Bone, 11, 205-210 (1990)

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Copyright © 1990 Pergamon Press plc

An Investigation of Vanishing Bone DiseaseG. R . DICKSON,' A. HAMILTON, 2 D. HAYES,3 K. E. CARR,3 R. DAVIS 3 and R . A. B . MOLLAN2

' Department ofAnatomy, Medical Biology Centre, The Queen's University ofBelfast, N. Ireland2 Department ofOrthopaedic Surgery, The Queen's University ofBelfast, Musgrave Park Hospital, Belfast, N . Ireland3 Department of Histopathology, The Belfast City Hospital, Belfast, N . Ireland

Address for correspondence and reprints : Dr. G . R. Dickson, Department of Anatomy, Medical Biology Centre, The Queen'sUniversity of Belfast, 97 Lisburn Road, Belfast BV) 7BL, N. Ireland .

Abstract

Vanishing bone disease is a rare condition producing localdeformity and instability . Fibrovascular tissue replaces bonecompletely but the mechanism of bone destruction and re-sorption is unknown and there is controversy regarding thepresence or absence of osteoclasts in the disease . Radiog-raphy, clinical chemistry, light microscopy, transmissionelectron microscopy (TEM) and cytochemistry were used toinvestigate the condition of a young woman presenting earlyin the disease process . We detected atypical ultrastructure inosteoblasts and endothelial cells . The rare osteoclasts, nu-merous mononuclear phagocytes and vascular endotheliumfound in the condition reacted positively for the enzyme acidphosphatase . Aggressive local excision of diseased tissue andinsertion of a free vascularized bone graft at an advancedstage of the disease, accompanied by subsequent radio-therapy for residual disease only were successful in rehabili-tating the affected forearm and hand .

Key Words: Vanishing bone disease -Radiography-Ultra-structure-Cytochemistry-Bone cells .

Introduction

Vanishing bone disease is descriptive of an osteolytic con-dition associated with a benign osseous angiomatosis andcharacterized by a progressive localized bone resorption(Johnson and McClure 1958) . However, there is a strikingdifference between the angioma of bone and vanishingbone disease . The former may destroy bone but never dis-solves it completely (Gorham et al . 1954) .

Over 70 cases of vanishing bone disease have appearedin the literature since Jackson's (1838) report of a bonelessarm. The disorder has also been described as disappearingbone disease (Heyden et al . 1977), massive osteolysis(Cannon 1986), phantom bone (Gorham and Stout 1955)and extensive idiopathic osteolysis (Picault et al . 1984) .Virtually all these cases were assessed radiologically andabout half by light microscopy . In only one patient waselectron microscopy applied as an investigative tool(Caulet et al . 1968) even though Heyden et al . (1977) calledfor further information on the disease, with particular ref-erence to its pathological diagnosis and treatment .

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Materials and Methods

Radiography and clinical assessment

A total skeletal survey of the patient, a 44-year-old house-wife, was undertaken, and subsequent radiographs of heraffected left forearm were recorded at 4, 7, 10, 14, 19 and24 months postpresentation . The patient's nervous andcardiovascular systems were assessed together with liverand thyroid function tests . Other investigations includedfull blood picture, urea, electrolytes, calcium phosphate,alkaline phosphatase, plasma proteins, Erythrocyte Sedi-mentation Rate (ESR) and screening for pathogenic or-ganisms .

Light microscopy, TEM and cytochemistry

Specimens for light microscopy were fixed in bufferedformaldehyde, demineralized using formic acid, dehy-drated in methanol, cleared in toluene and embedded inparaffin wax . Sections, 8 p.m in thickness, were stainedwith haematoxylin and eosin .

For TEM samples of diseased bone and control "graftbone" tissues were prepared mineralized and demincral-ized by conventional methods (Dickson 1984) . Specimens1 .5 mm 3 x 1 .5 mars were fixed immediately in 3% glutaral-dehyde in 0 .1 M sodium cacodylate buffer at pH 7 .2-7 .4containing 2 mM magnesium chloride . Fixation time was4-6 h at a temperature of 4°C. This was followed by anovernight buffer wash at 4°C in 0 .1 M sodium cacodylatebuffer, at which stage some specimens were demineralizedin 4.3% ethylene diaminetetraacetic acid (Warshawsky andMoore 1967). Secondary fixation was effected using 1%osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 h(mineralized specimens) or 4 h (demineralized specimens)at 4°C . Samples were dehydrated in a graded series of ace-tone prior to immersion in propylene oxide before infiltra-tion and embedding in Epon 812 substitute resin .

Ultrathin sections, silver-gold interference colors, wereprepared on a Reichert OMU2 ultramicrotome and liftedon copper support grids . These sections were stained withalcoholic uranyl acetate and Reynolds' lead citrate prior toexamination in a Jeol 1000XII transmission electron mi-croscope operated at 80 kV.

