J. Anat. (1990), 173, pp. 69-75 69 With 3 figures Printed in Great Britain Quantitative histology of cartilage vascular canals in the human rib. Findings in normal neonates and children and in achondrogenesis H-hypochondrogenesis* HELEN E. GRUBERtt, RALPH S. LACHMAN§ AND DAVID L. RIMOINtf t Medical Genetics-Birth Defects Center, and The Ahmanson Pediatric Center, Cedars-Sinai Medical Center, Los Angeles, CA, $ Department of Pediatrics, U.C.L.A., Los Angeles, CA and § Department of Radiology, Harbor- U.C.L.A. Medical Center, Torrance, CA (Accepted 10 May 1990) INTRODUCTION Relatively little is known about the vascular supply to cartilage in the long bones of human neonates and children. Although the avascular nature of adult cartilage is well recognised, resting cartilage in the developing and maturing limb and rib contains vascular canals through which course arterioles, venules and sinusoidal capillaries that are important for nutrition, and also connective tissue which serves as a source of stem cells. By the third month ofhuman fetal development, cartilage canals are recognisable, coming in from the perichondrium into the uncalcified epiphyses (Chappard, Alexandre & Riffat, 1986). Studies in developing mammals (Kugler, Tomlinson, Wagstaff & Ward, 1979; Wilsman & van Sickle, 1970, 1972) and more limited studies in the developing human fetus (Chappard et al. 1986; Agrawal, Kulkarni & Atre, 1986; Whalen, Parke, Mazur & Stauffer, 1985; Agrawal, Atre & Kulkarni, 1984) have revealed some information about the timing of appearance of canals and their physiological significance, but this remains a controversial field with respect to species variability and canal origin (Chappard et al. 1986; Cole & Madison, 1989). Most studies of human tissues have focused upon the role of cartilage canals in osteogenesis in the calcaneus (Agrawal et al. 1986) or femur (Chappard et al. 1986) or upon a role in nutrition ofthe disc in the vertebral end plate (Whalen et al. 1985). Oxygen tension and tissue vascularity are crucial factors in the regulation of calcification in the growth plate (Shapiro & Boyde, 1987). Data on cartilage vascular canals in normal paediatric subjects are scarce. Achondrogenesis II-hypochondrogenesis is a neonatally lethal skeletal dysplasia with a wide spectrum of phenotypic expressivity (van der Harten et al. 1988; Borochowitz, Ornoy, Lachman & Rimoin, 1986). Radiological findings include short trunk, extreme micromelia, a normal cranium, short ribs with cupped, flared ends, deficient spinal ossification, short long bones with metaphyseal irregularity, and absence of calcification in the pubis, calcaneus and talus. Histological abnormalities include cartilage hypercellularity and hypervascularity, enlarged chondrocyte lacunae and an irregular growth plate (Borochowitz et al. 1986; Maroteaux, Stanescu & Stanescu, 1983; Stanescu, Stanescu & Maroteaux, 1977). * Reprint requests to Dr Helen E. Gruber, Skeletal Dysplasia Morphology Lab, ASB Third Floor, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA. 70 HELEN E. GRUBER AND OTHERS Studies by Eyre et al. (1986), Horton (1984) and Horton, Machado, Chou & Campbell (1987) showed that cartilage from patients with the Langer-Saldino variant of achondrogenesis contained mainly Type I collagen in place of Type II. Godfrey & Hollister (1988) reported the presence of abnormal overmodified Type II collagen in a case of hypochondrogenesis and suggested the possibility of a mutation in one allele of the COL2A1 gene. Recent work by Vissing et al. (1989) has demonstrated that the mutation in the Type II procollagen gene in this latter patient is a single base change converting the codon for glycine at amino acid 943 of the alpha-i(II) chain into a codon for serine. Thus our current understanding of the aetiology of achondrogenesis IT-hypochondrogenesis is heterozygosity for a mutant allele of the COL2A1 gene; this results in a structurally abnormal procollagen chain which becomes overmodified. The purpose of the present study was to establish normative morphometric data for cartilage canal area and incidence and to investigate vascular canal features in achondrogenesis II-hypochondrogenesis. MATERIALS AND METHODS Specimens of the costochondral junction were obtained from newborn and older normal children, either at autopsy from subjects who were aborted, stillborn, had died from accidents or non-skeletal-related diseases, or during cardiac surgery. This group of normal subjects was utilised in our previous study of cartilage cell columns (Gruber & Rimoin, 1989). Specimens are part of the International Skeletal Dysplasia Registry collection. Specimens were embedded in glycol or methyl methacrylate, sectioned longitudinally at 2-55,m, and stained with 'Stains