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Editorial: Cell Number Versus Cell Vigor—What Really Matters to a Regenerating Skeleton?

Division of Endocrinology & Metabolism and Center for Osteoporosis & Metabolic Bone Diseases, University of Arkansas for Medical Sciences, and the Central Arkansas Veterans Health Care System, Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Stavros C. Manolagas, M.D., Ph.D., University of Arkansas for Medical Sciences, Division of Endocrinology & Metabolism, 4301 West Markham, Mail Slot 587, Little Rock, Arkansas 72205.

	Introduction

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"I agree with those African tribes who decorate themselves with bones. It is more to my taste than diamonds, which are a cold and soulless shine. Whilst bone, ah bone, is the pit of a man after the cumbering flesh has been eaten away. Bone is power. It is bone to which the soft parts cling, from which they are, helpless, strung and held aloft to the sun, lest man be but another slithering earth-noser ... What is this tissue that has double the strength of oak? One cubic inch of which will stand a crushing force of two tons? This substance that refuses to dissolve in our body fluids, but remains intact and solid through all vicissitudes of temperature and pollution? We may be grateful for this insolubility, for it is what stands us tall."—Richard Selzer, Mortal Lessons, Harcourt-Brace, San Diego, 1974, pp. 51–52.

The fact that the mineralized component of the skeleton remains intact millions of years after death "through all vicissitudes of temperature and pollution"—to the delight of paleontologists, archeologists, and anthropologists—all but overshadows an even more remarkable fact regarding the living human skeleton: it regenerates continuously throughout life and is completely renewed about every 10 yr in adults (1). The principles of skeletal regeneration were first proposed in the 1960s, thanks to the amazing perspective of Harold Frost and his apostles. Regrettably, four decades later, many investigators still attempt to interpret in vitro or in vivo data with little or no regard to the fact that bone does regenerate; and that involutional changes like menopause or old age, or acquired conditions like glucocorticoid-excess do not cause loss of bone mass by turning on a completely new process, but rather by deranging the existing process of bone regeneration.

During development and growth, the skeleton is sculpted to achieve its shape and size by the removal of bone from one site and deposition at a different one; this process is termed modeling. Once maturity has been reached, regeneration continues in the form of a periodic replacement of old bone with new at the same location; this process is termed remodeling. Removal of bone (resorption) is accomplished by osteoclasts, whereas formation of new bone is the task of the mature osteoblasts. However, in the uninjured adult skeleton, bone resorption and bone formation are not separate, independently regulated processes. Instead, all osteoclasts and osteoblasts belong to a temporary structure known as the basic multicellular unit (BMU). The BMU comprises a team of osteoclasts in the front and a team of osteoblasts in the rear. Osteoclasts adhere to bone and subsequently remove it by acidification and proteolytic digestion. As the BMU advances, osteoblasts move in to the previously excavated area and begin the process of new bone formation by secreting and subsequently mineralizing a collagen-rich proteinaceous matrix—the osteoid. In this manner, the BMU excavates and replaces a tunnel in cortical bone or a trench on the trabecular surface of cancellous bone. In healthy adult humans, three to four million BMUs are born every year and one million BMUs operate at any moment. At any one time, about 10–20% of the bone surface is involved, but the entire surface is involved at least once every 10 yr (albeit there are considerable regional variations to meet the different functions of different parts of the skeleton).

Both osteoclasts and osteoblasts are derived from precursors originating in the bone marrow. The precursors of osteoblasts are multipotent mesenchymal stem cells, which also give rise to bone marrow stromal cells, chondrocytes, muscle cells, and adipocytes. The precursors of the osteoclasts are hematopoietic cells of the monocyte/macrophage lineage. The genesis and differentiation of either cell type is regulated by growth factors and cytokines produced in the local microenvironment as well as by systemic hormones that control the production and/or action of growth factors and cytokines, and adhesion molecules (2). In spite of the fact that millions of small packets of bone are continually remodeled, bone mass is preserved in the healthy skeleton thanks to a remarkably tight balance between the amount of bone resorbed and formed during each cycle of remodeling. While many details responsible for the orchestration of this balance remain unknown, several fresh insights have emerged.

