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Osteogenesis imperfecta congenita: A connective tissue diathesis
OSTEOGENESIS
IMPERFECTA CONGENITA: TISSUE DIATHESIS
A CONNECTIVE
RICHARD H. FOLLIS, JR., M.D. BALTIMORE, 1V[D. T I S an honor and a pleasure to be asked to c o n t r i b u t e to this anniv e r s a r y n u m b e r in h o n o r of Dr. P a r k . F u r t h e r m o r e , it is g r a t i f y i n g to be able to m a k e a small contribution to a subject in which Dr. P a r k excels. He first i n t r o d u c e d me to diseases of bone a n u m b e r of y e a r s ago and has continued to stimulate and teach me ever since. A t the p r e s e n t time, w h e n so m u c h a t t e n t i o n is being focused on the connective tissues and their diseases, a n y new observations in this field would seem w o r t h y of recording. We should t h e r e f o r e like to p r e s e n t a t y p i c a l case of osteogenesis imp e r f e c t a congenita and to detail and discuss certain heretofore unrep o r t e d morphological aspects of this syndrome.
I
CASE REPORT
Autopsy (22803): The body was that of a N e g r o i n f a n t weighing 1,400 grams. The b a b y was delivered a f t e r a prolonged labor (fourteen "hours) and was p r e s u m a b l y n e a r term. The child died a f t e r twenty-seven rainutes. The parents were n o r m a l as were three siblings, one of whom was delivered ten months a f t e r the b i r t h of the present patient. External Appearance: There was marked symmetrical shortening of the four extremities, which tended to t a p e r off at the ends in characteristic fashion a n d give the a p p e a r a n c e of micromelia (Fig. 1, A ) . The proportions of the b o d y a n d head did not a p p e a r abnormal. The scleras were From the Department of Pathology. The Johns Hopkins University School of Medicine.
d a r k in color. The eyes did not app e a r enlarged. The skin seemed unusually thin. Abdominal Cavity- The contents of the abdominal cavity a p p e a r e d norreal. The liver weighed 115 grams (normal for 3,000 g r a m s is 135 grams). The spleen weighed 10 grams (normal f o r 3,000 g r a m s is 10 grams). Thoracic Cavity: The contents of the thoracic cavity were entirely normal. Cranial Cavity: On cutting t h r o u g h the c a l v a r i u m there a p p e a r e d to be no bone whatsoever. There was extensive subdural and subarachnoid hemorrhage. Extremities: On cross section the epiphyseal cartilages a p p e a r e d norreal in size f o r a fnll-term infant. There was a well-developed center of ossification in the u p p e r end of each tibia. There was great shortening of the diaphyses with n u m e r o u s fractures of all extremities as well as the ribs. The costochondral a n d epip h y s e a l - d i a p h y s e a l junctions w e r e straight. I n the f r a c t u r e areas there was thickening of the cortex. Elsewhere it was thin and in places appeared not to be present at all. Microscopic Findings.--T i s s u e s f r o m all organs were fixed in Zenkerformol and f o r m a l i n alone, embedded in paraffin, cut, and stained with hematoxylin and eosin. Certain tissues were stained with 2 per cent silver nitrate, the Gomori 1 ammoniacal silver procedure, the McManus~-Lillie TM Hotchkiss 8 periodic acid-Schiff reaction, and toluidine blue. Skin: I n hematoxylin~eosin preparations the epidermis a p p e a r e d normal when compared with t h a t of children of weights r a n g i n g f r o m 1,300
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Fig.. I.---A, External appearance of infant, showing marked deformity of the extremities. B and C, Roentgeno~rams of humerus of case being described (B) and normal (C). Note diiierenee in length and similarity in size of epiphyseal cartilages. Fractures and bony proliferation in diaphysis are striking.
F i g . 2.--A, G o m o r i s i l v e r s t a i n 1 of s k i n of p a t i e n t in c o m p a r i s o n w i t h n o r m a l s k i n of i n f a n t w e i g h i n g 1,500 g r a m s ( B ) . N o t e t h i n a r g y r o p h i l i c f i b e r s m a k i n g up t h e n a r r o w c o r i u m (A) in comparison with thicker dermis (B) made up of stouter nonargyrophilic fibers.
FOLLIS:
OSTEOGENESIS
to 3,000 grams. The fibers of the eorium, however, were much thinner in the case being described. W h e n stained specially the thin fibers were argyrophilic (Fig. 2), conforming with this criterion for retieulum, were metaehromatic, and exhibited a red color with the periodic aeid-Sehiff rea g e n t s . The fibers of m a t u r e corium, of course, were not argyrophilie or metachromatic.
IMPERFECTA
CONGENITA
715
fecta was thinner t h a n t h a t f r o m normal individuals of the same weight or weighing 3,000 grams. The fibers were more delicate (Fig. 3, C and D ) and gave a positive silver staining reaction a n d were metaehromatic. I n addition they were positive to the periodic acid-Sehiff stain, exhibiting a red color. Skeletal Tissues: Sections of eostochondral junctions, a n u m b e r of the
F i g . 3 . - - A a n d B, C o r n e a s f r o m i n f a n t b e i n g d e s c r i b e d a n d c o n t r o l s t a i n e d w i t h p e r i o d i c acid-Sehiff technique. I n a d d i t i o n to m a r k e d d i f f e r e n c e in w i d t h t h e f o r m e r s t a i n s r e d w h i l e t h e l a t t e r is a h n o s t c o l o r l e s s . C and D, Scleras from infant with osteogenesis imperfeeta and normal infant. T h e s e e x e m p l i f y d i f f e r e n c e in t h i c k n e s s a n d t h i n r e t i e u l u m f o u n d in f o r m e r (silver stain).
