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Long Bone Growth: A Case of Crying Wolff?
Long Bone Growth: A Case of Crying Wolff?
The trajectory theory presents idealized structures formed in response to pure forces by an isolated bone whose only function is to resist those mechanical forces. Theoretical approaches which treat bones as idealized, isolated units … simply fall short of reality. A particular bone’s response to altered mechanical stress might be compromised by the simultaneous response of the attached muscles or connective tissue … by altered blood flow, by associated mineral requirements, etc … Murray foresaw this interactive view and elegantly expressed it in his concluding chapter (1936, pp. 177–180): ‘Every bony structure is a compromise and no compromise is perfect … It [the trajectorial theory] requires, not rejection, but dilution’ (p. 179). (Hall, 1985a, pp. xxvi–xxvii)
WOLFF, VON MEYER OR ROUX The reality of history is not always reflected in modernday accounts of how the architecture of bone reflects the forces imposed upon it. Wolff (1870, 1892) is credited with originating the trajectory theory relating bone structure to the mechanical forces imposed upon it, a theory now known as Wolff’s Law. But Julius Ward (1838) had already compared the architecture of the proximal portion of the femur with a bracket, and it was von Meyer not Wolff, who first proposed the theory in 1867 (Fig. 2.7). Wolff was to von Meyer’s trajectory theory as Huxley was to Darwin’s theory of evolution – advocate, defender and popularizer – although Lee and Taylor (1999) argue that Wolff misinterpreted the mechanical data and rejected any role for bone resorption in determination of bone structure (Fig. 32.1). Recognition of the strength of bone and of correlations between strength and the size and shape of bones is, of course, even older than Ward’s comparison of cranes and femora. Galileo (1638) was well aware of the
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additional strength that came from bones as tubes rather than as boxes. Following Ward’s observation that the architecture of the proximal portion of the human femur resembles a bracket (Fig. 31.1) studies of the mechanical properties of intact bone were carried out in the mid-1800s by Wertheim (1847), von Meyer (1867) and Rauber (1876). When Meyer compared the trabecular structure within bone with the stress to which the bone had been subjected, he found a correlation: the orientation of the trabeculae follows lines of stress (Figs 2.6 and 32.1). Meyer was aided in this analysis by the Swiss engineer Culmann (1866) but Wolff compared the architecture of the femur to the design used in constructing the Fairbairn steam crane.1 Wolff explicitly related bone structure to bone function in terms of the forces and loads imposed on living active bone. Development and dissemination of Wolff’s Law owes much to Roux (1885) and Koch (1917). In fact, it has been argued that Roux was the first to accurately describe the adaptation of bone to altered load, and that consequently ‘Wolff’s law’ should really be ‘Roux’s law.’2 Two 20th century restatements of Wolff’s Law follow. ●
●
‘Wheresoever stresses of pressure and tension are caused in a bone … formation of bone takes place’ (Jansen, 1920, p. 5). ‘The form of a bone being given, the bone elements place or displace themselves in the direction of functional pressure and increase or decrease their mass to reflect the amount of functional pressure’ (Bassett, 1968, p. 260).
But trabecular structure of cancellous bone is not merely a set of actualized trajectories and the trajectory theory has been modified in various ways, mostly to account for factors other than mechanical history that leave their
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Figure 32.1 Julius Wolff (1870) compared the stress trajectories in the Culmann crane (left) with the pattern of trabeculae in the proximal end of the human femur (right). See text for details. Modified from Wolff (1870) and Lee and Taylor (1999).
mark on bone structure. As one example, microcracks of the order of 50 10 m in length are an important stimulus for remodeling of loaded bone (Lee et al., 2002). Results from studies on CAM-grafted embryonic bones were used to argue that blood vessels and vascular influences, rather than mechanical factors, determine trabecular architecture (Box 12.3). From studies on patterns of force transmission in cat skulls, it was proposed that stresses were best visualized in functional units within the skull rather than in individual elements, one element compensating for forces on another. Venous stasis, which is known to accelerate both fracture repair and periosteal osteogenesis, has no effect on longitudinal growth of long bones in rabbits, other than a decline one or two days after surgery.3 I wrote the epigraph to this chapter in an overview introducing a reprinting of Bones by P. D. F. Murray. As Murray himself saw it, ‘every bony structure is a compromise and no compromise is perfect … [the trajectorial theory] requires, not rejection, but dilution’ (Murray, 1936, p. 179). Four detailed papers by Howard Frost (1990a–d) set out a major review of skeletal adaptation to mechanical usage and discuss modeling and remodeling of bone, cartilage and fibrous tissue. The most insightful recent analysis of Wolff’s Law also appeared in the early 1990s (Bertram and Swartz, 1991). A major concern and source of confusion concerning the differing responses of bone to what appear to be similar mechanical conditions is the use of different models and the underlying assumption that what is true for one bone/situation will be true for all – the universal and over-riding primacy of Wolff’s Law. John Bertram is especially concerned with differences between: load applied during bone development (when
the effect is primarily on differentiation and morphogenesis rather than on growth), load applied during growth, and load applied to adult, non-growing animals/bones. John considers that changes in adults – especially during fracture repair – are more properly attributed to inflammatory mediated processes than to Wolff’s law.4
RESPONSE TO PRESSURE There was much debate in the latter part of the 19th century over whether pressure stimulates or retards bone growth (Fig. 31.5). Because of the responsiveness of epiphyseal cartilage to pressure, Wolff maintained that growth was stimulated by pressure. Hueter and Volkmann took the opposite view. From both medico-surgical experience and anthropological studies, it was clear that pressure could initiate bone formation in situations where no cartilage was present, as in the production of Wormian bones in skulls deformed by binding.5 More recent studies from three groups take some of the strain out of the system by resolving the apparent paradox. Hert and Liskova, Denis Carter and his colleagues, and Lanyon and Rubin all provide compelling theoretical and experimental verification that the effects of pressure on bone growth vary with the magnitude of the pressure applied. Pressure beyond normal physiological limits inhibits bone growth. Relaxation of pressure stimulates growth. Perhaps more importantly, bone is – and bones are – especially adapted to respond to intermittent loading. Rapid transformation from quiescence to bone formation and increased synthetic activity can be recorded in
Long Bone Growth: A Case of Crying Wolff? osteocytes in adult periostea following a single brief period of bone loading.6
CONTINUOUS OR INTERMITTENT MECHANICAL STIMULI We stimulate our bones with every breath, bite or step we take. Respiration and locomotion produce cycles of bone deformation as tension and compression are applied alternately to vertebrae, ribs and long bones.7 In their studies on the responses of rabbit tibiae to mechanical stress, J. Hert and his colleagues emphasized the importance of intermittent loading if skeletal cells are to be activated. Because mechanical loading of the skeleton
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is normally intermittent, Hert and Zalud (1971) argued that piezoelectric models based on differential responses of cells to pressure or tension are inadequate. Instead, they proposed that skeletal cells sense local changes in electrical potential and ionic fluxes. A contrary view came from C. A. L. Bassett’s laboratory. Using in-vitro studies, Bassett et al. (1964) demonstrated that mechanical deformation of the apatite–collagen piezoelectrical junctions in bone matrix produces an electrical signal, which in turn elicits a cellular response, resulting in synthesis and oriented deposition of newly formed collagen fibrils.8 See Box 29.1 for a discussion of the efficacy of intermittent mechanical stimulation in promoting chondrogenesis in fracture repair, and Box 32.1 for the use of pulsed electromagnetic fields (PEMF) in initiating repair in persistent non-unions.
