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Induction of Dentin Formation on Canine Amputated Pulp by Recombinant Human Bone Morphogenetic Proteins (BMP)-2 and -4 J DENT RES 1994 73: 1515 DOI: 10.1177/00220345940730090601 The online version of this article can be found at: http://jdr.sagepub.com/content/73/9/1515

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J Dent Res 73(9):1515-1522, September, 1994

Induction of Dentin Formation on Canine Amputated Pulp by Recombinant Human Bone Morphogenetic Proteins (BMP)-2 and -4 M. Nakashima Department of Conservative Dentistry, Faculty of Dentistry, Kyushu University, 61, Fukuoka 812,Japan

Abstract. Dental pulp cells have the potential to differentiate into odontoblasts. The molecular mechanisms underlying differentiation are not clear. Demineralized dentin matrix is osteoinductive and contains bone morphogenetic protein (BMP) activity. BMPs have been implicated in embryonic odontogenic differentiation and hence may play a role in the differentiation of adult pulp cells into odontoblasts during pulpal healing. This study examined the hypothesis that BMPs induce dentin formation on amputated canine pulp. Recombinant human BMP-2 and BMP-4 were capped with inactivated dentin matrix on amputated pulp. At two months, the amputated pulp was filled with tubular dentin in the lower part and osteodentin in the upper part. The amount of dentin formed was markedly diminished when dentin matrix alone was implanted. These findings imply that recombinant human BMP-2 and BMP-4 induce differentiation of adult pulp cells into odontoblasts. Thus, BMPs may have a role in dentistry as a bioactive pulp-capping agent to induce dentin formation. Key words. Bone Morphogenetic Proteins-2 and -4, Dentin, Dental Pulp, Odontoblasts, Differentiation, Dental Pulp Capping, Pulpotomy.

Received September 3,1993; Accepted May 11, 1994

Introduction Dental pulp tissue retains considerable potential for regeneration and repair (Fitzgerald, 1979; for a review, see Yamamura, 1985). The precise cellular and molecular mechanisms underlying this functional repair are, however, still unknown. Similarly, bone is also capable of complete repair. The molecular basis of bone repair has now been attributed to a family of bone morphogenetic proteins (BMPs) based on de novo induction of bone into extraskeletal sites. Eight BMPs-namely, BMP-1-BMP-7 and osteogenic protein (OP)-2-have so far been cloned and expressed (Wozney et al., 1988; Celeste et al., 1990; Ozkaynak et al., 1990; Wang et al., 1990; Hammonds et al., 1991; Ozkaynak et al., 1992; for a review, see Reddi, 1992). All BMPs except BMP-1 belong to the transforming growth factor-beta (TGF-j) superfamily, a large group of signaling proteins with multiple biological activities controlling the differentiation of a variety of cell types (Rizzino, 1988). A BMP-like activity which induces bone formation is also present in dentin matrix (Butler et al., 1977; Conover and Urist, 1982; Katz and Reddi, 1988; Mera, 1988; Kawai and Urist, 1989; Bessho et al., 1990, 1991). The precise BMPs in dentin have not been identified. It is likely that BMPs may play a role in dentinogenesis, and in fact Lyons et al. (1990) have shown that BMP-2 mRNA expression is evident in dental papilla and odontoblasts in developing tooth bud. In organ culture of dental papilla from 17-day-old mouse embryos, BMP-2 stimulates matrix secretion. When combined with inactive total EDTA-soluble dentin proteins, BMP-2 stimulates odontoblast differentiation (Begue-Kirn et al., 1992). Recently, it has been shown that BMP-4 mRNA expression is first detected in the presumptive dental epithelium and is shifted thereafter to the condensed dental mesenchyme. The message is then restricted to the pre-odontoblasts during bell stage and is

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ech t Rcs 73(9)) 1094 Nakcishinici 1516JI5

Figure 1. [IMP2 aftr te 2 mo'th Note the or-

deletiii anid ostcorgdentin in the Lower and im ide ar it son the am putatcd (ar rows) pulp 1In the upper part, pieces ol irnplantetd cletiti

