THE ANATOMICAL RECORD 235312-318 (1993) Morphometric Changes in Growth of the Rat Pelvis After Papain Ad minist ration ANDREW D. DIXON AND PETER T. GAKUNGA School of Dentistry and Dental Research Institute, University of California Los Angeles, Los Angeles, California ABSTRACT The proteolytic enzyme, papain, was given systematically to evaluate the short- and long-term effects of inhibition of endochondral bone formation on pelvic growth, with emphasis on the innominate bone. Ninety-eight Lewis-strain male rats, used concurrently for craniofacial growth studies, were divided into two groups. Thirty rats from Group I (n = 48) received 2% crude papain i.p. daily from 25-40 days-of-age and were euthanized at 40, 54, and 70 days-of-age. Thirty-five rats from Group I1 (n=50) were given papain at the same dose from 25-70 days-of-age and were euthanized at 26, 40, 54, 70, and 120 days. The remaining animals in both groups were the controls. Standardized dorsoventral pelvic radiographs were taken of all 98 animals. Ten linear dimensions were measured on each and the data evaluated statistically. A reduction both in size and rate of growth of the bony pelvis was found. All the anteroposterior and most of the transverse pelvic dimensions were significantly shorter, to a greater extent in the prolonged papain group. Bi-ischial width was increased, perhaps to compensate for pelvic shortening and to accommodate the pelvic contents. The findings may contribute to our better understanding of abnormal endochondral bone growth in the pelvis. 0 1993 Wiley-Liss, Inc. Key words: Rats, Pelvic growth, Parenteral papain, Radiographs, Morphometrics The morphology and growth of the pelvis in animal passed their peak growth velocity before the nutrimodels has been the subject of a number of studies tional insult became severe, recovered and caught up. (Payton, 1953; Harrison, 1958,a,b, 1961, 1968; Hughes Retardation of growth after 2 months-of-age may have and Tanner, 1970, 1973; Hughes, 1982). Harrison had some permanent effects (Dickerson, et al., 19721, (1958a, 1968) showed that the length of the rat innom- although animals undernourished from 49-70 days-ofinate bone, and the ilium and ischium as individual age were better able to recover fully from the same bones, increased rapidly up to 80 days-of-age, with degree of growth deficit (Hughes, 1982). Following the demonstration by Thomas (1956) that some growth still being detectable at the end of 1year. a single intravenous injection of crude papain in rabGrowth of the acetabular region decelerated by 60-70 days-of-age, coincident with the disappearance of the bits produced a reversible collapse of the ears associpubo-ischial flange of the acetabular, epiphysial carti- ated with depletion of cartilage matrix, i t soon was lage complex that lies between the ilium, ischium, and established that the enzyme inhibited endochondral pubic bones. Ossification of the acetabular complex be- bone formation at epiphysial growth plates, particugins 70 to 80 days after birth in the epiphysial cartilage larly in young, growing animals. Radiographically, adjoining the ilium. Synostosis of the three pelvic longitudinal growth was reduced, epiphysial plates bones begins about 100 days-of-age, with persisting is- closed prematurely, the pelvic bones were shorter, and lands of cartilage seen along their margins up to 170 their synchondroses disappeared (Hulth, 1958a). All days-of-age (Harrison, 1958). Hughes and Tanner cartilaginous growth plates did not display the same (1970, 1973) reported t h a t pelvic length in male rats level of susceptibility. Wider ones, such a s those of the was greater a t all ages and significantly so after 70 distal end of the femur and proximal tibia, showed a days-of-age. The velocity of growth in length peaked at more rapid longitudinal growth and higher resistance 50 days-of-age and the major sex difference was one of size and not of shape. The effects of undernutrition and subsequent rehabilitation on skeletal growth revealed that the length Received January 10, 1992; accepted June 24, 1992. of the skull and limbs recovered their normal size, but Address reprint requests to Dr. Andrew D. Dixon, Hilgard Avenue, the length of the spine and the dimensions of the pelvis School of Dentistry, 63-090 CHS, University of California Los Angedid not (Dickerson and Hughes, 1972). Bones that had les, Los Angeles, CA 90024-1668. 