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Mesenchymal stem cells (MSCs) are used for regenerative therapy. Dental pulp MSCs make extracted wisdom teeth a useful resource in humans. Preclinical validation of regenerative therapies requires large animal models such as the sheep. Since stem cells can be retrieved from the dental pulp of ovine incisors, the best age to extract a maximal volume of dental pulp needs to be defined. The objective of this ex vivo study was to quantify incisors dental pulp volume, in sheep of various age. Three jaws were dedicated to histology (one per age group); the others were imaged with a computed tomography scanner [3 years-old (n = 9), 4 (n = 3) and 6 (n = 5)]. The incisors dental pulp volume was measured after 3D reconstruction. Multiple linear regression showed that dental pulp volume of ovine incisors decreases with age (beta-estimate = -3.3; p < 0.0001) and teeth position from the more central to the more lateral (beta-estimate = -4.9; p = 0.0009). Weight was not a relevant variable in the regression model. The dental pulp volume ranged from 36.7 to 19.6 mm3 in 3-year-old sheep, from 23.6 to 11.3 in 4-year-old sheep, and from 19.4 to 11.5 in 6-year-old sheep. The pulp volume of the most central teeth (first intermediate) was significantly higher than the most lateral teeth (corner). Haematoxylin-Eosin-Safran of the whole incisors, and of isolated dental pulps demonstrated a similar morphology to that in humans. The first intermediate incisor of 3-year-old sheep should be selected preferentially in preclinical research to retrieve the highest volume of dental pulp. This work was supported by the FNRS—Fonds de la Recherche Scientifique (CR40000921, 2020-2023) as Dr. Hontoir Fanny is a FNRS post-doctoral researcher. We acknowledge Lucie Rendez, Florent Houbaille, and Nicolas DeSmet, imaging technicians of the CHU UCL NAMUR—Godinne (Centre Hospitalier UniversitaireUniversité Catholique de Louvain-Namur) for their precious technical help in this project. We acknowledge Narilis Institute for their support.

Organoid models provide powerful tools to study tissue biology and development in a dish. Presently, organoids have not yet been developed from mouse tooth. Here, we established tooth organoids (TOs) from early-postnatal mouse molar and incisor, which are long-term expandable, express dental epithelium stem cell (DESC) markers, and recapitulate key properties of the dental epithelium in a tooth-type-specific manner. TOs display in vitro differentiation capacity toward ameloblast-resembling cells, even more pronounced in assembloids in which dental mesenchymal (pulp) stem cells are combined with the organoid DESCs. Single-cell transcriptomics supports this developmental potential and reveals co-differentiation into junctional epithelium- and odontoblast-/cementoblast-like cells in the assembloids. Finally, TOs survive and show ameloblast-resembling differentiation also in vivo. The developed organoid models provide new tools to study mouse tooth-type-specific biology and development and gain deeper molecular and functional insights that may eventually help to achieve future human biological tooth repair and replacement. We are grateful to Evelyne Van Kerckhove (UHasselt), Marc Jans (UHasselt), Jeanine Santermans (UHasselt), and Veerle Vanslembrouck (KU Leuven) for valuable technical help. We thank Dr. Diether Lambrechts’ group (VIB,KULeuven) for technical assistance in 103 Genomics. Computational resources for transcriptome analyses were provided by the ‘‘Vlaams Supercomputer Centrum’’ (VSC), managed by the Fund for Scientific Research (FWO), Flanders (Belgium).We are also grateful to the ImagingCore (VIB,KULeuven) and the CIC (KU Leuven) for use ofmicroscopes and the Center for Brain & Disease Research (CBD) Histology unit (VIB, KU Leuven) for use of histology equipment. We acknowledge the use of the TEMplatforms at VIB-KU Leuven, UHasselt, and Tohoku University. The authors also thank Dr. Adrian Ranga (KU Leuven), Dr. Ronald Driesen(UHasselt), and other non-coauthor members of the Laboratory of Tissue Plasticity in Health and Disease (KU Leuven) for their input. Certain figures were created using BioRender.com or GraphPad Prism (v9.3.1) for macOS. This work was supported by grants from KU Leuven (Research Fund) and Fund for Scientific Research (FWO) Flanders. L.H. was an FWO PhD Fellow (1S84718N). C.B. is an FWO PhD Fellow (1129323N), while F.H. was supported by an FWO project grant (G061819FWO). Use of the Zeiss LSM 780 – SP Mai Tai HP DS is supported by Hercules AKUL/11/37 and FWO G.0929.15 funding to Dr. Pieter Vanden Berghe (CIC, KU Leuven), while the application of the JEOL FLASH 1400 TEM was supported by Hercules FWO funding I000220N to I.L.