Enamel ultrastructure in pigmented hypomaturation amelogenesis imperfecta

J. Timothy Wright', Valerie Lord', Coiin Robinson' and Roger Shore^ Departments of 'Pediatric Dentistry, University ot North Carolina at Chapel Hill, USA; 'Children's Dentistry, 'Orat Biology, University of Leeds, England

Wright JT, Lord V, Robinson C, Shore R; Enamel ultrastructure in hypomaturation amelogenesis imperfecta. J Oral Pathol Med 1992; 21; ,190 ,194. Hypomaturation amelogenesis imperfecta (Al) is a hereditary condition of enamel that is presumed to result from defects during the maturation stage of enamel development. This study characterized the enamel ultrastructure and enamel crystallite morphology, as well as the distribution of organic material in enamel affected with pigmented hypomaturation AI. Hnamcl exhibiting autosomal recessive pigmented hypomaturation Al was sectioned or fractured and examined using light microscopy, scanning electron microscopy and transmission electron microscopy. Enamel samples were treated with 30"Ai NaOCI or 8 M urea to remove organic components and determine the effect of deproteinization on crystallite morphology. These were compared with untreated normal enamel samples. The enamel crystallites in hypomaturation Al exhibited considerable variability in size and morphology. Examination of deproteinized tissue indicated that the AI crystallites had a thick coating, presumably of organic or partially mineralized material, which was not visible in normal enamel. The results of this investigation provide further evidence that hypomaturation AI is associated with the retention of organic material that is most probably enamel protein. Enamel protein retention is likely to be involved in the inhibition of normal crystallite growth resulting in the morphological crystallite abnormalities associated with this disorder.

Hereditary conditions that mainly affect the enamel of teeth, the amelogenesis imperfectas, are divided into multiple subtypes based on their clinical, hereditary and histologic features. Three main groups are recognized including hypoplastic, hypocalcified and hypomaturation types. The most widely accepted classification of AI lists four hypomaturation Al subtypes and two hypomaturation/hypoplastic subtypes which exhibit different clinical, hereditary, and histologic features (I). There remains considerable controversy as to the mode of inheritance and most appropriate classification of specific AI types. It is considered that abnormal enamel maturation, occurring after matrix secretion and initial mineralization, results in the hypomaturation types of Al (2). Impairment of the maturation process is thought to result in small enamel crystallites which fail to grow the same extent as those of normal enamel and may be associated with retention of excessive organic material. The clinical and histologic features of the dilTcrent hypomaturation AI types

are diverse with the snow capped subtype reportedly affecting only the outer enamel while other types show marked histologic abnormalities throughout the tissue (3, 4). Histologic studies have shown the presence of material within the enamel of teeth affected by pigmented hypomaturation AI which has been postulated to be retained enamel matrix protein (4, 5). This material, which appeared to be amorphous and is thought to be enamel protein, was distributed throughout the enamel structure, intermixed with the crystallites (5). There have been few histological studies of the specific hypomaturation Al subtypes and no quantitative investigations of enamel crystallite size. In addition, the biochemistry of these defects remains essentially unknown for all these disorders. Biochemical analysis of fully developed autosomal recessive pigmented hypomaturation Al enamel has shown an increased organic content. Normal mature human enamel contains between 0,1 and ^^ protein with great site to site variability in the protein content and composition (6, 7).

Key words: amelogenesis imperfecta; enamel crystallite; enamel protein; hypomaturation; teeth. Dr. Tim Wright. Department of Pediatric Dentistry. School of Dentistry. Brauer Hall /i7450. University of North Carolina at Chapel Hill. Chapel Hill. NC Accepted for publication May 14. 1992.

