lack of apical constriction. The signal was slightly discontinuous. Bar scale 1 cm.

Figure 36 -

 36 Figure 36-Segmentation of mandible soft tissues blood supply. a) Input image of the hemimandible; b) manual bone segmentation; c) volume of interest selection followed by bone extrusion; d) vessels selection. Bar scale 1 cm.

Figure 37 -

 37 Figure 37-Example of morphometric analysis of mandibular soft tissue vascular network. Average diameter of blood vessels in the volume of interest.

Figure 38 -

 38 Figure 38-Volume rendering of mandibular blood supply. Sagittal view of blood supply of soft tissues surrounding the hemimandible alone (a) and superimposed on bone (b). Axial view of blood supply of soft tissues surrounding the hemimandible alone (c) and superimposed on bone (d). Bar scale 1 cm.

Figure 39 -Figure 40 -

 3940 Figure 39-Maxillary and mandibular blood supply in mouse. Intracardiac injection of iodine contrast agent.Bar scale 2mm

Figure 41 -

 41 Figure 41-Maxillary and mandibular blood supply in mouse. Intracardiac injection of barium contrast agent.Bar scale 2 mm.

Figure 42 -

 42 Figure 42-Maxillary and mandibular blood supply in mice. Intracardiac injection of barium contrast agent. Absence of radiological signal into dental pulp. Bar scale 2 mm.

Figure 44 -

 44 Figure 44-Maxillary and mandibular blood supply in rat. Injection into carotid of small animals adapted contrast agent (Exitron 12000). Bar scale 2 mm.

Figure 45 -

 45 Figure 45-Maxillary and mandibular blood supply in rat. Injection into carotid of small animals adapted contrast agent (Exitron 12000). Presence of radiological signal into the incisor. Bar scale 2 mm.

Figure 1 .

 1 Figure 1. Porcine dental pulp cell (pDPC) proliferation in 0.2% PuraMatrix in vitro. pDPC proliferation has been tested in vitro; 2.10 6 cells were seeded in 100 μL of 0.2% PuraMatrix hydrogel in a 96-well plate and cultured for 7 d. Cell proliferation was tested at days 1, 3, and 7. For each time point, pDPCs/scaffolds were made in triplicate. Cell proliferation was assessed at days 1, 3, and 7 with the Vybrant MTT cell proliferation assay kit (Invitrogen). Absorbance measurements showed increasing cells proliferation from day 1 to 7. Triangle and error bars indicate the mean values of absorbance and SDs, respectively.

Figure 2 .

 2 Figure 2. Micro-computed tomography analysis of reparative dentinogenesis in teeth implanted with and without porcine dental pulp cells/scaffold. (A) Cross-sections focusing on the reparative dentin bridge, 3 wk after implantation in third premolars. A thick well-defined calcified tissue was present at the entrance of each root perfectly matching the endodontic anatomy and forming a canal obstruction in both conditions (without or with cells in the scaffolds). Coronal views of tooth implanted without cells (i) and with cells (v). Sagittal views of tooth implanted without cells (ii and iii) and with cells (vi and vii). Axial views of tooth implanted without cells (iv) and with cells (viii). (B) Teeth volume renderings showing the precise location of the dentin bridges at each root entrance. (i-iv) The red arrows indicate mesiolingual root; the yellow ones, the mesiovestibular root; and the blue ones, the distovestibular root. (C) Volume renderings of segmented dentin bridges in the different roots: mesiovestibular (i and iv), mesiolingual (ii and v), distovestibular root (iii and vi). Scale bar: 0.5 mm.

Figure 3 .

