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

Local application of Usag-1 siRNA can promote tooth regeneration in Runx2-deficient mice

Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis

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

Increased Risk of Temporomandibular Joint Closed Lock: A Case-Control Study of ANKH Polymorphisms

Objectives: This study aimed to carry out a histological examination of the temporomandibular joint (TMJ) in ank mutant mice and to identify polymorphisms of the human ANKH gene in order to establish the relationship between the type of temporomandibular disorders (TMD) and ANKH polymorphisms.\ud \ud Materials and Methods: Specimens from the TMJ of ank mutant and wild-type mice were inspected with a haematoxylin and eosin staining method. A sample of 55 TMD patients were selected. Each was examined with standard clinical procedures and genotyping techniques.\ud \ud Results: The major histological finding in ank mutant mice was joint space narrowing. Within TMD patients, closed lock was more prevalent among ANKH-OR homozygotes (p = 0.011, OR = 7.7, 95% CI 1.6–36.5) and the elder (p = 0.005, OR = 2.4, 95% CI 1.3–4.3).\ud \ud Conclusions: Fibrous ankylosis was identified in the TMJ of ank mutant mice. In the human sample, ANKH-OR polymorphism was found to be a genetic marker associated with TMJ closed lock. Future investigations correlating genetic polymorphism to TMD are indicated

BMP-7とUSAG-1との相互作用による歯数制御に関する機能解析

Kiso H, Takahashi K, Saito K, Togo Y, Tsukamoto H, et al. (2014) Interactions between BMP-7 and USAG-1 (Uterine Sensitization-Associated Gene-1) Regulate Supernumerary Organ Formations. PLoS ONE 9(5): e96938.京都大学0048新制・課程博士博士(医学)甲第18545号医博第3938号新制||医||1006(附属図書館)31445京都大学大学院医学研究科医学専攻(主査)教授 斎藤 通紀, 教授 戸口田 淳也, 教授 妻木 範行学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Id2 acts downstream of BMP signaling in chondrogenesis

Objectives: This study aimed to conduct a case-control investigation in maxilofacial morphogenesis, using a sample of Id2 homozygous knockout (KO) and wild-type (WT) mice. A special interest was to establish a relationship among Id2, BMP signals and chondrogenesis.\ud \ud Materials and Methods: Appropriate ethics approval has been received. Crania collected from mice at the age of 0 day, 2 weeks and 12 weeks were assessed with a Euclidean Distance Matrix Analysis method. Cultured synchondroses of the murine cranial base were inspected with a histological approach and examined with a reverse transcription polymerase chain reaction technique.\ud \ud Results: A shorter nasofrontal profile was seen in Id2 KO 12-week-olds but not 0-day-olds. The WT-KO gap in the skull length instead of the width increased with age. KO 2-week-olds showed a narrower hypertrophic zone and an inhibited proliferative zone in the presphenoid and the spheno-occipital synchondroses. Id2 expression in WT synchondroses was identified. Distribution of Type X collagen other than osteopontin was downregulated in Id2 KO samples. The WT murine cranial base showed a wider hypertrophic zone, a higher degree of ectopic hypertrophy and a larger number of proliferative chondrocytes in the presence of exogenous BMP2, BMP4 and BMP7, respectively. Such acquired growth was not detected in KO subjects. A 5-fold upregulation of Smad7 transcripts and a decrease in phosphorylation of Smad1-Smad5-and-Smad8-positive cells were identified in Id2 KO cartilage.\ud \ud Conclusions: Postnatal chondrogenesis was related to Id2 that acted downstream to enhance BMP signals by inhibiting Smad7 expression

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

ANKH polymorphisms and clicking of the temporomandibular joint in dental residents

Aim\ud \ud This study aimed to carry out a case-control research study to assess occurrence of clicking of the temporomandibular joint (TMJ) in order to establish the relationship between TMJ clicking and the genotype of "ANKH inorganic pyrophosphate transport regulator" (ANKH) polymorphisms.\ud \ud Materials and Method\ud \ud A sample of 41 first-year dental residents was selected. Each was examined using standard\ud clinical procedures and genotyping techniques.\ud \ud Results\ud \ud The participation rate was 91.8 %. The prevalence of TMJ clicking was 51.2 % (95 % CI:\ud 35.7–66.7 %). Occurrence of TMJ clicking was not related to age, gender and genotypes of ANKH-OR as well as ANKH-TR polymorphisms (p ≥ 0.165).\ud \ud Conclusion\ud \ud A similar distribution of ANKH genotypes in TMJ clicking and asymptomatic individuals has been\ud demonstrated by this study. A high percentage of TMJ clicking has been confirmed. Future investigations are indicated

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