Enamel directly adjacent to the dentinenamel junction is typically highest in protein content compared with the other eiiatnel (7). Hypomaturation AI enamel shows a much higher protein content that averages about 5% (8), The amino acid composition of hypomaturation AI enamel protein appears similar to that seen in early maturation/transition stage hutnan enamel being rich in proline and therefore amelogenin-like. Whether the presence of this material results in alteration of crystallite growth remains unknown. Taken together the results of previous histologic and biochemical investigations of pigmented hypomaturation AI appear to support the hypothesis of defective maturation with abnormal enaiTiel crystallite formation and increased retention of enatTiel protein. The purpose of this investigation was to evaluate the histologic and ultrastructural features of a previously unreported kindred affected with autosomal recessive hypomaturatioti Al and examine the enamel crystallite morphology.

Hypomaturation amelogenesis imperfecta 391 ronapthalene. Enamel particles were dissected from thin sections for examination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Areas of enamel fractured from thin sections were mounted on aluminum stubs and coated with gold for examination with SEM. High resolution SEM analysis was conducted using a field emission electron microscope. Other particles were fixed in a 0.1 M phosphate buffer pH 7.4; 2.5"Mi glutaraldehyde solution, then serially dehydrated in ethanol and embedded in resin prior to sectioning for TEM ,inalysis. Several TEM sections were stained with lead citrate and uranyl acetate. To detemiinc the cITcct of removing organic material from the enamel, fractured particles of AI and normal enamel llow brown discoloration. t I'robuiuK Luc mixed dentition displayed a geiK-i were subjected to two different extraction procedures. Enamel particles were treated with either 30% NaOCl or an 8 from unalTected healthy individuals M urea in metastable calcium phosiMateriai and methods were obtained for comparison and phate solution for 30 min. The urea soExperimental material served as controls for all analyses. lution was saturated with respect to Ca Two erupted, noncarious, maxillary preand P to reduce the potential for minermolars were extracted for orthodontic Histologic examination al loss during protein removal. After purposes from a lO-yr-old girl with hypoimmersion in the solution the particles maturation AL The dentition was mor- The Al and control teeth were obtained were rinsed with H.O pH 7.2 and dried phologically normal but showed a gener- immediately after extraction and pre- prior to mounting for examination alized brown discoloration of the enamel pared for histologic evaluation. One Al using SEM. (Fig. 1). Radiographic analysis showed tooth and two control teeth were serially Cross-sectional crystallite dimensions that all the permanent teeth were present sectioned using a diamond blade. The were detennined in the AI enamel using and that the enamel had a radiodensity remaining AI tooth was retained for computerized image analysis. Measuresimilar to that of the dentin. There was biochemical studies. Sections were hand ments of 30 AI enamel crystallites were no evidence of taurodontism. Examina- ground to approximately 100 (im in taken from TEM specimens for comparition of the immediate family members re- thickness for analysis using light micro- son with known normal crystallite divealed an affected younger sister who scopy. Thin sections were examined mensions. Only those crystallites apalso exhibited a generalized brown pig- both drv and imbibed in H^O and chlo- proximating a hexagonal shape indicamentation of the primary dentition. None of the other immediate family members exhibited any evidence of AI and there was no report of other affected family members. Pedigree analysis showed that the children were the result of a consanguineous relationship (Fig. 2). Fully developed, noncarious teeth

6' 6' • 9

EXAMINED AFFECTED FEMALE

Fig. 2, Examination of family history and clinical examinations revealed aflected younger sibling and consanguineous relationship ol parents indicative oi aulosomal recessive inheritance.

Eig. 3. Thin section imbibed with H;O and viewed with LM illustrates the generalized pigmentation seen throughout W enamel and pronounced Hunter Schreger bands.

