Does static precede dynamic osteogenesis in endochondral ossification as occurs in intramembranous ossification.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 288A:1158–1162 (2006) Does Static Precede Dynamic Osteogenesis in Endochondral Ossiﬁcation as Occurs in Intramembranous Ossiﬁcation? MARZIA FERRETTI, CARLA PALUMBO, LAURA BERTONI, FRANCESCO CAVANI, AND GASTONE MAROTTI* Dipartimento di Anatomia e Istologia, Sezione di Anatomia Umana, Università di Modena e Reggio Emilia, Modena, Italy ABSTRACT Endochondral ossiﬁcation takes place with calciﬁed cartilage cores providing a rigid scaffold for new bone formation. Intramembranous ossiﬁcation begins in connective tissue and new bone formed by a process of static ossiﬁcation (SO) followed by dynamic ossiﬁcation (DO) as previously described. The aim of the present study was to determine if the process of endochondral ossiﬁcation is similar to that of intramembranous ossiﬁcation with both a static and a dynamic phase of osteogenesis. Endochondral ossiﬁcation centers of the tibiae and humeri of newborn and young growing rabbits were studied by light and transmission electron microscopy. The observations clearly showed that in endochondral ossiﬁcation, the calciﬁed trabeculae appeared to be lined ﬁrst by osteoclasts. The osteoclasts were then replaced by ﬂattened cells (likely cells of the reversal phase) and ﬁnally by irregularly arranged osteoblastic laminae, typical of DO. This cellular sequence did not include osteoblasts seen in the phase of SO. These ﬁndings clearly support our working hypothesis that SO only forms in soft tissues to provide a rigid framework for DO, and that DO requires a rigid mineralized surface. The presence of osteocytes in contact with the calciﬁed cartilage also suggests the existence of stationary osteoblasts in endochondral ossiﬁcation. Stationary osteoblasts did not appear to be a unique feature of SO. The presence of stationary osteoblasts may appear to provide the initial osteocytes during osteogenesis that may function as mechanosensors throughout the bone tissue. If this is the case, then bone would be capable of sensing mechanical strains from its inception. Anat Rec Part A, 288A:1158–1162, 2006. Ó 2006 Wiley-Liss, Inc. Key words: endochondral ossiﬁcation; intramembranous ossiﬁcation; static osteogenesis; dynamic osteogenesis In a series of investigations carried out in recent years on intramembranous ossiﬁcation centers (Ferretti et al., 2002; Palumbo et al., 2004), we pointed out the existence of two types of bone deposition we respectively named static osteogenesis (SO) and dynamic osteogenesis (DO). SO always occurs ﬁrst; it is characterized by stationary osteoblasts, arranged in cords, which form at a rather constant distance from the network of blood capillaries and, without moving, they transform into osteocytes in the same site where they differentiated. DO is the more familiar mechanism of bone deposition. DO is characterÓ 2006 WILEY-LISS, INC. Grant sponsor: 2004 MURST Coﬁnancing; Grant number: 2004067411_001. *Correspondence to: Gastone Marotti, Dipartimento di Anatomia e Istologia, Sezione di Anatomia Umana, Via del Pozzo 71, 41100 Modena, Italy. Fax: 39-059-4224861. E-mail: firstname.lastname@example.org Received 6 March 2006; Accepted 26 July 2006 DOI 10.1002/ar.a.20386 Published online 9 October 2006 in Wiley InterScience (www.interscience.wiley.com). 1159 EARLY OSTEOGENESIS IN OSSIFICATION CENTERS Fig. 2. LM micrograph of an undecalciﬁed cross-section of the epiphysial plate in the humerus of an 8-day-old rabbit. The arrow points to spindle-shaped cells of the reversal phase lining the cartilage surface previously eroded by osteoclasts. CC, calciﬁed cartilage; B, bone. Scale bar ¼ 15 mm. Fig. 1. LM micrograph of an undecalciﬁed cross-section of the epiphysial plate in the tibia of a newborn rabbit. Note an osteoclast (OCL) resorbing the calciﬁed cartilage (CC) in an osteocartilaginous trabecula. B, bone. Scale bar ¼ 15 mm. ized by movable osteoblasts arranged in pseudoepithelial laminae, which move away from the mineralizing surface during preosseous matrix secretion and osteocyte transformation. In the case of intramembranous ossiﬁcation, we observed that DO takes place only after SO on the surface of the bony trabeculae laid down by SO. The main speculation we drew from these investigations was that SO is needed in soft mesenchymal tissue to provide a more rigid framework for DO and DO can only occur on a preexisting rigid mineralized surface. To verify the validity of our working hypothesis, the process of endochondral ossiﬁcation was studied to determine the sequence of ossiﬁcation. Speciﬁcally, to compare the SO and DO previously described in intramembranous ossiﬁcation. Contrary to intramembranous ossiﬁcation, endochondral ossiﬁcation begins with a rigid scaffold of calciﬁed cartilage. MATERIALS AND METHODS Metaphyseal plates from the tibiae and humeri of three