Biol.

Cell (2008) 100, 291302 (Printed in Great Britain)

doi:10.1042/BC20070092

Research article

Dentin non-collagenous proteins (dNCPs) can stimulate dental follicle cells to differentiate into cementoblast lineages
Junjie Wu*1 , Fang Jin*1 , Liang Tang, Jinhua Yu, Lin Xu*, Zhenhua Yang*, Gang Wu, Yinzhong Duan*2 and Yan Jin2

www.biolcell.org Biology of the Cell

*Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xian, Shaanxi 710032, Peoples Republic of China, Department of Oral Histology and Pathology, School of Stomatology, Fourth Military Medical University, Xian, Shaanxi 710032, Peoples Republic of China, Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xian, Shaanxi 710032, Peoples Republic of China, Department of Endodontics, School of Stomatology, Nanjing Medical University, Nanjing, Jiangsu 210029, Peoples Republic of China, and Department of Endodontics, School of Stomatology, Fourth Military Medical University, Xian, Shaanxi 710032, Peoples Republic of China

Background information. Although the mechanism of cementogenesis is an area full of debate, the DFCs (dental follicle cells) are thought to be the precursors of cementoblasts. At the onset of cementogenesis, DFCs come into contact with the root dentin surface and undergo subsequent differentiation. But the exact effects of dentin or dentin matrix on DFCs remain an open question. In the present study, we hypothesized that dNCPs (dentin non-collagenous proteins) extracted from dentin could stimulate DFCs to differentiate into cementoblast lineages. Results. DFCs were isolated from tooth germs of SD (SpragueDawley) rats and then co-cultured with dNCPs. Treated DFCs presented several features of cementoblast lineages in vitro, as indicated by morphological changes, decreased proliferation, enhanced ALP (alkaline phosphatase) activity and increased matrix mineralization. The expression of mineralization-associated proteins and genes were up-regulated after induction, whereas the expression of specic markers of odontoblast were not detected. Incubation of treated DFC pellets in vivo revealed that a large amount of cementum-like tissues was formed within the novel dentin carriers, which were quite distinct from the newly formed osteodentin secreted by DPSCs (dental pulp stem cells). The negative expression of DSP (dentin sialoprotein) also excluded the possibility of producing dentin matrix by treated DFCs. Conclusions. dNCPs can stimulate DFCs to differentiate into cementoblast lineages. The present study provides new insights into the mechanism of cementogenesis.

authors contributed equally to this work. whom correspondence should be addressed (email fmmuwu@fmmu.edu.cn or yanjin@fmmu.edu.cn). Key words: cell differentiation, cementogenesis, dental follicle cell (DFC), dentin non-collagenous protein, periodontal development. Abbreviations used: ALP, alkaline phosphatase; BMP, bone morphogenetic protein; BrdU, bromodeoxyuridine; BSP, bone sialoprotein; Col I, type I collagen; DFC, dental follicle cell; DMP-1, dentin matrix protein 1; dNCP, dentin non-collagenous protein; DPP, dentin phosphoprotein; DPC, dental pulp cell; DPSC, dental pulp stem cell; DSP, dentin sialoprotein; DSPP, dentin sialophosphoprotein; EMD, enamel matrix derivative; EMSC, ectomesenchymal stem cell; HERS, Hertwigs epithelial root sheath; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H -tetrazolium bromide; OCN, osteocalcin; ON, osteonectin; RER, rough endoplasmic reticulum; RT, reverse transcription; SD, SpragueDawley; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
2 To

1 These

Introduction Cementogenesis is one of the most important processes in periodontal development and regeneration. Cementum matrix is secreted by highly differentiated cells called cementoblasts. However, the origin of cementoblasts remains a matter of debate (Hammarstr om et al., 1996). To date, several theories have been proposed about this intriguing issue. The classical theory suggests that DFCs (dental follicle cells) give birth to cementoblasts and secrete cementum matrix after penetrating through the

www.biolcell.org | Volume 100 (5) | Pages 291302

291

J. Wu and others

barrier of HERS (Hertwigs epithelial root sheath) (Furseth et al., 1986; Schroeder, 1986a; Provenza, 1988). Another hypothesis considers that cementoblasts are derived from HERS cells by epithelial mesenchymal transformation (Stahl and Slavkin, 1972; Slavkin, 1976; Bosshardt and Nanci, 1997, 2004; Bosshardt, 2005). Besides the above, it is assumed that the proteins (including enamel matrix protein and others) secreted by HERS cells affect cell migration, attachment and function leading to cementogenesis (Gestrelius et al., 2000; Zeichner-David, 2001, 2006). Regardless of the different theories, it has been well recognized that DFCs penetrate disintegrated HERS and come into contact with the root dentin surface prior to any cementum formation (see Supplementary Figure 1 at http://www.biolcell.org/boc/100/ boc1000291add.htm). Therefore it would be of great interest to investigate the effects of dentin or dentin matrix on DFCs. dNCPs (dentin non-collagenous proteins), the mixed proteins extracted from dentin, comprise glycoproteins/sialoproteins, phosphoproteins, proteoglycans and growth factors (Smith and Leaver, 1979), which have been proved to be involved in the nucleation process and hydroxyapatite growth during dentin formation (Butler, 1998; Butler et al., 2002). Moreover, dNCPs can act as an inducing agent in promoting cell differentiation; for instance, they can induce DPCs (dental pulp cells), DPSCs (dental pulp stem cells) and even EMSCs (ectomesenchymal stem cells) to differentiate into odontoblast-like cells (Deng et al., 2005; Liu et al., 2005; Nie et al., 2006). Based on the direct dentincell contact and the inducing ability of dNCPs, we have proposed in the present study that dNCPs could stimulate DFCs to differentiate into cementoblasts. To test this hypothesis, we investigated the biological effects of dNCPs on the proliferation and differentiation of DFCs through in vitro and in vivo experiments. The present study provides a better understanding of the regulation mechanism of cementogenesis in periodontal development and regeneration.

Figure 1 Effect of dNCPs on the morphological changes of DFCs (A, B) Phase-contrast microscopy of DFCs. (C, D) SEM of DFCs. (A, C) Untreated DFCs at day 6 were polymorphic in shape, and the predominant cells were broblastic in shape, elongated with processes at each pole. (B, D) Treated DFCs at day 6 became at, cuboidal or polygonal in shape. (E) TEM of untreated DFCs at day 6. (F) Enlargement of the boxed area in (E). Arrows indicate the electron-dense granules, which have been regarded as one of the markers for identication of DFCs. (G) TEM of treated DFCs at day 6. (H) Enlargement of the boxed area in (G). Arrows indicate a lot of distended RER within the cytoplasm; the arrowhead indicates mitochondrion adjacent to RER. Abbreviation: N, nucleus.

