Osteocyte Apoptosis and Absence of Bone Remodeling in Human ...

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Jan 31, 2012 - Abstract Considering the pivotal role as bone mecha- nosensors ascribed to osteocytes in bone adaptation to mechanical strains, the present ...
Calcif Tissue Int (2012) 90:211–218 DOI 10.1007/s00223-012-9569-6

ORIGINAL RESEARCH

Osteocyte Apoptosis and Absence of Bone Remodeling in Human Auditory Ossicles and Scleral Ossicles of Lower Vertebrates: A Mere Coincidence or Linked Processes? Carla Palumbo • Francesco Cavani • Paola Sena • Marta Benincasa • Marzia Ferretti

Received: 7 December 2011 / Accepted: 5 January 2012 / Published online: 31 January 2012 Ó Springer Science+Business Media, LLC 2012

Abstract Considering the pivotal role as bone mechanosensors ascribed to osteocytes in bone adaptation to mechanical strains, the present study analyzed whether a correlation exists between osteocyte apoptosis and bone remodeling in peculiar bones, such as human auditory ossicles and scleral ossicles of lower vertebrates, which have been shown to undergo substantial osteocyte death and trivial or no bone turnover after cessation of growth. The investigation was performed with a morphological approach under LM (by means of an in situ end-labeling technique) and TEM. The results show that a large amount of osteocyte apoptosis takes place in both auditory and scleral ossicles after they reach their final size. Additionally, no morphological signs of bone remodeling were observed. These facts suggest that (1) bone remodeling is not necessarily triggered by osteocyte death, at least in these ossicles, and (2) bone remodeling does not need to mechanically adapt auditory and scleral ossicles since they appear to be continuously submitted to stereotyped stresses and strains; on the contrary, during the resorption phase, bone remodeling might severely impair the mechanical resistance of extremely small bony segments. Thus, osteocyte apoptosis could represent a programmed process devoted to make stable, when needed, bone structure and mechanical resistance.

The authors have stated that they have no conflict of interest. C. Palumbo (&)  F. Cavani  P. Sena  M. Benincasa  M. Ferretti Dipartimento di Scienze Biomediche, Sezione di Morfologia umana—Istituti Anatomici, Universita` di Modena e Reggio Emilia, Via del Pozzo 71 (area Policlinico), 41125 Modena, Italy e-mail: [email protected]

Keywords Osteocyte death  Apoptosis  Bone remodeling  Auditory ossicle  Scleral ossicle

Apoptosis is a process of programmed cell death involved in the development and turnover of tissues and organs [1, 2] in both normal and pathologic conditions, as well as in the pathogenesis of tumor processes [3–5] and in the evolution of many diseases [6, 7]. Moreover, it has also been recognized as playing a pivotal role in tissue and organ formation during fetal development [8–10]. The importance of apoptosis in the histophysiology of skeletal tissues and, in particular, its functional relationship with bone turnover have been investigated by various authors [11–13] in order to understand whether and how the mechanism of programmed cell death interferes with bone modeling and remodeling. In normal conditions, cells of osteogenic lineage, mostly osteoblasts and osteocytes, as well as those of osteoclastic lineage have been shown to be affected by apoptosis [11, 14–16]; and the potential role of apoptosis in the control of cell populations, in particular those that form or destroy bone tissue, is irrefutable. Bone turnover of skeletal segments is due to the bone remodeling cycle, i.e., the process that continuously renews bone tissue through a balance between the two phases of bone erosion and bone deposition, which in turn depends on the interaction in bone of mechanical (gravity, loading) and nonmechanical (hormones, cytokines, and factors) agents. These skills have been attributed to osteocytes inside the bone that should trigger both bone deposition in response to mechanical demands and bone erosion in response to metabolic requests [17–19]. In particular, it was suggested that osteocytes are arranged in a functional syncytium together with osteoblasts/bone-lining cells (according to whether the bone surfaces are in growing or

