Feasibility of Molecularly Targeted Therapy for Tooth Regeneration

[Extract] The tooth is a complex organ that consists of enamel, dentin, cementum, and pulp. Missing teeth is frequently occurring problem in aging populations. To treat these defects, the current approach involves prostheses, autotransplantation, and dental implants. The exploration of new strategies for tooth replacement has become a hot topic. Using the foundations of experimental embryology, developmental and molecular biology, tooth regeneration is becoming realistic possibility. Several different methods have been proposed to achieve biological tooth replacement. These include scaffold-based tooth regeneration, cell pellet engineering, stimulation of the formation of a third dentition, and gene-manipulated tooth regeneration. The idea that a third dentition might be locally induced to replace missing teeth is an attractive concept (Young et al., 2005; Edward & Mason, 2006; Takahashi et al., 2008, 2013). This approach is generally presented in terms of adding molecules to induce de novo tooth initiation in the mouth. Tooth development is the result of reciprocal and reiterative signaling between oral ectoderm-derived dental epithelium and cranial neural crest cell-derived dental mesenchyme under genetic control (Thesleff, 2006). More than 200 genes are known to be expressed during tooth development (http://bite-it.helsinki.fi/). A number of mouse mutants are now starting to provide some insights into the mechanisms of supernumerary tooth formation. Multiple supernumerary teeth may have genetic components in their etiology and partially represent the third dentition in humans. Such candidate molecules might be those that are involved in embryonic tooth induction, in successional tooth formation, or in the control of the number of teeth. This means that it may be possible to induce de novo tooth formation by the in situ repression or activation of a single candidate molecule. In this review, we provide an overview of the collective knowledge of tooth regeneration, especially regarding the control of the number of teeth for molecularly targeted therapy by the stimulation of a third dentition

Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization

RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20 S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20 S637A mouse as a good model for in vivo study

Effects of Usag-1 and Bmp7 deficiencies on murine tooth morphogenesis

[ackground]Wnt5a and Mrfzb1 genes are involved in the regulation of tooth size, and their expression levels are similar to that of Bmp7 during morphogenesis, including during the cap and early bell stages of tooth formation. We previously reported that Usag-1-deficient mice form supernumerary maxillary incisors. Thus, we hypothesized that BMP7 and USAG-1 signaling molecules may play important roles in tooth morphogenesis. In this study, we established double genetically modified mice to examine the in vivo inter-relationships between Bmp7 and Usag-1. [Results]We measured the volume and cross-sectional areas of the mandibular incisors using micro-computed tomography (micro-CT) in adult Bmp7- and Usag-1-LacZ knock-in mice and their F2 generation upon interbreeding. The mandibular incisors of adult Bmp7+/− mice were significantly larger than those of wild-type (WT) mice. The mandibular incisors of adult Usag-1−/− mice were the largest of all genotypes examined. In the F2 generation, the effects of these genes were additive; Bmp7+/− was most strongly associated with the increase in tooth size using generalized linear models, and the total area of mandibular supernumerary incisors of Usag-1−/−Bmp7+/− mice was significantly larger than that ofUsag-1−/−Bmp7 +/+ mice. At embryonic day 15 (E15), BrdU assays demonstrated that the labeling index of Bmp7+/− embryos was significantly higher than that of WT embryos in the cervical loop. Additionally, the labeling index of Usag-1−/− embryos was significantly the highest of all genotypes examined in dental papilla. [Conclusions]Bmp7 heterozygous mice exhibited significantly increased tooth sizes, suggesting that tooth size was controlled by specific gene expression. Our findings may be useful in applications of regenerative medicine and dentistry

歯の発生におけるUSAG-1とRUNX2の拮抗作用

京都大学0048新制・課程博士博士(医学)甲第20078号医博第4171号新制||医||1018(附属図書館)33194京都大学大学院医学研究科医学専攻(主査)教授 妻木 範行, 教授 開 祐司, 教授 松田 秀一学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Morphological analysis of supernumerary teeth in CEBP/beta knock-out mice