 3 Figure 3. Morphometric analysis of dentin bridge structure in comparison with native dentin control and influence of porcine dental pulp cells on dentin bridge microstructure. The mineralized volume of the dentin bridges (i.e., mineralized volume/total volume of the dentin bridge) (a) was lower but denser (b) and more compact (c) in comparison with control dentin: mineralized volume fraction (BV/TV), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0020); mineralized surface density (BS/TV), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0016); specific mineralized surface (BS/BV), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0008). The dentin bridge presented more nonmineralized areas (d and e) separated by thicker mineralized tissue (f) than in dentin control: total porosity percentage (porosity), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0020); mineralized areas thickness (Tb.Th.), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0016); mineralized areas separation (Tb.Sp.), cells vs. control (P = 0.0020) and no cells vs. control (P = 0.0010). Nonmineralized areas of dentin bridge, corresponding to cellular inclusions, showed a higher volume than the dentin control, and these areas were highly interconnected (g and h): mean volume of pores, cells vs. control (P = 0.0098) and no cells vs. control (P = 0.0342); connectivity of pores, cells vs. control (P = 0.0195) and no cells vs. control (P = 0.010). When the influence of cells on the microstructure of the bridges was analyzed, it was found that the mineralized component in the entire volume of dentin bridge was reduced when cells were seeded in the hydrogel (a). Moreover, in the presence of cells, the reparative dentin was less dense (b) but similar in thickness (f) and compactness (c): mineralized volume fraction (BV/TV), no cells vs. cells (P = 0.0061); mineralized surface density (BS/TV), no cells vs. cells (P < 0.0017); mineralized areas thickness (Tb.Th.), no cells vs. cells (P = 0.2541); specific mineralized surface (BS/BV), no cells vs. cells (P = 0.3751). The dentin bridge, in the presence of cells, presented more numerous and interconnected nonmineralized areas (d, e, h) whose volume, however, did not differ from reparative dentin of samples treated without cells (g): total porosity percentage, no cells vs. cells (P < 0.0001); mineralized areas separation (Tb.Sp.), no cells vs. cells (P < 0.0001); mean volume of pores, no cells vs. cells (P = 0.8110); connectivity of pores, no cells vs. cells (P < 0.0001). *P < 0.05. N = 144 dentin bridges. BS/BV, specific mineralized surface; BS/TV, mineralized surface density; BV/TV, mineralized volume fraction; Tb.Sp. mineralized areas separation, ; Tb.Th. mineralized areas thickness,.

Figure 4 .

 4 Figure 4. Masson's trichrome staining and labeling for dentin sialoprotein (DSP) and bone sialoprotein (BSP) in the pulp of teeth, at day 21 after surgery. At day 21, a dentin bridge was detected at the root canal entries apically to the pulpotomy limit, as shown in the micro-computed tomography sections (a and d). Masson's trichrome confirmed the presence of a large reparative dentin bridge in both groups (b and e), in contact with dentin walls.Hyperemia was observed in the radicular pulp. At higher magnification (c and f), cell inclusions appeared within the bridge in both conditions, with or without cells in the scaffold. Characterization of the reparative dentin bridge was performed by DSP and BSP immunostaining. BSP and DSP labeling were observed associated to the cells adjacent to the reparative bridge as well as in cell inclusions, in both conditions (g, i, k, and m). At higher magnification of specific areas (squares), BSP-and DSP-labeled cells presented a polarized morphology (arrows) and were organized as a palisade at the dentin bridge-pulp interface as well as inside the inclusions, in both conditions (h, j, l, and n). N = 48 teeth. Scale bar: 50 μm. DB, dentin bridge; P, pulp.

Table 1 -Immunohistochemical analysis of human DPSCs and BMSCs in vitro. The immunostaining

 1 of DPSCs demonstrated the markers in common with BMSCs.

	immunohistochemistry and DPSCs are able to differentiate in vitro into numerous types
	of cells (tab 1 and fig 2)

Table 2 -Techniques for blood supply visualization

 2

. ✓ possible ; -not possible ; + high ; -low ; +⁄-mean.

  Computed Tomography angiography is the main used technique for blood supply visualization, because it is less time consuming, economical and it has sufficient spatial resolution. Nevertheless, it implicates ionising radiation and injection of iodinated contrast media. The latter, mostly if injected in high volume, can induce nephropathy.

