Evolution and development of Hertwig's epithelial root sheath

Abstract

Periodontal regeneration and tissue engineering has re-awakened interest in the role of Hertwig's Epithelial Root Sheath (HERS), an epithelial tissue layer first discovered in amphibians more than a century ago. Using developmental, evolutionary, and cell biological approaches, we have, therefore, performed a careful analysis of the role of HERS in root formation and compared our data with clinical findings. Our developmental studies revealed HERS as a transient structure assembled in the early period of root formation and elongation and, subsequently, fenestrated and reduced to epithelial rests of Malassez (ERM). Our comparative evolutionary studies indicated that HERS fenestration was closely associated with the presence of a periodontal ligament and a gomphosis-type attachment apparatus in crocodilians and mammals. Based on these studies, we are proposing that HERS plays an important role in the regulation and maintenance of periodontal ligament space and function. Additional support for this hypothesis was rendered by our meta-analysis of recent clinical reports related to HERS function.

Fig. 1. Sagital section of a tooth organ in the lower jaw of Salamandra maculata from the original drawing by Oscar Hertwig (1874)

The epithelial root sheath (“Epithelhülse”, H) has been enhanced using blue color. Note Hertwig’s intricate differentiation of tissues of the attachment apparatus, including Processus dentalis (F), Os dentale (Od), Cement (C), undecalcified zone between Os dentale and tooth crown (h), and tooth socket (So). Other tissues are labeled enamel (S), dentin (d), basement membrane (B), and enamel membrane (MS).

Fig. 2. Immunostaining of epithelial cells along the developing root surface using a polyclonal anti-keratin wide-spectrum screening antibody

Epithelial cells positively labeled with anti-keratin antibody were stained in brown color. Representative images of first mandibular molars of the following stages were selected: Fig. 2A: 1 day postnatal, Fig. 2B: 5 days postnatal, Fig. 2C: 10 days postnatal, and Fig. 2D: 20 days postnatal. For orientation purposes, the following cell layers were labeled: stratum intermedium (si), ameloblasts (am), enamel (en), dentin (de), pre-dentin (pd), odontoblasts (od) and HERS (hers). In Fig. 2A the ameloblast cell layer (am) and HERS were not separated. HERS developed as a cervical loop extension of the outer enamel epithelium (oee) and ameloblast (am) cell layers. From Fig. 2B (5 days postnatal) to Fig. 2C (10 days postnatal), the distance between the cervical margin of the ameloblast cell layer (am) and the most cervical HERS (hers) cell had increased more than five-fold. Between Fig. 2C (10 days postnatal) and Fig. 2D (20 days postnatal), the distance between single epithelial cells (ep) had significantly increased. The distance mark (d) in Figs. 2C and 2D demonstrates the two-fold increased distance between two epithelial cells (ep) proximal to the apical margin of the ameloblast cell layer (d₁=50μm, d₂=100μm). Note the fibers (fib) inserting at the cervical portion of the developing root surface in Fig. 2D (20 days postnatal).

Fig. 3. Three-dimensional reconstruction of anti-keratin labeled serial sections of a 10 days postnatal developing mouse first mandibular molar

The reconstructed image consists of images of nine adjacent 5μm thick paraffin sections that were stained using anti-keratin antibodies and immunoperoxidase detection methods. Green (dentin, de) and dark blue (enamel, en) colors represent the mineralized tissues. The light blue color marks the epithelial cells layers ameloblasts (am), HERS (hers), and the epithelial cells between HERS and ameloblasts. Fig. 3A is a 30° horizontal rotation of the montage shown in Fig. 3B. Note the fenestrated network of epithelial cells covering the root surface in Fig. 3A and the space (arrows) between root surface and epithelial cells demonstrated in Fig. 3B.

