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An electron microscope study on primary periosteal bone
© 1967 by Academic Press Inc.
J. ULTRASTRUCTURE RESEARCH 18, 605--618
(1967)
605
An Electron Microscope Study on Primary Periosteal Bone A. ASCENZI, E. BONUCCI AND D. STEVE BOCCIARELLI1
Institute of Morbid Anatomy, University of Pisa and Istituto Superiore di Sanitgt, Rome, Italy Received June 29, 1966 Our specially devised dissection technique for electron microscope examination of bone units in which the degree of calcification has been previously established was applied to investigate primary nonlamellar periosteal ox bone. The circumferential systems forming this type of bone tissue are made up of collagen bundles running concentrically in a shell-like arrangement. Each bundle is in close contact with the next, but the direction of fibrils changes in successive bundles. Not all collagen fibrils are totally calcified. Along fibrils totally obscured by a large amount of needle-shaped apatite crystallites, there are only a few areas in which calcium deposition involves only a portion 400 Zk wide of the main cross-banding of fibrils. Whereas along the very highly calcified cementing bands interposed between the circumferential systems the organic matrix is composed of collagenous fibrils irregularly oriented in all directions and heavily flanked by a very large amount of apatite crystallites totally covering the collagen. The present results are thoroughly discussed and compared with the findings obtained for other types of bone tissue in order to furnish further data on general problems concerned with ossification. In long ox bones, newly formed periosteal tissue during the first year of postnatal life reveals some peculiarities as regards structural features as well as type and degree of calcification. Cross sections show no lamellar, circular, or circumferential systems coaxial to the external surface of the bone. The systems are in close connection with a three-dimensional vascular tree. The bone tissue is of the type k n o w n as finely bundled bone or shell-like bone, according to Weidenreich's classification (18). Fiber bundles are fine and regularly arranged in almost parallel sheets, but the tissue does not have the evidently stratified appearance it has in lamellar bone. N a r r o w isotropic bands, ranging f r o m 20 to 60 # in thickness, are interposed between the circular systems, and in some respects are similar to large cement lines. Osteocytes are regular in size and shape. M o r e o v e r they are fairly regularly but relatively widely spaced and oriented according to the fiber direction as a whole. The 1 This work was supported by a grant of the National Research Council of Italy.
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processes of cells and canaliculi in which they lie are extremely numerous and have a predominantly radial or transverse orientation with respect to the fiberbundles as a whole, whereas within the isotropic bands osteocytes are large, irregular in shape and size, and have comparatively few processes. Historadiography furnishes evidence that the degree of calcification is uniform, the latest-formed subperiosteal layers absorbing nearly as m u c h X-ray as the inner zones built m u c h earlier. This fact indicates that periosteal bone very quickly reaches a high degree of calcification and then remains relatively constant. Its calcium content is always somewhat higher than that of secondary bone, e.g., Haversian systems (1, 2). W h e n the X-ray absorption of periosteal bone outside isotropic cement bands is c o m p a r e d with absorption within those bands, the latter are f o u n d to be more highly calcified (Fig. 1). These features show that primary periosteal bone is an interesting tissue which is fundamentally different f r o m secondary bone, e.g., Haversian systems. W o r k i n g f r o m such a premise, the features of periosteal bone calcification were investigated using the electron microscope. As a result new data were produced on the general problems of ossification. MATERIAL AND METHOD The technique used is quite similar to those already applied in examining osteons (5, 6). Samples were prepared in which the amount of calcium salts as well as the orientation of anisotropic components had been previously established. Longitudinal and cross sections 2-3 m m thick from ox femoral shafts were fixed in neutral formol and further ground down on a glass plate to a thickness of about 30-40 #. In order to estimate the degree of calcification of the tissue, the sections, dried at room temperature, were microradiographed according to the procedure described by Engstr6m and Wegstedt (11) (see also 9). The X-rays were generated (at 6-12 kV) by a AEG-50 T Machlett tube and filtered through 1 mm Be; current intensity, 15 mA ca. The focus-to-emulsion distance was 8 cm. The microradiographs were made on Eastman Kodak high resolution plates, and the exposed emulsions were developed in Kodak D 19 at 18°C for 5 minutes. After microscopic examination of sections both in ordinary and polarized light and after the collection of microradiographic data, samples corresponding to periosteal bone systems outside and inside the highly calcified bands were chosen with great care. At this stage wedgeshaped bone fragments with a portion of the chosen sample on top were dissected from the section according to the technique described by Ascenzi and Fabry (7) and Ascenzi and Bonucci (3, 4). In this way it was easy to prepare from each periosteal system outside and
FI~. 1. Micrographs illustrating some material and specimens used in this investigation. (a) Microradiogram of a longitudinally sectioned periosteal primary bone. The arrows indicate two highly calcified bands, x 50. (b and c) Two dissected samples having on top a portion of periosteal bone outside the highly calcified bands. Polarizing microscope, x 115. (d and e) Two dissected samples having on top a portion of highly calcified band, as seen under polarizing microscope, x 115. In b e, the arrows indicate the orientation of the ultrathin sections.
