Growth Hormone Injections Improve Bone Quality in a Mouse Modelof Osteogenesis Imperfecta

Donna King,1 David Jarjoura,1,2 Heather A McEwen,1 and Michael J Askew3

ABSTRACT: Systemic growth hormone injections increased spine and femur length in a mouse model of OI.Femur BMC, cross-sectional area, and BMD were increased. Smaller gains were produced in vertebral BMCand cross-sectional area. Biomechanical testing showed improvements to structural and material properties inthe femur midshaft, supporting expanded testing of growth hormone therapy in children with OI.

Introduction: Osteoblasts in heterozygous Cola2oim mutant mice produce one-half the normal amounts of the�2 strand of type I procollagen. The mice experience a mild osteogenesis imperfecta (OI) phenotype, withfemurs and vertebrae that require less force than normal to break in a biomechanical test.Materials and Methods: Subcutaneous injections of recombinant human growth hormone (rhGH) or salinewere given 6 days per week to oim/+ mice between 3 and 12 weeks of age, in a protocol designed to simulatea trial on OI children.Results: rhGH injections promoted significant weight gain and skeletal growth compared with saline-treatedcontrol animals. Femur and spine lengths were increased significantly. Significant increases at the femurmidshaft in cortical BMD (2.2%), BMC (15.5%), and cross-sectional area (13%) were produced by rhGHtreatment. Increases in the same cortical bone parameters were measured in the metaphyseal region of thefemur and in tail vertebrae, but lumbar vertebrae showed significant increases in BMC (9.6%) and cross-sectional area (10.1%) of trabecular bone. Three-point bending testing documented functional improvementsto the femur midshafts. GH treatment produced significant increases in bone stiffness (23.7%), maximum load(30.8%), the energy absorbed by the femurs to the point of maximum load (44.5%), and the energy to actualfracture (40.4%). The ultimate stress endured by the bone material was increased by 14.1%.Conclusions: Gains in bone length, cross-sectional area, BMD, BMC, structural biomechanical properties, andstrength were achieved without directly addressing the genetic collagen defect in the mice. Results supportexpanded clinical testing of GH injections in children with OI.J Bone Miner Res 2005;20:987–993. Published online on January 18, 2005; doi: 10.1359/JBMR.050108

Key words: osteogenesis imperfecta, oim, growth hormone, biomechanics, BMD

INTRODUCTION

OSTEOGENESIS IMPERFECTA (OI) is a dominant skeletaldisorder caused by genetic defects in type I collagen

expression. Severity in humans runs from mild increasedrisk of fracture to prenatal lethality, depending on the mu-tation. Mutations primarily create one of two effects: theyreduce the output of a procollagen chain, thus reducing theabundance of proper collagen I heterotrimers in the matrix,or they create an altered procollagen structure that distortsthe function in the matrix of the collagen I rods that incor-porate the mutant chain. There is no cure for OI and fewtreatment options. Short stature is a common side effect.(1)

Injections of recombinant growth hormone (rhGH) andclonidine, a stimulator of GH release, have been tested byMarini et al.(2,3) and Vieira et al.(4,5) in a limited number of

moderate to severely affected children with OI. Some ofthose children, labeled as “responders,” grew at a fasterrate and experienced decreased fracture frequency in con-junction with their treatment.(2,3) Serum calcium kineticsimplied that the half-life of calcium bound in their bone wasincreased, and more calcium was available for bone miner-alization.(4,5) Genetically matched controls and extensivehistological and biomechanical analysis were not possiblewith the children, so details of the nature of their improve-ment are lacking.

Because of the need to confirm details of GH therapyoutcomes in a well-controlled study, we used the Cola2oim

mouse (a widely used animal model of OI, hereafter calledoim) to evaluate growth hormone effects. The Col1a2 genein oim mice sustained a single base deletion that altered theC-terminal reading frame.(6) The aberrant collagen is notsecreted to the matrix, altering the normal ratio of �1(I)and �2(I) procollagens; excess �1(I) chains in the matrixThe authors have no conflict of interest.