Fig. 4. Hertwig’s root sheath as visualized in Keratin-14 transgenic mice

The K14 transgene was detected using the lacZ reporter gene and stained with β-galactosidase. Fig. 4A is a sagital section through the mouse molar region (m₁, m₂, m₃) of the lower jaw of a one week postnatal mouse. Oral epithelium (oe), enamel organ (eno), and Hertwig’s epithelial root sheath (hers) were intensely labeled via the K14 transgene. Note the clearly outlined position of the epithelial diaphragm (dia) in the second molar (m₂). In the further developed first molar (m₁), HERS continuity was already interrupted, while HERS was continuous in the second molar (m₂). Fig. 4B illustrates the network or HERS in two weeks postnatal mice (hers) covering the developing root surfaces of the first molar (m₁). The position of the second molar (m₂) and of the enamel layer (en) are marked for the purpose of orientation. This and the preparation in Fig. 4C are whole mount sections, in which the superficial aspect of the tooth and jaw had been removed to allow visual access toward the root surface. Fig. 4C. Whole mount preparation of a four weeks postnatal mouse jaw carrying the K14 transgene. At this stage, root formation was almost complete and a network of Hertwig’s root sheath (hers) was outlining the circumference of the tooth roots. The three molars (m₁, m₂, m₃) and the enamel layer of the second molar (en) are marked for orientation purposes. Fig. 4D is a higher magnification of the apical tip of the enamel organ of a one week postnatal mouse carrying the K14 transgene. Hertwig’s epithelial root sheath (hers) and ameloblasts (amel) were labeled at this point, while dentin (de), odontoblasts (od), dental follicle (df), and alveolar bone (ab) were not. At this stage of initial root formation, Hertwig’s root sheath was continuous. Fig. 4E. Sagital section through the anterior root of a second lower jaw molar at six weeks postnatal. At this stage, HERS had been reduced to Epithelial rests of Malassez (M), which reacted positive for the K14 transgene. Alveolar bone (ab), dentin (de), root cementum (cem), pulp (plp), and periodontal ligament (lig) were negative. Note the greatly interspersed distribution of the epithelial rests of Malassez (M) allowing for periodontal ligament cells to attach to the root surface.

Fig. 5. Histochemical analysis of epithelial and mesenchymal tissues in chondrichthyan and actiopterygian teeth

Figs. 5A,B, and C were from a guppy fish (Poecilia reticularis). 5D–G were from a Shovelnosed Guitarfish (Rhinobatos productus), Fig. 5H was from a Pacific Hornshark (Heterodontus franciscii). The enamel organs (eo) of developing guppy teeth (Figs. 5A–C) were attached to the oral epithelium (oe) via a general lamina. The root dentin (de) between individual teeth and Os dentale (Od) were connected by a cementoid bridge (cem). The Shovelnosed Guitarfish (Rhinobatos productus)(Fig. 5D–G) and the Pacific Hornshark (Heterodontus franciscii)(Fig. 5H) demonstrated a clear separation between enamel organ epithelial cells (cl, cervical loop) and mesenchymal cells of the periodontal ligament (pl) in chondrichthyan teeth. The root surface apical of the cervical loop (cl) was devoid of any epithelial cells. Periodontal ligament and cervical loop were separated from each other by a basement membrane (arrowheads). Tooth enameloid (en), oral epithelium (oe) and root dentin (rd) were labeled for orientation purposes.

Fig. 6. Immunohistochemical staining of epithelial tissues in amphibian teeth using anti-keratin wide-spectrum screening antibodies

Figs. 6A and B were from a Leopard Frog (Rana pipiens), Figs. 6C–E were from a Mexican Axolotl (Ambystoma mexicanum). In the frog jaw (Figs. 6A and B) Hertwig’s epithelial root sheath (hers) measured less than a quarter in length compared to the entire length of the tooth root. The remaining root surface was covered with cementoid of attachment between adjacent tooth roots and mandibular bone. Borders between root dentin (de), cementoid of attachment (cem), and Os dentale (bone) were difficult to distinguish. The anti-keratin antibody labeled Hertwig’s epithelial root sheaths (hers) as well as oral epithelium (oe). The axolotl jaw (Figs. 6C–E) was similar to the frog jaw in that a cementoid tissue (cem) connected teeth amongst each other as well as individual teeth with the Os dentale (Od). In comparison to the frog, the cementoid tissue (cem) of the axolotl was less prominent. Hertwig’s epithelial root sheath (hers) covered between one half and two thirds of the root dentin (rd) surface. Note the presence of islands of epithelial cells (isl) in the interdental region and distant from HERS. The apical portion of the root dentin surface was occupied by mesenchymal cells. The anti-keratin antibody recognized oral epithelium (oe), epithelial islands (isl), and Hertwig’s epithelial root sheath (hers).

