Periosteal response in translation-induced bone remodelling

J. Anat. (1990), 171, pp. 69-84 69With 14 figures

Printed in Great Britain

Periosteal response in translation-induced bone remodelling

SOPHIE A. FEIK, GRAHAM ELLENDER, DANIEL M. CROWEAND SUSAN M. RAMM-ANDERSON

School of Dental Science, University of Melbourne, 711 Elizabeth Street,Melbourne 3000, Australia

(Accepted 29 December 1989)

INTRODUCTION

Mechanical loading influences bone remodelling although the nature of the load-related stimulus is unknown (Pead et al. 1988). Some workers consider that thestimulus most likely arises from strains engendered by the load within the bone tissue(Pead et al. 1988). However, loading experiments have shown that local strainmagnitude is not directly related to the site of new bone formation (Rubin & Lanyon,1985). The living skeleton is purported to monitor its loading history and adapt itsarchitecture to this rather than to one-time events but definite proof of this is lackingand how it could be achieved remains obscure (Frost, 1988).Our earlier histological and radiographic studies using the rat caudal vertebral

model demonstrated that not only strain within the bone tissue per se but also strainin the soft tissues enveloping bone must be considered when seeking an explanationfor the remodelling which occurs when mechanical stress is applied to bones in vivo.Two models were used, one employing a bending stress (Storey & Feik, 1982, 1985)and the other a translation-induced stress (Pollard, Feik & Storey, 1984). In both, asthe vertebrae move within the encasing soft tissues, new bone forms under soft tissuetension and resorbs when soft tissues are compressed against bone.When bones are bent, internal bone strain is induced whereas with simple

translation bone strain is virtually eliminated and the influence of the soft tissues onremodelling can therefore be determined. Since caudal vertebrae in young ratsnormally remodel symmetrically with periosteal formation and endosteal resorption(Hammond & Storey, 1974) remodelling changes can be observed easily. The modelfor the ultrastructural study now undertaken has additional advantages. Very youngbones are used, hence, rapid and marked changes can be expected and the specimensare suitably small so that the leading and trailing sides can be examined in the onevertebra.

Previous histological and ultrastructural studies have shown that the periosteumin rapidly growing caudal vertebrae is a three-layered structure (Feik, Storey &Ellender, 1987; Ellender, Feik & Carach, 1988). When mechanical stress is applied bybending, changes occur in all three layers and differ on the pressure and tension sides.There is a lag period before the osteogenic layer is affected, with the greatest changeoccurring in the mid-zone (Ellender, Feik & Ramm-Anderson, 1989). Thisultrastructural study, using a model where internal bone strain is virtually eliminated,studies the cellular and matrix changes which occur in the periosteum under thefollowing conditions: (i) when tension is applied to the bone surface and boneformation is accelerated and (ii) when pressure is exerted on the bone surface and areversal from formation to resorption occurs. More evidence is coming to light of the

SOPHIE A. FEIK AND OTHERS

Fig. 1. Radiograph of the loaded appliance, with tail segments in place, prior to transplantation. x 3.

coupling of formation and resorption and of the pivotal role played by osteoblasts inboth processes (Meikle, Heath & Reynolds, 1986). Our model is ideally suited to studythis problem in an in vivo situation where both processes are occurring in the one bonein an already defined model.

MATERIALS AND METHODS

Segments of three consecutive caudal vertebrae from 10-12 g inbred Sprague-Dawley donor rats were threaded onto the arms of a helical torsion spring appliancewhich was prestressed to unwind when a connecting ligature was released. Theappliance was transplanted subcutaneously into 50 g female hosts and the transplantsallowed a period of stress-free organisation and development after which they weremoved through the host tissues by a defined force of 15-20 g for a deflection ofapproximately 20 mm (Pollard et al. 1984).

Appliance designThe design and dimensions of the expansion appliances are illustrated and described

in detail in a previous publication (Pollard et al. 1984). The appliances (Fig. 1),approximately 20 mm in length, were constructed of 0-23 mm diameter stainless steelElgiloy wire (Rocky Mountain Corporation, USA). Tail segments were threaded onthe spring arms which were connected by a single helical coil (diameter 2 mm). Theappliance was loaded by approximating the arms with a C-clasp constructed of0-25 mm soft stainless steel ligature wire (Unitek Corporation, USA). In the controls,the C-clasp was left intact while in the experimental animals the appliance wasactivated by cutting the C-clasp in situ.

