Craniofacial growth in growth hormone-deficient rats after growth hormone supplementation

ORIGINAL ARTICLE

Craniofacial growth in growth hormone-deficient rats after growth hormone supplementation Douglas A. Singleton,a Peter H. Buschang,b Rolf G. Behrents,c and Robert J. Hintond Acworth, Ga, Dallas, Tex, and St Louis, Mo Introduction: Growth hormone (GH) supplementation is an established therapy to increase stature in GH-deficient or short-for-age children, but comparatively little is known of its effects on the craniofacial skeleton. Methods: Using a mutant strain of Lewis rats (dw/dw) in which GH levels were 6% to 10% of normal, but other trophic hormones were unaffected, we investigated the differential susceptibility of craniofacial measures to GH supplementation, characterized their potential for partial or complete catch-up growth, and compared their growth changes with those in long bones. At 24 days of age and for 3 subsequent weeks, radiographs of the lateral head, upper limb, and lower limb were obtained from 3 groups of growing rats (n ⫽ 8-9 in each group): dwarf (experimental) with GH injection, dwarf (sham) with vehicle injection, and wild type (control) with vehicle injection. The x-ray images were scanned, standardized points digitized, and linear distances measured. Absolute growth curves were generated for each group by using multilevel modeling procedures and iterative generalized least-squares curve fitting. Results: For every measure, growth differences were evident between the experimental and the sham groups, but the treatment effect varied inversely with relative maturity of the measure. Although all craniofacial measures showed some catch-up growth, only 31% of craniofacial measures had complete catch-up compared with all limb measures. The percentage of catch-up varied inversely with relative maturity of the measure. Our results suggest that the effects of GH supplementation vary considerably, so that measures with the lowest relative maturity (greatest baseline potential) show the greatest treatment effect and catch-up, whereas more mature measures show less growth response to GH replacement. Conclusions: These results suggest that, depending on the timing of GH supplementation, there is potential for change in proportions or shape of the craniofacial complex. (Am J Orthod Dentofacial Orthop 2006;130:69-82)

C

hildren with various forms of growth hormone (GH) deficiency or other defects in the GH signal-transduction pathway (eg, GH receptor insensitivity or Laron syndrome) typically do not reach their full potential of size and stature.1 Untreated GH-deficient children end up some 6 SD below the a

Private practice, Acworth, Ga. Professor and director of research, Department of Orthodontics, Baylor College of Dentistry, Texas A&M University System Health Sciences Center, Dallas, Tex. c Chair and director, Center for Advanced Dental Education, St Louis University, St Louis, Mo. d Professor and vice chair, Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M University System Health Sciences Center, Dallas, Tex. Winner of the 2004 Milo Hellman Award. Supported by a grant from the American Association of Orthodontists Foundation to the fourth author and by intramural funds from Baylor College of Dentistry and the Department of Orthodontics to the first author. Reprint requests to: Robert J. Hinton, Department of Biomedical Sciences, Baylor College of Dentistry, 3302 Gaston Ave, Dallas, TX 75246; e-mail, [email protected]. Submitted, July 2004; revised and accepted, February 2005. 0889-5406/$32.00 Copyright © 2006 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2005.02.016 b

population mean for height.2,3 Since GH has become more abundant via recombinant DNA techniques, GH supplementation is now an accepted and efficacious therapy to increase stature in GH-deficient children.4,5 Because GH supplementation for short normal children is also becoming more common,6-8 it has been estimated that there are more than 1 million potential GH-therapy candidates in the United States alone.9 Whereas the effects of GH on long-bone growth and stature are relatively well understood, appreciably less is known of the effect of GH supplementation on bone growth in other regions of the body, such as the craniofacial skeleton. Although an association between craniofacial and somatic development has been clearly established,10,11 it has been shown that GH has a less pronounced effect on craniofacial growth than on height or skeletal maturation.12 Understanding the differential effects of GH on craniofacial and somatic growth is crucial, because growth of the craniofacial region is much more complex than that of most long bones. Unlike long bones, growth of the craniofacial region involves the interaction of a mosaic of adjacent 69

70 Singleton et al

growth sites, each with a different pattern and timing of growth. Nanda10 suggested that the splanchnocranium and neurocranium follow skeletal and neural growth curves, respectively, with the cranial base a composite of the 2. Building on the concept of growth maturity gradients in the extremities proposed by Tanner,13 Buschang et al14 postulated that the craniofacial complex expresses a continuous pattern of variation in maturity, or differential growth, and called this a craniofacial growth maturity gradient. This variation in maturity is manifest between structures as relative maturity (ie, percentage of adult status attained) for a particular structure at a given time. In this context, less mature structures would be expected to grow more rapidly at a given time than more mature structures. Likewise, insults or therapy might produce differing results based on the relative maturity of the structure. This concept, although frequently mentioned in the literature, has not been extensively evaluated in an experimental context.13,15 Children with GH deficiency exhibit overall reductions in cranial base size and small mandibles,12,16-19 with steep mandibular plane angles, a result of smaller posterior face heights.19,20 Several studies have shown that craniofacial measures differ in their potential for retardation with GH deficiency and in their potential for catch-up after GH therapy.12,15 Because human studies can be limited by small sample sizes, cross-sectional designs, uncontrolled variables, and often retrospective nature, animal models have been used to yield more rigorous analyses. Recently, our laboratory showed that the growth of various craniofacial dimensions in mutant (dwarf) Lewis rats with constitutively low levels of GH was inversely correlated with the relative maturity of the dimension.21 Despite the relatively widespread use of GH to augment stature, no study to date has systematically examined the interaction of GH supplementation with the growth potential of various craniofacial measures. Building on the findings of our previous work, we used the Lewis dwarf rat to analyze the differential susceptibility of craniofacial measures to stimulation by GH, characterized their potential for partial or complete catch-up growth, and compared their growth changes with those in long bones.21 MATERIAL AND METHODS

Female GH-deficient (dw/dw) Lewis rats were obtained from a colony at Harlan-Olac Ltd. (Blackthorn, Bicester, United Kingdom); female outbred Lewis rats from Harlan (Indianapolis, Ind) served as age-matched controls. The mutation, an autosomal recessive that arose spontaneously in a breeding colony of Lewis rats

