Inactivation of Fam20C in Cells Expressing Type I Collagen Causes Periodontal Disease in Mice

Background

FAM20C is a kinase that phosphorylates secretory proteins. Previous studies have shown that FAM20C plays an essential role in the formation and mineralization of bone, dentin and enamel. The present study analyzed the loss-of-function effects of FAM20C on the health of mouse periodontal tissues.

Methods

By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20C^fl/fl mice, we created 2.3 kb Col 1a1-Cre;Fam20C^fl/fl (cKO) mice, in which Fam20C was inactivated in the cells that express Type I collagen. We analyzed the periodontal tissues in the cKO mice using X-ray radiography, histology, scanning electron microscopy and immunohistochemistry approaches.

Results

The cKO mice underwent a remarkable loss of alveolar bone and cementum, along with inflammation of the periodontal ligament and formation of periodontal pockets. The osteocytes and lacuno-canalicular networks in the alveolar bone of the cKO mice showed dramatic abnormalities. The levels of bone sialoprotein, osteopontin, dentin matrix protein 1 and dentin sialoprotein were reduced in the Fam20C-deficient alveolar bone and/or cementum, while periostin and fibrillin-1 were decreased in the periodontal ligament of the cKO mice.

Conclusion

Loss of Fam20C function leads to periodontal disease in mice. The reduced levels of bone sialoprotein, osteopontin, dentin matrix protein 1, dentin sialoprotein, periostin and fibrillin-1 may contribute to the periodontal defects in the Fam20C-deficient mice.     

Exposure to time varying magnetic fields associated with magnetic resonance imaging reduces fentanyl-induced analgesia in mice

The effects of exposure to clinical magnetic resonance imaging (MRI) on analgesia induced by the mu opiate agonist, fentanyl, was examined in mice. During the dark period, adult male mice were exposed for 23.2 min to the time-varying (0.6 T/sec) magnetic field (TVMF) component of the MRI procedure. Following this exposure, the analgesic potency of fentanyl citrate (0.1 mg/kg) was determined at 5, 10, 15, and 30 min post-injection, using a thermal test stimulus (hot-plate 50 degrees C). Exposure to the magnetic-field gradients attenuated the fentanyl-induced analgesia in a manner comparable to that previously observed with morphine. These results indicate that the time-varying magnetic fields associated with MRI have significant inhibitory effects on the analgesic effects of specific mu-opiate-directed ligands.     

Primary structure and phosphorylation of dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP) uniquely determine their role in biomineralization

The SIBLING (small integrin-binding ligand N-linked glycoproteins) family is the major group of noncollagenous proteins in bone and dentin. These extremely acidic and highly phosphorylated extracellular proteins play critical roles in the formation of collagenous mineralized tissues. Whereas the lack of individual SIBLINGs causes significant mineralization defects in vivo, none of them led to a complete cessation of mineralization suggesting that these proteins have overlapping functions. To assess whether different SIBLINGs regulate biomineralization in a similar manner and how phosphorylation impacts their activity, we studied the effects of two SIBLINGs, dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP), on mineral morphology and organization in vitro. Our results demonstrate distinct differences in the effects of these proteins on mineralization. We show that phosphorylation has a profound effect on the regulation of mineralization by both proteins. Specifically, both phosphorylated proteins facilitated organized mineralization of collagen fibrils and phosphorylated DMP1-induced formation of organized mineral bundles in the absence of collagen. In summary, these results indicate that the primary structure and phosphorylation uniquely determine functions of individual SIBLINGs in regulation of mineral morphology and organization.     

