Apical Papilla Cells Are Capable of Forming a Pulplike Tissue with Odontoblastlike Cells without the Use of Exogenous Growth Factors.

INTRODUCTION: Dental pulp is a complex tissue with highly differentiated cells, which makes its reconstruction a challenging task. The apical papilla is an undifferentiated tissue considered as the remnant of the dental papilla that forms the dentin-pulp complex. Aiming to analyze morphologic features of the tissue formed in an in vivo pulp model, we used human apical papilla as a cell source without the use of exogenous growth factors. METHODS: A construct was built using newborn mice molar crowns treated with TrypLE (Fisher Scientific, Loughborough, UK) and EDTA. The crowns were filled with PuraMatrix (Corning Inc, Corning, NY) and a pool population of human apical papilla cells. As a control, we used crowns filled only with PuraMatrix and empty crowns. The constructs were transplanted under severe combined immunodeficient mice kidney capsules. Immunohistochemistry for lamin A, dentin sialophosphoprotein, and dentin matrix protein 1 was performed. RESULTS: Morphologic analysis of all transplanted crowns showed the formation of a loose connective tissue of variable cellularity with the presence of well-formed functional vessels. In the study group, lamin A-positive cells represented the majority of cells within the pulp chamber and a few cells in the vessel lining. We also found positivity for dentin sialophosphoprotein and dentin matrix protein 1, an indicator of odontoblast differentiation. CONCLUSIONS: In our study model, human transplanted apical papilla cells mixed with the host cells and formed a vascularized viable tissue, and these cells were able to differentiate into odontoblastlike cells without the use of exogenous growth factors.

Dental Apical Papilla as Therapy for Spinal Cord Injury.

Stem cells of the apical papilla (SCAP) represent great promise regarding treatment of neural tissue damage, such as spinal cord injury (SCI). They derive from the neural crest, express numerous neurogenic markers, and mediate neurite outgrowth and axonal targeting. The goal of the present work was to investigate for the first time their potential to promote motor recovery after SCI in a rat hemisection model when delivered in their original stem cell niche-that is, by transplantation of the human apical papilla tissue itself into the lesion. Control groups consisted of animals subjected to laminectomy only (shams) and to lesion either untreated or injected with a fibrin hydrogel with or without human SCAP. Basso-Beattie-Bresnahan locomotor scores at 1 and 3 d postsurgery confirmed early functional decline in all SCI groups. This significant impairment was reversed, as seen in CatWalk analyses, after transplantation of apical papilla into the injured spinal cord wound, whereas the other groups demonstrated persistent functional impairment. Moreover, tactile allodynia did not develop as an unwanted side effect in any of the groups, even though the SCAP hydrogel group showed higher expression of the microglial marker Iba-1, which has been frequently associated with allodynia. Notably, the apical papilla transplant group presented with reduced Iba-1 expression level. Masson trichrome and human mitochondria staining showed the preservation of the apical papilla integrity and the presence of numerous human cells, while human cells could no longer be detected in the SCAP hydrogel group at the 6-wk postsurgery time point. Altogether, our data suggest that the transplantation of a human apical papilla at the lesion site improves gait in spinally injured rats and reduces glial reactivity. It also underlines the potential interest for the application of delivering SCAP in their original niche, as compared with use of a fibrin hydrogel.

Enriched trimethylation of lysine 4 of histone H3 of WDR63 enhanced osteogenic differentiation potentials of stem cells from apical papilla.

INTRODUCTION: Dental tissue-derived mesenchymal stem cells (MSCs) are a reliable cell source for dental tissue regeneration. However, the molecular mechanisms underlying their directed differentiation remain unclear, thus limiting their use. Trimethylation of lysine 4 of histone H3 (H3K4Me3) correlates with gene activation and osteogenic differentiation. We used stem cells from apical papilla (SCAPs) to investigate the effects of genomic changes in H3K4Me3 modification at gene promoter regions on MSC osteogenic differentiation. METHODS: ChIP-on-chip assays were applied to compare the H3K4Me3 profiles at gene promoter regions of undifferentiated and differentiated SCAPs. Alkaline phosphatase activity assay, alizarin red staining, quantitative analysis of calcium, the expressions of osteogenesis-related genes, and transplantation in nude mice were used to investigate the osteogenic differentiation potentials of SCAPs. RESULTS: In differentiated SCAPs, 119 gene promoters exhibited >2-fold increases of H3K4Me3; in contrast, the promoter regions of 21 genes exhibited >2-fold decreases of H3K4Me3. On the basis of enriched H3K4Me3 and up-regulated gene expression on the osteogenic differentiation of SCAPs, WDR63 may be a potential regulator for mediating SCAP osteogenic differentiation. Through gain-of-function and loss-of-function studies, we discovered that WDR63 enhances alkaline phosphatase activity, mineralization, and the expression of BSP, OSX, and RUNX2 in vitro. In addition, transplant experiments in nude mice confirmed that SCAP osteogenesis is triggered by activated WDR63. CONCLUSIONS: These results indicate that WDR63 is a positive enhancer for SCAP osteogenic differentiation and suggest that activation of WDR63 signaling might improve tissue regeneration mediated by MSCs of dental origin.