American Journal of Orthodontics and Dentofacial Orthopedics July 2006

in Oxford, United Kingdom, causes GH to be secreted at 6% to 10% of normal.22 Levels of all other pituitary hormones are normal. The probable cause of this reduced GH synthesis is a defect in signal transduction in the GH-releasing pathway.23 These mutants show retarded growth so that at 3 months of age their weight is approximately 40% less than that of their wild-type littermates. No radiographically visible skeletal abnormalities have been documented in these rats. Supplementation with exogenous GH or insulin-like growth factor-1 (IGF-1) corrects the growth deficiency.24 In recent years, this strain of GH-deficient rat has been used to study the effects of GH on bone strength,25 structure and contractility of the heart muscle,26 organ growth,27 and growth and fiber type composition of skeletal muscle.28 All animals were housed in the Baylor College of Dentistry Animal Resource Unit in climate-controlled rooms equipped with a light-dark cycle and an automatic watering system. The mutants were randomly assigned to the following experimental groups: dwarf plus vehicle (n ⫽ 9, sham), dwarf plus recombinant human growth hormone (rhGH) (n ⫽ 8, experimental), and wild type plus vehicle (n ⫽ 9, control). Beginning at approximately 24 days of age, all animals were weighed and x-rayed weekly for 3 weeks. The experimental group was given subcutaneous injections of rhGH (Genentech, San Francisco, Calif), and the sham and control groups received injections of vehicle (bacteriostatic water containing 0.9% benzyl alcohol) based on body weight. To simulate pulsatile release that occurs in vivo, the rhGH was administered twice daily (0700 and 1630) in equal doses,29-31 for a total dose of 2 mg per kilogram per day of body weight subcutaneous.26,27 This regimen continued for 21 days. The doses of rhGH ranged from 0.092 mg per day at the beginning of the experiment to 0.316 mg per day at the end based on daily monitoring of body weight. At the end of the experimental period the rats were killed by intraperitoneal injections of Nembutal (Abbott Laboratories, Chicago, Ill). At baseline (T0) and after days 7, 14, and 21(T1, T2, and T3), rats were anesthesized with intramuscular injections of 1 mL per kilgram of 1:10 xylazine: ketamine and radiographs were taken. At each time, a lateral head radiograph, an upper limb radiograph, and a lower limb radiograph were taken. All radiographs were taken with the same standard dental unit, with machine settings of 10 mA at 55 kV for 18/60 seconds at a distance of 10 cm from the film for the lateral cephalometric films with reductions to 8/60 seconds and 7.5 cm for the limb films. A 10-mm steel calibration rod was incorporated into the clear acrylic table on which the animals were positioned for the radiographs.

American Journal of Orthodontics and Dentofacial Orthopedics Volume 130, Number 1

Table I.

Cephalometric and limb-point definitions

Sagittal radiograph N: most anterior point on nasal bone E: most inferior point of frontal bone at location of frontal sinus Po: most posterior and superior point on skull Ba: most posterior and inferior point on occipital condyle Co: most posterior and superior point on the mandibular condyle Go: most posterior point on mandibular ramus Mn: junction of mandibular ramus and corpus Gn: most inferior point on ramus that lies on perpendicular bisector of line Go-Mn Il: most anterior and superior point on mandibular corpus superior to mandibular incisors So: intersection of most anterior tympanic bulla and superior border of sphenoid bone CB1: most anterior point on occipital bone at spheno-occipital synchondrosis CB1=: most posterior point on sphenoid bone at spheno-occipital synchondrosis CB2: most anterior point on sphenoid bone at sphenobasispheno synchondrosis CB2=: most posterior point on basisphenoid bone at sphenobasispheno synchondrosis Ml: junction of alveolar bone and mesial surface of mandibular first molar Mu2: junction of alveolar bone and distal surface of maxillary third molar Mu1: junction of alveolar bone and mesial surface of maxillary first molar Iu: most anterior-inferior point on maxilla posterior to maxillary incisors Tibia Tp: most proximal point on tibial epiphysis Td: midpoint of width on distal surface of tibia Femur Fp: deepest midpoint of proximal head and lesser trochanter Fd: most distal point of femur Humerus Hp: most proximal point on humeral epiphysis Hd: midpoint of width on distal surface of humerus Radius Rp: midpoint of width on medial head Rd: midpoint of styloid process Ulna Up: most prominent point of olecranon Ud: midpoint of styloid process

The lateral films used a 3-point positioner for the skull, and fixed holders and midlimb rubber bands were used for the limbs to ensure complete film contact, orientation, and position. Ultra-speed DF-50 occlusal film (Kodak, Rochester, NY) was used for all radiographs. All radiographs were developed and scanned at high resolution (300 dpi) by the same operator (D.A.S.). The computer software used for digitization and measurement was Viewbox (D. Halazonetis, Hellas, Greece). Three custom analyses were constructed for this experiment: lateral cephalometric, forelimb, and hindlimb. The cephalometric landmarks (Table I, Fig 1)

Singleton et al 71

were derived from previous studies on rodents.21,32,33 To ensure measurement reliability and replicability, all radiographs were digitized twice at a minimum of 3 days apart, and the average measurement value was used for data analysis. From each radiograph type, subsets of 10 films were randomly chosen and digitized (10 days later) an additional 2 times. These last digitizations were averaged and compared with the average value of the first digitizations. Method error was then computed for each measurement and is given with the measurement in Table II. With the MLwiN software (Centre for Multilevel Modeling, London, England), multilevel statistical models were used to describe longitudinal absolute growth changes and statistically evaluate group differences.34-36 The multilevel models were composed of 2 parts: a fixed part and a random part. The fixed part of the model estimated the polynomial parameters describing the growth curves of the various craniofacial measures. The random part of the model estimated variation at 2 hierarchical levels, with 1 level nested within the preceding level. The 2 levels pertained to random variations between animals and between repeats within animals. Iterative generalized least-squares methods were used to estimate the parameters. To assess differences in relative maturity among measures at the start of the experiment, ranked relative growth curves (Fig 2) were constructed for each measurement by calculating the percentage of adult status at each time interval, with adult status defined as the measurement size at the final time interval (about 42 days). Relative maturity of each measurement was defined as (T0/T3) ⫻ 100—ie, percentage of T3 growth completed by the start of the experiment (T0) (Table III). Measures with low relative growth for the experiment had high relative maturity, and vice versa. RESULTS Deficiency effect: control (wild type) v sham (dwarf)

Most (69%) of the craniofacial growth curves (derived from Table IV) in the control group were linear, increasing steadily from T0 to T3. All mandibular and neurocranial curves except total skull length (Po-N) and total cranial base length (Ba-E) were linear. Among the craniofacial control measures (Table III), absolute linear growth over the 21 days ranged from a low of 0.34 mm (M1-I1, anterior corpus length) to a high of 61 mm (Po-N, total skull length). Over the same time period, absolute limb increases ranged from a high of 7.49 mm (tibial length) to a low of 4.19 mm in radial length. Twelve measures, including all limb measures, body weight, and all viscerocranial measures except

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American Journal of Orthodontics and Dentofacial Orthopedics July 2006

Fig 1. Location of cephalometric points on radiographs: A, sagittal; B, hindlimb; C, forelimb. Table II.

Measurements of craniofacial skeleton and limbs (with method error)

