Kei Nakajima

Functional tooth restoration by next-generation bio-hybrid implant as a bio-hybrid artificial organ replacement therapy

Bio-hybrid artificial organs are an attractive concept to restore organ function through precise biological cooperation with surrounding tissues in vivo. However, in bio-hybrid artificial organs, an artificial organ with fibrous connective tissues, including muscles, tendons and ligaments, has not been developed. Here, we have enveloped with embryonic dental follicle tissue around a HA-coated dental implant, and transplanted into the lower first molar region of a murine tooth-loss model. We successfully developed a novel fibrous connected tooth implant using a HA-coated dental implant and dental follicle stem cells as a bio-hybrid organ. This bio-hybrid implant restored physiological functions, including bone remodelling, regeneration of severe bone-defect and responsiveness to noxious stimuli, through regeneration with periodontal tissues, such as periodontal ligament and cementum. Thus, this study represents the potential for a next-generation bio-hybrid implant for tooth loss as a future bio-hybrid artificial organ replacement therapy.

Saliva secretion in engrafted mouse bioengineered salivary glands using taste stimulation

PURPOSE The aim of this study was to compare saliva flow and protein composition induced using five basic taste stimulations between natural and bioengineered salivary glands. MATERIALS AND METHODS We developed a mouse saliva secretion model using taste stimulation and analyzed the saliva secretion from natural and bioengineered salivary glands using an assay. The protein components and alpha-amylase in the natural and bioengineered saliva were analyzed by gel electrophoresis and Western blotting. RESULTS The salivary flow responses induced by sour (citric acid) and bitter (quinine-HCl) stimuli were significantly high in the natural and bioengineered salivary glands. Although the protein concentrations in the natural and bioengineered saliva induced using five basic taste stimulations were similar, the protein composition and the amylase concentration in the natural saliva after taste stimulation had different profiles. Sympathetic and non-sympathetic nerves were observed around the acini and ducts in the natural and bioengineered salivary glands. However, the frequency of neuropeptide Y-positive sympathetic nerves in the bioengineered gland was relatively high compared to that in the natural gland. CONCLUSIONS These results suggest that the signal balance between the sympathetic and parasympathetic components of the efferent nerves in an engrafted bioengineered salivary gland may differ from that in a natural salivary gland.

Development of a Functional Biohybrid Implant Formed from Periodontal Tissue Utilizing Bioengineering Technology.

Current osseointegrated dental implants have been widely used for the rehabilitation of tooth loss. Although dental implants are considered an available treatment in the paradigm shift from traditional dental therapies, such as fixed dental bridges and removable dentures, the fundamental problems must be overcome before their clinical use in young patients who are still undergoing jawbone growth. Here, we show a novel bioengineering method for a functional biohybrid implant that is combined with adult-derived periodontal tissue and attached with bone tissue as a substitute for cementum. This biohybrid implant was successfully engrafted using the bioengineered periodontal ligament, and it restored physiological function, such as orthodontic movement through bone remodeling and appropriate responsiveness to noxious stimuli. Thus, this study represents the functional biohybrid implants potential for clinical use as a next-generation dental implant using adult-derived tissues.

Functional tooth restoration utilising split germs through re-regionalisation of the tooth-forming field

The tooth is an ectodermal organ that arises from a tooth germ under the regulation of reciprocal epithelial-mesenchymal interactions. Tooth morphogenesis occurs in the tooth-forming field as a result of reaction-diffusion waves of specific gene expression patterns. Here, we developed a novel mechanical ligation method for splitting tooth germs to artificially regulate the molecules that control tooth morphology. The split tooth germs successfully developed into multiple correct teeth through the re-regionalisation of the tooth-forming field, which is regulated by reaction-diffusion waves in response to mechanical force. Furthermore, split teeth erupted into the oral cavity and restored physiological tooth function, including mastication, periodontal ligament function and responsiveness to noxious stimuli. Thus, this study presents a novel tooth regenerative technology based on split tooth germs and the re-regionalisation of the tooth-forming field by artificial mechanical force.

Dental pulp cells promote the expression of receptor activator of nuclear factor-κB ligand, prostaglandin E2 and substance P in mechanically stressed periodontal ligament cells

