Anat Embryol (2002) 206:21–29 DOI 10.1007/s00429-002-0265-6
O R I G I N A L A RT I C L E
Marzia Ferretti · Carla Palumbo · Miranda Contri Gastone Marotti
Static and dynamic osteogenesis: two different types of bone formation Accepted: 27 May 2002 / Published online: 25 September 2002 © Springer-Verlag 2002
Abstract The onset and development of intramembranous ossification centers in the cranial vault and around the shaft of long bones in five newborn rabbits and six chick embryos were studied by light (LM) and transmission electron microscopy (TEM). Two subsequent different types of bone formation were observed. We respectively named them static and dynamic osteogenesis, because the former is characterized by pluristratified cords of unexpectedly stationary osteoblasts, which differentiate at a fairly constant distance (28±0.4 µm) from the blood capillaries, and the latter by the well-known typical monostratified laminae of movable osteoblasts. No significant structural and ultrastructural differences were found between stationary and movable osteoblasts, all being polarized secretory cells joined by gap junctions. However, unlike in typical movable osteoblastic laminae, stationary osteoblasts inside the cords are irregularly arranged, variously polarized and transform into osteocytes, clustered within confluent lacunae, in the same place where they differentiate. Static osteogenesis is devoted to the building of the first trabecular bony framework having, with respect to the subsequent bone apposition by typical movable osteoblasts, the same supporting function as calcified trabeculae in endochondral ossification. In conclusion, it appears that while static osteogenesis increases the bone external size, dynamic osteogenesis is mainly involved in bone compaction, i.e., in filling primary haversian spaces with primary osteons.
M. Ferretti · C. Palumbo · G. Marotti (✉) Dipartimento di Anatomia e Istologia, Sezione di Anatomia Umana, Università di Modena e Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy e-mail:
[email protected] Tel.: +39-59-4224800, Fax: +39-59-4224861 M. Contri Dipartimento di Scienze Biomediche, Sezione di Patologia Generale, Università di Modena e Reggio Emilia, Modena, Italy
Keywords Intramembranous ossification · Static osteogenesis · Dynamic osteogenesis · Osteoblasts · Osteocytes · Gap junctions
Introduction According to the classical view, bone matrix deposition depends on the secretory activity of monostratified osteoblastic laminae, whose elements are synchronized by side-to-side gap junctions (Jeansonne et al. 1979; Palumbo et al. 1990a, 1990b) and are all polarized towards the same direction, i.e., the osteogenic surface. It is also generally admitted that, as osteoid seam secretion proceeds, the osteoblastic laminae move away from the osteogenic surface, and the osteoblasts selected to transform into osteocytes remain entrapped within the preosseus matrix by widening their secretory territory (Marotti et al. 1992). In fact, clear evidence has been provided that the rate of osteoblast movement is a function of the ratio V/ST, where V refers to the protoplasmic volume and ST to the secretory territory of the osteoblast. In other words, the osteoid appositional growth rate of each osteoblast depends on the amount of its protoplasm engaged on a given bone surface (Marotti 1976). In a preliminary report (Marotti et al. 1999), we referred to this type of bone formation, which involves osteoblast movement, as dynamic osteogenesis to distinguish it from a disregarded type of bone deposition occurring at the onset of intramembranous ossification. This we named static osteogenesis, because it is performed by immovable stationary osteoblasts that transform into osteocytes at the same site where they differentiate. In the present paper, an exhaustive structural and ultrastructural documentation of this new process will be provided. Moreover, the two types of osteogenesis will be discuss in relation to their time of appearance and functional meaning.
