Book - Oral Histology and Embryology (1944) 13

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Orban B. Oral Histology and Embryology (1944) The C.V. Mosby Company, St. Louis.

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Chapter XIII Temporomandibular Joint

1. Anatomic Remarks

The mandibular articulation (temporomandibular joint) is a diarthrosis between mandibular fossa and articular tubercle of the temporal bone, and capitulum (head, condyle) of the mandible. A fibrous plate, the articular disc, intervenes between the articulating bones.

The articulating surface of the temporal bone is concave in its posterior, convex in its anterior part. The 91131 concavity, articular fossa, extends from the squamotympanic and petrotympanic fissure in the back to the con vex articular tubercle in front. Th latter is strongly convex in a sagittal and slightly concave in a frontal plane. The convexity varies considerably, the radius ranging from 5 to 15 mm. The long axes of fossa and tubercle are directed medially and slightly posteriorly. The articular surface of the mandibular head is, approximately, part of a cylinder the axis of which is placed in the same direction as that of the articular surfaces on the temporal bone. The articulating parts of the temporomandibular joint are covered by a fibrous or fibrocartilaginous tissue and not by hyaline cartilage, as in most other articulations of the human body. The

hyaline cartilage in the mandibular condyle which is present during its growth period does not reach the surface.

The articular disc is an oval fibrous plate which is united around its margin with the articular capsule (Fig. 252). It separates the articular space into two compartments: a lower, between condyle and disc, and an upper, between disc and temporal bone. The disc appears biconcave in sagittal section. Its central part is thin, in rare cases perforated; the anterior and especially the posterior borders are thickened (Fig. 253). Fibers of the external pterygoid muscle are attached to its anterior border. The disc serves to adapt the bony surfaces to each other, especially in a forward position of the mandible when the convex condyle approaches the

aonvex articular tubercle. The disc is, at the same time, the movable socket for the mandibular head.

First draft subxpitted by Donald A; Kerr. 324 TEMPOROMANDIBULAR JOINT 325

The articular capsule consists of an outer fibrous sac which is loose. It IS strengthened on its lateral side by the temporomandibular ligament.‘

The inner synovial membrane is divided like the articular space. The superior part reaches from the margin of the articular surfaces on the tem poral bone to the disc; the inferior extends from the disc to the neck of the mandible.

2. HISTOLOGY

The condyle of the mandible is composed of typical cancellous bone Bony covered by a thin layer of compact bone (Fig. 253). The trabeculae are smmm

grouped in such a way that they radiate from the neck of the condyle and

Ma.ndibula.r ..

V‘

rossa. =. _ Articular tubercle

Mandibular head

Fig. 252.—Sagitta1 section through the temporomandibular joint. (Courtesy W. Bauer,‘ St. Louis University School oi‘. Dentistry.)

Fig. 253.—Sagitta1 section through the temporomandibular joint of a 28-year-old man. (Courtesy S. W. Chase. Western Reserve University.) 326 ORAL HISTOLOGY AND EMBRYOLOGY

reach the cortex at right angles, thus giving maximal strength to the condylar bone While still maintaining a light construction. In young

individuals the trabeculae are thin and may contain islands of hyaline cartilage near the surface (Fig. 254, A). In older individuals these car

- 3- Fibrous . covering

. - .. Cartilage V; , -I islands

. A! .-.1:-::. '

1 -§'Fibrous ‘ " covering

Fig. 254.—-Sections through the mandibular head. A. Newborn infant. R. Young adult.

tilaginous islands are resorbed and replaced by bone (Fig. 254, B). The marrow spaces are large at first, but decrease in size with progressing age by a marked thickening of the trabeculae. The marrow in the condyle TEMPOROMANDIBULAR JOINT 327

is of the myeloid or cellular type; in older individuals it is sometimes replaced by fatty marrow.

In young individuals the bone of the condyle is capped by a layer of hyaline cartilage which develops as a secondary growth center in three-month-old embryos. It is interposed between the fibrocartilage and the bone. It may still be present in the jaw of a person in his twenties (Fig. 254). The cartilage grows interstitially and by apposition from the deepest layer of the covering fibrous tissue; at the same time it is, gradually, replaced by bone on its inner surface.

. Ii-,,.’

, i . <: k'V.‘»} ' I,‘ I‘ .

Fig. 255.—Higher magnification of part of the mandibular condyle of Fig. 253.

The bone of the mandibular fossa varies considerably from that of the articular tubercle (Fig. 253). In the fossa it consists of a thin compact layer; the articular tubercle is composed of spongy bone covered with a thin layer of compact bone. In rare cases islands of hyaline cartilage are found in the articular tubercle.

The condyle as well as the articular fossa and tubercle are covered by a rather thick layer of fibrous tissue containing a. variable number of cartilage cells. The fibrous or fibrocartilaginous covering of the mandibular condyle is of fairly even thickness (Fig. 255). Its superficial layers consist of a network of strong collagenous fibers. Cartilage cells or chondrocytes may be present and have a tendency to increase in number with age. They can be recognized by their thin capsule which stains heavily with basic dyes. The deepest layer of the fibrocartilage is rich in

Bone

Articular Fibro cartilage Arflcularbisc

ORAL HISTOLOGY AND EMBRYOLOG3.

chondroid cells as long as hyaline cartilage is present in the condyle; it contains only a few thin collagenous fibers. In this zone the appositional growth of the hyaline cartilage of the condyle takes place.

The fibrous layer covering the articulating surface of the temporal bone (Fig, 256) is thin in the articular fossa and thickens rapidly on the posterior slope of the articular tubercle (Fig. 253). In this region the fibrous tissue shows a definite arrangement in two layers, with a small transitional zone between them; the two layers are characterized by the different course of the constituent fibrous bundles. In the inner zone the fibers are at right angles to the bony surface; in the outer zone they run parallel to that surface. As in the fibrous covering of the mandibular condyle, a variable amount of chondrocytes is also found in the tissue on the temporal surface. In adults the deepest layer shows a thin zone of

calcification.

Bone

Calcification

\ p " ~ - ' zone

v -- --s Inner fibrous layer

-— --——a Outer fibrous layer

Fig. 256.—Higher magnification of articular tubercle of Fig. 253

There is no continuous cellular lining on the free surface of the fibrocartilage. Only isolated fibroblasts are situated on the surface itself; they are, generally, characterized by the formation of long flat cytoplasmic processes.