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tive of a cross-sectional orientation were measured, Resuits Pedigree analysis of this kindred was consistent with an autosomal recessive mode of inheritance given the consanguineous relationship of the parents and distribution of affected family members. Expression of the disorder was similar in the two affected siblings. Histologic examination using light microscopy showed a prismatic enamel structure and a generalized pigmented discoloration extending from the dentin-enamel junction to the Al tooth surface. There were pronounced HunterSchreger bands across the enamel that appeared opaque compared with the adjacent enamel (Fig. 3). Imbibition of the Al enamel with either HiO or chloronapthalene failed to produce a significant reduction in the generalized opacity or change in the tissue's discolor-

ation. The dentin and cementum appeared to be nonnal. Examination of fractured Al enamel using SEM confirmed the presence of a prismatic structure, however, ultrastructural defects were seen throughout the tissue. There were marked decussations of the AI enamel prisms (Fig, 4), Although enamel crystallites were apparent in the Al enamel they were frequently obscured by areas of amorphous material. This amorphous material was seen throughout the Al enamel with some areas presenting as flat plates dispersed amongst the enamel crystallites (Fig. 5). Round globular material also was frequently seen intermixed with the Al enamel crystallites (Fig. 6) which was not seen in the control enamel (Fig, 7). The AI enamel crystallites appeared to have a much rougher surface that was granular in comparison with normal enamel crystallites (Fig, 6, 7), The AI enamel samples treated to remove organic material tended to disin-

tegrate during the extraction procedure while the normal enamel remained intact. Treated AI samples showed a disorganized surface with crystallites, apparently dislodged from the bulk of enamel, left lying randomly on the fractured enamel surface. Globular structures seen intermixed with the AI enamel crystallites prior to being treated were not present after extraction to remove organic material. Although the NaOCl caused a much greater degree of enamel disorganization, with some AI samples actually disintegrating, both NaOCl and 8 M urea with metastable calcium phosphate resulted in the removal of globular structures. Some amorphous material remained after treatment and was more prevalent in the urea treated particles. The normal enamel appeared unaffected by both extraction procedures. Examination of the AI enamel with TEM revelaed an altered prismatic and crystallite morphology with some areas

Hypomaturation amelogenesis imperfecta 393

Fig 4. Although prismatic structure was diseernable with SEM marked decussations o'( prisms were seen, x ,S00. Fig. 5. Amorphous material which lacked crystallite structure and sometimes presented as flat plates was intermixed with AI enamel crystallites (arrow), x .V^OOt). Eig ft. AI enamel crystalliles displayed rough granular surface with rounded globules of varying size appearing on and around crystallites (arrows). x500(K). Eig. 7. Crystallites of normal enamel display smooth surface and do not demonstrate globular structures. x40 0(KV Eig S. Heterogeneous and morphologically diverse population of AI enamel crystallites ranging from large flat plates to small thin disorganized crystallites were seen with TEM. x 1(X)(K)O. Eig. 9. Al enamel crystallites frequently showed irregular outline with areas protruding from crystallite surface (arrows), x 2(X>tX)0. Fig. It). Staining of AI enamel for TEM produced small radiodense granules scattered over crystallites and in adjecent amorphous material. X ltK)t)OO.

ot Al enamel showing a normal key hole type prism pattern while other regions showed no discernible prism pattern. Al enamel crystallites varied markedly in their morphology ranging from Hat plates to small thin needles. The crystallite orientation was very disorganized with areas of circular whorls of crystallites being seen (Fig, 8), Rounded protrusions were frequently seen proiecting from the larger AI crystallites that typically had a very irregular outline (Fig, 9), While the unstained samples showed translucent areas in the crystallites, stained sections had numerous dark globules associated with the crystallites and in the amorphous material surrounding them (Fig, 10), Untreated Al enamel crystallites apPeared wider than normal enamel crystallites with SEM. Treatment of the Al enamel using NaOCl and 8 M urea in metastable calcium phosphate both resulted in a reduction of the crystallite widths making them appear similar to

those of the normal enamel. Crystallite measurements taken using TEM (;; = 30) showed the well-formed AI crystallites to be 84 nM (± 10.7 nM) in width and 22 nM ( ± 2.5 nM) in thickness. Difficulty assessing the crystallite orientation in the very small and disorganized crystallites precluded meaningful quantification of their dimensions, therefore only those crystallites with a reasonable morphology were measured. Discussion

The hallmark features of hypomaturation Al reportedly consist of impaired crystallite growth and retention of organic material in the fully developed enamel (4). The case of hypomaturation AI evaluated in this investigation presented both of the characteristics having a markedly altered crystallite size and morphology as well as evidence that organic material, not seen in normal enamel, was present.

