Dentin dysplasia Type II: absenceofType III collagen in dentin

Helena Ranta\ Pirjo-LiisaLukinmaa^ and Janna Knif̂Departments of 'Carioiogy and ^DentalRadioiogy, 'Oral Pathology, Institute ofDenfisfry, University of Helsinki, Finland

Ranta H, Lukinmaa P-L, Knif J: Dentin dysplasia Type II: absence ofType IIIcollagen in dentin. J Oral Pathol Med 1990; 19: 160-5.

A three-generation family with dentin dysplasia (DD) Type II is presented.Affected family members share common radiologic features with elinically variedexpression of tooth discoloration and occlusal wear. Both the primary and thepermanent dentition appear to be affected. No generalized connective tissueinvolvement is found. The mode of inheritance is autosomal dominant. Histologi-cally, the findings are consistent with DD Type II. In indirect immunofluores-cence, the irregular radicular dentin of an affected permanent tooth failed to stainwith specific antibodies against Type III collagen and the N-terminal propeptideof Type III procollagen.

Key words: dentin dysplasia: Type III collagen.

H. Ranta, Department of Carioiogy, Institute ofDentistry, University of Helsinki,Mannerheimintie 172, SF-00300 Helsinki,Finland.

Accepted for publication January 20, 1990.

The current classification of heritablehuman dentin defects includes dentindysplasia (DD) Types I and II, and den-tinogenesis imperfecta (DI) Types I-III(1). In addition, focal odontoblasticdysplasia possibly representing DDType III has been suggested by EAST-MAN et al. (2).

Both the radicular (Type I) and thecoronal (Type II) dysplasias have beenwell characterized (1, 3). The majorradiographic findings in DD Type Iinclude defective root formation, pul-pal obliteration of the primary teeth,crescent-shaped remnants of pulpehambers with denticles in the perma-nent dentition and periapical radiolu-cent areas of unknown etiology. Afamily with an autosomal dominantdisorder, characterized by DD Type Iand progressive generalized corticalhyperostosis, has been described (4).Type II DD shares some common fea-tures with DD Type I in the primarydentition, but the permanent teeth inType II exhibit thistle-tube-shapedpulps with dentieles (1, 5-11). Defectsresembling DD Type II have alsobeen reported in patients with os-teogenesis imperfecta (01) but notwith DI Type I (12, 13).

Type III collagen is not a normalmatrix component of mineralized den-tin. Yet, it has been found to be pre-sent in dentin of selected patients withDI Type I associated with 01 (14, 15).To discover whether this is the case inDD Type II, the affected dentin ma-trix was studied by indirect immuno-

fluorescence using specific antibodiesagainst Type III collagen and the N-terminal propeptide of Type III pro-collagen.

Material and methods

The dental examination of selected fam-ily members was perfortTied at the Insti-

Fig. I. Pedigree of family with dentin dysplasia Type II. Generations (I-III), individuals(Arabic figures) and clinically examined members (arrows) are indicated.

Fig. 2. Father (II-I9) of proband (III-4) showing oeelusal wear.

Dentin dysplasia 161

Fig. 3. Thistle-tube pulps with pulp stones in father's perma-nent dentition (11-19) of proband (III^). A, panoramictomogram. B, bite-wing radiograph.

tute of Dentistry, University of Helsin-ki. The family pedigree is given in Fig. 1.Panoramic tomograms were taken andevaluated for characteristics of DDType 11. Clinical photographs weretaken to document of the color ofsclerae and the extent of occlusal wear.

Previously exfoliated primary teethwere obtained from one individual(11-19). His mother (1-16) had pre-served and kept them air-dried. An im-pacted permanent molar was extractedfrom another family tnetnber (1-7) be-cause of loeal inflatiimation. Two pri-

Fig. 4. Panoramic tomogram ofa 14-yr-old boy (II-5) demonstrating coincident hypodontia.

mary teeth were demineralized with 0.33M aqueous ethylenediaminetetra-aceticacid (EDTA) for 1 wk; the permanentmolar was fixed in 70% ethanol for 1 wkand demineralized with 0.33 M aqueousEDTA for about 3 wk. Conventionalserial paraffin sections were cut andstained with hematoxylin-eosin, vanGieson stain, and Schmorl's picrothi-onin.

