Responses of BrdU label-retaining dental pulp cells to allogenic tooth transplantation into mouse maxilla

Recently, we demonstrated that a pulse of BrdU given to prenatal animals reveals the existence of slow-cycling long-term label-retaining cells (LRCs), putative adult stem or progenitor cells, which reside in the dental pulp. This study aims to clarify responses of LRCs to allogenic tooth transplantation into mouse maxilla using prenatal BrdU-labeling, in situ hybridization for osteopontin and periostin, and immunohistochemistry for BrdU, nestin, and osteopontin. The upper-right first molars were allografted in the original socket between BrdU-labeled and non-labeled mice or between GFP transgenic and wild-type mice. Tooth transplantation caused degeneration of the odontoblast layer, resulting in the disappearance of nestin-positive reactions in the dental pulp. On postoperative days 5–7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in the successful cases. In BrdU-labeled transplanted teeth, dense LRCs were maintained in the center of the dental pulp beneath the odontoblast-like cells including LRCs, whereas LRCs disappeared in the area surrounding the bone-like tissue. In contrast, LRCs were not recognized in the pulp chamber of non-labeled transplants through the experimental period. Tooth transplantation using GFP mice demonstrated that the donor cells constituted the dental pulp of the transplant except for endothelial cells and some migrated cells, and the periodontal tissue was replaced by host-derived cells except for epithelial cell rests of Malassez. These results suggest that the maintenance of BrdU label-retaining dental pulp cells play a role in the regeneration of odontoblast-like cells in the process of pulpal healing following tooth transplantation.     

The Expression of GM-CSF and Osteopontin in Immunocompetent Cells Precedes the Odontoblast Differentiation Following Allogenic Tooth Transplantation in Mice

Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the expression of granulocyte macrophage colony-stimulating factor (GM-CSF) and osteopontin (OPN) in the process of reparative dentin formation by allogenic tooth transplantation using in situ hybridization for OPN and immunohistochemistry for GM-CSF and OPN at both levels of light and electron microscopes. Following the extraction of the mouse molar, the roots and pulp floor were resected and immediately allografted into the sublingual region. On days 1 to 3, immunocompetent cells such as macrophages and dendritic cells expressed both GM-CSF and OPN, and some of them were arranged along the pulp-dentin border and extended their cellular processes into the dentinal tubules. On days 5 to 7, tubular dentin formation commenced next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until day 14, bone-like tissue formation occurred in the pulp chamber, where OPN-positive osteoblasts surrounded the bone matrix. These results suggest that the secretion of GM-CSF and OPN by immunocompetent cells such as macrophages and dendritic cells plays a role in the maturation of dendritic cells and the differentiation of odontoblasts, respectively, in the regenerated pulp tissue following tooth transplantation.     

Influence of extended operation time and of occlusal force on determination of pulpal healing pattern in replanted mouse molars

The mechanism regulating the divergent healing processes following tooth replantation is unclear. This study clarifies the relationship between the healing pattern, the time taken for tooth replantation, and the influence of occlusal force. We investigated the pulpal healing process after tooth replantation by immunohistochemistry for 5-bromo-2′-deoxyuridine and nestin and by histochemistry for tartrate-resistant acid phosphatase. The upper right first molar of 3-week-old mice was extracted and repositioned in the original socket immediately or 30 min to 6 h after the operation. We divided the animals into a non-occluded group in which the lower right first molar was extracted and an occluded group without extraction of the counterpart tooth. In control teeth (upper left first molar), the periphery of the coronal dental pulp showed intense nestin-positive reaction. Tooth replantation weakened the nestin-positive reaction in the pulp tissue. On postoperative days 5–7, tubular dentin formation commenced next to preexisting dentin in which nestin-positive odontoblast-like cells were arranged in successful cases. In other cases, bone-like tissue formation occurred in the pulp chamber until day 14. The ratio of tertiary dentin formation was significantly higher in the non-occluded group. The intentionally prolonged time for the completion of tooth replantation induced bone-like tissue formation, expanded inflammatory reaction, or fibrous tissue formation in pulp tissue. Thus, the lack of a proper oxygenated medium is probably decisive for the survival of odontoblast-lineage cells, and occlusal force during and/or after operation worsens the fate of these cells. This work was supported in part by KAKENHI (B) from MEXT, Japan (no. 16390523 to H.O.) and by the Japan-Korea Joint Research Project from JSPS and KOSEF.     