0 1993 WILEY-LISS, INC MORPHOMETRIC CHANGES IN RAT PELVIC GROWTH 313 to the effects of papain (Hulth, 1958b). The generalized skeletal stunting was equated with human achondroplastic dwarfism (Hulth and Westerborn, 1959). The work on epiphysial cartilage has pertinence for the study of skull growth. Irving and Ronning (1962) showed similarities in the responses by skull base synchondroses in rats. Ronning (1971) analyzed the effects of papain on various dimensions of the rat skull. There was a lag in longitudinal growth so that the skull became brachycephalic. A coincident increase in neurocranial width was hypothesized as a compensatory mechanism for decreased cranial base length. The findings on skull growth suggested that a morphometric study of the effects of papain on major components of the pelvic skeleton, where growth cartilages are a prominent feature, would contribute to our knowledge of the role of cartilage in pelvic growth. MATERIALS AND METHODS Ninety-eight 21-day-old Lewis-strain male rats, which were used concurrently for the effects of papain on craniofacial growth, were divided randomly into two groups and maintained on standard laboratory chow and water ad libitum until the papain regimen was begun. Thirty animals from Group I (n = 48) were given daily intraperitoneal injections of 2% crude papain (Type 1, activity 1-2 units/mg solid, Sigma Chemical Co., St. Louis, MO) in normal saline, 100 mg/kg, with a n injection volume of 0.5 m1/100g, from 25 to 40 daysof-age. The remaining 18 animals served as untreated controls. Papain-treated and control animals were euFig. 1 . Linear dimensions measured in the study. They are defined thanized by pentobarbital sodium, 100 mg/kg i.p. a t 40, in the text. 54 and 70 days-of-age. Thirty-five animals from Group I1 (n = 50) received daily intraperitoneal injections of 2% crude papain (Sigma Chemical Co., St. Louis, MO, (caudal) point on the ischial tuberosity of the same Type 1)from 25 to 40,54, and 70 days-of-age. The other side. 15 animals were the controls. Group I1 animals were H. Obturator-acetabulum distance: The distance beeuthanized at 26, 40, 54, 70, and 120 days. tween the most posterior (caudal) point on the obtuStandardized dorsoventral pelvic radiographs of all rator foramen and the deepest point in the acetabu98 animals were taken. A portable radiographic unit lum of the same side. (Oralix 70, Philips Medical Systems, Inc., Shelton, I. Obturator foramen length: The maximum anteroCT) and dental occlusal film (Eastman Kodak, Rochesposterior (rostro-caudal) dimension of the obturator ter, NY) were used. Radiographic settings were conforamen. stant (70 kVp, 5 mA), with a target-film distance of J. Obturator-ischial distance: The distance between 24.5 cm. Photographic enlargements ( x 4) were made the most posterior (caudal) point on the obturator and ten linear dimensions measured on each print (Fig. foramen and the most posterior (caudal) point of the 1): ischial tuberosity of the same side. The means of the data for each dimension were anA. Bi-iliac width: The distance between the most latalyzed statistically using Student’s t test for differences eral, everted points on the iliac crests. B. Iliac length: From the anterior (rostral) border of between papain-treated and control groups. Dried pelthe iliac crest to the midpoint of the ilio-ischial junc- ves from both experimental and control rats were prepared for gross morphological comparison. tion of the same side. C. Bi-acetabular width: The distance between the RESULTS deepest point in the concavity of each acetabular By 40 days-of-age, papain-treated animals had both fossa. D. Interobturator width: The distance between the shorter and narrower pelves and this general reduction most posterior (caudal) points on the obturator fo- in pelvic size was maintained throughout the experiment (Fig. 2). Superimposition of the pelvic outlines of ramina. E. Bi-ischial width: The distance between the most papain-treated and control animals at the level of the acetabulum showed the shorter ilium and ischium, pelposterior (caudal) points on the ischial tuberosities. F. Acetabular diameter: The maximum antero-pos- vic narrowing, and a lateral inclination or flaring of the ischial region (Fig. 3). This appearance was more terior diameter of the acetabulum. G. Ischial len&h: The distance between the midpoint pronounced in Group I1 rats that had received papain of the ilio-ischial junction and the most posterior for 45 consecutive days (Fig. 4). The treated animals 314 A. D. DIXON AND P. T. GAKUNGA Fig. 3. Superimposition of short-term papain-treated (shaded) and control (dotted outline) pelves at 54 days-of-age. The papain-treated pelvis is shorter, narrower, and the ischial tuberosities are flared outwards. Fig. 2. Dried pelves of papain-treated (right) and control rats (left) at 40, 54, and 70 days-of-age. The pelves of papain-treated rats are smaller and show a lateral flaring of the ischial bones. had a concave-shaped ischium, unlike the typically convex form of control ischia. Other features were a lateral displacement of the ischial tuberosities, a concavity of the caudal border of the ischial ramus, a more acute suprapubic angle, and a n increase in the subpubic angle. Tables 1 and 2 give the percentage changes and levels