For itntnunofiuorescence, paraffinsections of the permanent tooth (1-7)were rehydrated and incubated at 37°Cwith pepsin (Merck, Darmstadt, FRG;0.04% in 0.01 M HCl) for 60 min, withovine testicular hyaluronidase (Boeh-ringer, Mannheim, FRG; 0.7% in phos-phate-buffered saline, PBS, pH 6.0) for60 tnin or with trypsin 1:250 (DifcoLaboratories, Detroit, MI; 0.05% in0.05% CaCU, pH 7.6). Enzymaticallyuntreated sections were also used. Thesections were washed with PBS and pre-treated with normal goat serutn (Dako,Glostrup, Denmark, Lot No. 628; dilu-

162 RANTA ef al

Figs. 5-7 illustrate demineralized longitudinal paraffin seetions of a permanent molar (1-7).Schmorl's picrothionin, x 202.

Fig. 5. Major part of coronal dentin (CD) exhibits regular tubular pattern; virtually atubulardentin zone (AD) is scon adjacent to pulp (P) occupied by denticle (D).

tion 1:5 in PBS, pH 7.4, containing 1%bovine serum albumin) to prevent non-specific adsorption of the antibodiesduring further stages of the procedure,and stained with immunoaffinity-puri-fied rabbit antibodies against calf skinType III collagen (cross-reactive withhuman tissues; dilution 1:40 in PBS)(16) and the N-terminal propeptide ofType III procollagen purified from as-citic fluid from cancer patients (20 [ig/ml PBS) (17). Fluorescein isothiocya-

i^Wl

nate (FITC)-conjugated goat antirabbitIgG (Cappel Laboratories, Malvern,PA, Lot No. 27223; dilution 1:40) wasused to label the primary antibodies.The sections were then stained and ex-amined as previously described (14).

To confirm the specificity of the stain-ing reactions, rabbit nonimmune serumand PBS were substituted for the pri-mary antibodies and PBS for both theprimary and the secondary antibodies.For comparison, sections of an unerupt-

•dS^t

Fig. 6. Transition from normal coronal dentin (CD) to irregular radicular dentin (RD) isabrupt.

ed third molar from a normal subjectwere treated identically.

Results

The pedigree shows an autosotnal domi-nant mode of inheritance (Fig, 1). Ofthe 41 members of the family 6 weredeceased. Thus far, one edentulous andnine dentulous metnbers have beenavailable for clinical and radiographicexaminations. Anamnestic data, pho-tographs and previous patioramic to-mograms have been available, too. Clin-ically, the color of the sclerae was nor-mal, but the shade of the teeth variedfrom normal to bluish-brown. The de-gree of occlusal wear also showed con-siderable variation (Fig. 2). Enamelcracking with extensive wear, character-istic to DI, was not found. Radiological-ly, pulp stones were present in thistle-tube pulp chambers in all affected per-manent dentitions (Fig. 3A, B). Thepulp chambers appeared partially oblit-erated. So far, true hypodontia has beenconfirmed in one patient (II-5) (Fig. 4).

In the preserved primary teeth, thehistologic examination revealed that thetubular pattern of the eoronal mantledentin was relatively regular, thoughdelicate, whereas in the circumpulpaldentin, only occasional tubules were ob-served (not shown).

In the permanent molar, with the ex-ception of the dentin zone facing thesmall pulp chatnber, the coronal dentinexhibited a regular tubular pattern. Thepulpal part of the dentin, virtually de-void of tubules, was sharply demarcatedfrom the normal-appearing dentin. Thesmall pulp chamber was partly occupiedby a dentiele (Fig. 5). At the level of theenamel-cementum junction, there wasan abrupt transition from the regularcoronal dentin to irregular radiculardentin exhibiting occasional tubulesonly (Fig. 6). Structures resembling ca-nals filled with hard tissue were numer-ous (Fig. 7). The whole root surface wascovered by an extremely thick layer ofcellular cementum.

The immunofiuorescence stainingpatterns with antibodies against TypeIII collagen and the N-terminal propep-tide of Type III procollagen were iden-tieal. In enzymatically untreated sec-tions of both the affected and the nor-mal tooth, staining in the pulp andperiodontal ligament, particularlyaround the blood vessels, was positive.Faint fiuorescence was seen in variedwidth of the predentin. This staining

Dentin dysplasia 163f/g-. 7. Canal-like structures (C) are seen inirregular radicular dentin.

reaction was somewhat intensified byhyaluronidase pretreatment and furtherintensified by incubation with pepsin ortrypsin, Odontoblasts and dentin ap-peared negative (Fig. 8A-D). No stain-ing was visible in the irregular radiculardentin of the affected tooth (Fig. 9A,B) or in the root dentin of the normalcontrol tooth. Faint fluorescence, inter-preted as staining of the embedded fi-bers of the periodontal ligament, wasobserved in the cementum of the norrnaltooth.