Fractured AI enamel specimens in this and previous studies have shown globular structures, presumed to be residual organic material, mixed with the crystallites (4, 5). Treatment of AI enamel with solutions solubilizing organic material removed these structures implying that they were indeed organic in nature. The granular staining pattern seen with TEM also suggested the presence of organic material in the AI enamel on and around the crystallites. Excess organic material could represent enamel proteins that are not removed during maturation and crystallite growth and are present as globular aggregates located on and/or between the crystallites. Treatment of the .\\ enamel with agents to remove protein (NaOCl, urea) resulted in substantial disorganization of the surface crystallites and in some areas disUxrated enamel prisms providing further evidence of the intimate association between mineral and organic

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components. Removal of organic material also resulted in an apparent reduction of the AI crystallite widths seen with SEM. This indicates that the crystallites are coated with organic material that is removed during treatment with either NaOCl or 8 M urea. Alternatively, treatment with these solutions could have removed mineralized material from the enamel crystallites, although the 8 M urea solution contained a saturated metastable calcium phosphate solution, which sould have reduced or prevented the removal of mineral from the crystallite. Despite this precaution it is possible that a poorly mineralized periphery of the AI crystallites was removed during extraction of organic material. When X M urea without metastable calcium phosphate was used to treat enamel samples the amount of phosphorus in the resulting solution was much greater in the AI sample compared with the solution from normal enamel (data not shown). This indicates that poorly mineralized or loosely bound mineralized tissue may have been removed from the AI enamel. Partial mineralization of the thick organic crystallite coating may also explain the slightly larger AI crystallite width determined using TEM in this study (84 nM) compared with those published for normal enamel crystallites (68.,1 nM) (9). It must be remembered that only well formed crystallites were measured and that a significant population of very small crystallites were present in the AI enamel which were not easy to quantify due to their abnormal morphology. There have been few previous TEM investigations of the crystallites in AI enamel and there remains insufficient information to determine the crystallite character in the different AI types. Several reports on the hypoplastic types of AI have shown significant morphologic variation ranging from flat plates to needle like crystallites similar to those seen in this case of hypomaturation AI (10, 11). KHRIHKL & DAC ULSi showed amorphous areas amongst the enamel crystallites that were frequently disordered in their arrangement and had widened intercrystallitc spacing in hypoplastic AI (11). Similar amorphous areas and diversity of crystallite morphology were observed in the hypomaturation AI enamel of this investigation indicating severe disruption of normal crystallite growth. Clusters of very small