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Materials and methods The present structural and ultrastructural study was carried out on the intramembranous perichondral center of ossification surrounding the mid-shaft level of various long bones, particularly the tibiae, of five newborn rabbits and six White Leghorn chick embryos aged 8–16 days (stages 34–42 according to Hamburger and Hamilton 1951). Intramembranous ossification centers in bones of the cranial vault of the same animals were also investigated. All specimens were fixed for 2 h with 4% paraformaldehyde in 0.13 M phosphate buffer pH 7.4, postfixed for 1 h with 1% osmium tetroxide in 0.13 M phosphate buffer pH 7.4, dehydrated in graded ethanol and embedded in epoxy resin (Durcupan ACM), and sectioned with a diamond knife mounted in an Ultracut-Reichert Microtome. The perichondral centers of ossification were cross-sectioned perpendicular to the longitudinal axis of the shaft; the ossification centers of the cranial vault were tangentially-sectioned with respect to the bone external surface. Thin sections (1 µm) were stained with toluidine blue and examined by Axiophot-Zeiss light microscope (LM). Ultrathin sections (70–80 nm) were mounted on Formvar- and carbon-coated copper grids, stained with 1% uranyl acetate and lead citrate and examined by a Zeiss EM109 transmission electron microscope (TEM). The distance between the vessels and the core of the bony trabeculae, i.e., the sites where the first stationary osteoblasts differentiate, was measured by means of a Videoplan-Zeiss image analyzer in the perichondral centers of ossification of the tibiae in two chick embryos and two newborn rabbits.
0.01% sodium azide (NaN3) and 1% Tween-20 for 30 min. The grids were than incubated on droplets of primary antibodies diluted 1:10 in PBS, pH 7.4, 1% BSA, 2.5% NaCl, 0.01% NaN3 and 1% Tween-20 overnight at 4°C. After extensive washing in 0.05 M Tris buffer, pH 7.4, and in 0.05 M Tris buffer, pH 7.4 containing 0.2% BSA, the grids were incubated on droplets of 0.05 M Tris buffer pH 8.4 containing 1% BSA for 15 min and then transferred to droplets of gold-labeled (10 nm) goat antirabbit IgG diluted 1:25 with 0.05 M Tris buffer, pH 8.4 containing 1% BSA, for 120 min at room temperature. Finally, the grids were rinsed with PBS, pH 7.4, and microfiltered bidistilled water and air dried. The specificity of the reaction was tested by omitting the primary antibody. Sections were stained with 1% uranyl acetate and lead citrate before examinations under a Zeiss EM 109 electron microscope.
Results In all intramembranous ossification centers studied, the mesenchyme, where bone formation is due to start, is a highly cellular and vascularized tissue. In perichondral centers of long bones, the mesenchymal cells are particularly elongated and flattened, with a high nucleus-to-cytoplasm ratio and few cytoplasmic organelles; they are connected by gap junctions and arranged in several con-
Immuno-gold reaction Thin sections (80 nm thick) of cords of stationary osteoblasts, mounted on nickel grids, were processed for immunoelectron microscopy using a rabbit anti-chicken collagen type I polyclonal antibody (Chemicon International). The sections were washed and rinsed on droplets of phosphate-buffered saline (PBS), pH 7.4, for 10 min at room temperature, then transferred to droplets of PBS, pH 7.4, containing 1% bovine serum albumin (BSA), 2.5% NaCl,
Fig. 1 Cross sections at the mid-shaft level of the cartilaginous bud of the tibia in a 7-day-old chick embryo. A LM (×350), B TEM (×17,500). The arrows in A point to vessels scattered in the mesenchymal tissue surrounding the perichondrium (arrowhead). Note in B the flattened aspect of the cells surrounding the outer surface of the perichondrium; the arrow points to a gap junctions
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Fig. 3 Frequency histogram of the distance between the sites of stationary osteoblast differentiation and blood capillaries. Horizontal axis: the distance in µm. Vertical axis: the frequency in absolute value. Data recorded from the perichondral centers of ossification of the tibiae in two chick embryos and two newborn rabbits. (m mean, s.d. standard deviation, s.e. standard error)
Fig. 2 Cross section at the mid-shaft level of the cartilaginous bud of the tibia in a 9-day-old chick embryo. Cords of stationary osteoblasts (arrows) are differentiating around the blood capillaries in the inner layer of the periosteum. Note that the mineralization (black areas) of the preosseous matrix inside the cords has already started. LM micrograph (×350) Fig. 4 LM micrograph (×430) showing cords of stationary osteoblasts in the intramembranous ossification center of the parietal bone in a newborn rabbit. The direction of bone growth is from left to right, as shown by the decrease in the amount of preosseous matrix (asterisks) laid down in between the osteoblasts. The arrow points to a blood capillary