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resting phase, respectively), stromal cells, and endothelial cells. In the syncytial network, osteocytes modulate the process of bone remodeling to allow adaptation of bone mass, architecture, and structure to mechanical and metabolic demands acting on the skeleton; in order to answer to such demands, osteocytes need to be inside the network. An intriguing question is whether or not bone deposition and erosion occur in the absence of living osteocytes. Some bone segments, once they reach their definitive size, are devoted only to mechanical stereotyped stress for their life. This is the case for human auditory ossicles of the middle ear, whose target is to transmit the deformation of the tympanic membrane produced by sound waves to the receptor structures in the inner ear. Also, scleral ossicles, dermal bones present in the eye of many lower vertebrates [20] such as teleosts, birds, and reptiles [21, 22], are devoted to peculiar functions. They form a sclerotic ring around the eye of such vertebrates with bulging eyes [23]; some of the proposed functions ascribed to these peculiar ossicles are to support and protect the eyeball during deformation while flying or diving [24] and to provide an attachment for the ciliary muscles, particularly in the anterior portion of the cornea, thus suggesting a role in visual accommodation [22, 25–29]. On the basis of the above considerations, it appears that the function of both human auditory ossicles and lower vertebrate scleral ossicles differs from that of the other skeletal segments. Both in scleral ossicles (unpublished data presented to recent meetings) and in human auditory ossicles [30] the presence of massive osteocyte death and the absence of bone remodeling were shown. The aim of the present morphological study was to investigate, both in auditory ossicles and in scleral bones, if osteocyte death and bone remodeling are casually coincident (i.e., independent each other) or functionally correlated. In the latter case, the death of osteocytes should be a programmed phenomenon that could be targeted by means of apoptosis, to avoid bone remodeling. The study was performed using both light microscopy (LM), by means of in situ end-labeling technique (TUNEL), and transmission electron microscopy (TEM). Fig. 1 Schematic drawing showing the location of scleral ossicles in the bird eyeball (a). Ossicles are located along the scleral–corneal boundary and form a bony ring consisting of 13–14 bone laminae, each overlapping adjacent ossicles. Ossicles are numbered clockwise (b): ossicle 1 is situated above the choroid fissure. C cornea, SO scleral ossicles, S sclera

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Materials and Methods Human stapes were taken from nine subjects who underwent stapedectomy, aged 22–54 years, and from eight cadavers, aged 18–72 years. All enrolled patients, who underwent surgery at the University Hospital of Modena, were asked to give informed written consent to the study protocol, which was approved by the local ethics committee. Scleral ossicles from two species of lower vertebrates were considered in the study, (chicken) and reptiles (lizard). Scleral ossicles (Fig. 1) were taken from the eyes of 18-day-old chicken embryos; 7-, 15-, and 30-day-old chicks; and adult lizard. All surgical procedures were performed according to the Bioethical Committee of the Italian National Institute of Health. Animal care was conducted in accordance with Italian law (D.L. 116/1992) and European legislation (EEC 86/609). Histological and Ultrastructural Analyses Stapes and scleral ossicles 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, embedded in epoxy resin (Durcupan ACM; Electron Microscopy Sciences, Hatfield, PA), and sectioned with a diamond knife mounted in an Ultracut microtome (Reichert-Jung, Wetzlar, Germany). Stapes were sectioned along the axial plane perpendicular to the base and the tranversal plane parallel to the base; scleral ossicles were transversely sectioned. Thin sections (1 lm) were stained with toluidine blue and examined under an Axiophot LM (Zeiss, Oberkochen, Germany). Ultrathin sections (70–80 nm) were mounted on formvar- and carboncoated copper grids, stained with 1% uranyl acetate and lead citrate, and examined under a Zeiss EM109 TEM. In Situ End-Labeling Analysis (TUNEL) Stapes and scleral ossicles were also prepared according to the following TUNEL method. Samples were fixed in 4%

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buffered paraformaldehyde (0.1 M phosphate buffer, pH 7.2) overnight at 4°C, embedded in paraffin via a standard method, sectioned on a microtome (5 lm thick), and mounted on silanized slides. After being deparaffinized and rehydrated, sections were treated in a humidified chamber with proteinase K (Boehringer, Mannheim, Germany) at 20 lg ml-1 for 15 min at room temperature, washed in distilled water, treated with 2% H2O2 in methanol for 10 minutes at room temperature, and then washed in distilled water. Slides were preincubated with terminal deoxynucleotidyl transferase (TdT) buffer and 1 mM CoCl2 for 5 min at room temperature and then incubated for 60 min in a humidified chamber at 37°C with 50 ll TdT and biotinylated deoxyuridine triphosphate (BiodUTP) (Boehringer) (TdT 0.3 U ll-1, BiodUTP 8 lM in TdT buffer, and CoCl2 1 mM). Sections were then washed four times in bidistilled apyrogen water (for 2 min each), twice in phosphate-buffered saline (PBS, 5 min each), in human serum albumin 2% (5 min), and in PBS (5 min), then covered with streptavidin-biotinylated peroxidase complex (Boehringer) diluted 1:100 in a humidified chamber for 45 min at room temperature, washed in PBS, and stained with diaminobenzidine 50 mM (0.05%). Slides were then washed in water and counterstained with 0.5% methyl green for 10 min. Positive and negative controls were included in each experiment. For positive controls, sections were treated with DNase I (1 lg ml-1, Boehringer) in DNase buffer for 10 minutes at room temperature before exposure to BiodUTP and TdT. For negative controls, sections were incubated without the TdT enzyme.

