Origi zal Contributions U L T R A S T R U C T U R E OF OSTEOID OSTEOMA Germa~ C. Steine~. M.D.*
Abstract T h e u l t r a s t r u c t u r e in five cases o f osteoid o s t e o m a is described. T h e osteoblasts generally h a d a m o r p h o l o g y similar to that o f n o r m a l osteoblasts with a few differences. T h e y c o n t a i n e d i r r e g u l a r i n d e n t e d nuclei, glycogen particles, a b u n d a n t fine intracytoplasmic fibrils, a n d rare iron containing iysosomes. In several osteoblasts in two cases t h e r e were atypical m i t o c h o n d r i a with a iobulated or " h o n e y c o m b " a p p e a r a n c e . T h e s e atypical m i t o c h o n d r i a were also o b s e r v e d in two osteoclasts; otherwise these cells r e s e m b l e d n o r m a l osteoclasts. O t h e r ceils p r e s e n t in osteoid o s t e o m a besides osteocytes included osteoprog e n i t o r ceils r e s e m b l i n g Scott type A a n d B cells a n d cells in transitional stages o f differentiation. T i l e osteoblasts most likely originated f r o m Scott type A cells or preosteoblasts. T h e areas o f mineralized matrix c o n f o r m e d to tile m o r p h o l o g y o f coarse woven bone. Var),ing a m o u n t s o f osteoid were noted. In two cases the osteoid contained, in addition to collagen, fine g r a n u l a r material, which p r o b a b l y r e p r e s e n t s polysaccharides. No n e r v e fibers were identified in the material studied. It is thottght that osteoid osteonm is a b e n i g n neoplastic lesion. A case o f o s t e o b l a s t o m a was studied for c o m p a r i s o n ; the o s t e o b l a s t o m a cells were f o u n d to h a v e basically the s a m e m o r p h o l o g y as the cells in osteoid osteoma, including the atypical mitochondria. O u r u h r a s t r u c t u r a l observations s u p p o r t tile idea that osteoid o s t e o m a a n d osteoblastoma are closely related lesions.
Osteoid o s t e o m a is a relatively comm o n b e n i g n osteoblastic lesion o f bone. J a f f C was the first to i n t r o d u c e the n a m e and c o n c e p t o f osteoid o s t e o m a as a clinicopathologic entity in 1935; he t h o u g h t that the lesion was not i n f l a m m a t o r y in origin but m o s t likely neoplastic.
Osteoid o s t e o m a occurs in y o u n g individuals, usually m e n , most frequently between 10 a n d 30 years o f age, and has a predilection for the f e m u r and tibia. H o w ever, it m a y o c c u r in any o t h e r bone. 2-8 Clinically the principal complaint in osteoid o s t e o m a is pain, which m a y be
*Assistant Professor of Clinical l'athology, Mount Sinai School of Medicine of the City University of New, Vork. Acting Director of Laboratories. Hospital for Joint Diseases and Medical Center, New York,'New York.
309
HUMAN I ' A T I t O L O G Y - - V O L U M E 7, NUMBER 3
May 1976
persisten.t and severe. Radiographic e x a m i n a t i o n characteristicall)' reveals a slnali radiolucent defect in tire bone, often surrounded by sclerosis. Tire nidus m e a s u r e s al)proximatel )' 1 cm.; lfistologicall)' it consists o f vascularized tissue with osteoblastic proliferation, osteoid and new b o n e f o r m a t i o n , a n d osteoclastic resorption.'-' T h e r e are only two r e p o r t s c o n c e r n i n g the u h r a s t r u c t u r e o f this lesion in tim literatm'e. ~'s In tiffs p r e s e n t a t i o n a detailed account o f the u h r a s t r u c t u r e in five cases o f osteoid o s t e o m a is presented. ht addition, a b r i e f c o m p a r i s o n is m a d e with tire ttltrastructure o f osteoblastoma.
MATERIALS AND METHODS T h e tissue was o b t a i n e d by curettage a n d en bloc resection. For light microscopy the tissue was fixed in 10 p e r cent formalin, d e h y d r a t e d , e m b e d d e d in paraffin, and stained with h e m a t o x y l i n a n d eosin, l ' a r t o f tim tissue was decalcified b)" the formic a c i d - c i t r a t e ntethod. For electron microscopy i m m e d i a t e iixation was carried out using d o u b l e fixation with 3 pet" cent glutaraldeh)"de followed b)" I p e r cent osntiunt tetroxide, both in l)ltosplutte buffer. T h e tissue was then d e h y d r a t e d a n d e m b e d d e d in E p o n 81 o. Sections were cut on a Huxle)' nticrotonte. In o r d e r to prevent demineralization o f the tissue d u r i n g sectioning, the n t e t h o d described by B o o t h r o ) ' d a was carefully followed. T h e sections were then m o u n t e d on coated c o p p e r grids and stained witlt uranyl acetate a n d lead citrate. Sonte sections o f osteoid o s t e o m a tlmt included calcified matrix were stained only with lead I h y d r o x i d e in o r d e r to p r e v e n t loss a n d translocation o f mineral d u r i n g the staining? ~ T h e sections were e x a m i n e d witlt a RCA E M U 31-I electron microscope at 50 kv.
Figure 1. Case I. Roougenograph shows an of bone destruction in tile neck of
irregular area
the left scalmla along the axillary border.
CASE REPORTS
310
Caw 1. A 21 year old white male complained of pain in the left shoukler fbr three weeks. The pain was constant and was relieved b)' aspirin. The results of laborato D" studies were within normal limits. Radiologic e.xaminatio0 revealed an irregtflar area of destruction of the 'l)one in the neck of the scapula, just
Figure 2. Case 2. A snmll circumscribed radiolucent area (arrow) is observed in the proximal end of tile left femur, with mininml perilflleral sclerosis.
O S T E O I D OSTEOMA--STmNER below the glenok! cavity (Fig. i). En bloc resection o f b o n e f i o m the neck o f the scapula was u n d e r t a k e n . Histologic sections revealed a nidus o f osteoid osteoma. Caw 2. A n i n e )'eat- old white boy had severe pain in the left groin for eight months. Initially he was d i a g n o s e d as h a v i n g arthritis of the left hip. Radiologic e x a m i n a t i o n revealed a small r a d i o h t c e n t area in the proximal e n d of the left f e m u r (Fig. 2). En bloc resection o f the lesion was done. Histologic study d e m o n strated a n osleoid osteoma. Case 3. A 22 year old white male complained o f pain ill the right leg of t h r e e years' duration. O n physical e x a m i n a t i o n there was swelling o f the u p p e r part o f the right leg. Radiologic e x a m i n a t i o n disclosed a small lyric lesion in the proximal region o f the tibia, with reactive s u r r o u n d i n g sclerosis (Fig. 3). At s u r g e r y a n excisional biopsy was carried Otlt alld the lesion p r o v e d to be a n ostcoid osteoma. Case 4. All 18 ) e a r old wllite male had noted the g r a d u a l onset of pain in the right wrist for the p r e c e d i n g 15 months. T h e patient was k n o w n to have had muhil)le here(limry
Figure 3. Case 3. l'roximal end of right tibia. A small area of radioluccncy is observed (arrow). A marked degrcd of bony sclerosis is present surrounding the lesion.
Figure 4. Case -t. A small ill defined area of radioluccncy is noted in the medial aspect of the distal end of the radius (arrow). There is associated bone sclerosis around the lesion. Additionally, there is shortening of the distal end of the tflna and a small area of exostosis.
Figure 5. Case 5. An ill defined area of radiohtcency in tile base of tile second nletacarpal bone is observed (arrow). There is cortical sclerosis distal to the lesion.
311
HUMAN I ' A T H O L O G Y - - V O L U M E 7, NUMBER 3 exostoses involving the right ulna and radius and the phalanges of the right hand. Radiologic examination revealed a small area of radiolucency in the distal end of the right radius (Fig. 4). The patient underwent curettement of the distal end of the radius, and pathologic examination of the tissue revealed an osteoid osteoma. 9 Case 5. A 68 year old white female noted pain in the left wrist for the preceding two )'ears. Radiologic examination revealed a lytic lesion in the proximal end of the second metacarpal, with cortical sclerosis distally (Fig. 5). The patient had undergone biopsy of that region three months before, and the tissue sections showed only sclerotic bone. Surgery was undertaken again and a partial resection of the base of the metacarpal bone was done, followed by fusion, l'athologic examination of the tissue revealed an osteoid osteoma.
H I S T O L O G I C A P P E A R A N C E OF THE OSTEOID OSTEOMA In all five cases tissue fl-om the lesion showed the s a m e histologic features. It consisted o f irregtalar trabeculae o f osseous
May 1976
tissue a n d osteoid separated by vascular tissue (Fig. 6). T h e a m o u n t o f calcified bone a n d osteoid varied f r o m case to case. Most trabeculae were outlined by osteoblasts, indicating active osteogenesis. T h e r e were also osteoclasts within the nkhts u n d e r g o i n g bone r e s o r p t i o n (Fig. 6).
U L T R A S T R U C T U R E OF OSTEOID OSTEOMA T i l e electron microscopic features observed in the five cases are described in the following p a r a g r a p h s . OSTEOBLASTS. T h e osteoblasts are usttally e l o n g a t e d and val-)" in size and shape, s o m e cells m e a s u r i n g u p to 50 microns in length. Most osteoblasts have an eccentric nucleus located t o w a r d one pole o f the cell. T h e nuclear m e n a b r a n e is often i r r e g u l a r with indentations a n d with the cln'omatin c o n d e n s e d t o w a r d the p e r i p h e r y (Figs. 7, 8). T h e cytoplasna has a b t t n d a n t r o u g h e n d o p l a s m i c reticuluna with occasional dilatation; in s o m e cells the r o u g h e n d o -
Figure 6. Case I. Photomicrograph of osteoid osteoma shows irregular trabeculae of osteoid and calcified Qsseolls tisst,e, separated b)" a vascular tissue rich in osteoblasts. Note several osteoclasts at lower right
3 1 2 corner. 320.)
