Archives of
Oto-Rhino-Laryngology 9 Springer-Verlag 1979
Arch. Otorhinolaryngol. 224, 301-311 (1979)
The Vascular Mantles of Labyrinthine Bone* A Comparative Anatomical Study Alexander Rauchfuss Anatomisches Institut der Universit~it Hamburg, Abteilung f/Jr Neuroanatomie, Universit~itskrankenhaus Eppendorf, MartinistraBe 52, D-2000 Hamburg 20, Federal Republic of Germany
Die Gef~iBmiintel des Labyrlnthknoehens Eine vergleichendeanatomisehe Studie Zusammenfassung. In der enchondralen Schicht des Labyrinthknochens von Hund (Canis f. intermedius Woldrich) und Affe (Pithecus fascicularis Ram.) werden ,,GeffiBm~intel" mittels der Licht- und der Transmissionselektronenmikroskopie untersucht und mit entsprechenden Strukturen beim Menschen verglichert. Bei der dreidimensionalen Rekonstruktion der Gef'~il3eund ihrer Mfintel ergibt sich, dab nur Teile des jeweiligen Gef/il3es von einer Mantelzone bedeckt sind. Im polarisierten Licht finder man M/intel, die Lamellenfragmente enthalten. Mittels der Transmissionselektronenmikroskopie 1/il3t sich eine Kanalisierung der perivaskul~iren Mantelmatrix nachweisen. Die Ultrastruktur der lichtmikroskopisch gering differenziert erscheinenden Zellen in den Mantelzonen zeigt, dab es sich um Osteoblasten, Osteozyten und Osteoklasten handelt; eine Osteozyten-Osteolyse ist nachweisbar. Die Bedeutung der Oxygenation via Kanalisierung der Mantelmatrix wird diskutiert. Desgleichen wird die Bedeutung fehlender mechanischer Beanspruchung in der enchondralen Schicht in Bezug auf die Mantelbildung er6rtert. Entsprechend dem ,,embryonalen Charakter" der Strukturen der enchondralen Schicht wird eine Analogie zwischen Entwicklungsformen des Osteons im Extremit/itenskelet und Geffil3m~inteln diskutiert. Sehliisselwiirter: Gef~il3m/intel - Labyrinthknochen - Vergleichende Anatomic Summary. The vascular mantles of the endochondral layer of labyrinthine bone in dog (Canis f. intermedius Woldrich) and monkey (Pithecus fascicularis Ram.) were examined by means of light and transmission electron microscopy and compared to those in man.
*
Dedicated to my father on the occasion of his 65th birthday
0302-9530/79/0224/0301/$ 02.20
302
A. gauchfuss A three-dimensional reconstruction of vessels and their vascular mantles is demonstrated. It is shown that these mantles only cover parts of the vessel. Using polarized light one can find vascular mantles containing lamellar fragments. By means of transmission electron microscopy a canalization of perivascular mantle matrix can be demonstrated. The ultrastructure of cells in mantle zones shows that these cells are osteoblasts, osteocytes, and osteoclasts. Signs of osteocytolysis are found. The importance of oxygenation via canalization of the vascular mantle matrix is discussed as well as the importance of lacking biomechanical stress in endochondral layer as to their influence on formation of vascular mantles. According to the "embryologic character" of endochondral layer an analogy between incomplete osteons which are to be found in developing woven bone and vascular mantles is considered. Key words: Vascular mantles - Labyrinthine bone - Comparative anatomy
Throughout life the endochondral layer of the labyrinthine bone exists of "embryonic structures" (Meyer, 1933): the interglobular spaces (Manasse, 1897) and the skein bone (Meyer, 1927). The interglobular spaces are remnants of calcified embryonic cartilage (Anson et al., 1948) which are the result of an incomplete endochondral ossification of the otic capsule (Bast, 1930). Meyer (1927) describes the skein bone as an alamellar, woven, fine-fibered embryonic type of bone. Comparative anatomical studies have given evidence that these characteristic structures are also visible in the labyrinthine bone of some adult mammals (Rauchfuss, 1978, 1979). The otic capsule was called to exist of too old, dead bone, lacking of remodelling processes (Lange, 1938; Marx, 1947). By means of histochemistry (Del Bo, 1961; Zechner, 1969, 1971a, b, c), autoradiography (Aliprandi et al., 1963; Rauchfuss, 1977; Hildmann, 1977), and fluorescence microscopy (Hawke and Jahn, 1975) remodelling processes in the skein bone on molecular level were demonstrated in labyrinthine bone of man, rabbit, and guinea pig. In skein bone one does not find lamellar osteons around the vessels. Resorption of perivascular skein bone matrix and replacement by another type of woven bone is described in man. The resulting structures are called "vascular mantles" which can appear blue or red according to their staining quality in hematoxylin eosin stained sections. Their color is related to their age (Zechner and Altmann, 1972). The socalled "mixed mantles" contain lamellar fragments (Manasse, 1914; Mayer, 1917; Meyer, 1931; Zechner and Altmann, 1972). .By means of comparative anatomy using the light and the transmission electron microscope vascular mantles are studied especially considering the fact that the endochondral layer of otic capsule exists of a special embryonic type of bone.
Materials and Methods
Healthy, non-otosclerotictemporalbones of dog (Canis f. intermediusWoldrich)and monkey(Pithecus fascicularis Raffi.) were taken. The animals were anesthetizedwith Nembutal and perfusedvia the left heart.