Box 32.1 Pulsed electromagnetic fields (PEMF) A substantial although not entirely concordant body of basic and clinical studies documents enhanced fracture repair following application of pulsed electromagnetic fields (PEMF), especially to initiate repair of persistent non-unions and the false joints (pseudarthroses, Fig. 29.2) that can form following prolonged misalignment and abnormal mechanical stresses (Box 29.2). The pioneering studies were initiated by the late Cal Bassett, then at Columbia University in New York.a It is unusual for clinical studies with humans to demonstrate more consistent enhanced healing than is found in studies with other animal models. For example, healing of fibula fractures in dogs was enhanced, and the mechanical quality of the bone improved, 28 days after application of 65-Hz pulses of PEMF. But when 15-Hz pulses were applied, there appeared to be no effect when the bones were examined two or six months later, nor did Law et al. (1985) see any improvement in sheep tibiae. In part this is because the wave form of PEMF is important: four different wave forms stimulate healing of fractures of the rodent radius (including increasing the tensile strength of the callus by up to 30 per cent), three other wave forms having no effect.b After Grace et al. (1998) applied 72-Hz square wave PEMF to an osteochondral defect in the patello-femoral groove of rats they observed enhanced early vascular invasion, early initiation of chondrogenesis, early initiation of osteogenesis and advanced restoration of normal trabecular architecture, concluding that PEMF advances the early phases of repair. However, they counsel against prolonged use – chondrogenesis can be prolonged to the stage where development of normal bone architecture is delayed – too much of a good thing too early. McLeod and Rubin (1989) determined that frequency bands of
75 Hz were optimal for bone adaptation, implying that bones are extremely sensitive to low-frequency electric fields. For comparison, the refresh rate of a good monitor operates at around 70–75 Hz/second and neural oscillators in the brain at 35–75 Hz. In an early clinical trial, Bassett reported the successful repair of 96 per cent of human tibial non-unions. In studies in other animals, pulsed electromagnetic fields applied to rats improved the effects of disuse osteoporosis and prevented congenital pseudarthroses; a pulse train of PEMF increased cellular calcium and promoted mineralization while a single pulse decreased cellular calcium but promoted bone formation, a desirable outcome in patients with osteoporosis or avascular necrosis.c Initially, many were skeptical about the efficacy of PEMF. Barker and Lunt (1983) cautioned about the total absence of controlled trials and the lack of experimental verification of effects caused by magnetic fields (see below). Smith and Nagel (1983) applied PEMF
to rabbit long bones for two hours/day for eight weeks and found no change in bone growth but did find a 22 per cent increase in articular cartilage glycosaminoglycans (GAGs). Of course, there was no fracture or non-union in these animals. These authors were unduly pessimistic. By 1982 Bassett could report that 5000 patients had been treated with PEMF, that PEMF was beginning to be used in dentistry, and that evidence had accumulated to show that PEMF provided a signal for chondrogenesis and for vascular invasion of cartilage, although not for osteogenesis per se. Studies with isolated cell populations bear this out: tibial chondroblasts from seven-day-old chick embryos react to continuous exposure to pulsed magnetic fields for seven days in vitro with reduced collagen synthesis, a less marked reduction in both acid mucopolysaccharide production and proliferation, but with enhanced mineralization. Rabbit costal chondrocytes respond to PEMF by enhanced responsiveness to parathyroid hormone and enhanced differentiation, while 16-day-old chick embryo tibial and sternal chondrocytes (and skin fibroblasts) respond to PEMF with enhanced synthesis of GAGs.d Ongoing investigations in both experimental and clinical situations continue to explore the efficacy of PEMF in such situations as: the healing of the flexor tendon in chickens, in which tension decreases and tendinous adhesions increase; a double-blind clinical trial in which 27 individuals with osteoarthritis of the knee were treated with PEMF over a month, with between 20 and 60 improvement in the parameters measured; and a study in which TGF-1 increased in cells from non-unions exposed to PEMF, while cell number, alkaline phosphatase activity, collagen synthesis, prostaglandin E-2 and osteocalcin production were unaffected.e a See Bassett et al. (1974a,b, 1977, 1978, 1979), Fitton-Jackson and Bassett (1980) and Goodman et al. (1983). For a review of electro-stimulation and bone-fracture repair, see Behari (1991). In what was perhaps their first study, Bassett and Herrmann (1968) showed that fibroblasts are sensitive to electrostatic fields, synthesis of collagen increasing by as much as 100 per cent. b See Bassett et al. (1974a,b) and Miller et al. (1984) for 65- and 15-Hz pulses, and Christel et al. (1980) for wave form. c See Bassett et al. (1978) for the early trial, L. S. Bassett et al. (1979) and C. A. L. Bassett et al. (1981a,b) for disuse osteoporosis and congenital pseudarthroses, and Goodman et al. (1983) for mineralization. d See Archer and Ratcliffe (1983) for tibial chondrocytes, Hiraki et al. (1987) for costal chondrocytes, and Norton et al. (1988) for enhanced GAG synthesis. e See Robotti et al. (1999) for chick flexor tendons, Trock et al. (1993) for the study of osteoarthritis, and Guerkov et al. (2001) for the study with non-union cells.
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SCALING AND VARIATION: WHEN WOLFF MEETS THE DWARFS Are rats ‘merely’ scaled-up mice and are dwarf (miniaturized) taxa ‘merely’ scaled down versions of their larger relatives, or does taxonomic diversification accompanied by size increase involve modification of the processes of development and growth? It has, for example, been argued that all members of the genus Australopithecus are variations of the same animal over a range of sizes, but here we are dealing with a single taxon. Similarly, an examination of the scaling of skeletal growth in six specimens of Archaeopteryx lithographica, all of which are sub-adult (Fig. 32.2), led to the twofold conclusions that these represent a single growth series of a single species.9 Going in the other direction, an allometric analysis of skull development in the common hippopotamus, Hippopotamus amphibius, and the pygmy hippopotamus, Hexaprotodon liberiensis, shows that the dwarf
species is not simply a scaled-down version of its larger relative (Weston, 2003). Regional variation in bone growth is related to local parameters of mechanical stress and strain, as demonstrated, for example, in the distal growth plates of rabbit femora, in which stress patterns correlate with growth rates, high compressive stress correlating with lower growth. These associations, however, explain only 15 per cent of the variation in growth rate. Responses to strenuous exercise also result from local loading effects, as determined in rat tibiae and metatarsals, just as total body size and regional variation within individual skeletal elements influence response to mechanical loading.10 van der Meulen and Carter (1995) modelled intrinsic longbone growth with extrinsic adaptive modeling to simulate allometric morphological relationships over the size range from mice to mammals. In this model, scaling is adaptive, not intrinsic. There are situations where Wolff’s law seems not to apply, especially in such small mammals as bats and shrews, where terrestrial locomotion is limited. For instance, no correlation between mechanical forces and bone structure was found in a sample of 94 femora from the short-tailed shrew, Blarina brevicauda, the little brown bat, Myotis lucifugus, and another bat, the eastern pipistrelle, Pipistrellus subflavus. All are small – the average weights of these animals are 8.6, 6.4 and 6.3 grams, respectively, very low for mammals; squirrels weigh around 900–1200 grams. As David Dawson concluded: ‘There is no evidence that bones in these diminutive mammals respond to mechanical forces, and the applicability of Wolff’s Law is not indicated … it is hypothesized that intrinsic tissue strength is sufficient to resist mechanical deformation, and femoral anatomy in these species is dictated by genetic and inherent physiologic conditions’ (Dawson, 1980, p. 1).11
GRAVITY
Figure 32.2 Archaeopteryx lithographica, approximately one-third natural size. From Romanes (1901).