Operative proceduresThe donors were anaesthetised with halothane (Fluothane. ICI Australia Operations

Pty. Ltd, Melbourne, Australia) and radiographed to identify the required vertebrae.The tails were severed, skinned, and segments comprising caudal vertebrae (CV) 7-9were excised and placed in a solution ofDMEM with antibiotics (Dulbecco's modified

70

Periosteum and translation-induced stress 71Eagle's medium with streptomycin 50 U/ml and penicillin 50 ,ug/ml). The segmentswere threaded through their long axes onto the prepared appliances and radiographedprior to transplantation. The hosts were weighed and anaesthetised with chloralhydrate (36 mg/100 g body mass). A 10 mm long transverse incision was made directlybelow the sternum and a subcutaneous pocket created by blunt dissection toaccommodate the transplants. The incisions were closed with 3/0 silk sutures (EthnorPty. Ltd, Sydney, Australia) and the hosts then radiographed with the ventral sidedown. After seven days hosts were anaesthetised, the C-clasp palpated and, in theexperimental animals, the clasp was cut through a small incision in the skin. Theanimals were radiographed at this stage and again immediately after termination,prior to removal of the appliance from the host.

RadiographyRadiographs were taken on a Faxitron 43805N X-ray System Unit (Hewlett-

Packard Co., McMinnville, Oregon) with a current of 2-5 mA and a film-to-sourcedistance of 610 mm. Kodak Industrex M film was used for radiographs of live,anaesthetised animals and Kodak Industrex R for dissected specimens; two exposureswere made of the latter so that the soft tissues and the bone could be visualisedoptimally.

Bone labellingA fluorescent marker was used to determine the remodelling pattern in this

translation model. The hosts received an intraperitoneal injection of 2-5 mgtetracycline hydrochloride (Calbiochem, San Diego, California, USA) at zero time, i.e.the time of activation of the appliance in the experimental animals, and two hoursbefore killing the animals with an overdose of sodium pentobarbitone (Nembutal60 mg/ml, Abbott Laboratories Pty. Ltd, Kurnell, NSW). The appliances weredissected out at 1, 3, 5, 7, 10 and 14 days, two experimental and one control specimenbeing obtained at each time interval. The tissues were fixed in 70% ethanol,dehydrated through graded ethanols and embedded in polyester clear embedding resin(R.F. Services, Melbourne, Australia). Transverse 40 ,tm sections were cut of the mid-diaphysis of CV8 on a Leitz 1600 saw microtome (Ernst Leitz, Wetzlar, Germany).Sections mounted on glass slides with DPX were examined under fluorescentillumination and photographed on a 400 ASA film (Kodak, Australasia, Melbourne,Australia). Histology

Specimens for histology were marked with India ink (Pelikan, Gunther Wagner,Germany) on the leading or outside edge and the appliances removed from the hostand radiographed. The segments were fixed in neutral buffered formalin, demineralisedin 3'4% sodium formate and 17-5 % formic acid and processed to paraffin. Sections,5 ,um thick, were cut longitudinally through the centre of the segment on one arm andtransverse sections, through the mid-diaphysis, obtained of the vertebrae on the otherarm of the appliance. Tissues from two experimental and one control appliance wereobtained at zero time, 1, 3, 5, 7, 10 and 14 days. Sections were stained withhaematoxylin and eosin (H & E), Masson's trichrome and picrosirius red (Junquiera,Bignolas & Brentani, 1979).