OSTEOID
OS'I'EOMA--STEINER
Figure 7. Case -t. Electron inicrograph of osteoblast adjacent to calcified matrix (C). Tile Golgi apparatus (G) shows dilated clear vesicles. There is al)undant rough endoplasniic reticuhnn with dilatation in lower portion of cell. Note predomi,lance of cell processes (1') oil side adjacent to the matrix. (x 11,3-10.)
313
HUMAN PATHOI.OGY--VOLUME 7, NUMBER 3 May 1976
Figure 8. Case 5. Two osteoblasts showing dense aggregates of gl)cogen (arrows). Note eccentric nuclei with indented menll)ranes. (• I 1,3-t0)Inset: lligh power view of glycogen from large osteoblast. (x 38,100.)
plasnfic reticuluna is disposed in dense parallel lines. T h e Golgi al)paratus is well d e v e l o p e d and quite p r o m i n e n t in most cells. It is located in the j u x t a n u c l e a r region and occupies a volume similar to r smaller than that o f the nucleus. It conisis o f many small vesicles, several llattened sacs, and occasional polyhedral vesicles similar to those described in normal osteoblasts (Fig. 7). H Rough endoplasmic reticulum is f o u n d between the Golgi structures. In a few osteoblasts the Golgl structnres are located close to tile cell m e m b r a n e . Rarely small coated vesicles are seen associated with the Golgi apparatus. T h e mitochondria are ovoid or r o u n d , their cristae e x t e n d i n g into the matrix; they have tile typical m o r p h o l o g y alre,3d)" "described} z Very rarely intramitochbndrial granules are seen.
~
314
In two o f o u r cases (3 and 5) t h e r e was in addition a different type o f mitochondria. T h e y vary in size and shape. Tile inner m e n t b r a n e forms cristae, which e x t e n d irregularly across the organelles. T h e s e atypical mitochondria have a Iobulated or "honeyconlb" appearance and o f t e n contain fine electron dense granules (Fig. 9). T h e matrix a n d the cristae o f t e n show areas o f electron lucenc)', which probably r e p r e s e n t material lost d u r i n g the processing o f the tissue (Fig. 9). Tile osteoblasts containing the atypical milochondria are located adjacent to calcified matrix and in the perivascular spa.ces. Occasionall)" typical m i t o c h o n d r i a may be fimnd in these cells. Except for the presence o f tile atypical mitochondria, these osteoblasts do not differ fi'om the o t h e r cells.
OSTEOI I) OSTEOM A--STEL~ER
Figure 9. Case 3. Atypical luitochondria in osteoblast. Note irregularity of the mitochondria's shape and cristae, resulting in a "honeycombed" or Iohulated al)pearance. (x 38,100.)
T h e osteoblasts also contain free ribosonles and fine intracytol)lasmic iliameats that p r e d o m i n a t e at the cell p e r i p h e r y and the cell processes. Some osteoblasts may have an a b u n d a n c e o f fine filaments. Lysosome-like bodies are observed in small numbers; occasionally they are large and xmmerous, h'on containing lysosomes are seldom seen. Glycogen particles are seen in several osteoblasts ill cases 4 and 5, disposed in dense aggregates o f rosette units at the poles o f the cells and infrequently t h r o u g h o u t the cytoplasm (Fig..8). *licrotulmles are o n l y rarely seen.
Tile cell naembrane contains occasional pinocytotic vesicles; coated vesicles are also rarely seen at the cell peripllery. T h e cell processes I)redominate on tile side adjacent to the matrix and may penetrate deep into the collagen and calcified matrix (Fig. 7). T h e osteoblasts form one or m o r e layers o f cells lilting the matrix. T h e cells are disposed in close apposition or are separated by collagen tibrils. Occasionally a zont, la a d h e r e n s is seen at the points o f contact with the cell naembl'ane. Not all osteoblasts show the typical m o r p h o l o g y (lescribe(l. Some cells ad-
315
HUMAN PATHOLOGY--VOLUME 7, NUMBER 3 May 1976
" ... " ~ ; .
."."?,~i : . ' . . ~ . - :
' " .::
L
*
Figure 10. Case 5. Superficial osteoc)te surrounded by calcified matrix. Note well developed Golgi apparatus and sparsity of rough endoplasmic reticulum. Glycogen particles are present in the c)toplasm (arrows); they are very rare ill osteocytes. The lacunar space contains grallu]al" material alld a few collagen fibrils. (x 14,200.)
316
jacent to the osteoid or calcified matrix are smaller, round, or elongated and have sparse organelles and rough endoplasmic reticulum; other cells resemble fibroblasts. T h e perivascular space also contains cells with the typical morphology of nsteoblasts (Fig. 8). OSTEOCYTES, As the osteoblasts are being incorporated into the calcified matrix, the cells develop fewer cell processes and the adjacent collagen undergoes patchy calcificatioll (Fig. 10). T h e deep osteocytes are completely s u r r o u n d e d by calcified matrix and are located in the osseous l a c u n a e . Tile osteocytes are smaller than osteoblasts and contain less rough endoplasmic reticululn. T h e Golgi apparatus is well developed; a few lysosome-like bodies and fine filaments are seen. Ver)' rarel)" glycogen is seen in the osteo'cyres (Fig. 10). T h e cell [Jrocesses of
the osteocytes penetrate into tile canaliculi of tile bone lacunae. T h e lacunar space a r o u n d the osteocytes contains finely granular material and a few collagen fibrils (Fig. 10). T h e margins of the osseous lacunae are well outlined, but the zone of the mineralization is often irregular, with patchy calcification. In case 3 the calcified niatnx contained laminated circumscribed areas of calcified material tile size of a bone lactma. These areas are interpreted as f o r m e r osteocyte lacunae that became mineralized after the death of the osteocyte. Examination of tissue f i o m an osteoid osteoma nidus that was previously decalcified with ,t pet" cent disodium ethylenediaminetetraacetate (case ,t) disclosed the presence of a distinct electron dense layer a r o u n d the osteocyte lacunae and canaliculi. This dense layer, recently called a - l a m i n a
OSTEOID
OSTEOMA--STElyER
Figure 11. Case 4. Area of mineralized matrix shows irregular pattern ofc~dcification ofcoarsc woven or immature I• (x 18,500.)
limitans: 3 is generally seen all a r o u n d the lacunae, althougli it is poorly defined in sonic areas.
~[INFRALIZED ~[ATRIX.
The
mineral-
ized matrix shows calcification o f the collagen fibrils, which are r a n d o m l y arranged and not disposed in a lamellar pattern. This indicates that the bone formec] in osteoid osteoma is coarse or fiber bbne r a t h e r than lamcllar (Fig. 11): 4 Along the surface o f the mil~eralized matrix, adjacent to the osteoblasts or osteoid, the mineralization f r o n t is not even and t h e r e is patchy calcification (Fig. 7). No clear-cut evidence is seen indicating mineralization o f individual fibrils. In a few areas o f the surface the mineralization is not p,ttchy but, rather, solid and granular. T h e s e areas are generally devoid o f surface osteoblasts. OSTFOID. t'k varying a n a o u n t of osteoid or : l n m i n e r a l i z e d collagen matrix is present in hll cases o f osteoid o s t e o m a .
It consists o f bundles or groups o f densely packed, parallel collagen fibrils. Tile bundles vary in size and are generally r a n d o m l y a r r a n g e d in tile osteoid (Fig. 12A). T h e direction o f the collagen is not related to tile position of tile surface osteoblasts. T h e a m o u n t o f osteoid seen in osteoid osteoma varies greatly, even within the same nidus. I f the matrix is heavily mineralized, t h e r e is little osteoid seen. On the o t h e r h a n d , the osteoid m a t r i x m a y be quite p r o m i n e n t in other areas where there is little or no mineralization. In cases I a n d '2, in addition to tile collagen, the osteoid contains finely granular, nonfibrillar material, interspersed irregularly between the collagen fibrils (Fig. 12). T h e r e is no evidence o f calcification in these areas (some sections were stained only with" lead h y d r o x i d e to prevent loss o f nfineral). T h e granular material p r e d o m i n a t e s in dense osteoid, a h h o u g h it may be seen adjacent to osteo-
317
ItUMAN
318
I'ATilOLOGY-VOLUME
7, N U M B F R 3
May 1976
Figure 12. A. Area ofosteoid (O) adjacent to ostcoblast (Ob) in case 2. Tile collagen fibrils arc seen in both cross ;tnd hingitudinal sections. "File osteoid in the lower left of tlle electron micrograph shows dense material interspersbd between the collagen. (• 18,500.) B, High power view ofosteoid cousisting of fine grantllar"material alld collagen. (• 37,900.)
OSTEOI D OSTEOMA--STEINER
F i g u r e 13. Case 4. Osteoclast adjacent to mineralized matrix (M). T h e r e a r e n u m e r o p s clear vacuoles p r e d o m i n a t i n g n e a r the ruffled b o r d e r at'ca (B) o f the cell. Also note sparsity o f organelles in ru|tled border.