Many have investigated the skeletons of numerous species under varied conditions of micro- or zero gravity, including space flight. If one can generalize from the data obtained, it would appear that bone formation is inhibited during space flight – there is complete cessation of periosteal bone formation in rats, for example – but that resorption is unchanged. Increased gravity increases resorption of mouse calvarial bone, and more Ca is released (Gazit, 1980). In a study on the effects of 18.5 days of space flight on trabecular bone in rats (Table 32.1), fat mass increased while mineralized tissue mass declined. Osteoblast numbers declined but osteoclast numbers remained unaffected, confirming the effect on formation, not resorption, and demonstrating an uncoupling of the normal tight association between formation and resorption, a coupling that is influenced by mechanical conditions. By 29 days
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Long Bone Growth: A Case of Crying Wolff? Table 32.1 Effects of spaceflight on periosteal formation, periosteal apposition, and length of arrest lines in rat tibiaea Rats
Mean periosteal formation rate (10 3 mm3/day)
Mean periosteal apposition rate (10 3 mm3/day)
Bone
Matrix
Bone
Matrix
Flight Flight controlb Vivarium controlc
9.4 2.8 15.8 1.5 16.0 1.4
Flight period 7.2 2.8 13.8 1.4 14.0 1.4
1.3 0.4 2.2 0.2 1.8 0.2
1.0 0.4 1.9 0.2 2.3 0.2
Flight Flight control Vivarium control
17.1 2.2
Post-flight period 17.8 2.1 2.2 0.2
2.3 0.2
11.3 1.4
11.2 0.4
1.4 0.2
1.4 1.4
Arrest line length (mm)
5.3 0.6 2.1 0.6 1.5 0.7
a
Based on data from Morey and Baylink (1978). Controls were housed in an identical, ground-based spacecraft for the duration of the experiment. c Controls were housed in standard cages and animal quarters. b
post-flight most parameters had returned to normal. Although mineral metabolism is disrupted in zero gravity, there is a six- to nine-month musculoskeletal ‘safety period’ for humans in space. Any bone or mineral loss during this period is reversed upon returning to earth’s gravity.12 A sample of studies follows, mostly representative of results obtained on Kosmos or NASA flights. (i) Rats maintained in prolonged hypogravity in the 1975 US–USSR Kosmos Space Laboratory suffered substantial bone loss (Asling, 1977). (ii) Growing rats given tetracycline to label bone growth, and then flown on board Kosmos 782 and Kosmos 936 for 19 days, showed defects at the periosteal surfaces of the tibial diaphyses, consisting of a 3 m arrest line, hypomineralized matrix and abnormal collagen orientation (Turner et al., 1985). (iii) Five days of weightlessness on Kosmos 1514 increased osteoclast numbers in pregnant rats and so decreased bone mass via excess resorption (Vico et al., 1987). (iv) Kosmos 2044 involved a 13.8-day flight, and a 10-hour recovery that proved to be sufficient time to replenish preosteoblasts in the rat maxillary periodontal ligament lost during the flight (Garetto et al., 1992). (v) Teeth are not immune to the effects of gravity. Rat incisor dentine showed a 10–15 per cent increase in Ca, a 20–30 per cent increase in PO43 and abnormal distribution of GAGs after 18.5 days on board Kosmos 1129 (Rosenberg et al., 1984). (vi) Growing rat bones were examined in NASA Space Lab 3 after one week in space. Levels of osteocalcin, calcium and hydroxyproline had all declined in vertebrae and humeri. A one-week space flight reduced the growth rates of humeri but not tibiae of growing rats, but resulted in tibiae that were weaker, as assessed by a three-point bending test (Patterson-Buckendahl et al., 1987; S. R. Shaw et al., 1988).13
Both weight-bearing and non-weight-bearing elements of the skeleton respond to weightlessness with decreased histogenesis, as demonstrated in a comparative study of periodontal ligament and tibial metaphysis in SpragueDawley rats in simulated weightlessness and in the nonweight-bearing portions of the rat mandible. Scleral ossicles, which are non-weight-bearing, developed normally in quail embryos flown on the Mir US–Russian joint project.14 Developing skeletons also are responsive to increased gravity. Mouse limb buds exposed in vitro to 2.6 g in a centrifuge show a proximo-distal gradient of sensitivity, the proximal mesenchyme being less sensitive. Indeed, proximal elements present at the beginning of the experiment disappeared, while the differentiation of distal elements was accelerated. Pre-metatarsal elements at an early developmental stage produced cartilage rods when flown in the space shuttle, suggesting normal differentiation.15 Studies on humans in space are relevant to the skeletal loss consequent to prolonged bed rest, menopause (one per cent and more loss of skeletal mass/year) or in middle to old age (during which 15 per cent of skeletal mass is lost).16 During the manned space flights, astronauts lost 3–15 per cent of their heel-bone density (Gemini) and 0.2 per cent of body calcium (Apollo) (Goode and Rambaut, 1985). In a review of the consequences of space flight published in 1986, Wheldon concluded that bone loss in space parallels bone loss during bed rest or in individuals with polio, and is of the order of 0.4 per cent of body calcium/month. Bone density does not return to normal if the duration of the space flight is prolonged, presumably because loss isolates bone spicules or trabeculae to the point of no recovery. Other approaches may enable us to address these issues; rats maintained under conditions of 2.76 or 4.15 g for prolonged periods (810 days in this case) show effects similar to those seen after immobilization (Table 32.2).
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Bones and Cartilage: Developmental and Evolutionary Skeletal Biology
Table 32.2 Rats maintained under increased gravity conditions (2.76 or 4.15 g) for 810 days show an effect similar to that seen after immobilizationa Parameters
Gravity of 2.76 g
Gravity of 4.15 g
Body weight Femur length Cross-section of femur
19.3 6.6 15.5
29.1 9 19.1
a
Results shown as % of control values.