In young individuals the articular disc is composed of dense fibrous tissue which resembles a ligament because the fibers are straight and

tightly packed (Fig. 257). Elastic fibers are found throughout the disc, but only in relatively small numbers. The fibroblasts in the disc are TEMPOROMANDIBULAR JOINT 329

elongated and send flat cytoplasmic wing-like processes into the interstices between the adjacent bundles. The mandibular disc does not show the usual fibrocartilaginous character of other interarticular discs. This

may be regarded as a functional adaptation to the high mobility and plasticity of this disc.

Articular tubercle

Superior articular space

__ Articular disc

--- Inferior articular space

- -—= Mandibular head

43

Fig. 257.—-Higher magnification of articular disc of Fig. 253.

With advancing age some of the fibroblasts develop into chondroid cells Which, later, may become real chondrocytes. Even small islands of hyaline cartilage may be found in the discs of older persons. Chondroid cells, true cartilage cells and hyaline ground substance develop in situ by difierentiation of the fibroblasts. In the disc as well as in the fibrous tissue covering the articular surfaces, this cellular change seems to be dependent upon mechanical influences. The presence of chondrocytes increases the resistance and resilience of the fibrous tissue. Articulal: capsule

330 ormr. HISTOLOGY AND EMBRYOLOGY

As in all other joints, the articular capsule consists of an outer fibrous layer which is strengthened on the lateral surface to form the temporamandibular ligament. The other parts of the fibrous capsule are thin and loose. The inner or synovial layer is a thin layer of connective tissue.

'It contains numerous blood vessels which form a capillary network close «to its inner surface. In many places larger and smaller folds or finger like processes, synovial folds and villi protrude into the articular cavity (Fig. 258). The former concept of a continuous cellular covering of the free synovial surface has been abandoned. Only a few fibroblasts of the synovial membrane reach the surface and, with some histiocyte and lymphatic‘ wandering cells, form an incomplete lining of the synovial membrane.

I ‘ T synovial villl

Fifi 258.—Villi on the synovial capsule of ternporomandibular joint.

A small amount of viscous fluid, synovial fluid, is found in the articular spaces. It is a lubricant and also a nutrient to the avascular coverings of the bones and to the disc. Its origin is not clearly established. It is possibly in part derived from the liquefied detritus of the most superficial elements of the articulating surfaces. It is not clear whether it is a product of filtration from the blood vessels or a secretion of the cells of the synovial membrane; possibly it is both. TEMPOROMANDJBULAR JOINT ' 331

3. CLINICAL CONSIDERATIONS

The thinness of the bone in the articular fossa is responsible for fractures if the mandibular head is driven into the fossa by a heavy blow. In such cases injuries of the dura mater and the brain have been reported.

The finer structure of the bone and its fibrocartilaginous covering depends upon mechanical influences. A change in force or direction of stress, occurring especially after loss of posterior teeth, will cause structural changes. These are brought about by resorption and apposition of bone, and by degeneration and reorganization of fibers in the covering of the articulating surfaces and in the disc.“

There is considerable literature on the disturbances after loss of teeth

or tooth substance due to changes in the mandibular articulation.“ The clinical symptoms are said to be: impaired hearing, tinnitus (ear buzzing), pain localized to the temporomandibular joint or irradiating into the region of ear or tongue. Many explanations have been advanced for

these variable symptoms: pressure on the external auditory meatus exerted by the mandibular condyle which is driven deeply into the articular fossa; compression of the auriculotemporal nerve; compression of the chorda tympani; compression of the auditory tube; impaired function of the tensor palati muscle. Anatomical findings do not substantiate any one of these explanations. Probably, all the diverse symptoms are but consequences of a traumatic arthritis in the mandibular joint.“ 2 It is caused by an increase and a change in direction of the forces of the masticatory muscles upon the structures of the joint.

References

1. Bauer, W.: Anatomische und mikroskopische Untersuchungen iiber das Kiefergelenk Anatomical and Microscopic Investigations on the Temporo-Mandibular oint), Ztschr. f. Stomatol. 80: 1136, 1932.

2. Bauer, W. H.: Osteo-Arthritis Deformans of the Temporo-Mandibular Joint, Am. J. Path. 17: 129, 1941.

3. Baecker, B.: Zur Histologie des Kiefergelenkmeniskus deg Menschen und der

Siiu er (Histology of the Temporo-Mandibular Disc in Man and Mammals), Zts . f. mikr.-anat. For-sch. 26: 223, 1931.

Breitner, 0.: Bone Changes Resulting From Experimental Orthodontic Treatment, Am. J. Orthodont. 26: 521 1940.

Cabrini, R., ‘and Erausquin, La. Articulacion Temporomaxilar de la Rata (Temporo-Mandibular Joint of the Rat), Rev. Odont. de Buenos Aires, 1941.

Cowdry, E. V.: Special Cytology, ed. 2, New York, 1932, Paul B. Hoeber, Inc., pp. 981-989, 1055-1075.

Hammer, J. Aug.: Ueber den feineren Bau der Gelenke (The Microscopic Architecture of the Joints), Arch. f. mikr. Anat. 43: 266, 1894.

Marquart, W.: Zur Histologie der Synovialmembran (Histology of the Synovial Membrane), Ztschr. f. Zellforsch. u. mikr. Anat. 12: 34, 1931.

Peterson, H.: Die Organe des Skeletsystems (Organs of the Skeletal System), Moel1endorf’s Handb. d. mikr. Anat. d. Menschen. Book 2, Part 2, Berlin, 1930, Julius Springer.

10. Schaefler, J. P.: Morris’ Human Anatomy, ed. 10, Philadelphia, 1942, The

Blakiston Co.

11. Schafler, J.: Ueber den feineren Bau und die Entwicklung dos Knorpelgewebes und iiber verwandte Formen der Stiitzsubstanz (On the Microscopic Structure and Development of Cartilage and Related Forms of Supporting Tissue), Ztschr. f. wissensch. Zoo]. 80: 155, 1905.