1Northeastern Ohio Universities College of Medicine, Rootstown, Ohio; 2Present address: Center for Biostatistics, Ohio State Uni-versity, Columbus, Ohio; 3Walter A. Hoyt, Jr. Musculoskeletal Research Laboratory, Summa Health System, Akron Ohio.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 20, Number 6, 2005Published online on January 18, 2005; doi: 10.1359/JBMR.050108© 2005 American Society for Bone and Mineral Research

987

JO410594 987 993 June

form homotrimers.(6) Homozygous oim mice have a mod-erate to severe brittle bone phenotype and can break theirown bones in normal cage activity.(6) Their skeletal fragilityand dwarfed body size reflect their inability to deposit anynormal collagen in their bone matrix.(6,7) The abnormalbone matrix of homozygous mutants affects mineral sizeand arrangement adversely.(8–10)

The genetic mutation in oim mice was originally thoughtto be recessive, because the brittle bone phenotype was notvisually evident in heterozygotes.(6) Genetically, however,heterozygous oim mice (oim/+) are actually more represen-tative of human OI patients, in that their osteoblasts arecapable of depositing some normal type I collagen fibrils.We have shown that oim/+ mice have bones that are patho-logically and biomechanically inferior to wildtype counter-parts.(11,12) This study used growing oim/+ mice to evaluatephysical, densitometric, and biomechanical outcomes ofGH therapy.

MATERIALS AND METHODS

Animals and tissues

Heterozygous oim mice were produced by breeding ho-mozygous stock from The Jackson Laboratory (Bar Har-bor, ME, USA) to C57BL/6J mice. All mice were fed For-mulab Rodent Diet (PMI Nutrition International) freechoice. This diet has 23.5% protein, 6.5% fat, and 6.8% ash,including supplementation with calcium (1%) and phos-phorus (1.07%). The mice were treated with rhGH (Dr AFParlow, National Hormone and Peptide Program, NIDDK,NIH) for 9 weeks, when they were between 3 and 12 weeksof age. They were injected subcutaneously 6 days per weekin the midpoint of their light cycle with 0.25 �g/g bodyweight (1 IU/kg/day) of rhGH. At 12 weeks, they werekilled, and their bones harvested for biomechanical anddensitometric analysis. Bones were stored frozen in salineuntil use, at which time they were thawed and kept moist.Femur length was measured from the ball joint to the me-dial condyle using an electronic digital caliper (Starrett,Athol, MA, USA). Dissected spines were radiographicallyimaged using a mammography unit. To account for slightcurvatures, a string was used to mark the lengths on theradiograph of lumbar vertebrae 1–6 and caudal (tail)vertebrae 1–7, and the string was measured with the cal-iper. The numbers of mice in each treatment group were asfollows: saline—male, 8; female, 11; rhGH—male, 10; fe-male, 6.

pQCT scanning

Femurs from male and female mice were analyzed forBMD, BMC, and cross-sectional area using pQCT (Nor-land XCT Research M; Orthometrix, White Plains, NY,USA). Total, trabecular, and cortical data were measuredfor the BMD, BMC, and area categories, and trabecularand cortical results are reported here. Loop parameters in-cluded contour mode 2 at 169.0 mg/cm3, peel mode 2 at 524mg/cm3 plus filter, and cortical mode 2 at 524.0 mg/cm3.Analysis was performed using a voxel size of 70 �m. Onetransverse slice was measured at the midpoint of the right

and left femurs. This was the same site tested in three-pointbending. Three transverse slices were measured proximal tothe growth plate at the distal metaphysis of each femur, andthese results were averaged. The middle of the three sliceswas 15.5% of the total distance from the distal end of thebone with 0.50 mm between scans. The second and thirdlumbar vertebrae were analyzed with two transverse scansmade in each vertebral body between the growth plates,and results of the four scans were averaged for each mouse.The third, fourth, and fifth caudal (tail) vertebrae were ana-lyzed similarly, with three scans through each vertebralbody, for a total of nine scans that were averaged for eachmouse tail.

Biomechanical testing

Femurs were tested in three-point bending using a servo-hydraulic, materials testing machine (Model 812; MTSCorp., Minneapolis, MN, USA). Each femur was tested inthe same orientation so that, during the bending test, theanterior cortex was placed in longitudinal compression andthe posterior cortex in tension. Each femur was centered ontwo supports spaced 10 mm apart, and the bending load wasapplied at the midpoint at a constant displacement rate of 5mm/minute until the bone fractured. Fracture was taken ascomplete loss of load carrying ability. During the bendingtest, load-displacement data were collected by a computer-ized data acquisition system at a sampling rate of 80 Hz.