Eye: The corneas of both eyes were thinner t h a n normal. There was no a b n o r m a l i t y of the epithelial layer. The u n d e r l y i n g substantia p r o p r i a was composed of thin fibers, much more delicate t h a n normal (Fig. 3, A and B). I n addition these fibers were argyrophilic and metachromatic and stained red with the periodic acid-Schiff procedure. The sclerotic coat of the eyes f r o m the infant with osteogenesis imper-
epiph yseal-diaphyseal junctions of the long bones a n d the skin, subcutaneous tissues of the scalp, and the d u r a of the head were examined. As noted grossly, in the epiphyseal cartilage of the u p p e r humerus a center of ossification was found. I I e r e the cartilage cells a p p e a r e d to be growing in normal fashion and spicules of cartilage matrix, i m p r e g n a t e d with inorganic materials, were being produced as a r e s u l t of disappearance
9"ig. 4.--A, C a r t i l a g e s h a f t j u n c t i o n of u p p e r t i b i a to s h o w m a t u r a t i o n of c a r t i l a g e cells and bone matrix beneath. S i l v e r n i t r a t e t r e a t m e n t of u n d e c a l c i f i e d section, B, s h o w s n o r m a l C, H e r u a t o x y l i n a n d e o s i n - s t a i n e d s e c t i o n of d e p o s i t i o n of i n o r g a n i c m a t e r i a l s in m a t r i x . m e t a p h y s e a l r e g i o n of u p p e r t i b i a to s h o w r i m of d e e p b l u e m a t e r i a l a b o u t s p i c u l e s of c a r t i l a g e m a t r i x . D, P e r i o d i c a c i d - S c h i f f s t a i n to s h o w i n t e n s e s t a i n i n g o f m a t e r i a l n o t e d a b o v e . E; A r e a in d i a p h y s i s w h e r e h e a l i n g of f r a c t u r e h a s o c c u r r e d . T h r e e z o n e s f r o m ~ v i t h i n - o u t a r e s e e n : original cartilage matrix, peculiar basophilic and periodic acid-Sehiff positive material, and n o r m a l l y a p p e a r i n g bone.
FOLLIS:
OSTEOGENESIS IMPERFECTA CONGENITA
of the hypertrophic cartilage cells. However, at this point any similarity to normal growth sequences ceased. 0steoblasts were not to be found and no osteoid (or bone) was therefore present on the framework of calcified cartilage matrix. A similar situation existed at the cartilage shaft junctions (Fig. 4, A) ; that is, the growth
717
deposition of inorganic salts was demonstrated conclusively by staining undecalcified sections with 2 per cent silver nitrate (Fig. 4, B). Another abnormality not so prominent in the slower growing center is also clear]y made out; at the cartilage-shaft junction the spicules of calcified cartilage matrix did not appear to be under-
F i g . 5.---A, M i c r o s c o p i c loci of m e m b r a n o u s b o n e f o r m a t i o n in s c a l p . B, S e c t i o n of w a l l of p u l m o n a r y artery" to s h o w t h i c k e n i n g a n d c a l c i f i c a t i o n . 6', F o c u s of c a l c i f i c a t i o n a n d g l i a l P r o l i f e r a t i o n in b r a i n .
of cartilage, its invasion by blood yessels, and the deposition of inorganic materials in the m a t r i x between the hypertrophic cells whieh are being invaded were entirely normal. The
going destruction. More prominent was the absence of osteoblasts; consequently there was a lack of osteoid or b.one on the framework of calcified cartilage matrix. Therefore, just as
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in scurvy, numerous fractures of this framework were found. In such areas there were sometimes red blood cells but even more prominent was a pinkstaining eosinophilic material such as one encounters in seLirvy 5 or in rarefying" diseases of bone such as leu-
kemia2 F u r t h e r down in the lower part of the metaphysis there was another abnormality. In partially decalcified hematoxylin-eosin preparations light blue-staining spicules of calcified cartilaginous matrix were coated with a deep blue-staining layer of a substance which stains also intensely red with the periodic aeid-Schiff reagents (Fig. 4, C and D). The red color was more intense than one ever encounters in bone or cartilage. It was not afleered by saliva; hence it was not free polysaecharide. Its nature could not be f u r t h e r characterized by histochemical procedures save to say that it was metachromatic. I n undecalcified sections it was not darkened by silver nitrate. F u r t h e r down in the shaft where fractures of the calcified matrix and cortex of the diaphysis occurred there was bone formation, both periosteal and endosteal, going on in normal fashion. Hence, trabeculae consisting of three layers, calcified cartilage matrix, the zone of blue-staining material described above, and bone, were founr (Fig. 4, E ) . I n sections of skin and dura, which were thought to contain no bone grossly, small islands of membranous bone were present (Fig. 5, A ) . The insufficient osseous tissue which was present appeared normal. The degree of development was comparable to that which might be expected in an embryo of 2 to 3 months of age. Lungs: In s e v e r a l large pulmon a r y arteries there was marked proliferation of the endothelium (Fig. 5, B) and loci of calcification in the underlying media. The remainder of the tissues was normal. Brain: Scattered through the brain there were numerous small vessels with deep blue-staining refractile
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PEDIATRICS
walls. In addition similar material was found as extravascular granules in the parenchyma (Fig. 5, C). In such loci the glial cells had proliferated. This material gave positive silver and iron reactions. Other Tissues: P a r t i c u l a r attention was directed to the connective tissue of all other organs. No abnormality was demonstrated. DISCUSSION
Osteogenesis imperfecta congenita would a p p e a r to be fairly easy to diagnose f r o m the gross appearance: deformities of the extremities, thin skin, and d a r k scleras. The only possible skeletal abnormality which might be a cause for confusion is achondroplasia. However, w h en compared gr0ss]y, microscopically, and roentgcnographically, these two