Capacity of dental pulp differentiation after tooth transplantation

Under pathological conditions, dental pulp elaborates both bone and dentin matrix in which the contribution of periodontal tissue cannot be excluded. This study has aimed to clarify the capability of dental pulp to deposit bone matrix in an auto-graft experiment by using (1) immunohistochemistry for 5-bromo-2′-deoxyuridine (BrdU) and nestin and (2) histochemistry for tartrate-resistant acid phosphatase (TRAP). Following the extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately transplanted into the sublingual region. On Days 5–7, tubular dentin formation commenced next to the pre-existing dentin at the pulp horn in which nestin-positive odontoblast-like cells were arranged. Up until Day 14, bone-like tissue formation occurred in the pulp chamber in which intense TRAP-positive cells appeared. These results suggest that odontoblast- and osteoblast-lineage cells reside in the dental pulp. Overall, specific dental pulp regeneration should provide fundamental knowledge for the realization of human tooth regeneration in the near future.This work was supported in part by a grant from MEXT to promote the 2001-multidisciplinary research project (in 2001–2005), KAKENHI (B) (no. 16390523 to H.O.) from MEXT, and the Japan-Korea Joint Research Project from JSPS and KOSEF (no. F01-2005-000-10212-0).     

Differentiation and regenerative capacities of human odontoma-derived mesenchymal cells

Regenerating human tooth ex vivo and biological repair of dental caries are hampered by non-viable odontogenic stem cells that can regenerate different tooth components. Odontoma is a developmental dental anomaly that may contain putative post-natal stem cells with the ability to differentiate and regenerate in vivo new dental structures that may include enamel, dentin, cementum and pulp tissues. We evaluated odontoma tissues from 14 patients and further isolated and characterized human odontoma-derived mesenchymal cells (HODCs) with neural stem cell and hard tissue regenerative properties from a group of complex odontoma tissues from 1 of 14 patients. Complex odontoma was more common (9 of 14) than compound type and females (9 of 14) were more affected than males in our set of patients. HODCs were highly proliferative like dental pulp stem cells (DPSCs) but demonstrated stronger neural immunophenotype than both DPSCs and mandible bone marrow stromal cells (BMSCs) by expressing higher levels of nestin, Sox 2 and βIII-tubulin. When transplanted with hydroxyapatite/tricalcium phosphate into immunocompromised mice, HODCs differentiated and regenerated calcified hard tissues in vivo that were morphologically and quantitatively comparable to those generated by DPSCs and BMSCs. When transplanted with polycaprolactone (biodegradable carrier), HODCs differentiated to form new predentin on the surface of a dentin platform. Newly formed predentin contained numerous distinct dentinal tubules and an apparent dentin–pulp arrangement. HODCs represent unique odontogenic progenitors that readily commit to formation of dental hard tissues.     

The role of preameloblast-conditioned medium in dental pulp regeneration

Pulp regeneration using human dental pulp stem cells (hDPSCs) maintains tooth vitality compared with conventional root canal therapy. Our previous study demonstrated that preameloblast-conditioned medium (PA-CM) from murine apical bud cells induces the odontogenic differentiation of hDPSCs and promoted dentin formation in mouse subcutaneous tissue. The purpose of the present study is to evaluate the effects of PA-CM with human whole pulp cells on pulp regeneration in an empty root canal space. Human pulp cells were seeded in the pulp cavities of 5 mm-thick human tooth segments with or without PA-CM treatment, and then transplanted subcutaneously into immunocompromised mice. In the pulp cell-only group, skeletal muscle with pulp-like tissue was generated in the pulp cavity. A reparative dentin-like structure with entrapped cells lined the existing dentin wall. However, in the PA-CM-treated group, only pulp-like tissue was regenerated without muscle or a reparative dentin-like structure. Moreover, human odontoblast-like cells exhibited palisade arrangement around the pulp, and typical odontoblast processes elongated into dentinal tubules. The results suggest that PA-CM can induce pulp regeneration of human pulp cells with physiological structures in an empty root canal space.     