The structural parameters of stiffness, maximum load,and stored energy were determined from the load displace-ment data. The stiffness of each femur was determined asthe slope of the initial linear portion of its load displace-ment curve. The maximum load was determined as thehighest load magnitude experienced by the femur during itstest. Some femurs fractured in near-brittle fashion, whereasothers showed ductile behavior before fracture. The post-yield displacement (the displacement represented by theinelastic or nonlinear portion of the load displacementcurve before fracture) can be used as a measure of ductility,or conversely, as an indicator of the “brittleness” of thefemurs. Postyield displacements and the stored energy (theareas under the load displacement curve) were calculated atboth maximum load and fracture. The modulus of the bonematerial (the ratio of elastic stress to elastic strain) and theultimate stress (the longitudinal stress at the cortex at pointof maximum load) were calculated, using standard flexureformulae for the three-point bend test, from the stiffnessand the maximum load, and the cross-sectional geometry ofthe midpoint transverse slice obtained previously by pQCTscanning.

Statistics

A linear mixed model statistical analysis was performedon the biomechanic and densitometric data to account forboth fixed and random effects.(13) Random effects wereused to model the correlated (nested) observations of leftand right femurs from the same mouse. In other words, weavoided any assumptions about the independence of obser-vations from the same mouse. The modeling provided ameasure of the degree of correlation of the two femurs. A

KING ET AL.988

hypothesis testing strategy was used to protect against typeI error. Variables tested in each analysis are indicated inTables 1–3. Related variables were grouped into six catego-ries: bone lengths, femur biomechanics, and densitometryof lumbar vertebrae, caudal vertebrae, femur diaphyses,and femur metaphyses. A significant global test across therelated variables was required before the individual vari-ables in a category were tested. Densitometry data wereadditionally subjected to an interaction test to determinewhether there was a difference in treatment effect on tra-becular and cortical bone. In three of the four bone sites,separate global tests were subsequently required for trabec-ular and cortical measurements. This hypothesis testingstrategy protected against type I error (keeping it under0.05 for each of the categories of variables), given a nulltreatment effect for all variables in a category. Statisticalanalysis was accomplished using the SAS Proc Mixed (ver-sion 9.1; SAS, Cary, NC, USA). In addition to significancetests, estimates of rhGH effects are provided along withtheir SE. To interpret effect sizes across variables, rhGHeffects are also reported as a percentage of the controlgroups mean, and SE of these converted values is also pro-vided. Any apparent discrepancies in the tables are ex-plained by rounding of the group values. Models were alsoused to test for sex and sex-by-treatment interactions. Theexperimental design was slightly unbalanced with regard tothe numbers of male and female animals in each treatmentgroup. Adjustments for sex effects were applied in obtain-ing the model estimates of the effect of rhGH. In addition,as noted below, we found no significant sex effects; there-fore, the adjustments for them were negligible.

RESULTS

GH promoted growth

Oim/+ mice have a functional GH axis and were able torespond to rhGH injections by growing larger. By the thirdweek of treatment, at 6 weeks of age, both female and malemice weighed significantly more than their sex-matchedcontrols (Fig. 1). The significant difference was maintaineduntil the 12-week endpoint, at which time females andmales had gained an extra 14% and 12%, respectively, inbody weight. The similar growth curves are representativeof the kinds of parallel responses the two sexes showed inother analyses where we do not report data subdivided bysex.

The rhGH mice grew longitudinally. There were signifi-cant increases in lumbar spine and tail length and a signifi-cant increase in femur length (Table 1). The average in-crease per growth plate within the spine (0.3%) wascomparable between lumbar and caudal sites. The twogrowth plates of the femur each contributed more (0.8% onaverage) than the individual vertebral growth plates to theoverall skeletal growth of the rhGH-treated mice.

