1000291-N01

Biol. Cell (2008) 100, 291–302 (Printed in Great Britain) doi:10.1042/BC20070092 Research article

Dentin non-collagenous proteins(dNCPs) can stimulate dentalfollicle cells to differentiate intocementoblast lineagesJunjie Wu*†‡1, Fang Jin*1, Liang Tang†‡, Jinhua Yu†‡§, Lin Xu*†‡, Zhenhua Yang*†‡, Gang Wu†‡¶,Yinzhong Duan*2 and Yan Jin†‡2

*Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi 710032, People’s Republic

of China, †Department of Oral Histology and Pathology, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi 710032,

People’s Republic of China, ‡Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi’an, Shaanxi

710032, People’s Republic of China, §Department of Endodontics, School of Stomatology, Nanjing Medical University, Nanjing, Jiangsu

210029, People’s Republic of China, and ¶Department of Endodontics, School of Stomatology, Fourth Military Medical University, Xi’an,

Shaanxi 710032, People’s Republic of China

Background information. Although the mechanism of cementogenesis is an area full of debate, the DFCs (dentalfollicle cells) are thought to be the precursors of cementoblasts. At the onset of cementogenesis, DFCs comeinto contact with the root dentin surface and undergo subsequent differentiation. But the exact effects of dentinor dentin matrix on DFCs remain an open question. In the present study, we hypothesized that dNCPs (dentinnon-collagenous proteins) extracted from dentin could stimulate DFCs to differentiate into cementoblast lineages.

Results. DFCs were isolated from tooth germs of SD (Sprague–Dawley) 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. Theexpression of mineralization-associated proteins and genes were up-regulated after induction, whereas the ex-pression of specific markers of odontoblast were not detected. Incubation of treated DFC pellets in vivo revealedthat a large amount of cementum-like tissues was formed within the novel dentin carriers, which were quite distinctfrom 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 providesnew insights into the mechanism of cementogenesis.

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

IntroductionCementogenesis is one of the most important pro-cesses in periodontal development and regeneration.Cementum matrix is secreted by highly differenti-ated cells called cementoblasts. However, the originof cementoblasts remains a matter of debate(Hammarstrom et al., 1996). To date, several the-ories have been proposed about this intriguing is-sue. The classical theory suggests that DFCs (dentalfollicle cells) give birth to cementoblasts and secretecementum matrix after penetrating through the

www.biolcell.org | Volume 100 (5) | Pages 291–302 291

Bio

log

y o

f th

e C

ell

w

ww

.bio

lcel

l.org

J. Wu and others

barrier of HERS (Hertwig’s epithelial root sheath)(Furseth et al., 1986; Schroeder, 1986a; Provenza,1988). Another hypothesis considers that cemento-blasts 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, itis assumed that the proteins (including enamelmatrix protein and others) secreted by HERScells affect cell migration, attachment and func-tion leading to cementogenesis (Gestrelius et al.,2000; Zeichner-David, 2001, 2006). Regardlessof the different theories, it has been well recog-nized that DFCs penetrate disintegrated HERS andcome into contact with the root dentin surfaceprior to any cementum formation (see Supplement-ary Figure 1 at http://www.biolcell.org/boc/100/boc1000291add.htm). Therefore it would be of greatinterest to investigate the effects of dentin or dentinmatrix on DFCs.

dNCPs (dentin non-collagenous proteins), themixed proteins extracted from dentin, compriseglycoproteins/sialoproteins, phosphoproteins, pro-teoglycans and growth factors (Smith and Leaver,1979), which have been proved to be involved inthe nucleation process and hydroxyapatite growthduring dentin formation (Butler, 1998; Butler et al.,2002). Moreover, dNCPs can act as an inducing agentin promoting cell differentiation; for instance, theycan induce DPCs (dental pulp cells), DPSCs (dentalpulp stem cells) and even EMSCs (ectomesenchymalstem cells) to differentiate into odontoblast-like cells(Deng et al., 2005; Liu et al., 2005; Nie et al., 2006).

Based on the direct dentin–cell contact and theinducing ability of dNCPs, we have proposed inthe present study that dNCPs could stimulate DFCsto differentiate into cementoblasts. To test this hy-pothesis, we investigated the biological effects ofdNCPs on the proliferation and differentiation ofDFCs through in vitro and in vivo experiments. Thepresent study provides a better understanding of theregulation mechanism of cementogenesis in period-ontal development and regeneration.

ResultsEffect of dNCPs on the morphological changes ofDFCsObserved under a phase-contrast microscope, the un-treated DFCs of the 3rd passage at day 6 appeared

Figure 1 Effect of dNCPs on the morphological changesof 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 fibroblastic in shape,

elongated with processes at each pole. (B, D) Treated DFCs

at day 6 became flat, 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 identification 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.

polymorphic, but the predominant cells were fibro-blastic in shape, elongated with two processes at eachpole (Figure 1A). In the treated group, after the in-duction of dNCPs for 6 days, DFCs became flatterand most of them were cuboidal or polygonal (Fig-ure 1B). To examine the ultramicrostructure of DFCs,a scanning electron microscope and a transmission

292 C© The Authors Journal compilation C© 2008 Portland Press Ltd

Differentiation of DFCs induced by dNCPs Research article

electron microscope were used for the observation.The SEM (scanning electron microscopy) results werein agreement with the above light-microscope find-ings, showing that most of the untreated DFCswere elongated, spindle-shaped and displayed abund-ant microvilli on the cell surface (Figure 1C),whereas the treated cells closely adherent to the sub-strate appeared cuboidal or polygonal in shape withmultiprocesses (Figure 1D). In addition, comparedwith the control, the cell-surface microvilli weregreatly decreased in the treated cells around whichsome extracellular matrix was secreted (Supplement-ary Figure 2A at http://www.biolcell.org/boc/100/boc1000291add.htm). As to the TEM (transmissionelectron microscopy) observations, the untreated cellsdemonstrated the typical ultramicrostructure of amesenchymal fibroblast except that electron-densegranules could be occasionally observed adjacent tothe RER (rough endoplasmic reticulum), which hasbeen regarded as one of the markers for identifyingDFCs (Wise et al., 1992) (Figures 1E and 1F). How-ever, inside the treated cells, the RER frequently ap-peared distended and contained flocculent material,implying the potent function of protein synthesisand secretion (Figures 1G and 1H). The extracellularmatrix could be found around the treated cells byTEM (Supplementary Figure 2B).