of significance for the ten pelvic dimensions measured in Group I and 11, respectively. Anteroposterior Dimensions By 40 days-of-age, iliac length was significantly shorter in the papain-treated animals (Figs. 2-4). Iliac length plotted against age showed a steady increase in the control length until 54 days-of-age, when i t began to level off (Fig. 5a). At 54 days-of-age iliac length was 12.34% shorter in Group I and more than 22% shorter in Group 11. The growth pattern for ischial length was very similar (Fig. 5b). By 54 days-of-age it was shorter by 7.47% in Group I and by about three times as much in Group I1 (Table 2). The antero-posterior diameter of the acetabulum in the papain groups was shorter a t first but then showed a small increase (2.65%) in Group I by 54 days-of-age, when the diameter was shorter by over 20% in Group 11. Obturator foramen length also was shorter at first but by 54 days-of-age was greater in Group I1 compared to the controls. At 70 days-of-age the length was shorter again by 10%. Transverse Dimensions Bi-iliac width in papain-treated rats was less than in controls a t all stages of the experiment; for example, by 4.86% in Group I and by 10% in Group I1 at 54 daysof-age (Tables 1, 2; Fig. 4). At 70 days-of-age, the narrowing was statistically significant in Group I1 (Table 2). Bi-acetabular width was affected significantly by papain only in Group 11, where it was 26% less than in the control a t 54 days-of-age (Fig. 5c). This was particularly evident when the superimpositions of dried pelves were compared (Figs. 3, 4). Interobturator width had increased by 5.67% and 6.48% in Group I a t 54 and MORPHOMETRIC CHANGES IN RAT PELVIC GROWTH , I ,--, \ 315 was a mirror image of the control curve, with a sharp drop in velocity between 40 and 54 days-of-age. Bi-ischial width growth rate peaked a t 54 days-ofage both in papain-treated and control animals (Fig. 5f). This was followed by deceleration in both groups up to 70 days-of-age, much more so in the controls. The velocity curve for bi-acetabular width in control rats was similar to the curve for iliac length, peaking at 54 days-of-age. The papain-treated animals showed a large drop in velocity a t this age but recovered almost to the control rate by 70 days-of-age. DISCUSSION Fig. 4. Superimposition of long-term papain-treated (shaded) and control (dotted outline) pelves a t 54 days-of-age. The papain-treated pelvis is considerably smaller than the Group I pelvis shown in Figure 3 and the subpubic angle is flattened. 70 days-of-age, respectively. The width was less in Group I1 rats a t these ages. In the short-term papain Group I, bi-ischial width increased significantly, by 42.32% and 35% at 54 and 70 days-of-age, respectively (Table 1,Fig. 5d). In Group I1 the increase was not significant a t 54 days-of-age but had increased by 18.51% at 70 days-of-age (Table 2). These changes were matched by the ischial flaring seen in dried specimens (Figs. 3 and 4). Other Dimensiohs At 40 days-of-age, the obturator-acetabulum distance was shorter in both experimental groups. The decrease at 70 days-of-age was 18.13% in Group I and 6.94% in Group 11. But in Group I1 this dimension was about 20% shorter at 120 days-of-age. The obturator-ischial distance increased by 59% in Group I and by about one third in Group I1 (Tables 1 and 2). At 70 days-of-age, the increase was 21.40% in Group I. In contrast, there was a significant decrease of 16.95% in Group 11. Rates of Growth Iliac length growth rate peaked at 54 days-of-age both in the controls and the papain-treated groups, with the control animals having the higher growth velocity (Fig. 5e). Ischial length rate peaked earlier, at 40 days-of-age, and the curve for papain-treated animals Dimensions were selected for measurement that would give maximum information about changes in length of the individual components of the innominate bone and alterations in pelvic width. A number of the dimensions related to the inner pelvis and were indicators of any altered morphology in and around the acetabular region, such a s might result from the effects of papain on the Y-shaped, triradiate growth cartilage, which unites the three parts of the hip bone and is synchondrosal in nature. We were aware that papain affects the length of the cranial base through interference with endochondral bone formation at its synchondroses (Dixon et al., 1987). Both the amount and rate of stunting of pelvic dimensions were clearly dependent on whether papain was given over the shorter or longer period, with the more pronounced effects usually resulting from prolonged administration. Comparative information from previous studies is limited. Hulth (1958a) gave papain intravenously to rabbits up to four times over a 2-week period and observed in whole-body radiographs that the synchondroses between the separate