(x 5700.)
blasts where the collagen is loose. T h e areas o f osteoid containing the granular material are devoitl of collagen (l:ig. 12). Mqst of the osteoid sm'ihce is covered by ostebblasts and cells resembling fibroblasts. Some osteoblasts are quite large, with multiple lysosome-like bodies and abundant fine filaments. T h e r e are also large g r a n u l a r bodies that probably represent iron containing lysosomes. In the areas of osteoid not covered b)' osteoblasts, the osteoid border is irregular and ill defined, and collagen fibrils can be seen to end abrtq~tl)'. T h e osteoid also contains osteoblasts that have become incorporated into the matrix and, are therefore designated as osteoid osteoc'ytes, t~ T h e y are similar to
osteocytes o f mineralized matrix. Sonm osteoid osteocytes are s u r r o u n d e d by dense collagen matrix and granular material. Others have a 1)ericellular lacuna, which contains fine granular material, few fibrils, and cell processes. Frequently the cell processes penetrate into the canaliculi of the s u r r o t m d i n g osteold. Occasional pinocytotic vesicles and coated vesicles are seen in the osteoid osteocytes. No glycogen is identified. Although most of the osteoid is not mineralized, it may show isolated patches of calcification. This occurs in the areas of osteoid adjacent to mineralized matrix. OSTr:OCCASTS. These cells are present in varying numl)ers in the osteoid o s t e o l l l a nidi. Most ceils are large and con~
319
IIUMAN I'ATIIOLOGY--VOLUME 7, NUMBER 3 tain nm.ltii)le nuclei, whiclt are ovoid a n d i n d e n t e d a n d are located at tire center or 1)eriphery o f the cells (Fig. 13). T h e Golgi a p p a r a t u s consists o f llatt e n e d sacs and small vesicles disposed a r o u n d the individual nuclei. O f t e n small coated vesicles are seen associated with tire Golgi a p p a r a t u s . Free ribosomes are seen scattered througl~out the cytoplasm. T h e r o u g h endoplasnaic reticulunt is not p r o n f i n e n t a n d shows occasional dilation. Fine intracytoplasmic fibrils are also present, as well as occasional lysosome-like bodies. Most osteoclasts have clear m e m b r a n e b o u n d vacuoles, s o m e the size o f m i t o c h o n d r i a attd o t h e r s larger. T h e s e large vacuoles p r e d o m i n a t e at tim site o f the rullled b o r d e r (Fig. 13). S o m e o f these vacuoles m a y r e p r e s e n t d e g e n e r ated mitochondria. A large n u m b e r o f nfitochondria are distributed througi~out the cytoplasm except f o r tlm areas n e a r the ruffled b o r d e r (Fig. 13). Only in two osteoclasts (fi'om cases 3 and 5) are tlm m i t o c h o n d r i a quite different f r o m those in o t h e r cells; they vary in size a n d slmpe a n d reselnble the atypical nfitochondria
320
May 1976
described in osteoblasts. T h e cristae f o r m small c o m p a r t m e n t s and the mitochondrial matrix has areas o f electron lucency (Fig. 14). T h e ruffled or brush b o r d e r is a specialized a r e a o f the osteoclast c o m p o s e d of folds a n d irregtdar projections o f the plasma m e m b r a n e , in contact with or adjacent to calcified matrix (Fig. 15). T h e folds are devoid of organelles, lmve a g r a n u l a r c)'toplasm, anti often contain coated pits. In the areas o f close apposition with the calcified matrix, loose b o n e salt crystals are seen within the spaces between the lolds (Fig. 15). Not infi'equently collagen fibrils are seen d e n u d e d o f mineral. T h e r e is no evidence o f b o n e crystals within the cytoplasm of the osteoclasts. Not all the surfaces o f osteoclasts adjacent to calcified matrix have a ruffled b o r d e r ; in these areas the cell m e m b r a n e is smootlt. Ruffled b o r d e r s are not o b s e r v e d in all osteoclasts, a h h o u g h serial sections o f these cells m a y d e m o n s t r a t e their presence. H o w e v e r , several giant cells are seen surr o u n d e d by osteoblasts and at a distance f r o m the b o u e matrix; this ntay indicate
Figure 14. A, Atypical initochondria from osteoclast in case 3. Tile mitochondrial matrix shows electron luceut areas and a few granules. (• 38,000.) B, At)'pical mitochondria from oste,,blast in case 5 shows irregular configur'ation of the cristae that divide the oiganelles into small compartments. (• 38,000.)
OSTEOID O S T E O M A - STr:I,~rCR
Figure 16. Case 1. I'rimitive perivascular cell (P) witfi scanty cytoplasm and sparse organelles. It resembles the Scott t)pe A cell or preosteoblast. A capillary is seen at the left upper corner. (x 10,500.)
Figure 1,5. Case 4. Ruffled border of osteoclast. Note the tmmerous projections adjacent to calcified matrix. (• 32,400.)
that some o f these giant cells probably are in a resting state and not u n d e r g o i n g bone resorption. ~5 VFSSELS
AND
PERIVASCULAR
CELLS.
matrix. Most o f these cells are identified as perivascular osteoblasts o f varying size, with eccentric nuclei (Fig. 8). T h e p r o p o r tion o f organelles varies fi-om cell to cell. Glycogen particles are identified in several osteoblasts although not in all five cases studied (Fig. 8). O t h e r cells present in the perivascular spaces inchtde a few ehmgated or ovoid cells with scanty cytoplasm and sparse organelles. Some cells contain glycogen toward the p e r i p h e r y . T h e s e l)oorly differentiated cells resemble the type A cell described by Scott ~Gin fetal bone (Fig. 16). Few o t h e r cells resemble the type B cell o f
Vascular structures al'e often seen in osteoid osteomas. T h e vessels wwy from small cal~illaries to large vascular spaces, which ntay be s u r r o t m d e d by pericytes or smooth muscle cells. Arterioles are often identilied. Most capillaries have a continuous e n d o t h e l i u m , a h h o u g h occasionally fenestrated capillaries are seen. Most o f these vessels and their cells have the same morphoh)gy as normal vessels (Fig. 16). v' (The exception was an arteriole o f case 5 in which the endothelial and smooth nmscle cells were t b u n d to contain atypical mitochondria similar to the ones described in osteoblasts.) A large n u m b e r o f cells s u r r o u n d the Figure 17. Case I. Perivascular cell resembling vessels and occtq))' the spaces between the - Scott type B cell.-(x 11,500.)
321
IIUMAN I'ATIIOLOGY-VOI.UME 7, NUMBER 3 May 1976 Scott. "I'hey have an u n d u l a t e d cell membrane, ~r developed (;olgi apparatus, free ribosonms, and iysosome-like bodies (Fig. 17). T h e r e are a few o t h e r elongated cells with the n m r p h o l o g y o f fibroblasts, occasionally containing glycogen. T h e r e are still o t h e r cells with morphologic features between poorly differentiated cells, osteoblasts, and fibroblasts. T h e y may r e p r e s e n t transitional stages. T h e presence o f nerve fibers a r o u n d the blood vessels was also investigated. O u r ultrastructural observations failed to find nerve fibers in osteoid osteoma. An intensive search was carried out in cases 4 and 5, lint the results were negative. l'araffin sections o f cases 4 and 5 were stained for nerve fibers and onl)' a few nerve axons were identified in the tissue) 7 OSTEOBLASTOMA. T h e patient was a I,t year old female with a destructive lesion in the midshaft o f the right llu111el"tlS
322
measuring apl)roxinmtely 3 cm. in diameter. She lind had surgery in that region one year previously. Curettage was done. In general tim ultrastructure o f the osteoblasts resembles that o f the osteoid ostcoma. T h e cells have eccentric, often i n d e n t e d nuclei (Fig. 18). T h e r o u g h endoplasmic reticuhmi is l n o m i n e n t . T h e Golgi a p p a r a t u s is well developed and is located in the j v x t a n u c l e a r region. In some osteoblasts there are abnormal mitoc h o n d r i a similar to the ones described in the cases o f osteoid osteoma (Fig. 18). T h e osteoblasts are quite p r o m i n e n t and a b t m d a n t along the margins o f the bone matrix and in the perivascular spaces. T h e osteocytes show fewer organelles than osteoblasts, although rarely they also contain atyl)ical initochondria. T h e osteoclasts show a m o r p h o l o g y similar to that o f the osteoclasts in osteoid osteolna. A few poorly differentiated cells are seen in the perivascular spaces.
Figure,18. Ostcol)lastoma.TWOosteotflastsshow indented nuclei, abundant rough cndoplasmic reticuhml, ;m,i at~.pioalmitochondria similar to those described in osteoid ostcoma (see Fig. 9). (x I-t,200.)