Mutant DLX 3 disrupts odontoblast polarization and dentin formation

Tricho-dento-osseous (TDO) syndrome is an autosomal dominant disorder characterized by abnormalities in the thickness and density of bones and teeth. A 4-bp deletion mutation in the Distal-Less 3 (DLX3) gene is etiologic for most cases of TDO. To investigate the in vivo role of mutant DLX3 (MT-DLX3) on dentin development, we generated transgenic (TG) mice expressing MT-DLX3 driven by a mouse 2.3 Col1A1 promoter. Dentin defects were radiographically evident in all teeth and the size of the nonmineralized pulp was enlarged in TG mice, consistent with clinical characteristics in patients with TDO. High-resolution radiography, microcomputed tomography, and SEM revealed a reduced zone of mineralized dentin with anomalies in the number and organization of dentinal tubules in MT-DLX3 TG mice. Histological and immunohistochemical studies demonstrated that the decreased dentin was accompanied by altered odontoblast cytology that included disruption of odontoblast polarization and reduced numbers of odontoblasts. TUNEL assays indicated enhanced odontoblast apoptosis. Expression levels of the apoptotic marker caspase-3 were increased in odontoblasts in TG mice as well as in odontoblastic-like MDPC-23 cells transfected with MT-DLX3 cDNA. Expression of Runx2, Wnt 10A, and TBC1D19 colocalized with DLX3 expression in odontoblasts, and MT-DLX3 significantly reduced expression of all three genes. TBC1D19 functions in cell polarity and decreased TBC1D19 expression may contribute to the observed disruption of odontoblast polarity and apoptosis. These data indicate that MT-DLX3 acts to disrupt odontoblast cytodifferentiation leading to odontoblast apoptosis, and aberrations of dentin tubule formation and dentin matrix production, resulting in decreased dentin and taurodontism.In summary, this TG model demonstrates that MT-DLX3 has differential effects on matrix production and mineralization in dentin and bone and provides a novel tool for the investigation of odontoblast biology.     

FAM20C Plays an Essential Role in the Formation of Murine Teeth

FAM20C is highly expressed in bone and tooth. Previously, we showed that Fam20C conditional knock-out (KO) mice manifest hypophosphatemic rickets, which highlights the crucial roles of this molecule in promoting bone formation and mediating phosphate homeostasis. In this study, we characterized the dentin, enamel, and cementum of Sox2-Cre-mediated Fam20C KO mice. The KO mice exhibited small malformed teeth, severe enamel defects, very thin dentin, less cementum than normal, and overall hypomineralization in the dental mineralized tissues. In situ hybridization and immunohistochemistry analyses revealed remarkable down-regulation of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein in odontoblasts, along with a sharply reduced expression of ameloblastin and amelotin in ameloblasts. Collectively, these data indicate that FAM20C is essential to the differentiation and mineralization of dental tissues through the regulation of molecules critical to the differentiation of tooth-formative cells.     

Immunodetection of osteoadherin in murine tooth extracellular matrices

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.     

The isolation and partial characterization of a rat incisor dentin matrix polypeptide with in vitro chondrogenic activity

In vivo implants of demineralized dentin matrix into muscle induce the formation of bone within the muscle. As with bone matrix implants, the bone induction appears to follow a chondrogenic pathway. Outgrowth cells from explants of neonatal rat muscle respond to bone matrix, in vitro, by expressing a heightened synthesis of sulfated proteoglycans and type II collagen, phenotypic of cartilage. The in vitro cell culture system has been used as an assay to monitor the isolation of the factor responsible for expression of this phenotypic transformation. Soluble proteins extracted from rat incisor dentin matrix during demineralization with EDTA, and not precipitable with 1.0 M CaCl2, were active in the in vitro system. The active extract was fractionated by Sephacryl S-100 chromatography in 6 M guanidine HCl, isoelectric focusing in Immobilines, and by reverse phase high performance liquid chromatography. All fractions were assayed for activity at every stage. The final active fraction from the reverse phase chromatography on a Zorbax Poly-F column was purified to homogeneity, and yielded a single spot on two-dimensional gel electrophoresis. The component, RP-4, had pI 5.4-5.5, and an apparent Mr 6,000-10,000, based on globular protein standards. Maximal activity with respect to both sulfate incorporation into proteoglycan and production of type II collagen was in the 1.0-10 ng/ml range. The RP-4 had a unique amino-terminal amino sequence and was rich in Gly, Pro, Glx, and Ala residues. It was different from transforming growth factor-beta and the bone morphogenetic protein family of proteins in these essential features.     