Fig. 7. Anti-keratin immunohistochemical staining of epithelial tissues in mammalian and reptilian teeth

Figs. 7A and F are micrographs of a developing tooth organ of a Texas Banded Gecko (Coleonyx brevis) immunostained with anti-keratin antibodies. Hertwig’s root sheath (HERS), ameloblasts (amel), and general lamina (general lamina) were labeled by the anti-keratin antibody. A typical characteristic of reptilian jaws was the position of bone of attachment (cem) connecting adjacent teeth among each other. The body of the jaw bone (Od, Os dentale) was in distinct distance to the cementum-derived tooth-carrying bone. Note that the epithelial cell layer was limited to the tooth crown allowing for cementoid tissue and bone of attachment (cem) forming between adjacent teeth. Figs. 7B, C, and D are anti-keratin immunoreactions in the epithelial tissues of erupting iguana teeth. Fig. 7C is an overview; Figs. 7B and 7D are higher magnifications. The anti-keratin antibody labeled oral epithelium (oe) and Hertwig’s epithelial root sheath (hers). The cementum layer (cem) covered the entire root area and facilitated an ankylotic attachment between tooth root and alveolar bone (alv). Adjacent teeth were attached to a basal Os dentale (Od) instead of being directly connected with each other via a cementoid bridge as seen in the gecko and frog. In Fig. 7F, the anti-keratin antibody was applied to sagital paraffin sections through a caiman tooth organ (Caiman crocodilus). Here, the antibody discretely labeled the coronal ameloblasts (am) and remnants of Hertwig’s root sheath (hers) interspersed along the root surface. Note the wide spaces between individual HERS cell rests allowing for access of the caiman periodontal ligament (pdl) to attach to the root surface. Fig. 7G is a micrograph from a mouse first mandibular molar prior to eruption. Oral epithelium (oe), ameloblast cell layer (amel), and papillary layer (pap) were distinctly stained with anti-keratin antibodies. While the entire tooth crown (crown) was surrounded by keratin-positive epithelial cells, the root surface (root) and the alveolar bone (alv) were not labeled. Only the most apical portion of HERS (ap, arrow) was recognized by the anti-keratin antibody.

Fig. 8. Hertwig’s Epithelial Root Sheath in human teeth (specimens from the Gottlieb Collection, Dallas, Texas)

Fig. 8A. Network of Hertwig’s root sheath in humans in en face orientation toward the root surface. Note the wide open regions containing periodontal ligament fibroblasts (lig) between individual cords of HERS (hers) cells. Fig. 8b. Sagital section through the network of HERS cells in perpendicular orientation to Fig. 8A. The network of HERS cells (hers) formed a defined layer adjacent to the root surface and embedded in periodontal ligament fibroblasts (lig). Fig. 8c. Close relationship between HERS and nerve cells. Note the presence of nervous plexus (pl) and ganglion cells (gg) within knots of the HERS network. Fig. 8d. Another demonstration of the close association between HERS cells and nervous plexus within the fibroblast-rich (fib) periodontal ligament space. These preparations are from the historic Gottlieb collection at Baylor University. Bar = 100μm.

Fig. 9. Demonstration of apoptotic cells in the periodontal ligament using the TUNEL technique

Figs. 9A and 9C were DAPI labeled fluorographs of the same areas as in the TUNEL stainings in Figs. 9B and 9D. The position of alveolar bone (ab), dental follicle (df), ameloblast nuclei (am), and odontoblast nuclei (od) was indicated for orientation purposes. Ameloblast mitochondria (am) provided orientation marks in the TUNEL stained Fig. 9B. Using the TUNEL technique, numerous apoptotic cell nuclei in the dental follicle were visualized (arrows, Figs. 9B and D). Figs. 9E and 9F are double exposures of the same area using both propidium iodide staining and ApopTag Red apoptosis labeling. Apoptotic nuclei were marked by arrows. Note the two adjacent apoptotic nuclei in the proximity of the root surface (Fig. 9F). Alveolar bone (ab), dental follicle (df), root dentin (rd), and enamel (en) were labeled for orientation purposes.