Transmission electron microscopy

As for histology the segments were marked with India ink prior to removal from theanaesthetised hosts. Tissues from two experimental and one control appliance wereremoved at each of the following time intervals: zero time, 1, 3, 5, 7 and 10 days. For


primary fixation of tissues the appliances were immersed for one hour in Karnovsky'sfixative (Karnovsky, 1965), modified to 2-5 % glutaraldehyde and 2% paraformal-dehyde in 0-1 M sodium cacodylate buffer. They were then radiographed, the segmentsslipped off the arms and a transverse incision made through the middle of CV8. Theblocks were postfixed in 2% osmium tetroxide in 0-1 M sodium cacodylate buffer atpH 7-3 for 2 hours. They were block-stained in 0-5 % uranyl acetate, dehydratedthrough graded acetones and processed through propylene oxide into the hardformulation of Spurr's resin (Spurr, 1969) via a protracted infiltration sequence.Transverse survey sections through the mid-diaphysis of the vertebrae were obtainedand the leading and trailing sides (or the equivalent outer and inner sides in thecontrols) identified. Double mesas were produced on the block face and, by means ofa 2° horizontal rotation of the knife, thin sections of both regions obtained in the sametissue plane (Ellender, 1990). Sections were collected on fine bar copper grids(Gilder Grids, Grantham, UK), stained with uranium and lead and examined in aPhilips EM 300.

RESULTS

RadiographyThe caudal vertebrae on the control appliances develop normally and remain

symmetrical, whereas, when the bones are translated, eccentric remodelling ensues(Fig. 2). The arrangement of the soft tissues surrounding the caudal vertebrae varies;the tissues may form a connecting sheath between the two arms (Fig. 3) or a separatecapsule around each arm. In either case, when the arms move apart, traction is exertedon the soft tissue envelope and results in bone remodelling. Long, thin trabeculae,orientated in the line of soft tissue tension, form on the trailing side while the shaft onthe leading edge becomes flatter as a result of resorption. When translation ceases theexperimental bones remodel back in the opposite direction to translation, as wasshown also by Pollard et al. (1984).

Bone labellingTransverse sections through the mid-diaphysis of CV8 show that, in the control,

new bone forms on all periosteal surfaces. In the translated bones the leading andtrailing sides of the caudal vertebrae differ markedly. Three days after release a single,narrow, labelled line is visible on the periosteal margin of the leading side, confirmingthat little new bone has formed on this surface during translation (Fig. 4a). On thetrailing side, after the same time interval, the labelled area is wider and diffuse and thetwo lines denoting tetracycline injections are not clearly resolved since the new boneformed is trabecular in nature (Fig. 4b).

HistologyThe periosteum, as shown in a previous developmental study (Ellender et al. 1988),

can be distinguished from the adjacent soft tissues and, at the optical level, appears tohave two layers - an inner osteogenic and an outer fibrous layer in which the majorityof cells and fibres lie parallel to the shaft.One day after translation there is a difference between the control and the leading

and trailing sides of the experimental CV. On the leading side the orientation of thecompressed connective tissues and periosteum is predominantly parallel to the bonesurface while on the trailing side the soft tissues are aligned approximatelyperpendicular to the bone surface. Picrosirius red-stained sections illustrate clearly thedifference in orientation of the fibres (Fig. 5).

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Periosteum and translation-induced stress

Fig. 2. Control (C) and experimental (E) CV at 7 days. The arrow indicates the direction oftranslation. The control CV show relatively normal, symmetrical development unlike theeccentrically remodelling experimental CV. x 2 5.Fig. 3. A soft radiograph at 7 days of an activated appliance, with the C-clasp cut, shows theconnective tissue sheath that frequently forms between the tail segments on the arms. x 4 5.Fig. 4 (a-b). Tetracycline-labelled experimental bones at 3 days. (a) A single, narrow, labelled lineis visible on the periosteal surface (P) of the leading side (L). (b) On the corresponding trailing side(T) a wide band of trabecular bone shows labelling. x 90.Fig. 5 (a-b). Histology of mid-diaphyseal transverse sections ofCV8 3 days after activation showingthe difference in the orientation of the periosteal (marker) fibres on the leading (a) and trailing (b)sides. Picrosirius red. x 330.

73


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Fig. 6. TEM of control periosteum at 7 days shows the well-ordered cellular periosteum (CPo),mid (MZ) and fibrous (FPo) zones. x 4700.

With time, trabeculae of bone form on the trailing side and resorption cavitiesappear on the leading periosteal surface. The entire surface does not resorbsimultaneously: the process commences opposite an area of maximum soft tissuecompression followed by resorption in adjacent sites as these become subject topressure. When translation ceases, the direction of remodelling changes, with theleading periosteal surface becoming formative again while the corresponding periostealsurface on the trailing side shows osteoclastic resorption.