Adenosine A2B receptors play an important role in bone homeostasis

Bone homeostasis is a finely regulated mechanism involving different molecular pathways including adenosine signaling. The aim of this study is to determine the bone phenotype of adenosine A2B receptor knockout (A2BRKO) mice and to measure their ability to form new bone. Moreover, we analyzed the functionality of osteoclasts and osteoblasts from A2BRKO mice. Microcomputed tomography (μCT) analysis revealed a decrease of bone substance, bone mineral density, and trabecular number in A2BRKO mice compared to the WT mice at the same age. We measured the new bone formation by injecting fluorescent markers: it was reduced in femur and tibia of A2BRKO mice compare to the WT. A2BRKO young mice have fewer osteoblasts and an increase of osteoclasts was measured in the hind limbs of young and adult mice. A2BRKO osteoclasts are also more active in vitro, showing an increase of pit formation in dentin discs. Surprisingly in mature osteoblasts from A2BRKO mice, we measured an increase of calcified matrix production, collagen deposition, and alkaline phosphatase activity. These results demonstrate that A2BR on osteoblasts and osteoclasts regulate bone homeostasis.     

Anti-cancer activity of nitrones in the ApcMin/+ model of colorectal cancer

Abstract

The nitrones of α-phenyl-tert-butyl nitrone (PBN) and 4-hydroxyl-PBN (4-OH-PBN) that have anti-cancer activity in models of liver cancer and glioblastomas were tested in the ApcMin/+ mouse model. Mice were administered PBN and 4-OH-PBN in drinking water and intestinal tumour size and number assessed after 3–4 months. Throughout the experiment, contrast-enhanced magnetic resonance imaging (MRI) was used to monitor colon tumours. MRI data showed a time-dependent significant increase in total colonic signal intensity in sham-treated mice, but a significant decrease for PBN-treated mice and slight decrease for 4-OHPBN treated mice, probably due to the limited water solubility of 4-OH-PBN. Final pathological and percentage survival data agreed with the MRI data. PBN had little effect on oxaliplatin-mediated killing of HCT116 colon cancer cells and caused only a slight decrease in the amount of active fraction caspase 3 in oxaliplatin-treated cells. PBN has significant anti-cancer activity in this model of intestinal neoplasia.     

Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization, and expanded cavities of pulp and root canal during postnatal tooth development

The dentin matrix protein-1 (DMP-1) gene is identified in odontoblasts during both embryonic and postnatal development. In vitro study suggests that this noncollagen acidic phosphoprotein plays a role in mineralization. However, deletion of the Dmp-1 gene has little effect on tooth development during embryogenesis. To address the role of DMP-1 in tooth during postnatal development, we analyzed changes of dentinogenesis in Dmp-1 null mice from 3 days after birth to 1 year. Here we show that Dmp-1 null mice postnatally develop a profound tooth phenotype characterized by a partial failure of maturation of predentin into dentin, enlarged pulp chambers, increased width of predentin zone with reduced dentin wall, and hypomineralization. The tooth phenotype of these mice is strikingly similar to that in dentin sialophosphoprotein (Dspp) null mice and shares some features of the human disease dentinogenesis imperfecta III. We have also demonstrated that DSPP levels are reduced in Dmp-1 null mice, suggesting that DSPP is probably regulated by DMP-1 during dentinogenesis. Finally, we show the absence or delayed development of the third molar in Dmp-1 null mice, which is probably secondary to defects in Dmp-1 null bone. Taken together, these studies suggest that DMP-1 is essential for later dentinogenesis during postnatal development.     

A role for magnetic susceptibility in synthetic computed tomography

PurposeRadiotherapy treatment planning based on magnetic resonance imaging (MRI) benefits from increased soft-tissue contrast and functional imaging. MRI-only planning is attractive but limited by the lack of electron density information required for dose calculation, and the difficulty to differentiate air and bone. MRI can map magnetic susceptibility to separate bone from air. A method is introduced to produce synthetic CT (sCT) through automatic voxel-wise assignment of CT numbers from an MRI dataset processed that includes magnetic susceptibility mapping.MethodsVolumetric multi-echo gradient echo datasets were acquired in the heads of five healthy volunteers and fourteen patients with cancer using a 3 T MRI system. An algorithm for CT synthesis was designed using the volunteer data, based on fuzzy c-means clustering and adaptive thresholding of the MR data (magnitude, fat, water, and magnetic susceptibility). Susceptibility mapping was performed using a modified version of the iterative phase replacement algorithm. On patient data, the algorithm was assessed by direct comparison to X-ray computed tomography (CT) scans.ResultsThe skull, spine, teeth, and major sinuses were clearly distinguished in all sCT, from healthy volunteers and patients. The mean absolute CT number error between X-ray CT and sCT in patients ranged from 78 and 134 HU.ConclusionSusceptibility mapping using MRI can differentiate air and bone for CT synthesis. The proposed method is automated, fast, and based on a commercially available MRI pulse sequence. The method avoids registration errors and does not rely on a priori information, making it suitable for nonstandard anatomy.     

