Fully functional bioengineered tooth replacement as an organ replacement therapy

Abstract

Current approaches to the development of regenerative therapies have been influenced by our understanding of embryonic development, stem cell biology, and tissue engineering technology. The ultimate goal of regenerative therapy is to develop fully functioning bioengineered organs which work in cooperation with surrounding tissues to replace organs that were lost or damaged as a result of disease, injury, or aging. Here, we report a successful fully functioning tooth replacement in an adult mouse achieved through the transplantation of bioengineered tooth germ into the alveolar bone in the lost tooth region. We propose this technology as a model for future organ replacement therapies. The bioengineered tooth, which was erupted and occluded, had the correct tooth structure, hardness of mineralized tissues for mastication, and response to noxious stimulations such as mechanical stress and pain in cooperation with other oral and maxillofacial tissues. This study represents a substantial advance and emphasizes the potential for bioengineered organ replacement in future regenerative therapies.

Fig. 1.

Eruption and occlusion of a bioengineered tooth. (A) Schematic representation of the transplantation technology used for the generation of reconstituted tooth germ. (B) Phase contrast image of bioengineered tooth germ on day 5 of an organ culture. (Scale bar, 200 μm.) (C) Oral photographs of a bioengineered tooth during eruption and occlusion processes, including before eruption (Left), immediately after eruption (Center), and full occlusion (Right). (Scale bar, 200 μm.) (D) Histological analysis of the bioengineered tooth during the eruption and occlusion processes, including before eruption (Left), immediately after eruption (Center), and full occlusion (Right). (Scale bar, 100 μm.) (E) Oral photograph of a bioengineered tooth reconstituted using a combination of epithelial cells from normal mice and mesenchymal cells from GFP-transgenic mice (GFP bioengineered tooth). A merged image is shown. (Scale bar, 200 μm.) (F) A sectional image of a GFP bioengineered tooth. Fluorescent and DIC images are merged. (Scale bar, 100 μm.) (G) Oral photographs showing occlusion of normal (Upper) and bioengineered (Lower) teeth. (Scale bar, 200 μm.) (H) MicroCT images of the occlusion of normal (Left) and bioengineered (Right) teeth. External (Left) and cross section (Right) images are shown. The bioengineered tooth is indicated by the arrowhead.

Fig. 2.

Assessment of the hardness of the bioengineered tooth. Knoop microhardness values of the enamel (Left) and dentin (Right) of the bioengineered tooth at 11-weeks post transplantation were compared with those of normal teeth from 3- and 9-week-old mice. Error bars show the standard deviation (n = 3). P < 0.001 (*) and <0.0001 (**) was regarded as statistically significant (t test).

Fig. 3.

Experimental tooth movement. (A) Horizontal sections of the root of a normal tooth (Upper) and a bioengineered tooth (Lower) were analyzed by hematoxylin-eosin staining (HE) at days 0 (Left), 6 (Center), and 17 (Right) of experimental orthodontic treatment. (Scale bar, 100 μm.) (B) Sections of a normal and bioengineered tooth were analyzed by TRAP staining and in situ hybridization of Ocn at day 6 of the orthodontic treatment. TRAP-positive cells (arrow) and Ocn mRNA-positive cells (arrowhead) are indicated. (Scale bar, 100 μm.) (C) The root of the bioengineered tooth was analyzed for bone formation. The image in the box in Left is shown at higher magnification in Right. Tetracycline (arrowhead) and calcein (arrow) labeling was detectable on the tension side. (Scale bar, 50 μm.)

Fig. 4.

Pain response to mechanical stress. (A) Nerve fibers in the pulp and PDL in the normal (Upper) and bioengineered (Lower) tooth were analyzed immunohistochemically using specific antibodies for the combination (left 2 columns) of NF (green) and NPY (red) and the combination (right 2 columns) of NF and CGRP (red). (Scale bar, 25 μm.) (B) Analysis of galanin immunoreactivity in the PDL of a normal (Upper) and bioengineered (Lower) tooth for the assessment of orthodontic force. No galanin expression was evident in the untreated tooth (Left). Galanin expression (arrowhead) was detected in the PDL of a normal and bioengineered tooth after 48 h of orthodontic treatment (Right). (Scale bar, 25 μm.) (C) Analysis of c-Fos-immunoreactivity in the medullary dorsal horn of mice with a normal tooth (Upper) or a bioengineered tooth (Lower) after 0 h (Left), 2 h (Center), and 48 h (Right) of orthodontic treatment. c-Fos expression (arrowhead) was also detected. (Scale bar, 50 μm.) (D) Analysis of c-Fos immunoreactivity in the medullary dorsal horn of mice with a normal tooth (Upper) or a bioengineered tooth (Lower) after 0 h (Left), 2 h (Center), and 48 h (Right) of stimulation by pulp exposure. c-Fos expression (arrowhead) was evident in the medullary dorsal horn after 2 and 48 h of pulp exposure. (Scale bar, 50 μm.) D, dentin; P, pulp; B, bone; PDL, periodontal ligament; T, spinal trigeminal tract.