disorders are entirely different. Before commenting on the significance of the findings in the autopsy described above it is necessary to say something concerning current concepts of the n a t u r e of reticulum and collagen. The f o r m e r term was applied by MalF to the e x t r e m e l y fine fiber networks f o u n d in such organs as the spleen and lungs. Some years later as a result of Maresch's modification of Bielschowsky's silver impregnation method it was found s t h a t these fine fibers were argyrophilie while the stouter, broader ones were not. As a result argyrophilia became a criterion for reticulum, and a r g y r o p h o b i a a characteristic of collagen. There has been p r e t t y general agreement t h a t retieulum is a precursor of collagen. This concept has been based on studies of the corium in healing skin wounds, in infectious granulomas, and particularly in the organization of blood
FOLLIS
:
OSTEOGENESIS
clots in scorbutic guinea pigs treated with vitamin C2 More recently further evidence has been brought forth by the use of the electron microscope and x-ray diffraction techniques. Employing the former in studying the transition of argyrophilic reticulure in newborn rat skin to argyrophobic collagen in the adult animal, Gross ~~ has found that both exhibit the same characteristic average periodicity of 640 X and that only the fiber width varies, i.e., newborn animal, 500 to 600 A versus 60-day-old rat, 13,000 to 15,000 A. Both types of fibers have the same x-ray diffraction pattern. = The basis for the difference in silver staining, has not been clearly defined. Gross ~ has determined that electron photomicrographs of silver-stained reticulum fibers show irregular surface adsorbtion of metal particles of colloidal dimensions in random fashion. Purplish staining collagen fibers, on the other hand, appear to adsorb particles varying in size and shape. Gross 1~ points out that skin containing retieulum is more gelatinous than adult collagenous skin. Could such gelatinous material be implicated in argyrophilia? Such a possibility must not be overlooked since reticulure in the embryo is metaehromatie and newly formed argyrophilic fibers in healing scurvy also stain red with toluidine blue2 Metaehromasia is a characteristic of tissues containing mueopolysaceharides though the specific chemical reactions are not understood. Another stain of interest, the periodic acid-Schiff reaction first introduced by McManus, 2 was shown by Lillie ~2 to demonstrate retieulum.
ISIPERFECTA
CON~ENITA
719
The presence of thin skin in individuals with osteogenesis imperrecta has been commented upon in previous descriptions of autopsicd eases.14, 15, ~ However, no studies employing more specialized stains have been reported. It is therefore of great interest to find that in this supposedly full-term infant argyrophilic, metachromatie, and periodic acid-Sehiff positive reticu]um fibers make up the corium rather than argyrophobic, nonmetachromatic, a n d periodic acid-Schiff negative fibers. It would appear that the corium is greatly retarded in development and resembles that seen in the early embryo. 4 It must be pointed out at this time that although this infant weighed only 1,400 grams and should therefore be classed as a premature in~ant (e.g., under 2,500 grams) according to the menstrual history, it was full term and furthermore a center of ossification was present in the upper epiphysea] cartilage of the tibia, smaething virtually never seen in a premature infant. From the great decrease in skeletal mass and size of the extremities one might expect a marked effect on body weight. A similar situation has been noted in a case reported by Weber. 17 The thin scleras and corneas noted above are, at least, in the case of the former, not unexpected. " A bluish tinge of the eyebal l " as a part of the osteogenesis imperfecta syndrome was noted by Spurway in 1896 ~ and four years later Eddowes ~9 related the blue color to a lack of fibrous tissue. However, no special stains have been applied to the selera so t hat it is of interest to find that the thin seleras are composed of argyrophilic, meta-
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chromatic, and periodic acid-Schiff positive reticulum, rather t h a n thicker, mature collagen fibers. The thin corneas which also stain intensely with the periodic acid-Schiff procedure f u r t h e r indicate a disturbance in connective tissue maturation. The ocular tissue disturbances in this and other cases will be detailed elsewhere by Dr. Albert D. Ruedemann, Jr.
0
So-called membranous bone formation which takes place normally in certain bones of the calvarium and a portion of the clavicle is quite similar to matrix production by the osteoblast and periosteal cells. The basic morphological change is the appearance of organic matrix consisting of fibers and interfibrillar material in the vicinity of specialized mesenchyreal ce]]s (osteoblasts). It is quite likely that such cells promote the appearance of this matrix but how t h e y mediate the synthesis its two prominent constituents, the protein, collagen, and the polysaccharide moiety, consisting of hexose amine and hexuronic acid, are questions that have yet to be answered. Failure to p r o d uc e bone matrix is the fault in osteogenesis imperfecta. This, of course, has been commented upon in a number of reports. ~5, ~7, 2~, 22,2~,~4 So, too, the absence of any abnormality in the growth and maturation of epiphyseal cartilage and in the deposition of inorganic materials in cartilage matrix has been well recognized. The similarity of the morphological changes in osteogenesis imperfecta and scurvy, for one cannot look at sections of bone from the former without thinking of the latter, has also been commented upon elsewhere.