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Results LM observations of toluidine blue-stained sections of both stapes and scleral ossicles showed bony lacunae mostly containing nonviable osteocytes, often with pyknotic nuclei, or empty lacunae (Figs. 2, 3). Concerning the latter (i.e., scleral ossicles), they appeared as thin bone laminae whose thickness increased from the embryonic stage to postnatal life: at the beginning they included a single layer of osteocytes and their surfaces were lined by active prismatic osteoblasts (Fig. 3a), after which they appeared thicker and outlined by flattened osteoblasts or bone-lining cells (Fig. 3b). In adult chicken (not shown) as well as in adult lizard (Fig. 3c) they included various generations of empty osteocyte lacunae or lacunae containing dead osteocytes. Another finding from LM observations was the absence inside the bone of both auditory and scleral ossicles of cement lines, i.e., the reversal lines corresponding, during bone remodeling, to the surface on which osteoclasts stop bone resorption and osteoblasts start bone deposition. TEM ultrastructural analysis of the same samples showed the presence of osteocytes mostly in apoptosis (Fig. 4). The ultrastructure of such osteocytes is different, depending on the different degree of chromatin condensation and fragmentation. The aspect of the chromatin masses is variable: it ranges from a low degree of condensation and fragmentation to a much higher one. In some cells we observed good preservation of the organelles, whereas in others we observed a large degree of alterations with considerable vacuolization. Apoptotic bodies were also often visible inside the osteocyte lacunae (Fig. 5).

Fig. 2 LM micrographs of a toluidine blue-stained slice of a human stape, sectioned according to the axial plane perpendicular to the base. b Enlarged field of the stape head, boxed in a. Note the abundance of empty osteocyte lacune (representative areas between red arrows) and the absence of cement lines. Scale bars a = 20 lm, b = 100 lm (Color figure online)

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Fig. 3 LM micrographs of toluidine blue-stained transverse sections of scleral ossicles from 18-day-old chicken embryo (a), 15-day-old chick (b), and adult lizard (c). Note laminae of active prismatic osteoblasts (arrowheads) outlining the bone surfaces in a and flattened osteoblasts or bone lining cells (arrowheads) in b. Note also dead osteocytes inside the chick bone (b) and the abundance of empty lacunae in adult lizard ossicle (c). Cement lines were never observed. Scale bars a, b = 45 lm, c = 90 lm

The TUNEL method, which labels cells with DNA fragmentation with a brown color, revealed the presence of apoptotic phenomena affecting most osteocytes, both in human stapes and in scleral ossicles of the chick and lizard (Fig. 6).

Discussion Several preliminary considerations are to be made in order to discuss our results. It has long been believed that cells of osteogenic lineage participate in the transmission of mechanical and biochemical signals, which trigger and/or exert control over bone formation as well as bone resorption during bone growth, bone modeling, and bone remodeling [17]. In particular, it was suggested that osteocytes are arranged in a functional syncytium together with the other cells of osteogenic lineage and endothelial cells and that they modulate the process of bone remodeling to allow adaptation of bone mass, architecture, and structure to mechanical (skeletal homeostasis) and metabolic (mineral homeostasis) demands acting on the skeleton. Such bone adaptation to the mechanical and metabolic demands occurs both during growth (childhood and adolescence) and after the end of skeletal growth (adulthood and aging). This notwithstanding, some bone segments, once they reach their definitive size, are devoted only to mechanical stereotyped

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stress (e.g., auditory ossicles) for their lifetime; and in such bone segments the process of bone remodeling is to be avoided. The need to avoid bone remodeling is present not only in the case of human auditory ossicles but also in the evolutionary ladder; scleral ossicles, in fact, for their location and proposed functions (see above), represent very peculiar bony segments whose morphofunctional implication is completely differ from the other skeletal segments. Another point concerns osteocyte death during the development of the ossicles of the middle ear. Human auditory ossicles derive from cartilaginous buds, whose ossification begins in the third to fourth month of prenatal life; however, their rate of growth is such that at birth they have practically achieved their final shape and dimensions since they stop growing soon after birth (data confirmed by clinical–radiological evidence as well as by studies of comparative anatomy [31]). Osteocyte death in ear ossicles, though gradually increasing with age, as occurs in other skeletal segments, starts abruptly during the perinatal period soon after they cease to grow: the process of osteocyte death is very rapid and widespread so that over 40% of cells are dying within the second year of life [30]; this percentage is incomparably higher than those estimated in other bones of the human skeleton, for which values range between 1% in the newborn to 40% in the elderly [32]. In the cited work, however, the authors were not able to establish the cause of osteocyte death, i.e., degeneration or