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A. ASCENZI, E. BONUCCI AND D. S. BOCCIARELLI
inside the highly calcified bands, two specimens shaped as described in a previous paper (5). The top of the specimens are illustrated in Fig. 1 b-e. They were dehydrated and embedded in Araldite. In the resin blocks, specimens were oriented in such a way as to obtain ultrathin sections whose orientation is indicated by arrows. Obviously, by choosing suitable specimens from longitudinal and cross sections of bone, it was possible to obtain ultrathin sections running longitudinally, transversally, and radially in relation to the circumferential systems. Thus, for instance, ultrathin sections from the sample shown in Fig. 1 b ran tangentially along a circumferential system outside the highly calcified line whereas the ultrathin sections obtained from the specimen illustrated in Fig. 1 c were oriented radially. From the specimens shown in Figs. 1 d and 1 e, having on top a portion of an isotropic and highly calcified band ultrathin sections oriented tangentially and radially were prepared. The bone specimens were sectioned with a Porter-Blum microtome fitted with a diamond knife at an angle of 45-50 degrees. The sections were mounted on Formvar and carboncoated copper grids. The exposure time of the ultrathin sections to water was in no case longer than 3 minutes, so that decalcification according to Boothroyd's suggestion (10) could not occur. A Siemens Elmiskop I was used to examine the sections, using both condensors and welldefined optical conditions to make possible an exact correlation of findings on samples calcified to different degrees. For particular purposes the specimens were decalcified by ethylenediaminetetraacetic acid (EDTA) before embedding. Decalcification was also carried out on ultrathin sections using phosphotungstic acid, which is also a good staining agent for collagen fibrils.
RESULTS The present results deal with calcified bone matrix, which is the only c o m p o n e n t of bone tissue excellently preserved. Bone cells, when treated by the m e t h o d described above, become basically changed, or artifacts appear in them, rendering them unsuitable for further investigation. Moreover, as the ultrathin sections were not artificially stained, the structures observed under the electron microscope correspond to calcium salts, given the relatively high atomic weight of this element. Their distribution is closely related to the collagen matrix so that the m o r p h o l o g y and orientation of fibrils are indirectly revealed by apatite crystallites. The submicroscopic features of periosteal bone outside and inside the isotropic and highly calcified bands will be considered separately. In the course of the present exposition, bone tissue outside the highly calcified bands will be referred to as periosteal bone or periosteal bone tissue. In agreement with light microscopy, periosteal bone tissue consists of rather orderly arranged fiber bundles (Fig. 2). In these bundles, fibrils are usually parallel to
FIG. 2. Periosteal bone tissue consisting of rather orderly arranged fiber bundles. The holes are artifacts produced by the processes of the osteocytes, x 17,000.
2
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each other although fibrils interweaving at small angles can also often be seen. Unlike lamellar bone, the cementing zones resulting from bridging fibrils are lacking. The limits between successive bundles are therefore indicated only by the changing orientation of fibrils. The arrangement resulting from the different directions taken by fiber bundles may vary greatly, and the fibrils in one bundle may make an angle of nearly 90 degrees with the fibrils of the next. At some points fibrils show a cross, or a strongly oblique, orientation and go through from one bundle to the next. Fiber bundles are only a few microns wide. Generally, osteocyte processes run within fiber bundles and have the same orientation as the bundles. In this bone type, calcium salts appear in two different arrangements. The commoner arrangement consists of needle-shaped crystallites of considerable length. Owing to their length they may cover m a n y major collagen periods (Fig. 3). In ultrathin cross sections crystallites penetrate collagen fibrils deeply and are closely packed. They are rod-like and m a y be as much as 40 ~ thick. In the zones where crystallites are not superimposed, interspaces are visible, these probably being occupied by filaments belonging to the organic matrix. The second arrangement of calcium salts found in fiber bundles is a series of parallel-oriented bands which are separated by clear interbands and run exact y perpendicular to the main direction of fibrils. The bands have an average width of about 400/~ and the interbands reach 250/~. Therefore each band with its successive interband covers about 650 ~ , i.e., about the length of a major collagen period. When micrographs are well focused and sections are so thin as to permit high resolution, small spots can be made out in bands 400 ~ wide. They are preferentially distributed along lines parallel to the long axis of the collagen fibrils and frequently fuse in linear or needle-shaped crystallites. The spots appear to measure no more than 10/~ across (Fig. 3). Transitional figures are found intermediate between the spots and crystallites covering the bands 400 A wide and bundles of elongated needle-shaped crystallites. Usually the latter have a very fine structure consisting of small spots closely fused together. To estimate the percentage of bone matrix showing calcification only or preferentially on bands 400 A wide and that of bone matrix totally covered by elongated crystallites, the surface showing these two features were measured separately in 28 micrographs. As the boundaries between the two types of areas were often u n s h a r p - as shown by our previous investigation (5)--the ambiguous zones were considered
FIG. 3. Structure at the level of the periosteal bone tissue. Explanation in text. x 170,000. Fie. 4. Ultrathin section of an isotropic and highly calcified band showing a very irregular orientation of the calcified matrix. × 90,000.