,

rouIIded by pulp tissue cani he see n

Figure 2. BMPI-2 with carrier, the supeirior part of the caxvitv Red blood cells and spindle-shapdeelcls canie scen airound imlplanted dentin inatrix (M)

s0

correlated with tooth induction (Vainio et of., 1993). BMP-2 mRNA expression is l'irst seen in the inner enamel epitheliurn about 3 days later thani BMP-4. Then it is shifted to the mesenchymal cells of dental papilla and appears in pre-odontoblasts during their terminal differentiation, suggesting a role in odontoblast differentiation Recombinanit BMI'-2 or BMP 4 containing agarose beads is able to induce BMp-4 expression in the mesenchyme in the absence of epithelium, suggesting that BMP-4 is involved in the biochemical pathway mediating early inductive interactions between the epithelial and mesenchymal tissues (Vainio ct al, 19c)3). In view of this, BMPs play an integral role in dentin development and morphogenesis. Using bovine pulp cell cultures, we have shown that BMP 4 rnRNA is expressed on day 14 during the increased expression of the extracellular matrix protein (Nakashima et f., L[994). BMP-2 imRNA is expressed on day 28, when pulp cel Is have already dilflerentiated into pre odontoblasts. Genes lor BMP 2 have been cloned from the cells in pulp cell culture on day 28 (Nakashima, unpublished data). In vitro, recombinant human BMP-2 increased osteocalcin

synthesis, BMI-4 increased exprcssion of rIl(l) collagen mRNA, and both 13MI'-2 and B3Mp-4 stimulated allkaline phosphatase activity. These indinigs suggest a rcgulatory role l'or BMPs on the diitctfcitiationi of pulp cells into preodontoblasts (Nakashima et al., 19)94). Partially purilfied BMP from bone has been showni to inclutce dentini on the amputated pulp (Nakashirna, 1990b). It is possible that BMP 2 and BLMP-4 mRNA may be expressed in pulp mesenchymal cells duriing pulpal wound hacaling and resultant Iuorination ol dentin. Bio-active pulp-capping agents need to be dcveloped. Agents that enhance healing poteitial of pulp tissue and induce the formation of' a large amounit ol dentini ovexc exposed pulp which protects 1tromi microleakage anid pulp infection could be clinically usel'ul f-or direct pulp cappilg. In this communication, we havc examined the hypothiesis that BMPs in dentin matrix may induce denitn feormation. The results revealed that BMLps may be used as potent bioactive pulp-capping agents.

Materials and methods Preparation of samples Inactivated dernineralized

dentini matrix powdci- withi a Guanidine-hydrochloridecextracted particle (200-500 p.m in size) was prepared from bovine permanent incisors as previously described (Nakashima, 1989). Two p.g olf recombinant humnan BMP-2 (mol wt. 30.000) and recombinant humnan BMP-4 (rnol wt, 32,000) wer-e added.

respectively, to carriers: 10 trig ol inactivated dcntinl matrix powder, 0.5 mg of chondroitini 6-sulfate sodiuLim salt (Seikagaku Kogyo Co., Nagoya, Japan), and 250 p.g of acidsoluble type I rat tail tendon collagen (Muthiukumiaran cl ofl., 1988). The samples were mixed and lelt f'or I h at roomn temperature before the proteins wei dried under vacuum.

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'] Dc nt Rcs 73(9) 1994

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Figure 3. BMP 2 w ith carrier, the uppei- part, Both undiflerentiated large cells (arrowheacds) around implanitcd dentin matrix (M) and attached to mnatrix (M) and spindle-shaped cells (arrows) synthesizing matrix airound them can he seen

Pulp-capping procedures Twelve teeth from 2 young adult dogs weighing I5 kg were used. Surgical anesthesia was obtained by intravenous injection ol' 20 mg pentobarbital sodium per kg of body weight. The puips ol' the upper third incisor and caninie and ol the lower caninle were amputated as described previously (Nakashima. 1989). BMPI-2 with carrier, BMP-4 with carrier, and carrier alone were capped into the cavities ol' four teeth on the amnputated pulp. The dry samnples were moistened with saline and transl'erred to the amputated pulp by a smnall brush. The cavities over the implanted mnaterials were filled with Elite" cement (zinc phosphate cement, G.C. Dental Industrial Corporation, Tokyo, Japan) and Clearfil' (resin composite, Kurare Company. Okayaina, Japan). Histological examination All ol' the anirnals were killed alter 2 mnonths. The apical two-thirds ol the root was imnmediately removed, and the teeth were ixed in 10%'/,, I'orinalin They were then demineralized in 10k', lformnic acid, embedded in paralLin, sectioned at 4-5 p.m, and stained with hematoxylin and eosin lor routine light microscopy. The extracted teeth were evaluated by contact radiography by soft x ray apparatus, type SRO M50 (Sofron Company Limited, Tokyo), at 40kV, 5mA. for I mnin belore demineralization. Quantitative analyses of newly formed dentin We examined relative amrounts of newly induced dentin in each sample by capturing, through the microscope, video images of the histological preparations 5 sections [1rom one tooth, at 150 p.n intervals. Irmage Grabber 1.15 (Neoteck l-imnited. Fastleigh, Hampshire, UK), installed in a Macintosh Quadra 700 (Apple Comnputer. Cupertino, CA, USA) connected to the video camera, was used to digitize the images. Using Canvas 3-Jz soltware (Deneba Systems Inc.,