Because organomegaly, particularly of the liver, occurs intransgenic mice overexpressing GH (D King, unpublisheddata, 1994),(14,15) livers, spleens, and kidneys were weighedat the endpoint. Livers and kidneys grew proportionally tothe increased body weights, but spleens were increased sig-

nificantly beyond that: 17% (females) to 31% (males) oversex-matched control spleen weights after normalization tobody weight (t-test, p < 0.005; data not shown).

Densitometric analysis

Lumbar vertebrae: Table 2 presents densitometric scan-ning data from four skeletal sites. BMC is expressed asmilligrams quantitated in a slice of 1 mm thickness. For thelumbar vertebrae, significant increases caused by rhGH

FIG. 1. Growth curves. Means are plotted with SD. Significanceis indicated by superscripts: ap < 0.05; bp < 0.005; cp < 0.001.

TABLE 1. BONE LENGTH COMPARISONS OF OIM/+ MICE

IN MILLIMETERS

Treatment nLumbarvertebrae

Caudalvertebrae Femur

Saline 19 19.32 ± 0.10 27.39 ± .26 15.65 ± 0.05rhGH 16 20.07 ± 0.12 28.67 ± .29 15.90 ± 0.06Percent change ↑3.9 ± 0.8% ↑4.7 ± 1.4% ↑1.6 ± 0.5%Individual p value <0.0001 0.0028 0.0036

Lengths of six lumbar vertebrae (L1–L6) and seven caudal vertebrae(Ca1–Ca7) are represented.

Data are mean ± SE.n, number of mice.The global p value < 0.0001.

GROWTH HORMONE IMPROVES OI MOUSE BONE 989

stimulation were found for trabecular area and BMC. Theglobal test for the multiple measures was significant (p �0.0015), and no overall significant difference between cor-tical and trabecular rhGH effects was found (interactionp � 0.2). This result allowed us to interpret the p values foreach of the measures. Differences between male and femalevalues were not significant in lumbar bone (p � 0.087), andno significant difference in the effect of rhGH was foundbetween the two sexes. Therefore, we reported the resultsfor both sexes combined.

Caudal vertebrae: The GH response in caudal vertebraewas essentially within the cortical compartment, with sig-nificant increases in cortical BMC and cortical area thatwere also reflected in a cortical BMD increase that ap-proached significance. This conclusion was based on a sig-nificant interaction between rhGH effect and bone type(interaction p � 0.048) and a significant global test forcortical measures only. Again, no significant difference wasfound in rhGH effect between the two sexes.

Femurs: Femurs were analyzed at the midpoint of thediaphysis. Cortical BMD, BMC, and cross-sectional areawere significantly increased for rhGH-injected mice.Whereas GH treatment increased the size of the bone, aswas predicted, it also increased the BMC to such a degreethat BMD was increased significantly. The gains in corticalbone positively impacted the biomechanical performanceof the femurs when tested at the midshaft. Note that tra-becular bone is a minor component of midshaft architec-ture. Significant treatment effects in the distal femur me-taphysis were noted as gains in cortical BMD and BMC,and a weak trend was found toward more cortical cross-sectional area. These results were again based on significant

interactions between bone type and rhGH effect and globaltests that were significant only for cortical bone. Again, nosignificant difference in the rhGH effect was found for thetwo sexes.

Biomechanics

The results of the three-point bending test indicated thatthere were significant increases in stiffness, maximum load,energy stored to maximum load, energy stored to fracture,and ultimate stress for the treatment group relative to con-trol (Table 3). The data did not indicate any changes in themodulus.

There were no significant differences of the means ofvalues measured for left femurs compared with right femursin any of the femur data sets. Correlations between left andright femurs varied greatly depending on measure. Theywere high for densitometric measurements. Correlationsfor left and right femur biomechanical measures werelower, however, ranging from −0.38 to 0.19.

The postyield displacement data were highly skewed. Alogarithmic transformation produced normally distributeddata for the postyield displacements to fracture. Analyses(t-test) of both transformed and untransformed data failedto indicate significance for mean postyield displacement tomaximum load or postyield displacement to fracture (datanot shown). Thus, we cannot draw a conclusion regardingthe ductility or brittleness of the treated femurs.