S°9°.“‘.°’S"'." 332 omu. HISTOLOGY AND EMBRYOLOGY

12. Schaffet, J.: Die Stiitzgewebe (Supporting Tissues), Moe11endorf’s Handb. f. mikr. Anat. d. Menschen, Book 2, Part 2, Berlin, 1930, Julius Springer.

13. Shapiro, H. IL, and Ti-uex, R. 0.: The Temporo-Mandibular Joint and the Auditory Function, J. A. D. A. 30: 1147 1943.

14. Sicher, Harry: Temporomandibufar Articulation in Mandibular Overclosure, J. A. D. A. 36: 131, 1948.

15. Sicher, Harry: Some Aspects of the Anatomy and Pathology of the Temporamandibular Articulation, New York State D. J. 14: 451, 1948.

16. Steinhardt Gr.: Die Beanspruchun der Gelenkfliichen bei versehiedenen Bissarten ( vestigations on the tresses in the Mandibular Articulation and Their Structural Consequences), Deutsche Zahnh. in Vortr. 91: 1, 1934.

Chapter XIV - The Maxillary Sinus

IN'1'RODUC'.|'.'ION DEVELOPMENT

ANATOMIG REMARKS FUNCTION

HISTOLOG-Y

CLINICAL CONSIDERATIONS

9‘S"'."9°!°'."

1. INTRODUCTION

The relation of the maxillary sinus to the dentition was first recognized by Nathaniel Highmore. In his Work Carports Humawi Disquisitio Anatomicafi (1651) he described the adult state of the cavity in detail, and pointed out that his attention had been called to it because a patient had an abscess there which was drained by the extraction of a cuspid tooth. This proved to be one of those misleading first observations, since it is now known that the cuspid‘ root seldom is related to this space in such a way that its simple extraction would drain it. However, the erroneous idea still persists that this relationship is generally true. The molar roots most often, and the bicuspid roots less frequently, are the dental structures which lie closest to the sinus (Fig. 259). Individual variations are great and can be determined only by careful interpretation of good roentgenographs.’

2. DEVELOPMENT

The maxillary sinus begins its development in about the third monthof fetal life. It arises by a lateral evagination of the mucous membrane of the middle nasal meatus, forming a slitlike space. In the newborn its measurements are about 8 x 4 x 6 mm. (Fig. 260); thereafter, it gradually expands by pneumatization of the body of the maxilla. The sinus is well developed when the second dentition has erupted, but it may continue to expand, probably throughout life.5

3. ANATOMIG REMARKS

The maxillary sinus, or antrum of Highmore, is situated in the body of the maxilla. It is pyramidal in shape; the base of the pyramid is formed by the lateral wall of the nasal cavity; the apex extends into the zygomatic process; the anterior wall corresponds to the facial surface of the maxilla, and the roof to its orbital surface. The posterior wall is formed by the infratemporal surface of the maxilla; the floor, usually, reaches into the alveolar process (Fig. 261).

First draft submitted by Paul C. Kitchin in collaboration with L. F. Edwards. Department of Anatomy, Ohio State University.

333 334 ORAL msronoev AND nmsnvonocv

There is a considerable variation in size, shape and position of the maxillary sinus, not only in different individuals, but also on the two sides of the same individual. Its average capacity in the adult is about one-half of one fluid ounce (14.75 c.c.) with average dimensions as follows: anteroposteriorly, 3.4 cm.; transversely, 2.3 cm.; and vertically, 3.35 cm. The maxillary sinus communicates with a recess of the middle meatus of the nasal cavity (semilunar hiatus) by means of an aperture, the ostium maxillare, which is located high on its nasal or medial wall and is, therefore, unfavorably situated for drainage (Fig. 261). An accessory ostium may occur which is, usually, lower and thus more advantageously placed for drainage than is the normal ostium.

Bony floor of sinus

Buccal « alveolar plate

.2 , :3

Fig. 259.—Bucco1ingua1 ection throu h n t b‘ ‘d. ' ; fzéom the sinusg by is thiirgpgfatenbisliione. The apex ls iepamted

Variations in the size of the maxillary sinus are explained on the basis of the degree or extent of pneumatization of the body of the maxilla (ho11owing—out by an air-filled pouch of the nasal cavity). In genera], the greater the pneumatization the thinner the walls of the sinus will be, since pneumatization occurs at the expense of bone. During THE MAXILLARY sums 335

enlargement of the sinus various recesses or accessory fossae may form. Thus, subcompartments or recesses may be present in the palatine, zygomatic, frontal and alveolar processes. The floor of the sinus may extend downward not only between adjacent teeth but also between the roots of individual teeth so that their apices cause elevations in the floor and appear to protrude into the sinus. The type and number of teeth whose

,1. : ,,,

.7!

Nasal septum

Maxillary sinus

"y Inferior nasal concha.

Fig. 260.—Fronta1 sections through the head. A. Newborn infant

B. Nine-month-old child.

Compare the size of maxillary sinus.

apices indent the floor of the space depend upon the degree and shape ofpneumatization. In the majority of cases the roots are covered by a. thin layer of bone (Fig. 259). In some instances, they are covered only by the mucous membrane which lines the cavity and by the periodontal membrane of the root of the tooth. The floor of the sinus may be on the 336 omu. HISTOLOGY AND EMBRYOLOGY

Ethmoidal cell ‘

Aperture of ,- .lnus

1

Maxillary sinus - —-—‘

Nasal septum

Inferior nasal concha

Maxtllary sinus

~ 3'. ‘;bhéiF'w.;\?L( $-or‘ '

Fig. 261.—Rela.tion or the maxillary sinus and its opening into the nasal cavity. A. Frontal section showing marked asymmetry between right and left sinus. B. Relation of sinus to root apicea. rm: MAXILLARY sINUs 337

same level with that of the nasal cavity, or higher or lower than that. In some cases the sinus may be incompletely divided by osseous and membranous ridges, commonly known as septa.

Unilateral supplemental maxillary sinuses have been observed.‘ They occur posteriorly to the sinus proper and are, from the standpoint of origin, overdeveloped posterior ethmoid cells. Clinically, they must be considered as maxillary sinus.