Figure 4. Bml4 2 with carriei, thc mniddlc part (a, top) Notc the odontoblast-likc cells (arrowheads) cxtending, t lwici cytoplasin ic processcs into newly foiined tubular dc tti (TD)) (h hottomn) Note osteodentiniocytes (arrows) entrapped in osteodenitinl

Miami, Fl, USA), we hand-traced the oin-sci-enl image outlines of' pulp tissue and implanted dentini matrLix in the cavity on the amnputated pulp, and the total areas ol these were determined by Ultimage/24+ sol'tware (Image & Measurement Inc., Mirmande France).

Results Histological observations Two months after implantation ol 13MIl-2 withi caLicrie theie is a gradient of cellular response I rom the bottom ol the cavity on the amnputated pulp to the top (iFig. 1). The cavitv on the am?putated pulp was divided into l'our parts designated lower, middle, upper, aiid supeirior- at I-mill increments I'rom the amputated site. In the supel-ioi pait ol the cavity, red blood cells, rnonocytes. and spiindle shaped mesenchymal cells with slender piocesses containing elongated rod-shaped nuclei weie observed (Fig 2). The upper part olf the cavity was f'illed with the pulp tissuc alnl consisted ol loose coniective tissue contaiiiilg capillaries, spindle-shaped cells, and undilfI eenitiated laigc oval, or polygonal cells whose nuclei were weakly stainccd Somc ol

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Figure 5. BMP 2 with carrier, the lower part A few odontoblasts and a capilLary i-emain in tubulat dctitiii (TD). Figure 6. 3MP 4 with carrier, alter 2 riToniths. Note a large aTiroun t of in-

discontinuous zones. Radiography

duced dcentiti c o ii

and a few odontoblasts. osteodentitiocytes, and capillaries remained in the mnineralized matrix (Fig. 5). Soic of implanted dentin mnatrix was resoirbed. Teeth capped with B3MP-4 plus carn-icr showed histological results sinilar to those ol BMIM-2 plus carcrier as described above (Fig. 6). Considerable amounits ol osteodentini surrounded osteodentinocyte like cells (Fig. 7a). Occasionally, tubular dentin was also seen adjaccit to the osteodentin (Fig. 7b). In the superior part ol the cavity, spindle-shaped cells deposited extracellular mnatrix around the cells themselves (Fig. 8). In teeth which were capped with cat-rier alone, all parts ol the cavity were Ililled with pulp tissue (Fig. 9). TLbulaidentiin formation was minuscule compared w ith I3MI'-2and BMP 4-enriched carrier, although odontoblast-like cells lined the implanted matrix in the lower and mniddle parts ol the cavity (Fig 10). Osteodentin f'orrnationxwas irarely seen. In all irnplanted teeth, there was no sign of inl laimationi or dystrophic mrineralization in the remaininig pulIp tissue. Some of the newly induced dentini appeared in

si s t i n g

Contact-radiographic observation (Fig. I) showed that optimal mineralization had occurred in the cavities in teeth implanted with BMP-2 and BMPI-4. Whel cairiiei- alone w as implanted, there was a signif icantly larger radiolucent area compared with that when BMP-2 and BMI' 4 were capped.

mTiai nlIyof tubular deititi in the lower part and osteodentin

part Remaining pulp tissue uncder the amputated site (arrows) is normal.

Quantitative analyses Since there was no sign of if laminationl the area excluding pulp tissue and implanted dentin mati-ix was regarded as newly formed dentin Therelore, the pulp tissue and implanted dentin matrix were traced in definite area lfroim lower and middle parts of the cavity on the amputated pulp, and the relative areas of newly formed denitnxwere calculated as follows: [1 (total area ol pulp tissue aiid implanted matrix)/delinite area)l x 100"'. The peicent relative area ol BMP-2- and BMIP-4-enriched carrier was 8082% compared with 42째/. in carrier alone (Table 1).