centric layers around the perichondrium of the cartilaginous shaft (Fig. 1). In all centers, the onset of osteogenesis is morphologically recognized by the appearance of variously shaped (cuboidal, polygonal and globous) plump cells that differentiate at about midway between adjacent blood capillaries (Fig. 2). The mean distance between the capillaries and the core of the bony trabeculae, where such plump cells differentiate, was found to be fairly constant (Fig. 3). These cells soon take on the ultrastructure of typical osteoblasts, with a highly developed rough endoplasmic reticulum and a large Golgi apparatus. These osteoblasts never form typical monostratified osteogenic laminae; they are irregularly arranged in cords of 2–3
24 Fig. 5 Cord of stationary osteoblasts, enclosing a trabecular core of preosseous matrix, in the perichondral center of a newborn rabbit tibia. The direction of bone growth is from left to right. A LM (×1,090); B TEM enlargement (×3,500) of the squared area in A
Fig. 6 A, B Cords of stationary osteoblasts whose secretory activity appears to be polarized in different directions (arrows); note newly secreted collagen fibrils (asterisks). C, D High magnifications, taken from thin sections processed for immuno-gold reaction, showing intercellular spaces between stationary osteoblasts filled with type I collagen fibrils labeled by immunoreactivity (black 10 nm particles) for rabbit anti-chicken collagen type I policlonal antibody. TEM micrographs (A, B ×5,800; C ×60,000; D ×100,000)
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layers of cells (Fig. 4 and Fig. 5), and each osteoblast, with respect to the adjacent ones, is connected by gap junctions and appears to be polarized in a different, often opposite, direction, as clearly revealed by the position of their organellar machinery with respect to the nucleus and the presence, along their secretory territory, of newly secreted type I collagen fibrils, as shown by immunogold reaction (Fig. 6).
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Fig. 7 TEM micrographs (A ×10,000; B, C, D ×6,000) of immature osteocytes, differentiating from stationary osteoblasts. They contain an ill-defined nucleus (B and D) and a well-developed or-
ganellar machinery similar to those of the original osteoblasts and radiate very short cytoplasmic processes (arrows), connecting them by means of simple contacts and gap junctions (arrowhead in A)
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Fig. 9 TEM micrograph (×5,800) of the same specimen in Fig. 8, showing in the lower right corner a typical lamina of movable osteoblasts, lining a mineralized bony trabecula laid down by static osteogenesis and containing osteocytes (OC) within confluent lacunae
Fig. 8 Cross section at the mid-shaft level of the tibia in a 16-dayold chick embryo; the direction of bone growth is from left to right. The bony trabeculae laid down by static osteogenesis appear to be carpeted by typical movable osteoblastic laminae. The arrows point to the sites of differentiation of cords of stationary osteoblasts along the periosteal surface. LM micrograph (×430)
Additionally, these osteoblasts appear to be stationary since they directly transform into osteocytes at the same site where they differentiate: they secrete all around their cellular cord a preosseous matrix that soon undergoes mineralization. The osteocytes to which they give origin are irregularly grouped inside confluent lacunae and display a globous cell body with an ill-defined nucleus and a well-developed organellar machinery similar to those of the original osteoblasts; also, they radiate very short cytoplasmic processes, which are connected by means of simple contacts and gap junctions (Fig. 7). The further expansion of the intramembranous ossification centers proceeds following the same sequence of events: (1) differentiation of stationary osteoblasts irregularly arranged in cords around the blood vessels, (2) secretion of preosseous matrix and in situ transformation of immovable osteoblasts into osteocytes, (3) mineralization of preosseous matrix and formation of very thin
(10–15 µm thick) bony trabeculae usually containing no more than 2–3 layers of osteocytes. As these processes of static osteogenesis are in progress at the periphery of the ossification centers, the compaction of the formerly laid down trabecular spaces (the so-called primary haversian spaces) takes place by dynamic osteogenesis. Typical osteogenic laminae, made up of movable osteoblasts all polarized in the same direction, differentiate along the surface of the trabeculae previously laid down by stationary osteoblasts. These movable osteoblastic laminae lay down concentric layers of bone, thus filling the primary haversian spaces with primary haversian systems (or osteons) (Fig. 8 and Fig. 9).
