Optic and Electron Microscope Observation of the Tissue Composition of Enamel Lamella (Part 4)

Yasuyuki AWAZAWA*

In the previous reports of this serial study, the author described in detail the tissue structure of enamel lamella. Also in this research, the lamella wasfurther investigated to determine its histology. As mentioned in Part 3, lamella is one of the most highly disputed tissues in enamel. This problem should be principally considered histologically from various viewpoints. The author's following opinion was previously published: Genuine lamella can bealways found on enamel surfaces or in the inner layers of enamel of every tooth. In addition,lamella must be identified by its own peculiar so-called lamellar form. Its general direction runs from the enamel surface to the dentino-enamel junction. Lamella, of course, can be decisivelydistinguished mor- phologically from other hypocalcified areas often found in enamel. This genuine lamella is so commonly found in every tooth that it can be considered a normally existing structure in enamel. The tissue composition of genuine lamella is (1) hypomineralized enamel rods and (2) organic interrod substance. As a result of this research, the author has again reached the conclusion that lamellae consist of hypocalcified enamel rods and interrod substance. The observation of optic and electron micrographs are described below.

I Materials and Methods

As materials normal fresh permanent teeth of human beings and cows were em- ployed for observation. The human teeth were extracted with slight force from alveoli afflicted with advanced acute periodontal disease and then were stained immediately after extraction. The bovine teeth were stained in the jaw bones also immediatelyafter the cows were decapitated. The staining method is described as follows : The teeth were placed in a 0.5 % silver nitrate solution from half an hour to 10 hours and stored in an incubator during this period. The silver nitrate solution was renewed several times during this incubation period. Following this, they were placed in an 2 % hydroquinone solution from half an hour to 10 hours to fully reduce the silver nitrate that had permeated the lamellae tissue. The hydroquinone solution was also changed several times during silver reduction. The incubator temperature was always kept at 36℃. The enamel surface was decalcified by washing the teeth in a 5 to 10 % nitric acid solution for several minutes. This washing exfoliated the black reduced silver residue from the enamel surface so that silver-stainedlamellae could be clearly seen in contrast

* 粟沢靖之: Dept. of Pathology, Nihon Univ. School of Dentistry (Chief: Prof Yozo KOBAYAKAWA)

145 146 to the milky-white enamel with either the naked eye or a magnifying glass. Lamellae were then selected for observation. Observations were itemized into the following four categories: 1. Morphologic observation of lamella's superficial layers 2. Location of lamella in enamel 3. Electron microscopy of portions of argyrophilelamella's ends 4. Electron microscopy of silver-unstainable lamellae The procedures for observation are described below. For observation of a lamella's surface, some of the teeth were stained with silver nitrate for half an hour and the silver nitrate that had permeated the lamella tissue was reduced with hydroquinone for half an hour. Following this, the enamel surface was decalcified, as described above. For other observations, the other teeth were stained for 10 hours and then reduced for another 10 hours so that the inner layers of lamellae could be fully stained. To observe the lamella's surface, the enamel surfaces of silver-stained teeth were surveyed where silver-blackenedlamellae were seen with either the naked eye or a magnifying glass as shown in Figs. 39-54. To investigate a lamella's location in enamel, silver-stained lamellae were longitudinally exposed as illustrated in Figs. 55 and 56, and ground cross-sections were made of tooth-crowns in which stained lamellae were found as seen in Figs. 57-65. For the electron microscope examination of lamellae seen on thesurface oftooth- crowns, the outer surface of the enamel including the lamella was slightly abraded and etched with 0.5 % hydrochloric acid for 5 seconds to better expose selected lamellae. Argyrophile lamellae, portions of argyrophile lamella's ends, and silver-unstainable lamellae were chosen for this observation. The silver-unstainable lamellae were identified by the so-called lamellar ridges remaining on the enamel surface which were theun- stainable lamellae themselves. These lamellae strongly resisted the acid solution, as clearly observed in Fig. 54. Tofurther prepare for electron microscopy of these lamellae, the outersurface of the enamel and the lamella was again slightly abraded and etched with a0.5 % hydrochloric acid solution for 4 more seconds to better expose selected lamellae. The other procedures for electron microscopy were all the same with those described in Part 1. As a result of this research, the author has again reached the conclusion that lamella consists of hypocalcified enamel rods and interrod substance. The results are described below with their individual micrographs.

II Individual Micrograph Descriptions

1. Morphologic Observation ofLamella's Superficial Aspects Figs. 39-53 show silver-stained lamellae in teeth. In Fig. 54 there are about 9 silver-unstainable lamellae. One of the lamellae is seen partly blackened by silver. Fig.