TRANSDUCTION OF MECHANICAL STIMULI Despite the studies discussed above and others, we do not fully understand how the skeleton responds to mechanical stimulation, although cAMP as a mediator of prechondroblast proliferation to conform with changing mechanical condition represents an important feedback control system (Chapter 31). Muscles can and do insert onto resorptive surfaces on bone so that mechanical action can stimulate resorption and deposition directly (Figs 35.2 and 35.4). One potential mechanism receiving wide support in the 1970s was translation of mechanical stimuli into bioelectrical activity to which skeletal cells could respond. I discuss this approach and then turn to three other approaches.17 There is no doubt but that electrical currents in the amp range influence the proliferation, differentiation and activity of skeletal cells, although a mechanistic relationship between mechanical stimulation and bioelectrical potential is elusive. Empirical evidence for the efficacy of exposure to electric current/fields includes: ●
●
●
●
●
●
enhanced chondrogenesis and acceleration of the growth of epiphyseal cartilage and bone following direct application of an electrical field; acceleration of the proliferation of induced cartilage following application of a 5 A current to muscle cultured with Bmp – the cartilage having been induced by Bmp; promotion of precocious initiation of mineralization by hypertrophic chondrocytes following 10 A DC current applied in vitro to femora from nine-day-old chick embryos, at which age the cartilage is unmineralized; enhancement of osteogenesis in chick tibiae and mouse calvariae exposed in vitro to as little as 10 5 V/m for 30 minutes/day; promotion of osteogenesis in foetal rat long bones in response to direct or pulsating current in vitro; and alteration of oxygen tension in the immediate vicinity of skeletal cells.18
Bone is deposited in regions of electro-negativity. Healing fractures respond to low-voltage electric fields with enhanced repair. For example, new bone is deposited around an active electrode implanted in the medullary canal of rabbits, but not around an inactive
electrode. Indeed, bone disappears around the inactive electrode but can be regained if current is allowed to flow. The new bone forms from polymorphic perivascular cells associated with invading blood vessels. Mineral is differentially deposited around the electrodes – mineralizing collagen around both active and inactive electrodes, and degenerating cells around the inactive electrode (Brighton and Hunt, 1986). In studies testing skeletal responsiveness to electrical stimuli, as in all experimental studies, adequate controls must be used. Inserting the wire into medullary canals or marrow in vivo can enhance osteogenesis, even with no current flowing through the wire. The effect is not simply the result of trauma – inserting and removing a wire does not enhance osteogenesis – indicating an effect of the wire itself. Furthermore, the authors of these studies found that electrical and mechanical stimuli were additive. Not all mechanical stimuli are perceived by cells as electrical change.19
NOTES 1. The Fairbairn steam crane built in 1875 for the dockyard at Bristol, designed by the Scottish engineer William Fairbairn (1789–1874) to lift 35 tons from a height of 115 feet, was in continuous operation at the dockyards until 1973. Now refurbished, the crane can be visited as part of the Bristol dockyard restoration. Fairbairn operated a shipyard in London where the first iron-hulled steamship, the Lord Dundas, was built. He conceived using tubular steel for construction when assisting Robert Louis Stephenson in the construction of the Conway and the Menai Strait bridges in North Wales. 2. Wolff’s classic 1892 study was reprinted in translation by Maquet and Furlong (1986). See Lee and Taylor (1999) for Roux’s Law. Benninghoff (1925, 1930) performed some of the more detailed analyses on trabecular fibre structure (Fig. 2.6); Murray (1936) and Altmann (1964) provide thorough evaluation of these studies. Glücksmann (1938) used in-vitro cultivation of skeletal elements to demonstrate the influence of pressure on structural orientation, while Murray and Selby (1930), Murray (1936) and Altmann (1964) provide what are still among the best accounts of the early literature on the response of the skeleton to mechanical forces. For later studies, see Pauwels (1973), and see Oxnard (1991) for an insightful analysis of the three-dimensional architecture – the morphanalysis – of bone. 3. See Hancox (1947) for the CAM-grafts, Buckland-Wright (1978) for the functional unit approach, and Hansson et al. (1975) for venous stasis. Under conditions of prolonged venous status, as when the inferior vena cava is ligated, osteocytes dedifferentiate to chondrocytes as areas of bone cells transform from acidophilic to basophilic and are replaced by an island of cartilage cells (Abdalla and Harrison, 1966). 4. See also Bertram (2001) for an analysis of how the trabecular bone immediately beneath the articular cartilage of mammalian long bones may attenuate loading of both bone and cartilage. I am grateful to John Bertram for his analyses and his
Long Bone Growth: A Case of Crying Wolff?
5.
6.
7. 8.
9.
10.
11.
12.
13.
comments on this chapter. John considers that growth ‘appears to have a strong element of determination (however that is mediated), whereas the modifications of mature bone appear to have some stringent limitations’ (pers. comm.). See Wolff (1892), Hueter (1862, 1863) and Volkmann (1862) for pressure and epiphyseal cartilages, and Dorsey (1897) for Wormian bones. See Hert (1964a–c, 1969), Hert and Liskova (1964), Lanyon and Rubin (1984), Carter (1987), Carter et al. (1987), Rubin and Lanyon (1987), Carter and Wong (1988), Rubin et al. (1989), Wong and Carter (1990), Carter et al. (1991, 1996) and Carter and Orr (1992). See Pead et al. (1988a,b) for rapid response to brief loading. See Lanyon (1972), Lanyon et al. (1975) and Piekarski and Munro (1977) for data. See Hert et al. (1969, 1971a,b, 1972a,b), Liskova and Hert (1971) and Chamay and Tschantz (1972). For the contrary view, see Bassett and Becker (1962), Becker et al. (1964), Bassett (1968), Bassett and Pawluk (1972) and Becker (1978). For mechanical loading of tibial periostea stimulating rapid changes in periosteal gene expression, see Raab-Cullen et al. (1994). See Nonaka et al. (1993) and Sasaki et al. (1994a,b, 1995) for uterine transplants, Pilbeam and Gould (1974) for australopithecines, and Houck et al. (1990) for Archaeopteryx. See Lerner et al. (1998) and Li et al. (1991) for these rat and rabbit studies, and see Felts and Spurell (1965, 1966) for analyses of long-bone structure in cetaceans. The selective pressure for evolution of the distinctive interdigital membrane in bat wings seems to have been elongation of the metacarpal bone (Kovtun, 1985). See Adams (1998) for the developmental and functional integration of the elements of bat wings. See Morey and Baylink (1978) for the general statement, Whedon et al. (1976) for mineral metabolism, Jee et al. (1983) for trabecular bone, and Whedon and Heaney (1993) for an overview of the effects of weightlessness, paralysis and inactivity on bone growth. John Bertram commented on a ‘rat-in-space’ study in which there was a decrease in the deposition of periosteal bone in the dentary (which is not a weight-bearing bone) and that
14. 15.
16.
17.
18.
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the decrease occurred in areas not associated with muscle attachment as well as where muscles attach, the implication being that some effects of zero gravity may be systemic (Bertram, pers. comm.). See Fielder et al. (1986), Simmons et al. (1983a) and Barrett et al. (2000) for these three studies. See Duke (1983) for excess gravity, Klement and Spooner (1994) for the space shuttle, and Klement et al. (2004) for subsequent studies using the NASA rotating wall vessel which simulates microgravity by randomizing the direction of the gravitational forces on cells. This is because immobilization, more or less whatever the cause, leads to hypoplasia of bone. As one example, four weeks after unilateral resection of the sciatic nerve to the rat femur, periosteal (cortical) bone and ash content are both reduced, the latter indicative of reduced mineral content (Pennock et al., 1972). See Brash (1924, 1934) and Hoyte and Enlow (1966) for stimulation of resorption and deposition, and see the reviews by Bassett (1972b), Marino and Becker (1977), Spadaro (1977), Hall (1983f), and chapters in the volumes edited by Liboff and Rinaldi (1974), Attinger and Parakkal (1977), Brighton et al. (1979) and Connolly (1981). See Watson et al. (1975), Brighton et al. (1976) and Norton et al. (1977) for cartilage, Norton and Moore (1972), Spadaro (1982) and Noda and Satoh (1985b) for bone, Nogami et al. (1982) for proliferation of induced cartilage, Noda and Sato (1985a) for precocious mineralization, Fitzsimmons et al. (1986) for tibiae and calvariae in vitro, Theharne et al. (1980) for direct or pulsating current, and Brighton and Friedenberg (1974) and Brighton et al. (1975) for enhanced local O2 tension. Electrical stimulation enhances bone and muscle mass in paraplegics (Pacy et al., 1988). See Spadaro et al. (1986) and Schaberg et al. (1985) for these studies, and see the volumes edited by Brighton et al. (1979), and Connolly (1981) and Hall (1983f) for the electrical properties of bone and cartilage as seen in both experimental and clinical studies. Becker (1972b) applied electrical currents of 3–6 nA to the amputated limbs of 15 rats and obtained a high percentage of regeneration and formation of epiphyseal growth centres.