4. FUNCTION

In the past, various functions have been ascribed to the maxillary sinus and the other accessory nasal sinuses. It has been claimed by some, for instance, that they aid in warming and moistening inhaled air, thus acting as air-conditioning chambers. Others believe that the sinus plays an important role in vocalization. However, the most probable explanation of the development of all nasal sinuses is that bone which has lost its mechanical function is resorbed. An example is the marrow cavity in long bones where fatty tissue develops in the place of the disappearing bone. The disappearance of useless bony substance in the neighborhood of the air-filled nasal cavity leads to development of air-filled pouches which grow into the bone and occupy the place of bony tissue which is no longer needed to withstand mechanical stresses. The supporting function of bone is maintained but with a minimum of material. This is in accord with principles of economy which exist in the animal body.

5. HISTOLOG-Y

The maxillary sinus is lined by a mucosa covered with an epithelium typical of the respiratory passages. It is thinner and more delicate than that of the nasal cavity.

The lamina propria of the mucosa is fused to the periosteum of the underlying bone and consists of loose bundles of collagenous fibers with very few elastic fibers; it is only moderately vascular (Fig. 262, A). Glands of the mucous and serous type are confined largely to that part of the tunica propria which is located around the opening, or openings, into the nasal cavity.

The epithelium is pseudostratified ciliated columnar, rich in goblet cells (Fig. 262, B). The nuclei of the individual columnar cells are‘located at different distances from a delicate basement membrane. Actually, each columnar cell rests upon the basement membrane, but not all the cells reach the surface. The goblet cells secrete mucus which moistens the surface of the sinus mucosa. The cilia beat in such a way as to move any surface material toward the opening communicating with the nasal cavity, and hence act to clear the sinus cavity of inhaled substances, and

mucus. 6. GLINIGAI. CONSIDERATIONS

Pulpal infection in teeth whose root apices are in close approximation to the floor of the sinus are dangerous because it can be a cause of sinus Maxillary sinus Epithelium

- —"- ‘ “ ‘ - Mucous membrane

‘T’ --- “F” and perlosteum

Incomplete bony floor of sinus

_ _ ,_,......\‘. 7»;-w

Fig. 262.——Mucous membrane and epithelium of maxillary sinus.

A. Apical region of :1 second bicuspid. The lining oi.’ the sinus is continuous with the periapicsl tissue through openings in the bony floor of the sinus.

B. High magnification of the epithelium of maxillary sinus. (Courtesy W. 11. Bauer.‘ St. Louis University School of Dentistry.)

.- - -j-—-A .g.g3;‘.I“&' ‘S

-2‘Q

es.

F'iE- 263.—Roentgenogrs.m or upper jaw. Maxillary sinus extends toward alveolar crest after loss of flrst molar. Tl-IE MAXILLARY SINUS 339

infection.‘= 3 Thus, the prevention of the dental type of sinusitis is possible by prevention or elimination of pulpal infection. Any root canal operation in maxillary bicuspid or molar areas should be carried out with particular care, in order to prevent infection of the sinus.

The dentist should always keep in mind that disease of the maxillary sinus may produce referred dental pain. The superior alveolar nerves run in narrow canals in the thin wall of the sinus and, frequently, these canals are partly open toward the sinus. When this happens the nerves which supply the teeth are in contact with the lining of the sinus where they may become involved in an inflammation affecting the mucosa. In such cases, the pain resembles pulpal pain but involves a group of teeth or even all the teeth in one maxilla. If apices of some roots are in contact with the lining of the sinus the affected teeth may show symptoms of periodontitis during sinus infection. In cases where there is doubt whether the teeth or sinus are the cause of pain, the patient should be referred to a rhinologist before an extraction is performed.

In the course of an extraction a root may be forced into the sinus. If it cannot be easily removed through the socket the patient should be informed of the circumstances and be referred to a rhinologist. Even if it is possible for the dentist to remove the root of the tooth, subsequent treatment by the sinus specialist is advisable. Any invasion of the field of sinus surgery by the dentist operating through the alveolar wall should be discouraged by both dental and medical professions.

After loss of a single maxillary molar or, more rarely, bicuspid, the bony scar is, sometimes, hollowed out by the sinus (Fig. 263). The risk of opening the sinus during extraction of a tooth adjacent to such an extension has to be recognized. If a single molar remains in the maxilla for a long time after loss of the neighboring teeth, downward extensions,‘ of the maxillary sinus may occur mesially and distally to this tooth. If [ greater force is applied in extracting such a tooth, tooth and socket are removed together rather than extracting the tooth from its socket. To minimize the necessary force the crown should be removed, the roots separated and extracted singly. The expansion of the maxillary sinus (and other sinuses) in old individuals should not be considered a process of growth. It is rather the consequence of progressive disuse atrophy of the bones, especially after loss of teeth, or of senile osteoporosis. The senile expansion of sinuses strengthens the belief that they develop as fill-ins in bones whose core is under reduced mechanical stress.

References

1. Bauer. W. ‘EL: Maxillary Sinusitis of Dental Origin, Am. J. Orthodont. & Oral Surg.

29: 133, 1943. _ 2. Ennis, L. M., and Batson, 0.: Variations of the Maxillary Sinus as Seen in the

Roentgenogram, J. A. D. A. 23: 201, 1936. 3. Hofer, 0.: Dental Diseases and Their Relation to Maxillary Antrum, J. Dent.

Research 17: 321, 1938 (Abstract). 340 omu. HISTOLOGY AND EMBRYOLOGY

4. MacMilla.n, H. W.: The Relationship of the Teeth to the Maxillary Sinus; Anatomic Factors glnderlying the Diagnosis and Surgery of This Region, J‘. A. D. A. 14.: 1635, 19 7.

5. Schaefier, J. I’.: The Sinus Maxillaris and Its Relations in the Embryo, Child and Adult Man, Am. J. Anat. 10: 313, 1910.

6. Schaefler, J. P.: The Nose, Paranasal Sinuses, Nasolacrymal Passageways and Olfactory Organ in Man, Philadelphia, 1920, P. B1a.kiston’s Son & Co.

7. Sedwick, H. .1'.: Form, Size and Position of the Maxillary Sinus at Various Ages Studied by Means of Roentgenograms of the Skull, Am. J. Roentgenol. 32: 154, 1934.