Hippuric acid and 3-(3-hydroxyphenyl) propionic acid inhibit murine osteoclastogenesis through RANKL-RANK independent pathway

Nutritional factors influence bone development. Previous studies demonstrated that bone mass significantly increased with suppressed bone resorption in early life of rats fed with AIN-93G semi-purified diets supplemented with 10% whole blueberry (BB) powder for 2 weeks. However, the effects of increased phenolic acids in animal serum due to this diet on bone and bone resorption were unclear. This in vitro and in ex vivo study examined the effects of phenolic hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on osteoclastic cell differentiation and bone resorption. We cultured murine osteoclast (macrophage) cell line, RAW 264.7 cells, and hematopoietic osteoclast progenitor cells (isolated from 4-week-old C57BL6/J mice) with 50 ng/ml of receptor activator of nuclear factor κ-Β ligand (RANKL). Morphologic studies showed decreased osteoclast number with treatment of 2.5% mouse serum from BB diet–fed animals compared with those treated with serum from standard casein diet–fed mice in both RAW 264.7 cell and primary cell cultures. HA and 3-3-PPA, but not 3–4-PPA, had dose-dependent suppressive effects on osteoclastogenesis and osteoclast resorptive activity in Corning osteo-assay plates. Signaling pathway analysis showed that after pretreatment with HA or 3-3-PPA, RANKL-stimulated increase of osteoclastogenic markers, such as nuclear factor of activated T-cells, cytoplasmic 1 and matrix metallopeptidase 9 gene/protein expression were blunted. Inhibitory effects of HA and 3-3-PPA on osteoclastogenesis utilized RANKL/RANK independent mediators. The study revealed that HA and 3-3-PPA significantly inhibited osteoclastogenesis and bone osteoclastic resorptive activity.     

Sequence of protein expression of bone sialoprotein and osteopontin at the developing interface between repair cementum and dentin in human deciduous teeth

Experimental periodontal regeneration studies have revealed the weak binding of repair cementum to the root surface, whereas attachment of cementum to dentin preconditioned by odontoclasts appears to be superior. The aim of this study has been, therefore, to analyze the structural and partial biochemical nature of the interface that develops between resorbed dentin and repair cementum by using human deciduous teeth as a model. Aldehyde-fixed and decalcified tooth samples were embedded in acrylic or epoxy resins and sectioned for light and transmission electron microscopy. Antibodies against bone sialoprotein (BSP) and osteopontin (OPN), two noncollagenous proteins accumulating at hard tissue interfaces in bone and teeth, were used for protein A-gold immunocytochemistry. Light microscopy revealed a gradually increasing staining intensity of the external dentin matrix starting after the withdrawal of the odontoclast. Labeling for both BSP and OPN was first detected among the exposed collagen fibrils and in the intratubular dentin matrix when odontoclasts had withdrawn but mesenchymal cells were present. Subsequently, collagen fibrils of the repair cementum were deposited concomitantly with the appearance of labeling for BSP and OPN over the intratubular, intertubular, and peritubular dentin matrix. Labeled mineralization foci indicated the advancing mineralization front, and the collagenous repair matrix became integrated in an electron-dense organic material that showed labeling for BSP and OPN. Thus, no distinct planar interfacial matrix layer lies between the resorbed dentin and the repair cementum. The results suggest that odontoclasts precondition the dentin matrix such that the repair cementum becomes firmly attached.This study was supported by the Clinical Research Foundation (CRF) for the Promotion of Oral Health, University of Berne, Berne, Switzerland.     