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TABLE I AREAS(%) COVEREDBY ELONGATEDNEEDLE-SHAPEDCRYSTALLITESIN 56 MICROPHOTOGRAPHSFROMBOTHFULLYCALCIFIEDOSTEONSAND PRIMARYPERIOSTEALBONE Fully Calcified Osteons
Primary Periosteal Bone
71.5 87.4 74.7 81.2 83.0 82.3 80.9 88.0 81.8 85.6 85.7 82.8 66.6 82.6 76.1 91.0 70.2 92.0 87.0 89.8 80.1 88.3 78.8 85.4 88.7 93.6 88.8 85.0 Mean 83.2 %
93.2 98.2 93.2 93.5 91.6 95.1 93.1 91.0 89.6 98.2 87.1 88.5 92.0 88.9 88.5 90.6 97.0 97.3 96.9 98.0 93.0 92.3 87.1 97.6 97.4 94.2 88.9 96.4 Mean 93.1%
as being covered by neeele-shaped crystallites only. The results are given in Table I together with those obtained applying the same method to fully calcified ox osteons. They show that in primary periosteal bone the areas covered by elongated needleshaped crystallites are greater than those in fully calcified osteons, the mean values being 93.1% and 83.2%, respectively. Moreover the statistical analysis given by applying the t test for significance, according to Student, shows that the differences between the two mean values were significant, t being 7.184 for P<0.05. The ultrathin sections which were decalcified using phosphotungstic acid, show regularly banded collagen fibrils closely packed in bundles (Fig. 5). In spite of their generally parallel orientation, fibrils frequently interweave at small angles. Usually the major collagen period is clearly reduced, probably as a consequence of the shrinking of ultrathin sections after removal of apatite crystallites. Ultrathin sections from specimens decalcified by E D T A before embedding reveal at some points fibrils having a delicate structure consisting of filaments oriented along the axis and separated by thin transparent interstices (Fig. 6). It appears probable that the interstices correspond not only to spaces previously occupied by crystallites,
FIG. 5. Bone matrix of periosteal bone tissue. The fibrils were decalcified and stained with phosphotungstic acid. x 180,000. FIG. 6. Bone matrix of periosteal bone tissue decalcified by EDTA before embedding. Ultrathin section stained with phosphotungstic acid. Transparent interstices between the longitudinally oriented filaments can be seen. x 170,000.
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but also to technical artifacts, EDTA performing extraction of material from organic matrix (17). A careful examination of decalcified ultrathin sections made in an attempt to recognize structural differences between fibrils previously calcified along their whole length and fibrils only incompletely calcified on bands 400/~ wide furnished no conclusive data. The submicroscopic features of isotropic and highly calcified bands are those of a tissue having crystallites irregularly distributed and oriented in all directions and forming at some points whorled figures. The crystallites are long and needle-shaped. Their maximum width is about 40 •. Figures of incomplete calcification in bands are not observable or are very exceptional (Figs. 4 and 7). The absence or scarcity of these figures in which the calcium amount is obviously low, is in agreement with the very high calcium level present in this bone type as revealed by microradiographic techniques. After decalcification bone matrix appears as thin fibrils irregularly oriented in all directions. Their transverse banding is sometimes not clearly appreciable and the elementary filaments are obviously dissociated. Moreover the spaces between the fibrils are rather large. This feature is especially evident when ultrathin sections were obtained from specimens treated by EDTA before embedding (Fig. 8). DISCUSSION First of all the present electron microscope investigation furnishes useful analytic data concerning the differences between primary nonlamellar periosteal bone and lamellar osteonic bone. As shown in our previous study on osteonic ultrastructure (5), lamellae are to be regarded as stratified entities whose individuality depends on two main conditions: orientation of the anisotropic structures found in each lamella; and interposition of a thin interlamellar cementing sheet or zone between successive lamellae. As regards the anisotropic components of each lamella, i.e., collagen fibrils and apatite crystallites, they lie parallel. This is a statistical result, because each bundle of fibrils shows some irregularity as well as a more or less pronounced deviation from the main direction. In successive lamellae, fiber-bundle orientation may change through any angle from 0 to 90 °, stratification being most clearly apparent when the angle is large. Moreover, fiber bundles frequently leave one lamella and pass into the next through discontinuities in the interlamellar cementing zone. The organic matrix in the latter structure looks as though it is composed almost completely of collagenous fibrils, irregularly oriented in every direction and heavily flanked by a very large amount of apatite crystallites. F~G. 7. Different types of orientation and structure at the boundary with an isotropic, highly calcified band (top right corner), x 175,000.
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FIG. 8. Ultrathin section of an isotropic band. Decalcification by E D T A before embedding and staining of section with phosphotungstic acid. Very irregular orientation and dissociation of collagen fibrils, x 240,000.
Primary nonlamellar periosteal bone, on the other hand, consists of stratified fiber bundles running in different directions in neighboring strata, with cementing zones completely lacking. In successive fiber bundles the fibril direction may change through any angle from 0 to 90 °. In each bundle fibrils are parallel oriented, although they also frequently interweave at small angles. The fine structure of calcium deposition is essentially similar in fully calcified osteons and in primary nonlamellar periosteal bone. In both bone types a large percentage of the matrix is deeply penetrated by elongated needle-shaped crystallites totally covering the main periodic banding of collagen, while the remaining matrix fixes calcium only or preferentially along bands 400 A wide. However, in primary periosteal bone this incompletely calcified collagen fraction is found in rather smaller quantities than in fully calcified osteons. This finding is in agreement with the results of microradiographic analysis which showed (1) that the calcium content of primary bone is higher than that of Haversian systems having reached maximum calcification.