Aims: Recently, relationships between CEBP/beta and hard tissue formation such as osteogenesis, chondrogenesis, and odontogenesis have been reported. Regarding osteogenesis, CEBP/beta regulates IGF1, Runx2, osteocalcin and Cdknlc. As for odontogenesis, CEBP/beta is associated with DSPP which influences differentiation of dentinoblasts. Here we report supernumerary teeth in mice with CEBP/beta deficiency, with an approach of morphological analysis.\ud \ud Materials and Methods: The maxilla and mandible of 12-week-old CEBP/beta deficient mice were examined with micro-CT and HE stain methods.\ud \ud Results: Under micro-CT, supernumerary teeth were identified in CEBP/beta deficient mice. The supernumerary teeth were located near the radicular area of upper incisors. Under microscopy, the supernumerary teeth located near upper incisors were also confirmed. The supernumerary teeth showed an evidence of dentine and pulp tissue. This implied to formation of normal dental tissues.\ud \ud Conclusions: Formation of supernumerary teeth was related to CEBP/beta mutation. Because the cause of formation of supernumerary teeth remained unclear, future investigation in mechanisms of CEBP/beta on tooth formation is indicated. In addition, succeeding studies into supernumerary teeth of humans are required

Gene Therapy

The Gene Therapy field is living exciting times after more than 20 years of poor results. Scientist and clinicians working in the gene therapy field have encountered many problems in the past that are now starting to be solved. The development of safer and more efficient gene transfer vectors and the advances on the cell therapy field have open new opportunities to tackle different diseases. The aim of this book is to bring together information about the different gene therapy tools, the clinical successes of gene therapy and the future applications

Antagonistic Functions of USAG-1 and RUNX2 during Tooth Development

Supernumerary teeth and tooth agenesis are common morphological anomalies in humans. We previously obtained evidence that supernumerary maxillary incisors form as a result of the successive development of the rudimentary maxillary incisor tooth germ in Usag-1 null mice. The development of tooth germs is arrested in Runx2 null mice, and such mice also exhibit lingual epithelial buds associated with the upper molars and incisors. The aim of this study is to investigate the potential crosstalk between Usag-1 and Runx2 during tooth development. In the present study, three interesting phenomena were observed in double null Usag-1-/-/Runx2-/- mice: the prevalence of supernumerary teeth was lower than in Usag-1 null mice; tooth development progressed further compared than in Runx2 null mice; and the frequency of molar lingual buds was lower than in Runx2 null mice. Therefore, we suggest that RUNX2 and USAG-1 act in an antagonistic manner. The lingual bud was completely filled with odontogenic epithelial Sox2-positive cells in the Usag-1+/+/Runx2-/- mice, whereas almost no odontogenic epithelial Sox2-positive cells contributed to supernumerary tooth formation in the rudimentary maxillary incisors of the Usag-1-/-/Runx2+/+ mice. Our findings suggest that RUNX2 directly or indirectly prevents the differentiation and/or proliferation of odontogenic epithelial Sox2-positive cells. We hypothesize that RUNX2 inhibits the bone morphogenetic protein (BMP) and/or Wnt signaling pathways regulated by USAG-1, whereas RUNX2 expression is induced by BMP signaling independently of USAG-1

Aldehyded dextran and ε-poly(l-lysine) hydrogel as nonviral gene carrier

The expression term of the gene transfected in cells needs to belong enough inorder to make a gene therapy clinically effective. The controlled release of the transfected gene can be utilized. The new biodegradable hydrogel material created by 20 w/w% aldehyded dextran and 10 w/w% ε-poly(L-lysine) (ald-dex/PLL) was developed. We examined whether it could be as a nonviral carrier of the gene transfer. Methods. A plasmid (Lac-Z) was mixed with ald-dex/PLL. An in vitro study was performed to assess the expression of Lac-Z with X-gal stain after gene transfer into the cultured 293 cells and bone marrow cells. As a control group, PLL was used as a cationic polymer. Results. We confirmed that the transfection efficiency of the ald-dex/PLL had a higher transfection efficiency than PLL in 293 cells (plasmid of 2 μg: ald-dex/PLL 1.1%, PLL 0.23%, plasmid of 16 μg: ald-dex/PLL 1.23%, PLL 0.48%). In bone marrow cells, we confirmed the expression of Lac-Z by changing the quantity of aldehyded dextran. In the groups using ald-dextran of the quantity of 1/4 and 1/12 of PLL, their transfection efficiency was 0.43% and 0.41%, respectively. Conclusions. This study suggested a potential of using ald-dex/PLL as a non-carrier for gene transfer

