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.     

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.     

Stem cells and tooth tissue engineering

The notion that teeth contain stem cells is based on the well-known repairing ability of dentin after injury. Dental stem cells have been isolated according to their anatomical locations, colony-forming ability, expression of stem cell markers, and regeneration of pulp/dentin structures in vivo. These dental-derived stem cells are currently under increasing investigation as sources for tooth regeneration and repair. Further attempts with bone marrow mesenchymal stem cells and embryonic stem cells have demonstrated the possibility of creating teeth from non-dental stem cells by imitating embryonic development mechanisms. Although, as in tissue engineering of other organs, many challenges remain, stem-cell-based tissue engineering of teeth could be a choice for the replacement of missing teeth in the future.     

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.     

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).     

Collagenase synthesis in clonal rabbit odontoblast-like cells (RP cells)

Post-confluent cultures of cloned rabbit odontoblast-like cells (RP: Rabbit Pulp cells) produced latent collagenase, which was isolated from serum-containing culture media by heparin-Sepharose affinity chromatography. RP collagenase was purified 4,420-fold from serum-containing medium to a specific activity of 1,313 units/mg with a yield of 14% by heparin-Sepharose affinity chromatography, carboxymethyl-cellulose ion-exchange chromatography, and by immunoadsorption chromatography. The RP cell culture appears to be a suitable model to use for studying collagenase synthesis in mineralized tissue-forming cells and for elucidating the mechanism of collagen degradation in pulp tissue and dentin matrix.     

Evaluation of rabbit auricular chondrocyte isolation and growth parameters in cell culture

Auricular cartilage is an attractive potential source of cells for many tissue engineering applications. However, there are several requirements that have to be fulfilled in order to develop a suitable tissue engineered implant. Animal experiments serve as important tools for validating novel concepts of cartilage regeneration; therefore rabbit auricular chondrocytes were studied. Various parameters including isolation procedures, passage number, rate of proliferation and gene expression profile for major extracellular matrix components were evaluated in order to assess the potential use of elastic chondrocytes for tissue engineering. Chondrocytes were isolated from rabbit ear cartilage and grown in monolayer cultures over four passages. Yields of harvested cells and proliferation were analysed from the digestion step to the fourth passage, and changes in phenotype were monitored. The proliferation capacity of cell cultures decreased during cultivation and was accompanied by enlargement of cells, this phenomenon being especially evident in the third and fourth passages. The expression of cartilage specific genes for collagen type II, aggrecan and cartilage non-specific collagen type I was determined. The mRNA levels for all three genes were obviously lower in the primo culture than immediately after isolation. During subsequent cultivation the expression of collagen type II decreased further, while there were only slight changes in expression of aggrecan and collagen type I. This study provides a valuable basis for testing of different tissue engineering applications in rabbit model, where auricular chondrocytes are considered as cell source.     

Mesenchymal progenitor cells in adult human dental pulp and their ability to form bone when transplanted into immunocompromised mice

The technique of tissue engineering is developing for the restoration of lost tissues. This new technique requires cells that fabricate tissue. Mesenchymal stem cells in bone marrow have been used as the cell source for this technique; however, dental pulp cells have recently been shown to possess stem-cell-like properties. We earlier demonstrated that dental pulp cells proliferate and produce an extracellular matrix that subsequently becomes mineralized in vitro. We now report that such dental pulp cells (first to eighth passage) produced bone instead of dentin when those cells were implanted into subcutaneous sites in immunocompromised mice with HA/TCP powder as their carrier. This evidence shows that dental pulp cells are the common progenitors of odontoblasts and osteoblasts, or dental pulp cells are mesenchymal stem cells themselves. It is expected that dental pulp cells can be a useful candidate cell source for tissue engineering, and contain the potential of new therapeutic approaches for the restoration of damaged or diseased tissue.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

Isolation,Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells^1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation^6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers & also their differentiation capacity.Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,^11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 & CD44), hematopoietic/endothelial Markers (CD34, CD45 & CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) & Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE & dentin sialophosphoprotein; DSPP).¹⁴     

In vitro differentiation and mineralization of human dental pulp cells induced by dentin extract

Summary In this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], β-glycerophosphate [β-GP]) and MS+DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS+DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, β-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.     