Bone specimens for cytochemical investigation of alka-hne phosphatase and acid phosphatase activity were incu-bated using sodium (3 glycrophosphate as substrate ac-

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cording to the methods outlined by Doty and Schofield(1984) .

Results

Clinicalhistory (radiography,clinicalchemistryandtreatment)

The patient was treated for four months for an apparentlyminor wrist injury. During that period she developedmarked radiological changes in her lower radial metaphysicand there was involvement of the surrounding radius andcarpal bones. By the time of referral to the orthopaedicservice the cortex of the radial metaphysis had been par-tially eroded . The only serious clinical condition undertreatment was idiopathic adrenal failure which was presentfor some seven years . In the absence of any obvious causefor the destructive bone lesion she was admitted for furtherinvestigation in 1981 .

There was no lymphadenopathy or pyrexia, the chestwas clinically clear and nothing of relevance was foundwith respect to the nervous and cardiovascular systems orthe abdomen . Subsequent investigations indicated normalparameters for full blood picture, urea and electrocytes,calcium phosphate and alkaline phosphatase, plasma pro-tein electrophoresis and thyroid and liver function tests .Immunoglubulins were normal except for a slight rise inIgM, urine was negative for Bence Jones protein and theESR 5 mm in the first hour. No pathogenic organisms weredetected .

A chest radiograph and skeletal survey were bothnormal . A biopsy of the area did not establish a definitivediagnosis. Treatment was continued with a short armplaster cast to protect the forearm from pathological frac-ture . In the months that followed, a complete discontinuitydeveloped in the lower radial metaphysis with tapering ofthe bone ends followed by early erosion of the radialcortex of the ulna. Clinically vanishing bone disease wassuggested and a further biopsy was considered compatablewith that condition .

Activity continued with further bony dissolution as faras the upper third of the radius and with loss of the ulnarhead by May 1982. The patient was readmitted for bonescan and angiography. Both were normal . Repeat bonebiopsies were taken at the disease front for transmissionelectron microscopy and cytochemical analysis . After thisthe patient was treated empirically with a course ofelectro-stimulation to the involved area for three months .

Radiography demonstrated that the process continuedunabated with almost total dissolution of the radius leavingno distal stability at the wrist (Figs . la, lb, 1c) . In April1984 surgery was undertaken to excise all the diseasedtissue and to restore bony continuity with a free vascular-ized fibular bone graft between the normal upper ulnarshaft and the carpus . During 1985 serial radiographsshowed nonunion and progression in the carpal bonesaround the lower end of the fibular graft . The patient wasthen given local radiotherapy in a dose of 2500 cGys, in 12fractions over 19 days to the affected area . By April 1986 itwas evident that the proximal fixation was secure despitethe nonunion at the distal end (Fig . 1d) . As there had beenno further radiological evidence of disease activity, a fur-ther operation was performed in which a plate was appliedbetween the lower fibular graft and the metacarpal bones .It was surrounded by autogenous iliac bone graft . This re-

G. R . Dickson et al . : Vanishing bone disease

suited in carpal/fibular fusion (Figs . le, lt) and restorationof function to the hand .

Lightmicroscopy

The classical lamellar arrangement of cortical bone wasdemonstrated at sites on the shaft bone away from the dis-ease front (Fig . 2a). In affected bone the cortex was greatlythinned with expanded Haversian canals, and cancelloustrabeculae were sparse . The endosteal surface was gener-ally rimmed by a layer of osteoblasts, and occasional os-teoclasts were identified in Howship's lacunae (Fig . 2b) .Fibrous tissue of varying density containing numerousthin-walled capillary-like spaces replaced the cortical bone(Fig. 2c). The fatty marrow cavity exhibited a similarprominent ramifying pattern of dilated vessels togetherwith fibrous tissue . At the active front of the disease thevascular pattern of the fibrous tissue was less evident andseemed altered by the remodeling changes in the residualshaft bone (Fig. 2d). Some metachromatically stained f-brocartilage was found adjacent to the remaining bone .Minimal evidence of bone remodeling was suggested by alittle woven bone, marginated by osteoblasts, which wasfound adjacent to the original cortical bone and was sur-rounded by fibrovascular tissue containing sparse lympho-cytes. In the marrow fat, thinly scattered aggregates oflymphocytes were present . There was nothing to indicatean angiomatous tumour. Histological examination of fib-ular bone tissue selected for grafting purposes revealed noabnormality.