Autotransplantation of an unerupted wisdom tooth germ without its follicle immediately after removal of an impacted mandibular second molar: a case report.

An impacted left mandibular second molar (tooth 37) was extracted, but most of its dental follicle remained in the socket. The crown and dental papilla of an unerupted, left mandibular third molar (tooth 38) were then placed into the socket of tooth 37. Successful interaction between the dental follicle of tooth 37 and the crown and dental papilla of tooth 38 led to the formation of a new tooth 37 at the recipient site. This suggests that the dental follicle may function non-specifically with the crown and dental papilla of other tooth germs.

Recubrimiento radicular mediante un colgajo de doble papila con injerto de tejido conectivo: caso clínico / Root coverage by means of a double papilla flap with a connective tissue graft

Una de las terapias cosméticas periodontales más usuales es el cubrimiento de recesiones radiculares. En las últimas décadas se han desarrollado numerosas técnicas con este fin. En este caso clínico mostramos la utilización de una técnica de doble papila compbinada con un injerto de tejido conectivo para solucionar un inconveniente estético en un canino superior. Presentamos resultados y seguimiento a 3 meses

Cell pellets from dental papillae can reexhibit dental morphogenesis and dentinogenesis.

We isolated dental papilla mesenchymal cells (DPMCs) from different rat incisor germs at the late bell stage and incubated them as cell pellets in polypropylene tubes. In vitro pellet culture of DPMCs presented several crucial characteristics of odontoblasts, as indicated by accelerated mineralization, positive immunostaining for dentin sialophosphoprotein and dentin matrix protein 1, and expression of dentin sialophosphoprotein mRNA. The allotransplantation of these pellets into renal capsules was also performed. Despite the absence of dental epithelial components, dissociated DPMCs with a complete loss of positional information rapidly underwent dentinogenesis and morphogenesis, and formed a cusp-like dentin-pulp complex containing distinctive odontoblasts, predentin, dentin, and dentinal tubules. These results imply that DPMCs at the late bell stage can reexhibit the dental morphogenesis and dentinogenesis by themselves, and epithelial-mesenchymal interactions at this stage may not be indispensable. Furthermore, different DPMC populations from the similar stage may keep the same developmental pattern.

Cultured human and rat tooth papilla cells induce hair follicle regeneration and fiber growth.

The mesenchymal-epithelial interactions that characterize the early stages of tooth and hair follicle morphogenesis share certain similarities, and there is increasing evidence that mesenchymal cells derived from both mature structures retain interactive and stem cell-like properties. This study aimed to gauge the cross-appendage inductive capabilities of cultured tooth dental papilla (or pulp) cells from different species and ages of donor. Adult human and juvenile rat tooth papilla cells were implanted into surgically inactivated hair follicles within two different microenvironments. The human cells interacted with follicle epithelium to regenerate new end bulbs and create multiple differentiated hair fibers. Rodent tooth dental cells also induced new epithelial matrix structures and stimulated de novo hair formation. However, in many instances they also elicited mineralization and bone formation, a phenomenon that appeared to relate to their donor's age; the type of tooth of origin; and the host environment. Taken together, this study reveals that cultured dental papilla cells from postnatal mammals (adult, juvenile, and newborn) retain inductive molecular signals that must be common to both hair and teeth follicles. It highlights the stem cell-like qualities and morphogenetic abilities of tooth and hair follicle cells from mature humans, and their capacity for cross-appendage and interspecies communication and interaction. Besides the developmental implications, the present findings have relevance for stem cell biology, hair growth, tissue repair, and other biotechnologies. Moreover, the critical importance of considering the local microenvironment in which different cells/tissues are naturally or experimentally engineered is firmly demonstrated.