Overall the direction of remodelling during translation is thus in an oppositedirection to the movement of the bones. By 14 days a normal remodelling pattern isresumed with periosteal formation and endosteal resorption on both leading andtrailing sides.

Transmission electron microscopyThe structure of the periosteum in the control transplants resembles that of the

periosteum in situ at a comparable developmental stage (Ellender et al. 1988).Similarly, it decreases slightly in thickness but shows greater organisation with time.By three days a clear delineation into three zones is evident and by seven days (Fig.6) the periosteum is indistinguishable from the 50 g control (23-24 days old) describedpreviously (Ellender et al. 1989). The one to two cells thick osteoblastic layer consistsof active, closely-packed and predominantly cuboidal osteoblasts. The mid-zone,containing blood vessels, mononuclear phagocytes and undifferentiated cells, has a

74

Periosteum and translation-induced stress 75

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Fig. 7. TEM of the leading side at one day. The periosteum is narrowed, drawn out parallel to thebone and shows reduced activity in all three layers - cellular (CPo), mid (MZ) and fibrous (FPo). Anosteoblast (Ob) undergoing degeneration is visible. x 4500.Fig. 8. (a-b). TEM of the trailing side in the 'tail' area at one day. (a) Active osteoblasts (Ob) arein a rosette-like arrangement, and many widely-spaced mid-zone cells of irregular shape containglycogen (arrows). x 2600. (b) The fibrous periosteum (FPo) merges with the adjacent connectivetissue. Fibroblasts are roughly stellate and spaces (arrow) are present in the matrix betweenirregularly arranged collagen bundles. x 2300.


Fig. 9. TEM of the leading side at 3 days. The mineralisation front is discrete, the osteoblasts (Ob)dense and the mid- (MZ) and fibrous (FPo) zones appear relatively inactive. x 5600.

looser structure but cell connections are maintained via gap junctions. The collagenousmatrix in this zone is sparse and the collagen diameters more uniform than those inthe osteoid; they resemble the more densely packed matrix in the fibrous zone. Thefibroblasts, more ordered than the cells in the mid-zone, are aligned parallel to thebone surface and most appear to be actively synthesising. In the pseudopodalextensions of some of these cells are membrane-limited compartments containingcollagen profiles. Gap junctions between processes are seen occasionally.On the leading side at one day, compared with the appropriate control, the

periosteum is narrower and overall shows reduced metabolic activity (Fig. 7). Themineralisation front is not as wide as in the control, with fewer osteoblastic processesentering the osteoid. The osteoblastic layer is generally one cell thick and the cellselongated rather than cuboidal, the nuclei contain more heterochromatin, the roughendoplasmic reticulum (RER) is generally not as dilated nor the Golgi as prominent.Occasionally 'pale' cells with grossly dilated RER, swollen mitochondria and clumpednuclear chromatin are seen. Compared with the control there are fewer cells in themid-zone and the collagen appears to be more closely packed. In the fibrousperiosteum the cells are elongated and have long, thin processes.On the trailing side at one day the reverse phenomenon is occurring in that

periosteal activity is increased in all three layers. In transverse sections the periosteum

76

Periosteum and translation-induced stress 77

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Fig. 10. TEM of the leading side at 7 days. Transition from formation to osteoclastic resorption (0c)is seen, with cells of a heterogenous nature along the intervening bone margin. The mid- (MZ) andfibrous (FPo) zones appear to be reduced. x 4400.

Fig. I I (a-b). TEM of the leading side at 7 days. (a) Fibroblasts with cytosegregated collagen profiles(arrows) he adjacent to a 'spent' osteoclast (0c). x 7000. (b) A cell in mitosis adjacent to a bonemargin (arrow) which is covered by a granular coat. x 5500.

