Online Submissions: http://www.journaltcm.com J Tradit Chin Med 2012 September 15; 32(3): 1-2info@journaltcm.com ISSN 0255-2922

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JTCM |www. journaltcm. com September 15, 2012 |volume 32 | Issue 3 |

BASIC INVESTIGATION

Amomum villosum induces longitudinal bone growth in adolescentfemale rats

Sun Haeng Lee, Ji Young Kim, Hocheol Kim, Seul Ki Park, Cho Young Kim, Sun Yong Chung, Gyu Tae Changaa

Sun Haeng Lee, Seul Ki Park, Cho Young Kim, Gyu TaeChang, Department of Pediatrics of Korean Medicine,Kyung Hee University Hospital at Gang-dong, 892Dongnam-ro, Gangdong-gu, Seoul 134-727, Republic ofKoreaJi Young Kim, Hocheol Kim, Department of HerbalPharmacology, College of Korean Medicine, Kyung HeeUniversity, 26 Kyungheedae-ro, Dongdaemoon-gu, Seoul130-701, Republic of KoreaSun Yong Chung, Department of Neuropsychiatry ofKorean Medicine, Kyung Hee University Hospital atGang-dong, 892 Dongnam-ro, Gangdong-gu, Seoul 134-727, Republic of KoreaSupported by the Fund of Amomum villosum in bonegrowthCorrespondence to: Prof. Gyu Tae Chang, Department ofPediatrics of Korean Medicine, Kyung Hee UniversityHospital at Gang-dong, 892 Dongnam-ro, Gangdong-gu,Seoul 134-727, Republic of Korea. gtchang@khu.ac.krTelephone: +82-2-440-7127Accepted: April 24, 2012

AbstractOBJECTIVE: This study was conducted to evaluatethe effect of Amomum villosum on longitudinalbone growth.

METHODS: Adolescent female Sprague-Dawleyrats were divided into 3 groups and treated for 4days: control (distilled water, p.o.), recombinanthuman growth hormone (rhGH; 100 μg/kg, s.c.),and A. villosum (500 mg/kg, p.o.) groups. On day 3,tetracycline (20 g/kg, i.p.) was injected for growthplate identification. On days 2, 3 and 4,5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg, i.p.)was injected to label proliferating cells. On day 5,

tibias were dissected and fixed in 4%paraformaldehyde, dehydrated, and sectioned forimmunohistochemistry and histomorphometry.

RESULTS: The rate of bone growth in the A.villosum and rhGH groups increased to (410 ± 44)and (389 ± 46) μm/day (P<0.01), respectively, ascompared with the control (330.7 ± 34.7) μm/day.The thickness of the growth plates also increasedto (591 ± 37) and (598 ± 32) μm, respectively, ascompared with the control (524±89) μm (P<0.001).The number of BrdU-positive cells in thechondrocytes of the A. villosum and rhGH groupswas also significantly higher (126±24) and (143±18)cells/mm2, respectively) than in the control (109 ±25) mm2 (P<0.05). Insulin-like growth factor-1 andbone morphogenetic protein-2 in the A. villosumand rhGH groups were highly expressed in thegrowth plate as compared with the controlsamples, indicating increased bone formation.

CONCLUSIONS: A. villosum could be used to treatgrowth retardation during adolescence.

© 2012 JTCM. All rights reserved.

Keywords: Amomum villosum; Growth plate; BoneDevelopment; Recombinant human growthhormone

INTRODUCTIONA major factor controlling height is bony growth,which stimulates other tissue growth depending on theframework of the body. Bones in different parts of theskeleton develop through two distinct processes,intramembranous ossification and endochondral

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JTCM |www. journaltcm. com September 15, 2012 |volume 32 | Issue 3 |

Lee SH et al. Amomum villosum induces longitudinal bone growth in adolescent female rats

ossification. Endochondral ossification in the growthplate leads to longitudinal bone growth, involvingreplacement of a cartilage model with bone tissue.After birth, much of bony elongation occurs throughthe activity of chondrocytes within the growth plate,which are continually replaced by bone-forming cellsthrough a combination of proliferation, cartilageextracellular matrix secretion, and hypertrophy. Animportant stimulator of chondrocyte proliferation inthe growth plate is growth hormone (GH), which isactivated through stimulation of insulin-like growthfactor (IGF)-1. Furthermore, chondrocyte proliferationis also regulated through the activation of bonemorphogenetic protein (BMP) signaling in the bonegrowth plate.1 Observations of chondrocyteproliferation, IGF-1, and BMP concentrations areuseful indicators of the degree of growth.The Amomum plant is one of the most ancient andhighly valued spices in the world. Amomum villosum(Zingiberaceae), native to Guangdong province, is aperennial herb that occurs in the understory of tropicaland subtropical forests2 and is an important medicinalplant for improvement of gastrointestinal motility.3