OSTEOI D OSTEOMA--STEINER DISCUSSION
A review of the literature reveals tlmt there are only two reports concerning the ultrastructure o f osteoid osteoma, only one of wlfich appeared in the English literature. 8 In that report, however, very few observations a l l d COllllllelltS w e l ' e made by the authors concerning their ultrastructural findings. The other report appeared in the Italian literature, and the authors described the presence of viruslike particles, as well as pseudocrystalline structures within the cells of the nidus.; In the present study the predominant cells in osteoid osteoma are the osteoblasts. They lmve a morphology similar to that of normal osteoblasts, with only a few differences. In osteoid osteoma the osteoblasts Imve irregular and indented nt,clei, atypical mitocim,ldria, occasional glycogen, a larger numbcr of intracytoplasmic fine [ibrils, and occasional iron contai,fing lysosomcs. In additiou, although the Golgi apparatus is well developed, it is usually more irregularly disposed i,a the juxtanuclear region of the cells, often intermingled with the rough endoplasmic retict,lum. "l'he abundance of rough endoplasmic reticulum in most of these cclls is an iudication that the osteoblasts in osteoid osteoma are actively engaged i,a matrix production. T h e presence of more than one layer of osteoblasts lining the matrix and the haphazard arrangement of the cells indicate that, at least in some areas, the bone formation takes place rapidly. ~ The areas of mineralized matrix conform to the morphology of coarse woven bo,ae. The bundles of calcified collagen are irregular in size and are randomly arranged, ~vith no lamellar orientation of the fibrils. 14 The osteoid present in ostcoid ostcoma shows irregular layers of collagen that in general intersect each other randomly. Some osteoid osteocytes h,tve l)ericelhtlar lacunae. The degree of osteoid tbrmation without mineralization in the nidus is abnormal. It is known, however, that in growi,ag bone there is a variable amount ofosteoid formation prio," to calcification,TM aud in fracture callus there is abundant osteoid formation with a marked delay in c.tlcificationYa It" appears that in osteoid
osteoma the osteoblasts lay down an osteoid matrix, which may remain unmineralized at least temporarily. The lack of mineralization of the osteoid matrix may be due to local factors tlmt include the composition of the osteoid, the availability of mineral, and the vascularity of the lesion. Although most of the osteoid is unmineralized, it shows patchy calcification in a few areas near the calcified matrix. These findings have raised doubts tlmt collagen alone provides the site tot initial calcification,va Bernard and Pease '-'~ found polysaccharide cores in the osteoid during early ossification; they believe that this material might be the initial calcification locus. An interesting observation in two cases of osteoid osteoma was that in addition to the collagen the osteoid matrix contained finel)" granular material in a focal distribution. This material has not been previously described in unmineralized bone matrix. However, granular material similar to this Ires been observed in decalcified sections of bone matrix during early mineralization and was shown to be polysacclmride?s'-~~ We do not know the signilicance of this granular material in the osteoid matrix; it is possible timt it represents polysaccharides. The osteoid with this material may thus represent the matrix ready for mineralization. In addition to osteoblasts, osteocytes, and osteoclasts there are primitive cells identified in the perivascular spaces. These are osteoprogenitor ceils tlmt resemble the type A (preosteoblasts) and type B (preosteoclasts) cells described by Scot0 ~' in fetal bone. Our observations indicate timt the osteoblasts most likely are derived from Scott type A cells or preosteoblasts. No infl)rmation was obtained concerning the origin of the osteoclasts. Glycogen was found in two cases of osteoid osteoma, disposed in the form of rosette units. It was observed mainly in the osteoprogenitor cells undergoing osteoblastic transfornmtion and less fiequently in developed osteoblasts ar to bone matrix. Glycogen was quite rare in osteocytes. It is known that during fetal development all osteogenic cells contain varying amounts of glycogen.-"~ In postnatal bone, on the other Imnd, glycogen was demonstrated in differentiating osteo-
323
HUMAN I'ATHOLOGY--VOLUME 7, NUMBER 3 May 1976
324
blasts o r preosteoblasts, but was absent in d e v e l o p e d osteoblasts.""" ":~ In fracture callus, ahhougl~ some authors have f o t m d glycogen in i)reosteoblasts, 22 others fail to find it in any bone cell. "3'za T h e significance o f glycogen in osteogenic cells is not known. Pritchard ''~ believes that the presence o f glycogen indicates a special adaptation o f the osteogenic cells to conditions o f fetal life and not a typical feature o f these cells tln'ouglmut life. T h e significance o f the atypical mitoc h o n d r i a in some osteoblasts and a few osteoclasts is not known. T h e y have not been previously described in these cells. Some o f the cristae bear a resemblance to the villi described in the mitochondria o f the giant ameba, l'elomyxa carolinensis. "-'~ It is possible that the morphologic changes ' in these mitochondria may be the resuh o f pathologic conditions not yet known "-'n T h e intramitochondrial granules most likely r e p r e s e n t depots or binding sites for divalent cations, particularly calcium. "G N e r v e fibers can be identified in osteoid osteoma by use o f the light microscope; their presence has been d o c u m e n t e d by using silver staining techniques. ~r T h e pain, so characteristic o f osteoid osteoma, is probably transmitted by a u t o n o m i c nerve fibers, which are sensitive to changes in vascular pressure. ~r In o u r ultrastructural studies we failed to find nerve fibers in osteoid osteoma. An intensive search was u n d e r t a k e n in cases 4 and 5, but the results were negative. T h e difficulty in identifying nerve fibers at the uhrastructural level is probably related to the sampling of the tissue and the fact that nerve fibers al'e not a b u n d a n t in the nidus. T h e origin o f osteoid osteoma is not known. Some authors imve identified ~firus-like particles in the cells o f the nidus find believe that osteoid osteoma is a dysplastic disease: We were not able to find virus-like particles in o u r cases. Jaffe l was the first a u t h o r to favor the idea tlmt osteoid osteoma is a benign neoplasm, and this is now accepted by most authors and by the present writer.'-" a,.~, ,, ,.,r Althougil the osteoblasts in osteoid osteoma have i n d e n t e d nuclei, atypical mitochondria, and an a b u n d a n c e o f fine fi]alnents, basically they do not differ from normal osteoblasts. Except for the presence o f atypical lnitochondria in a few-cells, the
osteoclasts resemble nornlal osteoclasts. T h e osteocytes o f osteoid osteoma are morplmlogically sitnilar to normal osteocytes. We believe, as do others~ s that in the nidus t h e r e is evidence o f a dynamic process o f rapid bone formation and destruction in a small volume o f tissue. T h e bone r e s o r p t i o n is exerted by the action o f the osteoclasts on the mineralized matrix; they a p p e a r to Imve no affinity for o s t e o i d . 28
As to the relationslfip between osteoid osteoma and osteoblastoma, most authors consider tlmt these lesions are closely related altlmugll with some differences in r e f e r e n c e to size, localization, and reactive sclerosis, e-G Most o f the osteoid osteomas measure approximately I cm. in diameter; the osteoblastomas are larger.'-' Histologically both lesions show basically the same morplmlogy, although the osteoblastoma may be more vascular and lmve a larger a m o u n t o f osteoid than the osteoid o s t e o i l l a . '2''~ O l . l r
uhrastructural
obsel'va-
tions s u p p o r t the idea that osteoid osteoma and osteoblastoma are closely related lesions. In general, cells o f the latter have features quite similar to those o f osteoid osteoma, including the presence o f atypical mitochondria. ACKNOSVLEDGMENT
T h e a u t i m r expresses Iris gratitude to Carol Schreck for teclmical assistance. REFERENCES 1. Jaffe, ! I. I..: "Ostctfid ostconla": :l bcnign ostcoI)l;istic t u m o r COml)oscd o f ostcoid and atypical b,me. Arch. Surg., 31:709, 1935. 2. Jalt'e, H. L.: T u m o r s and T u , u o r o u s Conditions o f the l~,oucs andJoints, lqlila(lcll)hia, Lea and Febigcr, 1958. 3. I)ahlin, C.: Bone T v m o r s . Springfield, Illinois, Charles C T h o m a s , 1973. 9t. Byers, 1'. I).: Solitary benign osteoblastic lesions o f bone. Ostcoid osteoma and ostcoblastoma. Cancer, 22:-t3, 1968. 5. Schajowicz, F., and I.emos, ('.: Osteoid osteoma and ostcoblastoma. Acta Orthop. Stand., 41: 272, 1970. 6. l.ichtcnstein, I..: Bone T u m o r s . St. l.otfis, T h e ('. V. Mosby Cgmpany, 1972. 7. I)e Giuli, C., and Fromino, G.: Rilicvo al microscopio clcttronico di partlco, lari strutture endocelh,lari in un caso di osteoxna ostcoidc. Arch. Ital. i'at. Cliq. Tumori, X/:35, 1968.
OSTEOID 8. lliroh;ua, K., and Mnrimoto, K.: Ultrastructtn-e of Bone and J'oint Discascs. Tokyo, Igaku Shoin l.td. (Grune & Stratton, Inc.), 1971. 9. Boothroyd, IL: T h e prnblcms of dcmincralisation in thin sections of fully calcified bone. J. Cell Biol., 20:165, 196-t. 10. Karnovsky, M. J.: Simple methods fi)r "staining with lead" at high pH in electron microscopy. .l. l~,iophys, lfiochem. Cytol., 11:729, 1961. 11. Cameron, 1). A.: T h e uhrastructure of bone. In Bourne, G. il. (Editor): T h e Biochemistry and I'hysiology of Bone. New York, Academic Press, Inc., 1972, Vol. !. 12. ttam, A. W.: llistology. Ed. 7. Philadelldfia, J. B. Lipplncott Co., 197-t. 13. Scherft, J. 1'.: T h e lamina limitans of the organic nmtrix of calcified cartilage and b o n c . . l Uhrastruct. Res., 38:318, 1972. 14. Dudley, tt. R., and Spiro, I).: T h e fine structure of bone cells. J. l~iol)h)'s. Biochcm. Cytol., I1: 627, 1961. 15. I lancox, N. M.: Biology of Bone. Cambridge, Cambridge University Press, 1972. 16. Scott,. B. L.: Th)'midine-3ti electron microscope radioautography of ostcogenic cells in the fetal rat. J. Cell Biol., 35:115, 1967. 17. Schuhnan, 1.., and I)orfman, It. D.: Nerve fibers in ostcoid osteoma. J. Bone Joint Surg., 52:1: 1351, 1970. 18. Scott, B. L., and Pease, D. C.: Electron microscopy of the epiphyseal al~paratus. Anat. Rec., 126:-t65, 1956. 19. Cameron, D. A.: T h e fine StltlCttlle Of bone and
OSTEOMA--STEINER
calcified cartilage. Clin. Orlhop., 26:199, 1963. 20. l~,el'nard, (,. W., and I'ease, I). C.: An electron microscopic study of ilfitial intranlembranotls osteogencsis. Amer. J. Anat., 125:271, 1969. 21. Soon, B. I.., and Glimcher, M.J.: l)istribution of glycogcn in ostcol)lasts of tllc fetal rat. J. Uhrastruct. Res., 36:565, 1971. '2'2. Schajowicz, F., and (:abrini, R.: llistnchemical studies on glycogcn in normal ossification and calcification. J. Bone Joint Surg., 40..1:1081, 1958. 23. l'ritchard, J. J.: T h e osteoblast. In Bourne, G. H. (Editor): T h e Biochemistry and l'hysiology of Bone. New York, Academic Press, Inc., 1972, Vol. 1. 2.t. Gothlin, G.: Electron microscopic observations on fracture repair in the rat. Acta l'ath. Microbiol. Scand., Section A. 81, 507, 1973. 25. Pal)pas, G. D., and Brandt, 1'. W.: Mitochondria. I. Fine structure of the complex patterns in the mitochondria of l'elomyxa carolinensis Wilson (Chaos chaos, L.). J. Biophys. Biochem. C)tol., 6:85, 1959. '26. "lalldlcr, B., and llol)pcl, C. L.: Mitochondria. New York, Academic I'rcss, Inc., 1972. 27. Spjut, ll.J., I)orfinan, II. I)., Fechner, R. E., and Ackerman, 1.. V.: Atlas of "l'lllnOl" Pathology, Second Series, Fascicle 5. Tumors of Bone and Cartilage. Washington, D.C., Armed Forces Institute of I'athoh>gy, 1971. 28. Johilston, A. D.: Clinical pnoblcnns in ostcoid osteoma. Evidence of osteoclastic aversi(m to osteoid. Bull. Hosp. Joint Dis., 23:81), 1962.