The trajectory theory presents idealized structures formed in response to pure forces by an isolated bone whose only function is to resist those mechanical forces. Theoretical approaches which treat bones as idealized, isolated units … simply fall short of reality. A particular bone’s response to altered mechanical stress might be compromised by the simultaneous response of the attached muscles or connective tissue … by altered blood flow, by associated mineral requirements, etc … Murray foresaw this interactive view and elegantly expressed it in his concluding chapter (1936, pp. 177–180): ‘Every bony structure is a compromise and no compromise is perfect … It [the trajectorial theory] requires, not rejection, but dilution’ (p. 179). (Hall, 1985a, pp. xxvi–xxvii)
WOLFF, VON MEYER OR ROUX The reality of history is not always reflected in modernday accounts of how the architecture of bone reflects the forces imposed upon it. Wolff (1870, 1892) is credited with originating the trajectory theory relating bone structure to the mechanical forces imposed upon it, a theory now known as Wolff’s Law. But Julius Ward (1838) had already compared the architecture of the proximal portion of the femur with a bracket, and it was von Meyer not Wolff, who first proposed the theory in 1867 (Fig. 2.7). Wolff was to von Meyer’s trajectory theory as Huxley was to Darwin’s theory of evolution – advocate, defender and popularizer – although Lee and Taylor (1999) argue that Wolff misinterpreted the mechanical data and rejected any role for bone resorption in determination of bone structure (Fig. 32.1). Recognition of the strength of bone and of correlations between strength and the size and shape of bones is, of course, even older than Ward’s comparison of cranes and femora. Galileo (1638) was well aware of the
Chapter
32
additional strength that came from bones as tubes rather than as boxes. Following Ward’s observation that the architecture of the proximal portion of the human femur resembles a bracket (Fig. 31.1) studies of the mechanical properties of intact bone were carried out in the mid-1800s by Wertheim (1847), von Meyer (1867) and Rauber (1876). When Meyer compared the trabecular structure within bone with the stress to which the bone had been subjected, he found a correlation: the orientation of the trabeculae follows lines of stress (Figs 2.6 and 32.1). Meyer was aided in this analysis by the Swiss engineer Culmann (1866) but Wolff compared the architecture of the femur to the design used in constructing the Fairbairn steam crane.1 Wolff explicitly related bone structure to bone function in terms of the forces and loads imposed on living active bone. Development and dissemination of Wolff’s Law owes much to Roux (1885) and Koch (1917). In fact, it has been argued that Roux was the first to accurately describe the adaptation of bone to altered load, and that consequently ‘Wolff’s law’ should really be ‘Roux’s law.’2 Two 20th century restatements of Wolff’s Law follow. ●
●
‘Wheresoever stresses of pressure and tension are caused in a bone … formation of bone takes place’ (Jansen, 1920, p. 5). ‘The form of a bone being given, the bone elements place or displace themselves in the direction of functional pressure and increase or decrease their mass to reflect the amount of functional pressure’ (Bassett, 1968, p. 260).
But trabecular structure of cancellous bone is not merely a set of actualized trajectories and the trajectory theory has been modified in various ways, mostly to account for factors other than mechanical history that leave their
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Figure 32.1 Julius Wolff (1870) compared the stress trajectories in the Culmann crane (left) with the pattern of trabeculae in the proximal end of the human femur (right). See text for details. Modified from Wolff (1870) and Lee and Taylor (1999).
mark on bone structure. As one example, microcracks of the order of 50 10 m in length are an important stimulus for remodeling of loaded bone (Lee et al., 2002). Results from studies on CAM-grafted embryonic bones were used to argue that blood vessels and vascular influences, rather than mechanical factors, determine trabecular architecture (Box 12.3). From studies on patterns of force transmission in cat skulls, it was proposed that stresses were best visualized in functional units within the skull rather than in individual elements, one element compensating for forces on another. Venous stasis, which is known to accelerate both fracture repair and periosteal osteogenesis, has no effect on longitudinal growth of long bones in rabbits, other than a decline one or two days after surgery.3 I wrote the epigraph to this chapter in an overview introducing a reprinting of Bones by P. D. F. Murray. As Murray himself saw it, ‘every bony structure is a compromise and no compromise is perfect … [the trajectorial theory] requires, not rejection, but dilution’ (Murray, 1936, p. 179). Four detailed papers by Howard Frost (1990a–d) set out a major review of skeletal adaptation to mechanical usage and discuss modeling and remodeling of bone, cartilage and fibrous tissue. The most insightful recent analysis of Wolff’s Law also appeared in the early 1990s (Bertram and Swartz, 1991). A major concern and source of confusion concerning the differing responses of bone to what appear to be similar mechanical conditions is the use of different models and the underlying assumption that what is true for one bone/situation will be true for all – the universal and over-riding primacy of Wolff’s Law. John Bertram is especially concerned with differences between: load applied during bone development (when
the effect is primarily on differentiation and morphogenesis rather than on growth), load applied during growth, and load applied to adult, non-growing animals/bones. John considers that changes in adults – especially during fracture repair – are more properly attributed to inflammatory mediated processes than to Wolff’s law.4
RESPONSE TO PRESSURE There was much debate in the latter part of the 19th century over whether pressure stimulates or retards bone growth (Fig. 31.5). Because of the responsiveness of epiphyseal cartilage to pressure, Wolff maintained that growth was stimulated by pressure. Hueter and Volkmann took the opposite view. From both medico-surgical experience and anthropological studies, it was clear that pressure could initiate bone formation in situations where no cartilage was present, as in the production of Wormian bones in skulls deformed by binding.5 More recent studies from three groups take some of the strain out of the system by resolving the apparent paradox. Hert and Liskova, Denis Carter and his colleagues, and Lanyon and Rubin all provide compelling theoretical and experimental verification that the effects of pressure on bone growth vary with the magnitude of the pressure applied. Pressure beyond normal physiological limits inhibits bone growth. Relaxation of pressure stimulates growth. Perhaps more importantly, bone is – and bones are – especially adapted to respond to intermittent loading. Rapid transformation from quiescence to bone formation and increased synthetic activity can be recorded in
Long Bone Growth: A Case of Crying Wolff? osteocytes in adult periostea following a single brief period of bone loading.6
CONTINUOUS OR INTERMITTENT MECHANICAL STIMULI We stimulate our bones with every breath, bite or step we take. Respiration and locomotion produce cycles of bone deformation as tension and compression are applied alternately to vertebrae, ribs and long bones.7 In their studies on the responses of rabbit tibiae to mechanical stress, J. Hert and his colleagues emphasized the importance of intermittent loading if skeletal cells are to be activated. Because mechanical loading of the skeleton
411
is normally intermittent, Hert and Zalud (1971) argued that piezoelectric models based on differential responses of cells to pressure or tension are inadequate. Instead, they proposed that skeletal cells sense local changes in electrical potential and ionic fluxes. A contrary view came from C. A. L. Bassett’s laboratory. Using in-vitro studies, Bassett et al. (1964) demonstrated that mechanical deformation of the apatite–collagen piezoelectrical junctions in bone matrix produces an electrical signal, which in turn elicits a cellular response, resulting in synthesis and oriented deposition of newly formed collagen fibrils.8 See Box 29.1 for a discussion of the efficacy of intermittent mechanical stimulation in promoting chondrogenesis in fracture repair, and Box 32.1 for the use of pulsed electromagnetic fields (PEMF) in initiating repair in persistent non-unions.
