Remodeling in bone without osteocytes: Billfish challenge bone structure–function paradigms Ayelet Atkinsa,1, Mason N. Deanb,1, Maria Laura Habeggerc, Phillip J. Mottac, Lior Ofera, Felix Reppb, Anna Shipova, Steve Weinerd, John D. Curreye, and Ron Shahara,2 aKoret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel; bDepartment of Biomaterials, Max Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany; cDepartment of Integrative Biology, University of South Florida, Tampa, FL 33613; dDepartment of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel; and eDepartment of Biology, University of York, York YO10 5DD, United Kingdom Edited by David B. Burr, Indiana University School of Medicine, Indianapolis, IN, and accepted by the Editorial Board September 19, 2014 (received for review July 3, 2014) A remarkable property of tetrapod bone is its ability to detect and borders, heavily remodeled tissue, which forms particularly in remodel areas where damage has accumulated through prolonged situations of large load magnitudes and/or cyclic loading, bears use. This process, believed vital to the long-term health of bone, is a characteristic morphology of overlapping osteons (Fig. 1 C and D), considered to be initiated and orchestrated by osteocytes, cells a structural record of vigorous remodeling (ref. 2; SI Text). within the bone matrix. It is therefore surprising that most extant Considering the pivotal role of osteocytes in remodeling and fishes (neoteleosts) lack osteocytes, suggesting their bones are not that remodeling is believed to be essential for the proper long- constantly repaired, although many species exhibit long lives and term function of bone, it is surprising that the bones of almost all high activity levels, factors that should induce considerable fatigue members of the huge group of extant neoteleost fish—close to damage with time. Here, we show evidence for active and intense 50% of all vertebrates—are completely devoid of osteocytes remodeling occurring in the anosteocytic, elongated rostral bones (anosteocytic) (Fig. 1E) (5, 6). According to the current tenets of of billfishes (e.g., swordfish, marlins). Despite lacking osteocytes, bone biology, because local internal strains are believed to be this tissue exhibits a striking resemblance to the mature bone of sensed by osteocytes alone (3, 7), remodeling in anosteocytic fish large mammals, bearing structural features (overlapping second- species would either not occur at all, be driven by something EVOLUTION ary osteons) indicating intensive tissue repair, particularly in areas other than damage, or be stochastic in nature (i.e., “undi- where high loads are expected. Billfish osteons are an order of rected”). The former option, lack of remodeling, is supported magnitude smaller in diameter than mammalian osteons, how- by the fact that the bone of most fish species examined to date ever, implying that the nature of damage in this bone may be has been shown to be featureless (i.e., to lack traces of remod- different. Whereas billfish bone material is as stiff as mammalian eling; e.g., Fig. 1E), particularly in comparison with mammalian bone (unlike the bone of other fishes), it is able to withstand much bone (e.g., Fig. 1 A–D) (5, 6, 8–11). From a functional point of greater strains (relative deformations) before failing. Our data view, however, there are many reasons to expect that both show that fish bone can exhibit far more complex structure and osteocytic and anosteocytic fishes should need to remodel their physiology than previously known, and is apparently capable bones: Remodeling occurs in a wide assortment of aquatic and of localized repair even without the osteocytes believed essential terrestrial amniote species (4, 12, 13); fish experience large loads for this process. These findings challenge the unique and primary on their skeletons (e.g., bite forces can exceed 300 N, and fish role of osteocytes in bone remodeling, a basic tenet of bone bio- logy, raising the possibility of an alternative mechanism driving this process. Significance anosteocytic bone | osteon | bone toughness | A fundamental paradigm of bone biology is that the remod- damage-driven remodeling | fish skeleton eling process—by which bones detect and repair damage—is orchestrated by osteocytes. The bones of most extant fish, one forms the stiff, load-bearing framework of the body of however, lack these cells and should be unable to repair Bvertebrates. The current paradigm of bone biology is that the damage in their bones. We provide evidence for intense remod- microdamage that accumulates in bone—the result of repeated eling in the anosteocytic bone of billfishes, such as swordfish loading over long periods of time—is prevented from reaching and marlin. Our observations challenge the central axiom that failure levels by “remodeling,” a physiologic maintenance pro- osteocytes alone are responsible for remodeling, suggesting al- cess by which packets of damaged bone material are removed, ternate