Thomas G.H. Diekwisch

Evolution and Development of Hertwig’s Epithelial Root Sheath

Periodontal regeneration and tissue engineering has re‐awakened interest in the role of Hertwigs 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. Developmental Dynamics 235:1167–1180, 2006.

Initial enamel crystals are not spatially associated with mineralized dentine

During epithelial-mesenchymal interactions associated with mammalian tooth development, epithelially-derived and mesenchymally-derived extracellular matrix molecules form a discrete dentine-enamel junction. The developmental and molecular processes required to form this junction are not known. To address this problem we designed studies to test the hypothesis that ectodermally-derived epithelial cells synthesize and secrete enamel proteins which function to nucleate and regulate the growth of enamel calcium phosphate crystals. Initial enamel crystals were detected separate from the adiacent dentine. Electron-microprobe analyses revealed that early enamel crystals were octacalciumphosphate or tricalciumphosphate rather than hydroxyapatite. Thereafter, enamel crystals became confluent with the adjacent, albeit significantly smaller hydroxyapatite crystals associated with mineralized dentine. Therefore, we interpret our data to indicate that de novo enamel crystal nucleation and growth are independent from the mineralization processes characterized for dentine. We further argue that gene expression of enamel protein appears to have a constitutive function during early enamel formation and that supramolecular aggregates of amelogenin and enamelin provide the microenvironment for the nucleation and crystal growth of the initial enamel matrix.

Extracellular matrix-mediated differentiation of periodontal progenitor cells

The periodontal ligament (PDL) is a specialized connective tissue that connects the surface of the tooth root with the bony tooth socket. The healthy PDL harbors stem cell niches and extracellular matrix (ECM) microenvironments that facilitate periodontal regeneration. During periodontal disease, the PDL is often compromised or destroyed, reducing the life-span of the tooth. In order to explore new approaches toward the regeneration of diseased periodontal tissues, we have tested the effect of periodontal ECM signals, fibroblast growth factor 2 (FGF2), connective tissue growth factor (CTGF), and the cell adhesion peptide Arg-Gly-Asp (RGD) on the differentiation of two types of periodontal progenitor cells, PDL progenitor cells (PDLPs) and dental follicle progenitor cells (DFCs). Our studies documented that CTGF and FGF2 significantly enhanced the expression of collagens I & III, biglycan and periostin in tissue engineered regenerates after 4 weeks compared to untreated controls. Specifically, CTGF promoted mature PDL-like tissue regeneration as demonstrated by dense periostin localization in collagen fiber bundles. CTGF and FGF2 displayed synergistic effects on collagen III and biglycan gene expression, while effects on mineralization were antagonistic to each other: CTGF promoted while FGF2 inhibited mineralization in PDL cell cultures. Incorporation of RGD peptides in hydrogel matrices significantly enhanced attachment, spreading, survival and mineralization of the encapsulated DFCs, suggesting that RGD additives might promote the use of hydrogels for periodontal mineralized tissue engineering. Together, our studies have documented the effect of three key components of the periodontal ECM on the differentiation of periodontal progenitor populations.

The Role of Dust, Grit and Phytoliths in Tooth Wear

The threat of wear to dental enamel from hard particles of silica or silicates may have exerted great selective pressure on mammals. Increasing the hardness of enamel helps to forestall this, but capacity for variation is small because the tissue is almost entirely composed of hydroxyapatite. Hard though it is, enamel also displays considerable toughness, which is important in setting the sharpness of particles (defined as an attack angle) necessary to wear it. Added to the threat from environmental silica(tes) are phytoliths, particles of opaline silica embedded in plant tissues. We show here that phytoliths have very different properties to grit and dust and are unlikely to wear enamel. However, phytoliths would tend to fracture between teeth under similar conditions, so resembling natural agents of wear. In this context, we suggest that phytoliths could represent an example of mimicry, forming an example of a feeding deterrent operating by deceit.

Successful periodontal ligament regeneration by periodontal progenitor preseeding on natural tooth root surfaces.

The regeneration of lost periodontal ligament (PDL) and alveolar bone is the purpose of periodontal tissue engineering. The goal of the present study was to assess the suitability of 3 odontogenic progenitor populations from dental pulp, PDL, and dental follicle for periodontal regeneration when exposed to natural and synthetic apatite surface topographies. We demonstrated that PDL progenitors featured higher levels of periostin and scleraxis expression, increased adipogenic and osteogenic differentiation potential, and pronounced elongated cell shapes on barren root chips when compared with dental pulp and dental follicle cells. When evaluating the effect of surface characteristics on PDL progenitors, natural root surfaces resulted in elongated PDL cell shapes, whereas PDL progenitors on synthetic apatite surfaces were rounded or polygonal. In addition, surface coatings affected PDL progenitor gene expression profiles: collagen I coatings enhanced alkaline phosphatase and osteocalcin expression levels and laminin-1 coatings increased epidermal growth factor (EGF), nestin, cadherin 1, and keratin 8 expression. PDL progenitors seeded on natural tooth root surfaces in organ culture formed new periodontal fibers after 3 weeks of culture. Finally, replantation of PDL progenitor-seeded tooth roots into rat alveolar bone sockets resulted in the complete formation of a new PDL and stable reattachment of teeth over a 6-month period. Together, these findings indicate that periodontal progenitor cell type as well as mineral surface topography and molecular environment play crucial roles in the regeneration of true periodontal anchorage.

Pathways and fate of migratory cells during late tooth organogenesis.