Fig. 10. Fate of Hertwig’s root sheath in six weeks old mice visualized in Keratin-14 transgenic mice

In this study, the K14 transgene was detected using the lacZ reporter gene and stained with β-galactosidase. Micrographs illustrate the incorporation of epithelial rests of Malassez (M)(Figs. 10A, B) as well as individual cells of Hertwig’s epithelial root sheath (hers) into the mineralizing cementum layer (cem)(Fig. 10C). Tissues labeled for orientation are periodontal ligament (pdl), alveolar bone (ab), and root dentin (den). Note the strong staining intensity for the K14 transgene in Malassez’ rests of 6 weeks old mice. Figs. 10A and 10B illustrate the incorporation of K14 Malassez’ rests into the cementum matrix of the root shaft at multiple sites. Fig. 10C documents the encapsulation and incorporation of individual HERS cells in the mineralizing cementum mass of the root apex.

Fig. 11. Schematic relationship between Hertwig’s root sheath, periodontal ligament, and mineralized tissues of tooth anchorage and attachment throughout vertebrate evolution and development

This sketch illustrates the position of mineralized tissues of attachment basal of HERS in tooth-bearing gnathostomes as well as the relationship between HERS and periodontal ligament anchorage in thecodont vertebrates. Fig. 11A summarizes the ligamenteous anchorage of teeth in chondrichthyans. The ligament (pdl) is positioned basal of the enameloid organ (eo). In Fig. 11B (many teleosts), the Os dentale (Od) of the jaw is surrounded by two other, closely related mineralized tissues, the cementum (cem) which interconnects individual tooth units, and the pedestal (ped) on which the tooth crown is anchored. Mineralized tissues of anchorage are all positioned basal of the enameloid organ (eo). The amphibian tooth attachment apparatus (Fig. 11C) features an elongated Hertwig’s root sheath (hers) which covers part of the tooth root. The apical tip of HERS coincides with the coronal border of the cementoid of attachment (cem). Similar as in teleosts, individual amphibian tooth units are supported by pedestals (ped). All three mineralized tissues of attachment, cementum, pedestal, and Os dentale, are fused together via ankylosis. In amphibians, the extension of mineralized tissues in coronal direction is defined by HERS and other epithelial rests. The acrodont and pleurodont dentitions of many reptiles (Fig. 11D) are characterized by ankylotic attachment of tooth roots to the Os dentale (Od) via cementum (cem) and the pedestal (ped). While the entire root surface apical of HERS is covered by cementum (cem), adjacent teeth and gingival tissues are also interconnected by ligamenteous tissues (pdl) in the cervical region of the tooth. In contrast, Crocodilian dentitions (Fig. 11E) are truly thecodont. Their teeth are embedded in sockets provided by alveolar bone (ab). Alveolar bone (ab) and root cementum (cem) are separated by Hertwig’s root sheath and by a periodontal ligament (pdl). In contrast to mammals, the crocodilian periodontal ligament is partially mineralized (MacIntosh et al. 2002). Fig. 11F illustrates a cross section through a mammalian tooth. The mammalian periodontal ligament (pdl) is well organized into parallel fiber bundles and lacks the mineral deposits found in crocodilians. During mammalian root development, HERS stretches along the entire root surface (hers) and extends into an epithelial diaphragm (dia) at the root apex. A theca formed by ankylotic fusion of alveolar (alv) and mandibular/maxillary bone (mand) provides anchorage for the tooth root. Figure legends: enamel/enameloid (en, yellow), dentin (de, green), enamel organ (eo, red), cementum (cem, blue dots), pedestal (ped, mauve), Os dentale/mandible (Od, blue graticule), periodontal ligament (pdl, blue interrupted lines), Hertwig’s Epithelial Root Sheath (hers, red dots), Epithelial Diaphragm (dia, red oval). The orientation of illustration patterns in the periodontal ligament space does not follow physiological fiber orientation.

Fig. 12. Schematic phylogeny of species involved in the current study

Note that the thecodont archosaurs are more distant from the thecodont mammals than the pleurodont/acrodont squamates (Lepidosauromorpha) suggesting convergent evolution of mammals and crocodilians in respect to tooth morphogenesis.