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Dentonin, a MEPE Fragment, Initiates Pulp-healing Response to Injury
1 Laboratoire de Réparation et Remodelage des Tissus Orofaciaux, EA 2496, Groupe Matrices Extracellulaires et Biominéralisation, Faculté de Chirurgie Dentaire, Université Paris 5, 1, rue Maurice Arnoux, 92120 Montrouge, France; Correspondence: * corresponding author, MgoldOd{at}aol.com
Phosphorylated extracellular matrix proteins, including matrix extracellular phosphoprotein (MEPE), are involved in the formation and mineralization of dental tissues. In this study, we evaluated the potential of Dentonin, a synthetic peptide derived from MEPE, to promote the formation of reparative dentin. Agarose beads, either soaked with Dentonin or unloaded, were implanted into the pulps of rat molars, and examined 8, 15, and 30 days after treatment. At day 8, Dentonin promoted the proliferation of pulp cells, as visualized by PCNA-labeling. RP59-positive osteoblast progenitors were located around the Dentonin-soaked beads. PCNA- and RP59-labeling were decreased at day 15, while osteopontin, weakly labeled at day 8, was increased at 15 days, but dentin sialoprotein was undetectable at any time. At 8 days, precocious reparative dentin formation occurred in pulps containing Dentonin-soaked beads, with formation slowing after 15 days. These results suggest that Dentonin affects primarily the initial cascade of events leading to pulp healing.
Key Words: Dentonin • MEPE • reparative dentin • pulp
The SIBLING family (Small Integrin-Binding LIgand, N-linked Glycoprotein) includes Dentin Sialophosphoprotein, cleaved immediately after secretion into Dentin Sialoprotein (DSP), and Dentin Phosphoprotein, Dentin Matrix Protein-1, Osteopontin (OPN), Bone Sialoprotein, and Matrix Extracellular Phosphoglycoprotein (MEPE), also called osteoblast/osteocyte factor 45 (OF45) (Fisher and Fedarko, 2003). In humans, the genes coding for all the SIBLINGs are located within a contiguous region on chromosome 4q21 (Petersen et al., 2000; Rowe et al., 2000; MacDougall et al., 2002). Because this is the critical locus for the dentin defects including dentinogenesis imperfecta types II and III, and dentin dysplasia type II, the SIBLINGs are suggested to play a role in dentin formation and mineralization (Qin et al., 2004). The exact roles of MEPE are still a matter of debate. This molecule has been shown to promote skeletogenesis and regeneration of bone in tibial fractures (Lu et al., 2004). MEPE is expressed in bone during the proliferation and early-maturation phases by fully differentiated osteoblasts (Argiro et al., 2001), with maximal expression during mineralization (Siggelkow et al., 2004). Other studies have suggested that MEPE is a mineralization inhibitor. MEPE is expressed at high levels in tumor-induced osteomalacia (Rowe et al., 2000; Bresler et al., 2004). Targeted disruption of the rodent homologue (OF45) results in increased bone formation and bone mass (Gowen et al., 2003). MEPE (OF45) is increased in Hyp mice, a murine model for X-linked hypophosphatemia (S Liu et al., 2005), and in human pathology as well (Quarles, 2003). The MEPE-derived C-terminal ASARM peptide is a candidate for defective mineralization (Rowe et al., 2005). In addition, MEPE has been implicated in phosphate homeostasis and bone metabolism (Jain et al., 2004; Berndt et al., 2005). It should also be noted that MEPE is also found at low levels in non-mineralized tissues, such as brain, lungs, human placenta, salivary glands, and in the proximal tubules of the adult kidney (Rowe et al., 2000; Ogbureke and Fisher, 2004, 2005). In dental tissues, MEPE is expressed in odontoblasts during odontogenesis (MacDougall et al., 2002), but is down-regulated as dental pulp stem cells differentiate (H Liu et al., 2005). The central portion of MEPE (residues 242-264) includes an integrin-binding triplet (RGD sequence, residues 247–249), a glycosaminoglycan-attachment (SGDG, residues 256–259), and a putative calcium-binding motif. The synthetic peptide corresponding to this central fragment is known as Dentonin, or AC-100 (TDLQERGDNDISPFSGDGQPFKD). Dentonin stimulates the proliferation of human bone marrow stromal cells and their differentiation into osteoblast precursors (Nagel et al., 2004). This peptide stimulates new bone formation both in vitro and in vivo (Hayashibara et al., 2004), and enhances dental pulp stem cell proliferation in vitro (Liu et al., 2004). Therefore, the peptide may have some unique biological activities compared with those of the whole molecule. Several bioactive molecules have been studied in vivo by means of direct implantation with appropriate carriers into the dental pulp of rats, for the evaluation of their potential in the formation of reparative dentin (Goldberg et al., 2001; Goldberg and Smith, 2004). Since Dentonin has been shown to promote the proliferation of dental pulp stem cells, a first step in pulp repair (Liu et al., 2004), we investigated the effects of this peptide in our pulp implantation model.