8. Zuckerhandl, E.: N ormale und pathologische Anatomie der N asenhiihle und ihrer pneumatischen Anhiinge (Anatomy of the Nasal Cavity), Leipzig, 1893. CHAPTER XV TECHNICAL REMARKS

1. INTRODUCTION

2. PREPARATION OF EISTOLOGIC SPI:GIM:E:N S

a. Dissection

b. Fixation

c. Decalciflcation

d. Embedding

e. Sectioning

f. staining g. Altmann-Gersh Technique

3. PREPARATION OI‘ G-ROUND SECTIONS 4. PREPARATION OI‘ ORGANIC STRUCTURES IN THE ENAMEL 5. PEOTOMICROGRAPHY

1. INTRODUCTION

This chapter is intended to give the student a general idea of the preparation of microscopic slides, rather than to cover fully the subject of microscopic technique. For detailed information specialized textbooks should be consulted." 11’ 12' 1’ The various processes to which a tissue is subjected from the time it is taken from the body until it is ready to be examined under the microscope, are termed microtechnique. Its object is to prepare the specimen for examination of its microscopic structure.

2. PREPARATION OF I-IISTOLOGIC SPECIMENS

Dissection is the first step in the preparation of a specimen; the material may be secured by a biopsy (excision during life) or at an autopsy (postmortem examination). Pieces of tissue are cut as small as possible to insure satisfactory fixation and impregnation. A very sharp knife should be used to prevent tissue structures from being distorted and squeezed.

Immediately after the specimen is removed and the surface washed free of blood, it is placed in fixing solution. The object of fixing is to preserve the tissue elements in the same condition in which they are at the moment the reagent acts upon them, and harden or so affect them that they will not be altered by the processes of dehydration, embedding, staining, clearing and mounting. The amount of the fixing solution should be at least 20 times the volume of the tissue. The fixing tissue coagulates the protein content of the cells, thus preventing decomposition.

First draft submitted by Joan Launspach, research technician or the Foundation for Dental Research, Chicago College of Dental Surgery.

341

Dissection

Fixation Lciflcation

342 ORAL msronocr AND EMBRYOLOGY

There are several fixing agents in general use, the most common of which are formalin, formalin-alcohol, Zenker-formol solution, and Bouin’s fluid. A good and rapidly penetrating fixative for small specimens is Zenker-formol solution, a mixture of 9 parts of potassium bichromate and bichloride of mercury with 1 part of neutral formalin. Formalin (5 to 10 per cent) is used for large pieces of tissue, e.g., jaws. It does not deteriorate and it penetrates very rapidly. Formalin-alcohol fixes and dehydrates simultaneously, and is used mostly for surgical specimens. Bouin’s fluid is a solution of picric acid and formalin, and is especially applicable in studying cell outlines, but is rather slow to penetrate.

The length of time necessary for a fixing agent to act upon a tissue varies according to the size of the specimen and penetrating power of the fixative. Generally, it should be just long enough for the agent to saturate the piece thoroughly without allowing it to become brittle. Small pieces of tissue, e.g., gingiva, are left in Zenker-formol solution only 4 to 8 hours, while larger pieces such as jaws may be left in formalin for days. In order to obtain good fixation of pulp in an intact tooth, the surface of enamel and dentin is ground away to a thin layer of dentin around the pulp. This process not only insures rapid and thorough penetration of the fixing agent but also reduces the time of decalcification and permits a thorough impregnation with cellodin. When fixing biopsy specimens, the solution should be at approximately body temperature.

After the specimens are thoroughly fixed, they are washed in running water for twenty-four to forty-eight hours to remove all acids and reagents. Occasionally, however, special treatment is required to remove the precipitates caused by certain agents. Example: specimens fixed in Zenker-formol solution are treated with Lugol’s (iodine) solution and sodium thiosulfate, and specimens fixed in formalin are placed in a mixture of potassium hydroxide before staining. However, there is no need of this if neutral formol is used.

Animal tissues may be classified as hard and soft, or calcified and noncalcified. The dental histologist is particularly interested in the hard tissues, namely, the enamel, dentin, cementum and bone. These are impregnated with a variable quantity of calcium salts and cannot be seetioned on the microtome unless decalcified.

Decalcification of a tissue is the removal of its mineral content by an acid such as nitric, hydrochloric, trichloracetic, formic, or sulfosalycilic acid. The length of time a specimen remains in the decalcifying agent is influenced by the choice and concentration of the acid, and the size of the specimen; however, the shorter the time the better is the staining. A 5 per cent solution of nitric acid seems to be most satisfactory and is, therefore, widely used. It acts quickly without causing swelling of the tissue or any other undue changes in its elements; it does not interfere with the staining process to any marked degree.

While tissues are being decalcified they should be suspended in a large quantity of the fluid in order that the salts dissolved may sink to the TECHNICAL REMARKS 343

bottom of the jar. Occasional stirring or gentle agitation of the specimen and heating of the acid may hasten the process of decalcification, but great care should be taken not to injure the tissues.

To ascertain whether the inorganic salts have been completely removed, the specimen can be pierced with a sharp needle or pin: when no gritty substance is detected, the decalcification is sufficient. Roentgenographic check-up can also be employed. After decalcification a tooth should be as pliable as a piece of cartilage. The enamel disappears almost entirely owing to its low percentage of organic matter. The decalcifying agent may also be tested for calcium; the acid is changed periodically until the test is negative.

Following decalcification the specimen is washed thoroughly in running water for at least 24 hours. From this point it is treated as a soft tissue and is ready for the embedding process. It is possible, however, to embed hard tissues first and decalcify them later: the specimen is run through the solutions in routine fashion and, after it is blocked, the excess celloidin is cut away and the tissue is suspended in acid until decalcified. This takes much longer than the usual procedure and the results are often uncertain.

In order that a tissue may be sectioned on the microtome it has to have a certain rigidity to offer sufficient resistance to the cutting edge of the knife. This may be accomplished by freezing the tissue or, as is more commonly done, by using an embedding medium which fills the interstices of the tissue. The freezing technique is employed where immediate investigation of the specimen is required, as in the course of a surgical operation. Some substances (fat, lipoids, etc.) are dissolved during embedding: tests for such substances can be made only in frozen sections.