Neurocranium Po-N: total skull length (0.184) Po-E: cranial vault length (0.196) Ba-E: total cranial base length (0.121) So-E: anterior cranial base length (0.123) Ba-CB1: occipital bone length (0.148) CB1=-CB2: sphenoid bone length (0.130) Ba-So: posterior cranial base length (0.171) Po-Ba: posterior neurocranium height (0.185) Viscerocranium E-N: nasal length (0.162) Mu2-Iu: palate length (0.132) Cb2-Iu: midface length (0.183) E-Mu1: viscerocranial height (0.136) Mandible Go-Mn: posterior corpus length (0.166) Ml-Il: anterior corpus length (0.124) Co-Il: total mandibular length (0.201) Co-Gn: ramus height (0.199) Other Tibia length (0.104) Femur length (0.111) Humerus length (0.110) Radius length (0.101) Ulna length (0.104) Body weight (NA)

palate length (Mu2-Iu), showed varying expressions of deceleration. Total skull length (Po-N) and nasal length (E-N) had the greatest initial increases followed by a clear plateau (deceleration) in the last 2 intervals. In the

remaining measures (total cranial base length [Ba-E], midface length [Cb2-Iu], viscerocranial height [E-Mu1], and all limb measures), growth velocity began to decrease only slightly by T3. Absolute linear increases occurred in all sham measurements, yet in every instance absolute growth in the sham group was less than in the control group. Additionally, differences in growth velocity between the sham and control groups were found for all measurements (Table IV). Three basic patterns characterized the differences with the control group: size, growth, and size ⫹ growth. Samples of each type are shown in Figure 3. Five measurements showed only size differences, so that the growth curves between sham and control were essentially parallel (size difference). The size difference measures (Fig 3, A) showed no significant effect of GH on growth velocity, but a significant size difference was evident at the first time interval. The size difference measures included: ramus height (Co-Gn), anterior corpus length (Ml-Il), occipital bone length (Ba-Cb1), posterior neurocranial height (Po-Ba), and total mandibular length (Co-Il). The second pattern was characterized by growth curves of the sham and control groups that became increasingly divergent over time (growth difference). For these measures (Fig 3, B), there was a significant effect of GH on growth velocity but no difference in size at the first interval. The growth difference measures included nasal length (E-N), sphenoid length (Cb1=-Cb2), humerus and femur lengths, and body weight. Roughly

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American Journal of Orthodontics and Dentofacial Orthopedics Volume 130, Number 1

Ranked Relative Grow th Curves (Control)

P ercent M aturity

85

75

65 T0

T1

T2

T3

P e rc e n t M a t u rit y

95

95

85

75

B

65 T0

T1

T2

T3

Po-E Po-Ba M1-Il So-E Mu2-Iu Ba-E E-Mu1 Ba-So Po-N Co-Il Cb2-Iu Cb1-Cb2 Ba-Cb1 Humerus Radius E-N Ulna Go-Mn Tibia Femur Co-Gn

Interval

Figure 2c. Ranked Rela tive Grow th Curves (Expe rime nta l) 105

Po-E M1-Il Po-Ba So-E Mu2-Iu Ba-E Ba-Cb1 Ba-So E-Mu1 Po-N Cb2-Iu Co-Il Cb1-Cb2 Radius Co-Gn Humerus Ulna Tibia E-N Femur Go-Mn

95

Percent Maturity

Po-Ba M1-Il Po-E So-E Co-Il Ba-E Mu2-Iu Po-N Ba-Cb1 Cb2-Iu E-Mu1 Ba-So Cb1-Cb2 Humerus Co-Gn Ulna Radius E-N Tibia Go-Mn Femur

105

A

Ranked Relative Growth Curves (Sham) 105

85

75

65

C

T0

T1

T2

T3

Interval

Interval

Fig 2. Relative growth curves for craniofacial variables: A, control group; B, sham group; C, experimental group. Relative maturity was calculated as (value at T0 divided by value at T3) x 100.

half of the craniofacial measurements exhibited both size and growth differences (size ⫾ growth difference). These measurements (Fig 3, C) displayed significant group differences in growth velocity and significant size differences at the first time interval. The size ⫹ growth measures included cranial vault length (Po-E), total cranial base length (Ba-E), anterior cranial base length (So-E), total skull length (Po-N), posterior cranial base length (Ba-So), midface length (Cb2-Iu), viscerocranial height (E-Mu1), palate length (Mu2-Iu), posterior corpus length (Go-Mn), and radius, ulna, and tibia lengths. Considerable variation in the relative maturity of the control rats was evident at the first interval (Table III; Fig 2, A), ranging from 27% (weight) to 94% (posterior neurocranial height, Po-Ba) of adult size. Measures above 90% were considered mature, whereas those from 65% to 89% were considered immature. Craniofacial measures had attained an average relative maturity of 84% with regional differences (neurocranial, 87%; mandibular, 83%; viscerocranial, 83%). All craniofacial measurements were more mature than

those of body weight and limbs. All craniofacial measurements had relative maturity of at least 81% with 3 notable exceptions: ramus height (Co-Gn) (77%), nasal length (E-N) (75%), and posterior corpus leng th (Go-Mn) (71%). Weight was the least mature (27%), whereas limb measurements ranged from 68% in the femur to 79% in the humerus, with a limb composite relative maturity of 74%. For craniofacial measures, there was an overall trend of higher maturity for cranial vault/cranial base (neurocranial) measures and lower maturity for facial (viscerocranial and mandible) measures. Most notably, 3 neurocranial measures (cranial vault length [Po-E], anterior cranial base length [So-E], and posterior neurocranial height [Po-Ba]) were more than 90% mature, whereas the average facial measurement was relatively immature at 83%. Nonetheless, clear exceptions to this pattern were evident in the low maturity (81%) in sphenoid length (Cb1=-Cb2) and the relatively high maturity (94% and 88%) in 2 facial measurements: anterior corpus length (Ml-Il) and total mandibular length (Co-Il), respectively. Without exception, for all

74 Singleton et al

Table III.

American Journal of Orthodontics and Dentofacial Orthopedics July 2006

Absolute growth and relative maturity for all measures Absolute growth (mm)

Neurocranium Po-N Po-E Ba-E So-E Ba-Cb1 Cb1=-Cb2 Ba-So Po-Ba Average Viscerocranium E-N Mu2-Iu Cb2-Iu E-Mu1 Average Mandible Go-Mn M1-I1 Co-I1 Co-Gn Average Limb Radius Ulna Humerus Tibia Femur Average Weight (grams)

Relative maturity (T0/T3)*100

Control

Exper

Sham

Pattern of difference

Control

Exper

Sham

6.10 2.24 3.29 1.27 1.33 1.29 1.85 0.48

7.73 2.88 3.77 1.48 1.25 1.56 1.82 1.07

4.43 1.35 2.35 0.86 1.24 0.86 1.10 0.55

S S S S

85.07 91.18 88.05 90.33 84.60 80.80 83.13 94.53 87.21

81.21 88.47 85.87 87.92 84.77 76.97 82.32 88.13 84.46

88.33 94.22 90.58 92.63 84.69 86.20 88.67 93.53 89.86

3.94 2.20 3.45 1.65

4.91 2.39 4.19 1.72

3.05 1.37 2.70 0.97

G S⫹G S⫹G S⫹G

75.19 88.00 84.38 83.34 82.73

70.21 86.50 80.29 81.66 79.66

79.60 91.79 86.29 88.85 86.63

2.82 0.34 2.48 2.12

3.57 0.59 4.15 2.62

1.98 0.34 2.48 1.97

S⫹G S S S

71.39 93.70 88.22 77.31 82.65

65.01 88.48 80.22 71.40 76.28

76.21 93.11 87.33 75.78 83.11

4.19 4.82 4.21 7.49 6.88

4.21 5.80 5.57 8.08 6.91

2.98 4.05 3.47 5.84 5.05

S⫹G S⫹G G S⫹G G

95.16

50.15

G

75.89 76.41 79.13 71.96 68.02 74.28 27.85

76.15 71.77 73.04 70.34 67.49 71.76 33.96

81.61 78.76 81.79 75.64 74.02 78.36 47.23

112.27

⫹ ⫹ ⫹ ⫹ S G S⫹ S

G G G G

G

Exper, Experimental; S, size; G, growth.

groups, all forelimb measurements were clearly more mature than any hindlimb measurement. In the sham group, a similar craniofacial maturity gradient of neurocranial (90%), viscerocranial (87%), and mandible (83%) was again seen, although the range of values was much smaller than in the control group. The relative maturity values ranged from 94% (Po-E) to 76% for ramus height (Co-Gn), compared with 95% to 71%, respectively, in the control group (Fig 2, A). In the sham group, relative maturity was greater for almost every variable compared with the control group. The growth curves clustered between 85% and 95%; the only 3 outliers were nasal length (80%), posterior corpus length (76%), and ramus height (76%) (Fig 2, B). A regression of deficiency effect (degree of separation of paired slopes of control and sham over time) on relative maturity (Fig 4) clearly shows a negative correlation (r ⫽ ⫺0.58, P ⬍.01). As the relative maturity of a measurement increased, the degree of deficiency effect decreased, and vice versa.