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78 SOPHIE A. FEIK AND OTHERS

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Periosteum and translation-induced stress 79assumes a 'V' conformation with the fibrous layer at the point of the 'V' becomingcontinuous with the connective tissue sheath. Greatest activity is seen at this point withmultilayered stacking of actively synthesising osteoblasts often in a rosette-likearrangement (Fig. 8 a). In the centre of the 'V' the cells in the mid-zone, some ofwhichcontain glycogen deposits, are more widely-spaced and of irregular form, while thosein the arms are elongated and closer together. The fibrous periosteum shows a similararrangement (Fig. 8 b).At three days, on both the leading and trailing sides, the periosteal changes are

essentially a progression of those described at one day. On the leading side there is afurther reduction in the width of the mineralisation front and the osteoid seam (Fig.9) which in some areas has almost disappeared.By five days, and more so at seven days, there is a change from formation to

resorption on the leading side in some areas. The entire sequence from activation,resorption, reversal and back to formation (A-R-Rev-F) can be observed along thebone margin. The osteoblasts become elongated and quiescent and the osteoiddisappears; cells of a heterogenous nature including mononuclear phagocytes arefound along the intervening bone margin before frank osteoclastic resorption isevident (Fig. 10). Fibroblasts with many cytosegregated collagen profiles are foundnear the osteoclasts, particularly those that appear to be 'spent' (Fig. 11 a). During thereversal phase a granular coat covers the bone surface and cells with phagosomes,dense bodies and many cytoplasmic processes are found adjacent to bone. Furtheralong the bone margin pre-osteoblasts and/or osteoblasts, some undergoing mitosis,make their appearance (Fig. 11 b). The mid- and fibrous zones resemble those at threedays. The trailing side at five days is still highly cellular and the osteoblastic layermoderately active (Fig. 12) but many cells in the mid- and fibrous zones are involutingor reducing their activity and have an increased number of phagosomes and 'empty-looking' cytoplasm. By seven days only a few, widely-spaced cells remain in the mid-zone along with a number of vessels (Fig. 13). The fibrous periosteum is similarlyreduced.By ten days the leading side has again reverted to a forming surface with an active

osteoblastic layer and well-developed mid- and fibrous zones. The trailing side nowexhibits sequential resorption like that observed on the leading side at 5-7 days withsimilar periosteal changes. By 14 days on both the leading and trailing sides theperiosteum resembles the control.

DISCUSSION

This in vivo study of vascularised whole bones, where soft tissue/bone interrela-tionships are maintained, confirms and extends our previous findings (Pollard et al.1984); with the elimination of internal bone strain the nature and rate of boneremodelling can be altered by inducing strain in the enveloping soft tissues throughtranslation. During the translation phase the rate of formation on the trailing side ismarkedly accelerated and, following a lag period, resorption occurs on the leadingside, i.e. eccentric remodelling is occurring in a direction opposite to translation. Whenmovement of the bones ceases the direction of remodelling is reversed, i.e. the trailingside now becomes resorptive and the leading is again formative. By this mechanism thebones are relocated into a position of equilibrium within the enveloping soft tissues.A normal remodelling pattern is resumed when this is achieved (Fig. 14). In this paperthe overall changes are reported; aspects of interest will be covered in detail in a laterpublication. Events at the bone surface reflect the changes observed in the coveringconnective tissues so that through an understanding of the cell-cell and cell-matrix


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-o ----- Direction of translation _o* Direction of growth- Formation or quiescence -rResorption

Fig. 14. Diagram of the bone remodelling occurring in the control (C) and experimental (E) bonesthroughout the duration of the study. L-leading, T-trailing. Numbers indicate days afteractivation of the appliance. See text for further details.

interactions in these tissues, and more specifically in the periosteum, we may elucidatehow mechanical stress influences the local remodelling response.The connective tissue sheath or capsule forming around the CV on the arms is