Discussion the large cells attached to the iiiinplan-ted dentin matrix, and of- the spindle-shaped cells formTed mnatrix arounid them (Fig. 3) In the middle part of the cavity, regularly arranged odonitoblast-like, polarized cells with long cytoptasmic processes had formed irregular tubular dentin (Fig. 4a). Darkly stained osteodentinocyte-like cells with round or oval nuclei were em-bedded in irregular oval lacunae, synthesizing extracellular mineralized iniatrix, osteodentiii (Yamnamiiura, 1985) (Fig. 4b). The lower part of the cavity wxas f'illed mostly with irregular tubular dentin, some

This investigation has demonstrated that both recoimbiniatit human BMPI-2 and BMP 4 plus dentini matiix carrier induced the formation of a large amounlt ol dentinl. The newly induced dentin was mostly tubular dentini in the lower part of the cavity and osteodentin in the upper part. There was a graded cellular response torn the bottom ol the cavity on the amputated pulp to the top, as in previous observations from I week to 2 months post-operatively (Nakashima, 1989). Reparative dentin is del mied as a localized zone of dentin that is deposited in non exposed pulps in responise to some external stimulation (Cox et al., 1992). Deposition of

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j Dcnt Rcs 73(9) 1994

Dentin Formation with BMP-2 and -4

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Table. Relative areas ol induced dentin in response to BMPs Percent Relative Areas lrnplanted Materials Mean + SD Carrier alone

42.4 + 4.4

80.7 + 4.1 i3MI 2 with carrier 82.1 + 5.5, BMP-4 v.ith carrier P < 0.001 (t test); n 4 rnean ol 5 sections used as one sample)

reparative dentin has been considered to be a consequence of either naturally occurring physiologic factors, such as occlusal attrition, erosion, abrasion, and aging, or pathologic effects, such as caries, periodontal disease, orol'acial infections, and instrumentation trauma during tooth preparation (Beust, 1931). "Dentin bridge" has been used in the literature to describe that deposition of a new matrix either directly adjacent, or subjacent, to some sort of' material, such as the pulp-capping agent. Cox et al. (1987, 1992) have shown, however, that healing of exposed pulp is dependent not on the effect of a particular type of' medicament but rather on the capacity of the capping agents to prevent bacterial leakage. The inherent healing capacity of' the pulp tissue has been suggested by several authors (Berman and Massler, 1958; Schroder and Granath, 1971; Schroder and Sundstrom, 174; Yamamura, 1985). In this manuscript, I have chosen to describe BMP 2- or BMP-4stimulated dentin as "induced dentin" formation. I wish to distinguish this I rom the termns "reparative dentin" formation and "dentin bridge" formation, used by restorative dentists such as Cox et al (1992). BMP-2 and BMP-4 do not af fect proliferation of pulpal mesenchymal cells in vitro (Vainio et al., 1993; Nakashima et al. 1994), but stimulate differentiation of pulp cells into odontoblasts in vitro (Beque-Kirn et al, 1992; Nakashima et al., 1994). BMP-2 and BMP-4 induce the expression of Msx-I and Msx-2 genes, which function as transcription factors controlling the transcription of other genes, suggesting the widespread signaling functions of' BMP-2 and BMP-4 in morphogenesis and organogenesis (Vainio et al., 1993). Albumin does not have any effect on proliferation and differentiation of pulp cells in vitro (Nakashima, 1992). The cavity on the amputated pulp was filled with putp tissue, with little or no induced dentin in vivo in response to albumin placement (Nakashima, 1990b). The amount of newly induced dentin was much smaller in teeth with inactivated dentin matrix than in teeth with BMP-2 and BMP 4 enriched carrier (Table 1). BMI'-2 with collagen carrier induced dentin in a dose dependent manner (unpublished data). These findings suggest that BMP has a stimulatory eflf'ect on dentin formation. It is clear that the cavity was free from bacterial infection due to aseptic instrumentation, capping, and double-sealing with zinc phosphate cement and Clearfil"', with enamel etching and

Figure 7. BMP-4 with carrier, the upper part. (a, top) Repiescnting osteodentinl Many osteodentillocytes entrappedhby mi neralized matrix within irregular oval lacunac and capillaries can be secn (b. bottom) Representing tubular dentini (T D) sutrounlided by osteodentin (OD) Cytoplasmic process (arrosvs) ol odontoblast like cell extending into tubular dentin (ED).

Figure 8. BMP-4 with carnier, the superior part Note spindle shaped cells (arrows) and osteodentinoblasts (arrowheads) synthesizing extracellular matrix.