mechanisms in bone physiology and/or variation in the then replaced by new, undamaged tissue (1). The most crucial roles of bone cells. In addition, billfish bone exhibits an array of role in the remodeling sequence is thought to be played by the striking material properties that distinguish it from mammalian osteocytes, the cells residing within the bone matrix, which sense bone despite having similar composition, underlining that skele- local increases in strain (or strain energy density) caused by tal biology concepts are limiting when based on mammalian microcrack accumulation, then initiate and coordinate remod- tissues alone. eling once a defined threshold is reached (2, 3). First, surface- Author contributions: A.A., M.N.D., M.L.H., P.J.M., S.W., J.D.C., and R.S. designed research; residing osteoclasts (bone-resorbing cells) are recruited to invade M.L.H. acquired animals; A.A., M.L.H., L.O., A.S., J.D.C., and R.S. performed research; F.R. and erode packets of damaged tissue within the bone material, contributed new reagents/analytic tools; A.A., M.N.D., and R.S. analyzed data; and M.N.D. a process that creates visible resorption cavities. Osteoblasts and R.S. wrote the paper. (bone-building cells) are then recruited to these eroded areas, The authors declare no conflict of interest. and fill them concentrically from the periphery inwards with new This article is a PNAS Direct Submission. D.B.B. is a guest editor invited by the bone, leaving only a small central canal for the passage of blood Editorial Board. vessels and nerves (4). This “repair by replacement” process 1A.A. and M.N.D. contributed equally to this work. alters bone’s ultrastructural morphology, leaving behind lamel- 2To whom correspondence should be addressed. Email: [email protected]. “ ” – late, nearly circular secondary osteons (Fig. 1 A D). Because This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. subsequent secondary osteons do not respect each other’s 1073/pnas.1412372111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1412372111 PNAS | November 11, 2014 | vol. 111 | no. 45 | 16047–16052 Downloaded by guest on September 26, 2021 (Fig. 2 and Fig. S1). Billfish rostra therefore represent an ex- treme example of anosteocytic bone, loaded heavily and cycli- cally in cantilever bending over the animals’ long lifespans, and likely in need of regular remodeling to avoid failure. The five species examined provide a broad palette of body sizes and bill lengths in a closely related group of fishes (Fig. 2 and Fig. S1) and, therefore, are an ideal system to investigate correlations among body/bill size, reported activity level, and evidence for remodeling. Structural Properties of the Bone of the Billfish Rostrum The internal architecture of the rostral bone of all billfish species examined here bears striking resemblance to mammalian sec- ondary osteonal bone, in stark contrast to the bone of fish pre- viously examined, and bone from billfish opercula (Figs. 1–3 and SI Text). Bone cross-sections are densely populated by “osteon- like” structures that are lamellate and nearly circular, possess a central canal, and are surrounded by a slightly crenulated border (a “cement line”). However, whereas mammalian sec- Fig. 1. Mammalian bone ultrastructure, showing morphological evidence ondary osteons are heavily populated with osteocytes and be- for remodeling, compared with relatively “featureless” fish bone. (A) Cross- lieved to depend on them for their formation (7, 20, 21), billfish section of dog femoral midshaft with localized areas showing primary and “osteons” are entirely devoid of these cells, thus presenting remodeled bone (secondary osteons). (B) Higher magnification of the upper a striking variation on the classical appearance of mammalian boxed region in A, showing isolated, nonoverlapping osteons (borders in- secondary osteons (compare osteons in Figs. 1 and 3). The ce- dicated by dashed lines) present in otherwise lamellar (layered) primary ment lines of “anosteocytic osteons” (a term we will use to dis- bone tissue. Every osteon is organized around a central Haversian canal (Ca); tinguish them from tetrapod osteons) are much brighter than the smaller black dots are osteocyte lacunae. (C) Higher magnification of the lower boxed region in A, showing much higher osteonal density, with many bone matrix they surround when viewed in back-scattered scan- overlapping osteons (secondary osteons, shown by arrows, and in more ning electron (BSE) microscopy (Fig. 3B and insets in Fig. 2 and detail in the inset image), indications of tissue remodeling. (D) Horse third Fig. S1), suggesting they are comprised of a material with higher metatarsal bone showing heavy remodeling, evidenced by extensive osteo- mean mineral density (22). Cement lines are a prominent feature nal overlap. Inset polarized microscopy image illustrates how osteonal la- of tetrapod secondary osteons and are also reported to be more mellae are “interrupted” by lamellae of new osteons (white arrows).