The induction of enamel and dentin complexes by subcutaneous implantation of reconstructed human and murine tooth germ elements.

Tooth induction by xenogenic graft of reconstructed human tooth germ components has never been attempted. Here we report our first attempt at a transplantation of human tooth germ components, heterologously recombined with mouse dental epithelia, into immunocompromised animals. Human third molar tooth germs enucleated from young patients as prophylactic treatment for orthodontic reasons were collected. The whole or minced human dental papilla was reconstructed with human- or mouse molar enamel epithelium, and transplanted in the dorsal aspect of C.B-17/Icr-scid Jcl mice. The transplant of human dental papilla reconstructed with human enamel epithelium formed thin dentin and immature enamel layers by 3 to 4 weeks, but remained extremely small in quantity due to a shortage of epithelial components in the graft. The addition of E16 mouse molar enamel organs (n=10-12) to each graft augmented the formation of tooth germ-like structures, but the differentiation of mouse molar ameloblasts was suppressed. However, once a solid layer of mineralized dentin was established, mouse ameloblasts accelerated their differentiation, and completed the enamel matrix formation and maturation within the following 4 weeks, whereas human ameloblasts, which had interacted with human dental papilla, remained in the stage of matrix formation during the same period. These data imply that, in reconstructed transplants, the differentiation of mouse dental epithelia is restrained by putative suppressive factors derived from human dental papilla until they are separated by mineralized dentin layers that serve as a diffusion barrier. The mouse enamel organ nevertheless retains its own phenotypic characteristics and intrinsic timing of cell differentiation and function.

Response of odontoblast-like cells to hydroxyapatite ceramic granules.

In this study the inductive influence of hydroxyapatite ceramic (HAC) granules on preodontoblast differentiation was investigated. Dental papilla cells harvested from upper molar tooth germs were implanted intramuscularly in a pouch created for this purpose. Six months after surgery tooth-root-like bodies had developed with pulp-like cavities in the specimens in which dental papilla cells had been implanted with and without HAC. These bodies consisted of regular tubular dentine in the central part, fibrodentine peripherally and sometimes osteodentine. HAC was haphazardly enclosed in these root-like bodies, whereas the implantation of HAC alone had no effect.

[Dental lamina as presumptive source of odontogenic cyst].

The possibility of the dental lamina as a source of odontogenic cyst was investigated. The mandibular first molar tooth germs with the dental lamina and surface oral epithelium were cut from 17.5-day-old C3H mouse embryos. The following 5 kinds of grafts were prepared: (I) recombinant of the dental lamina and dental papilla, (II) dental lamina, (III) dental papilla, (IV) recombinant of the oral epithelium and dental papilla and (V) oral epithelium. After the renal subcapsular transplantation to the 3-month-old syngenic male mice, each graft was harvested at timed sequences from 2 to 24 weeks and was examined histopathologically. The recombinant of the dental lamina and dental papilla (1) grew into a cyst lined by para-keratinized stratified squamous epithelium. The cyst enlarged gradually and might be compared to the odontogenic keratocyst of the human being. The recombinant of the oral epithelium and dental papilla (IV) and the oral epithelium (V) developed into a cyst lined by orthokeratinized stratified squamous epithelium which differed from the epithelium seen in Experiment (I). The dental papilla (III) grew to be a bone tissue while nothing developed from the dental lamina (II). These results suggest that the dental lamina is one of the sources of the odontogenic keratocyst and the dental papilla plays an important role in its histogenesis.

Calcification capacity of dental papilla mesenchymal cells transplanted in the isogenic mouse spleen.

The capacity of the dental pulp to form calcified tissue was examined in papilla cells dissociated from first molar tooth germs of the neonatal mouse and isografted in the spleen for up to 7 days. To obtain papilla cell populations without odontoblasts, pulpal mesenchyme was isolated mechanically from the enamel organ after 0.1% trypsin treatment and rolled on a membrane filter. On day 3 after transplantation, the grafted papilla cells had changed into large, spindle-shaped cells, and initial calcification with needle-like crystals began in association with the collagenous matrix surrounding those cells. On day 7 after transplantation, the spindle cells transformed into odontoblast-like cells containing well-developed secretory organelles, and irregular, but nontubular, calcified tissues were commonly observed surrounding the extracellular collagenous matrix. The calcified tissue matrix with cellular inclusions displayed a structure similar to that of osteodentin. During this period, an intense positive reaction for alkaline phosphatase (ALPase) activity was demonstrated along the cell membranes of the odontoblast-like cells aligned at the periphery of forming calcified tissue. Enzymatic activity could not be detected on the cells incorporated completely into osteodentin-like matrix. The present results show that the papilla cell population transplanted into the spleen formed osteodentin-like material, thus demonstrating the capacity of papilla cells to produce calcified tissue.