	Thus, catheter angiography is more invasive and less used; in fact literature reports an
	important percentage of indwelling catheter malposition and vascular damage, resulting in
	a high risk of bleeding, thrombosis, embolism and infection. However, intra-arterial

angiography requires a reduced volume of contrast medium with a satisfactory blood concentration and it provides good image quality. This modality is mainly used in the oncology field, because of the possibility to obtain a detailed map of the local vascular net

  created an ovine periodontal defect model: oPDLSC seeded into a Gelfoam ® scaffold were implanted in surgically created zero-wall dehiscence periodontal defects. At 4 weeks after implantation, periodontal tissues regeneration has been histologically showed.[START_REF] Menicanin | Periodontal-ligamentderived stem cells exhibit the capacity for long-term survival, self-renewal, and regeneration of multiple tissue types in vivo[END_REF] used an ovine preclinical periodontal defect model to test the self-renewal potential of autologous oPDLSCs using serial xenogeneic transplants of PDLSCs onto Gelfoam ® scaffold from mice. PRP) in bone healing; finally,[START_REF] Gallego | Repair of segmental mandibular bone defects in sheep using bone marrow stromal cells and autologous serum scaffold: a pilot study[END_REF] studied BMSCs regenerative potential when associated with autologous serum scaffold and they concluded that these procedure improve rate and quality of bone regeneration. Just one study focused on temporo-mandibular condyle joint regeneration in sheep, by using BMCS and a CAD-CAM customized porous scaffold (HA)[START_REF] Ciocca | Mesenchymal stem cells and platelet gel improve bone deposition within CAD-CAM custom-made ceramic HA scaffolds for condyle substitution[END_REF].More recently, onlyAltaii et al., have developed an ovine in vivo dental pulp regeneration/revascularization model. In a previous study they selected three dental ages

	This study confirmed, at 8 weeks after transplantation, the survival of oPDLSCs in
	association with periodontium-related tissues regeneration, including cementum and bone-
	like structures.
	Regarding bone defects regeneration, sinus lift procedures using ovine bone marrow stem
	cells (oBMSC) and different types of scaffold have been extensively assessed. Gutwald et al.

and Sauerbier et al., in 2010 demonstrated the bone regeneration potential of mesenchymal stem cells harvested from ovine iliac crest and combined with bovine bone mineral (BBM) in sinus lift augmentation in 6 adult sheep. Berardinelli et al., in 2013 showed that RegenOss®, which is a MgHA/collagen-based scaffold used in bone regeneration, provided better outcomes when ovine amniotic fluid mesenchymal cells (oAFMC) were added in a sinus lift model. In 2015, Ardjomandi et al. described lower bone regeneration in sinus lift in sheep when mesenchymal stem cells, seeded onto BMM scaffold, were obtained with bone marrow aspirate centrifugation (BMAC) in comparison with FICOLL density centrifugation. Oshima et al., in 2014 tested the new bone formation enhancement by novel gabapentinlactam novel (GBP-L) combined with BMM and oMSCs in sinus lift procedures in sheep and they didn't find a faster bone regeneration in comparison with sinus treated with just BMM and oMSCs. Many sheep preclinical models have been developed also for segmental bone defects regeneration. In fact, in 2001, Schliephake et al. showed that oBMSCs seeded into cylindrical calcium phosphate scaffolds increased bone regeneration; Hosgor et al., in 2013 demonstrated the effectiveness of Osteoblast Cell Culture (OCC) associated with platelet rich plasma (

  , periodontal ligament and Sharpey's fibres after implantation of PDLSCs in the periodontal defect area.[START_REF] Ding | Allogeneic periodontal ligament stem cell therapy for periodontitis in swine[END_REF] demonstrated that allogeneic PDLSCs could induce bone defects healing without activation of humoral immunity in vivo in miniature pigs. These outcomes were in line with those of[START_REF] Liu | Periodontal ligament stem cells regulate B lymphocyte function via programmed cell death protein 1[END_REF].