ELECTRON MICROSCOPE STUDY ON PRIMARY PERIOSTEAL BONE
617
On the other hand, primary periosteal bone and osteons fix apatite in different ways. According to Amprino and Engstr6m (2), in the former type of bone tissue calcium is laid down quickly and ossification is accomplished in one stage only; osteons, however, calcify as soon as they are laid down, but their calcium content increases slowly and progressively afterward. In this connection our previous electron miscroscope investigations have demonstrated that after a sudden initial penetration of a large percentage of the matrix by elongated needle-shaped crystallites, apatite is laid down, starting with calcified bands measuring 400 ~ in width, at the major collagen period. Here small spots corresponding to loci of crystal inception are distributed along the axis of the collagen and fuse in needle-shaped crystallites. Later the crystallites become long enough to span all the major periods, completely obscuring the fibril structure. Of course as osteon calcification increases, the areas covered by needle-shaped crystallites get larger. We are not in a position to give an explanation of the difference in rates of matrix calcification between osteons and primary periosteal bone. All that it is possible to establish is that in primary periosteal bone with a very rapid rate of ossification the completely calcified collagen fraction is found in rather higher quantities than in fully calcified osteons in which calcium is laid down slowly in two stages. At the moment the true significance of the difference between fibrils penetrated by crystallites totally covering the main periodic banding and fibrils fixing calcium only or preferentially along bands 400 ~ wide is also a matter for speculation. As regards the latter, Hodge's theory (14, 15) on the manner of alignment of tropocollagen macromolecules into "quarter-stagger" fibrous arrays indicates that "holes" or '"pores" about 400 A in length occur at precise points on macromolecular aggregation and the position of these "holes" coincides with localization of crystallites (see also 12). On the other hand, the recent investigations carried out by Grant, Horne, and Cox (13, 16) in order to explain the 640 ~ periodicity of native collagen indicates that each unit contains a light band of length about 265 N, seen as a bonding zone, and a dark band of length about 375 ~, seen as a nonbonding zone. According to this view, one could suggest that penetration of fibrils by apatite crystallites is possible only (or principally) in nonbonding zones. Another point to be discussed is the problem concerning the significance of the high degree of calcification along isotropic bands pertaining to primary periosteal bone. Here the organic matrix looks as though it is composed of collagenous fibrils, irregularly oriented in all directions, and spaces between fibrils are rather large. Moreover, fibrils are heavily flanked by a very large amount of apatite crystallites totally covering the collagen. These findings explain why, in spite of the structural anisotropy of its components (collagen fibrils and crystallites), highly calcified bands show an apparent isotropy under the polarizing microscope due to the random often-
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tation of these components themselves. Furthermore, the deep penetration of the matrix by a large amount of apatite crystallites and the absence of fibrils partially calcified in bands, together furnish a satisfactory explanation for the higher calcium content found along isotropic bands than in other periosteal bone tissue. S u c h a thorough penetration of matrix by crystallites could depend on the p o o r state of aggregation of collagen, as suggested by figures of dissociated fibrils and by the existence of rather large spaces between fibrils in ultrathin sections obtained from specimens treated by E D T A before embedding. Indeed the latter feature supports the view that poorly aggregated material is removed by E D T A along with apatite crystals (17). There is agreement between these results and those concerning the fine structure and biochemical behavior of bone induced by estrogens in birds, such bone becoming very highly calcified (8). The authors gratefully acknowledge the excellent technical assistance of G. Arancia, F. Castellano, P. Crateri, and F. Piccirilli. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
AMPRINO, R., Z. Zellforsch. 37, 144 (1952). AMPRINO,R. and ENGSTROM,A., Acta Anat. 15, 1 (1952). ASCENZI,A. and BONUCCI, E., Acta Anat. 58, 160 (1964). ASCENZI, A., BONUCCI,E. and CHECCUCC~,A., in EVANS, F. G. (Ed.), Studies on the Anatomy and Function of Bone and Joints, p. 121. Springer, New York. 1966. ASCENZI,A., BONUCCI,E. and STEVEBOCCIARELLI,D., J. Ultrastruct. Res. 12, 287 (1965). -Etude au microscope 61ectronique de la matrice ossifiable; Syrup. sur l'ost~omalaeie, Artigny, 1965. Masson, Paris, in press. ASCENZI,A. and FABRY, C., J. Biophys. Biochem. Cytol. 6, 139 (1959). ASCENZI,A., FRAN(~OIS,C. and STEVEBOCCIARELEI,D., J. Ultrastruct. Res. 8, 491 (1963). ASCENZI,A., STEVEBOCCIARELLI,D. and MARINOZZI,V., Rend. Ist. Super. Sanit& 21, 843 (1958). BOOTHROYD,B., J. Cell Biol. 20, 165 (1964). ENGSTR6M,A. and WEGSTEDT,L., Acta Radiol. 35, 345 (1951). FITTONJACKSON,S., in BRACKET,J. and MmsKY, A. E. (Eds.), The Cell, Vol. VI, p. 387. Academic Press, New York, 1964. GRANT, R. A., HORNE, R. W. and Cox, R. W., Nature 207, 822 (1965). HOD~E, A. J. and SCHMITT,F. O., Proc. Natl. Acad. Sci. U.S. 46, 186 (1960). HOD~E, A. J. and PETRUSKA,J. A., in RAMACHANDRAN,G. N. (Ed.), Aspects of Protein Structure, p. 289. Academic Press, New York, 1963. HORN~, R. W., Lab. Invest. 14, 1054 (1965). VmHAN~, F. and MAROTTr, F., Clin. Ortop. 15, 521 (1963). WEIDENREICH, F., in VON MOLLENDORFF, W. (Ed.), Handbuch der mikroskopischen Anatomie des Menschen, Vol. II, Part 2, p. 416. Springer, Berlin, 1931.
