The short rib polydactyly syndromes (SRP) are lethal neonatal skeletal dysplasias with a narrow chest, short limbs, and other abnormalities. Type II (Majewski) short rib polydactyly can be distinguished from the Type I/III (Saldino-Noonan) type on the basis of radiographic and histologic changes. Our previous transmission electron microscopic studies suggested unusual patterns of cartilage calcification in these syndromes. We evaluate calcification in the present study using scanning electron microscopy and quantitative morphometry of calcification regions digested to expose calcospherite nodules (calcification nodules), distinctive morphologic structures which form during cartilage calcification. Mean calcospherite area of the Majewski Type II SRP (3.5 ± 0.24 x 10-6 mm2 (3) (mean ± sem (n)) did not differ from normal control means (3.1 ± 0.5 x 10-6 mm2, (3)). The mean area for Type I/III, however, was significantly larger than both the control and Type II means (8.9 ± 1.16 x 10-6 mm2 (7), p=.001). This difference in calcospherite size adds a further differentiating feature between these two dysplasias

Gruber, Helen E.

Lachman, Ralph S.

English

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Scanning Microscopy Volume 4 Number 3 Article 23 8-22-1990 Calcospherite (Calcification Nodule) Size in the Short Rib Polydactyly Syndromes Helen E. Gruber Medical Genetics-Birth Defects Center Ralph S. Lachman Harbor-UCLA Medical Center Follow this and additional works at: https://digitalcommons.usu.edu/microscopy  Part of the Life Sciences Commons Recommended Citation Gruber, Helen E. and Lachman, Ralph S. (1990) "Calcospherite (Calcification Nodule) Size in the Short Rib Polydactyly Syndromes," Scanning Microscopy: Vol. 4 : No. 3 , Article 23. Available at: https://digitalcommons.usu.edu/microscopy/vol4/iss3/23 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact digitalcommons@usu.edu. Scanning Microscopy, Vol. 4, No. 3, 1990 (Pages 775-780) 0891-7035/90$3.00+.00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA CALCOSPIIBRITK (CALCIFICATION ODULB) SIZB IN TBB SHORT RIB POLYDACTYLY SYNDROMES * 1 Helen E. Gruber, Ralph S. Lachman, and David L. Rimoin *Medical Genetics-Birth Defects Center and Ahmanson Pediatric Center, Cedars-Sinai Medical Center and Department of 1 Pediatrics, u.c.L.A., Los Angeles, CA Department of Radiology, Harbor-UCLA Medical Center, Torrance, CA (Received for publication April 11, 1990, and in revised form August 22, 1990) Abstract The short rib polydactyly syndromes (SRP) are lethal neonatal skeletal dysplasias with a narrow chest, short limbs, and other abnormalities. Type II (Majewski) short rib polydactyly can be distinguished from the Type 1/111 (Saldino-Noonan) type on the basis of radiographic and histologic changes. Our previous transmission electron microscopic studies suggested unusual patterns of cartilage calcification in these syndromes. We evaluate calcification in the present study using scanning electron microscopy and quantitative morphometry of calcification regions digested to expose calcospherite nodules (calcification nodules), distinctive morphologic structures which form during cartilage calcification. Mean calcospherite ar~g of 2 the Majewski Type II SRP (3.5 ± 0.24 x 10 mm (3) (mean± sem (n)) did not ~iffe 2 from normal control means (3.1 ± 0.5 x 10 mm, (3)). The mean area for Type I/III, however, was significantly larger than bo!g th~ control and Type II means (8.9 ± 1.16 x 10 mm (7), p=.001). This difference in calcospherite size adds a further differentiating feature between these two dysplasias. KEY WORDS: Mineralization, calcification, short rib polydactyly, skeletal dysplasias, quantitative morphometry, scanning electron microscopy * Address for correspondence: Helen E. Gruber Medical Genetics-Birth Defects Center, ASB Third Floor, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048 Phone: 213/855-6451 Fax: 213/967-0112 775 Introduction The short rib polydactyly syndromes (SRP) are lethal neonatal skeletal dysplasias in which the chest is narrowed, limbs shortened and in which other congenital abnormalities may be present (Taybi and Lachman, 1990). The reason these two syndromes were given the same name derives solely from the combination of the features of short ribs and polydactyly, each of which is present in other skeletal dysplasias. The names of these syndromes in no way suggest a common pathogenetic mechanism. Our previous transmission electron microscopic examinations of growth plate specimens from patients with SRP revealed unusual calcification sites (Gruber et al, 1989). Because of this