Effect of dNCPs on the proliferation of DFCsThe MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyl-2H-tetrazolium bromide] assay, BrdU (bromod-eoxyuridine) incorporation and cell-cycle analysiswere performed to investigate whether dNCPs couldaffect cell proliferation in vitro. As shown in Fig-ure 2(A), the treated DFCs exhibited significantlylower attenuance (D) values than the control cellsfrom day 5, suggesting that dNCPs may exert a sup-pressive effect on the proliferation of DFCs. The resultof BrdU incorporation, which is representative of asignificant portion of cells in the S-phase, indicatedthat the number of BrdU-positive cells in the con-trol group was higher than that in the treated group(Figure 2B).

Cell-cycle distributions were analysed by flow cyto-metry in the form of proliferation index (PI; per-centage of cells in the S- and G2/M-phases). In thepresent study, it was revealed that untreated DFCspresented a significantly higher percentage of cellsin S+G2/M phases (50.7%; Figure 2D) when com-

pared with treated cells (25.9%; Figure 2C), indic-ating that dNCPs could inhibit the proliferation ofDFCs (χ2 = 13.01, P < 0.01).

Effect of dNCPs on ALP (alkaline phosphatase)activity and mineralization behaviour of DFCsTo quantify the differentiation ability, ALP activ-ity and matrix mineralization were analysed at thedefined time points. ALP activity was significantlyhigher in the treated group than in the control groupfrom day 5, reaching a peak at day 9, then declin-ing gradually (Figure 3A). The mineralization wasquantified by calculating the nodule area and nod-ule number after von Kossa staining. After treatmentwith dNCP for 21 days, the mineralized nodule areawas 3.82 +− 0.26 mm2 per well and the nodule num-ber reached 86.17 +− 4.79 nodules per well; however,a little mineralized nodule was formed after 21 daysof routine culture with the corresponding data as0.16 +− 0.06 mm2 per well and 12.00 +− 3.00 nodulesper well (Figures 3B–3E).

Effect of dNCPs on protein and gene expressionof DFCsUsing immunocytochemical staining and RT (reversetranscription)–PCR, the expression of factors relatedto cementoblast differentiation of DFCs were ex-amined at the protein and mRNA levels. Our resultsshowed that treated cells, after a 6-day induction,stained positively for mineralization markers suchas BSP (bone sialoprotein), ON (osteonectin), OCN(osteocalcin) and Col I (type I collagen) and stainednegatively for DSP (dentin sialoprotein) and DMP-1(dentin matrix protein 1), the specific markers ofodontoblast lineages. Meanwhile, negative stainingfor all of the above proteins were detected in un-treated cells (Figures 4A–4L).

To confirm the cementoblast phenotype of treatedDFCs, RT–PCR was performed to investigate theexpression of DMP-1, DSPP (dentin sialophosphop-rotein), ON, OCN, BSP and Col I genes. The geneexpression of DSPP and DMP-1 could not be detec-ted in both groups; however, ON, OCN, BSP andCol I genes could be clearly identified after dNCP in-duction (Figure 4M), thereby indicating that DFCscan differentiate along cementoblast lineages but notodontoblasts. The markers have been investigated intriplicate with identical results.

www.biolcell.org | Volume 100 (5) | Pages 291–302 293

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 differentiationof DFCsIn order to evaluate the cementogenic capacity ofDFCs in vivo, a novel cell carrier consisting of hardtissue (enamel outside and dentin inside) from thetooth germ was adopted (Figure 5A). After trypsin-ization, any remaining cells were not found to adhereto the carrier before loading of the cell pellet (Fig-ure 5B). All of the cell pellets could be very easilyloaded into the inside chamber of carriers and havea direct contact with predentin, imitating the devel-opmental event during cementogenesis.

After 4 weeks’ incubation under renal capsulesof adult rats, all explants were harvested and ana-

lysed by means of histological examination. In thetreated group, a large amount of mineralized tis-sues was formed within the chamber of the carrier,which could be apparently distinguished from theoriginal dentin (Figures 5C–5E). The newly min-eralized tissues occupied the most space within thecarrier, and only a limited central area was left forthe growth of connective tissues and blood vessels.Some of the formative cells were entrapped withinthe newly formed tissues. However, in the controlgroup, DFCs proliferated well, and only limited ce-mentoid tissues were formed along the inner sur-face of dentin. There was a distinct demarcation linebetween the original dentin and the newly formed

294 C© The Authors Journal compilation C© 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, King–Armstrong 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 significantly larger area per well compared with the control. (C) Com-

parison of mineralized nodule number between treated (Exp) and untreated (Cont) DFCs. The nodule number per well in the exper-

imental group was significantly 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 µm.

mineralized tissues (Figures 5F and 5G). In orderto quantify these findings, the relative amount ofnewly formed cementum is expressed as the percent-age of the area of the newly formed cementum againstthe chamber area of the dentin carrier (cross-section),and the ratio for the experimental group is signific-antly higher than that for the control (Supplement-ary Figure 3 at http://www.biolcell.org/boc/100/boc1000291add.htm). The results of fluorescent tra-cing analyses further confirmed that the trans-planted DFCs participated in mineralized tissue

formation rather than in angiogenesis (Supple-mentary Figure 4B at http://www.biolcell.org/boc/100/boc1000291add.htm). Furthermore, no positivestaining against DSP could be detected in the newlyformed mineralized tissues (Figure 5I).

DPSCs were used as another control group be-sides untreated DFCs. Previous studies suggestedthat DPSCs had the ability to differentiate into odon-toblasts (Yu et al., 2006a, 2007; Liu et al., 2007;Sloan and Smith, 2007). The results of DPSC trans-plantation reconfirmed this idea by finding the fact

www.biolcell.org | Volume 100 (5) | Pages 291–302 295

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 µm. (M) Electrophoresis of RT–PCR

products: Exp, treated DFCs; Cont, untreated cells. β-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. Significant 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 C© The Authors Journal compilation C© 2008 Portland Press Ltd

Differentiation of DFCs induced by dNCPs Research article

that amorphous osteodentin was secreted within thecarrier and was immunopositive against DSP (Fig-ures 5H and 5J).