crystallites in the AI enamel show that References localized groups of crystallites attained 1. WiTKop CJ JR. Amelogenesis imperfecta, little growth after their initial mineralidentinogenesis imperfecta and dentin zation. The presence of rounded protudysplasia revisited, problems in classifiberances extending from the crystallite cation. J Oral Pathot 1989; 17: 547 53. surface as seen in this investigation may 2. WiTKOP CJ JR, SAUK JJ JR. Heritable delects in enamel. In; STKWAKT RE, represent areas of crystallite fusion as PRISCOIT GH. Oral facial genetics. St. shown previously in normal mature Louis: C.V Mosby Co., 1976: 151 226. enamel (9). Alternatively, these crystal3. EiSCOHAR V H , GOLDBAI.IT LI, BlXI.KR lite protuberances my result from localDA. Clinical, genetic, and ultrastructural ized areas where the enamel protein study of snow capped teeth: ameloglobules were at least partially mineralgenesis imperiecta, hypomaturation ized. We feel this later explanation as type. Oral Surg Oral Med Oral Pathol being more likely given the size and dis1981; 52: 607 14. tribution of protuberances seen on the 4. WiTKOP CJ JR, SAUK JJ. Autosomal reAI crystallites. cessive pigmented hypomaturation amelogenesis imperfecta. Oral Surg Oral Extracellular processing of the Med Oral Patlwl 1973; 36: 367 82. enamel proteins is thought to play an 5. WRKiirr JT. Anaiysis of a kindred with integral role in the regulation of enamel amelogenesis imperfecta. J Oral Pathol crystallite development (12, 13). Re1985; 14; 366 74. moval of the enamel proteins is felt to 6. RoniNSON C, Lowi: NR, WEATHERKLL be a prerequisite for normal crystallite JA. Amino acid composition distribution growth. Studies designed to determine and origin of tuft protein in human and the effect of retaining or removing the bovine dental enamel. Arch Oral Biol enamel proteins during the maturation 1975; 20: 29 42. 7. ROBINSON C \ WiATiiLRiit JA, HAI i.sof enamel crystallites have shown that woRrii AS. Variation in composition ol the presence of enamel protein results in dental enamel within thin ground tooth inhibition of crystallite growth whereas sections. Caries Res 1971; 5; 44 57. extraction with either enzymes or solu8. WRKJHT JT, BUTLER WT. Alteration of tions that extract organic material allow enamel proteins in hypomaturation continued crystallite growth (14, 15). amelogenesis imperfecta. J Dent Res The results of this study indicate that 1989; 68: 1328 330. organic material remains on the enamel 9. DA( tJi.si G, KHREBEL B. High-resolution crystallites as well as in globular strucelectron microscope study of human tures between the crystallites. It seems enamel crystallites: Size, shape, growth. ,/ Ultrastruct Res 1978; 65: 163 72. likely that this material is an enamel 10. HoHi.iNCi HJ, ERPINSTIIN H . Elektroprotein since hypomaturation AI nemikroskopische IJntersuchungen erblenamel has a very high protein content icher Schmebhypoplasien. Deutsche which, in one kindred, has been shown Zahnaerzt /. 1967; 22: 50i 13. to be amelogenin like in amino acid KiRiBEi. B, DA(t)Lsi G. Ultrastructural composition (8). Impairment of crystal- 11. study of amelogenesis imperfecta. CalciJ lite growth may therefore be a direct Tis.iue Res \911: 24: 191 7. result of incomplete removal of matrix 12. ROBINSON C , KIRKHAM J, STONKHOUSE protein. NJ, SHORI: RC. Extracellular processing of enamel matrix and origin and function Collectively the results of this histoof tuft protein. In: Hi ARNIU AD RW, ed. logic evaluation and characterization of Tdiith enamel V. Japan: I'lorence Pubcrystallite morphology are consistent lishers, 1989: 59 68. with a hypothesis that defective enamel 13. FiNciiAM AG, Hti Y, LAti EC, Si AVKIN maturation, in this AI type, is associated HC\ SNI Al) Ml.. Amelogenin post-secrewith retention of organic material that tory processing during biomineralization may be responsible for the impairment in the postnatal mouse molar tooth. Arch Oral Biol 1991: 36: .305 17. of crystallite growth. There appears to 14. AoBA T, FtJKAti M, TANABI: 1. SHIMI/.L' be excess organic material located on M, MORENO EC. Selective adsorption of and between the crystallites that may porcine amelogenins onto hydroxyapainterfere with the normal crystallite tite and their inhibitory activity on growth process. Given the complexity seeded crystal growth of hydroxyapatite. and as yet poorly understood processes Calcid Ti.s.me Int i987; 41: 28i 9. of enamel maturation, processing of 15. ROBINSON C , SHORE RC, KIRKHAM Jenamel proteins, and crystallite growth SroNEHOtiSE NJ. (Extracellular processthe specific defect or defects responsible ing of enamel matrix proteins and the for this AI type remains elusive. controi of crystal growth. J Biol Buccale i99l; 18: 355 6 i .

Enamel ultrastructure in pigmented hypomaturation amelogenesis imperfecta.

Hypomaturation amelogenesis imperfecta (AI) is a hereditary condition of enamel that is presumed to result from defects during the maturation stage of...
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