Sequential use of human-derived medium supplements favours cardiovascular tissue engineering

For clinical application of tissue engineering strategies, the use of animal-derived serum in culture medium is not recommended, because it can evoke immune responses in patients. We previously observed that human platelet-lysate (PL) is favourable for cell expansion, but generates weaker tissue as compared to culture in foetal bovine serum (FBS). We investigated if human serum (HS) is a better human supplement to increase tissue strength. Cells were isolated from venous grafts of 10 patients and expanded in media supplemented with PL or HS, to determine proliferation rates and expression of genes related to collagen production and maturation. Zymography was used to assess protease expression. Collagen contraction assays were used as a two-dimensional (2D) model for matrix contraction. As a prove of principle, 3D tissue culture and tensile testing was performed for two patients, to determine tissue strength. Cell proliferation was lower in HS-supplemented medium than in PL medium. The HS cells produced less active matrix metallo-proteinase 2 (MMP2) and showed increased matrix contraction as indicated by gel contraction assays and 3D-tissue culture. Tensile testing showed increased strength for tissues cultured in HS when compared to PL. This effect was more pronounced if cells were sequentially cultured in PL, followed by tissue culture in HS. These data suggest that sequential use of PL and HS as substitutes for FBS in culture medium for cardiovascular tissue engineering results in improved cell proliferation and tissue mechanical properties, as compared to use of PL or HS apart.     

Cell aggregation and neurite growth in gels of extracellular matrix molecules

Components of the extracellular matrix are believed to guide both nerve cells and neurites to their targets during embryogenesis and, therefore, might be useful for controlling regeneration of nervous tissue in adults. To study the influence of extracellular conditions on neurite outgrowth and cell motility, PC12 cells were suspended in three-dimensional gels containing (i) collagen (0.4 to 2 mg/mL), (ii) collagen (1 mg/mL) with added fibronectin or laminin (1 to 100 mug/mL), and (iii) agarose (7 mg/mL) with added collagen (0.001 to 1 mg/mL). Neurite outgrwoth was stimulated with nerve growth factor (NGF) and both the extent of neurite outgrowth ad cell aggregation were quantitated over 10 to 12 days in culture. The extent of neurite outgrowth was greatest at the lowest collagen concentration tested (0.4 mg/mL) and decreased with increasing concentration. The addition of laminin or fibronectin altered the extent of neurite outgrowth in collagen gels, but the differences were small. Although no neurite growth was observed in pure agarose gels, considerable neurite outgrowth occurred with the addition of small amounts (>/=0.01 mg/mL) of collagen. Mean aggregate size increased more quickly in gels with lower concentrations of collagen. For cells in 1.0 mg/mL collagen, a four- to fivefold increase in aggregate volume was seen between days 2 and 10 o the culture period, whereas the increase in DNA content during this same period was less than twofold, suggesting that the cells were aggregating, not multiplying. These results suggest that the composition of the matrix supporting nerve cells has a significant effect on both neurite outgrowth and cell motility. (c) 1994 John Wiley & Sons, Inc.     

Three-dimensional culture of leech and snail ganglia for studies of neural repair

Three-dimensional (3D) collagen gels provide a stable matrix in which isolated regenerating ganglia from leech and snail can be maintained for studies of the molecular and cellular mechanisms underlying the regenerative process. Segmental ganglia from leech, or supraoesophageal, suboesophageal or buccal ganglia from snail were maintained for up to 3 weeks in 3D matrices of mammalian Type I collagen. The collagen matrix supports the regenerative outgrowth of axon tracts as well as the migration of microglial cells, important elements in the repair process. Proteins or soluble factors or target tissue may be added to the basic collagen matrix to manipulate the environment of the regenerating tissue. We describe techniques for immunostaining of regenerating axons and microglial cells within the gel matrix in combination with staining of cell nuclei, and the use of intracellular labelling to distinguish axons of identified neurons within the regenerative outgrowth.E.J. Babington and J. Vatanparast contributed equally     

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.     

Type I collagen regulated dentin matrix protein-1 (Dmp-1) and osteocalcin (OCN) gene expression of rat dental pulp cells

In this study, we investigated the effect of type I collagen on dentin matrix protein-1 (Dmp-1) and osteocalcin (OCN) gene expression of dental pulp cells. The mRNA level of Dmp-1 gene was down-regulated; however, OCN gene expression was up-regulated by the culture of dental pulp cells with type I collagen. These findings imply that type I collagen regulates mRNA level of Dmp-1 and OCN gene that are predominantly expressed in active odontoblasts. The change of gene expression by type I collagen was suppressed by the blocking of collagen-integrin interaction. We could conclude that the effect of type I collagen was mediated via binding of collagen to integrin receptors.     