	created mandibular defects in minipig. They reported synthesis of dental, periodontal and
	bony tissues.
	Pieri et al. (2008, 2009) demonstrated enhancement of bone formation in edentulous ridge
	and periimplant defects in minipigs by using MSCs and PRP integrated to a
	Wei et al. (2012) highlighted a higher regenerative potential of PDLSCs sheets in fluorohydroxyapatite (FHA) scaffold. Zheng et al. (2009) achieved bone repair of critical
	comparison with dissociated PDLSCs when they were transplanted in periodontal defects in size mandibular defects in swine transplanting autologous pSHED.
	swine.
	They demonstrated bone healing with penetration of bone and blood vessels in the
	engineered construct (Abukawa et al., 2004). Moreover, in 2006 Abukawa et al. showed that
	interferon could induce porcine MSCs differentiation into odontoblasts but not improved
	the bone rate formation of defects healing in minipig (Abukawa et al., 2006).

porcine periodontal ligament fibroblastic cell lines showing endothelial and osteoblastic phenotypes, able to reconstruct periodontium.

[START_REF] Liu | Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine[END_REF]

showed periodontal defects regeneration by using PDLSCs. In particular, they reported formation of new bone, cementumThese outcomes were in agreement with those of

[START_REF] Cao | Adenovirus-mediated transfer of hepatocyte growth factor gene to human dental pulp stem cells under good manufacturing practice improves their potential for periodontal regeneration in swine[END_REF]

and

[START_REF] Hu | Periodontal regeneration in swine after cell injection and cell sheet transplantation of human dental pulp stem cells following good manufacturing practice[END_REF]

reporting higher bone defects healing in swine, using PDLSCs sheets than PDLSCs injection.

In 2013, Yang et al. induced experimental periodontitis in minipig and transplanted porcine embryonic stem cells (ES) in bony defects. In 3 months, after surgery, they demonstrated periodontal regeneration. Allogenic SHEDs in swine periodontits models have been reported to induce periodontium regeneration

(Fu et al., 2014)

. With regard to cell-based tissue engineering of jawbone defects, many studies have been developed in porcine models. In 2003, Abukawa et al. achieved condyle regeneration by combining biodegradable synthetic polymer (PLGA) and MSCs in minipig. The same team used the (PLGA) scaffold seeded with MSCs to regenerate minipig segmental bone defects. It has been demonstrated by Zhang et al. (2008) and Abukawa et al. (2009) that DPSCs and BMMSCs seeded into a hybrid tooth-bone construct are able to regenerate surgically

Table 3 -Example of morphometric analysis of mandibular soft tissue vascular network.

 3

ResuméLa pulpe dentaire est un tissu connectif innervé et vascularisé, contenu dans une structure minéralisée inextensible formée par l'email, la dentine et le cément. Ce tissu assure l'homéostasie et la sensibilité de la dent. Il est sujet à des lésions sévères faisant suite à une carie ou à un traumatisme. La thérapeutique conventionnelle préconisée alors est le traitement endodontique, qui consiste en l'exérèse de la totalité du tissu pulpaire et le comblement de l'espace évidé par un matériau synthétique bioinerte. Malgré les résultats cliniques satisfaisants, cette thérapeutique induit une fragilisation de la dent et une plus grande susceptibilité aux infections, qui peuvent conduire à terme à la perte de la dent.En se basant sur la présence de cellules souches mésenchymateuses dans la pulpe dentaire, des stratégies de régénération alternatives au traitement endodontique traditionnel sont à l'étude, afin de permettre le maintien des fonctions de nutrition et de sensibilité de la pulpe, garantes de la pérennité de la dent sur l'arcade.Elles s'inscrivent dans deux approches: la régénération de novo, en cas de nécrose du tissu pulpaire et la régénération partielle, lorsque seul le tissu pulpaire endommagé est éliminé et régénéré. Nos travaux portent sur la faisabilité de cette dernière approche dans un modèle préclinique. En effet, dans la perspective d'un transfert vers la clinique humaine, des modèles chez le gros animal doivent être développés afin de tester la faisabilité et le succès de cette thérapie, dans des conditions proches de la clinique.Du fait de ses similitudes avec l'homme en termes d'anatomie et de physiologie, le miniporc représente un modèle de choix pour les études précliniques d'ingénierie pulpaire.