Five milligrams of Dentonin (Acologix, Hayward, CA, USA) were dissolved in a 0.5-mL solution of PBS. Affi-gel agarose microbeads (70–150 µm in diameter, BioRad, Hercules, CA, USA) were incubated in the Dentonin-PBS solution for 1 hr at 37°C. Each bead absorbed approximately 1 µg Dentonin.
Operative Procedure and Bead Implantation Fifty maxillary molars from 25 Sprague-Dawley rats, aged 6 wks, were used. The experiment was carried out according to regulations for animal care, and was approved by the Scientific Committee of the Dental Faculty. The control group included 12 teeth implanted with agarose beads alone. Sham preparations, calcium hydroxide, and implantation of other bioactive molecules, according to these same methods, and on the same strain of rats in our laboratory, have been previously reported and therefore were not repeated (Decup et al., 2000; Six et al., 2002a,b, 2004). The experimental group of 32 teeth was implanted with Dentonin-soaked agarose beads, and the cavities were filled with a light-cured glass-ionomer cement (Fuji II, GC Corporation, Tokyo, Japan). From 4 to 6 beads were implanted per pulp. Both groups were distributed into 3 subgroups, to be evaluated at 8, 15, and 30 days, respectively. The control and experimental molars were processed for light microscopy after demineralization. In addition, 6 undemineralized Dentonin-implanted molars (2 for each period of time) were prepared for EDS/x-ray microanalysis coupled to a SEM (EDAX, Ameteck, Mahwah, NJ, USA).
Specimen Preparation for Evaluation by Light Microscopy
Immunohistochemical Evaluation Proliferation was evaluated by immunodetection of the proliferating cell nuclear antigen (PCNA) (Celis and Celis, 1985; Robbins et al., 1987), with the use of a mouse monoclonal PCNA antibody (PC-10, Dako, Glostrup, Denmark), at 1/75 dilution in PBS-1% BSA. PCNA-positive cells were scored on 700-mm2 areas of photomicrographs printed at 250x magnification, from control and experimental pulps at 8 and 15 days. At day 30, reparative dentin areas masked cellular events, and therefore this period was not included in the immunohistochemical evaluation. Adjacent sections were labeled with the following primary antibodies: a purified rabbit polyclonal antibody raised against Dentonin (Pab 1480-2b, gift of Acologix, Hayward, CA, USA) at 1/100 dilution in PBS-1% BSA; rabbit anti-RP59, a marker for osteoblast progenitors, implicated in osteoblast recruitment (Wurtz et al., 2001) (kindly provided by Dr. T. Wurtz, Paris 7, France), at 1/500 dilution; and anti-osteopontin (OPN) (LF 153) and anti-dentin sialoprotein (DSP) (LF153), at 1/100 dilution (kindly provided by Dr. L. Fisher, NIDCR- NIH, Bethesda, MD, USA). The sections were further incubated with the secondary antibody using 1/100 dilution of peroxidase-conjugated goat anti-mouse IgG for the PCNA visualization, or a peroxidase-conjugated swine anti-rabbit IgG at 1/100 concentration for Dentonin, RP59, OPN, and DSP (Dako, Glostrup, Denmark). The antibody localization was visualized with a solution of 3-3' diamino-benzidine (DAB, Sigma, St. Louis, MO, USA) and H2O2 for 20 min. Controls consisted of samples where primary antisera were omitted from the labeling procedure.
Eight days after implantation, the Dentonin-implanted pulps showed a slight to moderate inflammatory reaction in the exposure area and around the dentin fragments, which were pushed inside the pulp by the steel probe during exposure (Figs. 1a, 1b  ). At this time interval, no significant difference in inflammatory responses was detectable between control and Dentonin-loaded beads. A thick ring of cells surrounded the Dentonin-loaded beads (Fig. 1c). Some distance away from the pulp exposure, precocious reparative dentin started as a thin layer deposited at the surface of dentin debris, or as dendritic bone-like structures within the pulp (Fig. 1b).
Dentonin was still detectable by immunostaining at the surfaces of the beads (Fig. 1d  ). In the experimental group, though immunopositive PCNA-labeled cells were seen throughout the coronal pulp, the high concentration of labeled cells was close to the beads (Fig. 1e). This is in contrast to the control pulp, where labeled cells were randomly distributed (APPENDIX). Relatively fewer of the pulp cells located around the Dentonin–soaked beads were positive for RP59 (Fig. 1f), as compared with the control pulps. Only a thin ring of cells located at the surfaces of the beads was labeled for OPN (Fig. 1g). The DSP antibody stained the odontoblast layer, but did not stain cells implicated in the reparative process (data not shown).