DiscussionIn contrast with enamel and dentin, the originof cementum still remains enigmatic (Slavkin andDiekwisch, 1996, 1997; Diekwisch, 2001). Cemen-tum formation begins when both epithelial cells ofHERS and mesenchymal cells of dental follicle are inproximity to the developing root surface. Althoughthe exact role of epithelial and mesenchymal com-ponents in cementogenesis is an open question, it hasgenerally been suggested that the disruption of HERSis the key event at the onset of root development (Choand Garant, 1988). Thus DFCs have access to the rootdentin surface and have a high probability of under-going cementoblastic differentiation to secrete theoriginal cementum matrix by dentinal induction.

In the present study, dNCPs extracted from dentin,as the representative of dentin matrix, were con-firmed to have the ability to stimulate DFCs intocementoblast lineages. Morphologically, the treatedDFCs appeared to have a flat, cuboidal or polygonalshape with a distended RER, showing an active func-tion of synthesis and secretion, which is similar tocementoblast phenotypes (Schroeder, 1986b; Faltinet al., 2001; Cattaneo et al., 2003). Interestingly, theelectron-dense granules within the cytoplasm, one ofthe markers of DFCs, were greatly reduced after in-duction, possibly attributed to cell maturity (Wiseet al., 1992; Martin, 1998). Cell proliferation, eval-uated by the MTT assay, BrdU incorporation andcell-cycle analysis, coincidentally demonstrated thatdNCPs could inhibit DFC proliferation during theprocess of differentiation, which was in accordancewith the previous concept that proliferation and dif-ferentiation are inversely correlated with each otherdue to the existence of dual-function regulators par-ticipating in controlling both the processes (Zhu andSkoultchi, 2001). Therefore it is believed that somecomponents of dNCPs could act not only as promotersfor the differentiation of DFCs but also as inhibitorsfor cell proliferation.

ALP plays a vital role in calcified tissue forma-tion by regulating phosphate transfer (Beck et al.,2000). Human cementoblasts have a high ALP activ-ity showing an active capacity for mineralization(Kitagawa et al., 2006). In the present study, the

increased ALP activity could imply that the treatedDFCs have been induced to mineral-forming cells. Itis also worth noting that mineralized nodule form-ation significantly increased after dNCP induction.Furthermore, in the investigated markers, DMP-1and DSPP/DSP are specific for identifying odonto-blasts, whereas ON, OCN, BSP and Col I are thecommon markers found in cementum and cemento-blasts (D’Errico et al., 1997). Previous studies havesuggested that dentin matrix could induce EMSCs,DPSCs or DPCs into odontoblast-like cells (Denget al., 2005; Liu et al., 2005; Nie et al., 2006).However, in the present study, treated DFCs coulddifferentiate into cementoblasts rather than odonto-blasts, as indicated by positive expression of ON,OCN, BSP and Col I, and negative expression ofDSP and DMP-1 at the mRNA and protein levels.Taken together, it was revealed by our in vitro studythat cementoblasts have possibly originated fromDFCs, a process in which dentin matrix is criticallyinvolved.

As to the in vivo experiments, the previous resultsof in vivo differentiation of DFCs were quite contro-versial. Bovine DFCs transplanted into immunodefi-ciency mice formed fibrous tissue and cementum-likematrix (Handa et al., 2002). However, murine DFCstransformed with SV40 (simian virus 40) seeded onto a PLGA [poly(D,L-lactide-co-glycolic acid)] scaffoldcould not mediate mineral formation in vivo and evenseemed to inhibit periodontal healing of artificialperiodontal defects in athymic rats (Zhao et al., 2002,2004). As we know, a better understanding of thefunction of cells can be attained by re-transplantationof cells from the formative tissues (Song et al., 2007).Thus we used a novel model (cell pellet loaded inthe chamber of the dentin carrier) and put it in therenal capsule of an adult rat, a transplantation site,which provides a suitable environment for DFC dif-ferentiation in vivo. It was found that, without theinduction of dNCPs, a limited amount of cementoidwas formed closely attached to the inner dentin sur-face, whereas after the induction, a lot of non-dentinmineralized tissues were produced by differentiatedDFCs, which was quantified as the percentage of thearea of newly formed mineralized tissues againstthe chamber area of the dentin carrier. Fluorescenttracing analyses further proved that it was the trans-planted DFCs that evidently formed such tissues.These phenomena can be explained as follows. First,

www.biolcell.org | Volume 100 (5) | Pages 291–302 297

J. Wu and others

once the cell pellet is loaded in the dentin carrier,the untreated DFCs make good contact with thepredentin surface of the carrier. Some dentin mat-rix might be diffused from predentin in the carrier toaffect proximate cells to form thin cementum, whichcould be termed as the ‘endogenous dNCPs effect’and is more likely a physiological situation since weknow that the acellular cementum layer in the liv-ing body is usually very thin. The treated cells haveundergone differentiation by the induction of exogen-ous dNCPs before loading, which could be regardedas the amplification of the endogenous effect, finallyleading to evident mineral formation. Secondly, thephysical property of the dentin substrate could exertan effect on DFC differentiation. It was revealed thatmatrix elasticity directs stem-cell lineage specifica-tion (Engler et al., 2006). Thus we can speculate thatthe dentin substrate has the preferential ability todrive DFCs into cementoblast lineages. In addition,we had proposed the concept of ‘cell pellet engineer-ing’, and successfully used dental papilla cell pelletto regenerate the dentin–pulp complex (Yu et al.,2006b). The pellet culture model, which can providethe three-dimensional growth environment necessaryfor cell differentiation, may help DFCs develop intocementoblasts.