With BMU progression, cell recruitment at each new cross-sectional location is successive, osteoblasts not arriving until the osteoclasts have moved on. But, during the longitudinal progression of the BMU as a whole, new osteoclasts and new osteoblasts are needed simultaneously, although not at the same location. The distinction between the cross-sectional and longitudinal events during BMU progression corresponds to the distinction that has been drawn between serial and parallel models of osteoblast recruitment (3). According to the serial model, osteoblast precursor proliferation and differentiation is stimulated by factors released from resorbed bone, or by the local increase in mechanical strain consequent on bone resorption (4). According to the parallel model, precursor cell proliferation and differentiation for both osteoblasts and osteoclasts are consequences of the same mechanism, which is probably initiated by whatever signals are needed for BMU origination, and whatever hormones prolong BMU progression (5). With either model, new osteoblasts must be directed to the right location. Osteoclast development cannot be accomplished unless mesenchymal progenitors are present to provide essential support. Moreover, mesenchymal cell differentiation toward the osteoblast phenotype and osteoclastogenesis are inseparably linked as both are stimulated by the same factors, proceed simultaneously, and the former event is a prerequisite for the latter (6, 7, 8, 9, 10).

In the last 2 yr, the discovery of three proteins related to the TNF receptor superfamily has greatly clarified the mechanistic basis of the dependency of osteoclastogenesis on mesenchymal cells (11). Two of these proteins are membrane bound cytokine-like molecules: the receptor activator of NF-B (RANK) and the RANK-ligand. Other names used for RANK are osteoprotegerin ligand (OPG-L) and TRANCE. RANK is expressed in hematopoietic osteoclast progenitors, whereas RANK-ligand is expressed in committed preosteoblastic cells and T-lymphocytes (12, 13, 14). RANK-ligand binds to RANK with high affinity. This interaction is essential and, together with M-CSF, sufficient for osteoclastogenesis. 1,25(OH)₂D₃, PTH, PTHrP, gp130 activating cytokines (e.g. IL-6, IL-11) and IL-1 induce the expression of the RANK-ligand in stromal/osteoblastic cells (11, 15). The third of the three proteins, osteoprotegerin (OPG), unlike the other two, is a secreted disulfide-linked dimeric glycoprotein with profound inhibitory effects on osteoclastogenesis and bone resorption in vitro and in vivo (16). OPG binds to RANK-ligand and, as a decoy, blocks the RANK-ligand/RANK interaction. OPG is produced by many tissues including bone, skin, liver, stomach, intestine, lung, heart, kidney, and placenta as well as hematopoietic and immune organs. Consistent with an important role of OPG in the regulation of osteoclast formation, OPG transgenic mice develop osteopetrosis, whereas OPG knockout mice exhibit severe osteoporosis (17). Interestingly, the two cell types that express high levels of RANK-ligand, T-lymphocytes and osteoblastic cells, are also the two cell types that express high levels of the osteoblast specific transcription factor Cbfa1 (18). More intriguingly, it has been recently determined that both the murine and human RANK-ligand genes contain two functional Cbfa1 sites, and mutation of these sites abrogates the transcriptional activity of the RANK-ligand gene promoter (19). Therefore, the cell-specific expression of RANK-ligand in cells of the stromal/osteoblastic lineage might be dictated, at least in part, by the expression of Cbfa1. Therefore, a Cbfa1 requirement for RANK-ligand gene expression may constitute the molecular mechanism of the linkage between osteoblastogenesis and osteoclastogenesis. Consequently, and in view of the fact that the bone morphogenetic proteins (BMP) 2 and 4 stimulate Cbfa1 expression, a BMPCbfa1RANK-ligand gene expression cascade in cells of the bone marrow stromal/osteoblastic lineage may well constitute the molecular underpinnings of the control of the rate of bone regeneration and the concurrent production of osteoclasts and osteoblasts. In this scenario, BMPs provide the tonic baseline control of both processes—and thereby, the rate of bone remodeling—upon which other inputs (e.g. biomechanical, hormonal, etc.) operate. A tight association between concurrent osteoblast and osteoclast production makes perfect teleologic sense as at least one of the means of assuring a balance between bone formation and resorption under normal conditions.