Embedding is a much more lengthy process but results are more satisfactory. Before the specimen is embedded, i.e, impregnated with a suitable substance such as paraffin or celloidin, the water has to be removed from the tissues. Paraffin embedding is more rapid and is used for small pieces, usually soft tissue, as decalcified pieces become brittle during the heating which is necessary in using this method. Celloidin embedding takes longer but causes less shrinkage. This technique is more commonly used in dental histology when large blocks of decalcified material have to be sectioned.

Dehydration is accomplished by placing the specimens in ascending alcohols (50, 70, 95, 100 per cent) for approximately one day each; the length of time depends on the size and permeability of the specimen. Two consecutive changes of absolute alcohol are used. As, however, paraifin or celloidin is not soluble in alcohol it has to be replaced by a fluid which is a solvent for the embedding medium.

When paraffin is selected as the embedding medium, the absolute alcohol is replaced by xylol or oil of cedarwood. The specimens are placed in the solvent 12 to 24 hours, and are then placed in liquid parafiin in the

Embedding Sectioning

344 ORAL HISTOLOGY AND EMBRYOLOGY

incubator (56 C.) for several hours. Finally the specimen is placed in a form filled with molten parafiin and quickly cooled. It is then ready to be sectioned.

If celloidin, a solution of nitrocellulose in absolute alcohol and ether, is selected as the embedding medium, a mixture of equal parts of ether and alcohol is used as a solvent in which the tissue remains for 12 hours. It is then carried through a thin (6 per cent) and medium (121/; per cent) into thick (25 per cent) solution of celloidin. The length of time which is necessary for each of the solutions to penetrate the specimen depends upon size and permeability of the tissue. Soft tissue is well infiltrated with celloidin after three weeks while decalcified specimens require at least six weeks; to insure thorough impregnation, it is wise to leave teeth longer in the lower concentration of celloidin and a somewhat shorter time in the stronger concentration. When it is necessary to “rush” a specimen the tissue in thin celloidin may be placed in a 50° C. oven in a tightly stoppered container; the embedding period is thus shortened to two or three days. This method causes considerable shrinkage. Small pieces of tissue may be placed directly on a fiber block and left to harden in a desiccator filled with chloroform vapor. Large pieces of tissue, e.g., jaws, are placed in an evaporating dish filled with celloidin which is allowed to harden down slowly. When the celloidin has reached the desired degree of hardness, blocks are cut out and, after being softened in thin celloidin a few minutes, are placed on fiber blocks,

allowed to air dry, and then placed in 70 per cent alcohol for storage or sectioning.

Tissues are sectioned by means of a microtome, ‘a machine equipped with a knife. There are three different types of microtomes: the freezing, rotary, and sliding, the use of which depends on the kind of tissue and embedding medium used. Each is a heavy specially designed machine precisely constructed, capable of slicing prepared tissues into exceedingly thin sections. The knife is wedge-shaped and made of heavy steel to aflord the greatest possible rigidity; it must have a very keen edge as the slightest nick would tear a section. Sharpening a microtome knife is one of the most important as well as the most difficult tasks of a technician. Larger nicks are removed on a coarsely grained stone, and a fine edge is achieved by grinding the knife on a fine hone. The final cutting edge is obtained by stropping on a finishing leather microtome strop. A much more rapid and just as satisfactory a method that has recently been developed is the use of a grinding machine, consisting of an ebony wheel mounted on a rotary motor. A strop, dusted with abrasive powder, is used to put the finishing edge on the knife. The possibility of making good sections depends upon the type of tissue, its preparation, and the condition of the knife. Sections of 5 to 15 microns (174000 millimeter equals 1 micron) are considered thin. TECHNICAL REMARKS 345

The importance of the freezing technique in preparing surgical specimens has been mentioned. It is well known that, by exposing tissues to an extreme degree of cold, they become hard and can be easily sectioned with the freezing microtome. The cold is generated by means of carbon dioxide which is sprayed onto the stage holding the specimen: rapid evaporation produces the required temperature.

The rotary microtome is used only for sectioning paraffin blocks; the knife is immovably fixed at a right angle to the block which is carried past the sharp edge of the knife by turning a wheel. With this machine it is possible to out long ribbons of serial sections. The ribbons are placed in lukewarm water where the wrinkles are removed as the paraffin becomes soft. The desired sections are then floated onto slides smeared with egg albumen and placed in a 37° C. oven for a few minutes. Before staining, the paraffin is dissolved in xylol. the slides rinsed in absolute alcohol, the sections are carried through descending concentrations of alcohols into distilled water; then they can be stained by water-soluble dyes.

A celloidin block is sectioned by a different method. For this purpose the sliding microtome, a heavy sledgetype instrument, is used. The longitudinal angle of the knife is adjusted to each specimen so that the entire cutting edge is used in sectioning. The angle of the cutting edge of the knife should be changed according to the hardness and density of the material. To obtain the most satisfactory results the knife should be in an almost horizontal position for large decalcified pieces, at an acute angle for soft tissue. During sectioning both specimen and knife are continually moistened with 70 per cent alcohol; the sections are placed in distilled water before staining. The celloidin is usually not removed from the section as the stain penetrates the tissues in spite of this embedding medium. However, it has to be removed from the section in the case of specific stains, i.e., Mallory, azure-eosin, etc. For this procedure the section is mounted on a slide smeared with egg albumen and is flooded with oil of cloves to dissolve the celloidin; the slide is rinsed in 95 per cent alcohol and placed in 70 per cent until ready for staining.“ When serial sections are desired, sections are mounted on glass slides which are then blotted and flooded with a very thin solution of collodion. After a coating is formed, the slides are marked with a diamond pencil or India ink, and stored in 70 per cent alcohol until ready for staining.“

Some special staining methods can be applied only to sections of undecalcified teeth and bone. To obtain such sections mature enamel has to be removed from the teeth. The tissue impregnated With celloidin is placed in a shallow dish and covered with celloidin. The solution is allowed to evaporate slowly until the celloidin is very hard (two to four weeks) . A very hard knife should be used which has been sharpened and stropped ; when checked under the microscope it has deep and even teeth and should be clamped in the microtome at a 13° angle. Staining

Altmann-Gersh Technique

346 orm. ursronoev AND nmasvonoer

Dyes used to stain specimens for microscopic examination may be classified as basic or acid, according to their affinity for different cellular elements. Basic dyes, sometimes called nuclear dyes, primarily stain nuclear chromatin, basic substance of cartilage and mucus; the more commonly used are hematoxylin, methylene blue, safranin, and carmin. Acid dyes color the cytoplasm of the cell, uncalcified bone and dentin matrix, some connective tissue fibers; eosin and phloxin are representative of this group. By using combinations of the two groups, due to their different affinities, a marked difierentiation of the cellular elements of the specimen is possible.