The specificity controls were nega-tive.

Discussion

The clinical attributes of the heritablehuman dentin defects have been thor-oughly discussed by SHIELDS (18). It isevident that the current classification assuch is not comprehensive. Clinical, ra-diographic and histopathologic char-acteristics are of diagnostic value, butfor the understanding of expression andphenotypic variance of mutant gene(s)other approaches are needed.

Thus far, no information as to a can-didate gene for dentin dysplasias hasbeen published. Type II DI occurring asa single trait has been assigned to theGc region of the long arm of humanchromosome 4 (19). This linkage hasbeen confirmed by CONNEALLY et al.(20) and CORNEY et al. (21). Interesting-ly, an autosomal dominant form of ju-venile periodontitis in association withDI Type II has also been linked to theGc region (22).

A number of inherited disorders areknown to involve tissues rich in Type Icollagen. During the last decade, severalmutations, including both deletions andinsertions, in the genes of proa 1(1) andproa2(I) chains have been identified inosteogenesis imperfeeta (for summary,see ref. 23). Bearing in mind the close

Fig. S. Immunofiuorescence for A: the N-tcrminal propeptide of Type III procollagenin permanent tooth with DD Type 11. B, TypeIII collagen in DD Type 11 and C, the N-terminal propeptide ofType III proeollagenin normal tooth, is positive in coronal pulp(P) and pulpal part of predentin (arrow),whereas odontoblastic layer (O) and dentin(D) are negative. Cleft between odontoblastsand predentin is artefactual. D, no stainingin DD Type II is seen with normal rabbitserum: odontoblasts are slightly autofiuores-cent. Hyaluronidase pretreatment, x 202.

164 RANTA et al

Fig. 9. Immunostaining of permanent tooth with DD Type 11 for A, N-terminal propeptideof Type III procollagen and B, Type III collagen is positive in radicular dental pulp (P) andnegative in irregular dentin (D). No enzymatic pretreatment; x 202.

relatedness of bone and dentin organicmatrices, a putative linkage to Type Iprocollagen genes could be expected ininherited dentin defects. Recently, alarge Finnish family with DI Type IIwas studied for restriction fragmentlength polymorphisms (RFLP) of theType I collagen gene (a2 chain) (24). Nodefinite linkage could be confirmed. Thepossibility of mutation(s) in genes fornoncollagenous protein components ofdentin matrix cannot be excluded.

The clinical, radiographic, and histo-logic dental characteristics of the familyreported here are consistent with DDType II. The histologic appearance ofthe radicular dentin of the permanentmolar closely resembled that seen in DIassociated with 01.

The differentiation of ameloblastsand odontoblasts and the subsequentsynthesis of organic matrices are deter-mined by complex epithelial-mesenchy-mal interactions (25, 26). The transitionof normal coronal dentin to irregularroot dentin at the enamel-cementum in-terface could be explained by the dis-tinction between the formation of thesetwo Types of dentin. In root dentin,Hertwig's epithelial root sheath degen-erates after differentiation of odonto-blasts and initial deposition of preden-tin. Subsequently, the undifferentiatedmesenchymal cells located close to den-tin will become cementoblasts (27). Anydisturbance in this cascade of cell/ma-trix interactions could result in defectiveexpression of odontoblasts depositingradicular dentin. However, it should be

borne in mind that except for mantledentin, the eoronal dentin of primaryteeth in DD Type II is abnormal.

The presence of Type III (pro)colla-gen in normal predentin is somewhatcontroversial (28-33). It has been de-monstrated that Type III collagen canbe present in the dentin matrix of pa-tients with Type I DI (14, 15, 34) andType II DI (15). Type III collagen tuole-cules retain the N-terminal propeptideas shown both in non-mineralizedtissues (35) and in the dentin matrix ofpatients with Type I DI (14).

Indirect immunofiuorescence micro-scopy has been applied to study thestructural proteins of human dentin ma-trix in patients with 01 (14, 36, 37). Thepresent study failed to demonstrate theoccurrence of Type III collagen in theirregular radicular dentin of a perma-nent tooth affected by DD Type II, evenafter unmasking enzymatic pretreat-ments. This suggests that the structuralirregularity of dentin is not necessarilyassociated with incorporation of TypeIII collagen in the matrix. Further stud-ies are needed to confirm whether theabsence ofType III collagen in the aber-rant dentin is a consistent findingand for the evaluation of the expres-sion of gene defect(s) in inherited den-tal disorders including dentin dys-plasias.