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Fixational procedures: 1. Bouin's solution, decalcification in EDTA 10%, rinsing, dehydration in ethanol of increasing concentration, embedding in paraffine; unstained sections of 18 ~m of thickness for use in polarized light, sections of 4--8 ~m stained with: eosin, hematoxylin, hematoxylin eosin, azan, and alcian blue. 2. Glutaraldehyde 6.25% in a 0.1 mol phosphate buffer, decalcificationin EDTA 10%, rinsing in a 0.1 mol Tris buffer, postfixation in OsO4 1% in a 0.1 mol phosphate buffer including saccharose 1% at 4~ C for 2 h, dehydration in ethanol of increasing concentration (Doty and Schofield, 1976), embedding in EPON 812 (Luft, 1961); semithin sections of 0.5--1.0 ~m for light microscopy on a Reichert OMU 2, staining: toluidine blue and pyronine red (Ito and Winchester, 1963), ultrathin sections on a Porter-Blum ultramicrotome, visualization of the sections in a Zeiss electron microscope EM 9. For comparison healthy, non-otosclerotic human temporal bones were studied also. Fixational procedure: fixation by immersion in Carnoy's or in Bouin's solution, treated likewise under 1.
Notes on Terminology The cartilage remnants are called interglobular spaces - "Interglobularr/iume"(Manasse, 1897) - and not globuli interossei or even globuli ossei as it is done in American literature. According to Meyer (1927), the bone of the endochondral layer is called skein bone - "Str/ihnenknochen" - and not endocbondral bone, a term which is used also in American literature and in our opinion is not exact. Whether this is an adequate term concerning phylo- and ontogenetic facts is discussed in detail by Weidenreich (1930). In the present study it is not explicitly distinguishedbetween "blue", "red", or "mixed" mantles. These structures are not identical with the "blue borders" ("blaue S~iume") which sometimes were interpreted as mantle bone (see Lempert and Wolff, 1945).
Results In the surroundings of the semicircular canals in the endochondral layer of healthy, non-otosclerotic labyrinthine bone in dog and monkey vascular mantles are to be found. In eosine stained sections one can easily visualize these dark blue colored structures whereas in hematoxylin eosin stained sections they appear dark blue or grey blue (see Nager und Meyer, 1931). Using polarized light or the m o n o c h r o m a t o r one can see that they exist of woven bone which is short-fibered and which sometimes contains lamellar fragments lying often parallel to the circumference of the vessel. Often the mantle matrix is demarcated from the skein bone matrix by a tiny cement line. Stained with alcian blue the mantles appear dark blue which is significant for the presence of acid mucopolysaccharides. The vascular mantles are arranged asymmetrically around the vessels. Sometimes two or even more vessels can be surrounded by only one mantle. It is possible that only one side or only one part of one side of the vessel wall m a y be covered with mantle matrix. The three-dimensional reconstruction of the vascular mantles using serial paraffine sections gives evidence that these mantles cover only parts of a vessel. The same vessel can have mantle parts and portions which are "only" surrounded by skein bone. Inside the mantles' matrix of dog and m o n k e y one observes a small n u m b e r of lacunae which is just the opposite in man. I n the periphery of the mantles by means of light microscopy "osteocytes" are to be found. Regarding their ultrastructure
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Fig. 1. A a) Incompletedevelopingembryonic osteon according to Knese and Titschak (1962). b) c) d) Vascular mantles. Woven bone grey. Skein bone white. Bone lamellaeblack lines. B Three-dimensional reconstruction of vascular mantles (grey) surrounding vessels
different types of cells can be distinguished. In the periphery of the mantle zone one finds mononucleated cells surrounded by a complete boundary. The pericellular space is small, without any formed structures. There are a few processes in the surrounding matrix. On the other hand one realizes mononucleated cells which are surrounded by an irregular inner border of the lacuna, forming insulas and peninsulas of bone matrix. The pericellular space is widened, containing fibrillar materials. The cytoplasm of these cells sometimes appears vacuolated. A third type of cells can be seen. Those are mononucleated smaller cells with a relatively large nucleus lying in a semicircle of dense matrix. This semicircle is open toward the perivascular space. The fourth type are cells which have to be distinguished from the vascular lining cells. They have two or more large nuclei and the cytoplasmic membrane has the characteristics of a ruffled border. The same matrix like in mantle regions can be observed in some skein bone areas surrounding the interglobular spaces. By means of transmission electron microscopy one also finds mononucleated cells in the periphery of these zones with signs of osteocytolysis. It is worth noticing that not the cartilage of interglobular spaces but the skein bone is resorbed. Studying healthy, non-otosclerotic human labyrinthine bones one can affirm the results achieved by the light microscope. One can also observe mantles containing lamellar fragments which are arranged asymmetrically around the vessels in endochondral layer. Multinucleated osteoclasts are not visible. Because of postmortal changes preparation of human temporal bones for a transmission electron microscopic study is not useful. Using the transmission electron microscope a canalization of interglobular spaces in mantle zones which is postulated by Zechner and Altmann (1972) in man
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using paraffine embedded sections is not to be found in monkey and dog though there is a canalization of mantle zones. There is a connection between vessels and canals. It seems to be evident that canalization starts from the vessels. The canals are already to be found in developing vascular mantles and their number increases with the expansion of the mantle zone. They do not contain cell processes from neighboring osteocytes.
