Toshiyuki Yoshida

Cell lineage in mammalian craniofacial mesenchyme

We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells.

Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model.

Cytotherapeutic approaches have been investigated to overcome the limitations of existing procedures for periodontal regeneration. In this study, cell sheet transplantation was performed using three kinds of mesenchymal tissue (periodontal ligament, alveolar periosteum, and bone marrow)-derived cells to compare the differences between cell sources in a canine severe defect model (one-wall intrabony defect). Periodontal ligament cells (PDLCs), iliac bone marrow mesenchymal stromal cells (BMMSCs), and alveolar periosteal cells (APCs) were obtained from each dog; a total of four dogs were used. Three-layered cell sheets of each cell source supported with woven polyglycolic acid were autologously transplanted to the denuded root surface. One-wall intrabony defects were filled with a mixture of β-tricalcium phosphate (β-TCP) and collagen. Eight weeks after the transplantation, periodontal regeneration was significantly observed with both newly formed cementum and well-oriented PDL fibers more in the PDLC group than in the other groups. In addition, nerve filament was observed in the regenerated PDL tissue only in the PDLC group. The amount of alveolar bone regeneration was highest in the PDLC group, although it did not reach statistical significance among the groups. These results indicate that PDLC sheets combined with β-TCP/collagen scaffold serve as a promising tool for periodontal regeneration.

Cell sheet engineering and its application for periodontal regeneration

Periodontitis is a inflammation induced by a bacterial infection that causes the destruction of the attachment apparatus of dental roots. Several materials, such as bone graft materials, barrier membranes and protein products have been developed and used to treat periodontal defects clinically; however, it is difficult to regenerate the complete periodontal tissue structure. Recently, cytotherapeutic approaches have been introduced to overcome the limitation of conventional procedures. The in vitro‐expanded autologous cells derived from several kinds of tissues have already been used in several clinical trials. These cytotherapeutic treatments have been shown to be safe and effective for the treatment of periodontitis. Our strategy has been to integrate stem cell biology and cell sheet engineering, in which a temperature‐responsive intelligent polymer is grafted onto the surface of cell culture dish to create a ‘cell sheet’, to achieve a novel treatment method for periodontitis. By simple reduction of the temperature to below 32°C, a contiguous cell sheet, which is capable of keeping extracellular matrix proteins and cell–cell interactions intact, can be harvested for transplantation without the use of scaffolds. This technology has already been employed in clinical trials, confirming the safety and efficacy of the treatment. In this review, we introduce recent progress in the engineering of cell sheets and review the potential of cell sheet technology for periodontal regenerative medicine. Copyright

Current status and future development of cell transplantation therapy for periodontal tissue regeneration.

It has been shown that stem cell transplantation can regenerate periodontal tissue, and several clinical trials involving transplantation of stem cells into human patients have already begun or are in preparation. However, stem cell transplantation therapy is a new technology, and the events following transplantation are poorly understood. Several studies have reported side effects and potential risks associated with stem cell transplantation therapy. To protect patients from such risks, governments have placed regulations on stem cell transplantation therapies. It is important for the clinicians to understand the relevant risks and governmental regulations. This paper describes the ongoing clinical studies, basic research, risks, and governmental controls related to stem cell transplantation therapy. Then, one clinical study is introduced as an example of a government-approved periodontal cell transplantation therapy.

Twist is required for establishment of the mouse coronal suture

Cranial sutures are the growth centres of the skull, enabling expansion of the skull to accommodate rapid growth of the brain. Haploinsufficiency of the human TWIST gene function causes the craniosynostosis syndrome, Saethre–Chotzen syndrome (SCS), in which premature fusion of the coronal suture is a characteristic feature. Previous studies have indicated that Twist is expressed in the coronal suture during development, and therefore that it may play an important role in development and maintenance of the suture. The Twist‐null mouse is lethal before the onset of osteogenesis, and the heterozygote exhibits coronal suture synostosis postnatally. In this study we investigated the function of Twist in the development of the mouse coronal suture, by inhibiting Twist synthesis using morpholino antisense oligonucleotides in calvarial organ culture. Decreased Twist production resulted in a narrow sutural space and fusion of bone domains within 48 h after the addition of the morpholino oligonucleotides. Proliferation activity in the sutural cells was decreased, and the expression of osteogenic marker genes such as Runx2 and Fgfr2 was up‐regulated in the developing bone domain within 4 h. These results suggest that during establishment of the suture area, Twist is required for the regulation of sutural cell proliferation and osteoblast differentiation.

Cooperation of nectin‐1 and nectin‐3 is required for normal ameloblast function and crown shape development in mouse teeth

Nectins are immunoglobulin‐like cell adhesion proteins and their interactions recruit various cell–cell junctions. Mutations in human NECTIN‐1 cause an ectodermal dysplasia syndrome, but Nectin‐1 null mice have only slight defects in teeth, suggesting compensation by other nectin(s). We observed overlapping expression of nectin‐3 with nectin‐1 and enamel abnormality in the nectin‐3 mutant. We, therefore, generated nectin‐1;nectin‐3 compound mutants. However, all teeth developed and no significant dental abnormalities were observed before birth. At postnatal day 10, the upper molars of compound mutants exhibited conical crown shape and retarded enamel maturation. Nectin‐1 was expressed in ameloblasts whereas nectin‐3 was expressed in neighboring stratum intermedium cells at this stage. The immunohistochemical localization and electron microscopical observations indicated that the desmosomal junctions between stratum intermedium and ameloblasts were significantly reduced. These results suggest that heterophilic interaction between nectin‐1 and nectin‐3 recruits desmosomal junctions, and that these are required for proper enamel formation. Developmental Dynamics 239:2558–2569, 2010.