Figure 20-Segmentation of pores in the native dentin control volume (Amira).Figure 21-Segmentation of pores in the native dentin control volume (Amira).

Acknowledgments

Acknowledgments

This work was supported by grants from University Paris Descartes, from the National French Agency for Research (Grant PulpCell ANR-14-CE16-0006-01) and Fondation des Maladies Rares (Grant Large Animal Experiment LAM-RD-U201140104). The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

special thank to Julie Lesieur for her teaching and helping as well as for her involvement in this project.

Thank you to all my colleagues and friends from « la salle des étudiants » and from the hospital Albert Chenevier.

I am grateful to University Paris Descartes, National French Agency for Research (Grant PulpCell ANR-14-CE16-0006-01) and Fondation des Maladies Rares (Grant large animal experiment LAM-RD-U201140104) that supported this work with their grants.

Finally I would like to express all my sincere gratitude to my parents Luigi and Fiorella, my

reconstruction (MPR) by OsiriX software.

In order to test a more selective way of injection, the angiography protocol has been simulated by injection of Patent blue V stain in the inferior alveolar artery. Since the life staining has been detected into pulp chambers of mandibular teeth and soft tissues, the protocol has been validated and the inferior alveolar artery angiography was performed.

MICRO-COMPUTED TOMOGRAPHIC IMAGING AND ANALYSIS

Image acquisition and export

All harvested teeth were scanned ex vivo at day 21 after surgery using an X-ray microCT device (Quantum FX Caliper, Life Sciences, Perkin Elmer, Waltham, MA). The microCT parameters were: 10x10 mm field of view, 20 µm voxel size, 90 kVp, 160 mA, 360° rotation, 3 min exposure time. All data sets were exported using the DICOM format with the isotropic 20 µm3 voxel. Acquired volumes were analyzed with Osirix dicom viewer software (v.5.8.5. 64-bit) in multiplanar reconstruction (MPR) mode.

Image segmentation, 3D reconstruction and quantitative morphometric characterization of hard tissue formation

All scanned samples were imported into MeVisLab (MeVis Research, Bremen, Germany).

Newly formed hard tissue (dentine bridge) within the pulp space was extracted from the surrounding tissue (tooth structure) using a dedicated tool developed in MeVisLab and validated for accurate tooth/root canal space segmentation (EzEldeen et al., 2015). Briefly, Due to the similar radiopacity of bone and contrast agent and the lack of signal into the pulp of treated teeth, morphometric characterization of blood supply was possible only for soft tissues surrounding mandible (fig 35-38).

Thus the cortical bone could be segmented from the acquired volume of the hemimandible (fig . 36a-b). After the selection of soft tissues' vessels in the volume of interest (fig 36c -d), blood supply could be morphometrically analysed. selective injection of Patent blue V, the staining was detected in the pulp of all treated teeth.

Further investigations are still required to set contrast agent density by maintaining a high radiological signal.

A detailed knowledge of oro-facial and dental blood supply can be the key factor to achieve positive results not only in regenerative translational endodontics, but also in bone healing/regeneration, mostly in cases of BRONJ.

Thus this work opens interesting perspectives for further developments of imaging techniques able to characterize in a quali-quantitative manner the vascular network of this anatomic area. In order to set a 3D subtraction angiography protocol allowing the visualization and the morphometric analysis of blood vessels within mineralized tissues, our team in collaboration with the department of OMFS-IMPATH of the University of Leuven, already performed this technique in mouse and rat models. The increasing size of animals allowed to test ever more selective ways of injection and different contrast mediums. An in vivo microCT scan has been used for images acquisition.