Discussion
In this present study healthy, non-otosclerotic labyrinthine bones are examined. Vascular mantles are to be found in dog (Canis f. intermedius Woldrich), monkey (Pithecus fascicularis Raft.), and man. It was pointed out by Weber (1933) that these vascular mantles are a previous stage in otosclerotic bone formation. Using polarized light one can visualize almost identical structures in otosclerotic loci and in vascular mantles (Weber, 1960; Zechner and Altmann, 1972). The findings of NylOn (1949) and Chevance et al. (1969, 1970) indicate that vascular mantles are not a result of pathological reaction but signify "physiological" remodelling in endochondral layer. Typical localizations of otosclerotic foci are to be found in the cochlear region of labyrinthine bone (Sch~itzle and Haubrich, 1975). Vascular mantles prefer the surroundings of the semicircular canals (Zechner and Altmann, 1972). Zechner and Altmann (1972) did not find constant correlations between occurrence of otosclerotic foci and presence of vascular mantles though in labyrinthine bone of individuals suffering from otosclerosis there is a larger number of vascular mantles. Using hematoxylin eosin stained sections the different staining qualities of the mantle zones indicate different stages of remodelling and different age. According to this, Gussen (1968) and Zechner and Altmann (1972) describe depolymerization and repolymerization of mantle matrix which is related to the amount of acid mucopolysaccharides. In Gussen's (1968) opinion mantle bone per se is not an abnormal bone but might perhaps signify extensive degeneration of bone. Some of the vascular mantles contain fragments of lamellar bone. In adult long bones asymmetric osteons can be observed and in developing long bones one can find incomplete osteons being embedded in woven bone (Knese and Titschak, 1962). These incomplete osteons are the result of incomplete resorption of woven bone followed by deposition of lamellar bone. These structures look similar to vascular mantles in endochondral layer. By three dimensional reconstruction of vessels and their vascular mantles one finds that the vessel is only partly covered by mantle matrix. The expansion of the vascular mantle in a defined area might depend on specialization of bone cells in this region which differs from cells in other regions. Such specialization is reported in embryonic and growing bones (Amprino, 1955; Jande, 1971). It depends on sufficient canalization and an O2-optimum in the area supplied (Anderson and Parker, 1966; Bohatirchuk, 1969; Hall, 1970, 1972). A relation between capillary density and persistence of embryonic tissue in endochondral layer was demonstrated by means of morphometry concerning the vessels and the interglobular spaces (Rauchfuss, 1978). Oxygenation via canalization seems to be an important factor for occurrence of vascular mantles for only the canalized
Fig. 2. A Endochondral layer dog. Vascular mantle (m), vessels (1~),skein bone (s). Semithin section. Toluidine blue and pyrortine red. • 580. B Endoehondral tayer dog, Vascular mantle (m), vessels (v), skein bone (s). 8emithin section. Toluidine blue and pyronine red. x 380. C Endochondral layer man. Otosclerotic focus (O), skein botle (s), vessel (v). Hematoxylin eosine. Monochromator. x 380. D Endochondral layer monkey. Mixed mantle (x) contaitting lamellae (arrows), blue mantles (b), vessels (v), skein bone (s). Azan. Monochromator. x 360
matrix of skein bone can be replaced by another type o f woven bone (Zechner, 1971a, b, c). The noncanalized interglobular spaces remain. This corresponds to resorption of cartilage in developing long bones (Wilsman and van Sickle, 1970; Miller et al., 1972). O n the other hand biomechanical factors are important for canalization of the tissue block (Altmann, 1964) as to their influence on maturation of bone from woven
Fig. 3. A Endochondral layer dog. Formation of perivascular mantle bone. Osteoblast (b) lying in a semicircle of newly produced matrix (arrows), vascular lining cells (v), endothelial cells (e), skein bone (s). x 4800. B Endochondral layer dog. Periphery of vascular mantle (m), skein bone matrix (s), osteocyte (o) with complete lacunar wall, osteocyte undergoing osteocytolysis (x), canalization of mantle matrix (arrows), endothelial cells (e). x 4800
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Fig. 4. A Endochondral layer dog. Osteocytie osteolysis. Irregular lacunar border forming insulas and peninsulas of bone matrix (arrows), skein bone (s), mantle bone (m), nucleus (n), vacuolization of dedifferentiated ergastoplasm (x). x 9800. B Endochondral layer dog. Osteocytic osteolysis. Detail of irregular lacunar border. The insulas and peninsulas are parts of unmineralized bone matrix (X) containing collagen fibrils of skein bone (arrows). This indicates that these structures are not artifacts resulting from the fragmented cytoplasmic membrane, x 48000
to lamellar bone (Pauwels, 1965). It is feasible that the lacking of biomechanical stress in endochondral layer which is responsible for constancy of many parameters of labyrinth throughout life (Rauchfuss, 1978) is one reason for asymmetry of vascular mantles. Meyer (1927) pointed out that skein bone is an embryonic type of bone. According to this and according to Knese and Titschak's (1962) classification of osteons one should call the vascular mantles "developing" or "embryonic" osteons. Using the transmission electron microscope the ultrastructure of cells in mantle zones gives evidence that there are mononucleated osteocytes being surrounded by a complete boundary. On the other hand there are cells undergoing osteocytolysis
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which is characterized by an irregular inner border. Their pericellular space contains fibrillar material. Their cytoplasm sometimes appears vacuolated (Jande and Belanger, 1971). Osteocytic osteolysis is an important factor in bone remodelling (Belanger, 1969). Another type of cells is lying in a semicircle of newly produced matrix. These cells have a large nucleus and they are identical with osteoblasts which are found in the periphery of developing osteons (Cooper et al., 1966). The fourth type of cells is a typical osteoclast having two or more large nuclei and a cytoplasmic membrane with the characteristics of a ruffled border (Hancox and Boothroyd, 1961). All these cells seem to originate from vascular mesenchymal cells (Cooper et al., 1966). Considering the embryonic character of the endochondral layer and its structures one can affirm Zechner and Altmann's (1972) opinion that vascular mantles are the result of physiological remodelling. Mantle bone per se is not an abnormal bone (Gussen, 1968). These remodelling processes depend on oxygenation via canalization. It should be mentioned that oxygenation as well osteocytolysis are important factors for enzymatic activities of cells in otosclerotic foci. Considering the findings of Chevance et al. (1970) the similarities between bone changes in young otoscleroric foci reported by these authors and matrix alterations in developing and developed mantle bone might indicate that the beginning of otosclerotic bone formation is governed by the same morphological principles as formation of mantle bone.