Under the same protocol, DPSC implants demon-strated the formation of DSP-immunopositive osteo-dentin, implying that DPSCs could differentiate intoodontoblasts within the chamber of the carrier as pre-dicted. However, even in the inducing circumstance,treated DFCs could not differentiate into odonto-blasts, because the resulting mineralized tissues wereimmunonegative against DSP. The reason for this dif-ference may lie in cell-fate determination. It is knownthat, although both DPSCs and DFCs are derivedfrom one ancestor, namely EMSCs, different parts ofthe mesenchymal cells may have been determinedas different precursors for specific tissue formativecells by the intrinsic programmed fate during de-velopment. And here, DFCs and DPSCs only act asdetermined precursors for periodontal tissue and thedentin–pulp complex respectively in order to ensurethe harmony of tooth development and regeneration.

During the last decade, many studies have provedthat EMD (enamel matrix derivative) has obviouseffects on periodontal regeneration, including ce-mentogenesis (Gestrelius et al., 1997; Venezia et al.,2004; Bosshardt et al., 2005; Rincon et al., 2005).

As to DFCs, EMD could regulate in vitro differ-entiation of mouse follicle cells towards the osteo-cementoblastic phenotype (Hakki et al. 2001). Al-though similar effects of EMD and dNCPs couldbe achieved, there are obvious differences betweenthem. First of all, the application of these two mixedagents is based on respective hypotheses – EMDhas been proposed to mimic the inducing role ofHERS products involved in cementogenesis. How-ever, direct dentin–DFC contact as an importantdevelopmental event, and the inducing ability ofdNCPs, have reasonably supported our present study.Secondly, the compositions of the EMD and dNCPsare quite different – the presence of three matrix pro-teins (amelogenin, enamelin and sheathlin) and BMP(bone morphogenetic protein)-like growth factors(BMP-2 and BMP-7) have been confirmed in EMD. Arecent report indicates that BMPs present in EMDare probably crucial for the differentiation of hu-man DFCs into cementoblasts, in which Smad-1 andMAPK (mitogen-activated protein kinase) pathwaysseem to be involved (Kemoun et al., 2007). Besides,amelogenin, as the major fraction of EMD, has beenproved to be an effective molecule (Viswanathan et al.,2003; Boabaid et al., 2004; Swanson et al., 2006). Incomparison, DSP, DPP (dentin phosphoprotein) andDMP-1 secreted by odontoblasts are the major con-stituents of dNCPs, which have been suggested to beinvolved in the nucleation process and hydroxyapat-ite growth during dentin formation (Butler et al.,1998, 2002). However, the potential of these pro-teins as signalling molecules has been seldom pro-posed. DSP, which lacks the RGD sequence, is notable to promote the attachment and spreading ofcells (Butler et al., 1992), and mainly plays a rolein dentin mineralization (Goldberg et al., 2004). Incontrast, DPP up-regulates osteoblast marker genesin several kinds of mesenchymal cells by binding tointegrin receptors on the surface via its RGD domainand subsequently activating the MAPK signallingpathways (Jadlowiec et al., 2004). Also, DMP-1 wasshown to stimulate rat DPSCs (Almushayt et al.,2006) and mouse embryonic mesenchymal cells todifferentiate into odontoblast lineages (Narayananet al., 2001). Furthermore, we hypothesized thatdentin matrix proteins produced by odontoblastscould migrate through and diffuse from newly depos-ited predentin, which is similar to the well-knownphenomenon of diffusion of enamel matrix proteins

298 C© The Authors Journal compilation C© 2008 Portland Press Ltd

Differentiation of DFCs induced by dNCPs Research article

through the pre-mineralizing mantle dentin into theodontoblast layer (Inai et al., 1991). Collectively, itwas speculated that DPP and DMP-1 might be theeffective molecules in dNCPs for driving DFCs intocementoblast lineages in the present study as well asroot development. Besides, dentin represents a signi-ficant storage of various growth factors such as BMP-2, -3 and -7 and TGF-β (transforming growth factor-β). And even amelogenin slices could be identifiedfrom dentin extracts, as the remains of ameloblastsecretions (Veis et al., 2000). Thus these moleculescan be proposed to act in concert with other factorsto promote cementogenesis by affecting DFCs just inthe same way as they are in EMDs.

As we know, periodontal tissue is a typical ‘sand-wich structure’ consisting of cementum, periodontalligament and alveolar bone. However, there was noevident periodontal structure, but cementum formedin the present in vivo study if DFCs were stimulatedby dNCPs alone. Actually, periodontal developmentand regeneration involve cascades of numerous sig-nals. In a recent report, PLAP-1 (periodontal liga-ment associated protein-1)/asporin has been identi-fied to inhibit the mineralization of periodontal lig-ament cells, which is also intensively expressed bydental follicle (Yamada et al., 2007). Since DFCs arecommonly regarded as precursors of the formativecells for the above three kinds of tissues in the peri-odontium, molecules other than dNCPs might exertnegative effects on the differentiation of DFCs intocementoblasts as well as positive effects on the fateof DFCs into fibroblasts or osteoblasts, leading to thebalance of periodontal development.

In conclusion, the in vitro and in vivo evidence ac-cumulated in the present study showed that dNCPscould promote the cementogenic process by stimu-lating DFCs to differentiate into cementoblasts. Al-though the effective components of dNCPs shouldbe analysed in the future, the present study affirm-atively provides a better understanding of the under-lying mechanisms of cementogenesis, which may behelpful for regenerative periodontal therapies.