Results
Effect of dNCPs on the morphological changes of DFCs

Observed under a phase-contrast microscope, the untreated DFCs of the 3rd passage at day 6 appeared

polymorphic, but the predominant cells were broblastic in shape, elongated with two processes at each pole (Figure 1A). In the treated group, after the induction of dNCPs for 6 days, DFCs became atter and most of them were cuboidal or polygonal (Figure 1B). To examine the ultramicrostructure of DFCs, a scanning electron microscope and a transmission

292

The Authors Journal compilation

2008 Portland Press Ltd

Differentiation of DFCs induced by dNCPs

Research article
pared with treated cells (25.9%; Figure 2C), indicating that dNCPs could inhibit the proliferation of DFCs (2 = 13.01, P < 0.01).
Effect of dNCPs on ALP (alkaline phosphatase) activity and mineralization behaviour of DFCs

electron microscope were used for the observation. The SEM (scanning electron microscopy) results were in agreement with the above light-microscope ndings, showing that most of the untreated DFCs were elongated, spindle-shaped and displayed abundant microvilli on the cell surface (Figure 1C), whereas the treated cells closely adherent to the substrate appeared cuboidal or polygonal in shape with multiprocesses (Figure 1D). In addition, compared with the control, the cell-surface microvilli were greatly decreased in the treated cells around which some extracellular matrix was secreted (Supplementary Figure 2A at http://www.biolcell.org/boc/100/ boc1000291add.htm). As to the TEM (transmission electron microscopy) observations, the untreated cells demonstrated the typical ultramicrostructure of a mesenchymal broblast except that electron-dense granules could be occasionally observed adjacent to the RER (rough endoplasmic reticulum), which has been regarded as one of the markers for identifying DFCs (Wise et al., 1992) (Figures 1E and 1F). However, inside the treated cells, the RER frequently appeared distended and contained occulent material, implying the potent function of protein synthesis and secretion (Figures 1G and 1H). The extracellular matrix could be found around the treated cells by TEM (Supplementary Figure 2B).
Effect of dNCPs on the proliferation of DFCs

To quantify the differentiation ability, ALP activity and matrix mineralization were analysed at the dened time points. ALP activity was signicantly higher in the treated group than in the control group from day 5, reaching a peak at day 9, then declining gradually (Figure 3A). The mineralization was quantied by calculating the nodule area and nodule number after von Kossa staining. After treatment with dNCP for 21 days, the mineralized nodule area 2 was 3.82 + 0.26 mm per well and the nodule number reached 86.17 + 4.79 nodules per well; however, a little mineralized nodule was formed after 21 days of routine culture with the corresponding data as 2 0.16 + 0.06 mm per well and 12.00 + 3.00 nodules per well (Figures 3B3E).
Effect of dNCPs on protein and gene expression of DFCs

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, BrdU (bromodeoxyuridine) incorporation and cell-cycle analysis were performed to investigate whether dNCPs could affect cell proliferation in vitro. As shown in Figure 2(A), the treated DFCs exhibited signicantly lower attenuance (D) values than the control cells from day 5, suggesting that dNCPs may exert a suppressive effect on the proliferation of DFCs. The result of BrdU incorporation, which is representative of a signicant portion of cells in the S-phase, indicated that the number of BrdU-positive cells in the control group was higher than that in the treated group (Figure 2B). Cell-cycle distributions were analysed by ow cytometry in the form of proliferation index (PI; percentage of cells in the S- and G2 /M-phases). In the present study, it was revealed that untreated DFCs presented a signicantly higher percentage of cells in S+G2 /M phases (50.7%; Figure 2D) when com-

Using immunocytochemical staining and RT (reverse transcription)PCR, the expression of factors related to cementoblast differentiation of DFCs were examined at the protein and mRNA levels. Our results showed that treated cells, after a 6-day induction, stained positively for mineralization markers such as BSP (bone sialoprotein), ON (osteonectin), OCN (osteocalcin) and Col I (type I collagen) and stained negatively for DSP (dentin sialoprotein) and DMP-1 (dentin matrix protein 1), the specic markers of odontoblast lineages. Meanwhile, negative staining for all of the above proteins were detected in untreated cells (Figures 4A4L). To conrm the cementoblast phenotype of treated DFCs, RTPCR was performed to investigate the expression of DMP-1, DSPP (dentin sialophosphoprotein), ON, OCN, BSP and Col I genes. The gene expression of DSPP and DMP-1 could not be detected in both groups; however, ON, OCN, BSP and Col I genes could be clearly identied after dNCP induction (Figure 4M), thereby indicating that DFCs can differentiate along cementoblast lineages but not odontoblasts. The markers have been investigated in triplicate with identical results.

www.biolcell.org | Volume 100 (5) | Pages 291302

293

J. Wu and others

Figure 2 Effect of dNCPs on the proliferation of DFCs (A) Growth curves of treated and untreated DFCs analysed by the MTT assay. The proliferation of treated DFCs was inhibited compared with untreated cells. (B) The BrdU incorporation assay. The percentage of BrdU-positive cells in the experimental group was less than that in the control. (C) Representative cell-cycle distribution of treated DFCs. Here, 74.1% of the cells were arrested in G0 /G1 -phase, indicating the inhibitory effect on the treated group. (D) Representative cell-cycle distribution of untreated DFCs. Only 49.3% of the cells were arrested in G0 /G1 -phase. P < 0.01 compared with the untreated group.

Effect of dNCPs on the in vivo differentiation of DFCs

In order to evaluate the cementogenic capacity of DFCs in vivo, a novel cell carrier consisting of hard tissue (enamel outside and dentin inside) from the tooth germ was adopted (Figure 5A). After trypsinization, any remaining cells were not found to adhere to the carrier before loading of the cell pellet (Figure 5B). All of the cell pellets could be very easily loaded into the inside chamber of carriers and have a direct contact with predentin, imitating the developmental event during cementogenesis. After 4 weeks incubation under renal capsules of adult rats, all explants were harvested and ana-

lysed by means of histological examination. In the treated group, a large amount of mineralized tissues was formed within the chamber of the carrier, which could be apparently distinguished from the original dentin (Figures 5C5E). The newly mineralized tissues occupied the most space within the carrier, and only a limited central area was left for the growth of connective tissues and blood vessels. Some of the formative cells were entrapped within the newly formed tissues. However, in the control group, DFCs proliferated well, and only limited cementoid tissues were formed along the inner surface of dentin. There was a distinct demarcation line between the original dentin and the newly formed