Few reports have been published the effects of A.villosum on longitudinal body growth. However, inDongeuibogam, the traditional book of Koreanmedicine, malnutrition associated with growthretardation was treated using an herbal mixturecontaining A. villosum.In this study, the effects of A. villosum on longitudinalbone growth were determined in the epiphyseal platesof Sprague-Dawley rats. BrdU (5-bromo-2'-deocyuridine), a thymidine analogue incorporated intoDNA during replication,4 was used to determineproliferation, and tetracycline labeling for growth plateidentification. Furthermore, the expressions of BMP-2and IGF-1 were studied using immunohistochemistry.

METHODS

AnimalsFour-week-old female Sprague-Dawley rats, weighing(80±10)g, were used in this study (Samtako Co., Osan,Korea). The experimental procedures were performedin accordance with the animal care guidelines ofKyung Hee University's Institutional Animal Care andUse Committee [protocol number KHUASP(SE)2011-008). Animals were housed 5/cage undercontrolled temperature (23 ± 2)° C, relative humidity[55 ± 10)% ], and lighting (lights on 07:00-19:00 h),with food and water made available ad libitum. After 1week of acclimatization, the rats were split into theirrespective groups for treatment.

Preparation of amomum villosum water extractThe fruit of Amomum villosum was collected in

Guangdong, China (KH Herb Pharm., Seoul, Korea).The dried plant (200 g) was extracted twice withdistilled water in 3 h of reflux heating. The filtrateswere evaporated by rotary evaporator and lyophilizedby a freeze-dryer (Operon ™, Seoul, Korea). Thepowder was stored at -20°C until it was needed. Theyield of the freeze-dried product of A. villosum wasabout 10.71%.

Treatment groupsAnimals were randomly allocated into 3 groupsaccording to supplement regimen. Distilled water(DW; 10 rats) or A. villosum (500 mg/kg; 10 rats) wasadministered orally twice daily at 8:00 and 20:00 h ina fixed dose volume of 10.0 ml/kg. Recombinanthuman growth hormone (rhGH) (100 μg/kg; 10 rats)(LG Life Science, Seoul) was subcutaneously injectedonce daily. Treatment was continued for 4 consecutivedays. On day 3, all animals were injected intra-peritoneally with tetracycline hydrochloride (20 mg/kg,Sigma, St. Louis, MO, USA) in saline for themeasurement of bone growth. On days 2, 3, and 4,5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg, Sigma)was intraperitoneally injected to label proliferatingcells. Twenty-four h after the final injection, animalswere sacrificed, and then the tibias were dissected freefrom the soft tissues.5

Tissue preparation and detection of longitudinalbone growthThe dissected tibias were fixed in 4% para-formaldehyde for 48 h and dehydrated by immersionin 30% sucrose for 1 day.6 Dehydrated bone waslongitudinally sectioned at a thickness of 40 μm with amicrotome (Leica, Berlin, Germany).7 The sectionswere mounted onto gelatinized glass slides andobserved by fluorescence microscopy (Olympus,Tokyo, Japan). The longitudinal bone length betweenthe fluorescent line and the epiphyseal end line of thegrowth plate at three different locations using Image Jversion 1.43 u (National Institutes of Health, USA). 8

Data were averaged to calculate the longitudinal bonegrowth rates.