OF P E D I A T R I C S
One of the most interesting, yet difficult, phenomena to explain is the prompt stimulation of osteoblastic activity when fractures occur. W h a t the stimulus m~y be is, of course, an extremely important matter to determine. Likewise the activity of fibroblasts in the corium does not appear to be impaired in this syndrome since wounds heal in normal fashion in children and adults. 13 The eosin0philic material about the fractures o f calcified cartilaginous matrix is quite similar to that which is encountered in scurvy. This pinkstaining substance has not as yet .been characterized histochemically. ~ The blue-staining material which colors so red with the periodic acidSchiff reaction is unique in our experience with bone disease. Cartilage matrix and osteoid may stain pale pink but never as red as this peculiar material, which we have noted above is not impregnated with inorganic substances. The studies of Heller-Steinberg 25 have not revealed such intense red-staining matrix even in t h i c k s e c t i o n s (20 micra). It is to be regretted that the teeth in this infant were not saved for microscopic examination. It would appear that osteogenesis imperfecta is a systemic disturbance of connective tissue in certain areas. This is not a new concept, since, based on the failure of bone matrix to develop, it is one which has been advanced by other~. 16, 17, ~3, 2~ However, the histochemical studies described above which demonstrate a disturbance in the differentiation of reticulum into adult collagen fibers, place this concept on a much firmer foundation.
FOLLIS:
OSTEOGENESIS
IYIPERFECTA
SUMlYIAEY
A case of osteogenesis i m p e r f e c t a in a n e w b o r n i n f a n t has been reported. The failure of r e t i c u l m u to differentiate into m a t u r e collagen has been d e m o n s t r a t e d in the corium, sclera, and cornea. The presence of a m a t e r i a l in the bones which stains intensely w i t h the periodic acidSehiff technique and which m a y be an i m m a t u r e f o r m of bone m a t r i x has been described. REFERENCES
1. Gomori, G. : Silver Impregnation of Reticulum in Paraffin Sections, Am. J. Path. 13: 993, 1937. 2. McManus, J. F. A.: The Histological Demonstration of Mucin A f t e r Periodic Acid, N a t u r e 158: 202, 1946. 3. Hotchkiss, R. D.: A Microchemical Reaction Resulting in t h e S t a i n i n g of Polysaeeharide Structures in Fixed Tissue Preparations, Arch. Bioehem. 16: 131, 1948. 4. Follis, R. H., Jr.: P e r s o n a l observations. 5. Follis, tl.. tt., Jr.: tIistochemieal Studies on Cartilage a n d Bone. 1I. Aseorbic Acid Deficiency, Bull. J o h n s Hopkins Hosp. 89: 9, 1951. 6. Pollis, R. H., Jr., a n d Park, E. A.: Some Observations on the Morpho]ogic B a s i s for the l%entgenographie Changes in Childhood Leukemia, Bull. ttosp. J o i n t Dis. 12: 67, 1951. 7. Mall, F. P.: R e t i c u l a t e d Tissue and I t s l~elatlon to the Connective Tissue Fibrils, J o h n s Hopkins Hosp. Rep. 1: 171, 1896. 8. Sehmidt, E.: Ueber die S t u t z s u b s t a n z der Leber im normalen und pathoIogischen Zustand, Beitr. z. path. A n a t . u. z. allg. P a t h . 42: 606, 1907. 9. Wolbaeh, S. B." Controlled F o r m a t i o n of Collagen and Reticulum. A S t u d y of the Source of I n t e r c e l l u l a r Substance
10.
11. 12:
13. 14.
15. 16.
17. 18. 19. 20. 21. 22. 23.
24.
25.
CONGENITA
721
in Roco~,ery From Experimental Scorbutus, Am. J. Path. 9: 689, 1933. Gross, J.: A g i n g of Collagenous Connective Tissue of R a t Skin as Studied with the E l e c t r o n Microscope, Am. J. Path. 26: 708, 1950. Gross, J.: Connective Tissue Fine Structure and Some Methods for Its Analysis, J. Gerontol. 5: 343, 1950. Lillie, R . D . : Retieulum S t a i n i n g W i t h Schiff Reagent A f t e r Oxidation by Acidified Sodium Periodate, J. Lab. & Clin. Med. 32: 910, 1947. Johnson, R. W.: Personal communication. Blegvd, O., and H a x t h a u s e n , ~ . : Blue Sclerotics and B r i t t l e Bones W i t h Macular A t r o p h y of the Skin a n d Zonular Cataract, Brit. M. J. 2: ]071, 1921. Siedorf, K. S.: Osteogenesis Impertecta, 1949, U n i v e r s i t e t s f o r l a g e t , Arhus, Denmark. Gruber, G. B.: Zur JAenntnis und Kritik der Osteogenesis i m p e r f e c t a congenltalis, Arch. path. Anat. 316: 317, 1949. Weber, M.: Osteogenesis I m p e r f e c t a Congenita. A Study of I t s Histopathogenesis, Arch. P a t h . 9: 984, 1930. Spurway, J.: H e r e d i t a r y Tendency to Fracture, Brit. M. J. 2: 844, 1896. Eddowes, A.: D a r k Sclerotics and F r a g i l i t a s Ossium, Brit. M. J. 2: 222, 1900. Ruedemann, A. D.: To be published. H a r b i t z , F.: U e b e r Osteogenesis imperfecta, Beitr. z. path. A n a t . u. z. al]g. P a t h . 30: 605, 1901. K a r d a m a t i s , C.: Anatomische Befunde der Osteogenesis imperfecta, Arch. path. Anat. 212: 436, 1913. Bauer, K . H . : U e b e r Osteogenesis imperfecta. Zugleich eln B e i t r a g zur F r a g e elner a]lgerneinen E r k r a n k u n g samtlicher Stutzgewebe, D e u t s c h e Ztsehr. f. Chir. 154: 166, 1920. Jeckln, E.: System gebundene meseachymal Erschopfung. Eine neue Begriffsfassung der Osteogenesls impertecta, Arch. path. A n a t . 280: 351, 1931. Heller-Steinberg, M.. Ground Substance, Bone Salts, and Cellular Activi t y in Bone F o r m a t i o n and Destruction, Am. J. A n a t . 89: 347, 1951.