294

The Authors Journal compilation

2008 Portland Press Ltd

Differentiation of DFCs induced by dNCPs

Research article

Figure 3 Effect of dNCPs on ALP activity and mineralization behaviour of DFCs (A) Comparison of ALP activity between treated (Exp) and untreated (Cont) DFCs. The experimental group showed an increased ALP activity. K.A. units, KingArmstrong units (Yu et al., 2006a). (B) Comparison of the mineralized nodule area between treated (Exp) and untreated (Cont) DFCs. After a 21-day induction, von Kossa staining was carried out to determine mineralized nodule formation. The nodule formed by treated DFCs occupied a signicantly larger area per well compared with the control. (C) Comparison of mineralized nodule number between treated (Exp) and untreated (Cont) DFCs. The nodule number per well in the experimental group was signicantly increased. (D) Representative mineralized nodule formed by treated DFCs (von Kossa staining). (E) Representative mineralized nodule formed by untreated DFCs (von Kossa staining). P < 0.01 compared with untreated group. Scale bar, 50 m.

mineralized tissues (Figures 5F and 5G). In order to quantify these ndings, the relative amount of newly formed cementum is expressed as the percentage of the area of the newly formed cementum against the chamber area of the dentin carrier (cross-section), and the ratio for the experimental group is signicantly higher than that for the control (Supplementary Figure 3 at http://www.biolcell.org/boc/100/ boc1000291add.htm). The results of uorescent tracing analyses further conrmed that the transplanted DFCs participated in mineralized tissue

formation rather than in angiogenesis (Supplementary Figure 4B at http://www.biolcell.org/boc/ 100/boc1000291add.htm). Furthermore, no positive staining against DSP could be detected in the newly formed mineralized tissues (Figure 5I). DPSCs were used as another control group besides untreated DFCs. Previous studies suggested that DPSCs had the ability to differentiate into odontoblasts (Yu et al., 2006a, 2007; Liu et al., 2007; Sloan and Smith, 2007). The results of DPSC transplantation reconrmed this idea by nding the fact

www.biolcell.org | Volume 100 (5) | Pages 291302

295

J. Wu and others

Figure 4 Effect of dNCPs on protein and gene expression of DFCs (A, C, E, G, I, K) Treated DFCs were immunopositive for BSP, ON, OCN and Col I, whereas they were negative for DMP-1 and DSP. (B, D, F, H, J, L) Untreated DFCs were negative for the above proteins. Scale bar, 50 m. (M) Electrophoresis of RTPCR products: Exp, treated DFCs; Cont, untreated cells. -Actin was used as an internal control for each group.

Figure 5 Effect of dNCPs on the in vivo differentiation of DFCs (A) Three components of molar tooth germ isolated from a 6-day-old SD rat. (a) Dental follicle and enamel organ; (b) hard tissue from tooth germ including developing dentin and enamel; (c) dental papilla. (B) Histological evaluation of the dentin carrier after trypsinization. The arrow indicates the developing dentin along the inner surface of hard tissue from tooth germ; the arrowhead indicates the remaining enamel along the outer surface. (C) Cross-section of explants of treated DFCs within the chamber of the dentin carrier after 4 weeks of in vivo incubation. Signicant cementum-like mineralized tissues were formed within the carrier. (D) Enlargement of the solid-boxed area in (C). The closed arrow indicates newly formed blood vessel; arrowheads indicate the mineral-forming cells differentiated from DFCs. (E) Enlargement of the dash-boxed area in (C). The closed arrow indicates newly formed blood vessel; open arrows indicate the formative cells residing in the lacunae. (F) Histological evaluation of explants of untreated DFCs within the chamber of the dentin carrier after 4 weeks of in vivo incubation. Limited mineralized tissues were formed along the dentin surface. (G) Enlargement of the boxed area in (F). (H) Histological evaluation of explants of DPSCs within the chamber of the dentin carrier after 4 weeks of in vivo incubation. Amorphous osteodentin (OD) was formed within the carrier. (I) Immunohistological evaluation of explants of treated DFCs within the chamber of the dentin carrier after 4 weeks of in vivo incubation. The dentin carrier was positive for DSP, whereas the new cementoid was negative; arrowheads indicate formative cells negative for DSP. (J) Immunohistological evaluation of explants of DPSCs within the chamber of the dentin carrier after 4 weeks in vivo incubation. Both the dentin carrier and osteodentin were positive for DSP. Abbreviations: E, enamel; D, dentin; NC, newly formed cementum.

296

The Authors Journal compilation

2008 Portland Press Ltd

Differentiation of DFCs induced by dNCPs

Research article
increased ALP activity could imply that the treated DFCs have been induced to mineral-forming cells. It is also worth noting that mineralized nodule formation signicantly increased after dNCP induction. Furthermore, in the investigated markers, DMP-1 and DSPP/DSP are specic for identifying odontoblasts, whereas ON, OCN, BSP and Col I are the common markers found in cementum and cementoblasts (DErrico et al., 1997). Previous studies have suggested that dentin matrix could induce EMSCs, DPSCs or DPCs into odontoblast-like cells (Deng et al., 2005; Liu et al., 2005; Nie et al., 2006). However, in the present study, treated DFCs could differentiate into cementoblasts rather than odontoblasts, as indicated by positive expression of ON, OCN, BSP and Col I, and negative expression of DSP and DMP-1 at the mRNA and protein levels. Taken together, it was revealed by our in vitro study that cementoblasts have possibly originated from DFCs, a process in which dentin matrix is critically involved. As to the in vivo experiments, the previous results of in vivo differentiation of DFCs were quite controversial. Bovine DFCs transplanted into immunodeciency mice formed brous tissue and cementum-like matrix (Handa et al., 2002). However, murine DFCs transformed with SV40 (simian virus 40) seeded on to a PLGA [poly(D,L-lactide-co-glycolic acid)] scaffold could not mediate mineral formation in vivo and even seemed to inhibit periodontal healing of articial periodontal defects in athymic rats (Zhao et al., 2002, 2004). As we know, a better understanding of the function of cells can be attained by re-transplantation of cells from the formative tissues (Song et al., 2007). Thus we used a novel model (cell pellet loaded in the chamber of the dentin carrier) and put it in the renal capsule of an adult rat, a transplantation site, which provides a suitable environment for DFC differentiation in vivo. It was found that, without the induction of dNCPs, a limited amount of cementoid was formed closely attached to the inner dentin surface, whereas after the induction, a lot of non-dentin mineralized tissues were produced by differentiated DFCs, which was quantied as the percentage of the area of newly formed mineralized tissues against the chamber area of the dentin carrier. Fluorescent tracing analyses further proved that it was the transplanted DFCs that evidently formed such tissues. These phenomena can be explained as follows. First,

that amorphous osteodentin was secreted within the carrier and was immunopositive against DSP (Figures 5H and 5J).