Materials and methodsCell isolation and cultureHarlan SD (Sprague–Dawley) rats were obtained from the Labor-atory Animal Research Centre of Fourth Military Medical Uni-versity. All surgical procedures and care administered to theanimals were approved by the University Animal Care Com-mittee and performed according to institutional guidelines. The

DFCs were isolated and cultured using a previously describedmethod (Wise et al., 1992) as detailed below. SD rats of 6 or7 days of age were killed by cervical dislocation. The mandibleswere aseptically dissected and placed in PBS solution where thedental follicle and enamel organ were dissected from the firstmolars with the aid of a dissecting microscope. The isolated tis-sues were placed in 1% trypsin/l mM EDTA solution for 10 minat room temperature (22◦C) and the follicle was separated by mi-crodissection from the stellate reticulum. The follicles were thenincubated in a 0.25% trypsin/1 mM EDTA solution for 10 minat 37◦C to partially dissociate the follicle cells. Then, the abovetissues were transferred to 35 mm tissue-culture dishes and in-cubated in DMEM (Dulbecco’s modified Eagle’s medium)/F12(Gibco BRL, Grand Island, NY, U.S.A.) containing 15% (v/v)fetal calf serum, 0.1 g/l sodium pyruvate, 100 µg/ml strepto-mycin and 100 units/ml penicillin and cultured at 37◦C in ahumidified atmosphere of 5% CO2. Within 15–20 days afterdissection, the cells were confluent and conventionally passaged.In the present study, the 3rd passage of DFCs was investigatedand divided into a treated group with 250 µg/ml dNCP medium(Deng et al., 2005) and an untreated group with conventionalculture medium (DMEM/F12 containing 10% fetal calf serum).The fresh medium was changed every 2 days.

Morphological observationAfter 6 days of culture, treated and untreated DFCs of the3rd passage were routinely observed and photographed undera phase-contrast inverted microscope to evaluate their pheno-types (Olympus Optical, Tokyo, Japan).

SEMDFCs of the 3rd passage were cultured in dNCP medium or con-ventional medium for 6 days and then digested in order to seedthem on to coverslips. DFCs were then incubated in the respect-ive medium for a further 24 h. After washing, the cell coverslipswere fixed with 2.5% glutaraldehyde at 0◦C, dehydrated anddried in a critical-point dryer. The cells were observed under aHitachi S-3400N scanning electron microscope (Hitachi, Tokyo,Japan).

TEMAfter being treated for 6 days, the DFCs (3rd passage) weretrypsinized and centrifuged (3000 g, 5 min, 4◦C) to form pel-lets, then fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer(pH 7.3) for 1 h at room temperature and postfixed in aq. 2%(v/v) osmium tetroxide for a further 1 h. The cells then weredehydrated in a series of ethanols (50, 70, 95 and 100%) andembedded in Epon 812 resin. Ultrathin sections were madeand stained with uranyl acetate and lead citrate. The sampleswere viewed with a JEM 100 SX electron microscope (Jeol,Tokyo, Japan).

Cell proliferation assayDFCs of the 3rd passage were seeded on to 96-well plates at adensity of 1000 cells per well. After incubation for 24 h, to half ofthe wells dNCP medium was added, and conventional mediumwas added to control wells. The MTT assay was performed at day1, 3, 5, 7, 9, 11 and 13 after induction. Briefly, 20 µl of MTT(5 mg/ml; Sigma–Aldrich, St. Louis, MO, U.S.A.) was added toeach well, which was converted into an insoluble blue formazanproduct by mitochondria of living cells but not of dying cells

www.biolcell.org | Volume 100 (5) | Pages 291–302 299

J. Wu and others

or debris. After incubation at 37◦C for 4 h, the supernatant wasremoved and the formazan crystals were dissolved in 150 µl ofDMSO. Attenuance values for each well were measured spec-trophotometrically at 490 nm and the assay was repeated threetimes.

BrdU incorporationDFCs of the 3rd passage were seeded on to 24-well platesat a density of 5 × 103 cells per well. After the induction ofdNCPs for 6 days, all cells were incubated with 10 µM BrdU(Sigma–Aldrich) for 1 h and fixed with 4% polyoxymethylene.Then, the cultures were processed for immunostaining accordingto the manufacturer’s instructions. Cells containing denselybrown-stained nuclei with clear morphology were consideredBrdU-positive, and they were counted in five fields per well(centre and at 03:00, 06:00, 09:00 and 12:00 h). Results wereexpressed as the percentage of BrdU-positive cells against totalcells counted.

Cell-cycle analysisAfter being treated for 6 days, DFCs were collected for flow cyto-metry analysis. Briefly, cells were digested and cell precipitateswere washed twice with 0.01 M PBS and resuspended in 1 ml ofphysiological saline by repeated vibration to ensure a single cellsuspension. Then, 2 ml of cold dehydrated alcohol was mixedquickly with the cell suspension to fix cells at 4◦C for 24–48 h.Finally, the cells were washed twice again with PBS, stained with100 mg/ml propidium iodide at 4◦C for 30 min and subjectedto cell-cycle analysis using Elite ESP flow cytometry (BeckmanCoulter, Fullerton, CA, U.S.A.).

ALP activity assayAt defined time points, the ALP activity of treated and untreatedDFCs was detected using an ALP assay kit (JianCheng, Nanjing,China). In brief, cells (1 × 103 per well) were placed in a 96-wellplate. After culture, cells were washed three times with 0.01 MPBS and 50 ml of cold 10 mM Tris/HCl buffer (pH 7.4) con-taining 0.1% Triton X-100 added prior to incubation at 4◦Covernight. A 100 µl portion of ALP substrate solution (2 mMMgCl2 and 16 mM p-nitrophenyl phosphate) was then mixedwith each sample. After incubation at 37◦C for 30 min, the re-action was stopped by the addition of 50 ml of 0.2 M NaOH andthe liberated p-nitrophenol was measured spectrophotometric-ally at 410 nm. The results were described in King–Armstrongunits (Yu et al., 2006a).

von Kossa staining for mineralized nodule formationDFCs of the 3rd passage were seeded on to 24-well plates at adensity of 5 × 103 cells per well. After being treated or untreatedwith dNCPs for 21 days, von Kossa staining was carried out todetermine mineralized nodule formation. In brief, the cells werefixed with cold absolute acetone for 30 min, then incubated in5% AgNO3 for 30 min, washed with distilled water, treatedwith a 5% Na2CO3 and 25% formalin solution for 2 min andthen washed with distilled water again. Finally, the cells werecounterstained with Nuclear Fast Red. The nodule area andnodule number per well were measured quantitatively with animage analysis system Image-Pro Plus 5.0 (Media Cybernetics,Baltimore, MD, U.S.A.).