1919 Madison Avenue New York, New York 10035
325
Abstract T h e u l t r a s t r u c t u r e in five cases o f osteoid o s t e o m a is described. T h e osteoblasts generally h a d a m o r p h o l o g y similar to that o f n o r m a l osteoblasts with a few differences. T h e y c o n t a i n e d i r r e g u l a r i n d e n t e d nuclei, glycogen particles, a b u n d a n t fine intracytoplasmic fibrils, a n d rare iron containing iysosomes. In several osteoblasts in two cases t h e r e were atypical m i t o c h o n d r i a with a iobulated or " h o n e y c o m b " a p p e a r a n c e . T h e s e atypical m i t o c h o n d r i a were also o b s e r v e d in two osteoclasts; otherwise these cells r e s e m b l e d n o r m a l osteoclasts. O t h e r ceils p r e s e n t in osteoid o s t e o m a besides osteocytes included osteoprog e n i t o r ceils r e s e m b l i n g Scott type A a n d B cells a n d cells in transitional stages o f differentiation. T i l e osteoblasts most likely originated f r o m Scott type A cells or preosteoblasts. T h e areas o f mineralized matrix c o n f o r m e d to tile m o r p h o l o g y o f coarse woven bone. Var),ing a m o u n t s o f osteoid were noted. In two cases the osteoid contained, in addition to collagen, fine g r a n u l a r material, which p r o b a b l y r e p r e s e n t s polysaccharides. No n e r v e fibers were identified in the material studied. It is thottght that osteoid osteonm is a b e n i g n neoplastic lesion. A case o f o s t e o b l a s t o m a was studied for c o m p a r i s o n ; the o s t e o b l a s t o m a cells were f o u n d to h a v e basically the s a m e m o r p h o l o g y as the cells in osteoid osteoma, including the atypical mitochondria. O u r u h r a s t r u c t u r a l observations s u p p o r t tile idea that osteoid o s t e o m a a n d osteoblastoma are closely related lesions.
Osteoid o s t e o m a is a relatively comm o n b e n i g n osteoblastic lesion o f bone. J a f f C was the first to i n t r o d u c e the n a m e and c o n c e p t o f osteoid o s t e o m a as a clinicopathologic entity in 1935; he t h o u g h t that the lesion was not i n f l a m m a t o r y in origin but m o s t likely neoplastic.
Osteoid o s t e o m a occurs in y o u n g individuals, usually m e n , most frequently between 10 a n d 30 years o f age, and has a predilection for the f e m u r and tibia. H o w ever, it m a y o c c u r in any o t h e r bone. 2-8 Clinically the principal complaint in osteoid o s t e o m a is pain, which m a y be
*Assistant Professor of Clinical l'athology, Mount Sinai School of Medicine of the City University of New, Vork. Acting Director of Laboratories. Hospital for Joint Diseases and Medical Center, New York,'New York.
309
HUMAN I ' A T I t O L O G Y - - V O L U M E 7, NUMBER 3
May 1976
persisten.t and severe. Radiographic e x a m i n a t i o n characteristicall)' reveals a slnali radiolucent defect in tire bone, often surrounded by sclerosis. Tire nidus m e a s u r e s al)proximatel )' 1 cm.; lfistologicall)' it consists o f vascularized tissue with osteoblastic proliferation, osteoid and new b o n e f o r m a t i o n , a n d osteoclastic resorption.'-' T h e r e are only two r e p o r t s c o n c e r n i n g the u h r a s t r u c t u r e o f this lesion in tim literatm'e. ~'s In tiffs p r e s e n t a t i o n a detailed account o f the u h r a s t r u c t u r e in five cases o f osteoid o s t e o m a is presented. ht addition, a b r i e f c o m p a r i s o n is m a d e with tire ttltrastructure o f osteoblastoma.
MATERIALS AND METHODS T h e tissue was o b t a i n e d by curettage a n d en bloc resection. For light microscopy the tissue was fixed in 10 p e r cent formalin, d e h y d r a t e d , e m b e d d e d in paraffin, and stained with h e m a t o x y l i n a n d eosin, l ' a r t o f tim tissue was decalcified b)" the formic a c i d - c i t r a t e ntethod. For electron microscopy i m m e d i a t e iixation was carried out using d o u b l e fixation with 3 pet" cent glutaraldeh)"de followed b)" I p e r cent osntiunt tetroxide, both in l)ltosplutte buffer. T h e tissue was then d e h y d r a t e d a n d e m b e d d e d in E p o n 81 o. Sections were cut on a Huxle)' nticrotonte. In o r d e r to prevent demineralization o f the tissue d u r i n g sectioning, the n t e t h o d described by B o o t h r o ) ' d a was carefully followed. T h e sections were then m o u n t e d on coated c o p p e r grids and stained witlt uranyl acetate a n d lead citrate. Sonte sections o f osteoid o s t e o m a tlmt included calcified matrix were stained only with lead I h y d r o x i d e in o r d e r to p r e v e n t loss a n d translocation o f mineral d u r i n g the staining? ~ T h e sections were e x a m i n e d witlt a RCA E M U 31-I electron microscope at 50 kv.
Figure 1. Case I. Roougenograph shows an of bone destruction in tile neck of
irregular area
the left scalmla along the axillary border.
CASE REPORTS
310
Caw 1. A 21 year old white male complained of pain in the left shoukler fbr three weeks. The pain was constant and was relieved b)' aspirin. The results of laborato D" studies were within normal limits. Radiologic e.xaminatio0 revealed an irregtflar area of destruction of the 'l)one in the neck of the scapula, just
Figure 2. Case 2. A snmll circumscribed radiolucent area (arrow) is observed in the proximal end of tile left femur, with mininml perilflleral sclerosis.
O S T E O I D OSTEOMA--STmNER below the glenok! cavity (Fig. i). En bloc resection o f b o n e f i o m the neck o f the scapula was u n d e r t a k e n . Histologic sections revealed a nidus o f osteoid osteoma. Caw 2. A n i n e )'eat- old white boy had severe pain in the left groin for eight months. Initially he was d i a g n o s e d as h a v i n g arthritis of the left hip. Radiologic e x a m i n a t i o n revealed a small r a d i o h t c e n t area in the proximal e n d of the left f e m u r (Fig. 2). En bloc resection o f the lesion was done. Histologic study d e m o n strated a n osleoid osteoma. Case 3. A 22 year old white male complained o f pain ill the right leg of t h r e e years' duration. O n physical e x a m i n a t i o n there was swelling o f the u p p e r part o f the right leg. Radiologic e x a m i n a t i o n disclosed a small lyric lesion in the proximal region o f the tibia, with reactive s u r r o u n d i n g sclerosis (Fig. 3). At s u r g e r y a n excisional biopsy was carried Otlt alld the lesion p r o v e d to be a n ostcoid osteoma. Case 4. All 18 ) e a r old wllite male had noted the g r a d u a l onset of pain in the right wrist for the p r e c e d i n g 15 months. T h e patient was k n o w n to have had muhil)le here(limry
Figure 3. Case 3. l'roximal end of right tibia. A small area of radioluccncy is observed (arrow). A marked degrcd of bony sclerosis is present surrounding the lesion.
Figure 4. Case -t. A small ill defined area of radioluccncy is noted in the medial aspect of the distal end of the radius (arrow). There is associated bone sclerosis around the lesion. Additionally, there is shortening of the distal end of the tflna and a small area of exostosis.
Figure 5. Case 5. An ill defined area of radiohtcency in tile base of tile second nletacarpal bone is observed (arrow). There is cortical sclerosis distal to the lesion.
311
HUMAN I ' A T H O L O G Y - - V O L U M E 7, NUMBER 3 exostoses involving the right ulna and radius and the phalanges of the right hand. Radiologic examination revealed a small area of radiolucency in the distal end of the right radius (Fig. 4). The patient underwent curettement of the distal end of the radius, and pathologic examination of the tissue revealed an osteoid osteoma. 9 Case 5. A 68 year old white female noted pain in the left wrist for the preceding two )'ears. Radiologic examination revealed a lytic lesion in the proximal end of the second metacarpal, with cortical sclerosis distally (Fig. 5). The patient had undergone biopsy of that region three months before, and the tissue sections showed only sclerotic bone. Surgery was undertaken again and a partial resection of the base of the metacarpal bone was done, followed by fusion, l'athologic examination of the tissue revealed an osteoid osteoma.
H I S T O L O G I C A P P E A R A N C E OF THE OSTEOID OSTEOMA In all five cases tissue fl-om the lesion showed the s a m e histologic features. It consisted o f irregtalar trabeculae o f osseous
May 1976
tissue a n d osteoid separated by vascular tissue (Fig. 6). T h e a m o u n t o f calcified bone a n d osteoid varied f r o m case to case. Most trabeculae were outlined by osteoblasts, indicating active osteogenesis. T h e r e were also osteoclasts within the nkhts u n d e r g o i n g bone r e s o r p t i o n (Fig. 6).
U L T R A S T R U C T U R E OF OSTEOID OSTEOMA T i l e electron microscopic features observed in the five cases are described in the following p a r a g r a p h s . OSTEOBLASTS. T h e osteoblasts are usttally e l o n g a t e d and val-)" in size and shape, s o m e cells m e a s u r i n g u p to 50 microns in length. Most osteoblasts have an eccentric nucleus located t o w a r d one pole o f the cell. T h e nuclear m e n a b r a n e is often i r r e g u l a r with indentations a n d with the cln'omatin c o n d e n s e d t o w a r d the p e r i p h e r y (Figs. 7, 8). T h e cytoplasna has a b t t n d a n t r o u g h e n d o p l a s m i c reticuluna with occasional dilatation; in s o m e cells the r o u g h e n d o -
Figure 6. Case I. Photomicrograph of osteoid osteoma shows irregular trabeculae of osteoid and calcified Qsseolls tisst,e, separated b)" a vascular tissue rich in osteoblasts. Note several osteoclasts at lower right
3 1 2 corner. 320.)