Discussion In contrast with enamel and dentin, the origin of cementum still remains enigmatic (Slavkin and Diekwisch, 1996, 1997; Diekwisch, 2001). Cementum formation begins when both epithelial cells of HERS and mesenchymal cells of dental follicle are in proximity to the developing root surface. Although the exact role of epithelial and mesenchymal components in cementogenesis is an open question, it has generally been suggested that the disruption of HERS is the key event at the onset of root development (Cho and Garant, 1988). Thus DFCs have access to the root dentin surface and have a high probability of undergoing cementoblastic differentiation to secrete the original cementum matrix by dentinal induction. In the present study, dNCPs extracted from dentin, as the representative of dentin matrix, were conrmed to have the ability to stimulate DFCs into cementoblast lineages. Morphologically, the treated DFCs appeared to have a at, cuboidal or polygonal shape with a distended RER, showing an active function of synthesis and secretion, which is similar to cementoblast phenotypes (Schroeder, 1986b; Faltin et al., 2001; Cattaneo et al., 2003). Interestingly, the electron-dense granules within the cytoplasm, one of the markers of DFCs, were greatly reduced after induction, possibly attributed to cell maturity (Wise et al., 1992; Martin, 1998). Cell proliferation, evaluated by the MTT assay, BrdU incorporation and cell-cycle analysis, coincidentally demonstrated that dNCPs could inhibit DFC proliferation during the process of differentiation, which was in accordance with the previous concept that proliferation and differentiation are inversely correlated with each other due to the existence of dual-function regulators participating in controlling both the processes (Zhu and Skoultchi, 2001). Therefore it is believed that some components of dNCPs could act not only as promoters for the differentiation of DFCs but also as inhibitors for cell proliferation. ALP plays a vital role in calcied tissue formation by regulating phosphate transfer (Beck et al., 2000). Human cementoblasts have a high ALP activity showing an active capacity for mineralization (Kitagawa et al., 2006). In the present study, the

www.biolcell.org | Volume 100 (5) | Pages 291302

297

J. Wu and others

once the cell pellet is loaded in the dentin carrier, the untreated DFCs make good contact with the predentin surface of the carrier. Some dentin matrix might be diffused from predentin in the carrier to affect proximate cells to form thin cementum, which could be termed as the endogenous dNCPs effect and is more likely a physiological situation since we know that the acellular cementum layer in the living body is usually very thin. The treated cells have undergone differentiation by the induction of exogenous dNCPs before loading, which could be regarded as the amplication of the endogenous effect, nally leading to evident mineral formation. Secondly, the physical property of the dentin substrate could exert an effect on DFC differentiation. It was revealed that matrix elasticity directs stem-cell lineage specication (Engler et al., 2006). Thus we can speculate that the dentin substrate has the preferential ability to drive DFCs into cementoblast lineages. In addition, we had proposed the concept of cell pellet engineering, and successfully used dental papilla cell pellet to regenerate the dentinpulp complex (Yu et al., 2006b). The pellet culture model, which can provide the three-dimensional growth environment necessary for cell differentiation, may help DFCs develop into cementoblasts. Under the same protocol, DPSC implants demonstrated the formation of DSP-immunopositive osteodentin, implying that DPSCs could differentiate into odontoblasts within the chamber of the carrier as predicted. However, even in the inducing circumstance, treated DFCs could not differentiate into odontoblasts, because the resulting mineralized tissues were immunonegative against DSP. The reason for this difference may lie in cell-fate determination. It is known that, although both DPSCs and DFCs are derived from one ancestor, namely EMSCs, different parts of the mesenchymal cells may have been determined as different precursors for specic tissue formative cells by the intrinsic programmed fate during development. And here, DFCs and DPSCs only act as determined precursors for periodontal tissue and the dentinpulp complex respectively in order to ensure the harmony of tooth development and regeneration. During the last decade, many studies have proved that EMD (enamel matrix derivative) has obvious effects on periodontal regeneration, including cementogenesis (Gestrelius et al., 1997; Venezia et al., 2004; Bosshardt et al., 2005; Rincon et al., 2005).

As to DFCs, EMD could regulate in vitro differentiation of mouse follicle cells towards the osteocementoblastic phenotype (Hakki et al. 2001). Although similar effects of EMD and dNCPs could be achieved, there are obvious differences between them. First of all, the application of these two mixed agents is based on respective hypotheses EMD has been proposed to mimic the inducing role of HERS products involved in cementogenesis. However, direct dentinDFC contact as an important developmental event, and the inducing ability of dNCPs, have reasonably supported our present study. Secondly, the compositions of the EMD and dNCPs are quite different the presence of three matrix proteins (amelogenin, enamelin and sheathlin) and BMP (bone morphogenetic protein)-like growth factors (BMP-2 and BMP-7) have been conrmed in EMD. A recent report indicates that BMPs present in EMD are probably crucial for the differentiation of human DFCs into cementoblasts, in which Smad-1 and MAPK (mitogen-activated protein kinase) pathways seem to be involved (K emoun et al., 2007). Besides, amelogenin, as the major fraction of EMD, has been proved to be an effective molecule (Viswanathan et al., 2003; Boabaid et al., 2004; Swanson et al., 2006). In comparison, DSP, DPP (dentin phosphoprotein) and DMP-1 secreted by odontoblasts are the major constituents of dNCPs, which have been suggested to be involved in the nucleation process and hydroxyapatite growth during dentin formation (Butler et al., 1998, 2002). However, the potential of these proteins as signalling molecules has been seldom proposed. DSP, which lacks the RGD sequence, is not able to promote the attachment and spreading of cells (Butler et al., 1992), and mainly plays a role in dentin mineralization (Goldberg et al., 2004). In contrast, DPP up-regulates osteoblast marker genes in several kinds of mesenchymal cells by binding to integrin receptors on the surface via its RGD domain and subsequently activating the MAPK signalling pathways (Jadlowiec et al., 2004). Also, DMP-1 was shown to stimulate rat DPSCs (Almushayt et al., 2006) and mouse embryonic mesenchymal cells to differentiate into odontoblast lineages (Narayanan et al., 2001). Furthermore, we hypothesized that dentin matrix proteins produced by odontoblasts could migrate through and diffuse from newly deposited predentin, which is similar to the well-known phenomenon of diffusion of enamel matrix proteins