Discussion The structural and ultrastructural observations described in the present paper give, for the first time, the demonstration of the existence of two mechanisms of bone formation, namely, static and dynamic osteogenesis, occurring in sequence during intramembranous ossification. The former process is performed by stationary osteoblasts and allows the formation of a trabecular bony framework, enclosing blood vessels. These appear to be essential for the subsequent bone apposition by typical movable osteoblasts. In fact, these trabeculae have the same supporting function as those made up of
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calcified cartilage in endochondral ossification. It also appears from our findings that static osteogenesis is mainly devoted to the expansion of the ossification center and consequently to increasing bone size, whereas dynamic osteogenesis is mainly involved in bone compaction or, at least, in thickening the primitive trabeculae. No substantial differences were found in the structure and ultrastructure between stationary and movable osteoblasts. Both display an ill-defined euchromatic nucleus and a highly developed organellar machinery, characteristically ordered as in polarized secretory cells. This means that stationary osteoblasts also secrete preosseous matrix from one cellular surface (i.e., secretory territory) only, and not all around them. The differences observed between the two types of osteogenesis concern osteoblast arrangement and polarization: stationary osteoblasts are irregularly arranged in cords of 2–3 layers of cells, and each cell is polarized in a different direction with respect to the adjacent ones. In contrast, movable osteoblasts form monostratified laminae and are all polarized in the same direction. In other words, while movable osteoblastic laminae share the same osteogenic surface, stationary osteoblastic cords have different osteogenic surfaces, thus allowing stationary osteoblasts to be surrounded completely by bone matrix. This means that in static osteogenesis the osteoblasts become osteocytes by a mechanism of “self-burial,” whereas in dynamic osteogenesis the osteoblasts selected to transform into osteocytes are embedded within the bone by the secretory activity of the adjacent movable osteoblasts (Marotti et al. 1992). This fact explains why clusters of osteocytes within “lacunae confluentes” can only form during static osteogenesis. Typically, these osteocytes appear to be immature since they retain many features of the original osteoblasts. This suggests that they are still involved in secreting the bony septa aimed at partitioning the confluent lacunae. The existence of gap junctions among stationary osteoblasts accounts for the simultaneous transformation of groups of them into clusters of osteocytes. Hence, osteoblast activity inside each stationary osteogenic cord appears to be synchronized as seems to occur in movable osteogenic laminae. In the latter, however, synchronization is probably needed to coordinate the rate of osteoblast secretion and movement rather than osteoblast-into-osteocyte differentiation. Since during static osteogenesis bone is laid down without preexistent osteocytes, we believe that the irregular arrangement and polarization of the osteoblasts in stationary osteogenic cords probably depend on the lack of osteocyte guidance. It is interesting to note that, contrary to what was asserted in some early descriptions of intramembranous ossification, mineralization only occurs in the preosseous matrix secreted by stationary osteoblasts (and later on by movable osteoblasts). Mineralization never appears to include the collagenous matrix of the surrounding preexistent mesenchymal tissue. Therefore, the term “direct ossification,” which is also used to define intramembra-
nous ossification and which implies the concept of a direct transformation into bone of the preexistent mesenchyme, should be dismissed. Another intriguing problem is the differentiation of stationary osteoblasts at a fairly constant distance of 28±0.4 µm from the vessels. It has already been shown that the vessels in the inner layer of the periosteum represent a sort of framework around which periosteal bone formation takes place (Marotti and Zambonin Zallone 1980). However, the reason why osteoblasts differentiate at such a distance from blood capillaries remains a mystery. It might be argued that, if osteoblasts were to differentiate closer to the vessels, bone deposition would be eccentric and not concentric with respect to the vascular framework; but this suggestion by no means explains the mechanism by which preosteoblasts “sense” the position of the vessels. Several factors may be involved, such as O2 tension (Brighton et al. 1991; Lennon et al. 2001), endothelial-cell-derived cytokines (i.e., endothelin-1) (Inoue et al. 2000; Kasperk et al. 1997; Sasaki and Hong 1993) and growth factors (ECGF) (Canalis et al. 1989; Guenther et al. 1986; Streeten and Brandi 1990; Villanueva and Nimni 1990) and dendritic stromal cells connecting vascular endothelium to the bone surfaces (Palazzini et al. 1998), but their precise role and mechanism of action have yet to be to defined. In conclusion, the intramembranous ossification appears to occur by two subsequent processes of osteogenesis, differing in arrangement, movement and polarisation of the osteoblasts. SBF is characterised by cords of pluristratified stationary osteoblasts, each polarized in a different direction, whereas DBF occurs by monostratified laminae of movable osteoblasts all polarized in the same direction. The two processes also have different functional meaning. The former appears mainly devoted to the formation of the core of the primary trabeculae, which form around the vascular framework during periosteal bone growth. The latter process seems to be mainly involved in the deposition of primary osteons inside the primary haversian spaces, i.e., in bone compaction. Acknowledgements This study was supported by 2001 MURST Cofinancing. The authors wish to thank Dr. Marta Benincasa for her valid technical cooperation.