Detection of chondrocyte proliferationFor the detection of cell proliferation in the growthplate, BrdU-specific immunofluorescence was per-formed.9 Dehydrated tibial sections were pretreated in2 M HCl at 37°C for 1 h and rinsed twice in 0.1 Msodium borate (pH 8.5). The sections were incubatedin phosphate-buffered saline (PBS), including 3%bovine serum albumin and 0.3% Triton X-100 for 1hour and incubated with BrdU-specific mousemonoclonal antibody (Santa Cruz Biotechnology,Santa Cruz, CA, USA) (diluted 1∶100) overnight at4° C. The sections were then washed 3 times with0.1 M PBS before incubation with fluoresceinisothiocyanate-conjugated mouse secondary antibody

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Lee SH et al. Amomum villosum induces longitudinal bone growth in adolescent female rats

(Jackson Imm- unResearch Laboratories, Bar Harbor,ME, USA) (diluted 1∶ 200) for 4 h at roomtemperature. Finally, the sections were washed withPBS and mounted onto gelatinized glass slides. Tomeasure the number of BrdU-positive cells,fluorescence images of the sections were collected usingfluorescence microscopy (Olympus, Tokyo, Japan).The mean density of BrdU-labeled cells in each rat wascalculated as the number of BrdU-labeled nuclei perarea (in mm2) in the growth plate, as measured by twoindependent observers.10

Measurement of BMP-2 and IGF-1 in the growthplatesFor the detection of BMP-2 and IGF-1 in the growthplate, the dehydrated tibial sections were incubated in1% Triton X-100 at room temperature and incubatedwith goat BMP-2 antibody or rabbit IGF-1 antibody(Santa Cruz Biotechnology) (diluted 1:200) overnightat 4° C. The sections were then incubated with anti-goat and anti-rabbit antibodies (Jackson Immun-oResearch Laboratories) (diluted 1:200) for 60 min,respectively, and then stained with 0.05% 3,3-diaminobenzidine.7

Statistical analysisResults are expressed as the mean±SD of the differentexperiments under the same conditions. Statisticallysignificant values were compared using Student's t testfor comparisons between groups. P-values<0.05 wereconsidered statistically significant.

RESULTS

Daily body weight changeOn days 1-5, the daily body weights of the rats in thecontrol, rhGH and A. villosum group are described inTable 1. From the 1st day to the 5th day, the bodyweight gain was (18.8±1.5) g (control), (19.1±1.7) g(rhGH), and (17.5 ± 1.7) g (A. villosum). The weightfor the rats in the A. villosum treatment groupincreased slower than the other two groups, but thisdifference was not statistically significant (Figure 1).To evaluate the rate of longitudinal bone growth,

tetracycline was used to label the newly formed bone(Figure 2). The longitudinal bone growth of thecontrol adolescent female rats was (331±35) μm/day.The rhGH group showed a significantly greater rate ofbone growth of (389 ± 46) μm/day as compared withthe control group (P=0.0050). The A. villosum groupalso showed a significantly greater rate of bone growthof (410 ± 44) μm/day as compared with the controlgroup (P=0.0003) (Figure 3).The proximal tibial growth plate in the control groupwas approximately (524 ±89) μm thick. Following A.villosum administration, the growth plate thicknessincreased to (591±37) μm (P=0.0007). For rats in therhGH group, the growth plate thickness increased to(598±32) μm (P=0.0002) (Figure 4).BrdU-labeled cells were observed in the chondrocytesto determine the proliferation levels in the growthplates (Figure 5). The number of BrdU-positive cells inthe control group was (110 ± 25) cells/mm2. In therhGH group, the number of BrdU-positive cellsincreased to (143 ± 18) cells/mm2 (P<0.0001). Thenumber of BrdU-positive cells in the A. villosum groupalso increased to (126±24) cells/mm2 (P=0.02) (Figure6).To investigate the expression of BMP-2 and IGF-1,

25

20

15

10

5

0Control rhGH HP349

100 μg/kg 500 mg/kgs.c. p.o.

Body

weight

gain

(g)

Figure 1 Effect on weight gain over 5 daysControl: DW group. rhGH: recombinant humangrowth hormone (100 μg/kg, s.c.) group. A. villosum:Amomum villosum (500 mg/kg, p.o.) group. Eachvalue is the mean±SD of 10 rats. Statistical significancewas determined using a t test: no significant differencescompared with the control.

Group

Control

rhGH

A. villosum

Body weight (g)

1st day

79±3

80±4

80±3

2nd day

86±3

86±5

86±4

3rd day

93±3

93±6

92±4

4th day

99±4

98±5

98±4

5th day

105±4

106±5

103±4

Table 1 The body weight changes in adolescent female rats

Notes: Control: distilled water group. rhGH: recombinant human growth hormone (100 μg/kg, s.c.) group. A. villosum: Amomumvillosum (500 mg/kg, p.o.) group. Each value is the mean±SD of 10 rats. Statistical significance was determined using a t test: nosignificant differences compared with the control.

