Hypoxia-inducible factors in postnatal mouse molar dental pulp development: insights into expression patterns, localisation and metabolic pathways.

Hypoxia is relevant to several physiological and pathological processes and this also applies for the tooth. The adaptive response to lowering oxygen concentration is mediated by hypoxia-inducible factors (HIFs). Since HIFs were shown to participate in the promotion of angiogenesis, stem cell survival, odontoblast differentiation and dentin formation, they may play a beneficial role in the tooth reparative processes. Although some data were generated in vitro, little is known about the in vivo context of HIFs in tooth development. In order to contribute to this field, the mouse mandibular first molar was used as a model.The expression and in situ localisation of HIFs were examined at postnatal (P) days P0, P7, P14, using RT-PCR and immunostaining. The expression pattern of a broad spectrum of hypoxia-related genes was monitored by customised PCR Arrays. Metabolic aspects were evaluated by determination of the lactate level and mRNA expression of the mitochondrial marker Nd1.The results show constant high mRNA expression of Hif1a, increasing expression of Hif2a, and very low expression of Hif3a during early postnatal molar development. In the examined period the localisation of HIFs in the nuclei of odontoblasts and the subodontoblastic layer identified their presence during odontoblastic differentiation. Additionally, the lower lactate level and higher expression of mitochondrial Nd1 in advanced development points to decreasing glycolysis during differentiation. Postnatal nuclear localisation of HIFs indicates a hypoxic state in specific areas of dental pulp as oxygen demands depend on physiological events such as crown and root dentin mineralization.

Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

Repurposing Metformin for periodontal disease management as a form of oral-systemic preventive medicine.

BACKGROUND: Despite the improvements in treatment over the last decades, periodontal disease (PD) affects millions of people around the world and the only treatment available is based on controlling microbial load. Diabetes is known to increase the risk of PD establishment and progression, and recently, glucose metabolism modulation by pharmaceutical or dietarian means has been emphasised as a significant modulator of non-communicable disease development. METHODS: The impact of pharmaceutically controlling glucose metabolism in non-diabetic animals and humans (REBEC, UTN code: U1111-1276-1942) was investigated by repurposing Metformin, as a mean to manage periodontal disease and its associated systemic risk factors. RESULTS: We found that glucose metabolism control via use of Metformin aimed at PD management resulted in significant prevention of bone loss during induced periodontal disease and age-related bone loss in vivo. Metformin also influenced the bacterial species present in the oral environment and impacted the metabolic epithelial and stromal responses to bacterial dysbiosis at a single cell level. Systemically, Metformin controlled blood glucose levels and age-related weight gain when used long-term. Translationally, our pilot randomized control trial indicated that systemic Metformin was safe to use in non-diabetic patients and affected the periodontal tissues. During the medication window, patients showed stable levels of systemic blood glucose, lower circulating hsCRP and lower insulin levels after periodontal treatment when compared to placebo. Finally, patients treated with Metformin had improved periodontal parameters when compared to placebo treated patients. CONCLUSION: This is the first study to demonstrate that systemic interventions using Metformin in non-diabetic individuals aimed at PD prevention have oral-systemic effects constituting a possible novel form of preventive medicine for oral-systemic disease management.

Ift88 regulates enamel formation via involving Shh signaling.

OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

Enhanced bone tissue regeneration with hydrogel-based scaffolds by embedding parathyroid hormone in mesoporous bioactive glass.