In Vitro Reparative Dentin: a Biochemical and Morphological Study

In this study, starting from human dental pulp cells cultured in vitro, we simulated reparative dentinogenesis using a medium supplemented with different odontogenic inductors. The differentiation of dental pulp cells in odontoblast-like cells was evaluated by means of staining, and ultramorphological, biochemical and biomolecular methods. Alizarin red staining showed mineral deposition while transmission electron microscopy revealed a synthesis of extracellular matrix fibers during the differentiation process. Biochemical assays demonstrated that the differentiated phenotype expressed odontoblast markers, such as Dentin Matrix Protein 1 (DMP1) and Dentin Sialoprotein (DSP), as well as type I collagen. Quantitative data regarding the mRNA expression of DMP1, DSP and type I collagen were obtained by Real Time PCR. Immunofluorescence data demonstrated the various localizations of DSP and DMP1 during odontoblast differentiation. Based on our results, we obtained odontoblast-like cells which simulated the reparative dentin processes in order to better investigate the mechanism of odontoblast differentiation, and dentin extracellular matrix deposition and mineralization.Key words: dental tissue, in vitro differentiation, DMP1, DSP, type I collagen     

Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo.

Articular cartilage exhibits little intrinsic repair capacity, and new tissue engineering approaches are being developed to promote cartilage regeneration using cellular therapies. The goal of this study was to examine the chondrogenic potential of adipose tissue-derived stromal cells. Stromal cells were isolated from human subcutaneous adipose tissue obtained by liposuction and were expanded and grown in vitro with or without chondrogenic media in alginate culture. Adipose-derived stromal cells abundantly synthesized cartilage matrix molecules including collagen type II, VI, and chondroitin 4-sulfate. Alginate cell constructs grown in chondrogenic media for 2 weeks in vitro were then implanted subcutaneously in nude mice for 4 and 12 weeks. Immunohistochemical analysis of these samples showed significant production of cartilage matrix molecules. These findings document the ability of adipose tissue-derived stromal cells to produce characteristic cartilage matrix molecules in both in vitro and in vivo models, and suggest the potential of these cells in cartilage tissue engineering.     

TGF-beta3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development

Several members of the transforming growth factor (TGF)-beta superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-beta1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-beta3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-beta3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-beta3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-beta3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-beta3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-beta3 soaked beads in vivo. These results indicate for the first time that TGF-beta3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts.     

Matrix and TGF-β-related gene expression during human dental pulp stem cell (DPSC) mineralization

We have recently reported the induction of dental pulp stem cells (DPSCs) into dentin-secreting odontoblast-like cells after stimulation by isolated dentin matrix components, thus mimicking the nature of tissue regeneration seen after tooth disease and injury. After confluency, the cells were further cultured for 21 d in the 10% fetal bovine serum (FBS) Dulbecco’s modified Eagle’s medium (DMEM) (control), and in this medium, with the addition of dentin extract (DE) and the mineralization supplement (MS) of ascorbic acid and β-glycerophosphate (treatment). To identify genes associated with this process, specimens were analyzed with a HG-U133A human gene chip and Arrayassist software. A total of 425 genes, among them 21 matrix and eight TGF-β-related genes, were either up- or downregulated in the experimental group in which the cells showed odontoblast-like differentiation and mineralization. Expression of selected genes was further confirmed by real-time polymerase chain reaction (PCR) analysis. Of the extracellular matrix (ECM)-related genes, two types of collagen genes were upregulated and seven others downregulated. Other ECM-related genes, for example fibulin-1, tenascin C, and particularly thrombospondin 1, were upregulated, and fibulin-2 was downregulated. Most noticeably, the matrix metalloproteinase 1 was induced by the treatment. In the TGF-β superfamily, upregulation of the type II receptor, endoglin, and growth/differentiation factor 5 was coordinated with the downregulation of activin A, TGF-β2, and TGF-β1 itself. This study identifies the matrix and TGF-β-related gene profiles during the DPSC cell mineralization in which several genes are reported for the first time to be associated with this process, thus greatly expanding our molecular knowledge of the induced disease repair process.