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Lee SH et al. Amomum villosum induces longitudinal bone growth in adolescent female rats

immunohistochemistry in the growth plate wasperformed. BMP-2 was highly expressed in theproliferative and hypertrophic zones in the rhGHgroup but was minimally expressed in the resting zones(Figure 7). IGF-1 expression was relatively higher inthe proliferative and hypertrophic zones than in theresting zones (Figure 8). BMP-2 and IGF-1 expressionof the A. villosum group was lower than thoseexpressions in the rhGH group, but higher than thoseexpressions in the control group.

DISCUSSIONAmomum villosum increases longitudinal bone growth

in adolescent female rats. A. villosum significantlyincreased the tibial growth rate by 24.07% comparedto normal rats (P<0.001). This rate was more than the17.60% of the rhGH (P<0.01), which has been knownto enhance tibial growth in peripubertal female rats.11

In addition, the daily body weight changes over 5 dayswere not significantly different among the groups. A.villosum is thought to be effective for bone growth andis not associated with extra weight gain.The thickness of the growth plate is direct evidence oflongitudinal bone growth.12 In the A. villosum group,the thickness of the growth plate was significantlyincreased as compared with the controls (P<0.001). A.

500

400

300

200

100

0

Long

itudi

nalb

one

grow

thra

te(μm

/day

)

Control rhGH HP348100 μg/kg 500 mg/kg

s.c. p.o.

b c

Figure 3 Effect on longitudinal bone growthControl: DW group. rhGH: recombinant human growthhormone (100 μg/kg, s.c.) group. A. villosum: Amomumvillosum (500 mg/kg, p.o.) group. Each value is the mean±SD of 10 rats. Statistical significance was determined using ttest: bP<0.01, cP<0.001, compared with the control.

800

600

400

200

0Control rhGH A. villosum

100 μg/kg 500 mg/kgs.c. p.o.

Gro

wth

plat

eth

ickn

ess

(μm)

bb

Figure 4 Effect on growth plate thicknessControl: DW group. rhGH: recombinant human growthhormone (100 μg/kg, s.c.) group. A. villosum: Amomumvillosum (500 mg/kg, p.o.) group. Each value is the mean±SD of 10 rats. Statistical significance was determined using at test: bP<0.001, compared with the control.

Figure 5 Representative images of 5-bromo-2'-deoxyuridine (BrdU)-labeled chondrocytes in the growth plates of the proximal tibiaControl: DW group. rhGH: recombinant human growth hormone (100 μg/kg, s.c.) group. A. villosum: Amomum villosum (500 mg/kg, p.o.) group. Green, BrdU-labeled chondrocyte. Scale bar = 100 μm.

Figure 2: Fluorescent photomicrographs of longitudinal sections at the proximal tibia in the proximal tibia growth plateControl: DW group. rhGH: recombinant human growth hormone (100 μg/kg, s.c.) group. A. villosum: Amomum villosum (500 mg/kg, p.o.) group. The fluorescent line corresponds to the injection of tetracycline hydrochloride (20 mg/kg), which binds withcalcium and can be detected by ultraviolet illumination. The arrow between the fluorescent line formed by tetracycline and theepiphyseal end line of the growth plate indicates the bone growth during the 24-hour period. Scale bar=200 μm.

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Lee SH et al. Amomum villosum induces longitudinal bone growth in adolescent female rats

villosum increased the growth plate thickness,demonstrating the growth potential.13

BrdU is incorporated into DNA during the S-phaseand can be detected by immunohistochemistry.4 Ahigher number of BrdU-labeled cells, the greater thenumber of proliferating chondrocytes. The number ofBrdU-positive cells in the rhGH and A. villosumgroups was 1.31-fold (P<0.001) and 1.15-fold (P<0.05) greater than that of the control group. This resultsuggests that A. villosum induces cell proliferation inchondrocytes.The growth plate can be divided into threehistologically distinct layers—the resting zone, theproliferative zone, and the hypertrophic zone. Thetopmost layer, the resting zone, contains chondrocytesthat are scattered in a cartilage matrix bed and that

divide infrequently in postnatal life.14 The resting zonechondrocytes serve as stem-like cells for the growthplate, with the potential to generate clones of rapidlyproliferating chondrocytes, which are located in theproliferative zone in the second layer of the growthplate.15 In the proliferative zone, these clones ofchondrocytes are arranged in columns parallel to thelong axis of the bone. Over time, as longitudinalgrowth proceeds, proliferative cells close to thehypertrophic zone undergo terminal differentiation.During this process, they stop proliferating andphysically enlarge to become hypertrophicchondrocytes, composing the third, hypertrophic layerof the growth plate. Simultaneously, the bottom of thehypertrophic zone is invaded by blood vessels,osteoclasts, and differentiating osteoblasts, whichremodel the newly formed cartilage into bone tissue.16