OBJECTIVES: To evaluate hydrogel-based scaffolds embedded with parathyroid hormone (PTH)-loaded mesoporous bioactive glass (MBG) on the enhancement of bone tissue regeneration in vitro. MATERIALS AND METHODS: MBG was produced via sol-gel technique followed by PTH solution imbibition. PTH-loaded MBG was blended into the hydrogels and submitted to a lyophilisation process associated with a chemical crosslinking reaction to the production of the scaffolds. Characterisation of the MBG and PTH-loaded MBG scaffolds, including the scanning electron microscope (SEM) connected with an X-ray detector (EDX), Fourier transform infrared (FTIR), compression strength, rheological measurements, swelling and degradation rates, and PTH release analysis, were performed. Also, bioactivity using simulated-body fluid (SBF), biocompatibility (MTT), and osteogenic differentiation analyses (von Kossa and Alizarin Red stainings, and µ-computed tomography, µCT) of the scaffolds were carried out. RESULTS: SEM images demonstrated MBG particles dispersed into the hydrogel-based scaffold structure, which was homogeneously porous and well interconnected. EDX and FTIR revealed large amounts of carbon, oxygen, sodium, and silica in the scaffold composition. Bioactivity experiments revealed changes on sample surfaces over the analysed period, indicating the formation of carbonated hydroxyapatite; however, the chemical composition remained stable. PTH-loaded hydrogel-based scaffolds were biocompatible for stem cells from human-exfoliated deciduous teeth (SHED). A high quantity of calcium deposits on the extracellular matrix of SHED was found for PTH-loaded hydrogel-based scaffolds. µCT images showed MBG particles dispersed into the scaffolds' structure, and a porous, lamellar, and interconnected hydrogel architecture. CONCLUSIONS: PTH-loaded hydrogel-based scaffolds demonstrated consistent morphology and physicochemical properties for bone tissue regeneration, as well as bioactivity, biocompatibility, and osteoinductivity in vitro. Thus, the scaffolds presented here are recommended for future studies on 3D printing. CLINICAL RELEVANCE: Bone tissue regeneration is still a challenge for several approaches to oral and maxillofacial surgeries, though tissue engineering applying SHED, scaffolds, and osteoinductive mediators might help to overcome this clinical issue.

Stem cell contributions to cementoblast differentiation in healthy periodontal ligament and periodontitis.

Loss of tissue attachment as a consequence of bacterial infection and inflammation represents the main therapeutic target for the treatment of periodontitis. Cementoblasts, the cells that produce the mineralized tissue, cementum, that is responsible for connecting the soft periodontal tissue to the tooth, are a key cell type for maintaining/restoring tissue attachment following disease. Here, we identify two distinct stem cell populations that contribute to cementoblast differentiation at different times. During postnatal development, cementoblasts are formed from perivascular-derived cells expressing CD90 and perivascular-associated cells that express Axin2. During adult homeostasis, only Wnt-responsive Axin2+ cells form cementoblasts but following experimental induction of periodontal disease, CD90+ cells become the main source of cementoblasts. We thus show that different populations of resident stem cells are mobilized at different times and during disease to generate precursors for cementoblast differentiation and thus provide an insight into the targeting cells resident cells for novel therapeutic approaches. The differentiation of these stem cells into cementoblasts is however inhibited by bacterial products such as lipopolysaccharides, emphasizing that regeneration of periodontal ligament soft tissue and restoration of attachment will require a multipronged approach.

Expression of periodontitis susceptibility genes in human gingiva using single-cell RNA sequencing.

OBJECTIVE: Single-cell transcriptomics was used to determine the possible cell-type specificity of periodontitis susceptibility genes. BACKGROUND: The last decade has witnessed remarkable advances in the field of human genomics. Despite many advances, the genetic factors associated with or contributing to the periodontitis pathogenesis have only been identified to a limited extent and are often poorly validated. Confirming whether a given single nucleotide polymorphism has an association with periodontitis requires a robust mechanistic explanation on the functional consequences of a given genetic variant. METHODS: We globally assessed the expression of 26 disease-associated genes identified by GWAS within the gingival mucosa. A total of 12 411 cells from 4 different donors were analysed. Differentially expressed genes were analysed using Seurat, a non-parametric Wilcoxon rank sum test. The minimum threshold for significance was defined as p < .05. RESULTS: This exploration at a cellular-level suggests diverse populations contributing to disease pathogenesis, with macrophages expressing a higher number of the analysed disease-associated genes. IL1B, PTGS2, FCGR2A, IL10 and IL1A specifically showed a more restricted expression in the myeloid lineages. CONCLUSION: This short report combines human genetics and single-cell genomics to better understand periodontitis by mapping variants to predict their cells of action and putative functions. These findings seem to suggest that innate cell dysfunction is linked to disease susceptibility.

Weight loss outcomes achieved by adults accessing an online programme offered as part of Public Health England's 'Better Health' campaign.