Superficial aspects of lamellae

Fig. 39. lingual surface of ∟8 Fig. 43. ling ual surf ace of [8 Fig. 40. lingual surface of ∟7 Fig. 44. buccal surface of (8 Fig. 41. mesio-proximal surface of [8 Fig. 45. mesio-proximal surface of ∟6. Fig. 42 buccal surf ace of ∟8 Fig. 46. mesio-proximal surface of 8∟ 147 148 149

54 is takenfrom the disto-proximal surface of a bovine permanent right central incisor. All figures except Fig. 54 are taken from human permanent teeth. Silver-unstainable lamellae are barely seen in Figs. 39, 41, and 49. Generally speaking, the cervical portion of the crown usually contains a large number of narrow lamellae, while longer and wider lamellae are found on the outside surfaces of theocclusal portions of crown enamel. A few number of lamellae are seen extending from the outer lateral surfaces to the occlusal surface. Lamellae are also seen lying almost parallel with the long axis of tooth.

2. Location of Lamella in Enamel Fig. 55 schematically illustrates various locations of lamellae. Relations are illustrated amonglamella's surface aspects, a longitudinally exposedlamella is shown, and

Fig.55

A D G

Fig: 55. Each tooth was stained by silver, and the enamel surface was B E slightly decalcified with hydrochloric acid, as described in'Method'. La- mellae were then selected for observation, as shown in each illustration of this figure. The lamellae were longitudinally exposed by grinding the teeth along the chosen lamella. And then theselamella's locations in enamel were determined in each ground cross-section by the same method to determine the location of longitudinal lamellae. C F

Superficial aspects of lamellae Fig. 47. disto-proximal surface of ∟8 Fig. 57. lingual surface of 3∟ of 2∟ Fig. 48. buccal surface of7∟ Fig. 52. mesio-proximal surface of∟2 Fig. 49. mesio-proximal surface of 7∟ Fig. 53. mesio-proximal surface of I1∟ Fig. 50. disto-proximal surface. of 7‡˜ Fig. 54. disto-proximal surface 150 also a lamella's cross-section. We can quite easily find suchlamellae as seen in A, B, E. F, and G. Occasionally lamellae are found which are illustrated in C, and D. Particularly lamellae as seen in C are always mistaken for enamel tufts. We should note with much care thislamella's location.

Fig. 56. The lamellae were investigated in bovine teeth by the same method described for Fig. 55.

Figs. 57-65show bovine lamellae seen in ground cross-sections. We often observe such lamellae as shown in Figs. 57-63.Lamellae seen in Figs. 64-65 are seen rather often as a rule. Bovine lamellae are seen running from the enamel surface towards the dentin. This finding is more commonly seen in bovine teeth than in human teeth, andquite possibly explains thatlamellae fundamentally differ from enamel tufts. A and B in in Fig. 56 are, of course, equally significant for this explanation.

Optic microphotographs taken from lamellae seen on bovine ground cross-sections L-lamella, S-enamel fracture caused by preparation of ground section, J-dentin-enamel junction Figs.57-65.show lamellae in bovine permanent teeth. Thelamellae look as though they run from the enamel surface to the inner layer of enamel. Fig. 57. lamella seen in lower right 2nd premolar, ×100 Figs. 58 and 59.lamellae in lower right 1st premolar ,×100 Fig. 60, lamella in lower right 2nd molar, ×100 Fig. 61.lamella in lower left 1st premolar×100 Fig. 62. lamella in lower left 3rd molar, ×100 Fig. 63.lamellae seen in lawer right 1st incisor,×40 Fig. 64. lamellae in lower right 1st molar, ×50 Fig. 65.lamella in lower right 3rd premolar ,×100 151 152

Fig. 66 153

Fig. 67 154

Fig. 68 155

Fig. 69 156

Fig. 70

Fig 72 157

Fig 71 158

Fig. 73 159

3. Electron Microscopy of Portions of the Ends of Argyrophile Lamellae Figs. 66-68 show portions of silver-stained lamella's ends in bovine teeth. In each figure, a clear lamella is seen which appears like a chain of islets. These islet-like portions surround themselves with moderately hypocalcified enamel tissue which intercon- nects the islets. These lamella's islets are clearly seen composed of hypocalcified enamel rods and interrod substance. Fig. 66 clearly shows a lamella seen on the buccal surface of bovine M11, in which the enamel is so hypocalcified that it seems completely homogenous. The lamella's hypomineralized portions look like a chain of islets. The lamella is seen divided into several parts which appear like a chain of islets, and these parts are interconnected with slightly hypocalcified enamel. These parts are diffuse around the islets of lamella. This moderately hypocalcified enamel, of course, is apparently involved with lamella. The slightly hypomineralized enamel divides the lamella into several portions in which we can clearly recognized organic enamel rods and interrod substance. On the contrary, we can hardly distinguish enamel structures in the islet-like portions since these portions are homogenous. This figure of course shows a part of the end of a lamella. From this point of view, Figs. 67 and 68 are the same. Fig. 67 also shows a portion of the end of a lamella seen on the lingual surface of bovine M1. This lamella is a single array of islet-like homogenous enamel. These islet-like enamel portions are interconnected with moderately hypocalcified enamel. Inter isletspaces are comparatively wider. Fig. 68 shows lamella arrayed with homogenous enamel zonules. A part of the end of a lamella found on the buccal surface of bovine M21is shown in the figure.The lamella's homogenous rods and interrod substance are seen almost parallel with those seen in normal enamel. In the homogenous portions we can barely differentiate enamel rods from interrod substance. Fig. 69 shows a portion near the end of a lamella found on the labial surface of human 11 . The lamella is running vertically in the figure. The lamella looks very clear and smooth. This finding indicates that the lamella consists of both organicrods and interrod substance. This figure shows that several rods cross the border bet ween the lamella and normal enamel. That is, portions of these rods compose parts of the lamella, while the other portions compose normal enamel. Some residual enamel structures are found in the lamella tissue.