Endothelial cells enhance the in vivo bone-forming ability of osteogenic cell sheets

Addressing the problem of vascularization is of vital importance when engineering three-dimensional (3D) tissues. Endothelial cells are increasingly used in tissue-engineered constructs to obtain prevascularization and to enhance in vivo neovascularization. Rat bone marrow stromal cells were cultured in thermoresponsive dishes under osteogenic conditions with human umbilical vein endothelial cells (HUVECs) to obtain homotypic or heterotypic cell sheets (CSs). Cells were retrieved as sheets from the dishes after incubation at 20 °C. Monoculture osteogenic CSs were stacked on top of homotypic or heterotypic CSs, and subcutaneously implanted in the dorsal flap of nude mice for 7 days. The implants showed mineralized tissue formation under both conditions. Transplanted osteogenic cells were found at the new tissue site, demonstrating CS bone-inductive effect. Perfused vessels, positive for human CD31, confirmed the contribution of HUVECs for the neovascularization of coculture CS constructs. Furthermore, calcium quantification and expression of osteocalcin and osterix genes were higher for the CS constructs, with HUVECs demonstrating the more robust osteogenic potential of these constructs. This work demonstrates the potential of using endothelial cells, combined with osteogenic CSs, to increase the in vivo vascularization of CS-based 3D constructs for bone tissue engineering purposes.

Critical role for αvβ6 integrin in enamel biomineralization

Summary Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with the ameloblast plasma membrane. Although it is known that integrins mediate cell–matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. We hypothesized that &agr;v&bgr;6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both &bgr;6 integrin mRNA and protein. The maxillary incisors of Itgb6−/− mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6−/− mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6−/− enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6−/− mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, in which &agr;v&bgr;6 integrin was not an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin &agr;v&bgr;6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition and/or turnover and subsequent enamel biomineralization.

Allogeneic Transplantation of Periodontal Ligament-Derived Multipotent Mesenchymal Stromal Cell Sheets in Canine Critical-Size Supra-Alveolar Periodontal Defect Model

Abstract Periodontitis is a chronic inflammatory disease that induces the destruction of tooth-supporting tissues, followed by tooth loss. Although several approaches have been applied to periodontal regeneration, complete periodontal regeneration has not been accomplished. Tissue engineering using a combination of cells and scaffolds is considered to be a viable alternative strategy. We have shown that autologous transplantation of periodontal ligament-derived multipotent mesenchymal stromal cell (PDL-MSC) sheets regenerates periodontal tissue in canine models. However, the indications for autologous cell transplantation in clinical situations are limited. Therefore, this study evaluated the safety and efficacy of allogeneic transplantation of PDL-MSC sheets using a canine horizontal periodontal defect model. Canine PDL-MSCs were labeled with enhanced green fluorescent protein (EGFP) and were cultured on temperature-responsive dishes. Three-layered cell sheets were transplanted around denuded root surfaces either autologously or allogeneically. A mixture of β-tricalcium phosphate and collagen gel was placed on the bone defects. Eight weeks after transplantation, dogs were euthanized and subjected to microcomputed tomography and histological analyses. RNA and DNA were extracted from the paraffin sections to verify the presence of EGFP at the transplantation site. Inflammatory markers from peripheral blood sera were quantified using an enzyme-linked immunosorbent assay. Periodontal regeneration was observed in both the autologous and the allogeneic transplantation groups. The allogeneic transplantation group showed particularly significant regeneration of newly formed cementum, which is critical for the periodontal regeneration. Serum levels of inflammatory markers from peripheral blood sera showed little difference between the autologous and allogeneic groups. EGFP amplicons were detectable in the paraffin sections of the allogeneic group. These results suggest that allogeneic PDL-MSC sheets promoted periodontal tissue regeneration without side effects. Therefore, allogeneic transplantation of PDL-MSC sheets has a potential to become an alternative strategy for periodontal regeneration.

Promotion of mouse ameloblast proliferation by Lgr5 mediated integrin signaling

Rodent incisors grow throughout the animals lives, and the tooth‐forming cells are provided from proximal ends of the incisors where the tooth epithelium forms a stem cell niche called cervical loop. The committing cells in a cervical loop actively begin to proliferate (pre‐ameloblasts), and differentiating into ameloblasts. This study showed that the lower incisors of mice null for CD61 (CD61−/−), also known as integrin β3, were significantly shorter than those of the wild‐type mice at 8‐week‐old. The protein and mRNA expressions levels of Fgfr2, Lgr5, and Notch1, which are known to be involved in pre‐ameloblastic cell proliferation and stem cell maintenance, were reduced in the cervical loop of 2‐week‐old CD61−/− mice. The proliferation of pre‐ameloblasts was reduced in CD61−/− ameloblasts. The siRNA‐mediated suppression of CD61 (siCD61) reduced the proliferation of pre‐ameloblastic cell line ALC, and the expression levels of Lgr5 and Notch1 were reduced by the transfection with siCD61. The suppression of Lgr5 by transfection with siLgr5 suppressed the proliferation of the ALC cells. These results suggested that CD61 signaling is required for the proper growth of the cervical loop and for the promotion of the proliferation of pre‐ameloblastic cells through Lgr5. J. Cell. Biochem. 114: 2138–2147, 2013.