IMPERFECTA CONGENITA: TISSUE DIATHESIS
A CONNECTIVE
RICHARD H. FOLLIS, JR., M.D. BALTIMORE, 1V[D. T I S an honor and a pleasure to be asked to c o n t r i b u t e to this anniv e r s a r y n u m b e r in h o n o r of Dr. P a r k . F u r t h e r m o r e , it is g r a t i f y i n g to be able to m a k e a small contribution to a subject in which Dr. P a r k excels. He first i n t r o d u c e d me to diseases of bone a n u m b e r of y e a r s ago and has continued to stimulate and teach me ever since. A t the p r e s e n t time, w h e n so m u c h a t t e n t i o n is being focused on the connective tissues and their diseases, a n y new observations in this field would seem w o r t h y of recording. We should t h e r e f o r e like to p r e s e n t a t y p i c a l case of osteogenesis imp e r f e c t a congenita and to detail and discuss certain heretofore unrep o r t e d morphological aspects of this syndrome.
I
CASE REPORT
Autopsy (22803): The body was that of a N e g r o i n f a n t weighing 1,400 grams. The b a b y was delivered a f t e r a prolonged labor (fourteen "hours) and was p r e s u m a b l y n e a r term. The child died a f t e r twenty-seven rainutes. The parents were n o r m a l as were three siblings, one of whom was delivered ten months a f t e r the b i r t h of the present patient. External Appearance: There was marked symmetrical shortening of the four extremities, which tended to t a p e r off at the ends in characteristic fashion a n d give the a p p e a r a n c e of micromelia (Fig. 1, A ) . The proportions of the b o d y a n d head did not a p p e a r abnormal. The scleras were From the Department of Pathology. The Johns Hopkins University School of Medicine.
d a r k in color. The eyes did not app e a r enlarged. The skin seemed unusually thin. Abdominal Cavity- The contents of the abdominal cavity a p p e a r e d norreal. The liver weighed 115 grams (normal for 3,000 g r a m s is 135 grams). The spleen weighed 10 grams (normal f o r 3,000 g r a m s is 10 grams). Thoracic Cavity: The contents of the thoracic cavity were entirely normal. Cranial Cavity: On cutting t h r o u g h the c a l v a r i u m there a p p e a r e d to be no bone whatsoever. There was extensive subdural and subarachnoid hemorrhage. Extremities: On cross section the epiphyseal cartilages a p p e a r e d norreal in size f o r a fnll-term infant. There was a well-developed center of ossification in the u p p e r end of each tibia. There was great shortening of the diaphyses with n u m e r o u s fractures of all extremities as well as the ribs. The costochondral a n d epip h y s e a l - d i a p h y s e a l junctions w e r e straight. I n the f r a c t u r e areas there was thickening of the cortex. Elsewhere it was thin and in places appeared not to be present at all. Microscopic Findings.--T i s s u e s f r o m all organs were fixed in Zenkerformol and f o r m a l i n alone, embedded in paraffin, cut, and stained with hematoxylin and eosin. Certain tissues were stained with 2 per cent silver nitrate, the Gomori 1 ammoniacal silver procedure, the McManus~-Lillie TM Hotchkiss 8 periodic acid-Schiff reaction, and toluidine blue. Skin: I n hematoxylin~eosin preparations the epidermis a p p e a r e d normal when compared with t h a t of children of weights r a n g i n g f r o m 1,300
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Fig.. I.---A, External appearance of infant, showing marked deformity of the extremities. B and C, Roentgeno~rams of humerus of case being described (B) and normal (C). Note diiierenee in length and similarity in size of epiphyseal cartilages. Fractures and bony proliferation in diaphysis are striking.
F i g . 2.--A, G o m o r i s i l v e r s t a i n 1 of s k i n of p a t i e n t in c o m p a r i s o n w i t h n o r m a l s k i n of i n f a n t w e i g h i n g 1,500 g r a m s ( B ) . N o t e t h i n a r g y r o p h i l i c f i b e r s m a k i n g up t h e n a r r o w c o r i u m (A) in comparison with thicker dermis (B) made up of stouter nonargyrophilic fibers.
FOLLIS:
OSTEOGENESIS
to 3,000 grams. The fibers of the eorium, however, were much thinner in the case being described. W h e n stained specially the thin fibers were argyrophilic (Fig. 2), conforming with this criterion for retieulum, were metaehromatic, and exhibited a red color with the periodic aeid-Sehiff rea g e n t s . The fibers of m a t u r e corium, of course, were not argyrophilie or metachromatic.
IMPERFECTA
CONGENITA
715
fecta was thinner t h a n t h a t f r o m normal individuals of the same weight or weighing 3,000 grams. The fibers were more delicate (Fig. 3, C and D ) and gave a positive silver staining reaction a n d were metaehromatic. I n addition they were positive to the periodic acid-Sehiff stain, exhibiting a red color. Skeletal Tissues: Sections of eostochondral junctions, a n u m b e r of the
F i g . 3 . - - A a n d B, C o r n e a s f r o m i n f a n t b e i n g d e s c r i b e d a n d c o n t r o l s t a i n e d w i t h p e r i o d i c acid-Sehiff technique. I n a d d i t i o n to m a r k e d d i f f e r e n c e in w i d t h t h e f o r m e r s t a i n s r e d w h i l e t h e l a t t e r is a h n o s t c o l o r l e s s . C and D, Scleras from infant with osteogenesis imperfeeta and normal infant. T h e s e e x e m p l i f y d i f f e r e n c e in t h i c k n e s s a n d t h i n r e t i e u l u m f o u n d in f o r m e r (silver stain).