At the conclusion of the remodeling cycle, all osteoclasts and the majority of osteoblasts (50–70%) disappear without a trace. The remaining osteoblasts become elongated "lining cells" that cover the newly formed bone surface or are entombed into the mineralized matrix as osteocytes—the most common (90%) cell type in bone. Osteocytes are believed to be the sensors of the local need for bone augmentation or reduction during functional adaptation and the transmitters of signals that lead to bone repair by remodeling. Like specialized cells of other regenerating tissues, both osteoclasts and osteoblasts die by apoptosis (20, 21). This evidence explains the fact that all osteoclasts and the majority of the osteoblasts present in the BMU vanish at the end of the remodeling cycle. Considering that the average lifespan of human osteoclasts is about 2 weeks and the lifespan of osteoblasts is 3 months, much shorter than the lifespan of the BMU (6 months), it is obvious that these cells must be continually replaced and the number present in bone depends not only on their birth rate, which reflects the frequency of cell division of the appropriate precursor cell, but also on the lifespan, which reflects the timing of death by apoptosis. In other words, the rate of supply of new osteoblasts and osteoclasts and the timing of the death of these cells by apoptosis are critical determinants of the initiation of new BMU’s and/or extension or shortening of the lifetime of existing ones. Evidence confirming this truism has been amassing with the elucidation of the pathogenetic mechanisms responsible for the development of postmenopausal, senile, and glucocorticoid-induced osteoporosis. Indeed, bone loss underlying all three forms of the disease can be now explained by changes in the birth and/or the death rate of osteoclasts and osteoblasts. Thus, a concurrent increase in osteoclastogenesis and osteoblastogenesis fully accounts for the increased rate of remodeling that ensues upon loss of estrogen or androgen (22, 23). Moreover, opposite effects of sex steroids on the lifespan of osteoblasts/osteocytes vs. osteoclasts, documented both in vitro and in vivo, explains the imbalance between formation and resorption and thereby the progressive bone loss that continues long after the high rate of remodeling has declined (24). Likewise, a decrease in osteoblastogenesis (accompanied by a reciprocal increase in adipogenesis in the bone marrow) can explain how, in old age, the amount of bone formed during each remodeling cycle decreases, whereas adipogenesis in the bone marrow increases (6). The cardinal histologic features of glucocorticoid-induced osteoporosis—decreased bone formation rate, decreased wall thickness of trabeculae, a strong indication of decreased work output by osteoblasts—and in situ death of portions of bone (osteonecrosis) can be also explained by evidence that glucocorticoid-excess has a suppressive effect on osteoblastogenesis in the bone marrow and also promotes the apoptosis of mature osteoblasts and osteocytes (25, 26). Loss of antiapoptotic effects of estrogen and proapoptotic effects of glucocorticoids on osteocytes may contribute independently to the increased bone fragility associated with the postmenopausal state and glucocorticoid-excess. In agreement with this body of evidence, bisphosphonates—an effective treatment for osteoporosis—exert their beneficial effects, at least in part, by stimulating osteoclast apoptosis and preventing osteoblast/osteocyte apoptosis (27, 28). Moreover, a 20-fold inhibition of osteoblast apoptosis by intermittent PTH administration—an effect reproduced in vitro on both osteoblasts and osteocytes—explains for the first time the anabolic effects of PTH on bone (29).