All' (Eastman Kodak Co., Rochester, N.Y; Green & Pastewka, 1974) or Goldner's stain (Goldner, 1938) as previously described (Sillence, Horton & Rimoin, 1979; Yang, Kitchen, Gilbert & Rimoin, 1986; Gruber et al. 1988). Mean age for normal subjects excluding newborns was 7-2 + 1 5 (mean + S.E.M). In the fetal-newborn group, subjects ranged in age from 36 weeks gestation to 3 days postnatal. Patients with achondrogenesis II-hypochondrogenesis ranged in age from 23-42 weeks gestational age. One patient expired at the age of 12 days. Diagnosis of achondrogenesis II-hypochondrogenesis was based on clinical findings and radiological features. Quantitative histomorphometry, performed using a Zeiss microscope, camera lucida, Summagraphics BitPad One interfaced with an IBM XT computer (IBM, Irving, TX), utilised programs prepared by BioMed Stats, Inc. (Tacoma WA). Resting cartilage area was determined at a magnification of x 31. Quantitation of vascular canals lying within the resting cartilage area was performed at a magnification of x 120. For the study reported here, canals were only measured if they were completely surrounded by cartilage matrix (thus canals which had not yet completely crossed the perichondrium were not scored). For normal newborns, measured resting cartilage sites ranged from 10-7 to 32-7 mm2 (21-0 + 3-7 mean + S.E.M). For paediatric subjects, resting cartilage tissue areas ranged from 9-3-96 1 mm2 (37-3 + 8-2). For patients with achondrogenesis II- hypochondrogenesis, resting cartilage tissue areas ranged from 7-3-43-2 mm2 (16-7+4 7). Statistical analyses utilised Student's t test for independent groups (Sokal & Rohlf, 1969). Relationships between variables were evaluated using Pearson's correlation coefficient (Sokal & Rohlf, 1969). Data are expressed as means+ S.E.M. (n). Cartilage vascular canals in human rib 71 6 ** 5~~~~~~~~~~~~~~~~~~~~~~~~~. -.. .,:. .:.:.v..:.... -4~ ~ ~ ~ ~ ~ -+ ::.::::..... :: :: _4 ..........:: 3~~ ~~~~~~~~~~~~~~. 0cl~ Nra Noma Achondrogenesi:..vv::: :,.:.:.. ::.:.:.:.:.:.:...... :.. X::::~~~~~~~~~~~~~~~~~~~~~~~~~~..:::::::..::.:...........::::::: neworn; _, P. < 000 coprdt oraebrs N orma6 Norma Achondrogenesis 11- newborn paediatric hypochondrogenesis 20 Fig. 1. The percentage of resting cartilage area occupied by vascular canals is shown here for normal newborn subjects (hatched bar), normal paediatric subjects (open bar), and newborn patients with achondrogenesis 11-hypochondrogenesis (shaded bar). (,P > 0-003 compared with normal newborns; **P < 0-001I compared to normal newborns.) E , 1.2 0 0-8 E Z 0.4 Normal Normal Achondrogenesis Il- newborn paediatric hypochondrogenesis Fig. 2. The number of canals/mm2 resting cartilage tissue area is shown here for normal newborn subjects (hatched bar), normal paediatric subjects (open bar) and newborn patients with achondrogenesis IT-hypochondrogenesis (shaded bar). (*, P > 0-02 compared with normal newborns; **, P < 0-001 compared to normal newborns.) RESULTS Findings in normal subjects Normal newborn subjects 'had a significantly greater proportion of resting cartilage occupied by vascular canals than did older children (paediatric subjects) (042% +0'15 mm2 (6) vs. 0-08 +004 (12), P = 0003) (Fig. 1). The number of canals per mm2 resting cartilage was also significantly greater in normal newborn compared to paediatric subjects (0 19 ± 0r09 (6) vs. 0f04 ±0f02 (12), P = 0f02) (Fig. 2). There was a weak, but significant relationship between percentage canal area and age in the normal subjects (percentage canal area = (- 026) (age) + 0f319, r =- 50, P = 0f03). 72 HELEN E. GRUBER AND OTHERS Fig. 3 (a-b). (a) Vascular canal in resting cartilage of a normal newborn subject. Goldner's stain. x 50. (b) Representative micrograph of vascular canal morphology from a patient with achondrogenesis II-hypochondrogenesis. Goldner's stain. x 50. Findings in achondrogenesis II-hypochondrogenesis Newborn patients with achondrogenesis TI-hypochondrogenesis had greatly increased canal area compared to normal newborns (522% + l01 (8), P < 0001) (Fig. 1). The incidence/mm2 of resting cartilage was also significantly elevated above normal (I 45+0-26 (8), P < 0 001) (Fig. 2). Cartilage vascular canals in human rib 73 Figure 3 contrasts the histological appearance of vascular canals in patients with achondrogenesis II-hypochondrogenesis (Fig. 3b) with the appearance of canals in normal resting cartilage (Fig. 3 a). Resting cartilage from these patients is hypervascular with many large canals containing fibrous tissue and sclerotic vessels. The perichondrial region in many instances showed superficial canals which could be interpretated as early canals entering the cartilage matrix. Deeper canals were seen to contain much more fibrous connective