OBJECTIVE This study investigated the expression of receptor activator of nuclear factor-κB ligand (RANKL) in periodontal ligament (PDL) cells co-cultured with dental pulp (DP) cells following mechanical stress in vitro. Furthermore, the expression of prostaglandin (PG) E2 and substance P (SP) by the PDL cells and by the DP cells were also examined. DESIGN PDL and DP cells were obtained from 10 rats. The experimental group consisted of PDL cells subjected to centrifugal force as mechanical stress and co-cultured with DP cells. The 3 control groups of PDL cells were: 1) PDL cells without mechanical stress, 2) PDL cells treated with mechanical stress and 3) PDL cells co-cultured with DP cells. The 2 control groups of DP cells were: 1) DP cells without mechanical stress and 2) DP cells co-cultured with PDL cells. In each group, both cells were examined at day 1 and day 3, and mRNA levels of RANKL by PDL cells were analyzed using Real time quantitative Reverse Transcription (RT)-PCR. Furthermore, RANKL expression was observed using Immunofluorescence staining. PGE2 and SP expression levels by PDL cells and DP cells were characterized by ELISA analysis. RESULTS The expression of RANKL by PDL cells under mechanical stress increased by co-culture with DP cells. PGE2 and SP expressions were increased in the group of PDL cells subjected to mechanical stress and co-cultured with DP cells. CONCLUSION DP cells may facilitate the expression of RANKL in PDL cells under mechanical stress via PGE2 and SP.

Congenital Epulis: A Case and Review of the Literature

Congenital epulis is an unusual benign oral mucosal lesion in newborns with no tendency to recur after excision. The histogenesis of the lesion is unknown, but it is believed to be of mesenchymal origin. We describe a case of congenital epulis (20×10 mm) in the mandibular gingiva of a newborn. The mass, which was smooth-surfaced and pedunculated with a healthy color, was surgically removed at 5 months post-birth. Histologically, the tumor consisted mainly of large eosinophilic granular cells. Immunohistochemical studies revealed intense staining for vimentin, STRO-1, and CD44, suggesting that it was derived from mesenchymal cells. The literature and immunohistochemical profile of congenital epulis are also discussed.

Differentiation behavior of iPS cells cultured on PLGA with osteoinduction medium

In the present report, we have generated osteoblast-like cells derived from mouse induced-pluripotent stem (iPS) cells on PLGA with osteoinduction medium in vitro and in vivo. The cell culture period was 2 weeks. At 2 weeks, mRNA level of type I collagen was significantly higher than at 1 week. Osteocalcin mRNA level at 2 weeks was tendency to increase compared with at 1 week. And the cells cultured on PLGA were positive for immunofluorescent staining of osteocalcin and alizarin red S staining. The scaffold and osteogenic-like cells induced in vitro were implanted subcutaneously into SCID mice. In resected teratoma, hard tissues resembling bone were observed mixed with other tissues on the scaffold. The sum of these findings suggests that PLGA does not disturb the osteogenesis of iPS cells.

Effects of CO2 Lasers on Dental Pulp Biology in Rats

OBJECTIVE The purpose of this study was to investigate the effects of CO2 lasers on the proliferation and differentiation of dental pulp cells, and their latent self-recovery in connection with their stemness using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. MATERIALS AND METHODS The first molars from male Sprague-Dawley rats, each weighing ∼150-200 g, were used for this study. The upper first molars were irradiated with a 10,600 nm wavelength CO2 laser under identical parameters (2 W CO2 laser, energy 4J, energy density 203.84 J/cm(2) for 8.8 sec) through the dentin of the occlusal surface. The molars were extracted immediately, or at 1, 3 or 5 days after the laser irradiation. RT-PCR analysis using primers specific for heat shock protein 70 (Hsp70), adenosine triphosphate (ATP)-binding cassette transporter G2 (ABCG2), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP1), and immunohistochemistry using antibodies specific for proliferating cell nuclear antigen (PCNA), ABCG2, CD34, and CD44 were performed. RESULTS RT-PCR analysis revealed that Hsp70 mRNA expression in the immediate group and ABCG2 mRNA expression at day 1 were the highest. DSPP and DMP1 mRNA expression in the laser-irradiated groups increased gradually, reaching its peak on the 5th day of the experiment, although no significant difference found among groups with regard to DMP1 expression. Immunohistochemically, PCNA-positive cells were observed at all times after the laser irradiation; however, they were most evident on day 3. CD44-positive cells were observed strongly on day 1 and day 3, while ABCG2-positive cells were the most evident on day 3. CONCLUSIONS These results demonstrate that CO2 laser irradiation induces degeneration in the pulp tissue, which is then repaired by newly formed odontoblast-like cells.

Whole Tooth Regenerative Therapy Using a Bioengineered Tooth Germ

Dental disorders, including dental caries and periodontal disease, can cause fundamental problems for oral functions, such as enunciation, mastication and occlusion, as well as general health issues. Tooth regenerative therapies for tissue repair and whole tooth replacement are currently being developed as novel treatment approaches. As a form of bioengineered organ replacement, whole tooth replacement therapy is considered an important model system for next-generation regenerative therapy. We recently reported bioengineered tooth replacements after transplantation of a bioengineered tooth germ or mature tooth unit comprising the bioengineered tooth and periodontal tissues. Whole tooth regenerative therapy has the potential to fully restore tooth function, including masticatory potential in response to mechanical stress and perceptive potential for noxious stimulation. In this review, we describe recent findings and technologies underpinning tooth regenerative therapy.