OSTEOID
OS'I'EOMA--STEINER
Figure 7. Case -t. Electron inicrograph of osteoblast adjacent to calcified matrix (C). Tile Golgi apparatus (G) shows dilated clear vesicles. There is al)undant rough endoplasniic reticuhnn with dilatation in lower portion of cell. Note predomi,lance of cell processes (1') oil side adjacent to the matrix. (x 11,3-10.)
313
HUMAN PATHOI.OGY--VOLUME 7, NUMBER 3 May 1976
Figure 8. Case 5. Two osteoblasts showing dense aggregates of gl)cogen (arrows). Note eccentric nuclei with indented menll)ranes. (• I 1,3-t0)Inset: lligh power view of glycogen from large osteoblast. (x 38,100.)
plasnfic reticuluna is disposed in dense parallel lines. T h e Golgi al)paratus is well d e v e l o p e d and quite p r o m i n e n t in most cells. It is located in the j u x t a n u c l e a r region and occupies a volume similar to r smaller than that o f the nucleus. It conisis o f many small vesicles, several llattened sacs, and occasional polyhedral vesicles similar to those described in normal osteoblasts (Fig. 7). H Rough endoplasmic reticulum is f o u n d between the Golgi structures. In a few osteoblasts the Golgl structnres are located close to tile cell m e m b r a n e . Rarely small coated vesicles are seen associated with the Golgi apparatus. T h e mitochondria are ovoid or r o u n d , their cristae e x t e n d i n g into the matrix; they have tile typical m o r p h o l o g y alre,3d)" "described} z Very rarely intramitochbndrial granules are seen.
~
314
In two o f o u r cases (3 and 5) t h e r e was in addition a different type o f mitochondria. T h e y vary in size and shape. Tile inner m e n t b r a n e forms cristae, which e x t e n d irregularly across the organelles. T h e s e atypical mitochondria have a Iobulated or "honeyconlb" appearance and o f t e n contain fine electron dense granules (Fig. 9). T h e matrix a n d the cristae o f t e n show areas o f electron lucenc)', which probably r e p r e s e n t material lost d u r i n g the processing o f the tissue (Fig. 9). Tile osteoblasts containing the atypical milochondria are located adjacent to calcified matrix and in the perivascular spa.ces. Occasionall)" typical m i t o c h o n d r i a may be fimnd in these cells. Except for the presence o f tile atypical mitochondria, these osteoblasts do not differ fi'om the o t h e r cells.
OSTEOI I) OSTEOM A--STEL~ER
Figure 9. Case 3. Atypical luitochondria in osteoblast. Note irregularity of the mitochondria's shape and cristae, resulting in a "honeycombed" or Iohulated al)pearance. (x 38,100.)
T h e osteoblasts also contain free ribosonles and fine intracytol)lasmic iliameats that p r e d o m i n a t e at the cell p e r i p h e r y and the cell processes. Some osteoblasts may have an a b u n d a n c e o f fine filaments. Lysosome-like bodies are observed in small numbers; occasionally they are large and xmmerous, h'on containing lysosomes are seldom seen. Glycogen particles are seen in several osteoblasts ill cases 4 and 5, disposed in dense aggregates o f rosette units at the poles o f the cells and infrequently t h r o u g h o u t the cytoplasm (Fig..8). *licrotulmles are o n l y rarely seen.
Tile cell naembrane contains occasional pinocytotic vesicles; coated vesicles are also rarely seen at the cell peripllery. T h e cell processes I)redominate on tile side adjacent to the matrix and may penetrate deep into the collagen and calcified matrix (Fig. 7). T h e osteoblasts form one or m o r e layers o f cells lilting the matrix. T h e cells are disposed in close apposition or are separated by collagen tibrils. Occasionally a zont, la a d h e r e n s is seen at the points o f contact with the cell naembl'ane. Not all osteoblasts show the typical m o r p h o l o g y (lescribe(l. Some cells ad-
315
HUMAN PATHOLOGY--VOLUME 7, NUMBER 3 May 1976
" ... " ~ ; .
."."?,~i : . ' . . ~ . - :
' " .::
L
*
Figure 10. Case 5. Superficial osteoc)te surrounded by calcified matrix. Note well developed Golgi apparatus and sparsity of rough endoplasmic reticulum. Glycogen particles are present in the c)toplasm (arrows); they are very rare ill osteocytes. The lacunar space contains grallu]al" material alld a few collagen fibrils. (x 14,200.)
316
jacent to the osteoid or calcified matrix are smaller, round, or elongated and have sparse organelles and rough endoplasmic reticulum; other cells resemble fibroblasts. T h e perivascular space also contains cells with the typical morphology of nsteoblasts (Fig. 8). OSTEOCYTES, As the osteoblasts are being incorporated into the calcified matrix, the cells develop fewer cell processes and the adjacent collagen undergoes patchy calcificatioll (Fig. 10). T h e deep osteocytes are completely s u r r o u n d e d by calcified matrix and are located in the osseous l a c u n a e . Tile osteocytes are smaller than osteoblasts and contain less rough endoplasmic reticululn. T h e Golgi apparatus is well developed; a few lysosome-like bodies and fine filaments are seen. Ver)' rarel)" glycogen is seen in the osteo'cyres (Fig. 10). T h e cell [Jrocesses of
the osteocytes penetrate into tile canaliculi of tile bone lacunae. T h e lacunar space a r o u n d the osteocytes contains finely granular material and a few collagen fibrils (Fig. 10). T h e margins of the osseous lacunae are well outlined, but the zone of the mineralization is often irregular, with patchy calcification. In case 3 the calcified niatnx contained laminated circumscribed areas of calcified material tile size of a bone lactma. These areas are interpreted as f o r m e r osteocyte lacunae that became mineralized after the death of the osteocyte. Examination of tissue f i o m an osteoid osteoma nidus that was previously decalcified with ,t pet" cent disodium ethylenediaminetetraacetate (case ,t) disclosed the presence of a distinct electron dense layer a r o u n d the osteocyte lacunae and canaliculi. This dense layer, recently called a - l a m i n a
OSTEOID
OSTEOMA--STElyER
Figure 11. Case 4. Area of mineralized matrix shows irregular pattern ofc~dcification ofcoarsc woven or immature I• (x 18,500.)
limitans: 3 is generally seen all a r o u n d the lacunae, althougli it is poorly defined in sonic areas.
~[INFRALIZED ~[ATRIX.
The
mineral-
ized matrix shows calcification o f the collagen fibrils, which are r a n d o m l y arranged and not disposed in a lamellar pattern. This indicates that the bone formec] in osteoid osteoma is coarse or fiber bbne r a t h e r than lamcllar (Fig. 11): 4 Along the surface o f the mil~eralized matrix, adjacent to the osteoblasts or osteoid, the mineralization f r o n t is not even and t h e r e is patchy calcification (Fig. 7). No clear-cut evidence is seen indicating mineralization o f individual fibrils. In a few areas o f the surface the mineralization is not p,ttchy but, rather, solid and granular. T h e s e areas are generally devoid o f surface osteoblasts. OSTFOID. t'k varying a n a o u n t of osteoid or : l n m i n e r a l i z e d collagen matrix is present in hll cases o f osteoid o s t e o m a .
It consists o f bundles or groups o f densely packed, parallel collagen fibrils. Tile bundles vary in size and are generally r a n d o m l y a r r a n g e d in tile osteoid (Fig. 12A). T h e direction o f the collagen is not related to tile position of tile surface osteoblasts. T h e a m o u n t o f osteoid seen in osteoid osteoma varies greatly, even within the same nidus. I f the matrix is heavily mineralized, t h e r e is little osteoid seen. On the o t h e r h a n d , the osteoid m a t r i x m a y be quite p r o m i n e n t in other areas where there is little or no mineralization. In cases I a n d '2, in addition to tile collagen, the osteoid contains finely granular, nonfibrillar material, interspersed irregularly between the collagen fibrils (Fig. 12). T h e r e is no evidence o f calcification in these areas (some sections were stained only with" lead h y d r o x i d e to prevent loss o f nfineral). T h e granular material p r e d o m i n a t e s in dense osteoid, a h h o u g h it may be seen adjacent to osteo-
317
ItUMAN
318
I'ATilOLOGY-VOLUME
7, N U M B F R 3
May 1976
Figure 12. A. Area ofosteoid (O) adjacent to ostcoblast (Ob) in case 2. Tile collagen fibrils arc seen in both cross ;tnd hingitudinal sections. "File osteoid in the lower left of tlle electron micrograph shows dense material interspersbd between the collagen. (• 18,500.) B, High power view ofosteoid cousisting of fine grantllar"material alld collagen. (• 37,900.)
OSTEOI D OSTEOMA--STEINER
F i g u r e 13. Case 4. Osteoclast adjacent to mineralized matrix (M). T h e r e a r e n u m e r o p s clear vacuoles p r e d o m i n a t i n g n e a r the ruffled b o r d e r at'ca (B) o f the cell. Also note sparsity o f organelles in ru|tled border.