Application of anti-BMP antibodies to immunohistochemical examination of fibrous dysplasia

Objective: Fibrous dysplasia of bone (FD) is a benign developmental disorder of bone in which normal bone is replaced by fibrous tissue, containing trabeculae of immature woven bone. This disease has been classified into three types: monostotic (MFD), polyostotic (PFD), and McCune–Albright syndrome (MAS). Occurring in multiple adjacent craniofacial bones (craniofacial FD) is considered to be an MFD lesion. Surgical reduction is performed for form revision after having observed the progress until adulthood, but postoperative recurrence cases are not rare, there is a report of malignant progression to osteosarcoma, and the clinical phenotypes are generally various. This study aimed to identify the relationship between clinical futures of FD and expression of bone morphogenetic protein (BMP) subtypes.\ud \ud Methods: We studied 10 cases (9 MFD cases and 1 McCune–Albright syndrome case) diagnosed with fibrous dysplasia in our hospital, and immunohistochemical examinations of the excised sample from each case with anti-BMP-2, 4, 6, and 7 antibodies were performed.\ud \ud Results: 5 MFD cases and 1 MAS case underwent reoperation, and the case that had the most number of operations was 9 times in the MFD case. Malignant transformations were not identified. BMP-4, 6, and 7 expressions were positive in all cases, but the expression of BMP-2 was positive only in 6 MFD cases. BMP-2 negative cases tended to undergo reoperation.\ud \ud Conclusions: The variable expression of BMP-2 demonstrated in the current study was suggested to be a useful indication for clinical activity and convalescence of the lesions

Prospective signs of cleidocranial dysplasia in Cebpb deficiency

Background: Although runt-related transcription factor 2 (RUNX2) has been considered a determinant of cleidocranial dysplasia (CCD), some CCD patients were free of RUNX2 mutations. CCAAT/enhancer-binding protein beta (Cebpb) is a key factor of Runx2 expression and our previous study has reported two CCD signs including hyperdontia and elongated coronoid process of the mandible in Cebpb deficient mice. Following that, this work aimed to conduct a case-control study of thoracic, zygomatic and masticatory muscular morphology to propose an association between musculoskeletal phenotypes and deficiency of Cebpb, using a sample of Cebpb-/-, Cebpb+/- and Cebpb+/+ adult mice. Somatic skeletons and skulls of mice were inspected with soft x-rays and micro-computed tomography (μCT), respectively. Zygomatic inclination was assessed using methods of coordinate geometry and trigonometric function on anatomic landmarks identified with μCT. Masseter and temporal muscles were collected and weighed. Expression of Cebpb was examined with a reverse transcriptase polymerase chain reaction (RT-PCR) technique.\ud \ud Results: Cebpb-/- mice displayed hypoplastic clavicles, a narrow thoracic cage, and a downward tilted zygomatic arch (p < 0.001). Although Cebpb+/- mice did not show the phenotypes above (p = 0.357), a larger mass percentage of temporal muscles over masseter muscles was seen in Cebpb+/- littermates (p = 0.012). The mRNA expression of Cebpb was detected in the clavicle, the zygoma, the temporal muscle and the masseter muscle, respectively.\ud \ud Conclusions: Prospective signs of CCD were identified in mice with Cebpb deficiency. These could provide an additional aetiological factor of CCD. Succeeding investigation into interactions among Cebpb, Runx2 and musculoskeletal development is indicated