References
Aliprandi, G., Pesenti, M., Villa, A.: Studio sull' incorporazione del radiozolfo (35S). Arch. Ital. Otol. 74, 526-532 (1963) Altmann, K.: Zur kausalen Histogenese des Knorpels. Berlin, G6ttingen, Heidelberg, New York: Springer 1964 Amprino, R.: Autoradiographic research on the 35S metabolism in cartilage and bone differentiation and growth. Acta Anat. (Basel) 24, 121--163 (1955) Anderson, C. E., Parker, J.: Invasion and resorption in enchondral ossification. J. Bone Joint Surg. [Am.] 48, 899-914 (1966) Anson, B. J., Bast, T. H., Cauldwell, E. W.: The development of the auditory ossicles, the otic capsule and the extraeapsular tissues. Ann. Otol. Rhinol. Laryngol. 52, 3, 603-632 (1948) Bast, T. H.: Ossification of the otic capsule in human fetusses. Contr. Embryol. Carneg. Inst. 121, 53-82 (1930) Belanger, L. F.: Osteocytic osteolysis. Calcif. Tissue Res. 4, 1-12 (1969) Bohatirchuk, F. P.: Metaplasia of cartilage into bone - a study by stain historadiography. Am. J. Anat. 126, 243-254 (1969) Chevance, L. G., Balslev, Jorgensen, M., Bretlau, P., Causs~, J.: Electronmicroscopic studies of the otosclerotic focus. Acta otolaryngol. (Stockh.) 69, 563-581 (1969) Chevance, L. G., Balslev Jorgensen, M., Bretlau, P., Causs6, J.: Otosclerosis. Acta Otolaryngol. [Suppl.] (Stockh.) 272, 1-44 (1970) Cooper, R., Milgram, J. D., Robinson, R. A.: Morphology of the osteon. J. Bone Joint Surg. [Am.] 48, 7, 1239-1217 (1966) Del Bo, M.: Rilievi istochimici sullo strato osseo encondrale della capsula labirintica. Arch. Ital. Otol. 72, 620-628 (1961) Doty, S. B., Schofield, B. H.: Enzyme histochemistry of bone and cartilage cells. Progr. Histochcm. Cytochem. Vol. 8, No. 1. Stuttgart: Fischer 1976 Gussen, R.: The labyrinthine capsule: Normal structure and pathogenesis of otosclerosis. Acta Otolaryngol. [Suppl.] (Stockh.) 235, 1-55 (1968)
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Hall, B. K.: The origin of cartilage and bone cells from common stem cells. Calcif. Tissue Res. 4 [Suppl.], 147 (1970) Hall, B. K.: Immobilization and cartilage transformation into bone in the embryonic chick. Anat. Rec. 173, 391-404 (1972) Hancox, N. M., Boothroyd, B.: Motion picture and electron microscopic studies on the embryonic avian osteoclast. J. biophys, biochem. Cytol. 9, 651-661 (1961) Hawke, M., Jahn, A. F.: Bone formation in the normal human otic capsule. Arch. Otolaryngol. 101, 462-468 (1975) Hildmann, H.: Autoradiographische tierexperimentelle Untersuchungen an der Schneckenkapsel des Kaninchens. Laryngol. Rhinol. Otol. (Stuttg.) 56, 508--512 (1977) Ito, S., Winchester, R. J.: The fine structure of the gastric mucosa in the bat. J. Cell Biol. 16, 541-578 (1963) Jande, S. S.: Fine structural study of osteocytes and their surrounding bone matrix with respect to their age in young chicks. J. Ultrastruct. Res. 37, 279-300 (1971) Jande, S. S., Belang6r, L. F.: Electron microscopy of osteoeytes and the pericellular matrix in rat trabecular bone. Calcif. Tissue Res. 6, 280-289 (1971) Knese, K. H., Titschak, S.: Untersuchnngen mit Hilfe des Lochkartenverfahrens fiber die Osteonstrukmr von Haus- und Wildschweinen, sowie Bemerkungen zur Baugeschichte des Knochens. Morph. Jb. 102 (3), 337-458 (1962) Lange, W.: Das Ohr des Kindes. In: Handbuch der Anatomic des Kindes. Peter et al. (Hrsg.). Mfinchen: Bergmann 138 Lempert, J., Wolff, D.: Histopathology of the incus and the head of the malleus in cases of the stapedial ankylosis. Arch. Otolaryngol. 42, 339-449 (1945) Luft, J. M.: Improvements in epoxy resin embedding methods. J. biochem, biophys. Cytol. 9, 404-414 (1961) Manasse, P.: Uber knorpelhaltige Interglobularr/iume in der menschlichen Labyrinthkapsel. Z. Ohrenheilk. 31, 1-10 (1897) Manasse, P.: ~ber Ossifikationsanomalien im menschlichen Felsenbein. Arch. Ohrenheilk. 95, 145-159 (1914) Marx, H.: Handbuch der Ohrenheilkunde. 2. Auflage. Jena: Fischer 1947 Mayer, O.: Untersuehungen fiber die Otosklerose. Wien, Leipzig: Holder 1917 Meyer, M.: f0ber eine eigentfimliche Art von Knochengewebe beim erwachsenen Menschen (den lamellenlosen, feinfaserigen - str/ihnenartigen - Markknochen) und fiber den embryonalen Markknothen. Z. Anat. Entwiekl.-Gesch. 83, 734-751 (1927) Meyer, M,: Die normale Anatomic der menschlichen Labyrinthkapsel. Z. Hals-, Nas.-, u. Ohrenheilk. 33, 1-72 (1933) Miller, M. L., Basom, C. R., McCuskey, R. S.: Temporary lobulation in cartilagenous models of long bones. Anat. Rec. 172, 523-528 (1972) Nager, F., Meyer, M.: Beitrgge zur normalen und pathologischen Histologie der knSchernen Labyrinthkapsel. 