J. ULTRASTRUCTURE RESEARCH 18, 605--618
(1967)
605
An Electron Microscope Study on Primary Periosteal Bone A. ASCENZI, E. BONUCCI AND D. STEVE BOCCIARELLI1
Institute of Morbid Anatomy, University of Pisa and Istituto Superiore di Sanitgt, Rome, Italy Received June 29, 1966 Our specially devised dissection technique for electron microscope examination of bone units in which the degree of calcification has been previously established was applied to investigate primary nonlamellar periosteal ox bone. The circumferential systems forming this type of bone tissue are made up of collagen bundles running concentrically in a shell-like arrangement. Each bundle is in close contact with the next, but the direction of fibrils changes in successive bundles. Not all collagen fibrils are totally calcified. Along fibrils totally obscured by a large amount of needle-shaped apatite crystallites, there are only a few areas in which calcium deposition involves only a portion 400 Zk wide of the main cross-banding of fibrils. Whereas along the very highly calcified cementing bands interposed between the circumferential systems the organic matrix is composed of collagenous fibrils irregularly oriented in all directions and heavily flanked by a very large amount of apatite crystallites totally covering the collagen. The present results are thoroughly discussed and compared with the findings obtained for other types of bone tissue in order to furnish further data on general problems concerned with ossification. In long ox bones, newly formed periosteal tissue during the first year of postnatal life reveals some peculiarities as regards structural features as well as type and degree of calcification. Cross sections show no lamellar, circular, or circumferential systems coaxial to the external surface of the bone. The systems are in close connection with a three-dimensional vascular tree. The bone tissue is of the type k n o w n as finely bundled bone or shell-like bone, according to Weidenreich's classification (18). Fiber bundles are fine and regularly arranged in almost parallel sheets, but the tissue does not have the evidently stratified appearance it has in lamellar bone. N a r r o w isotropic bands, ranging f r o m 20 to 60 # in thickness, are interposed between the circular systems, and in some respects are similar to large cement lines. Osteocytes are regular in size and shape. M o r e o v e r they are fairly regularly but relatively widely spaced and oriented according to the fiber direction as a whole. The 1 This work was supported by a grant of the National Research Council of Italy.
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A. ASCENZI, E. BONUCCI AND D. S. BOCCIARELLI
processes of cells and canaliculi in which they lie are extremely numerous and have a predominantly radial or transverse orientation with respect to the fiberbundles as a whole, whereas within the isotropic bands osteocytes are large, irregular in shape and size, and have comparatively few processes. Historadiography furnishes evidence that the degree of calcification is uniform, the latest-formed subperiosteal layers absorbing nearly as m u c h X-ray as the inner zones built m u c h earlier. This fact indicates that periosteal bone very quickly reaches a high degree of calcification and then remains relatively constant. Its calcium content is always somewhat higher than that of secondary bone, e.g., Haversian systems (1, 2). W h e n the X-ray absorption of periosteal bone outside isotropic cement bands is c o m p a r e d with absorption within those bands, the latter are f o u n d to be more highly calcified (Fig. 1). These features show that primary periosteal bone is an interesting tissue which is fundamentally different f r o m secondary bone, e.g., Haversian systems. W o r k i n g f r o m such a premise, the features of periosteal bone calcification were investigated using the electron microscope. As a result new data were produced on the general problems of ossification. MATERIAL AND METHOD The technique used is quite similar to those already applied in examining osteons (5, 6). Samples were prepared in which the amount of calcium salts as well as the orientation of anisotropic components had been previously established. Longitudinal and cross sections 2-3 m m thick from ox femoral shafts were fixed in neutral formol and further ground down on a glass plate to a thickness of about 30-40 #. In order to estimate the degree of calcification of the tissue, the sections, dried at room temperature, were microradiographed according to the procedure described by Engstr6m and Wegstedt (11) (see also 9). The X-rays were generated (at 6-12 kV) by a AEG-50 T Machlett tube and filtered through 1 mm Be; current intensity, 15 mA ca. The focus-to-emulsion distance was 8 cm. The microradiographs were made on Eastman Kodak high resolution plates, and the exposed emulsions were developed in Kodak D 19 at 18°C for 5 minutes. After microscopic examination of sections both in ordinary and polarized light and after the collection of microradiographic data, samples corresponding to periosteal bone systems outside and inside the highly calcified bands were chosen with great care. At this stage wedgeshaped bone fragments with a portion of the chosen sample on top were dissected from the section according to the technique described by Ascenzi and Fabry (7) and Ascenzi and Bonucci (3, 4). In this way it was easy to prepare from each periosteal system outside and
FI~. 1. Micrographs illustrating some material and specimens used in this investigation. (a) Microradiogram of a longitudinally sectioned periosteal primary bone. The arrows indicate two highly calcified bands, x 50. (b and c) Two dissected samples having on top a portion of periosteal bone outside the highly calcified bands. Polarizing microscope, x 115. (d and e) Two dissected samples having on top a portion of highly calcified band, as seen under polarizing microscope, x 115. In b e, the arrows indicate the orientation of the ultrathin sections.