Immunocytochemistry analysisThe DFCs in both groups were seeded on to coverslips at adensity of 2 × 105 cells/ml, maintained for another 2 days inrespective medium and then fixed with 4% polyoxymethyl-ene. Immunocytochemical analyses were performed with thestreptavidin–biotin complex method according to the manufac-turer’s recommended protocol. Antibodies included (i) affinity-purified polyclonal rabbit anti-rat BSP at a 1:500 dilution, (ii)polyclonal rabbit anti-rat ON at a 1:500 dilution, (iii) affinity-purified polyclonal rabbit anti-rat OCN at a 1:100 dilution, (iv)polyclonal rabbit anti-rat Col I at a 1:500 dilution, (v) polyclonalgoat anti-rat DMP-1 at a 1:100 dilution and (vi) polyclonal rab-bit anti-rat DSP at a 1:50 dilution. The antibodies for DSP, ONand Col I were gifts from Dr Larry W. Fisher [NIDR (NationalInstitute of Dental and Craniofacial Research), Bethesda, MD,U.S.A.], and the others were obtained from Santa Cruz Biotech-nology (Santa Cruz, CA, U.S.A.). The specimens were counter-stained with haematoxylin, and all the samples were examinedunder an Olympus compound microscope (Olympus Optical).

RT–PCRDFCs at 90% confluence were harvested after a 6-day induc-tion by dNCPs. Total RNA was isolated by means of TRIzol®

reagent (Invitrogen, Carlsbad, CA, U.S.A.) and was reverse-transcribed using the SuperScriptTM First-Strand Synthesis Sys-tem for RT–PCR (Invitrogen) following the manufacturer’s in-structions. Primer sequences for DMP-1 (GenBank® accessionno. 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 β-actin (GenBank® accession no. NM 031144) were asfollows: DMP-1 sense, 5′-CGGCTGGTGGTCTCTCTAAG-3′, and DMP-1 antisense, 5′-GTCCCTCTGGGCTATCTT-CC-3′; DSPP sense, 5′-TAAGGACAAGGACGAATC-3′, andDSPP antisense, 5′-ACTGCTGTCACTGCTTTC-3′; ON sense,5′-CTGCAGAAGAGATGGTGGCGG-3′, and ON antisense,5′-CAGGCAGGGGGCAATGTATTTG-3′; OCN sense, 5′-AT-GAGGACCCTCTCTCTGCTC-3′, and OCN antisense, 5′-CT-AAACGGTGGTGCCATAGAT-3′; BSP sense, 5′-CTGCTT-TAATCTTGCTCTG-3′, and BSP antisense, 5′-CCATCTCCAT-TTTCTTCC-3′; Col I sense, 5′-CGTGACCAAAAACCAA-AAGTGC-3′, and Col I antisense, 5′-GGGGTGGAGAAA-GGAACAGAAA-3′; β-actin sense, 5′-GAGACCTTCAAC-ACCCCAGCC-3′, and β-actin antisense, 5′-CATAGCACA-GCTTCTCTTTAA-3′, used as an internal control. After 30cycles, the PCR products were separated by using 2% agarose-gel electrophoresis and were stained with ethidium bromide, anddigital images were taken on UV background. This experimentwas repeated three times and PCR products were confirmed bysequencing (Sangon Biotechnology, Shanghai, China).

In vivo differentiation of DFC pelletsIn order to mimic the contact of DFCs with dentin, as well as tofacilitate the transplantation of cell pellets, we utilized a novelnatural dentin carrier in the present study to contain DFCs pel-lets. In fact, we removed dental papilla tissues from the remainsof tooth germs after dental follicle isolation, and only hard tissueswere left and further digested by 0.25% trypsin for 30 min to be

300 C© The Authors Journal compilation C© 2008 Portland Press Ltd

Differentiation of DFCs induced by dNCPs Research article

completely acellularized. The hard tissues showed a chamber-likeshape that consisted of enamel outside and dentin inside. Thecell pellet culture was performed as previously described withsome modifications (Yu et al., 2006a). Briefly, DFCs co-culturedwith dNCPs for 7 days were labelled with Hoechst 33342 (Sup-plementary Figure 4A) and then centrifuged in a 10-ml conicalpolypropylene tube (Asahi Techno Glass, Tokyo, Japan) at 800 gfor 5 min. Cell pellets in the tube were maintained in respect-ive medium at 37◦C for 5 h to make them well congregatedand favourable for transplantation procedures. The pellets weretransferred into the carrier chamber and then seeded directly intothe renal capsules on the left side of 8-week-old allogenic SD rathosts. As a control, the untreated DFC pellets within the carrierwere implanted into the other side of the same host. In addition,pellets of rat DPSCs were also used as a control. Transplantationprocedures were performed as described elsewhere (Colnot et al.,2004). Totally, 58 cell pellets (28 treated DFCs, 25 untreatedDFCs and 5 DPSCs) were implanted into the renal capsules of30 rats. After 28 days of incubation, the explants were isolatedand fixed in 4% polyoxymethylene, decalcified with buffered10% EDTA (pH 8.0) and processed for H&E (haematoxylin andeosin) staining and immunohistological staining against DSP.Besides, frozen sections were prepared and examined with afluorescent microscope to identify donor cells. Histological ana-lyses were quantified with an image analysis system Image-ProPlus 5.0 (Media Cybernetics).

Statistical analysisStatistical significance was assessed by a χ2 test and an independ-ent samples t test with SPSS v 12.0 software (SPSS, San Rafael,CA, U.S.A.). Statistical significance was determined at P < 0.05.All data acquisition and analyses were performed blindly.

AcknowledgmentsWe are grateful to Professor Anthony J. Smith(Oral Biology, School of Dentistry, University ofBirmingham, Birmingham, U.K.) for his kind pro-vision of dNCPs. We also thank Dr Juan Dai for hercritical reading of this paper. This work was suppor-ted by grants from the Nature Science Foundation ofChina (project no. 30572046).