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4Dand PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS. This work has been supported by FWO (12G0817N, 1S57521N, G041421N, and 12G0817N), Fondation Charctot Stichting (ID2020- 0019), Nationale Belgische Multiple Sclerose Liga (Charco18VT), MS Liga Vlaanderen and Stichting MS Research (18-1016 MS). MS, EP, JP and TV have a proprietary interest in selective PDE4D inhibitors for the treatment of demyelinating disorders and neurodegenerative disorders. JP has a proprietary interest in the PDE4 inhibitor roflumilast for the treatment of cognitive impairment as well as PDE4D inhibitors for the treatment of Alzheimer’s disease. We thank Prof. Dr. O.N. Viacheslav (University Medical Center Hamburg-Eppendorf, German Center for Cardiovascular Research) and Prof. Dr. M. Conti (University of California), for providing the PDE4B KO animals. Furthermore, we thank Rewind Therapeutics for providing the visual evoked potential equipment.

The use of Ti6Al4V in bone engineering is limited, due to the biological inertia of the surface. In this study, a porous Ti6Al4V scaffold with mechanical properties similar to cancellous bone was designed and 3D-printed. Under physiological conditions, the scaffold was immersed firstly in a 5-fold-concentrated sim-ulated body fluid, then in a supersaturated CaP solution containing BMP-2, to form a bone-like porous micro/nano structured biomimetic coating on the surface. Scaffolds were implanted in the muscle pouches created in six beagle dogs and were retrieved four weeks later for histologic and histomorpho-metric analysis. Results showed that BMP-2 integrated biomimetic CaP coating induced ectopic bone for- mation, which was absent in other two groups. Soft tissue infiltrated the scaffold’s outside 1 mm layer, while the new-formed bone was evenly distributed in the longitudinal and horizontal directions withinv the rest of the scaffold based on BA/TA, BIC and BA measurements. In conclusion, the BMP-2 incorporated biomimetic CaP coating creates a micro/nano surface structure on the Ti6Al4V scaffold, which helps to increase biocompatibility. The integrated BMP-2 is capable of inducing ectopic bone formation in vivo We acknowledged the contributions of all members of our research group to this article. This research was granted by National Natural Science Foundation of China(NSFC) No. 81670964, and Shandong Taishan Scholar Program to Dr. Yue-lian Liu. The authors appreciate Shanghai Rebone Biomaterials Co. for the supply of BMP-2

Insight into human tooth epithelial stem cells and their biology is sparse. Tissue-derived organoid models typically replicate the tissue's epithelial stem cell compartment. Here, we developed a first-in-time epithelial organoid model starting from human tooth. Dental follicle (DF) tissue, isolated from unerupted wisdom teeth, efficiently generated epithelial organoids that were long-term expandable. The organoids displayed a tooth epithelial stemness phenotype similar to the DF's epithelial cell rests of Malassez (ERM), a compartment containing dental epithelial stem cells. Single-cell transcriptomics reinforced this organoid-ERM congruence, and uncovered novel, mouse-mirroring stem cell features. Exposure of the organoids to epidermal growth factor induced transient proliferation and eventual epithelial-mesenchymal transition, highly mimicking events taking place in the ERM in vivo. Moreover, the ERM stemness organoids were able to unfold an ameloblast differentiation process, further enhanced by transforming growth factor-beta (TGF beta) and abrogated by TGF beta receptor inhibition, thereby reproducing TGF beta's known key position in amelogenesis. Interestingly, by creating a mesenchymal-epithelial composite organoid (assembloid) model, we demonstrated that the presence of dental mesenchymal cells (i.e. pulp stem cells) triggered ameloblast differentiation in the epithelial stem cells, thus replicating the known importance of mesenchyme-epithelium interaction in tooth development and amelogenesis. Also here, differentiation was abrogated by TGF beta receptor inhibition. Together, we developed novel organoid models empowering the exploration of human tooth epithelial stem cell biology and function as well as their interplay with dental mesenchyme, all at present only poorly defined in humans. Moreover, the new models may pave the way to future tooth-regenerative perspectives. This work was supported by grants from KU Leuven (Research Fund) and Fund for Scientific Research (FWO) Flanders. L.H. is an FWO PhD Fellow (1S84718N). Acknowledgements We are grateful to all staff members of the Oral and Maxillofacial Surgery (MKA) of