Effective use of health technology may offer a scalable solution to the obesity pandemic. Online digital programmes provide a convenient and flexible way for more people to access regular support. This service evaluation aims to determine whether adults accessing an online weight management programme via a national campaign are successful in losing weight.Data was analysed for adults registering with Slimming World's online programme using a discounted membership offered as part of PHE's 'Better Health' campaign between July and December 2020. Last-weight carried forward was used to calculate weight outcomes for participants who had the opportunity to complete 12-weeks and recorded ≥ one weight besides baseline. Engagement was determined using number of online weekly weights recorded with high engagers having weight data for ≥ 9 occasions. Socioeconomic status was assessed using postcode data. Resubscription and uploaded weight data were used to determine numbers who continued beyond the offer period.Twenty-seven thousand two hundred forty-eight adults (5.3% males) with mean age 41.0 ± 11.4 years met inclusion criteria. Mean baseline BMI was 33.4 ± 6.8 kg/m2 (29.2% 30-34.9, 18.3% 35-39.9 and 15.1% > 40 kg/m2). Mean weight loss at 12 weeks was 2.7 (± 3) kg representing a mean loss of 3% (± 3.1) body weight with 42.3% achieving ≥ 3% and 22.1% weight loss ≥ 5%. Median number of weigh-ins was six. Men had greater weight losses compared to women (p < 0.001). High engagers, both men and women, achieved greater weight losses (p < 0.001). Absolute weight loss was associated with joining BMI (rs = -0.15, p < 0.001) but for % weight change only small differences were seen (max effect size = 0.03) with no differences in weight change for high engagers between different baseline BMI categories (p > 0.05). 30.9% were in the lowest two IMD quintiles and absolute and percentage weight change did not differ across deprivation quintiles (p > 0.05). 34.9% continued to access the online support after the offer period.This service evaluation shows that an online programme, offered as part of a national campaign, can offer effective support to a large number of people with different starting BMIs and from different socioeconomic backgrounds. An increased level of engagement leads to better weight losses.

Developmental plasticity of epithelial stem cells in tooth and taste bud renewal.

In Lake Malawi cichlids, each tooth is replaced in one-for-one fashion every ∼20 to 50 d, and taste buds (TBs) are continuously renewed as in mammals. These structures are colocalized in the fish mouth and throat, from the point of initiation through adulthood. Here, we found that replacement teeth (RT) share a continuous band of epithelium with adjacent TBs and that both organs coexpress stem cell factors in subsets of label-retaining cells. We used RNA-seq to characterize transcriptomes of RT germs and TB-bearing oral epithelium. Analysis revealed differential usage of developmental pathways in RT compared to TB oral epithelia, as well as a repertoire of genome paralogues expressed complimentarily in each organ. Notably, BMP ligands were expressed in RT but excluded from TBs. Morphant fishes bathed in a BMP chemical antagonist exhibited RT with abrogated shh expression in the inner dental epithelium (IDE) and ectopic expression of calb2 (a TB marker) in these very cells. In the mouse, teeth are located on the jaw margin while TBs and other oral papillae are located on the tongue. Previous study reported that tongue intermolar eminence (IE) oral papillae of Follistatin (a BMP antagonist) mouse mutants exhibited dysmorphic invagination. We used these mutants to demonstrate altered transcriptomes and ectopic expression of dental markers in tongue IE. Our results suggest that vertebrate oral epithelium retains inherent plasticity to form tooth and taste-like cell types, mediated by BMP specification of progenitor cells. These findings indicate underappreciated epithelial cell populations with promising potential in bioengineering and dental therapeutics.

Modulators of Wnt Signaling Pathway Implied in Dentin Pulp Complex Engineering: A Literature Review.

The main goal of vital pulp therapy (VPT) is to preserve the vitality of the pulp tissue, even when it is exposed due to bacterial invasion, iatrogenic mechanical preparation, or trauma. The type of new dentin formed as a result of VPT can differ in its cellular origin, its microstructure, and its barrier function. It is generally agreed that the new dentin produced by odontoblasts (reactionary dentin) has a tubular structure, while the dentin produced by pulp cells (reparative dentin) does not or has less. Thus, even VPT aims to maintain the vitality of the pulp. It does not regenerate the dentin pulp complex integrity. Therefore, many studies have sought to identify new therapeutic strategies to successfully regenerate the dentin pulp complex. Among them is a Wnt protein-based strategy based on the fact that Wnt proteins seem to be powerful stem cell factors that allow control of the self-renewal and proliferation of multiple adult stem cell populations, suitable for homeostasis maintenance, tissue healing, and regeneration promotion. Thus, this review outlines the different agents targeting the Wnt signaling that could be applied in a tooth environment, and could be a potential therapy for dentin pulp complex and bone regeneration.

OFCD syndrome and extraembryonic defects are revealed by conditional mutation of the Polycomb-group repressive complex 1.1 (PRC1.1) gene BCOR.