finding we were especially interested in visualizing and quantitatively measuring calcospherites in SRP. In the present study we apply digestive procedures to growth plate tissues to remove soft and cellular tissue; the exposed calcospherites are then examined with scanning electron microscopy and quantitatively studied with morphometric analysis. The calcospherite calcification nodule is a distinctive morphologic structure which develops at the loci of calcification in the growth plate. In the past it has been studied under a variety of names ("initial calcification nodules", Bernard, 1972; large aggregates of "crystal ghosts", Bonucci, 1987; "crystal clusters", Ali, 1983 and Bonucci', 1971; "calcospherites", Boyde and Jones, 1983, and Ornoy et al, 1980). As chondro-osseous growth progresses, these sites of calcospherite clusters merge and coalesce forming the individual septum upon which the primary trabeculae form at the growth plate. Materials and Methods Specimens were obtained from SRP patients at autopsy and from normal subjects at autopsy from abortuses, stillbirths or individuals who died from non-skeletal-related diseases. This group of normal subjects has been utilized in our previous study of cartilage cell columns (Gruber and Rimoin, 1989). Specimens are part of Helen E. Gruber, Ralph S. Lachman and David L. Rimoin the Cedars-Sinai International Skeletal Dysplasia Registry collection. Tissue specimens at growth plate regions of long bones (femur, radius), iliac crest, and rib (at the costo-chondral junction) were fixed in glutaraldehyde, digested in 5% solutions of NaOCl for one hour at room temperature and rinsed thoroughly with distilled water to remove soft tissues and bleach (Boyde, 1984). Specimens were critical-point dried, coated with gold/palladium and exa mined with an Hitachi S405 A/2 scanning electron microscope. Original micrographs at a magnification of 1500 x were selected for print s in which clear, distinct calcos pherite profiles were imaged. Calcospherite areas were traced onto a Summagraphics Bit Pat One interfaced with an IBM XT computer. Software utilized was "Morphom General" produced by BioMed Stats, Inc. (Tacoma, VIA). Statistical analyses test for independent Cochran, 1973). Dat a are s.e.m. (n). utilized Student's t-groups (Snedecor and expressed as means± For quantitati ve study of calcospherites, the following mean numbers of indi vidual nodules were measured: controls, 33 ± 10 (range 18-51); SRP Type I/III, 39 ± 6 (range 16-64); SRP Type II, 50 ± 9 (range 37-66). Case Summaries Two types of SRP were studied: SRP syndrome Type I/III (Saldino-Nonan/Verma-Naumoff) and SRP syndrome Type II (Majewski). Clinical and radiologic feature s of these autosomal recessive syndromes have recently been reviewed by Taybi and Lachman (1990). Types I and III appear to represent one disorder with variable expressivity, Type I being the most severe. Radiographically Type I/III is characterized by shortened horizont al ribs, small iliac bones with flattened acetabular roofs, markedly shortened long tubular bones with irregular metaphyses with spurs, incomplete metacarpal, metatarsal and phalangeal ossification and postaxial polydactyly (Figure 1) . Histologic features of I/III include shortened or absent proliferative zone, absence of chondrocyte columns and irregularly dispersed hypertrophic cel ls with areas of acellular cartilage abutting directly on bony trabeculae. SRP Type II radiologically exhibits underdeveloped mandible, short horizontall y oriented ribs, shortened tubular bones (especially the tibiae) with rounded metaphyseal ends, precocious ossification of the proximal femoral epiphysis and pre and/or post- axial polydactyly (Figur e 2). His tol ogically, the growth plate i s short with irregular columnization. Case summaries of cases 1-10 are outlined in Table 1. 776 Figure 1. Representative skeletal features of SRP illustrated. radiograph of the Type I/III. Case #4 is Results The special digestion techniques used here allow direct visualization of the calcospherite domains in the matrix septal regions surrounding hypertrophic chondrocy tes (Figure 3). Calcospherites line elongated cup-shaped depressions (Figure 4). They are closel y packed but, with our preparative techniques, appear round and not angled as would be expected in objects which show hexagonal close packing. Calcospherites fr~~ n2rmal su bjects had a mean area of 3.07 x 10 mm ± 0.52 (3) (Figures 4 and 5). Mean calcospherite area from patients with Type -~I 2RP did not differ from normal (3.46 x 10 mm ± 0.24 (3)) (Figures 5 and 6). The mean calcosp herite area from