4. Electron Microscopy of Silver-Unstainable Lamellae Fig. 70 is the electron micrograph of the replica attached to the sheet-mesh of a portion of the lamella seen on the labial surface of bovine I11. This lamella was barely stained with silver, and was selected by the above described method. In the figure, the lamella is marked by Fig. 71 is the magnification of a part of the lamella seen in Fig. 70 the upper part indicated by The lamella was hardly stained by silver and looks remarkably smooth and clear. It seems so homogenous that we can hardly distinguish the lamella's organic rods from interrod substance. In the left portion of the lamella, we can observe comparatively clearly the lamella's rod outlines. This evidence supports the belief that 160 lamellae are composed of organic rods and interrod substance. Fig. 72 is another portion of the lamella shown in Fig. 70, but this portion is not seen in Fig. 70. This portion is the lower part of the lamella marked by x. This lamella's portion also is so highly homogenous that we can not differentiate organic rods from interrod substance. In Fig. 71 and 72 artefacts are found in the lamella which occurred from the grinding of the enamel surface. Fig. 73 shows a part of the lamella seen on the labial surface of bovine Mil,. Macroscopically this lamella is included in the category of silver-unstainable lamellae. The lamella is so homogenous that organic rods and interrod substance can not be distinguished.

III Discussion and summary

The following facts concerning enamel lamellae were established in this research. First most lamellae are nothing but hypocalcified enamel tissue. Macroscopic enamel fracture appearances are actually lamellae since traumatic fractures in the mouth are rare in human beings and practically non-existent in other animals deduced from the observation of teeth from cows and rabbits). The electron microscope revealed that these lamellae that look like fractures are actually composed of hypocalcified enamel rods and interrod substance. This clarification of the lamella's composition indicates that enamel is highly adaptable to its living conditions. Only extreme physical stresses probably have a detrimental effect. In fact the author could find no suspected traumatic fracture lines in any teeth, however of course we can assume that they would rarely exist. Animals, except human beings, instinctively avoid physical stresses that cause traumatic enamel fractures in their teeth, as mentioned in " Discussion " of Electron Microscope Studies of the Tissue Structure of Enamel Lamella — Part II appeared in Vol. 1, No. 4 of this Journal. The author previously failed to find enamel lamellae or traumatic enamel fractures in the incisors of six hundreds and seventy one (671) domesticated rabbits. That is, fractures and lamellae in enamel proved quite negative in rabbit incisors. The results of this rabbit experiment will appear in the next issue of this Journal. The author judges from the rabbit experiment that animals have simple circumstances in the oral cavity, while people have a wider variety of stresses in the oral cavity because of their tastes. We find lamellae on all surfaces of crown enamel. The cervical areas, however, contain most lamellae. These lamellae are short and fine as a rule. Longer and larger lamellae are found on the outer surfaces of crown enamel towards the occlusal. Lamellae are seen almost parallel with the long axis of tooth : i. e. from the cervical area towards the occlusal surface. Lamellae seen on the occlusal surface run in various directions. The author could detect most of these lamellae with silver-impregnation. A few, however, were detected without silver-stain, since they appeared as enamel traumatic fractures of yellowish-white, yellowish-brown, brownish-yellow, whitish-yellow, orbrilliant milky-white colors. In spite of these fracture line appearances, these lamellae showed the typical tissue composition of so-called genuine lamella hypocalcified rods and interrod substance. Cross-sectioning and longitudinal grinding of tooth-crowns indicate that lamellae Tun from the enamel surface towards the dentin. This is more clearly seen in bovine 161 teeth than in human teeth. This observation indicates that genuine lamellae are fundamentally different from enamel tufts, particularly their locations in enamel. Electron microscopy of portions of the ends of argyrophile lamella and silver- imstainable lamellae has clearly revealed in detail that lamellae are all composed of hypocalcified enamel rods and interrod substance. The author observed that lamellae vary in proportion of organic substance in lamella's rods and interrod substance.

References

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