BCOR is a critical regulator of human development. Heterozygous mutations of BCOR in females cause the X-linked developmental disorder Oculofaciocardiodental syndrome (OFCD), and hemizygous mutations of BCOR in males cause gestational lethality. BCOR associates with Polycomb group proteins to form one subfamily of the diverse Polycomb repressive complex 1 (PRC1) complexes, designated PRC1.1. Currently there is limited understanding of differing developmental roles of the various PRC1 complexes. We therefore generated a conditional exon 9-10 knockout Bcor allele and a transgenic conditional Bcor expression allele and used these to define multiple roles of Bcor, and by implication PRC1.1, in mouse development. Females heterozygous for Bcor exhibiting mosaic expression due to the X-linkage of the gene showed reduced postnatal viability and had OFCD-like defects. By contrast, Bcor hemizygosity in the entire male embryo resulted in embryonic lethality by E9.5. We further dissected the roles of Bcor, focusing on some of the tissues affected in OFCD through use of cell type specific Cre alleles. Mutation of Bcor in neural crest cells caused cleft palate, shortening of the mandible and tympanic bone, ectopic salivary glands and abnormal tongue musculature. We found that defects in the mandibular region, rather than in the palate itself, led to palatal clefting. Mutation of Bcor in hindlimb progenitor cells of the lateral mesoderm resulted in 2/3 syndactyly. Mutation of Bcor in Isl1-expressing lineages that contribute to the heart caused defects including persistent truncus arteriosus, ventricular septal defect and fetal lethality. Mutation of Bcor in extraembryonic lineages resulted in placental defects and midgestation lethality. Ubiquitous over expression of transgenic Bcor isoform A during development resulted in embryonic defects and midgestation lethality. The defects we have found in Bcor mutants provide insights into the etiology of the OFCD syndrome and how BCOR-containing PRC1 complexes function in development.

Ift88 is involved in mandibular development.

The mandible is a crucial organ in both clinical and biological fields due to the high frequency of congenital anomalies and the significant morphological changes during evolution. Primary cilia play a critical role in many biological processes, including the determination of left/right axis patterning, the regulation of signaling pathways, and the formation of bone and cartilage. Perturbations in the function of primary cilia are known to cause a wide spectrum of human diseases: the ciliopathies. Craniofacial dysmorphologies, including mandibular deformity, are often seen in patients with ciliopathies. Mandibular development is characterized by chondrogenesis and osteogenesis; however, the role of primary cilia in mandibular development is not fully understood. To address this question, we generated mice with mesenchymal deletions of the ciliary protein, Ift88 (Ift88fl/fl ;Wnt1Cre). Ift88fl/fl ;Wnt1Cre mice showed ectopic mandibular bone formation, whereas Ift88 mutant mandible was slightly shortened. Meckel's cartilage was modestly expanded in Ift88fl/fl ;Wnt1Cre mice. The downregulation of Hh signaling was found in most of the mesenchyme of Ift88 mutant mandible. However, mice with a mesenchymal deletion of an essential molecule for Hh signaling activity, Smo (Smofl/fl ;Wnt1Cre), showed only ectopic mandibular formation, whereas Smo mutant mandible was significantly shortened. Ift88 is thus involved in chondrogenesis and osteogenesis during mandibular development, partially through regulating Sonic hedgehog (Shh) signaling.

Glial origin of mesenchymal stem cells in a tooth model system.

Mesenchymal stem cells occupy niches in stromal tissues where they provide sources of cells for specialized mesenchymal derivatives during growth and repair. The origins of mesenchymal stem cells have been the subject of considerable discussion, and current consensus holds that perivascular cells form mesenchymal stem cells in most tissues. The continuously growing mouse incisor tooth offers an excellent model to address the origin of mesenchymal stem cells. These stem cells dwell in a niche at the tooth apex where they produce a variety of differentiated derivatives. Cells constituting the tooth are mostly derived from two embryonic sources: neural crest ectomesenchyme and ectodermal epithelium. It has been thought for decades that the dental mesenchymal stem cells giving rise to pulp cells and odontoblasts derive from neural crest cells after their migration in the early head and formation of ectomesenchymal tissue. Here we show that a significant population of mesenchymal stem cells during development, self-renewal and repair of a tooth are derived from peripheral nerve-associated glia. Glial cells generate multipotent mesenchymal stem cells that produce pulp cells and odontoblasts. By combining a clonal colour-coding technique with tracing of peripheral glia, we provide new insights into the dynamics of tooth organogenesis and growth.