BMP-2 and IGF-1 were highly expressed in theproliferative and hypertrophic zones after A. villosumtreatment. BMP is a member of the transforminggrowth factor (TGF)-β family, acting as both a growthand differentiation factor.17 Of the various forms ofBMP, BMP-2 plays an important role in thedevelopment of the epiphyseal growth plate.18WhileTGF-β is unable to initiate the entire osteoinductioncascade by itself, BMP-2 uniquely exhibits an ectopicbone formation property.19 IGF-1 is a growth factorstructurally related to insulin that induces subsequentcellular activities, particularly those related to bonegrowth. Involved in the expression mechanism ofgrowth hormone, IGF-1 regulates cell differentiation,proliferation, and maturation in most parts of the bodyby autocrine and paracrine activities. The mechanismof IGF-1 action in promoting structural growthinvolves 'insulin-like' anabolic effects, supporting the

200

150

100

50

0 Control rhGH A. villosum100 μg/kg 500 mg/kg

s.c. p.o.

BrdU

-pos

itive

cells

(N/m

m2 )

ca

Figure 6 Effect on BrdU-positive cellsControl: DW group. rhGH: recombinant human growthhormone (100 μg/kg, s.c.) group. A. villosum: Amomumvillosum (500 mg/kg, p.o.) group. Each value is the mean±SD of 10 rats. Statistical significance was determined using ttest: aP<0.05, cP<0.001, compared with the control.

Control rhGH A. villosum

RZ

PZ

HZ

Figure 7 Immunohistochemical localization of bonemorphogenetic protein-2 in the growth plateControl: DW group. rhGH: recombinant human growthhormone (100 μg/kg, s.c.) group. A. villosum: Amomumvillosum (500 mg/kg, p.o.) group. RZ: resting zone, PZ:proliferative zone, HZ: hypertrophic zone. Scale bar=100 μm.

Control rhGH A. villosum

RZ

PZ

HZ

Figure 8 Immunohistochemical localization of insulin-likegrowth factor-1 in the growth plateControl: DW group. rhGH: recombinant human growthhormone (100 μg/kg, s.c.) group. A. villosum: Amomumvillosum (500 mg/kg, p.o.) group. RZ: resting zone, PZ:proliferative zone, HZ: hypertrophic zone. Scale bar=100 μm.

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Lee SH et al. Amomum villosum induces longitudinal bone growth in adolescent female rats

extraordinary biosynthetic activity, somatic growth,and matrix production that characterize hypertrophicchondrocytes.20 The expressions of BMP-2 and IGF-1directly represent the proliferative activity of cartilage.Tibial growth rate of A. villosum was greater than thatof rhGH, but the thickness of the growth plate, thenumber of BrdU-positive cells, and BMP-2 and IGF-1expressions of A. villosum did not exceed those ofrhGH. Thus, A. villosum cannot be an alternative toGH treatment for short stature. However, long-termGH treatment is expensive, and the final height gainsare small as compared with those of non-GH-deficientchildren.21 GH appears to be relatively safe, but itslong-term effects are not yet fully known, and someadverse effects have been reported, including edemaandpseudotumor cerebri, gynecomastia, hyperinsulinismor elevated blood glucose level, and a potential increasein nevi.22 A. villosum might be an alternative treatmentwhich meets the cost-benefit qualification whileavoiding the potential risks of GH therapy.A. villosum stimulated chondrocyte proliferation andchondrocyte hypertrophy in the growth plate throughthe generation of BMP-2 and IGF-1, as well as directlyincreasing the longitudinal tibial length. Therefore, A.villosum could be helpful for increasing bone growthin children with growth retardation. Further studies areneeded to identify the active components of A.villosum that can provide confirmation of growthpromotion, and to better understand the mechanismsin this process.

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