(x 5700.)
blasts where the collagen is loose. T h e areas o f osteoid containing the granular material are devoitl of collagen (l:ig. 12). Mqst of the osteoid sm'ihce is covered by ostebblasts and cells resembling fibroblasts. Some osteoblasts are quite large, with multiple lysosome-like bodies and abundant fine filaments. T h e r e are also large g r a n u l a r bodies that probably represent iron containing lysosomes. In the areas of osteoid not covered b)' osteoblasts, the osteoid border is irregular and ill defined, and collagen fibrils can be seen to end abrtq~tl)'. T h e osteoid also contains osteoblasts that have become incorporated into the matrix and, are therefore designated as osteoid osteoc'ytes, t~ T h e y are similar to
osteocytes o f mineralized matrix. Sonm osteoid osteocytes are s u r r o u n d e d by dense collagen matrix and granular material. Others have a 1)ericellular lacuna, which contains fine granular material, few fibrils, and cell processes. Frequently the cell processes penetrate into the canaliculi of the s u r r o t m d i n g osteold. Occasional pinocytotic vesicles and coated vesicles are seen in the osteoid osteocytes. No glycogen is identified. Although most of the osteoid is not mineralized, it may show isolated patches of calcification. This occurs in the areas of osteoid adjacent to mineralized matrix. OSTr:OCCASTS. These cells are present in varying numl)ers in the osteoid o s t e o l l l a nidi. Most ceils are large and con~
319
IIUMAN I'ATIIOLOGY--VOLUME 7, NUMBER 3 tain nm.ltii)le nuclei, whiclt are ovoid a n d i n d e n t e d a n d are located at tire center or 1)eriphery o f the cells (Fig. 13). T h e Golgi a p p a r a t u s consists o f llatt e n e d sacs and small vesicles disposed a r o u n d the individual nuclei. O f t e n small coated vesicles are seen associated with tire Golgi a p p a r a t u s . Free ribosomes are seen scattered througl~out the cytoplasm. T h e r o u g h endoplasnaic reticulunt is not p r o n f i n e n t a n d shows occasional dilation. Fine intracytoplasmic fibrils are also present, as well as occasional lysosome-like bodies. Most osteoclasts have clear m e m b r a n e b o u n d vacuoles, s o m e the size o f m i t o c h o n d r i a attd o t h e r s larger. T h e s e large vacuoles p r e d o m i n a t e at tim site o f the rullled b o r d e r (Fig. 13). S o m e o f these vacuoles m a y r e p r e s e n t d e g e n e r ated mitochondria. A large n u m b e r o f nfitochondria are distributed througi~out the cytoplasm except f o r tlm areas n e a r the ruffled b o r d e r (Fig. 13). Only in two osteoclasts (fi'om cases 3 and 5) are tlm m i t o c h o n d r i a quite different f r o m those in o t h e r cells; they vary in size a n d slmpe a n d reselnble the atypical nfitochondria
320
May 1976
described in osteoblasts. T h e cristae f o r m small c o m p a r t m e n t s and the mitochondrial matrix has areas o f electron lucency (Fig. 14). T h e ruffled or brush b o r d e r is a specialized a r e a o f the osteoclast c o m p o s e d of folds a n d irregtdar projections o f the plasma m e m b r a n e , in contact with or adjacent to calcified matrix (Fig. 15). T h e folds are devoid of organelles, lmve a g r a n u l a r c)'toplasm, anti often contain coated pits. In the areas o f close apposition with the calcified matrix, loose b o n e salt crystals are seen within the spaces between the lolds (Fig. 15). Not infi'equently collagen fibrils are seen d e n u d e d o f mineral. T h e r e is no evidence o f b o n e crystals within the cytoplasm of the osteoclasts. Not all the surfaces o f osteoclasts adjacent to calcified matrix have a ruffled b o r d e r ; in these areas the cell m e m b r a n e is smootlt. Ruffled b o r d e r s are not o b s e r v e d in all osteoclasts, a h h o u g h serial sections o f these cells m a y d e m o n s t r a t e their presence. H o w e v e r , several giant cells are seen surr o u n d e d by osteoblasts and at a distance f r o m the b o u e matrix; this ntay indicate
Figure 14. A, Atypical initochondria from osteoclast in case 3. Tile mitochondrial matrix shows electron luceut areas and a few granules. (• 38,000.) B, At)'pical mitochondria from oste,,blast in case 5 shows irregular configur'ation of the cristae that divide the oiganelles into small compartments. (• 38,000.)
OSTEOID O S T E O M A - STr:I,~rCR
Figure 16. Case 1. I'rimitive perivascular cell (P) witfi scanty cytoplasm and sparse organelles. It resembles the Scott t)pe A cell or preosteoblast. A capillary is seen at the left upper corner. (x 10,500.)
Figure 1,5. Case 4. Ruffled border of osteoclast. Note the tmmerous projections adjacent to calcified matrix. (• 32,400.)
that some o f these giant cells probably are in a resting state and not u n d e r g o i n g bone resorption. ~5 VFSSELS
AND
PERIVASCULAR
CELLS.
matrix. Most o f these cells are identified as perivascular osteoblasts o f varying size, with eccentric nuclei (Fig. 8). T h e p r o p o r tion o f organelles varies fi-om cell to cell. Glycogen particles are identified in several osteoblasts although not in all five cases studied (Fig. 8). O t h e r cells present in the perivascular spaces inchtde a few ehmgated or ovoid cells with scanty cytoplasm and sparse organelles. Some cells contain glycogen toward the p e r i p h e r y . T h e s e l)oorly differentiated cells resemble the type A cell described by Scott ~Gin fetal bone (Fig. 16). Few o t h e r cells resemble the type B cell o f
Vascular structures al'e often seen in osteoid osteomas. T h e vessels wwy from small cal~illaries to large vascular spaces, which ntay be s u r r o t m d e d by pericytes or smooth muscle cells. Arterioles are often identilied. Most capillaries have a continuous e n d o t h e l i u m , a h h o u g h occasionally fenestrated capillaries are seen. Most o f these vessels and their cells have the same morphoh)gy as normal vessels (Fig. 16). v' (The exception was an arteriole o f case 5 in which the endothelial and smooth nmscle cells were t b u n d to contain atypical mitochondria similar to the ones described in osteoblasts.) A large n u m b e r o f cells s u r r o u n d the Figure 17. Case I. Perivascular cell resembling vessels and occtq))' the spaces between the - Scott type B cell.-(x 11,500.)
321
IIUMAN I'ATIIOLOGY-VOI.UME 7, NUMBER 3 May 1976 Scott. "I'hey have an u n d u l a t e d cell membrane, ~r developed (;olgi apparatus, free ribosonms, and iysosome-like bodies (Fig. 17). T h e r e are a few o t h e r elongated cells with the n m r p h o l o g y o f fibroblasts, occasionally containing glycogen. T h e r e are still o t h e r cells with morphologic features between poorly differentiated cells, osteoblasts, and fibroblasts. T h e y may r e p r e s e n t transitional stages. T h e presence o f nerve fibers a r o u n d the blood vessels was also investigated. O u r ultrastructural observations failed to find nerve fibers in osteoid osteoma. An intensive search was carried out in cases 4 and 5, lint the results were negative. l'araffin sections o f cases 4 and 5 were stained for nerve fibers and onl)' a few nerve axons were identified in the tissue) 7 OSTEOBLASTOMA. T h e patient was a I,t year old female with a destructive lesion in the midshaft o f the right llu111el"tlS
322
measuring apl)roxinmtely 3 cm. in diameter. She lind had surgery in that region one year previously. Curettage was done. In general tim ultrastructure o f the osteoblasts resembles that o f the osteoid ostcoma. T h e cells have eccentric, often i n d e n t e d nuclei (Fig. 18). T h e r o u g h endoplasmic reticuhmi is l n o m i n e n t . T h e Golgi a p p a r a t u s is well developed and is located in the j v x t a n u c l e a r region. In some osteoblasts there are abnormal mitoc h o n d r i a similar to the ones described in the cases o f osteoid osteoma (Fig. 18). T h e osteoblasts are quite p r o m i n e n t and a b t m d a n t along the margins o f the bone matrix and in the perivascular spaces. T h e osteocytes show fewer organelles than osteoblasts, although rarely they also contain atyl)ical initochondria. T h e osteoclasts show a m o r p h o l o g y similar to that o f the osteoclasts in osteoid osteolna. A few poorly differentiated cells are seen in the perivascular spaces.
Figure,18. Ostcol)lastoma.TWOosteotflastsshow indented nuclei, abundant rough cndoplasmic reticuhml, ;m,i at~.pioalmitochondria similar to those described in osteoid ostcoma (see Fig. 9). (x I-t,200.)