Treatment effect: sham (dwarf) v experimental (GH-injected dwarfs)

In the experimental group, the number of craniofacial measures that showed varying expressions of a quadratic curve was similar to that in the controls (Table V), but the areas of expression were different. In the experimental group, most neurocranial measures were quadratics, whereas all viscerocranial and mandibular curves were linear except for midfacial length (Cb2-Iu); in the control sample, this trend was reversed. Among the craniofacial measures, absolute linear growth for the experimental measures ranged from 0.59 mm for anterior corpus length (Ml-Il) to 7.73 mm for total skull length (Po-N) (Table III). Over the same time period, the experimental absolute limb increases ranged from 4.21 mm (radius length) to 8.08 mm (tibia length). With only 2 exceptions— occipital length (Ba-Cb1) and posterior cranial base length (BaSo)—absolute growth in the experimental group exceeded that in the control group.

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American Journal of Orthodontics and Dentofacial Orthopedics Volume 130, Number 1

Table IV.

Multilevel models: control v sham Control estimates

Neurocranium Po-N Po-E Ba-E So-E Ba-Cb1 Cb1=-Cb2 Ba-So Po-Ba Viscerocranium E-N Mu2-lu Cb2-lu E-Mu1 Mandible Go-Mn M1-Il Co-Gn Co-I1 Limb measurements Radius Ulna Humerus Tibia Femur Weight

Sham group differences

Constant

Linear

Quadratic

Constant

Linear

Quadratic

34.769 (.233) 23.213 (.102) 24.19 (.146) 11.843 (.068) 7.33 (.103) 5.41 (.047) 9.104 (.106) 8.351 (.084)

3.160 (.203) .748 (.035) 1.379 (.112) .424 (.023) .441 (.031) .428 (.016) .62 (.034) .172 (.026)

–.375 (.065)

–1.354 (.0329) –1.250 (.145) –1.539 (.2) –1.083 (.096) –.505 (.032) –.069 (.066) –.445 (.149) –.332 (.106)

–1.125 (.291) –.299 (.05) –.312 (.063) –.131 (.034)

.188 (.094)

11.901 (.18) 16.192 (.135) 18.593 (.107) 8.27 (.052)

2.445 (.179) .709 (.063) 1.566 (.054) .699 (.048)

–.377 (.057)

–.028 (.253) –.9 (.192) –1.587 (.15) –.0536 (.07)

–1.244 (.257) –.255 (.09) –.248 (.03) –.217 (.027)

7.029 (.154) 5.061 (.034) 7.237 (.12) 18.578 (.134)

.938 (.058) .115 (.009) .683 (.03) .827 (.041)

–.695 (.218) –.457 (.045) –1.102 (.159) –1.45 (.169)

–.281 (.083)

13.091 (.127) 15.504 (.154) 15.825 (.149) 19.181 (.247) 14.613 (.231) 43.253 (1.794)

1.771 (.065) 2.067 (.085) 1.985 (.105) 2.956 (.176) 2.904 (.171) 5.899 (.132)

–.519 (.178) –.546 (.215) –.345 (.206) –1.056 (.341) –.286 (.318) 5.996 (2.464)

–.299 (.036) –.285 (.047) –.189 (.059) –.55 (.098) –.606 (.095) .3 (.177)

–.094 (.035)

–.139 (.017) –.053 (.015)

–.12 (.02) –.144 (.026) –.213 (.032) –.154 (.054) –.205 (.053) –.023 (.006)

–.143 (.023) –.251 (.05)

.315 (.083)

–.06 (.008)

Po-NControl ⫽ 34.769 ⫹ (3.160 ⫻ age) ⫹ (–3.75 ⫻ age ). Po-Nsham ⫽ (34.76 – 1.35) ⫹ [(3.160 – 1.125) ⫻ age] ⫹ [(–.375 ⫹ .188) ⫻ age2]. 2

In every instance, absolute growth was greater in the experimental group than in the sham group. At T0, there were insignificant size differences, yet throughout the experiment significant differences in growth velocities were seen for all (cranial and postcranial) measurements. In every case, but to varying degrees, growth differences were evident between the 2 groups (Table III). As with the control and sham groups, regional differences again were evident as average experimental relative maturity in the neurocranial measurements (84%) outpaced viscerocranial (80%) and mandibular (76%) averages (Table III). The range of maturity values was intermediate between the control and sham groups, but overall less mature than either group. Among craniofacial measures, relative maturity in the experimental group ranged from 88% (cranial vault length, Po-E) to 65% (posterior corpus length, Go-Mn). However, a striking exception to the high maturity rates in the neurocranium was sphenoid length (Cb1=-Cb2), which was 77% mature. With regard to neurocranial and mandibular measurements, nasal length (E-N) and posterior corpus length (Go-Mn) were much less mature (70% and 65%) than their respective regional

averages. Except for occipital length (Ba-Cb1), the experimental group was relatively less mature than the control group for every measurement (Fig 2, C ). A regression of experimental treatment effect (degree of separation of paired slopes experimental and sham over time) on relative maturity (Fig 5) clearly shows a negative correlation (r ⫽ ⫺0.45, P ⬍.05). As the relative maturity of a measurement increased, the treatment effect decreased, and vice versa. Catch-up growth: control (wild-type) v experimental (GH-injected dwarfs)

Only 5 (31%) of the experimental craniofacial measures displayed complete catch-up: ie, had attained or exceeded the control value by T3 (Fig 6, Tables VI and VII). By T2, sphenoid length (Cb1=-Cb2), posterior neurocranial height (Po-Ba), posterior corpus length (Go-Mn), and nasal length (E-N) reached control values, and, by T3, total skull length (Po-N) reached control values. However, all limb measures except femur length achieved complete catch-up, at times ranging from T0 (tibia length) to T3 (ulnar length). Body weight caught up in the initial stages of the

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American Journal of Orthodontics and Dentofacial Orthopedics July 2006

Total Mandibular Length Cont

Size Difference: Ba-Cb1 Occipital length Posterior neurocranial height Po-Ba Anterior Corpus length M1-I1 Total Mandibular length Co-I1 Co-Gn Ramus height

Sham

Co-I1 (mm)

23 21 19 17 15 0

1

2

3

Interval

A Sphenoid Length

Growth Difference: Sphenoid length Cb1’-Cb2 Nasal length E-N Humerus, femur Weight

Cont

Sham

7 6.5 6 5.5 5 0

2

3

I n te rv a l

B

Palate Length Cont Mu2-Iu (mm)