UZ Leuven, as well as the patients, for their invaluable help and contribution to collect freshly extracted third molars. We would also like to thank Dr. Reinhilde Jacobs and Dr. Elisabeth Tijskens for their help with sample collection. We thank Dr. Diether Lambrechts’ group for their technical assistance in 10× Genomics. Computational resources for scRNA-seq analysis were provided by ‘Vlaams Supercomputer Centrum’ (VSC), managed by the Fund for Scientific Research (FWO)—Flanders (Belgium). We acknowledge the use of the TEM platforms at VIB-KU Leuven, Biomedical Research Institute (BIOMED) UHasselt, and Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). We would also like to thank Dr. Ronald Driesen (UHasselt) for help with CTOF analysis, and Dr. Marianne Carlon’s group (particularly Liesbeth De Keersmaecker; KU Leuven) for guiding and assisting in lentiviral transduction. We are grateful to Jeanine Santermans and Marc Jans (UHasselt) for their technical assistance in TEM and staining analyses. We also would like to thank Reena Chinnaraj and Vera Dermesrobian (FACS Core, KU Leuven) for their help with FACS experiments.

Blood-based (BB) biomarkers for A beta and tau can indicate pathological processes in the brain, in the early pathological, even pre-symptomatic stages in Alzheimer's disease. However, the relation between BB biomarkers and AD-related processes in the brain in the earliest pre-pathology stage before amyloid pathology develops, and their relation with total brain concentrations of A beta and tau, is poorly understood. This stage presents a critical window for the earliest prevention of AD. Preclinical models with well-defined temporal progression to robust amyloid and tau pathology provide a unique opportunity to study this relation and were used here to study the link between BB biomarkers with AD-related processes in pre- and pathological stages. We performed a cross-sectional study at different ages assessing the link between BB concentrations and AD-related processes in the brain. This was complemented with a longitudinal analysis and with analysis of age-related changes in a small cohort of human subjects. We found that BB-tau concentrations increased in serum, correlating with progressive development of tau pathology and with increasing tau aggregates and p-tau concentrations in brain in TauP301S mice (PS19) developing tauopathy. BB-A beta 42 concentrations in serum decreased between 4.5 and 9 months of age, correlating with the progressive development of robust amyloid pathology in APP/PS1 (5xFAD) mice, in line with previous findings. Most importantly, BB-A beta 42 concentrations significantly increased between 1.5 and 4.5 months, i.e., in the earliest pre-pathological stage, before robust amyloid pathology develops in the brain, indicating biphasic BB-A beta 42 dynamics. Furthermore, increasing BB-A beta 42 in the pre-pathological phase, strongly correlated with increasing A beta 42 concentrations in brain. Our subsequent longitudinal analysis of BB-A beta 42 in 5xFAD mice, confirmed biphasic BB-A beta 42, with an initial increase, before decreasing with progressive robust pathology. Furthermore, in human samples, BB-A beta 42 concentrations were significantly higher in old (> 60 years) compared to young (< 50 years) subjects, as well as to age-matched AD patients, further supporting age-dependent increase of A beta 42 concentrations in the earliest pre-pathological phase, before amyloid pathology. Also BB-A beta 40 concentrations were found to increase in the earliest pre-pathological phase both in preclinical models and human subjects, while subsequent significantly decreasing concentrations in the pathological phase were characteristic for BB-A beta 42. Together our data indicate that BB biomarkers reflect pathological processes in brain of preclinical models with amyloid and tau pathology, both in the pathological and pre-pathological phase. Our data indicate a biphasic pattern of BB-A beta 42 in preclinical models and a human cohort. And most importantly, we here show that BB-A beta increased and correlated with increasing concentrations of A beta in the brain, in the earliest pre-pathological stage in a preclinical model. Our data thereby identify a novel critical window for prevention, using BB-A beta as marker for accumulating A beta in the brain, in the earliest pre-pathological stage, opening new avenues for personalized early preventive strategies against AD, even before amyloid pathology develops. This project was supported by Stichting Alzheimer Onderzoek (SAO-FRA Belgium) (SAO2020022) and Fonds Wetenschappelijk Onderzoek—Vlaanderen (FWO)—Research project No. G0C6819N and BOF funding UHasselt (Methusalem fnancing).

