Skull growth in achondroplasic (cn) mice ; a craniometric study

Inherited chondrodystrophies have been reported in a wide variety of vertebrate groups, including chickens, mice, rats, rabbits, dogs, sheep, cattle and man (for comparative descriptions and bibliography see Grüneberg, 1963). All are associated with disturbances of endochondral ossification that lead to disproportionate dwarfism in which the limbs are usually shortened to a greater extent than the trunk. The changes produced in the skull are generally less well documented than those occurring postcranially, but it appears that in the majority of the mammalian conditions the cranial base is shortened with compensatory broadening of the calvarium and retraction of the nasal skeleton (Crew, 1923; Stockard, 1941; Landauer and Chang, 1949; Grüneberg, 1963).

The complex changes in the skull are of particular interest in that this part of the skeleton contains, in addition to the endochondral elements of the cranial base and sense capsules, the intramembranously ossifying dermal bones of the calvarium and face. Moreover, certain of the dermal bones contain zones of secondary cartilage which appear after the initiation of intramembranous ossification, but subsequently undergo endochondral ossification to make a significant contribution to overall bony growth. The number of secondary cartilages varies from one vertebrate group to another but includes the condylar, coronoid and angular cartilages of the mandible in most mammalian groups (De Beer, 1971). Of these, the condylar cartilage has been the most intensively studied in many species, including the mouse, and its growth has been found to proceed by apposition on its upper surface as a result of the activity of chondroblasts differentiating in the intermediate cell zone (located between condylar and articular cartilages) rather than by interstitial proliferation of chondrocytes such as occurs in a typical epiphyseal cartilage (Blackwood, 1966; Frommer, Monroe, Morehead & Belt, 1968; Silbermann & Frommer, 1972). Unlike epiphyseal cartilage, no regular columns of hypertrophic chondrocytes are formed in the condylar cartilage (Durkin, Irving & Heeley, 1969) and the cells survive their passage through the cartilage to emerge, still vital, in the zone of ossification on its undersurface (Silbermann & Frommer, 1972). In view of the observations that the underlying defect in many of the mammalian forms of chondrodystrophy lies in the processes of interstitial cartilaginous growth that lead to the formation of the columns of hypertrophic chondrocytes (Grüneberg, 1963), it might be expected that these disorders would be associated with less disturbance of the appositionally growing condylar cartilage than of the interstitially growing septal cartilage of the nasal capsule or synchondrotic cartilages of the cranial base.

In this study, a craniometric analysis is made of the effects of one type of chondrodystrophy (cn/cri) occurring in the mouse upon the growth of the skull components, with special attention being given to possible differential effects at the various sites of endochondral ossification. This particular chondrodystrophy was chosen because it has been shown (Konyukhov & Paschin, 1970) that the cartilage defect consists of a reduction in the rate of chondrocyte division manifested by a diminution in the number of chondrocytes per isogenous group in zone II (the zone of cellular proliferation) of the epiphyseal cartilage. It might be expected, therefore, to provide a suitable model for the skull changes observed in other chondrodystrophies, including the classic form of human achondroplasia, in which there is a failure of interstitial cartilage growth.

The achondroplasic strain of mice arose as a spontaneous mutation in the AKR/J strain (Lane & Dickie, 1968). The body weight of enjen mice is reduced at birth compared with normal siblings (i.e. +/+ or cn/ + ) and growth retardation continues for the first month of life. From the fifth week onwards, the relative rates of growth of the cn/cn mice and their normal siblings are similar (Konyukhov & Paschin, 1967).

The cn/cn mice can be distinguished at birth by their dome-shaped skulls and short thick tails. Many of the affected animals die, but those that reach maturity have shortened trunks and tails, pronouncedly shortened limbs, and heads that retain their initial doming (Lane & Dickie, 1968). A malocclusion, in which the lower molars occlude anterior to the upper molars, present as soon as the teeth have erupted, becomes progressively more accentuated as growth proceeds.

As in most other forms of heritable chondrodystrophy, the fundamental defect of cartilage growth is unknown, but the work of Konyukhov & Paschin (1967), using subcutaneous implantation of limb bones between 7- or 14-day-old cn/cn mice and their normal litter-mates, suggests that there is a primary gene effect manifested in the chondrocytes and also a growth-inhibiting substance released into the blood in the cn/cn mice. In a later study (Konyukhov & Paschin, 1970) these authors found that the principal difference between the epiphyseal cartilages of the cn/cn and normal mice was a reduction in the thickness of the hypertrophic zone (zone III) and a reduction in the number of cells per isogenous group in the columnar zone (zone II). These differences became less pronounced after 21 days of postnatal life.

Ten cn/cn mice (9 female, 1 male), all more than 13 weeks old, were compared with 10 similarly aged normal siblings (all female). The animals were killed by ether inhalation and the skeletons macerated by immersion in papain solution at 56 °C. Lateral radiographs were taken of the crania, each being positioned so that the X-ray beam was directed at right angles to the mid-sagittal plane along the long axes of the external auditory meatuses, with constant tube-object-film distances.