Size + Growth Difference: So-E Anterior cranial base length Ba-E Total cranial base length Po-E Cranial vault length Mu2-Iu Palate length Go-Mn Posterior corpus length E-Mu1 Viscerocranial height Cb2-Iu Midface length Ba-So Posterior cranial base length Po-N Total skull length Radius, ulna, tibia

1

Sham

19 18 17 16 15 0

1

2

3

Interval

C

Deficiency Effect (mm)

Fig 3. Differences in absolute growth between control and sham samples for selected cephalometric measures: A, size difference; B, growth difference; C, size ⫹ growth difference. 2

r = -0.58, p< 0.01 1

0

-1 60

70

80

90

100

Relative Maturity (%)

Fig 4. Regression of deficiency effect on relative maturity. Deficiency effect was calculated as (control value at T3 ⫺ sham value at T3) ⫺ (control value at T0 ⫺ sham value at T0). It is estimate of extent of divergence of slopes over time. Both craniofacial and limb measures were included.

experiment, but T1 showed a deceleration that ultimately caused it to fall below the linearly increasing controls by T2. The remaining measures showed varying

extents of incomplete catch-up, with the least catch-up displayed by anterior cranial base (So-E), posterior cranial base (Ba-So), and anterior corpus length (Ml-Il). Most measures displaying incomplete catch-up were neurocranial measures and the very mature anterior corpus length. Percentage of catch-up varied by the relative maturity of the measure. This is illustrated by the negative correlation of percentage of catch-up on relative maturity in Figure 7 (r ⫽ ⫺0.50, P ⬍.05). In this regression, it is clearly apparent, as the relative maturity of a measurement increased, that the percentage of catch-up decreased, and vice versa. DISCUSSION

As previously demonstrated for humans, rodents also have a growth maturity gradient across craniofacial measurements, with the most mature components associated with neural growth (neurocranial), the least mature associated with somatic growth (mandible), and with viscerocranial growth somewhere between the 2.1

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Table V.

Multilevel models: experimental v sham Experimental estimates

Neurocranium Po-N Po-E Ba-E So-E Ba-Cb1 Cb1=-Cb2 Ba-So Po-Ba Viscerocranium E-N Mu2-lu Cb2-lu E-Mu1 Mandible Go-Mn M1-I1 Co-Gn Co-I1 Limb measurements Radius Ulna Humerus Tibia Femur Weight

Constant

Linear

33.485 (.214) 22.063 (.12) 22.904 (.168) 10.75 (.116) 6.893 (.094) 5.23 (.051) 8.44 (.139) 7.969 (.103)

2.579 (.068) 1.025 (.105) 1.35 (.175) .493 (.062) .707 (.101) .522 (.018) .933 (.103) .36 (.041)

11.702 (.167) 15.241 (.113) 17.101 (.107) 7.661 (.053)

1.634 (.054) .831 (.038) 1.521 (.08) .585 (.017)

6.635 (.2) 4.538 (.039) 6.546 (.132) 16.833 (.16)

1.189 (.086) .197 (.021) .874 (.044) 1.384 (.059)

14.742 (.122) 14.806 (.185) 15.101 (.162) 17.882 (.236) 14.38 (.235) 44.369 (1.811)

1.531 (.032) 1.937 (.063) 1.856 (.067) 3.145 (.153) 2.498 (.177) 3.783 (.086)

Sham group differences Quadratic

Constant

Linear

–.022 (.032) –.031 (.056)

.105 (.295) –.123 (.159) –.345 (.231) .021 (.158)

–1.01 (.094) –.51 (.058) .008 (.24) –.215 (.086)

.111 (.07) .117 (.186) .016 (.141)

–.237 (.025) –.239 (.057) –.163 (.057)

.228 (.229) .04 (.155) –.087 (.148) .125 (.073)

–.615 (.075) –.374 (.053) –.228 (.11) –.259 (.023)

–.301 (.275) .062 (.054) –.331 (.18) .296 (.22)

–.528 (.118) –.085 (.029) –.241 (.061) –.555 (.081)

–.818 (.171) .292 (.254) .587 (.222) .245 (.319) .084 (.315) 5.302 (2.534)

–.299 (.036) –.585 (.087) –.7 (.092) –.745 (.086) –.623 (.099) 2.289 (.044)

Quadratic

–.161 (.077)

–.107 (.032) –.11 (.032)

–.041 (.026)

–.12 (.02)

–.151 (.047) –.063 (.054) –.076 (.004)

–.09 (.035)

Treatment Effect (mm)

Po-NExp ⫽ 33.485 ⫹ (2.579 ⫻ age) ⫹ (0 ⫻ age2). Po-Nsham ⫽ (33.485 ⫹ .105) ⫹ [(2.579 – 1.01) ⫻ age]. 4

3

r = -0.45, p < 0.05 2

1

0 60

70

80

90

100

Relative Maturity (%)

Fig 5. Regression of treatment effect on relative maturity. Treatment effect was calculated as (experimental value at T3 ⫺ sham value at T3) ⫺ (experimental value at T0 ⫺ sham value at T0). It is estimate of extent of divergence of slopes over time. Both craniofacial and limb measures were included.

In this study, the maturity of an overall length measurement in a particular region appeared in several instances to be a hybrid of its (sometimes very different) constituent measurements. For example, total cranial

base length (88%) was composed of a relatively mature anterior segment (90%) and a less mature posterior segment (83%); total skull length (85%) reflected mature cranial vault length (91%) and immature nasal length (75%). Similarly, total midface length (84%) encompassed the more mature palate length (88%) and the less mature nasal length (75%). In the most extreme example, total mandibular length (88%) was a composite of the very mature anterior corpus length (94%) and the much less mature posterior corpus length (71%). In every group, except for nasal length (E-N), ramus height (Co-Gn), and posterior corpus length (Go-Mn), all craniofacial variables were more mature than any limb length or body weight. The comparison of the sham group with the control group reflects growth differences due to the deficiency of GH. Like those of VandeBerg et al,21 our results suggest that GH deficiency affects various parts of the craniofacial skeleton differentially, depending on the relative maturity of the particular part. This is well illustrated by the negative correlation between the deficiency effect (degree of divergence of paired slopes over time) and relative maturity shown in Figure 4. In

78 Singleton et al

American Journal of Orthodontics and Dentofacial Orthopedics July 2006

Po-Ba E-N Humerus Radius Go-Mn Tibia Cb1-Cb2 Po-N

Measurement

Ulna Co-Il Femur Co-Gn W eight Po-E Ba-E Cb2-Iu Mu2-Iu E-Mu1 Ba-So So-E M1-Il Ba-Cb1

0

25

50

75

100 125 150 175 200

Percentage Catch-Up

Fig 6. Percentage catch-up at T3 for craniofacial and limb measures. Percentage catch-up was calculated as (experimental value at T3 ⫺ sham value at T3) divided by (control value at T3 ⫺ sham value at T3) ⫻ 100. It is measure of extent to which T3 experimental value approximates (or exceeds) control value.