References Brighton CT, Schaffer JL, Shapiro DB, Tang JJ, Clark CC (1991) Proliferation and macromolecular synthesis by rat calvarial bone cells grown in various oxygen tensions. J Orthop Res 9: 847–854 Canalis E, McCarthy T, Centrella M (1989) The regulation of bone formation by local growth factors. In: Peck WA (ed) Bone and mineral research, vol 6. Elsevier, Amsterdam, pp 27–56 Guenther HL, Fleisch H, Sorgente N (1986) Endothelial cells in culture synthesize a potent bone cell active mitogen. Endocrinology 119: 193–201 Hambuger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morph 88: 49–92
29 Inoue A, Kamiya A, Ishiji A, Hiruma Y, Hirose S, Hagiwara H (2000) Vasoactive peptide-regulated gene expression during osteoblastic differentiation. J Cardiovasc Pharmacol 36 [Suppl 1]: S286–S289 Jeansonne BG, Feafin FF, McMinn RW, Scoemaker RL, Rehm VS (1979) Cell-to-cell comunication of osteoblasts. J Dent Res 58: 1415–1423 Kasperk CH, Borcsok I, Schairer HU, Schneider U, Nawroth PP, Niethard FU, Ziegler R (1997) Endothelin-1 is a potent regulator of human bone cell metabolism in vitro. Calcif Tissue Int 60: 368–374 Lennon DP, Edmison JM, Caplan AI (2001) Cultivation of rat marrow-derived mesenchimal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis. J Cell Physiol 187: 345–355 Marotti G (1976) Decrement in volume of osteoblasts during osteon formation and its effects on the size of the corresponding osteocytes. In: Meunier PJ (ed) Bone histomorphometry. Armour Montagu, Paris, pp 385–397 Marotti G, Zambonin Zallone A (1980) Changes in the vascular network during the formation of the haversian system. Acta Anat 106: 84–100 Marotti G, Ferretti M, Muglia MA, Palumbo C, Palazzini S (1992) A quantitative evaluation of osteoblast-osteocyte relationships
on growing endosteal surface of rabbit tibiae. Bone 13: 363–368 Marotti G, Ferretti M, Palumbo C, Benincasa M (1999) Static and dynamic bone formation and the mechanism of collagen fiber orientation. Bone 25: 156 Palazzini S, Palumbo C, Ferretti M, Marotti G (1998) Stromal cell structure and relationships in perimedullary spaces of chick embryo shaft bones. Anat Embryol 197: 349–357 Palumbo C, Palazzini S, Zaffe D, Marotti G (1990a) Osteocyte differentiation in the tibia of newborn rabbit: an ultrastructural study of the formation of cytoplasmic processes. Acta Anat 137: 350–358 Palumbo C, Palazzini S, Marotti G (1990b) Morphological study of intercellular junctions during osteocyte differentiation. Bone 11: 401–406 Sasaki T, Hong MH (1993) Endothelin-1 localization in bone cells and vascular endothelial cells in bone marrow. Anat Rec 237: 332–337 Streeten EA, Brandi ML (1990) Biology of the bone endothelial cells. Bone Miner 10: 85–94 Villanueva JE, Nimni ME (1990) Promotion of calvarial cell osteogenesis by endothelial cells. J Bone Min Res 5: 733–739