298

The Authors Journal compilation

2008 Portland Press Ltd

Differentiation of DFCs induced by dNCPs

Research article
DFCs were isolated and cultured using a previously described method (Wise et al., 1992) as detailed below. SD rats of 6 or 7 days of age were killed by cervical dislocation. The mandibles were aseptically dissected and placed in PBS solution where the dental follicle and enamel organ were dissected from the rst molars with the aid of a dissecting microscope. The isolated tissues were placed in 1% trypsin/l mM EDTA solution for 10 min at room temperature (22 C) and the follicle was separated by microdissection from the stellate reticulum. The follicles were then incubated in a 0.25% trypsin/1 mM EDTA solution for 10 min at 37 C to partially dissociate the follicle cells. Then, the above tissues were transferred to 35 mm tissue-culture dishes and incubated in DMEM (Dulbeccos modied Eagles medium)/F12 (Gibco BRL, Grand Island, NY, U.S.A.) containing 15% (v/v) fetal calf serum, 0.1 g/l sodium pyruvate, 100 g/ml streptomycin and 100 units/ml penicillin and cultured at 37 C in a humidied atmosphere of 5% CO2 . Within 1520 days after dissection, the cells were conuent and conventionally passaged. In the present study, the 3rd passage of DFCs was investigated and divided into a treated group with 250 g/ml dNCP medium (Deng et al., 2005) and an untreated group with conventional culture medium (DMEM/F12 containing 10% fetal calf serum). The fresh medium was changed every 2 days.
Morphological observation

through the pre-mineralizing mantle dentin into the odontoblast layer (Inai et al., 1991). Collectively, it was speculated that DPP and DMP-1 might be the effective molecules in dNCPs for driving DFCs into cementoblast lineages in the present study as well as root development. Besides, dentin represents a signicant storage of various growth factors such as BMP2, -3 and -7 and TGF- (transforming growth factor). And even amelogenin slices could be identied from dentin extracts, as the remains of ameloblast secretions (Veis et al., 2000). Thus these molecules can be proposed to act in concert with other factors to promote cementogenesis by affecting DFCs just in the same way as they are in EMDs. As we know, periodontal tissue is a typical sandwich structure consisting of cementum, periodontal ligament and alveolar bone. However, there was no evident periodontal structure, but cementum formed in the present in vivo study if DFCs were stimulated by dNCPs alone. Actually, periodontal development and regeneration involve cascades of numerous signals. In a recent report, PLAP-1 (periodontal ligament associated protein-1)/asporin has been identied to inhibit the mineralization of periodontal ligament cells, which is also intensively expressed by dental follicle (Yamada et al., 2007). Since DFCs are commonly regarded as precursors of the formative cells for the above three kinds of tissues in the periodontium, molecules other than dNCPs might exert negative effects on the differentiation of DFCs into cementoblasts as well as positive effects on the fate of DFCs into broblasts or osteoblasts, leading to the balance of periodontal development. In conclusion, the in vitro and in vivo evidence accumulated in the present study showed that dNCPs could promote the cementogenic process by stimulating DFCs to differentiate into cementoblasts. Although the effective components of dNCPs should be analysed in the future, the present study afrmatively provides a better understanding of the underlying mechanisms of cementogenesis, which may be helpful for regenerative periodontal therapies.

After 6 days of culture, treated and untreated DFCs of the 3rd passage were routinely observed and photographed under a phase-contrast inverted microscope to evaluate their phenotypes (Olympus Optical, Tokyo, Japan).
SEM

DFCs of the 3rd passage were cultured in dNCP medium or conventional medium for 6 days and then digested in order to seed them on to coverslips. DFCs were then incubated in the respective medium for a further 24 h. After washing, the cell coverslips were xed with 2.5% glutaraldehyde at 0 C, dehydrated and dried in a critical-point dryer. The cells were observed under a Hitachi S-3400N scanning electron microscope (Hitachi, Tokyo, Japan).
TEM

After being treated for 6 days, the DFCs (3rd passage) were trypsinized and centrifuged (3000 g, 5 min, 4 C) to form pellets, then xed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h at room temperature and postxed in aq. 2% (v/v) osmium tetroxide for a further 1 h. The cells then were dehydrated in a series of ethanols (50, 70, 95 and 100%) and embedded in Epon 812 resin. Ultrathin sections were made and stained with uranyl acetate and lead citrate. The samples were viewed with a JEM 100 SX electron microscope (Jeol, Tokyo, Japan).
Cell proliferation assay

Materials and methods

Cell isolation and culture

Harlan SD (SpragueDawley) rats were obtained from the Laboratory Animal Research Centre of Fourth Military Medical University. All surgical procedures and care administered to the animals were approved by the University Animal Care Committee and performed according to institutional guidelines. The

DFCs of the 3rd passage were seeded on to 96-well plates at a density of 1000 cells per well. After incubation for 24 h, to half of the wells dNCP medium was added, and conventional medium was added to control wells. The MTT assay was performed at day 1, 3, 5, 7, 9, 11 and 13 after induction. Briey, 20 l of MTT (5 mg/ml; SigmaAldrich, St. Louis, MO, U.S.A.) was added to each well, which was converted into an insoluble blue formazan product by mitochondria of living cells but not of dying cells

www.biolcell.org | Volume 100 (5) | Pages 291302

299

J. Wu and others
or debris. After incubation at 37 C for 4 h, the supernatant was removed and the formazan crystals were dissolved in 150 l of DMSO. Attenuance values for each well were measured spectrophotometrically at 490 nm and the assay was repeated three times.
BrdU incorporation

Immunocytochemistry analysis

DFCs of the 3rd passage were seeded on to 24-well plates at a density of 5 103 cells per well. After the induction of dNCPs for 6 days, all cells were incubated with 10 M BrdU (SigmaAldrich) for 1 h and xed with 4% polyoxymethylene. Then, the cultures were processed for immunostaining according to the manufacturers instructions. Cells containing densely brown-stained nuclei with clear morphology were considered BrdU-positive, and they were counted in ve elds per well (centre and at 03:00, 06:00, 09:00 and 12:00 h). Results were expressed as the percentage of BrdU-positive cells against total cells counted.
Cell-cycle analysis

The DFCs in both groups were seeded on to coverslips at a density of 2 105 cells/ml, maintained for another 2 days in respective medium and then xed with 4% polyoxymethylene. Immunocytochemical analyses were performed with the streptavidinbiotin complex method according to the manufacturers recommended protocol. Antibodies included (i) afnitypuried polyclonal rabbit anti-rat BSP at a 1:500 dilution, (ii) polyclonal rabbit anti-rat ON at a 1:500 dilution, (iii) afnitypuried polyclonal rabbit anti-rat OCN at a 1:100 dilution, (iv) polyclonal rabbit anti-rat Col I at a 1:500 dilution, (v) polyclonal goat anti-rat DMP-1 at a 1:100 dilution and (vi) polyclonal rabbit anti-rat DSP at a 1:50 dilution. The antibodies for DSP, ON and Col I were gifts from Dr Larry W. Fisher [NIDR (National Institute of Dental and Craniofacial Research), Bethesda, MD, U.S.A.], and the others were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The specimens were counterstained with haematoxylin, and all the samples were examined under an Olympus compound microscope (Olympus Optical).
RTPCR