ReferencesAlmushayt, A., Narayanan, K., Zaki, A.E. and George, A. (2006)

Dentin matrix protein 1 induces cytodifferentiation of dental pulpstem cells into odontoblasts. Gene Ther. 13, 611–620

Beck, Jr, G.R., Zerler, B. and Moran, E. (2000) Phosphate is a specificsignal for induction of osteopontin gene expression. Proc. Natl.Acad. Sci. U.S.A. 97, 8352–8357

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 moleculeduring cementogenesis. J. Periodontol. 75, 1126–1136

Bosshardt, D.D. (2005) Are cementoblasts a subpopulation ofosteoblasts or a unique phenotype? J. Dent. Res. 84, 390–406

Bosshardt, D.D. and Nanci, A. (1997) Immunodetection of enameland cementum-related (bone) proteins at the enamel-free area andcervical portion of the tooth in rat molars. J. Bone Miner. Res. 12,367–379

Bosshardt, D.D. and Nanci, A. (2004) Hertwig’s epithelial root sheath,enamel matrix proteins, and initiation of cementogenesis in porcineteeth. J. Clin. Periodontol. 31, 184–192

Bosshardt, D.D., Sculean, A., Windisch, P., Pjetursson, B.E. andLang, N.P. (2005) Effects of enamel matrix proteins on tissueformation along the roots of human teeth. J. Periodontal Res. 40,158–167

Butler, W.T. (1998) Dentin matrix proteins. Eur. J. Oral Sci. 106,204–210

Butler, W.T., Bhown, M., Brunn, J.C., D’Souza, 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 dentinsialoprotein (DSP). Matrix 12, 343–351

Butler, W.T., Brunn, J.C., Qin, C. and Mckee, M.D. (2002) Extracellularmatrix proteins and the dynamics of dentin formation. Connect.Tissue Res. 43, 301–307

Cattaneo, V., Rota, C., Silvestri, M., Piacentini, C., Forlino, A.,Gallanti, A., Rasperini, G. and Cetta, G. (2003) Effect of enamelmatrix derivative on human periodontal fibroblasts: proliferation,morphology and root surface colonization. An in vitro study.J. Periodont. Res. 38, 568–574

Cho, M.I. and Garant, P.R. (1988) Ultrastructural evidence of directedcell migration during initial cementoblast differentiation in rootformation. J. Periodont. Res. 23, 268–276

Colnot, C., Lu, C.Y., Hu, D. and Helms, J.A. (2004) Distinguishing thecontributions of the perichondrium, cartilage, and vascularendothelium to skeletal development. Dev. Biol. 269, 55–69

Deng, M.J., Shi, J.N., Smith, A.J. and Jin, Y. (2005) Effects oftransforming growth factor β1 (TGF-1) and dentin non-collagenousproteins (DNCP) on human embryonic ectomesenchymal cells in athree-dimensional culture system. Arch. Oral Biol. 50, 937–945

D’Errico, J.A., MacNeil, R.L., Takata, T., Berry, J., Strayhorn, C. andSomerman, M.J. (1997) Expression of bone associated markers bytooth root lining cells, in situ and in vitro. Bone 20, 117–126

Diekwisch, T.G.H. (2001) Developmental biology of cementum. Int. J.Dev. Biol. 45, 695–706

Engler, A.J., Sen, S., Sweeney, H.L. and Discher, D.E. (2006) Matrixelasticity directs stem cell lineage specification. Cell 126, 677–689

Faltin, R.M., Faltin, K., Sander, F.G. and Arana-Chavez, V.E. (2001)Ultrastructure of cementum and periodontal ligament aftercontinuous intrusion in humans: a transmission electronmicroscopy study. Eur. J. Othod. 23, 35–49

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. 131–202, Munksgaard, Copenhagen

Gestrelius, S., Andersson, C., Lidstrom, D., Hammarstrom, L. andSomerman, M. (1997) In vitro studies on periodontal ligament cellsand enamel matrix derivative. J. Clin. Periodontol. 24, 685–692

Gestrelius, S., Lyngstadaas, S.P. and Hammarstrom, L. (2000)Emdogain-periodontal regeneration based on biomimicry.Clin. Oral Investig. 4, 120–125

Goldberg, M., Septier, D., Bourd, K. and Menashi, S. (2004) Role ofmatrix proteins in signalling and in dentin and enamelmineralization. C. R. Palevol. 3, 573–581

Hakki, S.S., Berry, J.E. and Somerman, M.J. (2001) The effect ofenamel matrix protein derivative on follicle cells in vitro.J. Periodontol. 72, 679–687

Hammarstrom, L., Alatli, I. and Fong, C.D. (1996) Origins ofcementum. Oral Dis. 2, 63–69

Handa, K., Saito, M., Yamauchi, M., Kiyono, T., Sato, S., Teranaka, T.and Narayanan, A.S. (2002) Cementum matrix formation in vivo bycultured dental follicle cells. Bone 31, 606–611

Inai, T., Kukita, T., Ohsaki, Y., Nagata, K., Kukita, A. and Kurisuk, K.(1991) Immunohistochemical demonstration of amelogeninpenetration toward the dental pulp in the early stages of ameloblastdevelopment in rat molar tooth germs. Anat. Rec. 229, 259–270

www.biolcell.org | Volume 100 (5) | Pages 291–302 301

J. Wu and others

Jadlowiec, J., Koch, H., Zhang, X., Campbell, P.G., Seyedain, M. andSfeir, C. (2004) Phosphophoryn regulates the gene expressionand differentiation of NIH3T3, MC3T3-E1, and humanmesenchymal stem cells via the integrin/MAPK signaling pathway.J. Biol. Chem. 279, 53323–53330

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 cellsacquire cementoblast features under stimulation by BMP-2/-7 andenamel matrix derivatives (EMD) in vitro. Cell Tissue Res. 329,283–294

Kitagawa, M., Tahara, H., Kitagawa, S., Oka, H., Kudo, Y., Sato, S.,Ogawa, I., Miyaichi, M. and Takata, T. (2006) Characterization ofestablished cementoblast-like cell lines from humancementum-lining cells in vitro and in vivo. Bone 39, 1035–1042

Liu, J., Jin, T., Ritchie, H.H., Simth, A.J. and Clarkson, B.H. (2005)In vitro differentiation and mineralization of human dental pulp cellsinduced by dentin extract. In Vitro Cell Dev. Biol. Anim. 41,232–238