this regression, a low deficiency effect indicates a size difference (little divergence of control and sham growth curves over time), and a high deficiency effect characterizes a measure with a growth difference (marked divergence of control and sham growth curves over time). Thus, this regression demonstrates that the effect of GH deficiency during the period studied was positively correlated with the amount of growth remaining for a particular structure. Although this has been appreciated to some extent in previous work in humans, our study provides a more detailed understanding of the differential maturation of the craniofacial skeleton.12,37 Our data on the sham group reflect similar characteristics to other studies of the Lewis (dw/dw) dwarf strain. Body weight differences (initially insignificant) between controls and mutants were similar to the original description of the strain.22 Initial relative maturity values in our craniofacial control measures had a similar range as those in the study of VandeBerg et al.21

Moreover, most measurements fell into the same categories as in our previous study of GH-deficient rats as either size difference, growth difference, or both.21 Our sham results also closely approximated those in the Snell mice (devoid of all anterior pituitary hormones) used by Rice et al.38 They found that the values for 22-day-old dwarf Snell mice (approximately equal to our study age) that had the highest percentage of that in fully grown mice (“percent mature”) were cranial width (99%; not measured in our study), skull height (94%; 94% in our study), and cranial base length (87%; 90% in our study). Lower maturity percentages (74%-85%) were evident for viscerocranial and mandibular measures such as midface and mandibular length, in accord with our findings. Although there are obvious differences in craniofacial form between rats and humans, some pronounced differences in growth curves between control and sham rats occurred in measures that are strongly affected in GH-deficient children. Anterior cranial base length has been shown to be normal or much less affected than posterior cranial base in GH-deficient children.16,17,39 Although both anterior cranial base (So-E) and posterior cranial base (Ba-So) measures differed from the controls in initial size and rate of growth (Table IV), anterior cranial base length had a greater relative maturity (90%) and a lower percentage of catch-up growth (40%) than posterior cranial base length (83% mature; 48% catch-up). The comparisons of sham and experimental groups provide an estimate of the treatment effect produced by the supplemental GH. GH administration had an effect on growth velocity for virtually every measure, although the percentage of stimulation at T3 varied widely. As in the comparison between the control and sham animals, the measures (body weight, limb measures, posterior corpus length [Go-Mn], ramus height [Co-Gn], and nasal length [E-N]) with the greatest overall absolute and relative growth potential showed the best response to GH supplementation: ie, the least mature components had the greatest treatment effects. This is exemplified by the negative correlation of treatment effect (degree of separation of slopes during time studied) with relative maturity of experimental sample measures (Fig 5; r ⫽ ⫺0.45, P ⬍.05). Measures with lower relative maturity values had large treatment responses. It was interesting that GH altered the growth rate even in measures that had demonstrated only a size difference between controls and shams, and were presumed to have relatively little response to GH during this time span. In 3 measures (total mandibular length [Co-I1], ramus height [Co-Gn], and posterior neurocranial height [Po-Ba]), the stimulation was very great,

Singleton et al 79

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Table VI.

Multilevel models: control v experimental Control estimates

Neurocranium Po-N Po-E Ba-E So-E Ba-Cb1 Cb1=-Cb2 Ba-So Po-Ba Viscerocranium E-N Mu2-lu Cb2-lu E-Mu1 Mandible Go-Mn M1-I1 Co-Gn Co-I1 Limb measurements Radius Ulna Humerus Tibia Femur Weight

Experimental group differences

Constant

Linear

Quadratic

Constant

Linear

Quadratic

34.769 (.192) 23.212 (.118) 24.288 (.117) 11.853 (.105) 7.323 (.066) 5.411 (.032) 9.115 (.076) 8.299 (.074)

3.159 (.191) .748 (.039) 1.096 (.044) .422 (.057) .448 (.026) .429 (.011) .612 (.025) .386 (.085)

–.375 (.061)

–1.387 (.28) –1.127 (.172) –1.354 (.171) –1.102 (.154) .307 (.079) –.181 (.047) –.58 (.097) –.407 (.1)

–.264 (.283) .211 (.059) .161 (.066)

.269 (.09)

11.999 (.138) 16.116 (.124) 18.635 (.092) 8.248 (.041)

2.173 (.138) .76 (.047) 1.445 (.067) .699 (.048)

–.286 (.043)

–.582 (.191) –.789 (.149) –1.59 (.131) –.598 (.046)

.321 (.078)

7.029 (.162) 5.067 (.041) 7.171 (0.123) 18.578 (.145)

.94 (.071) .112 (.021) .727 (.040) .827 (.06)

–.395 (.237) –.529 (.059) –.625 (.179) –1.745 (.212)

.252 (.106) .085 (.031) .147 (.06) .556 (.089)

13.263 (.109) 15.719 (.19) 16.048 (.168) 19.254 (.146) 14.661 (.137) 42.697 (1.727)

1.73 (.077) 1.935 (.195) 1.458 (.086) 2.737 (.138) 2.73 (.157) 6.264 (.182)

–.921 (.14) –1.031 (.237) –.96 (.221) –1.303 (.204) –.345 (.159) 1.557 (2.353)

.257 (.043) .352 (.107) .401 (.114) .196 (.077)

–.079 (.026)

–.099 (.021) –.046 (.015)

–.117 (.023) –.119 (.059) –.08 (.043) –.144 (.05) –.068 (.019)

.092 (.017) .203 (.048)

.248 (.038)

–2.52 (.143)

–.047 (.007)

Po-NControl ⫽ 34.769 ⫹ (3.159 ⫻ age) ⫹ (–3.75 ⫻ age ). Po-NExp ⫽ (34.769 – 1.38) ⫹ [(3.159 – .264) ⫻ age] ⫹ [(–3.75 ⫹ .269) ⫻ age2]. 2

Table VII.

Incomplete Midface length (Cb2-Iu) Cranial vault length (Po-E) Total cranial base length (Ba-E) Anterior cranial base length (So-E) Posterior cranial base length (Ba-So) Occipital length (Ba-Cb1) Palate length (Mu2-Iu) Viscerocranial height (E-Mul) Anterior corpus length (M1-I1) Ramus height (Co-Gn) Total mandibular length (Co-I1)

Complete (intersect period) T2 Sphenoid length (Cb1=-Cb2) Posterior corpus length (Go-Mn) Posterior neurocranial height (Po-Ba) Nasal length (E-N) T3 Total skull length (Po-N)

whereas, for the other 2 (anterior corpus length M1-I1 and occipital length Ba-Cb1), it was much more modest. This might reflect a much earlier influence of GH on these measures with a “parallel” pattern of growth difference, since GH is thought to begin to influence growth by 9 months of age in humans.40

200

Percent Catch-up

Incomplete versus complete catch-up in experimental (GH-injected) rats: craniofacial measurements

r = -0.50, p < 0.05 100

0 60

70

80

90

100

Relative Maturity (%)

Fig 7. Regression of percentage of catch-up on relative maturity. Percentage of catch-up was calculated as (experimental value at T3 ⫺ sham value at T3) divided by (control value at T3 ⫺ sham value at T3) * 100. Both craniofacial and limb measures were included.