After being treated for 6 days, DFCs were collected for ow cytometry analysis. Briey, cells were digested and cell precipitates were washed twice with 0.01 M PBS and resuspended in 1 ml of physiological saline by repeated vibration to ensure a single cell suspension. Then, 2 ml of cold dehydrated alcohol was mixed quickly with the cell suspension to x cells at 4 C for 2448 h. Finally, the cells were washed twice again with PBS, stained with 100 mg/ml propidium iodide at 4 C for 30 min and subjected to cell-cycle analysis using Elite ESP ow cytometry (Beckman Coulter, Fullerton, CA, U.S.A.).
ALP activity assay

At dened time points, the ALP activity of treated and untreated DFCs was detected using an ALP assay kit (JianCheng, Nanjing, China). In brief, cells (1 103 per well) were placed in a 96-well plate. After culture, cells were washed three times with 0.01 M PBS and 50 ml of cold 10 mM Tris/HCl buffer (pH 7.4) containing 0.1% Triton X-100 added prior to incubation at 4 C overnight. A 100 l portion of ALP substrate solution (2 mM MgCl2 and 16 mM p-nitrophenyl phosphate) was then mixed with each sample. After incubation at 37 C for 30 min, the reaction was stopped by the addition of 50 ml of 0.2 M NaOH and the liberated p-nitrophenol was measured spectrophotometrically at 410 nm. The results were described in KingArmstrong units (Yu et al., 2006a).
von Kossa staining for mineralized nodule formation

DFCs of the 3rd passage were seeded on to 24-well plates at a density of 5 103 cells per well. After being treated or untreated with dNCPs for 21 days, von Kossa staining was carried out to determine mineralized nodule formation. In brief, the cells were xed with cold absolute acetone for 30 min, then incubated in 5% AgNO3 for 30 min, washed with distilled water, treated with a 5% Na2 CO3 and 25% formalin solution for 2 min and then washed with distilled water again. Finally, the cells were counterstained with Nuclear Fast Red. The nodule area and nodule number per well were measured quantitatively with an image analysis system Image-Pro Plus 5.0 (Media Cybernetics, Baltimore, MD, U.S.A.).

DFCs at 90% conuence were harvested after a 6-day induction by dNCPs. Total RNA was isolated by means of TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.) and was reversetranscribed using the SuperScriptTM First-Strand Synthesis System for RTPCR (Invitrogen) following the manufacturers instructions. Primer sequences for DMP-1 (GenBank accession no. NM 203493), DSPP (GenBank accession no. NM 012790), ON (GenBank accession no. NM Y13714), OCN (GenBank accession no. NM 340986), BSP (GenBank accession no. NM 012587), Col I (GenBank accession no. NM Z78279) and -actin (GenBank accession no. NM 031144) were as follows: DMP-1 sense, 5 -CGGCTGGTGGTCTCTCTAAG3 , and DMP-1 antisense, 5 -GTCCCTCTGGGCTATCTTCC-3 ; DSPP sense, 5 -TAAGGACAAGGACGAATC-3 , and DSPP antisense, 5 -ACTGCTGTCACTGCTTTC-3 ; ON sense, 5 -CTGCAGAAGAGATGGTGGCGG-3 , and ON antisense, 5 -CAGGCAGGGGGCAATGTATTTG-3 ; OCN sense, 5 -ATGAGGACCCTCTCTCTGCTC-3 , and OCN antisense, 5 -CTAAACGGTGGTGCCATAGAT-3 ; BSP sense, 5 -CTGCTTTAATCTTGCTCTG-3 , and BSP antisense, 5 -CCATCTCCATTTTCTTCC-3 ; Col I sense, 5 -CGTGACCAAAAACCAAAAGTGC-3 , and Col I antisense, 5 -GGGGTGGAGAAAGGAACAGAAA-3 ; -actin sense, 5 -GAGACCTTCAACACCCCAGCC-3 , and -actin antisense, 5 -CATAGCACAGCTTCTCTTTAA-3 , used as an internal control. After 30 cycles, the PCR products were separated by using 2% agarosegel electrophoresis and were stained with ethidium bromide, and digital images were taken on UV background. This experiment was repeated three times and PCR products were conrmed by sequencing (Sangon Biotechnology, Shanghai, China).
In vivo differentiation of DFC pellets

In order to mimic the contact of DFCs with dentin, as well as to facilitate the transplantation of cell pellets, we utilized a novel natural dentin carrier in the present study to contain DFCs pellets. In fact, we removed dental papilla tissues from the remains of tooth germs after dental follicle isolation, and only hard tissues were left and further digested by 0.25% trypsin for 30 min to be