DISCUSSION

In this experiment, the rhGH treatment duration andtiming was designed to begin at 3 weeks, the age when mice

TABLE 2. DENSITOMETRIC MEASUREMENTS AT FOUR SKELETAL SITES

Treatment

BMD (mg/cm3) BMC (mg) Cross-sectional area (mm2)

Trabecular Cortical Trabecular Cortical Trabecular Cortical

Lumbar vertebrae (*interaction p � 0.20; global p � 0.0015)Saline 210 ± 3 687 ± 4 0.81 ± 0.01 0.94 ± 0.03 3.87 ± .06 1.36 ± 0.04rhGH 208 ± 4 694 ± 5 0.89 ± 0.01 0.99 ± 0.04 4.26 ± 0.07 1.41 ± 0.05Percent change ↓0.7 ± 2.4 ↑1.0 ± 0.9 ↑9.6 ± 2.5 ↑4.8 ± 5.1 ↑10.1 ± 2.5 ↑3.6 ± 4.5Individual p value 0.76 0.29 0.0005 0.36 0.0003 0.44

Caudal vertebrae (†interaction p � 0.048; global for trab p � 0.57; global for cort p � 0.012)Saline 153 ± 3 998 ± 6 0.057 ± 0.003 0.89 ± 0.01 0.37 ± 0.02 0.90 ± 0.01rhGH 158 ± 3 1015 ± 7 0.053 ± 0.003 0.95 ± 0.01 0.34 ± 0.02 0.93 ± 0.01Percent change ↑3.4 ± 2.7 ↑1.7 ± 0.9 ↓6.7 ± 6.5 ↑6.2 ± 2.3 ↓9.4 ± 6.2 ↑4.4 ± 1.7Individual p value 0.21 0.071 0.31 0.010 0.14 0.018

Femur diaphysis (†interaction p < 0.0001; global for trab p � 0.060; global for cort p < 0.0001)Saline 152 ± 1 1121 ± 4 0.114 ± 0.003 1.16 ± 0.02 0.74 ± 0.02 1.03 ± 0.01rhGH 149 ± 1 1145 ± 5 0.108 ± 0.003 1.33 ± 0.02 0.72 ± 0.02 1.16 ± 0.02Percent change ↓2.4 ± 1.2 ↑2.2 ± 0.6 ↓5.2 ± 3.5 ↑15.5 ± 2.6 ↓2.5 ± 3.8 ↑13 ± 2.2Individual p value 0.054 0.0005 0.15 <.0001 0.53 <.0001

Femur metaphysis (†interaction p � 0.011; global for trab p � 0.83; global for cort p � 0.0024)Saline 247 ± 5 805 ± 5 0.46 ± 0.01 0.99 ± 0.02 1.81 ± 0.02 1.23 ± 0.03rhGH 242 ± 6 831 ± 5 0.46 ± 0.01 1.08 ± 0.03 1.85 ± 0.03 1.31 ± 0.03Percent change ↓2.1 ± 3 ↑3.1 ± 0.9 ↑0.2 ± 3.5 ↑9.6 ± 3.6 ↑2.2 ± 2.0 ↑5.9 ± 3.4Individual p value 0.050 0.001 0.96 0.012 0.27 0.096

Data are mean ± SE; n � 19 saline-treated oim/+ controls and 16 rhGH-treated oim/+ mice.* Test for different treatment effects on trabecular and cortical bone (“interaction”).† A significant interaction effect required separate global tests for cortical and trabecular data.

KING ET AL.990

are first capable of responding with growth to GH trans-gene expression (D King, unpublished data, 1994)(16) andGH injections.(17) GH-deficient mice begin to show stuntedgrowth at 2–3 weeks, also indicating that the growth-promoting function of GH becomes important at thattime.(18) The endpoint was selected as skeletal maturity (12weeks). Whereas mouse growth plates (and those of otherrodents) never completely close, histologically it can be rec-ognized that their activity slows considerably by this age,and multiple mechanisms retard further longitudinalgrowth.(19) Mouse growth curves are clearly flattening outby 12 weeks as well, and this is shown in Fig. 1. Our pro-tocol was designed as an aggressive test of the potentialosteogenic effects of rhGH during the full testable growingperiod of young OI mice.