seven well-defin ed cases of SRP Type I/III, however, was significan tl y larger than b~6h co2 trol and Type II SRP means (8.92 x 10 mm ± 1.16, p=.001) (Figures 5 and 7). No overlap was seen Morphometry of calcospherites Figure 2. Representative skeletal features of SRP (The structure above the umbilical cord clamp.) Case radiograph of the Type II (Majewski). right ilium is an #8 is illustrated. for the Type I/III mean value s with either control or SRP Type II mean values (Figure 8). Discussion Cartilage calcification is an intricate and complex process. The initial changes which occur in matrix near hypertrophic chondrocytes are many; one current model suggests involvement of the C-propeptide of the type II collagen fibril, proteoglycan aggregates and link protein (Poole et al, 1989). The morphologic structure studied here, the calcospherite, i s a later 777 Figure 3. Low magnification scanning electron micrograph shows the chondrocyte columns (cc, arrow), lacunae of hypertrophic chondrocytes, and primary spongiosa in the growth plate of a normal subject (bar= 1 µm). Figure 4. Scanning electron micrograph of calcospherite domain in a normal newborn (bar= 1 µm). < 10 w a: < w I-ii: -w"b ::c ... B; .... "" 5 o E o E ~-0 z < w :I Helen E. Gruber, Ralph S. Lachman and David L. Rimoin SRP Type I/III: Case Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 SRP Type II: Case 8 Case 9 Case 10 Table 1: Description of SRP Patients Age/Sex* F, 25 wks 23-24 wks M, newborn M, 28 wks Baby girl Baby girl M, 30 wks Baby boy F, 41 wks Baby boy History/Comments Short limbs detected on ultrasound at 24 weeks Short limbs detected on ultrasound at 18 weeks Siblings. Another male sibling reported to have died from "thanatophoric dysplasia". 1 normal sibling. Second affected sibling in 1989 had identical radiology at 21 weeks. Edema; situ s inversus (except for liver) . Ventricular septal defect. Bilateral urinary bladders; 2 spleens Par tial cleft lip and palate. Malrotation of GI tract, hydrocephalus, absence of external penis Short femora detected on ultrasound at 31 weeks . Multiple frenula in lips. 5 s pleens. Malrotation of large bowel, polycystic kidneys, bi cornate uterus. * Ages are weeks of gestation CONTROL TYPE II SRP * p< .001 TYPE I/ Ill SRP Figure 5. Mean calcospherite size is significantly larger in SRP Type I/III compared to either control subjects or SRP Type II (p=.001). (Open bar, mean of 3 control subjects; hatched bar, mean of 3 SRP Type II patients; solid bar, mean of 7 SRP Type I/III patients.) (Error bars are s.e.m.) Figure 6. Scanning electron micrograp.1 of calcospherite domain in SRP Type II (b1r = 1 µm). 778 Morphometry of calcospherites Figure 7. Scanning electron micrograph of calcospherite domain in SRP Type I/III (bar= 1 µm). developmental stage formed when these initial calcification sites have grown in size. Other hypotheses for the calcification process invoke mediation and initiation by matrix vesicles located in the longitudinal septae or origin directly from the 150 nm matrix vesicles (Ali, 1983; Bernard, 1972; Boyde and Jones, 1983; Bonucci, 1971). The SEM methods employed here are well-suited for morphologic studies of calcospherites. This study has provided new information on the quantitative morphology of calcospherites in the human term infant and in patients with SRP. One form of SRP, Type II, WRS characterized by calcospherites which were similar in size to those of the normal human. In Type I/III patients, however, calcospherites averaged twice as large as normal. One individual p~6ien 2 had a mean calcospherite size of 12.9 x 10 mm, more than four times normal. The cause of these enlarged calcospherites is not known. There are hypothetically at least two possible explanations: l) matrix alterations may be present which act to offer resistance to, or chemically inhibit, growing calcospherites; 2) there may be alterations in the matrix which result in fewer, more widely spaced initial sites of calcification. A third alternative hypothesis is that these bone dysplasias are due to defective endochondral ossification and the unusual calcospherite nodules are the result, but not the cause, of these changes. The calcospherites which develop may then grow larger in their less crowded domain. Since these disorders are inherited as autosomal recessive traits, an inborn error of metabolism which could result in increased calcospherite size is a likely cause of the bone dysplasia. 