Transmission electron microscopy and cytochemistry

Ultrastructurally, osteoblasts exhibited standard featuressuch as rough endoplasmic reticulum (RER) in variousstates of dilation, Golgi lamellae and vesicles, mitochon-dria, the occasional lysosome and cytoplasmic lipid inclu-sion body. However, many possessed only moderately di-lated RER and flattened Golgi lamellae . A prominent os-teoblastic characteristic in cells close to the disease frontwas an accumulation of cytoplasmic fine filaments (Fig . 3) .Mineralization of new woven bone was patchy and seemedto be matrix vesicle initiated . A heavily positive alkalinephosphatase reaction product was present at the plasmamembrane of the latter osteoblasts, and in association withextracellular matrix vesicles situated adjacent to collagenfibers (Fig . 4). Close to the disease front crenated osteo-cytes were found which had electron dense bodies in theircytoplasm and much heterochromatin in their pyknotic nu-clei .

Rare multinuclear osteoclasts and numerous mononu-clear cells were found at the erosion front of the bony ma-trix . The ruffled border region of these osteoclasts was ap-posed to mineralized and demineralized collagen . It dis-played a positive acid phosphatase reaction product, andthe osteoclast cytoplasm contained many vesicles and vac-uoles, numerous mitochondria and a perinuclear Golgi ap-paratus . Mononuclear phagocytes, probably macrophages(Fig . 5), were prominent close to the region of bone re-moval and here the collagenous matrix was clearly disorga-nized. These cells had an irregular cytoplasmic and nuclearoutline, RER was scantily represented, lysosomal bodieswere abundant, and numerous vesicles were found beneaththe plasma membrane. Their cytoplasm was rich in reac-tion product for acid phosphatase activity (Fig . 6) whichwas concentrated in Golgi lamellae, vesicles and vacuoles .

Numerous thin-walled endothelial vessels were located

G. R. Dickson et al . : Vanishing bone disease

Fig . 1. Radiographic illustrations of the disorder in the patient's radius and ulna at a) presentation, b) 7 months, c) 24 months, d) with thefree vascularized bone graft in place at the stage of non-union at the distal end and e, f) showing the free vascularized bone graft and platein situ at the final stage of carpal/fbular fusion .

in the unmineralized fibrous tissue matrix which substi-tuted for lost bone . A significant cytoplasmic feature of themany vessels found adjacent to the remaining bone was thepresence of fine filaments . Vesicles were abundant in theircytoplasm, and acid phosphatase reaction product waspresent in their Golgi lamellae (Fig. 7) and lysosomes(Fig. 8) .

Discussion

This is the first study to apply collectively radiography,clinical chemistry, light microscopy, TEM and cytochem-

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ical methods to an investigation of vanishing bone disease .The case manifests classical radiological changes withshelling out and tapering of the involved bones (Resnickand Niwayama 1981) . It demonstrates a painless, unstabledeformity of the involved limb with progressive atrophy ;all constituting a useful diagnostic sign of massive osteo-lysis. Also, the failure of clinical investigations such asblood chemistry to reveal any obvious abnormality reflectsthe localized nature of the condition (Sage and Allen 1974) .

In this patient, the substitution of lost bone by fibrovas-cular tissue and the presence of many mononuclear cellsat the resorption front but of few multinuclear osteoclastswere characteristic features of the condition . Also, there

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was no finding to indicate a true vascular tumour. Heydenet al . (1977) distinguished this disorder from a solitary ormultiple skeletal haemangioma by its more extensive de-struction of involved bones .

There is controversy concerning the presence or ab-sence of osteoclasts in vanishing bone disease . Gorhamand Stout (1955) did not find osteoclasts in areas of boneresorption . They concluded that the bone resorptive mech-anism was not evident by use of light microscopy and feltthat bone was apparently adsorbed and replaced by fibroustissue. Osteoclasts, while virtually absent, were detectedin investigations by Sage and Allen (1974) and observed insome cases reviewed by Cannon (1986) . The scarcity ofosteoclasts in the present study suggests, but does notprove, that these cells may not be responsible for the totalbone loss, even allowing for their efficiency as bone re-sorbers (Boyde and Jones 1979) .

Ultrastructurally, the flattened Golgi lamellae and mod-erate dilation of rough endoplasmic reticulum within manyosteoblasts might subjectively suggest decreased syntheticactivity. However, more significantly, osteoblasts showingdistinct signs of degeneration were found close to sites ofbone resorption. Their cytoplasm contained numerous finefilaments and reaction product for secretory acid phospha-tase activity (Dickson et al . 1987), findings which supportthe poor tendency towards bone formation . This observa-tion of many fine filaments in the cytoplasm of degenerate