tensed as translation occurs, thereby exerting pressure on the bone surface, as seen alsoin the looped tail model (Ellender et al. 1989). On the leading edge of the translatedbones, analogous to the outer side of the looped tail, resorption occurs firstapproximately opposite the ' tail ', i.e. at the point ofmaximum compression, and thenmoves to adjacent areas as the bony contour and hence pressure areas alter. Asimilar phenomenon was observed on the pressure side of a moving tooth (Rygh,1972) where less extensive cellular alterations were observed in protected locations andnew bone formation was evident in some areas, even on the pressure side (Rygh, 1973).The time lag before resorption occurs may be explained in terms of the time it takesfor a signal of sufficient magnitude to be transmitted to the cells on the bone surfaceto modify their activity. We hypothesise that the stimulus here is pressure on theosteoblastic layer caused by a combination of compression of the periosteum againstthe adjacent soft tissues and tension induced in the fibrous periosteum as the bonesgradually translate. A reduction iin all three layers is seen, withsigns of cellular damage in some osteoblasts similar to those observed in pressure areasadjacent to moving teeth (Rygh, 1972). This is followed by disappearance of osteoidbefore resorption becomes evident. The findings are consistent with the key roleassigned to osteoblasts in the local control of resorption (Marshall, Nisbet & Green,1986) through release of collagenolytic enzymes (Chambers, Darby & Fuller, 1985),possibly in response to physical forces (Sodek & Overall, 1988). The remodellingprocess, i.e. A-R-Rev-F, confours to that previously described (Tran Van, Vignery& Baron, 1982) but the entire sequence can be observed at different points along thebone surface simultaneously and it occurs more rapidly. This accelerated remodelling,which applies also to events on the trailing side, may be explained by the young ageof the transplants and the increased environmental temperature compared with thatof the tail in situ (Garrard, Harrison & Weiner, 1974). A similar enhancement ofmaturation compaere tionormay beported in transplanted CV anlagen (Feik& Storey, 1983). 0

Fibroblasts, with an increased number of cytosegregated collagen profiles, have

80

Periosteum and translation-induced stressbeen observed previously near osteoclasts (Garant, 1976) and a role for theseconnective tissue cells in the resorptive process has been postulated (Heersche &Kanehisa, 1988) through the release of factors enhancing collagenase activity (Heath,Meikle, Atkinson & Reynolds, 1984). The cells in mitosis observed at the bone surfaceare probably a 'transitory' osteoblast phenotype capable of dividing, unlike the fullydifferentiated osteoblasts usually found at bone margins (Bruder & Caplan, 1989), andprovide evidence of the resumption of formation at this site. Retention of patent bloodvessels, despite the overall reduction in the mid-zone, theoretically enables osteoclastprecursors of haematogenous origin (Marks, 1983) to arrive via this route rather thanvia the intramedullary vessels. Osteoclast progenitors in the periosteum of embryonicmouse long bones were shown to be seeded via the blood stream, appearing longbefore the multinucleated osteoclasts became visible (Scheven, Kawilarang-De Haas,Wasenaar & Nijweide, 1986). Periosteal rather than undermining resorption may thusbe occurring.The shape of the connective tissue sheath and the 'V' arrangement of the

periosteum on the trailing side resemble conditions found in in vitro studies of cell-containing collagen gels attached at both ends. Initial orientation of collagen fibresis followed by similar orientation of cells, the process commencing at the periphery ofthe gel with a looser, more random arrangement centrally: a situation very similar tothat observed in the 'tail' in this study. However, cell orientation occurs only in gelswhere tension develops (Bellows, Melcher & Aubin, 1982). In our model, tension,generated by stretching the sheath as the arms of the appliance move apart, may leadto reorientation of periosteal fibroblasts and subsequent alignment of newly secretedcollagen by fibroblast traction (Harris, Stopak & Wild, 1981) in the line of soft tissuetension. Results of our in vivo study are consistent with the hypothesis (Bellows et al.1982) that the development of tension between two points results in the orientation ofthe cells along an axis connecting the points of attachment. We postulate that thetension induced in the covering soft tissues is transmitted by the periosteum to thebone surface, resulting in acceleration of bone formation. A similar positive correlationbetween the osteogenic response and the magnitude of tensile forces was found byMiyawaki & Forbes (1987) in an in vivo/in vitro system. The effect of mechanicaltension on vertebrate cells is to speed up the cell cycle (Curtis & Seehar, 1978). In vitrostudies show that tension also induces biochemical changes in cells consistent withincreased synthetic activity (Harell, Dekel & Binderman, 1977; Meikle, Sellers &Reynolds, 1980; Binderman, Shimshoni & Somjen, 1984) and increases the number ofbone cells synthesising DNA (Hasegawa et al. 1985).The response of the periosteum to tension is seen in all three layers and occurs in

a sequential fashion similar to the conditions observed in the looped tail model(Ellender et al. 1989). As translation slows, the fibrous periosteum shows first signs ofa reduction in formative activity at three days by the appearance of an increasednumber of membrane-bound compartments containing collagen profiles; these aregenerally associated with increased collagen degradation (Bienkowski, 1984). Signs ofcellular involution are seen at five days and by seven days the fibrous periosteumappears to be relatively quiescent. The mid-zone similarly shows progressive changes.In the precursor cells at one day the numerous glycogen deposits, considered to be asource of potential energy for cell differentiation (Chen & Hardwick, 1977), maypresage the increased numbers of osteoblasts seen at three days. The associatedincrease in vessels reflects, and is an essential prerequisite for, accelerated formation,for a clear link between the degree of vascularisation and the rate of osteogenesis hasbeen established (Burnstein & Canalis, 1985). As in the fibrous zone, involution then