Increased growth was shown as possible in the oim/+mice, even in the presence of a fundamental bone matrixprotein defect. This was predicted because the heterozy-gous condition is not crippling in the mice. Oim mice haveno recognized defects in their GH-IGF axis, and untreatedoim/+ mice are not noticeably different in size from wild-type siblings. Wildtype and oim/+ mice cannot be distin-guished by phenotype and must have their genotypes de-fined by DNA analysis.(20) Camacho et al.(9) found nostatistical difference between weights or femur lengths ofoim/+ mice and their wildtype controls. rhGH treatmentproduced significant body weight gain in oim/+ mice andskeletal bone growth in both longitudinal and circumferen-tial directions. People with OI, even the mildest form, arefrequently shorter than normal.(1,21,22) Whereas the maingoal of rhGH therapy is to improve BMD and biomechan-ical quality, increased stature is accepted as a beneficialadditional effect.

In our limited pathological evaluation, organomegaly waslimited to the spleen. One possible explanation for the en-larged spleens is that the rhGH injections stimulated eryth-ropoiesis (both directly and by stimulating IGF produc-tion(23)). The spleen is a minor erythropoietic organ in ahealthy adult mouse but can become readily expanded tosupport increased erythropoiesis if necessary.(24) However,hematocrits were not measured, and spleens were not pre-served for histological testing of this hypothesis.

Densitometric analysis revealed significant changes atfour skeletal sites tested. Cortical BMD, BMC, and cross-sectional area were significantly increased at both testedsites in the femur and in caudal vertebrae (with trends to-ward significance in caudal cortical BMD and femur me-taphysis cortical area). There were no changes in the cor-

tical measurements in lumbar vertebrae, however. Unlikethe other sites, lumbar vertebrae showed a trabecular re-sponse, with rhGH producing significant increases in tra-becular BMC and area. These differential responses likelyreflect the relative composition of each bone site with re-spect to cortical and trabecular architecture. For example,even within the femur, the cortical bone response wasgreatest at the midshaft, which is almost exclusively corticalbone. We are presently studying the details using a histo-morphometric approach.

We offer several possible explanations for why or howrhGH treatment might have increased BMD in the oim/+mice. (1) The partially mineralized bone surfaces may havebecome more fully mineralized, as occurs in bisphospho-nate-treated bones.(25) (2) rhGH treatment increases min-eral absorption,(26) which may have led to increased min-eral incorporation in the bones of treated mice. (3) Theactivity of enzymes that process proteoglycans that can pro-mote or inhibit mineral nucleation in the matrix may havebeen altered by rhGH. Human OI bone has been shown tohave more proteoglycan particles.(27) (4) Levels of othernoncollagenous bone matrix proteins may have been nor-malized. For example, human OI bone has been shown toincorporate lower levels than normal of osteonectin andhigher levels of bone sialoprotein and osteocalcin.(28) Suchchanges may have enabled additional crystal formation orgrowth.

Densitometric values of left and right femurs from eachmouse were highly correlated, as expected, with r valuesbetween 0.7 and 0.9. In contrast, biomechanical datashowed very low correlations, not statistically differentfrom zero, between the left and right femurs of each mouse(r values of 0.13 for stiffness, 0.19 for maximum load, −0.38for energy to maximum load, −0.19 for energy to fracture,0.18 for ultimate stress, and 0.088 for modulus). We cautionothers to be aware of the possibility that performance mea-sures of bones may not necessarily be symmetrical, and theopposite limb of a test animal may not necessarily serve asa better control than another animal. A plausible explana-tion of the left versus right dichotomy in our oim/+ mice isthat one femur may have had more lamellar bone at themidshaft testing point, whereas the opposite femur from thesame mouse may have had correspondingly more wovenbone. This variation in microarchitecture is one hallmark ofOI bone in humans(29,30) and has been documented in theoim mouse line as well.(11)

rhGH treatment produced significant outcomes for thebiomechanical structural properties of energy to maximum

TABLE 3. STRUCTURAL AND MATERIAL PROPERTIES OF OIM/+ FEMURS TESTED IN THREE-POINT BENDING

Treatment nStiffness(N/mm)

Maximumload (N)

Energy to maximumload (Nmm)

Energy tofracture (Nmm)

Ultimate stress(N/mm2)

Modulus(N/mm2)