2. Mitteilung. Die Bedeutung der Farbreaktion f/Jr die Beurteilung des Baues des Knochengewebes in der Labyrinthkapsel mit besonderer Darstellung ihres periostalen Teiles. PassowSchaefer Beitr. 29, 157--170 (1931) Nyl6n, B.: Histopathological investigations on the localization, number, activity, and extent of otoselerotic foci. J. Laryngol. Otol. 63, 321-343 (1949) Pauwels, F.: Gesammelte Abhandlungen zur funktionellen Anatomie des Bewegnngsapparates. Berlin, Heidelberg, New York: Springer 1965 Rauchfuss, A.: Autoradiographische Untersuchungen am Labyrinthknochen. Inaug. Diss. Aachen (1977) Rauchfuss, A.: Neue Befunde zum Ban der enehondralen Schicht des Labyrinthknochens. Vortrag. 73. Vers. Anat. Ges. Innsbruck 1978. Verh. Anat. Ges. (im Druck) (1979) Rauchfuss, A.: Ein Beitrag zum Vorkommen von Str/ihnenknochen in der enchondralen Schicht des Labyrinthknochens. Z. Mikrosk. Anat. Forsch. (im Druck) (1979) Weber, M.: The blue mantles in otosclerosis. Ann. Otol. Rhinol. Laryngol. 42~ 438-452 (1933) Weber, M.: Neuere Erkenntnisse fiber histologische Anfangsstadien der Otosklerose. Z. Laryng. Rhinol. 39, 521--530 (1960) Weidenreich, F.: Das Knochengewebe. In: Handbuch der mikroskopischen Anatomic des Menschen II/2. MSllendorff (Hrsg.). Berfin: Springer 1930
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Wilsman, N. J., Van Sickle, D. C.: The relationship of cartilage canals to the initial osteogenesis of secondary centers of ossification. Anat. Rec. 168, 381-392 (1970) Zechrler, G.: Untersuchungen an der organischen Matrix der Labyrinthkapsel des Menschen. Arch. klin. exp. Ohr.-, Nas.-, u. Kehlk.-Heilk. 194, 576-579 (1969) Zechner, G.: Uber die blauen M/intel in der menschlichen Labyrinthkapsel. Mschr. Ohrenheilk. 104~ 319-320 (1970) Zechner, G.: fiber Knorpelreste in der menschlichen kn6chernen Labyrinthkapsel. Arch. klin. exp. Ohr.-, Nas.-, u. Kehlk.-Heilk. 198, 317-324 (1971a) Zechner, G.: Neue morphologische und histochemische Aspekte der kn6chernen Labyrinthkapsel des Menschen. Mschr. Ohrenheilk. 105, 160-161 (1971b) Zechner, G.: Aufbau und Umbau der kn~Schernen Labyrinthkapsel des Menschen. Acta Otolaryngol. (Stockh.) 71, 81-88 (1971c) Zechner, G., Altmann, F.: fdber blaue M/intel in der kn/Sehernen Labyrinthkapsel. Arch. klin. exp. Ohr.-, Nas.-, u. Kehlk.-Heilk. 201~ 172-180 (1972) Received November 16, 1978
Oto-Rhino-Laryngology 9 Springer-Verlag 1979
Arch. Otorhinolaryngol. 224, 301-311 (1979)
The Vascular Mantles of Labyrinthine Bone* A Comparative Anatomical Study Alexander Rauchfuss Anatomisches Institut der Universit~it Hamburg, Abteilung f/Jr Neuroanatomie, Universit~itskrankenhaus Eppendorf, MartinistraBe 52, D-2000 Hamburg 20, Federal Republic of Germany
Die Gef~iBmiintel des Labyrlnthknoehens Eine vergleichendeanatomisehe Studie Zusammenfassung. In der enchondralen Schicht des Labyrinthknochens von Hund (Canis f. intermedius Woldrich) und Affe (Pithecus fascicularis Ram.) werden ,,GeffiBm~intel" mittels der Licht- und der Transmissionselektronenmikroskopie untersucht und mit entsprechenden Strukturen beim Menschen verglichert. Bei der dreidimensionalen Rekonstruktion der Gef'~il3eund ihrer Mfintel ergibt sich, dab nur Teile des jeweiligen Gef/il3es von einer Mantelzone bedeckt sind. Im polarisierten Licht finder man M/intel, die Lamellenfragmente enthalten. Mittels der Transmissionselektronenmikroskopie 1/il3t sich eine Kanalisierung der perivaskul~iren Mantelmatrix nachweisen. Die Ultrastruktur der lichtmikroskopisch gering differenziert erscheinenden Zellen in den Mantelzonen zeigt, dab es sich um Osteoblasten, Osteozyten und Osteoklasten handelt; eine Osteozyten-Osteolyse ist nachweisbar. Die Bedeutung der Oxygenation via Kanalisierung der Mantelmatrix wird diskutiert. Desgleichen wird die Bedeutung fehlender mechanischer Beanspruchung in der enchondralen Schicht in Bezug auf die Mantelbildung er6rtert. Entsprechend dem ,,embryonalen Charakter" der Strukturen der enchondralen Schicht wird eine Analogie zwischen Entwicklungsformen des Osteons im Extremit/itenskelet und Geffil3m~inteln diskutiert. Sehliisselwiirter: Gef~il3m/intel - Labyrinthknochen - Vergleichende Anatomic Summary. The vascular mantles of the endochondral layer of labyrinthine bone in dog (Canis f. intermedius Woldrich) and monkey (Pithecus fascicularis Ram.) were examined by means of light and transmission electron microscopy and compared to those in man.