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s 73(9) 1994 JI)1 ic Nakashirna 1520IJ2c

(2) Inactivated dentintmatrix provides a local site for pulp cells suitable loar attachment, providing an optimal envir-onimilenit loir dif Ierentiationi. (3)BMP-2 and I3MP 4 stimulatc the difl erentiation of the attached cclls into odontoblasts.

Figure 9. C(arrier

alone, after 2 months. Note mnuch smaller amount ol induced dentin coinpared with that in Figs. I ancl 6.

In teeth which were capped with BMP 2- ort 13MP 4-cnrichecd carrier the lowei palt olf the cavity was I'illcd inostly with tubular dentin. The implanted 4M-guanidine-extr actcd demineralized dentin matrix is inactive hccausc it is dclaoid ol any growth factors and osteogenic proteinl as pi cxiousiV shoxx'n in Oitro (Somerman ct of. 1987) and ii xit xi (Katz and Rcddi 1988). In teeth whiich were capped witih cairici- alone tunular dentin formation was dim inished coLmpared xwitIi teeth implanted with BMPI-2 ancl BMI' 4 trcatcd implants A teccit work using osteogenic proteini-1 (BMI' 7) icConlstitLtcd witli type I collagen matrix fr-om cortical honic lor diircct pulp capping demonstirates not tubular dentinl but osteodentini lormation over exposed pulp (Rutherilord ct tl.u 1993). Thc authors attribute this to its short-termii application (6 xxccks). The adhesion of pulp cells to an appiopriate suilface niay he the critical requirement lot the appearanice ol clongatccl IPolarized odontoblast like cells (Veis 1985). Biomechanlical coriincctioil between the compoanents ol extracelfular- matirix and the intracellular cytoskeletal constituents ol periphicral papilla cells is critically iminportant in determi ining theii- iittogcicsis, shape, and ftunction (Ruch 1990). An uiniminrcializecl nati-ix ol pulpal ectoiriesencliyrial origiii synthiesizcd cithcr by originial odontoblasts or by pulp tissue cells-sutCI as ib odeiitinl osteodentin predentin and demincralized dentini is cssential l'or the initiation ol tubular dentinii ormationl in pulpal fialimi

41 'J"'

_1-11

Figure 10. Carriet alone, the middle part Note odontoblast-like cells fining ieplantcd dcnti a matn-ix (M), Ioatini ig a small amoulit

of tubular dentin(TD)

bonding which covered the whole buccal lace of the teeth. Therelorei the healitng sequcince in BMP '2 with carrier and BMP-4 with carrier implanted teeth is described as lollows:

(Baume 1980; Yamamura 1985; Naf<ashima, 1989, l990a,hb Tziafas and Kolokuris 1990; Tzial'as ct Al. 1992). The demineralized bone matirix is knowiv to inedLice osteodentini formnation on amputated pulp (Stlika et o1. 1979) and in pnulp tissue (Tziafas and Kolokuris 1990). Theircorc it is likele that the increased tubular dentini forimiationi in the pr'CSCilt study in contrast to osteodentin formation in tecthliwhich wxeie implanted with BIMP 7 with collagcnous bone mati-ix (Rutherford ct 1n[. 1993) might be cluc to the ca-rrici- dentin matrix rather than to the type ol' BMI' usecl. The present study demonistr-ated the Litilit) of recombinanit human BMP 2 and IBMI 4 as hio activc pu,lp

capping agents. These moi-phogenietic lactot-s caln inedLice a large amount of dentin on amputatecl pulp without neet of the alfecting the remainiiig pulp. Fuattic -r riefici delivery system may result in predictahle optitial dcntin induction

(I) Fibroblast-like cells migrate flrom underneath pulp tissue and prolilferate (Fitzgerald 1979), and pulp tissue regenerates on the amputated pulp under the proper environinent without bacterial inflectioni.

Acknowledgments I wish to thank lProf'essor Fl. Nagasaxwa for a ci-itical rcading ol this manuscript.

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Figure II. Contact radiographs after 2 months, showing mineralization of induced dentin much more dif'use and porous with carrier alonle (a. left) compared with BMl'-2 with carrier (b, center) and BMP 4 with carrier (c. right). Bairs represent 2 mm.

This investigation was supported by Scientific Grants from Japanese Ministry of Education, 04457082 and 04454473.

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Induction of dentin formation on canine amputated pulp by rhBMP2-4