After 15 days, inflammation was generally completely resolved in most Dentonin specimens (Fig. 2a
At 30 days, either the pulps implanted with Dentonin were at the same stage of reparative dentin formation as seen at 15 days (Fig. 3a ), or the exposure site was filled massively by amorphous reparative dentin (Fig. 3b). Residual agarose beads and dentin debris were trapped in this structure (Fig. 3b). In some specimens, the formation of reparative dentin was limited and seen only close to the beads (Fig. 3c), or x-ray microanalysis revealed mineralized tissue closing the exposure site by forming a complete bridge 30 days after implantation (Figs. 3d, 3e).
The present results show that the implantation of beads in the exposed pulp, both with and without Dentonin, initially induced a mild inflammatory reaction and cell proliferation. After 8 days, the density of PCNA-labeled cells was the same in the Dentonin and control groups. However, proliferation was seen randomly throughout the pulp in the absence of Dentonin (APPENDIX), whereas in the Dentonin-loaded beads, cells proliferated around or near the beads. More specifically, in the experimental day 8 pulps, the RP59 antibody labeled many cells, and OPN only a few. Most of the labeled cells were located at the surfaces of the Dentonin-soaked beads. The reverse occurred at day 15, with RP59 being decreased, whereas OPN labeling was increased, suggesting that committed progenitors are required to differentiate into cells of the osteoblast lineage. RP59 has been reported to be expressed by bone marrow cells, mesenchymal cells, nucleated erythroblasts, and megacaryocytes, and is mostly related to osteoblast recruitment (Wurtz et al., 2001; Krüger et al., 2002). Analysis of the present data suggests that Dentonin has the capacity to recruit progenitor cells. It is possible that Dentonin stimulates latent adult stem pulp cells to produce both PCNA-positive cell populations of proliferative inflammatory cells and RP59-positive progenitors involved in pulp repair. Alternatively, cells migrating from other reservoirs, such as CD-45-labeled cells (cells of the leukocyte lineage), may become reoriented by Dentonin to an osteo-chondrogenic lineage (Lacerda-Pinheiro et al., 2006). In adult rats, osteopontin is mostly a marker of the osteoblastic lineage. DSP is expressed mostly by odontoblasts, but also at a low rate by osteoblasts (Qin et al., 2004). At day 15, the DSP antibody did not stain the reparative cells, suggesting that cells stimulated to form by Dentonin are not of odontoblastic lineage. Indeed, in the Dentonin-treated teeth, bone-like trabeculae started to form as early as day 8, with additional trabeculae at day 15. Later, a heterogeneous reaction was observed, with some teeth displaying a thick dentinal bridge, while others remained unchanged after day 15. These results suggest that Dentonin primarily functions during the first steps of repair—the recruitment, proliferation, and early differentiation of osteoblast-like progenitors—and appears to exert less control over terminal differentiation into cells producing mineralized tissue. In our experimental model, we have used an approach to pulp repair that involves the use of bioactive molecules with the capacity to create a local microenvironment, or with chemo-attractive properties, leading undifferentiated latent progenitors toward terminal differentiation. Various molecules tested in this system have differing and unique mechanisms in the stimulation of pulp repair (Goldberg et al., 2006). In the initial response to pulp injury, Dentonin provoked a response faster than that of other bioactive molecules studied in our laboratory. However, after this initial response, Dentonin did not stimulate further mineralization, as evidenced by only a slight increase at day 30 as compared with day 15, in approximately half of our samples. This effect of Dentonin on dental pulp cell proliferation is consistent with results from in vitro studies that showed enhanced proliferation of dental pulp stem cells in culture (Liu et al., 2004). However, we did not find an effect of Dentonin on pulp cell terminal differentiation. This result is different from that found for bone, where Dentonin, identified as the synthetic central fragment of MEPE, increased proliferation of human marrow stromal cells, the expression of type I procollagen, alkaline phosphatase, and osteocalcin, and mineralized nodule formation (Nagel et al., 2004). The RGD and SGDG sequences may contribute to stimulate new bone formation (Hayashibara et al., 2004), and may be less effective in promoting reparative dentin formation. In conclusion, these studies showed that Dentonin stimulated the two initial steps of pulp repair: cell recruitment and cell proliferation. We did not find evidence for the role of Dentonin as a mineralization inhibitor. While it did not directly promote pulp cell late differentiation, early differentiation appeared to be enhanced, possibly in response to enhanced pulp cell proliferation. We suggest that this peptide may be useful in clinical settings that would benefit from enhanced pulp cell proliferation.
Dr. Ngampis Six was supported by le Fondation de la Recherche Médicale, research grants from Acologix, and from the French Institut for Dental Research (IFRO). Research support for P. DenBesten was from NIDCR grant #P60DE013058.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication April 21, 2006. Revision received March 17, 2007. Accepted for publication March 19, 2007.
Journal of Dental Research, Vol. 86, No. 8,
780-785 (2007) This article has been cited by other articles:
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