Acknowledgments - The authors wish tothank Drs. KAIJA LAIMGRUBER for carefuldiagnostic help and KARI RANTA, Institute of

Dentistry, for fruitful discussions. Antibodiesagainst Type III collagen were supplied byDr. LEENA PELTONEN, National Public HealthInstitute, Helsinki, and against the N-ter-minal propeptide of Type III procollagen byDrs. LEILA RISTELI and JUI-IA RISTELI, Col-

lagen Research Unit, University of Oulu,Finland.

References

1. SHIELDS ED, BIXLER D, EL-KAFRAWY

AM. A proposed classification for heri-table human dentine defects with a de-scription ofa new entity. Arch Oral Biol1973; 18: 543-53.

2. EASTMAN JR, MELNICK M , GOLDBUATT

LI. Focal odontoblastic dysplasia: dentindysplasia Type III. Oral Surg Oral MedOral Pathol 1977; 44: 909-14.

3. WITKOP CJ JR. Hereditary defects ofdentin. Dent Clin North Am 1975; 19:25-45.

4. MORRIS ME, AUGSUURGER RH. Dentine

dysplasia with sclerotic bone and skeletalanomalies inherited as an autosomal do-minant trait. Oral Surg Orat Med OratPathol \<)11: 43: 267-83.

5. GIANSANTI JS, ALLEN JD. Dentin dyspla-sia. Type II, or dentin dysplasia, coronalType. Orat Surg Orat Med Orat Pathol1974; 38: 911-7.

6. GRIMER P T . An atypical form of heredi-tary opalescent dentin. Br Dent J 1956;100: 275-8.

7. MELNICK M , EASTMAN JR, GOLDBLATT

LI, MicHAUD M, BIXLER D. Dentin dys-plasia. Type II: a rare autosomal domi-nant disorder. Oral Surg Oral Med OratPattiot 1977; 44: 592-9.

8. RAO SR, WITKOP CJ, YAMANE G M . Pul-

pal dysplasia. Oral Surg Oral Med OralPathol 1970; 30: 682-9.

9. RICHARDSON AS, FANTIN TD. Anoma-

lous dysplasia of dentine: report of case.,/ Can Deni A.ssoc 1970; 5: 189-91.

10. STEIDLER N E , RADDEN BG, READE PC.

Dentinal dysplasia: a clinieopathologicalstudy of eight cases and review of theliterature. Br J Oral Maxiltofac Surg1984; 22: 274-86.

11. WALD C , DINER H . Dysplasia of the den-

tal pulp: report ofa ease. ASDC J DentCtutd 1974; 41: 212-5.

12. LEVIN L S , YOUNG RJ, PYERITZ RE. Os-

teogenesis imperfecta Type I with un-usual dental abnormalities. Am J MedGenet 1988; 31: 921-32.

13. LUKINMAA P - L , RANTA H , RANTA K ,

KAITILA I, HiETANEN J. Dental findingsin osteogenesis imperfecta: 11. Dysplasticand other developmental defects. J Cra-niofae Genet Dev Biot 1987; 7: 127-35.

14. LUKINMAA P-L. Immunofluorescent lo-calization of Type III collagen and theN-terminal propeptide of Type III pro-collagen in dentin matrix in osteogenesisimperfecta. / Craniofae Genet Dev Biot1988; 8: 235-43.

15. SAUK JJ, GAY R , MILLER EJ, GAY S.

Immunohistochemical localization oftype III collagen in the dentin of patientswith osteogenesis imperfecta and heredi-tary opalescent dentin. / Oral Pathol1980; 9: 210-20.

16. PELTONEN L , MYLLYLA R , TOLONEN U ,

MYLLYLA VV. Changes in collagen me-tabolism in diseased muscle. II. Immuno-histochemical studies. Arch Neurol 1982;39: 756-9.

17. NiEMELA O, RISTELI L , PARKKINEN J, RIS-

TELI J. Purification and characterizationof the N-terminal propeptide of humantype III procollagen. Bioehem J 1985;232: 145-50.

18. SHIELDS ED. A new classification of heri-table human enamel defects and a discus-sion of dentin defects. Birth Defects1983; 19: 107-27.