Sections may be stained on the slide or floating in dishes; in the latter case better differentiation is afiorded. Although the steps of the various staining methods differ considerably, they may be arranged in the following order: staining, differentiating, decolorizing, dehydrating, clearing and mounting.

Hematoxylin and eosin is one of the most commonly used combinations of stains because it is the simplest to handle. For the differentiation of more specialized tissues the following are recommended: Mallory stain,“ or Heidenhain’s Azan“ (modification of Mallory’s stain) for connective tissue; the latter is more brilliant and has greater capacity for differentiation; Silver Impregnation (modification of Foot’s stain by Gomori) for connective tissue fibers and nerve elements ;’ Van Gieson’s stain, a counterstain to hematoxylin, for differentiation of white connective tissue," and Weigert’s stain for elastic tissue.“

After sections of tissues have been stained and differentiated, they are dehydrated and then passed through a medium that will mix with the dehydrating fluid as well as the reagent in which the sections are to be mounted. These intermediary fluids are called clearing agents because they have a high refractive index, thus rendering the sections more or less transparent.

For celloidin sections a variety of clearing agents is used: terpineol (Lilacine), carbol-xylol, oil of cloves, oil of cedar wood, oil of origanum and beechwood creosote; for paraffin sections usually only two are used: xylol or toluol.

After clearing, the sections have to be placed in some medium which will preserve the stain and prevent the tissue from drying. Such solutions are termed mounting agents: among the most common are Gum damar, Canada balsam, and clarite. Loose celloidin sections are floated onto the slide and straightened out with the aid of a fine camel’s hair brush; they are carefully blotted and covered with a drop of mounting medium and a coverslip. Weights are placed on the coverslips to prevent the formation of air bubbles. When dry, they are carefully cleaned with xylol and labeled with India ink.

The Altmann-Gersh freezing and drying technique for special microchemical studies should also be mentioned. The tissues are frozen inTECHNICAL REMARKS 347

stantaneously when placed in a tube of isopentane in a Liquidair container and are dehydrated under vacuum while still frozen, thus avoiding a redistribution of minerals. Fixation, alcohol dehydration, and clearing are omitted as the dehydrated tissue can be immediately infiltrated with paraffin and sectioned according to the usual methods. This technique has proved valuable in the preparation of tissues for micro-incineration, and for special micro-chemical reactions.

Many submicroscopic structures may be seen with the electron microscope, not visible in sections prepared in the routine manner.

3. PREPARATION OF GROUND SECTIONS

Ground sections are prepared by using abrasive stones upon a tooth or bone until the tissue is reduced to translucent thinness. It is the principal method of examining the enamel which has so little organic material that it disappears almost entirely when the teeth are decalcified by ordinary methods. Therefore, this technique should complement the decalcification method. i

To prepare a ground section of a tooth it is first ground down on one side on a carborundum stone which rotates at high speed on a laboratory lathe. It is important that the tooth be kept wet constantly with cold water to lessen the heat produced by friction and to prevent the section from drying. If it is allowed to dry its organic constituents will shrink and present a picture untrue to the conditions during life. The tissue is likewise more apt to crack and break up during preparation if it becomes dry. When the desired level is reached and the ground surface is perfectly plane, this surface is polished on wet ground glass and, finally, on an Arkansas stone. The other side of the specimen is then ground down until the section is sufficiently translucent. This second side is also polished in the above described manner, to remove the gross scratches produced by the carborundum stone. The finished ground sections should have an average thickness of 25 to 50 microns and, if desired, may be

stained before they are dehydrated, cleared and mounted.”

For surface staining of ground sections the surface is well polished and the section is covered with a 0.25 per cent H01 to decalcify it slightly; then it is stained lightly with hematoxylin.“ By this method only the surface of the ground section is stained and the stained layer can be viewed with high power lenses as it is only a few microns in thickness. This method, however, causes slight decalcification of the enamel making a marked differentiation of rods from sheaths and cementing substance,

3. condition not representative of normal enamel. Enamel that has been

partially decalcified by caries, or a poorly formed enamel has this appearance.

If it is necessary to investigate an undecalcified tooth with the surrounding soft tissue, ground sections can be made by using the petrification method. The specimen is embedded in Kollolith-chloroform solution 348 omu. HISTOLOGY AND EMBRYOLOGY

or in Canada balsam”, 15 where it is left until it is sufficiently hard before it is ground down to a desired thickness. Thin “serial” ground sections of teeth and jaws may be cut in one operation by infiltrating the specimen with a plastic material and using a cutting device made up of steel wheels set at various distances."

4. PREPARATION OF ORGANIC STRUCTURES IN THE ENAMEL

The routine decalcification of whole teeth in an aqueous solution of acid usually destroys the enamel completely. At most, merely shreds of the organic structures remain near the cervical areas in a tooth of a young person.

The organic structures may be demonstrated by C. F. Bodecker’s celloidin decalcifying method.‘ When dentin is included in the specimen sections are rarely satisfactory because this tissue becomes very brittle as a result of the many media through which it passes. It is necessary only for study of the organic structures in the enamel under high magnification. In general, this method is erratic and a high percentage of failures must be expected.

The Cape-Kitchin modification‘ 5 of Bodecker’s method is quite simple and gives satisfactory results if the structures of the matrix are not magnified more than about 500 diameters.