Tissue recombination experiments and cell lineage analyses of the developing neural crest have documented the role and central pathways of migratory cells during early craniofacial development. In the present study, regional pathways of cells during late peripheral morphogenesis were investigated using the crown stage tooth organ as a model. Homing targets during tooth integument formation were analyzed to understand the fate of migratory cells involved in late tooth organogenesis and the developmental origin of periodontal tissues. After surgical removal of the oral mucosa, the oral aspect of the dental follicle of lower first mouse molar teeth was labeled using a fluorescent contact dye. Following sacrifice after 0, 2, 4, and 6 days, labeled cells were detected in the dental follicle, in the alveolar bone, and in the periodontal ligament adjacent to the molar root. The distribution of labeled tissues was reconstructed three-dimensionally via confocal microscopy. Using a tooth molar organ culture system, labeled cells within the dental follicle were documented traveling in the apical direction. Our results indicated that cell migration during tooth organogenesis was following specific pathways and that cells within the circumference of the dental follicle were migrating in the apical direction. We speculate that migratory cells passing through the dental follicle connective tissue may contribute to the formation of the periodontium. The present documentation visualizes pathways, role, and dynamics of extensive cell movements during late tooth organogenesis.

IMMUNOHISTOCHEMICAL SIMILARITIES AND DIFFERENCES BETWEEN AMELOGENIN AND TUFTELIN GENE PRODUCTS DURING TOOTH DEVELOPMENT

Amelogenins and tuftelins are highly specialized proteins secreted into the developing enamel matrix during mammalian enamel formation. Both tuftelins and amelogenins have been associated with various functions during nucleation and maturation of the developing enamel matrix. In this study we conducted experiments to investigate whether tuftelins and portions of the amelogenin molecule were deposited and processed in spatially distinguished portions of the developing enamel matrix, using antibodies specific against tuftelin or amelogenins. The amelogenin antibodies were raised against recombinant and native amelogenins and also included an antibody against a polypeptide encoded by amelogenin exon 4. To compare spatial expression patterns of enamel protein epitopes, 3-day postnatal mouse molar tooth organs were processed for paraffin histology and cut into serial sections. Adjacent sections were exposed to antibodies against either tuftelin or various amelogenin epitopes. To investigate age-related changes of enamel protein expression, amelogenin and tuftelin antibodies were applied to tooth organs of developmental stages E19 and 1, 3, 5, 7, 9 and 11 postnatal days. Tuftelin was detected within the odontoblast processes during earlier stages of development (E19 and 1 day postnatal), whereas during later stages (3–11 days) it was recognized in a portion of the enamel layer adjacent to the dentine–enamel junction. In contrast, all four antibodies against amelogenins reacted with parts of the ameloblast cytoplasm and the entire enamel layer. Using immunohistochemistry, we were not able to detect any differences in the spatial distribution of the four amelogenin epitopes investigated. The spatial differences in the distribution of amelogenin and tuftelin as observed in this study may be intepreted as an indication of functional differences between both proteins during early enamel biomineralization.

Amelogenin Supramolecular Assembly in Nanospheres Defined by a Complex Helix-Coil-PPII Helix 3D-Structure

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four α-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 310 helices (∼P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal α-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule.

RANKL, osteopontin, and osteoclast homeostasis in a hyperocclusion mouse model.

The biological mechanisms that maintain the position of teeth in their sockets establish a dynamic equilibrium between bone resorption and apposition. In order to reveal some of the dynamics involved in the tissue responses towards occlusal forces on periodontal ligament (PDL) and alveolar bone homeostasis, we developed the first mouse model of hyperocclusion. Swiss-Webster mice were kept in hyperocclusion for 0, 3, 6, and 9 d. Morphological and histological changes in the periodontium were assessed using micro-computed tomography (micro-CT) and ground sections with fluorescent detection of vital dye labels. Sections were stained for tartrate-resistant acid phosphatase, and the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteopontin (OPN) was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). Traumatic occlusion resulted in enamel surface abrasion, inhibition of alveolar bone apposition, significant formation of osteoclasts at 3, 6 and 9 d, and upregulation of OPN and RANKL. Data from this study suggest that both OPN and RANKL contribute to the stimulation of bone resorption in the hyperocclusive state. In addition, we propose that the inhibition of alveolar bone apposition by occlusal forces is an important mechanism for the control of occlusal height that might work in synergy with RANKL-induced bone resorption to maintain normal occlusion.

Extracellular Matrix-mediated Tissue Remodeling Following Axial Movement of Teeth

Tooth eruption is a multifactorial process involving movement of existing tissues and formation of new tissues coordinated by a complex set of genetic events. We have used the model of the unopposed rodent molar to study morphological and genetic mechanisms involved in axial movement of teeth. Following extraction of opposing upper molars, lower molars supererupted by 0.13 mm. Labeled tissue sections revealed significant amounts of new bone and cementum apposition at the root apex of the unopposed side following supereruption for 12 days. Newly apposited cementum and alveolar bone layers were approximately 3-fold thicker in the experimental vs the control group, whereas periodontal ligament width was maintained. Tartrate-resistant acid phosphatase staining indicated bone resorption at the mesial alveolar walls of unopposed molars and provided in tandem with new bone formation at the distal alveolar walls an explanation for the distal drift of molars in this model. Microarray analysis and semiquantitative RT-PCR demonstrated a significant increase in collagen I, integrin β5, and SPARC gene expression as revealed by comparison between the unopposed molar group and the control group. Immunohistochemical verification revealed increased levels of integrin β5 and SPARC labeling in the periodontal ligament of the unopposed molar. Together our findings suggest that posteruptive axial movement of teeth was accomplished by significant formation of new root cementum and alveolar bone at the root apex in tandem with upregulation of collagen I, integrin β5, and SPARC gene expression.