Liu, J., Jin, T., Chang, S., Ritchie, H.H., Smith, A.J. and Clarkson,B.H. (2007) Matrix and TGF-beta-related gene expression duringhuman dental pulp stem cell (DPSC) mineralization. In Vitro CellDev. Biol. Anim. 43, 120–128

Martin, V.J. (1998) Development of nerve cells in hydrozoan planulae:I. Differentiation of ganglionic cells. Biol. Bull. 174, 319–329

Narayanan, K., Srinivas, R., Ramachandran, A., Hao, J., Quinn, B.and George, A. (2001) Differentiation of embryonic mesenchymalcells to odontoblast-like cells by overexpression of dentin matrixprotein 1. Proc. Natl. Acad. Sci. U.S.A. 98, 4516–4521

Nie, X., Tian, W., Zhang, Y., Chen, X., Dong, R., Jiang, M., Chen, F.and Jin, Y. (2006) Induction of transforming growth factor-β 1 ondentine pulp cells in different culture patterns. Cell Biol. Int. 30,295–300

Provenza, D.V. (1988) Attachment apparatus. Fundamentals of OralHistology and Embryology (Provenza, D.V., ed.), pp. 195–235, Leaand Febiger, Philadelphia, PA

Rincon, J.C., Xiao, Y., Young, W.G. and Bartold, P.M. (2005)Enhanced proliferation, attachment and osteopontin expression byporcine periodontal cells exposed to Emdogain. Arch. Oral Biol.50, 1047–1054

Schroeder, H.E. (1986a) The periodontium. In Handbook ofMicroscopic Anatomy (Oksche, A. and Vollrath, L., eds),pp. 170–232, Springer-Verlag, Berlin

Schroeder, H.E. (1986b) The periodontium. In Handbook ofMicroscopic Anatomy (Oksche, A. and Vollrath, L., eds),pp. 23–128, Springer-Verlag, Berlin

Slavkin, H.C. (1976) Towards a cellular and molecular understandingof periodontics. Cementogenesis revisited. J. Periodontol. 47,249–255

Slavkin, H.C. and Diekwisch, T.G.H. (1996) Evolution in toothdevelopmental biology: of morphology and molecules. Anat. Rec.245, 131–150

Slavkin, H.C. and Diekwisch, T.G.H. (1997) Molecular strategies oftooth enamel formation are highly conserved during vertebrateevolution. Ciba Found. Symp. 205, 73–80

Sloan, A.J. and Smith, A.J. (2007) Stem cells and the dental pulp:potential roles in dentine regeneration and repair. Oral Dis. 13,151–157

Smith, A.J. and Leaver, A.G. (1979) Non-collagenous components ofthe organic matrix of rabbit incisor dentin. Arch. Oral Biol. 24,449–454

Song, A.M., Shu, R., Xie, Y.F., Song, Z.C., Li, H.Y., Liu, X.F. andZhang, X.L. (2007) A study of enamel matrix proteins ondifferentiation of porcine bone marrow stromal cells intocementoblasts. Cell Prolif. 40, 381–396

Stahl, S.S. and Slavkin, H.C. (1972) In Development of gingivalcrevicular epithelium and periodontal disease. In DevelopmentalAspects of Oral Biology (Slavkin, H.C. and Bavetta, L., eds),pp. 326–350, 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 regulateexpression of genes associated with cementoblasts in vitro. Eur. J.Oral Sci. 114, (Suppl. 1), 239–243

Venezia, E., Goldstein, M., Boyan, B.D. and Schwartz, Z. (2004) Theuse of enamel matrix derivative in the treatment of periodontaldefects: a literature review and meta-analysis. Crit. Rev. OralBiol. Med. 15, 382–402

Veis, A., Tompkins, K., Alvares, K., Wei, K., Wang, L., Wang, X.S.,Brownell, A.G., Jengh, S.M. and Healy, K.E. (2000) Specificamelogenin gene splice products have signaling effects oncells in culture and in implants in vivo. J. Biol. Chem. 275,41263–41272

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, 1423–1431

Wise, G.E., Lin, F. and Fan, W. (1992) Culture and characterization ofdental follicle cells from rat molars. Cell Tissue Res. 267, 483–492

Yu, J.H., Deng, Z.H., Shi, J.N., Zhai, H.H., Nie, X., Zhuang, H. andJin, Y. (2006a) Differentiation of dental pulp stem cells intoregular-shaped dentin-pulp complex induced by tooth germ cellconditioned medium. Tissue Eng. 12, 3097–3105

Yu, J.H., Shi, J.N., Deng, Z.H., Zhuang, H., Nie, X., Wang, R.N. andJin, Y. (2006b) Cell pellets from dental papillae can reexhibit dentalmorphogenesis and dentinogenesis. Biochem. Biophys. Res.Commun. 346, 116–124

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 cellsversus dental pulp stem cells. Biol. Cell 99, 465–474

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 periodontalligament mineralization. J. Biol. Chem. 282, 23070–23080

Zhao, M., Xiao, G., Berry, J.E., Franceschi, R.T., Reddi, A. andSomerman, M.J. (2002) Bone morphogenetic protein 2 inducesdental follicle cells to differentiate toward a cementoblast/osteoblast phenotype. J. Bone Miner. Res. 17, 1441–1451

Zhao, M., Jin, Q., Berry, J.E., Nociti, F.H., Giannobile, W.V. andSomerman, M.J. (2004) Cementoblast delivery for periodontaltissue engineering. J. Periodontol. 75, 154–161

Zhu, L. and Skoultchi, A.I. (2001) Coordinating cell proliferation anddifferentiation. Curr. Opin. Genet. Dev. 11, 91–97

Zeichner-David, M. (2001) Is there more to enamel matrix proteinsthan biomineralization? Matrix Biol. 20, 307–316

Zeichner-David, M. (2006) Regeneration of periodontal tissues:cementogenesis revisited. Periodontol. 2000 41, 196–217

Received 30 July 2007/21 November 2007; accepted 28 November 2007

Published as Immediate Publication 28 November 2007, doi:10.1042/BC20070092

302 C© The Authors Journal compilation C© 2008 Portland Press Ltd