OSTEOI D OSTEOMA--STEINER DISCUSSION
A review of the literature reveals tlmt there are only two reports concerning the ultrastructure o f osteoid osteoma, only one of wlfich appeared in the English literature. 8 In that report, however, very few observations a l l d COllllllelltS w e l ' e made by the authors concerning their ultrastructural findings. The other report appeared in the Italian literature, and the authors described the presence of viruslike particles, as well as pseudocrystalline structures within the cells of the nidus.; In the present study the predominant cells in osteoid osteoma are the osteoblasts. They lmve a morphology similar to that of normal osteoblasts, with only a few differences. In osteoid osteoma the osteoblasts Imve irregular and indented nt,clei, atypical mitocim,ldria, occasional glycogen, a larger numbcr of intracytoplasmic fine [ibrils, and occasional iron contai,fing lysosomcs. In additiou, although the Golgi apparatus is well developed, it is usually more irregularly disposed i,a the juxtanuclear region of the cells, often intermingled with the rough endoplasmic retict,lum. "l'he abundance of rough endoplasmic reticulum in most of these cclls is an iudication that the osteoblasts in osteoid osteoma are actively engaged i,a matrix production. T h e presence of more than one layer of osteoblasts lining the matrix and the haphazard arrangement of the cells indicate that, at least in some areas, the bone formation takes place rapidly. ~ The areas of mineralized matrix conform to the morphology of coarse woven bo,ae. The bundles of calcified collagen are irregular in size and are randomly arranged, ~vith no lamellar orientation of the fibrils. 14 The osteoid present in ostcoid ostcoma shows irregular layers of collagen that in general intersect each other randomly. Some osteoid osteocytes h,tve l)ericelhtlar lacunae. The degree of osteoid tbrmation without mineralization in the nidus is abnormal. It is known, however, that in growi,ag bone there is a variable amount ofosteoid formation prio," to calcification,TM aud in fracture callus there is abundant osteoid formation with a marked delay in c.tlcificationYa It" appears that in osteoid
osteoma the osteoblasts lay down an osteoid matrix, which may remain unmineralized at least temporarily. The lack of mineralization of the osteoid matrix may be due to local factors tlmt include the composition of the osteoid, the availability of mineral, and the vascularity of the lesion. Although most of the osteoid is unmineralized, it shows patchy calcification in a few areas near the calcified matrix. These findings have raised doubts tlmt collagen alone provides the site tot initial calcification,va Bernard and Pease '-'~ found polysaccharide cores in the osteoid during early ossification; they believe that this material might be the initial calcification locus. An interesting observation in two cases of osteoid osteoma was that in addition to the collagen the osteoid matrix contained finel)" granular material in a focal distribution. This material has not been previously described in unmineralized bone matrix. However, granular material similar to this Ires been observed in decalcified sections of bone matrix during early mineralization and was shown to be polysacclmride?s'-~~ We do not know the signilicance of this granular material in the osteoid matrix; it is possible timt it represents polysaccharides. The osteoid with this material may thus represent the matrix ready for mineralization. In addition to osteoblasts, osteocytes, and osteoclasts there are primitive cells identified in the perivascular spaces. These are osteoprogenitor ceils tlmt resemble the type A (preosteoblasts) and type B (preosteoclasts) cells described by Scot0 ~' in fetal bone. Our observations indicate timt the osteoblasts most likely are derived from Scott type A cells or preosteoblasts. No infl)rmation was obtained concerning the origin of the osteoclasts. Glycogen was found in two cases of osteoid osteoma, disposed in the form of rosette units. It was observed mainly in the osteoprogenitor cells undergoing osteoblastic transfornmtion and less fiequently in developed osteoblasts ar to bone matrix. Glycogen was quite rare in osteocytes. It is known that during fetal development all osteogenic cells contain varying amounts of glycogen.-"~ In postnatal bone, on the other Imnd, glycogen was demonstrated in differentiating osteo-
323
HUMAN I'ATHOLOGY--VOLUME 7, NUMBER 3 May 1976
324
blasts o r preosteoblasts, but was absent in d e v e l o p e d osteoblasts.""" ":~ In fracture callus, ahhougl~ some authors have f o t m d glycogen in i)reosteoblasts, 22 others fail to find it in any bone cell. "3'za T h e significance o f glycogen in osteogenic cells is not known. Pritchard ''~ believes that the presence o f glycogen indicates a special adaptation o f the osteogenic cells to conditions o f fetal life and not a typical feature o f these cells tln'ouglmut life. T h e significance o f the atypical mitoc h o n d r i a in some osteoblasts and a few osteoclasts is not known. T h e y have not been previously described in these cells. Some o f the cristae bear a resemblance to the villi described in the mitochondria o f the giant ameba, l'elomyxa carolinensis. "-'~ It is possible that the morphologic changes ' in these mitochondria may be the resuh o f pathologic conditions not yet known "-'n T h e intramitochondrial granules most likely r e p r e s e n t depots or binding sites for divalent cations, particularly calcium. "G N e r v e fibers can be identified in osteoid osteoma by use o f the light microscope; their presence has been d o c u m e n t e d by using silver staining techniques. ~r T h e pain, so characteristic o f osteoid osteoma, is probably transmitted by a u t o n o m i c nerve fibers, which are sensitive to changes in vascular pressure. ~r In o u r ultrastructural studies we failed to find nerve fibers in osteoid osteoma. An intensive search was u n d e r t a k e n in cases 4 and 5, but the results were negative. T h e difficulty in identifying nerve fibers at the uhrastructural level is probably related to the sampling of the tissue and the fact that nerve fibers al'e not a b u n d a n t in the nidus. T h e origin o f osteoid osteoma is not known. Some authors imve identified ~firus-like particles in the cells o f the nidus find believe that osteoid osteoma is a dysplastic disease: We were not able to find virus-like particles in o u r cases. Jaffe l was the first a u t h o r to favor the idea tlmt osteoid osteoma is a benign neoplasm, and this is now accepted by most authors and by the present writer.'-" a,.~, ,, ,.,r Althougil the osteoblasts in osteoid osteoma have i n d e n t e d nuclei, atypical mitochondria, and an a b u n d a n c e o f fine fi]alnents, basically they do not differ from normal osteoblasts. Except for the presence o f atypical lnitochondria in a few-cells, the
osteoclasts resemble nornlal osteoclasts. T h e osteocytes o f osteoid osteoma are morplmlogically sitnilar to normal osteocytes. We believe, as do others~ s that in the nidus t h e r e is evidence o f a dynamic process o f rapid bone formation and destruction in a small volume o f tissue. T h e bone r e s o r p t i o n is exerted by the action o f the osteoclasts on the mineralized matrix; they a p p e a r to Imve no affinity for o s t e o i d . 28
As to the relationslfip between osteoid osteoma and osteoblastoma, most authors consider tlmt these lesions are closely related altlmugll with some differences in r e f e r e n c e to size, localization, and reactive sclerosis, e-G Most o f the osteoid osteomas measure approximately I cm. in diameter; the osteoblastomas are larger.'-' Histologically both lesions show basically the same morplmlogy, although the osteoblastoma may be more vascular and lmve a larger a m o u n t o f osteoid than the osteoid o s t e o i l l a . '2''~ O l . l r
uhrastructural
obsel'va-
tions s u p p o r t the idea that osteoid osteoma and osteoblastoma are closely related lesions. In general, cells o f the latter have features quite similar to those o f osteoid osteoma, including the presence o f atypical mitochondria. ACKNOSVLEDGMENT
T h e a u t i m r expresses Iris gratitude to Carol Schreck for teclmical assistance. REFERENCES 1. Jaffe, ! I. I..: "Ostctfid ostconla": :l bcnign ostcoI)l;istic t u m o r COml)oscd o f ostcoid and atypical b,me. Arch. Surg., 31:709, 1935. 2. Jalt'e, H. L.: T u m o r s and T u , u o r o u s Conditions o f the l~,oucs andJoints, lqlila(lcll)hia, Lea and Febigcr, 1958. 3. I)ahlin, C.: Bone T v m o r s . Springfield, Illinois, Charles C T h o m a s , 1973. 9t. Byers, 1'. I).: Solitary benign osteoblastic lesions o f bone. Ostcoid osteoma and ostcoblastoma. Cancer, 22:-t3, 1968. 5. Schajowicz, F., and I.emos, ('.: Osteoid osteoma and ostcoblastoma. Acta Orthop. Stand., 41: 272, 1970. 6. l.ichtcnstein, I..: Bone T u m o r s . St. l.otfis, T h e ('. V. Mosby Cgmpany, 1972. 7. I)e Giuli, C., and Fromino, G.: Rilicvo al microscopio clcttronico di partlco, lari strutture endocelh,lari in un caso di osteoxna ostcoidc. Arch. Ital. i'at. Cliq. Tumori, X/:35, 1968.
OSTEOID 8. lliroh;ua, K., and Mnrimoto, K.: Ultrastructtn-e of Bone and J'oint Discascs. Tokyo, Igaku Shoin l.td. (Grune & Stratton, Inc.), 1971. 9. Boothroyd, IL: T h e prnblcms of dcmincralisation in thin sections of fully calcified bone. J. Cell Biol., 20:165, 196-t. 10. Karnovsky, M. J.: Simple methods fi)r "staining with lead" at high pH in electron microscopy. .l. l~,iophys, lfiochem. Cytol., 11:729, 1961. 11. Cameron, 1). A.: T h e uhrastructure of bone. In Bourne, G. il. (Editor): T h e Biochemistry and I'hysiology of Bone. New York, Academic Press, Inc., 1972, Vol. !. 12. ttam, A. W.: llistology. Ed. 7. Philadelldfia, J. B. Lipplncott Co., 197-t. 13. Scherft, J. 1'.: T h e lamina limitans of the organic nmtrix of calcified cartilage and b o n c . . l Uhrastruct. Res., 38:318, 1972. 14. Dudley, tt. R., and Spiro, I).: T h e fine structure of bone cells. J. l~iol)h)'s. Biochcm. Cytol., I1: 627, 1961. 15. I lancox, N. M.: Biology of Bone. Cambridge, Cambridge University Press, 1972. 16. Scott,. B. L.: Th)'midine-3ti electron microscope radioautography of ostcogenic cells in the fetal rat. J. Cell Biol., 35:115, 1967. 17. Schuhnan, 1.., and I)orfman, It. D.: Nerve fibers in ostcoid osteoma. J. Bone Joint Surg., 52:1: 1351, 1970. 18. Scott, B. L., and Pease, D. C.: Electron microscopy of the epiphyseal al~paratus. Anat. Rec., 126:-t65, 1956. 19. Cameron, D. A.: T h e fine StltlCttlle Of bone and
OSTEOMA--STEINER
calcified cartilage. Clin. Orlhop., 26:199, 1963. 20. l~,el'nard, (,. W., and I'ease, I). C.: An electron microscopic study of ilfitial intranlembranotls osteogencsis. Amer. J. Anat., 125:271, 1969. 21. Soon, B. I.., and Glimcher, M.J.: l)istribution of glycogcn in ostcol)lasts of tllc fetal rat. J. Uhrastruct. Res., 36:565, 1971. '2'2. Schajowicz, F., and (:abrini, R.: llistnchemical studies on glycogcn in normal ossification and calcification. J. Bone Joint Surg., 40..1:1081, 1958. 23. l'ritchard, J. J.: T h e osteoblast. In Bourne, G. H. (Editor): T h e Biochemistry and l'hysiology of Bone. New York, Academic Press, Inc., 1972, Vol. 1. 2.t. Gothlin, G.: Electron microscopic observations on fracture repair in the rat. Acta l'ath. Microbiol. Scand., Section A. 81, 507, 1973. 25. Pal)pas, G. D., and Brandt, 1'. W.: Mitochondria. I. Fine structure of the complex patterns in the mitochondria of l'elomyxa carolinensis Wilson (Chaos chaos, L.). J. Biophys. Biochem. C)tol., 6:85, 1959. '26. "lalldlcr, B., and llol)pcl, C. L.: Mitochondria. New York, Academic I'rcss, Inc., 1972. 27. Spjut, ll.J., I)orfinan, II. I)., Fechner, R. E., and Ackerman, 1.. V.: Atlas of "l'lllnOl" Pathology, Second Series, Fascicle 5. Tumors of Bone and Cartilage. Washington, D.C., Armed Forces Institute of I'athoh>gy, 1971. 28. Johilston, A. D.: Clinical pnoblcnns in ostcoid osteoma. Evidence of osteoclastic aversi(m to osteoid. Bull. Hosp. Joint Dis., 23:81), 1962.
1919 Madison Avenue New York, New York 10035
325