Eye: The corneas of both eyes were thinner t h a n normal. There was no a b n o r m a l i t y of the epithelial layer. The u n d e r l y i n g substantia p r o p r i a was composed of thin fibers, much more delicate t h a n normal (Fig. 3, A and B). I n addition these fibers were argyrophilic and metachromatic and stained red with the periodic acid-Schiff procedure. The sclerotic coat of the eyes f r o m the infant with osteogenesis imper-
epiph yseal-diaphyseal junctions of the long bones a n d the skin, subcutaneous tissues of the scalp, and the d u r a of the head were examined. As noted grossly, in the epiphyseal cartilage of the u p p e r humerus a center of ossification was found. I I e r e the cartilage cells a p p e a r e d to be growing in normal fashion and spicules of cartilage matrix, i m p r e g n a t e d with inorganic materials, were being produced as a r e s u l t of disappearance
9"ig. 4.--A, C a r t i l a g e s h a f t j u n c t i o n of u p p e r t i b i a to s h o w m a t u r a t i o n of c a r t i l a g e cells and bone matrix beneath. S i l v e r n i t r a t e t r e a t m e n t of u n d e c a l c i f i e d section, B, s h o w s n o r m a l C, H e r u a t o x y l i n a n d e o s i n - s t a i n e d s e c t i o n of d e p o s i t i o n of i n o r g a n i c m a t e r i a l s in m a t r i x . m e t a p h y s e a l r e g i o n of u p p e r t i b i a to s h o w r i m of d e e p b l u e m a t e r i a l a b o u t s p i c u l e s of c a r t i l a g e m a t r i x . D, P e r i o d i c a c i d - S c h i f f s t a i n to s h o w i n t e n s e s t a i n i n g o f m a t e r i a l n o t e d a b o v e . E; A r e a in d i a p h y s i s w h e r e h e a l i n g of f r a c t u r e h a s o c c u r r e d . T h r e e z o n e s f r o m ~ v i t h i n - o u t a r e s e e n : original cartilage matrix, peculiar basophilic and periodic acid-Sehiff positive material, and n o r m a l l y a p p e a r i n g bone.
FOLLIS:
OSTEOGENESIS IMPERFECTA CONGENITA
of the hypertrophic cartilage cells. However, at this point any similarity to normal growth sequences ceased. 0steoblasts were not to be found and no osteoid (or bone) was therefore present on the framework of calcified cartilage matrix. A similar situation existed at the cartilage shaft junctions (Fig. 4, A) ; that is, the growth
717
deposition of inorganic salts was demonstrated conclusively by staining undecalcified sections with 2 per cent silver nitrate (Fig. 4, B). Another abnormality not so prominent in the slower growing center is also clear]y made out; at the cartilage-shaft junction the spicules of calcified cartilage matrix did not appear to be under-
F i g . 5.---A, M i c r o s c o p i c loci of m e m b r a n o u s b o n e f o r m a t i o n in s c a l p . B, S e c t i o n of w a l l of p u l m o n a r y artery" to s h o w t h i c k e n i n g a n d c a l c i f i c a t i o n . 6', F o c u s of c a l c i f i c a t i o n a n d g l i a l P r o l i f e r a t i o n in b r a i n .
of cartilage, its invasion by blood yessels, and the deposition of inorganic materials in the m a t r i x between the hypertrophic cells whieh are being invaded were entirely normal. The
going destruction. More prominent was the absence of osteoblasts; consequently there was a lack of osteoid or b.one on the framework of calcified cartilage matrix. Therefore, just as
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in scurvy, numerous fractures of this framework were found. In such areas there were sometimes red blood cells but even more prominent was a pinkstaining eosinophilic material such as one encounters in seLirvy 5 or in rarefying" diseases of bone such as leu-
kemia2 F u r t h e r down in the lower part of the metaphysis there was another abnormality. In partially decalcified hematoxylin-eosin preparations light blue-staining spicules of calcified cartilaginous matrix were coated with a deep blue-staining layer of a substance which stains also intensely red with the periodic aeid-Schiff reagents (Fig. 4, C and D). The red color was more intense than one ever encounters in bone or cartilage. It was not afleered by saliva; hence it was not free polysaecharide. Its nature could not be f u r t h e r characterized by histochemical procedures save to say that it was metachromatic. I n undecalcified sections it was not darkened by silver nitrate. F u r t h e r down in the shaft where fractures of the calcified matrix and cortex of the diaphysis occurred there was bone formation, both periosteal and endosteal, going on in normal fashion. Hence, trabeculae consisting of three layers, calcified cartilage matrix, the zone of blue-staining material described above, and bone, were founr (Fig. 4, E ) . I n sections of skin and dura, which were thought to contain no bone grossly, small islands of membranous bone were present (Fig. 5, A ) . The insufficient osseous tissue which was present appeared normal. The degree of development was comparable to that which might be expected in an embryo of 2 to 3 months of age. Lungs: In s e v e r a l large pulmon a r y arteries there was marked proliferation of the endothelium (Fig. 5, B) and loci of calcification in the underlying media. The remainder of the tissues was normal. Brain: Scattered through the brain there were numerous small vessels with deep blue-staining refractile
OF
PEDIATRICS
walls. In addition similar material was found as extravascular granules in the parenchyma (Fig. 5, C). In such loci the glial cells had proliferated. This material gave positive silver and iron reactions. Other Tissues: P a r t i c u l a r attention was directed to the connective tissue of all other organs. No abnormality was demonstrated. DISCUSSION
Osteogenesis imperfecta congenita would a p p e a r to be fairly easy to diagnose f r o m the gross appearance: deformities of the extremities, thin skin, and d a r k scleras. The only possible skeletal abnormality which might be a cause for confusion is achondroplasia. However, w h en compared gr0ss]y, microscopically, and roentgcnographically, these two