*
Dedicated to my father on the occasion of his 65th birthday
0302-9530/79/0224/0301/$ 02.20
302
A. gauchfuss A three-dimensional reconstruction of vessels and their vascular mantles is demonstrated. It is shown that these mantles only cover parts of the vessel. Using polarized light one can find vascular mantles containing lamellar fragments. By means of transmission electron microscopy a canalization of perivascular mantle matrix can be demonstrated. The ultrastructure of cells in mantle zones shows that these cells are osteoblasts, osteocytes, and osteoclasts. Signs of osteocytolysis are found. The importance of oxygenation via canalization of the vascular mantle matrix is discussed as well as the importance of lacking biomechanical stress in endochondral layer as to their influence on formation of vascular mantles. According to the "embryologic character" of endochondral layer an analogy between incomplete osteons which are to be found in developing woven bone and vascular mantles is considered. Key words: Vascular mantles - Labyrinthine bone - Comparative anatomy
Throughout life the endochondral layer of the labyrinthine bone exists of "embryonic structures" (Meyer, 1933): the interglobular spaces (Manasse, 1897) and the skein bone (Meyer, 1927). The interglobular spaces are remnants of calcified embryonic cartilage (Anson et al., 1948) which are the result of an incomplete endochondral ossification of the otic capsule (Bast, 1930). Meyer (1927) describes the skein bone as an alamellar, woven, fine-fibered embryonic type of bone. Comparative anatomical studies have given evidence that these characteristic structures are also visible in the labyrinthine bone of some adult mammals (Rauchfuss, 1978, 1979). The otic capsule was called to exist of too old, dead bone, lacking of remodelling processes (Lange, 1938; Marx, 1947). By means of histochemistry (Del Bo, 1961; Zechner, 1969, 1971a, b, c), autoradiography (Aliprandi et al., 1963; Rauchfuss, 1977; Hildmann, 1977), and fluorescence microscopy (Hawke and Jahn, 1975) remodelling processes in the skein bone on molecular level were demonstrated in labyrinthine bone of man, rabbit, and guinea pig. In skein bone one does not find lamellar osteons around the vessels. Resorption of perivascular skein bone matrix and replacement by another type of woven bone is described in man. The resulting structures are called "vascular mantles" which can appear blue or red according to their staining quality in hematoxylin eosin stained sections. Their color is related to their age (Zechner and Altmann, 1972). The socalled "mixed mantles" contain lamellar fragments (Manasse, 1914; Mayer, 1917; Meyer, 1931; Zechner and Altmann, 1972). .By means of comparative anatomy using the light and the transmission electron microscope vascular mantles are studied especially considering the fact that the endochondral layer of otic capsule exists of a special embryonic type of bone.
Materials and Methods
Healthy, non-otosclerotictemporalbones of dog (Canis f. intermediusWoldrich)and monkey(Pithecus fascicularis Raffi.) were taken. The animals were anesthetizedwith Nembutal and perfusedvia the left heart.
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Fixational procedures: 1. Bouin's solution, decalcification in EDTA 10%, rinsing, dehydration in ethanol of increasing concentration, embedding in paraffine; unstained sections of 18 ~m of thickness for use in polarized light, sections of 4--8 ~m stained with: eosin, hematoxylin, hematoxylin eosin, azan, and alcian blue. 2. Glutaraldehyde 6.25% in a 0.1 mol phosphate buffer, decalcificationin EDTA 10%, rinsing in a 0.1 mol Tris buffer, postfixation in OsO4 1% in a 0.1 mol phosphate buffer including saccharose 1% at 4~ C for 2 h, dehydration in ethanol of increasing concentration (Doty and Schofield, 1976), embedding in EPON 812 (Luft, 1961); semithin sections of 0.5--1.0 ~m for light microscopy on a Reichert OMU 2, staining: toluidine blue and pyronine red (Ito and Winchester, 1963), ultrathin sections on a Porter-Blum ultramicrotome, visualization of the sections in a Zeiss electron microscope EM 9. For comparison healthy, non-otosclerotic human temporal bones were studied also. Fixational procedure: fixation by immersion in Carnoy's or in Bouin's solution, treated likewise under 1.