G. R . Dickson et al . : Vanishing bone disease

Fig . 2 . a) Light micrograph of a portion ofradial shaft bone some distance back fromthe region of bone destruction . Note thetypical lamellar arrangement characteristicof normal cortical bone . An unstained sec-tion of a resin embedded specimen . x 106 .b) An osteoclast (arrow) active at the scal-loped endosteal bone surface and osteo-blasts on the bone surface . x 150 . c) Fibroustissue with thin-walled capillary-like spacesreplaced the cortical bone . x 106 . d) Frontof the disease process with shaft bone mar-ginated by osteoblasts (arrow) and adjacentfibrovascular tissue . X 120 . b, c, d. Demin-eralized preparations of paraffin wax em-bedded specimens, stained with haematox-

' ylin and eosin . (All figures shown 80% oforiginal size .)

osteoblasts is paralleled by the findings of Meachim andRoy (1967) for degenerate chondrocytes of adult human ar-ticular cartilage . The presence of crenated osteocytes withpyknotic nuclei and electron dense bodies in their cyto-plasm is comparable to the observations of Heyden et al .(1977) who reported osteocytes occupying enlarged la-cunae in osteolytic bone . Our investigation confirms thatwoven bone mineralization is initiated within membranebound matrix vesicles (Bernard and Marvaso 1981) and isassociated with alkaline phosphatase activity (Dickson1982) .

Strong activity for the enzymes acid phosphatase andleucine aminopeptidase was found in mononuclear perivas-cular cells that came in contact with remaining bone(Heyden et al . 1977) . They suggested that perhaps thesecells were important in the process of bone resorption . Al-though a histochemical examination of the enzyme leucineaminopeptidase proved unrewarding (Cannon 1986), theacid phosphatase positive lysosomes and heterophagic dis-estion vacuoles found in the macrophage cell cytoplasm, inthe present investigation, imply involvement of these cellsin the resorptive process . Similarly, the demonstration ofacid phosphatase activity in endothelial cells of the thin-walled vascular channels adjacent to the remaining bonesuggests that these vessels might also be involved in thedestructive process (Caulet et al . 1968; Dickson et al .1987) .

G. R . Dickson et al . : Vanishing bone disease

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Fig. 3. TEM micrograph showing fine filaments in the cytoplasm of an osteoblast (below) adjacent to collagenous fibers of bone osteoid(above) . Uranyl acetate and Reynolds' lead citrate staining . x 40,000 .

Fig. 4 . Alkaline phosphatase reaction product associated with the plasma membrane of an osteoblast and discretely concentrated in theextracellular matrix . Preparation demineralized in formic acid and incubated using p glycerophosphate as substrate ; uranyl acetatestaining . x60,000 .

Fig . S . A macrophage in the disaggregated fibrous region . Note the indented nucleus, lysosomal bodies, vesicles beneath the plasmamembrane and irregular cytoplasmic projections . Uranyl acetate and Reynolds' lead citrate staining- x 7800 .

Fig . 6. Non-specific acid phosphatase (substrate : A glycerophosphate) in association with vacuoles of a macrophage . Uranyl acetatestaining . x 60,000 . (Figures shown 80% of original size .)

Difficulty with early diagnosis and the possibility ofspontaneous regression and indefinite stabilization (Ed-wards et al. 1983) often lead to delay in the specific treat-ment of vanishing bone disease. We found no evidence ofregression . Anatomically normal bone was present awayfrom the disease front, so treatment choice involved ag-gressive local surgery combined with the insertion of a freevascularized bone graft (Weiland et al . 1979). As a variableresponse to radiation therapy has been reported and thepromotion of osteoblastic activity and successful reossifi-cation have only been claimed following radiotherapy (Pat-

rick 1976; Heyden et al . 1977), it was decided to apply thistreatment for residual disease only.

Our investigation confirms the need for multidiscipli-nary studies of the condition if earlier diagnosis is to beachieved. TEM demonstrated filament accumulation in en-dothelial cells and osteoblasts while cytochemistry re-vealed the ultrastructural location of alkaline and acidphosphatases . Reossification was facilitiated and to datethere is no further evidence of active disease. Although oursingle case study seems to support the use of resection,bone grafting and radiotherapy in the treatment of van-

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Fig . 7 . Non-specific acid phosphatase (substrate: /3 glycerophosphate) in association with the Gotgi apparatus of an endothelial cell .Uranyl acetate staining . x83,160 . (Figure shown 80% of original size .)

Fig . 8. Non-specific acid phosphatase (substrate : R glycerophosphate) in association with lysosomes in an endothelial cell . Uranyl acetatestaining. x 25,000 . (Figure shown 80% of original size .)

ishing bone disease, it would be adventerous to recom-mend the universal application of this treatment regime toa disease of known variable course .

Acknowledgments : We wish to acknowledge the help of Dr. P.Abraham of the Radiotherapy Centre, Bevoir Park Hospital, Bel-fast, and of Mr. M. D . Brennen, Plastic Surgery, Ulster Hospital,Dundonald, Belfast., N . Ireland .

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Received: February 16, 1989Revised: December 8, 1989Accepted: January 23, 1990