disorders are entirely different. Before commenting on the significance of the findings in the autopsy described above it is necessary to say something concerning current concepts of the n a t u r e of reticulum and collagen. The f o r m e r term was applied by MalF to the e x t r e m e l y fine fiber networks f o u n d in such organs as the spleen and lungs. Some years later as a result of Maresch's modification of Bielschowsky's silver impregnation method it was found s t h a t these fine fibers were argyrophilie while the stouter, broader ones were not. As a result argyrophilia became a criterion for reticulum, and a r g y r o p h o b i a a characteristic of collagen. There has been p r e t t y general agreement t h a t retieulum is a precursor of collagen. This concept has been based on studies of the corium in healing skin wounds, in infectious granulomas, and particularly in the organization of blood
FOLLIS
:
OSTEOGENESIS
clots in scorbutic guinea pigs treated with vitamin C2 More recently further evidence has been brought forth by the use of the electron microscope and x-ray diffraction techniques. Employing the former in studying the transition of argyrophilic reticulure in newborn rat skin to argyrophobic collagen in the adult animal, Gross ~~ has found that both exhibit the same characteristic average periodicity of 640 X and that only the fiber width varies, i.e., newborn animal, 500 to 600 A versus 60-day-old rat, 13,000 to 15,000 A. Both types of fibers have the same x-ray diffraction pattern. = The basis for the difference in silver staining, has not been clearly defined. Gross ~ has determined that electron photomicrographs of silver-stained reticulum fibers show irregular surface adsorbtion of metal particles of colloidal dimensions in random fashion. Purplish staining collagen fibers, on the other hand, appear to adsorb particles varying in size and shape. Gross 1~ points out that skin containing retieulum is more gelatinous than adult collagenous skin. Could such gelatinous material be implicated in argyrophilia? Such a possibility must not be overlooked since reticulure in the embryo is metaehromatie and newly formed argyrophilic fibers in healing scurvy also stain red with toluidine blue2 Metaehromasia is a characteristic of tissues containing mueopolysaceharides though the specific chemical reactions are not understood. Another stain of interest, the periodic acid-Schiff reaction first introduced by McManus, 2 was shown by Lillie ~2 to demonstrate retieulum.
ISIPERFECTA
CON~ENITA
719
The presence of thin skin in individuals with osteogenesis imperrecta has been commented upon in previous descriptions of autopsicd eases.14, 15, ~ However, no studies employing more specialized stains have been reported. It is therefore of great interest to find that in this supposedly full-term infant argyrophilic, metachromatie, and periodic acid-Sehiff positive reticu]um fibers make up the corium rather than argyrophobic, nonmetachromatic, a n d periodic acid-Schiff negative fibers. It would appear that the corium is greatly retarded in development and resembles that seen in the early embryo. 4 It must be pointed out at this time that although this infant weighed only 1,400 grams and should therefore be classed as a premature in~ant (e.g., under 2,500 grams) according to the menstrual history, it was full term and furthermore a center of ossification was present in the upper epiphysea] cartilage of the tibia, smaething virtually never seen in a premature infant. From the great decrease in skeletal mass and size of the extremities one might expect a marked effect on body weight. A similar situation has been noted in a case reported by Weber. 17 The thin scleras and corneas noted above are, at least, in the case of the former, not unexpected. " A bluish tinge of the eyebal l " as a part of the osteogenesis imperfecta syndrome was noted by Spurway in 1896 ~ and four years later Eddowes ~9 related the blue color to a lack of fibrous tissue. However, no special stains have been applied to the selera so t hat it is of interest to find that the thin seleras are composed of argyrophilic, meta-
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chromatic, and periodic acid-Schiff positive reticulum, rather t h a n thicker, mature collagen fibers. The thin corneas which also stain intensely with the periodic acid-Schiff procedure f u r t h e r indicate a disturbance in connective tissue maturation. The ocular tissue disturbances in this and other cases will be detailed elsewhere by Dr. Albert D. Ruedemann, Jr.
0
So-called membranous bone formation which takes place normally in certain bones of the calvarium and a portion of the clavicle is quite similar to matrix production by the osteoblast and periosteal cells. The basic morphological change is the appearance of organic matrix consisting of fibers and interfibrillar material in the vicinity of specialized mesenchyreal ce]]s (osteoblasts). It is quite likely that such cells promote the appearance of this matrix but how t h e y mediate the synthesis its two prominent constituents, the protein, collagen, and the polysaccharide moiety, consisting of hexose amine and hexuronic acid, are questions that have yet to be answered. Failure to p r o d uc e bone matrix is the fault in osteogenesis imperfecta. This, of course, has been commented upon in a number of reports. ~5, ~7, 2~, 22,2~,~4 So, too, the absence of any abnormality in the growth and maturation of epiphyseal cartilage and in the deposition of inorganic materials in cartilage matrix has been well recognized. The similarity of the morphological changes in osteogenesis imperfecta and scurvy, for one cannot look at sections of bone from the former without thinking of the latter, has also been commented upon elsewhere.