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Fig. 4 TEM micrographs showing osteocytes at different stage of apoptosis in scleral ossicles of 18-day-old chicken embryo (a), 30-day-old chick (c), and adult lizard (b) and in human stapes (d, e). Scale bars a = 2.5 lm, b = 5 lm, c = 1 lm, d = 1.5 lm, e = 3 lm

apoptosis, which widely differed from the point of view of both etiology and functional meaning. The fact that osteocyte death occurs by means of apoptosis represents a programmed phenomenon, rather than a simple coincidence, that leads to the absence of bone remodeling. Concerning scleral ossicles of lower vertebrates, those of birds and reptiles are morphologically similar and organized in the same manner [33], are well developed [34], and have a relevant role in preserving the shape of the eyeball [24]. The ossicles at the boundary of the scleral cartilage form a groove in the concavity of the eye, which plays an important role in accommodating the cornea, as well as in its protection [34]. They originate from neural crest–derived ectomesenchyme [35–37], and their ossification occurs in beds situated 70–100 lm away from the surface of the eye and beneath the original location of the

superficial condensations of ectomesenchymal cells [38], eventually forming a ring of overlapping, trapezoidal shaped bony plates surrounding the corneal margin, whose function is decoupled from that of all other bones in the skeleton, as inferable by the massive osteocyte death. Regarding the osteogenic cell network, forming the functional syncytium that allows bone adaptation, the cells communicate with each other by means of both volume and wiring transmission [18] and, thus, are able to react to both local signals (mechanical strains, cytokines, and growth factors) and systemic factors (hormones). Thanks to their location inside the bone matrix, osteocytes are likely the first cells to sense mechanical strains, whereas stromal cells should be the first to be activated by hormonal molecules which diffuse across the endothelial lining. Thus, it appears that the presence of viable osteocytes inside the bone is

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Fig. 5 TEM micrographs showing sections of a scleral ossicle from a 30-day-old chick (a) and a human stape (b). Apoptotic bodies are visible inside the osteocyte lacunae. Scale bars a = 2 lm, b = 1 lm Fig. 6 LM micrographs of in situ end-labeling (TUNEL): transverse sections of scleral ossicles of a 30-day-old chick (a) and an adult lizard (b); sections of the base of a human stape, cut on a tangential plane (c, d). Labeled cells exhibit a brownish color. Counter stain, methyl green. Scale bars a = 10 lm, b = 12 lm, c, d = 17 lm

necessary to sense the mechanical demands and trigger the proper answer by means of the activation of the bone remodeling cycle. We already suggested and supported the putative role of osteocytes in bone remodeling, not only in activating but also in arresting osteoclast activity [18, 19]; moreover, it was observed that bone with dead osteocytes does not undergo remodeling [30, 32]. Our present results clearly show the poorness of viable osteocytes due to apoptosis, both in human auditory ossicles and in scleral ossicles, thus suggesting the impossibility of osteocytes triggering or

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modulating the bone remodeling process. In line with this evidence are our previous morphometric investigations on the existence of a network of communicating living osteocytes, required to adapt bone mass and architecture to actual mechanical demands [39–42]. This fact is also in accordance with the mechanostat postulate of Frost [43] and the feedback theory of Turner [44], according to which if the physiological set points of bone deformation (50–2,500 microstrains) are exceeded (i.e., in the state of disuse or overuse), bone resorption or bone formation occurs, respectively. Other authors, indeed, have proposed

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that osteocyte apoptosis could be involved in triggering bone turnover [13, 45]. The present results are, however, in line with (1) the above-cited morphometric studies on the auditory ossicles of the human middle ear [30] demonstrating that osteocyte death occurs very rapidly (within the second year of age) and bone remodeling occurs only occasionally and (2) our recent unpublished histomorphometric investigations on the eye bony ring of lower vertebrates showing that about 60% of osteocyte lacunae are empty or full of mineral crystals or contain dead osteocytes and that bone remodeling never occurs. On the basis of the above considerations, it appears that auditory and scleral ossicles do not need to adapt to their loading environment after they have reached their final dimension. In both cases, the programmed osteocyte death, i.e., apoptosis, probably represents the way by which ossicles lose their ability to react to strains and stresses and achieve the structural stability they need to perform their peculiar stereotyped function. In conclusion, notwithstanding the mere morphological observations we recorded concerning the abundant presence of osteocyte apoptosis and the total absence of cement lines (of bone remodeling), the novelty of the present report lies in the fact that two very different ossicles, phylogenetically so distant and differing in functional meaning, share the need of avoiding bone remodeling by means of programmed osteocyte death. Moreover, the present results strongly support the hypothesis that in these peculiar ossicles programmed cell death makes stable in time both bone structure and mechanical resistance. Acknowledgment The authors dedicate the present paper to their mentor, Prof. Gastone Marotti, on the occasion of the conferment of the title ‘‘professor emeritus.’’ This study was supported by funds of the Fondazione of Vignola and Banca Popolare of Emilia Romagna.

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