Allogenic tooth transplantation inhibits the maintenance of dental pulp stem/progenitor cells in mice

Our recent study suggested that allogenic tooth transplantation may affect the maintenance of dental pulp stem/progenitor cells. This study aims to elucidate the influence of allograft on the maintenance of dental pulp stem/progenitor cells following tooth replantation and allo- or auto-genic tooth transplantation in mice using BrdU chasing, immunohistochemistry for BrdU, nestin and Ki67, in situ hybridization for Dspp, transmission electron microscopy and TUNEL assay. Following extraction of the maxillary first molar in BrdU-labeled animals, the tooth was immediately repositioned in the original socket, or the roots were resected and immediately allo- or auto-grafted into the sublingual region in non-labeled or the same animals. In the control group, two types of BrdU label-retaining cells (LRCs) were distributed throughout the dental pulp: those with dense or those with granular reaction for BrdU. In the replants and autogenic transplants, dense LRCs remained in the center of dental pulp associating with the perivascular environment throughout the experimental period and possessed a proliferative capacity and maintained the differentiation capacity into the odontoblast-like cells or fibroblasts. In contrast, LRCs disappeared in the center of the pulp tissue by postoperative week 4 in the allografts. The disappearance of LRCs was attributed to the extensive apoptosis occurring significantly in LRCs except for the newly-differentiated odontoblast-like cells even in cases without immunological rejection. The results suggest that the host and recipient interaction in the allografts disturbs the maintenance of dense LRCs, presumably stem/progenitor cells, resulting in the disappearance of these cell types.     

Responses of immunocompetent cells in the dental pulp to replantation during the regeneration process in rat molars

Responses of immunocompetent cells to tooth replantation during the regeneration process of the dental pulp in rat molars were investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6 antibody), monocyte/macrophage lineage cells (ED1 antibody) and protein gene product 9.5 (PGP 9.5), as well as by histochemical reaction for periodic acid-Schiff (PAS). Tooth replantation caused an increase in both the number of OX6- and ED1-positive cells and their immunointensity in the replanted pulp, but almost all PGP 9.5-immunoreactive nerves diminished in the initial stages. By postoperative day 3, many OX6- and ED -immunopositive cells had accumulated along the pulp-dentin border to extend their cytoplasmic processes into the dentinal tubules in successful cases. Once reparative dentin formation had begun after postoperative day 7, OX6- and ED1-immmunopositive cells became scattered in the odontoblast layer, while reinnervation was found in the coronal pulp. The temporal appearance of these immunocompetent cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defense reactions during the pulpal regeneration process. On postoperative day 14, the replanted pulp showed three regeneration patterns: (1) reparative dentin, (2) bone-like tissue formation, and (3) an intermediate form between these. In all cases, PAS-reactive cells such as polymorphonuclear leukocytes (PML) and mesenchymal cells occurred in the pulp space. However, the prolonged stagnation of inflammatory cells was also discernible in the latter two cases. Thus, the findings on PAS reaction suggest that the migration of the dental follicle-derived cells into the pulp space and the subsequent total death of the proper pulpal cells are decisive factors for eliciting bone-like tissue formation in the replanted pulp.     

The role of angiogenesis and pulpal healing in tooth replantation and allograft transplantation

Tooth transplantation is one of the treatment options for extracted teeth that can be considered before dental implantation. Although the success rate of tooth transplantation is lower than that of implantation, tooth replantation and transplantation have the great advantage of using natural teeth. Tooth replantation might be considered a promising option in some cases. In present study, the expression patterns of revascularization and pulpal healing, which are the most important for the pulp viability, were analyzed after tooth replantation and allograft in mice. The inflammatory response and root dentin resorption were observed and not different between replantation and allograft in initiation of healing process. However, bonelike tissue formation, pulp revascularization and pulp healing were faster in replantation. The difference of healing patterns between tooth replantation and allograft found in present study will be helpful to select the treatment option and to understand healing mechanism.     

Analysis of the odontogenic and osteogenic potentials of dental pulp in vivo using a Col1a1-2.3-GFP transgene