Box 32.1 Pulsed electromagnetic fields (PEMF) A substantial although not entirely concordant body of basic and clinical studies documents enhanced fracture repair following application of pulsed electromagnetic fields (PEMF), especially to initiate repair of persistent non-unions and the false joints (pseudarthroses, Fig. 29.2) that can form following prolonged misalignment and abnormal mechanical stresses (Box 29.2). The pioneering studies were initiated by the late Cal Bassett, then at Columbia University in New York.a It is unusual for clinical studies with humans to demonstrate more consistent enhanced healing than is found in studies with other animal models. For example, healing of fibula fractures in dogs was enhanced, and the mechanical quality of the bone improved, 28 days after application of 65-Hz pulses of PEMF. But when 15-Hz pulses were applied, there appeared to be no effect when the bones were examined two or six months later, nor did Law et al. (1985) see any improvement in sheep tibiae. In part this is because the wave form of PEMF is important: four different wave forms stimulate healing of fractures of the rodent radius (including increasing the tensile strength of the callus by up to 30 per cent), three other wave forms having no effect.b After Grace et al. (1998) applied 72-Hz square wave PEMF to an osteochondral defect in the patello-femoral groove of rats they observed enhanced early vascular invasion, early initiation of chondrogenesis, early initiation of osteogenesis and advanced restoration of normal trabecular architecture, concluding that PEMF advances the early phases of repair. However, they counsel against prolonged use – chondrogenesis can be prolonged to the stage where development of normal bone architecture is delayed – too much of a good thing too early. McLeod and Rubin (1989) determined that frequency bands of
75 Hz were optimal for bone adaptation, implying that bones are extremely sensitive to low-frequency electric fields. For comparison, the refresh rate of a good monitor operates at around 70–75 Hz/second and neural oscillators in the brain at 35–75 Hz. In an early clinical trial, Bassett reported the successful repair of 96 per cent of human tibial non-unions. In studies in other animals, pulsed electromagnetic fields applied to rats improved the effects of disuse osteoporosis and prevented congenital pseudarthroses; a pulse train of PEMF increased cellular calcium and promoted mineralization while a single pulse decreased cellular calcium but promoted bone formation, a desirable outcome in patients with osteoporosis or avascular necrosis.c Initially, many were skeptical about the efficacy of PEMF. Barker and Lunt (1983) cautioned about the total absence of controlled trials and the lack of experimental verification of effects caused by magnetic fields (see below). Smith and Nagel (1983) applied PEMF
to rabbit long bones for two hours/day for eight weeks and found no change in bone growth but did find a 22 per cent increase in articular cartilage glycosaminoglycans (GAGs). Of course, there was no fracture or non-union in these animals. These authors were unduly pessimistic. By 1982 Bassett could report that 5000 patients had been treated with PEMF, that PEMF was beginning to be used in dentistry, and that evidence had accumulated to show that PEMF provided a signal for chondrogenesis and for vascular invasion of cartilage, although not for osteogenesis per se. Studies with isolated cell populations bear this out: tibial chondroblasts from seven-day-old chick embryos react to continuous exposure to pulsed magnetic fields for seven days in vitro with reduced collagen synthesis, a less marked reduction in both acid mucopolysaccharide production and proliferation, but with enhanced mineralization. Rabbit costal chondrocytes respond to PEMF by enhanced responsiveness to parathyroid hormone and enhanced differentiation, while 16-day-old chick embryo tibial and sternal chondrocytes (and skin fibroblasts) respond to PEMF with enhanced synthesis of GAGs.d Ongoing investigations in both experimental and clinical situations continue to explore the efficacy of PEMF in such situations as: the healing of the flexor tendon in chickens, in which tension decreases and tendinous adhesions increase; a double-blind clinical trial in which 27 individuals with osteoarthritis of the knee were treated with PEMF over a month, with between 20 and 60 improvement in the parameters measured; and a study in which TGF-1 increased in cells from non-unions exposed to PEMF, while cell number, alkaline phosphatase activity, collagen synthesis, prostaglandin E-2 and osteocalcin production were unaffected.e a See Bassett et al. (1974a,b, 1977, 1978, 1979), Fitton-Jackson and Bassett (1980) and Goodman et al. (1983). For a review of electro-stimulation and bone-fracture repair, see Behari (1991). In what was perhaps their first study, Bassett and Herrmann (1968) showed that fibroblasts are sensitive to electrostatic fields, synthesis of collagen increasing by as much as 100 per cent. b See Bassett et al. (1974a,b) and Miller et al. (1984) for 65- and 15-Hz pulses, and Christel et al. (1980) for wave form. c See Bassett et al. (1978) for the early trial, L. S. Bassett et al. (1979) and C. A. L. Bassett et al. (1981a,b) for disuse osteoporosis and congenital pseudarthroses, and Goodman et al. (1983) for mineralization. d See Archer and Ratcliffe (1983) for tibial chondrocytes, Hiraki et al. (1987) for costal chondrocytes, and Norton et al. (1988) for enhanced GAG synthesis. e See Robotti et al. (1999) for chick flexor tendons, Trock et al. (1993) for the study of osteoarthritis, and Guerkov et al. (2001) for the study with non-union cells.
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SCALING AND VARIATION: WHEN WOLFF MEETS THE DWARFS Are rats ‘merely’ scaled-up mice and are dwarf (miniaturized) taxa ‘merely’ scaled down versions of their larger relatives, or does taxonomic diversification accompanied by size increase involve modification of the processes of development and growth? It has, for example, been argued that all members of the genus Australopithecus are variations of the same animal over a range of sizes, but here we are dealing with a single taxon. Similarly, an examination of the scaling of skeletal growth in six specimens of Archaeopteryx lithographica, all of which are sub-adult (Fig. 32.2), led to the twofold conclusions that these represent a single growth series of a single species.9 Going in the other direction, an allometric analysis of skull development in the common hippopotamus, Hippopotamus amphibius, and the pygmy hippopotamus, Hexaprotodon liberiensis, shows that the dwarf
species is not simply a scaled-down version of its larger relative (Weston, 2003). Regional variation in bone growth is related to local parameters of mechanical stress and strain, as demonstrated, for example, in the distal growth plates of rabbit femora, in which stress patterns correlate with growth rates, high compressive stress correlating with lower growth. These associations, however, explain only 15 per cent of the variation in growth rate. Responses to strenuous exercise also result from local loading effects, as determined in rat tibiae and metatarsals, just as total body size and regional variation within individual skeletal elements influence response to mechanical loading.10 van der Meulen and Carter (1995) modelled intrinsic longbone growth with extrinsic adaptive modeling to simulate allometric morphological relationships over the size range from mice to mammals. In this model, scaling is adaptive, not intrinsic. There are situations where Wolff’s law seems not to apply, especially in such small mammals as bats and shrews, where terrestrial locomotion is limited. For instance, no correlation between mechanical forces and bone structure was found in a sample of 94 femora from the short-tailed shrew, Blarina brevicauda, the little brown bat, Myotis lucifugus, and another bat, the eastern pipistrelle, Pipistrellus subflavus. All are small – the average weights of these animals are 8.6, 6.4 and 6.3 grams, respectively, very low for mammals; squirrels weigh around 900–1200 grams. As David Dawson concluded: ‘There is no evidence that bones in these diminutive mammals respond to mechanical forces, and the applicability of Wolff’s Law is not indicated … it is hypothesized that intrinsic tissue strength is sufficient to resist mechanical deformation, and femoral anatomy in these species is dictated by genetic and inherent physiologic conditions’ (Dawson, 1980, p. 1).11
GRAVITY
Figure 32.2 Archaeopteryx lithographica, approximately one-third natural size. From Romanes (1901).