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A. ASCENZI, E. BONUCCI AND D. S. BOCCIARELLI
inside the highly calcified bands, two specimens shaped as described in a previous paper (5). The top of the specimens are illustrated in Fig. 1 b-e. They were dehydrated and embedded in Araldite. In the resin blocks, specimens were oriented in such a way as to obtain ultrathin sections whose orientation is indicated by arrows. Obviously, by choosing suitable specimens from longitudinal and cross sections of bone, it was possible to obtain ultrathin sections running longitudinally, transversally, and radially in relation to the circumferential systems. Thus, for instance, ultrathin sections from the sample shown in Fig. 1 b ran tangentially along a circumferential system outside the highly calcified line whereas the ultrathin sections obtained from the specimen illustrated in Fig. 1 c were oriented radially. From the specimens shown in Figs. 1 d and 1 e, having on top a portion of an isotropic and highly calcified band ultrathin sections oriented tangentially and radially were prepared. The bone specimens were sectioned with a Porter-Blum microtome fitted with a diamond knife at an angle of 45-50 degrees. The sections were mounted on Formvar and carboncoated copper grids. The exposure time of the ultrathin sections to water was in no case longer than 3 minutes, so that decalcification according to Boothroyd's suggestion (10) could not occur. A Siemens Elmiskop I was used to examine the sections, using both condensors and welldefined optical conditions to make possible an exact correlation of findings on samples calcified to different degrees. For particular purposes the specimens were decalcified by ethylenediaminetetraacetic acid (EDTA) before embedding. Decalcification was also carried out on ultrathin sections using phosphotungstic acid, which is also a good staining agent for collagen fibrils.
RESULTS The present results deal with calcified bone matrix, which is the only c o m p o n e n t of bone tissue excellently preserved. Bone cells, when treated by the m e t h o d described above, become basically changed, or artifacts appear in them, rendering them unsuitable for further investigation. Moreover, as the ultrathin sections were not artificially stained, the structures observed under the electron microscope correspond to calcium salts, given the relatively high atomic weight of this element. Their distribution is closely related to the collagen matrix so that the m o r p h o l o g y and orientation of fibrils are indirectly revealed by apatite crystallites. The submicroscopic features of periosteal bone outside and inside the isotropic and highly calcified bands will be considered separately. In the course of the present exposition, bone tissue outside the highly calcified bands will be referred to as periosteal bone or periosteal bone tissue. In agreement with light microscopy, periosteal bone tissue consists of rather orderly arranged fiber bundles (Fig. 2). In these bundles, fibrils are usually parallel to
FIG. 2. Periosteal bone tissue consisting of rather orderly arranged fiber bundles. The holes are artifacts produced by the processes of the osteocytes, x 17,000.
2
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each other although fibrils interweaving at small angles can also often be seen. Unlike lamellar bone, the cementing zones resulting from bridging fibrils are lacking. The limits between successive bundles are therefore indicated only by the changing orientation of fibrils. The arrangement resulting from the different directions taken by fiber bundles may vary greatly, and the fibrils in one bundle may make an angle of nearly 90 degrees with the fibrils of the next. At some points fibrils show a cross, or a strongly oblique, orientation and go through from one bundle to the next. Fiber bundles are only a few microns wide. Generally, osteocyte processes run within fiber bundles and have the same orientation as the bundles. In this bone type, calcium salts appear in two different arrangements. The commoner arrangement consists of needle-shaped crystallites of considerable length. Owing to their length they may cover m a n y major collagen periods (Fig. 3). In ultrathin cross sections crystallites penetrate collagen fibrils deeply and are closely packed. They are rod-like and m a y be as much as 40 ~ thick. In the zones where crystallites are not superimposed, interspaces are visible, these probably being occupied by filaments belonging to the organic matrix. The second arrangement of calcium salts found in fiber bundles is a series of parallel-oriented bands which are separated by clear interbands and run exact y perpendicular to the main direction of fibrils. The bands have an average width of about 400/~ and the interbands reach 250/~. Therefore each band with its successive interband covers about 650 ~ , i.e., about the length of a major collagen period. When micrographs are well focused and sections are so thin as to permit high resolution, small spots can be made out in bands 400 ~ wide. They are preferentially distributed along lines parallel to the long axis of the collagen fibrils and frequently fuse in linear or needle-shaped crystallites. The spots appear to measure no more than 10/~ across (Fig. 3). Transitional figures are found intermediate between the spots and crystallites covering the bands 400 A wide and bundles of elongated needle-shaped crystallites. Usually the latter have a very fine structure consisting of small spots closely fused together. To estimate the percentage of bone matrix showing calcification only or preferentially on bands 400 A wide and that of bone matrix totally covered by elongated crystallites, the surface showing these two features were measured separately in 28 micrographs. As the boundaries between the two types of areas were often u n s h a r p - as shown by our previous investigation (5)--the ambiguous zones were considered
FIG. 3. Structure at the level of the periosteal bone tissue. Explanation in text. x 170,000. Fie. 4. Ultrathin section of an isotropic and highly calcified band showing a very irregular orientation of the calcified matrix. × 90,000.