newborn rabbits and four growing rabbits aged 8–10 days were used for light and electron microscopy. The care and use of animals were in accordance with the National Institutes of Health guidelines. All specimens were ﬁxed for 2 hr with 4% paraformaldehyde in 0.13 M phosphate buffer, pH 7.4, postﬁxed for 1 hr with 1% osmium tetroxide in 0.13 M phosphate buffer at pH 7.4, dehydrated in graded ethanol, and embedded in epoxy resin (Durcupan ACM). Decalciﬁed specimens were prepared by placing the tissue in 2.5% EDTA (0.13 M phosphate buffer, pH 7.2) until soft and then postﬁxing in osmium tetroxide prior to embedding in epoxy resin. The epoxy blocks were sectioned with a diamond knife using an Ultracut-Reichert Microtome. The methaphyseal plates were longitudinally and transversely sectioned with respect to the main axis of the shaft. Semithin sections (1 mm) were stained with toluidine blue and examined with a 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 with a Zeiss EM109 transmission electron microscope (TEM). RESULTS In all metaphyseal plates, the following sequence of events of endochondral ossiﬁcation were observed: columns of proliferating chondrocytes undergoing gradual hypertrophy towards the midshaft level; cartilage mineralization; chondrolysis and partial calciﬁed cartilage disruption by osteoclasts; and the formation of osteocartilaginous trabeculae. Our LM and TEM analyses were particularly focused on the surface of the trabeculae of calciﬁed cartilage reabsorbed by osteoclasts (Fig. 1) and the early stages of bone matrix deposition. Prior to the appearance of osteoblasts, a consistent observation was the presence of ﬂattened or spindle-shaped cells, similar to those of a reversal phase, lining the cartilage lacunae eroded by osteoclasts (Figs. 2 and 3). After the disappearance of the spindle-shaped cells, they were replaced by osteoblasts arranged in laminae from their inception (Fig. 4). Osteoblasts were sometimes irregularly grouped in such laminae, particularly where the eroded surface of calciﬁed cartilage was irregularly indented by small and narrow lacunae. Most osteoblasts appeared to be very active, being rounded in shape and displayed a well-developed endoplasmic reticulum and Golgi apparatus. All osteoblasts were functionally polarized toward the cartilaginous or the osteoid seam surface, as shown by the posi- 1160 FERRETTI ET AL. Fig. 3. TEM micrograph of a decalciﬁed specimen showing a ﬂattened cell of the reversal phase (asterisk) lining the remnant of an eroded cartilaginous trabecula. Scale bar ¼ 2.5 mm. Fig. 5. TEM micrograph of a decalciﬁed specimen showing a lamina of osteoblasts (OB) polarized toward the cartilaginous surface. CH, chondrocytes. Scale bar ¼ 2.5 mm. Fig. 4. LM micrograph of an undecalciﬁed cross-section of the epiphysial plate in the tibia of a newborn rabbit showing laminae of plump osteoblasts lining lacunae of calciﬁed cartilage. Scale bar ¼ 15 mm. tion of their organelles with respect to the nucleus (Fig. 5). Gap junctions were sometimes observed between adjacent osteoblasts (Fig. 6). The bone covering the remnants of calciﬁed cartilage contained osteocytes, whose cell body displayed a globous or ovoid shape, enclosed in single lacunae. Conﬂuent lacunae were never observed (Fig. 2). An interesting and fairly frequent ﬁnding was the presence of osteocyte cell bodies very close to the calciﬁed cartilage (Figs. 7 and 8; see also Figs. 1 and 2). Osteocyte dendrites showed an asymmetrical arborization with short mineral dendrites radiating toward the calciﬁed cartilage and longer vascular dendrites directed toward the osteoblastic laminae and the vessels (Fig. 8). Gap junctions or simple contacts were observed between the cytoplasmic Fig. 6. TEM micrograph showing a gap junction (arrows) between movable osteoblasts. Scale bar ¼ 0.25 mm. processes of adjacent osteocytes and between osteocyte dendrites and osteoblasts (Fig. 9). DISCUSSION In intramembranous ossiﬁcation centers that surround the developing shafts of long bones, transverse periosteal bone growth occurs by progressive extension of osteoblastic cords inside the surrounding mesenchyme at about midway between blood capillaries (mean distance of cords from vessels, 28 6 0.4 mm). The cords are EARLY OSTEOGENESIS IN OSSIFICATION CENTERS Fig. 7. TEM micrograph showing two osteocytes, derived from stationary osteoblasts, close to calciﬁed cartilage (asterisks). The three arrows point to osteocyte mineral dendrites radiating toward the calciﬁed cartilage. Decalciﬁed specimen. Scale bar ¼ 5 mm. 1161 Fig. 8. TEM micrograph of an osteocyte (OC), derived from stationary osteoblasts, with a short mineral dendrite directed toward the calciﬁed cartilage (top) and longer vascular dendrites extending toward an osteoblast (OB). Undecalciﬁed specimen. Scale bar ¼ 2.5 mm. Fig. 9. A: Gap junction between osteocyte dendrites in a cross-sectioned canaliculus. B: Gap junction between a