In mice, barium and iodine contrast medium via intracardiac injection were administrated.

Only in the first case maxillary blood supply could be visualized on microCT images. Dental vessels could not be displayed in any situations (fig 39-42). Subsequently, after surgical access, a contrast medium customized for small animals (Exitron 12000) was injected in the external carotid. Detailed images of the blood supply of all the entire area of internal maxillary artery were obtained.

The infraorbitary artery with its cutaneous, nasal and sinus branches have been displayed.

Superior alveolar artery's branches have been also highlighted.

The inferior alveolar artery and the incisor and mental branches have been visualized (fig

44).

Sciences, Perkin Elmer). The microCT parameters were as follows: 10 × 10 mm-field of view, 20-μm voxel size, 90 kVp, 160 mA, 360° rotation, 3-min exposure time. All data sets were exported with the DICOM format with the isotropic 20-μm 3 voxel. Acquired volumes were analyzed with Osirix DICOM viewer software (version 5.8.5, 64 bit) in multiplanar reconstruction mode.

Image Segmentation, 3-dimensional Reconstruction, and Quantitative Morphometric Characterization of Hard Tissue Formation. All scanned samples were imported into MeVisLab (MeVis Research). Newly formed hard tissue (dentine bridge) within the pulp space was extracted from the surrounding tissue (tooth structure) with a dedicated tool developed in MeV-isLab and validated for accurate tooth/root canal space segmentation (EzEldeen et al. 2015). Briefly, the imaging analysis tool applies semi-interactive livewire boundary extraction (Barrett and Mortensen 1997) to create a set of orthogonal contours, followed by a variational interpolation algorithm that reconstructs the surface of an object with energy-minimizing, smooth, and implicit functions [START_REF] Heckel | Interactive 3D medical image segmentation with energy-minimizing implicit functions[END_REF]. For each dentin bridge, a geometrically equivalent region was extracted from the native dentine by superimposing the contours created during the segmentation of the newly formed tissue, to be used as a control (Appendix Fig. 2).

The nature of the dentine bridges was characterized with specific bone-derived morphometric parameters commonly used for mineralized tissues analysis (Parfitt et al. 1983), with dedicated protocols in CTAn and Amira software (FEI Visualization Sciences Group).

For mineralized areas of the segmented volumes, the following parameters were calculated: mineralized volume fraction, mineralized surface density, specific mineralized surface, mineralized areas thickness. For nonmineralized areas, the following parameters were calculated: total porosity percentage, mineralized areas separation, mean volume of pores, and connectivity of pores.

After segmentation, the 3-dimensional triangle-based surface of the dentine bridges and the root canal were reconstructed and superimposed on the tooth structure of origin for spatial localization with 3matic (Materialise).

Histology and Immunohistochemistry

Teeth were decalcified in 10% nitric acid during 1 mo. After tissue embedding in Paraplast, serial sections (7 μm) were stained for Masson's trichrome.

For immunohistochemical analysis, the sections were incubated with primary antibodies, against bone sialoprotein (BSP; LF-84, kindly donated by Larry Fisher) diluted at 1/200 and against the dentin sialoprotein (DSP; LF-153, kindly donated by Larry Fisher) at 1/200. Tissue sections were then incubated with anti-rabbit immunoglobulin (DakoCytomation) diluted at 1/100. Color reaction was developed with 3,3′-diaminobenzidine tetrahydrochloride. A negative control was included with an equal concentration of rabbit preimmune serum as primary antibody.

Author Contributions

F. Mangione, S. Opsahl-Vital, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; M. EzEldeen, contributed to data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; C. Bardet, J. Lesieur, B. Salmon, R. Jacobs, contributed to data acquisition, analysis, and interpretation, critically revised the manuscript; M. Bonneau, F. Decup, contributed to data acquisition, critically revised the manuscript; C. Chaussain, contributed to conception and design, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.