Single-cell (sc) omics has become a powerful tool to unravel a tissue’s cell landscape across health and disease. In recent years, sc transcriptomic interrogation has been applied to a variety of tooth tissues of both human and mouse, which has considerably advanced our fundamental understanding of tooth biology. Now, an overarching and integrated bird’s-view of the human and mouse tooth sc transcriptomic landscape would be a powerful multifaceted tool for dental research, enabling further decipherment of tooth biology and development through constantly progressing state-of-the-art bioinformatic methods as well as the exploration of novel hypothesis-driven research. To this aim, we re-assessed and integrated recently published scRNAsequencing datasets of different dental tissue types (healthy and diseased) from human and mouse to establish inclusive tooth sc atlases, and applied the consolidated data map to explore its power. For mouse tooth, we identified novel candidate transcriptional regulators of the ameloblast lineage. Regarding human tooth, we provide support for a developmental connection, not advanced before, between specific epithelial compartments. Taken together, we established inclusive mouse and human tooth sc atlases as powerful tools to potentiate innovative research into tooth biology, development and disease. The maps are provided online in an accessible format for interactive exploration. Funding This work was supported by grants from the KU Leuven Research Fund and from the FWO (G061819N). LH is an FWO PhD fellow (1S84718N). Acknowledgments Computational resources for all sc transcriptome analyses were provided by the ‘Vlaams Supercomputer Centrum’ (VSC), managed by the Fund for Scientific Research (FWO) - Flanders (Belgium). Certain figures were created using BioRender.com or GraphPad Prism version 9.3.1 for macOS. Authors also thank the other members of the Laboratory of Tissue Plasticity in Health and Disease (KU Leuven) for their input.

The progressive nature of demyelinating diseases lies in the inability of the central nervous system (CNS) to induce proper remyelination. Recently, we and others demonstrated that a dysregulated innate immune response partially underlies failure of CNS remyelination. Extensive accumulation of myelin-derived lipids and an inability to process these lipids was found to induce a disease-promoting phagocyte phenotype. Hence, restoring the ability of these phagocytes to metabolize and efflux myelin-derived lipids represents a promising strategy to promote remyelination. Here, we show that ApoA-I mimetic peptide 5A, a molecule well known to promote activity of the lipid efflux transporter ABCA1, markedly enhances remyelination. Mechanistically, we find that the repair-inducing properties of 5A are attributable to increased clearance and metabolism of remyelination-inhibiting myelin debris via the fatty acid translocase protein CD36, which is transcriptionally controlled by the ABCA1-JAK2-STAT3 signaling pathway. Altogether, our findings indi-cate that 5A promotes remyelination by stimulating clearance and degradation of myelin debris. We thank M.P. Tulleners for excellent technical assistance. The work was financially supported by the Research Foundation of Flanders (FWO Vlaanderen; 1S15519N, G099618FWO, and 12J9119N) and the Interreg V-A EMR program (EURLIPIDS, EMR23). The funding agencies had no role in the design, analysis, or writing of the article.