In this issue of Endocrinology, Hofbauer and colleagues (30) report that glucocorticoids inhibit OPG and concurrently stimulate the expression of the RANK-ligand [the alternative term osteoprotegerin ligand (OPG-L) is used by the authors] in human osteoblastic, primary, and immortalized bone marrow stromal cells in vitro. They suggest that this could be a mechanism whereby glucocorticoids promote osteoclastogenesis. These interesting and provocative observations suggest a new set of potential culprits in the pathogenesis of glucocorticoid-induced, and perhaps other forms of osteoporosis. The cardinal feature of the bone disease caused by chronic glucocorticoid excess is unquestionably decreased bone formation. Nonetheless, with glucocorticoid treatment, the loss of bone is biphasic with a rapid initial phase of approximately 12% during the first few months, followed by a slower phase of about 2–5% annually. Even though there is a significant correlation between the severity of the bone loss and the extent of reduction in bone formation, some of the loss may be due to an early increase in bone resorption, as evidenced by an early increase in osteoclast perimeter of vertebral cancellous bone after 7 days of steroid treatment in mice (25). It is plausible, therefore, that there may be an early increase in osteoclastogenesis and that the increased RANK-ligand/OPG ratio are responsible for this. Even though increased osteoclast surfaces have been shown in some histologic studies, others have not confirmed this finding and more recent ones have even shown that the number of osteoclasts in chronic glucocorticoid-excess is decreased (31). These apparent discrepancies could reflect differences in the duration of glucocorticoid treatment. Be that as it may, can the extremely rapid bone loss caused by glucocorticoid-excess be due to a simultaneous combination of increased osteoclastogenesis and decreased osteoblastogenesis? Based on the principles of bone regeneration highlighted before, and in particular the dependency of osteoclastogenesis on RANK-ligand expression by stromal/osteoblastic cells, an increase in osteoclastogenesis occurring simultaneously with a decrease in osteoblastogenesis is very unlikely. The only possible exceptions to this, are the focal osteolytic disease associated with malignancy, where the normal process of bone regeneration in the immediate vicinity of invasion succumbs to the tumor dynamics, and immobilization. If changes in OPG and/or RANK-ligand mediate an increase in osteoclastogenesis with glucocorticoid treatment in vivo, this could only be an acute, transient event in the very early phases of the disease. Later on, with suppressed osteoblastogenesis, and thereby a decrease in the number of cells that make RANK-ligand, a RANK-ligand dependent increase in osteoclastogenesis will be inexorably attenuated. An alternative explanation of the rapid initial phase of bone loss with glucocorticoid treatment is that preexisting osteoclasts may go on resorbing bone while formation is severely compromised by the abrupt apoptotic death of mature osteoblasts—a phenomenon seen in an inducible osteoblast ablation mouse model (32).