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TABLE I AREAS(%) COVEREDBY ELONGATEDNEEDLE-SHAPEDCRYSTALLITESIN 56 MICROPHOTOGRAPHSFROMBOTHFULLYCALCIFIEDOSTEONSAND PRIMARYPERIOSTEALBONE Fully Calcified Osteons
Primary Periosteal Bone
71.5 87.4 74.7 81.2 83.0 82.3 80.9 88.0 81.8 85.6 85.7 82.8 66.6 82.6 76.1 91.0 70.2 92.0 87.0 89.8 80.1 88.3 78.8 85.4 88.7 93.6 88.8 85.0 Mean 83.2 %
93.2 98.2 93.2 93.5 91.6 95.1 93.1 91.0 89.6 98.2 87.1 88.5 92.0 88.9 88.5 90.6 97.0 97.3 96.9 98.0 93.0 92.3 87.1 97.6 97.4 94.2 88.9 96.4 Mean 93.1%
as being covered by neeele-shaped crystallites only. The results are given in Table I together with those obtained applying the same method to fully calcified ox osteons. They show that in primary periosteal bone the areas covered by elongated needleshaped crystallites are greater than those in fully calcified osteons, the mean values being 93.1% and 83.2%, respectively. Moreover the statistical analysis given by applying the t test for significance, according to Student, shows that the differences between the two mean values were significant, t being 7.184 for P<0.05. The ultrathin sections which were decalcified using phosphotungstic acid, show regularly banded collagen fibrils closely packed in bundles (Fig. 5). In spite of their generally parallel orientation, fibrils frequently interweave at small angles. Usually the major collagen period is clearly reduced, probably as a consequence of the shrinking of ultrathin sections after removal of apatite crystallites. Ultrathin sections from specimens decalcified by E D T A before embedding reveal at some points fibrils having a delicate structure consisting of filaments oriented along the axis and separated by thin transparent interstices (Fig. 6). It appears probable that the interstices correspond not only to spaces previously occupied by crystallites,
FIG. 5. Bone matrix of periosteal bone tissue. The fibrils were decalcified and stained with phosphotungstic acid. x 180,000. FIG. 6. Bone matrix of periosteal bone tissue decalcified by EDTA before embedding. Ultrathin section stained with phosphotungstic acid. Transparent interstices between the longitudinally oriented filaments can be seen. x 170,000.
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but also to technical artifacts, EDTA performing extraction of material from organic matrix (17). A careful examination of decalcified ultrathin sections made in an attempt to recognize structural differences between fibrils previously calcified along their whole length and fibrils only incompletely calcified on bands 400/~ wide furnished no conclusive data. The submicroscopic features of isotropic and highly calcified bands are those of a tissue having crystallites irregularly distributed and oriented in all directions and forming at some points whorled figures. The crystallites are long and needle-shaped. Their maximum width is about 40 •. Figures of incomplete calcification in bands are not observable or are very exceptional (Figs. 4 and 7). The absence or scarcity of these figures in which the calcium amount is obviously low, is in agreement with the very high calcium level present in this bone type as revealed by microradiographic techniques. After decalcification bone matrix appears as thin fibrils irregularly oriented in all directions. Their transverse banding is sometimes not clearly appreciable and the elementary filaments are obviously dissociated. Moreover the spaces between the fibrils are rather large. This feature is especially evident when ultrathin sections were obtained from specimens treated by EDTA before embedding (Fig. 8). DISCUSSION First of all the present electron microscope investigation furnishes useful analytic data concerning the differences between primary nonlamellar periosteal bone and lamellar osteonic bone. As shown in our previous study on osteonic ultrastructure (5), lamellae are to be regarded as stratified entities whose individuality depends on two main conditions: orientation of the anisotropic structures found in each lamella; and interposition of a thin interlamellar cementing sheet or zone between successive lamellae. As regards the anisotropic components of each lamella, i.e., collagen fibrils and apatite crystallites, they lie parallel. This is a statistical result, because each bundle of fibrils shows some irregularity as well as a more or less pronounced deviation from the main direction. In successive lamellae, fiber-bundle orientation may change through any angle from 0 to 90 °, stratification being most clearly apparent when the angle is large. Moreover, fiber bundles frequently leave one lamella and pass into the next through discontinuities in the interlamellar cementing zone. The organic matrix in the latter structure looks as though it is composed almost completely of collagenous fibrils, irregularly oriented in every direction and heavily flanked by a very large amount of apatite crystallites. F~G. 7. Different types of orientation and structure at the boundary with an isotropic, highly calcified band (top right corner), x 175,000.
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FIG. 8. Ultrathin section of an isotropic band. Decalcification by E D T A before embedding and staining of section with phosphotungstic acid. Very irregular orientation and dissociation of collagen fibrils, x 240,000.
Primary nonlamellar periosteal bone, on the other hand, consists of stratified fiber bundles running in different directions in neighboring strata, with cementing zones completely lacking. In successive fiber bundles the fibril direction may change through any angle from 0 to 90 °. In each bundle fibrils are parallel oriented, although they also frequently interweave at small angles. The fine structure of calcium deposition is essentially similar in fully calcified osteons and in primary nonlamellar periosteal bone. In both bone types a large percentage of the matrix is deeply penetrated by elongated needle-shaped crystallites totally covering the main periodic banding of collagen, while the remaining matrix fixes calcium only or preferentially along bands 400 A wide. However, in primary periosteal bone this incompletely calcified collagen fraction is found in rather smaller quantities than in fully calcified osteons. This finding is in agreement with the results of microradiographic analysis which showed (1) that the calcium content of primary bone is higher than that of Haversian systems having reached maximum calcification.