vascular dendrite of an osteocyte (OC) and a short cytoplasmic processes of an osteoblast (OB). Undecalciﬁed specimens. TEM micrographs. Scale bars ¼ 0.2 mm (A); 0.3 mm (B). made up of 2–3 layers of active plump osteoblasts, all functionally polarized, but in a different direction with respect to the adjacent ones. Additionally, these osteoblasts are stationary since they transform into osteocytes without moving from the site where they appeared to differentiate. It is from these observations that we proposed the term ‘‘static osteogenesis’’ to describe this process (Ferretti et al., 2002). As a consequence, the bony trabeculae, laid down in this manner, contain several globous-shaped osteocytes, often irregularly grouped inside conﬂuent lacunae and displaying short dendrites having about the same length all around the cell body (Palumbo et al., 2004). Afterward, typical monostratiﬁed laminae of movable osteoblasts differentiate along the surface of these trabeculae laid down by static osteogenesis. These osteoblasts form new bone by dynamic osteogenesis that results in thicker trabeculae that cause the narrowing of the enclosed primitive Haversian spaces (primary osteons). Osteocytes derived from movable osteoblasts generally are ovoid and display an asymmetrical dendrite arborization. It should be noted that the network of dendrites extending throughout all osteocytes, derived by both static and dynamic osteogeneses, forms a functional syncytium since they were found to be connected by gap junctions (Ferretti et al., 2002; Palumbo et al., 2004). 1162 FERRETTI ET AL. In endochondral ossiﬁcation, static osteogenesis never seems to take place. In fact, the osteoblasts in contact with the remnants of calciﬁed cartilage are directly arranged in movable laminae and all appear to be functionally polarized in the same directions, i.e., toward the calciﬁed cartilage. Additionally, the osteocytes inside the bone surrounding the calciﬁed cartilage are never grouped inside conﬂuent lacunae and all display an asymmetrical arborization of dendrites. Thus, in endochondral ossiﬁcation, dynamic osteogenesis is not preceded by static osteogenesis. We wish to stress, however, that one must not confuse static osteogenesis with stationary osteoblasts. In endochondral ossiﬁcation, the presence of osteocytes close to the calciﬁed cartilage clearly indicates that such osteocytes are derived from stationary osteoblasts that remain in the same site where they differentiated. The thin layer of bone matrix separating the osteocyte cell bodies from the calciﬁed cartilage generally contained osteocyte mineral dendrites. Therefore, this layer does not indicate that the parental osteoblasts moved; it instead reﬂects the diminution in size of osteoblast protoplasms and the radiation of cytoplasmic processes that occurred during the differentiation of the osteoblast into an osteocyte. Osteocytes derived from stationary osteoblasts can frequently be observed close to the reversal lines of secondary Haversian systems; hence, stationary osteoblasts are not a distinctive feature of static osteogenesis. At the onset of bone formation, it is necessary for some stationary osteoblasts to be present, independently of whether static or dynamic osteogenesis is taking place, to ensure the presence of osteocytes, thus providing a network of strain-sensitive cells throughout the developing bone tissue. Without these stationary osteoblasts, the ﬁrst layers of bone tissue would not contain osteocytes. We wish to stress that a continuous osteocyte functional syncytium was observed throughout the bone laid down in both intramembranous and endochondral ossiﬁcation and that such syncytium comes into contact with the osteoblasts covering the bone growing surfaces. Such a cellular organization suggests that the bone is capable of sensing and answering mechanical signals since from its inception. Osteoblast differentiation seems to depend on a variety of inductive factors; in intramembranous ossiﬁcation, it probably depends on endothelial cell-derived cytokines, such as endothelin-1 (Sasaki and Hong, 1993; Kasperk et al., 1997; Inoue et al., 2000) and growth factors (EDGF) (Guentheret al., 1986; Canalis et al., 1989; Streeten and Brandi, 1990; Villanueva and Nimni, 1990). Since static and dynamic osteogeneses were observed to occur in the same manner and sequence during bone repair (Marotti, 2004), osteoblast differentiation could also depend on platelet-derived growth factor (PDGF) (Zheng et al., 1992; Lind, 1998; Chaudhary et al., 2004) in bone healing. In endochondral ossiﬁcation, osteoblast differentiation can depend on endothelial-cell derived cytokines as in intramembranous ossiﬁcation; however, it seems likely that it might also depend on coupling factors released by the cells of the reversal phase, as has been suggested in the bone remodeling cycle (Baron, 1976, 1989). 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