Notes on Terminology The cartilage remnants are called interglobular spaces - "Interglobularr/iume"(Manasse, 1897) - and not globuli interossei or even globuli ossei as it is done in American literature. According to Meyer (1927), the bone of the endochondral layer is called skein bone - "Str/ihnenknochen" - and not endocbondral bone, a term which is used also in American literature and in our opinion is not exact. Whether this is an adequate term concerning phylo- and ontogenetic facts is discussed in detail by Weidenreich (1930). In the present study it is not explicitly distinguishedbetween "blue", "red", or "mixed" mantles. These structures are not identical with the "blue borders" ("blaue S~iume") which sometimes were interpreted as mantle bone (see Lempert and Wolff, 1945).
Results In the surroundings of the semicircular canals in the endochondral layer of healthy, non-otosclerotic labyrinthine bone in dog and monkey vascular mantles are to be found. In eosine stained sections one can easily visualize these dark blue colored structures whereas in hematoxylin eosin stained sections they appear dark blue or grey blue (see Nager und Meyer, 1931). Using polarized light or the m o n o c h r o m a t o r one can see that they exist of woven bone which is short-fibered and which sometimes contains lamellar fragments lying often parallel to the circumference of the vessel. Often the mantle matrix is demarcated from the skein bone matrix by a tiny cement line. Stained with alcian blue the mantles appear dark blue which is significant for the presence of acid mucopolysaccharides. The vascular mantles are arranged asymmetrically around the vessels. Sometimes two or even more vessels can be surrounded by only one mantle. It is possible that only one side or only one part of one side of the vessel wall m a y be covered with mantle matrix. The three-dimensional reconstruction of the vascular mantles using serial paraffine sections gives evidence that these mantles cover only parts of a vessel. The same vessel can have mantle parts and portions which are "only" surrounded by skein bone. Inside the mantles' matrix of dog and m o n k e y one observes a small n u m b e r of lacunae which is just the opposite in man. I n the periphery of the mantles by means of light microscopy "osteocytes" are to be found. Regarding their ultrastructure
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Fig. 1. A a) Incompletedevelopingembryonic osteon according to Knese and Titschak (1962). b) c) d) Vascular mantles. Woven bone grey. Skein bone white. Bone lamellaeblack lines. B Three-dimensional reconstruction of vascular mantles (grey) surrounding vessels
different types of cells can be distinguished. In the periphery of the mantle zone one finds mononucleated cells surrounded by a complete boundary. The pericellular space is small, without any formed structures. There are a few processes in the surrounding matrix. On the other hand one realizes mononucleated cells which are surrounded by an irregular inner border of the lacuna, forming insulas and peninsulas of bone matrix. The pericellular space is widened, containing fibrillar materials. The cytoplasm of these cells sometimes appears vacuolated. A third type of cells can be seen. Those are mononucleated smaller cells with a relatively large nucleus lying in a semicircle of dense matrix. This semicircle is open toward the perivascular space. The fourth type are cells which have to be distinguished from the vascular lining cells. They have two or more large nuclei and the cytoplasmic membrane has the characteristics of a ruffled border. The same matrix like in mantle regions can be observed in some skein bone areas surrounding the interglobular spaces. By means of transmission electron microscopy one also finds mononucleated cells in the periphery of these zones with signs of osteocytolysis. It is worth noticing that not the cartilage of interglobular spaces but the skein bone is resorbed. Studying healthy, non-otosclerotic human labyrinthine bones one can affirm the results achieved by the light microscope. One can also observe mantles containing lamellar fragments which are arranged asymmetrically around the vessels in endochondral layer. Multinucleated osteoclasts are not visible. Because of postmortal changes preparation of human temporal bones for a transmission electron microscopic study is not useful. Using the transmission electron microscope a canalization of interglobular spaces in mantle zones which is postulated by Zechner and Altmann (1972) in man
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using paraffine embedded sections is not to be found in monkey and dog though there is a canalization of mantle zones. There is a connection between vessels and canals. It seems to be evident that canalization starts from the vessels. The canals are already to be found in developing vascular mantles and their number increases with the expansion of the mantle zone. They do not contain cell processes from neighboring osteocytes.