pelvic bones had disappeared. Although he did not comment on it, shortening and flaring of the ischial region is obvious in one of his radiographs. He showed that pelvic length in papain-treated rabbits was significantly shorter, by 9.4-28.5%, depending on the initial age and length of the follow-up period (Hulth, 1958b). Our short-term data, when iliac and ischial lengths were summed, were in the same range. There was a conspicuous difference in the size of our dried pelvic specimens between the papain-treated and control animals. As expected, the reduction in the overall size of the pelves was more pronounced in Group 11. The retardation in growth caused by papain was evident not only in the anteroposterior direction, but in most transverse measurements as well. This emphasized the dramatic effects of papain on pelvic skeletal dimensions. Even though some measurements were affected more than others, in keeping with earlier reports of the effects of nutritional deficit on the skeleton (Hughes, 1982), systemically administered papain apparently influenced all the cartilaginous areas of the pelvis to some degree, contributing in toto to the size reduction. The ratio of ischial to iliac length was constant throughout the period of the experiment, a s were ratios for most of the transverse dimensions (bi-iliac to biacetabular width, bi-iliac to bi-ischial width), both for the papain-treated and control animals. This was consistent with results reported by Harrison (1958a1, who 316 A. D. DIXON AND P. T. GAKUNGA TABLE 1. Dimensional changes in Group I (P< 0.001, unless indicated), expressed as a percentage decrease or increase ( +) compared with the controls Iliac length Ischial length Acetabular diameter Obturator foramen length Bi-iliac width Bi-acetabular width Interobturator width Bi-ischial width Obturator-acetabular distance Obturator-ischial distance *P<0.05; n.s. **P<O.Ol. = 40 12.69 13.05 6.31** 6.37 7.17** 7.69 n s . 12.11 n.s. 3.82 n s . 9.16** 1.14* Days-of-age 54 12.34** 7.47 n.s. + 2.65 n.s. + 2.89 n.s. 4.86 n.s. 1.93 n.s. + 5.67 n s . +42.32** 9.45* + 59.00 70 11.76 10.08 + 1.97 n.s. 15.47 0.75 n.s. 1.01 n.s. + 6.48* + 35.00 18.13 +21.40 not significant. TABLE 2. Dimensional changes in Group I1 (P< 0.001, unless indicated), expressed as a percentage decrease or increase ( + ) compared with the controls Davs-of-aee Iliac length Ischial length Acetabular diameter Obturator foramen length Bi-iliac width Bi-acetabular width Interobturator width Bi-ischial width Obturator-acetabular distance Obturator-ischial distance *P<0.05; n.s. **P<0.01. = 40 12.69 13.05 6.31** 6.37 7.17** 7.69 n s . 12.11 n.s. 3.82 n.s. 9.16** 1.14* 54 22.66 23.12** 21.19* + 14.50** 10.00 26.04 3.25* 0.90 n.s. 14.31** +31.35 70 23.36** 18.28** 9.46** 10.50** 17.67 24.88 10.57** + 18.51 6.94* 16.95** 120 16.73 i9.76 + 8.29 44.40** 8.03* 18.35 18.35 + 4.70* 21.35* 3.50 n.s. not significant. opined that pelvic growth was regulated by general growth factors. Further experiments are required to differentiate the specific contributions to pelvic growth made by various cartilaginous growth sites, particularly in the acetabular region. Administration of papain directly into selected areas, such a s the hip joint cavity and the vicinity of the triradiate cartilage, would be two possibilities. Tingey and Shapiro (1982) injected papain unilaterally into the temporomandibular joint of growing rabbits and found a significant decrease in vertical growth of the mandibular ramus, as a result of inhibited cartilaginous bone growth at the condyle. Cuenin, Dixon, and Hoyte (1991) showed that localized injection of papain a t the septosphenoidal synchondrosis in the cranial base of young rats, a bone-cartilage junction whose structure is comparable to a pelvic growth cartilage, caused shortening of the snout. This demonstrated the importance of a specific region of the cranial base for facial growth. The outward displacement of the ischial tuberosities that gave the ischial rami a laterally concave form in papain-treated rats was confirmed by the measured increase in bi-ischial width. This consequence may have been a compensatory mechanism to maintain the normal volume of the lesser pelvis despite the reduction in ischial length. It is assumed that papain did not affect the development and growth of the soft tissues (e.g., the genito-urinary system) bounded by the ischial bones. If the normally expanding pelvic contents led to ischial widening, this has its parallel in the functional matrix concept used to explain growth of the craniofacial complex (Moss, 1962). Growth of soft tissues such as the brain and eyeball seem to dictate three-dimensional growth of surrounding bones. Similar conclusions have been drawn with respect to the growth of the neurocranium following papain administration. Volumetric requirements of the growing brain in