ELECTRON MICROSCOPE STUDY ON PRIMARY PERIOSTEAL BONE
617
On the other hand, primary periosteal bone and osteons fix apatite in different ways. According to Amprino and Engstr6m (2), in the former type of bone tissue calcium is laid down quickly and ossification is accomplished in one stage only; osteons, however, calcify as soon as they are laid down, but their calcium content increases slowly and progressively afterward. In this connection our previous electron miscroscope investigations have demonstrated that after a sudden initial penetration of a large percentage of the matrix by elongated needle-shaped crystallites, apatite is laid down, starting with calcified bands measuring 400 ~ in width, at the major collagen period. Here small spots corresponding to loci of crystal inception are distributed along the axis of the collagen and fuse in needle-shaped crystallites. Later the crystallites become long enough to span all the major periods, completely obscuring the fibril structure. Of course as osteon calcification increases, the areas covered by needle-shaped crystallites get larger. We are not in a position to give an explanation of the difference in rates of matrix calcification between osteons and primary periosteal bone. All that it is possible to establish is that in primary periosteal bone with a very rapid rate of ossification the completely calcified collagen fraction is found in rather higher quantities than in fully calcified osteons in which calcium is laid down slowly in two stages. At the moment the true significance of the difference between fibrils penetrated by crystallites totally covering the main periodic banding and fibrils fixing calcium only or preferentially along bands 400 ~ wide is also a matter for speculation. As regards the latter, Hodge's theory (14, 15) on the manner of alignment of tropocollagen macromolecules into "quarter-stagger" fibrous arrays indicates that "holes" or '"pores" about 400 A in length occur at precise points on macromolecular aggregation and the position of these "holes" coincides with localization of crystallites (see also 12). On the other hand, the recent investigations carried out by Grant, Horne, and Cox (13, 16) in order to explain the 640 ~ periodicity of native collagen indicates that each unit contains a light band of length about 265 N, seen as a bonding zone, and a dark band of length about 375 ~, seen as a nonbonding zone. According to this view, one could suggest that penetration of fibrils by apatite crystallites is possible only (or principally) in nonbonding zones. Another point to be discussed is the problem concerning the significance of the high degree of calcification along isotropic bands pertaining to primary periosteal bone. Here the organic matrix looks as though it is composed of collagenous fibrils, irregularly oriented in all directions, and spaces between fibrils are rather large. Moreover, fibrils are heavily flanked by a very large amount of apatite crystallites totally covering the collagen. These findings explain why, in spite of the structural anisotropy of its components (collagen fibrils and crystallites), highly calcified bands show an apparent isotropy under the polarizing microscope due to the random often-
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tation of these components themselves. Furthermore, the deep penetration of the matrix by a large amount of apatite crystallites and the absence of fibrils partially calcified in bands, together furnish a satisfactory explanation for the higher calcium content found along isotropic bands than in other periosteal bone tissue. S u c h a thorough penetration of matrix by crystallites could depend on the p o o r state of aggregation of collagen, as suggested by figures of dissociated fibrils and by the existence of rather large spaces between fibrils in ultrathin sections obtained from specimens treated by E D T A before embedding. Indeed the latter feature supports the view that poorly aggregated material is removed by E D T A along with apatite crystals (17). There is agreement between these results and those concerning the fine structure and biochemical behavior of bone induced by estrogens in birds, such bone becoming very highly calcified (8). The authors gratefully acknowledge the excellent technical assistance of G. Arancia, F. Castellano, P. Crateri, and F. Piccirilli. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
AMPRINO, R., Z. Zellforsch. 37, 144 (1952). AMPRINO,R. and ENGSTROM,A., Acta Anat. 15, 1 (1952). ASCENZI,A. and BONUCCI, E., Acta Anat. 58, 160 (1964). ASCENZI, A., BONUCCI,E. and CHECCUCC~,A., in EVANS, F. G. (Ed.), Studies on the Anatomy and Function of Bone and Joints, p. 121. Springer, New York. 1966. ASCENZI,A., BONUCCI,E. and STEVEBOCCIARELLI,D., J. Ultrastruct. Res. 12, 287 (1965). -Etude au microscope 61ectronique de la matrice ossifiable; Syrup. sur l'ost~omalaeie, Artigny, 1965. Masson, Paris, in press. ASCENZI,A. and FABRY, C., J. Biophys. Biochem. Cytol. 6, 139 (1959). ASCENZI,A., FRAN(~OIS,C. and STEVEBOCCIARELEI,D., J. Ultrastruct. Res. 8, 491 (1963). ASCENZI,A., STEVEBOCCIARELLI,D. and MARINOZZI,V., Rend. Ist. Super. Sanit& 21, 843 (1958). BOOTHROYD,B., J. Cell Biol. 20, 165 (1964). ENGSTR6M,A. and WEGSTEDT,L., Acta Radiol. 35, 345 (1951). FITTONJACKSON,S., in BRACKET,J. and MmsKY, A. E. (Eds.), The Cell, Vol. VI, p. 387. Academic Press, New York, 1964. GRANT, R. A., HORNE, R. W. and Cox, R. W., Nature 207, 822 (1965). HOD~E, A. J. and SCHMITT,F. O., Proc. Natl. Acad. Sci. U.S. 46, 186 (1960). HOD~E, A. J. and PETRUSKA,J. A., in RAMACHANDRAN,G. N. (Ed.), Aspects of Protein Structure, p. 289. Academic Press, New York, 1963. HORN~, R. W., Lab. Invest. 14, 1054 (1965). VmHAN~, F. and MAROTTr, F., Clin. Ortop. 15, 521 (1963). WEIDENREICH, F., in VON MOLLENDORFF, W. (Ed.), Handbuch der mikroskopischen Anatomie des Menschen, Vol. II, Part 2, p. 416. Springer, Berlin, 1931.