Our results have several similarities to those describing GH injections in normal Wistar mice41 and Snell mice.38 Although Miura et al41 observed absolute growth changes only in the viscerocranium of younger animals and no change in the neurocranium, we found

80 Singleton et al

small but significant changes in most neurocranial measures. This might be a consequence of a species difference or their injection of control (nondeficient) animals rather than GH-deficient animals. Using multigroup cross-sectional data over a 20-day period, Rice et al38 noted that the effect produced by the GH treatment varied inversely with the “percent mature” (relative maturity) of the measurement, ranging from a slight gain of 3% to 4% for mature cranial width and height to 22% and 32% for immature humerus length and body weight measures, respectively. Our results compare nicely with human studies of differential growth after GH replacement therapy. While studying GH-deficient children, Cantu et al12 showed differential responses to GH replacement therapy, with significant effects in posterior cranial base length and facial height. Similar to our findings, Poole et al19 demonstrated that GH therapy increased mandibular length and lower face height while minimally affecting cranial base length. In our study, cranial base measurements (So-E, Ba-So, Ba-E) had smaller percentages of increases in the experimental sample (7%-7.2%; average, 7.1%) than the mandibular measurements CoGn, Go-Mn, and Co-Il (7.7%-22.9%; average, 14.7%). The comparisons between the control and the GHsupplemented experimental groups allow for assessment of the potential for catch-up in each measure. Catch-up growth is generally thought of as a phase of recovery after removal of a stressor42—in this case, GH deficiency. The potential for catch-up is inherent in an immature measurement; ie, completely mature measures cannot catch up. Tanner,3 describing types of catch-up with GH deficiency (complete v incomplete), assigned the ability to attain complete catch-up to earlier treatment of the child—ie, while the structure is relatively immature. We found varying degrees of catch-up, with only 5 experimental craniofacial variables (Cb1-Cb2, Go-Mn, Po-Ba, E-N, and Po-N) demonstrating complete catch-up. Whereas all craniofacial measures showed some catch-up, most craniofacial measures (63%) demonstrated incomplete catch-up. With the exception of posterior neurocranial height (88%), those with complete catch-up averaged 76% relative maturity; those with incomplete catch-up averaged a more mature 84%. If the very mature anterior corpus length is left out, cranial base measures displayed considerably less catch-up growth (40%-65%) than mandibular measures (87%-122%). Overall, most (63%) craniofacial (more mature) variables showed incomplete catch-up, whereas all limb measures (more immature) except femur length showed complete catchup. The negative correlation of percentage of catch-up with relative maturity underscores this relationship.

American Journal of Orthodontics and Dentofacial Orthopedics July 2006

Because GH is known to be a potent stimulator of cartilage-mediated growth,43-45 most previous studies have attributed its growth-promoting effects to its action on interstitial growth at cartilaginous growth centers.15,38 Although this effect is undoubtedly important, our results suggest that GH also stimulates growth of measures that primarily involve intramembranous or sutural growth. In particular, 3 of the 5 craniofacial measures that had complete catch-up (sphenoid length (Cb1=-Cb2), posterior corpus length (Go-Mn), and posterior neurocranial height (Po-Ba)) involved intramembranous bone growth and were not associated with cartilaginous components. In a study of craniofacial growth of GH-treated children born small for gestational age, high-dose GH supplementation over a 2-year period led to significant craniofacial catch-up growth, particularly in posterior total facial height, cranial base length, and overall mandibular length.15 These measurements also demonstrated an age-dependent effect on velocity: ie, the younger the age, the more pronounced the effect. Although catch-up was found to be significant for all craniofacial variables except maxillary length and lower anterior facial height, most variables in treated small-for-gestational-age children failed to normalize and remained small in comparison with Bolton standards (P ⬍.01). Cantu et al12 also showed a significant effect for both treatment duration and age at start of treatment. Their study showed the greatest effects of therapy in height and skeletal age, whereas posterior facial height showed greater improvement than either anterior facial height or posterior cranial base length. They concluded that craniofacial measures in GHdeficient children have different catch-up potentials after GH therapy based on their initial growth potentials (relative maturity). CONCLUSIONS

Our results show that GH has clearly a less positive effect on more mature neurocranial values than on viscerocranial, mandibular, and somatic (limb/weight) values. This study provides quantitative support for the concept of a craniofacial growth maturity and differential susceptibility gradient: the greatest relative growth was seen for most mandibular measures (least mature region), followed closely by the viscerocranium, and the least relative growth for the neurocranium (most mature region). Because older children have a greater percentage of structures with high maturity, this relative maturity effect in a person could underlie the age-dependent effect noted in supplemented GH-deficient children. Because our study involved comparatively larger doses than normally pre-

American Journal of Orthodontics and Dentofacial Orthopedics Volume 130, Number 1

scribed therapeutically to GH-deficient children (2 mg per kilogram per day subcutaneous v 0.16 to 0.23 mg per kilogram per week46), the differential effect on growth of various craniofacial measures might have been exaggerated in our study. However, our results show the potential importance of the timing of GH replacement therapy. Most children are treated primarily for stature reasons, with little thought to craniofacial growth. If therapy begins early at a mean age of 5.1 years,15 then craniofacial growth, and particularly growth of the neurocranium, would be affected differently than if therapy begins during or before puberty.47 In the latter instance, viscerocranial measures would be expected to show the greatest increase in the head region. Our results suggest that, depending on the timing of the therapy, there is potential for change in proportions or shape of the craniofacial complex. We thank David Farnsworth for his technical assistance and Genentech (San Francisco, Calif) for donating the rhGH used in this study.

REFERENCES 1. Albertsson-Wikland K, Rosberg S. Analyses of 24 hour growth hormone profiles in children: relation to growth. J Endocrin Metab 1988;67:493-500. 2. Rimoin DL, Merimee TJ, McKusick VA. Growth hormone deficiency in man: an isolated, recessively inherited defect. Science 1966;152:1635-7. 3. Tanner JM. Growth as a target-seeking function: catch-up and catch-down in man. Human Growth 1986;1:167-79. 4. Kaplan SI, Fenno J, Stebbing N, Hintz R, Swift R. The biological effectiveness of pituitary-derived and biosynthetic methionylhGH in animals and man. In: Schultz SW, editor. From gene to protein: translation into biotechnology. New York: Academic Press; 1982. p. 419-28. 5. Grumbach MM, Bin-Abbas BS, Kaplan SL. The growth hormone cascade: progress and long-term results of growth hormone treatment in growth hormone deficiency. Horm Res 1998;49:4157. 6. van Vliet G, Styne DM, Kaplan SL, Grumbach MM. Growth hormone treatment for short stature. New Eng J Med 1983;309: 1016-22. 7. deZegher F, Maes M, Gargosky SE, Heinrichs C, Du Caju MV, Thiry G, et al. High-dose growth hormone treatment of short children born small for gestational age. J Clin Endocrinol Metab 1996;81:1887-92. 8. Finkelstein BS, Imperiale TF, Speroff T, Marrero U, Radcliffe DJ, Cuttler L. Effect of growth hormone therapy on height in children with idiopathic short stature. Arch Pediatr Adolesc Med 2002;156:230-40. 9. Finkelstein BS, Silvers JB, Marrero U, Neuhauser D, Cuttler L. Insurance coverage, physician recommendations, and access to emerging treatments: growth hormone therapy for childhood short stature. J Am Med Assoc 1998;279:663-8. 10. Nanda RS. The rates of growth of several facial components measured from serial cephalometric roentgenograms. Am J Orthod 1955;41:653-73.