The following measurements were taken on the dried skulls using a measuring microscope (Fig. 1).

• (1) Overall length of cranium, opisthion (a) to most anterior point on nasal bones (b).

• (2) Length of neurocranium, opisthion (a) to nasion (f).

• (3) Width of neurocranium, between posterior roots of zygomatic arches (c-c′).

• (4) Height of neurocranium, mid-point between fronto-parietal and parieto-interparietal sutures (d) to spheno-occipital synchondrosis (e).

• (5) Width of cranial base, between lateral borders of carotid canals (g-g′).

• (6) Length of viscerocranium, nasion (f) to most anterior point on nasal spine (b).

• (7) Width of viscerocranium, between lateral surfaces of upper second molar teeth (h-h′).

• (8) Height of viscerocranium, nasion (f) to midline of palate at a transverse axis through the anterior borders of the first molar teeth (j).

• (9) Length of mandible (left), condyle (k) to symphysis (l).

• (10) Length of condylar process (left), condyle (k) to masseteric notch (m).

• (11) Volume of endocranial cavity ; the external surface of the cranium was coated with wax to render it watertight and the volume of water required to fill the endocranial cavity was then determined using a tuberculin syringe.

As a measure of the changes in the limb bones, the length of the humerus was taken between the intertubercular sulcus and intercondylar notch.

Both linear and angular measurements were taken on enlargements of the radiographs. Since the aim of the study was to compare the normal and achondroplasic mice, rather than to obtain absolute measurements, no correction was made for magnification, which was identical for both groups. The linear measurements were related to the three components of the cranial base and included the following (Fig. 2).

• (1) The basicranial axis, presphenoid-ethmoidal synchondrosis (n) to the Bolton point (p).

• (2) The anterior extension, presphenoid-ethmoidal synchondrosis (n) to the fronto-ethmoidal suture (q).

• (3) The posterior extension, Bolton point (p) to opisthion (a).

The angular measurement was taken between the basicranial axis and its anterior extension, as determined by drawing lines tangential to the outlines of the superior surfaces of the ethmoid bone and of the basicranial axis and measuring the superior angle at the point of intersection (Fig. 2). To make it comparable with the spheno-ethmoidal angle as used in man, the angle was subtracted from 360°.

Means and standard errors were computed for each dimension in the cn/cn and normal mice. The means for the two groups were compared by t tests, P values below 0 · 05 being taken as statistically significant.

The total length of the cranial base was reduced in the cn/cn mice compared to the normal animals but the amount of shortening was not uniform in the three subdivisions. The greatest reduction was in the basicranial axis which was shortened by almost 25 %, while the posterior extension was shortened by 14 %. The reduction in the anterior extension was not statistically significant. The width of the cranial base was 9 % less in the cn/cn than in the normal animals. The angle between the anterior extension and the basicranial axis was decreased in the cn/cn mice by some 3 %. The spheno-occipital synchondrosis did not appear to undergo premature closure, being still open in the oldest (36 weeks) achondroplasic mouse reared in our colony.

The length of the neurocranium was reduced by some 19 % in the cn/cn mice compared to the normal animals. Neither the width nor the height of the neurocranium showed statistically significant differences between the two groups.

The length of the viscerocranium was shorter by almost 18 % in the cn/cn mice. The height of the viscerocranium was reduced by just over 8 %. There was no statistically significant difference between the two groups in the width of the viscerocranium.

Both the total length of the mandible and the length of the condylar process were reduced by almost 11 % in the cn/cn mice compared to the normal mice.

The shortening of the humerus in the cn/cn animals was some 38 %.

None of the measurements of the skull in the cn/cn mice was reduced to the same extent as the length of the humerus, which was taken to provide an indication of the effect of achondroplasia on the growth of epiphyseal cartilage. The greatest reduction in the cranial dimensions was observed in the basicranial axis of the cranial base. This finding is not unexpected, since elongation of the basicranial axis occurs entirely at synchondrotic cartilages (between the sphenoid and occipital and pre- and post-sphenoid bones) where the processes of endo-chondral ossification are similar, if not identical, to those occurring in epiphyseal cartilages (Baume, 1961 a, 1968).-The flattening of the cranial base in the cn[cn mice, as indicated by the decrease in the angle between the basicranial axis and its anterior extension, is attributable to the expansion of the growing brain and meninges within the shortened endocranial cavity. The doming of the skull and the tendency towards an increase in the height and width of the neurocranium (the data were insufficient to establish the latter two trends as statistically significant) may also be attributable to the same effect. As a result, the volume of the endocranial cavity was reduced less than might have been expected from the degree of shortening of the neurocranium (allowing for the cube relationship between volume and linear dimensions).

The extensions of the basicranial axis were reduced by much smaller amounts than the axis itself. So far as the posterior extension is concerned, this finding is not surprising in that this component of the skull contains no cartilaginous growth sites. The anterior extension, however, is coterminous with the endo-chondrally ossifying mesethmoid. Why this part of the ethmoid complex should show no statistically significant reduction in length in the achondroplasic animals is not apparent from craniometric data alone (see below).