Frisbie, Nuckolls, and Saunders’ have recently developed a technique for the successful recovery of the enamel matrix. The fresh specimen is immediately fixed in neutral formalin for a long period of time (six months) ; most of the dentin is then removed with a dental bur and the tooth is placed in the fixative again for a shorter period of time, depending on the penetrability of the specimen. The completely fixed enamel is decalcified by placing the specimen on a gauze stretched over a platinum wire frame, and immersing it in a 5 per cent solution of nitric acid in 80 per cent alcohol for 24 to 48 hours. Dehydration is begun with 70 per cent alcohol without preliminary washing. The specimen is infiltrated with celloidin at 56 C. for two weeks and then allowed to harden down slowly at room temperature until the block is very hard. Sectioning is done with a sliding microtome at 3 to 4 microns.

An aqueous decalcification of enamel under a cover-glass is the simplest but the least satisfactory method for its study. It is sufficient to show enamel lamellae, cuticle, tufts, and can be used to demonstrate gross differences in quantity of organic structures of enamel in recently erupted teeth and in teeth of old persons. However, the disadvantages are that only low magnifications up to 100 diameters are possible and that the specimens are not durable.

Another method of differentiating the organic from the inorganic content of the enamel is by incineration. It has been shown that the heating of sections of human adult enamel up to 800° 0. causes a destruction of the organic content but leaves enamel rods intact. TECHNICAL REMARKS 349

5. PHOTOMICROGRAPHY

Photomicrographs are photographs of small microscopic objects, made with the aid of a microscope. Most of the illustrations in this book are such pictures. Transmitted light is the most commonly used method of illumination as it permits the sharpest differentiation of details and the highest magnification of tissue structures in stained decalcified sections.

Refiected light is used in oral histology in photographs of ground sections of enamel and dentin. These sections should be ground perfectly smooth. There is no need for extreme thinness because the specimen is viewed only from the surface from which the light is reflected.

Polarized light also is useful in the study of the dental tissues. It vibrates in a single known plane and requires special equipment and technique. By this means it is possible to determine details of the submicroscopic structure of tissues, due to the differences in optical properties of various elements. Polarized light is particularly useful in the study of calcified tissues, but is not confined to these, because fibrous and keratinized structures also yield information when studied by this method.

Grenz rays are a form of exceedingly soft roentgen rays. When ground sections of teeth are photographed in this way, slight variation in calcification may be defined thus rendering this method useful in the study of calcified structures." 13

A method has recently been developed by Gurney and Rapp” for studying the fine structural details of tooth surface by adapting the Fax Film technique used for study of metallographic surfaces. Micro-impressions are made of the specimen, using a plastic film which may then be mounted on a glass slide for a permanent preparation. Scott and Wyckoff“ obtained similar results by shadowing collodion replicas with vaporized metal in a high vacuum, a more complicated method. The eifect of chemical agents on tooth structure and the changes in tooth surfaces (caries) may be observed using these methods. When examined under the electron microscope, many submicroscopic structures are visible.

Ultraviolet light technique‘ and fluorescence light microscopy, likewise, have been applied in special studies of dental tissues, but have not yet attained wide use.

References

1. Applebaum, E.. Hollander, F., and Bodecker. C. F.: Normal and Pathological Variations in Calcification of Teeth as Shown by the Use of Soft X-rays, Dental Cosmos 75: 1097, 1933. _

Bensley, R. R., and Bensley, S. 8.: Handbook of Histological and Cytological Technique, Chicago, University of Chicago Press. _ _

Bodecker, C. F.: Cape-Kitchin Modification of Celloidin Deoalcifymg Method for Dental Enamel, J. Dent. Research 16: 143, 1937. _ _ _ '

Bodecker, C. F.: Enamel of Teeth Decalcifled by Celloidin Decalcifying Method and Examined by Ultra Violet Light, Dental Review 20: 317, 1906.

Cape, A. T., and Kitchjn, P. 0.: Histologic Phenomenon of Tooth Tissues Observed Under Polarized Light, With a Note on Roentgen Ray Spectra of Enamel and Dentin, J. A. D. A. 17: 193, 1930.

S"l“9°.l‘-" 350 ORAL HISTOLOGY AND EMBRYOLOGY

6. Cowdry, E. V.: Microscopic Technique in Biology and Medicine, Baltimore, 1943, Williams & Wilkins Co.

7. Frisbie, H. E., Nuckolls, J., and Saunders, J. B. de G. M.: Distribution of Organic Matrix of Enamel in Human Teeth and Its Relation to Histopathology of Caries, J. Am. Coll. Dent. 11: 243, 1944.

8. Gurney, B. R, and Rapp, G. W.: Technic for Observing Minute Changes on Tooth Surfaces, J. Dent. Research 25: 367, 1946.

9. Guyer, M. F.: Animal Micrology, Chicago, 1943, University of Chicago Press.

10. Hotchkiss, R. D.: Microchernical Reaction Resulting in Staining of Polysaccharide Structures in Fixed Tissue Preparation, Arch. Biochem. 16: 131, 1948.

11. Loosli, C. G.: Outline of Histological Methods, Chicago, University of Chicago Press.

12. Mallory, F. B.: Pathological Technique, Philadelphia, 1938, W. B. Saunders Co.

13. McClung, C. Microscopic Technique, New York, 1937, Paul B. I-Ioeber, Inc., pp. 353-401.

14. McLean, F. 0., and Bloom, W.: Calcification and Ossification. Calcification in Normal Growing Bone, Anat. Rec. 78: 133, 1940.

15. Meyer, W.: Die Anfertigung histologicher Schlifie (Preparation of Histologic Ground Sections), Vrtljschr. f. Zahnheilk. 41: 111, 1925.

16. Scott, D. B., and Wyckofi, R. W. G.: Shadowed Replicas of Tooth Surfaces, Pub. Health Rep. 61: 697, 1946.

17. Sognaecs, R. F.: Preparation of Thin Serial Ground Sections of Whole Teeth and Jaws and Other Highly Calcified and Brittle Structures, Anat. Rec. 99: 133, 1947.

17a. Sognnaes, R. F.: The Organic Elements of the Enamel. I, II, III, IV, J. Dent. Research 27: 609, 1948; 28: 549, and 558, 1949; 29: 260, 1950.

18. Willman, M.: Technique for Preparation of Histological Sections Through Teeth and Jaws for Teaching and Research 16: 183, 1937.

19. Wolf, J.: Plastische Histologie der Zahngewebe (Plastic Histology of Dental Tissues), Deutsche Zahn-, Mund- und Kieferheilkunde 7: 265, 1940.

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