300

The Authors Journal compilation

2008 Portland Press Ltd

Differentiation of DFCs induced by dNCPs

Research article
Bosshardt, D.D. and Nanci, A. (2004) Hertwigs epithelial root sheath, enamel matrix proteins, and initiation of cementogenesis in porcine teeth. J. Clin. Periodontol. 31, 184192 Bosshardt, D.D., Sculean, A., Windisch, P., Pjetursson, B.E. and Lang, N.P. (2005) Effects of enamel matrix proteins on tissue formation along the roots of human teeth. J. Periodontal Res. 40, 158167 Butler, W.T. (1998) Dentin matrix proteins. Eur. J. Oral Sci. 106, 204210 Butler, W.T., Bhown, M., Brunn, J.C., DSouza, R.N., Farach-Carson, M.C., Happonen, R.P., Schrohenloher, R.E., Seyer, J.M., Somerman, M.J., Foster, R.A. et al. (1992) Isolation, characterization and immunolocalization of a 53-kDa dentin sialoprotein (DSP). Matrix 12, 343351 Butler, W.T., Brunn, J.C., Qin, C. and Mckee, M.D. (2002) Extracellular matrix proteins and the dynamics of dentin formation. Connect. Tissue Res. 43, 301307 Cattaneo, V., Rota, C., Silvestri, M., Piacentini, C., Forlino, A., Gallanti, A., Rasperini, G. and Cetta, G. (2003) Effect of enamel matrix derivative on human periodontal broblasts: proliferation, morphology and root surface colonization. An in vitro study. J. Periodont. Res. 38, 568574 Cho, M.I. and Garant, P.R. (1988) Ultrastructural evidence of directed cell migration during initial cementoblast differentiation in root formation. J. Periodont. Res. 23, 268276 Colnot, C., Lu, C.Y., Hu, D. and Helms, J.A. (2004) Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development. Dev. Biol. 269, 5569 Deng, M.J., Shi, J.N., Smith, A.J. and Jin, Y. (2005) Effects of transforming growth factor 1 (TGF-1) and dentin non-collagenous proteins (DNCP) on human embryonic ectomesenchymal cells in a three-dimensional culture system. Arch. Oral Biol. 50, 937945 DErrico, J.A., MacNeil, R.L., Takata, T., Berry, J., Strayhorn, C. and Somerman, M.J. (1997) Expression of bone associated markers by tooth root lining cells, in situ and in vitro. Bone 20, 117126 Diekwisch, T.G.H. (2001) Developmental biology of cementum. Int. J. Dev. Biol. 45, 695706 Engler, A.J., Sen, S., Sweeney, H.L. and Discher, D.E. (2006) Matrix elasticity directs stem cell lineage specication. Cell 126, 677689 Faltin, R.M., Faltin, K., Sander, F.G. and Arana-Chavez, V.E. (2001) Ultrastructure of cementum and periodontal ligament after continuous intrusion in humans: a transmission electron microscopy study. Eur. J. Othod. 23, 3549 Furseth, R., Selvig, K.A. and Mjor, I.A. (1986) The periodontium. Human Oral Embryology and Histology (Mjor, I.A. and Fejerskov, O., eds), pp. 131202, Munksgaard, Copenhagen Gestrelius, S., Andersson, C., Lidstrom, D., Hammarstrom, L. and Somerman, M. (1997) In vitro studies on periodontal ligament cells and enamel matrix derivative. J. Clin. Periodontol. 24, 685692 Gestrelius, S., Lyngstadaas, S.P. and Hammarstrom, L. (2000) Emdogain-periodontal regeneration based on biomimicry. Clin. Oral Investig. 4, 120125 Goldberg, M., Septier, D., Bourd, K. and Menashi, S. (2004) Role of matrix proteins in signalling and in dentin and enamel mineralization. C. R. Palevol. 3, 573581 Hakki, S.S., Berry, J.E. and Somerman, M.J. (2001) The effect of enamel matrix protein derivative on follicle cells in vitro. J. Periodontol. 72, 679687 Hammarstrom, L., Alatli, I. and Fong, C.D. (1996) Origins of cementum. Oral Dis. 2, 6369 Handa, K., Saito, M., Yamauchi, M., Kiyono, T., Sato, S., Teranaka, T. and Narayanan, A.S. (2002) Cementum matrix formation in vivo by cultured dental follicle cells. Bone 31, 606611 Inai, T., Kukita, T., Ohsaki, Y., Nagata, K., Kukita, A. and Kurisuk, K. (1991) Immunohistochemical demonstration of amelogenin penetration toward the dental pulp in the early stages of ameloblast development in rat molar tooth germs. Anat. Rec. 229, 259270

completely acellularized. The hard tissues showed a chamber-like shape that consisted of enamel outside and dentin inside. The cell pellet culture was performed as previously described with some modications (Yu et al., 2006a). Briey, DFCs co-cultured with dNCPs for 7 days were labelled with Hoechst 33342 (Supplementary Figure 4A) and then centrifuged in a 10-ml conical polypropylene tube (Asahi Techno Glass, Tokyo, Japan) at 800 g for 5 min. Cell pellets in the tube were maintained in respective medium at 37 C for 5 h to make them well congregated and favourable for transplantation procedures. The pellets were transferred into the carrier chamber and then seeded directly into the renal capsules on the left side of 8-week-old allogenic SD rat hosts. As a control, the untreated DFC pellets within the carrier were implanted into the other side of the same host. In addition, pellets of rat DPSCs were also used as a control. Transplantation procedures were performed as described elsewhere (Colnot et al., 2004). Totally, 58 cell pellets (28 treated DFCs, 25 untreated DFCs and 5 DPSCs) were implanted into the renal capsules of 30 rats. After 28 days of incubation, the explants were isolated and xed in 4% polyoxymethylene, decalcied with buffered 10% EDTA (pH 8.0) and processed for H&E (haematoxylin and eosin) staining and immunohistological staining against DSP. Besides, frozen sections were prepared and examined with a uorescent microscope to identify donor cells. Histological analyses were quantied with an image analysis system Image-Pro Plus 5.0 (Media Cybernetics).
Statistical analysis

Statistical signicance was assessed by a 2 test and an independent samples t test with SPSS v 12.0 software (SPSS, San Rafael, CA, U.S.A.). Statistical signicance was determined at P < 0.05. All data acquisition and analyses were performed blindly.

Acknowledgments We are grateful to Professor Anthony J. Smith (Oral Biology, School of Dentistry, University of Birmingham, Birmingham, U.K.) for his kind provision of dNCPs. We also thank Dr Juan Dai for her critical reading of this paper. This work was supported by grants from the Nature Science Foundation of China (project no. 30572046). References
Almushayt, A., Narayanan, K., Zaki, A.E. and George, A. (2006) Dentin matrix protein 1 induces cytodifferentiation of dental pulp stem cells into odontoblasts. Gene Ther. 13, 611620 Beck, Jr, G.R., Zerler, B. and Moran, E. (2000) Phosphate is a specic signal for induction of osteopontin gene expression. Proc. Natl. Acad. Sci. U.S.A. 97, 83528357 Boabaid, F., Gibson, C.W., Kuehl, M.A., Berry, J.E., Snead, M.L., Nociti, Jr, F.H., Katchburian, E. and Somerman, M.J. (2004) Leucine-rich amelogenin peptide: a candidate signaling molecule during cementogenesis. J. Periodontol. 75, 11261136 Bosshardt, D.D. (2005) Are cementoblasts a subpopulation of osteoblasts or a unique phenotype? J. Dent. Res. 84, 390406 Bosshardt, D.D. and Nanci, A. (1997) Immunodetection of enamel and cementum-related (bone) proteins at the enamel-free area and cervical portion of the tooth in rat molars. J. Bone Miner. Res. 12, 367379

