Biological synthesis of tooth enamel instructed by an artificial matrix

Abstract

The regenerative capability of enamel, the hardest tissue in the vertebrate body, is fundamentally limited due to cell apoptosis following maturation of the tissue. Synthetic strategies to promote enamel formation have the potential to repair damage, increase the longevity of teeth and improve the understanding of the events leading to tissue formation. Using a self-assembling bioactive matrix, we demonstrate the ability to induce ectopic formation of enamel at chosen sites adjacent to a mouse incisor cultured in vivo under the kidney capsule. The resulting material reveals the highly organized, hierarchical structure of hydroxyapatite crystallites similar to native enamel. This artificially triggered formation of organized mineral demonstrates a pathway for developing cell fabricated materials for treatment of dental caries, the most ubiquitous disease in man. Additionally, the artificial matrix provides a unique tool to probe cellular mechanisms involved in tissue formation further enabling the development of tooth organ replacements.

Figure 1. Characterization of peptide amphiphile assembly

Chemical structures of (A) the branched RGDS-bearing peptide amphiphile (bRGDS PA) and (B) a scrambled control molecule (Scr bRGDS PA) that has a charge matched, but altered amino acid sequence to disrupt bioactivity. Cryogenic TEM images of the (C) bRGDS PA and the (D) Scr bRGDS PA demonstrate the formation of cylindrical micelles at 1% (w/v) in phosphate buffered saline.

Figure 2. Animal model for injection of peptide amphiphiles into mouse incisors

Peptide amphiphiles are injected adjacent to the ameloblasts of 1-day postnatal dental incisors, explanted to the renal capsule for selected times and recovered for analysis. (A) 1-day postnatal dental incisor injected with bRGDS PA mixed with a rhodamine containing PA to fluorescently label the site of injection. The injection site (arrow) can be varied along the rostral-caudal gradient of incisor development. The number of injections within a single incisor can also be varied from one to multiple sites. Following injection of the incisors, they are (B) surgically placed under the kidney capsule for selected periods of time. (C) Incisors are explanted and dissected free of the capsule for further analysis. (D-G) show incisors recovered from the renal capsule after three days’ culture. (D, F) Phase contrast images of the incisor injected with the control scrambled peptide amphiphile and the bioactive bRGDS PA, respectively. (E, G) Calcium salts were identified using von Kossa staining with Kohler illumination of incisors injected with the control scrambled peptide amphiphile and the bioactive bRGDS PA, respectively. Mineral deposits only formed in the bRGDS PA injected incisors.

Figure 3. Regenerated enamel pearls form at the site of peptide amphiphile injection

(A) SEM reveals the presence of pearls along the anterior enamel surface at sites of injected bRGDS PA. (B) Microcomputed tomography reveals the presence of enamel pearls (arrows) adjacent to authentic enamel in a sagittal view of the PA-injected incisors. Multiple bRGDS PA injection sites result in multiple sites of ectopic enamel formation. A line is introduced to demarcate the enamel (E) from the underlying dentin (D) and the soft-tissue containing pulp chamber (P).

Figure 4. The ultrastructure of the enamel pearl reveals aligned rod-like structures consisting of aligned hydroxyapatite crystals

Bright field STEM images of FIB milled samples revealed rod structures sectioned longitudinally (arrows) and in cross section (arrowheads). Images of enamel pearl lift-outs located in a rostral (A) segment of development and a caudal (B) section of the developing incisor. (C) Selected area for electron diffraction of the pearls indicate that they are made of hydroxyapatite with texture along the c-axis in the direction of the rod imaged in (B), identical to authentic enamel. 1: (002), 2: (004), 3: (211), 4: (310) (D) A representative EDS spectrum showing that the pearl is composed of calcium and phosphorus, as expected. The copper X-ray emission lines are due to the copper grid on which the sample was mounted.

Figure 5. Incisors injected with bRGDS PA forms regenerated enamel nodules in the shape of a pearl, complete with ameloblasts after 56 days in the kidney capsule

(A) shows a representative tissue section of a bRGDS PA injected incisor stained with hematoxylin and eosin in Kohler illumination. (B, C) show laser excited fluorescence images of the hematoxylin and eosin (H & E) section shown in (A) captured by confocal microscopy of the nodule at lower (B) and higher (C) magnification. The cells contacting the nodule are polarized and resemble authentic ameloblasts with the specialized secretory-end of the ameloblast know as Tomes’ processes (arrowheads). (D) Amelogenin immunostaining of a representative section containing an enamel pearl detected enamel matrix protein production and accumulation. To identify host cells that could contribute to pearl formation, a transgenic mouse strain constitutively expressing a green fluorescent protein (E) was used as a host for a wild type incisor primordia injected with a combination of rhodamine conjugated PA and bioactive bRGDS PA (F). The merged image (G) shows several GFP-marked cells (arrowheads) surrounding the bRGDS PA injection site and within the pulp (P). None of the ameloblast cells contributing to the formation of the pearl are GFP-labeled suggesting that they originate from the incisor tooth and not from the host. Abbreviations: P, pulp; D, dentin; E, enamel.

Figure 6. The artificial matrix displaying a high density of RGDS epitopes serves as a signal to induce changes in gene expression in the odontogenic epithelia that it contacts

The artificial matrix provides cell-matrix interactions similar to that of the dental mesenchyme during authentic enamel formation. The responding cells differentiate along an ameloblast lineage and enamel matrix proteins accumulate to produce an extracellular matrix capable of guiding the habit and orientation of hydroxyapatite crystals yielding a pearl of enamel analogous to authentic enamel.