brachycephaly induced by papain seemed to dictate a compensatory increase in skull width and height (Ronning, 1971; Schramm et al., 1974; Kvinnsland, 1974a,b). Further study to compare the volume of the lesser pelvis before and after papain injection would help clarify this hypothesis for pelvic growth. There are other possibilities. The divergence of the ischial bones may have been a direct result of disruption of the triradiate cartilage by papain that, for example, could prompt a reduction in bone formation on the lateral aspect of the acetabular region. If such a restriction in cartilage growth occurred it conceivably could lead to “constriction” of acetabular growth, by inhibition of either the triradiate or cup-shaped parts of the acetabular cartilage, or both. Atypical differential growth within its different parts could lead to a n everted ischium. 317 MORPHOMETRIC CHANGES IN RAT PELVIC GROWTH 30 22 1 T h E E v 5 m 20 - C 4 --O- 1 0 1 . I . I . I . I 80 60 40 Controls Papain . I 100 120 . I 20 140 40 60 80 100 120 140 Age (days) 22 7 m- - 18 1 20 - 18 h h E 16V 5 22 - - 12 lo 8 1 16 - 5 14- is Controls Papain . E v . - d P 14: * Controls 1 20 . 1 40 . I 60 . I . 80 I 100 Papain . , 120 12 . , 140 - 101 30 . , . I 40 '. 50 Age (days) I . . I 70 60 I 80 Age (days) 500 500 f * Controls 400 mx U h s - Papain 300 a, m U r, 2W s 100 0 I 20 40 60 80 100 120 140 Age (days) 1 0 40 60 80 100 120 140 Age (days) Fig. 5. Dimensional changes and growth rates in papain-treated and control rats over time. a, iliac length; b, ischial length; c, bi-acetabular width; d, bi-ischial width (short-term, Group I); e, iliac length growth rate; f, bi-ischial growth rate. In a d , vertical bar ? S.E. Again, the influence of papain would not have excluded the cartilage of the fermoral head. If it were smaller in papain-treated rats, then this in turn would lead to a smaller acetabulum, with the ultimate effect on ischial position we found. While we did not measure the femoral head, our data showed that the diameter of 318 A. D. DIXON AND P. T. GAKUNGA the acetabulum actually increased by a small amount initially, then decreased and increased again by more than 8% at the end of the long-term (Group 11) experiment. However, the femoral head still may be a prime contributing factor, for Harrison (1961) removed one or both heads in rats and found the innominate bones became laterally concave and medially rotated around their long axes. He concluded that the phenomenon was due to the collapse of the acetabular cup, possibly leading to a directional change in the growth of the more medially placed triradiate cartilage. Our initial histological findings show significant changes both in the thickness and internal structure of the acetabular cartilage, which indicate its probable demise as a growth site after papain is administered. The superimposition of the outlines of short-term papain-treated and control pelves at 54 days-of-age is remarkably similar to a text-figure in Harrison’s (1961) study, which showed superimposed tracings of normal and experimental pelves 144 days after bilateral removal of the femoral heads. The flaring and shortening of the ischia is virtually identical in both studies. Thus, the overall effects of papain may equate with acetabular dysplasia and lend support to Harrison’s contention that normal acetabular development and growth depends on a normally placed and growing femoral head. Finally, our results suggest that besides giving clues to normal growth processes, the papain model provides another valuable method for study of the effects of cartilage dysplasia a t the hip joint, devoid of the variables that surgical trauma may introduce (Kuba et al., 1985). ACKNOWLEDGMENTS We wish to thank Ms. Irene Petravicius for the line drawings and Mr. Richard Friske and Ms. B. J. Coburn for their photographic skills. The study was supported by a grant from the USPHS, RR-05304. LITERATURE CITED Cuenin, R.M., A.D. Dixon, and D.A.N. Hoyte 1991 The septosphenoidal junction and midface growth in young rats. J . Dent. Res., 70:360 (Abstract). Dickerson, J.W.T., and P.C.R. Hughes 1972 Growth of the rat skeleton after severe nutritional intrauterine and post-natal retardation. Resuscitation 1:163-170. Dickerson, J.W.T., P.C.R. Hughes, and P.A. McAnulty 1972 The growth and development of rats given a low-protein diet. Br. J . Nutr., 27:527-536. Dixon, A.D., M.R. Ricupito, P.J. Styrt, and M. Bjornbach 1987 Effects of prolonged papain administration on craniofacial growth of rats. In: Proceedings of the VIIth European Anatomical Congress. Acta Anat., 130t26 (Abstract). Harrison, T.J., 1958a The growth of the pelvis in the rat: A mensural and morphological study. J . Anat., 92:236-260. Harrison, T.J. 195813 An experimental study of pelvic growth in the rat. J. 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