779 TYPE I/ Ill SRP TYPE II SRP CONTROL ti&6 [II] 0 0 0 2 4 6 8 10 12 14 MEAN CALCOSPHERITE AREA (mm 2 x 106) Figure 8. Distribution of mean calcospherite areas in normal subjects (circles), SRP Type II (squares) and SRP Type I/III (triangles). Acknowledgements The authors wish to thank Mrs. L. Nolasco for technical assistance, Mrs. M. Priore for assistance in coordinating the International Skeletal Dysplasia Registr y and Ms. G. Reyburn for assistance in typing the manuscript. This study of SRP was possible due to the generous referral of cases from a number of physicians and investigators: Dr . J. Bass (Torrance, CA); Dr. A. Uri (Philadelphia, PA); Dr. K. Lay (Glendale, CA); L. Suslak and Dr. L-Y Shih (Newark, N.J.); Dr. R. Bachman (Oakland, CA); Dr. D. Flannery (Richmond, VA); Dr. P. Bowen (Alberta, Canada); Dr. J. Mann (San Jose, CA); Dr. L. Karp (Seattle, WA). This work was supported, in part, by a USPHS-NIH program project grant 5 POI HD22657-04. References Ali SY. (1983). Calcification of cartilage. In: Cartilage Vol . 1. Structure, Function and Biochemistry, BK Hall (ed.) . Academic Press, N.Y., pp. 343-378. Bernard GV. (1972). Ultrastructural observations of initial calcification in dentine and enamel. J. Ultrastruct. Res. 41: 1-17. Boyan BD, Schwartz z, Swain LD, Khare A. (1989). Role of lipids in calcification of cartilage. Anat. Rec. 224: 211-219. Boyde A. (1984). Methodology of calcified tissue specimen preparation for scanning electron microscopy. In: Methods of Calcified Tissue Preparation, GR Dickson (ed.). Elsevier Scientific Publishers, pp. 251-307. Boyde A, Jones SJ. (1983). Scanning electron microscopy of cartilage. In: Cartilage, Vol. 1, Structure, Function and Biochemistry, BK Hall (ed.). Academic Press, N.Y., pp. 105-148. Bonucci E. (1971). The locus of initial calcification in cartilage and bone. Clin. Orthoped. 78: 108-139. Helen E. Gruber, Ralph S. Lachman and David L. Rimoin Bonucci E. (1987). Is there a calcification factor common to all calcifying matrices? Scanning Microsc. 1: 1089-1102. Gruber HE, Lachman R, Rimoin DL. (1989). Ultrastructural abnormalities in bone and calcifying cartilage in the short rib polydactyly syndromes. J. Bone Mineral Res. 4 (suppl 1): 242 (abstract). Gruber HE, Rimoin DL. (1989). Quantitative histology of cartilage cell columns in the human costochondral junction: Findings in newborn and pediatric subjects. Pediatr. Res. 25: 202-204. Ornoy A, Atkin I, Levy J. (1980). Ultrastructural studies on the origin and structure of matrix vesicles in bone of young rats. Acta Anat. 106: 450-461. Poole AR, Matsui Y, Hinek A, (1989). Cartilage macromolecules calcification of cartilage matrix. 224: 167-179. Lee ER. and the Anat. Rec. Snedecor GV, Cochran VG (1973). Statistical Methods. 6th ed. Iowa State Univ. Press, Ames, Iowa, pp. 91-119. Taybi H, Lachman RS. (1990). Radiology of and Skeletal Book Medical Syndromes, Metabolic Disorders, Dysplasias. 3rd ed. Year Publishers, Chicago, pp. 787-793. 780 Discussion with Reviewers K. Bonucci: Abnormalities of calcospherites might reflect abnormalities of HV (matrix vesicles). Have you any information about numbers, morphology, and distribution of HV in SRP? s.c. Marks: Can your data be correlated with anything known about matrix vesicles in the syndrome? Authors: There are no previously published data on HV abnormalities in SRP. Ve have begun to collect transmission EM data on this but have not yet examined enough cases to formulate a sound description. This aspect also demands very well-preserved specimens, and not all of our autopsy cases are well suited for such studies. s.c. Hiller: Are there any clues concerning themolecular defect(s) or the gene loci for these disorders? Authors: Type I/III SRP is quite different from Type II. It was only the shared features of short ribs and polydactyly that first caused them to be combined into one group. Their nomenclature should not suggest they share a common pathogenetic mechanism. To date, there are no molecular data on the etiology of these syndromes. B. Bonnucci: Because structure and organization of the growth zone appear deeply altered in SRP type I/III, so that acellular cartilage abuts directly on osseous trabeculae, could the enlargement of calcospherites be due simply to the fact that they derive from bone in type I/III and from cartilage in type II? Authors: The size difference we found cannot be due to differences in origin of the calcospherites since all calcospherites measured were in the calcifying cartilage site. 

Calcospherite (Calcification Nodule) Size in the Short Rib Polydactyly Syndromes

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Calcospherite (Calcification Nodule) Size in the Short Rib Polydactyly Syndromes

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