Discussion
In this present study healthy, non-otosclerotic labyrinthine bones are examined. Vascular mantles are to be found in dog (Canis f. intermedius Woldrich), monkey (Pithecus fascicularis Raft.), and man. It was pointed out by Weber (1933) that these vascular mantles are a previous stage in otosclerotic bone formation. Using polarized light one can visualize almost identical structures in otosclerotic loci and in vascular mantles (Weber, 1960; Zechner and Altmann, 1972). The findings of NylOn (1949) and Chevance et al. (1969, 1970) indicate that vascular mantles are not a result of pathological reaction but signify "physiological" remodelling in endochondral layer. Typical localizations of otosclerotic foci are to be found in the cochlear region of labyrinthine bone (Sch~itzle and Haubrich, 1975). Vascular mantles prefer the surroundings of the semicircular canals (Zechner and Altmann, 1972). Zechner and Altmann (1972) did not find constant correlations between occurrence of otosclerotic foci and presence of vascular mantles though in labyrinthine bone of individuals suffering from otosclerosis there is a larger number of vascular mantles. Using hematoxylin eosin stained sections the different staining qualities of the mantle zones indicate different stages of remodelling and different age. According to this, Gussen (1968) and Zechner and Altmann (1972) describe depolymerization and repolymerization of mantle matrix which is related to the amount of acid mucopolysaccharides. In Gussen's (1968) opinion mantle bone per se is not an abnormal bone but might perhaps signify extensive degeneration of bone. Some of the vascular mantles contain fragments of lamellar bone. In adult long bones asymmetric osteons can be observed and in developing long bones one can find incomplete osteons being embedded in woven bone (Knese and Titschak, 1962). These incomplete osteons are the result of incomplete resorption of woven bone followed by deposition of lamellar bone. These structures look similar to vascular mantles in endochondral layer. By three dimensional reconstruction of vessels and their vascular mantles one finds that the vessel is only partly covered by mantle matrix. The expansion of the vascular mantle in a defined area might depend on specialization of bone cells in this region which differs from cells in other regions. Such specialization is reported in embryonic and growing bones (Amprino, 1955; Jande, 1971). It depends on sufficient canalization and an O2-optimum in the area supplied (Anderson and Parker, 1966; Bohatirchuk, 1969; Hall, 1970, 1972). A relation between capillary density and persistence of embryonic tissue in endochondral layer was demonstrated by means of morphometry concerning the vessels and the interglobular spaces (Rauchfuss, 1978). Oxygenation via canalization seems to be an important factor for occurrence of vascular mantles for only the canalized
Fig. 2. A Endochondral layer dog. Vascular mantle (m), vessels (1~),skein bone (s). Semithin section. Toluidine blue and pyrortine red. • 580. B Endoehondral tayer dog, Vascular mantle (m), vessels (v), skein bone (s). 8emithin section. Toluidine blue and pyronine red. x 380. C Endochondral layer man. Otosclerotic focus (O), skein botle (s), vessel (v). Hematoxylin eosine. Monochromator. x 380. D Endochondral layer monkey. Mixed mantle (x) contaitting lamellae (arrows), blue mantles (b), vessels (v), skein bone (s). Azan. Monochromator. x 360
matrix of skein bone can be replaced by another type o f woven bone (Zechner, 1971a, b, c). The noncanalized interglobular spaces remain. This corresponds to resorption of cartilage in developing long bones (Wilsman and van Sickle, 1970; Miller et al., 1972). O n the other hand biomechanical factors are important for canalization of the tissue block (Altmann, 1964) as to their influence on maturation of bone from woven
Fig. 3. A Endochondral layer dog. Formation of perivascular mantle bone. Osteoblast (b) lying in a semicircle of newly produced matrix (arrows), vascular lining cells (v), endothelial cells (e), skein bone (s). x 4800. B Endochondral layer dog. Periphery of vascular mantle (m), skein bone matrix (s), osteocyte (o) with complete lacunar wall, osteocyte undergoing osteocytolysis (x), canalization of mantle matrix (arrows), endothelial cells (e). x 4800
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Fig. 4. A Endochondral layer dog. Osteocytie osteolysis. Irregular lacunar border forming insulas and peninsulas of bone matrix (arrows), skein bone (s), mantle bone (m), nucleus (n), vacuolization of dedifferentiated ergastoplasm (x). x 9800. B Endochondral layer dog. Osteocytic osteolysis. Detail of irregular lacunar border. The insulas and peninsulas are parts of unmineralized bone matrix (X) containing collagen fibrils of skein bone (arrows). This indicates that these structures are not artifacts resulting from the fragmented cytoplasmic membrane, x 48000
to lamellar bone (Pauwels, 1965). It is feasible that the lacking of biomechanical stress in endochondral layer which is responsible for constancy of many parameters of labyrinth throughout life (Rauchfuss, 1978) is one reason for asymmetry of vascular mantles. Meyer (1927) pointed out that skein bone is an embryonic type of bone. According to this and according to Knese and Titschak's (1962) classification of osteons one should call the vascular mantles "developing" or "embryonic" osteons. Using the transmission electron microscope the ultrastructure of cells in mantle zones gives evidence that there are mononucleated osteocytes being surrounded by a complete boundary. On the other hand there are cells undergoing osteocytolysis
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which is characterized by an irregular inner border. Their pericellular space contains fibrillar material. Their cytoplasm sometimes appears vacuolated (Jande and Belanger, 1971). Osteocytic osteolysis is an important factor in bone remodelling (Belanger, 1969). Another type of cells is lying in a semicircle of newly produced matrix. These cells have a large nucleus and they are identical with osteoblasts which are found in the periphery of developing osteons (Cooper et al., 1966). The fourth type of cells is a typical osteoclast having two or more large nuclei and a cytoplasmic membrane with the characteristics of a ruffled border (Hancox and Boothroyd, 1961). All these cells seem to originate from vascular mesenchymal cells (Cooper et al., 1966). Considering the embryonic character of the endochondral layer and its structures one can affirm Zechner and Altmann's (1972) opinion that vascular mantles are the result of physiological remodelling. Mantle bone per se is not an abnormal bone (Gussen, 1968). These remodelling processes depend on oxygenation via canalization. It should be mentioned that oxygenation as well osteocytolysis are important factors for enzymatic activities of cells in otosclerotic foci. Considering the findings of Chevance et al. (1970) the similarities between bone changes in young otoscleroric foci reported by these authors and matrix alterations in developing and developed mantle bone might indicate that the beginning of otosclerotic bone formation is governed by the same morphological principles as formation of mantle bone.
References
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