The viscerocranium was reduced in both length and height in the cn/cn mice. Part of this reduction may be due to the diminution in masticatory muscle function secondary to the malocclusion. That this is not the only factor involved is indicated by comparing the present findings with those of Moore (1967) where the reduction in viscerocranial growth in rats following bilateral ablation of the masseter muscles was only some 1–4 %. The much greater reductions in the cn/cn mice appear to be largely attributable to the chondrodystrophic effect on the septal cartilage which has been shown in primates (no equivalent information being available for the mouse) to bear a close structural resemblance to an epiphyseal cartilage in the region of the septo-ethmoidal junction (Baume, 1961 b), there being no reason to assume any diminution in the impetus of sutural growth except as a secondary effect. The reductions in overall mandibular length and length of the condylar process were much smaller than those in the basicranial axis and viscerocranium, and may be more completely attributable to the diminution of muscle function consequent upon the malocclusion, since the mandible appears to have a greater susceptibility than the cranium to changes in muscle activity -in the rat, bilateral ablation of the masseter muscles led to a 7 % shortening of the mandible and an 11 % diminution in the length of the condylar process (Moore, 1967, 1973). The finding that the reduction of condylar growth in the cn/cn mice was much less than the corresponding reductions in the basicranial axis and viscerocranium supports the suggestion (Grüneberg, 1963) that appositional cartilage growth is less affected than interstitial cartilage growth in achondroplasia.

Organ culture and transplantation studies of the condylar cartilage (Petrovic, 1972) indicate that this structure probably possesses less inherent growth potential than do septal (Petrovic, Charlier & Herrmann, 1968) or epiphyseal and synchondrotic cartilages (Koski & Ronning, 1965, 1966, 1969, 1970). Numerous extirpative procedures have been performed to determine whether the septal and condylar cartilages are primary determinants of viscerocranial and mandibular growth respectively, but the results are inconclusive (see, for example, Moss, Bromberg, In Chul Song & Eisenman, 1968; Sarnat & Muchnic, 1971). The findings of the present study appear, at first sight, to support the view that the synchondrotic and septal cartilages, if not the condylar cartilage, are such primary growth determinants, but an alternative possibility is that their reduced growth in the cn/cn mice results in their acting as ‘ties’ which physically restrain growth at other sites (see Moore & Lavelle, 1974 for a full discussion of this problem).

Craniometric data for chondrodystrophies in other mammalian species are sparse and usually qualitative. The most comprehensive accounts, which include metrical data, have been given by Stockard (1941), who describes the skull in dog breeds of disproportionate body form in which the growth defect is believed to be of a chondrodystrophic nature. He deals most fully with the Bulldog in which the disproportion is confined to the axial skeleton. Although Stockard’s measurements were not designed specifically to determine the extent of growth reductions in the various cranial sites of cartilage growth, it is clear that the deformities in the Bulldog skull are similar to those in the cn/cn mouse. As compared to a dog of normal proportions (e.g. Alsatian), the cranial base is shortened, the vault is domed, the upper facial skeleton is reduced in length and the mandible is also reduced but to a lesser degree than the upper face. Similar deformities occur in the skulls of the French Bulldog, Boston Terrier, Pekinese and Brussels Griffon.

In man, cranial deformities have been described as a constant feature of the classic form of achondroplasia, helping to distinguish it from other chondrodystrophic conditions such as spondylo-epiphyseal dysplasia (Maroteaux & Lamy, 1964). The cranial deformities bear many similarities to those just described in the cn/cn mouse. The cranial base is shortened, the angle between the basicranial axis and its anterior extension is reduced (to about 90° compared with a normal value of about 120°), the calvarium is domed and the nose retracted (Maroteaux & Lamy, 1964). The human achondroplasic skull differs from that in the mouse in that the endocranial cavity is enlarged, but whether due to megalocephaly or hydrocephaly is uncertain (Dennis, Rosenberg & Alvord, 1961; Bergstrom, Laurent & Lundberg, 1971), and in the early closure (or replacement by fibrous tissue) of the spheno-occipital synchondrosis (Benda, 1947). We have been unable to locate any quantitative descriptions of the human mandible in achondroplasia, but from the numerous accounts of Angle Class III malocclusions (i.e. prognathous mandibles) in subjects with this condition (e.g. Weinmann & Sicher, 1955), it appears that the growth of the lower jaw is affected less than that of the upper jaw, as in the achondroplasic mouse.

An investigation of the growth of the skull of the achondroplasic mouse is now being carried out using intravital staining of newly formed bone and cartilage. This will enable a direct comparison to be made of (1) the rates of endochondral ossification at the individual cartilaginous growth sites of the cranial base, to help elucidate the differential effects of this form of chondrodystrophy on the basicranial axis and its anterior extension, and (2) the rates of ossification at synchondrotic, septal and condylar cartilages.