Many have investigated the skeletons of numerous species under varied conditions of micro- or zero gravity, including space flight. If one can generalize from the data obtained, it would appear that bone formation is inhibited during space flight – there is complete cessation of periosteal bone formation in rats, for example – but that resorption is unchanged. Increased gravity increases resorption of mouse calvarial bone, and more Ca is released (Gazit, 1980). In a study on the effects of 18.5 days of space flight on trabecular bone in rats (Table 32.1), fat mass increased while mineralized tissue mass declined. Osteoblast numbers declined but osteoclast numbers remained unaffected, confirming the effect on formation, not resorption, and demonstrating an uncoupling of the normal tight association between formation and resorption, a coupling that is influenced by mechanical conditions. By 29 days
413
Long Bone Growth: A Case of Crying Wolff? Table 32.1 Effects of spaceflight on periosteal formation, periosteal apposition, and length of arrest lines in rat tibiaea Rats
Mean periosteal formation rate (10 3 mm3/day)
Mean periosteal apposition rate (10 3 mm3/day)
Bone
Matrix
Bone
Matrix
Flight Flight controlb Vivarium controlc
9.4 2.8 15.8 1.5 16.0 1.4
Flight period 7.2 2.8 13.8 1.4 14.0 1.4
1.3 0.4 2.2 0.2 1.8 0.2
1.0 0.4 1.9 0.2 2.3 0.2
Flight Flight control Vivarium control
17.1 2.2
Post-flight period 17.8 2.1 2.2 0.2
2.3 0.2
11.3 1.4
11.2 0.4
1.4 0.2
1.4 1.4
Arrest line length (mm)
5.3 0.6 2.1 0.6 1.5 0.7
a
Based on data from Morey and Baylink (1978). Controls were housed in an identical, ground-based spacecraft for the duration of the experiment. c Controls were housed in standard cages and animal quarters. b
post-flight most parameters had returned to normal. Although mineral metabolism is disrupted in zero gravity, there is a six- to nine-month musculoskeletal ‘safety period’ for humans in space. Any bone or mineral loss during this period is reversed upon returning to earth’s gravity.12 A sample of studies follows, mostly representative of results obtained on Kosmos or NASA flights. (i) Rats maintained in prolonged hypogravity in the 1975 US–USSR Kosmos Space Laboratory suffered substantial bone loss (Asling, 1977). (ii) Growing rats given tetracycline to label bone growth, and then flown on board Kosmos 782 and Kosmos 936 for 19 days, showed defects at the periosteal surfaces of the tibial diaphyses, consisting of a 3 m arrest line, hypomineralized matrix and abnormal collagen orientation (Turner et al., 1985). (iii) Five days of weightlessness on Kosmos 1514 increased osteoclast numbers in pregnant rats and so decreased bone mass via excess resorption (Vico et al., 1987). (iv) Kosmos 2044 involved a 13.8-day flight, and a 10-hour recovery that proved to be sufficient time to replenish preosteoblasts in the rat maxillary periodontal ligament lost during the flight (Garetto et al., 1992). (v) Teeth are not immune to the effects of gravity. Rat incisor dentine showed a 10–15 per cent increase in Ca, a 20–30 per cent increase in PO43 and abnormal distribution of GAGs after 18.5 days on board Kosmos 1129 (Rosenberg et al., 1984). (vi) Growing rat bones were examined in NASA Space Lab 3 after one week in space. Levels of osteocalcin, calcium and hydroxyproline had all declined in vertebrae and humeri. A one-week space flight reduced the growth rates of humeri but not tibiae of growing rats, but resulted in tibiae that were weaker, as assessed by a three-point bending test (Patterson-Buckendahl et al., 1987; S. R. Shaw et al., 1988).13
Both weight-bearing and non-weight-bearing elements of the skeleton respond to weightlessness with decreased histogenesis, as demonstrated in a comparative study of periodontal ligament and tibial metaphysis in SpragueDawley rats in simulated weightlessness and in the nonweight-bearing portions of the rat mandible. Scleral ossicles, which are non-weight-bearing, developed normally in quail embryos flown on the Mir US–Russian joint project.14 Developing skeletons also are responsive to increased gravity. Mouse limb buds exposed in vitro to 2.6 g in a centrifuge show a proximo-distal gradient of sensitivity, the proximal mesenchyme being less sensitive. Indeed, proximal elements present at the beginning of the experiment disappeared, while the differentiation of distal elements was accelerated. Pre-metatarsal elements at an early developmental stage produced cartilage rods when flown in the space shuttle, suggesting normal differentiation.15 Studies on humans in space are relevant to the skeletal loss consequent to prolonged bed rest, menopause (one per cent and more loss of skeletal mass/year) or in middle to old age (during which 15 per cent of skeletal mass is lost).16 During the manned space flights, astronauts lost 3–15 per cent of their heel-bone density (Gemini) and 0.2 per cent of body calcium (Apollo) (Goode and Rambaut, 1985). In a review of the consequences of space flight published in 1986, Wheldon concluded that bone loss in space parallels bone loss during bed rest or in individuals with polio, and is of the order of 0.4 per cent of body calcium/month. Bone density does not return to normal if the duration of the space flight is prolonged, presumably because loss isolates bone spicules or trabeculae to the point of no recovery. Other approaches may enable us to address these issues; rats maintained under conditions of 2.76 or 4.15 g for prolonged periods (810 days in this case) show effects similar to those seen after immobilization (Table 32.2).
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Table 32.2 Rats maintained under increased gravity conditions (2.76 or 4.15 g) for 810 days show an effect similar to that seen after immobilizationa Parameters
Gravity of 2.76 g
Gravity of 4.15 g
Body weight Femur length Cross-section of femur
19.3 6.6 15.5
29.1 9 19.1
a
Results shown as % of control values.