OF P E D I A T R I C S
One of the most interesting, yet difficult, phenomena to explain is the prompt stimulation of osteoblastic activity when fractures occur. W h a t the stimulus m~y be is, of course, an extremely important matter to determine. Likewise the activity of fibroblasts in the corium does not appear to be impaired in this syndrome since wounds heal in normal fashion in children and adults. 13 The eosin0philic material about the fractures o f calcified cartilaginous matrix is quite similar to that which is encountered in scurvy. This pinkstaining substance has not as yet .been characterized histochemically. ~ The blue-staining material which colors so red with the periodic acidSchiff reaction is unique in our experience with bone disease. Cartilage matrix and osteoid may stain pale pink but never as red as this peculiar material, which we have noted above is not impregnated with inorganic substances. The studies of Heller-Steinberg 25 have not revealed such intense red-staining matrix even in t h i c k s e c t i o n s (20 micra). It is to be regretted that the teeth in this infant were not saved for microscopic examination. It would appear that osteogenesis imperfecta is a systemic disturbance of connective tissue in certain areas. This is not a new concept, since, based on the failure of bone matrix to develop, it is one which has been advanced by other~. 16, 17, ~3, 2~ However, the histochemical studies described above which demonstrate a disturbance in the differentiation of reticulum into adult collagen fibers, place this concept on a much firmer foundation.
FOLLIS:
OSTEOGENESIS
IYIPERFECTA
SUMlYIAEY
A case of osteogenesis i m p e r f e c t a in a n e w b o r n i n f a n t has been reported. The failure of r e t i c u l m u to differentiate into m a t u r e collagen has been d e m o n s t r a t e d in the corium, sclera, and cornea. The presence of a m a t e r i a l in the bones which stains intensely w i t h the periodic acidSehiff technique and which m a y be an i m m a t u r e f o r m of bone m a t r i x has been described. REFERENCES
1. Gomori, G. : Silver Impregnation of Reticulum in Paraffin Sections, Am. J. Path. 13: 993, 1937. 2. McManus, J. F. A.: The Histological Demonstration of Mucin A f t e r Periodic Acid, N a t u r e 158: 202, 1946. 3. Hotchkiss, R. D.: A Microchemical Reaction Resulting in t h e S t a i n i n g of Polysaeeharide Structures in Fixed Tissue Preparations, Arch. Bioehem. 16: 131, 1948. 4. Follis, R. H., Jr.: P e r s o n a l observations. 5. Follis, tl.. tt., Jr.: tIistochemieal Studies on Cartilage a n d Bone. 1I. Aseorbic Acid Deficiency, Bull. J o h n s Hopkins Hosp. 89: 9, 1951. 6. Pollis, R. H., Jr., a n d Park, E. A.: Some Observations on the Morpho]ogic B a s i s for the l%entgenographie Changes in Childhood Leukemia, Bull. ttosp. J o i n t Dis. 12: 67, 1951. 7. Mall, F. P.: R e t i c u l a t e d Tissue and I t s l~elatlon to the Connective Tissue Fibrils, J o h n s Hopkins Hosp. Rep. 1: 171, 1896. 8. Sehmidt, E.: Ueber die S t u t z s u b s t a n z der Leber im normalen und pathoIogischen Zustand, Beitr. z. path. A n a t . u. z. allg. P a t h . 42: 606, 1907. 9. Wolbaeh, S. B." Controlled F o r m a t i o n of Collagen and Reticulum. A S t u d y of the Source of I n t e r c e l l u l a r Substance
10.
11. 12:
13. 14.
15. 16.
17. 18. 19. 20. 21. 22. 23.
24.
25.
CONGENITA
721
in Roco~,ery From Experimental Scorbutus, Am. J. Path. 9: 689, 1933. Gross, J.: A g i n g of Collagenous Connective Tissue of R a t Skin as Studied with the E l e c t r o n Microscope, Am. J. Path. 26: 708, 1950. Gross, J.: Connective Tissue Fine Structure and Some Methods for Its Analysis, J. Gerontol. 5: 343, 1950. Lillie, R . D . : Retieulum S t a i n i n g W i t h Schiff Reagent A f t e r Oxidation by Acidified Sodium Periodate, J. Lab. & Clin. Med. 32: 910, 1947. Johnson, R. W.: Personal communication. Blegvd, O., and H a x t h a u s e n , ~ . : Blue Sclerotics and B r i t t l e Bones W i t h Macular A t r o p h y of the Skin a n d Zonular Cataract, Brit. M. J. 2: ]071, 1921. Siedorf, K. S.: Osteogenesis Impertecta, 1949, U n i v e r s i t e t s f o r l a g e t , Arhus, Denmark. Gruber, G. B.: Zur JAenntnis und Kritik der Osteogenesis i m p e r f e c t a congenltalis, Arch. path. Anat. 316: 317, 1949. Weber, M.: Osteogenesis I m p e r f e c t a Congenita. A Study of I t s Histopathogenesis, Arch. P a t h . 9: 984, 1930. Spurway, J.: H e r e d i t a r y Tendency to Fracture, Brit. M. J. 2: 844, 1896. Eddowes, A.: D a r k Sclerotics and F r a g i l i t a s Ossium, Brit. M. J. 2: 222, 1900. Ruedemann, A. D.: To be published. H a r b i t z , F.: U e b e r Osteogenesis imperfecta, Beitr. z. path. A n a t . u. z. al]g. P a t h . 30: 605, 1901. K a r d a m a t i s , C.: Anatomische Befunde der Osteogenesis imperfecta, Arch. path. Anat. 212: 436, 1913. Bauer, K . H . : U e b e r Osteogenesis imperfecta. Zugleich eln B e i t r a g zur F r a g e elner a]lgerneinen E r k r a n k u n g samtlicher Stutzgewebe, D e u t s c h e Ztsehr. f. Chir. 154: 166, 1920. Jeckln, E.: System gebundene meseachymal Erschopfung. Eine neue Begriffsfassung der Osteogenesls impertecta, Arch. path. A n a t . 280: 351, 1931. Heller-Steinberg, M.. Ground Substance, Bone Salts, and Cellular Activi t y in Bone F o r m a t i o n and Destruction, Am. J. A n a t . 89: 347, 1951.