Singleton et al 81

11. Hunter CJ. The correlation of facial growth with body height and skeletal maturation at adolescence. Angle Orthod 1966;36:44-54. 12. Cantu G, Buschang PH, Gonzalez JL. Differential growth and maturation in idiopathic growth-hormone-deficient children. Eur J Orthod 1997;19:131-9. 13. Tanner JM. Growth at adolescence. Oxford: Blackwell Scientific Publications; 1962. 14. Buschang PH, Baume R, Nass GG. A craniofacial growth maturity gradient of males and females between 4 and 16 years of age. Am J Phys Anthropol 1983;61:373-81. 15. van Erum R, Mulier M, Carels C, Verbeke G, de Zegher F. Craniofacial growth in short children born small for gestational age: effect of growth hormone treatment. J Dent Res 1997;76: 1579-86. 16. Spiegel RN, Sather AH, Hayles AB. Cephalometric study of children with various endocrine diseases. Am J Orthod 1971;59: 362-75. 17. Konfino R, Pertzelan A, Laron Z. Cephalometric measurements of familial dwarfism and high plasma immunoreactive growth hormone. Am J Orthod 1975;68:196-201. 18. Edler RJ. Cephalometric parameters in hypopituitary patients. Br J Orthod 1979;6:19-22. 19. Poole AE, Greene IM, Buschang PH. The effect of growth hormone therapy on longitudinal growth of the oral facial structures in children. In: Dixon AD, Sarnat BG, editors. Factors and mechanisms influencing bone growth. New York: Alan R. Liss; 1982. p. 499-516. 20. Takano K, Ogiuchi H, Hizuka N, Sangu Y, Shizume K. Oromaxillofacial development in patients with GH deficiency and in normal short children. Endocrinol Japon 1986;33:655-64. 21. VandeBerg JR, Buschang PH, Hinton RJ. Craniofacial growth in growth hormone-deficient rats. Anat Rec 2004;278A: 561-70. 22. Charlton HM, Clark RG, Robinson CAF, Goff AE, Cox BS, Bugnon C, et al. Growth hormone-deficient dwarfism in the rat: a new mutation. J Endocrinol 1988;119:51-8. 23. Downs TR, Frohman LA. Evidence for a defect in growth hormone-releasing factor signal transduction in the dwarf (dw/dw) rat pituitary. Endocrinol 1991;29:58-67. 24. Skottner A, Clark RG, Fryklund L, Robinson ICAF. Growth responses in a mutant dwarf rat to human growth hormone and recombinant human insulin-like growth factor I. Endocrinol 1989;124:2519-26. 25. Martinez DA, Orth MW, Carr KE, Vanderby R, Vailas AC. Cortical bone growth and maturational changes in dwarf rats induced by recombinant human growth hormone. Am J Physiol 1996;270:E51-9. 26. Cittadini A, Stromer H, Vatner DE, Grossman JD, Katz SE, Clark R, et al. Consequences of growth hormone deficiency on cardiac structure, function, and beta-adrenergic pathway: studies in mutant dwarf rats. Endocrinology 1997;138:5161-9. 27. Tei TM, Kissmeyer-Nielsen P, Christensen H, Flyvbjerg A. Growth hormone treatment increases transmural colonic growth in GHdeficient dwarf rats. Growth Horm IGF Res 2000;10:85-92. 28. Daugaard JR, Luastsen JL, Hansen BS, Richter EA. Growth hormone induces muscle fibre type transformation in growth hormone-deficient rats. Acta Physiol Scand 1998;164:119-26. 29. Clark RG, Jansson J-O, Isaksson O, Robinson ICAF. Intravenous growth hormone: growth responses to patterned infusions in hypophysectomized rats. J Endocrinol 1985;104:53-61. 30. Isgaard J, Carlsson L, Isaksson OGP, Jansson J-O. Pulsatile intravenous growth hormone (GH) infusion to hypophysectomized rats increases insulin-like growth factor I messenger

82 Singleton et al

31.

32.

33.

34.

35. 36. 37.

38.

39.

ribonucleic acid in skeletal tissues more effectively than continuous GH infusion. Endocrinol 1988;123:2605-10. Jansson J-O, Albertsson-Wikland K, Eden S, Thorngren KG, Isaksson O. Effect of frequency of growth hormone administration on longitudinal bone growth and body weight in hypophysectomized rats. Acta Physiol Scand 1982;114:261-5. Kiliaridis S, Engstrom C, Thilander B. The relationship between masticatory function and craniofacial morphology. I. A cephalometric longitudinal analysis in the growing rat fed a soft diet. Eur J Orthod 1985;7:273-83. Engstrom C, Jennings J, Lundy M, Baylink DJ. Effect of bone matrix-derived growth factors on skull and tibia in the growing rat. J Oral Path 1988;17:334-40. Strenio J, Weisberg HI, Bryk AS. Empirical bayes and estimation of individual growth curve parameters and their relationship covariates. Biometrics 1983;39:71-86. Goldstein H. Multilevel mixed linear model analysis using iterative generalized least squares. Biometrika 1986;73:43-56. Goldstein H. Multilevel methods in educational and social research. New York: Oxford University Press; 1987. VanErum R, Mulier G, Carels C, deZegher F. Craniofacial growth and dental maturation in short children born small for gestational age: effect of growth hormone treatment— own observations and review of the literature. Horm Res 1998;50:141-6. Rice DPC, Roberts GJ, Thomas ML. Catch-up growth induced by growth hormone in the craniofacial skeleton of the Snell strain of the hypopituitary dwarf mouse. Eur J Orthod 1997;19:141-50. Kjellberg H, Beiring M, Wikland KA. Craniofacial morphology, dental occlusion, tooth eruption, and dental maturity in boys of

American Journal of Orthodontics and Dentofacial Orthopedics July 2006

40.

41.

42. 43.

44.

45.

46. 47.

short stature with or without growth hormone deficiency. Eur J Oral Sci 2000;108:359-67. Karlberg J, Albertsson-Wikland K. Infancy growth pattern related to growth hormone deficiency. Acta Paediatr Scand 1988; 77:385-91. Miura F, Nunota E, Hanada K, Ohyama K, Noguchi K. Effect of growth hormone on growth and development of the dentofacial complex in the young rat: a study by means of longitudinal roentgenogrphic cephalometrics. Bull Tokyo Med Dent Univ 1969;16:109-22. Prader A, Tanner JM, von Harnack GA. Catch-up growth following illness or starvation. J Pediatr 1963;62:646-59. Petrovic AG, Stutzmann JJ, Oudet CL. Control processes in the postnatal growth of the condylar cartilage of the mandible. In: McNamara JA Jr, editor. Determinants of mandibular form and growth. Ann Arbor: Center for Human Growth and Development; University of Michigan; 1975. p. 101-54. Maor G, Hochberg Z, Silbermann M. Growth hormone stimulates the growth of mouse neonatal condylar cartilage in vitro. Acta Endocrinologica 1989;120:526-32. Isaksson OGP, Lindahl A, Nilsson A, Isgaard J. Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocrin Rev 1987;8:426-38. Wit JM. Growth hormone therapy. Best Pr Res Clin Endocr Metab 2002;16:483-503. Burns EC, Tanner JM, Preece MA. Final height and pubertal developent in 55 children with idiopathic growth hormone deficiency, treated for between 2 and 15 years with human growth hormone. Eur J Pediatr 1981;137:155-61.