19. BALL SP, COOK P J L , MARS M , BUCKTON

KE. Linkage between dentinogenesis im-perfecta and Gc. Ann Hum Genet 1982;46: 35-40.

20. CONNEALLY P M , BIXLER D , HORTON-

KELLY S, DAUGHERTY L . Confirmation

of linkage between dentinogenesis imper-fecta and Gc. Cytogenet Cell Genet 1984;37: 438 (only).

21. CORNEY G , BALL S, NOADES JE: Linkage

studies on dentinogenesis imperfecta(DGIl). Cytogenet Cell Genet 1984; 37:439-40.

22. BOUGHMAN JA, HALLORAN S L , ROULSTON

D, et al. An autosomal-dotninant form ofjuvenile periodontitis: its localization tochromosome 4 and linkage to dentinogen-esis imperfecta and Gc. J Craniofae Genet

Z»ev.S/o/1986; 6: 341-50.23. SYKES B . Genetics cracks bone disease.

Nature 1987; 330: 607-8.24. RANTA H , PELTONEN L. Dentinogenesis

imperfecta as a single trait. In: 3rd Intern-ational Conference on Osteogenesis im-perfecta. Pavia 1987: 10.

25. RUCH J-V. Determinisms of odontogen-esis. Cell Biol Rev RBC 1987; 14: 1-122.

26. THESLEFF I, HURMERINTA K. Tissue inter-actions in tooth development. Differenti-ation 1981; 18: 75-88.

27. SHAWARY M , BHUSSRY BR. Develop-

ment and growth of teeth. In: BHASKARSN, ed. Orban's Oral Histology andEmbryology. St. Louis: The C. V. MosbyCompany, 1986: 24-44.

28. ANJUDAR MB, HARTMANN D J , EM-

ONARD H, MAGLOIRE H . Distributionand synthesis of type I and type III colla-gens in developing mouse molar toothroot. Histoehemistry 1988; 88: 131-40.

29. BECKER J, SCHUPPAN D , BENZIAN H , et

al. Immunohistochemical distribution ofcollagens types IV, V, and VI and of pro-collagens types I and III in human alveo-lar bone and dentine. / Histochem Cyto-chem 1986; 34: 1417-29.

30. CouRNiL I, LEBLOND CP, POMPONIO J.

HAND AR, SEDERLOF L , MARTIN G R .

Immunohistochemical localization ofprocoUagens. I. Light microscopic distri-bution of procollagen I, III and IV anti-genicity in the rat incisor tooth by theindirect peroxidase-antiperoxidase meth-od. J Histochem Cytochem 1979; 27:1059-69.

Dentin dysplasia 165

31. TAKITA K , OHSAKI Y, NAKATA M , KURI-

su K. Immunofiuorescence localizationof type I and type III collagen and fibro-nectin in mouse dental tissues in late de-velopment and during molar eruption.Arch Oral Biol 1987; 32: 273-9.

32. THESLEFE I, STENMAN S, VAHERI A, TIMPL

R. Changes in the matrix proteins, fibro-nectin and collagen, during differentia-tion of mouse tooth germ. Dev Biol 1979:70: 116-26.

33. WRIGHT GM, LEBLOND CP. An attempt

at quantitation of procoUagens I and IIIand of collagen IV in tooth sections byexposure to the corresponding antibod-ies followed by '-'I-protein A and radio-autography. J Histochem Cytochem1980; 28: 1355-8.

34. GAGE JP. Dentinogenesis imperfecta. Anew perspective. Aust Dent J 1985; 30:285-90.

35. FLEISCHMAJER R , PERLISH JS, TIMPL R .

Collagen fibrillogenesis in human skin.In: FLEISCHMAJER R , OLSEN BR, KUHN

K, eds. Annals of the New York Academyof Seiences. New York: The New YorkAcademy of Sciences, 1985; 460: 246-57.

36. LUKINMAA P-L, RANTA H , RANTA K ,

PELTONEN L , HIETANEN J. Demineraliza-

tion of dentin with EDTA in organicsolvent: immunofiuorescence of collagenin osteogenesis imperfecta and normalteeth. Colt Retat Res 1985; 5: 505-12.

37. LUKINMAA P-L, RANTA H , VAHERI A. Os-

teogenesis imperfecta: fibronectin in den-tin matrix. J Craniofae Genet Dev Biot1988; 8: 75-82.