Burgess and colleagues (33), some of whom are also co-authors in the paper of Hofbauer et al. (30), have recently published elegant in vitro and in vivo studies suggesting that RANK-ligand may directly activate mature osteoclasts. Specifically, they have shown that RANK-ligand, acting via receptors present on mature rat osteoclasts, stimulated these cells to undergo more frequent cycles of resorption and induced cytoskeletal rearrangements in vitro; and iv administration of RANK-ligand to mice rapidly (within 1 h) increased blood ionized calcium. Along with the results of Hofbauer et al. (30), the logical extension of these observations is that glucocorticoids, by increasing the RANK-ligand/OPG ratio, may not only increase osteoclastogenesis, but also stimulate bone resorption by mature osteoclasts. Several reasons make this very unlikely. First, in contrast to the findings of Burgess et al. (33), Dempster and colleagues (34) have shown that glucocorticoids inhibit bone resorption by isolated rat osteoclasts by enhancing apoptosis; a finding consistent with evidence that, in sharp difference with their effects on the human skeleton, glucocorticoids inhibit bone resorption and increase bone mass in rats in vivo. Second, both in mice and humans the resorption surface is decreased and the erosion depth is shallow in glucocorticoid-excess. Moreover, even at the early stages of glucocorticoid administration, biochemical markers of bone resorption do not change (35). Third and more important, there is no evidence that human osteoclasts in the adult skeleton are in need of "activation." This notion was originally derived from studies of cortical bone modeling in young rapidly growing rats. Continuous endocortical resorption needs so many osteoclasts that keeping some in reserve could make sense. Osteoclast "activation" also occurs in the cyclical resorption of medullary bone in birds, but apart from these situations, osteoclast "activation" is either an artifact of in vitro conditions or, more likely, the result of the prolongation of the precariously short lifespan of isolated osteoclasts in vitro. Osteoclast "activation" is sometimes referred to erroneously as the first step in the initiation of bone remodeling. Frost defined "activation" as the initial stimulus to cell proliferation. More recently, this term has been extended to include all the processes involved in converting a quiescent bone surface to a site of bone resorption. These processes definitely do not include osteoclast "activation" because multinucleated osteoclasts waiting to be activated are never observed adjacent to quiescent bone surfaces. All morphologic evidence indicates that adult human osteoclasts need to assemble at the right place and time on a bone surface, but once there, they simply get on with the job.

The absence of in vivo evidence for osteoclast "activation" notwithstanding, it is very difficult to reconcile the finding that iv RANK-L administration increased rapidly blood ionized calcium in mice with a pathogenetic role of RANK-L in glucocorticoid-induced osteoporosis in humans. The most likely purpose of bone remodeling is to prevent accumulation of old bone, and the primary purpose of osteoclasts and osteoblasts is to subserve the mechanical rather than the metabolic functions of the skeleton by means of growth, modeling, remodeling, and repair. It is possible, albeit hypothetical, that the remodeling system may have a short-term contribution to calcium homeostasis by a concertina-like relationship between resorption and formation within each remodeling unit so as to accommodate the normal circadian changes in external calcium balance (36). However, by and large, the regulation of bone volume and mass and the regulation of plasma calcium are independent, as low, normal, or high levels for plasma calcium can each coexist with low, normal or high values for bone resorption and turnover. Ionic exchange between bone and the extracellular fluid is most likely facilitated by a nonvascular transport system formed by the syncytium of osteocytes and lining cells. If one were to accept a relationship between RANK-ligand, osteoclast "activation," hypercalcemia, and glucocorticoid-induced osteoporosis, one would have to explain the fact that hypercalcemia is not a feature of glucocorticoid-excess; and for this matter, not a feature of Paget’s disease, a condition where osteoclast size, number, and erosion depth are dramatically increased. To the contrary, before the advent of more powerful agents, glucocorticoids were routinely used for the treatment of hypercalcemia. Finally, even if true, for the same reasoning described above for the putative effect of RANK-ligand/OPG in glucocorticoid-induced osteoclastogenesis, increased resorption by mature osteoclasts could only be a transient event.

In conclusion, the findings of Hofbauer et al. (30) suggest a new and intriguing set of putative players (RANK-ligand/OPG) in the list of biochemical culprits responsible for the pathogenesis of glucocorticoid-induced osteoporosis. The onus is now upon these workers to test what seems to be a testable hypothesis. The existing evidence indicates that the principal change in the osteoporosis resulting from chronic glucocorticoid-excess is decreased bone formation, caused by a suppressive effect on osteoblastogenesis and promotion of osteoblast and osteocyte apoptosis; and that if RANK-ligand and/or OPG play a role, this can only be a transient one. While, conceptually, changes in cell vigor cannot be dismissed a priori, to date there is no evidence for, nor is there a need to invoke, such changes in the pathogenesis of common involutional or acquired metabolic bone diseases, such as osteoporosis. For a regenerating organ such as the skeleton, changes in the cell number—determined by the rate of birth and death—are evidently what really matters.

Received July 21, 1999.

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