Saline 19 56.2 ± 3.6 12.5 ± 0.4 2.00 ± 0.09 3.37 ± 0.21 129 ± 4 7408 ± 409rhGH 16 69.5 ± 4.0 16.4 ± 0.5 2.89 ± 0.10 4.73 ± 0.23 147 ± 4 7809 ± 459Percent change ↑23.7 ± 9.5 ↑30.8 ± 5.3 ↑44.5 ± 6.5 ↑40.4 ± 9.1 ↑14.1 ± 4.6 ↑5.4 ± 8.3Individual p value* 0.018 <0.0001 <0.0001 0.0001 0.0044 0.52

Data are mean ± SE.* The global p value <0.0001.

GROWTH HORMONE IMPROVES OI MOUSE BONE 991

load (also called energy to failure) and energy to fracture.These two measures are representative of performance ofthe bone in a living animal if bending force should be ap-plied around a fulcrum at the midshaft. It is concluded thatthe average rhGH treated oim/+ femur could successfullyresist a force that would break saline control mouse femurs.

When additional bone material was deposited in re-sponse to the rhGH treatment, increasing the cross-sectional area, BMD, and BMC as observed in these testresults, the structural and material properties of the femurswould be expected to increase. About a 40% increase instiffness would be predicted from the 18% increase in di-aphyseal cross-section: a 27% increase was observed. A30% increase in maximum load would be expected, as wasobserved (16.4N for the rhGH group versus 12.5N for thecontrols). The increased BMD and BMC would predict in-creases in the modulus and the ultimate stress. A significantincrease in ultimate stress was observed, but there was nochange seen in the modulus. The increase in BMD wassmall, however. Stimulation of production of bone proteinsor GH-stimulated metabolic changes may have facilitatedmineral nucleation, crystal growth, and/or enhanced incor-poration of mineral into the matrix as discussed above.This, in turn, may have resulted in a bony microarchitecturein which load-induced microstructural derangements couldoccur more easily. This would explain the near constantmodulus and the significant increases seen in the storedenergies.

The rhGH dose of 0.25 mg/kg/day that the oim/+ micereceived was 10-fold higher than the dose used by Antoni-azzi et al.(31) in their small trial of OI type I patients. Theyreported no increase in fracture frequency and significantincreases in linear growth velocity, bone turnover, andBMC of trabecular bone in the lumbar spine. Our rhGHdose was 5- to10-fold higher than doses used by Marini etal.(2,3) in the trial involving type III and type IV children.Some of those children responded with faster linear growthand decreased fracture frequency. The BMD in the lumbarvertebrae of the responders increased a significant 5–7% ofthe z score. For general comparison, rhGH doses used inclinical trials for other non-OI growth disorders rangedfrom 2.5- to 5.6-fold lower than the dose we used on themice.(32–36) It must be acknowledged, however, that ex-trapolations between human and mouse dosages fail to con-sider all implications of their different metabolic rates andpotential drug clearance rates.

A purported increased risk of leukemia in childrentreated with standard rhGH doses was recently dis-pelled.(37,38) At present, we do not know whether short-term rhGH treatment of OI adults could produce a similarbone anabolic response or whether gains could be main-tained once the GH treatment is ended. The treatment ismost easily justified for children who still have activegrowth plates.

The extra linear growth stimulated by rhGH injections inthe oim/+ mice could produce lifestyle advantages if extrap-olated to growing children with OI. BMC of lumbar trabec-ular bone was increased significantly. Significant increasesin BMD, BMC, and cross-sectional area contributed to im-proved biomechanical performance of the femurs, as well as

the strength of the bone material at the midshaft. Thesefindings support expanded clinical testing of rhGH therapyin children with mild OI.

ACKNOWLEDGMENTS

This work was supported by a grant from the Osteogen-esis Imperfecta Foundation. We thank Keding Hua andVoula Androulakakis for assistance with the statistics andMaria Serrat and Philip Reno for valuable discussion.

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Address reprint requests to:Donna King, PhD

NEOUCOMDepartment of Biochemistry and Molecular Pathology

4209 State Route 44Rootstown, OH 44272, USA

E-mail: dk@neoucom.edu

Received in original form October 12, 2004; revised form January5, 2005; accepted January 12, 2005.

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