Recently, transgenic mice that carry a Green Fluorescent Protein (GFP) reporter gene fused to 2.3 kb fragment of rat Col1a1 regulatory sequences (pOBCol2.3GFPemd) were generated. In the present study, we have examined the patterns of expression of Col1a1-2.3-GFP during odontoblast differentiation in this transgenic line. We report that Col1a1-2.3-GFP is expressed in newly differentiated odontoblasts secreting predentin and fully differentiated odontoblasts. The pattern of expression of Col1a1-2.3-GFP in odontoblasts is correlated with that of dentin sialophosphoprotein (DSPP). Col1a1-2.3-GFP is also expressed in the osteoblasts and osteocytes of alveolar bone. The pattern of expression of Col1a1-2.3-GFP in osteocytes is correlated with the expression of Dmp1. These observations indicate the 2.3 kb rat Col1a1 promoter fragment has sufficient strength and specificity to monitor the stage-specific changes during both odontoblast and osteoblast differentiation. We also used coronal pulp tissues isolated from postnatal pOBCol2.3GFPemd transgenic animals to follow their differentiation after transplantation under the kidney capsule. Our observations provide direct evidence that the dental pulp contains competent progenitor cells capable of differentiating into new generations of odontoblast-like cells which express high levels of Col1a1-2.3-GFP and DSPP and secrete tubular containing reparative dentin. We also report that the dental pulp is capable of giving rise to atubular bone-like tissue containing osteocytes expressing high levels of Col1a1-2.3-GFP and Dmp1. Our studies indicate that pOBCol2.3GFPemd transgenic animals provide a powerful tool for direct examination of the underlying mechanisms and the signaling pathways involved in dentin regeneration and repair, stem cell properties and heterogeneity of the dental pulp.     

Bone morphogenetic proteins in dentin regeneration for potential use in endodontic therapy

The human dentition is indispensable for nutrition and physiology. The teeth have evolved for mastication of food. Caries is a common dental problem in which the dentin matrix is damaged. When the caries is deep and the dental pulp is exposed, the pulp has to be removed in many cases, resulting ultimately in loss of the tooth. Therefore, the regeneration of dentin-pulp complex is the long-term goal of operative dentistry and endodontics. The key elements of dentin regeneration are stem cells, morphogens such as bone morphogenetic proteins (BMPs) and a scaffold of extracellular matrix. The dental pulp has stem/progenitor cells that have the potential to differentiate into dentin-forming odontoblasts in response to BMPs. Pulpal wound healing consists of stem/progenitor cells release from dental pulp niche after noxious stimuli such as caries, migration to the injured site, proliferation and differentiation into odontoblasts. There are two main strategies for pulp therapy to regenerate dentin: (1) in vivo method of enhancing the natural healing potential of pulp tissue by application of BMP proteins or BMP genes, (2) ex vivo method of isolation of stem/progenitor cells, differentiation with BMP proteins or BMP genes and transplantation to the tooth. This review summarizes recent advances in application of BMPs for dentin regeneration and possible use in endodotic therapy.     

Cell autonomous requirement for TGF-beta signaling during odontoblast differentiation and dentin matrix formation

TGF-β subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-β signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-β signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2^fl/fl;Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-β2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-β signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.     

Histochemical and immunocytochemical study of hard tissue formation in dental pulp during the healing process in rat molars after tooth replantation

Dental pulp is assumed to possess the capacity to elaborate both bone and dentin matrix under the pathological conditions following tooth injury. This study was undertaken to clarify the mechanism inducing bone formation in the dental pulp by investigating the pulpal healing process, after tooth replantation, by micro-computed tomography (μ-CT), immunocytochemistry for heat-shock protein (HSP)-25 and cathepsin K (CK), and histochemistry for both alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). Under deep anesthesia, the upper right first molar of 4-week-old Wistar rats was extracted and immediately repositioned in the original socket. In control teeth at this age, the periphery of the coronal dental pulp showed intense ALP-positive and HSP-25-positive reactions, whereas there were no TRAP-positive or CK-positive cells. Tooth replantation weakened or terminated ALP-positive and HSP-25-positive reactions in the pulp tissue at the initial stages. At 3–7 days after operation, the ALP-positive region recovered from the root apex to the coronal pulp followed by HSP-25-positive reactions in successful cases showing tertiary dentin formation. In other cases, TRAP-positive and CK-positive cells appeared in the pulp tissue of the replanted tooth at postoperative days 5–10 and remained associated with the bone tissue after 12–60 days. Immunoelectron microscopy clearly demonstrated that CK-positive osteoclast-lineage cells made contact with mesenchymal cells with prominent nucleoli and well-developed cell organelles. These data suggest that the appearance of TRAP-positive and CK-positive cells is involved in the induction of bone tissue formation in dental pulp.This work was supported in part by a grant from MEXT to promote 2001-multidisciplinary research project (in 2001–2005) and by KAKENHI (B) from MEXT, Japan (no. 16390523 to H.O.).     

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.     

Establishment of in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells

We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel’s method. We succeeded in establishing an in vitro culture system for evaluating dentin–pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1–3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp–dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.     

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.