Osteopontin negatively regulates parathyroid hormone receptor signaling in osteoblasts

Systemic hormonal control exerts its effect through the regulation of local target tissues, which in turn regulate upstream signals in a feedback loop. The parathyroid hormone (PTH) axis is a well defined hormonal signaling system that regulates calcium levels and bone metabolism. To understand the interplay between systemic and local signaling in bone, we examined the effects of deficiency of the bone matrix protein osteopontin (OPN) on the systemic effects of PTH specifically within osteoblastic cell lineages. Parathyroid hormone receptor (PPR) transgenic mice expressing a constitutively active form of the receptor (caPPR) specifically in cells of the osteoblast lineage have a high bone mass phenotype. In these mice, OPN deficiency further increased bone mass. This increase was associated with conversion of the major intertrabecular cell population from hematopoietic cells to stromal/osteoblastic cells and parallel elevations in histomorphometric and biochemical parameters of bone formation and resorption. Treatment with small interfering RNA (siRNA) for osteopontin enhanced H223R mutant caPPR-induced cAMP-response element (CRE) activity levels by about 10-fold. Thus, in addition to the well known calcemic feedback system for PTH, local feedback regulation by the bone matrix protein OPN also plays a significant role in the regulation of PTH actions.     

Divergent Resorbability and Effects on Osteoclast Formation of Commonly Used Bone Substitutes in a Human In Vitro-Assay

Bioactive bone substitute materials are a valuable alternative to autologous bone transplantations in the repair of skeletal defects. However, clinical studies have reported varying success rates for many commonly used biomaterials. While osteoblasts have traditionally been regarded as key players mediating osseointegration, increasing evidence suggests that bone-resorbing osteoclasts are of crucial importance for the longevity of applied biomaterials. As no standardized data on the resorbability of biomaterials exists, we applied an in vitro-assay to compare ten commonly used bone substitutes. Human peripheral blood mononuclear cells (PBMCs) were differentiated into osteoclasts in the co-presence of dentin chips and biomaterials or dentin alone (control) for a period of 28 days. Osteoclast maturation was monitored on day 0 and 14 by light microscopy, and material-dependent changes in extracellular pH were assessed twice weekly. Mature osteoclasts were quantified using TRAP stainings on day 28 and their resorptive activity was determined on dentin (toluidin blue staining) and biomaterials (scanning electron microscopy, SEM). The analyzed biomaterials caused specific changes in the pH, which were correlated with osteoclast multinuclearity (r = 0.942; p = 0.034) and activity on biomaterials (r = 0.594; p = 0.041). Perossal led to a significant reduction of pH, nuclei per osteoclast and dentin resorption, whereas Tutogen bovine and Tutobone human strikingly increased all three parameters. Furthermore, natural biomaterials were resorbed more rapidly than synthetic biomaterials leading to differential relative resorption coefficients, which indicate whether bone substitutes lead to a balanced resorption or preferential resorption of either the biomaterial or the surrounding bone. Taken together, this study for the first time compares the effects of widely used biomaterials on osteoclast formation and resorbability in an unbiased approach that may now aid in improving the preclinical evaluation of bone substitute materials.     

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.     

Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-beta, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-beta stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-beta, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-beta on bone cell populations are likely to be important in bone remodeling and fracture repair.     

Formation of bone-like tissue by dental follicle cells co-cultured with dental papilla cells

During tooth root formation, dental follicle cells (DFCs) differentiate into osteoblasts/cementoblasts when they are in contact with pre-existing dentin. Since some factors of dentin matrix were also produced by dental papilla cells (DPCs) and could induce DFCs differentiation, we hypothesized that DPCs can directly promote DFCs differentiation and that differentiation could occur in a co-culture model. To test this hypothesis, we investigated the characteristics of DFCs that are influenced by DPCs in an in vitro co-culture and in vivo heterotopic transplant model. One week into the co-culture, there were significant increases in the mRNA level of bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), bone sialoprotein (BSP) and osteocalcin (OCN), and a decrease of the receptor activator of nuclear factor κB ligand (RANKL). Additionally, the number of BMP2-, OPG-, BSP- and OCN-positive DFCs increased whereas RANKL-positive DFCs decreased. Three weeks after co-culture, DFCs produced calcified nodules, accompanied with increased sub-cellular organelles for protein synthesis and secretion. In the heterotopic transplant model, the adult male rats were used as hosts, DFCs were transplanted into the omentum. In vivo 5-week growth of DFCs in the presence of DPCs led to the formation of bone-like tissues, positive for BSP, OCN and BMP2. In contrast, DFCs alone led to fibrous-like tissues. These results indicated that in the absence of pre-existing dentin, DPCs can stimulate osteogenesis and inhibit osteoclastogenesis in DFCs and suggested a novel strategy to promote DFCs differentiation.