Overactivation of the NF-κB pathway impairs molar enamel formation.

OBJECTIVE: Hypohidrotic ectodermal dysplasia (HED) is a hereditary disorder characterized by abnormal structures and functions of the ectoderm-derived organs, including teeth. HED patients exhibit a variety of dental symptoms, such as hypodontia. Although disruption of the EDA/EDAR/EDARADD/NF-κB pathway is known to be responsible for HED, it remains unclear whether this pathway is involved in the process of enamel formation. EXPERIMENTAL SUBJECTS AND METHODS: To address this question, we examined the mice overexpressing Ikkß (an essential component required for the activation of NF-κB pathway) under the keratin 5 promoter (K5-Ikkß). RESULTS: Upregulation of the NF-κB pathway was confirmed in the ameloblasts of K5-Ikkß mice. Premature abrasion was observed in the molars of K5-Ikkß mice, which was accompanied by less mineralized enamel. However, no significant changes were observed in the enamel thickness and the pattern of enamel rods in K5-Ikkß mice. Klk4 expression was significantly upregulated in the ameloblasts of K5-Ikkß mice at the maturation stage, and the expression of its substrate, amelogenin, was remarkably reduced. This suggests that abnormal enamel observed in K5-Ikkß mice was likely due to the compromised degradation of enamel protein at the maturation stage. CONCLUSION: Therefore, we could conclude that the overactivation of the NF-κB pathway impairs the process of amelogenesis.

Characterization of progenitor/stem cell population from human dental socket and their multidifferentiation potential.

Dental stem cells have many applications in medicine, dentistry and stem cell biology in general due to their easy accessibility and low morbidity. A common surgical manoeuvre after a tooth extraction is the dental socket curettage which is necessary to clean the alveolus and favour alveolar bone healing. This procedure can cause very low morbidity compared to bone marrow collection procedures and the collected material is normally discarded. In order to investigate if the tissue obtained by dental socket curettage after a tooth extraction was a feasible alternative source to isolate human stem cells, we isolated and characterized two different stem cell populations based on STRO-1 and CD146 expression. We were able to collect and grow cells from dental socket of vital and non-vital teeth. Both populations were proliferative, clonogenic and expressed STRO-1, CD146, CD90, NG2, PDGFR-ß, which are markers found in stem cells, presented in vitro multiline-differentiation into osteogenic, chondrogenic, and adipogenic tissue, and in vivo transplanted cells formed mineralized tissue. Interestingly, STRO-1+ clonogenic cells presented better multidifferentiation than CD146+ cells. Our results showed that mesenchymal stem cells can be isolated from the tiny tissue collected by dental socket curettage after vital and non-vital tooth extraction and suggest that STRO-1 is an important marker to be used to sort cells with multidifferentiation capacity.

Drug Repurposing in Dentistry; towards Application of Small Molecules in Dentin Repair.

One of the main goals of dentistry is the natural preservation of the tooth structure following damage. This is particularly implicated in deep dental cavities affecting dentin and pulp, where odontoblast survival is jeopardized. This activates pulp stem cells and differentiation of new odontoblast-like cells, accompanied by increased Wnt signaling. Our group has shown that delivery of small molecule inhibitors of GSK3 stimulates Wnt/ß-catenin signaling in the tooth cavity with pulp exposure and results in effective promotion of dentin repair. Small molecules are a good therapeutic option due to their ability to pass across cell membranes and reach target. Here, we investigate a range of non-GSK3 target small molecules that are currently used for treatment of various medical conditions based on other kinase inhibitory properties. We analyzed the ability of these drugs to stimulate Wnt signaling activity by off-target inhibition of GSK3. Our results show that a c-Met inhibitor, has the ability to stimulate Wnt/ß-catenin pathway in dental pulp cells in vitro at low concentrations. This work is an example of drug repurposing for dentistry and suggests a candidate drug to be tested in vivo for natural dentin repair. This approach bypasses the high level of economical and time investment that are usually required in novel drug discoveries.

MicroRNAs control eyelid development through regulating Wnt signaling.

BACKGROUND: The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. RESULTS: It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing; Dicer fl/fl ;Wnt1Cre) leads to the EOB phenotype with full penetrance. Here, we identified that the up-regulation of Wnt signaling resulted in the EOB phenotype in Dicer mutants. Down-regulation of Fgf signaling observed in Dicer mutants was caused by an inverse relationship between Fgf and Wnt signaling. Shh and Bmp signaling were down-regulated as the secondary effects in Dicer fl/fl ;Wnt1Cre mice. Wnt, Shh, and Fgf signaling were also found to mediate the epithelial-mesenchymal interactions in eyelid development. CONCLUSIONS: miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.