Hormone-responsive cells derived from human dental papilla: characterization in vitro and in vivo in diffusion chambers.

Cells of the dental papilla are capable of odontoblastic, fibroblastic, and endothelial differentiation and formation of dentin and the dental pulp. In the present study dental papilla cells, obtained from human tooth buds (HDP cells), were cultured in vitro through 3 to 7 passages. After exposure to prostaglandin E2 there was a marked decrease in intracellular cyclic AMP (cAMP) levels as compared to hormone-free controls. Parathyroid hormone and calcitonin had stimulatory effects with 1 and 2 log increases in cAMP, respectively. The HDP cells showed moderate activity of alkaline phosphatase, 1 log higher than that of hamster kidney fibroblasts (BHK 13) and 1 log lower than that of osteoblastic osteosarcoma cells (ROS 17/2). When cultured for 4 or 8 wk in diffusion chambers (DC) implanted in athymic mice, many of the HDP cells underwent odontoblastic morphodifferentiation with very long, single processes extending into the matrix. This matrix contained banded and unbanded collagen fibers. Neither light nor electron microscopy of the DC content revealed mineral deposits. These results suggest that HDP cells have an intrinsic potential for partial odontoblastic differentiation; inductive signals like those originating from odontogenic epithelium are probably essential for the completion of hard tissue formation.

Epithelial-derived basal lamina regulation of mesenchymal cell differentiation.

The mechanisms by which epithelial-mesenchymal interactions result in differentiation are not known. A number of recombinations between vertebrate tissues associated with epidermal organs (e.g. skin, feather, mammary gland, salivary gland, tooth organ) indicate that regional mesenchymal specificity is instructive for determination and differentiation of epithelial phenotypes. In epidermal organs within which mesenchyme becomes determined and differentiates into a unique phenotype, such as during tooth organogenesis and odontoblast differentiation. Does the epithelial-derived basal lamina regulate mesenchymal differentiation into odontoblasts and the expression of dentine extracellular matrix? Experiments were designed to test the hypothesis that murine or avian epithelial-derived basal lamina possess information which is instructive for determined dental mesenchyme to differentiate into odontoblasts. The strategy was to examine homologous and heterologous tissue recombinants between Theiler stage 25 C57BL/6 molar tooth organs and Hamburger-Hamilton equivalent stage 22-26 Japanese Pharoah quail mandibular processes. Trypsin-dissociated molar epithelium and mesenchyme, reconstituted, secreted a basal lamina within 8 hours and mesenchyme differentiated into odontoblasts and formed dentine matrix within 3 days. Isolated trypsin-dissociated mesenchyme did not differentiate in vitro, whereas heterologous recombinants between odontogenic mesenchyma and quail epithelia resulted in odontoblasts and dentine production. Mouse tooth or quail mandibular epithelia served to regulate odontogenic mesenchyme differentiation. EDTA-dissociated mouse molar mesenchyme, in the absence of epithelium but with adherent basal lamina, routinely differentiated into odontoblasts. Control tooth organs routinely formed both dentine and enamel extracellular matrices within 7-10 days in our serumless, chemically-defined organ culture system. Regulation of determined mesenchymal cells to differentiate into functional and highly specialized odontoblasts appears to be mediated by epithelial-derived basal lamina and is not species or organ-specific.

Xenografts of recombined bovine odontogenic tissues and cultured cells to hypothymic mice.

It was demonstrated in this study that recombined tissues of bovine dental papilla and reduced enamel epithelium, when transplanted to the subcapsular kidney site of hypothymic mice, elaborated tissues resembling osteodentin and dentin matrices with which latter tissues were closely associated cells resembling odontoblasts. In xenografts of cultured cell populations of these tissues, osteodentin-like matrices occurred in the absence of dentin-like matrices.