www.biolcell.org | Volume 100 (5) | Pages 291302

301

J. Wu and others

Jadlowiec, J., Koch, H., Zhang, X., Campbell, P.G., Seyedain, M. and Sfeir, C. (2004) Phosphophoryn regulates the gene expression and differentiation of NIH3T3, MC3T3-E1, and human mesenchymal stem cells via the integrin/MAPK signaling pathway. J. Biol. Chem. 279, 5332353330 Kemoun, P., Laurencin-Dalicieux, S., Rue, J., Farges, J.C., Gennero, I., Conte-Auriol, F., Briand-Mesange, F., Gadelorge, M., Arzate, H., Narayanan, A.S. et al. (2007) Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro. Cell Tissue Res. 329, 283294 Kitagawa, M., Tahara, H., Kitagawa, S., Oka, H., Kudo, Y., Sato, S., Ogawa, I., Miyaichi, M. and Takata, T. (2006) Characterization of established cementoblast-like cell lines from human cementum-lining cells in vitro and in vivo. Bone 39, 10351042 Liu, J., Jin, T., Ritchie, H.H., Simth, A.J. and Clarkson, B.H. (2005) In vitro differentiation and mineralization of human dental pulp cells induced by dentin extract. In Vitro Cell Dev. Biol. Anim. 41, 232238 Liu, J., Jin, T., Chang, S., Ritchie, H.H., Smith, A.J. and Clarkson, B.H. (2007) Matrix and TGF-beta-related gene expression during human dental pulp stem cell (DPSC) mineralization. In Vitro Cell Dev. Biol. Anim. 43, 120128 Martin, V.J. (1998) Development of nerve cells in hydrozoan planulae: I. Differentiation of ganglionic cells. Biol. Bull. 174, 319329 Narayanan, K., Srinivas, R., Ramachandran, A., Hao, J., Quinn, B. and George, A. (2001) Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1. Proc. Natl. Acad. Sci. U.S.A. 98, 45164521 Nie, X., Tian, W., Zhang, Y., Chen, X., Dong, R., Jiang, M., Chen, F. and Jin, Y. (2006) Induction of transforming growth factor- 1 on dentine pulp cells in different culture patterns. Cell Biol. Int. 30, 295300 Provenza, D.V. (1988) Attachment apparatus. Fundamentals of Oral Histology and Embryology (Provenza, D.V., ed.), pp. 195235, Lea and Febiger, Philadelphia, PA Rincon, J.C., Xiao, Y., Young, W.G. and Bartold, P.M. (2005) Enhanced proliferation, attachment and osteopontin expression by porcine periodontal cells exposed to Emdogain. Arch. Oral Biol. 50, 10471054 Schroeder, H.E. (1986a) The periodontium. In Handbook of Microscopic Anatomy (Oksche, A. and Vollrath, L., eds), pp. 170232, Springer-Verlag, Berlin Schroeder, H.E. (1986b) The periodontium. In Handbook of Microscopic Anatomy (Oksche, A. and Vollrath, L., eds), pp. 23128, Springer-Verlag, Berlin Slavkin, H.C. (1976) Towards a cellular and molecular understanding of periodontics. Cementogenesis revisited. J. Periodontol. 47, 249255 Slavkin, H.C. and Diekwisch, T.G.H. (1996) Evolution in tooth developmental biology: of morphology and molecules. Anat. Rec. 245, 131150 Slavkin, H.C. and Diekwisch, T.G.H. (1997) Molecular strategies of tooth enamel formation are highly conserved during vertebrate evolution. Ciba Found. Symp. 205, 7380 Sloan, A.J. and Smith, A.J. (2007) Stem cells and the dental pulp: potential roles in dentine regeneration and repair. Oral Dis. 13, 151157

Smith, A.J. and Leaver, A.G. (1979) Non-collagenous components of the organic matrix of rabbit incisor dentin. Arch. Oral Biol. 24, 449454 Song, A.M., Shu, R., Xie, Y.F., Song, Z.C., Li, H.Y., Liu, X.F. and Zhang, X.L. (2007) A study of enamel matrix proteins on differentiation of porcine bone marrow stromal cells into cementoblasts. Cell Prolif. 40, 381396 Stahl, S.S. and Slavkin, H.C. (1972) In Development of gingival crevicular epithelium and periodontal disease. In Developmental Aspects of Oral Biology (Slavkin, H.C. and Bavetta, L., eds), pp. 326350, Academic Press, New York Swanson, E.C., Fong, H.K., Foster, B.L., Paine, M.L., Gibson, C.W., Snead, M.L. and Somerman, M.J. (2006) Amelogenins regulate expression of genes associated with cementoblasts in vitro. Eur. J. Oral Sci. 114, (Suppl. 1), 239243 Venezia, E., Goldstein, M., Boyan, B.D. and Schwartz, Z. (2004) The use of enamel matrix derivative in the treatment of periodontal defects: a literature review and meta-analysis. Crit. Rev. Oral Biol. Med. 15, 382402 Veis, A., Tompkins, K., Alvares, K., Wei, K., Wang, L., Wang, X.S., Brownell, A.G., Jengh, S.M. and Healy, K.E. (2000) Specic amelogenin gene splice products have signaling effects on cells in culture and in implants in vivo. J. Biol. Chem. 275, 4126341272 Viswanathan, H.L., Berry, J.E., Foster, B.L., Gibson, C.W., Li, Y., Kulkarni, A.B., Snead, M.L. and Somerman, M.J. (2003) Amelogenin: a potential regulator of cementum-associated genes. J. Periodontol. 74, 14231431 Wise, G.E., Lin, F. and Fan, W. (1992) Culture and characterization of dental follicle cells from rat molars. Cell Tissue Res. 267, 483492 Yu, J.H., Deng, Z.H., Shi, J.N., Zhai, H.H., Nie, X., Zhuang, H. and Jin, Y. (2006a) Differentiation of dental pulp stem cells into regular-shaped dentin-pulp complex induced by tooth germ cell conditioned medium. Tissue Eng. 12, 30973105 Yu, J.H., Shi, J.N., Deng, Z.H., Zhuang, H., Nie, X., Wang, R.N. and Jin, Y. (2006b) Cell pellets from dental papillae can reexhibit dental morphogenesis and dentinogenesis. Biochem. Biophys. Res. Commun. 346, 116124 Yu, J.H., Wang, Y.J., Deng, Z.H., Tang, L., Li, Y., Shi, J.N. and Jin, Y. (2007) Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells. Biol. Cell 99, 465474 Yamada, S., Tomoeda, M., Ozawa, Y., Yoneda, S., Terashima, Y., Ikezawa, K., Ikegawa, S., Saito, M., Toyosawa, S. and Murakami, S. (2007) PLAP-1/asporin: a novel negative regulator of periodontal ligament mineralization. J. Biol. Chem. 282, 2307023080 Zhao, M., Xiao, G., Berry, J.E., Franceschi, R.T., Reddi, A. and Somerman, M.J. (2002) Bone morphogenetic protein 2 induces dental follicle cells to differentiate toward a cementoblast/ osteoblast phenotype. J. Bone Miner. Res. 17, 14411451 Zhao, M., Jin, Q., Berry, J.E., Nociti, F.H., Giannobile, W.V. and Somerman, M.J. (2004) Cementoblast delivery for periodontal tissue engineering. J. Periodontol. 75, 154161 Zhu, L. and Skoultchi, A.I. (2001) Coordinating cell proliferation and differentiation. Curr. Opin. Genet. Dev. 11, 9197 Zeichner-David, M. (2001) Is there more to enamel matrix proteins than biomineralization? Matrix Biol. 20, 307316 Zeichner-David, M. (2006) Regeneration of periodontal tissues: cementogenesis revisited. Periodontol. 2000 41, 196217

Received 30 July 2007/21 November 2007; accepted 28 November 2007 Published as Immediate Publication 28 November 2007, doi:10.1042/BC20070092

302

The Authors Journal compilation

2008 Portland Press Ltd