Dental mesenchymal stem cells.

Mammalian teeth harbour mesenchymal stem cells (MSCs), which contribute to tooth growth and repair. These dental MSCs possess many in vitro features of bone marrow-derived MSCs, including clonogenicity, expression of certain markers, and following stimulation, differentiation into cells that have the characteristics of osteoblasts, chondrocytes and adipocytes. Teeth and their support tissues provide not only an easily accessible source of MSCs but also a tractable model system to study their function and properties in vivo In addition, the accessibility of teeth together with their clinical relevance provides a valuable opportunity to test stem cell-based treatments for dental disorders. This Review outlines some recent discoveries in dental MSC function and behaviour and discusses how these and other advances are paving the way for the development of new biologically based dental therapies.

Molecular Programming of Perivascular Stem Cell Precursors.

Pericytes have been shown to act as precursors of resident adult stem cells in stromal tissues in vivo. When expanded in vitro these cells are capable of giving rise to multiple mesenchymal cell types, irrespective of their tissue of origin. This phenomenon of multi-lineage differentiation is only observed in culture, whereas in vivo, stromal stem cell differentiation is restricted to tissue-specific cell types. An important unanswered question is how a single, widely distributed cell type (a pericyte) gives rise to stem cells with tissue-specific functions and attributes. Using a combination of transcriptomics and epigenomics we have compared the molecular status of two populations of stromal stem cell precursors. Using a LacZ transgene insertion that is expressed in pericytes but not in stem cells, we were able to compare pericyte populations from two different tissues, mouse incisors and bone marrow. Pericytes, freshly isolated from mouse incisors and bone marrow, exhibited transcriptomes and epigenetic landscapes that were extensively different, reflecting their tissue of origin and future in vivo differentiation potential. Dspp, an odontoblast differentiation gene, as well as additional odontogenic genes, are shown to be expressed in dental pulp-derived pericytes. These genetic loci are also decorated with histone modifications indicative of a transcriptionally active chromatin state. In bone marrow pericytes, a major osteogenic differentiation gene, Runx2, is not expressed but is marked by both active and repressive histones and therefore primed to be expressed. Polycomb repressor complex 1 analysis showed that key genes involved in the induction of adipogenesis, chondrogenesis, and myogenesis are targeted by Ring1b and therefore stably repressed. This indicates that pericyte populations are molecularly obstructed from differentiating down certain lineages in vivo. Stem Cells 2018;36:1890-15.

Disrupted mitochondrial function in the Opa3L122P mouse model for Costeff Syndrome impairs skeletal integrity.

Mitochondrial dysfunction connects metabolic disturbance with numerous pathologies, but the significance of mitochondrial activity in bone remains unclear. We have, therefore, characterized the skeletal phenotype in the Opa3L122P mouse model for Costeff syndrome, in which a missense mutation of the mitochondrial membrane protein, Opa3, impairs mitochondrial activity resulting in visual and metabolic dysfunction. Although widely expressed in the developing normal mouse head, Opa3 expression was restricted after E14.5 to the retina, brain, teeth and mandibular bone. Opa3 was also expressed in adult tibiae, including at the trabecular surfaces and in cortical osteocytes, epiphyseal chondrocytes, marrow adipocytes and mesenchymal stem cell rosettes. Opa3L122P mice displayed craniofacial abnormalities, including undergrowth of the lower mandible, accompanied in some individuals by cranial asymmetry and incisor malocclusion. Opa3L122P mice showed an 8-fold elevation in tibial marrow adiposity, due largely to increased adipogenesis. In addition, femoral length and cortical diameter and wall thickness were reduced, the weakening of the calcified tissue and the geometric component of strength reducing overall cortical strength in Opa3L122P mice by 65%. In lumbar vertebrae reduced vertebral body area and wall thickness were accompanied by a proportionate reduction in marrow adiposity. Although the total biomechanical strength of lumbar vertebrae was reduced by 35%, the strength of the calcified tissue (σmax) was proportionate to a 38% increase in trabecular number. Thus, mitochondrial function is important for the development and maintenance of skeletal integrity, impaired bone growth and strength, particularly in limb bones, representing a significant new feature of the Costeff syndrome phenotype.