81


occurs so that by seven days few mid-zone cells are present although some vesselsremain. This presumably is to provide nutrition to the osteoblastic layer which stillappears to be active at this stage. The findings support: (i) our previous propositionthat the periosteum functions as an integrated unit with the outer layers buffering theosteogenic zone which is the last to show structural changes and (ii) the concept thatcells are sensitive and responsive to physical load (Merrilees & Flint, 1980).A pivotal role has been ascribed to osteoblasts in the control of bone remodelling

not only through regulation of resorption, as discussed previously, but through theirability to regulate the function and activity of other osteogenic cell types in theperiosteum (Van der Plas & Nijweide, 1988). The interaction, however, is dependenton direct cell-cell contact, with communication possibly occurring via gap junctionalcomplexes. These structures are present between osteoblasts (Doty, 1981), mid-zonecells, as shown in this study, and fibroblastic cells of the periosteum (Doty, 1988;Ellender et al. 1988). So it appears that a mechanism exists whereby a signal can betransmitted through the periosteum to the cells at the bone surface and that theperiosteum may modify or regulate the transmission of this message. Strain inducedin periosteal cells both in vivo (Skerry, Bitensky, Chayen & Lanyon, 1989) and in vitro(Jones & Scholuebbers, 1989) produces metabolic changes and appears to do so atlower strain values than in cells within bone (Jones & Scholuebbers, 1989). Theconsideration of strain-related events in the periosteum, rather than in the bone perse,may be of greaterisnportance, therefore, in surface remodelling and may explain thelack of correlation between local strain magnitude within bone and the sites of newbone formation.

SUMMARY

Translation of transplanted bones induces strain in the periosteum and subsequentbone remodelling. This study examines the periosteal response on the leading andtrailing sides of translated bones using an in vivo model where internal bone strain isvirtually eliminated. Caudal vertebrae from 4 days old rats were threaded onto thearms of prestressed helical torsion springs and transplanted subcutaneously. In theexperimental rats, the appliances were activated seven days later causing the bones totranslate. Tissues were examined both optically and by transmission electronmicroscopy.A connective tissue sheath or capsule forms around the bones and, as the arms of

the appliance move apart, traction on the enveloping soft tissues produces compressionof the periosteum on the leading side and tension on the trailing side with remodellingoccurring in a direction opposite to translation. The control periosteum has anordered structure with well-delineated osteogenic, mid- and fibrous zones. Duringtranslation the periosteum on the leading side is consistently narrower than on thetrailing side and shows a gradual reduction in formative activity followed byresorption in select areas. Cells and fibres are aligned predominantly parallel to thebone surface. Accelerated formation characterises the trailing side during thetranslation phase with increased activity and widening of all three periosteal layers.The fibrous layer merges with the connective tissue sheath which frequently is orientedapproximately perpendicular to the bone surface. The direction of remodelling isreversed when translation ceases with corresponding changes visible in the periosteum,the osteoblastic layer being the last to show changes. A normal periosteal structureand remodelling pattern is regained when equilibrium of the bones within the softtissues is attained.

This study shows that the enveloping soft tissues profoundly influence the natureand rate of bone remodelling. The changes are reflected in the periosteum which

82

Periosteum and translation-induced stress 83functions as an integrated unit modulating the signal transmitted to the osteoblastswhich play a key role in events occurring at the bone surface. Changes are notattributable to internal bone strain.

This study was funded by Project Grant 88/0504 from the National Health andMedical Research Council of Australia. We thank Chris Owen, Steve Cleal andClaudia Gaviria for aid with the illustrations and Dr K. Ham for advice andencouragement.

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Periosteal response in translation-induced bone remodelling