TRANSDUCTION OF MECHANICAL STIMULI Despite the studies discussed above and others, we do not fully understand how the skeleton responds to mechanical stimulation, although cAMP as a mediator of prechondroblast proliferation to conform with changing mechanical condition represents an important feedback control system (Chapter 31). Muscles can and do insert onto resorptive surfaces on bone so that mechanical action can stimulate resorption and deposition directly (Figs 35.2 and 35.4). One potential mechanism receiving wide support in the 1970s was translation of mechanical stimuli into bioelectrical activity to which skeletal cells could respond. I discuss this approach and then turn to three other approaches.17 There is no doubt but that electrical currents in the amp range influence the proliferation, differentiation and activity of skeletal cells, although a mechanistic relationship between mechanical stimulation and bioelectrical potential is elusive. Empirical evidence for the efficacy of exposure to electric current/fields includes: ●
●
●
●
●
●
enhanced chondrogenesis and acceleration of the growth of epiphyseal cartilage and bone following direct application of an electrical field; acceleration of the proliferation of induced cartilage following application of a 5 A current to muscle cultured with Bmp – the cartilage having been induced by Bmp; promotion of precocious initiation of mineralization by hypertrophic chondrocytes following 10 A DC current applied in vitro to femora from nine-day-old chick embryos, at which age the cartilage is unmineralized; enhancement of osteogenesis in chick tibiae and mouse calvariae exposed in vitro to as little as 10 5 V/m for 30 minutes/day; promotion of osteogenesis in foetal rat long bones in response to direct or pulsating current in vitro; and alteration of oxygen tension in the immediate vicinity of skeletal cells.18
Bone is deposited in regions of electro-negativity. Healing fractures respond to low-voltage electric fields with enhanced repair. For example, new bone is deposited around an active electrode implanted in the medullary canal of rabbits, but not around an inactive
electrode. Indeed, bone disappears around the inactive electrode but can be regained if current is allowed to flow. The new bone forms from polymorphic perivascular cells associated with invading blood vessels. Mineral is differentially deposited around the electrodes – mineralizing collagen around both active and inactive electrodes, and degenerating cells around the inactive electrode (Brighton and Hunt, 1986). In studies testing skeletal responsiveness to electrical stimuli, as in all experimental studies, adequate controls must be used. Inserting the wire into medullary canals or marrow in vivo can enhance osteogenesis, even with no current flowing through the wire. The effect is not simply the result of trauma – inserting and removing a wire does not enhance osteogenesis – indicating an effect of the wire itself. Furthermore, the authors of these studies found that electrical and mechanical stimuli were additive. Not all mechanical stimuli are perceived by cells as electrical change.19
NOTES 1. The Fairbairn steam crane built in 1875 for the dockyard at Bristol, designed by the Scottish engineer William Fairbairn (1789–1874) to lift 35 tons from a height of 115 feet, was in continuous operation at the dockyards until 1973. Now refurbished, the crane can be visited as part of the Bristol dockyard restoration. Fairbairn operated a shipyard in London where the first iron-hulled steamship, the Lord Dundas, was built. He conceived using tubular steel for construction when assisting Robert Louis Stephenson in the construction of the Conway and the Menai Strait bridges in North Wales. 2. Wolff’s classic 1892 study was reprinted in translation by Maquet and Furlong (1986). See Lee and Taylor (1999) for Roux’s Law. Benninghoff (1925, 1930) performed some of the more detailed analyses on trabecular fibre structure (Fig. 2.6); Murray (1936) and Altmann (1964) provide thorough evaluation of these studies. Glücksmann (1938) used in-vitro cultivation of skeletal elements to demonstrate the influence of pressure on structural orientation, while Murray and Selby (1930), Murray (1936) and Altmann (1964) provide what are still among the best accounts of the early literature on the response of the skeleton to mechanical forces. For later studies, see Pauwels (1973), and see Oxnard (1991) for an insightful analysis of the three-dimensional architecture – the morphanalysis – of bone. 3. See Hancox (1947) for the CAM-grafts, Buckland-Wright (1978) for the functional unit approach, and Hansson et al. (1975) for venous stasis. Under conditions of prolonged venous status, as when the inferior vena cava is ligated, osteocytes dedifferentiate to chondrocytes as areas of bone cells transform from acidophilic to basophilic and are replaced by an island of cartilage cells (Abdalla and Harrison, 1966). 4. See also Bertram (2001) for an analysis of how the trabecular bone immediately beneath the articular cartilage of mammalian long bones may attenuate loading of both bone and cartilage. I am grateful to John Bertram for his analyses and his
Long Bone Growth: A Case of Crying Wolff?
5.
6.
7. 8.
9.
10.
11.
12.
13.
comments on this chapter. John considers that growth ‘appears to have a strong element of determination (however that is mediated), whereas the modifications of mature bone appear to have some stringent limitations’ (pers. comm.). See Wolff (1892), Hueter (1862, 1863) and Volkmann (1862) for pressure and epiphyseal cartilages, and Dorsey (1897) for Wormian bones. See Hert (1964a–c, 1969), Hert and Liskova (1964), Lanyon and Rubin (1984), Carter (1987), Carter et al. (1987), Rubin and Lanyon (1987), Carter and Wong (1988), Rubin et al. (1989), Wong and Carter (1990), Carter et al. (1991, 1996) and Carter and Orr (1992). See Pead et al. (1988a,b) for rapid response to brief loading. See Lanyon (1972), Lanyon et al. (1975) and Piekarski and Munro (1977) for data. See Hert et al. (1969, 1971a,b, 1972a,b), Liskova and Hert (1971) and Chamay and Tschantz (1972). For the contrary view, see Bassett and Becker (1962), Becker et al. (1964), Bassett (1968), Bassett and Pawluk (1972) and Becker (1978). For mechanical loading of tibial periostea stimulating rapid changes in periosteal gene expression, see Raab-Cullen et al. (1994). See Nonaka et al. (1993) and Sasaki et al. (1994a,b, 1995) for uterine transplants, Pilbeam and Gould (1974) for australopithecines, and Houck et al. (1990) for Archaeopteryx. See Lerner et al. (1998) and Li et al. (1991) for these rat and rabbit studies, and see Felts and Spurell (1965, 1966) for analyses of long-bone structure in cetaceans. The selective pressure for evolution of the distinctive interdigital membrane in bat wings seems to have been elongation of the metacarpal bone (Kovtun, 1985). See Adams (1998) for the developmental and functional integration of the elements of bat wings. See Morey and Baylink (1978) for the general statement, Whedon et al. (1976) for mineral metabolism, Jee et al. (1983) for trabecular bone, and Whedon and Heaney (1993) for an overview of the effects of weightlessness, paralysis and inactivity on bone growth. John Bertram commented on a ‘rat-in-space’ study in which there was a decrease in the deposition of periosteal bone in the dentary (which is not a weight-bearing bone) and that
14. 15.
16.
17.
18.
19.
415
the decrease occurred in areas not associated with muscle attachment as well as where muscles attach, the implication being that some effects of zero gravity may be systemic (Bertram, pers. comm.). See Fielder et al. (1986), Simmons et al. (1983a) and Barrett et al. (2000) for these three studies. See Duke (1983) for excess gravity, Klement and Spooner (1994) for the space shuttle, and Klement et al. (2004) for subsequent studies using the NASA rotating wall vessel which simulates microgravity by randomizing the direction of the gravitational forces on cells. This is because immobilization, more or less whatever the cause, leads to hypoplasia of bone. As one example, four weeks after unilateral resection of the sciatic nerve to the rat femur, periosteal (cortical) bone and ash content are both reduced, the latter indicative of reduced mineral content (Pennock et al., 1972). See Brash (1924, 1934) and Hoyte and Enlow (1966) for stimulation of resorption and deposition, and see the reviews by Bassett (1972b), Marino and Becker (1977), Spadaro (1977), Hall (1983f), and chapters in the volumes edited by Liboff and Rinaldi (1974), Attinger and Parakkal (1977), Brighton et al. (1979) and Connolly (1981). See Watson et al. (1975), Brighton et al. (1976) and Norton et al. (1977) for cartilage, Norton and Moore (1972), Spadaro (1982) and Noda and Satoh (1985b) for bone, Nogami et al. (1982) for proliferation of induced cartilage, Noda and Sato (1985a) for precocious mineralization, Fitzsimmons et al. (1986) for tibiae and calvariae in vitro, Theharne et al. (1980) for direct or pulsating current, and Brighton and Friedenberg (1974) and Brighton et al. (1975) for enhanced local O2 tension. Electrical stimulation enhances bone and muscle mass in paraplegics (Pacy et al., 1988). See Spadaro et al. (1986) and Schaberg et al. (1985) for these studies, and see the volumes edited by Brighton et al. (1979), and Connolly (1981) and Hall (1983f) for the electrical properties of bone and cartilage as seen in both experimental and clinical studies. Becker (1972b) applied electrical currents of 3–6 nA to the amputated limbs of 15 rats and obtained a high percentage of regeneration and formation of epiphyseal growth centres.